14
15
672
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/9dcaded8d2bbcd39c95c18efd31ca478.pdf
9f28a744696fc3e19a2e7c3e0c04a12a
PDF Text
Text
item D Number
0576
Author
Young Alvin L
°
Not Scanned
Corporate Author
Report/Article Title
Tn
'P ReP°rt - s - °- TA-1578 (21 sep 79)
Journal/Book Title
Year
™™
Month/Day
October 17
Gotor
D
Number of Images
°
DeSCriptOU NOtOS
Attachments 2 (VACO Agenda) and 5 (proposed questions
for position papers) are missing. Attachments 1, 3, and 4
were not scanned with this item, but were scanned for item
5756.
Thursday, March 28, 2002
Page 5760 of 5780
�DEPARTMENT OF THE AIR FORCE
USAF SCHOOL OF AEROSPACE MEDICINE (AFSC)
BROOKS AIR FORCE BASE, TEXAS 78235
REPLY TO
ATTNOF:
, H
.
EKS
'
1 «
SUBJECT: Trip Report - S.O. TA-1578 (21 Sep 79).
EK
CEA
CE
CD
CC
TS
IN TURN
'!, Places visited
a. Veterans Administration Central Office (VACO),
Washington DC, 24 Sep 79
b. HQ USAF/SGES and HQ USAF/CVAH (Air Force History),
Boiling AFB DC, 25 Sep 79
c. The Environmental Sciences Laboratory, Mount Sinai School
of Medicine, New York, NY
d. VA Conference on the Health-Related Effects of Exposure
to Agent Orange, 27-28 Sep 79, Hospitality House,
Arlington, VA
2. Personnel contacted
a. VACO
Participants in VA Advisory Committee on Health-Related
Effects of Herbicides (Attachment 1)
b. HQ USAF
Maj Phillip Brown, HQ USAF/SGES
Capt D. Rightmyer, HQ USAF/CVAH
c. Mount Sinai School of Medicine
Irving J. Selikoff, M.D., Director, The Environmental
Sciences Laboratory
Henry A. Anderson, M.D., Assistant Professor of Community
Medicine
Marion Moses, M.D., Resident in the Department of Community
Medicine
�d. VA Conference
Speakers listed on Program Agenda (Attachment 2)
Lyndon Lee, M.D., VACO, Washington DC
Lawrence B. Hobson, VACO, Washington DC
Paul DeGovin, M.D., VACO, Washington DC
3. Topics Discussed
a. VACO
The purpose for visiting the VACO was to attend and participate
in the second meeting of the VA Advisory Committee on Health-Related
Effects of Herbicides. The objectives and scope of the Committee
is described in the Committee's Charter (Atch 3). The agenda for
the Committee meeting is provided in Attachment 4. Items of .special
interest included:
(1) Reports of the VA Steering Committee
(a) As of 31 Aug 79, 3,100 Vietnam Veterans alleging
exposure to Herbicide Orange have been examined in VA Hospitals.
Approximately 2,900 of these have been found to be clinically
asymptomatic.
(b) As of 31 Aug 79, 625 claims for compensation have
been filed by Vietnam Veterans alleging exposure to Herbicide Orange.
Approximately 435 have claimed specific disabilities, while 190 have
claimed only herbicide exposure. All cases claiming only herbicide
exposure were denied, while 416 of the 435 claiming a specific
disability were denied. The remaining 19 claims were allowed: one (1)
allowed for chloracne and 18 allowed for disabilities originating
during or 1 year after service, but unrelated to VA criteria for
herbicide exposure.
(c) Fat biopsy has been performed on 34 volunteers.
Twenty-one (21) of these individuals were Vietnam Veterans alleging
exposure to Herbicide Orange; 3 were active duty Air Force personnel
associated with the Herbicide Orange disposal project (PROJECT
PACER HO); and 10 were controls (veterans not assigned in Vietnam).
Twenty-two of the fat samples have been analyzed for TCDD at the
parts per trillion level by the Department of Chemistry, University
of Nebraska, Lincoln NE. The remaining samples are to be analyzed
by 12 Oct 79. The VA is planning to have an "in-house" committee
review the analytical results on 21 Oct 79 in Chicago.
�(d) The VA has established a tissue registry with the
Armed Forces Institute of Pathology (AFIP), Washington DC. Any
pathological tissues removed from Vietnam Veterans alleging exposure
to Herbicide Orange are sent to AFIP for examination, diagnosis and
retention. A report from AFIP is then filed with the VA. AFIP will
monitor for any unusual or unique tumors, or tumors occurring at an
unusually young age in the Veterans or for an increase in tumors
in a specific military unit that served in Vietnam. As of Jul 79,
13 cases have been examined, including 7 tissues removed during
surgical procedures, 5 during autopsy and 1 seminal fluid.
(e) VACO is computer coding all medical data from the
examination of all Vietnam Veterans alleging exposure to Herbicide
Orange. When the coding is completed (^ 1 month), a printout of
symptom frequencies, diagnoses, clinical results, etc. will be
available.
(2) Discussion of Position Papers
The Advisory Committee was asked on 11 Jun 79 to answer
13 questions prepared by the VA Steering Committee, Accordingly, at
that, time, Dr. Haber asked selected Advisory Committee members to
prepare a draft position paper on each of the questions. Attachment
5 is a list of the proposed questions and the individual responsible
for preparing a draft. On 24 Sep 79, draft position papers were
discussed on 10 of the 13 questions. No drafts were available
on questions 3, 10, and 13.
(3) Presentations by L.t Col Wolfe and Major Young
At the request of HQ USAF/SG, two presentations were
given to the Advisory Committee to assist them to better understand the way in which herbicides were used in Vietnam, what data
are available in developing exposure criteria, and what diagnostic
approaches might be used to evaluate herbicide toxicity. Vu-graph
outlines for both presentations are provided as Attachments 6
(Major Young) and 7 (Lt Col Wolfe).
b. HQ USAF/SGES, HQ USAF/CVAH
On 25 Sep 79, a visit was made to HQ USAF, Boiling AFB.
Discussions on the RANCH HAND II protocol were held with Major Brown,
The visit to the Air Force History Office was made in order to examine
RANCH HAND historical records maintained by Capt Rightmyer. Eleven
names, dates of tours and number of herticide missions were obtained
�on RANCH HAND personnel. Only two of twelve boxes of RANCH HAND
historical data were examined.
t
c. Environmental Sciences Laboratory, Mount Sinai
On 26 Sep, the Environmental Sciences Laboratory was visited.
Brief discussions were held with Drs Selikoff, Anderson, and Moses
concerning the RANCH HAND protocol. Dr. Selikoff emphasized (a) that
criteria for the mortality study should be established prior to
initiation of study; (b) matched group analysis could be employed
to prevent loss of data; (c) when a subject is contacted, the name
of a referral should be obtained so that if the subject were to move
during course of study, the referral would have the change of
address; (d) more detailed information should be obtained on
reproduction, fertility and miscarriages; and (e) matching of controls with the RANCH HAND personnel should also be done on a geographic
basis. Dr. Moses briefly discussed the status of her study on the
Monsanto Workers at Nitro West Virginia. No definitive conclusions
are available at this time.
d. VA Conference, Arlington VA
This Conference was held to inform physicians from each of
the 160 VA Hospitals of the VA Central Office program for studying
health-related effects of herbicide exposure in Vietnam Veterans.
Mr. Max Cleland, VA Administrator, set the tone of the conference by
emphasizing VA's concern for Veterans alleging exposure to Herbicide
Orange. He stated that "at the very least, the Agent Orange Syndrome
is characterized by FEAR, and it should be every VA physician's
responsibility to show concern and understanding to ey_ery_ Vietnam
Veteran that contacts or visits a VA hospital claiming exposure to
Agent Orange". The agenda for the conference is provided in
Attachment ?.. Dr. Wolfe and I presented the same presentations as
were presented 24 Sep at the VA Advisory Committee meeting. We
received enthusiastic responses from not only the conference attendees
but also from the VA Central office staff.
4. Items of interest to procurement and USAFSAM programming: None
5. Information of interest to other USAFSAM divisions or branches: BR9
NG.
6. This TOY was performed in support of EPI ranch project.
�7. This report contains information worthy of filing in TSK,
ALVIN -LVYCfUNG, Hlaj, USAI
Consultant, Environmental Sciences
7 Atch
1. Advisory Committee Membership
2. VACO Agenda
3. Charter of VA Advisory
Committee
4. Committee Meeting Agenda
5. Proposed Questions for
Position Papers
6. Vu-Graph Outlines
(Major Young)
7. Vu-Graph Outlines
(Dr. Wolfe)
��FACTORS TO BE CONSIDERED
TIME BETWEEN THE EXPOSURE AND TH!
DURATION OF THE EXPOSURE
LAG TIME/INCUBATION PERIOD
CONFOUNDING EXPOSURES
�PROBLEMS UNIQUE TO
PHENOXY
MIXTURE OF TWO ACTfVE COMPOUNDS
AND ONE CONTAMINANT
LACK OF CLEARLY DEFINED AND OBJECTIVE
END POINTS
WIDE RANGE OF PRESUMED EFFECTS
CONFUSION- BETWEEN ACUTE, SUBACUTE
AND CHRONIC EFFECTS
DIFFICULTIES IN VERIFICATION OF EXPOSURE
�SUMMARY CHARACTERISTICS OF
AS OF 30
1979
8F
381
SEX: 1-08% MALE [84%
MEAN AGE; 34
MEAN
OF
PER
2.3
OF
US
US
• US AR
US NAVY
.
66,4%
17,4%
11.2%
5.0%
�PERCENT OF
CLAIMS BY
.
CATEGORY
23
15
.
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13
1
6
EAR/NOSE.THROAT
'
5
N - 417CLAI
190
ALLEGED "EXPOSURE"
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�COMPONENTS OF SELECTED HUMAN SYMPTOM/SIGNS F31 LOWING
f
EXPOSURE TO PHENOXY HERBICIDES AND/OR TCDD
NEURO-PSYCHiATmC ABNORMALITIES
PERIPHERAL
ASTHENIA
ANXIETY
DEPRESSION
FATIGUE
HYPOREFLEXIA
WEAKNESS
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LOSS OF
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MYALGIA
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END POINTS
DE RANGE OF PRESUMED EFFECTS
CONFUSOST BETWEEN ACUTE, SUBACUTE
AND CHRONIC EFFECTS
DIFFICULTIES IN VERIFICATION OF EXPOSURE
J
�UMFTED EXAMINATION
OF THE PATIENT
COMPREHENSIVE STUDY
OF THE PATIENT AND
FAMILY
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EXAMINATION
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NEURO-PSYCHIATRIC EVALUATION
LIVER FUNCTION EVALUATION
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GENERAL PHYSICAL EXAMINATION
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NEURO-PSYCHiATRIC EVALUATION
LIVER FUNCTION EVALUATION
REPRODUCTFVE EVALUATION
IMMUNOLOGiC EVALUATION
ENDOCRINE EVALUATION
�PHENOXY HERBICIDES
RESULT IN
VERSE HEALTH EFFECTS?
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�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
204
Folder
The folder containing the original item.
5760
Series
The series number of the original item.
Series VIII Subseries III
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young Alvin L.
Date
A point or period of time associated with an event in the lifecycle of the resource
October 17 1979
Title
A name given to the resource
Trip Report - S. O. TA-1578 (21 Sep 79)
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/7f082c6bcfbaa96fb14ce2ddbd3b7b0a.pdf
d424c5c3a2dfcf338c9d6248a930bf72
PDF Text
Text
itemDNumbBr
°5274
Author
D
Young, Alvin L.
Not Scanned
Corporate Author
RODOrt/ArtiClB TitlB Handwritten notes about a call received from Mr.
Schroeter, April 14, 1979
Journal/Book Title
Year
000
°
Month/Day
Color
DOSCriptOD NOtOS
n
Young is referring to hearings on EPA's Emergency
Suspension Order for 2,4,5-T and Silvex.
Tuesday, March 05, 2002
Page 5274 of 5363
�___
.xYer^
I .
L Vvdt
\%XA^V^_C
V-J
M^UU-
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
180
Folder
The folder containing the original item.
5274
Series
The series number of the original item.
Series VIII Subseries I
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Handwritten notes about a call received from Mr. Schroeter, April 14, 1979
ao_seriesVIII
-
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
184
Folder
The folder containing the original item.
5343
Series
The series number of the original item.
Series VIII Subseries I
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Letter from Alvin L. Young to William J. Carter, 1980
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/6120d57649c8ba8db8b570075d209158.pdf
651f91607242ddd9ef5ae98075ea89f1
PDF Text
Text
Item D Number
05«2
Author
D MotScannBfl
Young, Alvin L
United States District Court, Eastern District of New Yor
RODOrt/ArtlOlO Tltlfl Declaration of Alvin L. Young, In re: "Agent Orange"
Product Liability Litigation, All Cases, August 30,1984
Journal/Book Title
Year
000
°
Month/Day
Color
Number of bnagos
D
°
Descrlpton Notes
Friday, March 15, 2002
Page 5482 of 5571
�UNITED STATES DISTRICT COURT
EASTERN DISTRICT OF NEW YORK
I-» "RE T|fAGENT ORANGE
)
PRODUCT LIABILITY LITIGATION
)
ALL CASES
)
fc
DECLARATION OF ALVIN L. YOUNG
I, Alvin L. Young, being duly sworn, depose and state that
based on the records, files and documents to which I have access,
control and supervision, the following is true to the best of my
knowledge, information and belief:
(1)
I am a Senior Staff Scientist (AFSC 2616) with the United
States Air Force, currently detailed as Senior Policy Analyst,
Office of Science and Technology Policy, Executive Office
of the President, Washington, D.C.
Since 1968, I have
served as an expert for the government in areas of science
related to the military herbicide Agent Orange.
Specific
areas of expertise include formulation, equipment design,
application, military use, dioxin contamination, environmental
fate, toxicology and human risks to exposure.
(2)
Agent Orange, as formulated and procurred for the Department
of Defense, consisted of approximately a 50:50 mixture of
the normal-butyl esters of 2,4-dichlorophenoxyacetic
and 2,4,5-trichlorophenoxyacetic acid.
acid
The product was
formulated to contain approximately 8.6 pounds active
ingredient (acid equivalents of 2,4-D and 2,4,5-T) per
gallon of liquid.
Thus the product contained roughly
4 pounds of each herbicide in the acid
form.
�(3)
The herbicides 2,4-D and 2,4,5-T have been commercially
available and widely used since the late 1940's.
Numerous
formulations including the iso-butyl and normal-butyl
formulations were used commercially
ester
in the United States
prior to, during, and after the use of Agent Orange in
Vietnam.
In the 1960 Farm Chemicals Handbook, formulations
of the normal-butyl ester of 2,4-D containing 4 and 6 pounds
per gallon active ingredient were recommended
control
in wheat and other field grains.
for weed
In the same
handbook, formulations of the normal-butyl esters of both
2,4-D
and 2,4,5-T (as a mixture) were recommended for brush
control on rangelands and in reforestation.
Typically these
latter formulations contained the active ingredient at 4
and 6 pounds per gallon.
As late as 1980 normal-butyl
esters of 2,4-D were commercially available (DOW ESTERON
76BE) containing 6 pounds (acid equivalents) per gallon.
4.
Analyses in the early 1970's of archived samples of commercial
formulations of 2,4,5-T and archived samples of Agent Orange
showed similar levels of dioxin (2,3,7,8-tetrachlorodibenzo-pdixoin) contamination (See Young, A.L. et al. 1978.
The
Toxicology, Environmental Pate, and Human Risk of Herbicide
Orange and Its Associated Dioxin, Air Force Technical
Report OEHL - 78-92).
I declare under penalty of perjury that the ^above ijs true and
correct.
�CD
». ( / >
ACTIVE INGREDIENT:
2,4-Dichlorophenoxyacetic Acid, Butyl Esters
INERT INGREDIENTS:
2,4-D Acid Equivalent 63.2%-6 pounds per gallon
E.P.A. Registration No. 464-279 ^
SB 8
tug
USE DIRECTIONS
Use ESTERON 76 BE herbicide to control bitterweed, croton, dandelion, docks, galinsoga,
henbit, kochia, lambsquarters, morshelder, mustards, peppergrass, pigweed, plantains,
ragweed, shepherdspurse, thistles, vetch, wild radish, and many other broadleaf weeds,
without injury to most established grasses; also for control of 2,4-0 susceptible woody
plants such as coastal sage, sandsage, elderberry, hazel, locust, poison oak, sumac and
willow. Use in small grains, corn, sorghum, grass seed crops, pastures, rangeland and in
non-crop areas.
Apply ESTERON 76 BE as a water or oil spray during warm weather when weeds or brush
are actively growing. Application under drought conditions often will give poor results.
Use low spray pressure to minimize spray drift. On cropland and along roadsides, do not
exceed 20 psi pressure. Apply enough spray volume to provide uniform coverage of weeds
and brush, usually 5 to 20 gallons per acre by ground equipment and 3 to 5 gallons by aircraft. Higher gallonage may be used if desired to improve spray coverage and to reduce the
hazard from spray drift.
Generally, the lower dosages recommended on this label will be satisfactory for young,
succulent growth of sensitive weed species. For less sensitive species and under conditions
where control is more difficult, the higher dosages will be needed. For crop uses, do not mix
with oil or other adjuvants unless specifically recommended on this label. Deep-rooted
perennial weeds such as Canada thistle and field bindweed and many woody plants usually
require repeated applications for maximum control. Do not apply ESTERON 76 BE where
spray drift may contact nearby susceptible crape or other desirable plants or may contaminate water for irrigation or domestic use. Do not apply in the vicinity of 2,4-D sensitive crops or ornamental plants since vapors from this product may cause injury to such
crops or plants. Read and follow all Use Precautions given on this label.
NOTE: If there are uncertainties concerning special local use situations or specific crop
variety tolerances to 2,4-D, consult local Extension Service or University Specialists for advice.
TO PREPARE THE SPRAY: (1) Fill the spray tank about half full with water, then add the required amount of ESTERON 76 BE, with agitation, and finally the rest of the water. NOTE:
ESTERON 76 BE in water forms an emulsion which tends to separate unless the mixture is
kept agitated. (2) If oil is added, first mix the ESTERON 76 BE and the oil and then add this
mixture to the water with agitation. However, with adequate agitation, the oil can be added
after the ESTERON 76 BE is mixed in the water if strong agitation is provided. (3) If straight
oil is used, a solution is formed and separation does not occur. Do not allow any water to get
into the oil-herbicide mixture to avoid formation of an invert emulsion.
WEED CONTROL IN SMALL GRAINS NOT UNDERSEEDED WITH A LEGUME: NOTE: Water is
recommended to make up the spray. If oil is used, there is a greater risk of crop injury and
of spray drift. Do not permit dairy animals or meat animals being finished for slaughter to
forage or graze treated grain fields within 2 weeks after treatment.
Spring Wheat and Barley: Apply Va to % pint per acre by air or ground equipment. A '/2
pint per acre rate of ESTERON 76 BE is an average dosage, effective on many weeds. Spray
when grain is in full tiller stage (usually 4 to 8 inches tall) but before the boot stage and
when weeds are small. Do not apply before the tiller stage nor from early boot to the dough
stage. Higher rates (up to IVs pints per acre) may be required to control certain weeds but
crop injury may result.
Winter Wheat and Rye: Apply >/3 to Vz pint per acre in the spring at the full tiller stage
but before the early boot stage. See more complete use directions under Spring Wheat
and Barley.
Spring Seeded Oats: Apply '/3 pint per acre at the full tiller stage but before the early
boot stage. Oats are less tolerant to 2,4-D than wheat or barley and are more likely to suffer
some injury, especially if higher rates (Vi to 2/3 piAt) are used to control difficult weeds.
Preharvest Treatment: Apply 3/3 to l'/3 pints per acre when grains are in the hard dough
stage to control large weeds that may interfere with harvest. Best results will be obtained
when soil moisture is sufficient to cause succulent weed growth. NOTE: Do not feed treated
straw to livestock.
WEED CONTROL IN CORN: Use one of the following three programs: Preemergence: Apply
1'/3 to 22/3 pints per acre to soil anytime after planting but before corn emerges. Do not
use on light sandy soil. Emergence: Apply 2/3 pint per acre just as corn plants are breaking
ground. Postemergence: After emergence of corn, use '/3 pint per acre. Application of '/2
to 2/3 pint per acre may be needed for maximum control of some weeds but such rates are
more likely to injure the corn. If corn is over 8 inches tall, use drop nozzles to keep the spray
off the corn foliage as much as possible. Do not apply from the tasseling to dough stage. Do
not use with oil, atrazine or adjuvants. Crop injury is more likely to occur if corn is growing
rapidly under high temperature and high soil moisture conditions. To reduce breakage of
stalks from temporary brittleness caused by 2,4-D, delay cultivation for 8 to 10 days after
treatment. NOTE: Hybrids vary in response to 2,4-D and some are easily injured. Spray only
varieties known to be tolerant to 2,4-D. Contact seed company and Extension Service Weed
Specialists for this information.
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Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
194
Folder
The folder containing the original item.
5482
Series
The series number of the original item.
Series VIII Subseries I
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Description
An account of the resource
<strong>Corporate Author: </strong>United States District Court, Eastern District of New York
Title
A name given to the resource
Declaration of Alvin L. Young, In re: "Agent Orange" Product Liability Litigation, All Cases, August 30, 1984
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/5fd6587209638a281cdc8803d14645b4.pdf
2b553900c0bc0093d5c1f652854f1468
PDF Text
Text
ItemDNumber
05483
Author
Young, Alvin L.
Corporate Author
RBpOPt/ArtlClB TItiB Typescript: Hazardous Wastes: Problems,
Perspectives and Policy
Journal/Book Tttla
Yoar
000
°
Month/Day
Color
D
Descrlpton Notes
Friday, March 15, 2002
Page 5483 of 5571
�HAZARDOUS WASTES;
PROBLEMS, PERSPECTIVES AND POLICY
ALVIN L- YOUNG, PH.D.
SENIOR POLICY ANALYST FOR LIFE SCIENCES
OFFICE OF SCIENCE AND TECHNOLOGY POLICY
EXECUTIVE OFFICE OF THE PRESIDENT
WASHINGTON, D.C.
PRESENTATION
TO:
HAZARDOUS WASTE LITIGATION SYMPOSIUM
GARDEN CITY, NEW YORK
SEPTEMBER 24, 1984
THE PAST TWO DECADES HAVE USHERED IN A SHIFT OF ALMOST
REVOLUTIONARY PROPORTIONS
IN THE STUDY OF HEALTH AND DISEASE.
THE PREOCCUPATION WITH INFECTIOUS DISEASE HAS BEEN REPLACED WITH
A HEAVY COMMITMENT TO THE STUDY OF CHRONIC, LIFE THREATENING
ILLNESSES.
INCREASINGLY, MORE INVESTIGATORS HAVE
RECOGNIZED
THAT BEHAVIORS THAT PLACE AN INDIVIDUAL AT R I S K FOR DEVELOPING
PHYSICAL
I L L N E S S SELDOM
ACTIVITIES.
ALTHOUGH
OCCUR
IN ISOLATION FROM OTHER P A T H O G E N I C
THE PUBLIC ACKNOWLEDGES THAT LIFE STYLE
AND DISEASE ARE RELATED
(E'G« SMOKING MAY BE DETRIMENTAL TO
YOUR HEALTH), THE FEAR OF M A L I G N INFLUENCES IN OUR E N V I R O N M E N T
IS SO WIDESPREAD TODAY THAT THE GENERAL PUBLIC BELIEVES THAT
IT IS THOSE FACTORS THEY CANNOT CONTROL
THEIR EARLY DEMISE*
THAT WILL B R I N G ABOUT
THE PRESENCE OF ANY TRACE OF DIOXIN,
PESTICIDES, R A D I A T I O N (EVEN BACKGROUND RADIATION), OR INDUSTRIAL
WASTE PRODUCT IS TAKEN AS AN OMEN OF DEATHSTATING
PUBLIC
THIS
PERHAPS, I'M OVER-
ISSUE - HOWEVER AS A SCIENTIST NOW CONCERNED WITH
POLICY ITS BECOMING I N C R E A S I N G L Y A P P A R E N T TO ME THAT WE
ARE F A C I N G A C R I S I S AS TO W H E R E WE EXPEND R E S O U R C E S FOR GENERATING S C I E N T I F I C DATA
HEALTH
RISKS-
USEFUL IN THE M A N A G E M E N T OF E N V I R O N M E N T A L AND
�As AN E N V I R O N M E N T A L SCIENTIST, I AM ACUTELY
PUBLIC'S CONCERN
A W A R E OF THE
OVER THE POSSIBLE DEVELOPMENT OF CANCER
BECAUSE
OF COMMUNITY EXPOSURE TO C A R C I N O G E N S IN LOW DOSES FROM A W I D E
V A R I E T Y OF SOURCES SUCH AS HAZARDOUS
WASTE DEPOSITS, PESTICIDE
RESIDUES, AIR POLLUTANTS AND FOOD ADDITIVESREPORTS
TWO RECENT
P R E P A R E D BY THE N A T I O N A L RESEARCH COUNCIL
(NRC) OF THE
N A T I O N A L ACADEMY OF SCIENCES ARE NOTEWORTHY-
THE F I R S T REPORT "GROUNDWATER C O N T A M I N A T I O N "
EMPHASIZED
THAT T H E W I D E S P R E A D U S E O F C H E M I C A L PRODUCTS, C O U P L E D W I T H T H E
DISPOSAL OF LARGE VOLUMES OF WASTE M A T E R I A L S
POSES THE
FOR WIDELY DISTRIBUTED GROUNDWATER CONTAMINATIONPOTENTIAL SOURCES OF C O N T A M I N A T I O N OF G R O U N D W A T E R
OF SOLID AND L I Q U I D WASTESPROTECTION
AGENCY
I N 1980, T H E U - S -
(EPA) ESTIMATED
POTENTIAL
THE LARGEST
IS THE DISPOSAL
ENVIRONMENTAL
THAT THERE W E R E 200,000 LANDFILLS
AND DUMPS R E C E I V I N G 150 M I L L I O N TONS PER Y E A R OF M U N I C I P A L SOLID
W A S T E S A N D 240 M I L L I O N S T O N S P E R Y E A R O F I N D U S T R I A L S O L I D W A S T E S I N A D D I T I O N T O L A N D F I L L S , 176,000 S U R F A C E
IMPOUNDMENTS RECEIVE
10 T R I L L I O N GALLONS PER YEAR OF L I Q U I D I N D U S T R I A L WASTESNOT ALL WASTES ARE HAZARDOUS, THE EPA HAS
ALTHOUGH
I D E N T I F I E D AN ESTIMATED
50,000 S I T E S T H A T H A V E B E E N U S E D , A T S O M E T I M E , F O R T H E D I S P O S A L
OF H A Z A R D O U S W A S T E S -
OF T H E S E , 1200 TO 2000 ARE T H O U G H T TO P O S E
THREATS TO THE E N V I R O N M E N T W H E R E WASTE-DISPOSAL
SOME OF THESE LATTER SITES ARE A R E A S
P R A C T I C E S H A V E RESULTED
C O N T A M I N A T I O N OF G R O U N D W A T E R .
IN I R R E V E R S I B L E
THE E X A M P L E S OF LOVE CANAL, NEW YORK
AND THE ROCKY M O U N T A I N A R S E N A L , COLORADO,
I N D I C A T E THE M A G N I T U D E
OF THE R E S U L T I N G G R O U N D W A T E R C O N T A M I N A T I O N PROBLEMS WITH P O S S I B L E
HEALTH HAZARDS
AND OTHER D E L E T E R I O U S E F F E C T S -
THE R E P O R T NOTED
�3
THAT THE COST ESTIMATED FOR D E C O N T A M I N A T I O N OF JUST THE ROCKY
M O U N T A I N A R S E N A L R A N G E FROM $800 M I L L I O N TO $1 BILLION.
THE
QUESTION OF COST/BENEFIT TRADEOFFS TO SOCIETY IN THESE CASES
NEEDS TO BE E X A M I N E D C A R E F U L L Y j SOME SITES MAY PROVE TO BE SO
E X P E N S I V E TO RESTORE THAT THEY MAY HAVE TO BE DESIGNATED AS
PERMANTLY CONTAMINATED-
THE SECOND REPORT RECENTLY RELEASD BY THE NRC WAS "TOX1CITY
TESTING - STRATEGIES TO DETERMINE NEEDS AND PRIORITIES."
N A T I O N A L TOXICOLOGY P R O G R A M ASKED
(1)
NRC:
To C H A R A C T E R I Z E THE TOXIC ITY'TESTING NEEDS
TO W H I C H THERE
FEDERAL A G E N C I E S R E S P O N S I B L E FOR THE
To DEVELOP
AND VALIDATE U N I F O R M L Y APPLICABLE AND W I D E - R A N G I N G
To D E F I N E TOXICITY-TESTING NEEDS
E N V I R O N M E N T , IT WAS
NECESSARY
FOR THE
OF THE U N I V E R S E THAT WOULD
THE C O N S T R U C T I O N OF THIS
THAT W O U L D
SEARCH
BE THE
FOR SUBSTANCES
IN THE HUMAN
NRC TO SELECT A M A N A G E A B L E
I N C L U D E MOST OF THE
SUBSTANCES
IN THE U N I T E D STATES-
"SELECT U N I V E R S E " OF SUBSTANCES"A
R E F E R E N C E FOR THE S T U D Y " R E L I ED ON A
FOR LISTS OF SUBSTANCES
POTENTIAL AND COMPUTERIZED
LISTS USED
SUBSTANCES
IMPACT.
TO WHICH H U M A N S ARE L I K E L Y TO BE EXPOSED
CORE
HEALTH
NEEDS-
POTENTIALLY ADVERSE PUBLIC-HEALTH
SUBSET
SO THAT
NEEDED TO ANTICIPATE THE
C R I T E R I A BY W H I C H TO SET P R I O R I T I E S FOR RESEARCH ON
WITH
SUBSTANCES
PROTECTION OF PUBLIC
WILL HAVE THE APPROPRIATE INFORMATION
(2)
FOR
IS K N O W N OR A N T I C I P A T E D H U M A N EXPOSURE,
EXTENT OF TESTING
THE
PRESELECTED
FOR REASONABLY
INCLUDED THE TOXIC SUBSTANCES
FOR HUMAN
EASY
EXPOSURE
ACCESS-
CONTROL
ACT
THE
(TSCA)
�INVENTORY OF 48,523 CHEMICAL SUSTANCES IN COMMERCE; A LIST OF
3,350 PESTICIDES
(ACTIVE
AND INERT I N G R E D I E N T S ) R E G I S T E R E D FOR
USE BY THE EPAj A LIST OF 1,815 PRESCRIPTION
AND NONPRESCRIP'
TION DRUGS A P P R O V E D
BY THE FOOD AND DRUG A D M I N I S T R A T I O N (FDA)
AND E X C I P I E N T S USED
IN DRUG F O R M U L A T I O N S ; AND A LIST OF 3,410
COSMETIC
I N G R E D I E N T S FROM THE COSMETIC, TOILETRY
AND F R A G R A N C E
ASSOCIATION-
THIS SELECT U N I V E R S E DID NOT SYSTEMATICALLY I N C L U D E
ENVIRONMENTAL
D E C O M P O S I T I O N PRODUCTS, M A N U F A C T U R I N G
OR N A T U R A L SUBSTANCES
( E - G - , PLANT
CONTAMINANTS,
POLLENS AND FOODS).
THE SUM
OF THE ABOVE, 65,725 E N T R I E S FROM THE LISTS, WAS T A K E N AS THE
SELECT U N I V E R S E FOR THE PURPOSES
MENT
FOR D U P L I C A T I O N S
OF STUDY-
STATISTICAL
ADJUST-
I N D I C A T E D THAT THE SELECT U N I V E R S E
CONTAINED ABOUT 53,500 DISTINCT ENTITIES-
OBVIOUSLY,
IT WAS NOT POSSIBLE
TO E X A M I N E I N F O R M A T I O N ON
ALL 53,500 SUBSTANCES, T H E R E F O R E U S I N G A S T R A T I F I E D R A N D O M
PROCESS, 675 SUBSTANCES WERE SELECTED-
A RANDOM
SUBSAMPLE OF
100 SUBSTANCES WAS F U R T H E R SELECTED THAT R E P R E S E N T E D
C A T E G O R I E S OF S U B S T A N C E S ACCEPTED STANDARDS
IN THIS
W H E N JUDGED
SEVEN
AGAINST CURRENTLY
FOR TOXICITY TESTING, ONLY 8% OF THE TESTS
F I N A L SUBSAMPLE MET THE STANDARDS OF THE R E F E R E N C E
PROTOCOL G U I D E L I N E S AND A N O T H E R 19% OF TESTS P E R F O R M E D W E R E
JUDGED TO BE A D E Q U A T E -
ON THE GREAT M A J O R I T Y OF THE SUBSTANCES,
DATA C O N S I D E R E D TO BE E S S E N T I A L
FOR C O N D U C T I N G A HEALTH-HAZARD
ASSESSMENT W E R E L A C K I N G -
SO WHAT
IS THE
POINT OF THE TWO
WE H A V E G E N E R A T E D V E R Y LITTLE
REPORTS THAT I JUST CITED?
S C I E N C E ABOUT THE C H E M I C A L S IN
�OUR E N V I R O N M E N T AND WE H A V E L I T T L E OR NO IDEA W H A T R I S K S THEY
POSE!
0
THE S I T U A T I O N is COMPLEX:
THERE ARE 4-2 M I L L I O N COMPOUNDS REGISTERED
THAT N U M B E R
TOXIC
IS G R O W I N G BY 10% A Y E A R .
EFFECT
INFORMATION
TODAY, AND
OF T H E S E , S O M E
IS A V A I L A B L E ON 100,000-
IS L I M I T E D I N F O R M A T I O N ON LONG-TERM
TOXIC
EFFECTS
THERE
FOR
ALMOST 4000 COMPOUNDS.
0
THERE ARE 220 SEPARATE
TOXIC WASTES.
A RECENT C O O R D I N A T I N G EFFORT R E Q U I R E D
18 D I F F E R E N T DATABASES.
ON TOXIC
0
I N F O R M A T I O N SYSTEMS C O N C E R N I N G
SUBSTANCES
STATE AND LOCAL
THERE
IS NO C E N T R A L DATA BANK
TO O R G A N I Z E THIS
A G E N C I E S PRESENTLY
INFORMATION-
H A V E NO WAY TO Q U I C K L Y
OBTAIN
INFORMATION AND ASSISTANCE TO RESPOND IN AN OPTIMAL
*
FASHIONTHE T I M E R E Q U I R E D FOR P U B L I C POLICY D E C I S I O N
AND THE TIME R E Q U I R E D FOR G E N E R A T I N G S C I E N T I F I C INFORMATION
ARE R A R E L Y THE SAME.
0
THE FEDERAL EFFORT TO COPE WITH THE PROBLEMS POSED BY
HAZARDOUS
WASTE
HAS, H I S T O R I C A L L Y , BEEN DEVELOPED
ALMOST C R I S I S - B Y - C R I S I S B A S I S R A T H E R THAN
EFFICIENT MANNER.
IN AN
ON AN
ORDERLY,
As A RESULT THERE ARE O V E R 30 S E P A R A T E
STATUTES, A D M I N I S T E R E D BY 10 FEDERAL A G E N C I E S T H R O U G H
AT LEAST
0
30 B U R E A U S , I N S T I T U T E S , AND CENTERS.
COORDINATION,
NIGHTMARE
MUCH
IS ALMOST
LESS MANAGEMENT, OF THIS
BUREAUCRATIC
I M P O S S I B L E AS E V I D E N C E D BY THE 80
I N T E R A G E N C Y COMMITTEES A N D F I V E SEPARATE,
HIGH-LEVEL,
C O O R D I N A T I O N ATTEMPTS M A D E IN THE LAST DECADE.
�0
OF THE ALMOST B I L L I O N DOLLARS W H I C H GOES INTO RESEARCH ON
T O X I C S U B S T A N C E S , C O N D U C T E D BY 12 D I F F E R E N T A G E N C I E S , 55%
GOES TO THE D I S C O V E R Y OF POSSIBLE NEW PROBLEMS R E S U L T I N G
F R O M C H E M I C A L E X P O S U R E , 45% T O T H E D I S C O V E R Y O F T H E M E C H A N I S M
OF HOW THESE C H E M I C A L S ACT AND ALMOST N O T H I N G TO S O L V I N G
ALREADY
0
THERE
IDENTIFIED PROBLEMS-
is LITTLE
OR NO G O V E R N M E N T A L EFFORT DIRECTED
AT THE
DEVELOPMENT OF A C O O P E R A T I V E APPROACH C O M B I N I N G THE
OF
INDUSTRY, ACADEMIA, PUBLIC
TO R E S O L V E THE
INTEREST GROUPS, AND
EXPERTISE
GOVERNMENT
P R O B L E M S POSED BY THE D E L E T E R I O U S EFFECTS OF
TOXIC SUBSTANCES.
0
T H E R E is LITTLE OR NO PREPARATION AT FEDERAL,
STATE OR LOCAL
LEVELS FOR THE RATIONAL TREATMENT OF PERSONS I N V O L U N T A R I L Y
EXPOSED TO TOXIC WASTES, AND THE FOLLOWUP OF THESE PERSONS
IS H A P H A Z A R D AT BEST-
PERHAPS
THE GREATEST
F R U S T R A T I O N TO THE S C I E N T I S T is THAT
THE SCARCE RESOURCES A V A I L A B L E FOR PROBLEM S O L V I N G GO INTO CHEMICALS W H E R E THE R I S K TO THE POPULATION IS M I N I M A L , NOT BECAUSE THESE
C H E M I C A L S ARE NOT TOXIC BUT R A T H E R BECAUSE H U M A N EXPOSURE JJ5 M I N I
SUCH AN EXAMPLE
DIOXIN)'
IS 2,3,7,8-TETRACHLORODIBENZQ-p-DIOXIN
D l O X I N IS A HIGHLY TOXIC CHEMICAL AND
(TCDU OR
IS A CONTAMINAN
OF PRODUCTS MADE FROM T R I C H L O R O P H E N O L AND FROM LOW T E M P E R A T U R E
I N C I N E R A T I O N O F WASTES
C O N T A I N I N G CHLORINATED PRECURSORS*
IS AT THE CENTER OF C O N T R O V E R S I E S OVER
ORANGE
DlOXIN
(1) THE USE OF A G E N T
IN V I E T N A M AND THE HEALTH OF V E T E R A N S , (2) THE USE OF
2,4,5-T H E R B I C I D E IN A G R I C U L T U R E , AND (3) THE DEVELOPMENT OF
TECHNOLOGY
FOR THE
I N C I N E R A T I O N OF M U N I C I P A L AND I N D U S T R I A L
�7
WASTES.
IT HAS BEEN ASSOCIATED WITH THE E V A C U A T I O N OF HUMAN
POPULATIONS
IN SEVESQ, iTALYj TlMES BEACH,, M I S S O U R I ; AND
LOVE CANAL, NEW Y O R K -
THE UNITED
STATES G O V E R N M E N T
HAS
I N I T I A T E D AN EXTENSIVE LONG'TERM RESEARCH PROGRAM ON THE
CHEMISTRY, TOXICOLOGY, AND E N V I R O N M E N T A L FATE OF D I O X I N OF THIS WORK
IS INTENDED TO HELP E V A L U A T E THE
ALL
IMPACT OF D I O X I N
ON H U M A N HEALTH-
T H I S P R O G R A M ENCOMPASSES
FEDERAL A G E N C I E S -
S C I E N T I F I C RESOURCES FROM TEN
WHAT ARE THE SPECIFIC ACCOMPLISHMENTS TO
DATE:
0
S I N C E 1970,
THE UNITED STATES G O V E R N M E N T HAS SPONSORED
RESEARCH RESULTING
IN MORE THAN 500 M A J O R P U B L I C A T I O N S
ON TOXICOLOGY, E N V I R O N M E N T A L FATE AND H U M A N R I S K S OF
DIOXIN.
0
THE F E D E R A L G O V E R N M E N T HAS U N D E R T A K E N A $130
RESEARCH P R O G R A M ON H U M A N HEALTH
EFFECTS
MILLION
I N C L U D I N G STUDIES
ON B I R T H DEFECTS, MORTALITY, M O R B I D I T Y , C A N C E R , AND D I O X I N
IN H U M A N TISSUE.
°
IN A S S O C I A T I O N WITH STATE G O V E R N M E N T S , FEDERAL A G E N C I E S
HAVE
I N I T I A T E D E X T E N S I V E PROGRAMS TO DEVELOP
FOR CLEAN-UP
WITH D I O X I N .
OF HUNDREDS
TECHNOLOGY
AND D E C O N T A M I N A T I O N OF A R E A S C O N T A M I N A T E D
CLEAN-UP
EFFORT MAY WELL
INVOLVE E X P E N D I T U R E S
OF M I L L I O N S OF DOLLARS.
HOW HAS ALL OF THIS " G E N E R A T I O N OF SCIENCE" CONTRIBUTED
THE RESOLUTION OF THE D I O X I N C O N T R O V E R S Y HAVE SHOWN THAT ACTUAL
FlRST, THE
TO
STUDIES
E X P O S U R E TO D I O X I N S IS M I N I M A L , THAT IS,
�8
HUMAN
EXPOSURE TO DIOXIN OCCURS P R I M A R I L Y IN "HOT SPOTS", AND
THE N U M B E R OF I N D I V I D U A L S EXPOSED IS V E R Y LOW.
SECONDLY, THE
DATA COLLECTED TO DATE N E I T H E R IMPACTS
ACTIONS ON
PROPOSED
CLEAN-UP (E.G., TIMES BEACH) NOR IMPEDES CONGRESSIONAL
TO LEGISLATE P R E S U M P T I V E C O M P E N S A T I O N OBTAINED
DIOXIN
FROM COMBUSTION
ACTION
THIRDLY, THE DATA
STUDIES SHOW THAT THE PRODUCTION
IS M I N I M A L AND E A S I L Y CONTROLLED
YET THE FEAR OF D I O X I N HAS PARALYZED
WITH
OF
PRESENT TECHNOLOGY,
INCINERATION
TECHNOLOGY
AND ITS A P P L I C A T I O N .
IT IS A P P A R E N T THAT OUR F A I L U R E TO PROPERLY APPLY
SCIENCE
HAS CREATED A C R I S I S OF CONFIDENCE IN OUR ABILITY TO NOT ONLY
DEAL WITH PROBLEMS BUT ALSO TO
B E L I E V E THAT SCIENCE,
IDENTIFY THEM-
NEVERTHELESS, I
GOOD S C I E N C E , MUST BE USED BY THE D E C I S I O N
MAKERS••..
*.•
BBSO HOW DO WE PROVIDE R A T I O N A L E G U I D E L I N E S FOR H A N D L I N G THE
ISSUE OF TOXIC C H E M I C A L S
MUST S I M U L T A N E O U S
IN OUR
ENVIRONMENT?
I S U G G E S T THAT WE
I N I T I A T E AND ENCOURAGE THE DEVELOPMENT OF
RESEARCH AND TECHNOLOGY
INTO THE M A N A G M E N T
OF TOXIC WASTES WHILE
AT THE SAME TIME E D U C A T I N G THE PUBLIC AND LAW M A K E R S ON THE
ASSOCIATED ISSUES°
LET ME ADDRESS THE LATTER A R E A FIRST:
THE P U B L I C M U S T U N D E R S T A N D THAT USE OF THE P R O D U C T S OF
TECHNOLOGY C A R R I E S WITH IT THE R E S P O N S I B I L I T Y FOR SAFE
D I S P O S A L OF THAT T E C H N O L O G Y ' S W A S T E WITH WASTE DISPOSAL
A PERVASIVE DIFFICULTY
IS THAT I N D I V I D U A L S DO NOT WANT THE
W A S T E S , E S P E C I A L L Y T O X I C WASTES, D I S P O S E D O F I N T H E I R
NEIGHBORHOODOCEAN
I AM R E M I N D E D OF THE
INCINERATION
RECENT ACTION
IN THE tULF OF M E X I C O -
WlTHIN
INVOLVING
THE
�PAST FEW MONTHS, EPA REFUSED TO ISSUE THE NECESSARY
B U R N I N G PERMITS FOR THE DISPOSAL OF 80 M I L L I O N GALLONS
OF HAZARDOUS WASTE BY OCEAN
6000 PEOPLE PRESENTED
I N C I N E R A T I O N P R I M A R I L Y BECAUSE
INTENSE OPPOSITION TO THE B U R N I N G -
THIS IN SPITE OF THE O V E R S H E L M I N G S C I E N T I F I C DATA SHOWING
THAT THE
I N C I N E R A T O R SHIP WAS CAPABLE OF DESTROYING THE
WAST AT AN E F F I C I E N C Y OF 99-9999%, AND HAD DONE SO ON MANY
OCCASIONS
IN THE SAME A R E A -
THE PERCEPTION THAT WASTES
CANNOT BE SAFELY HANDLED HAS BEEN AN IMPORTANT C O N S I D E R A T I O N
IN WASTE M A N A G M E N T -
IN THE CASE OF HIGHLY TOXIC WASTES, T R A N S P O R T A T I O N BY
R A I L OR ROADWAY I N V O L V E S R I S K S THAT CANNOT BE
N A T I O N S NEED TO MOVE TOWARD
IGNORED-
P O L I C I E S R E Q U I R I N G STATES
OR R E G I O N S TO DISPOSE OF WASTES W I T H I N THE REGION/STATE
IN W H I C H THEY ARE G E N E R A T E D -
THUS G O V E R N M E N T A L AND
I N D U S T R I A L O R G A N I Z A T I O N S NEED TO A G R E E THAT V A R I O U S
CLASSES OF WASTES SHOULD BE DISPOSAL OF LOCALLY
OR IN
REGIONALLY DESIGNATED REPOSITIES°
PUBLIC C O N F I D E N C E IN WASTE DISPOSAL T E C H N I Q U E S W I L L BE
ENHANCED IF WASTES ARE R E S P O N S I B L Y AND SAFELY HANDLED, IF
THE LAWS ARE STRICTLY A D H E R E D TO AND IF THE C O M P A N I E S / A G E N C I E S
INVOLVED IN THE O P E R A T I O N S KEEP THE PUBLIC A W A R E OF THE
ACTIVITIES.
WE CANNOT DELETE THE PUBLIC'S OVERSIGHT OF
WASTE DISPOSAL-
THE FEAR OF THE " M I D N I G H T HAULERS" IS
STILL FRESH IN THE M I N D S OF M A N Y P E O P L E -
�10
LET ME NOW ADDRESS AREAS OF RESEARCH AND TECHNOLOGY DEVELOPMENTTHE CHALLENGE
IS BEFORE
US'
IN THE UNITED STATES, WE HAVE
I D E N T I F I E D AND P R I O R I T I Z E D MORE THAN
1800
ABANDONED
OR I N A C T I V E
SITES THAT HAVE BEEN USED TO IMPROPERLY DISPOSE OF TOXIC WASTESVIE ARE NOW
I N I T I A T I N G AN EFFORT WHICH WILL EXTEND OVER THE NEXT
10 YEARS AND W I L L COST M O R E THAN
$9-7 M I L L I O N PER SITE-
$10-5 B I L L I O N DOLLARS - ROUGHLY
IN ORDER TO SUCCESSFULLY
TASK WE WILL NEED TREMENDOUS SUPPORT
COMPLETE THIS
FROM THE S C I E N T I F I C COMMUNITY.
NEW METHODS OF CLEAN'UP TECHNOLOGY WILL BE R E Q U I R E D *
NEW METHODS
OF M O N I T O R I N G AND M O D E L L I N G E N V I R O N M E N T A L POLLUTANTS WILL BE
NEEDED.
FOR EXAMPLE, M O V E M E N T OF TOXICS THROUGH
THE GROUND
A Q U I F E R S IS EXTREMELY DIFFICULT, IF NOT IMPOSSIBLE,
OR IN
TO PREDICT-
MOREOVER, HOW DO WE D E C O N T A M I N A N T AN A Q U I F E R -
WE WILL NEED TO ESTABLISH
GREATEST
HAZARDS.
OBVIOUSLY
P R I O R I T I E S ON WHAT CHEMICALS
THE INTENSE C O N C E R N OVER THE
OF C H E M I C A L S IN THE E N V I R O N M E N T CENTERS ON PUBLIC HEALTHI D E N T I F Y E N V I R O N M E N T A L POLLUTANTS
POSE THE
IMPACT
WE CAN
W I T H I N C R E D I B L E A C C U R A C Y BUT
AS TO T H E I R S I G N I F I C A N C E AS POTENTIAL THREATS TO H U M A N HEALTH,
WE H A V E ONLY VAGUE S U G G E S T I O N S -
A C C O R D I N G L Y WE MUST
BETTER
DEVELOP
THE CONCEPT OF R I S K ASSESSMENT'
LET ME FOCUS ON THIS SUBJECT FOR A M O M E N T IS IMPORTANT THAT WE B R I N G SCIENCE BACK
RISKS?
HOW DO WE DO THIS?
OF R I S K ASSESSMENT-
As
I NOTED B E F O R E IT
INTO THE MANAGEMENT OF
THE A N S W E R HAS TO BE THROUGH
THE USE
�11
0
WE MUST STREN6HTEN
THE QUALITY OF S C I E N C E TO THE POINT
THAT R I S K C H A R A C T E R I Z A T I O N S ARE R E L I A B L E AND C R E D I B L E *
THIS IN T U R N WILL
I N C R E A S E THE DEGREE TO WHICH DECISION
M A K E R S ARE W I L L I N G TO ACCEPT THESE CHARACTERIZATIONS AS
THE P R I N C I P A L BASIS FOR REGULATORY D E C I S I O N S THE BEST WAYS OF S T R E N G T H E N I N G THE SCIENCE
THE USE OF CONSENSUS
WORKSHOPS-
IS AN EVOLVING PROCESS WHERE
ONE OF
IS THROUGH
THE CONSENSUS
WORKSHOP
SCIENTISTS FROM ACADEMIA,
GOVERNMENT,, INDUSTRY, AND PUBLIC INTEREST GROUPS CAN
MEET AND R E S O L V E S C I E N T I F I C QUESTIONS ON THE BASIS OF
THE BEST S C I E N C E C U R R E N T L Y A V A I L A B L E -
RECENTLY THIS OFFICE SPONSORED A CONSENSUS WORKSHOP ON
FORMALDEHYDE.
MORE THAN 70 I N T E R N A T I O N A L L Y RENOWNED SCIENTISTS
MET AND DISCUSSED
IN AN OBJECTIVE AND NON'ADVERSIAL F A S H I O N THE
RELEVANT S C I E N T I F I C E V I D E N C E ON FORMALDEHYDE IN THE G E N E R A L A R E A S
OF EPIDEMIOLOGY, EXPOSURE, TOXICOLOGY AND RISK ESTIMATIONEMPHASIS OF THE WORKSHOP WAS TO DEVELOP CONSENSUS
ON THE S C I E N T I F I C
ISSUES C O N C E R N I N G FORMALDEHYDEj H O W E V E R , THE RESULTS
USED TO ASSIST
PLEASED
THE
MAY ALSO BE
IN F U T U R E REGULATORY D E C I S I O N M A K I N G -
TO A N N O U N C E THAT THE REPORT OF THE WORKSHOP,
I AM
ENTITLED
D E L I B E R A T I O N S OF THE CONSENSUS WORKSHOP ON FORMALDEHYDE. WILL BE
PUBLISHED THIS
FALL
IN THE S C I E N T I F I C J O U R N A L E N V I R O N M E N T A L HEALTH
PERSPECIVIES-
0
WE MUST DEVELOP U N I F O R M G U I D E L I N E S FOR USE IN THE R I S K
ASSESSMENT
PROCESS-
A C C O R D I N G L Y , THE NAS 1983
REPORT,
�12
"RISK ASSESSMENT IN THE FEDERAL GOVERNMENT:
MANAGING
THE PROCESS/' is A START IN THE RIGHT D I R E C T I O N MAJOR
ISSUE LEFT UNADDRESSED IN THIS REPORT
A
IS HOW WE
ESTABLISH THE A P P R O P R I A T E WAY OF OVERSEEING THE
GOVERNMENT'S
USE OF RESEARCH IN THE "EVOLVING* FIELD OF R I S K ASSESSMENT.
THIS R E M A I N S TO BE DETERMINED:
FOUR AGENCIES ARE ACTIVELY
R E S E A R C H I N G "RlSK ASSESSMENT" METHODOLOGY*
°
IN A S I M I L A R V E I N , REGULATORY A G E N C I E S U S I N G R l S K
ASSESSMENT
MUST USE THE SAME S C I E N C E IN THE D E T E R M I N A T I O N
OF THE R I S K S *
MANY OF YOU ARE ONLY TOO A W A R E THAT THIS
IS ESPECIALLY A PROBLEM
IN THE
E S T I M A T I O N OF CANCER R I S K S -
A C C O R D I N G L Y , THE OFFICE OF SCIENCE AND TECHNOLOGY
A NEW DOCUMENT
TITLED "CHEMICAL
SCIENCE AND ITS ASSOCIATED
CARCINOGENS!
PRINCIPLES-"
A R E V I E W OF THE
THE T H I R D DRAFT W H I C H
IN THE MAY 22, 1984 ISSUE OF THE FEDERAL REGISTER
UPDATE OF THE
I N F O R M A T I O N AND
A C H E M I C A L COMPOUND
IDEAS USED
IS TO H U M A N S -
HAS RELEASED
APPEARED
PROVIDES AN
IN E S T A B L I S H I N G HOW D A N G E R O U S
IT DEVELOPS FROM THESE DATA A
C U R R E N T AND BALANCED SET OF P R I N C I P L E S TO BE R E F E R R E D TO IN
E S T A B L I S H I N G THE POTENTIAL
C A N C E R - C A U S I N G D A N G E R FROM C H E M I C A L S -
LOOKING TO THE FUTURE, THE D O C U M E N T ALSO
OF SCIENCE EXPECTED TO
PLANNED
PREDICTS THE E M E R G I N G AREAS
IMPACT ON REGULATORY ACTIONS, A L L O W I N G
I N C O R P O R A T I O N OF THESE DEVELOPMENTS R A T H E R THAN MERELY A
REACTION TO T H E M -
THE REPORT REFLECTS
THE C O M B I N E D EFFORTS OF S E N I O R
FROM N I N E FEDERAL HEALTH-RELATED
D I R E C T I O N OF MY O F F I C E -
SCIENTISTS
U N I T S O P E R A T I N G UNDER T H E
THE PUBLIC C O M M E N T S ARE NOW B E I N G
�13
INCORPORATED
INTO A F I N A L REPORT TO BE RELEASED LATE THIS
FALL0
INDEED, TO R E I N T E R A T E , IT is THE PUBLIC'S P A R T I C I P A T I O N
AND EDUCATION THAT CONSTITUTE MY C O N C E R N U N D E R S T A N D I N G AND CONFIDENCE
PUBLIC
IN E N V I R O N M E N T A L R E G U L A T I O N S
R E Q U I R E S THAT S C I E N T I F I C L I T E R A C Y IS ESSENTIAL
CITIZEN
IS TO CHOOSE
WISELY.
THE EDUCATIONAL
IF THE
PROCESS
MUST ALSO EXTEND TO THE NEWS MEDIA AND LAW M A K E R S °
ON ISSUES OF SCIENCE THAT HAVE SOCIETAL CONCERNS
AND E M O T I O N A L ) WE MUST DEVISE A BETTER
THEM THAN
IN THE COURTROOM-
THAT C U R R E N T L Y EXISTS
(REAL
WAY OF R E S O L V I N G
THE ADVERSAL R E L A T I O N S H I P
W I T H I N THE WHOLE SYSTEM
IS A
H I N D E R A N C E TO THE RESOLUTION OF PROBLEMS-
IN SUMMARY, THE SCIENCE O F F I C E OF THE PRESIDENT
IS
DEDICATED
TO THE TASKS OF P L A C I N G THE VERY BEST S C I E N C E INTO THE HANDS OF
DECISION MAKERS-
THESE TASKS I N V O L V E THE
I D E N T I F I C A T I O N OF
RESEARCH NEEDS, THE DEVELOPMENT OF THE R I S K ASSESSMENT
CONSENSUS
PROCESS, THE
WORKSHOP AND THE EFFORTS OF A G R O U P OF DEDICATED
PUBLIC
SERVICE-ORIENTED SCIENTIST-
THE NEXT TEN YEARS, W I L L BE C R I T I C A L YEARS TO THOSE OF US
CHARGED WITH THE R E S P O N S I B I L I T Y OF D E T E R M I N I N G THE D I R E C T I O N AND
SUPPORT
FOR THE PROPER H A N D L I N G OF TOXIC M A T E R I A L S BY OUR SOCIETIES
�THE C H A L L E N G E S ARE GREAT!
A GREAT EFFORT WILL BE REQUIRED 1 ,
THIS REMINDS ME OF THE TWO MEN WHO FIND THEMSELVES FACING A WIDE
R A V I N E WITH SOME DANGER RAPIDELY A P P R O A C H I N G FROM BEHIND*
THEY
HAVE THE CHOICE EITHER OF ATTEMPTING TO LEAP THE R A V I N E OR OF
T U R N I N G TO FACE THE DANGER.
A GOOD CASE CAN BE MADE FOR BOTH,
BUT THEY CANNOT A G R E E W H I C H TO CHOOSE'
tACH F A I L I N G TO C O N V I N C E
THE OTHER, THEY RESOLVE THEIR DIFFERENCES BY DECIDING TO JUMP
HALFWAY*
TASK-
WE MUST NOT DO THE JOB HALFWAY-
WE MUST COMPLETE THE
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
194
Folder
The folder containing the original item.
5483
Series
The series number of the original item.
Series VIII Subseries I
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Typescript: Hazardous Wastes: Problems, Perspectives and Policy
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/9bb9d9a34e82843d8fddb8cc95e99020.pdf
0b87e9207e8d9b01c2efca48c486bde5
PDF Text
Text
Item D Number
°5552
Author
Young, Alvin L.
D not scanned
Corporate Author
Report/Article Title Handwritten Notes: Testimony to 2,4,5-T Hearings
Journal/Book Title
Year
000
°
Month/Day
Color
Number of Images
D
°
Descrtpton Notes
Tuesday, March 19, 2002
Page 5552 of 5611
�i
'
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
197
Folder
The folder containing the original item.
5552
Series
The series number of the original item.
Series VIII Subseries I
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Handwritten Notes: Testimony to 2,4,5-T Hearings
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/bc954d5aecbc16ca436bf74ceeacf154.pdf
100e6761144f1183ede1aad0e6cf4457
PDF Text
Text
Item n Number
Author
Young, Alvin L.
Corporate Author
Report/Article Titlfl Handwritten Notes: Cu CHi Base Camp, 1969
Journal/Book Title
Year
Month/Day
Color
Number of Images
D
°
Alvin L. Young filed these documents together with others
under the label, "Agent Orange Exposure Project."
Tuesday, March 19, 2002
Page 5556 of 5611
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Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
197
Folder
The folder containing the original item.
5556
Series
The series number of the original item.
Series VIII Subseries II
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Handwritten Notes: Cu CHi Base Camp, 1969
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/7d688575442ef67331f7383d47262704.pdf
8e9b36fb46a2781c2f19d8641718ecf8
PDF Text
Text
item D Number
°5591
Author
Young, Alvin L.
D Not Scanned
Corporate Author
RBDOrt/ArtiGlB TltlB Outline Report of the Agent Orange Working Group
Science Panel Subcommittee on Exposure, November
1982
Journal/Book Title
Year
000
°
Month/Day
Color
Number of Images
D
°
Desorlpton Notes
Tuesday, March 19, 2002
Page 5591 of 5611
�Outline Report of the
Agent Orange Working Group
Science Panel
Subcommittee on Exposure
Alvin L. Young
VA/AOPO
November 1982
Factors that must be considered m the development of an exposure index
model include nature of the chemical and target, time after application of
the herbicide and distance from application site.
I. Factors that Influenced Exposure to Herbicide:
0
Herbicide Formulation
Chemical/Physical Characteristics
Solubility
Vapor Pressures
8
Dissemination of Herbicides
Application Parameters
Target Vegetation
0
Route of Exposure
Dermal
Inhalation
Ingestion
0
Environmental Fate
In
On
In
In
Air
Vegetation
Soil
Water
II. Dissemination System
1. RANCH HAND
KC 123/A/A45Y-1 Dispenser Internal Defoliant
2. UH-1B/D Helicopter Spray System (Agrinautics)
3. Buffalo Turbines
�4. Power-Driven Decontamination Apparatus (PDDA)
5. Back-Pack Sprayers ( 2 and 5 gallons)
6. "Jerry-Rigged" or Field Expedient Devices.
III. Factors described by Weighted Systems
A.
Toxicity of Herbicide
B.
Fate of TCDD
Location of Mission
Jungle
Mangrove
Crop
Other
C. Type of Mission
RANCH HAND
Perimeter
Herbicide Abort occurrence
D. Time After Exposure
Days
e
0
0
within 1
within 2
within 3
E. Distance from Exposure Type
(Application Site)
0
0
0
0
within 0.5 km
within 1.0 km
within 1.5 km
within 2.0 km
IV. Assignments of Weights
0
Toxicity of Herbicides
Orange
Weight Score
600 mgAg
5
Blue
2600 mgAg
2
White
3100 mg/kg
1
�Location of Mission
Defoliation
Jungle (Heavy Canopy)
Mangrove (Light Canopy)
Crop/Abort Missions
Perimeter Applications
10% Penetration 1
32%
3
100% '
10
70%
7
Time/Distance Matrix*
Distance (km)
Time
Days
<0.5
1.0
1.5
2.0
70
15
10
5
i
1
,l
'
2
35
8
5
3
3
18
4
3
RANCH HAND
Application
1
t. t
Perimeter
Application
* Values in Time/Distance matrix established in percent of herbicide
particles falling within specified distance from height of release (RH
Mission) and impact of environmental degradation or binding over specified
time intervals.
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
198
Folder
The folder containing the original item.
5591
Series
The series number of the original item.
Series VIII Subseries II
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Outline Report of the Agent Orange Working Group Science Panel Subcommittee on Exposure, November 1982
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/d6e49ce9e0a641e5b22f0b159a09e5f3.pdf
31bea5e58a70823473226d7992e89157
PDF Text
Text
item D Number
°5727
Author
Young, Alvin L.
Ll Not Scanned
Corporate Author
Report/Article Tltte
Use of
Herbicides in South Vietnam, 1961 -1971
Journal/Book Title
Year
000
°
Month/Day
Color
Number of hnagee
n
°
Descrlpton Notes
Tuesday, March 26, 2002
Page 5727 of 5743
�USE OF HERBICIDES IN SOUTH VIETNAM, 1961-1971*
Alvin L. Young
MAJOR, USAF, PHD
Epidemiology Division
USAF School of Aerospace Medicine
Brooks AFB, San Antonio TX 78235
Herbicides used in support of tactical military operations in South Vietnam from
1961 to 1971 are today, ten years after the last herbicide mission, the center of
intense scientific debate involving not only medical but also legal, political and
ecological issues. This paper reviews the historical and operational concepts and
some potential human exposure considerations involving the military use of herbicides
in the Southeast Asian Conflict.
Herbicides Used in South Vietnam
Synthesis technology, efficacy data, and field application techniques were developed
for the two major phenoxy herbicides, 2,4-dichlorophenpxyacetic acid (2,4-D) and
2,4,5-trichlorophenoxyacetic acid (2,4,5-T) during World War II at Fort Detrick,
Frederick, Maryland. Following World War II, the iAcoij^rcial use of these two
"synthetic" organic herbicides revolutionized Ameri.cJiKagri culture. In 1950, more
than 10 million pounds of these materials were used annually for weed and brush
control in the United States. By 1960, in excess of 36 million pounds were used.
In May 1961, the Office of the Secretary of Defense requested the Fort Detrick
personnel to determine the technical feasibility of defoliating jungle vegetation
in the Republic of Vietnam. By early fall, 1961, 18 different aerial spray tests
(defoliation and anticrop) had been conducted with various formulations of
commercially-available herbicides. The choice of these herbicides was based upon
the chemicals that had had considerable research, proven performance, and practical
background at that period in time. Also, such factors as availability in large
quantity, costs and known or accepted safety in regard to their toxicity to humans
and animals was considered. The results of these tests were that significant
defoliation and anticrop effects could be obtained with two different mistures of
herbicides. The first was a mixture of the n-butyl esters of 2,4-D and 2,4,5-T
and the iso-butyl ester of 2,4,5-T. This mixture was code-named "Purple." The
second "military" herbicide was code-named "Blue" and consisted of the acid and
sodium salt of cacodylic acid. The colored bands which were painted around the
center of the 55-gallon drums served as aid to the identification by support
personnel.
The first shipment of Herbicides Purple and Blue was recieved at Tan Son Nhut
Air Base, Republic of Vietnam, on 9 January 1962. These were the first military
herbicides used in Operation RANCH HAND, the tactical military project for the
aerial spraying of herbicides in South Vietnam. Two additional phenoxy herbicide
formulations were received in limited quantities in South Vietnam and evaluated
during the first two years of Operation RANCH HAND. These were code-named Pink
and Green. By January 1965, two additional military herbicides, code-named Orange
and White, had been evaluated and brought into the spray program. Herbicide Orange
*A synopsis of Information from Chapters I and III of The Toxicology, Environmental
Fate, and Human Risk of Herbicide Orange and Its Associated Dioxin, Air Force
Technical Report OEHL-TR-78-92, USAF Occupational and Environmental Health Laboratory,
Brooks Air Force Base, Texas. (Authors: A. L. Young, J. A. Calcagni, C. E. Thalken,
and J. W. Tremblay.) 1978.
�Herbicides used in support of tactical military operations in South Vietnam from
1961 to 1971 are today, ten years after the last herbicide mission, the center of
intense scientific debate involving not.only medical but also legal, political and
ecological issues. This paper reviews the historical and operational concepts and
some potential human exposure considerations involving the military use of herbicides
in the Southeast Asian Conflict.
Herbicides Used in South Vietnam
Synthesis technology, efficacy data, and field application techniques were developed
for the two major phenoxy herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D) and
2,4,5-trichlorophenoxyacetic acid (2,4,5-T) during World War II at Fort Detrick,
Frederick, Maryland. Following World War II, the commercial use of these two
"synthetic" organic herbicides revolutionized American*agriculture. In 1950, more
than 10 million pounds of these materials were used annually for weed and brush
control in the United States. By 1960, in excess of 36 million pounds were used.
In May 1961, the Office of the Secretary of Defense requested the Fort Detrick
personnel to determine the technical feasibility of defoliating jungle vegetation
in the Republic of Vietnam. By early fall, 1961, 18 different aerial spray tests
(defoliation and anticrop) had been conducted with various formulations of
commercially-available herbicides. The choice of these herbicides was based upon
the chemicals that had had considerable research, proven performance, and practical
background at that period in time. Also, such factors as availability in large
quantity, costs and known or accepted safety in regard to their toxicity to humans
and animals was considered. The results of these tests were that significant
defoliation and anticrop effects could be obtained with two different mistures of
herbicides. The first was a mixture of the n-butyl esters of 2,4-D and 2,4,5-T
and the iso-butyl ester of 2,4,5-T. This mixture was code-named "Purple." The
second "military" herbicide was code-named "Blue" and consisted of the acid and
sodium salt of cacodylic acid. The colored bands which were painted around the
center of the 55-gallon drums served as aid to the identification by support
personnel.
The first shipment of Herbicides Purple and Blue was recieved at Tan Son Nhut
Air Base, Republic of Vietnam, on 9 January 1962. These were the first military
herbicides used in Operation RANCH HAND, the tactical military project for the
aerial spraying of herbicides in South Vietnam. Two additibnal phenoxy herbicide
formulations were received in limited quantities in South Vietnam and evaluated
during the first two years of Operation RANCH HAND. These were code-named Pink
and Green. By January 1965, two additional military herbicides, code-named Orange
and White, had been evaluated and brought into the spray program. Herbicide Orange
*A synopsis of Information from Chapters I and III of The Toxicology, Environmental
Fate, and Human Risk of Herbicide Orange and Its Associated Dioxin, Air Force
Technical Report OEHL-TR-78-92, USAF Occupational and Environmental Health Laboratory,
Brooks Air Force Base, Texas. (Authors: A. L. Young, J. A. Calcagni, C. E. Thalken,
and J. W. Tremblay.) 1978.
�replaced all uses of Purple,.Pink, or Green, and eventually became the most widely
used military herbicide in South Vietnam. The composition of the three major
herbicides used in South Vietnam were as follows:
1. Herbicide Orange
Orange was a reddish-brown to tan colored liquid soluble in diesel fuel and
organic solvents, but insoluble in water. One gallon of Orange theoretically contained
4.21 pounds of the active ingredient of 2,4-D and 4.41 pounds of the active ingredient
of 2,4,5-T. Orange was formulated to contain a 50:50 misture of the n-butyl esters
of 2,4-D and 2,4,5-T. The percentages of the formulation typically were:
n-butyl ester of 2,4-D
free acid of 2,4-D
n-butyl ester of 2,4,5-T
Free acid of 2,4,5-T
inert ingredients (e.g.,
butyl alcohol and ester
moieties)
49.49
0.13
48.75
1.00
0.62
2. Herbicide White
White was a dark brown viscous liquid that was soluble in water but insoluble
in organic solvents and diesel fuel. One gallon of White contained 0.54 pounds of
the active ingredient of 4-amino-3,5,6-trichloropicolinic acid (picloram) and 2.00
pounds of the active ingredient of 2,4-D. White was formulated to contain a 1:4
mixture of the triisopropanoamine salts of picloram and 2,4-D. The percentages of
the formulation were:
triisopropanolamine salt of picloram
triisopropanolamine salt of 2,4-D
inert ingredient (primarily the
solvent triisopropanolamine)
10.2
39.6
50.2
3. Herbicide Blue
Blue was a clear yellowish-tan liquid that was soluble in water, but insoluble
in organic solvents and diesel fuel. One gallon of Blue contained 3.10 pounds of
the active ingredient hydroxydimethyarsine oxide (cacodylic acid). Blue was
formulated to contain cacodylic acid (as the free acid) and the sodium salf of
cacodylic acid (sodium cacodylate). The percentages of the formulation were:
cacodylic acid
sodium cacodylate
surfactant
sodium chloride
water
antifoam agent
4.7
26.4
3.4
5.5
59.5
0.5
As previously noted, not all of the herbicides used in South Vietnam were used
throughout the entire 10 years (1962-1971) encompassed by the Department of Defense
defoliation program. In addition, 2,4,5-T formulations used early in the program
are believed to have contained higher levels of the toxic contaminant TCDD
(2,3,7,8-tetrachlorodibenzo-p-dioxin or "dioxin") than did the formulations used in
the later years. The three time periods shown in Table 1 can be differentiated on
the basis of specific herbicides used and the mean dioxin content.
10
�TABLE 1
THE DIFFERENTIATION OF THREE TIME PERIODS DURING THE US MILITARY DEFOLIATION
PROGRAM IN SOUTH VIETNAM AND MEAN DIOXIN CONTENT
PERIOD
HERBICIDES USED
(CODE NAMES)
MEAN DIOXIN CONTENT
(PARTS PER MILLION)*
January 1962 June 1965
Purple, Pink, Green
Blue
July 1965 June 1970
Orange
White, Blue
2+
0
July 1970 October 1971
White, Blue
0
32**
0
*Found only in 2,4,5-T containing formulations.
**Value based on analyses of five samples.
•fValue based on the analyses of 488 samples.
Herbicide Orange was the most extensively used herbicide in South Vietnam. Orange
accounted for approximately 10.7 million gallons of the total 17.7 million gallons
of herbicide used (Table 2). It was used from mid-1965 to June 1970. However, as
noted in Table 2, Orange was not the only 2,4,5-T containing herbicide used in the
defoliation program. Small quantities of Purple, Pink, and Green, all containing
2,4,5-T were used from 1962 through mid-1965. In subsequent sections of this
document, the term "herbicide Orange" will refer to all of the 2,4,5-T containing
herbicides used in Vietnam (Purple, Pink, Green, and Orange).
TABLE 2 NUMBER OF GALLONS OF MILITARY HERBICIDE PROCURED BY THE US DEPARTMENT OF
DEFENSE AND DISSEMINATED IN SOUTH VIETNAM DURING JANUARY 1962 - OCTOBER 1971
Code Name
Quantity
Herbicide
Period of Use
Orange
2,4-D; 2,4,5-T
10,646,000
1965-1970*
White
2,4-D; Picloram
5,633,000
1965-1971**
Blue
Cacodylic Acid
1,150,000
1962-1971**
Purple
2,4-D; 2,4,5-T
145,000
1962-1965
Pink
2,4,5-T
123,000
1962-1965
Green
2,4,5-T
8,200
1962-1965
Total
17,705,200
*Last fixed-wing mission of Orange 16 April 1970; last helicopter mission of
Orange 6 June 1970.
**Last fixed-wing mission 9 January 1971; all herbicides under US control stopped
31 October 1971.
11
�Use Patterns of Individual Herbicides
Each of the three major herbicides (Orange, White, and Blue) had specific uses.
Ninety-nine percent of Herbicide White was applied in defoliation missions. It was
not recommended for use on crops because of the persistence of Picloram in soils.
Because the herbicidal action on woody plants was usually slow, full defoliation
did not occur for several months after spray application. Thus, it was an ideal
herbicide for use in the inland forests in areas where defoliation was not
immediately required, but where it did occur it would persist longer than if the
area were sprayed with Orange or Blue.
Herbicide Blue was the herbicide of choice for crop destruction missions
involving cereal or grain crops. Approximately 50 percent of all Blue was used
in crop destruction missions in remote or enemy controlled areas with the remainder
being used as a contact herbicide for control of grasses around base perimeters.
Ninety percent of all Herbicide Orange was used for forest defoliation and it
was especially effective in defoliating mangrove forests. Eight percent of
Herbicide Orange was used in the destruction of broadleaf crops (beans, peanuts,
ramie, and root or tuber crops). The remaining 2 percent was used around base
perimeters, cache sites, waterways, and communication lines.
Table 3 shows the number of acres sprayed with herbicides in South Vietnam
within the three major vegetational categories.
TABLE 3 THE NUMBER OF ACRES TREATED IN SOUTH VIETNAM, 1962-1971, WITH MILITARY
HERBICIDES WITHIN THE THREE MAJOR VEGETATIONAL CATEGORIES
Vegetational Category
Areas Treated*
Inland forest
2,670,000
Mangrove forests
318,000
Cultivated crops
260,000
Total
3,248,000
*Areas receiving single or multiple coverage.
Certain portions of South Vietnam were more likely to have been subjected to
defoliation. Herbicide expenditures for the four Combat Tactical Zones of South
Vietnam are shown in Table 4. These data were obtained from the HERBS tape (a
computer listing of all herbicide missions in South Vietnam from 1965 through 1971).
Total volume is in close agreement with the actual procurement data shown in
Table 2.
12
�TABLE 4 US HERBICIDES EXPENDITURES IN SOUTH VIETNAM, 1962-1971; A BREAKDOWN
BY COMBAT TACTICAL ZONE*
Herbicide Expenditure
(gallons)
Orange
White
Blue
Combat Tactical Zones
CTZ I
2,250,000
363,000
298,000
CTZ II
2,519,000
729,000
473,000
CTZ III
(includes Saigon)
5,309,000
3,719,000
294,000
CTZ IV
1.227.000
435.000
62.000
11,305,000
5,246,000
1,127,000
Subtotals
Grand Total
17.678.000
*Source: HERBS tape
In addition to the herbicides, numerous other chemicals were shipped to South Vietnam
in 55-gallon drums. These included selected fuel additives, cleaning solvents,
cooking oils, and a variety of other pesticides. The insecticide Malathion was
widely used for control of mosquitoes and at least 400,000 gallons of it were used
from 1966 through 1970. In addition, much smaller quantities of Lindane and DDT
were used in ground operations throughout the war in Southeast Asia. The
distribution of the herbicides within Vietnam after their arrival did not occur
randomly. About 65 percent was shipped to the 20th Ordnance Storage Depot, Saigon,
and 35 percent was shipped to the 511th Ordnance Depot, Da Nang.
Military Aircraft and Vehicles Used in the Dissemination of Herbicides
Numerous aircraft were used in the air war in Vietnam, but only a few of these
aircraft were used for aerial dissemination of herbicides. The "work horse" of
Operation RANCH HAND was the C-123, "Provider." This cargo aircraft was adapted
to receive a modular spray system for internal carriage. The module (the A/A 45 Y-l)
consisted of a 1,000-gallon tank pump, and engine which were all mounted on a frame
pallet. Ah operator's console was an integral part of the unit, but was not mounted
on the pallet. Wing booms ( . inches in diameter, 22 feet long) extended from the
15
outboard engine nacelles toward the wing tips. A short tail boom (3 inches in
diameter, 20 feet long) was positioned centrally near the aft cargo door. Each
aircraft normally had a crew of three men: the pilot, co-pilot (navigator), and
flight engineer (console operator). During the peak activity of RANCH HAND
operations ( 9 8 1 6 ) approximately 30 U C-123K aircraft were employed. However,
16-99,
many other squadrons of non-RANCH HAND C-123 aircraft were routinely used throughout South Vietnam in transport operations.
13
�The control of malaria and other mosquito-borne diseases in South Vietnam
necessitated an extensive aerial insecticide application program in order to control
these vector insects. From 1966 through 1972, three C-123 aircraft were used to
spray Malathion, an organophosphate insecticide. These aircraft could be
distinguished from the Herbicide-spraying aircraft because they were not camouflaged.
These aircraft routinely sprayed insecticide adjacent to military and civilian
installations, as well as in areas where military operations were in progress, or
about to commence.
Approximately 10 to 12 percent of all herbicides used in South Vietnam was
disseminated by helicopter or ground application equipment. Generally, helicopter
crews were not assigned to herbicide spray duties on a full-time basis and rotated
the spraying duties with other mission requirements. The military UH-1 series of
helicopters, deployed by the Air Force, the Army, and Navy units, generally sprayed
the herbicides. The most common spray system used was the AGRINAUTICS unit. This
unit was installed in or removed from the aircraft in a matter of minutes because
it was "tied down" to installed cargo shackles and aircraft modifications were not
required for its use. The unit consisted of a 200-gallon tank and a collapsible
32-foot spray boom. The unit was operated by manual -controls to control the flow
valve and a windmill brake. Generally, each helicopter had three crew members.
A summary of the aircraft used in herbicide and insecticide operations is
shown in Table 5.
TABLE 5 US MILITARY AIRCRAFT USED IN THE DISSEMINATION OF HERBICIDES AND
INSECTICIDES IN SOUTH VIETNAM
Aircraft
Camouflaged
Chemical Disseminated
UC-123/UC-123K
Yes
All Herbicides
UC-123K
No
Malathion
Yes
Orange, Blue
Helicopter
Air Force UH-1
Army UH-1B/UH-1D
Navy UH/1E
Various ground delivery systems were also used in South Vietnam for control of
vegetation in limited areas. Most of these units were towed or mounted on vehicles.
One unit that was routinely used was the Buffalo-turbine. It developed a wind
blast with a velocity up to 150 MPH at 10,000 ft /minute volume. When the
herbicide was injected into the air blast, it was essentially "shot" at the
foliage. The buffalo turbine was useful for roadside spraying and applications of
perimeter defenses. The herbicides of choise in these operations were Blue and
Orange.
�Exposure Considerations; Applications and Environmental Parameters
There were relatively few military operations that involved the handling of herbicides
by military personnel. A review of operations involving Herbicide Orange in South
Vietnam from January 1962 to April 1970 revealed that there were essentially three
groups of US military personnel potentially exposed to Herbicide Orange and its
associated dioxin contaminant. These three groups were:
*
1. "Operation RANCH HAND" personnel actively involved in the defoliation
program. This group included aircrew members and maintenance and support personnel
directly assigned to the RANCH HAND squadrons.
2. Personnel assigned to selected support functions that may have resulted in
exposure to Herbicide Orange. This group included, for example, personnel who
sprayed herbicides, using helicopters or ground application equipment; personnel who
may have delivered the herbicides to the units performing the defoliation missions;
aircraft mechanics who were specialized and occasionally provided support to
RANCH HAND aircraft; or, personnel who may have flown contaminated C-123 aircraft,
but were not assigned to RANCH HAND (e.g., during the Tet Offensive, all RANCH HAND
aircraft were reconfigured to transport supplies and equipment, and were assigned
to non-RANCH HAND squadrons).
3. Ground personnel who may have been inadvertently sprayed by defoliation
aircraft or who, during combat operations, may have entered an area previously
sprayed with Herbicide Orange.
The total number of US military personnel exposed to Herbicide Orange is not
known. Approximately 1,200 RANCH HAND personnel were exposed in direct support of
the defoliation operations; however, there are no data on the number of non-RANCH
HAND personnel who may have been exposed. The actual number of people may be in the
thousands since at least 100 helicopter spray equipment units were used in South
Vietnam, and most military bases had vehicle-mounted and backpack spray units
available for use in routine vegetation control programs. The number of military
ground personnel who may have inadvertently been sprayed by RANCH HAND aircraft,
or who may have entered areas recently sprayed with Herbicide Orange during combat
operations is not known. Approximately 10 percent of South Vietnam was sprayed
with herbicides, and most of this area was contested and/or controlled by enemy
forces. Most areas sprayed were remote, unpopulated and forested. Because of the
dense canopy cover, the target of the defoliation operation, the amount of
herbicide penetrating to the forest floor would have been small. The exposure
of personnel could have occurred by essentially three routes:
1. Percutaneous absorption and inhalation of vapors/aerosols by direct
exposure to sprays.
2. Percutaneous absorption and inhalation of vapors by exposure to treated
areas following spray application, and
3. Ingestion of foods contaminated with the material.
The chemical and physical characteristics of Herbicide Orange and the spray,
as it would have occurred following dissemination from a C-123, are important
factors in assessing relative exposure to the Herbicides and TCDD.
15
�Table 6 reviews the pertinent chemical and physical characteristics of Herbicide
Orange. Table 7 reviews both the application parameters, of the spray system used
in the UC-123K aircraft and the characteristics of the spray itself. Generally,
herbicides were sprayed in the early morning or late afternoon, so as to minimize
the effects of air movement on particle dispersion.
TABLE 6
PERTINENT CHEMICAL AND PHYSICAL CHARACTERISTICS OF HERBICIDE ORANGE
Formulation Concentrated
( . Ib ai/gal)*
86
Water Insoluble
Density « 1.28
-4
o
3.6 x 10
mm Hg at 30 C
Vapor Pressure
NBE** 2,4-D
:
NBE 2,4,5-T
:
1.2 x 10~A
-4
0.4 x 10
TCDD
:
1 x 10~4
Viscous
40 centipoises at 20 C
4
Noncorrosive to metal
Deleterious to paints, rubber, neoprene
Long Shelf life
*Pounds active ingredient (2,4-D and 2,4,5-T) per gallon.
**NBE - Normal Butyl ester.
16
�TABLE 7. APPLICATION PARAMETERS AND SPRAY CHARACTERISTICS OF THE
C-123 MODULAR INTERNAL SPRAY SYSTEM
Aircraft speed
130 KIAS*
Aircraft altitude
150 feet
Tank -volume
1,000 gallons
Spray time
3.5-4 minutes
Particle size:
100 microns: 1.9%
100-500 microns: 76.2%
500 microns: 21.9%
87% impacted within 1 min
13% drifted or volatilized
Mean particle volume
0.61 microliters
Spray swath
260 + 20 feet
Mean deposition
3 gallons/acre
Total area/tank
340 acres
*Knots indicated air speed
17
�Ground combat forces norjnally would not have been expected to have enter.ed a
previously treated area for'several weeks after treatment, during which
time numerous environmental factors would have reduced the potential for exposure to
military personnel. An indepth review of the environmental fate of Herbicide Orange
and TCDD concluded that the vast majority of the phenoxy herbicides would have
impacted forest canopy, the intended target.
Rapid uptake (e.g., within a few hours) of the ester formulations of 2,4-D and
2,4,5-T would have occurred. Most of the herbicide probably would have undergone
rapid degradation (weeks) within the cellular matrix of the vegetation. However, some
of the herbicide may have remained unmetabolized and would have been deposited on the
forest floor at the time of leaf fall.. Soil microbial and/or chemical action would
likely have completed the degradation process. Herbicide droplets that impacted
directly on soil or water would have probably hydrolyzed rapidly (within hours).
Biological and nonbiological degradative processes would have further occurred to
significantly reduce these residues. Some volatilization of the esters of 2,4-D and
2,4,5-T would have occurred during and immediately after application. The volatile
material most likely would have dissipated within the foliage of the target area.
Photodecomposition of TCDD would have minimized the amount of biologically active
volatile residues moving downwind of the target area.
Accumulation of phenoxy herbicides in animals may have occurred following
ingestion of treated vegetation. The magnitude of this accumulation would have likely
been at nontoxic levels. Herbicide residues in animals would have rapidly declined
after withdrawal from treated feed.
Most TCDD sprayed into the environment during defoliation operations would have
probably photodegraded within 24 hours of application. Moreover, recent studies
suggest that even within the shaded forest canopy, volatilization and subsequent
photodecomposition of TCDD can occur. Since translocation into vegetation would be
minimal, most TCDD that escaped photodegradation would probably have entered the soilorganic complex on the forest floor following leaf fall. Soil chemical and microbial
processes would have further reduced TCDD residues. Bioconcentration of the remaining
minute levels of TCDD may have occurred in liver and fat of animals ingesting
contaminated vegetation or soil. However, there are no field data available that
indicate that the levels of TCDD likely to have accumulated in these animals would
have had a biological effect.
The environmental generation of TCDD from 2,4,5-T residues, through thermal or
photolytic processes, would have been highly unlikely and of no consequence.
SUMMARY
The choice of herbicides used in South Vietnam in Operation RANCH HAND, 1962-1971,
was based upon those herbicides that had been widely used in world agriculture, shown
to be effective in controlling a broad spectrum of vegetation, and proven safe to
humans and animals. The major herbicides used in South Vietnam were by phenoxy
herbicides 2,4-D and 2,4,5-T. These two herbicides were formulated as the water
insoluble esters and code-named by the military as Purple, Orange, Pink and Green.
A water soluble amine formulation of 2,4-D was used in Herbicide White. Two other
herbicides were extensively used by the military, picloram (in White) and cacodylic
acid (in Blue).
18
�
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Alvin L. Young Collection on Agent Orange
Description
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<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
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202
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5727
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Series VIII Subseries II
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Use of Herbicides in South Vietnam, 1961-1971
ao_seriesVIII
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https://www.nal.usda.gov/exhibits/speccoll/files/original/e86931d45def0e5f1068220773166136.pdf
e4b48eb9e5c492bc858713558d52f20f
PDF Text
Text
Item D Number
°5761
Author
Young, Alvin L.
D Not Scanned
Corporate Author
Report/Article HUB Letter from Alvin L. Young to C. P. V. DeJesus,
November 6, 1979
Journal/Book Title
Year
Month/Day
Color
D
Number of Images
°
DOSGrlptOn Notes
Enclosures missing.
Thursday, March 28, 2002
Page 5761 of 5780
�EKS/Major Young/dd/3471/6 Nov 79
6 Nov 79
Or. C.P.V. DeJesus
Neurology Service
Audie L. Murphy Memorial Hospital
Veterans Administration
7400 Merton Mlnton
San Antonio, TX 78284
Dear Dr. DeJesus
In reference to your request for literature on human
and animal effects of 2,4,5-T and TCDD, please find enclosed a recent A1r Force Technical Report and two
bibliographies that may be of Interest to you.
If I can provide additional Information, please contact
me at 536-3471.
Sincerely
ALVIN L. YOUNG, Major, USAF, Ph.D.
Consultant, Environmental Sciences
3 Atch
1. OEHL-TR-78-92
2. Bibliography MP-1323
3. Bibliography MP-1343
�
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Title
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Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
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204
Folder
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5761
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Series VIII Subseries III
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Young, Alvin L.
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Letter from Alvin L. Young to C. P. V. DeJesus, November 6, 1979
ao_seriesVIII
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https://www.nal.usda.gov/exhibits/speccoll/files/original/780e6b7a563f6fc25c699dd3a0778fe8.pdf
b2960c1ad5b02d1bb2d4b1fe0cbeae42
PDF Text
Text
Item D Number
osyee
Author
Young, Alvin L.
n NfltSBannafl
Corporate Author
Report/Article Title
Tr
'P ReP°rt -TA'16 (3 Jan 8°)
Journal/Book Title
Year
198
Month/Day
January 23
Color
°
D
Number of Images
°
DeSCriptOR NOtBS
Attachments are included.
Thursday, March 28, 2002
Page 5766 of 5780
�(
DEPARTMENT OF THE AIR FORCE
USAF SCHOOL OF AEROSPACE MEDICINE (AFSC)
BROOKS AIR FORCE BASE, TEXAS
78235
RliPLY TO
ATTN OF:
_.,
C.K
SUBJECT:
Trip Report - TA-16 (3 Jan 80)
TO:
EK
CEA
CE
CD
CC
TS
IN TURN
1. Place Visited
Veterans Administration Central Office (VACO),
Washington DC, 7-8 Jan 80
2. Personnel Contacted
Dr Paul Haber, Assistant Chief Medical Director
for Professional Services, VACO
Dr Richard Levinson, Deputy for Clinical Support
Services, VACO
Mr. Alex Kutner, Assistant for Clinical Support
Services, VACO
Dr Larry Hobson, Assistant for Research and
Development, VACO
Dr John Castellot, Deputy for Medical Services,
VACO
Dr Jack Walsh, Office of Emergency Planning,
VACO
Dr Abraham Lilienfeld, Professor of Epidemiology,
The John Hopkins School of Hygiene, Baltimore, MD
Dr Raymond Suskind, Director, Department of Environmental
Health, Kettering Laboratory, Cincinnati, OH
�3. Topics Discussed
a. The "Veterans Health Programs Extension and improvement Act of
1979" (Atch 1), enacted 20 Dec 79 stated that:
The Administrator of Veterans' Affair shall cjesign a
protocol for and conduct an epidemiological study of
persons who, while serving in the Armed Forces of the
United States during the period of the Vietnam conflict,
were exposed to any of the class of chemicals known as
"the dioxins" produced during the manufacture of the
various phenoxy herbicides (including the herbicide known
as "Agent Orange") to determine if there may be longterm adverse health effects in such persons from such
exposure.
Accordingly, the present TOY was taken to VACO at the request of
Dr Paul A. Haber, M.D., Assistant Chief Medical Director for
Professional Services, VACO. Dr Haber requested that I brief
members of his staff and two special consultants (Drs Lilienfeld
and Suskind) on the use of herbicides in South Vietnam and on the
environmental fate of the phenoxy herbicides and TCDD. Drs
Lilienfeld and Suskind have been asked to design the protocol for
the epidemiologic study of veterans potentially exposed to Herbicide
Orange during combat operations during the Vietnam conflict. Information on how the herbicides were used and how persistent they
may have been in the environment, is critical in designing an appropriate epidemiologic protocol.
b. Immediately after my briefings, the group discussed the
following problems associated with preparing a protocol:
1. The study should focus on ground troopi; preferably
on both a group of Army personnel and a group of Mafine personnel.
Dr Haber, his staff, and Drs Lilienfeld and Suskind felt strongly
that the VA study should in no way conflict with the Air Force
study on RANCH HAND personnel (I was surprised that everyone
at the meeting thought that the Air Force study had already
received White House and Air Force clearance for sjtudy initiation,
and was in fact, underway). Dr Lilienfeld had high praise for the
Air Force protocol and felt that it was the only st[jdy where a
realistic exposure index could be developed.
2. The study should be prospective in nature. The VA would
make available 15 hospitals strategically located throughout the
United States for use in conducting physical examinations.
�3, The VA had recently completed examinations of 10,000 veterans
who alleged exposure to Herbicide Orange. Drs Lilienfeld and Suskind
were asked to consider (if possible) whether these people and their
exam data could be incorporated into an epidemiologic study.
4. Dr Haber stated that the VA was required to present the
protocol to the Director of the Office of Technology Assessment no
later than 20 Jun 1980. The protocol should have been reviewed by
at least a Committee appointed by the National Academy of Science,
and the VA Advisory Committees on Health-Related Effects of Herbicides
and the Cooperative Studies Guidance Group.
c. I was specifically asked to assist the VA in four areas:
1. Provide Dr Lilienfeld with pertinent literature on TCDD
toxicology and environmental fate (see Atch 2).
2. Obtain data from the Herbs tape on use of herbicides
in South Vietnam by Province, type, quantity, and month.
3. Prepare a literature update on human exposure to the
phenoxy herbicides and TCDD.
4. Prepare a model on the environmental fate of TCDD that may
provide an estimate of potential exposure, given that ground troops
entered a defoliated area at various times after aerial treatment
with Herbicide Orange.
The last three items will be officially requested from the Air Force
by VA. A letter from Dr Haber to HQ USAF/SG should be forthcoming.
A letter has been received by SAM/EK from Dr Haber related to my
assistance at the meeting of 7-8 Jan 80 (Atch 3).
4. Items of interest to procurement and USAFSAM programming: None
5. Information of interest to other USAFSAM divisions or branches: BRM
6. This TOY was performed in support of EPI Ranch project and at the
request of the VA.
7. This report jJo,es not contain information worthy of filing in TSK.
ALVIN L. YOUNG,'Major, USAF,"Ph.D.
Consultant, Environmental Sciences
3 Atch
1. PL 96-151
2. Ltr to Dr Lilienfeld
3. Ltr from Dr Haber
3
Cy to: HQ USAF/SGES
HQ AFSC/SG
�Pi,
H.E.3892
Binetpftth Congress of the United States of America
AT THE FIRST SESSION
Begun and held at the City of Washington on Monday, the fifteenth day of January,
one thousand nine hundred and seventy-nine
an act
To amend title 38, United States Code, to extend the authorizations of appropriations for certain grant programs and to revise certain provisions regarding such
programs, to revise and clarify eligibility for certain health-care benefits, to revise
certain provisions relating to the personnel system of the Department of Medicine
and Surgery, and to assure that personnel ceilings are allocated to the Veterans'
Administration to employ the health-care staff for which funds are appropriated;
to require the Veterans' Administration to conduct an epidemiologies! study
regarding veterans exposed to Agent Orange; and for other purposes.
Be it enacted by the Senate and House of Representatives of the
United States of America in Congress assembled, That (a) this Act
may be cited as the "Veterans Health Programs Extension and
Improvement Act of 1979".
(b) Whenever in this Act (except in section 306) an amendment or
repeal is expressed in terms of an amendment to, or repeal of, a
section or other provision, the reference shall be considered to be
made to a section or other provision of title 38, United States Code.
TITLE I—EXTENSION AND IMPROVEMENT OF CERTAIN EXPIRING VETERANS' ADMINISTRATION HEALTH PROGRAMS
GRANTS TO STATE HOME FACILITIES
SEC. 101. (a) Section 5033(a) is amended by striking out "and a like
sum for the succeeding fiscal year" and inserting in lieu thereof "a
like sum for each of the two succeeding fiscal years, and such sums as
may be necessary for the fiscal years ending September 30, 1981, and
September 30, 1982".
(bXD Section 641(a) is amended by striking out "$5.50", "$10.50",
and "$11.50" and inserting in lieu thereof "$6.35", "$12.10", and
"$13.25", respectively.
(2) The amendments made by paragraph (1) shall take effect on
January 1, 1980, but, with respect to fiscal year 1980, shall take effect
only to such extent and in such amounts as may be specifically
provided for such purpose in appropriation Acts.
EXCHANGE OF MEDICAL INFORMATION
SEC. 102. (a) Section 5054 is amended by adding at the end the
following new subsection:
"(c) The Administrator is authorized to enter into agreements with
public and nonprofit private institutions, organizations, corporations,
and other entities in order to participate in cooperative health-care
personnel education programs within the geographical area of any
Veterans' Administration health-care facility located in an area
remote from major academic health centers.".
(b) Section 5055(cXD is amended by inserting "and for each of the
three succeeding fiscal years" after "fiscal year 1979".
A^
�a R 8892-2
AMBIANCE TO HEALTH MANPOWER TRAINING mBTITOTIONS
SBC. 108. (a) Subsection (b) of section 5070 is amended to read as
follows:
"(b) The Administrator may not enter into any agreement under
•ubchapter I of this chapter after September 30,1979. .
(bXD Subsection (a) of section 6082 is amended to read as follows:
"(a) There is authorized to be appropriated for carrying out
programs authorized under this chapter $50,000,000 for the fiscal
year ending June 30,1973; a like sum for each of the six succeeding
fiscal years; $15,000,000 for the fiscal year ending September 30,1980;
$25,000,000 for the fiscal year ending September 30, 1981; and
$30,000,000 for the fiscal year ending September 30,1982.".
(2) Clause (1) of section 5083(b) is amended by striking out "and will
result" and all that follows in such clause through "at such school".
TITLE H—MODIFICATION OF VETERANS HEALTH CARE
AND RELATED BENEFITS
BENEFICIARY TRAVEL REIMBURSEMENT
SEC. 201. (a) Section lll(eX2XA) is amended byCD striking out "based on an annual declaration and certification by such person" and inserting in lieu thereof "pursuant to
regulations which the Administrator shall prescribe"; and
(2) striking out "a veteran" and all that follows through "title"
and inserting in lieu thereof "a person receiving benefits for or in
connection with a service-connected disability under this title, a
veteran receiving or eligible to receive pension under section 521
of this title, or a person whose annual income, determined in
accordance with section 503 of this title, does not exceed the
maximum annual rate of pension which would be payable to such
person if such person were eligible for pension under section 521
of this title".
(b) Section 601 is amended byCD striking out "transportation" in paragraph (5XA) and
inserting in lieu thereof "travel";
(2) striking out subclause (ii) of paragraph (5XO and inserting
in lieu thereof "(ii) travel and incidental expenses for such
dependent or survivor under the terms and conditions set forth
in section 111 of this title"; and
(3) striking out "necessary expenses of travel and subsistence"
in paragraph (6XB) and inserting in lieu thereof "travel and
incidental expenses".
(c) Section 614 is amended byCD striking out "necessary travel expenses" in subsection (a)
and inserting in lieu thereof "travel and incidental expenses
(under the terms and conditions set forth in section 111 of this
title)"; and
(2) striking out "all necessary travel expenses" in subsection
(b) and inserting in lieu thereof "travel and incidental expenses
(under the terms and conditions set forth in section 111 of this
title)".
(d) Section 628(a) is amended by striking out "the necessary travel"
and inserting in lieu thereof "travel and incidental expenses under
the terms and conditions set forth in section 111 of this title".
�H.R. 8892-3
CONTRACT HOSPITAL CARE
SEC. 202. Section 601(4XCXiii) IB amended byCD striking out "hospital care" the second place it appears and
inserting in Beu thereof "medical services"; and
(2) inserting "until such time as the veteran can be safely
transferred to any such facility" after "of this paragraph .
LIMITATION ON FURNISHING CONTRACT CARE DENTAL TREATMENT
SEC. 203. Section 612(b) is amended by adding at the end below the
last clause the following new sentence: "The total amount which the
Administrator may expend for furnishing, during any twelve-month
period, outpatient dental services, treatment, or related dental appliances to a veteran under this section through private facilities for
which the Administrator has contracted under clause (i), (ii), or (y) of
section 60K4XC) of this title may not exceed $500 unless the Administrator determines, prior to the furnishing of such services, treatment, or appliances and based on an examination of the veteran by a
dentist employed by the Veterans' Administration (or, in an area
where no such dentist is available, by a dentist conducting such
examination under a contract or fee arrangement), that the furnishing of such services, treatment, or appliances at such cost is reasonably necessary.".
HEALTH BENEFITS FOR VETERANS OF MEXICAN BORDER PERIOD AND
WORLD WAR I AND FOR CERTAIN SEVERELY DISABLED VETERANS
SEC. 204. Section 612(g) is amended by—
(1) striking out "Where any veteran" and inserting in lieu
thereof "In the case of any veteran who is a veteran of the
Mexican border period or of World War I or who"; and
(2) adding at the end thereof the following new sentence: "The
Administrator may also furnish to any such veteran home health
services under the terms and conditions set forth in subsection (f)
of this section.".
AMENDMENTS TO CHAMPVA PROGRAM
SEC. 205. (aXl) Section 613(a) is amended by—
(A) striking out "wife" in clause (1) and inserting in lieu
thereof"spouse";
(B) striking out "and" at the end of clause (1);
(C) striking out "widow" in clause (2) and inserting in lieu
thereof "surviving spouse";
(D) inserting "and at the end of clause (2); and
(E) inserting after clause (2) the following new clause:
"(3) the surviving spouse or child of a person who died in the
active military, naval, or air service in the line of duty and not
due to such person's own misconduct,".
(2) Section 613 is further amended by adding at the end the
following new subsection:'
"(c) For the purposes of this section, a child between the ages of
eighteen and twenty-three (1) who is eligible for benefits under
subsection (a) of this section, (2) who is pursuing a full-time course of
instruction at an educational institution approved under chapter 36
of this title, and (3) who, while pursuing such course of instruction,
incurs a disabling illness or injury (including a disabling illness or
injury incurred between terms, semesters, or quarters or during a
�H.R. 8892-4
vacation or holiday period) which is not the result of such child's own
willful misconduct and which results in such child's inability to
continue or resume such child's chosen program of education at an
approved educational institution shall remain eligible for benefits
under this section until the end of the six-month period beginning on
the date the disability is removed, the end of the two-year period
beginning on the date of the onset of the disability, or the twentythird birthday of the child, whichever occurs first.".
(b) The amendments made by subsection (a) shall take effect with
respect to fiscal year 1980 only to such extent and for such amounts as
may be specifically provided for such purpose in appropriation Acts.
DATE
SEC. 206. Except as otherwise provided in section 205(b), the
amendments made by this title shall take effect on January 1,1980.
TITLE HI—VETERANS' ADMINISTRATION MEDICAL PERSONNEL AMENDMENTS AND MISCELLANEOUS PROVISIONS
MEDICAL PERSONNEL STAFFING LEVELS
SEC. 301. (a) Section 5010(a) is amended by adding at the end the
following new paragraph:
"(4XA) With respect to each law making appropriations for the
Veterans' Administration, there shall be provided to the Veterans'
Administration the funded personnel ceiling defined in subparagraph (D) of this paragraph and the funds appropriated therefor.
"(B) In order to carry out the provisions of subparagraph (A) of this
paragraph, the Director of the Office of Management and Budget
shall, with respect to each such law (i) provide to the Veterans'
Administration for the fiscal year concerned such funded personnel
ceiling and the funds necessary to achieve such ceiling, and (ii)
submit to the appropriate committees of the Congress and to the
Comptroller General of the United States certification that the
Director has so provided such ceiling. Not later than the thirtieth day
after the enactment of such a law or, in the event of the enactment of
such a law more than thirty days prior to the fiscal year for which
such law makes such appropriations, not later than the tenth day of
such fiscal year, the certification required in the first sentence of this
subparagraph shall be submitted, together with a report containing
complete information on the personnel ceiling that the Director has
provided to the Veterans' Administration for the employees
described in subparagraph (D) of this paragraph.
"(C) Not later than the forty-fifth day after the enactment of each
such law, the Comptroller General shall submit to the appropriate
committees of the Congress a report stating the Comptroller General's opinion as to whether the Director of the Office of Management
and Budget has complied with the requirements of such subparagraph in providing to the Veterans' Administration such funded
personnel ceiling.
"CD) For the purposes of this paragraph, the term 'funded personnel ceiling' means, with respect to any fiscal year, the authorization
by the Director of the Office of Management and Budget to employ
(under the appropriation accounts for medical care, medical and
prosthetic research, and medical administration and miscellaneous
operating expenses) not less than the number of employees for the
�H.R. 3892-5
employment of which appropriations have been made for such fiscal
(b) The amendment made by subsection (a) shall take effect with
respect to Public Law 96-103, but, with respect to such Public Law,
the certification and report required by subparagraph (B) of paragraph (4) of section 5010 of title 38, United States Code (as added by
sucn amendment), and the report required by subparagraph (C) of
such paragraph (as added by such amendment) shall be submitted to
the appropriate committees of the Congress not later than January 15,1980, and February 1,1980, respectively.
QUALIFICATIONS OF CERTAIN HEALTH PROFESSIONALS EMPLOYED IN THE
DEPARTMENT OF MEDICINE AND SURGERY
SEC. 302. (a) Section 4104(2) is amended by inserting "psychologists," after "Pharmacists,".
(bXD Subsection (a) of section 4105 is amended by—
(A) striking out the period at the end of clause (9) and inserting
in lieu thereof a semicolon; and
(B) adding at the end the following new clause:
"(10) Psychologist—
"hold a doctoral degree in psychology from a college or
university approved by the Administrator, have completed
study for such degree in a specialty area of psychology and
an internship which are satisfactory to the Administrator,
and be licensed or certified as a psychologist in a State,
except that the Administrator may waive the requirement of
licensure or certification for an individual psychologist for a
period not to exceed two years on the condition that such
psychologist provide patient care only under the direct
supervision of a psychologist who is so licensed or certified,".
(2) Subsection (b) of such section is amended by inserting "podiatrist, optometrist," after "dentist,".
(c) The amendment made by subsection (bXD to require that a
psychologist appointed to a position in the Department of Medicine
and Surgery of the Veterans Administration be licensed or certified
as a psychologist in a State shall not apply to any person employed as
a psychologist by the Veterans' Administration on or before December 81,1979.
REDUCTION OF PROBATIONARY PERIOD FOR CERTAIN HEALTH PROFESSIONALS EMPLOYED IN THE DEPARTMENT OF MEDICINE AND SURGERY
SEC. 303. Section 4106(b) is amended by striking out "three years"
and inserting in lieu thereof "two years".
COOPERATIVE USE AGREEMENTS FOR SPECIALIZED MEDICAL RESOURCES
SEC. 304. Section 5053(a) is amended by inserting "or organ banks,
blood banks, or similar institutions" after "facilities".
SPECIAL MEDICAL ADVISORY GROUP AMENDMENTS
SEC. 305. Section 4112(a) is amended by—
(1) inserting in the first sentence "and a disabled veteran"
after "professions"; and
(2) inserting in the second sentence "and, not later than
February 1 of each year, shall submit to the Administrator and
�H.R. 3892-6
the Congress a report on its activities during the preceding fiscal
year" after "Administrator".
TECHNICAL AMENDMENT
SEC. 306. (a) Section 601(aX2) of the Veterans' Disability Compensation and Survivors' Benefits Amendments of 1979 (Public Law
96-128) is amended by striking out "clause (1)" and inserting in lieu
thereof "clause (11)".
(b) The amendment made by subsection (a) shall take effect as of
November 28,1979.
AGENT ORANGE STUDY
SEC. 307. (aXl) The Administrator of Veterans' Affairs shall design
a protocol for and conduct an epidemiological study of persons who,
while serving in the Armed Forces of the United States during the
period of the Vietnam conflict, were exposed to any of the class of
chemicals known as "the dioxins" produced during the manufacture of
the various phenoxy herbicides (including the herbicide known as
"Agent Orange") to determine if there may be long-term adverse
health effects in such persons from such exposure. The Administrator
shall also conduct a comprehensive review and scientific analysis of
the literature covering other studies relating to whether there may
be long-term adverse health effects in humans from exposure to such
dioxins or other dioxins.
(2XAXi) The study conducted pursuant to paragraph (1) shall be
conducted in accordance with a protocol approved by the Director of
the Office of Technology Assessment.
(ii) The Director shall monitor the conduct of such study in order to
assure compliance with such protocol.
(BXi) Concurrent with the approval or disapproval of any protocol
under subparagraph (AXi), the Director of the Office of Technology
Assessment shall submit to the appropriate committees of the Congress a report explaining the basis for the Director's action in
approving or disapproving such protocol and providing the Director's
conclusions regarding the scientific validity and objectivity of such
protocol.
(ii) In the event that the Director has not approved such protocol
during the one hundred and eighty days following the date of the
enactment of this Act, the Director shall (I) submit to the appropriate
committees of the Congress a report describing the reasons why the
Director has not given such approval, and (II) submit an update
report on such initial report each sixty days thereafter until such
protocol is approved.
(C) The Director shall submit to the appropriate committees of the
Congress, at each of the times specified in the second sentence of this
subparagraph, a report on the Director's monitoring of the conduct of
such study pursuant to subparagraph (AXii). A report under the
preceding sentence shall be submitted before the end of the six-month
period beginning on the date of the approval of such protocol by the
Director, before the end of the twelvemonth period beginning on
such date, and annually thereafter until such study is completed or
terminated.
(3) The study conducted pursuant to paragraph (1) shall be continued for as long after the submission of the report under subsection
(bX2) as the Administrator may determine reasonable in light of the
possibility of developing through such study significant new information on the long-term adverse health effects of exposure to dioxins.
�H.R. 3892-7
(bXD Not later than twelve months after the date of the enactment
of this Act, the Administrator shall submit to the appropriate
committees of the Congress a report on the literature review and
analysis conducted under subsection (aXl).
(2) Not later than twenty-four months after the date of the approval
of the protocol pursuant to subsection (aX2XAXi) and annually thereafter, the Administrator shall submit to the appropriate committees
of the Congress a report containing (A) a description of the results
thus far obtained under the study conducted pursuant to such
subsection, and (B) such comments and recommendations as the
Administrator considers appropriate in light of such results.
(c) For the purpose of assuring that any study carried out by the
Federal Government with respect to the adverse health effects in
humans of exposure to dioxins is scientifically valid and is conducted
with efficiency and objectivity, the President shall assure that—
(1) the study conducted pursuant to subsection (a) is fully
coordinated with studies which are planned, are being conducted, or have been completed by other departments, agencies,
and instrumentalities of the Federal Government and which
pertain to the adverse health effects in humans of exposure to
dioxins; and
(2) all appropriate coordination and consultation is accomplished between and among the Administrator and the heads of
such departments, agencies, and instrumentalities (that may be
engaged, during the conduct of the study carried out pursuant to
subsection (a), in the design, conduct, monitoring, or evaluation
of such dioxin-exposure studies.
(d) There are authorized to be appropriated such sums as may be
necessary for the conduct of the study required by subsection (a).
Speaker of the House of Representatives.
Vice President of the United States and
President of the Senate.
�DEPARTMENT OF THE AIR FORCE
USAF SCHOOL OF AEROSPACE MEDICINE (AFSC)
BROOKS AIR FORCE BASE, TEXAS
76235
11 Jan 80
Dr Abraham Lilienfeld, M.D.
Professor Epidemiology
The John Hopkins School of Hygiene
615 North Wolfe Street
Baltimore MD 21205
Dear Dr Lilienfeld
It was a pleasure to have again met with you on the Herbicide
Orange issue. I hope my discussions with you on the use of
herbicides in South Vietnam, and on the environmental fate of
dioxin, were of value in developing ideas for designing the
epidemiologic study. I have attached the following source
material for potential use in the protocol:
Atch 1. IRAC Report of PCDD and PCDF.
Atch 2. Chapter III, Effects of Herbicides in South
Vietnam.
Atch 3. Position Paper on Criteria for Estimating Exposure.
Atch 4. Technical Report OEHL-TR-78-92 (3 copies).
If I can provide you further assistance in the development of a
protocol, please contact me.
Sincerely
ALVIN L. YOUNG, Major, USAF, Ph.D.
Environmental Sciences Consultant
4 Atch
1. IRAC Report
2. Effects of Herbicides
3. Position Paper
4.
OEHL-TR-78-92
Cy to: Dr. R. Levinson
U t.
�Department of Medicine
and Surgery
Washington, D.C. 20420
Veterans
Administration
January 9, 1980
Col. George B. Lathrop, USAF, MC
C h i e f , Epidemiology
Division
USAFSAM/EK
Brooks Air Force Base, TX 78235
Dear Colonel Lathrop:
I wish to express my deep appreciation to you for permitting Major Alvin Young to participate in our January 7th
meeting on the design of our Agent Orange epidemiology
s tudy.
Major Young's detailed knowledge about the scope and
purpose of the Agent Orange spraying missions and on the
environmental fate of dioxins was invaluable to our
deliberations. In fact, I can unequivocally state that as
a result of Major Young's consultations, we will be able
to significantly expedite the study's development.
I sincerely hope that we might be able to make use of
Major Young's services again in the future. In addition,
I would like to invite you to give us the benefit of your
personal epidemiological expertise when we have proceeded
further in the study's preliminary design.
I confidently predict that the epidemiological studies
being planned by the Air Force and the Veterans
Administration will resolve the question of Agent Orange
effects on human health. By combining resources, we
should be enabled to meet our respective goals sooner.
Best wishes.
Sincerely yours,
PAUL A. L. HABER, M.D.
Assistant Chief Medical Director
for Professional Services
In Reply Refer To:
A.4, flu
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
205
Folder
The folder containing the original item.
5766
Series
The series number of the original item.
Series VIII Subseries III
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Date
A point or period of time associated with an event in the lifecycle of the resource
January 23 1980
Title
A name given to the resource
Trip Report - TA-16 (3 Jan 80)
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/742fb1feee1a8c20cef0b6e448ebec28.pdf
3884496b05eb23656880f4b3169be2f3
PDF Text
Text
item D Number
°5773
Author
Young, Alvin L.
D
Corporate Author
Report/Article Title Handwritten Notes on VA Meeting, Tuesday 3 February
Journal/Book Title
Year
Month/Day
Color
Number of Images
u
°
Descriptor) Notes
Thursday, March 28, 2002
Page 5773 of 5780
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Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
205
Folder
The folder containing the original item.
5773
Series
The series number of the original item.
Series VIII Subseries III
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Young, Alvin L.
Title
A name given to the resource
Handwritten Notes on VA Meeting, Tuesday 3 February 1981
ao_seriesVIII
-
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
Box
The box containing the original item.
201
Folder
The folder containing the original item.
5681
Series
The series number of the original item.
Series VIII Subseries II
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Creator
An entity primarily responsible for making the resource
Zamichow, Nora
Source
A related resource from which the described resource is derived
Washington Post
Date
A point or period of time associated with an event in the lifecycle of the resource
April 30 1986
Title
A name given to the resource
Agent Orange and the Noncombatants: Women of the USO and Red Cross Seek to Prove Injury from the Chemical
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/275a1f49f7c80beedcc0cd2c68b09c01.pdf
e89f4b65ea59039880296f174f6c5cf3
PDF Text
Text
Item D Number
05168
D
Author
Corporate Author
Report/Article Title
Chemical-Biological Warfare: U. S. Policies and
International Effects: Hearings before the
Subcommittee on National Security Policy and
Scientific Developments of the Committee on Foreign
Affairs, House of Representatives, Ninety-First
Congress, First Session
Journal/Book Title
Year
1969
Month/Day
November/December
Color
D
Number of Images
°
November 18, 20; December 2, 9, 18, and 19, 1969.
Friday, March 01, 2002
Page 5168 of 5263
�05. POLICIES ..AND INTERNATIONAL EFFECTS
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of the downed men. This protects the
well us the downed airmen from aimed
wearing protective masks then extracts
are many documented examples of such
ivscne crew and aircraft as
enemy lire. The rescue team
the downed crewmen. There
successful operations.
srSIMAKV
The riot control agent, CS, has liccomc n lifesaving part of military
o|H>nitions in Vietnam. C\, the older agent, IKVHUSC of its relative
ineffectiveness, is now seldom used. The use of CS in combat ojorations clearly reduces casualties among friendly troops, permits extraction of civilians who mav be under enemy control often without casualties, and frequently allows the enemy the option of capture rather
t h a n casualties. Perhaps the most valid indication of the effectiveness
of CS in combat operations is that U.S. ]>ersonnel continue to carry
('S grenades to the Held in lieu of some of their normal high explosive
ammunition, and ground commanders often call for CS rather than
high explosives. ]{iot control agents arc a valuable aid in accomplishing our mission and in protecting our forces.
IIKRIUCIUK.K
Turning now to the use of herbicides in Vietnam, one of the mosr
difficult problems of military oj>erationri in South Vietnam is the
inability to observe the ene.my in the dense forest and jungle. Defoliating herbicides introduced in l!Mi:i are capable of producing n significa,nt improvement in vertical and horizontal visibility in the ty]>e
of jungle, found in South Vietnam. As viewed by an aerial < observer,
it. is often ini]K>ssihlc to see through the canopy to detect ^"(' or XVA
o|»erationp. In (> to > we»>k>, a f t e r spraying with a herbicide, the
observer will have jjood observation throujrh the canopy. rFor ground
obser\-ation. defoliation is highly elfe<'tive in impro\"iii^ horixontal
visibility.
The herbicide program in terms of effects produced has required
an unusually small investment of military effort. The entire program
has |K*'II accompli shed with an average of alx>ut 17 C-1'23 spray aircraft and several smaller helicopter sprayers plus some improvised
"round-spray equipment.
Herbicide, operations are conducted under a program directed by the
Government of South Vietnam. Keqncsts for these operations <renerally originate at the district or provincial level and are submitted
through territorial administration command channels. The herbicide
spray plan, includes as a m i n i m u m the area requested to l>e treated
with herbicide, the public information, civil affairs, and intelligence
annexes, along with a statement by the province chief tiiat he will see
that just and legal claims are paid for any accidental damage. The
ARVX corps commanders and their U.S. corps senior advisers have
been delegated authority to approve small scale defoliation by groundbased spray and by helicopters. All requests for crop destruction and
larger scale defoliation by T-12:? aircraft are forwarded to the Vietnamese. Joint (Jeneral Staff. Upon approval of the request by the. Chief
of Joint General Staff it is forwarded to the MACV staff for final
review.
�The M A t ' V ;-f:ti! po>iiion is developed as the result of coordination
with ('OKI'S (Civil Operations and Revolutionary Development
Support). 1'>A1D. and political representatives at each level where
they exisl. An aerial reconnaissance is conducted as the next step to
insure that all populated areas and friendly crops have been excluded
from the, target ami. Having determined from this aerial survev and
an aiuilvsii- "! i!i»' military worth that the project is a valid herbicide
target, 'the pi->i*vt is forwarded to the tl.S. Amhassador and
( O.M1 "S.MA( V for ajiproval. The Ambassador personally approves
all C-}-2'-\ d e f o l i a t i o n projects and all enemy crop destruction projects.
" O M K SIT.ril-MC I SKS OK IIKUIIICHIKS
Some s|>e< me MM'- of hei'hici«les are :
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A portion of t h e -mall-scale ground ha«ed or the helicopter spray
mi-sioiis ar* 1 'j'Ctl in improving the defense of base camps and fire
bases, llei-birdie- aiv a great help in keeping down the growth of
high jungle .LTV:;—. bu>hes, and weeds which will prow in cleared areas
near these camp-. This clearance opens fields of tire and affords observ a t i o n for oiui'o-ts to prevent surprise a t t a c k and as such is truly a
life-sax ing mea-urc for our forces and our allies. Without the use
of herbicide- around our lire bases, adequate defense is difficult and ill
many pla«v* impossible.
'. Jtl fltl'ilhi//'
n ' :nn > t>l
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'I here are m a n y inMances of ambush sites lx?ing defoliated for
bi'ltcr acriiil "ii~ri-> MI ion and inipi-oved visibility along roads and
trails. I n l i n - 7 t l i c i - . - \\ere also many requests for defoliation of VC
tax collection | , o . i i f - , . I n otherwise friendly territory theiv were points
alonjr w e l l - t ] - a \ I'lrd routes where the enemy could hide under cover
and inteivepi i r a u ' l e i x to demand taxes. Defoliation along these roads
\\ a< very effect \\\- in opening these areas so that they can be seen from
observation a i r - r a f t , and w i t h few exceptions these roads were opened
ro free t r a v e l . Tin 1 n-e of aircraft, to spray alongside lines of communication proved valuable in clearing these areas and preventing
costly a m l i n - h of a r m y convoys with resulting friendly casualties.
3. 1'tcftiHiitii,,, i,< 'nti!>fii!ioii r<ni/<'>-'
Areas used b\ t ; i c enemy for routes of approach, resupply or ino\ - ement are target^ for herbicide spray. 1'robably the most valuable use of
herbii-ide-s for d e f o l i a t i o n i s t o ] > e r m i t aerial observation in such areas.
TJiis is puiri' !i];:riy (rue in areas near the border so t h a t we can detect
movement of rii'-my u n i t s and t h e i r resupply.
!). ])cit>l'i<ttmn <•/' < in a, i/ IHIXI' fitmfix
We. know fi-oin pi-isoners of war and from observation that the
enemy w i l l mo\v from areas t h a t have lx>en sprayed. Therefore, enemy
base camp* or mot headquarters are sprayed in order to mnke him
move to m o i d exposing himself to aerial observation. If he does move
back in w h i l e '!."' area is s t i l l defoliated, lie w i l l be ol(served and can
be engagt'd.
�230
5. Crop destruction
Ci-ops in areas remote from the friendly population and known to
belong to the enemy and which cannot bo captured by ground operations are sometimes sprayed. Such targets are carefully selected so as
to attack only those crops known to be grown by or from the VC or
NYA. The authorization to attack crops in specific areas has been
made by the U.S. Embassy, Saigon, MACV and South Vietnamese
Government.
Frequent reviews have been conducted of the herbicide program.
Tlip most recent one was personally directed and reviewed by COMUSMACV in October 1968 to assure himself that the program was militarily effective. Prior to that, the U.S. Ambassador had directed a
review which looked at the political and economic aspects of the
program. The Embassy report was released in August 1968. The crop
destruction program was also received by the CINCPAC scientific adviser in December 1967. Each of these reports concluded that the progium should be continued.
Mr. ZABLOCKI. At this point, will you supply the Embassy report
and the OINCPAC scientific advisor report for the fiscal year 1967 if
you have it?
Admiral LEMOS. I do not have them with me, but I will supply them.
(The reports, which are classified, were provided to the subcommittee for its records.)
The requests for defoliation and crop destruction have always exceeded our capability to spray. The requirement continues although a
tapering off should develop if enemy activity subsides. A recent review
by MACV indicated that operations for 1970 will be less than in 1969.
In addition, Rome plows are replacing defoliation for clearing along
many lines of communication.
THE USE OF 2,4.r>-T IN VIETNAM
With regard to the recent publicity of the herbicide agent, 2,4,5-T,
which is a component of orange, a herbicide mixture, the bionetics research laboratories conducted a study of the carcinogenic, teratogenic
and mutagenic activity of selected pesticides and industrial chemicals
for the National Cancer Institute during the period 1965-1968. The
studv indicated that a largo dose of 2,4,5-T administered orally to
specific strains of mice during the central portion of the gestation
period produced abnormal fetuses.
However, on October 29,1969, Dr. DuBridge, science advisor to the
President, stated, "It seems improbable that any person could receive
harmful amounts of this chemical from any of the existing uses of
2,4.5-T."
Nevertheless, Deputy Secretary of Defense David Packard has issued instructions to the Joint Chiefs of Staff to reemphasize the
already existing policy that 2,4,5-T be utilized only in areas remote
from population.
When the American Embassy conducted the political economic review of the herbicide program, it requested that a disinterested expert
be sent from the United States to assess any ecological consequences
of the program. Dr. Fred Tschirley, Agricultural Research Service,
�231
Department of Agriculture, was sent over in March 1968 for a 1-nionth
period. Arrangements wore made which permitted him to fly over any
area of Vietnam he wished to inspect plus on-the-ground visits to any
safe area. Ho concluded that the defoliation program had caused some
ecological changes. Although single treatment on semideciduous forest
would cause iiK-onseguential changes, repeated treatments could kill
enough trees to permit invasion of many sites by bamboo. The presence
of bamboo would then retard regeneration of the forest.
The Army supports the need for a more detailed investigation of the
ecological effects of herbicides used in Vietnam. Such im investigation
should bo conducted in coordination with other interested agencies. In
being
hostilities phase.
EXAMPLES OF DEFOLIATION SUCCESS IN VIETNAM
In the final analysis the sole purpose of the herbicide program is to
protect friendly forces and conserve manpower. The following examples should demonstrate the success of the defoliation effort in
Vietnam:
1. Major defoliation has been accomplished in war zone C. Prior to
defoliation, seven brigades were necessary to maintain U.S./GVN
presence. During 1%7, after defoliation only three brigade; were
required.
'2. The commander of naval forces in Vietnam in a report to General
Abrams stated—
As you know, a major ooucern is the vegetation along the main shipping ehininel. Your continuing efforts under difficult and hazardous flying conditions, in
keeping this area and the adjacent inland areas devoid of vegetation have contributed considerably in denying the protective cover from which to ninbush the
slow-moving merchant ships and U.S. Navy craft.
3. In 1968, the commanding general of the 1st Field Force reported—
Defoliation has been effective in enhancing the success of allied combat operation*. Herbicide operations using C-12.H aircraft, helicopters, truck mounted and
hand sprayers have become un integral part of the II CTZ operations against
VC/NVA. The operations are normally limited to areas under VC/NVA control
remote from population centers. The defoliation program has resulted in the
reduction of enemy concealment and permitted increased use of supply routes by
friendly unit*. Aerial surveillance of enemy areas has Improved and less *«>curlt'y
forces are required to control areas of responsibility. An overall result of the
herbicide program 1ms been to increase friendly security and to assist in returning
civilians to QVN control.
4. The U.S. commander in the III CTZ related:
Herbicide operations have contributed significantly to allied combat operations
In the III Corps. Defoliation is an important adjunct to target acquisition.
Aerial photographs cau often be taken from which interpreters can "see the
ground" in areas that previously were obscured. Defoliation also aids visual
reconnaissance. U.S. Air Force FAC's (forward air controllers) and U.S. Army
aerial observers have discovered entire VC base camps in defoliated ureas
thnt had previously been overlooked.
5. In the south in the IV CTZ, C-123 herbicide operations are
limited. This is because of the vast areas of valuable crops which are
not to lie destroyed, even though they may be in enemy hands. There-
�232
fotv, the commander of the I V Corps area in presentinir his cvahiiition cited tlu>, value of helicopter operations as follows—
A significant helicopter defoliation mission was conducted in 'lie vicinity of
S A I » K < ! in August IIHIS. Tin- target area consisted of three ;u;iin canals which
converged and formed a strong VC base. The dense, vegetation permitted viability of only ]ft-in meters horizontally and nil vertically. Tin1 area was sprayed
with approximately "•".."> gallons of herbicide white and over 'HI percent of Hie
!ire:i V M S defoliated. As Hie result of the defolintion. an A K V N l i n i t n l i i n i Mas
able to remain overnight in I lie area for the first time iu ."> yi»jir« Many enemy
hnnk'Ts were open to observation. Since the defoliation, the VC presence hii-decreased to the point that only UF/1'K forces are now i.e.v-ary for local
security.
(i. As a part of (lit 1 1!K'»S evaluation report of her'>i.-idf operation*.,
the. 1T.S. senior adviser in the IV ('orps tactical /one .-HIM reported—
A section of Nntiomil Highway 4 in 1'hong Pinh 1'rovince v > . i - the site for a
defoliation o)K>ration on June 'J4. T.KiS. Since .lauuary ]!MN. :> »,>rii's of iiiiibusli"<
wns conducted against SVN convoys and troop movement*. B-v> :iu-e of the total
Inability of ground troojis to ket-p the area clear of VC. thN .-ir.'a was sprayed
lining 8W gallons of herbicide while. The target area was primarily coconut
piilni and banana trees that had been abandoned by their owneri for several
years. 1 luring the period of abandonment the vegetation had liec.mie so dense
that convoy security elements were not nble to wee more than r, meters Into the
underbrush and had to rely on reconnaissance by fire to dix'ovi-r the hidden
enemy. This method of protection had proven Ineffective. Three HK/I'F companies with I'.S. advisers were used to secure the target for :!it» helicopter operation in addition to an armored cavalry troop. Since the det'oliari.ni mission was
completed, convoys have used the highway two or three times a week without
attack or harassment. Only one RP plat<»on 1ms remained ia tli>- area to provide
local security to the hamlet and highway.
7. In certain iiir-lanci's, we know the VI 1 have IHVI-. I'mvctl to divert
tactical units from combat missions to food-piwmviuiMit operation?
and food-transportation tasks, attesting to the ertVtivencss of the
crop destruction program. In local areas where extensive crop destruction missions were conducted, VC/NVA defections to GVX increased as a result of low morale resulting p r i n c i p a l l y from food
shortages.
The most highly valued item of equipment to tield nmmanders in
Vietnam is the helicopter. There was some question when the helicopter spray equipment was first procured whether tield commander?
would divert the use, of helicopters from combat operations for herbicide spray operations. The very fact that the commanders have used
their helicopter spray equipment to the fullest and have asked for
more is certainly proof that herbicide oix'ratious h.ive been helpful
in protecting the, American soldier ami contributing to successful
accomplishment of the ground combat mission.
roxn.vsiox
Gentlemen, we have presented to you in as complete rind candid a
manner as possible the lifesavin«r usage that we have made of riot
control agents and herbicides in South Vietnam and the policies under
which this usage has taken place. We believe this usa^e has been wise
and has been accomplished with restraint. The result is that our forces
have been better able to accomplish their mission v / i t h Hgnilicantly
reduced U.S. and Viet namese casualties.
�233
ADVERSE EFFECTS (IK J IKRlllClDK I'SK'. OAMACE CLAIMS
Mr. XAHI.OCKI. Thank you for your statement, Admiral Lemos.
You have outlined the list's and the benefits of chemical agents in
Vietnam. Certainly there, must be adverse political and economic, results Ix'cunso of the use of herbicides and tear gases like CS.
Jlsive there not been instances of the misuse of herbicides and tear
ga>? Could yon give a view of those for the record of the subcommittee '.
For example, I understand Cambodia has a suit against the United
States for damage done because of the use, of herbicides. It is also my
understanding some South Vietnamese are going to seek damages because of loss of income or loss of property. I understand banana plantat ions have l>cen destroyed. That, would indie-ate then; are uses of
chemicals which have been adverse to our interests.
Admiral LEMOS. There have been some such instances.
Mr. ZAIJLOCKI. Could you tell the committee ulioiit how many? You
have given all the good side of it. I wouldn't expect yon to dwell at
length on the reasons why chemicals should not have been used, or
where errors were committed, but I think in fairness you ought to tell
us a little bit of both sides.
Admiral LEMOS. There have been some few instances and these
have been very carefully explored in the reviews that I have mentioned and mute clearly from the results of the reviews it has l>een
determined that in the overall context, the disadvantages and the few
abuses were very greatly outweighed by the very real advantages
provided to our forces and to the, Vietnamese Forces and the very
real difficulties they provided the VC and North Vietnamese Forces.
With respect to the Cambodian incident, this was a border incident.
As yon know, sir, there are stretches of the Viefnamese-Cambodian
border that are under dispute. They are not clearly marked in any way.
In situations such as this where you know there are, enemy base
camps, enemy forces operating in the jungle, and from which heavylire may be coming, spray operations close to the border have been
conducted. It would appear that in one of these cases either the spray
plane was in an area that was in dispute or was not clearly marked
and this situation was reviewed and as I understand it observers were
sent to the area and a report has been prepared. Whether that report
has been yet released, 1 do not know. I have not seen it.
HOW CLAIMS AUK PROCESSED AXI) PAID
Mr. ZABLOCKI. In your testimony, Admiral, you say that the province, chiefs supply the basic intelligence as to what areas herbicides
should be used in.
Admiral LEMOS. Yes.
Mr. ZARI.OCKI. Realizing the political problems Iwtween Caml>odia
und South Vietnam, could this have entered into the, selection of the
defoliation of areas along the lx>rder of Cambodia?
Admiral LEMOS. I am certain it has. As 1 have indicated, all C-12"
spray operations must have the clearance of the (rovernment of Viet-
�234
nam, of COMTTSMACV and of the Ambassador. All three must
approve C-123 operations.
Mr. ZABLOCKI. You also stated that the province chief must state
that he will see that just and legal claims are paid for any accidental
damage. Where is he going to get the money?
Admiral LEMOS. To the extent there are damages—-—
Mr. ZABLOCKI. From my knowledge of the South Vietnamese anything they have is received from us.
Admiral LEMOS. We do provide substantial support to the South
Vietnamese. The payments, however, are made by the Government
of Vietnam.
Mr. ZABLOCKI. But we pick up the tab?
Admiral LEMOS. Not entirely.
Mr. ZABLOCKI. Could you advise the Congress if we ultimately are
going to have war damage claims like we had in the Philippines
because of the damage to crops and industry? Do you anticipate
there will be such war damage claims?
Admiral LEMOS. I am not able to anticipate that kind of claim.
Mr. ZABLOCKI. In view of the fact that we were the defoliator and,
our U.S. Ambassador has been consulted and must give the order for
the use of herbicides, wouldn't it be logical that South Vietnamese
owners of plantations will be filing claims through the U.S. Embassy
the first opportunity they have ?
.
Admiral LEMOS. Thev have that opportunity now, sir.
Mr. ZABijOCKi. And they are doing so ?
Admiral LEMOS. J presume
Mr. ZABLOCKI. And when hostilities end we will have millions of
dollars in claims thrown at us. Has this been taken into consideration
by Defense Department when the decision was made to use herbicides ?
Admiral LEMOS. I am certain it was taken into consideration.
Part of the reason for the reviews was to consider specifically these
points.
INTEL1JGENCE ON AREAS DEFOLIATED
Mr. ZART-OCKI. In outlining the elaborate procedures which you have
taken in the use of herbicides, in the past, we were impressed—certainly
I was—with the lack of accurate ana hard intelligence in the Vieteonpheld areas.
How sure can the military be that fields that are selected by the
Vietnamese province chiefs are in Vietcong-held areas, and how can
the province chief know for sure that the crop is going to feed Vietcong soldiers? If we have such excellent intelligence where their Vietcong rice fields are, we should have better intelligence in other areas.
Admiral LEMOS. Intelligence is improving daily, as we move out
in the countryside and get more and more hamlets and villages under
the control of the Government of Vietnam and more and more people
willing to risk their lives to give information that points out where
the Vietcong are and where it is difficult to go.
DOD DECISION ON 2,4,B-T
Mr. ZABLOCKI. On page 14, you said that the Deputy Secretary of
Defense, David Packard, has issued instructions to the Joint Chiefs
�235
of Staff to reemphosize the already existing policy that 2,4,5-T shall
be used in areas remote from population. I thought there was a decision
not to employ 2,4,5-T in the future.
Admiral LEMOS. I am told there is no such decision.
'
Mr. ZABLOCKI. The use of 2,4,5-T is however, under review.
On page 15 you say a review of the ecological effects of the military
use of herbicides is now being initiated.
Admiral LEMOS. Yes. sir, that is the whole herbicide program and the
ecological effects of all herbicides.
Mr. ZABLOCKI. 2,4,5-T, it is my understanding, is the most potentially
dangerous to people, resulting hi the greatest political problems. Is that
not a fact?
Admiral LEMOS. That is my understanding, yes, sir.
DOD HERBICIDE STUDY
Mr. ZABLOCKI. The herbicide investigation project that you referred
to on page 15, could you give the subcommittee more details on this
project? It will be conducted by the Department of Defease or contracted out?
Admiral LEMOS. Initially in-house and then go on contract.
Mr. ZABLOCKI. Wouldnx it be preferable to reverse the order and
have some uninvolved group study the ecological effects of the military use of herbicides in Vietnam, and then have the military review
the report?
Admiral LT.MOS. Mr. Hnyward yon have any comment on that?
Mr. ZABLOCKI. Wouldn't that be advisable ?
STATEMENT OF ALBERT E. HAYWARD, ASSISTANT DIRECTOR,
CHEMICAL TECHNOLOGY, DEFENSE RESEARCH AND ENGINEERING, DEPARTMENT OF DEFENSE
Mr. HAYWARD. Mr. Chairman, some time ago in our discussions with
the American Association for the Advancement of Science, the Department of Defense agreed in principle that this type of survey should be
done, and we have every intention of inviting this association, and also
of inviting the National Academy of Sciences and other scientific
groups, if they would participate in this survey.
The project which Admiral Lemos referred to is being started or
initiated now. I believe that our activities during the remainder of this
fiscal year will be largely in planning and in reviewing the things that
have been done. I can assure you that uninvolved scientific bodies will
be invited to participate in this.
Mr. ZABLOCKI. At a later date.
Mr. HAYWARD. Within the next few months.
They should be involved in the planning as well as the discussion,
sir. So it will be done within a reasonably snort time period.
Mr. ZABLOCKI. Some are hopeful that by then it may be a moot
question.
I have a call, will you excuse me ? Mr. Fraser . . .
Admiral LEMOS. Surely.
-70
10
�253
all circumstances to live up to the Geneva Convention if it didn't
prove convenient, satisfactory, or necessary ?
Admiral LKMOS. All I can say is that all of the. efforts against
North Vietnam, including our planning of it, were subjected to
the very highest review in all responsible agencies of the Government. I cannot conceive of the approval of plans in direct violation
of that convention.
Mr. FULTON. But you also said, when I brought up the word "consideration," that there may have l>een alternative plans under consideration although not used for the. bombing of North Vietnam dikes
that are for the main purpose of food production.
Admiral LF.MOS. Mr. Forman correctly analyzed my problem when
he said I misinterpreted what you are saying. There are a number of
dams in North Vietnam not directly related to the problem you
propose.
Mr. FULTON. We are really trying here to get the basis for the policy
that is in effect and the limitations on the policy under international
law. So my question is based really on finding out whether the policy
just "growed" like Topsy or it was an express lilting the policy in, the
U.S. military, under the Geneva Convention.
That is all, Mr. Chairman.
Admiral LEMOS. I don't know how to answer that question except to
say that (he military do not of themselves do things in violation of
U.S. policy.
Mr. FULTON. No, I agree with you thoroughly, but that's the other
side of (he coin. I am saying, how did we in the United States as a
military decision, arrive at this particular policy? How was the policy
arrived at?
Admiral LF.MOS. The policy not to bomb ?
Mr. FULTON. The dikes of North Vietnam that have to do with food
production?
Admiral LEMOS. I am very certain it was arrived at on the basis of
consideration of this convention.
Mr. FFI.TOX. So then there were, no plans ever us an alternative, by
the U.$. military, even considered for the bombing of dikes of North
Vietnam that are used primarily for food production ?
Admiral LF.MOS. In my capacity I can say clearly that I am unaware
of any such plans
Mr. FULTON. May we have (he information supplied for the record,
Mr. Chairman?
Mr. ZAULOCKI. Very well.
(The information follows:)
Quention. ffistorji of plan* for Vomiting of dnnin in Kortli Vietnam ax an anticroi> operation f
Answer. (Statement for record). The Department of Defense policy Is not to
fllvuljre drtnils of military plans.
A , ,i
HOW MANY DETOLIATTON CLAIMS?
Mr. ZAIILOCKI. I would like to clarify something. On page 10, Admiral, you list the procedure under which herbicide spray is used. T
refer again to the payment of legal claims. "The province chief will
sec that claims are paid for unjust and illegal damage."
�254
I nni not sure, when you answered my question wirlier, you understood what J meant /<> ask. On whose behalf was he making >hat
pledge? His Government? Our Government?
Admiral LKMOS. His Government.
Mr. ZAIIUM-KI. T believe I did ask whether you would supply for the
record, Admiral, how many claims have l>cen made.
Admiral LKMOS. I don't'recall.
Air. ZA»I,OCKI. And what will be the procedure for paying them?
I tun personally satisfied that if the claims are made to the province
chief he will somehow pot it to the Saigon Government, and they will
pet to the tLS. Government and we will be picking 1 up the tab. Please
supply for the. cximmittee this particular answer. It may be a very
controversial issue some day.
Admiral LKMOS. Yes, sir.
(The information follows:)
Question. Jivmltrr of claim* submitted for damage from herbicide and crop
damagct Procedure* lor paying these claimsT
Answer (statement for record). The US Foreign Claims Commission in RVN
has received 41) claims from residents of the RVN in the nmouiit of $6G,81C (US
dollars) for herbicide damages. The 43 claims adjudicated so far have been
denied cither hwaiisc such diiningc wan combat related or <l,>iiiinge observe*! was
not caused by dcfolianls. Claims ugainst the US Government may be payable
for certain accidental defoliation damage resulting from u spray aircraft
mishap.
Herbicide claims from Vietnamese and foreign nationals residing in the RVN
are normally made against the GVN and are handled in GVN channels. Prior
to the initiation of herbicide operations, the respective GVN province chief
requests such o|>erations and pledges indemnification for accidental damage to
croi»s and trees of friendly citizens. Procedures require that such claims be
initiated at District level and processed to Province headquarters for adjudication up to an amount of 100,000 piasters. (One US dollar equals 118 plasters).
Claims of an amount greater thnn 100,000 piasters are processed to Headquarters,
Joint General Stuff and the Ministry of I>efense for adjudication on a case by
case basis. Maximum compensation payment is 500,000 piasters. The GVN programs handle the great bulk of crop damage claims in RVN'; however, if a local
national insists on filing a claim against the United States because of defoliation
damages to his projierty. his claim is accepted and investigated. The claim and
report of investigation are referred to a US Foreign Claims Commission appointed by COMUSMACV for adjudication under the authority of the Foreign
Claims Act, 10 USC 2734 and PL 00-521.
Since the defoliation claims program is being kept in GVN channels and an
active claims program is being kept in operation by the. GVN, an influx of
compensable claims against the US Government generated by the potential
termination of hostilities is not expected.
Mr. ZABLOOKI. If there are no further questions, on behalf of myself
and the entire snlx'-ommittee, Admiral, I want to thank you for your
excellent, presentation and for the direct answers you have given to
the questions that were asked of you.
In view of the fact that certain matters are classified which you
will supply to the committee for the record, I see no reason to go
into executive session.
Therefore, this concludes the hearings of the sul>c.ommittee on the
international aspects of chemical-biological warfare. The subcommittee will stand adjourned until further notice.
Thankjou, Admiral.
(Whereupon, at 12:35 p.m., the subcommittee was adjourned until
further notice.)
�
Dublin Core
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Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
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176
Folder
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5168
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Series VIII Subseries I
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November/December 1969
Title
A name given to the resource
Chemical-Biological Warfare: U. S. Policies and International Effects: Hearings before the Subcommittee on National Security Policy and Scientific Developments of the Committee on Foreign Affairs, House of Representatives, Ninety-First Congress, First S
ao_seriesVIII
-
https://www.nal.usda.gov/exhibits/speccoll/files/original/f10d95c8bf5c82674a141435ef70ba84.pdf
8604d2ceb8051a862e612ef5991a4bcc
PDF Text
Text
Item D Number
°5169
D
Author
Corporate Author
Report/Article Tltto
Effects of
2,4,5-T on Man and the Environment:
Hearings Before the Subcommittee on Energy, Natural
Resources, and the Environment of the Committee on
Commerce, United States Senate, Ninety-First
Congress, Second Session, on Effects of 2,4,5-T on
Man and the Environment.
Journal/Book Tltto
Year
197
Month/Day
A
Color
°
P"'
D
Number of manes
°
Descrtaton Notes
ltems 475 612 629 795 8S5 1074 1142 and 2926 are
>
-
-
-
-
each pieces of this full report.
Friday, March 01, 2002
'
>
Page 5169 of 5263
�EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT •>
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SUBCOMMITTEE ON ENERGY, NATURAL
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EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT
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�CONTENTS
Opening statement by the chairman
1
CHRONOLOGICAL LIST OF WITNESSES
APRIL 7, 1970
Bayley, Dr., Ned D., director, Science and Education, Department of
Agriculture; accompanied by Dr. T. C. Byerly, assistant director
Letter of January 7, 1970
Prepared statement
Kotin, Dr., Paul, director, National Institute of Environmental Health
Sciences
Turner, James, Center for Study of Responsive Law, Washington, D.C_.
Letter of April 30. 1970
Wellford, Harrison, Center for Study of Responsive- Law, Washington,
D.C
Letter of April 30, 1970
Westing, Dr. Arthur II., chairman, Biology Department, Windham College, Putney, Vt
COMMITTEE ON COMMERCE
WARREN G. MAGNUSON, Washington, Chairman
NORHIS COTTON, New Hampshire
JOHN 0. PASTORE, Rhode Island
HUGH SCOTT, Pennsylvania
VANCE IIAKTKK, Indiana
WINSTON rilOUTY, Vermont
1'IIILir A. HART, Michigan
JAMES B. PEARSON, Kansas
HOWARD W. CANNON, Nevada
ROBERT P. GRIFFIN, Michigan
RUSSELL B. LONG, Louisiana
HOWARD H. BAKER, JR., Tennessee
FRANK E. MOSS, Utah
CHARLES E. GOODELL, New York
ERNEST F. HOLLINGS, South Carolina.
MARLOW W. COOK, Kentucky
DANIEL K. INOUYE, Hawaii
JOSEPH D. TYDINGS, Maryland
WILLIAM B. SPONG, Ja., Virginia
FRBDKRICK J. LOHDAN, Staff Director
M I C H A E L PEHISCHDK, Chief Counsel
LEONARD BICKWIT, Jr., Staff Counsel
AiiTiimi I'ANKOPF, Jr., Minority Staff Director
J. PAUL MOLLOV, Minority Staff Counsel
32
38
74
87
18
468
6
468
76
APRIL 15, 1970
Epstein, Dr. Samuel S., Children's Cancer Research Foundation, Inc.,
and Harvard Medical School, Boston, Mass
Appendix I
Appendix II
Johnson, Dr. Julius E., vice president and director of research, the Dow
Chemical Co.; accompanied by Etcyl Blair, director Dow Agricultural
Chemical Research, V. K. Rowe, director, Dow Toxicological Laboratory,
arid George Lynn, director, Government Regulatory Relations, tlie
Dow Chemical Co
Steinfeld, Dr. Jesse, Surgeon General, Department of Health, Education,
and Welfare; accompanied by Dr. David Gaylor, Dr. Diane Courtney,
and Dr. Dale Lindsay
_~_
Verrett, Dr. Jacqueline, Food and Drug Administration, Department of
Health, Education, and Welfare
SUIICOMMITTEE ON ENERGY, NATURAL RESOURCES, AND THE ENVIRONMENT
PHILIP A. HART, Michigan, Chairman
FRANK E. MOSS, Utah, Vice Chairman
JOHN O. PASTORE, Rhode Island
HOWARD H. BAKER, JR., Tennessee
KURSKLL B. LONG, Louisiana
CHARLES E. GOODELL, New York
JOSEl'H D. TYDINGS, Maryland
HUGH SCOTT, Pennsylvania
WILLIAM B. SPONQ, JB., Virginia
MARLOW W. COOK, Kentucky
I
I
1
|
|
i
ADDITIONAL ARTICLES, LETTERS, AND STATEMENTS
A reporter at large: Defoliation, article from the New York Times
Byerly, Dr. T. C., assistant director, Science and Education, Department
of Agriculture, letter of April 21, 1970
Chemical and Toxicological Evaluations of Isolated and Synthetic Chloro
Derivatives of Dibenzo-p-dioxin, article from Nature
Chick Edema Factor, article.
Chick Edema Factor: Sonic Tissue Distribution Data and Toxicologic
Effects in the Rat and Chick, article
-.
Clinical Picture and Etiology of Chloracne, article
Collabotative Bioassay for Cluck Edema Factor, article
Gushing, R. L., on behalf of the Hawaiian Sugar Planters' Association,
Honolulu, Hawaii, statement
Decontamination of Pesticides in Soils, article from Residue Reviews
Defoliants, Deformities: What Risk? article
Determination of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in 2,4,5-Trichlorophenoxyacctic Acid by Gas-Liquid Chromatography, letter of June 22,
1965
(Hi)
405
419
431
360
167
190
107
467
326
204
308
330
227
469
384
104
367
�DuBridge, Or. Lee A., Director, Office of Science and Technology, DepartIlcallh, Education, and Welfare, statement
452
l Persistence of Herbicides Applied to Soil in Puerto llican Forests,
from Weed Science
47
Rlectron Microscopic Alterations in the Liver of Chicken Fed Toxic Fat,
article
303
'Growth of Crops in Soils After Hcrbicidal Treatments for Brush Control in
the Tropics, article from Agronomy Journal
45
Hays, Harry W., Ph. D., Director, Agricultural Research Service, Pesticides
Regulation Division, U.S. Department of Agriculture, letter of November (I, 190G
12
'Herbicides in Soils, article from Agricultural Research Service
57
Identification and Crystal Structure of a llydropericardium-Produeing
Factor: 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin, article from Acta Crystallngrapilica
.
330
Injection of Chemicals Into the Yolk Sac of Fertile, Kggs Prior to Incubation as a Toxicity Test, article from Toxicology and Applied Pharmacology
278
Internal Preliminary Report Analysis of Commercial Chlorophenols for
Trace Amounts of Their Condensation and Polymerization Products,
article
328
Lawrence, J. F., Brig. Gen., U.S. Marine Corps, Deputy Assistant to the
Secretary for Legislative Affairs, Office of the Secretary of Defense, letter
of Aprif'21, 1970
467
Light and Electron Microscopic Observations in Macaco, mulaUa Monkeys
Fed Toxic Fat, article
314
Lipsnn, Dr. Steven, chief. Division of Epidemiology and Surveillance,
Montgomery County Health Department, letter of March 17, 1970
• 17
McCarthy, Hon. Richard D., U.S. Representative from New York, statement
2
Probe Into Use of Herbicide, hearings
128
Metcalf, Hon. Lee, U.S. Senator from Montana, statement
466
Note on an Improved Cleanup Method for the Detection of Chick Edema
Factor in Fats and Fatty Acids by Electron Capture Gas Chromatography, article
:
321
Nutritional Adjuncts, Chick Edema Factor. III. Application of Microcoiuoinetric Gas Chromatography to Detection of Chick Edema Factor
in Fats or Fatty Acids, article
265
'Occupational Intoxication Occurring in the Production of Chlorophcnol
Compounds, article
241
Occurrence of the Chick Pericardial Edema Factor in Some Oleic Acids
and Products Derived Therefrom, article
219
Persistence of 2,4-D, 2,4,5,5-T, and Dicamba in Range Forage Grasses,
article from Weeds
53
Pierovich, John M., assistant regional forester, Albuquerque, N. Mex.,
letter of February 2(i, 1970
153
Progress in the. Chick Edema Problem, article
235
Report on Methodology for Chlorinated Aromatics in Fats, Oils, and
Fatty Acids, article
341
Response of Rabbit Skin to Compounds Reported to Have Caused Acneform Dermatitis, article
362
Role of "Toxic Fat" in the Production of Hydropericandium and Ascites
in Chickens, article
294
Ryan, M. J., acting director. Office of Legislative Services, Department of
'Health, Education, and Welfare, letter of March 12, 1970-.
126
Soil Persistence of 2,4,5-T, article from Chemical Fallout
41
Stcinfcld, Dr. Jesse L., Surgeon General, Department of Health, Education,
and Welfare, letter of January 21, 1970
38
Studies of the Chick Edema Disease, article from Poultry Science
255
Studies of the Chick Edema Factor. II. Isolation of a Toxic Substance,
article
223
Studies of the Chicken Edema Disease Factor, article from the Journal of
the Association of Official Agricultural Chemists
207
Studies on the Metabolism of Chick Edema Factor: Distribution in Chick
TUsui's, nrticlo
_ __
311
Tochnic for Tolling Arnegmic Potency in 'Rabbits'~ Applied" to "the" Potent
AciifKrn, '^.T.H-Tetrnchlorodibenzo-p-Dioxin, article
249
Teratogenic Evaluation of 2,4,5-T, article
U.S. Shows Signs of Concern Over Effect in Vietnam of 9-year ,
Program, article from New York Times
_" _ ' ^^
Use of the Chicken Embryo in the Assay of Adatoxi'n Toxicit^artiYle
from the Journal of the Association of Official Agricultural Chemists
Whiteside, Thomas, Department of Amplification, letter of March 5 1970'
98
102
290
123
�EFFECTS OF 2,4,5-T ON MAN AND THE
ENVIRONMENT
TUESDAY, APRIL 7, 1970
U.S. SENATE,
COMMITTEE ON COMMERCE,
SUBCOMMITTEE ON ENERGY, NATURAL RESOURCES,
AND THE ENVIRONMENT,
Washington, D.C.
The subcommittee met, pursuant to notice, at 10 a.m., in room
1318, New Senate Office Building, Hon. Philip A. Hart, presiding.
Present: Senators Hart, Inouye, and Baker.
OPENING STATEMENT BY THE CHAIRMAN
Senator HART. The committee will be in order.
Permit me a brief opening statement. For the Subcommittee on
Energy, Natural Resources, and the Environment, I welcome those
present for tho first of these 2 days of hearings we are holding to
examine the effect of the herbicide known as '2,4,5-T on man and
the environment. I suggest that what is at stake at these hearings is
virtually impossible to evaluate at this moment, in light of the
uncertainty about this frequently used pesticide.
The questions which have been raised recently concerning the hazards of 2,4,5,-T and related chemicals may in the end appear to be
much ado about very little indeed.
On the other hand, they may ultimately be regarded as portending
the most horrible tragedy ever known to mankind.
What does emerge clearly from this uncertainty is that we must
take steps to eliminate it. In view of the potential disaster that
could befall us—or conceivably has insidiously already befallen us
—absolutely no delay is tolerable in the search for answers to the
questions posed.
It is with the hope that Congress will be able to play an active
role in that search that these hearings have been scheduled.
Although the title of the hearing refers to the effects of 2,4,5,-T
alone, it should be made clear early in the game that other similar
chemicals may give rise to similar problems for the environmentWitnesses should feel free, therefore, to address themselves to any
such chemical to the extent that it bears upon the central problems
at issue.
Our first witness was to have been Congressman Richard
McCarthy, of New York, but unfortunately he will not be able to be
Staff member assigned to this hearing: Tveonard Bickwit, Jr. '.
(1)
�ay. We will accordingly, if there is no proxy, submit his
t e s t i i r \ y for the record.
(The statement follows:)
STATEMENT OF HON. RICHARD D. MCCARTHY,
U.S. REPRESENTATIVE FROM NEW YORK
Mr. CHAIRMAN: I appreciate the opportunity to appear before this distinguished committee in order to comment on tho policies controlling the regulation mid use of the herbicide commonly known as 2,4,5-t. Your examination is
most timoly, for the effects of this defoliant on plant, mid animal life suggest
t h a t more stringent regulations regarding its application are called for.
As YOU may know, I have spent much of the past year investigating the
government's chemical and biological warfare program. I am happy to report
that several significant changes have taken place in U.S. policies: changes
which 1 have advocated for many months. These include a promise to resubmit
the 1025 Geneva Protocol outlawing chemical and biological agents in warfare,
and u bun on any further biological warfare development, including toxins.
These lire small steps forward, hut the chemical warfare prograin still contains many features which are of questionable value and safety. One of these
is the defoliation program in Southeast Asia. There is evidence that one of the
compounds sprayed on a widespread basis is teratogenic, or birth-deforming.
Without, objection, I wish to insert an article from the New York Timcn of
March 15 which examines possible birth defects in babies horn in areas where
compounds containing the chemical 2,4,5-trichlorophenoxyacetic acid are used.
This spray, containing an agent known as 2,4,5-tricholrophenoxyacetio acid,
has also been in common use as a brush killer in the United States. The commercial compound is known as Silvex, and is produced commercially by Dow
Chemical Company under the trade name, Kuron. It is used by the U.S. Forest
Service to kill brush as part of watershed projects in the Southwest United
States.
Last year, 1 learned some startling facts about 2,4,5-t. As I have, noted
before, laboratory tests conducted by the Bionetics Laboratories for the
National Cancer Institute indicated that the compound is teratogenir. A recent
discussion of these laboratory tests appears in the Medical "IVor/rf News of
February 25, 1!)70. Without objection, I wish to insert the article, "Defoliants,
Defonnatii'K: What. Risk?" in the Record at this point.
1 was therefore relieved when the White House, on October 29, I960,
announced that the Agriculture Department was to terminate its use around
food crops after the lirst of the year. The new year came, hut instead of
receiving word of compliance, I was informed that the Department of Agriculture had no intention of restricting its use. In their words:
"We are awaiting advice from DIIEW as to whether or not. they intend to
establish tolerances for 2,4,5-t before we decide wliether to cancel or extend
uses of 2,4,5-t on food crops. Our January 1, 1970, date was based on DIIIOW's
expectation that they would have reached a decision by that time. That agency
believes that the public interest, would best be served by waiting for additional
research data which will be available shortly. We concur in their judgment."
1 tried in vain to find out why the White House directive had not been put
into force. The explanations from all agencies involved, the White House, the
Department of Agriculture, and the Food and Drug Administration, simply
ignored the original directive, and I was informed that the spray would be
authorized until further laboratory tests were completed sometime this Spring.
The best, analysis of this confusing situation appeared in the AVir Yorker
Mmjnxinc of February 4, 1970. Without objection, 1 wish to insert the article
entitled "A Reporter at Large: Defoliation", by Thomas Whiteside in the
Kccnnl at this point.
1 have not had the opportunity to examine the effects of defoliation in Vietnam f i r s t h a n d , but I am happy to know that Dr. Matthew Messolson, a distinguished biologist and expert on chemical and biological warfare, is in the
process of initiating a thorough investigation on this matter under the auspices
of the American Association for the Advancement of Science.
I can only say that I am shocked by the unregulated manner in which the
Departinent of Defense permits its widespread application, and I urn appalled
t h a t no field investigation regarding its long-range effects has been conducted
by the military. 1 have, however, had the opportunity to review the policies
and examined the effects of 2,4,5-t spraying operations in this country. I hope my
observations on this matter will be of interest to the members of^^is committee.
Last year, I learned about a controversy over one spray p^Bt carried out
by the Forest Service in Eastern Arizona. Reports from GloS^Arizona indicated that the Chapparel Management Program, a part of the Salt Hiver
Watershed Project had been suspended because residents of the area had complained of irregularities in the program. •
Because of the difficulties I had in obtaining adequate information from
Administration officials in Washington, and because of the nature of the controversy in Globe, Arizona, I made arrangements to conduct two days of public
hearings in that town to learn first-hand about the spray program and its
problems.
The Department of Agriculture plays a unique role in a State like Arizona.
It has jurisdiction over approximately 80 percent of the land area, most of
which is administered by the U.S. Forest Service.
Originally established to preserve forest regions in their natural state of wilderness, the Forest Service now participates in a series of commercial projects
which attempt to increase the water supply for human consumption. One step
is to kill unwanted flora—in this case chapparel trees, which absorb what
officials believe is too much water. Traditionally, burning accomplished this
mission. In the past few years, however, chemical sprays, including Silvex
Kuron, have been used extensively.
According to Forest Service officials, Kuron has been sprayed on four occasions since 1965. Following the latest spraying operation in June 1969, residents complained of illnesses to themselves and to their livestock, including
respiratory problems and deformed offspring to their animals.
My purposes in conducting hearings on February 12 and 13, 1970 were the
following:
(1) To learn the regulations under which the Department of Agriculture
authorizes the use of the spray, and how they are enforced. In addition, I
wanted to learn how new policies from Washington were transmitted and
implemented, and how each regional office of the U.S. Forest Service was
advised of the latest scientific information on chemicals which were in use.
(2) To ascertain the degree of scientific understanding of the ecological
effects of the herbicide 2,4,5-t, and
(3) To learn the scope and nature of citizen complaints, and what if any,
relation they have with the chapparel spraying operations.
Accordingly, I requested six witnesses to appear at the public hearing. They
included Dr. Arthur Galston, Department of Biology, Yale University; Dr.
John rieroVich, Assistant Regional Commissioner, National Forest Service,
Albuquerque, New Mexico; Dr. F. I. Skinner, Veterinarian, Globe, Arizona;
and Dr. Paul Martin, Department of Geochrouology, University of Arizona,
Tuscon, Arizona. In addition, two residents of Globe, Mrs. Billie Shoecraft and
Mr. Robert McKusiak gave accounts of their experiences following the June
I960 spraying.
Mr. Pierovich gave me <a full explanation of the chapparel management program, including the regulations to be followed while the compound is being
sprayed.
These include restrictions around crops, over water, and above certain wind
speeds.
Mr. Pierovich admitted that violations had occurred; the spray has found its
way into bodies of water, and the compound had drifted onto private property
as a result of wind speeds above the prescribed ten miles per hour. In addition, he informed me that new policies regarding the use of sprays containing
2,4,5-t were transmitted to the field office long after the fact. For example,
Forest Service officials in Albuquerque were furnished with a policy statement
from the Department, of Agriculture in December which referred to the
DuBridge statement. Mr. Pierovich also admitted that be has learned of the
1900 laboratory tests on 2,4,5-t from the press. Evidently no effort was made
by the Department of Agriculture to inform the field offices of the latest scientific evidence regarding the possible dangers of 2,4,0-t.
I therefore concluded that the U.S. Forest Service is negligent in enforcing
current regulations regarding herbicides; that it fails to transmit new policies
�quickly, and. that no adequate system exists to transmit new scientific information. yi^Plerovich himself stated:
healthy thing that could happen in this area would be a definite
r
of literature that our technicians could refer to. There are abstracts
available now, but the combinatipn of inputs from the universities and from
the various departments of government in one abstract bulletin would be helpful to us."
I intend to write the Secretary of Agriculture urging him to arrange such
an information system.
A second purpose of my visit was to duscuss with a competent scientist, the
ecological effects of herbicides on a semi-arid region.
. Dr. Arthur Galston assisted me in this effort. An experienced biologist who
has studied at length the impact of defoliants at home and abroad, he outlined
in his testimony the chemical reactions of these compounds and discussed certain government investigations which caution against untested use of herbicides and pesticides. He refers, for example, to the Report of the Secretary's
Commission on Pesticides and Their Relationship to Environmental Health,
prepared by the distinguished panel shared by Doctor Emil Mrak, the Chancellor lOmevitus of the University of California at Davis. He goes on to say:
"Thiis report to which .1 have alluded includes as its final statement, a chapter
on Teratology. I would like to read you the summary which emphasizes my concern : 'All currently used pesticides should be tested for teratogenicity in the '.
near future in two or more mammalian species chosen on the basis of the clos- '
est metabolic and pharmacologic similarity to human beings possible. Pesticides should he tested at various concentrations including levels substantially
higher than those to which the human population is likely to be exposed. Test
procedures should also reflect routes related to human exposures. Apart from
the obvious route of ingestion, attention should be directed to other routes of
exposure, including inhalation exposures from pesticide aerosols and vaporizing
pesticide strips used domestically and exposures from skin absorption. Parenteral administration is an appropriate test route for pesticides to which
humans are exposed by inhalation, or for pesticides which are systematically
absorbed following ingestion.
•
.
,,
"The use of currently registered pesticides to which humans are exposed and
which are found to be teratogenic by suitable test procedures in one or more
mammalian species should be immediately restricted to prevent risk of human
exposure." . . .
•
,
Dr. Galston continues:
"Here, then, is the Government's most distinguished panel saying that there
is evidence tliat 2,4,'o-t has produced teratogenic effects in one or more mammalian
species. To me, this means s/hould be restricted immediately. The committee also
said no new pesticide found to be teratogenic, should be used only in circumstances where risk of huninn exposure is minimal."
In spite of tills recent information, the Forest Service—in Arizona, at least
—was unaware of its contents. Dr. Galston's remarks about the overall purpose of the project are worth repeating. He states:
"My overall view after one day of looking around is one of puzzlement. I
wonder why anyone desired to initiate this kind of an operation in this kind of an
environment The stated objective is to improve water runoff, and water runoff
will benefit, I presume, the citizens of a nearby urban area, Phoenix, which is
Browing rapidly, and which has a lot of water requirements, and their water
requirements will grow as the years go by, and we know this is an arid area.
"Truly, water Is going to be .wilting in this area for all activities. So far as
I win w unlr-ws nuclear technology makes it available on a massive scale, taking
water from this iirt-n to plve to another area, is, in fact, robbing Peter to pay Paul.
If jr<W nn> removing water from this area you are going to partially change the
rogrtatlon. IVrhnpn you are going to denude some of the areas in order to increase
HJw runoff. Thin Involved a comparative set of valves. Whose ox (is going to be
***"«! bwnc? WtK«* interests are paramount? Clearly, cities are not going to be
> la ttow Indefinitely; we nre going to have to put some limit on them. We
"""'ill* ***t?'»1«'' Umt HM> °"y °f Las Angeles got into a 'lot of trouble with
I lira*** llwm are Jl»t too many people there. In the same way, ctities in the
mrm may haw to limit their size ultimately based on the number of
v j*»W« tteMr «*» <wip|*>rl on tlm Imxlx of the amount of water resources available.
.:.:;• *X«*r' that PmMifnt Nixon among others is calling for a campaign to
.;• tw»l«r* Ifc* ravirotuwnt. It might b» that we would want to look at this project
in the context of what we are doing to the enire Stae and to the entire
countryside."
1 cannot help but conclude that such land management piAuuus involve
much more than the immediate water needs of urban spra-M^The natural
regions of the Southwest are a great natural resource. They must not be sacrificed so recklessly.
My third interest was to determine whether any relationship existed
between the maladies of certain residents and their livestock and the spraying
of Silvex.
The Agriculture Department readily admitted that the spray drifted out to
private property. The owners have a rightful complaint regarding this fact
which has not yet been resolved. The local veterinarian, Dr. F. I. Skinner
informed me that he would not have recommended the use of 2,4,5-t compounds had he been familiar with the results of the Bionetics Laboratory's
tests.
It is also true that illnesses developed whose symptoms are similar to those
which are known to he associated with herbicides. In addition, I saw malformed animals who were born after the incidents of last June. Statements
were made regarding this fact by persons testifying in good faith, and should
not be dismissed outright. They obviously have some bearing because the
Forest Service has suspended further sprayings in the area. This decision followed the complaints, and while Federal investigation found no direct relationship between the spray operations and the illnesses, Forest Service officials
await further developments before resuming.
No one is being helped by the procrastination of officials in Washington.
Three agencies are now involved in the 2,4,5-t controversy, yet none have
assumed responsibility for regulating this herbicide. The Food and Drug
Administration, which under the 1954 amendments to the Cosmetic Act of
1938, has the obligation to establish safe tolerance levels before a chemical of
this kind is put on the market, has failed to enforce the Law. The Agriculture
. Department continues to ignore other agencies in administering the Federal
Insecticide, Fungicide and Rodenticide Act. The Pesticide Regulation Division
established by this act, was sharply condemned by the House Government
Operations Committee report of November 13, 1969 for not carrying out its
responsibility to police the licensing of herbicides. In addition to the charge
that no legal steps have ever been taken against firms which violate licensing
regulations, the Committee report brought to light repeated instances of conflict of interest among various officials of the Pesticide Regulation Division
and agro-chemical companies.
Finally, the White House has backed down from its assertive position of
last October. After reversing an earlier ban, I am now told boldly by Dr. Lee
A. DuBridge in a letter of March 2, 1970, that "we anticipate, indeed we will
insist upon final action of 2,4,5-t before its period of principal usage in late
spring."
I will not hold my breath.
Mr. Chairman, there are obvious irregularities in the regulation and management of herbicide compounds containing 2,4,5-t. It is clear that the National
Forest Service no longer regards preservation of lands in their natural state
as a primary responsibility. There is insufficient information regarding Its
risks and inadequate statistics on its effects to animal and plant life. Its use
must not be continued until its safety is assured.
Accordingly, I recommend an immediate five year ban on the use of herbicides containing 2,4,5-t. During this period of suspension, the Food and Drug
Administration should establish, once and for all, whether the chemical has a.
safe tolerance level. The latest word from FDA Is that the officials "are in no
position to say that the chemical 2,4,5-t or the dioxin is without hazardous
effects". A letter to me of March 12, 1970, reflects the inconclusive evidence
regarding its safety. It is worth repeating in full. Without objection, I wish to
insert the letter from M1. J. Ryan of the Food and Drug Administration, along
with an attached fact sheet in the Record at this point.
In addition, steps should be taken immediately to collect information on the
number of children born with birth defects, including those which might be
caused by herbicides.
Accordingly, I will be writing to the Secretary of Health, Education and
Welfare urging him to instruct the National Institute of Neurological Diseases
and Strokes of the National Institutes of Health to begin gathering information on these phenomena.
�Mr. Chairman, I am confident that these hearings will help resolve a most
important issue. There can be no more delay.
Jj^tehairman, without objection, I wish to insert various documents
ing^^ny hearings in Globe, Arizona for inclusion as an appendix to
hearings.1
Senator HART. We will move to the next scheduled witnesses,
James Turner and Harrison Wellford of the Center for Responsive
Law.
STATEMENT OF HARRISON WELLFORD, CENTER FOR STUDY OF
RESPONSIVE LAW, WASHINGTON, D.C.
Mr. WvH.LPcmi). Thank you, Mr. Chairman.
We appreciate A'ery much the invitation to appear at these hearings this morning. I would like, if I may, to road excerpts from my
testimony rather than reading the entire testimony.
In this testimony we wish to bring to your attention that herbicides containing 2,4,5,-T are widely marketed for casual use by the
individual consumer in residential and other populated areas. The
herbicide 2,4, 5-T even in extremely small doses, causes massive and
severe birth defects in tost animals, including mice, rats, and hamsters. The herbicide 2,4-1), which is often mixed with 2,4,5-T in the
popular herbicides such as Ortho Weccl-Be-Gone, is also teratogenic,
but at higher dose levels. We feel that these herbicides, as currently
used, may pose a grave and unnecessary danger to public health.
In a recent article, the biologists Arthur W. Galston, William
Cooke, and William Haseltine stated that 2,4,5-T "may represent the
ecological equivalent of thalidomide." (Congressional Record, Feb.
19, 1970, S. 1984). Professor John T. Edsall of Harvard University,
stated before the American Association for the Advancement of Science's Committee on Chemical and Biological Warfare that "the use
of these compounds is much more seriously questionable than the use
of cyclamates. If one applies the same criteria, one would consider
the risks quite unacceptable." (Quoted in the New York Times, Dec."
29, 1969). Tim tests performed by the Bionetics Laboratory, by the
Food and Drug Administration, National. Institute of Dental
Research, and the National Institute of Environmental Health Sciences clearly show that these chemicals are potentially harmful.
Whether or not human beings are more or less susceptible than test
animals to these chemicals, we do not yet know. But clearly on the
evidence now available, the burden of proof should be on the industry to demonstrate that they are not harmful. In the meantime, all
uses of these herbicides around the home and in populated areas
should be immediately suspended.
Wo wish to discuss this morning the nature of the clanger as we
see it and the serious failures of both the Federal Government and
private industry to reduce the unnecessary risks to public health of
exposure to 2,4,5-T and other suspicious weedkillers. Specifically I
am concerned about :
(1) The attempts by Government and industry to conceal the
facts about 2,4,5-T and 2,4-D from the public and from other scientists.
1
HCP p. 102.
(2) The hazards to public health which may res^^from widespread use of 2,4-D and 2,4,5-T in populated areas.
WP
My colleague, Mr. James Turner, will discuss the profound implications which the 2,4,5-T-2,4-D case has for the effectiveness of
Government efforts to protect the public from pesticide hazards.
2,4,5-T was developed from research done at the Chemical and
Biological Warfare Center at Fort Detrick during the 1940's. It has.
been massively applied to the human environment for 20 years, but
until very recently no studies have been conducted by any Government agency 011 the possible carcinogenic, mutagenic, or teratogonic
properties of this herbicide, or on the ecological consequences of its
use. In 1966 the Bionetics Laboratory was commissioned by the
National Cancer Institute to begin research into the birth defect
properties of a variety of pesticides and herbicides, including 2,4-D
and 2,4,5-T. By 1968 these tests had revealed substantial evidence
that 2,4,5-T caused birth defects in test animals. In February of
1969, the preliminary results from the Bionetics testing were to be
presented to the annual meeting of the American Society of Toxicology. The report included tests results Avhich showed that 2,4-D and
2,4,5-T cause gross abnormalities and birth defects in mice. 2,4-D
was termed ''potentially dangerous, but needing further study,"
while 2,4,5-T was labeled "probably dangerous." Tins report would
have provided an early warning on potential hazards from these
herbicides, but for reasons still unknown, the Bionetics presentation
was canceled at the last minute, although its paper was listed on the
printed agenda of the meeting. The Bionetics report thereafter
returned to obscurity. Only a few scientists in the Government knew
of its existence. Even members of the Mrak Commission, Panel on
Teratogenicity were initially rebuffed when they asked to see the
report. The report, however, had been presented to selected officials
in the Food and Drug Administration, the U.S. Department of
Agriculture, and the Defense Department. These delays contradict
the recommendation of the Mrak report that "any teratogenic pesticide to which the population is exposed should be promptly identified so that appropriate precautions can be taken to prevent risk of
human exposure "(p. 657).
On October 29, after the existence of the report finally became
public (it has still not been officially released), Dr. Lee Dubridge,
the Science Adviser to the President, announced that "a coordinated
series of actions" was being undertaken by several Federal agencies
to ban the use of 2,4,5-T on food crops and Government use of the
herbicide in populated areas. Dr. Dubridge stated that these actions •
we,re being taken to "assure the safety of the public while further
evidence is sought." The ban on use of 2,4,5-T on crops was to go
into effect on January 1, 1970 unless by that time the FDA had
found a means to establish safe residue tolerances. Apparently
because Dr. Dubridge estimated that "almost none (of 2,4,5-T) is
used by home gardeners, or in residential areas," nothing was said
about stopping individual consumers from using 2,4,5-T in their own
backyards.
The Department of Interior did follow Dr. Dubridge's recommendation and ceased the use of 2,4,5-T in its operations. The deadline
�of ^fcuary 1, 1970, passed without FDA setting a tolerance. By
JanBwy, it was clear that the Department of Agriculture and
Federal agencies (with the exception of Interior) had no intention
of restricting the use o:f 2,4,5-T. On February 6, the Department of
Agriculture.announced that the original 2,4,5-T used in the Bionetics test had been contaminated with a tetradioxin and that further
testing with a purer batch of 2,4,5-T had shown no adverse effect.
The Department relied on tests conducted by the Dow Chemical Co.,
a major manufacturer of 2,4,5-T. The Dow evidence was immediately contradicted by tests conducted at several other Federal agencies which clearly showed that even the purest 2,4,5-T, where the
dioxin contaminant was present at less than one part per million,
still produced birth defects in test animals at significant levels.
Recent tests on 2,4-D conducted by the FDA have confirmed that
this herbicide, which is very often mixed with 2,4,5-T, is also teratogenic. Many other questions with significance for public health
remain to be answered: How persistent are 2,4,5-T and 2,4-D once
applied? How persistent is the dioxin contaminant? Is it cumulative
in human tissue? How much dioxin is present in herbicides already
on the shelves of local hardware stores?
Therefore, Mr. Chairman, 5 months after Dr. Dubridge urged
that most uses of 2,4,5-T be banned to "assure the safety of the
public while further evidence is sought," these herbicides continue to
be teratogenic and are as widely used as ever. The most recent tests
provide the "further evidence" Dr. Dubridge asked for. The burden
of proof on those who wish to demonstrate the safety of these herbi. cides has greatly expanded since the first Bionetics test.
When tests indicate that a pesticide is teratogenic in animals, the
burden of: proving that it is safe should be placed on the manufacturers of the pesticide, not on its possible victims in the general population. If the manufacturers do not cooperate, the Federal Government has a statutory responsibility to minimize human exposure to
teratogenic pesticides by appropriate regulatory preventive action.
The Government must not fail in its trust, for nationwide statistics
on birth defects are so inadequate that even an increase of several
thousand deformities could probably go undetected. Therefore, if
Government and industry act irresponsibly, there will probably be
few complaints from the medical profession or the general public to
call them to task.
Present population monitoring techniques do not provide adequate
gages of the incidence of birth defects in the population. Federal
regulators charged with protecting the public from pesticide hazards
are being very irresponsible if they assume, as did Dr. Lindsey of
the Food anc^ Drug Administration in a recent interview that, "the
National Institute of Neurological Disease and Stroke has recorded
birth defects for some 15 years and would be telling us if they were
on the rise,'-'1 Dr. Hincs Berendes, Chief of NiNDS' Perinatal
Research Branch, has unhappily conceded that, "no nationwide data
are available on the frequency or incidence of malformation." * Even
in States whore birth certificates request that doctors record birth
defects, the completeness and accuracy of the reporting depends on
1
Medical World News, Feb. 27, 1070.
the interest and diligence of the physician and on tin onspicuousness of the abnormality. Nationally, no attempt
^^ made to
collect and evaluate all the data on birth defects tha^Tre presently
available on birth certificates. After careful study of this problem,
the writers of the Mrak report concluded that "epidemiologic data
on possible effects of pesticides on human reproduction and teratologf are grossly inadequate." 2
The Mrak Commission report states that when animal experiments ,
indicate that a pesticide is teratogenic, the effect should be retrospectively evaluated when possible by a study of pregnancies during
which the mothers were inadvertently exposed to the pesticide, such
as in farmwork and industrial exposure and through accidental
ingestion. As far as we know, this has not been done for 2,4,5-T and
other weedkillers. Because of the need to minimize human exposure,
it is not possible to test on human populations pesticides previously
shown to be teratogenic by experimental animal studies.
The Mrak report states unequivocally that there is little comfort
to be gained from the expectation that present epidemiological surveys of: pesticides in current use will discover in time chemical cornpounds causing birth defects. It states that no major teratogen
(term for sxibstance causing birth defects) has been discovered in
this way. The malformations induced by X-rays, german measles,
thalidomide, and mercury were each recognized by "an alert medical
practitioner who observed a cluster of cases and then traced the
cause to its source."3 Tracing observed defects to a specified cause is
much more difficult when the defect commonly occurs. In the case of
2,4,5-T, the most common defects produced in test animals are
kidney abnormalities and cleft palates, neither of which is unusual
in humans. Had thalidomide produced such ordinary malformations
instead of bizarre and unusual ones, it probably would never have
been discovered. Thus any birth defects produced by human exposure to 2,4,5-T are unlikely to be traced to the weedkiller because
they are already common in the population.
If 2,4,5-T and 2,4-D, as commonly used in populated areas, do
produce birth defects in humans, the birth defects will remain a
very private family tragedy. Because 2,4,5-T leaves no unique fingerprints on the fetus to indicate its specific role as the teratogen, the
parents of the deformed child would probably remain silent, with no
knowledge of the caiise of their distress, They would probably never
know that they belong to a class of victims of a preventable tragedy.
There is another reason why Government has a special responsibility here to protect the public. Because a 2,4,5-T or 2,4-D induced
birth defect is not unique, the parents of deformed children will
have great difficulty in using the courts to discipline the manufacturers of dangerous herbicides. The parents will gain no compensation for their loss. Moreover, there will be no lawsuits to force the
chemical companies to test more thoroughly their products for teratogenic effects before they are released on the market or to maintain
strict quality control standards which will keep the level of contami1
Report of the Secretary's Commission on Pesticides (the Mrak report, U.S. Department of Health, Education, and Welfare. December 1008, p. 674.
,
' Mrak report, p. 661.
�10
natiowof dangerous dioxins as ]ow as possible. In the absence of
legal remedies for private citizens, protection must come from the
Federal Government.
As a first step, we suggest that the committee consider the recommendation of the Mrak Commission that efforts must be made to
improve and use information on congenital malformations recorded
. on birth certificates and that new systems of collecting birth defect
data.be established.
POTENTIAL HAZARDS FROM CONSUMER USE OP TERATOGENIC
HERBICIDES IN RESIDENTIAL AREAS
Weed-killers containing 2,4,5-T are readily available to the home
gardener. These products come both as premised, ready-to-use
liquid, spray or dust, and as liquid preparations which the user
dilutes at home, or uses with the garden hose atomizer. The concentrated liquids are obviously the most dangerous.
Last week two of my assistants made a survey of herbicide products in 10 Washington area stores: Eight hardware stores, including
Meenehans, Sears, Hechingers, Mclntyre, Community Paint and
Hardware, Kresge's at 7th and Pennsylvania, Chevy Chase Hardware; one grocery store, the Giant at Western Ave. and Wisconsin;
and one gardening store, Sheridan Garden Store on Old Georgetown
Road.
My assistants found the following:
(1) Eight of the stores carried lawn and garden weedkillers containing 2,4,5-T, nine of the stores carried product lines which
included 2,4,5-T products. All of the stores carried weed-killers containing 2,4-D. They found nine product lines containing 2,4-D; six
with 2,4,5-T.
(2) In addition to 2,4-D and 2,4,5-T, at least seven of these stores
carried all but one of the other products cited in the Bionetics
Eeport' as at least potentially causes of birth defects: Captan,
Folpet, Sevin; as well as organo-mercurial products which have been
known to produce birth defects in humans since an epidemic in
Japan led to their'being banned in that country.4
(3) Some of the products, the Scotts line in particular, were very
badly packaged. Most Scotts products and some others are packaged
in flimsy paper bags; a number of the bags which my assistants
examined were in such bad repair that they could not be handled
without spilling dust on the handler. As a result, there was frequently a coating of chemical dust on products nearby. In an unintended irony, most of the packages bore warnings to the user to
"avoid contact with skin, eyes or clothing."
(4) A number of products and again the Scotts line in particular,
were fertilizers with various herbicides added, including 2,4-D, and
2,4,5-T (in Greenfield products.) Since the contents were not very
conspicuously displayed, many users might well assume that these
were only "super fertilizers," and therefore not handle them with
thp care that their contents warranted. These products, containing a
mixture of fertilizer and herbicide are heavily promoted on radio
and television, with no warning of potential danger. Their names,
1
Mrnlt report, p. 001.
11
for example, Scotts Turf Builder Plus-1, which contd^Ja mercurial
compound, often disguise or play down the herbicide content.
Mixing herbicides with fertilizer tends to identify potentially dangerous chemicals with innocuous fertilizers and promotes unnecessary, as well as careless use.
(5) The manner in which these herbicides are used magnifies their
potential risk. Their labels all read: "Avoid contact with skin, eyes
or clothes. Avoid inhaling dust," Yet these products are dispensed in
a manner which makes contamination of the user with either dust or
spray inevitable. Herbicides in dust or powder form are often
applied by dumping them into a wheelbarrow, with a hole in the
bottom and a propeller underneath which the user pushes across his
lawn raising a cloud of dust. Liquid herbicides are applied through
attachments to a garden hose or through hand applicators. In both
cases, contact with the spray is unavoidable. Many of these products
on their packages depict homeowners spraying with bare hands and
bare arms. On no products, even the most poisonous, was there any
suggestion that the user wear rubber gloves. Several of the herbicides did contain the difficult instruction that children and pets be
kept off the treated areas, sometimes for an unspecified amount of
time, until after the area had been watered and dried or it had
rained.
Whether applied in spray or dust form, the application of herbicides containing 2,4,5-T presents serious problems of drift. The
report of the Subcommittee on Weeds of the National Research
Council stated in 1968 that spray with "droplets of 10 microns in
diameter can drift up to 1 mile when released at a height of 10 feet
with a 3-mile per-hour wind." (p. 248). Even when kept in perfect
condition, few nozzles used for spray application would produce uniform droplets large enough to minimize drift and yet small enough
to provide even coverage. The hazards of drift, even when the herbicide is applied in dust or powder form is also great. The Department of Agriculture, in its caution suggested for use on weed-killers
containing 2,4,5-T and 2,4-D, warns that "this dust may drift for
miles even on quiet days." (Federal Register, May 21,1969).
It is a conservative estimate that even on a relatively calm day
children playing within 100 yards of an area where a yard is being
sprayed or dusted with 2,4,5-T are probably going to be exposed to
the chemical. Droplets and dust particles of 2,4,5-T can be carried
by the wind into open windows and onto screen porches. In heavily
populated residential areas, one simply cannot defoliate his backyard of chickweed and dandelions without running the risk of contaminating his neighbors or their children. The economic and horticultural .benefits of these herbicides in residential areas do not
outweigh their risks to those who wish to enjoy the outdoors without
being contaminated by teratogenic spray.
(6) A few products did not bear even the minimum federally
required warning, "Caution, Keep out of reach of children," which
must be displayed prominently on the front. For example, Scotts
Kansel Weed Killer, in a salt-shaker like container, had no warnings
at all on the front, and only "Avoid contact with skin, eyes, clothing, etc." written very inconspicuously on the back. The same was
true of an identically packaged Amchem Garden Weeder.
45-302—70
2
�12
The potential hazards of 2,4,5-T herbicides are increased
'act that consumers frequently do not read the label on the pes
ticide package, or if they do, do not usually understand it. The
Mrak report cites the 1969 study of home pesticide use in Charle'ston, S.C. which found that of the 83 percent white and 97 percent
nomvhite families using pesticides:
Both white and nonwhite families commonly ignored safety precautions in
the use of household chemicals. Locked storage was not employed by 88 percent of all families; 66 percent stored the pesticides within easy reach of
small children; 54 percent stored the chemicals near food or medicine; and 66
percent never wore protective gloves during use or washed their hands after
application. 0 ,
.
It is unlikely that the labels of the 2,4,5-T herbicides we examined
in local stores could be changed sufficiently to prevent users from
contaminating vulnerable individuals.
I might add while it is not included in my prepared testimony
that we did come across yesterday herbicides on local hardware
shelves which have disclaimers of liabilities which are clearly in violation of the notice to manufacturers, formulators, and distributors
issued by the Pesticide Eegulation Division of the Department of
Agriculture on November 16, 1966.
(The notice follows:)
, '
UNITED STATES DEPARTMENT OF AGRICULTURE
'
.
AGRICULTURAL RESEARCH SERVICE
PESTICIDES REGULATION DIVISION,
Washington, D.G., November 16, 1966.
13
There is no objection to statements that are aimed
against damages from careless or improper handling,
are not false or misleading.
the seller
so long as they
EXAMPLES OF DISCLAIMER STATEMENTS WHICH ARE NOT OBJECTIONABLE
(1) Seller makes no warranty, expressed or implied, concerning the use of
this product other than indicated, on the label. Buyer assumes all risk of use
and/or handling of this material when such use and/or handling is contrary to
label instructions.
(2) Follow directions carefully. Timing and method of application, weather
and crop conditions, mixtures with other chemicals not specifically recommended, and other influencing factors in the use of this product are beyond
the control of the seller. Buyer assumes all risks of use, storage or handling of
this material not in strict accordance with directions given herewith.
(3) Buyer assumes all risks of use, storage or handling of this material not
in strict accordance with directions given herewith.
(4) Seller's guarantee shall be limited to the terms of the label, and subject
thereto the buyer assumes any risk to persons or property arising out of use
or handling and accepts the product on these conditions.
(5) Our recommendations for use of this product are based upon tests
believed to be reliable. The use of this product being beyond the control of the
manufacturer, no guarantee, expressed or implied, is made as to the effects of
such or the results to be obtained if not used in accordance with directions or
established safe practice. The buyer must assume all responsibility, including
injury or damage, resulting from its misuse as such, or in combination with
other materials.
Mr. AVELLiroRD. This order stated that the following disclaimer
would be considered both false and misleading, and a product which
had this kind of disclaimer on it would be rendered misbranded :
NOTICE TO MANUFACTURERS, FOBMULATOHS, DISTRIBUTORS,
AND REGISTRANTS OF ECONOMIC POISONS
The information furnished hereon is provided gratuitously by the manufacturer who assumes no responsibility whatsoever for the effectiveness or safety
of this product regardless or whether or not it is used as directed.
STATUS OP DISCLAIMER STATEMENTS ON PESTICIDE LABELING
Attention : Person responsible for Federal Registration of Economic Poisons.
The Federal Insecticide, Fungicide, and Rodeuticide Act, and the regulations
promulgated thereunder, provide that an economic poison is misbranded if its
labeling hears any statement, graphic representation, or design which is false
or misleading in any particular.
Labeling disclaimers which negate or detract from labeling information
required under the Act and regulations are not acceptable on products proposed for registration.
,
. .
An example of a disclaimer which would render a product misbranded is as
follows:
"The information furnished hereon is provided gratuitously by the manufacturer who assumes no responsibility whatsoever for the effectiveness or safety
of this product regardless of whether or not it is used as directed."
Such disclaimer is both false and misleading, since adequate directions for
use, necessary warnings and cautions, and other essential information on the
safe handling and use of a product are required under the Federal Act.
Labeling for registered products bearing disclaimer statements which are
either false or misleading must be revised or deleted. Such revisions or deletions will not require reregistration by this Division. However, five copies of
such amended labeling should be submitted for our records.
HARRY W. HAYS, Ph. D.,
Director.
Here I have two identical cans of Ortho Poison Ivy Killer, both
purchased in the last 2 days. One of them has the proper disclaimer.
It says "Notice, buyers assume all responsibility for safety and use
not in accordance with directions."
Senator HART. Would you restate that ?
Mr. WKLLFOUD. Yes, sir. The buyer assumes all responsibility for
safety and use not in accordance with directions. That is a proper
, disclaimer. This other product was purchased at the same time. It is
the same substance, but it has the illegal disclaimer on it :
There are two major provisions of disclaimer statements which have been
used by many registrants that are considered to be false or misleading. These
are the claims "that the information is provided gratuitously," and disclaiming
on any responsibility "whether used in accordance with the directions or not."
' Mnik report, p. 148.
Because of critical, unforeseeable factors beyond the manufacturer's control
prevent it from eliminating all risks in connection with the use of chemicals
even though reasonably flt for such use, buyer and user acknowledge and
assume all risks and liability resulting from handling, storage, and use of this
material. These risks include, but are not limited to, damage to plants,- crops
and animals to which the material is applied, failure to control pests, damage
caused by drift to other plants or crops and personal injury. Buyer and user
accept and use this material on these conditions whether or not such use is in
accordance with directions.
Now, either these products Avere being marketed in violation of
the order of the Pesticide Regulation Division or they have been on
the shelves since 1966. The manager of the store at which we bought
the one with the illegal disclaimer stated that the spray had been
purchased 1 month ago and that the distributor' got it 4 months
before that,
So, we would assume that these products are still being manufactured with that disclaimer.
�14
percent of all the herbicides we examined in all the stores,
contained either 2,4-D or 2,4-,5-T or a combination of both.
By the way, if the product—Ortho Poison Ivy Killer—which you
have just examined has been on the shelves since 1966, it raises questions of when the new quality control standards introduced by Dow
Chemical Co. and by probably other chemical companies •which have
•reduced the level of the dioxin contaminant took effect, it may be
that many products that we are now able to buy off the shelves of
' local hardware stores were produced before the new quality control
standards were introduced.
If that label is not illegal, it was produced at least 3 years ago,
maybe more, and this raises one of the basic questions we want to
stress this morning, to bring to your attention this morning, and
that is we really do not know at all how much of the dioxin contaminant is in the herbicides which are so widely available at the local
hardware stores. Some may be. very old. We do not know whether
til] the chemical companies that produce 2,4,5-T are adhering to the
same quality standards that Dow uses. This is a major area that
needs investigation.
In short, the general population is being exposed to 2,4,5-T herbicides in use in residential areas. Over 60 percent of all herbicides we
examined in local stores contained either 2,4-D, 2,4,5-T or a combination of both.
Estimating the precise hazard posed by weedkillers is difficult due
to the scantiness of scientific information and the difficulty of
extrapolating whatever information there is from animals to
humans. For instance, the level of dioxins in these products is
totally unknown. In many of the products 2,4,5-T is slightly modified
chemically, although this modification would probably not effect the
dioxin. Therefore, without further data there is no good way to
extrapolate the effects of high doses on small numbers of animals to
krgo populations exposed to low doses. Moreover, human sensitivity
to 2,4,5-T probably varies greatly from person to person. Finally, it
is worth recalling that thalidomide proved 50 to 200 times more teratogenic in humans than in the test rats and animals which had been
used to demonstrate its safety.
The potential harmfulness of the 2,4,5-T products marketed for
individual consumer use depends in part on the amount of dioxin
contaminant they contain.
I have just made my point on that, the fact that we do not know
how much they do contain.
~r x
f~-t i
•
i
/~*
i
knows how much dioxin is in Dow's older products still on the
market, which may have been made before it instituted its present
quality controls. Moreover, no one knows how much dioxin is in
'2,4,5-T products produced by other chemical companies which may
not have the standards of quality control employed by Dow Chemical. Moreover,'even Dow's current production of 2,4,5-T contains
oyoi'^5 percent of other impurities, including some other kinds of
dioxins. The effects of these impurities are totally unkown. Dr.
Meselson, appointed last year by the American Association for the
Advancement of Science to head a 2,4,5-T evaluatioi^|oject, says:
Tlie tetrachlore dioxin represents just one of 12 or 13 wlW the chlorine
atoms can arrange themselves on a benzene ring to form dioxin molecules.
How do we know about the hexa, hepta, and octyclors, or about how persistent
the tetraehlor itself is? Moreover, I'm very concerned about the dioxins that
might be formed by unreacted trichlorphenol (2,4,5-T precursor) wht>n the
product is exposed to heat. If it were taken up by plants or wood and these
were burned, you'd get more dioxin. Finally, I'm bothered by the bizarre
mental effects suffered by German workers making 2,4,5-T. I say when in
doubt.stop it"
A mystery with disturbing implications for public ^health is
whether the' dioxin contaminant in 2,4,5-T accumulates in human
tissue. There is as yet very little scientific evidence on this point. If
the dioxins do accumulate, they pose a danger, not only to the children of women who were pregnant when exposed, but also to women
who become pregnant subsequent to exposure. If the dioxin or other
teratogenic substances in 2,4,5-T are cumulative, it becomes academic
whether a single or even several, exposures to herbicides used on residential lawns and gardens are of sufficient magnitude to cause birth
defects.
CLTNlCAIj EVIDENCE OF 2,4,5-T POISONING
The clinical evidence of injury to-persons coming into contact
with herbicides containing 2,4,5-T is difficult to compile because
techniques for monitoring 'pesticide poisonings are nearly as inadequate as those for measuring the incidence of birth defects. Poison
control centers in many cities regularly treat victims of pesticide
poisoning, but only rarely are their records of poisoning broken
down as to particular pesticides.
Nevertheless, there are enough case histories on record to substantiate the laboratory findings that the herbicide 2,4,5-T, in addition to
being a possible teratogen, can be severely toxic to human beings,
causing nausea, diarrhea, chloracne (a serious skin disease), mental
distress, kidney damage, and other maladies.
(1) Thomas Whiteside, in an article in the New York magazine,
reported that chloracne has been found among workers in plants
manufacturing 2,4,5-T. In the mid-1960's the Dow Chemical Co.
closed down part of a 2,4,5-T plant in Midland, Mich., because
workers there contracted chloracne apparently as a result of contact
Math the dioxin contaminant in 2,4,5-T. The symptoms of this disease included extensive skin erruption, central nervous system disorders, fatigue, lassitude, and depression. Similar symptoms were
reported in German workers in plants producing 2,4,5-T as early as
1955.
(2) Professor Arthur Galston, at a hearing on herbicides conducted by Congressman Richard McCarthy, in Globe, Ariz., discussed a 'scientific report entitled "Dermatitis and Kidney Damage
Ascribed to Weedkiller 2,4,5-T." This report told of < two girls, aged
4 and 6 years old, \vho had played for several hours in a yard which
had been sprayed heavily a short time before with Ortho Brush
Killer. The girls developed general reddening of the skin and swell' Medical World News, Feb. 27,1970, p. 17.
�ing oL the oval and vaginal mucous membranes. The limbs and eyclids^lro also slightly swollen. On the third day after exposiiro,
kidney damage was indicated by the discovery of albumen in the
urine which persisted for about 2 weeks.
(3) • Several"-suspected cases of 2,4,5-T and 2,4-D poisoning are
being studied in Globe, Ariz.
(4) The Montgomery County Health Department has received at
least one report from a Bethesda woman whose child suffered
nausea, diarrhea, swelling of: the lymph glands, and prolonged
mental distress, as a result of exposure to a spray containing 2,4-D,
2,4,5-T and petroleum distillates, which had drifted from a neighbor's lawn being treated with the herbicide.
More clinical evidence might easily be compiled if an cpidemiological study were made of particularly vulnerable groups. For example, it would be interesting to know if Dow and other companies
producing 2,4,5-T have surveyed their female workers to see if they <
have a higher incidence of birth abnormalities. Similar studies
should be made of farmworkers', especially those associated with rice
and sugar production where 2,4,5-T is used. A special concern should
be females who work in the timber industry, where 2,4,5-T spray is
used to kill trees.
I worked for several summers spraying trees myself, and my colleagues among the sprayers were very frequently young women who
carried spray cans on their backs and sprayed the spray into cuts on
the trees made by men.
There is simply no wav of, after using that spray for more than
30 minutes that you would not be literally drenched with it.
It would seem very likely that many of these women, being young,
were in the early stages of pregnancy at some point in their' work,
and that they may have received very heavy doses of herbicide from
vapors and wotting of the skin.
In this work, herbicides containing 2,4,5-T or 2,4-D plus 2,4,5-T in
concentrations ranging from 8 to 16 pounds of acid equivalent per
100 gallons of fuel oil are commonly used.7 These herbicides are
sprayed into cuts made from girdling trees. It is common practice in
Virginia, for example, for women to be hired to carry spray cans
and apply herbicides 011 the trees after they have been cut by men.
The sprayer is invariably heavily exposed to the herbicide, through
inhilation of the vapors and wetting of the skin. Scores of women,
many of them in the early stages of pregnancy, have probably been
exposed to heavy concentrations of 2,4,5-T and 2,4-D in this manner.
The Government should take immediate steps to warn women
engaged in this work of the risks they are running, especially since
many of them are working on projects sponsored or'advised'by the
Forest Service of the U.S. Department of Agriculture.
In conclusion, Mr. Chairman, it seems an unnecessary risk to
' Wood Control, National Acndemy of Sciences, 1968, p. 330.
public health that a herbicide with the dangerous potential of 2,4,5T should be widely available for casual use 'by the^Biividual consumer. In a recent letter, Dr. Steven Lipson of tr^^'Iontgomery
County Health Department, stated this view very well.
(The letter follows:)
MAUCII 17, 1970.
Mn. HARRISON WELLFORD,
Center -for Study of Responsive Law,
Washington, D.G.
DEAR MB. WELLFOIUI : In response to your request, I nm writing you wltli
regard to my personal and professional feelings about the use of 2,4-D, 2,4,5-T
and related herbicides. I should like to preface my remarks with the note that
these comments are made as an individual and not as an official of the Montgomery County Health Department
Over the past years this Department has received a number of inquiries as
to the possible toxicity of these chemicals. Our information relating to this is
drawn from standard sources and it appear,? that there is no question but that
these are dangerous substances when used improperly. As you know, exposure
to these chemicals may cause central nervous system depression, weakness, loss
of appetite, diarrhea, coma and sudden death. There Is also a risk of skin irritation and damage to peripheral nerves.
Extensive discussion of this problem within the Health Department leads us
to the conclusion that local control is impractical. One may pass any number
of regulations regarding the use of herbicides in suburban and residential
areas but enforcement is completely impractical. Public education appears to
be the only feasible local alternative but as the continuing smoking problem
demonstrates daily, this is ,a long and slow process. Our conclusion then was
that the only reasonable means of control is at the point of sale. While again
one could limit this locally, the movement of the consumer to different marketing areas is great, particularly in the Washington Metropolitan area, and limitation of sale of a substance by any single jurisdiction would have little effect.
Accordingly, as a parent, physician and public health worker, I would
strongly support the limitation of use of herbicides to those situations in
which it is economically and scientifically indicated. To my mind, this would
not include suburban residential use. If the question is taken even more
broadly, I would agree fully that no foreign substance, irrespective of whether
toxicity has been demonstrated, should be introduced into the environment
unless there is a clear and evident need for its use. This argument becomes
even stronger when we talk about the exposure of children to potential hazards.
I strongly support and concur in your attempt to remove 2,4-D and 2,4,5-T
from the individual consumer market.
Sincerely yours,
STEVEN LIPSON, If./)., M.P.If.
Chief, Division of Epidemiology anil Surveillance,
Montgomery County Health Department.
Mr. WELLFOBD. The economic value of using these herbicides to
defoliate residential backyards of dandelions and chickweed is manifestly outweighed by their potential hazards. Until the manufacturers
establish that exposure of the population to 2,4,5-T and 2,4-D is not
dangerous, they should be banned from the individual consumer
market. We would also support efforts to ban the use of these herbicides on food crops. Thank you very much for inviting us to make
this statement.
My colleague, Mr. James Turner, now has a statement to make.
�18
19
EATEMENT OF JAMES TUENEE, CENTEE FOE STUDY OP
EESPONSIVE LAW, WASHINGTON, D.C.
Mr. TTJRNEH. I too would like to thank you for your kind invitation to be present hero today to discuss the way in which pesticide
safety is regulated by the Departments of Agriculture, and Health,
Education, and Welfare.
Mr colleague, Mr. Well ford, has outlined to you the details of the
very serious potential health problem presented by the widespread
use—particularly around the homes of many American families—of
the pesticides 2,4,5-T and 2,4-D.
There are possibly many other widely used pesticides which pose
health hn/imls to the public, because under the current pesticide
control system effectiveness is considered more important than
safety. Tt is also important to note that pesticides are only part of
the chemical problem.
An increasing number of scientists believe that many of the thousands of chemicals to which the average American is exposed—food
additives, drugs, industrial wastes, cosmetics and others, as well as
pesticides—present a massive threat to future health and well-being.
The potential health hazard presented by chemicals in the envi- .
ronmerit is difficult to exaggerate. Americans have been much too
secure in the belief that their health is the best in the world. In fact,
the health of Americans is not good. The life expectancy of an
American female ranks 21st in the world; that of an American male,
87th. That is for people who have reached the age of 20. Infant
mortality figures, called by many statisticians the best measure of a
nation's overall medical ability place the United States approximately 15th in the world, a drop from fifth in 1950.
Since 1900 .the average American reaching the age of 4-0 has had
only 4 years added to his life expectancy. The National Foundation
of the March of Dimes reports that one in seven births ends in death
or deformity of the infant.
An increasing number of scientists believe that much of this deterioration of American Health can at least in part be traced to the
strain placed on individuals by chemicals in the environment. Heart
disease, the major killer, may be made more likely by chemicals such
us caffeine. Cancer accounting for 20 percent of all deaths is being
increasingly related to chemical causes.
But more important is the potential for genetic damage that many
chemicals present. At present, the knowledge gained, about potential
mutagenic perils from research efforts by scientists in the field of
genetics has far outreached the efforts of the Federal Government to
establish procedures to insure that the general population is not
exposed to mutagenic chemicals.
The regulatory history of 2,4,5-T and 2,4-D serves as an example
of the governmental failure to protect the public from potentially
dangerous chemicals. There are some general antidotes that might
possibly begin to reverse the Government failure to deal with what
is potentially a very serious health hazard.
It might be of interest to this committee to examine the current
relationship and overlap between the Food and Drug Administra-
tion 'and the Department of Agriculture in the pest^^j field. Currently the law requires all pesticides to be certified by the Pesticide
Regulation Division of the Department of Agriculture,
The main criteria for registration is the effectiveness of the pesticide for its intended purpose. At one stage in the process of register- .
ing a new pesticide, the Agriculture Department is required to seek
an advisory opinion on safety of the pesticide product from the
Food and Drug Administration.
As the situation now stands the recommendations of the FDA that
certain products are unsafe and should not be marketed as intended
have been rejected nearly 100 percent of the time by the Agriculture
Department.
In addition, thousands of FDA recommendations concerning the
proper labeling of pesticide products have been rejected. As the law
now stands this procedure is perfectly legal. Agriculture is merely
required to seek advice. It is not required to act upon it. In the present time when the safety of pesticides has become a more important
concern than their economic usefulness, it might be wise to readjust,
the relationship between these two agencies.
Perhaps pesticide registration should be officially the responsibility of the Food and Drug Administration, with the Department of
Agriculture providing only an advisory opinion as to the potential
usefulness and effectiveness-of the chemical. Stich a readjustment
would direct Government attention toward the problem of safety
and environmental contamination which have become the major concerns in the pesticide field and away from the much simpler task of
determining the potential usefulness of pesticides in killing particular bugs and weeds.
It might aJso be of interest to this committee to explore the relationship between the producers of chemical pesticides and the agencies that must certify and offer opinions on them. Currently the
overwhelming majority of scientific information relied upon to
establish the effects of pesticide use is provided by industry sources.
This, by the way, is true in all chemical fields—foot additives, cosmetics, drugs, as well as pesticides.
Often the manufacturers of these chemicals work out relationships
with independent testing facilities which tend to seek the answer
most advantageous to their clients. An illustration of the situation is
what recently happened in the Food and Drug Administration
itself, to a scientist who sought some testing results from an independent laboratory. He waited for several weeks for the results of
what should have been a relatively easy experiment.-. Finally he
called the researcher and was told that there was trouble with the
experiment. The Food and Drug Administration official asked what
the problem was, and was told that as hard as he tried, the scientist
conducting the experiment could not find anything wrong with the
chemical he was testing. He was under the impression that he had to
find something wrong, or the Food and Drug Administration would
be dissatisfied with the work.
This kind of bias exists in both directions throughout-much of the
community that is currently testing the effects of chemicals. To solve
this problem in the drug field, Senator Nelson has proposed a'large^
Government-run testing facility that would establish the safety 6V
�20
dtu^B of all drugs submitted to the Food and Drug Administration^
for approval.
Such a program', if feasible in the drug area, could easily be
extended to pesticides, as well as the food additives and cosmetics.
Another approach to the problem of how to separate the manufacturer from the researcher would be the establishment of a Govern- •
mont referral board. Such a board would receive requests from the
manufacturer that wishes to have a certain pesticide or food additive certified. This board would refer these requests—by a prearranged lottery system—to one of a series of independent laboratories
previously certified by the board for such testing. The rules for such
referrals could include a prohibition against any laboratory testing
two or more chemicals from the same manufacturer in succession,
thereby breaking up the economic dependence that some laboratories
have on one manufacturer.
The rules could also outline in some detail the methods that must
be followed. And the laboratories could be inspected to insure that
they do in fact have the facilities and the expertise to conduct the
experiments necessary. Such a board could be useful in maintaining
the economic independence of scientific research facilities as well as
in upgrading the caliber of research done on environmental chemicals.
It might also be of interest to this committee to examine the interlocking relationships of the various groups that make up the scientific establishment in the pesticide field. Currently the FDA runs
three separate programs in the pesticide"field. It sets tolerances and
then conducts investigations to insure that tolerances once set are
not exceeded.
Second, it conducts a total diet survey which is the major program to determine whether or not too much pesticide is finding its
way into food.
Third, it advises the Agriculture Department on pesticide safety.
I have already indicated that the FDA program of advice to
Agriculture is not particularly effective. Unfortunately, the other
programs pursued by FDA are not much more effective. The total
diet program has been seriously criticized by one former FDA science advisor—"former," apparently as a direct result of the criticism
leveled at this program—because of its failures to be chemically relevant.
Apparently claims were made for scientific test methods that were'
scientifically untrue, operators were sloppy in the use of scientific
procedures and equipment, and officials did not wish to alarm the
public. As a result, it appears that the reports of the total diet study
which strongly suggest that pesticides in food present little danger
seem to be over optimistic.
Presently the FDA is conducting a review of the program based
on these criticisms. The tolerance setting program has been subject
to the twin problems of limited scientific ability and information
and a desire not to alarm.
What is most important about the FDA programs in these fields,
however, is that they tend to seek support for their effectiveness
from the Association of Official Agricultural Chemists and the
21
National Academy of Science's National Research C^fcil committee
on pesticides.
^F
Both of these groups are presented as independent evaluatprs of
FDA methods and programs. In fact, however, there is a significant
overlap between the groups. FDA officials are also officials of the
AOAC, and sit on the NAS-NRC committee. In fact, FDA itself is •
proud of their claim that it would be difficult to find any experts in
pesticide tolerance and residue monitoring techniques outside of the
agency. Interlocking relationships of this kind, however, are notorious for their ability to xmdermine scientific excellence. Without
vigorous and challenging debate, little scientific advance takes place.
The empirical evidence on pesticides suggests that a problem of
major magnitude threatening the health and well-being of thousands
of Americans may exist from exposure to pesticides.
The FDA, supported by the AOAC and NAS-NRC tend to discount this evidence. It would seem that information about these
groups and how they relate to each other would be useful in evaluating the meaning of their pronouncements.
More critical examination of official reassurances about the effect
of pesticides on the environment, an evaluation of the usefulness of
either greater Federal testing for safety of pesticides or the establishment of a Government referral board and the shifting of the
Government focus away from pesticide effectiveness and toward pesticide safety could begin to place the serious potential dangers from
pesticides into the proper focus.
Thank you for your consideration of these thoughts and the
opportunity to appear before you.
Senator HART. Gentlemen, thank you for expressing it so effectively.
Shortly after you began your testimony, Mr. Wellforcl, we were
joined by the Senator from Hawaii, Mr. Inouye, and the Senator
from Tennessee, Mr. Baker.
I have just a few questions.
First, you talk about two potential disastoi-s in your testimony.
The first is< one that may have already occurred, relating to past
birth deformities. You suggest that these perhaps are attributable to
the use of 2,4,5-T or related chemicals.
The second I think is implicit in your suggestion that 2,4,5-T and
2,4-D and the dioxin contaminant of these and other chemicals may
accumulate in the human tissue and so build up in each of us on sort
of a regular basis. The end result of an accumulation of such highly
toxic or teratogenic material is depressingly clear.
Now, there are many—if past experience is our guide—who will
criticize you, and perhaps me, for talking about these possibilities.
They will suggest that it is scare tactics, or some other label.
Anticipating that kind of reaction, what comment do you make?
I'm sure you have been exposed to it before.
Mr. WE^LFOIW. If people think we sound alarmist, they should
talk to the scientists we talk to, because they are very alarmed.
I think our main point is, there is just too much that we do not
know about these herbicides, and, secondly, we're saying that the
burden of proof should be placed on the mamifacturers to establish
their safety.
�22
TiffTm meantime, the public should not continue to bo the unwilling guinea pigs of their casual, and I think frequently unnecessary
:
use,
•
•
••
There is substantial evidence that these herbicides are potentially
dangerous. There will be many more tests to find but exactly hoty
dangerous they are, and maybe in the end there will be a determiria'tion that the amount of exposure that the average American receives
is nothing to worry about.
'
•'
But at this point, there is no evidence that we shouldn't be concerned, and in light of what^we feel that the use of these herbicides
should bo restricted.
Senator HART, In your prepared statement'—and I think as you
summarized it you repeated it—you make reference to the Whiteside *
articles in the New Yorker. If there is no objection, I would ask
that thov be made apart of the record at this time. .
'
_ Mr. WELI/FORD. I would also say, in further response to your question, that there has been a serious credibility gap on the part, of the
Government in the whole herbicide case. The Bionetics report was
withheld for a long time, even from scientists. Dr. DuBridge made a
statement in October. The public was reassured that something was
being done. And yet the statement was ignored.
I think in this situation there'is a particular necessity for people
to speak out. And that is one of the reasons we have appeared this
morning.
Senator HART. One specific reason I asked that the Whiteside articles go in is to acknowledge that they are responsible in major part
for our hearings,
The first of those two articles mentions the important part played
in the release of the Bionetics report by Nader's Raiders. Though
you were not introduced as such, can you respond by telling us the
story of the involvement of your organization in the release of the
information ?
Mr. TURNER. Yes, Senator. That activity regarding the Bionetics
paper revolved around the group that was investigating the activities of the Food and Drug Administration, of which I was the project director. Before going into the specific story, I would like to add
one comment to the statement in answer to your first question.
If anything, we have understated the scientific concern about the
problems of chemicals in the environment, of which pesticides is one
part. But what we have tried to talk about here is how to bring the
legal system into some kind of meaningful response to what We
admit to be a very unclear field of scientific endeavor.
We don't know, nor do many other people, what, exactly, is happening in the chemical environment. How, then, are we as'a society,
as the Congress and as people—how are we going to respond to the
legal challenge that scientific uncertainty presents ? And the kinds of
things we have been talking about today are designed to suggest
answers to these questions.
Concerning the Bionetics paper, to me it has represented since we
first came across it, oh, sometime in August of last summer, one of
the real tragedies and one of the sad parts of the kind of a job we
1
Sec p. 107.
23
try to do on a day to day basis. And that is, in our j^nties we constantly find scientists and regulatory officials in the Grovernment who
have very important concerns which they would like to bring to the
attention of someone, to have some kind of resolution made about
the issue that they are concerned with.
This was the case in the Bionetics paper. This, by the way, is not
the only paper of that nature that we have run across in the course
of the last year. We received the paper from two sources, basically,
that were unhappy scientists who saw a very serious potential problem and said that no one would really listen to what was being
said—internally, this is. We talked to the MEAK Commission and
asked them if they were aware of the report, and they had been
aware of the announcement and aware of the withdrawal, but had
not seen the report. So we did then try to make it known to certain
officials gradually, not knowing the full impact or not knowing the
full meaning of it. It is not all that clear, although it does raise
some serious problems. What was clear to us, though, were a number
of scientists in Government who were concerned about a specific,
problem that they wanted an answer to, or wanted attention focused
on, and could not find any way to bring such attention to focus.
Then we began to talk to people who we thought would raise the
issue in the proper circles. That is essentially the way the paper
began to emerge.
I would like to emphasize this is not the only one of those situations which we discovered.
Senator HART. You discovered other situations that bore on pesticides as potential dangers ?
Mr. TURNER. There are some other problems on pesticides which
we have alluded to which, are disturbing. The question of trying to
bring to the attention of regulatory officials the problems of Folpet
and Captan has been one that I have been concerned with for over
18 months, and have tried to alert various people in the regulatory
agency, the Food and Drug Administration, to. The scientists in
that agency are concerned about these two particular pesticides, and
they are asking why are they on. the market? The first time that
question was asked of me was in the summer of 1968, and wo are not
approximately—well, almost 2 years later, still selling these same
pesticides.
Senator HART. In your testimony you have talked about the tests
that several federal agencies have undertaken which clearly show in
Mr. Wellford's prepared statement that even the purest 2,4,5-T still
produces birth defects in test animals at significant levels.
You go on to say that a recent test on 2,4-D confirmed that this
herbicide which was very often mixed with 2,4,5-T, is teratogenic.
I have been told, however, that the tests which have been conducted are merely preliminary and that although they suggest certain conclusions they cannot confirm them. Is that correct? In other
words, as of now, we can't say that we know that the currently produced 2,4,5-T is teratogenic, can we ?
Mr. WELIJTORD. Certainly, as far as the effect on .human beings,
you are entirely right. There is no clear evidence—we haven't been
able to find evidence through epidemiological surveys for reasons
�24
described, that these herbicides are definitely a danger to/
hur
beings that come in contact with them.
"
It is also true that there has not been time for the tests, which
raise the suspicion to be checked and rechecked by many other scientists.
But I think one thing that is certain is the tests do raise very serious questions, and we can take no comfort whatsoever from them,
that these herbicides are not dangerous as they are not being used.
Again, it is a question of burden of proof.
Senator HART. You would turn it around and say—well, yes, you
can't say they are harmful, but you can't say they aren't?
Mr. WELLFORD. I would say the more recent tests have expended
the burden of proof upon those who wish to prove that they are not
harmful.
Senator HART. You suggest, or mention, that the advice on registration that Food and Drug has given the Department of Agriculture frequently, or sometimes, or however you phrase it, has been
ignored.
Mr. TURNER. What was said was 100 percent of the time, Senator.
Senator HART. Almost.
Mr. TURNER. Almost.
Senator HART. Almost 100 percent of the time. Isn't it the fact,
however, that Food and Drug could make its advice stick by setting
residual tolerances which a pesticide, if registered, would exceed ? Of
course, that is a shorthand way, and perhaps arbitrary.
Mr. TURNER. It is somewhat arbitrary. The problem is, the kind
of recommendations that are made are directed to products that
have some usefulness, and to arbitrarily say there should be no residue could seriously affect the use of these pesticides which—in themselves—may not be particularly dangerous.
The kinds of things we have been talking about, on a number of
occasions FDA recommended that certain marketed home use products that are used to kill rats should not be directed for use on bread
crumbs or bread particles to be left where rats can get them, because
then children can get them.
On a number of occasions this particular recommendation has
been ignored by Agriculture, and they have gone ahead with that
kind of a direction.
There, are others. For example, the Food and Drug Administration has recommended on a number of occasions that seeds which
will find their way back into the food supply, after they have been
treated with pesticide, be dyed with a color, so that you can see
them—blue or red. This is the general procedure in this kind of pesticide program.
But there are some products to which this is not done. When
FDA on several occasions has said this should be done before there
is any more marketing of that product, they have been rebuffed by"
Agriculture on those particular products.
Senator HART. In those situations where FDA has fixed a tolerance, do they have the facility to enforce adequately the tolerance
that is established ?
Mr. TURNER. It is my judgment that they do not. There are two
ways that the residue problem is dealt with by FDA. One is by
rs
trying to enforce established tolerances, and the otherjs by the general survey called the total diet study, which I
App
In the former program, the FDA conducts i^P
pproximately
7/100ths of 1 percent of an investigation process. That is how many
interstate shipments they examine for pesticides— seven-one hundredths of a percent.
_
.
The way that they do many of these investigations is by being
alerted beforehand that there is some kind of problem with the particular shipment. The situation, as the statistics emerge from FDA
—by the way, that is one-half of their announced program for the
years of 1963 to 1966; they did one-half of what they thought they
were going to do — shows that they find a 3 percent violation of tolerance in the seven hundredths of a percent investigation that they
conduct, and this would indicate— if the samples are statistically
sound— a minimum of 70,000 shipments going in interstate commerce with pesticide residues that are not detected each year.
My judgment is that that is not enough to provide the kind of
incentive to a person using pesticides, who must then ship his products, to be deterred from using too much.
I also would like to point out that the second program, the total
diet program, which is the one that is done to survey the entire
food supply, also has varieous weaknesses. The way the program
works is that the total diet of a 19-year-old boy— he is considered to be the major eater in the country — is bought at a supermarket at various areas in the country. Then it is in various ways
broken down and tested to see how much pesticide exists in the 2
weeks diet.
On the basis of this particular program, the Food and Drug
Administration has assured the American people for several years
tlmt there is no real problem of pesticide residue in food.
When the science advisor began to look closely at this particular
program, he came up with a series of very disturbing findings.
There were no verified test methods for evaluating what, in fact,
was being found by FDA when they conducted these tests. FDA had
reported residue amounts that were lower than the error that was
granted to the system. The system could find down to perhaps 10
parts per million without error, and they were finding residues as
low as five parts, or below, per million. The science adviser in Baltimore made a detailed presentation to the agency and he shortly
thereafter found that his contract for that year was not renewed.
It was at that point that I found another one of these tragic cases
in Government, and began talking to the Food and Drug Administration about this particular problem. And at our insistence and oitr
negotiation, a hearing within the agency was impaneled and a discussion was held, and a review of the program was decided upon.
As yet, I don't know what the results of their review are, but my
' feeling is we do not have good pesticide residue monitoring from
that program either, at this point.
Senator HART. I have indicated, and you have agreed, that for
FDA simply to fix a tolerance level not to be exceeded as a device to
keep an item off the market, or a particular use, would' be arbitrary.
And you indicate that FDA's ability to ride herd on tolerances that
are established is inadequate.
�2u •
Avind up, I tales it, by. making the case that the key to
that we are seeking to insure some protection involves
business of registration. You don't quite put it this way, but what
you are telling us is that registration is presently under the control
of the inappropriate agency.
Mr. TURNER. That is my feeling, Senator.
Senator HART. This is not necessarily to be critical of Agriculture,
because, as you point out, they have always been pretty occupied,
understandably, determining the potential usefulness of pesticides on
bugs, weeds and what have you, and FDA more likely would be concerned with the problems of environmental contamination and
safety.
My last question. You have told us how the lable content is regulated. What agency would have jurisdiction to require more adequate packaging to avoid breakage? Maybe I should say what
agency, if any ?
Mr. WELLFORIX It is cleai'ly a public health problem, and to be
perfectly frank, I do not know precisely who has direct jurisdiction
over packaging. I would presume, though, that' the first responsibility is the pesticide regulation division of the Department of Agriculture, but Mr. Bayley will be testifying and you can ask him that
question.
Senator HAKT. That does not require a great deal of scientific
interchange to figure that out.
Mr. WELIYFORD. No, it does not.
Senator HART. If we have given anybody the authority to fix the
package so it docs not bust, they ought to be able to figure it out,
and if we have not, we ought to fix the law.
Senator Inouye?
Senator INOUYE. Thank you very much, Mr. Chairman.
Gentlemen, are you convinced that the presently available data on
the potential dangers of 2,4,5-T and 2,4-D are sufficient to outlaw
the iise and sale of these herbicides and pesticides?
Mr. WELT/FOUD. I think that the data is sufficient to at the very
least suspend their use until the tests that need to be undertaken to
answer the questions we have raised have been performed. I think it
is^ au unnecessary risk for the public to have these substances so
widely used when we know so little about them.
Senator INOUYE. In other words, you are suggesting that further
and more intensive studies be made before a final decision be issued
by the government?
Mr. WELLFORD, No, I think that one decision can be taken by the
Government right away, and that is to suspend the use of these
products until more tests are done. I think to delay that, to wait
until tests and retests are performed might unnecessarily expose
people to hazards which you will eventually regret. That is the
point.
Senator INOUYE. I am convinced of the potential dangers of these
pesticides and herbicides, but I cannot help recalling the recent
cyclinnate scare that we had here in which our shelves were rid of
all these canned fruit cocktails with cyclamate. Now recently I recall
reading an article in which scientists indicated that in order to
sull'er the dangers involved in the use of cyclamate, a child would
.
,
,.
27
have to consume about 120 servings of fruit cockta^pr day for 10
years. As a result, I believe cyclamate fruits can be sold on the shelf
with a warning of some sort.
That is why I asked this question. Are you convinced that the
data are sufficient to suspend present use ?
Mr. TURNER. I would like to comment on the analogy to the cyclamate situation. There has been a great deal of misinformation circulated about cyclamates, and it should be brought back into perspective.
First of all, it is my belief that the present marketing of
cyclamates as it is being conducted is a violation of the law, and we
will proceed to undertake some kind of action to either bring that to
the attention of Congress or to try to do something in the courts
about it.
In fact, when the Food and Drug Administration gave its warning about cyclamates in April of 1969, before they knew that there
was any involvement with cancer, they announced that people, in
order to be safe, should consume only a certain amount of cyclamate. That amount for children was two-thirds of a package of presweetened Kool-Aid.
When the final cancer tests were done by Abbott Laboratories, the
ones that Secretary Finch relied on to take the substance off the
market, the National Academy of Sciences reported that applying
the hundredfold safety measures that is used generally in food additive areas and applying the general methods that are used scientifically to deal with food additives would show certain levels of cyclamate to be safe.
The amount that they were talking about as safe was the amount
that would go into three cups of coffee in a day. Subsequent to that
time, the Food and Drug Administration conducted experiments
which showed that the same kind of cancer problem that was found
by Abbott Laboratories was also found at FDA at one-sixth of the
level of intake over an 18-week period rather than a 2-year period,
thereby greatly increasing the amount of problem that a person is
perhaps subjected to when he takes cyclamate.
In addition to that, there is on file at the Food and Drug Administration or in the Health, Education, and Welfare Department a
memorandum which specifically points out that there is a serious
question that diabetics may be more susceptible to the dangers of
cyclamate than other people.
The question that you raise is very apropos to both of these chemicals. We cannot really say at this point that there is anything that
we can prove that will definitely say people are going to get these
results. The problem we have is how do we structure the legal
system to deal with the problems of uncertain science.
What we have done in the food additive area is to say that a food
additive must be proven to bo safe before it goes into the food
except for what was to be a very small loophole in the law. Unfortunately, as that law has been administered, that list has nearly
become as large as the additives proven safe.
In dealing with 2,4,5-T, we would like to provide, that is, those of
us testifying today, would like to provide the same kind of standard •
in the pesticide area that should be prevalent in the food additive
45-362 0—70
�28
29
area, and that is until the substance is proven to be not harwRil it
should not come on the market.
Once it is on the market, if there are questions raised and they
cannot be disposed of, then it should be removed from the market
until those questions are disposed of.
Senator INOTJYE. Your testimony indicates that you have done a
lot of study in this area, and I commend you for this. I am certain
you are aware that the State of Hawaii uses a lot of this herbicide.
In the year 1968 to cover an area of 120,000 acres we used 197,000
pounds of 2,4-D and 6,000 pounds of 2,4,5-T.
Obviously, it is an important part of our economy, because this
constitutes'the major portion of our income. In your studies, have
you come up with any substitute for 2,4,5-T or 2,4-D ?
Mr. WELLFORD. There are other herbicides on the market. I am not
really qualified to explain the virtues of one against the other as far
as the uses that 2,4-D and 2,4,5-T now have.
Again, I think that if there are not adequate substitutes that there
probably could be with more research and investigation. I think that
at the very least having these herbicides suspended for use in populated and residential areas could help bring that search about.
Mr. TURNER. I think there is another point to make in regard to
your question, and that is in much of this area of chemical environment we are presented with some minds set or misconceptions that
lead us down wrong tracks. One of the problems with, using pesticides such as 2,4,5-T and 2,4-D and many others is that they are
what we might call broad spectrum pesticides. They can be used on
many different pests.
If we are going to find alternatives for them we cannot think in
terms of finding a pesticide that can do as much as these will. We
have to think of what sort of pest is sought to be controlled and
find a method for it.
The Agriculture Department has found many methods of controlling problems, for example, alfalfa and cotton. These are nonchemical methods. Sterilization of pests is one of the approaches. It
depends on what kind of particular thing you happen to be directing
'the pesticide at.
There can be alternatives. I think we have not found them largely
because we thought in terms of these massively effective chemicals
rather than thinking in terms of how do we control this pest with
the least possible strain on the population around it.
Senator INOTJYE. Last week when I did my shopping to prepare
myself for the spring, I bought some of the items on your desk
there. Do you suggest that I get rid of them ?
Mr. WELLFORO. I suggest you be careful how you get rid of _ them.
I think one of the real problems with these substances is the
difficulty of knowing how to dispose of them if you decide you do
not want to use them. .You are not supposed to put them in a trash
can; you are not supposed to bury them; you cannot put them down
the sink.
I think if you call the various public health authorities to try to
find out the best way to dispose of them, they eventually tell you,
take them out to an incinerator on the outskirts of Washington, but
they do not really have any other answer.
I think that problem indicates the toxicity olWe substances that
we are dealing with, and in direct answer to your question, I would
not apply them to my lawn if there was any chance that pregnant
women or small children could come into contact with them.
Senator INOUYE. Thank you very much.
Thank you, Mr. Chairman.
Senator HART. Senator Baker?
Senator BAKER. Thank you, Mr. Chairman.
I must say I, too, commend you for a rather extensive and well
prepared presentation this morning on Mr. Wellford's part and Mr.
Turner's part.
There are two or three things that occur to me about which I
would like to inquire that relate to Senator Inouye's concern, and
that is what do we do if we discard these pesticides and herbicides?
I wonder, for instance, if there is a study or if you have any statistical evidence on what the economic loss would be, say, to the State
of Hawaii in the case of a suspension of 2,4,5-T and 2,4-D? Before
you answer, do not assume I am arguing against such suspension.
I am rather concerned about a complex life, and to be facetious
for a moment, none of us really will live through it.
Is there some way to judge the relative good or the relative bad
that flows from the use or nonuse of any of these pesticides?
Mr. WELLFORD. One quick point on that. There is a report commissioned by the Defense Department which is on the ecological effects
of continued herbicide use, and in that report they, state that in
many cases the chemical weed clearance methods are often no more
expensive and just as good as the use of herbicides. •
It has been my experience that herbicides or their massive and
popular use reflects the American penchant for technological gadgetry and marvels and perhaps we could find many more ordinary
ways to get the same job clone.
Senator BAKER. I agree with that. There are other ways. Take the
extreme case of the mosquito and malaria. Clearly there must be
some way to control the breeding grounds of the mosquitoes. It is
done in some areas by raising and lowering the lake levels. It is
done in other areas only by the use of pesticides. .
Once again there is a trade-off. In those cases and in remote areas
of Vietnam or the United States, it might be demonstrably better to
run whatever risk _there is of mutagenic danger in order to dispose
of the more immediate and more imperiling danger of malaria.
Is there any sort of study ? Is there any sort of weighing of these
equities by your group ?
Mr. TURNER. There is not anything done by our group and I do
not know if there has been anything done by any other group. Our
approach to this problem is this, and I think it bears directly on
this question: I am concerned about the cyclamate case, for example,
because of the tremendous economic impact that it had, but that economic impact came largely from the delay and the lack of effective
action over the period of 4 or 5 years when cyclamate was developing.
What concerns us in the area of 2,4,5-T, is that it was announced
its use would be curtailed. We in the public were under the impres-
,
�30
sion it was being curtailed. We began to look at what was happening, and it was not being curtailed.
We have urged specifically that the use in the home, in and
around the home be the first one to be specifically curtailed immediately.
My feeling is that since we have known for approximately 18
months that as a sprayed pesticide or herbicide it (2,4,5-T) has been
a problem,—that since someone has known for probably the last 15
years that it did present a problem, action should have already been
taken.
The workers at Dow Avere having serious occupational problems
from the manufacture of the substance. Now, admittedly when a
substance appears in the public eye for the first time it does create
the kind of crisis situation where you perhaps must balance economics versus the potential harm from the substance. However, what I
am concerned about is how we prevent that kind of crisis situation
from developing on other substances and on this particular one.
In my area, which is more directed at the food chemicals, I would
be more than willing to say that we should have an interim period
to solve the kinds of problems you are suggesting if there was any
kind of guarantee that that interim period would be lived up to by
the producers of these chemicals.
The problem is in any area where the interim period has been
allowed in the food area it has been used to erode the provisions of
the law, so at the end of the interim period there is not any kind of
authority or force to keep the product in control. That is what concerns me about trying to accommodate the economic situations which
you are raising, although I consider them to be rather serious.
Senator BAKER. They are not only economic conditions. What I am I
referring to is health. I am asking if there has been a study by
anyone in your group or any other group on the relative merit of
the discontinuance of the use of pesticides on the one hand and the
prevention of mosquitoes and malaria on the other.
Mr. TURNER. For example, you mentioned the mosquito problem. I
notice that one major company has just developed a nontoxic.
method of controlling mosquitoes. This would be an example. I
think with the proper kind of direction and the proper kind of
focus, these alternatives can be developed.
The reason I was talking about economic importance, is the real
impact of these particular pesticides we are talking about are economic. I do not believe they have use as a health control method.
Senator BAKER. Can you tell me whether FDA or anyone else is
conducting any extensive research on substitute agents for pest controls?
Mr. TURNER. As far as I know they are not, they are not conducting extensive research, but Agriculture does have a research program on this subject, but I do not believe it is extensive.
Senator BAKER. Are you familiar with the plan by the Forest
Service and, of a]] people, by the Atomic'Energy'Commission to,,
develop sterilization techniques for certain pests that attack forests?
Mr. TURNER. Presently, as I understand the Agriculture Department, it is dealing with this area and it believes at present they are
31
able to control about ten percent of the pest problelWvith those
kinds of methods.
Senator BAKER. To control completely ten percent of the pests
rather than ten percent control ?
Mr. TURNER. Eight.
Senator BAKER. Would it seem to you that further research in
these fields might very well produce acceptable substitutes, acceptable as we know them by these standards? I freely predict that 10
years from now they will not be the standards?
Mr. TURNER. That is right. Agriculture is working on research
methods whereby they can use much smaller amounts of pesticides
to achieve the same results that are achieved by the very large use of
chemical pesticides. In some cases more than a million or so pounds of
2,4-D is used on certain submerged weed areas to control the weeds
per acre. This is an incredible amount of pesticide use.
Now they are down in some cases to using chemicals to one one
hundredth of an ounce on an acre. This kind of a balance is going to
go a long way toward controlling some of the environmental hazards
that we have.
_ There are experiments being conducted at the University of Georgia which indicate that perhaps as much as 99 percent of all sprayed
pesticides come out of nozzles in droplets which are so large that
they are useless for this purpose and they are absorbed by the environment.
Senator BAKER. Are you familiar with the use of underground
watering for microporous piping?
Mr. TURNER. No, I am not.
Senator BAKER. That, of course, would stop air contamination by
pesticides and herbicides and it would increase the concentrations
and the effectiveness and eliminate the resin problem.
Mr. TURNER. Right.
Senator BAKER. I really commend that to further study.
Senator HART. Thank you, Mr. Baker. I think the committee is
fortunate to have Senator Baker on it. He either has done homework
or somehow or other is involved in the field.
Mr. Bickwit?
Mr. BICKWIT. I would just like to clear up one point on my own
mind. On several occasions in your testimony you have complained
that Dr. DuBridge's statement made in October of 1969 was not
complied with. However, in other parts of your testimony you have
suggested that mere compliance with that statement would not be '
sufficient in your view. Am I right in understanding that you feel
that Dr. DuBridge's statement does not go far enough?
Mr. WELLFORD. Absolutely. I think its biggest gap is the fact it
does not discuss pesticides which are used in residential lawns and
gardens. I think there he was under the misapprehension that
2,4,5-T was not widely used for this purpose. In fact it is. I suspect
if ho had known it, he would have included that in his1 statement.
Senator HART, Gentlemen, thank you very much, for a .very helpful presentation. You have raised some questions that clearly will
have to be resolved.
_ Next, we welcome Ned Bayley, the Director of Science and Education of the Department of Agriculture.
�32
'EMENT OF DR. NED D. BAYLEY, DIRECTOR OF SCIENCE A
EDUCATION, DEPARTMENT OF AGRICULTURE; ACCOMPANIED
BY DR. T. C. BYERLY, ASSISTANT DIRECTOR OF SCIENCE AND '
EDUCATION
Dr. BAYLEY. Mr. Chairman, I am Ned Bayley, Director of Science
and Education, Office of the Secretary, Department of Agriculture.
I have with me Dr. T. C. Byerly, Assistant Director of Science
and Education.
Before I proceed with the formal statement, I Avould like to
respond to some extent to the information Avhich has .been presented
already this morning.
Senator HART. Let me make it easier. Let me encourage you to do
it, and any succeeding Avitnesses, too.
To make the record as useful as possible, we would welcome
exchanges in the nature of reply.
Dr. BAYLEY. I appreciate in doing this I am putting myself in an
impromptu position and, therefore, would appreciate the privilege to
provide to the committee, for the record if they desire, fuller statements regarding the activities of the Department of Agriculture in
regard to pesticides.
Senator HART. Very well.
Dr. BAYLEY. I testified before this committee previously regarding
the broad policies and positions of the Department in the pesticide
area, and stated that we recognize that all pesticides are economic
poisons. They are only one group of the tremendous number of economic poisons which we use for a large number of useful reasons,
not only for economic purposes but also to take care of public
health.
We recognize also, as part of our civilization and as part of the
standard of living and the food supply that we already have, that
Avithout these economic poisons and their judicious use, AVC Avould be
in a very serious situation from the standpoint of our ability to produce food and fiber for this country.
Now, I also want to point out very briefly the references to the
activities of the Department of Agriculture regarding the registration of pesticides. I will be the first to agree that there have been
some problems in regard to these registration procedures and I will
be the first to agree that we haven't resolved all of them.
We have, hoAvever, particularly during the past year, taken a
number of steps toAvards eliminating some of the complexities and
bureaucratic difficulties which have existed in the area of registration.
The references which have been made here this morning primarily
reflect the relationship among the departments that did exist, but, I
think I am safe in saying, do not exist now.
Senator HART. I remember in those earlier hearings we discussed
this problem.
Dr. BAYLEY, Yes, AVB did.
Senator HART. And I had the impression that there was a transition period.
33
Dr. BAYLEY. I will very briefly refer to that. liRrux of the
problem was that there was an interagency agreement for resolving
differences in regard to registration. There was a procedure within
the agreement of bringing differences to the attention of the three
Secretaries involved.
Unfortunately, however, over the years this agreement was in
existence, not one of the departments ever used this procedure to
resolve their differences.
We in the Department of Agriculture must share the major
responsibility for not getting the differences resolved primarily
because the enforcement of FIFBA was primarily our responsibility. But I am glad to say that since that time, with the initiative of
Secretary Hardm and Secretary Finch and Secretary Hickel, there
has been a new agreement worked out between the departments. This
agreement specifically provides the basis whereby differences in
judgments regarding pesticide regulation can be brought up through
the decisionmaking procedure and the three Secretaries can share in
this as needed.
We believe this is a sound basis for increasing the interdepartmental relationships and providing a basis for all three departments
to have a rightful input into this.
I think you are also acquainted Avith the fact
Senator HART. Doctor, if you are going to leave that new agreement, I would like to ask one question. In the event of disagreement,
when the three Secretaries' attention is invited to the competing
claims, is the decision made by majority vote, or does the Secretary
of Agriculture retain the final voice ?
Dr. BAYLEY. It is my understanding that they will pursue the disagreement until they agree. The Secretary of Agriculture does retain
the final voice according to the law, however. We believe that based
on the way we are operating today this procedure can be effective.
Senator HART. A meeting in Paris ?
Dr. BAYLEY. Well, we are not dealing with that. I think Aye recognize that three Cabinet officers have the public interest in mind
when they get together and can make a decision along these lines.
Senator HART. Just to push you a little harder on it, and I suppose this is academic at the moment since no such dispute has yet
reached the three Secretaries ?
Dr. BAYLEY. This is correct.
Senator HART. If one does get there, it Avill involve the tricky balance that Senator Baker was talking about, the economic claims, the
public health claims, and the environmental concerns. HEW Avill
tend, I assume, to emphasize the health factor. Is Interior the third
department ?
Dr. BAYLEY. Yes.
Senator HART. They would I suppose, be concerned principally
with effects on fish and wildlife, and Agriculture would think primarily of the utility to the agricultural economy.
To put it harshly, why shouldn't the fellow who says it lias not
yet been established as safe for humans have the ultimate vote and
voice ?
�34
35
Jr. BAYLEY. ^ May I say this, that from the standpoint of the
Department of Agriculture, we recognize that issues involving
human health should have priority over all other issues.
The reference which was made earlier that the emphasis in USDA
had been primarily on effectiveness is not only incorrect regarding
the past but it is utterly incorrect regarding our position now.
Senator HAUT. Whatever the past, I would hope that human
health does have the overriding concern of three or any other nnmbers_of men that meet on this kind of claim.
I interrupted you.
Dr. BAYLEY. Surely.
With those preliminary comments, I will be glad to turn to the
issue of 2,4,5-T and the facts as we see them at the present time.
The _ herbicide 2,4,5-T has been recognized as the most effective
herbicide registered for use for control of certain weeds and brush
species for^more than 20 years. About four-fifths of the domestic use
.of 2,4,5-T is.for nonfarm use, the largest such use being for control
of brush on rights-of-way. It is also used extensively to control
brush on forest lands and certain weeds in turf. 2,4,5-T has been
used in the production of fruit crops, cereal grains, and sugarcane.
It is the most effective herbicide for control of brush on several million acres of rangeland in the Southwestern United States.
2,4,5-T is degraded in the environment within a few months after
application so that residues do not persist from one season to the
next. Residues on foods are unusual. Among 5,300 food samples analyzed by FDA for 2,4,5-T during the past 4 years, 25 were reported
to contain trace amounts; i.e., amounts less than the 0.1 p.p.m. limit
of accuracy of present analytical procedures for foods. Two samples ,
showed residues of 0.19 and 0.29 p.p.m., respectively.
No finite tolerance has been established for 2,4,5-T in food. In the
absence of such tolerances, any detectable amount-of 2,4,5-T in food
would make such food subject to seizure if found in the channels of
interstate commerce, From the data cited above
Senator PERCY. Do I understand your statement to mean, Doctor,
that in the absence of the establishment of human tolerances for
2,4,5-T, it is the present policy of the Department of Agriculture to
seize any shipments that show any measurable trace of 2,4,5-T on
food shelves ?
Dr. BAYLEY. It is the responsibility of the Food and Drug Administration to enforce the procedures and make the seizures.
Senator PERCY, I understood you to say there are no tolerances
established; therefore in the absence of any established human tolerances for 2,4,5-T, it is the present operation of the U.S. Department
of Agriculture to seize any food stuffs that contain any measurable
trace amounts of 2,4,5-T.
Dr. BAYLEY. It is the present obligation of the Food and Drug
Administration to do so.
Senator PERCY. Thank you very much.
Dr. BAYLEY. From the data cited above, it is apparent that contamination of food with 2,4,5-T is very infrequent and then only at
very low levels.
There is current concern over the continued use of 2,4,5-T arising
from the report of a research study completed under contract by the
National Cancer Institute by Bionetics, Inc. This sma\ was based
sWray
on a commercial lot of 2,4,5-T acquired for the study in 1965. It was
fed to pregnant mice and rats. Many of their developing young had
birth defects.
After review of this information and after consultation with Federal agencies concerned, Dr. Lee A. DuBridge, the President's Science Adviser, announced on October 29, 1969, a coordinated series of
actions being taken by those agencies with respect to the use of
2,4,5-T.
Among them was the announcement that: "The Department of
Agriculture will cancel registrations of 2,4,5-T for use on food crops
effective January 1, 1970, unless by that time the Food and Drug
Administration has found a basis for establishing a safe legal tolcranco in and on foods."
USDA was informed in January that the lot of 2,4,5-T used in
the Bionetics study contained significant amounts of a highly toxic
contaminant^ tetrachlorodibenzo paradioxin. The Department was
further informed that lots of 2,4,5-T of current and recent manufacture were reported to contain less than 1 p.p.m. of this contaminant
in contrast to the 27 p.p.m. reported for the lot used in the Bionetic
study.
Extensive studies are underway to determine whether 2,4,5-T is
itself teratogenic. Preliminary reports are consistent with the
hypothesis that the teratogenic results reported in the Bionetics
study were due to the contaminant dioxins or to interactions of such
contaminants with the 2,4,5-T rather than to 2,4,5-T per se.
The Department announced on February 6 that it would undertake examination of 2,4,5-T and 17 related compounds registered for
pesticidal use to determine whether or not they are contaminated
with dioxins. Preliminary results on 2,4,5-T show that those lots
examined of current manufacture and those now in channels of
trade gave the following results—I can summarize these quickly—
the amounts ranged from a trace to 2.9 parts per million, and they
were conducted both by the Department of Agriculture and the
Food and Drug Administration.
(The table follows:)
TABLE 1.—AMOUNTS OFTCDD FOUND IN COMMERCIAL 2,4,5-T BY TWO METHODS
TCDD content p.p.m.'
Sample
2.4, 5-T
2 4 S-T
2,4,5-T
2,4,5-T«
Manufacturer
-. Dow
.. -- Dow..
..
Lot
Grade i
120110
07-020
X-17394-21-5
MM-1 20449
TO
TG
TG
TG
Collected
2/70
2/70
2/70
2/70
FDA
USDA
trace
1.1 . .
NO.'
.48,
.
0.07
2.9
N.D
.47-. 52
i TG>=>Technical grade.
»TCDD Relers to the 2,3,7,8-tetrachloro-dibeiuo-p-dioxin (TCDD),
'N.D.^Levels of TCDD are below the limits of detection or below 0.05 p.p.m.
1
Sample supplied by Dow as a reference check and reported to contain about 0.5 p.p.m. TDCD.
These data are preliminary and are obtained from first drafts of
methods developed by chemists in the Crops Research Division of
the USDA and in the Pesticide Chemistry and Toxicology Division^
of the FDA. The dioxin values refer only to the 2,3,7,8-tetrachloro-
�37
nzo-p-dioxins (TCDD) and do not indicate levels of other
d dioxins (containing 5, 6, 7, or 8 chlorines) in the 2,
samples.
In view of all the information now available, we have not found
that registered use of 2,4,5-T without a finite tolerance on food crops
constitutes a hazard requiring cancellation or suspension of such
registered uses.
There has been and is concern over the ecological effects of 2,4,5-T
used as a defoliant in Vietnam. Dr. Fred Tschirley, Assistant Chief
of our Crops Protection Research Branch, has reported the results
of his examination of areas treated in Vietnam. He has reported no
evidence of irreversible ecological damage. Allegations that defoliation will lead to extensive laterization of Vietnamese soils, that
Mangrove areas will not recover, that fish production in wetland
areas will be reduced were not verified.
Dr. Tschirley also headed a team of scientists who investigated
allegations of injury to humans and animals due to herbicide treatment for control of Chapparal by the Forest Service on the Tonto
National Forest near Globe, Ariz. They found that apparent damage
consisted of damage to susceptible plants near the treated area from
drift of the herbicides used. The alleged injuries to a duck and a
goat were found to be groundless. Human illnesses were those
expected in a normal population with the possible exception of one
man with skin irritation on his eyelids. Clinical chemistry on specimens obtained during the investigation is in process.
Mr. Chairman, that is the completion of my formal statement. We
primarily presented it to provide you with the latest findings that
we have in the particular area.
Senator HART. Thank you very much, Doctor.
To summarize with respect to the sequence of events on the
DuBridge announcement of October 29,1969, the Department did not
in fact cleregister 2,4,5-T as DuBridge indicated would occur unless
these affirmed findings came along. But you tell ns your action was
based upon information that the tests by Bionetics used samples that
contained the contaminant dioxin, and that the current production
of that product was free of the dioxin; is that right?
Dr. BAYLEY. Not completely free. The dioxin was at a sufficiently
low level that Ave believed that
Senator HART. That the test material had sufficiently more dioxin
than the normal production amount ?
Dr. BAYLEY. Yes.
Senator HART. Are you aware that the preliminary results of tests
conducted by Food and Drug, Dow, and by the National Instititute
of Dental Research and by the National Institute of Environmental
Health Scientists all indicate that 2,4,5-T contaminated with no
more dioxin than is found in the currently produced 2,4,5-T is terato genie?
Dr. Bayley. We arc fully aware of this. The critical facts in
regard to those experiments is that those low level dioxin contaminated 2,4,5-T samples were fed at sufficiently high dosages that they
would be comparable to the dosages used for the 27 parts per million or nearly so.
_ Therefore, 'we do not believe this in any way changes the hypothesis that the IOAV level of dioxin is safe.
Senator HART. You say in your statement "Shouldi^ teratogenic
nature of 2,4,5-T be confirmed, registration for usd^P food crops
will be canceled."
I am attempting to establish what will confirm it. What events do
you look to to determine whether these preliminary indications
which you say resulted from the contamination in fact did? Is •
there something in particular that you look to ?
Dr. BAYLEY. Yes, the important considerations here are the usages
for which the 2,4,5-T are permitted or which in actual practice are
carried out.
The difference between the possibility of teratogenicity of the contaminate and the teratogenicity of the material that is used in the
field is based on the rates of application, the losses which occur. All
these effect the possibility of contamination of human beings.
Incidentally, my advice is that the one part per million level is at
least tenfold below what they would consider a safe level in terms of
allowances. In other words, that is the safety margin in this estimate.
Senator HART. In other words, there would have to be a finding of
10 times mo re?
Dr. BAYLEY. That is what I am told, yes. This is a statement
based on scientific information provided to me.
Senator HART. Do you have any opinion as to whether Food and
Drug might set a safe tolerance level in food ?
Dr. Bayley. The action we have taken is to extend the time in
which information can be provided or application made with Food
and Drug Administration regarding the establishment of tolerances
on food. I would not in any way want to prejudge .what their
actions should be, because it should be based on the data provided.
If I may elaborate on that, a petition was filed with the Food and
Drug Administration in December of 1967 requesting the establishment of tolerances of 0.2 parts per million for residues of 2,4,5-T on
apples, barley, blueberries, corn, oats, rye, sugarcane, and wheat.
Those were the only crops to which that petition would apply.
The petitioner withdrew his petition on December 29, 1969, as
provided under the pesticide regulations. We, have extended to
December 1970 the opportunity for him to provide the data needed
to reach a decision on this.
There is one thing I think is important here, and that is the earlier reference to the concept of first, the burden of proof, and secondly, that we should not believe their data. We have to watch out
for this paradox.
Wo in the Department of Agriculture, as you know, with the
cooperation of the Food and Drug Administration, are not simply
accepting the proof from industry in these cases. We are going out
to obtain samples and testing them ourselves in order to verify the
kind of information that is coming in.
Senator HART. I am not sure it is a paradox to say that the
burden of proof is on the fellow that wants to expose the public to a
product and some saying you cannot trust his data. They are two
separate problems.
Dr. BAYLEY. I recognize that.
�Senator HART. Let us be assured that the data is reliable and
octive, and let us insist that the burden of proof may be on,
low who may or may not be proposing the introduction of'
imical.
Dr. BAYLEY. I suspect I am sensitive to this because some people
challenge why we test products. We think it should be done in the
public interest when it is needed.
Senator HART. On the matter of the dioxin that was found to exist
in the samples in a much higher percentage than the normal production thereof
i
Dr. BAYLEY. You mean the samples that the Bionectics group had ?
Senator HART. Yes, that the Bionetics group used. That was the
reason, was it not, that led you not to follow through on the
DuBridge pronouncement of October? Is that the meat and potatoes
of it?
Dr. BAYLEY. This is an often misunderstood situation. I think it is
important to realize that the date we chose in regard to the possibility of taking such action was chosen because the Food and Drug
Administration had agreed to complete their action on the petition
by that time. When they had not completed their action, I wrote Dr.
lioger Egeberg a letter on January 7, asking for a statement regarding the status of their considerations on the petition.
On January 21, I received a response indicating that they had
further data which changed the position in regard to the need for
immediate cancellation.
We will be glad to provide these letters for the record.
Senator PlAirr. I think it may be helpful.
(Theletters follow:)
f
JANUARY 7, 1970.
DR. ROOER O. KOEDEKO, Assistant Secretary for Health and Scientific Affairs,
Office of the Secretary, Department of Health, Education, and Welfare,
Washington, D.G.
DEAR DR. EGEBEUQ : On December 13, 19C7, a petition was filed with the Food
and Drug Administration to establish tolerances for 2,4,5,T on specific food
crops. In accordance with the interdepartmental agreement reached in Dr.
DuJiridge's office on October 29, 1909, we announced that we would issue
notice of cancellation of the registered uses of 2,4,5,T on these crops unless the
Food and Drug Administration found a basis for establishing tolerances by
January 1, 1970. This date was chosen because the Food and Drug Administration agreed to complete action on the petition by that time.
We would appreciate receiving without delay a statement from the Food and
Drug Administration regarding the status of their considerations on the petition in order that we may take appropriate action. This request is made in
accordance with our mutual interest to take responsible action on this matter
and also in full cognizance of the exchange of letters between Secretary
Hardin and Secretary Finch regarding public health responsibilities in pesticide registration.
Sincerely,
NED D. BAYLEY, Director,
Science and Education.
SUBOEON GENEBAL OF THE PUBLIC HEALTH SERVICE,
DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE,
Washington, D.O., January 21, 1910.
Du. NED N. BAYLEY, Director of Science and Education, Office of the Secretary,
U.S. Department of Agriculture, Washington, D.G.
DEAR Dn. BAYLEY : In reply to your letter of January 7, 1970, inquiring as to
the status of Pesticides Petition 8F06G9 (2,4,5-T), submitted by the National
Agricultural Chemicals Association, the following is the current status.
Wo liuve been advised by Dow Chemical Company:
(1) That a sample of the 2,4,5,-T herbicide used in the Bion4k study contnlned 27±8 ppm of a highly active (biologically) contaminalHtetrachlordiiH'iizo-pftni-dioxiii. (This has been confirmed by the National~Institute for
Knvlronmentnl Health Sciences.)
(2) That tills material produced serious inflammation in rabbit ear tests and
Mint tho presence of the contaminant had been confirmed by chromatography.
(8) That standard production 2,4,5-T contains less than 1 ppm of this contninlmukt and does not produce inflammation in the rabbit ear test.
(4) That Sprague-Dawley female rats as clams have been under test in their
Inltonifory at Zionsville, Indiana, at five levels (25 rats each) of the standard
production line material. They came to term January 7, 8 and 9, 1970, at
wlilcli time they were killed by carbon dioxide inhalation and fetuses were
removed by cosarean section and subjected to standard examinations for malformations and anatomical anomalies of various kinds; further examination
will Include clearing, staining and histopathologie procedures.
Hr. Howard L. Richardson, Chief, Pathology Branch, FDA, participated in
tlio evaluation which related to full-term rat embryos subjected to 2,4,5-T
during gestation, as well as a number of full-term rabbit embryos. He reports
Unit no signs of malformations were found in gross and microscopic dissection
«f MICKC embryos, but that histologic examinations are yet to be made. PersonIH'I from the National Institute of Environmental Health Sciences were unable
to participate, but will be involved in the examination of this and other informiitlonnl material.
Further characterization of the contaminant tetrachlordibenzo-para-dioxin is
currently under way and Dr. Leo Friedman will welcome participation by your
rw'itrch start in this effort, We would point out the resemblance if not the
"practical identity" of the tetrachlordibenzo-para-dioxin with the "chick edema
fiictor," Tl>is substance is of extremely high toxicity to all species of animals
Mint have been exposed, and until now, its source in contaminated fatty materials has been a mystery.
Considering the- imminence of the availability of this additional information
and the legitimate question as to whether or not the teratology reported by
Mic Bionetics study was due to the 2,4,5-T or to the contaminant, we have
elected to delay action on the petition for a few more days. As you know, the
petitioner had requested, on December 5, 1969, an extension of 3 additional
months.
We will advise you of our decision as soon as our scientific staff assays the
results of this nearly completed test and considers them together with the
rtwilts of other current research on 2,4,5-T at the National Institute for
Dt'iitnl Research and in the Food and Drug Administration. Thus far, no one
has confirmed the Bionetics results although 2,4,5-T (with 27±8 ppm contniniunnt) has been found to be embryotoxic.
Sincerely yours,
JESSE L. STEINFELD, M.D.,
Surgeon General.
Senator HAKT. At the beginning of your testimony you say residues on food are unusual. AVhen they do occur, are they the result of
unauthorized use of 2,4,5-T or authorized use, or both?
Dr. BAYLEY. Mr. Chairman, I am going to ask Dr. Byerly to
respond to that.
Dr. BYEHLY. Sir, one of the two significant values, 0.19, was on
milk, and the other was on sugar beets. As far as sugar beet use, I
would have to verify whether or not there is a registered use on
sugar beets. There is on sugarcane. There is certainly none on milk.
This would be unauthorized use in the case of milk, certainly.
Senator HAHT. Is it authorized for use on grass?
Dr. BAYLKY. Yes, sir.
Senator HART. It is?
�40
41
r. BAYLKY. It is authorized for use on grass.
enator HART. And clearly not on milk?
Dr. BYERLY. No.
Senator HART. _ What do you say to the suggestion that if you
know that there is a regular unauthorized use of a pesticide go'ing
on you ought not permit it to be registered? How can you register a
pesticide even for safe usage when regularly it is used in an unsafe,
unauthorized manner ?
Dr. BAYLEY. The law provides, and I will not pretend to quote it
exactly, that if despite the registration restrictions, including use,
the Department finds there is injury to people and to the environment or desirable environmental organisms, that we can consider
tins as misbranded and cancel the registration.
So the pattern of the enforcement of that part of the law is for a
surveillance program to determine the extent to which their unauthorized. uses are providing injury and then we take action.
_ Phis is very clearly shown in the action Ave took recently involving a mercurial seed treatment program compound, It was this type
of action, where unauthorized use was creating an in-jury and we
immediately suspended it.
Senator HART. You say the use is authorized on grass, not milk.
Uould not cows cat grass, thus producing residues in our milk.
Dr. BAYLEY. This is based on the recommendations for use and
also based on the degradation properties of 2,4,5-T itself. Good practice would require withholding the grazing of cows from these pastures until such time as we can be sure there will be no residue in
the animal product.
The widespread use of this as herbicide on ranges and pastures
indicates that farmers are following these practices with the possible
one exception that we know of at this point.
Senator HART. What information can you add to this record substantiating the statement that 2,4,5-T degrades in a matter of— how
did you put it?
Dr. BAYIJCY. We will be glad to supply for the record the scientinc mi-ormation indicating the degradation time of 2,4,5-T as well'
as the circumstances under which this will vary.
Senator HART. That will be printed in the record.
(The information follows:)
There is a voluminous body of published literature on the degradation and
persistence of herbicides. Enclosed are five reprints availaWe to u , tha deal
a
ith 2 5 T F r a
K pesticides, we4'recommend m°re comprehensive fess on of 1,45-T and
' ' - °
other ,° ^ n
the following publications :
of Her-
of
(from: Chemical Fallout-Current Research on Persistent Pesticides Ed. by MorSTW. Miller & G G
Berg. Charles C. Thomas, Publisher. 1969]
Soil Persistence of 2,4,5-T
The persistence of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), when
applied at recommended rates (0.25 to 8 Ib/A) is 4 to 5 months as
compared to about 1 month for 2,4-dichlorophenoxyacetic acid (2,4-D)
»i shown in the figure below.
FHENOXY. TOIUIDINE ond NITRIU HERBICIDES
DcRose (3) found that a 3 Ib/A field application of 2,4,5-T was no
longer phytotoxic to soybeans 3 months later, However, rates of 10
and 20 Ib/A remained highly phytotoxic after 3 months. Newman, et al
(4) found that 2,4,5-T lost its phytotoxicity after 4 months when
•pplicd at rates up to 26 Ib/A. Burger, ct al (1) found a similar
loss of phytotoxicity to alfalfa 4 months after the application of
25 ppw (50 Ib/A). However, these were both laboratory studies in which
the soils were maintained moist and warm, two conditions that facilitate
the microbial inactivation of phenoxyacetic acid herbicides. The
6 month persistence of 2,4,5-T in soil, DeRosc and Newman ( ) appears
3,
to be one of the largest persistences reported.
Microorganisms are primarily responsible for degrading phenoxyacetic
acid herbicides in soils. The kinetics of 2,4-D, 2-methyl-4-chlorophcnoxyacetic acid (MCPA), and 2,4,5-T detoxification in soil-perfusion
experiments were exactly what would be expected if microorganisms were
tho detoxicating agents ( ) in addition, detoxication was blocked by
4;
the bacterial inhibitor sodium azide. The bacteria and actinomycetes
responsible for degrading phenoxyacetic acids are shown in Tables 1-2 ( )
4.
Tho metabolism of the phenoxyacetic acid herbicides has been studied
extensively (4). There appear to be two major pathways of degradation,
i.e., via a hydroxyphcnoxyncetic acid intermediate and degradation via
the corresponding phenol. Some of the important steps in microbial
BCtnbolism of representative phenoxyacetic acids arc shown in Figures
»-6 ( )
4.
�43
42
TABLE 1-2
CHjCOOH
Bacteria and Aclinomycclcs which Degrade Phouoxyaeclic Acids
o
JL.a
Phcnoxyacclic uoids niclabolucd
1
1 1 '-3
•£
| i
*
References
1 l.lI H i i'HIHI
UiMiBHjj
M
U
^
U
4
^
N"
^
«
«i
^
4
M'
a
^
a
(17)
1
i
***
(18)
Achrt)iii(>baftcr ip.
*
57
5<!
+*
fl-/,.Stf
+
W,.SP
+
+*
+
+*
+*
+
+
+
+
,
+ + +
+
+
+
\
V0
a•
\
CHjCOOH
* i r f
s
5-/.W
HOOC:
<«»
(»»)
IB)\
N^
1
Bacteria
f*tcinli>nitnias sp.
PieiKhittflHtis sp.
Myeopluna sp.
AcltruHiabftcter sp.
HOY^^y0
A*
1
Organism
|^Va
^.
*
g
K
+
*
3
+'
OH
J
L
?
QH
V\
'
OH
COOH
3.
°
^
>o
(-
a
do
*
£
g
'
a
(ID
a
(«>
a
c1-
(14)
CHjCOOH
F^'rbwn
f\ rfresrintin,
Cor)iirbaclcrittin sp.
organism
Ariltiobaeitrftltibt/ortnts
iBatttriiiiitfloittforme)
Arilirobdctcr sp.
Spororylupltfiga
•
99
P-^
+*
78,79
+'
57,95
95,96,100
+
+
+
+•
+*
0
OH
y
§
w
+
+
a
o
(15) '
(M)
fff/VVCfO/fl
( /"/oi obfiftcritiin
oi/ittitik)
AclmORiyjclCt
NocardM Sp.
Streptomycin
9
5J
101
SubjiMtc in enrichment and isolation media.
+
+.
+
4-
•
l;i|.. 1-6. Microbial dcyradation of phcnoxyacclic acids via the corr^ponding phenols.
( \] •> \-D (17) douradaiion via 2,-t-diclilorophonol (18), ?,5-dkl\lor.>GiUxliol (19), and
.,-chloroimiconic acid (20); (11) 2,4-D dcara^lalion via 2.4-diehlorophcnol, -1-chloroc.iu-ch..l (2.1), and /3-chloronuiconic acid (2-1); (C) -1-CI'A (21) dcgradaiion via .1-cliloroplivno! (22), 4<hloroca\cchol, and ^-chloroiiniconic acid ; and (O) MCTA (i?) douradalion via 2-moihyl-'-c!)lorophcnol (26). [Aficr (S3,S9).]
45-362 O - 10 - 4
�44
45
References
1.
Reprinted h u m A G R O N O M Y ) O U R N A I
Vol. (iO, Nov.-Deo 1%H. ]>. <i7H-ft7<l
Burger, K., I. C. MacRae, and M. Alexander (1962). Decomposition
of phenoxyalkyl carboxylic acids. Soil Sci. Soc. Amer. Proc.
. 26:243-246,
2. DeRose, H. R. (1946). Persistence of some plant growth regulators
when applied to the soil in herbicidal treatments, Bot. Gaz. 107:583.
3. DeRose, H. R. and A. S. Newman (1948). The comparison of the
persistence of certain plant growth-regulators when applied to soil.
Soil Sci. Soc, Amer. Proc. 12:222-226.
'"
4. Kearney, P. C, and D. D. Kaufman (Editors) (1969). Degradation of
. Herbicides, Marcel Dekker, Inc., New York, N. Y. 394 pp.
5. Newman, A. S., J. R. Thomas and R. L. Walker (1952). Disappearance
of 2,4-D and 2,4,5-T from soil. Soil Sci. Soc. Amer. Proc. 16:21.
Growth of Crops in Soils After Herbicidal Treatments for
Brush Control in the Tropics1
R. W, Bovey, F. R. Miller, and J. Diaz-Colon1
Herbicides 4-,imino-3,3,6-trlchloropicalinlc acid (picloram), a l-.t mixture of (he butyl eaters of 2,4'dlchlorc*
ph en ox y ace tic acid and 2,4,5-trichloropheiioxy acetic acid
l2,4>l}:2 t i(r5-T), and a 2:2:1 mixture of (he inooctyl ester*
of 2,4-n:2,4,5.T:pliloram at 6.7, 26,9, and 16.8 kg/ha (6,
24, and 15 lb/A) respectively, were applied as foliar
ipravs (o control guava (Psldnttn gitajtwa 1-), Six crop
i tpftltR were planted in soil collected from each plot, 1,
2, fl, GI/J, 9i/a, nnd i3iA month* alter treatment, to detect
herbicide residues nnd lo determine crop tolerance. Corn,
loighum, wheat, rice and cotton could be grown without
mlurtion in (rcsh weight ai early as S monthi after
application. Soybeans were the most susceptible crop to
herbicide rcnldues.
Additional index words: herbicide reilduei, plcloram,
2,4-D, 2,4,5-T, guava.
COMBINATION of 2,4-diclilorophenoxyacetic
acid (2,4-D) and 2,4,5-tiichlorophenoxyacetic
acid (2,4,5-T) is an established herbicide for woody
plant control (2,4,5). Herbicide 4-amino-3,5,6-trjthlorupicolinic acid (picloram) and combinations of
pidoram with 2,4,5-T show promise for control of
woody species which exhibit resistance to phenoxy
herbicides (1), Since high herbicide dosages are re<)uiic<l for adequate brush control in the tropics (10,
I I ) damage to agronomic and forage crops seeded following treatment could result from herbicide residues.
Disappearance of 2,4-D and 2,4,5-T from warm moist
soils in the temperate zone is rapid. Accumulation
of harmful residues is unlikely Irom year to year if recommended rates for weed control are followed (4,5).
Factors responsible for decomposition of phenoxy herbicides include microbial activity, leaching and volat i l i t y (9). It is assumed that phenoxy compounds
would have an accelerated rate of disappearance in
tropical soils compared to temperate climates because of higher temperatures and greater rainfall.
Available information suggests that picloiam remains
in soils much longer than 2,4-1) (3) (R. W. Hovey,
1968. Unpublished data), Decomposition by microotganisms is very slow (12). llioassay studies indicated
t h a t the half-life oC pidoram in soils throughout the
United States varied with location, but some persisted
I year after treatment (3). Similar studies in Texas
(fi) and Puerto Rico indicated that less than 0.05 ppm
was present I year niter treatment of picloram at 8
lb/A (8.96 kg/ha) and 9 lb/A (10 kg/ha), respectively (C. G. Howler, 19G8. Unpublished data). However,
the Texas determinations were made from sandy loam
soils and samples were taken to a depth of 24 inches
(61 cm). Additional work has indicated that the main
routes of picloram disappearance and decomposition
A
' C o n t r i b u t i o n horn Uic Ciops Rescaidi Division. ARS, USDA.
This research was sponsored by f u n d s by contract with Department of Anny. l-'oit Dcirick. Frederick, Mil. Received May 1, 19G8.
•Research Agronomist, Geneticist, and Agricultural Research
Technician, t'.iops Reseatch Division, ARS. USDA, Federal Experiment Station, Mnyagliez, I'ncrio Rico.
from soil profiles are by leaching and photo-decomposition (7).
Jungle areas in Hawaii were aerially treated with
2-niethoxy-S,6-dJchlorobenzoic acid (dicamba), 2- (2,4,
5-trichlorophcnoxy) propionic acid (silvex), combinai ion of paraquat plus dicamba and 2,4-D plus picloram
(8). When treated plots were planted to Monkey pod
trees (Samatiea toman (Jaccj.) Merrill) 1 month after
application, the trees developed no injury from herbicide residues in the soil.
This paper reports the growth of six crop species
in a tropical soil treated with picloram and combinations of 2,1-D:2,'I,5-T and 2l4,D:2,4,5-T:picloram collected from a guava control area, to determine the
longevity of herbicide residues and the tolerance of individual crop species to each herbicide.
MATERIALS AND METHODS
lietbiddal treatments were made on ma lure ttandi of guava
(I'sidiiim guajava L.) near Mayaguei, Puerto Rico. Characteristics of guavn, the physical environment, and responses to twrblcidcs have been described (11), Herbicide! were applied to
guava with a pole sprayer designed to cover a 12.16-tn (40-ft)
diameter circle (Dowler, C, C., and F. H. Tichirley. 1966. Defoliation Project Mayaguei, Puerto Rico. Ann. Rep., USDA).
Herbicides applied included the pouwium salt of picioram it
6.72 kg/ha (G Ib/A). a 1:1 mixture of the butyl «'«• o(
!M-D:2,4,5'T at 26-88 kg/ha (24 Ib/A and • 2:2:1 mixture of
the isooctyl esters of 2,4-D:2,4,5-T:picloram (M-3140) at 16.80
kg/ha (15 Ib/A).
Soil samples were taken trom herbicide treated ptou 1, 2,
3, 61/1. 9i/j amt l3 Vi month) alter treatment by collecting the
top ! (t (32.48 cm) ot soil at three or (our locations in each
plot. (Soil samples I and 2 monthi after treatment were not
taken (or M-3140.) To prevent contamination, a clean ihovel
was used for each plot, .Soil was placed in plastic bag*, tealed,
imrl immediately iiansported to the green home, Each toil wai
pulverized by ha ml and placed in 38 x '0.5 X I'-cm (15 X
12 x 5-incli) boxes lined with plastic to prevent leaching of
the heiliicide. Each worker was required to wash hii handi
thoioughly between each soil treatment. Untreated toil maple* were prepared tiirt, toUowed by longest field applied
ncauneni (13'/] months) to the shortest (I month). Four replications were prepared for each treatment. Soil from (he treated
area was n Mucarn silly clay-loam (11).
Twenty-live seeds n( corn (Zea may* L.) var. 'USDA-34,'
ioigluint (Sorghum bicotor L.) var. 'Combine Karir-60,* whnl
(Triticntn tieslivnnt I..) var, 'MeiUana,' rice (Oiyia Jafiiit L.)
var. 'Taichtmg Native No, 1,' colton (Go)iypiitm hirtutum L.)
var. 'Blightmaster,' nnd loybcans (Glycine max {L.) Merrill)
vnr. 'Clnik' were planted in each replicalinn, covered with a 0.6
to 1.2-cm (*/t to i/rinch) layer of soil, and watered. Th« oopi
wctc giotvn fov 21 days. Aerial portion! of all planti in each
icplication were weighed on an electronic balance ant) recorded
as fresh weight. Numerical values presented in the tablet that
follow arc pcircntagc of the control.
RESULTS
Rainfall is important in the decomposition and disappearance oi herbicides from soil profiles. Rainfall data arc presented in Table 1 for each of the
iifatnu'iu periods.
/'t(lomnt. Growth ol six crops in soil treated with
pidoram at (i./H kg/ha (G Hi/A) is gi\en in Table
�47
46
BOVEY ET AL.: CROP GROWTH IN SOILS AFTER HERBICIDAL TREATMENTS
lblc 1. Amount ot ralntall front each (life of herbicide
a|>j)llcmloa to date ot iniiiplliig Mil (Dec. 6, 1007),
679
iwiimcd (mm H'EKII SKIKKVH
Vol. 16, Nit. I. January, l!W8
Table 2. Percentage growth of nix crops in loih from foliar
applied herbicide* an RII.IVJI ], Z, 3, fiyv t>l/4 anil J.lt^, monlhl
nflcr trciilmcnt (growth of control pl;tim — Kin*),
•W 1 I . I B
r»h. I, u*
Pel. H. ll
2, AH crops showed a significant reduction in growlh .
when scccletl 1 ami 2 months after application of the
htrbicitlc. Rice, soybeans, and cotton were severely
affected ami showed essentially no growth when seeded in soils 1 mont|i after treatment. Soybeans and
corn continued to be adversely affected when seeded
in soils 5 months after treatment; while sorghum,
wheat, rice and cotto'n were not significantly affected.
Since the disappearance of picloram is much depenilcnl upon leaching the 58.4 cm (28 inches) o£ rainfall
received in the 8 months after treatment (Table 1)
undoubtedly removed; lethal amounts of picloram.
These data suggest that-, all crop species studied could
IK safely seeded in soils G months after picloram treatment without adversely'affecting growth.
1,4-D:2,4J-T, Crop growth was retarded in soil
treated with the 2,-l-D:2,4,5-T combination 1 and in
some cases 2 months before seeding (Table 2). At 1
and 2 months this combination of herbicides was less
inhibitory than picloram although the rate of treatment was four times as great. This suggests that the
phenoxy compounds either dissipated more rapidly
than picloram or that the crops were more tolerant
to the herbicide combination or both. Wheat and cotton produced growth comparable to the control when
iceded in soils 2 months after treatment. Soybeans
were the most readily inhibited of all crops grown.
Reasons for reduced growth in soils treated (or 1S>/Z
months are unknown. That this may be a chance variation i.i indicated by'over 1009ip production in samples
of soil taken in plots having a shorter time after
these same treatments (excepting soybeans). Average
(means) for all crops did not differ significantly at the
5% level of Duncan's Multiple Range Test.
M-3HO. Blended rates of treatment for M-SHO
were 6.72, 6.72, and 8.86 kg/ha (6, 6, and 8 Ib/A)
of 2,4-D, 2,4,5-T, and picloram, respectively. No significant differences (5%) in growth occurred among
averages for months but did for crop species when
M-3HO was applied. Soybeans showed injury in soils
treated for 9i/j months or less before seeding (Table
2). Cotton seeded S months after treatment was slightly injured. Apparently, sufficient picloram persisted
'(or the described intervals to cause damage to soybeans and cotton; but other crops were unaffected,
DISCUSSION
These studies indicate that high rates of herbicides used to control vegetation in tropical and subtropical areas disappear rapidly, even though per1
sistciu compounds such as picloram are used. Hence,
most crops could be safely grown within 6 months after application with few if any adverse effects.
Soybeans were the crop most sensitive to herbicidal
residues in soil. Use of an alternative crop should
be considered, if seedings are made during the first
i l J 4-Pj Li l t 4,ft-TllIt'll"'l
i'
01 b
Mb
D< a
11 be
TO 0
VH bfl
1
jJMtj*/l'».
IN li
li >
HI h
ltd
Hfl
1Mb
Mb
71 b
lUtb
51 bo
ID lo
nt «b
t»b«
ua b
71 e
TOb
111
(1 •
71 t
14 t
i
IMt
i
H) nh
131
H*b
»I|M>
11H
ElEect and Persistence of Herbicides Applied
to Soil in Puerto Rican Forests'
•
111 ll>
» iM
71 be
AVJ (Crop)
M-JUIj .pplrod
1
A»I (Crep.)
111 *
lib
in ib
111
tSb
RBb
111 1
1M »
lb
117 ft
16 b
1ST
11
1
i
CLYDE C. DOWI.EB, WILSON FORESTIER, and F. H. TSCIIIRLET'
H.tkflA*
in *
•
PI >b
11 lib
(1 •
11T »
11 t
1
11 I
1
90 0
U at)
18 bo
10*
let*
101*
Mb
104
«
ll
ll
DH
101*
Dunoui'i tsulHple ruin I*'1-
few months after treatment. Graminae species such
as corn, sorghum or wheat may be used first in treated
areas, because these are more tolerant to the residues
of herbicides used for controlling brush.
Rainfall (Table 1) is important in leaching herbicides from soil profiles. In areas of more abundant .
precipitation one could expect more rapid disappear• ance than was encountered in this study, providing
that other soil and environmental factors are equal.
We have concluded that harmful residues of herbicides in the soil would not occur in tropical areas
after chemical brush removal if a reasonable deferment of land was observed after application.
thrttMI. M« hi'ihlciik-i wnc applied nl 3. 0. and 27 ib/A to the
•U in f««m nf lime li|ic. in I'urrln Itico. The forests clllfcrcd in
<Ht li|«. Jmnmii oral I nil nf ininfall. and »iKcira compinltion.
IVMiiOon .nmriril i l o w l t . bill tin- ciirrrlntion between pertnaif ililiilialioil and pliinli llllrcl wan sU,miliram at the 1%
h..l <if |«.li.iliitiiy. Sinne- ilcliillnllcm wa< oidcnl I inonlli after
Ifijlnnil mil Incri-aicd ilnwl) dm ing Hie next li lo R momlii.
llw lm».( clh-illvr Jinimiilc was .l-:iinino-3.ri,fi-tricilloropiC(iiinic
i,i»I (iikliiiim). Carnally, !>.iiirll»ixv-3A-dlcliloiiil>ciinile and
(•hilnilul 2 iiirlhnxi -IJI.|il<(i«ipiii|)) 1 lainino)-<-trin/illi.' (iiinmelone),
! . Vlmniin J.IIT l n i i ) l - f l • i i i e l l i ) l i n a r i l (Inomacil) were I™ cffeci»o
*!
Kkin iiMoMin iinib U.3 (i'liidilnioiihenvlaeellc acid (fenac) and
*<V«<lklil«i<>|ilirri)l).l.Uliiiiriliyluica (clluron) were incffeclivc.
IkMillkin it.it gittilnr and more plant! wctc killed In Hie driest
«hw mi wfirr U r a l nl, fenac wai (tic most persistent hcrbiIklr In llic .oil. Mrililcidn wei c found lo a 30 to 4S-in dcplli
•llhlli 1 I,i,i,nil, afn-l a j i p l i r a l i a n .
MHlunnm anil wikwh .WflliriRS wcic present at all site! willun
t* pirtiilit alicr hi'ihicitle applitanon. There was no delinile n'laffcwililit l*iwrni lii'ibicidal uciitiunit and secondary succession,
li<«K Iliai llu- nmnliri and fircpiency of Miccessional ipcciei wnc
f»r»i*l rm plnH basing llic highest petccntaRC of defoliation,
LITERATURE CITED
1, Bovcy, R. W., F. S. Davis, and H. L. Morton. 196B. Herbicide combinations for woody plant control. Weed Scl. 16:
332-335.
2. Crafts, A. S., and W, W. Robbing. 1962. Weed Control. .
McGraw-Hill Book Company, Inc., New York.
8.,Goring, C. A, I., C. R. Youngson, and J. W. Hamaker. 1965,
Picloram herbicide disappearance from soils. Down to
Earth 20:3-5.
4. King, L, J, 1966. Weeds of the world. In Biology and Control. Interscicnce Publishers, Inc,, New York.
8, Klinginan. G. C. 1961, Weed control: Ai a science. John
Wiley and Sons, Inc., New York,
6. Mcrklc, M. G., R. W. Bovcy, and R, Hall. 1966. The determination of picloram residues in soil) wing gat chroma tography, Weedi 14:161-164.
7. Mcrkle, M, G.', R, W. Bovey, nnd F. S. Davis. I9fi7. Factors
affecting the pertinence of picloram in toil. Agron. J.
59:413-415.
8, Motooka, P. S., D. F. Saiki, D. L, Pluclmett, O. R. Younge,
and R. E. Daehler. 1967. Control of Hawaiian jungle with
aerially applied herbicide*. Down to Earth 23:18-22. , ,
,
9, Sheets. T. J., nnd L. L. Danielson. 1960. Herbicides In
(oils, in The nature and fate of chemicals applied to ioU«,
plants, and animals. ARS 209, USD A.
10. Tschirley, F. H. 1967. Problems in woody plant control
evaluation in the Tropics. Weed! 15:233-237.
H.,Tschirley, F. H., Rene T, Hcrnander, and C, C. Dowler.
1%7, Seasonal susceptibility of guava to selected herbicides.
Wcedi 19:217-219,
12. Young&on, C. R., C. A. I. Goring, R. W. Meiklc, H. H,
Scott, and J. D. Griffith, 1967, Factors influencing the dei composition of picloram herbicide in toll). Down to Earth
23:3.n,
'
"<• IN'IROUUCTION
lir rflnl of herbicides applied to the soil for control
nf wpnilr plants in tropical forests lias not been
Uliilird imi'ii'ivrly or extensively. A short review by
M»)o Mcnrmlf* (7) indicaicd tbat frill treatment to
Urn ».n llic most ellective method of application.
ttnriiiliii! (2), SIMMS ( I I ) , and Wyatt-Smith (17, 18)
nvnl f i i i l iiraiiticnis surccssfiilly on a large number of
irit|iicil >|"'ri" but nuled differential susceptibility
«mMiK «!«•( io. llnwkini ('I) reported that a basal spraying
Of |Kimling was ;n eltoclivc as frill treatment.
Man) hniclwoodi can be controlled effectively with
jillrrinxiiui'iic or picolinic acids applied in basal frills
m .11 HIT injrnioni (li, 15). Many compounds arc effective
j. loliar ( i c n t m r n l i for controlling woody plants (3, 16).
Njtinil (')) irpnilai that 4-;imino-3,5,6-trichloropicolinic
»riil fpirlorani), when applied as a soil treatment, was
• in tffrciivc licrblridc for controlling woody plants. He
«Ui nolfd dilk'irntial susceptibility among species.
Inu-iurnhle from the biological effect of herbicides
>|i|ilinl lo the soil are their movement and persistence.
T
""•«.',7licil t..r iiiibllrallon February 15, 1907,
.
•Hivitcli ARtonoinlit, AgiIcullnral Research Technician, Re.
« i M b RJIIBI? Vlemisl. reiprclholy. Crops Research Division.
Vintilmul Kriraiih Seivicc. U.S. nepaitment of Agriculture,
Ittktll K\|»criinent Station. Mayaguez, Puerto Rico.
Factors such as rainfall, physical and dicmical iharacter.
istics of the soil, microorganisms, chcmiral chniaclcristio
of the herbicides, and method of application may influence herbicidal movement and persistence- (5, 8. 10, M).
Our objectives were (a) to determine the toxicity of
selected herbicides on tropical arboreal vegetation, (b)
to determine the movement and persistence of these
chemicals in the soil, and (c) (o observe the short-term
effects of these chemicnls on secondary plant succession.
MATERIAI-S AND METHODS
Our studies were located in Guanica Conimonwcallh
Forest, Maricao Commonwealth Forest, and I.uqudlo
National Forest. The soil type at the Oiunica Commonwealth Forest is |acana clay. It is an alluvial soil normally less than SCi in deep, with very low permeability.
The vegetation is xerophytic. There were 35 woody
species within the test area, but Lciicarna lr\iracrtihah
(Lam.) DeWit and Harm<i!axy!an ramtifrlnanum I.,
comprised more than 9070 of the'woody plant population.
lUcan height of the vegetation was 15 ft. Annual rainfall
in the Guanica area, estimated at approximately 30 in,
occurs liirgclv from J u l y to October. The recorded annin
rainfall at the site for ItHM ami IMS was 27.8R ami
25.00 in, respectively.
The soil type at the Maricao Commonwealth Foresi
site is Nipe clay, a permeable, well-drained, lulcritic soil
derived from serpentine. The vegetation on this site is
classified by Heard (1) as moist tropical forest. There were
100 woody species within the test area. The mean canopy
level was approximately 50 ft. Mean annual rainfall at
the Maric.no site is estimated to be about !)0 in. Rainfall
is normally distributed throughout the year, but Decembcr to May is the driest period. The recorded annual
rainfall at the site for 1964 and l!>r,5 was M.IM and
100.88 in, respectively.
The soil type at the Luqtiillo National Forest site is
I.os Guineos clay loam, a plastic clay with poor internal
drainage. The vegetation is a tropical rain forest (1) with
a mean canopy level ot about 60 ft. There were R8 tree
species on the test site. Mean annual rainfall is estimated
to be over 100 in. The highest.rninfall normally occurs
from July lo October, but droughts arc unknown. The
recorded annual rainfall near the site was 85.78 in for
�49
I9(i'l and 12fi.l2 in for I9fi5. Tschirley et at. (14) characterized the Maricno and Luquillo sites in greater detail.
The herbicides used in this study were 5-bromo-3-JCCbutyl-fi-niethyluracil (bromacil), dimethylamine salt of
2-metlioxy-S,G-dichlorobenzoic acid (dicamba), S-(3,4dichlorophenyl)-1,1 -dimethylurca (diuron), Na salt of
2,3,6-irichloropiienylacetic acid (fcnac), K salt of piclonuu, and 2-methoxy-4,f)-bis(isopropylarnino)-s-triazine
(promctone). Each herbicide was applied at 3, 9, and
27 lb/A.
A randomized complete block design was used at each
location. The treatments were replicated three times at
Guanica and Luquilio, but only twice at Maricao because of topographic limitations. Eacli plot was 60 by
80 ft. with A 20-ft buffer separating adjacent plots. Data
were collected from a centrally located 40 by fiO-ft subplot, Narrow walkways were cut through the long axis
of each plot to facilitate treatment and data collection.
All woody plants having a diameter breast high (hereinafter referred to as dhh) mciisurcment of 0,75 in or
greater were marked, mapped, and identified (at least to
genus) at the Guanica site. At Maricao and Lucjuillo, only
plants having a dbli greater than 1 in were marked,
mapped, and identified. All defoliation data were collectrd only from the marked plants.
The herbicides were applied with a cyclone hand
spreader as granules, pellets, wci table powder, or liquid
adsorbed on vcrmicnlitc, Vermiculite was used as a
carrier to provide additional bulk for all treatments. The
application dates were: Gunnica—October, 1963; Maricao—December, 1963; and I.uquillo—January, 1964. Percentiigc of defoliation of each marked plant was estimated
periodically after treatment. Plot defoliation, calculated
from i n d i v i d u a l plant defoliation, represents a mean
percentage weighted according to the relative prevalence
of various species.
The downward movement of herbicides and their
persistence in the soil were studied by sampling 3, 6,
and 12 months after treatment. Duplicate soil samples
were collected, at random from centrally located 20 by
'10-fi subplots. Soil from depths of 0 to 6, 6 to 12, 12 to
24, 24 to 3fi, and 36 to 48 in was analyzed separately, If
the soil was not 48 in deep, samples were collected to
the maximum possible depth, The samples were placed,
into 1-qt styrenc cups, then sealed and transferred to
the greenhouse where a bioassay was immediately initiated. Cucumber (Cucumis salivits L,, vsir, Puerto Rico 59)
was u'rtl as an indicator plant. Approximately six
cucumber seeds were planted in each sample. After
emergence, the cucumbers were thinned to two seedlings
per sample. A mean injury raling of each sample was
m.-idc lifter 28 days of growth and compared to an
established standard curve, Abnormal growth characteristic* were expressed on a 10-point scale, where 0 = no
cffcci and 10 K plants killed.
Succession data were collected from one replication at
each site. Observations on the species present and their
relative importance were made at Maricao, Guanica,
and Luquilio in April, May, and September, 19G5,
respectively. The successional species on each plot were
identified to family, genus, or .species and grouped
according to tree seedlings, grasses and sedges, or herbaceous plants and vines.
RESULTS
, '
Hcrbicidal effect on woody plants, The correlation co>
efficient of percentage defoliation and plants killed was
significant at the 1% level of probability; consequently,
results are based on defoliation only.
Guanica. Picloram was clearly the most effective herbicide (Table 1). Bromacil and prometone caused high
P I C L O R A M 37 LB/A
Table ). Percentage of defoliation of the nine principal specie* in
Maricao test site after herbicidal treatments to (he soil. Treated
December, 196S and rated October, 1065.
Hcrbtclde am)
DefolUtl
rale, lb/A
ling ordct ortmpoTlancc*
Picto ram
2
96
12
r»n
<M\
11
10
10
fl
4
3
Promctont
18
2
2
8
65
:
SO
I
I
10
0
5T
80
10
0
10
2
Table I. Percentage of'defoliation and plants killed of all arboreal
species ill tlie Gtianlca, Maricao, and Luqtiillo forests approximately 2 years alter her Wci da I treatments,
Herbicide nnd rule, lb/A
...' major ipeclti in dtcrca
(1
71
3
|
fi
4
2
t
•
100
100
7)
ir
ST
100
0
0
S3
Guanica*
PICLORAM
3S
2
9 LB/A
5
Check
2
...
4
0
S
(R. 'A P.) Afff .
1. Cait*,it-DyWtt. 3. r7<^< •/
OfWn frmiW**
4. A littMa «<ai7<na Chriit. S Ttrtti'a'i* r«'it*'« (V>hl) S£ra <,,r. A •«.,(.«,> (Urn.)
(Sw ) MCI. 7, Mittnio linltn, K Cogn. 8. C»ffW«(* ip. 9. „
Cfl»r,»VW"'""'<'**Jacq.
Kn
•Def - percentage defoliation] PK - percentage planti killed.
defoliation at the higher rates but were less effective when
only S lb/A was used, Dicamba, diuron, and fenac
caused only slight defoliation even at the highest rate,
Differential susceptibility of die five principal species
at Guanica is shown in Table 2. Picloram, considering all
Table 2. Percentage of defoliation of the five .principal species In
Giianka test site Z yeais after lierbicklal trc-iumcnis to tlic soil.
Herbicide and rate, lb/A
liation of major ipcclti
order nf importan
^:r
b.
l
and density of the seedling complex could not be
definitely associated with herbicidal treatment. The most
common succession species in the Maricao site were
Tcrebraria resinosa (Vahl) Sprague, CiutiVi spp., Ichnanthus pallens (Sw.) Munro, PaniVtim g/ufmcuum Sw.,
MONTHS FOL'.OWING APPLICATION
and Poly podium spp.
Lnquillo. Picloram caused the highest percentage of
figure I. Percentage of defoliation at specified intervals
after picloram was applied to the soil in three forested
defoliation at the Luquilio site (Table I). The highest
areas in Puerto Rico.
rates of bromacil and dicamba caused appreciable defoliation, but both herbicides were much less effective when
remained essentially constant for the rest of the 24-month
only 3 or 9 Ib/A were used. Prometone, diuron, and fenac
period.
caused only slight defoliation at all rates. 1'icloram wai
Total vegetation control was short-lived, even on plots
as effective at 9 lb/A as dicamba and bromacil at 27 lb/A.
that had been treated with 27 lb/A. No treatment preDifferential susceptibility was apparent among the
vented plant succession for more than 18 months. Two
eight most numerous species (Table 4). Tabfbuia heteroyears after treatment, Ifromoea spp., Indigofera sufphylia (DC.) Britton was the most resistant species in that
frulicosa Mill., and cacti (Oftuntia spp.) were abundant,
no treatment caused more than Sl% defoliation. PsychoGrass and tree seedlings were less numerous. Leucaena
tria berteriana DC. on the other hand, was completely deIcucocephala (Lam.) DeWit was the most abundant tree
seedling. Seedling density was directly related to percentage o£ defoliation.
Table 4. Percentage of defoliation of the eight principal spedei
In Luquilio t«t lite 21 months after heiblcldal treatments to
Marieao. Picloram caused the highest percentage plot
the soil.
defoliation (Table 1). Dicamba at 27 lb/A caused 50%
defoliation, but this was considerably lower than that
Defoliation of major iprcki In otdrr of
Herbicide and rate, lb/A
„
(^creating Impm-t.nce'
caused by picloram at 9 lb/A. Bromacil, prometone,
diuron, and fenac were considerably less effective.
Pick ram
Picloram affected the broadest spectrum of tree species
100
SI
IT
i
100
!
100 100 100
(Table S). Miconia sintenisii Cogn. was more susceptible
1
40
to picloram than were the other eight species. Rapanea
3
1
SO
Jermginca (R. & P.) Mez appeared to be most susceptible
2
i
i
1
0
Drd melt
13
0
1)
5
too
to hromadl. Ocotea leucoxylon (Sw.) Mez tolerated all
7
100
) too
: .. .
the herbicides except picloram at 9 and 27 lb/A.
Prometonc
j
a
:
SS
Maximum defoliation resulting from treatment with
0
0
0
c
0
I
on
picloram was obtained at Maricao about 1 year after
;
11
a
3
5
0
treatment (Figure 1). There was essentially no change
43
3
0
during the second year of observation.
: ...
No herbicidal treatment prevented the succession of
D
Cheelt.,..
tree seedlings, grasses and sedges, or vines and herbaceous
1 . TtMuia htirtpWlt (DC.} BrUton. 2. Cjatfc. „»,„• 1L.)1. T_ Smith. S:Cwi
plants at Maricao. Tree seedlings were sparse on plots
tori fitmu Utbati. 41 A.jlwj *I*»MII* (Gr*ham) Nicholwn. S Minttit *r«iiiM (5t
DC 6. C*nm»-D,fp<t,,. 7. PytWri* ttrtnitnt DC.' 8. ^f./*(i
treated with picloram at 9 and 27 lb/A, but composition
100
10
0
100
1J
«
M. Leuentna Itucttttittala (Lnm.) DeWil, 2, Haintatetrttn tambuhianum I, 3, Sinrialia
wntiant (DC.) Brluan & Ron, 4. Pniepiijulifora (Sw.) DC. !. TntMlit hirta L.
rates, had the broadest spectrum of herbicidal activity,
Prometone was more effective than was any other herbicide for defoliating Prosopis juliflom (Sw,) DC, and
Trichilin hiria L.
At Guanica, the maximum percentage of defoliation
resulting from treatments with picloram was obtained
about 3 months after treatment (Figure 1). Defoliation
f*iit (L.) Wild.
�50
foliated by 0 and 27 Ib'A of picloram, dicamba, and
brnmacil. Picloram had the broadest spectrum of hcrhieidal activity, At the 27 lb/A rate, six of (lie ciglit species
wore completely defoliated- and the other two species,
Tnbehitin ht'tcrofihylla (DC,) Ilritton and Conlin borinrittenfiix Urban, were p a r t i a l l y defoliated. The 27 l b / A
nitfi of dirnmba cnnsccl 100% defoliation of P.\yrJwhia
harteriaiia DC, and /n^n fngifolia (L.) Willd, but no
defoliation of Alirotiia Inwinn (.Sw,) DC. The 27 I b / A
rate of broninril caused 100% defoliation of Presioca
montfitin (Graham) Nicholson and Piychotria bcrteriann
DC, but no defoliation of Caxenria-Dryltctcs and Ingn
fatfjolia (L,) Willd.
The speed at which trees defoliated was essentially
the same for nil herbicides applied. Data for the rale
of defoliation caused by pirloram indicate that maximum
effectiveness was obtained about fi to H months after
t r e n l i n e n t (Figure 1). The m a x i m u m defoliation occurred
sooner from piclornm at 9 l b / A than pirloram at 27 lb/A.
Defoliation remained essentially constant for the remainder of the 24-month period.
The relative density of new seedlings at the Lucjuillo
site could not be correlated with the hcrbicidal treatment. The most common tree seedlings were Psychotria
ht'rlrriann DC., Orotrit lt'iirt)\yhn (.Sw.) Afez, and Pre.\tnca man Inn ft (Graham) Nicholson. Grasses were best
represented by P.nnteitw atklir.i'.unn Trin. and sedges by
Sclcria srcons (L.}, Many vines and herbaceous plants
were present, ffjomoen spp, being the most common.
There was n direct relation between percentage of defoliation and number of new seedlings. One year after
treatment, the forest floor of plots that had been completely defoliated was covered with vegetation. The density of new plants is shown in Figure 2.
ffcrhirnlfjl residue in the toil. Three months after application, the herbicides had moved downward in the soil
to the 3f> to 48-in depth. The bioassay data for all
sampling depths indicated persistence of the herbicides
in the soil 1 year after treatment for all locations was
in the order of fenac > promctone > piclornm > diiiron > bromacil > dicambn, An example of. the residue
d:\ta is shown in "Figure 3, Dicambn had almost completely disappeared 1 year a f t e r treatment, Two years
after treatment, fcnnc was still the most persistent herbicide, followed by promctone and picloram.
The persistence of the herbicides generally was greatest in the driest area (Guanicn) and least in the wettest
area (Uiqulllo) (Figure 4). One year after application,
the residue of picloram in plots treated at 27 lb/A remained in relatively high concentrations at all test sites,
as determined by the cucumber bioassay test. The presence of picloram in plots treated at 9 lb/A could be
easily detected 1 year after treatment, but the concentrations were about 10 times less than in plots treated
with 27 lb/A. The residue data for all locations indicated
a trend for all the herbicides to dissipate more rapidly
in the top 12 in of soil.
51
AGIO***1 ffCS'DUE >E MONTHS
and 5.94 in at Luquillo. Sufficient rain to leach the
herbicides into the soil fell at all three locations within
a few days after application.
The rapid increase in retaliation shown for dicamba
and check plots in the Guanica site is the result of refoliation occurring during the rainy season (Figure 5).
"YftOUETONL
\
87
i I C - M B A VI
i5
6
9
12
15
16
21
24
MONTHS FOLLOWING APPLICATION
GUANICA
figure 5. Percentage of woody defoliation in the dry, On an lea
Forest site of Puerto Rico alter treatment with three hcrbicidci
applied to the toil.
fkfMt 4. I'lilorain residue at various depths
In i h t t c (on'Kl aieas of Puerto Rico 12
m»nihi after application,
'[;"-
Figure 2. Fores! lloni nf pint treated w i t h nlcloifim nl 27 Hi/A *
J.iiqiiillo. Top: Kiglit months a CUT treatment; bottom: T«*
Jem's after treatment. Noio saonilary succession,
SOIL DEPTH liKCHril
''gnre 3, Concentration of six hnbiddrs I year after
to Jacana clay (Gtianica site) at 9 lb/A.
DISCUSSION
3^ '• Om 2«l tKtfxly *|irdc* were represented on the three
l^if'.iWw riirt, hut M'vcnil species were represented by only a
,;iX~ *kr* terfmdital*. Tschirlcy (13) enumerated some of the
sfey (MiMfiBi Imolvrd in evaluating herbicides in tropical
", ftwinafc Other workers (2, {I, 11, Ifi) have shown distinct
| i&frfffntul *iiMC|iiibility of woody plants to various
? IwrWsfclri. D t l f n c u t i a l species susceptibility also was
!
nttJwtl »i our lot utcv When all the treatments at all
n ate tnmideretl,, Tnbebtilt
, Cor din (tor nfiitmsis Urban, 7»g/i (tigifoJia (L.)
*(iit Ofntra Icucoxylon (Sw.) Me?, were most
and I'syclialrta bertcriann DC,, Miconia sinSrnr%alii\ wcstiana (DC.) Britton &: Rose,
letintrrfihnla (Lam.) DeWit were most
innt>nt-I)ryfietes, fdironin sinle.nixii Cogn.
/Hflim/t (Sw.) DC. were represented at both
nd LtK|tiillo. The reaction of these species to
[ollowcd the same general trend at both
,
drfntiiilion was greater at Maricao.
^^f '!fcr*ff«t of climatic and cdaphic factors on herbicidal
1;^*****&'I «antifit l« clearly elucidated in this study be.-'}!'***•» il ililfcrrnin in specie* composition. At all three
1
*w< i^ift, rainfall l>efort' application was sufficient to
in adcqunic plnnt growth, Rainfall for 2 months
aintfitt was 2.41 in at Guanica, 5.16 in at Maricao,
The rainy season \mtally occurs during July lo October,
Most o£ the woody plants grow vigorously during the
rainy season and are deciduous during the long dry season. Retaliation on plots treated with 27 lb/A of dicamba
attests to its herbicidal ineffectiveness at the Guanica site.
The lack of retaliation of woody plants on plots treated
with picloram and prometone is indicative of their
effectiveness.
In this study, sufficient rainfall occurred after treatment to leach the herbicides into the soil and prevent
large tosses from volatilization and photodecom position.
The highest concentration of a herbicide in the soil
profile was consistently found at the low r a i n f a l l Guanica
site. On the other hand, the lowest concentrations of
herbicides occurred at the continually moist Luquillo
site.
Persistence was related to the amount of herbicide
applied, but the effectiveness of a herbicide on woody
plants.was not related to its persistence. Fcnac, the most
persistent herbicide, was ineffective for defoliating woody
plants at all test sites. Prometone was more jwrsUtetu
than was picloram but effectively defoliated plants only
at the Guanica site, Picloram effectively defoliated woody
plants at all three test sites.
Although a high degree of woody plant defoliation
was obtained from several treatments, total vegetation
control was short-lived. Secondary succession occurred
within 18 months on all defoliated plots at nil test sites.
Grasses, herbaceous plants, and vines generally were
more numerous than were woody tree seedlings.
The amount of rainfall and increased light penetration
appeared to influence secondary succession more than
did the herbicidal treatment. The number and density
of successional species were greatest on the wet Luquillo
site and smallest at the dry Guanica-site. In general, the
number and density of successional species were greater
on plots that had been defoliated. This suggested that
�53
52
increased light penetration was one of the major factors
influencing secondary succession. There did not appear
to he any relation between herbicidal residue and invading species. For example, several species such as
Ptyrlwtria berteriana DC. were extremely susceptible
to initial application of herbicides, but they were found
on nil treated plots 18 months after application,
ACKNOWLEDGMENTS
This study was supported by the Advanced Research
Projects Agency, Department of Defense. The herbicides
were donnted by Amdicm Products, Inc., Ambler, Pa.;
Dow Chemical Co., Midland, Michigan; E. I. DuPont
de Nemours R: Co,, Wilmington, Delaware; Geigy Chemical Corp., Ardsley, New York; and Velsicol Chemical
Corporation, Chicago, 111,
LITERATURE CITED
!, llKAHn, J. S. J948. The natural vegetation in (he windward
and leeward islands. Oxford Forestry Mem, 21. 192 p,
2, 11 KVF.nl no E, A. E. 1057. Arboricide trials In lowland dipteroraip 'ail forest of Malaya. The Malayan Forester 20:211225.
3, llovEV, R. W,, F, S. DAVIS, M. 0. MUKLR. R. E. MEYER, H. L.
MOHTON, and L. F. HOUSK. l%5. Defoliation and control of
huifh. IVoc. SWC 18:288-202.
4, DAWKINS, R, C, 1957. Contact arboricides for rapid tree
weeding in tropical forests. Trop. Silvicult. FAO Collcclion No. 13(2}:109~112.
5, FRISSFL. M. J, and C, H. HOLT. 1952, Interaction between
certain lonlzable organic compounds (herbicides) and clay
minerals. Soil Sci, 94:2B4-29J.
6. Ginns, CARTER B, 1959. Amines of 2.4-D hold promise for
hardwood control. Down in Earth 15(3):6.
7. MAVO-MENENDEZ, ENRIQUE. 1964. Klltninadon de arbolcs
indcscabtes incdiantc agcntcs quimicos. Rev, Interanicr,
Cicnciai Agr, Turrialba MM); 199-202.
8. MKRKI.K, M. G., R. W. BOVBY, and K. HALL. 19(56. The
determination of picloram residues In soil gas chromalography. Weeds 14:161-164,
9. NATION, HOVT A. 1965. Woody plant control on utility
lichts-of-way with "Tordon" herbicide pellets. Proc. SWC
I ft: 387-391.
10. SciiwmEk, E. E. and J. T. HOUTUH, JR. 1966. Persistence of
live cotton herbicides in four southern soils, Weeds 14:22-26.
H, SPOSTA, J. W. i960. Elimination de espccics tropieales infcriores por tncdio de suhstanclas quimicas. Apuntes Forest.
No. 4. Trop, Forest Ren. Center, Rio Piedras, Rncrto Rico.
t p.
12. THIEGS, B, J. 1962. Microbial decomposition of herbicides.
Down to Earth 18(2);7-10.
13. TSCIIIRLKV, F. H. 1967, Problems in woody plant control
evaluation in the tropics. Weeds 15:283-237.
14. TSCIIIRLEV, F, II., CLYPE C. DOWI,ER, and J. A. DUKE. Species
diversity in two plant communities of Puerto Ulco. A Tropical Rain Forest (In Publication).
15. WATSON, A. J. and II. J, MESLF.R, JR. I96'l. Effect of tordon
herbicide as basal mil and tree injection treatments on
certain hardwood trees. Down to Earth 19(4):20-23.
16. WATSON, A. J. and M. G. WILTSE. 1963. Tordon . . . for brush
control on utility rlghts-ot-way in the eastern United States,
Down to Earth IO(1):11~M,
17. WYATT-SMITH, J. I960. Further arboricide trials in lowland
Dipterocarp rain forest of Malaya. The Malayan Forester
23:814-33].
18. WYATT-SMITH, J. 1961. Arboricide trials usin K animate X,
2,3-D, 2,4,5-T, and sodium arscnitc. The Malayan Forester
24:81-84.
Reprinted from WEEDS
Vol. 15, No. 3, July, 1967
Persistence of 2,4-D, 2,4,5-T, and Dicamba in Range Forage Grasses1
HOWARD L. MORTON, E. D. ROIHSON, and ROBERT E. MEYER*
Abstract, The herbicides 2,4,-dlchlrjiophcnoxyacetic acid (2,4-D),
2.<l,r>-tr(cl]Ioropliciioxyacetic acid • (2,4,5-T), and 2-mctlioxy-3,6-diUilorobcnzoic acid (dicamba) each labeled in the caiboxyl posilioit wore spiaycd on a pastme consisting of a mixture of silver
bcardgiass (Atidrofrogan saccliaroides Swart?.). Hllic blucstcm f/J.
scoftariiii Michx.), and dalli.igrass (Pasl/atum dilatalum Poir.) and
a sidcoais giama (Houtrlaita citrtifietiuula [Michx.] Torr.) pastuic
over a 3-ycnr ])criod. riant samples were harvested at Intervals
between 1 hr and Ifi weeks after tieatment and residues determined
hy raclionssny, No important differences were found in the persistence of herbicides or of different foundations of the same
herbicide. Rainfall was the moat important factor influencing
ihe peisistencc of ilie herbicides. The little blucstem-sllver beardKiass-diilliagiass samples haivcsted 1 hr after treatment with the
buioxyclhyl ester of 2,4,5-T contained holK this ester and the
acid of 2,4,5-T. One week after tiralmcnt, the acid of 2,4,5-T
and unknown metabolites were found but no ester,
INTRODUCTION
VARIF.TY of herbaceous and woody plants are con\ trolled by 2,4-dichlorophenoxyacetic acid (2,4-D),
2,4,5-trichlorophenoxyacetic acid (2,4,5-T), and 2-methoxy-3,6-dichlorobcnzoic acid (dicamba). Although the
phenoxy acids have been registered and are used for
weed control on lands devoted to forage production,
[he substituted benzole acids have restricted usage on
these areas. Little direct evidence of the persistence of
these compounds in forage grasses has been published.
Glastonbury ct at. (3) spiaycd peas (Pisum sativum L.
vnr. Onward) with the sodium salt of 4-(2-mcthyl-4-chloro.
phcnoxy)butyrir acid (MCPli) and found that the halflife o£ the retained chemical was 3 days. Gutenmann
and Lisk (4) sprayed the diethylamine salt of 4-(2,4dichlorophenoxy)butyric acid (2,4-DIl) on a pasture containing birdsfoot trefoil (Lotus cornicitlatus L.) and
timothy (Phleum f>ratense L.) and found a rapid decrease in herbicide concentration in the forage after
rainfall. Concentration! of 2,4-DB in the forage immediately after application of 1.5 and 3.0 Ib/A rates were
about 70 and 160 ppm, respectively, but were O.S2 and
0.80 ppm, respectively, after 48 days.
Klingman et al. (5) sprayed a Kentucky bluegrass (Pan
pratensis L.) pasture with either the butyl ester or the
2-elhylhexy! ester of 2,4-D and found that most of the
butyl and about 75% of the 2-ethylhexyl ester were
hydrolyzed to the 2,4-D acid within i/2 hr after spraying. Total concentrations of 2,4-D residues from the
butyl and 2-cthylhcxyl esters dropped from 58.4 and
A
'Received for publication November 7, 1906. Cooperative In.
vcsllgalfons of the Crops Research Division, Agricultural Research
•Service and Texas A&M University,
'Research Agronomist, Crops Research Division, ARS, U, S,
Department of Agriculture, College Station, Texas; Assistant Range
Scientist, Rolling I'lains Livestock Research Station, Texas A&M
University, Spur, Texas; and Plant Physiologist, Crops Research
Division^ ARS, U. S. Department of Agriculture, College Station,
Texas, respectively, '
48.4 ppm i/2 hr after treatment to 5.0 and 15.1 ppm,
respectively, 7 days after treatment.
The investigation reported herein was conducted to
determine the persistence of 2,4-D, 2,4,5-T, and dicamba
in range forage grasses, to compare the persistence of
attune and acid formulations of 2,4,5-T, ami to determine
the influence of rate of application on the persistence of
2,4-D and 2,4,5-T.
MATERIALS AND METHODS
Two field sites were fenced for the study. One was at
College Station, Texas, in a pasture in which silver
beardgrass (Andropoeon saccharoides Swartz.), little bluestem (A, scoparius Michx.), and clallisgrass (Paspalum
dilalatnm Poir.) were the dominant species. The other
was at Spur, Texas, in a pasture in which sideoats
grama (Roitleloua curtipendula [Michx.] Torr.J was the
dominant species, Different areas were treated at each
site each year.
Herbicides labeled in the carboxyl position with carbon-14 were mixed with technical grade herbicides in
the proportions necessary to give the specified radioactive levels as well as the specified rate of herbicide per
acre. In all experiments, sprays were applied at volumes
equivalent to 20 gpa with a compressed air sprayer. Two
replications of each treatment were used. In 1962, the
plots were 2 by 10 ft and they were 2 by 12 ft in 1963
and 1964.
Jn 1962, we applied butoxyethyl ester of 2,4,5-T al
rates equivalent to yz and 2 Ib/A. Sprays were applied
June I I and _|une 19 at Spur and College Station, respectively, which contained 5 u,c of radioactivity per
plot. The carrier consisted of 7 parts water and 1 part
diesel fuel (v/v).
In 19G3, we applied 2,4-D and 2,4,5-T acids to the silver beardgrass-little bluestem-dallisgrass pasture June 14.
Each solution contained 50 |»c of radioactivity and sufficient herbicide to provide i/j, or 2 Ib/A rate. The
carrier was acetone-water (1:1) containing 0.5% (v/v)
surfactant 3 .
In 19G4, we applied dimcthylamine salt of 2,4-D, dime thyla mine salt of dicamba, and triethylaminc salt of
2,4,5-T to the silver beardgrass-little bluestcm-d.illisgrass
pasture July I, We applied both the ainme and acid
of 2,4,5-T and dicamba to the sideoats grama pasture
July 7. The carrier was water containing 0.5% (v/v) surfaciant* for amine salt formulations and acetone-water
(1:1 v/v) for the acid of 2,4,5-T. Each solution contained GO uc of radioactivity and sufficient herbicide to
x
provide a rate of 1 Ib/A.
•.Surfactant contained alkylarylpolyox veiny ten e glycols, free fatty
acids and isopropanol.
\ \
�54
In all years, we sampled the treated plots 1 hr (0
week), ], 2, 4, and 8 weeks after treatment. An additional
sampling was obtained on the fourteenth and sixteenth
weeks in 1003 and 1964, respectively. We harvested
2-sej-ft subplots from each main plot by clipping the
priiss plants at ground level, The clipped plants were
separated into those tissues produced during the current year, designated green tissues, and those tissues produced during previous seasons, designated litter tissues.
P a r t i a l l y decomposed plant tissues were gathered from
the soil surface of the subplots and were added to the
Utter tissues. After separation, the samples were placed
in polyethylene bags, sealed with rubber bands, weighed,
and stored at —10 C. .Samples harvested as Spur were
transported to College Station in an ice chest for analysis.
The harvested samples were shredded, and a 20-g
portion was homogeni/.ed in a blender with 80% ethanol
and filtered, The liomogenization \vas repeated until the
radioactivity of the residue was less than two times
background. The filtrates were combined, reduced in
volume under vacuum in a rotary evaporator, and
brought to volume in a 25-nil volumetric flask. Duplirate I-ml samples of each concentrated filtrate were dried
in I-in planchets, weighed, and the radioactivity assayed
with a Geiger-Miillcr tube. Counts were converted to
weight of herbicide from standard curves with appropriate corrections for background and self-absorption,
file q u a n t i t y of herbicide recovered on and in the forage was calculated for each subplot and converted to
parts per million equivalents of fresh weight.
In 1962 and I9f>8, identification and characterization
of the radioactive compounds in the concentrated ethanolic extracts were made by descending chromatography
on W h a t m a n No, 1 filter paper. An isopropanoliammnnium hydroxide:water (10:1:1 v/v/v) developer was
used. After development and drying, each chromatogram
was scanned with an autoscanner to determine the location of radioactive substance or substances on the chromatogram, Chromatograms of the ester of 2,4,5-T treating solulions contained radioactive butoxyethyl ester
o[ 2/1,5-T and small amounts of arid. Identifications
of the butoxyethyl ester of 2,'1,5-T, and acids of 2,4,5-T
and 2,4-1) were made by co-chromatography of the ethanolic extracts and standard solutions of these compounds.
RF.SIU.TS AND DISCUSSION
Recovery of herbicidrs from sprayed plots. The amount
of heibicidc recovered from grass tissues harvested 1 hr
a f i c r treatment, calculated as a percentage of the amount
applied, varied from 28% (20% green tissue and 8%
litter tissue) to 102% (42% green tissue and 60% litter
tissue) (Table I). In all but two plots, greater quantities of
the herbicides were recovered from the green tissues
t h a n frnni the litter tissues. Although the silver beardgrassl i t t l e bluestem-dallisgrass stands were relatively uniform,
the plants and litter did not cover all of the plot areas.
The low recovery percentages were due to sparse stands
and the higher recoveries to dense plant and litter
rover.
Exprriments in 1962, Figure IA presents a semilogarithmic graph of the concentrations of the ester of 2,4,5-T
in green tissues of silver beardgrass, little hint-stem and
55
Table I. Percentage of herbicides recoveied fn green and lid«
1 Issues of silver beardgrais, little uhieslem, and dallisgraw ind
sldeoats grama harvested I hr after treatment.*
Acirt or I,*-])..',
Aci.i of 2,4-n
Acid of 2,4,5-T
Add or2,4,5-T
Amine t«l( of 2,4-D, ...
A mini! milt of 2,4,5-T....
Acid of2,4,5.T
Diramba
»», liitlr liIiiMiem, and dallisgtass. Disappearance
*•« (twxi T»tild during the second week after treatment.
'IV HI* of [1ki|i]icarance in litter tissues was slower
"Hivwl tn ihtr KTCCII tissues. Tlic apparent half-life of ester
«l £,t.*T In ilie liner tissues was about 4 weeks under
*fl# ffwwfifiont of this experiment. Two factors were imjr*'**.!'** in OH" slower jalc of disappearance, first, growth
•i it* K»«II tinues would have diluted the herbicide,
• ftirt »h» lilltr umplrs were com|)oscd of non-living tis;','«!« tnd Riowih was not a factor in lowering the conceni In these samples. Second, conditions for micro-
lideoats grama. The ester oE 2,4,5-T disrapidly from sidcoats grama than from
dallisgrass harvested at five dates after treatment. The
lines for the two rates are essentially parallel, indicating
that the rate of disappearance was not affected by
rate of application. Concentrations of the ester of 2,4,5-T
residues decreased rapidly during the second week after
treatment when 2.18 in of rainfall occurred. The ap2,0 Lfl/A
,. 2.0 LB/A
..0.5 LB/A
O.9 LB/A
WKS.AFTER TREAT.
I. ONMrmralUini of ester of 2,4,5-T residues found in sideaim puna llwuri hancslcd at live dates after treatment June 11,
' WHIT., M OS mil J.I) Ili/A at Spur, (A) Green tissues, (B) litter
«*•**«. fcaU tedical lines indicate rainfall which occurred durb*H Ilk MwlkltH Interval nftcr treatment,
0
1
2
4
0
1
2
4
a
WKS. AFTER TREAT.
WKS. AFTER TREAT.
Figuie I. Concciilintioiis of t-slcr S/J.S-T residues found in lilver
iK'nidgi.iss-litllr hhiesIcm-d;Ulisgms!i (issues haivested a( five rlatrt
aficr treatmrnt June 19, 1962 nt 0.5 and 2,0 Ib/A at Co\\tyf
.Station. (A) Green tissues, (H) titter .issues. Solid vertical lino
indicate rainfall which occurred during (lie indicated interval
after h'catmcnt,
parent half-life of the ester of 2,4,5-T (half-life equal*
average length of time necessary for one-half of herbicidal residue to disappear) under the conditions of tliii
experiment averaged 2.6 weeks. Concentrations of csler
of 2,4,5-T in the green tissues 8 weeks after treatment
were 25 and 7 ppm, respectively, at the 2.0 and 0.5
Ib/A rates.
Figure lli is a semilogarithmic plot of the ester of
2,4,5-T concentrations in the litter tissues of silver
iin, liitlc bluestcm, and diillisjjrass. More
Tft&H foil mt thr sidcoats grama than on the silver beardwMt liitle liliifMctn, and dullisgrass, The apparent half, Mr <4 ilw hcrl>iciilc aveiitged 1.6 weeks in the green
•
»mt 1.7 weeks in the litter tissues. The amount
liwiurnty ol the r a i n f a l l were conducive to leachCTlrmbi.il decomposition of the herbicide, and
i al liilnuit grama plants. All of these factors con> * rapid reduction in herbicide concentrations.
f»ff>imtnt in 1961, The concentrations of 2,4-D and
• t.f,*-T roliliics found in green and litter tissues of
,!,"«***» IvjitlgraM, l i t t l e blucstcm and dallisgrass har,,-i»w*n) «t u\ djtci alter treatment June 18 are shown in
„ Itfw* J. A (MiO-in rain occurred during the first week
•fwr liMlmrnl. and the concentrations of both herbi<J4« In ftitrn and litter tissues decreased rapidly. No
!4«i!il| oriiiirril during the second week after treatment
ftstel id* lair tif herbicide disappearance was slower in
K<*i*t n( ihr plot* than it was during the first week. Dur(*m Itw ihiid and [otirth weeks after treatment, 1.58
, in ti r»ln octuircd and the rate of herbicide disa]>pear-
O.SLI/A t.«.S-T
«"I.OLB« M.S-T
— aate/A M-O
f.Ot»/A M-D
figure 3. Concentration! of 2,4-1) ami 2.4.5-T Kilduei found in silver
tcantarass-Httlc blucjtem-ilallisgrajl tissues harvcsltil al lix dat»
aficr irealmenl June 14. I%J al OS and 2,0 Ib/A at College
Station. (A) Green tissues, (B) litter tissues. Solid \emcal Imel
indicate rainfall which occurred during the Indicated interval
after treatment.
ance was more rapid in roost of the plots than during
the second week. During the fourth through the eighth
weeks after treatment, only 0.08 in of rainfall occurred
and relatively small decreases in herbicide concentrations
were found. The 2.78 in of rainfall which occurred
during the eighth through the fourteenth weeks after
treatment probably was the primary factor responsible
for the rapid rate of herbicide disappearance during
this interval. The average half-life for 2,4-D in green
and litter tissues was 2.3 and 2.8 weeks, respectively.
' The average half-life of 2,4,5-T in green and litter tissues was 2.9 and 3.4 weeks, respectively.
Experiments in 1961, Residues of amine salts of 2,4-D,
2,4,5-T, and dicamba disappeared from silver beardgrass,
little bluestem, and dallisgrass tissues at about the same
rate (Figure 4). The apparent average half-life for each
Figure 4. Concentrations of amine of 2,4-D, amine of 2,4,5-T, and
dicamba residues found In silver beardgrass-little blueslcm-dallisgrass tissues harvested at six dates after treatment July I.
11164 at 1 Ib/A at College Station. (A) Green tissues, (B) litter
tissues. Solid vertical lines indicate rainfall which occurred
during the indicated interval after treatment.
of the three compounds in green tissues was 2.0 weeks
under the conditions which existed during the experiment. Because of heavy rainfall during the fifteenth
week after treatment, the concentrations of the three
compounds were reduced to 1 or 2 ppm in the green
�56
57
HERBICIDES IN SOILS *
T. J. SHEETS AND L. L. DANIELSON '•
[issues. Concentrations o£ the three herbicides decreased
rapidly in the Iitier tissues during the first 8 weeks
after treatment (Figure 4B) when frequent rainfall kept
the soil and litter tissues moist. The average half-lives
of 2,4-D, 2,'i,5-T, and dicamba in the litter tissues were
2.8, 2.7, and 2.6 weeks, respectively.
A relatively slow disappearance rate was found for
all three herbicides in the green and litter tissues of
sideoats grama (Figure 5). This slow disappearance oc-
Figure S. ConccnMittionj of ncid of 2,4,5-T, nminc of 2,4,5-T, nnd
dicamba residue* found in sidcoats grama tissues harvested nt
six dates after treatment July 7, 1064, nt 1 Ib/A at Spur. (A)
Green tissues, (B) litter tissues. Solid vertical lines Indicate
rainfall which occurred during the indicated interval after treatment,
rui-rcd during a period of low rainfall. Although considerable variability occurred in the concentrations of
the three herbicides nt each sampling date, all herbicides
had essentially the same rate of disappearance during
the Ifi-week duration of the experiment. Concentrations of the acid of 2,4,5-1', amine of 2,4,5-T, and dicamba
were 6, 5, and 4 ppm, respectively, in the green tissues
at the time of final sampling 16 weeks after treatment.
The concentrations of the three herbicides in litter tissues of sideoats grama are shown in Figure 5B, The
acid of 2,4,5-T disappeared more slowly than the amine
of 2,4,5-T and dicamba in litter tissues during weeks
2 to 4, but all three compounds were present after 8
weeks in approximately equal concentrations.
Data indicate that formulation had no significant effect upon the persistence of 2,4,5-T in the tissues of silver bcnrdgrass. little bluestem, dallisgrass, and sideoats
grama. While there were minor differences in the rates of
disappearance of the three herbicides applied at College Station and Spur, their persistence in forage tissues
appears to be essentially the same after several weeks.
"1 he most important factor influencing the persistence
of these herbicides was rainfall. Both amount and frequency oE rainfall were important.
Even when rainfall did not occur, there was a gradual
reduction in the herbicide concentrations in the green
tissues, particularly if rainfall had occurred prior to the
interval when herbicide concentration was being measured. Dilution of the herbicides by plant growth was
an important factor during the intervals after rainfall
had occurred and soil moisture was adequate for growth
of the plants. Important reductions in the concentra-
(Reproduced from ARS 20-9 :170-181, Sept. 1960)
tions of the herbicides were not found in the litter tissues when no rainfall occurred. This is evident in Figures
111, 31i, and 5H, The exception to this statement is found
in Figure 411 when a reduction in the concentration of
the amine of 2,4,5-T from 146 ppm to 78 ppm occurred
during the first week after treatment.
It is not surprising that formulations had no influence
on the persistence of 2,4,5-T. Phenoxy herbicides deposited on the surfaces of plant leaves as ester formulations are hydroly/ed to the acid in a relatively short
period of time (1, 2, 5).
Identification of herbicide residues. Attempts to identify
the radioactive components in the ethanolic extracts by
paper chromatography were only partly successful. All
extracts from silver beardgrass, little bluestem, and dallisgrass green tissues harvested 1 hr after treatment witli
ester of 2,4,5-T contained the applied herbicide find the
acid of 2,4,5-T. The Rf values ranged from 0.59 to
0.75 and 0.85 to 0.89, respectively, for the acid and ester
of 2,4,5-T. Approximately 10% of the radioactivity was
attributed to the ester and 90% to the acid of 2,4,5-T.
These data confirm the results of Klingman et al (5)
who found rapid hydrolysis of the ester of. 2,4-D by Kentucky blucgrass. Extracts of green tissues of silver beardgrass, little bluestem, and dallisgrass harvested 1 week
after treatment contained the acid of 2,4,5-T and unidentified metabolites which had Rf values ranging
from 0.10 to 0.30 but no ester of 2,4,5-T. Approximately
50% of the radioactivity was attributed to the acid of
2,4,5-T and 50% to the unknown metabolites.
The extracts 6f green'tissues of silver bdardgrass,' little
bluestem, and dallisgrass harvested I hr after treatment
with acid of 2,4-D or acid of 2,4,5-T yielded only the
acids of 2,4-D or 2,4,5-T. Tissues harvested 1 week after
treatment contained both the acid and unknown metabolites. The metabolites of 2,4,5-T had Rf values ranging
from 0.10 to 0.30, and those of 2,4-D had Rf values
ranging from 0.07 to 0.25.
,>
ACKNOWLEDGMENT
The butoxyethyl ester of 2,4,5-T used in this study
was provided by Amchem Products, Inc. and the dicamba was provided by Velsicol Chemical Corp. The
authors are grateful for the technical assistance of Gloria
C. Taylor and T. O. Flynt.
LITF.RATIIPE CITRD
1. CRAFTS, A. S. 1PGO. Evidence for hydrolysis of esters of 2.4-D
during absorption by plan is. Weeds 8:19-25.
2. ERICKSON, LOUIS C., B. L, H R A N N A M A N , and CiMRi.rs W. COCGINS,
In. 1003,
Residues In stored lemons treated willi various
formulations of 2,4-0. J. Agr. Food Chem, 11:437-440.
3. Gi,AsroNnuRV, H, A., MARIJARCT D. SIFIVKNSON, and R. W, E,
DAM,. 1959. The persistence of 4-(2-mcthyl-4-chloropIienoxy)
butyric add in peas. Weeds 7:3(12-36,1.
4. GDTF.NMANN, WALTIR H. and DONALD J. LISK. 1963. Rapid
determination of 4(2,4-DB) and a metabolite, 2,4-D, In treated
forage by electron affinity spectroseopy. J, Agr, Food Chem.
11:304-300..
5. KLINGMAN, DAYTON L., CHiatFR II. GORDON, GEORCK YIP, and
H. P. BURCI(FIELD. IPG6. Residues in the forage and in
m i l k from cows grazing forage treated with esters of 2,4-D.
Weeds 14:164-167,
INTRODUCTION
Many herbicides are applied directly to the soil surface as selective preemergence sprays and as nonselective soil sterilants. Other chemicals are applied subsurface or are thoroughly mixed with the soil after surface application.
Residues remaining on leaves after foliar applications are carried to the soil
In rainwater or fall to the soil when injured leaves abscise and fall. Therefore
at least part of all herbicidal sprays eventually reach the soil.
Soils vary greatly in composition and reactivity. Many complex nnd everchanging processes occur continuously in most soils. Soils are composed of
mineral matter, organic matter, water, and air. The mineral fraction varies in
amounts of sand, silt, and clay, and in types and amounts of clay minerals.
The hydration and base saturation of the clay minerals also vary. The organicmatter fraction consists of decaying plant and animal -residues and active soil
flora and fauna. The organic and mineral colloids present in the soil contribute directly and indirectly to the extremely active nature of soil systems.
This mixture of mineral and organic matter is permeated by pore spaces of
various sixes. These spaces are filled with water and air in a more or less
reciprocal relation. The soil water contains many soluble compounds and serves
as an essential medium for many physical and chemical processes. The soil
atmosphere is composed of oxygen, carbon dioxide, nitrogen, and several minor
gases. The composition of the soil atmosphere varies, particularly the oxygen
and carbon dioxide contents. The complexity and variation of soil systems
' make the study of the fate of herbicides therein complicated and time consuming.
METHODS OF ASSAYING HERBICIDE RESIDUES IN SOILS
In most investigations on the persistence of herbicide in soils, researchers
determine the presence of the active entity of the herbicide by growing sensitive plants. The influence of time on herbicidal residues has been measured by
the growth of successive crops of test plants. This method, which has been used
in both field and greenhouse experiments, is qualitative only.
Quantitative bioassays have been developed for some herbicides. Holstun and
Loomis (85) measured the elongation of young shoots of germinated millet
seeds to determine the concentration of the sodium salt of 2,2-dichloropropionic
acid [dalapon] in soils. Burschei and Freed (11) used heights and weights
of seedling oats to determine the concentrations of isopropyl N-phenylcarbamate
[IPO], isopropyl N-(3-chlorophenyl) carbamate [GIPC], and 3-amino-l,2,4~
triazolo [amitrole] in soils. Rahn and Baynard (45) used weight of oat seedlings to assay quantitatively 3-(p-chlorophenyl)-l,l-dimethylurea [monuron] in
soils. Biological assays developed for solutions and vapors of herbicides could
be adapted for use with soils 11, 46, 55).
Some herbicides have been extracted from soils and their concentrations
determined by physical or chemical methods. Methods are available for monuron, amitrole, CIFC, and pentachlorophenol [PGP] (10, 11, 30, 62). Whiteside
and Alexander (61) followed the breakdown of several chlorinated phenoxy
aliphatic acid herbicides in solutions inoculated with soil by the disappearance
of the specific ultraviolet absorption.
A physical or chemical assay may be most suitable in one situation and a
biological assay in another. Both types of analyses are useful in some cases.
Rahn and Baynard (45) reported that the chemical method for the determination of monuron in soils was accurate if the assay was made within a few
weeks after application. If soils were chemically assayed more than 1 month
after treatment, values for monurou concentration were greater than those
obtained by bioassay. Rahn and Baynard (45) suggested that this apparent
disagreement could be explained since the chemical assay for monuron was
based on p-chloroanaline, a nonphytotoxic, hydrolytic product of monuron.
1
A contribution from the Crops Rosenrch Division, AgrlcnUnrnl Resonrnh Service, U.S.
Department of Agriculture, nnd the Mississippi Agricultural Experiment Station.
3
Plant PliyRlologlntfl, Crops Research Division, Agricultural Repenrch Service, U.S.
Department of Agriculture, Stonevllle, Miss., and Beltsville, Md.( respectively.
�58
*•:,.••••
fey
? '
FACTORS ALTERING HERBICIDES IN SOILS
Factors affecting the movement and persistence of herbicides in soils hnve
been reviewed by several workers (1, 8, 25, 20, 32, 41, 43). Leaching, fixation
by soil colloids, chemical and microbial decomposition, and volatilization were
stressed in one or more of these papers. In this discussion microbial action,
volatilization, adsorption, leaching chemical reaction, photodecompositiou, nnd
absorption by plants will be considered for their significance in the performance and fate of soil-applied herbicides.
Microbial action.—Most organic herbicides subjected to appropriate tost*
have been inactivated more rapidly in soil under conditions favoring growtli
and proliferation of soil microorganisms. Absorption by microorganisms is one
of the major pathways by which organic herbicides are detoxified. Perhaps
2-(2,4-dichlorophenoxy) propionic acid [2-(2,4-DP) ], 2,4,5-trichlorophenoxyacetlu
acid [2,4,5-T], 2-(2,4,5-trichlorophenoxy) propionic acid [silvex], and 4-(2,4,5trichlorophenoxy) butyric acid [4-(2,4,5-TB) ] are exceptions (7, 61). Optimum
oxygen, moisture, temperature, and nutrients favor microbial activity and nlsn
herbicidal detoxication. Numbers of soil microorganisms capable of inactivating
2,4-dichlorophenoxy acid [2,4-D] apparently increase when 2,4-D is present In
the soil (8, 42, 48, 61}. Thus repeat applications of 2,4-D were less persistent
in soil and therefore may be less effective herbicidally than the initial application. With the phenylureas and s-triazines such an increase in microbinl
activity apparently does not occur, because soils appear to exhibit about tlie
same capacity for inactivation for long periods. Therefore it seems that witli
the phenylureas and the s-triazines the action of soil microorganisms utilize
them but not selectively or preferentially. Another explanation of this effect
is that inactivation of these chemicals is catalyzed by heat-sensitive substances
occurring in the soil as products of microbial activity and that the herbicides
are not utilized directly by microorganisms as energy sources.
;
Bacteria, Bacterium globiforme and Flaviobacteriiim aquatilc, which were .
capable of inactivating 2,4-D were isolated from soil and grown in pure culture (G, 8, 86). Evans and Smith (27) isolated a small, Gram-negative, motile
soil organism which grew freely in a mineral-salt medium containing pchlorophenoxyacetic acid as the only organic-carbon source. They separated
2-hydroxy-4-chlorophenoxyacetic acid and 4-chlorocatechol from the culture.
The same investigators isolated a Gram-negative, motile rod which grew on ;
a mineral-salt, 2,4-D medium. From this culture they separated a phenolic acid
and presented evidence to suggest that the compound was C-hydroxy-2,-1dichlorophenoxy-acetic acid. They hypothesized that hydroxylation of the ring
was followed by ring cleavage.
Whiteside and Alexander (61) presented evidence suggesting that 4-(2,4tlichloroplienoxy) butyric "acid [4-(2,4-DB)] was converted to 2,4-D by micro-;
organisms in the soil and that a microflora capable of. (illicitly inactivating
both 2,4-D and 4-(2,4-DB) was present in soils which had received 4-(2,4-DB)
previously.
•
" "Hill, et al. (33) reported that a soil bacterium of the Pseudomonas group'
was capable of oxidizing monuron particularly in the presence of yeast extract.
One group of herbicides, the esters of clilorophenoxy alcohols, becomes herblcidally active only on contact .with the soil. In warm, moist soil sodium, 2,4dichlorophenoxyethyl sulfate [sesone] is hydrolyzed to 2,4-dichlorophenoxyethanol in the presence of either microorganisms or acids (12, 13). Tlie
hydrolysis by microorganisms was attributed to acids secreted during their
metabolism. The ethanol product is oxidized in the soil to 2,4-D, the active
entity.
In experiments conducted by the senior author, 2-ehloro-4,6-bis(diethylamino)s-triazine [chlorazine] mixed in the soil became more toxic to seedling onts
with time. This trend reversed after several months, the time depending on
the soil type and concentration, and thereafter the herbicidal activity of cultures containing chlorazine decreased with time. The increase in toxicity could
not be accounted for completely as a response to growing conditions iu the
greenhouse. Autoclaving the soil prior to treatment retarded the rate of onset
of increased toxicity. If one ethyl group was lost from either or both of tlie
amino substitutions, compounds much more toxic than chlorazine would be
formed. Perhaps formation of one of these compounds did occur in the soil.
The rates of inactivation of IPO, OIPO, and amitrole in the soil depended
on the Initial concentration of the herbicides'; and the inactivation of these
59
| bwtilcldes appeared to follow afirst-orderreaction ^F$. The rate of dis|
|
6j|!*pf»ttirnnca of monuron from soil was proportional to the concentration (53).
ppllil, Cl al. (33) concluded that although soil moisture and temperature often
|I|:*ll*rwl the rates of inactivation of monuron and 3-(3,4-dichlorophenyl)-l,lrilBMrtliyluren [Uluron], the first-order equation was probably applicable under
*wu»l field conditions. When monuron and diuron were applied at rates of 1
:**>A a wounds per acre in more humid regions of the United States, major
pmrU ot tho herbicides were inactivated each year. Accumulation from application* on the same soil 2 years in succession was negligible. Hahn and Bayi *»«! (48) found that monuron applied at 3.6 pounds per acre in two applicaIfeMW for 3 years in succession did not persist from one year to the next. Wheu
•iMIwl lit 0.4 pounds per acre, monuron toxicity persisted from one year to
; th* nwxt, but no accumulation occurred. Research conducted in the arid South|r;;'i***t; (luring the last 7 years indicated that monuron and diuron did not
: ,«mtmtilnti> significantly from successive annual applications at rates used for
wH*rtl«ro weed control in cotton. Some carryover often occurred, and the
., amount of carryover appeared to be related to weather conditions.
,; Can-out research indicates that the solvent used in the application of an
* Iforrlvfrlde may have a profound influence on the persistence of herbicidal
•j MllrKjr ( 1 8 ) . Ethel N,N-di-n-propylthiolcarbamate [EPTC] was applied in
:
.*if*ral Holvents and incorporated into the soil. At weekly intervals up to 6
!,y,***k* nfter treatment, the soils were assayed by the used of oat plants as Indl:-:.'<r»li»rw of toxicity. When the commercial formulation of EPTO was applied in
:j »'»l<-r, growth of oat plants seeded 6 weeks after treatment were markedly
'/; toMMtnl on flats which received 1 and 2 Ib./A, The persistence of technical
:•'.JBITO applied in acetone was comparable to that of the commercial forinlation
•f (*IH>'!tHl in water. However the rate of inactivation of technical EPTO applied
4 ftt tK>rvMw>np was much more rapid than that of the commercial formulation
;•:• iHT'lwl In water. Four weeks after treatment the 2 Ib./A rate of technical
:.; SITO Applied in kerosene did not inhibit growth of oats.
:!-•- IVrKlwtom.'o of several groups of herbicides in the soil is related to halogena.; Uwi of (he benzene ring. This relation was demonstrated for certain chlorinated
''p&aMjoxyncPtlc acids, carbamates, and phenylureas (21, 22, 23, 50). The results
~. <tf Alexander nnd Aleem (3) indicated that resistance of chlorinated phenoxy'i'ftlkjl rnrnoxllc acid herbicides or their derivatives to microbial degradation
:;w!i(i ftivoriicd by the position of the halogen rather than by the number of
tfr, fctJtofww on the ring and that the linkage of aliphatic side chain also Influenced
||,**»wr»tlt)lllty to microbial breakdown.
j|.<"-' ValnHlizatlon.—All compounds are volatile to some degree. Volatility of some
| bwtt)l(?|<j<vH IB 7ery iow and. of little significance. However, measurable loss of
|
||:(jMwr* occurs from soil surfaces by vaporizations.
j&f The volatilities of formulations of the same basic herbicide structure may be
jjfe,<wH«» different. The isopropyl ester of 2,4-D is more volatile than the octadecyl
||{.*§««•, which in turn is more volatile than the sodium salt (39). Many otber
^. ****** nm i wlltft of 2>4-D exhibit various rates of vaporization.
S,.» Vajwrs^of soil-applied herbicides have caused severe injury to treated crop
S**nt* tn Home instances. Vapors of 4,6-dinitro-o-seo-butylphenol [DNBP] after
|MM>AOTi!«mcQ applications caused extensive injury to cotton in the Mississippi
l*»tw In 1952 (25, 26, 34). DNBP injury was associated with high tempera,t*m*. lIolllnRsworth and Ennls (84) found that vapor injury to young cotton
ftoiil* Increased ns soil moisture increased.
-"; IWBl* Injury to cotton was reduced by application of lime and other basic
;
Hi ***<('HfllH to the treated soil surface (9, 19). Upon addition of a base, the
IJf.fcSwttfll-plipnnto equilibrium was probably shifted to the phenate, which Is less
||:*o*«illi» than the phenol ( 0 ) .
P:.;,-•'TatatHUnHon of the carbamates has been related to their effectiveness as
j*|i; fr*wro»nrimce herbicides. IPO and CIPO volatilized rapidly from tinfoil and
||pj(tftMHf mirfnces at high temperatures (4). IPO volatilized more rapidly than
l/l.CtPO, particularly at temperatures of 60° to 85° F. The most volatile carJit: MttmtM were found to be most phytotoxic (38) ; however, loss by volatility
|;; TBlte-wliiR prceuiergence application reduced the concentration of the more
Jit : ***<Mtl0 compounds to nonherbicidal levels more rapidly than the less phytoisj£ ..***'*'<r|1|HW volatile compounds.
Siif'! '*** Jp l herbicides are formulated on granular carriers to reduce loss by
;|.|i;.1HgllHllltj nnd leaching after application to the soil surface. The vapor and
tf'-frfcwrtwt activities of OlPO-impregnated granular carriers were investigated by
!—70
6
�60
nielson (17). The vapor activity of CIPO was related to the physical sn^e-ure and adsorptive capacity of the granular carriers. When the physical struc-"
ture' of attapulgite granules was changed by moistening with water, vapor
activity increased. Carriers that did not change in physical structure on contact with water exhibited unchanged or reduced vapor activity. Danielson
proposed the use of impervious granular carriers to obtain immediate 'shortterm activity of CIPC and more adsorptive carriers for long-term activity.
Adsorption.—The activity of most herbicides varies with soil composition.
Since many herbicides are adsorbed by colloidal particles and since the
amounts of mineral and organic colloids vary among soils, much of the variation in herbicidal activity is attributed to differences in the adsorptive
capacity.
The adsorption of six, growth-regulator herbicides by several ion-exchange
resins was demonstrated by Weaver (50). Weaver (60) and Smith and Ennis
(53) used activated charcoal as a soil amendment to protect germination seeds
from 2,4-D applied to the soil surface.
In a greenhouse experiment the initial toxicity of 2,4-D was greater in sandy
soils than in most clay soils (1G). A butylester of 2,4-D was fixed in a claysand mixture more strongly than a triethanolamine salt form ( 2 ) . Both amine
and polypropylene ester formulations of 2,4-D were adsorbed by montmorillonite, illite, and kaoliuite clays (32). The adsorption of 2,4-D increased as the
cation-exchange capacity and specific surface increased.
CIPC was adsorbed by activated charcoal and certain other materials (17).
In laboratory and greenhouse experiments EPTC was adsorbed least by those
soils in which it was most phytotoxic (5).
Shorburne and Freed (51) demonstrated adsorption of monuron by activated
charcoal, sawdust, straw, and soil. The amount of monuron adsorbed by soils
was correlated with organic matter and clay content. Studies by Hill (32)
showed that the clay content, type of clay, and organic matter of soils influenced the amount of monuron adsorbed. Adsorption increased na clay content
or organic matter increased. Approximately 150 p.p.m, wns required on- a.
bentonite clay to give 1 p.p.m. in the soil solution whereas less than 1 p.p.m
was required on a kaolinitic clay to give 1 p.p.m. in solution.
The herbieidal activity of the phenylureas was correlated inversely with soil
organic matter, total clay, and cation-exchange capacity (47, 5G). Multipleregression analyses suggested that soil organic matter was most important
in toxicity redxiction of monuron, diuron, 3-phenyl-l, 1,'1-dimethylurea [fenuron]j
and 3-(3,4-dichlorophenyl)-l-methylurea [DMU].
Variations in the effective dosage ranges among several soils suggested
greatest adsorption of diuron and DMU-and least adsorption of monuron and'
fenuron (47). Coggins and Crafts (.75) showed that clay suspended in solu<
tions of the phenylurea herbicides reduced the toxicity to barley. The toxicity
of l-w-butyl-3-(3|4-dichlorophenyl)-l-methylurea [neburon] was altered most,,
and alteration of. toxicities of DMU, diuron, monuron, and fenuron followed.
In general, water solubility and adsorption were inversely related. In a recent
report Leopold, et al. (87) demonstrated on inverse relation between solubility
of several chlorinated phenoxyacetic acids and their adsorption on charcoal.
In an aqueous medium 2-chloro-4,6-bis(ethylamino)-s-triazine [simazine] was
adsorbed to a cation exchanger and to activated charcoal but not to an anion1
exchanger (48). Soil toxicity tests with the s-triazines suggested considerable
variation in soil adsorption of these compounds. The effect of soil organic
matter, clay content, cation-exchange capacity, and pH on the phytotoxicity of
simazine was investigated in detail. Soil organic mater appeared to alter the
initial toxicity of simazine in soils most.
In soil systems adsorbed herbicides are probably gradually desorbed as leaching, chemical and biological degradation, and absorption by plants reduce the
concentration in the soil solution. However, the adsorption-desorption relations
of herbicide molecules in soils and the importance of these phenomena in the
movement of herbicides in soils have not been adequately investigated.
Leaching.—The movement of herbicides in soils depends on or is influenced
by several factors. Upchurch and Pierce (57, 58) indicated that at least two
i
stops are involved in the movement of an herbicide downward in soil: (a) En"
trance of the herbicide into solution and (b) adsorption of the herbicide to
soil particles. Entrance into solution could occur from solid particles of the
61
herbicide or from colloidal particles with adsorbed herbicide
cules. These
two processes, solution and adsorption, may be affected by several variables.
The solubilities of herbicides and of salts of herbicides that may form in the
soil are important properties affecting leaching (32, 41, ^3). Minarik (41) discussed the teachability of 2,4-D and its salts. He pointed out that the calcium,
magnesium, potassium, sodium, and ammonium salts of 2,4-D are more soluble
in water than the acid, whereas salts of heavy metals such as iron and copper
are less soluble than the acid. The equilibrium status of the several forma of
2,4-D in the soil probably affects the leaching rate of 2,4-D. However, Smith
and Ennis (53) did not measure a difference in the movement of the acid, the
triethanolamine salt, and the sodium salt of 2,4-D in soils. Hill (32) concluded
that the lower water solubility of diuron compared to monuron resulted in
slower movement of diuron that monuron in soils. Diuron is adsorbed morestrongly than monuron and differences in adsorption probably contribute to.
differential leaching of these two compounds.
An herbicide that is strongly fixed in soils should leach less readily than one
that is not so tenaciously fixed (B). The adsorptive capacity of soil is influenced by soil organic matter and the amounts and types of c.lay minerals. The
adsorption process is influenced by temperature and the nature of the solvent.
The adsorptive characteristics of a compound are influenced by pH of the
solution. Since these factors influence adsorption, they must influence movement of herbicides in soils. The organic matter content and soil texture are
known to influence leaching (20, 2/f, 25, SO, 32, 35, 44, 47, 5g, 53, 58).
The leachability of DNBP appeared to be influenced by pH. Dowler, et «?.
(&4) concluded that movement of DNBP in soils was as much a function of
soil type and soil reaction as of the amount of rainfall. In experiments by .
Upchurch and Pierce (58) soil temperatures of 50° to 45° C. had little effect
on the monuron leached from the upper 2-inch layer of soil columns. However,
greater amounts of monuron were retained by the 2- to 8-inch layer at 25°, 35°,
and 45°C. than at 5° arid 35° C.
That the amount of rainfall or of water applied as irrigation influences the
movement of herbicides has been demonstrated by many research workers. Sherburne, et al. (52) compared the movement of monuron in soil columns to the
movement of compounds in chromatography and concluded that the depth of
the highest concentration of the herbicide in soil columns was a function of the
amount of water added to the soil surface. Upchurch and Pierce (57) studied
the effect of amount, intensity, and frequency of simulated rainfall on the
leaching of mouuron. The greater the amount of simulated rain the greater
the movement of monuron. Rainfall intensities of 1/16 to 4 inches per application had little influence on the amount of monuron retained in the top 2-inch
layer. In the 2- to 8-inch zone greater accumulation of monuron occured with
low intensities than with high. A greater movement of monuron from the
upper soil layers occurred as frequency of rainfall increased. Approximately
half of the frequency effect was attributed to evaporation of water from the
surface of soil columns that received less frequent applications. These workers
concluded that of the three variables studied, the amount of rainfall would
be most directly correlated with the . distribution of herbicides in soil profiles
under field conditions although they maintained that intensity and frequency
might also be of practical importance.
The effect of the a.mount of monuron applied on the amount moved by simulated rainfall was also investigated by Upchurch and Pierce (5S). Monuron.
was applied to surfaces of soil columns at rates of 0.5, 1, 2, 4, 8, 16, 32, (VI.
128, and 250 Ib./A, and the applications were followed by 4 inches of simulated rainfall. The lowest percent retention (34 percent) in the 0- to 2-inch
layer was found in columns treated with 32 Ib./A. Retention by the 2-iuclt
layer was increasingly greater as the rate increased and decreased from 8Ib./A. These workers suggested that the increasing percent retention when the
rate of application was increased from 32 to 256 Ib./A was attributable to the
low solubility of monuron.
Molecular size may also be a factor in the movement of herbicides in soils
US, 44).
The exact pattern of movement of an herbicide in a particular soil would
be impossible to predict presently. However, relative rates, of movement can
be predicted for many herbicides.
>
Chemical reaction. — The formation of salts of 2,4-D and DNBP and a possible reaction scheme for inactivation of 2,4-D in soils have been discussed.
�63
Relatively little is known of the chemical reactions that
underdo in soils. Hydrolysis, oxidation, and formation of comple:Bire known
reactions for certain herbicides.
Amitrole forms stable complexes with cobalt, copper, nickel, iron, and
magnesium, Sund (54) suggested that complexing with metal ions in the soil
solution was one mechanism by which amitrole was detoxified.
The 2-chloroacetamides, for example 2-chloro-N,N-diallylacetamide [ODAA],
can be hydrolyzed in the soil (31). The chlorine atom and the amide linkage
are sites on the molecule where hydrolysis may occur. Begardless of the site
where hydrolysis begins, the end products are glycolic acid and secondary
amines.
The dithiocarbamates, for example 2-chloroallyl diethyldithiocarbamate
[CDEG], can be broken down in the soil by oxidation and hydrolysis (31).
If hydrolysis of CDEO precedes oxidation, allyl alcohol is an'intermediate;
•vwhereas if oxidation precedes hydrolysis, 2-(diethyldithiocarbamyl)acetice acid
"is an intermediate. The two reactions can occur separately or concurrently
-In the soil. End products of breakdown are formic acid, glycolic acid, carbon
••tlisulfide, and secondary amines for both reaction pathways. The breakdown
x products may undergo further reaction in the soil.
Freed, et al. (89) suggested that EPTO was hydrolyzed in water; and
; according' to the reaction scheme which they proposed, a secondary amine,
• carbon dioxide, and ethylmercaptan were end products.
Soil treated with 3,5-dimethyltetrahydro-l,3,5,2 .ff-thiadiazine-2-thione [DMTT]
• evolves formaldehyde, which is thought to be the first product of DMTT break•down in the soil. (40). Methylaminomethyldithiocarbamate forms next and
activation continues by forming monoethylamine, methyl isothiocyanate, and
hydrogen sulfide. Monoethylamine and hydrogen sulflde react with formaldehyde and form methylamirioethanol, dimethylarninorneUinne, and 1,3,5trithiocyclonexane. Eventually this reaction proceeds to carbon dioxide, ammonia, sulfur dioxide, and water. The methyl isothiocyanate and water react
to give carbon dioxide, hydrogen sulflde, and methylarnine; and the methylamine degrades into carbon dioxide and ammonia.
Monuron is thought to be hydrolyzed slowly in the soil to p-chloroamiline (4$)- '"'
In the presence of moisture tris-(2,4-dichlorophenoxyethyl)phosphite [2,4DEP] is slowly hydrolyzed step-wise to form one mole of phosphorous acid
and three moles of 2,4-dichlorophenoxyethanol (28).
The reactions which herbicides and agricultural pesticides in general undergo
in soils and the products formed are important with respect to residues in soils.
Weed research scientists should- emphasize this phase of herbicide research.
PhotodeeomposiUon.—Less is known about the direct effect of light on the
breakdown of herbicides than other factors suspected of being involved. However, photodecomposition of monuron was demonstrated by Hill, et al. (SS).
When a solution containing 88.3 p.p.m. of monuron sealed in quartz tubes was
exposed for 48.days to sunlight, an 83-percent loss of monuron occurred. Hill,
et al. .(S3) concluded that in dry areas of the Western United States monuron
may be inactivated by ultraviolet irradiation. They suggested that this factor
would account for disappearance of only a small part of the herbicide in
humid regions where frequent rains move it into soils.
Neburon, diuron, monuron, fenuron, and DMU were applied as alcohol solutions to filter paper ( H ) . After the paper dried, it was exposed to ultraviolet
light for several hours. The herbicides were not visible prior to exposure, but
in white light tiejr were readUj- T'isiMe after erpwGrrw as light fan spots. The
compounds were apparently changed during1 exposure.
The effects of shade, moisture, and position in the soil on the residual activity of monuron, diuron, and simazine were investigated in cooperation with
the California Agricultural Experiment Station. The activity of monuron and
diuron disappeared more rapidly from soil exposed to the sun from shaded
soil. The activity of monuron and simazine disappeared more rapidly from
moist soil than from dry soil. Monuron, diuron, and simazine were not affected
to the same degree by these variables. Soil temperature was measured but not
controlled in this experiment, and soil temperatures varied considerably among
the treatments during the day. Temperature markedly Influences vapor pressure and chemical reactions. Therefore the difference in the rate of disappearance in shaded soil and soil exposed to the sun cannot be attributed unquestionably to light inactivation,
&;/ plants.—Herbicides are absorbed ^Aplant roots and areHWimtly translocated to the aerial parts. Within thewmt the herbicide moleWiraJwi nro subjected to various physical and chemical processes. Crop plants may
:"rfc» removed from the land or they may be returned to the soil along with weed
:,*twwUi. Most plant roots remain in the soil, Therefore a portion of herbicides
'.,'vinMwrbed by plants and the metabolic products of herbicides in plant tissues
!:;!tt*jr«>vi»ntwilly be returned to the soil.
II IUIB been stressed previously that soils reduce the initial effectiveness of
whlcldra nrnl that the degree of effect varied among soil types and herbicides.
Uii|iijl<llx)jpd data showed that at least four times as much simazine was re;j:'^iiln«d In a clay loam soil as in solution culture to produce the same weight
.-h.'NMlucllonN of seedling oats. In another experiment the dry-weight increase of
;>-w»t tf»(w following simazine treatment through the roots was reduced 50 per; ft-nt by 7.2 ing. of the herbicide (C1* expressed as simazine) per gram of dry
ItaoiKt lit Imrvest 0 days after initial exposure (49). The amount of simazine
'•••.'1C*' expressed as simazine) required in seedling oat plants to reduce plant
'•'" wwiBlit wns less than 2 percent of that present in 400 ml. of the 0.05 p.p.m. by
nMKlit culture solution initially. Although the conditions of this experiment
]•-'• «»«!•*• markedly different from those which occur in the field, plants probably
x'. •Afawb only a small fraction of the total amount of an herbicide applied to the
HOll.
',,-"
CONCLUSIONS
:
S '" Alftiiiiiijti considerable progress has been made, much additional information
on (he fate of herbicides in soil Weed research scientists need to
• About the persistence of herbicides in soils under varying environMjcriinl conditions so that they can establish safe rotational practices. Mam(rtnllnn toxlclty of some soil degradation products should be determined, befl*uj*< fliww products can also be absorbed by plants. Information on adsorptiontfwwirplUm relationships: on the interrelationships of adsorption, volatility,
M'llillll.v, mil leaching of herbicides; on the nature and extent of microbial
flipinicnl innctlvntion ; on the importance of photodecomposition; and on
Inllwm-e of various environmental factors on these processes is essential
understanding of the behavior of herbicides in soils.
Ml scientists should determine the component or components of the soil
Jff'sj:nwrt whleli dosage requirements of soli-applied herbicides can be predicted.
|§y'!J*ww«r of this information is available (48, 56), but more is necessary. Even0fSi!"*"' H|MwUlc recommendations of rates, times, and methods of application of
4
r£':K"^'''>Ip'<'''« nmy be based on weather forecasts and analyses of soil samples
pi^Jfmin fanners' fields (25).
fei^'s"* *)"1* "f "I0 "1(>st ur ^ent needs for research on the fate of herbicides in soils
i||4;M* MiofhwlHan< Isolation and identification of herbicides and breakdown products.
of
S!fJ'"'°'°'{'f'n'
^ chemical assays must be improved and new ones derived. Radio|i;i|.pf««illv(» lHnfo])es bnve lieen used very little to study the decomposition of soil;t f pl!wl
s'iif':* f *
herbicides. Soil samples could be treated with different lots of an
*V;.! IwrWoUle with each lot tagged with C" at different positions in the molecule.
•5 HJT known analytical techniques the unchanged herbicide and many reaction
•;,.:." fwxlurfu could be separated and identified. Radioisotopes should be most useful
j,'.-ilO«l* In future research of this nature.
i"
An new herbicides are developed, their behavior in soils in response to vari*Wi« soil characteristics, weather conditions, and cultural practices must be
t the awns
T.ITEKATUKE til til
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257-260. 1953.
(g)
a nd Willard, C. J. Factors affecting the pre-emergence use of
2,4-D in corn. Weeds 1: 338-345. 1952.
(S) Alexander, M. and Aleem, M.I.H. Effect of chemical structure on microbial decomposition of aromatic herbicides. (Manuscript submitted to
Agric. and Food Chem,) 1960.
(//) Anderson, W. P., Linder, P. J., and Mitchell, J. W. Evaporation of some
plant growth regulators and its possible effect on their activity. Science
116: 502-503.1952.
�64
65
per presented
(38) Hill, G. D., Soil factors as related to herbicides actio
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before The Weed Society of America. New York. 1956.
of EPTC to oats injury in various soil types. Weeds 7: 88-90.
Bingeman,
(S3) .- , McGahen, J. W., Baker, H. M,, Finnerty, D.
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Agron. Journ. 47 : 93-103. 1955.
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(S)
23-31. 1953.
(0) Barrens, K. 0., Lynn, G.'' E., and Eastman, J. D. Experiments on the
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dichloropropionic acid in several Iowa soils. Weeds 4: 205-217. 1956.
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Bleidner, W. 0., Baker, H. M., Levitsky, M., and Lowen, W. K. Detersoil bacteria. Nature 170 : 39-40. 1952.
mination of 3-(ip-chlorophenyl)-l,l-dimethylurea in soils and plant
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tissue. Agric. and Food Chem. 2 : 476-479. 1954.
herbicide adsorption. Weeds 8: 48-54. 1960.
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Weeds 7 : 157-161. 1959.
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trib. Boyce Thomp. Inst. 16 : 409-417. 1952.
(!>(>) McKenzie, R. E. Activation of mylone, a temporary soil sterilant. Res.
Coggins, 0. W. Department of Horticulture, University of California,
Prog. Rpt., WWCO, p. 105. 1957.
Riverside, California, personal communication. Nov. 0, 1957.
(1,1) Minarik, C. E. Pre-emergence herbicides and their behavior. Proc.
(15) - and Crafts, A. S. Substituted urea herbicides : Their electroNEWCC (sup.) 5: 29-39. 1951.'
phoretic behavior and the influence of clay colloid in nutrient solution
(42) Newman, A. S., and Thomas. R. J. Decomposition of 2,4-dichlorophenoxyon their phytotoxicity, Weeds 7 : 349-358. 1959.
acetic acid in soil and liquid media. Soil Sci. Soc. Am. Proc. 14: 160(1G) Crafts, A. S. Toxicity of 2,4-D in California soils. Hilgardia 19: 141-158.
164. 1949.
1049.
(43) Norman, A. G., and Newman, A. S. The persistence of herbicides in soils.
(-17) Danielson, L. L. Mode and rate of release of isopropyl N-(S-chlorophenyl)Proc. NEWCC 4: 7-12. 1950.
carbaumte from several granular carriers. Weeds 7 : 418-420. 1959.
(18) -, Genrner, W. A., and Jansen, L. L, Besearch in progress, Agri- (44) Ogle, R. E., and Warren, G. F. Fate and activity of herbicides in soils.
Weeds 3: 257-273. 3954. •
cultural Research Service, U. S. Department of Agriculture, Beltsville,
(45) Rahn, E. M., and Baynard, R. E., Jr. Persistence and penetration of
Maryland. I960.
monuron in asparagus soils. Weeds 6: 432-440. 1958.
(19) Davis, D. JO. Some factors that, affect the phytotoxicity of water soluble
(46) Ready, D., and Grant, V. Q. A rapid sensitive method for determination
DNBP. Weeds 4 : 227-234. 1956.
of low concentrations of 2,4-dichlorophenoxyacetic acid in aqueous solu(20) Davis, F, TJ. and Selman, F.' L. Effects of water upon the movement of
tions. Bot. Gaz. 109: 39-44. 1947.
dinitro wood killers in soils. Weeds 3 : 11-20. 1954.
Sheets, T. J. The comparative toxicities of four phenylurea herbicides in
DeRose, H. H. Persistence of some plant growth regulatoi-s when applied
several soil types. Weeds 6: 413-424. 1958.
to the soil in herbicidal treatments. Bot. Gaz. 107: 583-589. 1946.
•. The uptake, distribution, and phytotoxicity of 2-chloro-4,6-bis (ethyl(48)
-. Crabgrass inhibition with 0-isopropyl N-(3-chloropheny])-carbaamino)-s-triazine. Doctoral thesis, Univ. of Calif., Davis, California.
mate. Agron. Journ. 43: 139-143. 1951.
1959.
,
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and Newman, A. S. The comparison of the persistence of certain
The toxiclty of simazane to seedling oat plants (Manuscript sub(40)
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mitted to Weeds). 1960.
12:222-220.1948.
and Crafts, A. S. The phytotoxicity of four phenylurea herbicides
(50)
(24) Dowler, 0., Baughman, N. M., and Veatch, C. The effect of soil type,
in soil. Weeds 5 : 93-101. 1957.
soil pH, and simulated rainfall on the distribution of DNBP in the
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dimethylurea as a function of soil constituents. Agric. and Food Chem.
(25) Ennis, W. B., Jr. Some soil and weather factors influencing usage of pre2: 937-939. 1954.
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(2C>)
. Weed control in principal crops of the southern United States.
3(p-chlorophenyl)-l,l-dimethylurea in a leaching study. Weeds 4: 50-54.
Adv. in Agron. 7: 251-297. 1955.
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(27) Evans, W. C., and Smith, B. S. W. The photochemical inactivation and
'(53) Smith, R. J., Jr., and Ennis, W. B., Jr. Studies on the downward movemicrobial metabolism of the chlorophenoxyacetic acid herbicides. Bioment of 2,4-D and 3-chloro-IPC in soils. Proc. SWG 6: 63-71. 1953.
cliem. Jour. Vol. 57. 1954.
(54) Sund, K. A. Residual activity of 3-amino-l,2,4-triazole in soils. Agric. and
(£8) Feldman, A. W., Technical summary on Falone. Naugatuck Chemical
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Undated.
N-(3-chloropheuyl) carbamate and an alkanolamine salt on germinating
(29) Freed, V. H., Montgomery, M., and Traegde, S. C. Physical properties of
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S-ethyl-di-»-propylthiolcarbamate. Res. Prog. Rpt., WWCC, pp. 89-90.
(56) Upchurch, R. P. The influence of soil factors on the phytotoxicity and
1058.
plant selectivity of dim-on. Weeds 6: 161-171. 1958.
(30) Gnrd, L. N., and Rudd, N. G. Herbicides determination: isopropyl Nand Pierce, W. C. The leaching of monuron from Lakeland sand
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(3-chlorophenyl) carbamate (CIPC) in soil and crops. Agric. and Food
soil. Part I. The effect of amount, intensity, and frequency of simulated
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rainfall. Weeds 5: 321-330. 1957.
(3t) Hannah, L. H., Field studies with two new classes of herbicidaL'chemi(58) —'— and Pierce, W. C. The leaching of monuron from Lakeland sand
cals; Proc. SWC 8: 316-321. 1955.
soil. Part II. The effect of soil temperature, organic matter, soil moisture, and amount of herbicide. Weeds 6: 24-33. 1958.
�67
Weaver, E. J. Reaction of certain plant growth regulators with iol^Kchangers. Bot, Gaz. 109 : 72-84. 1047.
(60) - . Some uses of activated carbon in contratoxiflcation of plant
growth regulators. Bot. Gaz. 110 : 300-312. 1948.
(61) Whiteside, Jean S., and Alexander, M. Measurement of microbiological
effects of herbicides. Weeds 8: 204-213. 3960.
(62) Young, H. 0., and Carroll, J. 0. The decomposition of pentachlorophenol
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604-507i 1951.
Senator HART. Do you have any comparable information about
dioxin ?
Dr. BAYLBY. The answer at this point is no.
Senator HART. Are any tests being run?
Dr. BAYLBY. Not yet. One of the problems we had here is developing the methodology for testing these in products. As soon as this is
settled, we will be able to expand our efforts and find this information.
Senator HART. And you hope you will find that it has not been
building up in our bodies every day ?
Dr. BAYLEY. We sincerely do yes, sir. But that will not bias our
results, I assure you.
Senator HART. Perhaps Dr. Byerly might help us with this. I am
told by my notes here that dioxins are chlorinated hydrocarbons and
that these tend to be stable, and more significantly, in view of the
fact that other dioxins are known to be absorbed and retained in the
tissue of animals, isn't it likely that there is a build-up in the human
of the dioxins found in 2,4,5 ?
Dr. BYERLY. I will give you an opinion first. You stated since they
are chlorinated hydrocarbons, they therefore would be persistent.
Tli is does not necessarily follow. There is a very wide range from
almost complete persistence, if you like, long-time persistence, to a
very short-time persistence on the part of some other chlorinated
hydrocarbons.
With respect to these which are in the family of chlorinated
hydrocarbons, of which 2,4,5-T is a member, the time of disappearance substantiated by empirical evidence, it is a matter of a few
months; With respect to the dioxins themselves, Dr. Bayley
answered you quite frankly, we do not have the information. We
will seek it when the methods are complete. '
Senator HART. In your report you state that you agree that more
rigorous tests on teratogenicity should be imposed before registration. If you favor such test as a requirement for registration, isn't
there an inconsistency in allowing 2,4,5-T, which is a pesticide with
suspected teratogenicity to be allowed to continue in a registered
status pending the outcome of the tests? In other words, should you
not deregister it now and then if the tests prove negative, register
it?
Dr. BAYLBY. The basis on which we would take such action would
have to be on the consideration that we believe a hazard now exists.
Based on the information which has been provided to us from the
Department of Health, Education, and Welfare, and on our own
analyses of the levels of the containment we do not believe such a
hazard exists at this time.
in terms
Senator HART. You cite the content of four lots of
producers
of the content of, as you put it, TCDD. How many •
of that product are there ?
Dr. BYERLY. There are only three primary producers, sir. There
are many f ormulators.
Senator HART. Is a formulator engaged in a process which
changes the generic business ?
Dr. BYERLY. No.
Senator HART. Almost the total of production is from three
sources, right?
Dr. BYERLY. Primary production. I understand the check has been
made and the importation is very small.
Senator HART. After you have registered the product, how frequently do you check up on the amounts of dioxins in the products
that are being produced ?
Dr. BAYLEY. Mr. Chairman, one of the requests which we are
making of the Congress this year is to strengthen our law so that we
can have plant inspection and insist on quality control within these
particular plants. We have asked the Congress to help strengthen
our activity in this area. It is not adequate, at the present time. We
very definitely need legislation to improve it.
Senator HART. Is that sort of a way to say that you do not check
the dioxins ?
Dr. BAYLEY. This is the first time. One of the reasons, of course, is
that the methodology has only been newly developed. The results
presented here were developed through check procedures with our
laboratories, the Food and Drug Administration, and the industry
group to see that our methodology was technically correct. So we are
just getting started.
Senator HART. You will include in your budget request moneys to
provide what ?
Dr. BAYLEY. The President's budget includes an increase of
approximately $2.4 million for the pesticide regulatory division.
This is between 50-and 100-percent increase in the funding for that
organization.
Senator HART. Do you believe that with that sum you would be in
a position to have plant inspections on a regular basis ? Would you be
able to have an enforcement staff which would be able to move in
the event a violation was discovered? Is this the sort of thing that
you visualize ?
Dr. BAYLEY. Yes. We based that estimate in our budget request OB
what we currently thought was necessary to do this.
Now, we all recognize that the problems of concern about chemicals are expanding, and I would not want to suggest that is the final
request that we would make in order to improve our operations. We
put those in believing they currently were adequate from the standpoint of the enforcement and registration procedures.
Senator HART. Is the table which shows the amount of dioxin of
this particular type—is there a test and do you have, facility and
personnel to attempt to identify the existence of any of the other
seven possible dioxins ? ,
Dr. BYERLY. This, sir, is in the process of development in coopera-.
tion with the Department of Health, Education, and Welfare and
�69
dustry. These methods are in the process of development. Tor
some of them the methods are quite adequate; for others, the methods have to be developed.
Senator HART. For some you feel you can ?
Dr. UTTERLY. Yes, sir.
;
Senator HART. In that case you do.
Dr. BYBRLY. We will.
Senator HART. Hinged on the money problem ?
Dr. BYERLY. No, it depends primarily on the development of the
competence of personnel. This is the thing that has to grow. We
have to have a cadre of people who are highly competent. These are
most sensitive methods and we cannot just create people who can
handle them overnight.
Senator HART. In the testimony of the first two witnesses, and I
do not recall whether it was that of Mr. Wellford or Mr. Turner, it
is my impression there was a reference to a chick embryo study. Do •
you recall the comment they made?
Dr. UYERLY. Mr. Chairman, I do not recall the specific comment.
There is a chicle embryo test. It is highly sensitive. I would point
out that it lias, as a screening test, possibilities. Again, its sensitivity '
would make me want to suggest that we be very careful with respect
into whose hands the tests were put.
With respect to the teratogenicity, with respect to the chick
embryo, the application in this test' is hardly like the applicationj
either to the skin or in the food of a mammal. So a direct compari
son of the effects in the chick embryo with rats and mice, the tradi-r
tional ones, would require a great deal of review.
Dr. BAYLEY. It would really require a correlated study to ascertain the relationship.
Dr. BYERLY. It would indeed.
Senator HART. I think just from what we have heard this
morning, everyone on this subcommittee will be'eager to assist you '
in obtaining the additional moneys, whether it is 2.4 million or more
to insure the development just as rapidly as possible of the technical
data on which to base tests and the human hands to administer
them.
Let me get into this burden of proof again—we sort of dismissed
it—very briefly.
The basic conclusion of your testimony is you have not found that
registered uses of 2,4,5-T without a finite tolerance on food crops
constitutes a hazard requiring cancellation or suspension of such
registered use.
Dr. BAYIYEY. That is correct.
Senator HART. And yet this morning we have heard testimony
that preliminary tests suggest that 2,4,5-T when contaminated by
dioxin comparable to that found in currently produced 2,4,5-T is
tcratogenic in three species; that the Mrak Commission or a panel
advisory to it said that the teratogenic effects in one or more such
species should be grounds for immediate restriction of pesticide use;
that residues of 2,4,5-T arc now found on approximately one out of
every 200 food samples analyzed by FDA; that we can't be sure of
(lie amounts of totradioxin 'in 2,4^5-T now being sold, nor do we
have as yet clear ideas on the amount of other dioxiraWn the pesticide, some of which may be more potent than tetra; that no evidence
suggests that these dioxins are not persistent or cumulative in
human tissue, and that some evidence which would indicate perhaps
they are.
If you accept that as a premise, in view of all of this, would you
say that you are sure that registration of 2,4,5-T for use directly on
food crops does not constitute a hazard to man ?
Dr. BAYLEY. I would say that the information we have does not
give us indication that it is a hazard to man in accordance with the
registered uses.
I think we have to recognize that—and I am sure the committee
and we are in agreement—these are all economic poisons, and the
purpose of registration is to provide for their use in such a way that
they are not a hazard. That is the basis on which we make our judgment.
Senator HART. Your position is that they do not constitute a
hazard ?
Dr. BAYLEY. Yes, sir. And our position is based not only on our
own data but, that provided to us from the medical authorities of the
Department of Health, Education, and Welfare, and to add, 20
years of safe use.
Senator HART. The first two witnesses described the difficulty of
finding the brand name on a deformed infant.
There are lots of birth defects. How can anyone say over 20 years
that this has not been a factor in some of these private tragedies.
Dr. BAYLEY. I do not in any way want to be facetious, but I think
we have to recognize that one of the compounds closely related to
this contaminant is lysol, a rather common household disinfectant,
and I hear no suggestion that we take this off the market. We in the
Department of Agriculture recognize that there is a large group of
chloropheuols that we are going to have to examine to find out
whether there is a real hazard or not. I am not here to raise a scare,
but I think we recognize that in. dealing with these compounds we
must have evidence that they are a hazard or .we will bo dealing :
with emotional conjecture based on inference from various scattered
data.
Senator HART. To make clear what is meant, what is your position
with respect to lysol ?
Dr. BAYLEY. Wo have no reason to take action at this time.
Senator HART. Do you have in process studies or evaluations to see
whether you modify that ?
Dr. BAYLEY. Within our capability, for example, we have already
moved out to ascertain the dioxin content of 17 other pesticides in
this area, and we recognize that this is a field in which we want to
make intensive study.
I do not single out this particular product as one which I would
consider as more hazardous than any of the rest, but merely as an
example of the total problem that we have in meeting these pesticide
issues.
It seems to me that from the standpoint of protecting the public
health, the important thing for us to do is to take those which, based
�70
^Bn scientific data appear to have the greatest potential hazaro^md
put our resources on evaluating these as we go ahead.
This is true of the mercurials. We are looking very closely at all
•of these compounds. I think we must recognize 5iat we are going to
have to do this on a priority basis as we go. We are giving attention
to 2,4,5-T, but wo need to have the appropriate facts as we proceed.
Mr. BIOKWI.T. I think what has come out here is that really it all
boils down to a question of burden of proof.
While you say there is no evidence that 2,4,5-T is hazardous, I would
have to dispute that. Assuming that you are right, that there is no
evidence that it is hazardous, and yet it cannot be shown that it is not
hazardous; on what do you base your inaction ?
Dr. BAYLEY. Let's recognize first of all that these are economic
poisons. We should all agree to that to start with. And when I USB
the word "hazardous" I use it in terms of sufficiently hazardous to
take action. This is bound to be a judgment based on scientific—
including medical scientific data. If there is a disagreement between
us, then it is in this judgment, not in anything else.
Mr. BICKWIT. I recognize my inability to make adequate scientific
judgments and, as a result, defer to those who I regard are .capable
of making such judgments.
The panel which reported to the Mrak Commission has recommended whenever teratogenic effects of a given pesticide are shown
in one or more mammalian species, that immediate steps should be
taken to restrict the use of that pesticide.
Are you rejecting that advice ?
Dr. BAYLEY. No. The use of this pesticide is already restricted
because of the registered uses.
Mr. BTCKWTT. I suspect that the thrust of their statement would
require that it be further restricted but perhaps we would have somo
difficulty pursuing what the exact intention of their statement was.
You say that you must believe that a hazard exists before you can
take a pesticide off the market and that in the case of 2,4,5-T you do
not believe that a hazard exists.
Dr. BAYLEY. We do not believe that a hazard exists which would
authorize us to take it off the market, yes.
Mr. BICKWIT. Are you sure that a hazard does not exist?
Dr. BAYLEY. One can never be absolutely sure that a hazard does
not exist, even if we are talking about table salt. ,
In fact, we know that table salt is hazardous if taken improperly,
and we don't even register it.
Mr. BICKWIT. There is a distinction from table salt in this case,
and that is that there has been evidence that suggests, and to my
mind rather strongly suggests, that there is a hazard here.
Dr. BAYLEY. I do not see the difference that you are trying to
point out.
Mr.- BICKWIT. You do not think that the studies that have been
done by FDA, by NIEHS, although preliminary, establish that
there is any greater hazard than the hazard of table salt?
Dr. BAYLEY. No; I did not say that.
Mr. BICK.WIT. I misunderstood you.
Dr. BAYLEY. I did not say that. But again I come back to the
point that we are dealing with economic poisons. There are hazards
•
"
A
use of all of them. The decision that has to be WGe is are the
^.ww^ls sufficiently great to take action at this particular time. That
pii!$a, * hn di (Terence.
F&-*w\t4t ]jiOKwm you do say issues involving human health should
priority over all other issues.
.*•!* 1 » -A,FfV A. l l l J It
J. UO* OJ.J. •
?«.; Mr. BICKWIT. What I am not clear on is whether you have to
||t*«tively believe there is a hazard before you take a pesticide off the
Jjfgiwarket,
J,: ;1» Uiat a legal requirement?
;* Dr. BAYLEY. I don't know what you mean by actively believe.
£::.Mr. BiCKwtT. I conceive of relative states of mind as being belief,
«t*loof suspension, and state of disbelief.
ij1". Do you think you have to be on the belief side of state of suspenjiion in* order to take a pesticide off the market?
•;''•,3f you are in a state of suspension, would that authorize you
J|epilly to take it of! the market?
|r Dr. BAYI,KY. The information provided to us has not shown that
ilhere "m sufficient hazard for us to take action, and the information
,,-!f»!widcd to us from the Department of Health, Education, and
||;W«lfare is the information primarily that we have used.
fel'Mv. BICKWIT. And you do not feel that if you are not sure one
„,-.-,- or the other that that would authorize you to take it off the
*;Wrket?
,*iy, Dr. BAYLEY. The data that we have at this time are not adequate
P$tO show us that there is a hazard, and the data to the contrary are
p|!»wflki<mtlv adequate to suggest that there is no hazard as 2,4,5-T
^li presently registered.
!' Mr. BICKWIT. I guess what it all does boil down to are two differ•ttees between us: one, in evaluation of the evidence
th Dr. BAYLKY. Yes, sir; and these types of judgments are inherent
to tho decisions being made.
f^Mr. BICKWIT. (continuing). And, two, differences in feelings about
itwrtlcn of proof?
ISf'''Dr. BAYLEY. And we believe that the relationships between the
H|td*jMirtinents have been fully utilized in working out. this type of a
^p^twuiis of position.
E$t":':'Mr. BICKWIT. At least we have emphasized what the differences
3S a
'-' -ro. '
Senator Baker earlier asked whether any studies have been run to
Utgh the benefits of poisons as against the detriments.
feVIfavo any actually been run?
'•?' Dr. BAYLEY. Are you talking about specific compounds or about all
^of them?
;<; ;:Mr. BICKWIT. All of them.
g•;. Dr. BAYLEY. Are there any data on it, Dr. Byerly ?
si,-';1";' Dr. BYEULY. There is one'study done by Velmar Davis.
#!*'! Whether it would be suspended without the suspension of other
K : I»hcnoxy herbicides or whether only it would be suspended makes a
|:sw»lwtftiitial difference.
•vV If nil of them were suspended, it might amount to more than^100
ftfmillion of added cost. If other phenoxys remained available to us, it
£.'.nould only be a fraction of that amount.
�72
73
Lcfc me emphasize that this kind of study is very treachero^prn
•awing any conclusion at all, because if you make a substantial difference in the amount produced or the quality produced, that which
remains may sell at a higher price, and this again brings in a matter
of value judgments.
What wo can say, I think, with respect to all herbicides and all
pesticides in general, is that if we had to do without them and had
available the hoes and, the people to do the hoeing and the other
things to produce our same crop, you would add a cost of production of more than $2 billion a year.
Let me emphasize, however, that our primary concern is not the
economic cost in the aggregate.
'
"
' '
Important as this may be, our primary concern, as Mr. Bayley has
said, is that'we shall control pests and we shall do it safely and
without hazard to human health or the public welfare.
Mr. BICKWIT. We have emphasized that we have some differences
in evaluation of the evidence. I am trying to discern what evidence
would convince you that this pesticide Avas in fact hazardous.
The one thing that you have said about what would convince you
is that you specify that should the teratogenic nature of 2,4,5-T. be
confirmed, registration for use on food crops will be canceled.
My understanding, correct me if I am wrong, is that the four
studies cited earlier; although all preliminary, demonstrate that
2,4,5-T is teratogenic.
Dr. BYERLY. We do not accept that statement.
Mr. BICKWIT. In what way do you not accept it ?
Dr. BYERLY. I think the statement that Dr. Bayley read is correct,
that all of the evidence known to me is compatible with the hypothesis that these results were due to contaminant dioxin or the
interaction of that dioxin and 2,4,5-T.
Mr. BICKWTT. The evidence which I have read shows that 2,4,5-T
when contaminated with dioxin in amounts similar to or less than
those in currently produced 2,4,5-T does produce teratogenic effects.
Now, I am on the basis of that evidence, unwilling to say it is
.because of the dioxin or the 2,4,5-T or the relationship between the
two. Are you ?
Dr. BYERLY. I believe that our previous dialogue indicated that
these are preliminary results, so preliminary, sir, that I have not
seen the published figures nor have I seen all of the figures to which
you allude in the record.
Mr. BICKWIT. I agree they are preliminary. What I am asking
you is if they are confirmed, will you deregister 2,4,5-T for use on
food products ?
Dr. BYERLY. This depends upon the dosage at which they are
effective.
Mr. BICKWIT. You will have to modify your statement then.
Dr. BYERLY. In what way ?
Mr, BICKWIT. Well, you have said that should the teratogenic
mature of 2,4,5-T be confirmed, you would deregister the pesticide.
Dr. BYERLY. I do not modify the statement, I said if 2,4,5-T.
Mr. BICKWIT. What does it matter whether pure 2,4,5-T is teratogenic if there is no such product as pure 2,4,5-T on the market? I
assumed that by your reference to "2,4,5-T," you mea^Ppure 2,4,5-T
as currently produced on the market.
Dr. BAYLEY. I think you ought to recognize that he was answering
your question precisely.
Dr. BYERLY. I believe good manufacturing practice can restrict
the amount of contaminant dioxin in the product. I believe it should
be done; I believe it is now being done and that it, will be done.
Mr. BICKWIT. Then, I take it that your statement 'was not in reference to the teratogenic nature of 2,4,5-T when contaminated with
any dioxin whatsoever ?
Dr. BAYLEY. No.
Mr. BICKWIT. Have we ever produced 2,4,5-T without any dioxin
whatsoever ?
Dr. BYERLY. This is not a statement that can be answered absolutely, but it can be answered within the limits of the method in Dr.
Bayleys statement. It indicates there was one in which there was no
detectable amount of dioxin.
Mr. BICKWIT. Would you be willing to say that if the teratogenic
nature of 2,4,5-T with the amount of dioxin that is contained in currently produced 2,4,5-T is found to be teratogenic that you would
deregister it for food use ?
Dr. BYERLY. I would be willing to say, sir, if the 2,4,5-T with no
detectable amount of dioxin, of tetrachlorodibenzo paradioxin,
would prove to be teratogenic, I would recommend to the Department that actions to cancel uses on food crops be taken.
Mr. BICKWIT. Yet, what is really relevant here is the effect of currently produced 2,4,5-T. Why then are you basing your decision on
the effects of 2,4,5-T in a form that we do not know it commercially?
Dr. BYERLY. I think you are misconstruing my reply. Again,
pending the fact that neither you nor I have before us published
figures which would sustain your statement that all four of these
things do in fact show teratogenic effects, if we accept what you say
may be true, but it has not been published nor publicly disclosed,
then let me say further that so far as I know, the dosage at the current level of 150 milligrams per kilo is equivalent to the amount of
the dioxin therein contained at one part per million which would be
expected to give a teratogenic effect if there were no 2,4,5-T present,
and 150 milligrams per kilo is astronomically higher than any
amount to which any person would normally be exposed in the
normal course of usage.
Mr. BICKWIT. How long is the usage to which you refer ?
Dr. BYERLY. I did not make a limit.
Mr. BICKWIT. In one's lifetime?
Dr. BYERLY. In a lifetime.
Mr. BICKWIT. You are willing to say this is more dioxin than one
is likely to be exposed to in his entire lifetime?
Dr. I3YERLY. That is my opinion.
Mr. BICKWIT. You will have to admit it is not based on much.
Dr. BYKHLY. You are forcing me into the realm of conjecture and
I do not choose to go into that very far. We do not have the empirical evidence on which to state whether or not it is degradable. We
have no evidence, sir, that it is not degradable.
t
�74
Mr. BICKWIT. Here we are again; we do not know whether it is or
is not.
Dr. BTERLY. We intend tofindout.
Dr. BAYLEY. Mr. Chairman, this dialogue is the same type of dialogue that we have with industry people who come in and want to
know exactly what evidence we have to have in order to assure them
that they have provided us with sufficient proof. These judgments
are not so simple that you can conjecture ahead of time of seeing the
data exactly what position you are going to take. It is characteristic
of them; it is inherent to them. And I suggest this is characteristic
not only when we are dealing with industry but when we are also
concerned about the public health.
Senator HART. Gentlemen, did you have anything you would like
to add?
Dr. BAYLEY. We do not, sir. We would be glad to enclose the additional statements for the record that we have discussed.
Senator HART. Thank you very much.
I had hoped we could continue through the lunch hour, but I am
stuck with a Policy Committee lunch.
(The statement follows:)
STATEMENT BY NED D. BAYLEY, DIBECTOB OF SCIENCE AND EDUCATION,
U.S. DEPARTMENT OF AGRICULTTJBE
MB. CHAIRMAN: I am Ned Bayley, Director of Science and Education, Office
.of the Secretary, USDA. I have with me T, C. Byerly, Assistant Director of
Science and Education.
We are pleased to be here to comment on the current state of knowledge
with respect to the herticide 2,4,5-T. We will be glad to respond to questions
relevant to its usage as fully as information available to us enables us to do
so.
The herbicide 2,4,5-T has been recognized a the most effective herbicide registered for use for control of certain weeds and brush species for more than 20
years. About four-fifths of the domestic use of 2,4,5-T is for nonfarm use, the
largest such use being for control of brush on rights-of-way. It is also used
extensively to control brush on forest lands and certain weeds in turf. 2,4,5-T
lias been used in the production of fruit crops, cereal grains, and sugarcane. It
is the most effective herbicide for control of brush on several million acres of
rangeland in the Southwestern United States.
2,4,5-T is degraded in the environment within a few .months after, application
so that residues do not persist from one season to, the next. Residues on foods
are unusual. Among 5800 food samples analyzed by FDA for 2,4,6-T during the
past four years, 25 were reported to contain trace amounts; i.e., amounts less
than the 0.1 pprn limit of accuracy of present analytical procedures for foods.
Two samples showed residues of 0.19 ppm and 0.29 ppm, respectively.
No finite tolerance has been established for 2,4,5-T in food. In the absence of
such tolerances, any detectable amount of 2,4,5-T in food would make sucl)
food subject to seizure if found in the channels of interstate commerce. From
the data cited above, it is apparent that contamination of food with 2,4,5-T is
very infrequent and then only at very low levels.
There is current concern over the continued use of 2,4,5-T arising from tlic
report of a research study completed under contract by the National Cancer
Institute by Bionetics Inc. Tliis study was based on a commercial lot of 2,4,5-T
acquired for the study in 1905. It was fed to pregnant mice and rats'. Many of
their developing young had birth defects.
After review of this information and after consultation with Federal agencies concerned, Dr. Lee A. DuBridge, the President's Science Advisor,
announced on October 29, 1969, a coordinated series of actions being taken by
those agencies with respect to the use of 2,4,5-T.
Among them was the announcement that: "The Department of Agriculture
will cancel registrations of 2,4,5-T for use on food crops effective January \,
j'WO. tinlriw by that time the Food and Drug Administration has found a basis
! far **tabllHhing a safe legal tolerance in and on foods."
was Informed in January that the lot of 2,4,5-T used in the Bionetics
contained significant amounts of a highly toxic contaminant, tetrachloroiwriuHoxin, The Department was further informed that lots of 2,4,5-T
;|«if ettrrvnt and recent manufacture were reported to contain less than 1 ppm
f;|*f Hil» contaminant in contrast to the 27 ppm reported for the lot used in the
tltenHJwi study.
?Y Sltteiwlvp studies are under way to determine whether 2,4,5-T is itself teratoti»»h». Preliminary reports are consistent with the hypothesis that the terato.fl»«k» rvHUltH reported in the Bionetics study were due to the contaminant
;E <t|rMtti» or to Interactions of such contaminants with the 2,4,5-T rather than to
':'3y.&-T JUT "e.
>' The lh»imrtmont announced on February 6 that it would undertake examina'**•*• Of 2,4,5-T nnd 17 related compounds registered for pesticidal use to deter,; M!IN> tthi'lhei- or not they are contaminated with dioxins. Preliminary results
. «•* 3M.&-T dhow that those lots examined of current manufacture and those
; V&* In clmnnelB of trade gave the following results:
i
TABLE 1.—AMOUNTS OF TODD FOUND IN COMMERCIAL 2,4,5-T BY TWO METHODS
TCDD Content p.p.m.'
Lot
Grade 1
Collected
USDA
120110
07-020
X-17394 21-5
MM-120449
Manufacturer
TG
TG
TG
TG
2/70
2/70
2/70
2/70
trace
1.1
W D» .
48
?•;
SKJIUM . . .
...
l**~— - . .
Dow
FDA
0.07
2.9
N.D.
. 47- 52
1S"*t«hnlt«l grade.
ttaOlMm lo the 2,3,7,8-tetraohloro-dibenzo-p-dioxin (TCDD).
"""• •- - 1 - I TCDO are below the limits of detection or below 0.05 p.p.m.
I by Dow as a reference check and reported to contain about 0.5 p.p.m. TCDD.
data are preliminary and are obtained from first drafts of methods
<l by chemists in the Crops Kesearch Division (TJSDA) and in the Pes' CtiowlHtry and Toxicology Division (FDA). The dioxin values refer only
"to* 2^l,7,a-tetrachloro-dibenzo-p-dioxins (TCDD) and do not indicate levels
! «*b*r Jinlogennted dioxins (containing 5, 6, 7, or 8 chlorines) in the 2,4,5-T
kteo.
tlew of all the information now available, we have not fonnd that regis1 n*e of 2,4,{i-T without a finite tolerance on food crops constitutes a
yMtSiknl requiring cancellation or suspension of such registered uses.
flfWTVre turn been and is concern over the ecological effects of 2,4,5-T used as a
glMbttant In Viet Nam. Dr. Fred Tschirley, Assistant Chief of our Crops ProftWrtiWi Ilfwnrch Branch, has reported the results of his examination of areas
lu Viet Nam. He has reported no evidence of irreversible ecological
jpMWKxi. Allegations that defoliation will lead to extensive laterization of Vietsolls, that Mangrove areas will not recover, that flsh production In
...
I nrons will be reduced were not verified.
|j|•'< J>r, Ttachlrley also headed a team of scientists who investigated allegations'
|tf'toinry to humans and animals due to herbicide treatment for control of
by the Forest Service on the Tonto National Forest near Globe, ArlTlioy found that apparent damage consisted of damage to susceptible
W nour the treated area from drift of the herbicides used. The alleged
rlwi lo a cluck and a goat were found to be groundless. Human illnesses
»thone expected in a normal population with the possible exception of one
:wlth skin irritation on his eyelids. Clinical chemistry on specimens
to«l during the investigation is in process,
HART. I think in fairness to all we should recess to resume
|ff'^(Whereupon, at 12:45 p.m., the subcommittee was recessed, to. rei nt 2:15 p.m., this same day.)
4ft-362—70
0
�76
77
AFTERNOON SESSION
lar scrutiny because of their potential ability to dSBlate certain
classes- of living organisms, and thereby to upset the balance of
nature—to disrupt what the ecologist refers to as the "ecosystem."
I shall here limit my remarks to the potential dangers to the environment that might be expected from the excessive or otherwise
incorrect use of one such substance: 2,4,5-T.
As we have hoard this' morning, this compound has recently
gained a degree of notoriety owing to its massive military use in
Vietnam despite the suspected ability of it, or an associated impurity, to cause birth defects.
I need not refer you to Thomas Whiteside's article- on this :subject
•- •>.•>
, 1
1 _ ji
_:_*
Senator HART. The committee will be in order.
Resuming this afternoon, our first witness is Dr. Arthur H.
Westing. Dr. Westing is chairman of the Biology Department of
Windham College in I3utney, Vt.
STATEMENT OF DR. ARTHUR H. WESTING, CHAIRMAN, BIOLOGY
DEPARTMENT, WINDHAM COLLEGE, PUTNEY, VT.
Dr. WESTING. Senator Hart, I consider it a privilege to be able to
testify before your committee. Actually, I am very pleasantly surprised that you and your staff show such tolerance toward me
despite a fairly questionable record with respect to your state,
First of all, most of the year I lived in Michigan, I devoted to
spraying your forests with 2,4,5-T, and perhaps even worse, when I
left Michigan I took with me one of your most desirable natives as
my wife.
Senator HART. I don't know whether that makes an even trade or
not.
Dr. WESTINO. AlLthe time I was listening to this morning's testimony and realizing*^how muddled the situation was with respect to
the medical and public health aspects and the legal and administrative aspects, I kept thinking that those aspects were crystal-clear in
relation to the aspects that I am going to try to talk about, and that
is the impact of 2,4,5-T on the environment.
It is such a difficult field to cope with because ecology is still
really in its infancy, particularly when it comes to the study of the
full impact of a toxic introduction to the environment.
To judge from the popular press, our nation is on the brink of
environmental disaster. Ecology has achieved some sort of a mystical
significance to many people, and a whole new vocabulary has
emerged overnight utilizing that wonderful avant-garde prefix
"eco."
Over and over again we are being reminded of our collision
course with "ecocatastrophe" leading to "ecocleath."'Wc are told that
we have to really use drastic "ecotactics;" a Senator like you should
be using aggressive "ecopolitics." The whole world is being challenged to accept a protocol on "ecocide." And I suspect that psychiatrists are very soon going to be diagnosing "eeoneuroses."
Actually, the unhappy truth of the matter is that there may well
be good cause for'Concern over the future of man's environment. It
is being assaulted from all quarters with a gusto that is hard to
grasp. Man has habitually ignored the impact that lie has had on
the environment, the environment that all of us depend upon for our
well-being and survival. Western man has always considered himself
master of his surroundings. Until the present, with far smaller numbers and very ineffectual technology, this self-delusion made very
little difference.
P>iit today we are introducing a great diversity of chemicals into
our environment in vast, continuing, and exponentially increasing
quantities. Among these chemicals, pesticides are worthy of particu-
it is one of a class of potent herbicides or plant killers, the one
preferred by utility employees, foresters, range managers, and farmers, and by our armed forces in Vietnam for the destruction of
unwanted woody vegetation. It is one of a class of growth-hormonemimicking herbicides whose close chemical relatives include 2,4-D,
MCPA, and Silvex. 2,4,5-T was developed during the early 1940's
(as a possible chemical warfare agent) and came into widespread
domestic use during the mld-1950's. In 1964, some 13 million pounds
of 2,4,5-T were manufactured in the United States. About a million
of these pounds were applied to about 3 million acres of U.S. croplands, another million pounds were sprayed on perhaps 80,000 acres
of forest lands in Vietnam, and most of the remaining 11 million
pounds wore presumably used domestically on an undetermined
number of acres of noncroplands. This morning we were vividly
reminded that a small portion of this is also used by home gardeners.
».
Now, these are the 1964 figures. Although I am not sure of the
current ones, I understand that the domestic use of herbicides in
general has been increasing at a compounded growth rate of 10 percent per annum.
2,4,5-T is commercially available in a number-of formulations of
which the most important are the oil-soluble esters and the slightly
less effective water-soluble amines. Whereas the amine formulations
are very low in volatility, some of the ester formulations are relatively high and others are relatively low. The low volatility esters
are actually somewhat more effective than the high volatility ones,
but they arc also slightly more expensive. 2,4,5-T is also available in
combination with 2,4-D, a mixture which is known domestically as
"brush killer" and by the military as "agent orange."
The 2,4,5-T is effectively applied either to the foliage of unwanted
woody vegetation from ground- or aircraft-mounted spray rigs, or to
their stems by a variety of techniques.
Domestically, it is very often applied highly diluted by oil or
water, although some domestic teclmiques of individual application
call for strong concentrations.
In Vietnam, it is aerially applied in totaly undiluted form.
Recommended broadcast dosages—these are domestic recommenclations-^-rango* from one-half to three pounds of active ingredient per
�78
re. At these levels of one-half to three pounds per acre, the
quite 'selective, killing many species of broad-leafed woody plants
•and sparing most grasses and conifers. At the high rates the military use in Vietnam—which is about 13 pounds per acre, together
with as much again of 2,4-D—it becomes far less selective and kills
- a high proportion of the vegetation.
In their silvicultural applications, foresters do some aerial spraying, but often resort to individual application to unwanted trees.
However, in range improvement and in the control of vegetation on
rights-of-way, and in Vietnam, application is mainly or entirely
from the, air.
Overall, the domestic applications average out to about one-third
to one-half pound per acre treated.
That is a very brief summary of the use of 2,4,5-T.
Now I would like to spend a few minutes on the potential dangers
from the use of 2,4,5-T. I am limiting my remarks, by and large, to
the dangers to the environment since the medical and public health
aspects were covered previously, and I understand will be covered
by subsequent speakers.
Senator HART. Doctor, as you leave the use section and before you
get into these potential dangers, can you describe for the record—I
think it has not yet been stated in layman's language for the record—
what the bush or tree or grass or area of earth surface looks like
when this is applied to it, you say 1 to 3 pounds an acre.
Dr. WESTING. That is right.
Senator HART. If you can in language describe for the reader and
me what it looks like. I frankly have not seen it.
Dr. WESTING. Stretching my memory back to the Upper Peninsula.
Senator HART. The beautiful Upper Peninsula.
Dr. WESTING. I might interject here that a lot of pioneering work
in aerial forest spraying was actually done in Michigan. The leaves
on unwanted oaks or maples very rapidly turn brown, within a
matter of 3 or 4 days. In 5 days they start showing signs of shriveling up. They usually hang on that way for 6 to 8 weeks, and perhaps longer; so, one sees a lot of trees that have brown, shriveled up
leaves. If conifers are intermingled, they show no damage so they
stand out like green thumbs, and a good bit of the forest floor stays
green; grasses and so on stay green, ferns and so on will turn
brown; some plants stay green and others do not, depending upon
the type. What it looks like really in this country is as if fall had
just decided to come a few months early.
Senator HART, flow would you describe the same scene if there was
applied to it the 13 pounds per acre which you say is the current
application on the average in Vietnam?
Dr. WESTING. It is actually about 25 or 26 pounds. It is 13 of
2,4,5-T plus another 13 of 2,4-D.
Senator HART. The picture you described
Dr. WESTING. Was for one to two pounds.
Senator HART. Of 2,4,5-T only ?
Dr. WESTING. Right.
I have not seen an area myself that has been, hit this heavily, but
I have seen pictures. Within a very short period of time, all the.
79
leaves look brown and shriveled up and within a matd^P>f perhaps
two to three weeks most of the leaves drop off the trees, vines, and
shrubs.
_ This, of course, is the reason why the military spray these herbicides and sprays them in such heavy dosages, in order to get as
rapid a leaf defoliation as possible. But in the process of getting
rapid defoliation, there is a high degree of kill, which is an
unhappy corollary. I am not sure if this is really intended: it happens, particularly in certain types of vegetation.
Subsequently, grasses, bamboos, and a variety of other weeds grow
hack fairly rapidly. So, after several months you see lots of large
•dead trees and then a very heavy new undergrowth.
Senator HART. You say the tree does die ?
Dr. WESTING. Well, it depends upon the species, Senator Hart.
Mangroves would be killed by one application in Vietnam whereas
some other trees might not be killed unless they were sprayed a
second time. A single spraying seems to kill about 10 percent of the
trees. There is a great diversity of tree species there.
I have flown over areas in southeast Asia that have been sprayed
•once and it seems that roughly one tree in eight or 10 is dead.
If these were sprayed a second time 6 months later, perhaps two out
•of three trees would be dead, or maybe even more.
Senator HART. Thank you. .
Dr. WESTING. I wish now to touch upon some of the potential
dangers to the environment from the use of 2,4,5-T, and I am speaking again primarily domestically. The dangers can arise not only
from the 2,4,5-T itself, but also from its contaminants, (such as were
discussed at great length this morning), from its additives, (and
'there are endless kinds of additives: wetting agents, emulsifiers,
•stickers, penetrants, thickeners, humectants, spreaders, etc.), from its
•carriers or diluents, (such as fuel oil, kerosene, seal oil), and from
its degradation products (or perhaps degradation products arising
from subsequent burning). All of these various possibilities I shall
lump together for purposes of my comments here, just calling them
:2,4,5-T.
_ The dangers from the use of 2,4,5-T need not be confined to the
site of application, but can be carried elsewhere by wind, either as
liquid or as vapor, or carried elsewhere by water, either surface
'water or ground water. Moreover, the potential dangers are not confined to the time of application, but last, of course, until the 2,4,5-T
'degrades to the level of insignificance. Under wet and warm field,
•conditions, one of the advantages of 2,4,5-T is that it breaks down
within a matter of several weeks, 6 or 8 weeks perhaps. But under
dry and cool conditions, this may take well over a year. Furthermore, the rate of degradation in the groundwater may also be very
slow.
The dangers from the use of 2,4,5-T can result from damage to
plants, damage to animals, both higher and lower, possibly from
damage to microorganisms, and from direct and indirect combina'tions of these effects.
I shall elaborate very briefly on some of these possibilities.
The most spectacular effect of 2,4,5-T—when used as recommended
•domestically—is, of course, on certain classes of plants, particularly
�80
81
F
but not exclusively the broadleafed woody vegettat,ion. In selectively
destroying such plants and sparing others, the species composition of
the treated area is altered, the overall diversity of species is reduced,
and the total mass of living things is probably diminished. And such
changes are considered by ecologists to be an unstabilizing and
therefore detrimental influence on an ecosystem. In other words,
they make the balance of nature more precarious.
A properly functioning, relatively undisturbed ecosystem owes its
stability—indeed, its very integrity—to a highly complex set of
interactions amongst all of its many living and nonliving components. Nutrients cycle and recycle from the soil up through the
interlocking food chains and back again to the soil. Population
levels of the many component plants, animals, and microorganisms
are kept in balance by a staggering multitude of predator/prey,
host/parasite, and other long-established interactions of mutual
dependcncjr.
As soon as a toxic factor such as 2,4,5-T intrudes upon this highly
complex, totally interacting system, a certain amount of the so-called
ecological buffering action (of the many inherent checks and balances) is lost, and things start going wrong. Erosion may bo accelerated, particularly in hilly terrain and even more particularly when
streamside vegetation is killed. This effect, together with a reduction
in the total mass of .the living component of an ecosystem inevitably
leads to a loss to the area of vital nutrient materials. Especially following heavy or repeated applications, the result is a steady decline
in the productivity of the treated ecosystem—something that may
take it centuries from which to recover.
Ou top of this there are all sorts of subtle things that can go
wrong. For example, a continuing supply of available nitrogen—one
of the elements.essential to all life, and often in short supply—depends to a large extent on. the presence of certain 2,4,5-T sensitive
plants, whose roofs play host to various microorganisms crucial to
this process.
Actually, there has been some evidence of this occurring in the
Pacific Northwest, where ponderosa pines are the crop tree and
alders are being removed by 2,4,5-T as weeds, with a resulting loss
to the area of available nitrogen.
Additionally, the birds and other animals that depend upon the
2,4,5-T decimated plants for food or cover are placed at a groat disadvantage and may be partially or even completely eliminated from
a treated area.
•
The direct toxicity of 2,4,5-T to most higher animals is known not
to be very severe, particularly at the recommended rates of application. However, that there is also potential danger in this regard is
suggested by its known effects on humans. The U.S. Department of
Agriculture categorize 2,4,5-T as "mildly" irritating to the skin in a
standard dermal response rating, and as "moderately" toxic when
ingested. In fact, one can onote the following precaution from the
product label: "Do not contaminate irrigation ditches or water used
for domestic purposes;" and also the following warning: "Causes
irritation of skin nnd eyes."
'Moreover, in aquatic habitats, the death of trout find some other
fish has been reported when 2,4,5-T is applied at recommended rates
for weed killing. Certain crabs, shrimps, and mollusks are also
harmed by low concentrations of 2,4,5-T.
The adverse effects on wildlife are not limited to the ones already
alluded to. Some plants exposed to sublethal doses of 2,4,5-T (or
2,4-D) start producing abnormally high levels of nitrates (and in
some cases there has been a suggestion of even cyanide). It has been
noted with livestock that when such plants are ingested, the excess
nitrates are converted ,to nitrites, toxic or even lethal to the animals.
Another occasional result of 2,4,5-T application is that naturally
poisonous, and usually avoided, plants are made attractive to animals as a result of 2,4,5-T spraying; and then the animals feed on
these newly attractive plants and are poisoned.
The known ability of 2,4,5-T to cause chromosomal damage in
some plants and the fact that in some animals it, or an associated
impurity, results in deformed offspring when ingested during pregnancy, suggest that the plant and animal populations thus affected
will be less able to cope with their environment.
All of these debilitations that I have been cataloging, and additional ones that I have not, do harm not only to the affected species,
but, of course, thereby also to the ecosystems of which they are a partSince man is also a part of nature, I can bring out here once
again for emphasis that there is strong reason to suspect that 2.4.5-T
or an unavoidably associated impurity, the dioxin we have been
hearing about this morning, 2,3,7,8-tetrachlorodibenzo-p-dioxin (or,
by the way, a dozen or so closely related compounds all coming
under the name of dioxin), are now known to be highly teratogcnic.
In other words, they result in malformed offspring when ingested
during pregnancy. Until this issue is clarified, I think it should go
without saying that the use of 2,4,5-T both domestically and in Vietnam be restricted to locations and amounts that would preclude its
possible human ingestion.
Well, let me now make a few concluding remarks.
Senator HART. Doctor, I think it would be wise if we interrupt
briefly for a recess. That was a signal that sounded for a vote. I
thin]?: this is the time to suspend.
(Short recess.)
Senator HART. Doctor, with luck we will finish before there is
another vote. '
You were just about to begin with your conclusions.
Dr. WESTING. It is possible that I have been painting somewhattoo grim a picture of the domestic use of 2,4,5-T. But I have no particular fears that detailed exposition of its safety and benefits can be
left to the herbicide manufacturers and others. So, I figure that
what I am describing here from the environmental standpoint is one
side of the picture, and let the manufacturers tell us the other side.
Senator HART. Let me react to that, but very briefly. It is not
inappropriate or a matter of surprise, nor in my book, should it be
the basis of criticism, if the manufacturer of the product describes it
in glowing terms if society and its government permits him to
market it. If those responsible for the protection of the health of the
society conclude that he can market the product with those claims,
then why get mad at him? Why don't we get mad at the society's
institutions? • •
�82
83
Dr. WESTING. I agree.
> -. '
The low volatile formulations are not only more effelWe as herbicides, but they are also much safer with respect to the problem of
Senator HART. You can't have it both ways, if I make myself
clear.
drift and volatilization.
Aerial spraying should be confined to relatively windless periods,
• Dr. WESTING. Yes, I certainly am in full sympathy with this. I
(wind speeds of less than 5 mph) and to air temperatures of less
think the burden falls upon our regulatory agencies.
Senator HART. Clearly.
| than 85 degrees. Only nozzles equipped with course sprays should be
Dr. WESTING. I don't think Dow is the culprit here at all. It is [' used. The cleaning of spray equipment or the dumping of excesses
FDAandUSDA, and soon.',
'
•
near lakes or streams must be avoided; and getting rid of the empty
cans and so on should be limited to sanitary land-fill dumps or simiSenator HART. This goes beyond the immediate product line we
are talking about. This goes to the marketplace and the role of
larly safe locations.
To insure all of the above, State and Federal regulations should
society in protecting itself, establishing regulations where needed,
and enforcing them as established.
be tightened both for manufacturers and users, and educational
efforts increased with the aim of minimizing unnecessary or excess,
Dr. WESTING. I would certainly have to admit that the vast sucapplication. Our flagrant misuse in Vietnam should be halted immecesses of productivity upon which our nation's current affluence
diately (see, e.g., my article in the Friends Journal of 1 April 1970).
hinges, depend to a large extent' upon the use of pesticides such as
Finally, I wish to stress once again the complex and as yet little
2,4,5-T. And it seems clear that the use of pesticides will continue,
understood nature of our environment. The study of ecosystems asperhaps even unabated, without a highly unlikely downward trend in
such is still in its infancy. And since hormonal herbicides have been
our population, and, even more particularly, in our collective desires
and demands.
in general use now for only two decades or less, we simply are not
yet able to predict the full range of potential disasters that their
Plowever, the time seems to be fast arriving when certain precauunrestricted use may inflict upon us and all other living creaturestions must be taken so as not to overload our environment with
such potent pollutants. A number of suggestions are thus in order to
with which we share this small world.
Senator HART. Doctor, for all its brevity, this is a very helpful
forestall the need for a basic change in our way of life.
•' ,
First of all, research efforts' should be expanded on several fronts^ | statement.
I have a couple of questions that I would like for you to react to.
Effective cultural and biological controls of pest species should be
You tell us in dry and cool conditions it may take well over a
sought and developed with renewed vigor. With respect to the pestiyear for 2,4,5-T to degrade. I think you were here this morning. The
cides themselves, highly selective and rapidly degrading ones should
be aimed for.
Department of Agriculture is not in agreement with that statement.
Can you give us some evidence for your statement, or refer us to
In the light of the current 2,4,5-T affair, I must add here that all
pesticides, existing and potential, must be rigorously tested prior to
sources that are in agreement with your statement ?
Dr. WESTING. To my knowledge, there has been precious little
their general release for possible toxicity, carcinogenicity, teratogenresearch done on the life of 2,4,5-T in the environment. I am aware
icity, and mutagcnicity to humans; and additionally, for possible
of one study that was done in a forest environment in which it was
adverse effects on livestock, on wildlife, on game, on fish, and on
other components of the ecosystem.shown that 2,4,5-T degraded to insignificance in a matter of several
With respect to 2,4,5-T, its use—in my considered opinion—must
months, as I recall.
On the other hand, it has been well established, and it is clearly
be limited to areas remote from human habitation. Control of vegeknown, that 2,4-1)—a compound similar to 2,4,5-T—degrades much
tation on rights-of-way must be regulated with particular care since
more rapidly than 2,4,5-T. It has been demonstrated a number of
utility, transportation, and other rights-of-way are by their very
times that under dry conditions, 2,4-D can persist in the environnature frequently close to civilization. I want to emphasize here,
ment and have detrimental effects for as long as a year or a year
Senator Hart, that one of the major uses of 2,4,5-T—one of its preand a half after application. From this I infer that 2,4,5-T?>which is
ferred uses—is in woody vegetation control along rights-of-way.
more persistent than 2,4,5-D, would have at least a similar life under
This is a major place where 2,4,5-T is likely to impinge upon human
habitation, to come in contact with civilization.
dry conditions.
Senator HART. Then, adopting your reasoning, it would mean that
Broadcast applications, where safely remote from human habitaunder those conditions, 2,4,5-T might be found on food that istion, should not exceed 3 pounds per acre; and where spraying covers
served months after the spraying of the crop; is that correct?
•extensive areas, unsprayed zones should be left as oases for wildlife,
.and so forth.
Dr. WESTING. I have no direct information, but one could surmise
that this could happen. This is a possibility.
Repeat applications should be controlled, perhaps to intervals of
Senator HART. What would you think the possibility of 2,4,5-T's3 years or more. Aerial broadcast spraying should be avoided where
capacity is to persist within the organism, plant or animal, which
possible, and always avoided near bodies of water, in favor of spot
applications, or individual applications.
had ingested it, including the humans
Dr. WESTING. I have no first-hand knowledge on this whatsoever f
In those areas where aerial spraying is permissible, the highly volso I prefer not to try to answer it. ,
atile (though cheaper) formulations should be banned completely.
�Senator HART. You would agree that it is impossible to
t possible?
Dr. WESTING. The likelihood is there. As far as I know, it may
persist, or even build up in the human body. Some other chemicals
that are fat soluble (as are the ester formulations of 2,4,5-T) are
known to deposit and be stored in the fatty tissue of humans; so, it
is highly possible that 2,4,5-T does this, but I simply do not know
whether it does or not.
Senator HAIIT. You suggest that nso of 2,4,5-T be limited to areas
remote from human habitation, and that it should be restricted in
.other respects. -Does that mean that you would feel that Dr.
DuBridge's suggestion that pesticides be deregistered for food use,
assuming there can be lio tolerance level set by FDA doesn't go far
enough?
Dr. WESTING. '2,4,5-T as it is commercially available with its
impurities, is a substance that should not have any food tolerance at
all. It should haA^e zero tolerance, 'at least given the current state of
knowledge.
'
• . . . - , >
Certainly, the suggestion made this morning by—I think it was
Mr. Well ford—that its use should be curtailed severely, or suspended until Ave clarify this whole issue is one that I fully support.
I think that 2,4,5-T is probably a safe chemical to. use at, the Iqw^ t
recommended doses in areas remote from human habitation. I don't
think it need be banned under such conditions in the forest environment, or on range lands.
On the' other hand, along power line rights-of-way, railroad
rights-of-way, and so on, that get near houses, I think there should
be severe restrictions. '
Senator HART. What about proximity to crops?
Dr. WESTING. Food crops?
Senator HART. Yes.
Dr. WESTING. I think that certainly for the time being, it should
not be registered for use on food crops and not be used near them.
Senator HART. In these areas that you have described where 2,4,5-T
has been applied you have said that some of the birds and animals
that depend on the plants that have been destroyed may be eliminated. Which birds and which animals are likely to be affected ?
'
One way to answer that I suppose is any that are in that area, but
I am trying to find out if some are and others are not affected.
Dr. WESTING. I wish I could give you some spectacular answer
about bird X or T having become extinct as a result of the use of
2,4,5-T, but I cannot. I can quote a recent statement made by a British authority on pesticides, Dr. N. W. Moore, director of tlie Monks
Wood Experimental Station in England:
'
The vi.se of 2,4-D and 2,4,5-T to control scrub by roads and in woods reduces
the essential habitnt of almost; all British land birds, which, because they are
mirvivnvs of the original forest fauna, are still dependent on trees and bushes.
(Advances in Ecological Research 4:108; 1907)
To judge from this statement Dr. Moore is concerned over the fate
of the native British birds as a result of the routine use of these herbicides.
In this country there is an extensive program over many tens of
thousands of acres in the West of sagebrush control in which herbi-
cides of this nature are used, primarily 2,4-D. There is some evi•dence that the sage grouse population has been deplelj^fc at least the
hunters are not as happy as they used to be.
^^
I have to warn you, Senator Hart, this is one area where the herbicide proponents will jump up and say that there are a number of
clear cases where the use of herbicides has actually benefittecl wildlife populations.
Senator HART. I made an interjection earlier to say that if we are
going to get mad at somebody let's get mad'at ourselves first of all
as a people for not recognizing dangers and setting down the laws
that will prohibit the marketing of certain things, but equally true,
•of course, is that the producer is obligated, absent any explicit regulation, to make truthful representation about its product—again, I
am thinking not of chemicals alone but anything—and report factually the experience that has come to his attention to whatever
public agency there is that is expected to make the judgment for all
of us as to whether that product in fact should be marketed. So, if
they jump up and explain it is good for us, I hope, they will not do
so unless they can explain why/
. ••
You noted, among other things, in your conclusion that we should
expand research, attempting to develop other controls of pests. What
development do you imagine would be fruitful?
Dr. WESTING. Well, the main thrust of alternatives to the use of
insecticides has been to introduce predators or diseases of the insect
pests. This same approach can also be used with herbicides such as
2,4,5-T. Plant pests are a little less amenable to this sort of an
approach, but one could push ahead on research on. possible virus
diseases or fungus diseases or insect enemies of weed species.
I am familiar with one success story in this regard. A serious
weed in the Northwest is St. Johnswort, and a beetle (chrysolina)
has been introduced from Australia that feeds on the St. Johnswort,
in a highly successful alternative to. chemical herbicides. This general type of approach should be exploited to the greatest extent possible.
There are all kinds of other possibilities. Just in forestry, for
example, closer spacing of crop trees shades out certain weeds. You
can go back to a greater emphasis on some of the mechanical methods that are now avoided because of the high cost of labor: mowing,
weeding (pulling out the weeds or cutting them doAvn), burning.
Flamethrowers are used in certain instances and even controlled
fires. These methods have a much more selective effect on the actual
weed and a minimum of lasting untoward side effects.
If chemicals are to be used, the forester's approach of individual
application is far preferable to the utility and range manager's
approach of broadcast spraying from the air.
So, there are a variety of alternatives available. With just the
slightest amount of urging,' the slightest realization that there is a
necessity to worry, these alternatives would at least be explored. In
the past it had never even been realized that there were possible ill
side effects to the use of herbicides.
Senator HART. Now, you have lectured us quite thoroughly on the
dangers inherent in changing the ecological pattern. Yet every one
�86
Bf these alternatives that you talk about suggests similar '
and some additional ones.
The Australian beetle is not native to the Northwest, I take it, but
you are going to bring Australian beetles in. The flamethrower is
not really an altogether acceptable
Dr. WESTING. There is a history of introducing something to<
combat a pest and thereby introducing a worse pest, so there has tobe some very careful preliminary testing and evaluating before this
approach is iise_d. With this in mind, it is safer to use something like'
a virus than it is to use something like an insect or a fungus because
the virus one will be far more host-specific and therefore will not
switch to an alternate host after it does its job and then become a
pest in its own right. This is a danger that has to be kept in mind.
With regard to the pesticides, I suggested that we keep searching
for much more highly selective ones. The problem with 2,4,5-T is.
that although selective in a certain sense it still is relatively unselective and kills lots of things that you do not want it to kill. This isthe sort of thing that has to be watched out for.
Senator HART. I must admit that I got the impression clearly this.
morning that the existence of a realistic alternative to some of these
things might help to convince the Department of Agriculture totake action; that is if they knew they had a realistic alternative,.
maybe the evidence which the Department now says is not sufficient
to alarm them might have higher credibility.
I don't know whether I make myself clear.
Dr. WESTING. Yes; that:is why it is important to mention that,
there are possible alternatives or at least that a goodly researcheffort should be aimed in that direction, to provide possible alternatives. We have come to depend upon the chemicals to such an extent
that I think other possible control methods have become less interesting.
Senator HART. I think it should be said, and not necessarily as a
direct criticism of anybody, but humans are humans and if there is;
some acceptable alternative for what would otherwise be a decision
that would put a lot of heat on the fellow making the decision, it
would be much easier to make and somewhat unconsciously perhaps
the existence of an alternative might change the attitude of some of
these individuals.
Mr. Bickwit.
Mr. BICKWIT. Part of your evidence for the persistence of 2,4,5-T
under certain conditions for over a year stems from experiments;
establishing the persistence of 2,4-D. I think for the record we ought
to have some reason why you can jump from evidence of the persistonce of 2,4-D to conclusions about the persistence of 2,4,5-T. Can
you meet the argument that the 2,4-D evidence might show that 2,4D is just more persistent than 2,4,5-T?
Dr. WESTING. No, I think one could be on completely safe ground'
in saying that 2,4-D is considerably less persistent than 2,4,5-T. 2,4D will degrade under normal, moist environmental conditions in a-,
matter of weeks. 2,4,5-T is perhaps twice as persistent. There are a
lot of studies to show that 2,4-D degrades more readily than 2,4,5-T;
lots of short-term experiments have shown this. I am not familiar
87
•with any definitive long-term 2,4,5-T studies. It is yerj^pisonable to
assume that as long as 2,4-D will, under dry conditions such as you
find in Idaho, have harmful effects on crops a year or more after use
that 2,4,5-T would also.
Senator HART. Doctor, thank you very much. It was a helpful •
paper.
The signal a few moments ago indicated another roll being called
in the Senate. I apologize to Dr. Kotin, but we will have to take
another recess, and I will be back just as soon as I can get on the
roll.
(Recess.)
Senator HART. The Committee will be in order.
Our concluding witness on this first day of hearing is the Director
•of the National Institute of Environmental Health Sciences, Dr.
Paul Kotin.
-STATEMENT OF DR. PAUL KOTIN, DIRECTOR, NATIONAL INSTITUTE
OF ENVIRONMENTAL HEALTH SCIENCES
Dr. KOTIN. Mr. Chairman, I am privileged to be here today
•engaging in the practice of one of my most pleasant responsibilities—that of discussing the programs and plans of the National Institute of Environmental Health Sciences of which I am Director.
Our Institute is a newcomer in the executive branch; we have been
in existence since 1966, achieving the status of National Institute of
Environmental Health Sciences only in January 1969.
This activity started as a small segment of the Department of
Health, Education, and Welfare's effort in environmental health in
response to recommendations made by several public advisory committees during the late 1950's and early 1960's. These committees—
starting with one chaired in 1958 by Dr. Stanhope Bayne-Jones and
concluding with one headed in 1965 by Dr. Detlev W. Bronk—repeatedly emphasized the necessity of establishing within the Public
Health Service an organization dedicated to performing fundamental research into the real and potential effects, of human health
wrought by a rapidly changing environment.
The decision of the Surgon General in 1966 that this research program be located within the National Institutes of Health—that Federal agency responsible for building the Nation's base of fundamental biomedical, health-related research—made clear the mission
envisioned for our program. That mission was and is:
First, to determine the magnitude and significance of-the hazard
to man's health from long-term exposures to low-level concentrations
of chemical, physical, and biological agents in the environment; and
second, to elucidate the underlying mechanisms of adverse response
with the hope that principles and generalizations would be identified
to provide a scientific base for criteria upon which control agencies
could set standards for protective and preventive measures.
During the present (1970) fiscal year, Congress and the President
have authorized $17,730,000 to be expended in the conduct of this
program.
Since you may be familiar with other programs of the National
Institutes of Health, I would like to take just a moment to point out
�• to you some ways in which we are similar to other part^^f NIH I uiul critical review of other researchers who have an opportunity to
and some ways in which we differ. I might preface this l^fcsaying question our methods and conclusions by usual strujpit^ standards.
I hope that the preceding discussion has placed tl^pational Instithat we are similar to other NIH research components in inore ways i
that we are different.
[ tvito of Environmental Health Sciences in perspective for you.
I would now like briefly to tell you in somewhat greater detail
Like the other research institutes of NIH, our mission reflects twovery important principles of operation: (l)We are in business prisome of the things we are doing, why we are doing them, and how
marily to add to the fundamental knowledge and understanding of
we come to be involved in the resolution of the problem which is the
environmental agents which as biomedical hazards immediately or
subject of these hearings.
ultimately affect human health.
Speaking quite broadly, the NI.EHS program attempts to employ
In other words, we are concerned about the what and how of
u wide spectrum of scientific disciplines and bring them to bear on
health effects first and foremost in human beings. That we must also
real and potential human health problems resulting from:
1. Changes in the makeup of the environment in consequence of
understand that what and how of the complex constituents of our
environment in order to perform the primary task is obvious.
technological progress and industrialization;
2. Changes in the size and characteristics of the population; and
Nevertheless, it is the results in humans which is of overriding
concern to us. (2) The responsibility for taking direct action to con3. Cha.nges in the character of interactions between those two.
In order to best understand the significance of changes in the
trol or eliminate the hazards which we must identify resides in other
components of HEW.
makeup of the environment, we employ the disciplines of analytical
I hasten to emphasize that we do not consider our job done until
and synthetic chemistry, pharmacology, and of biophysics.
our findings are made available to the appropriate components of
In order to better understand our changing population and the
Government. To accomplish this, we maintain effective, close, and
subtle interactions of new and changing environments on people, we
continuing relationships with the Environmental Health Service, the
employ the sciences of epidemiology, biometry, pathology, and toxiFood and Drug Administration, the Department of Interior, the
cology.
Department of Agriculture, the Federal Trade Commission, .and
In order to establish the mode and mechanisms of interactions, we
other agencies with control responsibilities.
employ all categorical divisions of scientific inquiry with special
The reasons for the distinction between fundamental research and
emphasis on comparative-biology to assure maximum relevance of
control powers are, I think, important. First, the urgency in the
research data to man/
need for control measures requires research directed to answering
These varied resources and methods have so far been brought to
today's questions witli today's techniques.
bear in programs studying the potential health hazards of:
There is, however, an equally, or perhaps more, important need
Natural products including fungal contaminants of food; fibers
for research directed to questions having long-range implications
and polymer dusts, asbestos and fiberglas; alpha radiation; trace
extending for decades and perhaps even generations into the future.
metals (such as lead) and their compounds; hydrocarbons and their
It is in response to this need that our Institute's program, is
reaction products; tobacco smoke; and pesticides and pesticide
designed.
flyncrgists (includingherbicides).
:
While techniques frequently used in attacking these two sets of
In all of these studies we are concerned with the effects of longquestions are similar, 'the orientation and end points stand in sharp
term exposures to low levels of concentration-because these are the
contrast.
usual characteristics of exposure during life in the environment we
Second, freedom from control activities permits us to devote our
have created for ourselves.
total effort to research.
Effects are likely to be gradual in appearance, and most commonly
Third, control activities are performed by experts in an environthe result of interactions of numberous agents combining in addiment in which the guidelines for operation are completely dedicated
tive, synergistic, or antagonistic manners. •
to this responsibility.
To dissect these complexities we must identify interactions at all
_ Fourth, our relationship with industries, communities, and indilevels from the intracellular organelle to the whole organism.
viduals is one based exclusively on scientific grounds rather than one'
Our goals include determinations of threshold for response, effects
of regulation, monitoring, and enforcing.
of repetitive exposures, effects of storage of the agents in living
Finally^ our inputs to control agencies are objective and provide
organisms, and the roles of such host factors as age, sex, antecedent
an impartial basis for the very real practical considerations which
or concurrent illness, nutrition, behavioral characteristics, and genemust be faced in formulating and inaugurating control measures.
He make-up.
As noted, the fruits of our work are promptly forwarded,. toIt may seem that our approach is somewhat complex, but it must
appropriate Government agencies for use in the pursuit of their misbo so in order to resolve the complex problems wrought by the
sion with virtual simultaneous publication in professional journals,
changes in our environment intrinsic to technological progress.
rather than in the popular press.
We have attempted, in. the process of establishing, the program of
This practice assures that our findings are subject to the scrutiny
(lie Institute during the past 3 years, to maintain a measure of flexibility amid this essential complexity to provide for response to
�90
91
nanticipated problems. Our current efforts in response to
over the widening use of herbicides is in a way a case in point.
You are aware, I am now certain, that the recently completed
study which revealed information about the toxicity of the herbicide
2,4,5-T, in fact, was initiated by the National Cancer Institute in
19G3. _
As indicated earlier, our Institute was not in existence at that
time. However, I was the scientific director for etiology in the
'Cancer Institute at that time, and along with members then and now
.on my staff played a leading role in the initiation of the research
.contract with Bionetics Research Laboratories, Inc., which yielded
the information under discussion.
Very briefly, that study was undertaken primarily to identify any
•potential carcinogenic (cancer causing) or teratogenic (birth defect
•causing) agents in a wide variety of pesticides and allied compounds
in commercial use.
We also anticipated that the study would provide data on which
•to develop improvements in pur methods for identifying carcinogenic agents and hopefully identify any correlations that might
exist between the carcinogenic and teratogenic capabilities of single
•specific compounds.
Pesticides were selected for inclusion in the study on either of two
bases; First, a projection of the potential extent of their use in
terms of their utility in the community; and; second, a judgment as
to potential carcinogenicity by virtue of chemical structure or.meta-,
bolic fate.
_ In consequence, some 86 pesticidal products—including insecti.cides, fungicides, and-herbicides—were subjected to controlled, longterm studies on mice. As had been intended from the start, the study
•continued through the 1960's.
In the interim, the then Division of Environmental Health
Sciences was^ established, and I was asked to become its first dir'ec•tor. In agreeing, I was granted approval to take with me one or two
key staff members—scientists, as it happened—who had also been
associated with the Bionetics contract.
Since intensive programing and developmental responsibilities
faced my staff and me during the first years of our Institute, we '
were quite satisfied to leave the. management of the Bionetics pesticide study in the able hands of our successors in the Cancer Institute. Furthermore, it should be recalled that the one major basis for
•the study was quite clearly related to the mission of the Cancer
Institute, the identification of cancer-causing agents in the environment.
Upon completion of the study in early 1969, the Cancer Institute
released the results of the study. The results of the teratogenic studies were released to the Mrak Commission immediately as they
became available. The popular press took intense interest in the findings reported, and pressures developed for more complete information on several of the pesticides included in the study.
The herbicide 2,4,5-T came under special scrutiny because its use
is especially widespread, particularly in military operations in Viet:nam. Word that the Bionetics study had shown this chemical com-
pound as "causing significantly more deformities thai^cpected" was
especially alarming in some quarters.
^^
Dr. Endicott, then director of National Cancer Institute, requested
that NIEHS staff familiar with the study in question, and also
familiar with teratogenicity and pesticide chemistry generally, be
assigned to data analysis and interpretation. NIEHS assumed sole '
responsibility for the statistical analysis of the very large volume of
data.
During the early stages of .the now public discussion, it was
pointed out by the Dow Chemical Co., a major supplier of 2,4,5-T,
that the materials used in the Bionetics study were significantly different than those which had been supplied by DOAV since 1965.
It is certainly true that the 2,4,5-T used in the study contained
significantly Jarger amounts of an impurity, dioxin. This impurity is
highly toxic" and its presence occurs incidental to minor alterations
in the reaction conditions during the manufacture of'2,4,5-T.
DOAV Chemical Co. scientists contended that it was the dioxin
derivative rather than the 2,4,5-T which had caused the deformities
in test animals. A sample of the original 2,4,5-T used in the Bionetics study was analyzed and was found to contain 30 parts per million of this dioxin compound.
In consequence, it became necessary to restudy the sitxiation to see
whether the virtually no-longer-existing impurity in 2,4,5-T could be
-held responsible for the adverse effects.
In order to verify the possible role of dioxin, NIEHS brought its
available resources to bear and undertook an accelerated program of
research.
Pure 2,4,5-T—and by pure, I mean that which is now in the marketplace with a dioxin concentration of less than one tenth of a
part per million—has been made available to us and recently we
received the dioxin in pure state so that experiments can be repeated
with the pure material, as well as with a combination of the two
ingredients.
These studies are now underway. As indicated in prior discussions
with the subcommittee staff, the results of this research are not yet
complete. At such time as they are, in the very, near future, we will
be pleased to supply them to this committee.
I would be happy at this time to answer any questions of the committee regarding the mission of NIEHS or the circumstances leading to our current study of 2,4,5-T.
Senator HART. Thank you, Doctor. It was thoughtless of me—I
should have suggested, since you commented on having a sore throat
before, that you not read the statement, but merely put it into the
record.
But I think as long as you were able to get through it, it helps all
of us to hear it, rather than waiting for the printed record.
On this business of the study, do you know when the National
Cancer Institute received its first data from Bionetics suggesting
that 2,4,5-T was teratogenic?
Dr. KOTIN. I can't tell you offhand, but I would be. very happy to
get it for the record, sir.
(The information was subsequently received for the record:)
45-362-JTO
7
�92
93
p
.Tune of 10GC, we received the first data indicating that 2,4,5-T adij
tored by injection at a dose of 113 mg/kg of body weight produced terato|BIc
effects, In May of 1968, data indicated teratogenic results from oral administration of 2,4,5-T at a dose of 113 mg/kg of body weight."
sis of data from a series of experiments in which mij^fcple species
were used, multiple doses were used, were terribly time-Wlsuming. _
So that all I can do is vouch for the commitment of resources it
took from the National Institute of Environmental Health Sciences
to do its little share, provide its little share of the total.
Senator HART. Doctor, I am going to ask Mr. Bickwit to continue
with these questions. We have reviewed them prior to the hearing,
and I will remain, using the time to road a memorandum that
explains what this vote that was just signaled is all about. I hope by
the time he finishes, and I finish this, we will have the answers.
Dr. KOTIN. I hope I don't disturb you.
Mr. BICKWIT. I'm frankly not clear on the major dates that arc
involved here, the dates that you received the Bionetics information,
the date that you came out with your first report on it, and the date
that you came out with your final report on it.
Now, if I'm right in thinking that those are relevant dates, could
you tell mo what those dates are?
Dr. KOTIN. Right. Well, the dates are relevant. I think it was,
again, the date I offered for the record, which I don't remember
offhand, is the date the Cancer Institute received the Bionetics
report.
You will recall Dr. Falk and our associates instigated the Bionetics study, and it wasn't a personal contract with vis. It was with the
Cancer Institute.
So the report went to the Cancer Institute and I don't know when
they received that.
Fundamentally, the only reason I suspect that we would have
gotten involved at all in terms of the Bionetics report, as distinct
from our own commitment by virtue of our mission in this, was the
fact that Dr. Endicott did have a need for a tremendous amount of
statistical and chemical analytical competency, and it was more than
he had available in the Cancer Institute.
So I can give the date at which the material was forwarded to us.
This was in 1968, and again, I will get the date for the record. But
it was—actually, the material was forwarded to us coincidental with
the request to get involved with some of the analyses.
Mr. BICKWIT. About when in 1968 ?
Dr. KOTIN. I will be happy to give you the exact date for the
record, sir.
(The information was subsequently received for the record:)
NIEHS performed analyses of the raw data between January and
June 1969.
Senator HART. We would appreciate that, and it will be made a
part of the record. I am under the impression that it was sometime
in 1966. In a sense I guess that's about the time you departed the
promises ?
Dr. KOTIN. Exactly.
Senator HART. Let's assume that the date is June 1.966, that being
the time the first data was received from Bionetics by the Institute.
Do you recall \vhen the final report came out?
Dr. KOTIN. Yes, the final report, in 1969—late 1968 and early
1969, as I recall. A little over a year ago, as I recall.
Again, I can't be sm-e of that, but I would be pleased to get the
exact date. I had left the Institute.
(The information was subsequently received for the record:)
"Bionotics supplied a draft "final" report in September of 1908. Questions
raised by NIH required additional work by Bionetics and subsequent revisions
of the report. Bionetics completed this work and submitted a truly "final"
report in September of 1969."
Senator HART. The NIEHS report—when did that come out?
Dr. KOTIN. The final report was last fall, when we were providing
the results of our statistical analysis, and the data on the teratogenicity to the Mrak Commission.
Senator HART. If it develops that the June 1966 date is the time
that the National Cancer Institute got its first data from Bionetics,
and the final report by NIEHS came out in the fall of 1969, why in
the world did it take so long to come up with the information for
that final report ?
Dr. KOTIN. I really can't answer that, other than to say that at
the time the National Institute of Environmental Health Sciences
was asked by Dr. Endicott to provide the statistical and analytical
competency for the review of the data, the work Avas done very
promptly. In fact, Aye didn't even wait until the end of the report to
make the information available to the Mrac Commission.
As each little increment of information that represented a part of
the total became available, this was made immediately available, to
the Mrak Commission, and the Food and Drug Administration.
Senator HART. I am trying to get these dates clearly fixed, if I
- can. You state that NCI released the results of the study in early
1969. Was this the preliminary report of Bionetic's findings?
Dr. KOTIN. No, sir; this, I think, represented the first report in
which conclusions were published, both in the scientific literature
and in the Journal of the National Cancer Institute, as well as made
available to the various responsible government agencies.
The really important aspects of the conclusions, the necessity for
voluminous work—there were some 86 compounds—the National
Cancer Institute justifiably felt that in-house staff should at least on
a random basis review the data. There was much, much new information that heretofore had been unknown. And just the histological
review of the slides from the autopsied animals, the statistical analy-
Mr. BICKWIT. Then you released reports periodically ?
Dr. KOTIN. To the Mrak Commission only, and to the relevant
Government agencies.
Mr. BICKWIT. About how many reports were there ?
Dr. KOTIN. These were not formal reports, but they were presented quite informally—we finished the analysis of the White Swiss
Mouse data, the C-57 black data, the DBA data.
We checked the statistical significance of the differences between
test and controls, between the various dose levls, between the various modes of admission. So that, rather than adorn the data with
�94
95
sc, we just gave them the statistical material with the listing^B
conclusions.
^^
Mr. BICKAVIT. So, whenever you had anything of any importance,
it went to Mrak.
Dr. KOTIN. Promptly.
•
>
,
,
Mr. BicicAvrr. Your final report came out in the fall of 1969, is'
that right?
Dr. KOTIN. Yes, we have submitted a paper for publication in the
journal Science which relates pur analysis on the teratogenicity of
2,4,5-T, and it should be appearing shortly.
Again I would be happy to make a preprint copy of the manuscript available for the Committee if you desire.1
Mr. BICKAVIT. Thank you. That would bo fine.
Now, if you got your information sometime in 1968, and we don't
know when, let's assume it was late 1968, and it took until the fall
of 1969 to como up with a final report, why did it take that length
of time ?
Dr. KOTIN. Just the difference.between the magnitude of the job
and the availability of professional resources within our institute.
At that time, our Biometry branch consisted of two professional
biometricians at the doctorate level. This staff was involved in a
series of studies including one on the relationship of asbestos to lung
cancer, and another on a quantification of the hazard to uranium
miners. This limited staff had to be literally redeployed in order to
perform the necessary analyses of the Bionetics data.
Mr. BICKWIT. On the carcinogenicity studies, when did you get
the information from Bionetics?
Dr. KOTIN. We really didn't, other than as information. It came
as part of the same report. But the analysis of the carcinogenicity
study remained entirely within the Cancer Institute, since it was
clearly relevant to their mission and responsibility as the National
Cancer Institute.
Mr. BICKWIT, You were not responsible for analyzing that?
Dr. KOTIN. No, sir.
Mr. BICKWIT. You have stated the results of' the toratogenicity.
studios were released to the Mrak Commission immediately when
they became available. I am sure you are familiar with Mr. Whiteside's allegation that Dr. Samuel Epstein of the Mrak Commission
had a great deal of difficulty acquiring information on the studies.
I Avonder if you could reply to, this allegation? If you are not
familiar Avith it
Dr. KOTIN. I am familiar with the allegation. I read it in the
story in the New Yorker, of course.
No, I think that we are probably speaking of two different things.
There was, at no time, the necessity for the requesting of any information from us. There was a mechanism for the fonvarding of the
information to the Mrak Commission; the best evidence that this
allegation is not so in another sense is that the head of our Biometry
Branch, Dr. David Gaylor, was on the Mrak Teratogonicity Committee, the very committee to which the data were being supplied.
Avn cornSo, essentially it would be denying his own data to
mittee if this were so.
Do you follow me.
Mr. BICKAVIT. I am sorry, I don't.
Senator HART. I am going to have to interrupt again, I am sorry.
I hoped Ave could avoid the necessity of holding you, but I will miss
the vote.
I will not be able to return as promptly as I like, because I must
remain on the floor to get something done, a matter that will be
voted on tomorroAv.
So, Ave will have to recess in the very unhappy condition of not
knowing exactly when I will get back, but as quickly as I can.
(Recess.)
Senator HART. We Avill resume, and Avith better luck than AVG have
been having in the last hour or so, maybe we can conclude before the
next vote is signaled.
Mr. BICKAVIT. I believe the last statement which you made I had
some difficulty with.
Dr. KOTIN. What I Avas saying Avas that Dr. Epstein and Dr.
Gaylor Avere on the same teratogenicity panel of the Mrak Commission, and each meeting they held Dr. Gaylor brought the data
"P_So the only information Dr. Epstein might have asked for that he
did not got Avere data that just were not complete. But certainly in
relation to the teratogenicity, I cannot conceive of any aAvailable
data that would not have been made available.
Mr. BICKAVIT. Was the final Bionetics report made available to the
Mrak Commission when they asked for it?
Dr. KOTIN. It is my impression that it Avas. And again they would
not have come to us, because the final report Avas the property of the
National Cancer Institute, as the contracting institution.
Mr. BICKAVIT. If they did come to' you, would you haA'e had
authority to give it to them ?
Dr. KOTIN. Actually, I suspect I Avould have picked up the phone
and asked Dr. Endicott Avho was responsible, and I would have
gotten authority for it because the information contained in it was
germane to itho Mrak Commission. But again I would emphasize
that the final report of any contractor Avould not include the interpretation and the analysis of the data. This was not part of the purchase.
Mr. BICKAVIT. I realize that, but if Dr. Epstein of the Mrak Commission had asked you for the final Bionetics report, Avithout an analysis from NIEHS, you would have furnished i t x to him
immediately ?
Dr. KOTIN. I would have furnished it to the Mrak Commission.
Mr. BICKAVIT. Would you not have furnished it to Dr. Epstein ?
Dr. KOTIN. The data itself ?
Mr. BICKWIT. Yes.
Dr. KOTIN. Uninterpreted?
Mr. BICKWIT. Yes.
Dr. KOTIN. Oh, I probably would not have, no.
Mr. BICKWIT. Why not ?
t
�96
'Dr. KOTIN. Essentially the data are crude data that require imKprotation, and essentially the implications, the results of the report,
arc the conclusions, and the responsibility for those conclusions
would have been ours,—that is, the responsibility of the NIH.
Mr. BicitAvrr. These data, I understand, did raise doubts, about
the toratogenicity of 2,4,5-ff.
{
Dr. KOTIN. You mean, rather than raise doubts, established the J
experimental teratogenicity of this. After the data were analyzed, jj
yes.
|
Mr. BIOKAVET. You are saying that you do not believe that a J
member of the teratology panel of the Mrak Commission should
have the right to examine those data unalyzed?
Dr. KOTIN, Oh, not at all. All I am trying to say is the data
themselves, short of total package, once the data were analyzed, and
conclusions made, then by no stretch of the imagination would the
date bo withheld from anybody.
Mr. BIGKWIT. But unanalyzcd, he should not be entitled to look at
them ?
. Dr. KQTIN. I do not think so, no, sir.
Mr. BICKAVIT. Should anybody other than the organization entrusted with the analysis of the data be entitled to look at them?
Dr. KQTTN. Oh, surely. Mr. Hart's Committee, or there are a
whole spectrum of responsible agencies.
Mr. BICKAVIT. Could you list those agencies that would be entitled
to look at this data?
Senator HAKT. You are inquiring about before analysis?
Mr. BICKWIT. Yes.
Dr. KOTIN, The hierarchy above me, as a lowly director of an
institute, the director of NIH, The Surgeon General, the Secretary
of HEW, all of the way up, any member of the legislature, any
member of the executive branch, with the authority, surely. '
Mr. BICKWIT. But you would not want to allow a nongovernmental scientist with, some expertise in the field to look at this data?
Dr. KOTIN. Again, there is no flat yes and no. There are many
instances when we call people in nongovernmentally to look.
Mr. BICKWIT. What I am asking you to do is draw the line. I
know it is hard, but you have excluded one nongovernmental scientist. I would like to know how you formulate your opinion in deciding who should be excluded and who should be included.
Dr. KOTIN. That is a matter of judgment. How much help I think
we can get from them, how much help we can provide them.
Mr. BICKWIT. Is that the only basis for your decision?
Dr. KOTIN. I would have to think. I suspect that is the major one.
We have crude data a.nd what we try to do is get the best expertise.
We have everything from advisory committees to councils to study
sections to consultants to the institutes, who are not Government
employees, who are on call at all times and who are used rather consistently, particularly by a young institute like our own, (we are 3
years old; our $17 million budget, when contrasted with the $150plus million budget of the larger well established institutes is probably as good an indication of our size as anything.)
I think a corollary of our small size is the great consistency with
which we get outside help in terms of consultation. We just had a
97
task force that spent 8 weeks preparing a consultatiA^puide, as it
were, for the Institute. So there is no tendency on pur part at all to
treat anything that we got as either clandestine or in any way not
open to scrutiny. In fact, as I said in my testimony, I made a special
point that scientific scrutiny is something that we insist on in all of
our data before wo accept it as fact.
As our critical mass at NIEHS enlarges, we will probably be
more certain. But we are a small outfit and we use outside consultants a lot. So in answer to your question specifically do I feel categorically that data should not be seen by outside scientists, not at all.
There are instances where you call them in and they see it initially
with us, as it were, around the table for the first time.
Mr. BICKAVTT. On the pro side yon are weighing the potential
helpfulness of the scientist who would be asking to see the data.
Dr. KOTIN. Oh, no. Also what he can contribute to the maximum
utilization of the data. In the years I have been in NIH when there
are implications of the data that aflect other executive branches, or
have socioeconomic implications, the people who yon try to get help
from and proxide help to are judged on an individual basis. This is
really so.
Mr. BICKWIT. What is on the other side? In formulating your
opinion what is it that would keep you from giving the information
out?
Dr. KOTIN. Number one, concern over data where the interpretation would be such that we would want our interpretation to be on
the record at the time the data were made available. That would be
one example.
Another example, where there is some question we have about the
data ourselves, so we want to go back and verify techniques, verify
the workbooks from which the reports were made. And in fact this
was done in this case. So there are lots of reasons. Not as many as on
the other side, but you just have to do it on an individual basis,
decide what is the best way to get maximum returns from the data.
Mr. BICKAVIT. With respect to Dr. Epstein, a member of the teratology panel of the Mrak Commission, would you rule out the possibility of his being able to contribute to the utilization of this material?
Dr. KOTIN. Yes.
Mr. BICKAVIT. Could you elaborate on that ?
Dr. KOTTN. Fundamentally it is a matter of judgment. I felt at
that time that the data themselves needed analysis for the reasons I
mentioned, that the conclusions Avere integral to the data because
again the mere fact that you had chi square indicated there that the
significance Avas in large measure determined by statistical methods.
It wasn't a situation where, as the data amply attest, an all or
none response occurred, Avhere all of the controls did one thing, all
of the test animals did the other. There Avere statistical differences.
There were differences in degree and intensity and in time. These
had to be determined by statistical techniques.
Senator HART. Doctor, I will be brief in my thanks,' since I am
under the compulsion of another vote signal.
(The information referred to on p. 94 folloAvs:)
�aa
TEKATOGENIO EVALUATION OF 2,4,5-T
ABSTRACT
The herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) has been shown to
be teviitogenic and fetocidal in two strains of mice using either subcutaneous
or oral routes of administration, and in one strain of rats by oral administration. The incidences of both cystic kidney and cleft palate were increased in
the G57BL/6 mice as well as the incidence of cleft palate in the AKR mice.
The incidence of cystic kidney was also increased in the rats. In addition, an
increase in liver to body weight ratio in the mouse fetus and the occurrence of
hcmorrhaglc gastrointestinal tract in the rat fetus suggest that this compound
also has fetotoxic properties.
The chlorinated.herbicide 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) is used
extensively for weed control (1). However, there have been relatively few
reports concerning its pharmacologic and toxicologic properties in animals
(2,3). Indeed, there are no data available concerning the effects of this compound on the developing embryo and fetus1. Therefore, this report evaluates the
teratogenic and fetotoxic potential of 2,4,5-T in mice and rats (4).
Breeding colonies of O57BIJ/6 and AKH strains of mice were established at
Bionetics Research Laboratories, Inc., to supply the mice. For the study, breeding was by random mating. Detection of a vaginal plug indicated day zero of
pregnancy. Rats were procured from the Holtzman Co., with knowu insemination dates. • Detection of sperm indicated day zero of pregnancy. All animals
received chow and water ad libitum.
2,4,5-T (5) was administered by one of two routes, subcutaneously or orally.
A solution of 2,4,6-T in 100% dimethylsulfoxide (DMSO) in a volume of 100
/il/inouse was used for each subcutaneous administration. For oral administration by gastric intubation, 2,4,5-T was suspended in a honey solution (honey:
water, 1:1) and volumes of 100 /jl/mouse and 200 /d/rat were used.
Jn the studies with the C57BL/6 strain, 2,4,6-T was administered daily
beginning on the sixth day of pregnancy and continuing through the 14th day
or from the Oth through the 17th day. The mice were sacrificed on the 18th
day of gestation for examination. In the studies with the AKR strain, 2,4,5-T
was administered daily beginning on the 6th day of pregnancy and continuing
through the 15th. These mice were sacrificed on the 19th day of gestation. The
rats were treated on the 10th through the 15th and sacrificed on the 20th day
of gestation.
Upon sacrifice both mothers and fetuses were examined carefully. In addition, about one-third of the mouse fetuses were stained with alizarin red S in
order to detect skeletal anomalies.
Tables 1 through 3 contain data on fetal mortality, abnormal litters, abnormal fetuses per litter, fetuses with cleft palate, fetuses with cystic kidney,
maternal weight gain, and maternal and fetal liver to body weight ratios. The
following conventions were observed in compiling these data. If a fetus was
either dead or resorbed, it was regarded as a dead fetus. A fetus was classified abnormal if it was alive and had at least one anomaly (regardless of
type). Similarly, a litter was classified ns abnormal if it contained one or more
abnormal retuses. A fetus was said to have a cystic kidney if at least one of
its kidneys was affected. In calculating the maternal liver/body weight ratio,
maternal body weight was defined as the difference between the weight of the
animal on the day it was sacrificed and the gravid uterus weight. Finally, the
maternal .weight gain was defined as the difference in the corrected maternal
body weight on the day it was sacrificed and its weight on day zero of pregnancy.
The percentages for fetal mortality, abnormal fetuses, fetuses with cleft
palate and fetuses with cystic kidney were computed by first, obtaining the percent for each litter and then calculating the average of these percentages.
' The percentage of abnormal litters provides a measure of the prevalence of
abnormal fetuses across litters, while the percentage of abnormal fetuses per
litter gives an indication of the prevalence of abnormal fetuses within litters.
In this report, the control animals are those that were on a large study
during the 3-year time period in which 2,4,5-T was evaluated. The data from
( h o DMSO and honey treated control groups were compared with the data for
the non-treated control group. Then the results from animals treated with
2,4,5-T in either DMSO or honey were compared to the appropriate control
data. Standard corrected 2x2 chi-square tests (6) were used to compare the
results of 2,4,5-T treated animals with the appropriate cont^^data for the
proportion of litters containing abnormal fetuses.
VP
The distribution of the percent of abnormal fetuses per litter for 2,4,5-T
treated litters was compared with the appropriate control distribution by use
of the non-parametric Mann- Whitney U-test (6). Also, this test was used for
comparing the percent fetal mortality, cleft palate, cystic kidney, and enlarged
ronal pelvis per litter. This test requires that the proportion of dead or abnormal fetuses per litter is independent from litter to litter, but requires no
assumption about the frequency distributions of these proportions.
Initial analyses of the data indicated that occurrences of anomalies among
fetuses within litters were correlated. That is, anomalies were not randomly
distributed across all litters but tended to cluster within litters. Many litters
possessed no anomalies whereas all of the fetuses in some litters were abnormal. Since fetuses within the same litter tend to be more alike, pooling the
data across Utters before performing statistical tests is not appropriate. The
experimental unit (7) is that entity to which treatments are applied, in this
case the pregnant animal. Hence, all calculations of averages and all statistical tests were performed on the independent responses of the experimental
units (litters).
The administration of DMSO or honey to mice or rats did not adversely
affect the development of the fetuses. The incidence and type of naturally
occurring anomalies observed in the DMSO and honey treated animals did not
show an increase compared to the non-treated group. The alizarin stained
fetuses of the control mice showed very few skeletal anomolies. No skeletal
anomalies were detected by staining in the treated mice. For both mice and
rats, there were no differences in the average number of implantations in the
control and experimental litters. A few values for treated animals were less
than those of their appropriate controls. None of these differences were statistically significant including the 8% fetal mortality observed in the C57BL/0
mice receiving a 21.5 mg/kg dose of 2,4,5-T reported in Table 1. This value of
3% reflects a period of low fetal mortality (9%) observed in the control mice
during the initial few mouths of the study. This difference in mortality is not
statistically significant. There were no other significant changes in these control data during the 3-year period.
As shown in Table 1, the administration of 2,4,5-T to C57BL/6 mice on days
0-14 at a dosage level of 113 mg/kg produced significant increases in percent
of abnormal litters and percent of abnormal fetuses per litter. The anomalies
produced by 2,4,5-T were almost exclusively cystic kidney and cleft palate.
Similar results were obtained regardless of whether the compound was administered subcutaneously or orally. A dosage level of 46.4 mg/kg administered
orally did not produce a significant increase in fetal mortality or an effect on
palatal development, but did cause a significant increase in the percentages of
fetuses with cystic kidney. Administration of 2,4,5-T subcutaneously at a
dosage level of 21.5 mg/kg did not affect the visability or development of the
fetuses. Thus, a dose-response relationship for the fetocidal and teratogenic
properties of 2,4,5-T in mice is suggested for both routes of administration.
It was also observed that in mice treated with 2,4,6-T on days 6 through 14,
there was a significant decrease in the incidence of naturally occurring anomalies. These consist of microphthaliuia followed by anophthalmia and are in
accord with other O57BI>/6 colonies (8). Although the fetuses from mice
treated on the C-14th days had fewer naturally occurring anomalies, the
fetuses from mice treated on the 9th to 17th days did exhibit these anomalies.
Thus, it appears that the interval of days 6 to 9 of gestation is one of the sensitive periods of development with respect to 2,4,5-T. Two other sensitive
periods are during development of the palate and kidney since they are so
highly affected. The occurrence of these two anomalies are statistically
unrelated.
In the study where 2,4,5-T was administered on the 9th to the 17th day of
gestation with the C57BL/6 mice, maternal and fetal liver weights were determined. As seen in Table 2, this treatment produced a significant increase in
maternal and fetal liver to body weight ratios. The significant increase in fetal
liver to body weight ratio reflects both an increase in fetal liver, weight and a
decrease in fetal body weight ,The significant increase in the liver to body
weight ratio suggests a change in activity of drug metabolizing enzymes of the
�101
100
|mdoplasmic retlculum which has been studied (9). Again, the Mann-'fl^Bey
J-test was used to compare the animals administered 2,4,5-T with the appropriate DMSO control mice.
Thus, in the C57BL/6 mice, 2,4,5-T is fetocidal, teratogenic and capable of
producing an increase in the liver to body weight ratios.
Treatment of mice of the AKR strain with 2,4,5-T in honey produced a significant increase in fetal mortality. The incidence of cleft palate was increased
with both routes of administration. However, 2,4,5-T did not produce an
increased incidence of cystic kidney in this strain. There was no effect of
2,4,5-T administration in this strain on the maternal weight gain with either
route of administration. However, the maternal liver to body weight ratio was
increased using either route of administration.
In addition, hybrid litters resulting from mating C57BL/6 females with
AKR males were evaluated. The administration of 113 mg./kg in DMSO from
days 6 through 14 of gestation produced a high incidence of both cystic kidney
and cleft palate. There was no effect on maternal weight gain.
The oral administration to rats of 2,4,5-T at a dosage level of 10.0 or 46.4
mg/kg on the 10th through the 15th day of gestation produced a significant
increase in fetal mortality (Table 3). The two lower dosage levels, 4.6 and 10.0
mg/kg produced a significant increase in the percentage of abnormal fetuses.
These fetuses displayed a high incidence of cystic kidney. At the highest dose
level, 46.4 mg/kg, the marked increase in fetal mortality reduced the population of live fetuses to a small sample. However, cystic kidneys were observed.
In a limited study, the administration of 2,4,5-T at dosage levels of 21.5 or
40.4 mg/kg from the 6th through the 15th day of gestation was highly fetocidal.
At all dosage levels studied in the rat, hemorrhagic gastrointestinal tracts
were observed in the fetuses. The percentages of fetuses per litter with hemorrhagic gastrointestinal tracts showed a dose-reponse relationship; i.e., 3%,
50%, and 83% at,doses of 4.6, 10.0 and 46.4 mg/kg, respectively. None were
observed in the fetuses from the control animals. Drill and Hiratzka (2) have
reported that dogs which received 2,4,5-T in the diet showed some necrosis and
inflammation of the intestinal mucosa. The hemorrhagic gastrointestinal tracts
observed in the rat fetuses is probably a toxic effect of 2,4,6-T on the fetal
organ as opposed to a developmental defect.
In conclusion, these studies show that 2,4,5-T adversely affects the development and viability of the mouse and rat fetus.
TABLE l.-TERATOGENIC EVALUATION OF 2,4,5-T IN MICE
Average
number
Compound
Vehicle
Dose
of
(mg/kg) litters
Percent
fetuses/ mortality/ abnormal
litter
litters
litter
Percent of fetuses per
Percent
litter with—
i~i
fetuses/ Cleft palate
Cystic
kidney
litter
C57VL/6 STRAIN TREATED DAYS 6-14
Nontreated..
Control
Control
2,4,5-T
2,4,5-T
2,4,5-T
2,4,5-T
,
. None...
. DMSO..
. Honey..
DMSO..,
DMSO.,
Honey...
...do....
Nontreated..
Control
2,4,5-T
. None..
None
None
None
72
21.5
106
32
6
46.4
18
6
1)3,0
113.0
12
5.8
5.5
?:i
7.7
4,4
8.5
4.8
26
29
15
3
42
41
50
42
8
147
i 100
> 100
38
»8S
11
12
14
12
" 57
237
"70
1
2
1
0
0
0
«22
2
"23
»4J
i 33
I 48
7
0
>60
C57BL/6 STRAIN TREATED DAYS 9-17
. DMSO..
DMSO..
None
None
113.0
10
10
5.1
6.1
7.7
36
23
11
! 100
!77
0
0
"29
19
24
0
5
4
0
329
3 55
<1
<1
0
»28
' 55
71
30
31
8
AKR STRAIN TREATED DAYS 6-15
Nontreated
Control
Control
2,4,5-T
2,4,5-T
None
DMSO
Honey
DMSO
Honey
None
None
. None
113.0
113.0
> Statistical Significance Levol=0.10;
58
72
12
14
7
7.1
6.9
8.8
6.9
5.3
16
15
9
23
M2
i Statistical Significance Level°»0.05;
3 71
» 100
<1
<\
(\
1
0
> Statistical Significance Level=O.OI.
iOUSLY IN DMSO
W8U Z.-LIVER WEIGHT STUDY: 2,4,5-T ADMINISTERED DAILY AT 113 MG/KG
FROM THE 9TH THROUGH THE 17TH DAY OF GESTATION IN
Maternal
Fetal
Treatment
Liver wt.
Body wt.
(8ms)
(8ms)
Nantiealed D«SO
M.S-T...
.810
.818
. 046
J.057
' Slatlslical significance level = 0.10.
(6ms)
Liver wt./
Body wt.
6.00
5.99
4.65
«.120
Wt. gain
.058
.056
1.738
.047
.
Liver wt./
Body wt.
".076
»Statistical significance level = 0.05.'
.069
.068
»Statistical significance level = 0.01.
TABLE 3.-TERATOGENIC EVALUATION OF 2,4,5-T IN RATS
Compound
Nontreated
Control
2,4,0-T
Z.45-T
M.5-T
1
Vehicle
None
. Honey
do
do.
do
Number
of
Dose
(mg/kg) litters
None
None
4.6
10.0
46.4
Statistical Significance Level = 0.10.
7
14
8
7
6
Average
number Percent
fetal Percent abnormal
live
fetuses/ mortality/ abnormal fetuses/
litter
litters
litter
litter
9.9
8.7
8.2
7.1
2.7
11
1
12
128
'59
s Statistical Significance Level = 0.05.
43
57
88
86
67
9
12
»36
'46
60
Percent of feluses per
litter with—
Enlarged
renal
pelvis
Cystic
kidney
9
12
18
17
27
0
<1
»21
»30
33
a Statistical Significance Level=0.01.
HEFERENCES AND NOTES
(1) Audus, Ii. ,T., The Physiology and Biochemistry of Herbicides, Academic
Tress, New York, 1964.
(2) Drill, V. A. and Hiratzka, T., Arch. Industrial Hygiene Occupational
Mca. 7, 61, 1953.
(3) Howe, V. 1C. and Hymas, T. A., Am, J. Vet. Res. 15, 622, 1954.
(4) These results are from a large study designed to screen selected compounds for teratogenic effects in mice which was performed at the Bionetics
Research Laboratories, Division of Litton Industries, under contract numbers
I'll 43-64-57 and PH 43-67-735 from the National Institutes of Health.
During the performance of this study, Dr. Courtney was a staff member of the
Bionetics Research Labs., Inc., and Dr. Falk was a member of the National
Cancer Institute.
(5) 2,4,5-T was produced by the Diamond Alkali Co., 98%, Tech., m.p. 149151°.
(6) Snedecor, G. W. and Cochrau, \V. G. Statistical Methods, 6th ed., Iowa
'State Univ. Press, Ames, Iowa, 1967.
(7) Kempthorne, O., The Design and Analysis of EsDperime^lts, Wiley, N.Y.,
1052.
(8) Kalter, H., Teratology 1, 193, 1968.
(9) Courtney, K. D. (In preparation).
Note added in proof:
The sample of 2,4,5-T used in this study contained approximately 30 ppm of
2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) (10). Dioxin as well as purified
2,4,5-T are currently being evaluated for their teratogenic and fetotoxic potential.
(10) We thank Dow Chemical Co., for the analysis of 2,4,5-T.
K. Diane Courtney
D. W. Gaylor
M. D. Hogan
H. L. Falk
National Institute of Environmental Health Sciences, National Institutes of
Health, Post Office Box 12233, Research Triangle Park, N.C..27709
R. R. Bates
I. Mitchell
National Cancer Institute
National Institutes of Health
Rethesda, Md. 20014
�102
103
Senator HAIIT. Are there any additional questions?
Mr. BICKWIT. No, Mr. Chairman.
Senator HAHT. If any arise we will submit them in writing and
receive the replies in the record.
I appreciate the cooperation of everyone through the day, and
apologize for the erratic scheduling this afternoon.
(The following was subsequently received for the record:)
Saigon's leading maternity hospital, Tudu, from which rum^Af an increase
of abnormal births emanate periodically, has not even compilerKnnual reports
of statistics for the last three years. Recent monthly figures show an average
oC about 140 miscarriages and 150 premature births among approximately 2,800
pregnancies, but the hospital is not prepared to say whether this represents nn
Increase and, if so, what the cause might be.
A high Agriculture Ministry official said: "I don't think the Aznericnns
would use the chemicals if they were harmful."
He conceded that his ministry had made no tests and asserted that his
experts had been unable to get any information about the defoliants from the
Defense Ministry, which considers such data secret The main defoliant compounds and some information about them are available in the United States.
Last Oct. 29, President Nixon's science adviser, Dr. Lee A. Du Bridge,
announced that as a result of a study showing that one of the defoliants used,
2,4,5-T, had caused an unexpectedly high incidence of fetal deformities in mice
and rats, the compound would henceforth be restricted to areas remote from
population.
That directive appears to be ambiguous in South Vietnam for military
spokesmen assert that 2,4,5-T continues to be used only in "enemy staging
areas"—by definition populated regions.
Appendix 1
U.S. SHOWS SIGNS OF CONCERN OVER EFFECT IN VIETNAM or 9-YEAR
DEFOLIATION PROGRAM
(By Ralph Blumenthal, special to the New York Times)
SAIGON, South Vietnam, March 14—Many South Vietnamese who live adjacent to areas that are being defoliated by spray from United States planes are
convinced that any ailments or misfortunes that they suffer are related to the
sprayings.
There is no proof that they are right about the effect of the chemical sprays
on the human body, but neither is there any assurance that they are wrong.
Although the defoliation program, organized and run by the United States,
has been in operation for nearly nine years the full effect of the chemicals on
. animal and human life remains largely undetermined.
The United States military command says the program, which is designed to
strip plant cover from areas occupied by the enemy and to destroy crops that
might yield him food, has covered about 5,000 of South Vietnam's 60,350
square miles.
U.S. TERMS IT VALUABLE
The United States command says the program has proved its military worth.
"It has contributed materially to the security of units operating in the field by
increasing their visibility from the ground as well as the air," the command
said.
About 13 per cent of the program has been directed against crops, presumably food grown by and for the enemy. Because of the drifting of defoliants
and the difficulty of assessing the results on the ground, it is virtually impossible to say how much of the crop has been destroyed by the chemicals, but it
would not appear to be a significant part of the country's capacity. It has
brought hardships, however, to individual farmers.
After yeiirs of assuring the South Vietnamese that this extensive spraying
was harmless to animals and humans, United States officials are showing signs
of concern over recent reports the the chemical sprays may have some littleunderstood and alarming effects.
, ,
,
PANEL STUDYING EFFECTS
In the last several months, reportedly on instruction from Washington, the
United States military command and the United States Embassy have formed
a special committee to review the effects of the defoliation program, especially
on humans.
The sensitivity of the issue has foreclosed official comment, but according to
informed sources the science advisory office of the command is responsible for
gathering data in interviews and tests that embassy officials will then evaluate,
The South A'ietnamese Government regards the entire subject as taboo. Vietnamese newspapers have been suspended for publishing articles about birth
defects allegedly attributed to the defoliants, and the public Health Ministry
declines to provide any statistics on normal and abnormal births.
However, the concern felt among the Americans is shared by many South
Vietnamese scientists, physicians, health officials and villagers interviewed in a
throe-week survey of the effects of the program.
Officers of the United States command are aware of the allegations of birth
defects but they generally discount the reports.
KoHponsible South Vietnamese scientists and officials say they know virtually
nothing about the effects of the chemical sprays.
DEFOLIANTS WERE CONCEALED
Don That Trinh, Minister of Agriculture from November, 1967 to May, 1008,
nnd for 10 years professor of agronomy at Saigon University, said that while
he was minister, the Defense Ministry "would try to conceal the defoliant
products from me."
"I did not believe in defoliation," he added.
According to one of the Vietnamese directors of a Government research laboratory in Saigon: "We didn't know anything before the United States started
spraying. It wsts only when we received complaints from the livestock people
that we started getting interested." But, he added, there are still no Vietnamese studies.
Even the village of Tanhiep, 20 miles north of Saigon, on which 1,000 gallons of defoliants were jettisoned on Dec. 1, 1968, has not been the object of
attention or study.
An American O-123 flying out of Bienhoa air base, Northeast of Saigon,
developed engine trouble shortly after takeoff. To lighten the craft, the pilot
sprayed the full load of chemicals over Tanhiep and nearby Binhtri in 30 seconds instead of the usual 4 minutes 30 seconds, which spreads the defoliant nt
the rate of three gallons an acre in unpopulated areas.
The defoliant involved, according to the United States command, was a 50-60
mixture of 2,4-Dichlorophenoxyacetate, or 2,4-D, and 2,4,5-Trichlorophenoxyacetate, or 2,4,5-T, in an oil base. It is one of three compounds the
' military says it uses here, the others being a Dow chemical product called
Tordon 101, a mixture of arnine salts of 2,4-D and Picloram, and an arsenic
compound of cacodylic acid.
No physicians visited Tanhiep to examine the people after their exposure,
which, like eight similar emergency dumpings since 1968—some over unpopulated forests—was not made public by the United States command.
A United States Air Force medical team visited Binhtri shortly after the
spraying and, according to American district officials, found the villagers had
suffered no ill effects. There was no later inquiry.
Mrs. Trail Thi Tien of Tanhiep, hwo says she has four normal children, is
convinced that the malfunction of her son, who still looks like a newborn at 14
months of age, "must be due to I ho, chemicals I breathed."
Her neighbors, Mrs. Nguyen Thi Hai and Mrs. Tong Thi An, blame the
spraying for the fact that their children, one year and 20 months old respectively, still crawl instead of walk.
Nguyen Van Nhap, a farmer, complains of suffering bouts of fever, sneezing
and weakness.
"I was working in the field when the spray came down," Mrs. Tien said
through an interpreter. "I felt dizzy, like vomiting and had to' stay in bed
three or four days."
i Many other villagers reported feeling the same sensations as Mrs. Tien,1 but,
except for the two children described as retarded in learning to walk, no other
abnormal children were described to visitors at the village of 1,200 residents.
�104
Tran Van Dang, a farmer in neighboring Binhtri, recalled that .,.,
after the spraying two villagers, Tarn Ten and Mrs. Hal Mua, died aOTr suffering respiratory difficulties and trembling. The next day, he said, a third villager, Mrs. Hai Nuc, died after showing similar symptoms. Mr. Ten was an old
man and could have been expected to die soon anyway but the two others, Mr.
Dang said, were middle-aged and seemed healthy.
Such complaints are not limited to Tanhiep and Binhtri, where villagers
were admittedly exposed to concentrated doses of defoliant—though just how
concentrated has not been established.
In Bienhoa city, 10 miles from Tanhiep, any defoliant in the air drifts down
from the heavily sprayed battle areas to the north.
Dr. Nguyen Son Oao says he finds a clear correlation between the days when
there is spraying and the number of patients who come in with respiratory ailments, mostly sneezing and coughing.
Dr. Cao, who has been practicing in Bienhoa for 21 years, said he had also
noticed that in the last two to three years the number of miscarriages among
his patients had doubled.
"These women are convinced they are the victims of the chemicals," he said.
"I only suspect there could be a relationship. This suspicion is very well
known. The increase in miscarriages is very obvious, very significant."
However, the manager of another clinic reported no increase in miscarriages
over the last several years.
Any increase in miscarriages has many possible explanations: perhaps tlie
deterioration of the daily diet, the cumulative effect of the hardships of war,
population and economic movements that register statistics of only certain
groups, or air pollution, of which the defoliant chemicals are a part.
Appendix 2
DEFOLIANTS, DEFORMITIES: WHAT RISK?
Dr. Jackie- Vorrctt is fascinated and horrified by what has now become an
everyday sight at her FDA toxicology lab in Washington, D.O.: several white
leghorn chicks struggling to get to their feet and then finally walking—on then
knees. Besides slipped tendons in their legs, some of the chicks have cleft palates
and beak deformities. All this has been wrought by injecting fertilized eggs with
an etlinnol solution containing just 2.5 micromicrograms (or 50 parts per trillion)
of 2,3,6,7-totrachlorodibenzo-p-dioxin, a contaminant in 2,4,5-trichlorophenoxyaceticacid (2,4,5-T).
Over the past nine years, 40 million pounds of this defoliating herbicide
have been sprayed in very heavy concentrations across at least five million
acres of Vietnam to destroy crops and expose the enemy. By MWN'S reckoning,
some 30 million pounds have been spewed out in lesser concentrations during
just the past five years across perhaps 30 million acres of range, forest, and
farmland (not to mention home gardens) in the TJ.S—an area three times the
size of Texas.
Thus, Dr. Verrctt's preliminary findings are not just of interest to poultrymen. The 11 crippled chicks in her study were among 15 survivors of a clutch
of 25 eggs. In the unhatched chicks, Dr. Verrett found pronounced evidence of
chick edema—swollen tissues, cysts on the back, necrotic livers, and the same
deformities the live birds have. The FDA researcher is diluting the dioxin content to try to find a "no effects" level. In another brood, she has produced a
similar pattern of birth defects with just 2% parts per trillion of dioxin,
1/400,000 the 1 ppm found in currently marketed products. Now she's experimenting with .25 parts per trillion. (The work is so politically sensitive that
she doesn't even know the origin of the 2,4,5-T involved and feels "like I'm in
the CIA.")
When told that HEW Secretary Robert Finch is doubtful about the applicability of the chick embryo work to human risk, Dr. Verrett snapped, "I know,
I know, but the only time Bob Finch sees eggs is when he eats them for
breakfast"
While Dr. Verrett labored in the lab early this month, Dr. Samuel Epstein,
• chief of toxicology at Children's Hospital Medical Center in Boston, was out in
Globe, a foothill town in southeastern Arizona, to evaluate reports of toxic and
&
sK
105
^
' tnTYitr>K<Miic effects attending the spraying of 2,4,5-T and its cj^Bcal cousin, 2: T.M,r>-Trichloroplienoxy) proprionic Acid (Silvea;) in adjacenWonto National
These reports have disturbed the nation and drawn experts to the
ODD EFFECTS AROUND GLOBE
la Globe, Dr. Epstein saw two goats and a duck with leg deformities similar •
,ln those in Dr. Verrett's chickens, and studied the histories of sick people.
"H's Impossible to say for certain whether the claimed symptoms and effects
art1 attributable to the spray," he said. But at the same time he lashed out at
the U.S. Forest Service for risking the contamination of water sources against
tl* own policy, for contributing to drift by using water as a 2,4,5-T solvent,
i mid for failing to post the area before spraying.
MWN found that the Department of Agriculture keeps such casual tabs of
fJM.fi-T spraying that it would take officials a week just to find which of the 33
national forests besides Tonto have been bombed with the two million pounds
Forest Service,has jetted out over the past six years. "But Interior uses more
tlum we do," said one official. Replies an Interior spokesman, "We used only
44,232 pounds last year."
In the Globe area, the Forest Service has sprayed 2,4,5-T and Silvea; four of
Hie past five years to promote growth of grass in a burned-over section and to
eliminate chaparral. But most 2,4,5-T use is unmonitored. The defoliant is
bought by ranchers and private foresters and it's pretty much up to them
what happens to it.
Human teratogenicity is the chief worry; it is fairly well known by now
that Dr. Verrett's work is not the first study to dramatize the risk. Yet MWN
leained that the U.S. doesn't keep nationwide birth-defect figures.
Dr. Edward Burger of the government's Office of Science and Technology
dons not seem worried by this absence of monitoring and supervision, nor,
Indeed, about the risk of 2,4,5-T teratogenicity. Dr. Burger, technical assistant
to Presidential science advisor Lee A. DuBridge, acknowledges that a study
done by Bionetics Research Laboratories for the National Cancer Institute
showed last March (it was suppressed for six months) that nearly all
offspring of mice and rats given 2,4,5-T early in gestation at the relatively
high levels of 21.5 mg/kg or 46.4 mg/kg were born dead or deformed—in some
cases with no eyes, with cleft palates, and cystic kidneys and enlarged livers.
Kven at 4.6 rng/kg dosage, 39% of the animals were born malformed.
The OST expert is more familiar than most with the high-level decisionmaking that went into Dr. DuBridge's declaration October 29 that on the basis
of the Bionetics study, the use of 2,4,5-T in populated areas would be
restricted. Dr. DuBridge said Agriculture would, by Jan. 1, 1970, withdraw
licenses for its use on crops (corn, bluberries, peaches, pears, and several leafy
vegetables) unless the FDA found that the residue was negligible and humans
were tolerant of it.
Dr. Burger explains that the FDA missed this deadline for a number of reasons. First, Dow Chemical Co., a major maker of 2,4,5-T, discovered last
December that the sample used by Bionetics contained 27 ppm of the tetrachloro dioxin instead of the "less than 1 ppm" Dow says is in its product. So the
study is now being re-run with a Dow sample at Dow labs in Zionsville, Ind.,
and Midland, Mich., and at the National Institute Environmental Health Sciences.
Next, says Dr. Burger, even after the teratogenic potential is re-evaluated in
a rodent model, the disappearance rate of the contaminant in the animal blood
stream must be determined and calibrated with that in human volunteers. He
concludes: "The possibility of exposure to 2,4,5-T, vis-a-vis the small teratogenic risk, is, certainly not sufficient at this time to justify wiping the chemical
off the market."
Comments Associate FDA Commissioner for Science Dale Lindsay: "Dr.
DuBridge had no damned business setting a tolerance deadline. Our marketbasket surveys for 1968 and 1969—thousands of samples of 120 foods and vegetables are constantly being assessed—show only five recoveries of 2,4,5-T—
three from leafy vegetables at negligible levels, plus one from, v
milk, and one
from meat at the .01-mg level.
"Yet if we Had to set a tolerance today it would be zero. The trouble with
this very active dioxin contaminant is that while it may be a known quantity
in a product, you can't extract it in the same quantity."
�107
106
w
Harvard microbiologist Matthew Meselson is worried for the same reasonand many others. Dr. Meselson—appointed last year by the American Associa
tion for the Advancement of Science to head a 2,4,5-T evaluation project—
says: "The tetrachloro dioxin represents just one of 12' or 13 ways the
chlorine atoms can arrange themselves on a benzene ring to form dioxin molecules. How do we know about the hexa, hepta, and octychlors, or about how
persistent the tetrachlor itself is? Moreover, I'm very concerned about the
dioxins that might be formed by vmreacted trichlorphenol [2,4,5-T precursor]
when the product is exposed to heat If it were taken up by plants or wood
and these were burned, you'd get more dioxin. Finally, I'm bothered by the
bizarre mental effects suffered by German workers making 2,4,5-T. I say when
in Dr. Julius Johnson, vice president and director of research for Dow, regards
doubt, stop it."
these concerns as speculative. "If we thought 2,4,5-T was harming anybody
we'd take it off the market tomorrow," he says. "We've been dedicated to
cleaning it up ever since 1964 [when the contaminant was linked to an outbreak of chlor-acne in Dow workers at Midland]." Dr. Johnson says it would
take a 200-degree jolt to produce reaction- of dioxin, and the contaminant disappears within hours under ultraviolet light. So far, he adds, Dow tests show
that its, 2,4,5-T has no teratogenic effect on rats at a dosage of 24 mg/kg and
on rabbits at 40 mg/kg. But how about Dr. Verrett's new findings in the chick
embryo test? The Dow executive confesses surprise. "But I'm confident," he
says, '"that we'll be safe when we propose a new specification for all 2,4,5-T
products of .1 ppm of dioxin." of teratogenicity in the population, however.
Safety also assumes gauges
FDA's Dr. Lindsay spoke with certitude when he told MWN that "the National
Institute of Neurological Diseases and Stroke has recorded birth defects for
some 15 years and would be telling us if they were on the rise." He's wrong.
Dr. Heinz Berendes, chief of KINDS' perinatal research branch, admits dolefully that "no nationwide data are available on frequency or incidence of malformation." biologist Arthur Galston: "It's shocking, but absolutely no studies
Adds Yale
have been made in Vietnam either. There have been reports of birth defects in
Saigon papers since last June but hospital records haven't been made available." Department' officials say they know of no policy whereby such data
State
would be classified or withheld. Significantly, however, Dr. Malcolm Phelps,
chief of the Vietnam medical section of the Agency for International Development, says he is acting on a recent White House request to collect figures on
tcratological occurrences in Vietnam civilian hospitals.
As for all the toxic effects reported by Globe residents after the June 8-11
spraying—a helicopter released 935 gallons of Silvem, 30 of 2,4,5-T, and 20 gallons of a combination called "Orange" over 1,900 acres of forest;—an MWN
reporter inquired into the histories of 18 patients with four of the five doctors
who treated them, and checked on the two crippled goats, the crippled duck, a
bleeding bull terrier, and two other dogs with pneumonia. Net result: two
strongly suspected herbicide poisoning cases linked to the spraying, and one
"definite." There's one-year-old Paul McOray, who lives on the edge of Tonto
National Forest and whose father drove the family right up to the 'copter
landing spot during spraying. The boy has had respiratory attacks and convulsions. Phoenix pediatrician W. Scott Ohisholm finds Paul has lymphositosis,
with a white coll count twice normal.
The second suspected case, a smeltery worker named James Andrews who
has complained of a number of symptoms associated with herbicide poisoning
—nausea, muscle weakness, vertigo, numbness, and stabbing pain—ia vouchsafed by Dr. Granville Knight of Santa Monica, Calif. In the third case, that
of Mrs, Billee Shoecraft, Dr. Knight says he has found 2-4-D in tissue.
Dr. Bernard Collopy would not label the muscle spasms and stabbing pain
suffered by potter Hobert McKusick, owner of the defective goats and ducks,
ns herbicide-related. Dr. William Bishop would not credit the chest pains of
^<* McCray, father of little Paul, or bis wife's tingling fingers and toes, as
1
i,' ' %C;^ ov SitDcoj poisoning. And veterinarian EVI. Skinner hadn't seen any of
*,*^'v\ cases.
*«*>' wn»vXi'lJl> ^ lR bop: "There's a good possibility some of the human cases
"raying, but symptomatic connections aren't connections and
I'm no toxicologist. People here are emotional and each mornlMlrake up with
new nails pounded into their palms. What's needed is solid scientific investigation. All I hope is they don't leave us hanging in the air for the next 20
years."
Appendix 3
[From the New Yorker, Feb. 7, 1070]
A REPOBTEB AT LARGE: DEFOLIATION
By Thomas Whiteside
Late in 1961, the United States Military Advisory Group in Vietnam began,
as a minor test operation, the defoliation, by aerial spraying, of trees along
the sides of roads and canals east of Saigon. The purpose of the operation was
to increase visibility and thus safeguard against ambushes of allied troops and
make more vulnerable any Vietcong who might be concealed under cover of
the dense foliage. The number of acres sprayed does not appear to have been
publicly recorded, but the test was adjudged a success militarily. In January,
1962 following a formal announcement by South Vietnamese and American
officials that a program of such spraying was to be put into effect, and that it
was intended "to improve the country's economy by permitting freer communication as well as to facilitate the Vietnamese Army's task of keeping these
avenues free of Vietcong harassments," military defoliation operations really
got under way. According to an article that month in the New York Times, "a
high South Vietnamese official" announced that a seventy-mile stretch of road
between Saigon and the coast was sprayed "to remove foliage hiding Communist guerrillas." The South Vietnamese spokesman also announced that defoliant chemicals would be sprayed on Vietcong plantations of manioc and sweet
potatoes in the Highlands. The program was gathering momentum. It was
doing so in spite of certain private misgivings among American officials, particularly in the State Department, who feared, first, that the operations might
open the United States to charges of engaging in chemical and biological warfare, and second, that they were not all that militarily effective; Roger Hilsman, now a professor of government at Columbia University, and then Director of Intelligence and Research for the State Department, reported, after a
trip to Vietnam, that defoliation operations "had political disadvantages" and,
furthermore, that they were of questionable military value, particularly in
accomplishing their supposed purpose of reducing cover for ambushes. Hilsman
later recalled in his book, "To Move a Nation," his visit to Vietnam, in March,
1062; "I had flown down a stretch of road that had been used for a tost and
found that the results were not very impressive. , . . Later, the senior Australian military representative in Saigon, Colonel Serong, also pointed out that
defoliation actually aided the ambushers—if the vegetation was close to the
road those who were ambushed could take cover quickly; when it was
removed the guerrillas had a better field of fire." According to Hilsman, "The
National Security Council spent tense sessions debating the matter."
Nonetheless, the Joint Chiefs of Staff and their Chairman, General Maxwell
Taylor, agreed that chemical defoliation was a useful military weapon. In
1962, the American military "treated" 4,940 acres of the Vietnamese countryside with herbicides, In 1963, the area sprayed increased five-fold to a total of
24,700 acres. In 1964, the defoliated area was more than tripled, In 1965, the
1964 figure was doubled, increasing to 155,610 acres. In 1966, the sprayed area
was again increased fivefold, to 741,247 acres, and in 1967 it was doubled once
again over the previous year, to 1,486,446 acres. Thus, the areas defoliated in
Vietnam had increased approximately three' hundredfold in five years, but now
adverse opinion among scientists and other people who were concerned about
the effects of defoliation on the Vietnamese ecology at last began to have a
braking effect on the program. In 1968, 1,267,110 acres were sprayed, and In
1969 perhaps a million acres. Since 1962, the defoliation operations have covered almost five million acres, an area equivalent to about twelve per cent of
the entire territory of South Vietnam, and about the size of the'state of Massachusetts. Between 1902 and 1967, the deliberate destruction of plots of rice,
manioc, beans, and other foodstuffs through herbicidal spraying—the 1 word
"deliberate" is used here to exclude the many reported instances of accidental
45-362—70
8
�108
'spraying of Vietnamese plots—increased three hundredfold, from an
741 acres to 221,312 acres, and by the end of 1909 the Vietnamese cropgrowing
area that since 1902 had been sprayed with herbicides totalled at least half a
million acres. By then, in many areas the original purpose of the defoliation
had been all but forgotten. The military had discovered that a more effective
way of keeping roadsides clear was to bulldoze them. But by the time of that
discovery defoliation had settled in as a general policy and taken on a life of
its own—mainly justified on the ground that it made enemy infiltration from
the North much more difficult by removing vegetation that concealed jungle
roads and trails.
During all the time since the program began in 1901, no American military
or civilian official has ever publicly characterized it as an operation of either
chemical or biological warfare, although there can be no doubt that it is an
operation of chemical warfare in that it involves the aerial spraying of chemical substances with the aim of gaining a military advantage, and that it is an
operation of biological warfare in that it is aimed at a deliberate disruption of
the biological conditions prevailing in a given area. Such distinctions simply
do not appear in official United States statements or documents; they were
long ago shrouded under heavy verbal cover. Thus, a State Department report,
made public in March, 1900, saying that about twenty thousand acres of crops
in South Vietnam had been destroyed by defoliation to deny food to guerrillas,
described the areas involved as "remote and thinly populated," and gave a
firm assurance that the materials sprayed on the crops were of a mild and
transient potency: "The herbicides used are nontoxic and not dangerous to
man or animal life. The land is not affected for future use."
However comforting the statements issued by our government during seven
years of herbicidal operations in Vietnam, the fact is that the major development of defoliant chemicals (whose existence had been known in the thirties)
and other herbicidal agents came about in military programs for biological
warfare. The direction of this work was set during the Second World War,
when Professor K. 3. Kraus, who then headed'the Botany Department of the
University of Chicago, brought certain scientific possibilities to the attention of
a committee that had been set up by Henry L. Stimson, the Secretary of War,
under the National Research Council, to provide the military with advice on
various aspects of biological warfare. Kraus, referring to the existence of hormone-like substances that experimentation had shown would kill certain plants
or-disrupt their growth, suggested to the committee in 1941 that it might be
interested in "the toxic properties of growth-regulating substances for the
destruction of crops or the limitation of crop production." Military research on
herbicides thereupon got under way, principally at Camp (later Fort) Detrick,
Maryland, the Army center for biological-warfare research. According to
George Merck, a chemist, who headed Stimson's biological-warfare advisory
committee, "Only the rapid ending of the war prevented field trials in an
active theatre of synthetic agents that would, without injury to human or
animal life, affect the growing crops and make them useless."
After the war, many of the herbicidal materials that had been developed
and tested for biological-warfare use were marketed for civilian purposes and
used by farmers and homeowners for killing weeds and controlling brush. The
most powerful of the herbicides were the two chemicals 2,4-dichlorophenoxyacetic acid, generally known as 2,4-D, and 2,4,5-trichlorpphenoxyacetic acid,
known as 2,4,5-T. The direct toxicity levels of these chemicals as they'affected
experimental animals, and, by scientific estimates, men, appeared then to be
low (although these estimates have later been challenged), and the United
States Department of Agriculture, the Food and Drug Administration, and the
Fish and Wildlife Service all sanctioned the widespread sale and use of both.
The chemicals were also reported to be Shortlived in soil after their application. 2,4-D was the bigger seller of the two, partly because it was cheaper, and
suburbanites commonly used mixtures containing 2,4-D on their lawns to control dandelions and other weeds. Commercially, 2,4-D and 2,4,5-T were used to
clear railroad rights-of-way and power-line routes, and, in cattle country, to
get rid of woody brush, 2,4,5-T being favored for the last, because it was considered to have a more effective herbicidal action on woody plants. Very often,
however, the two chemicals were used in combination. Between 1945 and 1903,
(lie production of herbicides jumped from nine hundred and seventeen thousand pounds to about a hundred and flfty million pounds in this country;
109
bercent—more
since 1903, their use had risen two hundred and seventy-c^^perc
than double the rate ~~ increase^ in the use of pesticides, thouiHpesl
i.imu wumj.c •,«« *..*..~ of
- -.
pesticides are
Htlll far more extensively used. By 1900, an area equivalent to more than three
per cent of the entire United States was being sprayed each year with herbicides.
Considering the rapidly growing civilian use of these products, it is perhaps ,
not surprising that the defoliation operations in Vietnam escaped any significant comment in the press, and that the American public remained unaware of
the extent to which these uses had their origin in planning for chemical and
biological warfare. Nevertheless, between 1941 and the present, testing and
experimentation in the use of 2,4-D, 2,4,5-T, and other herbicides as military
weapons were going forward very actively at Fort Detrick. While homeowners
were using herbicidal mixtures to keep their lawns free of weeds, the military
were screening some twelve hundred compounds for their usefulness in biological-warfare operations. The most promising of these compounds were testsprayed on tropical vegetation in Puerto Rico and Thailand, and by the time
fullscale defoliation operations got under way in Vietnam the U.S. military
had settled on the use of four herbicidal spray materials there. These went
under the names Agent Orange, Agent Purple, Agent White; and Agent Blue—
designations derived from color-coded stripes girdling the shipping drums of
each type of material. Of these materials, Agent Orange, the most widely used
as a general defoliant, consists of a fifty-fifty mixture of n butyl esters and of
2,4-D and 2,4,5-T. Agent Purple, which is interchangeable with Agent Orange,
consists of the same substances with slight molecular variations. Agent White,
which is used mostly for forest defoliation, is a combination of 2,4-D and
Picloram, produced by the Dow Chemical Company. Unlike 2,4-D or 2,4,5-T,
which, after application, is said to be decomposable by micro-organisms in soil
over a period of weeks or months (one field test of 2,4,5-T in this country
showed that significant quantities persisted in soil for ninety-three days after
application), Picloram—whose use the Department of Agriculture has not
authorized in the cultivation of any American crop—is one of the most persistent herbicides known. Dr. Arthur W. Galston, professor of biology at Yale, has
described Picloram as "a herbicidal analog of DDT," and an article in a Dow
Chemical Company publication called "Down to Earth" reported, that in field
trials of Picloram in various California soils between eighty and ninety-six
and a half per cent of the substance remained in the soils four hundred and
sixty-seven days after application. (The rate at which Picloram decomposes in
tropical soils may, however, be higher.) Agent Blue consists of a solution of
cacodylic acid, a substance that contains fifty-four per cent arsenic, and it is
used in Vietnam to destroy rice crops. According to the authoritative "Merck
Index," a source book on chemicals, this material is "poisonous." It can be
used on agricultural crops in this country only under certain restrictions
imposed by the Department of Agriculture. It is being used herbicidally on
. Vietnamese rice fields at seven and a half times the concentration permitted
for weed-killing purposes in this country, and so far in Vietnam something like
five thousand tons is estimated to have been sprayed on paddies and vegetable
fields.
Defoliation operations in Vietnam are carried out by a special flight of the
12th Air Commando Squadron of the United States Air Force, from a base at
Bien Hoa, just outside Saigon, with specially equipped C-123 cargo planes.
Bach of .these aircraft has been fitted out with tanks capable of holding a
thousand gallons. On defoliation missions, the herbicide carried in these tanks
is sprayed from an altitude of around a hundred and fifty feet, under pressure, from thirty-six nozzles on the wings and tail of the plane, and usually
several spray planes work in formation, laying down broad blankets of spray.
The normal crew of a military herbicidal-spray plane consists of a pilot, a copilot, and a technician, who sits in the tail area and operates a console regulating the spray. The equipment is calibrated to spray a thousand gallons of
herbicidal mixture at a rate that works out, when all goes well, to about three
gallons per acre. Spraying a thousand-gallon tankload takes five minutes. In
an emergency, the tank can he emptied in thirty seconds—a fact that has particular significance because of what has recently been learned about the nature
of at least one of the herbicidal substances.
The official code name for the program is Operation Hades, but a, more
friendly code name, Operation Ranch Hand, is commonly used. In similar fash-.
ion, military public-relations men refer to the herbicidal spraying of crops sup-
�110
111
dly grown for Vietcong use in Vietnam, when they refer to it at all
rt-denial program." By contrast, an American biologist who is less
enthusiastic about the effort has called it, in its current phase, "escalation to a
program of starvation of the population in the affected area." Dr. Jean Mayer,
the Harvard professor who now is President Nixon's special adviser on nutrition, contended in an article in Science and, Citizen in 1967 that the ultimate
target of herbicidal operations against rice and other crops in Vietnam was
"the weakest clement of the civilian population"—that is, women, children, and
tlm elderly—because in the sprayed area "Vietcong soldiers may . . . be
expected to get the fighter's share of whatever food there is." He pointed out
that malnutrition is endemic in many parts of Southeast Asia but that in wartime South Vietnam, where diseases associated with malnutrition, such as
beri-beri, anemia, kwashiorkor (the disease that has decimated the Biafran
population), and tuberculosis, are particularly widespread, "there can be no
doubt that if the (crop-destruction) program is continued, (the) problems will
grow."
AVhether a particular mission involves defoliation or crop destruction, American military spokesmen insist that a mission never takes place without careful
consideration of all the factors involved, including the welfare of friendly
inhabitants and the safety of American 'personnel. (There can be little doubt
that defoliation missions are extremely hazardous to the members oC the
planes' crews, for the planes are required to fly very low and only slightly
above stalling speed, and they are often targets of automatic-weapons flre from
the ground.) The process of setting up targets and approving specific herbicidal operations is theoretically subject to elaborate review through two parallel
chains of command; one chain consisting of South Vietnamese district and >
province chiefs—who can themselves initiate such missions—and South Vietnamese Army commanders at various levels; the other a United States chain,
consisting of a district adviser, a sector adviser, a divisional senior adviser, a
corps senior adviser, the United States Military Assistance Command in South
Vietnam, and the American Embassy in Saigon, ending up with the American
ambassador himself. Positive justification of the military advantage likely to
be gained from each operation is theoretically required, and applications with
such positive justification are theoretically disapproved. However, according to
one of a series of articles by Elizabeth Pond that appeared toward the end of
1907 in the Christian Science Monitor:
"In practice, [American] corps advisers find it very difficult to turn down
defoliation requests from province level because they simply do not have
sufficient specific knowledge to call a proposed operation into question. And
with the momentum of six years' use of defoliants, the practice, in the words
of one source, has long since been "set in cement."
"The real burden of proof has long since shifted from the positive one of
justifying an operation by its [military] gains to the negative one of denying
an operation because of [specific] drawbacks. There is thus a great deal of
pressure, especially above province level, to approve recommendations sent up
from below as a matter of course."
Miss Pond reported that American military sources in Saigon were "enthusiastic" about the defoliation program, and that American commanders and
spotter-plane pilots were "clamoring for more of the same." She was given
firm assurances as to the mild nature of the chemicals used in the spray operations :
"The defoliants used, according to the military spokesman contacted, are the
same herbicides . . . as those used commercially over some four million acres
in the United States. In the strengths used in Vietnam they are not at all
harmful to humans or animals, the spokesman pointed out, and in illustration
of this he dabbed onto his tongue a bit of liquid from one of ... three bottles
sitting on his desk."
As the apparently inexorable advance of defoliation operations in South
Vietnam continued, a number of scientists in the United States began to protest the military use of herbicides, contending that Vietnam was being used, in
effect, as a proving ground for chemical and biological warfare. Early in 1966,
a group of twenty-nine scientists, under the leadership of Dr. John Edsall, a
professor of biochemistry at Harvard, appealed to President Johnson to prohibit the use of defoliants and crop-destroying herbicides, and called the use of
these substances in Vietnam "barbarous because they are indiscriminate." In
the late summer of 1006, this protest was followed by a letter of petition to
President Johnson from twenty-two scientists, including seven Ncbel laureates.
The petition pointed out that the "large-scale use of auticroij^M 'nonlethal'
antipersonnel chemical weapons in Vietnam" constituted "danglBHs precedent"
in chemical and biological warfare, and it asked the President to order it
stopped. Before the end of that year, Dr. Edsall and Dr. Matthew S. Meselson,
a Harvard professor of biology, obtained the signatures of five thousand scientists to co-sponsor the petition. Despite these protests, the area covered by
defoliation operations in Vietnam in 1967 was double that covered in 1900, and
the acreage of crops destroyed was nearly doubled.
These figures relate only to areas that were sprayed intentionally. There is
no known way of spraying an area with herbicides from the air in a really
accurate manner, because the material used is so highly volatile, especially
under tropical conditions, that even light wind drift can cause extensive
damage to foliage find crops outside the deliberately sprayed area. Crops are
so sensitive to the herbicidal spray that it can cause damage to fields and gardens as much as fifteen miles away from the target zone, Particularly severe
accidental damage is reported, from time to time, to so-called "friendly" crops
in the III Corps area, which all but surrounds Saigon and extends in a rough
square from the coastline to the Cambodian border. Most of the spraying in
til Corps is now done in War Zones 0 and D, which are classified as free fire
zones, where, as one American official has put it, "everything that moves in
Zones C and D is considered Charlie." A press dispatch from Saigon in 1967
quoted another American official as saying that every Vietnamese farmer in
tint*- corps area knew of the defoliation program and disapproved of it. Dr.
(Jalston, the Yale biologist, who is one of the most persistent critics of American policy concerning herbicidal operations in Vietnam, recently said in an
interview, "We know that most of the truck crops grown along roads, canals,
and trails and formerly brought into Saigon have been essentially abandoned
because of the deliberate or inadvertent falling of these defoliant sprays;
many crops in the Saigon area are simply not being harvested." He also cited
reports that in some instances in which the inhabitants of Vietnamese villages
have been suspected of being Vietcong sympathizers the destruction of food
crops has brought about complete abandonment of the villages. In 1966, herbicidal operations caused extensive inadvertent damage, through wind drift, to a
very large rubber plantation northwest of Saigon owned by the Michelin
rubber interests. As the result of claims made for this damage, the South Vietnamese authorities paid the corporate owners, through the American military,
nearly a million dollars. The extent of the known inadvertent damage to crops
in Vietnam can be inferred from the South Vietnamese budget—in reality, the
American military budget—for settling such claims. In 1967, the budget for
this compensation was three million six hundred thousand dollars. This sum,
however, probably reflects only the barest emergency claims of the people
affected.
According to Representative Eichard D. McCarthy, a Democrat from upstate
Now York who has been a strong critic of the program, the policy of allowing
applications for defoliation operations to flow, usually without question, from
the level of the South Vietnamese provincial or district chiefs has meant that
these local functionaries would order repeated sprayings of areas that they
had not visited in months, or even years. The thought that a Vietnamese district chief can initiate such wholesale spraying, in effect without much likelihood of serious hindrance by American military advisers, is a disquieting one
to a number of biologists. Something that disquiets many of them even more is
what they believe the long-range effects of nine years of defoliation operations
will be on the ecology of South Vietnam. Dr. Galston, testifying recently
before a congressional subcommittee on chemical and biological warfare, made
these observations:
"It has already been well documented that some kinds of plant associations
subject to spray, especially by Agent Orange, containing 2,4-D and 2,4,5-T, have
been irreversibly damaged. I refer specifically to the mangrove associations
that line the estuaries, especially around the Saigon River. Up to a hundred
thousand acres of these mangroves have been sprayed. . . . Some (mangrove
areas) had been sprayed as early as 1961 and have shown no substantial signs
of recovery. . . . Ecologists have known for a long time that the mangroves
lining estuaries furnish one of the most important ecological niches for the
completion of the life cycle of certain shellfish and migratory fish. If these
plant communities are not in a healthy state, secondary effects on tbo whole
«
�112
interlocked web of organisms are bound to occur. . . . In the years^fcead the
Vietnamese, who do not have overabundant sources of proteins a^P&w, are
probably going to suffer dietarily because of the deprivation Of food in the
form of fish and shellfish.
"Damage to the soil is another possible consequence of extensive defoliation.
. . . We know that the soil is not a dead, inert mass but, rather, that it is a
vibrant, living community. . . . If you knock the leaves off of trees once, twice,
or three times . . . you change the quality of the soil. . . . Certain tropical
soils—and it lias been estimated that in Vietnam up to fifty per cent of all the
soils fall into this category—are laterizable; that is, they may be irreversibly
converted to rock as a result of the deprivation of organic matter. . . . If ...
you deprive trees of leaves and photosynthesis stops, organic matter in the soil
declines and laterization, the making of brick, may occur on a very extensive
scale. I would emphasize that this brick is irreversibly hardened; it can't be
made back into soil. . . .
"Another ecological consequence is the invasion of an area by undesirable
plants. One of the main plants that invade an area that has been defoliated is
bamboo. Bamboo is one of the most difficult of all plants to destroy once it
becomes established where you don't want it. It is not amenable to killing by
herbicides. Frequently it has to be burned over, and this causes tremendous
dislocations to agriculture."
Dr. Fred H. Tschirley, assistant chief of the Crops Protection Research
Branch of the Department of Agriculture, who made a month's visit to Vietnam in the spring of 1968 in behalf of the State Department to report on the
ecological effects of herbicidal operations there, does not agree with Dr. Galston's view that laterization of the soil is a serioxis probability. However, he
reported to the State Department that in the Bung Sat area, southeast of
Saigon, where about- a hundred thousand acres of mangrove trees had been
sprayed with defoliant, each single application of Agent Orange had killed
ninety to a hundred per cent of the mangroves touched by the spray, and he
estimated that the regeneration of the mangroves in this area would take
another twenty years, at least. Dr. Tschirley agrees with Dr. Galston that a'
biological danger attending the defoliation of mangroves is an invasion of virtually ineradicable bamboo.
A fairly well-documented example not only of the ecological consequences of
defoliation operations but also of their disruptive effects on human life was
provided last year by a rubber-plantation area in Kompong Cham Province,
Cambodia, which lies just across the border .from Vietnam's Tay Ninh Province. On June 2, 1969, the Cambodian government, in an angry diplomatic note
to the United States government, charged the United States with major defoliation damage to rubber plantations, and also to farm and garden crops in the
province, through herbicidal operations deliberately conducted on Cambodian
soil, It demanded compensation of eight and a half million dollars for destruction or serious damage to twenty-four thousand acres of trees and crops. After
some delay, the'State Department conceded that the alleged damage might be
connected with' "accidental drift" of spray over the border from herbicidal
operations in Tay Ninh Province. The Defense Department flatly denied that
the Cambodian areas had been deliberately sprayed. Late in June, the State
Department sent a team of four American scientists to Cambodia, and they
confirmed the extent of the area of damage that the Cambodians had claimed.
They found that although some evidence of spray drift across the Vietnamese
border existed, the extent and severity of damage in the tiren. worst affected
were such that "it is highly unlikely that this quantity could have drifted over
the border from the Tay Ninh defoliation operations." Their report added,
"The evidence we have seen, though circumstantial, suggests strongly that
damage was caused by direct overflight." A second report on herbicidal
damage to the area was made after an unofficial party of American biologists,
including Professor E: W. Pfeifl'er, of the University of Montana, and Professor Arthur H. AVesting, or Windham College, Vermont, visited Cambodia last
December at the invitation of the Cambodian government. They found that
about a third of all the rubber trees currently in production in Cambodia had
been damaged, and this had happened in an area that normally had the highest latex yield per acre of any in the world. A high proportion of two varieties
of rubber trees in the area had died as a result of the damage, and Dr. Westing estimated that the damage to the latex-producing capacity of some varieties might persist for twenty years. Between May and November of last year,
113
latex production in the affected plantations fell off by^^iverage of between
thirty-five and forty per cent. According to a report b^^e two scientists, "A
large variety of garden crops were devastated in the seemingly endless number
of small villages scattered throughout tiie affected area. Virtually all of the . . .
local inhabitants . . . depend for their wellbeing upon their own local produce. These people saw their crops . . . literally wither before their eyes." The
Cambodian claim is still pending.
Until the end of last year, the criticism by biologists of the dangers involved
in the use of herbicides centered on their use in what were increasingly construed as biological-warfare operations, and on the disruptive effects of these
chemicals upon civilian populations and upon the ecology of the regions in whicli
they were used. Last year, however, certain biologists began to raise serious
questions on another score—possible direct hazards to life from 2,4,C-T. On
October 29th, as a result of these questions, a statement was publicly issued
by Dr. Lee DuBridge, President Nixon's science adviser. In summary, the
statement said that because a laboratory study of mice and rats that had been
given relatively high oral doses of 2,4,6-T in early stages of pregnancy
"showed a higher than expected number of deformities" in the offspring, the
government would, as a precautionary measure, undertake a series of coordinated actions to restrict the use of 2,4,5-T in both domestic civilian applications and military herbicidal operations. The DuBridge statement identified the
laboratory study as having been made by an organization called the Bionetics
Research Laboratories, in Bethesda, Maryland, but gave no details of either
the findings or the data on which they were based. This absence of specific
information turned out to be characteristic of what has been made available to
the public concerning this particular.research project. From the beginning, it
seems, there was an extraordinary reluctance to discuss details of the purported ill effects of 2,4,5-T on animals. Six weeks after the publication of the
DuBridge statement, a journalist who was attempting to obtain a copy of the
full report made by Bionetics and to discuss its details with some of the government officials concerned encountered hard going. At the Bionetics Laboratories, an official said that he couldn't talk about the study, because "we're
under wraps to the National Institutes of Health"—the government agency
that commissioned the study. Then, having been asked what the specific doses
of 2,4,5-T were that were said to have increased birth defects in the fetuses of
experimental animals, the Bionetics official cut off discussion by saying,
"You're asking sophisticated questions that as a layman you don't have the
equipment to understand the answers to." At the National Institutes of
Health, an official who was asked for details of or a copy of the study on
2,4,5-T replied, "The position I'm in is that I have been requested not to distribute this information." He did say, however, that a continuing evaluation of
the study was under way at the National Institute of Environmental Health
Sciences, at Research Triangle Park, North Carolina. A telephone call to an
officer of this organization brought a response whose tone varied from wariness of downright hostility and made it clear that the official had no intention
of discussing details or results of the study with the press..
Tlie Bionetics study on 2,4,5-T was part of a series carried out under contract to the National Cancer Institute, which is an arm of the National Institutes of Health, to investigate more than two hundred compounds, most of
them pesticides, in order to determine whether they induced cancer-causing
changes, fetus-deforming changes, or mutation-causing changes in experimental
animals. The contract was a large one, involving more than two and a half
million dollars' worth of research, and its primary purpose was to screen out
suspicious-looking substances for further study. The first visible fruits of the
Bionetics research were presented in March of last year before a convention of
the American Association for the Advancement of Science, in the form of a
study of possible carcinogenic properties of the fifty-three compounds; the findings on 2,4,5-T were that it did not appear to cause carcinogenic changes in
the animals studied.'
By the time the report on the carcinogenic properties of the substances was
presented, the results of another part of the Bionetics studies, concerning the
teratogenic, or fetus-deforming, properties of the substances, were being compiled, but these results were not immediately made available to biologists outside the government. The data remained—somewhat frustratingly, in the view
of some scientists who had been most curious about the effects of herbicides—
out of sight, and a number of attempts by biologists who had heard about tlie
�JU<±
«
,tologieal study of 2,4,5-T to get at its findings appear to have been
arted by the authorities involved. Upon being asked to account fouflfcR
nrent delay in making this information available to biologists, an offlc^^E
the National Institute of Environmental Health Sciences (another branch of
the National Institutes of Health) has declared, with some heat, that the
results of the study itself and of a statistical summary of the findings prepared by the Institute were in fact passed on as they were completed to the
Commission on Pesticides and Their Relationship to Environmental Health, a
scientific group appointed by Secretary of Health, Education, and Welfare
Robert Finch and known—after its chairman, Dr. E. M. Mrak, of the University of California—as the Mrak Commission. Dr. Samuel S. Epstein, chief of
the Laboratories of Environmental Toxicology and Carcinogenesis at the Children's Cancer Research Foundation in Boston, who was co-chairman of the
Mrak Commission panel considering the teratogenic potential of pesticides,
tells a different story on the availability of the Bionetics study. He says that
he first heard about it in February. At a meeting of his panel in August, he
asked for a copy of the report. Ten days later, the panel was told that the
National Institute of Environmental Health Sciences would be willing to provide a statistical summary but that the group could not have access to the full
report on which the summary was based. Dr. Epstein says that the panel eventually got the full report on September 24th "by pulling teeth."
Actually, as far back as February, officials at the National Cancer Institute
had known, on the basis of a preliminary written outline from Bionetics, the
findings of the Bionetics scientists on the fetus-deforming role of 2,4,5-T. Dr.
Richard Bates, the officer of the National Institutes of Health who was in
charge of coordinating the Bionetics project, has said that during the same
month this information was put into the hands of officials of the Food and
Drug Administration, the Department of Agriculture, and the Department of
Defense. "We had a meeting with a couple of scientists from Fort Detrick,
find we informed them of what we had learned," Dr. Bates said recently. "I
don't know whether they were the right people for us to see. We didn't hear
from them again until after the DuBridge announcement at the White House.
Then they called up and asked for a copy of the Bionetics report."
At the Department of Agriculture, which Dr. Bates said had been informed
in February of the preliminary Bionetics findings, Dr. Tschirley, one of the
officials most intimately concerned with the permissible uses of herbicidal compounds, says that he first heard about the report on 2,4,5-T through the
DuBridge announcement. At the Food and Drug Administration, where appropriate officials had been informed in February of the teratogenic potential of
2,4,5-T, no new action was taken to safeguard the public against 2,4,5-T in
foodstuffs. I]] fact, it appears that no action at all was taken by the Food and
Drug Administration on the matter during the whole of last year. The explanation that F.D.A. officials have offered for this inaction is that they were
under instructions to leave the whole question alone at least until December,
because the matter was under definitive study by the Mrak Commission—the
very group whose members, as it turns out, had such extraordinary difficulty
in obtaining the Bionetics data. The Food Toxicology Branch of the1 F.D.A. did
not have access to the full Bionetics report on 2,4,5-T until after Dr . DuBridge
issued his statement, at the end of October.
Thus, after the first word went to various agencies about the fetus-deforming potential of 2,4,5-T, and warning lights could have flashed on in every
branch of the government and in the headquarters of every company manufacturing or handling it, literally almost nothing was done by the officials charged
with protecting the public from exposure to dangerous or potentially dangerous
materials—by the officials in the F.D.A., in the Department of Agriculture, and
in the Department of Defense. It is conceivable that the Bionetics findings
might still be hidden from the public if they had not been pried loose in midsummer through the activities of a group of young law students. The students
were members of a team put together by the consumer-protection activist
Ralph Nader—and often referred to as Nader's Raiders—to explore the labyrinthine workings of the Food and Drug Administration. In the course of their
investigations, one of the law students, a young woman named Anita Johnson,
happened to see a copy of the preliminary report on the Bionetics findings that
had been passed on to the F.D.A. in February, and its observations seemed
quite disturbing to her. Miss Johnson wrote a report to Nader, and in September she showed a copy of the report to a friend who was a biology student at
Harvard. In early October, Miss Johnson's friend, in a conversation with Professor Matthew Meselson, mentioned Miss Johnson's report onJjje preliminary
Bionetics findings. This was the first that Dr. Meselson had hj^B of the existence of the Bionetics study. A few days previously, he had^wceived a call
from a scientist friend of his asking whether Dr. Meselson had heard of certain stories, originating with South Vietnamese journalists and other South
Vietnamese, of an unusual incidence of birth defects in South Vietnam, which
were alleged to be connected with defoliation operations there.
A few days later, after his friend sent him further information, Mr. Meselson decided to obtain a copy of the Bionetics report, and he called up an
acquaintance in a government agency and asked for it He was told that the
report was "confidential and classified," and inaccessible to outsiders. Actually,
in addition to the preliminary report there were now in existence the full Bionetics report and a statistical summary prepared by the National Institute of
Environmental Health Sciences, and, by nagging various Washington friends,
Dr. Meselson obtained bootlegged copies of the two latest reports. What he
read seemed to him to have such serious implications that he got in touch
with acquaintances in the White House and also with someone in the Army to
alert them to the problems of 2,4,5-T, in the hope that some new restriction
would be placed on its use. According to Dr. Meselson, the White House people
apparently didn't know until that moment that the reports on the adverse
effects of 2,4,5-T even existed. (Around that time, according to a member of
Nader's Raiders, "a tremendous lid was put on this thing" within government
agencies, and on the subject of the Bionetics work and 2,4,5-T "people in government whom we'd been talking to freely for years just shut up and wouldn't
say a word.") While Dr. Meselson awaited word on the matter, a colleague
of his informed the press about the findings of the Bionetics report. Very shortly
thereafter, Dr. DuBridge made his public announcement of the proposed
restrictions on the use of 2,4,5-T.
In certain respects, the DuBridge announcement is a curious document. In
its approach to the facts about 2,4,5-T that were set forth in the Bionetics
report, it reflects considerable sensitivity to the political and international
issues that lie behind the widespread use of this powerful herbicide for civilian and military purposes, and the words in which it describes the reasons for
restricting its use appear to have been very carefully chosen :
"The actions to control the use of the chemical were taken as a result of
findings from a laboratory study conducted by Bionetics Research Laboratories
which indicated that offspring of mice and rats given relatively large oral
doses of the herbicide during early stages of pregnancy showed a higher than
expected number of deformities.
"Although it seems improbable that any person could receive harmful
amounts of this chemical from any of the existing uses of 2,4,5-T, and while
the relationships of these effects in laboratory animals to effects in man are
not entirely clear at this time, the actions taken will assure safety of the
public while further evidence is being sought."
These actions, according to the statement, included decisions that the
Department of Agriculture would cancel manufacturers' registrations of 2,4,5-T
for use on food crops, effective at the beginning of 1970, "unless by that time
the Food and Drug Administration has found a basis for establishing a safe
legal tolerance in and on foods," and that the Departments of Agriculture and
the Interior, in their own programs, would stop the use of 2,4,5-T in populated
areas and in all other areas where residues of the substance could reach man.
As for military uses of 2,4,5-T, the statement said, "The chemical is effective
in defoliating trees and shrubs and its use in South Vietnam has resulted in
reducing greatly the number of ambushes, thus saving lives." However, the
statement continued, "the Department of Defense will [henceforth] restrict the
use of 2,4,5-T to areas remote from the population."
All this sounds eminently fair and sensible, but whether it represents a
candid exposition of the facts about 2,4,5-T and the Bionetics report is debatable. The White House statement that the Bionetics findings "indicated that
offspring of mice and rats given relatively large oral doses of the herbicide
during early stages of pregnancy showed a higher than expected number of
deformities" is, in the words of one eminent biologist who has studied the Bionetics data, "an understatement." He went on to say that "if .the effects on
experimental animals are applicable to people it's a very sad and serious situa-
�116
t
." The actual Bionetics report described 2,4,5-T as producing "sufflciq
ninent effects of seriously hazardous nature" in controlled expei'ii
with pregnant mice to lead the authors "to categorize [it] as probably Aanfifirous." The report also found 2,4-D "potentially dangerous hut needing further
study." As for 2,4,5-T, the report noted that, with the exception of very small
subcutaneous dosnges, "all dosages, routes, and strains resulted in increased
incidence of abnormal fetuses" after its administration. The abnormalities in
the fetuses included lack of eyes, faulty eyes, cystic kidneys, cleft palates, and
enlarged livers. The Bionetics report went on to report on further experimental applications of 2,4,5-T to another species:
"Because of the potential importance of the findings in mice, an additional
study was carried out in rats of the Sprague-Dawley strain. Using dosages of
21.5 and 46.4 mg/kg [that is, dosages scaled to represent 21.5' and 46.4 .milligrams of 2,4,5-T per kilogram of the experimental animal's body weight] suspended in 50 per cent honey and given by the oral route on the 6th through
15th dnys of gestation, we observed excessive fetal mortality almost 80 per
cent) and a high incidence of abnormalities in the survivors. AVheu the beginning of administration was delayed until the 10th day, fetal mortality was
somewhat less but still quite high even when dosage was reduced to 4.6 mg/kg.
The incidence of abnormal fetuses was threefold that in controls even with the
smallest dosage and shortest period used. . . .
.It seems inescapable the 2,4,5-T is teratogenic in this strain of rats when
given orally at the dosage schedules used here."
Considering the fetus-deforming effects of the lowest oral dosage of 2,4,5-T
used in Bionetics work on rats—to say nothing of the excessive fetal mortality
—the White House statement that "relatively large oral doses of the herbicide
. . . showed a higher than expected number of deformities" is hardly an accurate description of the results of the study. In fact, the statistical tables presented as part of the Bionetics report showed that at the lowest oral dosage of
2,4,5-T given to pregnant rats between the tenth and fifteenth days of gestal.ion thirty-nine per cent of the fetuses produced were abnormal, or three, .times,,
the figure for control animals. At what could without much question be
described as "relatively large oral doses" of the herbicide—dosages of 21.5 and
46.4 milligrams per kilogram of body weight of rats, for example—the percentage of abnormal fetuses was ninety and a hundred per cent, respectively, or a
good bit higher than one would be likely to deduce from the phrase "a higher
than expected number of deformities." The assertion that "it seems improbable
that any person could receive harmful amounts of this chemical from any of
the existing uses of 2,4,5-T" also appears to be worth examining for this is
precisely what many biologists are most worried about in relation to 2,4,5-T
and allied substances.
It seems fair, before going further, to quote a cautionary note in the
DuBridge statement: "The study involved relatively small numbers of laboratory rats and mice. More extensive studies are needed and will be undertaken.
At best it is difficult to extrapolate results obtained with laboratory animals to
man—'Sensitivity to a given compound may be different in man than in animal
species. . ; ," It would be difficult to get a biologist to disagree with these
seemingly sound generalities. However, the first part of the statement does
imply, at least to a layman, that the number of experimental animals used in
the Bionetics study had been considerably smaller than the numbers used to
test commercial compounds other than 2,4,5-T before they are approved by
agencies such as the Food and Drug Administration and the Department of
Agriculture. In this connection, the curious layman could reasonably begin
with the recommendations, in 1963, of the President's Science Advisory Committee on the use of pesticides, which proposed that companies putting out pesticides should be required from then on to demonstrate the safety of their
products by means of toxicity studies on two generations of at least two
warm-blooded mammalian species. Subsequently, the F.D.A. set up new testing
requirements, based on these recommendations, for companies producing pesticides. However, according to Dr. Joseph McLaughlin, of the Food Toxicology
Branch of the F.D.A., the organization actually requires applicants for permission to sell pesticides to present the results of tests on only one species
(usually, in practice, the rat). According to Dr. Mclaughlin, the average
number of experimental animals used in studies of pesticides is between eighty
and a hundred and sixty, including animals used as controls but excluding litl-ci-H produced. The Bionetics studies of 2,4,5-T used both mice and rats, and
117
their total number was, in fact, greater, not less, than this average. Including
controls but excluding litters, the total number of animals u^Kn the 2,4,5-T
studies was two hundred and twenty-five. Analysis of th^^sults by the
National Institute of Environmental Health Sciences found them statistically
"significant," and this is the real purpose of such a study: it is meant to act
n s a coarse screen to shake out of the data the larger lumps of bad news.
Such a study is usually incapable of shaking out anything smaller; another
kind of study is needed to do that.
Thus, the DuBridge statement seems to give rise to this question: If the
Bionetics study, based on the effects of 2,4,5-T on two hundred and twenty-five
experimental animals of two species, appears to be less than conclusive, on the
ground that "the study involved relatively small numbers of laboratory rats
and mice," what is one to think of the adequacy of the tests that the manufacturers of pesticides make? If, as the DuBridge statement says, "at best it is
difficult to extrapolate results obtained with laboratory animals to man," what
is one to say of the protection that the government affords the consumer when
the results of tests of pesticidal substances on perhaps a hundred and twenty
rats are officially extrapolated to justify the use of the substances by a population of two hundred million people—not to mention one to two million
unborn babies being carried in their mothers' wombs?
The very coarseness of the screen used in all these tests—that is, the relatively small number of animals involved—means that the bad news that shows
up in the data has to be taken with particular seriousness, because lesser
effects tend not to be demonstrable at all. The inadequacy of the scale on
which animal tests with, for instance, pesticides are currently being made in
this country to gain F.D.A. approval is further indicated by the fact that a
fetus-deforming effect that might show up if a thousand test animals were
used is almost never picked up, since the studies are not conducted on that
scale; yet if the material being tested turned out to have the same effect,
quantitatively, on human beings, .this would mean that it would cause between
three and four thousand malformed babies to be produced each year. The teratogenic effects of 2,4,5-T on experimental animals used by the Bionetics people,
however, were not on the order of one in a thousand. Even in the case of the
lowest oral dose given rats, they were on the order of one in three.
Again, it is fair to say that what is applicable to rats in such tests may not
be applicable to human beings. But it is also fair to say that studies involving
rats are conducted not for the welfare of the rat kingdom but for the ultimate
protection of human beings. In the opinion of Dr. Epstein, the fact that the
2,4,5-T used in the Bionetics study produced teratogenic effects in botli mice
and rats underlines the seriousness of the study's implications. In the opinion
of Dr. Mclaughlin, this is even further underlined by another circumstance—
that the rat, as a test animal, tends to be relatively resistant to teratogenic
effects of chemicals. For example, in the late nineteen-fifties, when thalidomide, that disastrously teratogenic compound, was being tested on rats in oral
dosages ranging from low to very high, no discernible fetus-deforming effects
were produced. And Dr. McLaughlin says that as far as thalidomide tests on
rabbits were concerned, "You could give thalidomide to rabbits in oral doses fit
between fifty and two hundred times the comparable human level to show any
comparable teratogenic effects." In babies born to women who took thalidomide, whether in small or large dosages and whether in single or multiple dosages, between the sixth and seventh weeks of pregnancy, the rate of deformation was estimated to be one in ten.
Because of the relatively coarse testing screen through which compounds
like pesticides—and food additives as well—are sifted before they are
approved for general or specialized use in this country, the Food and Drug
Administration theoretically maintains a policy of stipulating, as a safety
factor, that the maximum amount of such a substance allowable in the human
diet range from one two-thousandth to one one-hundredth of the highest
dosage level of the substance that produces no harmful effects in experimental
animals. (In the case of pesticides, the World Health Organization takes a
more conservative view, considering one two-thousandth of the "no-effect" level
in animal studies to ,be a resaonsable safety level1 for human exposure.)
According to the standards of safety established by F.D.A. policy, then, no
human being anywhere should ever have been exposed to 2,4,5-T, because in
the Bionetics study of rats every dosage level produced deformed fetuses. A
"no-effect" level was never achieved.
�To make a reasonable guess about the general safety of 2,4,5-T for human
;s, as the material has been used up to now, the most appropriate p<^H« area to observe is probably not the relatively healthy and well-fed UlMerl
States, where human beings are perhaps better equipped to withstand the
assault of toxic substances, but South Vietnam, where great numbers of civilians are half-starved, ravaged by disease, and racked by the innumerable horrors of war. In considering any potentially harmful effects of 2,4,5-T on human
beings in Vietnam, some attempt has to be made to estimate the amount of
2,4,5-T to which people, and particularly pregnant women, may have been
exposed as a result of the repeated defoliation operations. To do so, a comparison of known rates of application of 2,4,5-T in the United States and in Vietnam is in order. In this country, according to Dr. Tschirley, the average recommended application of 2,4,5-T in aerial spraying for woody-plant control is
between three-quarters of a pound and a pound per acre. Therre are about five
manufacturers of 2,4,5-T in this country, of which the' Dow Chemical Company
is one of the biggest. One of Dow Chemical's best-sellers in the 2,4,5-T line is
Esteron 245 Concentrate, and the cautionary notes that a drum of Bsteron
bears on its label are hardly reassuring to someone lulled by prior allgeations
that 2,4,5-T is a substance of low toxicity:
"Caution—may cause skin irriation, avoid contact with eyes, skin, and clothing keep out of the reach of children." •
Under the word "warning" are a number of instructions concerning safe use
of the material, and these include, presumably for good reason, the following
admonition:
"Do not contaminate irrigation ditches or water used for domestic purposes
Then comes a "notice" :
"Seller makes no warranty of any kind, express or implied, concerning the
use of this product. Buyer assumes all risk of use or handling, whether in
accordance with directions or not,"
The concentration of Bsteron recommended—subject to all these warnings,
cautions, and disclaimers—for aerial spraying in, the United States varies with
the type of vegetation to be sprayed, but probably a fair average would be
three-quarters to one pound acid equivalent of the raw 2,4,5-T per acre. In
Vietnam, however, the concentration of 2,4,5-T for each acre sprayed has been
far higher. In Agent Orange, the concentrations of 2,4,5-T have averaged thirteen times the recommended concentrations used in the United States. The
principal route through which quantities of 2,4,5-T might be expected to enter
the human system in Vietnam is through drinking water, and in the areas
sprayed most drinking water comes either from rainwater cisterns fed from
house roofs or from very shallow wells. It has been calculated that, taking
into account the average amount of 2,4,5-T in Agent Orange sprayed per acre
in Vietnam by the military, and assuming a one-inch rainfall (which is quite
common in South Vietnam) after a spraying, a forty-kilo (about eighty-eightpound) Vietnamese woman drinking two litres (about 1.8 quarts) of contaminated water a day could very well be absorbing into her system a hundred and
twenty milligrams, or about one two-hundred-and-fiftieth of an ounce, of 2,4,5-T
a clay; that is, a daily oral dosage of three milligrams of 2,4,5-T per kilo of
body weight. Thus, if a Vietnamese woman who was exposed to Agent Orange
was pregnant, she might very well be absorbing into her system a percentage
of 2,4,5-T-only slightly less than the percentage that deformed one out of every
three fetuses of the pregnant experimental rats. To pursue further the question of exposure of Vietnamese to 2,4,5-T concentrations in relation to concentrations officially considered safe for Americans, an advisory subcommittee to
the Secretary of the Interior, in setting up guide-lines for maximum safe contamination of surface water by pesticides and allied substances some time ago,
recommended a concentration of one-tenth of a milligram of 2,4,5-T in one litre
of drinking water as the maximum safe concentration. Thus, a pregnant Vietnamese woman who ingested a hundred and twenty milligrams of 2,4,5-T in
two litres of water a day would be exposed to 2,4,5-T at six hundred times the
concentration officially considered safe for Americans.
Moreover, the level of exposure of Vietnamese people in sprayed areas is not
necessarily limited to the concentrations shown in Drt Meselson's calculations.
Sometimes the level may be far higher. Dr. Pfeiffer, the University of Mon- 1
tana biologist, says that when difficulties arise with the spray planes or the
spray apparatus, or when other accidents occur, an entire thousand-gallon load
of herbicidal agent containing 2,4,5-T may be dumped in one area by means of
the thirty-second emergency-dumping procedure. Dr. Pfeiffer has recalled going
along as an observer on a United States defoliation mission last March, over
the Plain of Eeeds area of Vietnam, near the Cambodian boi^B during which
the technician at the spray controls was unable to get the a^Ptatus to work,
and thereupon dumped his whole load. "This rained down a dose of 2,4,G-T
that must have been fantastically concentrated," Dr. Pfeiffer has said. "It was
released on a very watery spot that looked like headwaters draining into the
Mekong Elver, which hundreds of thousands of people use? In another
instance, he has recalled, a pilot going over the area of the supposedly
"friendly" Catholic refugee villages of Ho Nai, near Bien Hoa, had serious
engine trouble and dumped his whole spray load of herbicide on or near the
village. In such instance, the concentration of 2,4,5-T dumped upon an inhabited area in Vietnam probably averaged about a hundred and thirty times the
concentration reccommended by 2,4,5-T manufacturers as both effective and
safe for use in the United States.
Theoretically, the dangers inherent in the use of 2,4,5-T should have been
removed by means of the steps promised in the White House announcement
last October. A quick reading of the statement by Dr. DuBridge (who is also
the executive secretary of the President's Environmental Quality Council) certainly seemed to convey the impression that from that day onward there
would be a change in Department of Defense policy on the use of 2,4,5-T in
Vietnam, just as there would be a change in the policies of the Departments
of Agriculture and the Interior on the domestic use of 2,4,5-T. But did the
White House mean what it certainly seemed to be saying about the future military use of 2,4,5JT in Vietnam? The White House statement was issued on
October 29th. On October 30th, the Pentagon announced that no change would
be made in the policy governing the military use of 2,4,6-T in South Vietnam,
because—so the Washington. Post reported on October 31st—"the Defense
Department feels its present policy conforms to the new Presidential directive." The Post article went on:
"A Pentagon spokesman's explanation of the policy, read at a morning press
briefing, differed markedly from the written version given reporters later.
"When the written statement was distributed, reporters were told not to use
the spokesman's [previous] comment that the defoliant . . . is used against
enemy 'training and regroupment centers.'
"The statement was expunged after a reporter asked how use against such
centers conformed to the Defense Department's stated policy of prohibiting its
use in 'populated areas.' "
But the statement wasn't so easily expunged. A short time later, it was
made again, in essence, by Bear Admiral William E. Lemos, of the Policy
Plans and National Security Council Affairs Office of the Department of
Defense, in testimony before a subcommittee of the House Foreign Affairs
Committee, the only difference being that the phrase "training and regroupment centers" became "enemy base camps." And in testifying that the military
was mounting herbicidal operations on alleged enemy base camps Rear Admiral Lemos said:
"We know . . . that the enemy will move from areas that have been sprayed,
therefore, enemy base camps or unit headquarters are sprayed in order to
make him move to avoid exposing himself to aerial observation."
If one addsj;o the words "enemy base camps" the expunged words "training
.and regroupment centers"—centers that are unlikely to operate without an
accompanying civilian population—what the Defense Department seema
actually to be indicating is that the "areas remote from the population"
.against which the United States is conducting military herbicidal operations
are "remote from the population" at least in part because of these operations.
As for the Bionetics findings on the teratogenic effects of 2,4,5-T on experimental animals, the Department of Defense indicated that it put little stock in
the dangers suggested by the report. A reporter for the Yale Daily News who
telephoned the Pentagon during the first week in December to inquire about
the Defense Department's attitude toward its use of 2,4,5-T in the light of the
Bionetics report was assured that "there is no cause for alarm about defoliants." A week or so later, he received a letter from the Directorate for
Defense Information at the Pentagon which, described the Bionetics results as
liased on "evidence that 2,4,5-T, when fed in large amounts to highly imbred
and susceptible mice and rats, gave a higher incidence of birth defects than
was normal for these animals." After reading this letter, the Yale Daily News
�120
rter again telephoned the Pentagon, and asked, "Does [the Department
business of manufacturing 2,4,5-T. It appears that the presence of a dioxin conense] think defoliants could be affecting embryo growth in any way!
taminant in the process of manufacturing 2,4,5-T is a constant^ijpblem among
Vietnam?" The Pentagon spokesman said, "No." And that was that. The exf
all manufacturers. Three years ago, Dow was obliged to close^Hrn its 2,4,5-T
iiuental animals were highly susceptible; the civilian Vietnamese population,
plant in Midland, Michigan, for several months and partly rewmd it because
which even under "normal" circumstances is the victim of a statistically incalof what Dow people variously described as "a problem" and "an accident."
culable but clearly very high abortion and infant-mortality rate, was not.
The problem—or accident—was that workers exposed to the dioxin contamiNearly a month after Dr. DuBridge's statement, another was issued, this one
nant during the process of manufacture came down with an acute skin irritaby the President himself, on United States policy on chemical and biological
tion known as culor-aene. The Dow people, who speak with considerable pride
warfare. The President, noting that "biological weapons have massive, unpreof their toxicological work ("We established our toxicology lab the year Ralph
dictable, and potentially uncontrollable consequences" that might "impair the
Nader was born," a Dow public-relations man said recently, showing, at any
health of future generations," announcced it as his decision that thenceforrate, that Dow is keenly aware of Nader and his career), say that the chlorward "the United States shall renounce the use of lethal biological agents
acne problem has long since been cleared up, and that?' the current level of the
and weapons, and all other methods of biological warfare." Later, a White
dioxin contaminant in Dow's 2,4,5-T is less than one part per million, as
House spokesman, in answer to questions by reporters whether this included
opposed to the dioxin level in the 2,4,5-T used in the Biouetics study, which is
the use of herbicidal, defoliant, or crop-killing chemicals in Vietnam, made it
alleged to have been between fifteen and thirty parts per million. A scientist
clear that the new policy did not encompass herbicides.
at the DuBridge office, which has become a coordinating agency for informaSince the President's statement did specifically renounce "all other methods
tion having to do with the 2,4,5-T question, says that the 2,4,5-T used by Bioof biological warfare," the reasonable assumption is that the United States
netics was "probably representative" of 2,4,5-T being used in this country—aud
government does not consider herbicidal, defoliant, and crop-killing operations
presumably in Vietnam—at the time it was obtained but that considerably loss
against military and civilian populations, to be part of biological warfare. The
of the contaminant is present in the 2,4,5-T now being produced. Evidently, the
question therefore remains: What does, the United States government consider
degree of dioxin contamination present in 2,4,5-T varies from manufacturer to
biological warfare to consist of? The best place to look for an authoritative
manufacturer. What degree of contamination high or low, was present in the
definition is a work known as the Joint Chiefs of Staff Dictionary, an official
quantities of 2,4,5-T shipped to South Vietnam at various times this spokesman
publication that governs proper word usage within the military establishment,
didn't seem to know.
in the current edition of the Joint Chiefs of Staff Dictionary, "biological warThe point about the dioxin contamination of 2,4,5-T is an extremely imporfare" is defined as the "employment of living organisms, toxic biological prodtant one, because if the suspicions of the Dow people are correct and the cause
ucts, and plant-growth regulators to produce death or casulaties in man, aniof the fetus deformities cited in the Bionetics study is not the 2,4,5-T but the
mals, or plants or defense against such action." But the term "plant-growth
dioxin contaminant, then this contaminant may be among the most teratogeniregulators" is nowhere defined in the Joint Chiefs of Staff Dictionary, and
cally powerful agents ever known. Dr. McLaughlin has calculated that if the
since a certain technical distinction might be made (by weed-control scientists,
dioxin present in the Bionetics 2,4,5-T was indeed responsible for the teratofor example) between plant-growth regulators and defoliants, the question of
genic effects on the experimental -animals, it looks as though the contaminant
whether the Joint Chiefs consider military defoliation operations part of biowould have to be at least ten thousand times more teratogenically active in
logical warfare is left unclear, As for "defoliant agents," the Dictionary
rats than thalidomide was found to be in rabbits. Furthermore, it raises
defines such an agent only as "a chemical which causes trees, shrubs, and the
alarming questions about the prevalence of the dioxin material in our environother plants to shed their leaves prematurely." All this is hardly a surprise to
ment. It appears that under high heat the dioxin material can be produced in
anyone familiar with the fast semantic legerdemain involved in all official statea whole class of chemical substances known as trichlorophenols and p'entnchlorments on biological warfare, in which defoliation has the baffingly evanescent
ophenols. These substances include components of certain fatty acids used in
half-existence of a pea under a shell.
detergents and in animal feed.
To find that pea in the official literature is not easy. But it is resonable toAs a consequence of studies that have been made of the deaths of millions
assume that if the Department of Defense were to concede officially that,
of young chicks in this country after the chicks had eaten certain kinds of
"defoliant agents" were in the same category as "plant-growth regulators" that
chicken feed, government scientists are now seriously speculating on the possi"produce death . . . in plants," it would thereby also be conceding that it is in.
bility that the deaths were at the end of a chain that began with the spraying
fact engaging in the biological warfare that President Nixon has renounced.
of corn crops with 2,4,5-T. The hypothesis is that residues of dioxiu present in
And such a concession seems to have been run to earth in the current edition.
the 2,4,5-T remained in the harvested corn and wei'e concentrated into certain
oC a Department of the Army publication entitled "Manvial on Use of Herbibyproducts that were then sold to manufacturers of chicken feed, and that the
cides for Military Purposes," in which "antiplant agents" are defined as. | dioxin became absorbed into the system of the young chicks. One particularly
"chemical agents which possess a high offensive potential for destroying or
f;
disquieting sign of the potential of the dioxin material is the fact that bioseriously limiting the production of food and defoliating vegetation," and goes
assays made on chick embryos in another study revealed that- all the embryos
on "These compounds include herbicides that kill or inhibit the growth of
were killed by one twenty-millionth of a gram of dioxin per egg.
plants; plant-growth regulators that either regulate or inhibit plant growth,,
Perhaps an even more disquieting speculation about the dioxin is that 2.4,5-T
sometimes causing plant death. . . ." The admission that the Department of
may not be the only material in which it appears. Among the compounds
Defense is indeed engaging, through its defoliation and herbicidal operations
that several experienced biologists and toxicologists suspect might contain or
in Vietnam, in biological warfare, as this is defined by the Joint Chiefs and as;
produce dioxin are the trichlorophenols and pentachlorophenols, which are
it hns been formally renounced by the President, seems inescapable.
rather widely present in the environment in various forms. For example, A
Since the DuBridge statement, allegations, apparently originating in part1
number of the trichlorophenols and. pentachlorophenols are used 'as slime-killwith the Dow Chemical Company, have been made to the effect that the 2,4,5-T
ing agents in paper-pulp manufacture, and are present in a wide range of conused in the Bionetics study was unrepresentative of the 2,4,5-T generally prosumer products, including adhesives, water-based and oil-based paints, varduced in this country, in that it contained comparatively large amounts of a
nishes and lacquers, and paper and paper coatings. They are used to prevent
certain contaminant, which, according to the Dow people, is ordinarily present
slime in pasteurizers and fungus on vats in breweries and are also used in
in 2,4,5-T only in trace quantities. Accordingly, it has been suggested that the
Hair shnmpoo. Along with'the 2,4,5-T used in the Bionetics study, one trichlorreal cause oC the teratogenic effects of the 2,4,5-T used in Uie Bionntics study
ophenol and one pentachlorophenol were tested without teratogenic results.
may not have been the 2,4,5-T itself but, rather, the contaminant in the sample
But Dr. McLaughlin points out that since there are many such compounds put
used. The chemical name of the contaminant thus suspected by the Dow people
out by various companies, these particular samples might turn out to be —by
is 2,3,0,7-tetrachlorodibenzo-p-dioxin, often referred to simply as dioxin. The
the reasoning of the allegation that the 2,4,5-T used by Bionetics was unu2,4,5-T used by Bionetics was obtained in 1905 from the Diamond Alkali Comsually dirty—unusually clean.
pany, now known as the Diamond-Shamrock Company and no longer in the-
�Dr. McLaughlin tends to consider significant, in view of the now known
kxtreine toxicity and possible extreme teratogenicity of dioxin, the existej^^of
Iveu very small amounts of the trichloroplienols and pentachlorophenl^in
food wrappings and other consumers products. Since the production of dioxin
appears to be associated with high-temperature conditions, a question arises
whether these thermal conditions are met at any stage of production or subsequent use or disposal of such materials, even in minute amounts, One of the
problems here seems to be, as Dr. Epstein has put it, "The moment you introduce something into the environment it's likely to be burned sooner or later—
that's the way we get rid of nearly everything." And most of these consumer
products may wind up in municipal incinerators, and when they are burned,
the thermal and other conditions for creating dioxin materials may quite possibly be met If so, this could mean a release of dioxin material into the entire
environment through the atmosphere.
Yet so far the dioxin material now suspected of causing the fetus-deforming
effects in experimental animals has never been put through any formal teratological tests by any company or any government agency. If the speculation
over the connection between dioxin in 2,4,5-T and the deaths of millions of
baby chicks is borne out, it might mean that, quite contrary to the assumptions made up to now that 2,4,5-T is rapidly decomposable in soil, the dioxin
material may be extremely persistent as well as extremely deadly.
So far, nobody knows—and it is probable that nobody will know for some
time—whether the fetus deformities in the Bionetics study were caused by the
•2,4,5-T itself, by the dioxin contaminant, or by some other substance or substances present in the 2,4,5-T, or whether human fetuses react to 2,4,5-T in the
same way as the fetuses of the experimental animals in the Bionetics study.
However, the experience so far with the employment of 2,4,5-T and substances
chemically allied to it ought to be instructive. The history of 2,4,5-T is related
to preparations for biological warfare, although nobody in the United States
government seems to want to admit this, and it has wound up being used for
purposes of biological warfare, although nobody in the United States government seems to want to admit this, either. Since 2,4,5-T was developed, the
United States government has allowed it to be used on a very large scale on
our own fields and countryside without adequate tests of its effects. In South
Vietnam—a nation we are attempting to save—for seven full years the American military has sprayed or dumped this biological-warfare material on the
countryside, on villages, and on South Vietnamese mena and women in staggering amounts. In that time, the military has sprayed or dumped 'on Vietnam <
fifty thousand tons of heribicide, of which twenty thousand tons have apparently been straight 2,4,5-T. In addition, the American military has apparently
made incursions into a neutral country, Cambodia, and rained down on an
area inhabited by thirty thousand civilians a vast quantity of 2,4,5-T. Yet in
the quarter of a century since the Department of Defense first developed the
biological-warfare uses of this material it has not completed a single series
of formal teratological tests on pregnant animals to determine whether it has
an effect on their unborn offspring.
Similarly, officials of the Dow Chemical Company, one of the largest producers of 2,4,5-T, although they refuse to divulge how much 2,4,5-T they are and
have been producing, admit that in all the years that they had produced the
chemical before the DuBridge statement they had never made formal teratological tests on their 2,4,5-T, which they are now doing. The Monsanto Chemical
Company, another big producer, had, as far as is known, never made such
tests, either, nor, according to an official in the White House, had any other
manufacturer. The Department of Agriculture has never required any such
tests from manufacturers. The Food and Drug Administration has never
required any such tests from manufacturers. The first tests to determine the
teratogenic efforts of 2,4,5-T were not made until the National Institutes of
Health contracted for them with Bionetics Laboratories. And even then, when
the adverse results of the tests became apparent, it was, as Dr. Epstein said,
like "pulling teeth" to get the data out of the institutions involved. And when
the data were obtained and the White House was obliged, partly by outside
pressure and publicity, to act, the President's science adviser publicly presented the facts in a less than candid manner, while the Department of
Defense, for all practical purposes, ignored the whole business and announced
its intention of going on doing what it had been doing all along.
There have been a number of reports from. Vietnam both of animal abortions
nnd of malformed human babies that are thought to have resuttgl from spraying operations in which 2,4,5-T was used. But such seattered^^rts, however
well founded, cannot really shed much more light on the situi^Pn. The fact is
that even in this country, the best-fed, richest, and certainly most statisticsminded of all countries on earth, the standards for testing materials that are
put into the environment, into drugs, and into the human diet are grossly
inadequate. The screening system is so coarse that, as a teratology panel of
the Mrak Commission warned recently, in connection with thalidomide, "the
teratogenicity of thalidomide might have been missed had it not produced malformations rarely encountered." In other words, had it not been for the fact
that very unusual and particularly terrible malformations appeared in an
obvious pattern—for example, similarly malformed babies in the same hospital
at abo\it the same time—pregnant women might still be using thalidomide, and
lesser deformations would, so to speak, disappear into the general statistical
background. As for more subtle effects, such as brain damage and damage to
the central-nervous system, they would probably never show up as such at all.
If such risks existed under orderly, normal medical conditions in a highly
developed country, how is one ever to measure the harm that might be done to
unborn children in rural Vietnam, in the midst of the malnutrition, the disease, the trauma, the .poverty, and the general shambles of war?
DEPARTMENT OF AMPLIFICATION,
New York, March 5, 19W.
'
The EDITORS,
The New Yorker
DEAE Sins: In an article that appeared in The New Yorker on February 7th, I
wrote that Dr. Lee DuBridge, the President's science adviser, issued a statement last October at the White House saying that because a laboratory study
had shown a "higher than expected number of deformities" in the fetuses of
mice and rats exposed to the herbicide 2,4,5-T, agencies of the United States
government would take action to restrict the use of that substance in this
country and in Vietnam, where it was being used in extensive military defoliation operations. This action, Dr. DuBridge announced, would include the cancellation, by January 1st of this year, of Department of Agriculture permits
for the use of 2,4,5-T on some American food crops unless the Food and Drug
Administration had by then been able to determine a safe concentration o£ the
herbicide in foods. Dr. DuBridge further announced that the Department of
Defense would thenceforth "restrict the use of 2,4,5-T to areas remote from
the population" in Vietnam. His statement added that these actions and others
"will assure the safety of the public while further evidence [of the alleged
harmful effects of 2,4,5-T] is being sought"
Four months have passed, and 2,4,5-T is still being used as widely as ever.
The Department of Agriculture has yet to cancel its permits for the use of the
herbicide on food crops in this country, and the Department of Defense is continuing to use it in populated areas of Vietnam. In the meantime, officials o£
the Dow Chemical Company, which is one of the largest producers of 2,4,8-T,
have been maintaining that the samples of 2,4,5-T used in the study cited by
Dr. DuBridge, which was done by the Biouetics Research Laboratories, of,
Bethesda, Maryland, were uncharacteristic of the 2,4,5-T currently being produced, because the material tested by Bionetics—which did not come from
Dow—was contaminated to an unusual extent by a toxic substance identified
as symmetrical 2,3,6,7-tetrachlorodibenzo-p-dioxin. This contaminant, usually
called dioxin, was alleged by the Dow people to be present in the Bionetica
samples at a concentration of approximately twenty-seven parts per million)
and they claim that the 2,4,5-T that Dow is currently producing contains the
dioxin contaminant in concentrations of less than one part per million. The
Dow people maintain that their currently produced 2,4,5-T does not appear to
have the effect of deforming rat fetuses. In January, a Dow official told the
Department of Health, Education, and Welfare, "We strongly urge that actlou
concerning the status of 2,4,5-T be held in abeyance until [Dow's] testing program is completed [in] April." The United States government's .failure so fur
to place the promised restrictions on the use of 2,4,5-T in this country nmy in
part be attributed to this plea.
45-382—TO
8
�Because of the seriousness of the issues involved, it seems to m^hat the'
government's .failure to act on tlie use of 2,4,6-T here and in Vietn^^>alls for
much fuller public discussion. Even though the dioxin contaminarfBKay now
be present in 2,4,5-T in what the Dow Chemical Company apparently considers
to be no more than tolerable amounts, the substance is of such potency that its
release even in small concentrations must prompt deep concern. In the presumably more heavily dioxin-contazninated samples of 2,4,5-T that were used in
the Bionetics work, the smallest dosages of 2,4,5-T that the test animals were
given caused extensive deformities in fetuses. In more recent studies of the
dioxin contaminant, conducted by Dr. Jacqueline Verrett, of the Food and
Drug Administration (who earlier was responsible for revealing the carcinogenicity of cyclamates), extensive teratogcnic, or fetus-deforming, effects were
discovered iu chick embryos when the dioxin, or a distillate predominantly
consisting of it, was present at concentrations of little more than a trillionth
of a gram per gram of the egg. The magnitude of this effect on chick embryos
may be gathered from the fact that, according to Dr. Verrett's studies, the
dioxin appears to be a million times as potent a fetus-deforming agent as the
notorious teratogen thalidomide was found to be in tests on chicks. Of course,
chick embryos are far down the biological ladder from human fetuses, and
they a.re also extremely sensitive to many substances. But even if, for theoretical purposes, we reduced the teratogenie power of the dioxin, as shown in Dr.
Verrett's chick-embryo studies, approximately a million times, we would still
have to Consider that we were dealing with a substance as teratogenieally
potent as thalidomide. That the United States government permits the presence, even in minute amounts, of such a. substance in herbicide I mixtures to be
sold for spraying on food crops and on suburban lawns—where some of the
chemical may enter shallow wells and other drinking-water supplies—is hardly
reassuring. And it is particularly disturbing when one reflects that in the
quarter of a century in which 2,4,5-T was used prior to Dr. DuBridgc's
announcement not a single regulatory agency of the United States government,
not the Department of Defense—which has been spreading huge quantities oO
2,4,5-T on vast areas of Vietnam—and not, as far as is known, the researchers
for any one of the half-dozen large American chemical companies producing
the material had ever so much as opened up a pregnant mouse to determine
whether 2,4,5-T or the dioxin contaminant in it did any systemic or pathogenic
harm to the fetus. Several studies of the sort are now under way, but the
United States government still seems to take the position that the 2,4,5-T produced by Dow and other large chemical companies should be considered innocent until it is proved to be otherwise. Meanwhile, 2,4,5-T is being sprayed on
certain crops and on areas where it may come into contact with human beings,
cattle, and wildlife. In Vietnam, it is still being sprayed by the military in
concentrations that average thirteen times as great as those that the manufacturers themselves recommend as safe and effective for use in this country.
It is true that the teratogenicity of dioxin—as distinct from dioxiu-contaminatecl 2,4,5-T—has not yet been established in tests conducted on experimental
animals of mammalian species. However, the direct toxic, or body-poisoning,
effects—as distinct from fetus-deforming effects—of dioxin are known to be
very high both in animals and in human beings. In past studies on rats, dosages of forty-five millionths of a gram per kilo of the mother's hody weight
have been found to kill fifty per cent of the offspring. When dioxin was given
orally to pregnant rats in recent tests, it was found, on preliminary investigation, to kill all fetuses with dosages of eight millionths of a gram per kilo of
the mother's hody weight, and to damage fetuses with dosages of a half-millionth of a gram per kilo. '
•Further, the effects of dioxin on human beings, even in small dosages, are
known to be serious. In the past, in plants manufacturing 2,4,5-T an illness
called chloracne seems to have been widespread among the workers. In the
mid-sixties, Dow was obliged to close down part of a 2,4,5-T plant in Midland,
Michigan, for some time because about sixty workers contracted chloracne as a
result of contact with dioxin, which seems to be always present in varying
degrees during the process of manufacturing 2,4,5-T and in the finished ,2,4,5-T
itself. The symptoms of this disease include extensive skin eruptions, disorders
of the central nervous system, chronic fatigue, lassitude, and depression. Workers at a 2,4,5-T plant, in New Jersey run by another company suffered similar
symptoms in the mid-sixties, and six years later some of them were reported
to be still suffering from the effects of the disease. In Germany, since the
mid-fifties, workers in factory after factory producing 2,4,5-T ami nolychlow
phenolic compounds have been afflicted with chloracn^fcer absorbing apparently only minute amounts of the dioxin contaminan^Kieir symptoms have
been described in several medical papers as including liver damage, nervous
and mental disorders, depression, loss of appetite and weight, and markedly
reduced sexual drive.
'
A few weeks ago, when a reporter approached an official in Dr. DuBridge's
office for information on 2,4,5-T he was told that he would be given White
House cooperation "only to a certain extent," because the official didn't want
"wild speculation" stirred up. He cited as an example of "wild speculation"
the recent controversy over the birth-control pill, which, he said, had "caused
millions of women to get hysterical with worry." The reporter replied that he
didn't think the analogy between 2,4,5-T and the Pill was a particularly good
one, for the reason that a woman using the Pill could employ alternative
methods of contraception, whereas a Vietnamese woman exposed to herbicidai
spray put down by the American military had no choice in the matter.
But perhaps the comparison between 2,4,5-T (and its dioxin contaminant i
and commonly used pills is worth pursuing. Suppose that such a daugerou'substance as dioxin were found to be contained in a pill offered for human
consumption in this country, and suppose that the contaminant were present iii
,such minute amounts that an adult following the prescribed dosages nn.uli'
ingest a hundredth of a millionth of a gram of the contaminant per tla. v .
There is no doubt whatever that, according to existing Food and Drug Administration standards, the F.D.A. would imitunUately ban production and sale of
the pill on the ground that it was highly dangerous to public health; in f:ic(,
the amount of such a potent contaminant that the F.D.A. would permit in a
pill under the agency's present policy on toxicity would almost certainly be
zero.
While 2,4,5-T, with or without the dioxin contaminant, doesn't come in pill
form, it may be worthwhile to try to calculate, on the basis of a hypothetical
pill, how much 2,4,5-T (and dioxin) a Vietnamese woman living in an nre.i
sprayed by the American military might ingest in a day. It has already been
calculated by reputable biologists that, if one takes into account the average
amount of 2,4,5-T sprayed per acre in Vietnam, and also takes into account n
one-inch rainfall—such as is common there—after a spraying, a forty-kilo
(about eighty-eight-pound) Vietnamese woman drinking two litres (about two
quarts) of 2,4,5-T-contaminated water per clay could be ingesting about »
hundred and twenty milligrams (about a two-hundred-and-fiftieth of an ounce)
of 2,4,5-T a day. If the 2,4,5-T contained the dioxin contaminant at a level <>"
one part per million—which is what the Dow people say is the maximum
amount present in the 2,4,5-T they are currently producing—the Vietnamese
woman would be absorbing a little over a tenth of a microgram of dioxin per
(lay, or ten times the amount of dioxin entering the system of an adult from
the hypothetical pill that the F.D.A. would certainly find dangerous to humnn
health. .Further, if this Vietnamese woman were to conceive a child two weeks.
Say, after the spraying, the weight of the dioxin that hy these same calculations w'ould have then accumulated in her system (the evidence thus far is
that dioxin accumulates in mammalian tissue in the same manner as the chlorinated hydrocarbons, such as DDT) would be more than the weight of tinjust-fertilized ovum. Considering the existing evidence of the frightening
degree of teratogenicity of the dioxin in chick embryos and its highly toxic
effects on mammalian fetuses, the presence of this much dioxin in a mother's
body at the very beginning of a human life surely has ominous implications.
Now, what about the safety of 2,4,5-T itself? Admittedly, the dioxin contaminant seems to be a residue from one stage of its manufacture. But if by some
future chemical miracle the very last trace of dioxin could be removed froTn
the finished 2,4,5-T, would the resultant "pure" 2,4,5-T be harmless? The fact
seems to be that even then 2,4,5-T, as produced in this country, would have f<>
be viewed with suspicion, for the breakdown products of 2,4,5-T, when subjected to heat and other conditions, are themselves capable, according to »
number of responsible biologists, of producing dioxin. Given this potential, the
ultimate folly in our defoliation operations in Vietnam was possibly achieved
during 1965 and 1906, when the military made large-scale efforts in two defoliated areas to create fire storms—that is, fires so huge that all Hie oxygen in
those areas would be exhausted. The apparent intention was to render the coil
barren. (A fire storm would also, of course, have the result of burning or snf-
�126
any living beings remaining in the area.) Operation Sherwood
d in 1965, was an attempt to burn a defoliated section of the
Woods. In October, 1066, the military began Operation Pink Hose, a
project. Neither o£ the projects, in which tons of napalm were thrown down on
toil of the residue of tons of sprayed 2,4,5-T, succeeded in creating the desired
effect; whether they released into the atmosphere dioxin produced by the
breakdown products, of the 2,4,5-T will probably never be known.
There are also less spectacular ways in which conditions suitable for the
release of dioxin in Vietnam may have been created. For example, after areas
accessible by road have been defoliated, woodcutters move in to chop up the
cloud timber, which is then carted off to nearby towns'and 'Sold as'firewood. ,
Large quantities of it are said to have been entering Saigon for years. Since
the fires are customarily tended by Vietnamese women, and since many of
them are certainly pregnant, the hazards to health and to the lives of unborn
children surely cannot be ignored.
In the United States, the potential hazards from the present use of 2,4,5-T
are considerably less than they are in Vietnam. In the first place, the recommended concentrations of 2,4,5-T for spraying here are, as I have pointed out,
about a thirteenth of what the Vietnamese population is sometimes subjected
to. And, in the second place, a great deal, if not most, of the 2,4,5-T that
would otherwise have been sprayed on American crops and grazing areas has
for several years been sent to Vietnam. However, the shortage of 2,4,5-T in
this country does not necessarily mean that the potential hazards are at a
minimum. The substances known as the trichlorophenols and compounds of
pentachlorophenol, which officials of the F.D.A. believe may be chemical precursors of dioxiu under certain thermal and other conditions, are used widely
in the manufacture of a large variety of consumer products, ranging from
paper to laundry starch and hair shampoo. Dow Chemical puts out a whole
line of polychlorophenolic chemicals known as Dowicide Products. Monsanto
Chemical also puts out a line of pentachlorophenol substances, known as Penta
Compounds. Since a very great many consumer products wind up being burned
sooner or later, and since the polychlorophenolic compounds are suspected of
being capable, under particular thermal and other conditions, of releasing
dioxin, the alarming question arises whether, and to what extent, dioxin is
being released into the environment through the atmosphere. Pentachlorophenol, used in certain herbicides, is readily decomposed in sunlight, and in its
breakdown process a number of products, including chemical precursors of
chlorodibenzo-p-dioxin compounds, are produced. Because of these factors, a
whole range of pesticides, as well as of herbicides, now must come under suspicion of producing dioxin compounds'.
Although the chemical companies that manufacture 2,4,5-T have long taken
pride in pointing out that 2,4,B-T itself is quite readily decomposable in soil,
the crucial matters of how stable the dioxin contaminant is and to what
extent it is cumulative in animal tissue have apparently been neglected. Consequently, the fact that traces of compounds virtually indistinguishable from
dioxin have already been detected in this country in the human food chain—in
the livers of chickens and in edible oils—clearly indicates that dioxin should
be considered a hazard to man. Why, under all these inauspicious circumstances, the production and the use here and in Vietnam of 2,4,5-T has not
summarily been stopped by the United States government is hard to understand.
Sincerely,
THOMAS WHITESIDE.
Appendix 4
DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE,
PUBLIC HEALTH SERVICE,
FOOD AND DRUO ADMINISTRATION,
Roclcville, MA., March 1%, 1910.
Hon. RICHARD D. MCCARTHY,
House O'f Representatives,
Washington, D.G.
DEAR MR. MCCARTHY: The Secretary has asked us to reply to your letter of
February 3, 1070, requesting whether the B'ood and Drug Administration has
information indicating that 2,4,5-T is now safe to use.
No tolerances have been established for residues of 2,4,5-T in food or feed
crops. The whole matter of the safety of this herbicide, when its use results in
a residue in or on a food crop, is currently under evaluatioi^ljhis evaluation
will be completed as expeditiously as possible. We are enclasp a Fact Sheet
explaining the status of 2,4,5-T at this time.
We shall promptly inform you of our decision upon completion of the evaluation of 2,4,5-T.
Sincerely yours,
M. J. RYAN, Acting Director,
Office of Legislative Services.
FDA FACT SHEET
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE,
PUBLIC HEALTH SERVICE,
FOOD AND DHUQ ADMINISTRATION,
Washington, D.C.
* 2,4,5-T *
2,4,5-T (2,4,5-trichlorophenoxyacet.ic acid) has had extensive registered use
as a deColiant and weed killer. It has also been registered by the U.S. Department of Agriculture as a pesticide chemical (herbicide) on a no residue basis
on a few certain selected food crops for some years, primarily for weed control of pasture and rangeland.
TOXICITY
A research study recently completed under contract to the National Cancer
Institute on a commercial lot of 2,4,5-T showed that the feeding of this material to rats and mice produced abnormal birth effects on the embryos.
Further investigation of the 2,4,5-T used in the feeding studies established
that the material used contained a significant amount of one of more impurities called dioxins produced during the manufacture of 2,4,6-T. Improved manufacturing processes are claimed by one manufacturer to have reduced the
dioxiu impurities to insignificant amounts.
The dioxins are of concern because they are known to be extremely toxic to
poultry and to have produced severe skin irritation to workers in plants
exposed to dioxins inadvertently during the manufacture of other chemicals.
At present a number of research studies are. underway in both government and
commercial laboratories to determine if the reported birth defects of the earlier study are due to 2,4,5-T itself, the dioxin impurities, or a combination of
the 2,4,5-T and the dioxins.
Additional investigations are underway to improve our ability to detect very
small amounts of dioxins in samples of 2,4,5-T and to determine whether other
commonly used pesticides chemically related to 2,4,5-T contain significant
amounts of the dioxin contaminants. Drinking water supplies are being tested
for the presence of 2,4,5-T and other possible environmental sources of these
chemicals studied, but no results are available at this time.
The USDA announced on February 6, 1970, that it is investigating 17 commonly used pesticides chemically related to 2,4,5-T to determine whether they
contain hazardous amounts of these toxic contaminants.
FOOD IN THE UNITED STATES
The Food and Drug Administration is continually engaged in examining
samples of individual foodstuffs for residues of pesticides above the safe tolerances established under the Miller Pesticide Amendment In addition, FDA
purchases food in the markets of several cities, prepares the food in the quantities and combinations typical of the diet of an average 10-year-old male, and
determines the amounts of the several pesticides that might be actually ingested in the typical diet of a heavy eater.
Of 5300 food samples tested for 2,4,P-T residues during the last four-year
period, 25 samples indicated trace amounts '(less than the 0.1 p.p.m. limit of
accuracy of present analytical procedures) and 2 samples showed higher residues. 0.19 p.p.m. 2,4,5-T was detected iri one sample of milk taken in 1065 In
New England, and one sample of sugarbeets from Ohio in 1900 showed 0.29
�,m. 2,4,5-T. The milk had been distributed before analysis was complete and
icssing of the sugar-beets removes the chemical. If food is found to c
'te residues of 2,4,5-T, it is subject to removal from the market.
STATUS OF 2,4,5-T UNDEE THE FEDERAL FOOB, DBUG, AND COSMETIC ACT
No finite tolerances have been established for residues of 2,4,5-T or the dioxins in food. In the absence of established tolerances any detectable amount of
either chemical in food would make the contaminated food illegal and subject
to sei'/.uve if found in the channels of interstate commerce.
A. petition was filed in December, 1907 requesting the establishment of tolerances of 0.2 p.p.m. for residues of 2,4,5-T on apples, barley, blueberries, corn,
oats, rice, rye, sugarcane, and wheat. Neither the petition as originally submitted or as later supplemented provided data to support affirmative action and
the petitioner withdrew his petition on December 29, 1969, as provided for
under the pesticide regulations.
Petitions to establish a safe tolerance level for residues of 2,4,5-T in food
may again bo submitted to the FDA in the future. However, any such submission must include scientific research data to resolve the questions that have
been raised concerning toxicity of 2,4,5-T and the dioxins.
CONCLUSION
The Department of Health, Education, and "Welfare is continuing investigations to determine the potential hazards from the possible presence of residues
of 2,4,5-T and dioxius in foods, water, and other environmental sources to
which the public may be exposed.
It is to be emphasized that there is no tolerance for 2,4,5-T in food today;
the testing of food over the past several years has revealed no significant
problem of food contamination.
Appendix 5
PROBE INTO USE OF HERBICIDES BY CONGRESSMAN RICHARD D. MCCARTHY.
D-N.Y.
Globe, Ariz., February 13, 1070
Ladies and gentlemen, I think we should begin. I am Congressman Richard
D. McCarthy, and the hearings will coine to order.
For more than a decade scientists have had serious misgivings about the
widespread use of herbicides and, pesticides in the environment. The .late
Rnchael Carson warned of the risk of the use of herbicides, whose effects were
either harmful or unknown.
•
.
.
Tn the United States 120 million acres each year are sprayed with herbicides''
Cor the clearing of railroads, for brush control, for watershed management,
nud for other purposes. One of these is known as 2,4,5-T. It was developed and
perfected at Fort Detriclc, Md., the army's chief Biological Warfare Research
Center. The herbicide 2,4,5-T, and 2,4-D, a related herbicide, collectively
account for some 83 million pounds of production per year—that was the
figure in 1908.
I've long been concerned with the widespread use of these herbicides in Vietnam, Each day some 100 tons are dropped on South Vietnam, and scientists
for many months have been concerned about the adverse ecological effects of
this herbicidal inundation.
Last summer in the course of my inquiry into the Army's germ and gas warfare policies, I learned that a study, by the Bionetics Research Laboratories
for the National Cancer Institute showed that the herbicide 2,4,5-T produced
birth defects in rats and mice.
When the conclusions of this study were known, the President's science
adviser, in October, announced a ban on the herbicide beginning January 1,
1970, unless the F.D.A. had found safe legal tolerances. I was distressed 11
dnys ago to learn that contrary to the White House's announcement, the
Department of Agriculture continues to authorize the use of 2,4,5-T in the
United States, It's incredible to me that someone, or some people should have
succeeded in overruling the science adviser to the President of the United
States.
We know from the tlmlidomide experience Unit if we are going to -err, we
should err on the side of caution, and not on the side of danger. It is my firm
conviction that such chemicals should not be used unless fuU^fcets show that
they are safe. It is also incredible to me that this herbicide, ^Vh has been in
•existence since its development some 25 years ago at t h e u e r m Warfare
Research Center, still has not been fully tested for its teratogenic effects on
human beings—that is, its power to produce birth deformities.
We know that it produces birth deformities in test animals under laboratory
•conditions, and we continue to receive reports from Vietnam that civilian
women living in this heavily defoliated area are bringing forth deformed
•offsprings.
The Saigon Press has reported on these in considerable detail.
Now, we have the allegations, and complaints emanating from here, Globe,
Ariz. It is my hope that my investigation into these complaints and allegations
will assist me in continuing my inquiry into this whole matter. I wish to
determine how the White House was overruled, and why it is that we continue
to use this herbicide despite the warning signals that have arisen.
As the great French scientist physiologist, Claude Banard, once said, "True
science teaches us to doubt, and ignorance to refrain."
I want to welcome all the local State and Federal officials who are in
-attendance. I hope to have a chance to meet with you personally during our
"visit.
Our first witness is Prof. Arthur W. Galston, a professor of biology from
Yale University.
Doctor Galston.
Professor Galston, I wonder if, for the record, you would identify yourself,
and your background, and particular expertise in the matters under inquiry.
Dr, GALSTON. Very happy to do that, Congressman.
I'm currently a professor of biology at Yale University. I'm also lecturer in
forestry, and director of the March Botanical Gardens at Yale. I've been n professor of plant physiology for about 27 years. I was trained at the New York
State College of Agriculture at Cornell University.
I did my graduate work at the University of Illinois, where I earned a Ph.D.
•degree in 1943. 1 then went to work for the emergency rubber project for
the U.S. Government, located at Gal-Tech. During AVorld War I was agricultural officer for U.S. Navy Military Government on the Isle of Okinawa. I then
Tvorked at Gal-Tech for 10 years, and I've been at Yale for the last 15 years.
I've published books in the area of plant physiology, and I have over 100
articles in the subject.
Congressman MCCARTHY. For the record, Doctor Galston, I wonder if you
could give us a scientific information about the herbicide under investigation.
DR. GALSTON. Congressmen, what I'd like to do is to give you and the audience here some appreciation of the feeling of a large number of scientists as
exemplified in this report recently delivered to the Secretary of Health, Education, and Welfare, Finch.
It is culled, "The Report of the Secretary's Commission on Pesticides and
Their Relationship to Environmental Health." It's dated December 5, 1969, and
was prepared by the distinguished panel shared by Doctor Emil Mrak, the
•chancellor emeritus of the University of California at Davis.
It included many academic people, and also the vice presidents of two
important companies, Dow, and Eli Lilly, both of whom manufacture herbicides and other pesticides in wide use. •
The Commission takes note of the fact that there are now more than 400
•different kinds of chemicals which are being used as pesticides to combat
insects, fungi, weeds, and other predators.
Our modern agriculture and highly technicalized food production activities
•demand that we do use chemicals in agriculture.
I'd like to make it clear that I'm not alining myself with people that say,
"Stop nil chemicals." That's ridiculous in this day and age. We are dependent
'upon chemicals, and we have to keep using them.
Nonetheless, some of these chemicals are terribly noxious when introduced
into the environment.
All of us are now familiar with the fact that DDT may be more of a bane
•than a boom. It has become global. Even a penguin picked up on fin ice flow in
.Antarctica is full of DDT, and that was 400 miles from the'application of
�iOU
:>DT, and we know that DDT causes oversized livers, and alteration of the
(teroid metabolism in everyone's genes.
|^fc
This Commission agrees unanimously that DDT must be phased <^^as
quickly as possible as a pesticide.'
With that as a background, I think it's perfectly clear that as scientific
information develops, we are going to want to examine every pesticide for its
possible harmful effects on man and his domestic animals, and his environment.
Here I must digress to tell you about the changes that have occurred in our
concept of what constitutes adequate testing for a compound of this kind.
It used to be that simple toxicology tests were conducted. A laboratory
animal, such as a mouse or a rat was fed a certain amount of chemical. If
that animal showed serious' symptoms, the teratogenicity yas calculated on the
base of how many milligrams per kilogram of body weight of this material
produced the toxic effects.
We now have tables which tell us roughly how toxic given materials are.
Now, based on that kind of test, 2,4,5-T, for example, is not terribly toxic,
it's only a mildly toxic compound in the order of 2 to 700 kilograms milligram
of body weight cause toxicity.
If, however, you use more subtle tests, you find out that 2.4,5-T may be more
dangerous.
&mong these tests are: Does the compound cause cancer? That takes a much
more serious look than simply feeding and watching the dying of animals.
Secondly, do the compounds cause genetic effects, that is, does it break chromosomes, or cause mutations.
Thirdly, does the compound cause birth abnormalities. The word to describe
that is teratogenics; that is the formation of monsters.
Now, this report which I have alluded to has as its last chapter, a chapter
on teratology, and I'd like to read you just a little bit out of this chapter, and
out of the summary which is written here, which gives you my concern.
"All currently used pesticides should be tested for teratogenicity in the near
future in two or more mammalian species chosen on the basis of the closest
metabolic and pharmacologic similarity to human beings possible. Pesticides
should be tested at various concentrations including levels substantially higher
than those to which the human population are likely to be exposed. Test procedures should also reflect routes related to human exposures. Apart from the
obvious route of ingestion, attention should be directed to other routes of exposure, including inhalation exposures from pesticide aerosols and vaporizing ,
pesticide strips used domestically, and exposures from skin absorption. Parenteral administration is an appropriate test route for pesticides to which
humans are exposed by inhalation, or for pesticides, which are systemically
absorped following ingestion.
"The use of currently registered pesticides to which humans are exposed and
which are found to be teratogenic by suitable test procedures in one; or more
mammalian species should be immediately restricted to prevent risk of human
exposures."
I'd like to repeat that; "Currently registered pesticides to which humans are
exposed and which are found to be teratogenic by suitable test procedures in
one or more mammalian species should be immediately restricted to prevent
risk of human exposure. Such pesticides, in current use, include—" I'll skip a
lot. of names, 2,4-D and 2,4,5-T are listed.
Here's the Government's most distinguished panel saying that, there is evidence that 2,4,5-T has produced teratogenic effects in one or more mammalian
species, its use should be restricted immediately. They also said no new pesticide found to be teratogenic, should be used only in circumstances where risk
of human exposure is minimal.
Congressman MCCARTHY. AVhat's the date of that report, Professor?
Dr. GALSTON. December 5, 1969, it's now only 2 months old, Congressman,
and it says a scientific group, or commission should be charged with the
responsibility for continued surveillance of the whole problem of pesticide teraNow, the problem of determining whether a problem is teratogenic, whether
it's given rise to birth defects is terribly complicated. If you do a laboratory
test, whore you have one group of mice getting the chemical, and one grotip
not, there's no problem to determine teratogenicity. By this kind of test it has
been determined that 2,4,5-T as tested is one of the most teratogenic chemicals
131
weight have trebled
known. Even as little as 4% milligrams per kilo of body wei
the rate of abnormal production in mice and in rats a 113
.•ams per kilo
;rsWR 70 percent
of body weight lias produced 100 percent abnormal litters
abnormal individuals in those litters.
Congressman MCCARTHY. I wonder if you could translate those figures into
what a human being would be likely to receive in the United States, or in
Vietnam.
Dr. GALBTON. Well, if you take the lowest of those figures, 4% milligrams
per kilogram of body weight, and you say you have a 50-kilogram woman,
that's 110 pounds which is about the average weight of a Vietnamese woman,
then she needs to digest only about 200 milligrams total to have a teratogenic
dose, 100 milligrams per day. Now, we are spraying agent orange, which is a 1
to 1 mixture of 2,4-D, and 2,4,6-T, in Vietnam at the rate of 270 pounds per
acre. I should note that is 10 times what we used locally.
Congressman MCCARTHY. What would it be in Arizona?
Dr. GALSTON. I think our Forestry friends could tell us, it is in the order of
two pounds per acre.
Congressman MCCARTHY. We will get to that with them today or tomorrow,
but that's about the range?
Dr. GALSTON. At the Vietnam dose rate, if you assume a 27-pound per acre
sprayed, followed by a 1-inch rainfall, which is normal for that region
and you know that the rainwater is collected off the roof, or stored in cisterns,
or gotten from very shallow wells, then a woman need only consume less tlmn
3 quarts of water per day in combined drinking and cooking operations to
receive that teratogenic dose.
I have calculated on this basis that it's possible that in Vietnam people have
been given this kind of teratogenic dose.
Congressman MCCARTHY. Doctor, let me ask you this. Here we have the Bionetics Research Laboratory test which showed that 2,4,5-T is teratogenic in
test animals, mice and rats. Is it teratogenic in human beings—do we know?
Dr. GALSTON. One doesn't know for sure whether it's teratogenic in Imman
beings, one doesn't experiment with pregnant, women, feeding some of them
2,4,5-T, and not feeding others. That would be inhuman, we do not tolerate
that kind of experimentation, but the paragraph I was about to read here in
fact deals with this.
It says there are two ways that you can determine whether a chemical is
teratogenic. "First, chemicals or other agents may be administered to experimental animals to determine whether they induce prenatal damage. Secondly,
and on a post hoc basis, human populations may be epidemiologically surveyed
to detect geographical, or temporal clusters of unusual types of frequencies of
congenital malformaties. Combinations of these approaches are likely to insure
early detection and identification of teratogenic hazards."
Congressman MCCARTHY. Now, to your knowledge, has that been done in
Vietnam, or is it contemplated, is the American Association for the Advancement of Science going to do what you just read?
Dr. GALSTON. I think it's shocking that there are absolutely no studies on
the possible teratogenicity of these chemicals either in Vietnam or in this
country. That is why it's so important to gather data from places like globe,
and from places like the Saigon area to attempt to correlate, if it's possible to
do so, the use of any particular pesticide with the appearance of any birth
abnormalities, or any physiological malfunctions.
Congressman MCCARTHY : Doesn't the commission's study recommend that no
herbicides like this be used until we are sure that it doesn't produce effects inhuman beings?
Dr. GALSTON. That's correct, the Commission recommends that given the suspicion that these materials are teratogenic, given their widespread use, but
given also our wide dependency on these things in agriculture, we should
immediately restrict the use so that we only use these herbicides where it is
absolutely necessary to do so, and where there is no possibility of contact with
human organisms. I believe that is the safe policy when you think you may be
doing harm. You- stop until you find out whether you are in fact doing harm.
Congressman MCCARTHY. Do you have any information that you could give
for the record here, which would suggest why The White House ban never
went into effect? I have a letter here which I received just prior to leaving
Washington, which needs further clarification. It is from Mr. Ned D. Hnyley,
director of science and education for the Department of Agriculture in
�132
138
Onse to a letter I'd addressed to Secretary Hardin, asking why The Wl
n
....Snse ban 'didn't go into effect. Among other things, here's what he ss_
e
"Now, data submitted to D.H.E.W., Department of Health, Education, and
Welfare, relev'ant to this position is that the 2,4,5-0? used in the bionetics study
contained about 27ptm of
Dr. GALSTON. Dioxin is the way it's usually referred to. '
Congressman MCCARTHY, It's t-e-t-r-a-c-h-1-o-r-o-d-i-b-e-n-z-o p-a-r-a dioxin<
Dr. GALSTON, Tetrachlorodibenzo para dioxin.
Congressman MCCARTHY. A highly toxic contaminant.
Dr. GALSTON. Yes.
Congressman MOOABTHY. I'm going to seek further clarification that one of
the reasons the ban was lifted was this discovery. Now, do you know anything
about this in the course of your inquiry?
Dr. GALSTON. Yes, Congressman, I became aware of this new development—
2,4,5-T is a chemical synthesized from the reactants that are put together in a
vehicle. Depending on the method of synthesis, and the temperature of.syn- M
thesis, you may or may not get certain impurities formed in that reaction that
accompany the 2,4,5-T which is realized out of the reaction fixture. One of the
impurities is tetrachlorodibenzo-p-dioxin.
Now, there's previous information that this compound is a highly noxious
material. There have been several factory and laboratory accidents in which
people exposed to this compound have developed very severe blistering, loss of
sensation, and respiratory troubles. The Germans have had a similar experience.
So it's natural when you have a report of this kind about the toxicity of
2,4,5-T, to inquire whether the effect is due to the chemical itself, or to the
impurity.
Congressman MCCARTHY. Does it matter?
Dr. GALSTON. I'll mnke this statement.
I think it does matter in the long run, Congressman, because if it's theimpurity, then in the future we can learn possibly how to make the chemical
without the impurity, and continue its nse.
Congressman MCCAUTHY. I've read in the long article by Mr. Whiteside in*
the latest issue of New Yorker Magazine, at least he made the point that you
can't make 2,4,5-T without getting some dioxin.
Now, is that fight?
Dr. GALSTON. That's correct, I don't know if any sample that has less than a
part per million of dioxin, so all of the 2,4,5-T that has been sprayed both at
home and abroad has some dioxin.
The question is: Can you lessen the dioxin level down to the point where it
is no longer so dangerous?
Congressman MCCARTHY. Is there any other way that dioxin can be produced after it's sprayed?
Dr. GALSTON. Oh, yes, even if you sprayed 2,4,5-T without any dioxin it
might form chemicals in this Arizona sunshine. Putting all that light energy in1 could easily imagine compounds like the dioxin being formed.
If there were a little fire somewhere, that's just the condition which would
form the dioxin from 2,4,5-T. The only hard data on the terntogenicity of
2,4,5-T are right in this book that I have. There are no date which tell me, or
anybody else, that, it's the dioxin and not the 2,4,5-T that's responsible for
those teratogenic effects.
1 've had telephone conversations with people who have alleged this,
Congressman MCCARTHY. Who are they?
Dr. GALSTON. Well, one of them is a member of this Commission, DoctorJulius Johnson of Dow who is an old friend of mine, and 1 think he is very
terribly concerned about this development. Naturally, he would be since Dow
is tho manufacturer of some of this, and he told me that there are tests going
on now which are not finished. He said he would not care to quote the data as
of the present moment.
Congressman MCCARTHY. Mr. James Hansen of the Dow Chemical Co. visited"
uiy office last week and alluded to, I assume, the same tests.
Or. GALSTON. Yes.
Congressman MCCARTHY. That the Dow Co. itself was carrying out the following-up on this possibility that it is the dioxin.
Now, in this letter from Mr. Bayley he said new data submitted to D.H.B.W. ,
relevant to this position indicates that the 2,4,5-T contained the dioxin.
Well, it sounds as if it's the same thing. What I don't understand is how
the Dow Chemical Co. could, in effect, by intervening, countt^Bnd, or negate
White House orders.
^^
Now, have you discussed this with any other people in the Government, or
outside the Government?
Dr. GALSTON. I have not, Congressman. I don't have any information on how
this operation came about. I would only say that to me it's unthinkable that,
in absence of hard data, and to protect the lives and welfare of people in the
country, I don't see how this order could fail to be enforced.
We must be safe before we are sorry. I would say let's get the facts before
we resume spraying with this 2,4,5-T and at the present time there are no published data that I, or any other scientists have seen, that would say that
2,4,5-T is not the culpable agent. I think it's very peculiar that the orders of
Doctor DuBridge are not being followed by the Department of Agriculture and
the Department of Interior. The Department of Defense, said it announced
immediately it would not follow this directive.
Congressman MCCARTHY. That's right. The next clay on October 30th, the
spokesman for the Department of Defense contradicted the DuBridge order in
a verbal briefing to newsmen. He said that the 2,4,5-T would continue to be
sprayed in training and regroupment areas where obviously populated areas,
and of course as you know it has been sprayed in rubber plantations in Cambodia, which are also populated.
AVell, Professor Galston, I appreciate very much your testimony here.
Dr. GALSTON. Do you mind if I make one more brief statement?
Congressman MCCARTHY. No, please do.
Dr, GALSTON. As a biologist, I'm terribly concerned about this because I
believe in herbicides, I want to see that they continue to be used. I'm afraid
there may be overreaction on the part of the public. I would like to say that
there are probably ways that we can safely use these compounds, and the first
recommendation of this Commission—I would like to read you just two paragraphs, short ones, because they outline to me what would be a safe procedure.
It says: "A new interagency agreement is needed to strengthen cooperative
action among the Department of Health, Education and Welfare, U.S. Department of Agriculture, and the U.S. Department of Interior, to protect public
health, and the quality of environment from pesticides danger provided by the
Secretaries of I-I.E.W. and Interior, as well as Agriculture, should be required
for all pesticides registration, pesticide use determined by any of the three
Secretaries to be hazardous should be restricted, or eliminated.
"The agreement should further require the continuous review of new scientific information on pesticides now in use with the formal reviews made 2
years after initial registration, and subsequent formal reviews by the (hree
agencies at 5-year intervals."
That seems to be loiidly, essential for the continued safe use of pesticides
and it's coupled with the establishment of a national testing center for pesticides, which is also recommended, I would say that we .would be well on our
way for the safe use of pesticides.
Congressman MCCARTHY. Do you think it's proper to delegate to the manufacturer of such a chemical the responsibility for testing its teratogenicity and
carcinogenicity ?
Dr. GALSTON. Well, you can certainly accept the data that are contributed by
the manufacturer as relevant to the solution of the problem. I think those
people have shown necessary testing laboratories which give honest data, but I
would not depend on those alone. I would want to see the FDA or some other
agency independently test these same compounds aiso, under completely different conditions. That's only a scientific rule, you don't believe anything anybody
tells you, it has to be confirmed once or twice before you can believe it.
I would certainly hope the FDA, or some other agency, HEW would continue conducting further tests on these toxic chemicals.
Congressman MCCARTHY. And not really solely on the research of Dow, or
other manufacturers?
Dr. GALSTON. That's correct
Congressman MCCARTHY. Professor, I wonder if you would bo kind enough
to sit with us here, I'd like to use you as a resource person when, we have the
other witnesses.
Our next witness is Mr. John Pierovich, Assistant Regional Forester, from
Albuquerque.
«
•
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I Is he in the room?
If you would be seated and identify yourself for the record, anc
responsibilities in areas under scrutiny here.
Mr. PIEUOVICH. Yes, sir, I'm John Pierovich, Assistant Regional Forester in
Albuquerque, N. Mex. My responsibilities related to this matter are in connection with the complaints we've received here at Globe, and the overall evaluation of our Chaparral program, and our Chaparral program guidelines.
The primary reasons the Forest Service is here today is because this is a
Forest Service project. I think that we need to be cognizant of such hearings
as this, and we do try to keep informed through the literature of regulatory
rules and concerns.
In fact, we share quite deeply the concern of the people in this community
with their environment, we wouldn't want to do anything that would jeopardize their safety.
They're our neighbors, we also live here.
At the same time, we've been asked repeatedly to announce that we would
not spray again in the Globe area, and like Doctor Galston, I think that we
wouldn't want to overreact at this time. So we've said that such an announcement would be premature, we have our own studies going forward, and that
these studies must be resolved before f we can reach decisions on herbicide's
use, or on the Chaparral program.
In addition to that we believe that it would be also unwise to base decisions
on herbicides used particularly from the current allegations, or suspicions here
in this area.
These matters need to be studied deeply, and we hope to have them studied
deeply, and frankly welcome this inquiry because it will help to daylight some
of the areas of concern.
That's essentially our position, Mr. Congressman. I'd be glad to answer any
questions you might have.
Congressman MCCARTHY. Thank you very much.
In the course of my study, I have come into possession of documents that
have been exchanged between the Department of Agriculture and citizens in
the area. Here is one from John A. Williams for the Task Group, U.S. Department of Agriculture, Forest Service. Are you familiar with Mr. Williams?
Mr. PIEROVICH. Yes, I am.
Congressman MCCARTHY. Is he an associate of yours?
Mr, PIEROVICH. He works in our regional office.
Congressman MCCARTHY. Is he here today?
'Mr. PIEROVICH. No, he's not.
Congressman MCCARTHY, I'd like, to read you some of the things that he
says: "Paul Boffin (phonetic) called a Dow Chemical representative at Davis,
Calif., and requested information about Silvex. This man called Supervisor
Courtney later and indicated that a publicity release was being prepared for
submission to the news media concerning the known toxicity of Silvex. This if
accepted and used by the news media will go a long ways towards improving
the situation, and dispelling the fear of Silvex as a highly toxic, or poisonous
agent."
He then goes on to say in his conclusions, "We are fully convinced that^
many of the people in this area honestly believe they were being subjected to'
a highly toxic and extremely poisonous compound with a high degree of persistence and one which would increase in concentration in the water supplies,
and in the bodies of humans, and animals. These ideas are not in any way
supported by research findings."
Now, that is dated ,Tuly 22, 1969, and if I just would ask Professor Galston
when was the Bionetics study brought to light?
Dr. GALSTON. It was handed over to the Department of Health, Education,
and Welfare in December of 1968, to the best of our knowledge.
Congressman MCCARTHY. So that to the best of your knowledge, the Department of Agriculture
Dr. GAT,STON. Might have had access to that information.
Congressman MCCARTHY. Actually, the tests were run i7i 19(57. Now, Mr. Williams obviously either did not know about the Bionetics report, and I would—•
I would accept that, I don't think he did just from the tone of the letter, but
I'll ask you to comment.
Now, which do you think it was?
Mr. PIEHOVICH. First of all, Mr. Williams was heading a group for a general
survey of the effects here in the Globe area at the request of the Forest
Supervisor, and after the initial complaints. We've had si^equent studies go
forward, one of these coining out as a second task force >^B't which is somewhat more in depth. Mr. Williams' information was then^ra general nature
for an initial report for the forest supervisors. Williams himself is not an herbicide man. Mr. Boffin is, and his reason for talking with the Dow was to get
more information.
The second question you've asked regarding the Bionetics study was not
known to these people, and only known to a few people within the Forest
Service but the word of mouth communication that took place following the
review of the Bionetics study for publication.
This has precipitated a lot of discussion among the science community, and v
in the
Congressman MCCARTHY. Are you alluding to the Whiteside article in New
Yorker Magazine?
Mr. PIEKOVICH. No, that's the most recent and clarifying article, at'least I
found it very informative.
Congressman MCCARTHY. When did you first learn about the Bionetics findings on teratogenicity?
Mr. PIISROVICH. I personally learned about it in November when I was
assigned to this problem area, and I learned about it through reading in the
literature, seeing the discussions among others.
Congressman MCCARTHY. Was the present science advisors ordered ban ever
transmitted to you, or here in the area?
Mr. I'IISROVICH. We were furnished a policy statement from the Secretary of
Agriculture in December which referred to the DuBridge statement.
Congressman MCCARTHY. Did you take that as a directive not to continue
using 2,4,5-T?
Mr. PIEROVIOH. We understood it to be directed towards crops, and that }t
was not at that time being restricted in range-land use. However, we could
infer from this, and from discussions with our Washington counterparts, we
learned that there were other studies underway on this compound, and as you
perhaps have noted, we did defer our chaparral program in October. The last
spraying on this project was in June, and these events have unfolded since
that time.
It's currently our position here in this region not to use herbicides until
some of these matters are researched. The studies that are underway should
be most helpful to us in this regard.
Congressman MCCARTHY. I think there's a little confusion about just what
the DuBridge announcement banned, Doctor DuBridge said—this is October 20,
1960.
That 2,4,5-T would be prohibited for use on American agricultural products
after January 1, 1970, until the Food and Drug Administration could develop
information showing that it could be used with safety.
Dr. DuBridge also announced that the use of 2,4,5-T in Vietnam would be
restricted in areas remote from population.
Mr. PiioROVicn. This is where we found our references to the crop production
area, and the Secretary has interpreted this way. As I said the ban on crops
is in effect at this time, and as near as we can tell we are also examining the
future of the 2,4,5-T as it is compounded today.
Dr. GAT.STON. Congressman, could I make a comment here?
Congressman MCCARTHY. Yes.
Dr. GALSTON. I was unable to understand why when Dr. DuBridge issued
this statement he did not also take care to specify prohibition of use in
regions where 2,4,5-T might find its way into drinking water. For example,
supposing you are using 2,4,5-T to clear shrubs from under a power line, and
that power line is going through a town where people have wells, and they
draw water from these wells. Don't we need to know if the 2,4,5-T is going to
seep down in the water cable and get to these people? It seems to me applying
the ban to the food crops is only a halfway measure.
Mr. PIEROVICH. I think we need to be concerned by this, and this is why we
monitor water from treatment areas. It's significant in this Globe area. Our
reference—or the Federal water quality control criterion of one-tenth part per
million, this level has never been reached in any of the water, analyzed that
we've had run, or had been brought to our attention.
�Congressman MCCARTHY. You say yon received the directive NovemberMr. PIBBOVIOH. We received the Secretary's explanatory information in
ecember as I recall.
Congressman MCCARTHY. Were you ever advised that the bnn had
Mr. PIEROVICH. No, sir.
Congressman MCCARTHY, So the last you had was the DuBridge directive?
Mr. PIBHOVICH. Yes, and a statement from our Secretary to agriculture agencies o.C which we are, telling us that 2,4, 5-T was not to be used in crops, and
incidentally, the Secretary has added to his statement that we would use alternative methods whenever these are available and practical, and is stressing
within the department a use of noncheniical means where these are available
to us.
Now, this is all developments since the last spraying here1 at Globe, I hope
this is clear.
Congressman MCCARTHY. Are you spraying in other parts of your region?
Mr. PIEROVICI-I. No, sir, and we have no plans to spray during current, or the
coming fiscal year at this time.
Now, if we have some break-throughs, I'm sure we will be talking about
this. Again, it would be premature to say.
Congressman MCCARTHY. What's the basic rationale behind the spraying here
at Globe V
Mr. PiraiovicH. You meanCongressman MCCARTHY : AVhat's the purpose of it?
Mr. PIEIIOVIOH (continuing) : The purpose of the project. This is the part of
the region, and the Tonto National Forest chaparral management program.
This program has many objectives for —if I may take a minute— fire is a very
common ingredient in the life history of chaparral, and in trying to bring
management to Chaparral Forest, we have excluded fire, or we are using fire
by prescription, rather than have the chance of holocaust. In doing this, we
attempt to bring a break to the fuels in large continuous masses by developing
grassy ridge tops, or grassy openings. These have other advantages for people
who want to use the forest, and for game.
It happens that the project here in the area was a water-yield project. We
have learned through research at the 3-Bar experimental area, and particularly that we can substantially reach the flow of streams, particularly in the
winter months where the vegetation is not using the amounts of water that
chaparral vegetation does.
Now, herbicides were used here at Globe partially because of the known
flooding potential of these streams, and that they also know that fire over a
large area could cause floods. So rather than use prescribed fire as initial
treatment, herbicides were .used.
We have plans to use some small amount of fire to continue our work here.
Congressman MCCARTHY. Doesn't it say right on the container that this
should not be used over water?
Mr. PIEHOVICII. That's correct, and as the project instructions were followed
here, the applicator pilot was to interrupt his spray every time he passed a
major stream channel.
Congressman MCCARTHY. "Interrupt his spray," you mean from a helicopter?
Mr. PIEIIOVIOH.' From his helicopter, yes.
Congressman MCCARTHY. Do you think that is that the answer?
Mr. PIEHOVICH, Well, I think it's quite practical, sir.
Congressman MCCARTHY. Well, wind might carry. Aren't there restrictions
under the circumstances in which you use it?
Mr. PIISROVICH. First, let me explain in spraying this area the primary pattorn would be along, or parallel, or to a water course so that it isn't necessary
to turn valves off as you may each time he crosses at the creek, but he was
going to be crossing streams at the same time he has been spraying. So he
would be than instructed to interrupt the spray before making such a crossing.
Some drift did occur into the bayous, we have found some of the Sycamores in
the Kellner area, the tops have been hit. We don't feel that a substantial
amount of herbicide came to the water course, and the pilot was instructed
not to apply this over water.
Water residues again haven't indicated any great amount of the herbicide in
water.
I Congressman MCCARTHY. Are they instructed only to spray when the wind Is
blowing at a certain mile per hour?
Mr. PIEKOVIOH. Yes, that's right.
^_
Congressman MCCARTHY. What is it, eight ?
Am
Mr. PIEROVIOH. In some projects it's 5-miles per hour, in t^pjase it was 10.
Congressman MCCARTHY. Ten ?
Mr. PIEHOVICH. Yes.
Congressman MCCARTHY. Is that rigidly adhered to?
Mr. PIEROVICH. Well, I would hope that it is, here we are depending on other
people to do our work, hut we have a project area officer, aud this project had •
•u project area officer who works from the helispot where the copter is operating, using a pocket anemometer, aud as lie noticed the wind picking up he
would take the pocket anemometer out and keep track of the gusts. Whenever
it apnroaches 10-miles per hour, the project would be shut down.
i have records here with me of the shut-down on this project, if you are
interested.
Congressman MCCARTHY. You are undoubtedly aware that some of the residents in the area charge that spraying went on iu much stronger wind velocities?
Mr. PIEROVICH. Yes, sir, I am, and I am aware that there has been drifts,
and we are attempting to identify how far tin's drift went. In the task force 2
report, we identified a visual effects drift line, we are currently working on
infrared interpretation, and I would be very happy to furnish you with a map
which delineates how far the dead vegetation that shows up. That's not available to see by the naked eye.
Congressman MCCARTHY. That would be very good to fill out the record. I
would like to have that documentation very much.
Dr. GALSTON. Do you mind if I ask a question at this point?
As a scientist, I'm interested in following up one line of questioning here.
The benefits that one wishes to derive from this program has to do with
increased water ilow?
Mr. PIKHOVICH. In part.
Dr. GALSTON. And the other part is, I presume, to have a more accessible
and manageable terrain where the Chaparral vegetation is?
Mr. PIBKOVICH. That's a good generalization among other things. We would
like the esthetic qualities of the area to be an indication.
Dr. GALBTON. Do you see any deleterious consequences of partial denudation
of the hillsides where Chaparral is growing?
Mr. PiEttovion.,It's not our intent to denude the hillside.
Dr. GALSTON. I'said partial.
Mr. PIEKOVICJI. In the course of making a conversion, one often has to take a
compromise, and we do compromise to the extent that we will—say for example, in burning—taking out a.n area, we will burn only so long a slope here
because any more we would have an overflow of plants and water, and erosion
while it is bare from burning', it is an opportunity for a torrential thunderstorm, or wind to cause erosion. But this is also one of the compromises that a
farmer must make when he plows his field.
Dr. GALSTON. And this is something you think you can keep under pretty
good control with applied herbicides?
Mr. PIEROVICH. In this case we used herbicides for that reason, yes.
Dr. GALSTON. Was there any measurement for the relevant erosion rates
before and after herbicide use in a given area?
Mr. PIEROVICH. In the 3-Bar area this is being noted at this time. The studies have been in progress for some time, I don't have those data with me, but
I could find them for you.
Dr. GALSTON,. I, personally, would be very interested in having those data.
It's been my impression that some programs have been gone into fairly massively without the comfortable feeling that there's a lot of scientific data
behind the original studies to tell us that, this is really what we ousiht to do,
and in calculating returns per acre, in terms of where we've applied, I think
we have to have a negative quantity in there for possibly deleterious effects,
that possibly are not measured.
Congressman MCCARTHY. I'd be eager to see those.
Mr. PiEnoviCH. I'd be happy to furnish them for you. I think something we
have going right now, you may notice in the statement we've furnished you,
�e are looking at -alternatives, and tolerable levels, and we are approaching
very tiling using projects that have been installed as a basis for arrivi
Congressman MCCARTHY. On that I wonder if I could ask yon, are you
giving licenses for the use of Kuron?
Mr. TiEUovicH. We give no licenses for chemical uses. The answer would be
no.
Congressman MCCARTHY. I see. From whom do they get these licenses?
Mr. PIEROVICII, The use of chemicals is done by—in our case, the approval of
a project proposal by a regional and national pesticide committee. Once the
forest officer who has a project wants to apply a herbicide he prepares a ,
formal proposal, it's submitted to our regional committee, if they approve, to a
national committee. And I'll tell you right at this point, our committee won't
approve such a use, but we don't license,
Congressman MCCARTHY. Well, thank you very much.
Will you be available today and tomorrow?
Mr. PIEROVIOII. Yes, sir, I will, as will the ranger and the 'acting supervisor
here.
Congressman MCCARTHY. Thank you very much.
Our next witness is Dr. F. I. Skinner, veterinarian from Globe.
Is Dr. Skinner here?
Dr. Skinner, I'm pleased to have a veterinarian testify in light of recent
Indications that the use of 2,4,5-T spray may have had harmful effects on
animal fetuses, I wonder if you would, for the record, identify yourself, your
background and experience.
Dr. SKINNER. I am Dr. Skinner, local veterinarian, I've been in the area 14
years, graduate of Kansas State University with a degree of T.B.M..
Now, these are my people, and I've lived amongst them. Now, any questions
you'd like to ask I'll try to answer.
Congressman MCCARTHY. Would you recommend the use of this , Silvex
Kuron spray after tests have shown that is has teratogenic effects on animals?
Dr. SKINNER. No, I wouldn't recommend it without further study, further
research.
Congressman MCCARTHY, You think it should be stopped untilDr. SKINNER. Yes, sir.
Congressman MCCARTHY. You have some question about the Bionetics findings of the effects of this oh animals?
Dr. SKINNER. I'm a clinician, I'm not research. I have not seen any effects
of animals in this area—definitely, clinically. Now, as I say I'm not a
research, I'm a clinician. I don't set myself up to be an expert on it, but I've
not seen any abortions, malformations of fetuses in this area that I can clinically say it was caused by Silvex, or 2,4-D, or pesticides.
Congressman MCOABTHY. As I understand it, and we hope to hear from
others, that there have been allegations made that the 2,4,5-T sprayed did
cause malformation in animals.
Dr. SKINNEB. \ cannot speak for those, I have not seen them myself.
Congressman MCCARTHY. You did not. Were you ever asked to examine the
animals in question?
Dr. SKINNER. No, sir.
Congressman MCCARTHY. You were notDr. SKINNER. No, sir.
Congressman MCCARTHY. So that you just don't know?
Dr. SKINNER. I don't know, I don't pretend to know.
Congressman MCCARTHY. All right. Well, maybe they will be calling on you.
Dr. SKINNKR. I hope so.
Congressman MCCARTHY. Well, thank you very much, Doctor Skinner.
Dr. SKINNER. Thank you, Congressman McCarthy.
Congressman MCCARTHY. Our next witness we'd like to call is Mr. Eobert
Mc.Kuslak.
Mr. McKusiak?
Mr. SICOMP. Sir, I represent Mr. McKusiak as an attorney, and he's
requested that he be called later. Can you pass him at this time? He wants to
pass at this immediate time.
Congressman MCCARTHY. Surely.
In that event we'd like to call Mrs. Billee Shoecraft.
Mrs, Shoecraft, I wonder if you'd identify yourself for the record, and -
i
Mrs. SHOECRAFT. Billee Shoecraft, Ice House Canyon, Globe, Ariz.
Congressman MCCARTHY, And if you would tell us a little bit about how long
you've lived here, and your own experience with the chaparra^pray program?
Mrs. SHOECRAFT. We have been in the area since 1947—Mr^H>ecraft a little
longer than that,
^^
Congressman MCCARTHY, I wonder if you could tell us about your experiences with the spray program, nnd some of the correspondence you've had
with the various agencies of government in this connection.
Mrs. SHOECBAFT. I'd be glad to, thank you.
We first became aware that they were going to spray a chemical, which they
asserted was harmless
Congressman MCCARTHY. You say, "they"
Mrs. SHOECRAM. The Forest Service.
Congressman MCCARTHY. U.S. Forest Service?
Mrs. SHOEORAFT. Right, in 1965. They had published in the local paper a
news item dated August the 19th, 1965, in which they said the herbicide will
be 2,4-D, and 2,4,5-T mixed with diesel oil, and water. The diesel oil will serve
as a weight factor to insure against wind drift. Neither 2,4-D or 2,4,5-T is
harmful to birds, insects, fish, wildlife, or humans.
^
Congressman MCCARTHY. Do you have a date and name on that?
What was the publication, what newspaper is it?
Mrs. SHOECBAFT. From the Arizona Record.
Congressman MCCARTHY: Of what date?
Mrs. SHOECRAM', Of August the 19th, 1965.
I also have the typed-i\p version when he initiated at that time from which
he deleted the word. "1 anticipate honest inquiry from many individuals and
groups concerning the project! I also anticipate adverse criticism and harassment from those who devote their lives to criticizing and harassing."
I forgot to read the part where he invited the general public to come and
see them spray.
If you are as curious as I am, you will want to drive up and watch the
operation. I hope you will.
Again, I read from the report No. 16, Georgia Forest Research Counsel,
Macon, Ga., 1865. On page 28 it. says, "Possible harmful effects: 2,4-D and
2,4,5-T have a. low toxicity, although spray applications leave no toxic residue,
a tolerance of five parts per million has been established on or in apples,
citrus fruits, asparagus, pears, and quinces. We can find nothing in the
Department of Agriculture to back this up."
Then, they further said, "Since some persons may be allergic to the oil in
the herbicide mixture, skin contact should be avoided, and when treatments
are used a respirator is also a desirable piece of safety equipment
Congressman MCCARTHY. Who is saying this?
Mrs. SHOEORAFT. This is from the Southwestern Forest Experiment Station,
Forest Service, U.S. Department of Agriculture, Asheville, N.C.
Congressman MCCARTHY. And the day on that, please?
Mrs. SHOECRAFT. The date on this was 19651. It further says—after mentioning the respirator, the odor, or vapors may bring on a case of nausea. The
Forest Service Health and Safety cautioned that 2,4-D' and 2,4,5-T are mildly
poisonous, and flammable in an oil base. However, we were invited to come
and see the spray.
Congressman MCCARTHY. Do you have any more documents that cast some
Mrs. SHOECBAFT. Oh, I've many.
v
I have here this little item that was given to us, there were a few missing
pages, it only had four, so I got in touch with Dr. Holston (phonetic) at
Belleville, Md., because this is the U.S. Department of Agriculture, and I wondered where the rest of the pnges were. So Dr. Holston from Belleville mailed
me a package in which -was included the rest of it, it totaled 25 pages, and
this concerning the toxicity of some organic herbicide to cattle, sheep, and
chickens. It tells about some of the things that they found in relation to the
herbicides that we've been sprayed with. We don't know exactly because the
reports have varied, but they have said they used 2,4-D, 2,4,5-T, and Silvex.
They further said it one form, then the tests showed different forms. I quote:
"We concluded—that the enlargements were caused by the chemical reaction of
the diluted herbicide formulation. The ecropsy—the liver was enlarged and
viable. The kidneys were congested. A small abcess was found in the parotid
45-382—70
101
�lymph node. In one year that developed a swelling in the region related to the
chemical reaction. Associated other lymph nodes of the body were often
rged and hemologic."
Congressman MCCARTHY. Mrs. Shoecraft, I wonder if just for the
iight just interrupt you briefly. I would like to ask Professor
would explain the difference between Silvex Kuron, 2,4,5-T, and 2,4-D just for
the record.
Dr. GALBTON. These are very closely related materials, and I think from the
toxicology point oE view, and from the points of view—the presence of any o£
these impurities like, the dioxin we were talking about, they would all be in
the same bag.
2,4-D is 2,4-dichlorophenoxyacetic acid, 2,4,5-T has one more, that is 2,4,5tridilorophenoxyaectic acid, and Kuron is simply a trade name for a similar
preparation that I believe is a Dow product.
Is that correct, I don't whether the foresters here would
Mr. PIEROVICH. Yes, that's correct.
Congressman MCCARTHY. Is there anything significantly different between
2,4,5-T and Silvex?
Or. GALSTON. I would say none whatsoever from the point of view we are
talking about. The toxicity would not be due to the length of the chain, but
due to the fluorinated aromatic nucleus, as a chemist would call it.
Congressman MCCARTHY. Mrs. Shoecraft, I realize you have many documents, and wo would like if we could to have any of these you would care to
submit for the record.
Mrs. SHOECRAFT. I'd be glad to.
Congressman MCCARTHY. Would you, this would help very much.
Mrs. SHOECRAFT. Yes.
Congressman MCCARTHY. However, now, if there are any particularly salient
quotations that—without being overly lengthy, you think should go into the
record at. this point, we would like to have those.
Mrs. SHOECHAFT. May I submit Farmers Bulletin Number 2158, U.S. Department of Agriculture, issued April 1961, slightly revised, August 10C9, referring
to what their rules are on what the wind velocity should be.
Congressman MCCARTHY. What does that say?
Mrs. SHOEORAFT. It says, "Apply the spray when the wind velocity is less
than 6 miles per hour, and the air temperature is 90" or less. Again use a
coarse spray
"
They did not use a coarse spray, they used a fine spray. "Use a slowly
vaporizing formulation."
They did not use a slowing vaporizing formulation, they substituted water
for oil in a very small amount and released it at very high altitudes on a very
hot and windy day, and they kept no records—weather records on the job.
Congressman MCCARTHY. Can you substantiate those points ?
Sirs. SHOECRAFT. Yes, I can.
' '
'
' "
Congressman MCCARTHY, How?
Mrs. SnoECKAFT. I'm reading from file Noi 2520, and it states in this lefthand corner to the file, it's from William H, Moehn, district ranger.
Congressman MCCARTHY. How do you spell that?
Mrs. SnoiccuAFT. M-o-e-li-n, district ranger, date July 11, 1909, subject: Watershed protection, • Kellner Russell chemical maintenance, fiscal ywir' 3000.
This memo is a resume of the fiscal year 1909, maintenace project.
"The spraying- done on June 8, 9, 10, and 11, 1969, were started at 6:40 a.m.
on Sunday, June 8, and the hilltop located on the Icehouse Canyon Trail, at
(i ;51 a.m. after the third load was through, the pilot flew to the O.C.O. Camp
to check his spray. Wheu he landed Mrs. Shoecraft arrived and told him some
of tho spray had landed on her. The pilot returned at the hill at 7 :14 a.m. and
said someone should go talk to her.
"I left the spray job at that time and did not locate Mrs. Shoecraft."
In fact, I cnlled Washington on the third day, but they didn't find me, but
I hoy could have if they had looked.
"I left the spray job and we continued to spray from the hclispot until
30:57 a.m. when <we landed at the helispot the wind was coming out of the
Knst from 6:40 a.m. to 10:57 a.m., we left and went to the Pinnl Road helispot
and began to spray. We continued to spray until 15.05 a.m., at which time the
wind reached 10 miles per hour plus, and we shut down. We resumed spraying
• .at 5 :03 p.m. when the wind dropped below 10 miles per hour and continued on
until 7 :35 p.m.
continued to
"On July 9, the first load was off the ground at 5:35 p.m.
in excess
spray until 10:18 p.m., at which time we shut down because n
•of 10 miles per hour. We did not spray anymore on the 9th.
"We started at 6:02 a.m. on June 10, 3 days after Mrs. Shoecraft had notified, and flew until 11:15 a.m., when wind forced us to shut down. We did not
spray anymore on the 10th.
"On June 11, we started at 5:18 a.m., and flew until the project was completed. A total of 977 gallons of Silvex was used at a rate of 2 pounds acid,
•equivalent per acre. The total rate per acre was 8 gallons. 1,000 acres were
treated. We did not keep weather records on this project.
"The wind speed and direction at the Globe Ranger Station at 1 p.m., each
day of the spray job are listed on the next pages, and It shows on June 11, a
fipeed of 16 miles per hour southwest.
"Signed and stamped by William H. Moehn."
Congressman MCCARTHY. So that even in his own records he acknowledges
that he exceeded the limits that had been set?
Mrs. SHOECRAFT. Yes, he did. I refer further to the Department of the
~~'
~
' -
-
*-*-'
•>
•"
T>—^«Ar.xv«4.«4-4,,n
Qf^ifyrtT'c,
victims. I was informed by the Department Office that they sent it out to the
printer's. My suggestion was you either get it from the printer's, or you get a
copy, I need it now. I received it in 3 days.
In this it refers to the formulation which they call, Orange, and it says that
it is one part 2,4,5-T, and one part 2,4-D. I have before me a letter dated October 6, 1969, from the USDA, in Phoenix. The branch of the Forest Service, the
Tonto National forest Service, signed by Mr, Jenkins for Mr. R. B. Cortney,
Forest Service. He says:
Dear Mrs. Shoecraft, following is a list of chemicals purchased by the Tonto
Forest as requested by you. The mixture was two gallons chemical with seven
and one-half gallons per acre. In a few cases more water was used, and all of
them are 2,4-D and 2,4,5-T.
Since I was curious because there was no Silvex, I further proceeded to say
who bought the Silvex, and I was finally informed by Mr. Moore at Salt ttiver
Project they made the decision to purchase the Silvex. They did not purchase
it as they said in the Forest Service. They have lied, it's the only word I'd
like to use because it's lying when it covers things when they know better.
Congressman MCCARTHY. I wonder if you could submit those documents to
Mr. Riddleberger for our records?
Mrs. SHOECRAFT. All right.
Congressman MOCABTHY. And if you are available we hope to go out this
afternoon and tour the area.
Mrs. SHOICCRAFT. Be pleased to.
Congressman MCCARTHY. Thank you very much.
We would like to move on now and hear from Mr. McKusiak.
Mrs. SHOECRAFT. I had requested analysis that were clone on our plant back
in September before another task force is to arrive, which I understand is
next week, I've spoken with Mr. Tschirley this morning, he called, I told him
before I wanted anymore samples taken. I would like the reports of what they
took in September. They seem to be still evaluating these water samples we
sent in, and for your information I just learned this morning the samples
taken from our own drinking water last week are still highly contaminated,
and I suppose I'm the first human to go on record to be able to say that they
have now found 2,4-D in my pound of flesh, and that was as of this morning
from two different laboratories.
Congressman MCCARTHY. That's important, could you elaborate on that? Do
you have those laboratory findings?
Mrs. SHOECRAFT. These were found in the G.H.T. Laboratories in California,
the other laboratory I'm not even aware of the name where the samples were
sent.
Congressman MCCARTHY. What's that, G. H.
Mrs. SHOECRAFT. That's the laboratory where the Department of Agriculture,
Doctor Hemton (phonetic) had recommended that the samples be sent on the
plant life originally. There will be a longer report on it this afternoon.
�142
^Congressman MCCARTHY. We will check that out. Did you mean to
lat a biopsy has been applied on your tissues, and 2,4-D has been foil
your
Mrs. SIIOKCRAFT. As of this morning they were not complete.
Congressman MCCARTHY. Thank you very much.
We'd like to call Mr. McKusiak now.
Mr. McKusiak, do you care to be accompanied by counsel? If you do, it's
perfectly all right
Mr. SKOMP. We hnvp no objection,
Congressman Mc( AKTHY. All right. Mr. McKusiak, I wonder would you identify yourself for the record, please, your name and your background, and how
long you've resided here.
Mr. McKusiAK. I'm Robert McKusiak, and I've been an Artist in tile and
mosaic for somo 22 years. I have a background prior to that time, and since
that time also in science. I majored in chemistry in college,
Congressman MCCARTHY. What was that?
Mr. McKusiAK. University of Arizona, I do not hold a degree.
Congressman MCCARTHY. How long have you resided here?
Mr. McKusiAK. I've lived in this area since 1932 with the exception of the
time that I attended the University of Arizona.
Congressman MCCARTHY. Now, I wonder if you would verbally give us a generalization of your experience with the Forest Service spray program?
Mr. McKusiAK. My experience with the Forest Service spray program really
didn't come into, being fully until 1969 following the June spraying. Well, let
me back up, it came into being in about May 31, 1968. I was aware prior to
that time that they had been spraying, but I was not aware that the things
that they were 'spraying were particularly harmful. I had seen unusual effects
taking place, but. I didn't know what to attribute them to.
Congressman MCCARTHY. What unusual effects, could you cite a couple?
Mr. McKusiAK. Yes, one in particular which I would prefer that Mrs,
McKusiak documented for you because that's her field, and not mine, but specifically in 1066, in May of 1966, the brown pewee population, these are birds
Hint, live in our canyon area, suddenly started dying in great numbers in our
yard. We have a waterer that birds come to, and there were birds all over
during May which had matter in their eyes, and seemed to be having respiratory trouble, and were dying, and at that time we continued spraying it.
Congressman MCCARTHY. You don't happen to have any photographs of that,
do you?
Mr. McKusiAK. No, I don't, I would prefer on a discussion of birds to have
MA'S. McKusiak go into this because that.was her field. But, in 1968, on the
31st of May, I was up at my property where I get my clay, it's private land in
the area that wns sprayed, it was included in the area sprayed. I had my wife
and three children, and the two dogs up there, and the spraying was taking
place down canyon. The helicopter came up the canyon, we have a stock pond
that was between us and the edge of the property, so to speak, and the helicopter came up the canyon and made a turn southerly, in other words, it made
n right-angle turn toward the moxmtains, and it approached. We were waving
our arms because we didn't want to be sprayed. He made a turn and he was
so close to UK, and the spray descended upon us, and upon the pond, and upon
oar kids and dogs, and so forth. At that time we weren't really aware that
anything was wrong with it except we both rushed home, my wife and I have
both had headchcs from it.
Congressman MCCARTHY. The pond, is that drinking water?
Mr. McKusiAK. This is a pond which is used for livestock water, but it's on
private land.
Congressman MCCARTHY. Now, you heard undoubtedly the Forest Service say
that they stopped spraying 'when they would get over a stream, but they didn't
over a pond. I suppose that would be obviously important?
- Mr. McKusiAK. It's incorrect that they stopped over streams, they sprayed
directly over three different semipermanent streams that I know of, and one
permanent
Congressman MCCARTHY. Did you see that yourself?
Mr. McKusiAK, I saw them spraying in this area over it, and the devastation continues right down to tho edge of the stream, it's quite visible.
Congressman MCCARTHY. Will we be able to see that this afternoon?
Mr. McKusiAK. I'm sure you will.
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Congressman MCCARTHY. I think it's very important.
vaSsii;
Mr. McKusiAK. One canyon in particular in 1968 when I walHsprayed with
my family on our property, and we did have illnesses and have had illnesses
thereafter, continued since this time. This particular little canyon, when they
flew up toward us—which has a permanent stream in it, and they flew right
up the canyon to the pond, it's a stream that seeps out from the pond, and has
never been dry.
Congressman MCCARTHY. I wonder if you would, for the record, tell us about
changes in livestock, and other animal life on your farm, which you would
attribute to this spraying.
Mr. MoKusiAK. I really don't have a farm to correct the record, I have
many different animals, my wife keeps ornamental fowl, she is an archeoornithologist, and she works with archeologlcal birds, and she keeps flies of various types for comparative work, and also for our own enjoyment
We have 10 or 12 milking goats that we have had for 10, or 17 years. Wo'vo
kept a small population of them, and in the last 2 years we have had a
number of our milk goats bear kids, they have from two to three offspring a
year, each goat, and a number of these have borne deformed offspring. When I
say deformed, I'm referring generally to their heads, their heads were born
^alshaped, and malformed in some cases their bodies, but generally their
heads.
We have one goat which is already been covered by the news media, but we
have one goat which wasn't as malformed as the others. We have kept it alive
simply because people were denying such things happening. I would say most
of the offspring that were born were born either dead, or deformed, or both.
Most of them who were born deformed were born dead. In other words, the
animal miscarried deformed offspring.
Congressman MCCARTHY. Did you ever ask Dr. Skinner to come out and look
at these animals?
Mr. MoKusiAK. No, I don't believe I've ever discussed these animals with
Dr. Skinner until just recently, but Dr. Skinner and I are good friends, and
we have from, time to time called him to ask how much dosage to give an
animal if we were going to give them a shot. Some of our animals from time
to time have suffered from pneumonia, or things of this type. For- example,
many of our fowl in birth have died. I'm referring specifically to geese, and
ducks, and some chickens, and many of them have died, and we found by
giving them a shot of com-biotic, it's a penecillin streptomycin, I believe, combination, by giving them a shot, usually we could save them. These fowl would
come down with what seemed to be pneumonia. There are many other people
in the canyon whose fowl done the same thing.
We found by giving them a shot we could save them. We called Dr. Skinner
to find out what the correct dosage would be, and we generally didn't call
back telling him it came out.
Congressman MCCARTHY. Well, Mr. McKusiak, I know we could go on for
some time, but we have to adjourn shortly, but we will be with you this afternoon.
Mr. McKusiAK. I would like to make one other comment, if I could, for the
record.
Congressman MCCARTHY. Surely.
Mr. McKusiAK. I was talking about 1968 when we were sprayed on our own
property, and our own dogs following this spraying, we went home and
washed, but our own dogs that were with us, two of them became ill immediately with what we considered to be pneumonia, at that time we didn't associate it really with the spray, we didn't think about it, and we gave the dogs
—we tried to call Dr. Skinner and he was out of town, and we gave the dogs
com-biotics for this, and I believe it was the next day we called Dr. Skinner,
ho was hack, and, my wife checked with him and she cheeked the dosage she
had given them, and he said it was twice too much, and give them half as
much again, and we did, and the dog survived. It would have died if we had
not given him the medication.
Congressman MCCARTHY. You still have the two dogs?
Mr, McKcjsiAK. Neither are malformed or anything, one of them has never
been quite well, it's never been well, It wheezes a lot
Ono other thing, there are many families in the canyon and many families
in Globe and Miami who have dogs that are bleeding from all body openings.
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Wo have clogs of this type, and people who have had dogs die from this, we1
could put you in contact with.
Congressman MCCARTHY. We would like to have that information.
Well, thank you, Mr. McKusiak. We'll look forward to seeing you this afternoon.
This hearing will stand adjourned.
Congressman MCCARTHY. The hearings will come to order.
I've just received the following letter from the White House which I wish to
read into the record at this point. It's from the Science Adviser to the President of the United States, Dr. Lee A. DuBridge.
"The White House, February 10, 1970.
"Dear Mr. McCarthy: This will acknowledge your February 3rd letter cop-^
corning 2,4,5-T, the October 20th announcement that you referred to was'a
statement of the actions that were planned to be taken by the various units of
the Federal Government in relation to the 2,4,5-T. It was not a directive toagencies for the simple reason that statutory responsibility for these decisions
rest in the separate agencies.
"I'm sure that by now you have heard from the Department of Agriculture.
I appreciate your views on the desirability of an investigation of reports of
birth, of malformed children in Vietnam. By copy of this letter I'm calling:
your views to Secretary Laird's attention since this area is primarily his
responsibility.
"As to 2,4-D, this compound is being reviewed along with other compounds,
being singled out as requiring additional study in the Bionetics records towhich yon referred."
Signed, "Lee n DuBridge, Science Adviser to the President."
I'd like to contrast this with a statement as it was issued on October 20'
whore DuBridge said that the Defense Department will restrict use of 2,4,5-T
to tlie areas remote from population, that the Agriculture Department will
cancel registration of 2,4-D for food crops effective January 1, 1970. TheDepartment of Agriculture- and Interior will stop using 2,4,5-T in their own
programs in populated areas, or where the residues from use could otherwiseroach man. That the Department of Health, Education, and Welfare will complcfc action on a tolerance for 2,4,5-T, the residues on foods prior to January
1, 1970.
This is obviously a retreat from the position taken by the White House in
October 29. As I road the statement at that time it was in the form of a directive that the departments will do such and such, now we find that the White
House is backing off from this, and is saying that the statutory authority restswith tho agencies.
It snems to me that the President of the United States has authority—Hie
nlh'mato authority over these agencies, and I regret very much that the President's Science Adviser has seen fit to retreat from the decision of October 29,.
which 1 believe was the wise one. The use of this particular chemical should
bo banned pending tests.
On the plus side I'm delighted to be informed last night,.and it's reported'
today in the press, that the distinguished Senator from Michigan, Philip Hart
has announced he will hold hearings on 2,4,5-T. He asked Secretary Hardin,.
Secretary of Agriculture, Robert Finch, Secretary of Health, Education, and
Welfare, and DuBridge to testify on March 11, This is further evidence to methat the compound's effects require additional evaluation, and I expect that I
will testify myself before this Senate Subcommittee when they have hearings,
I will make that request.
I should also announce that a report on my investigation will bo prepared in1
consultation with Dr. Galston, and will be issued at the earliest practical
point,
Now, we would like to hear again from Mr. Pierovich of the Forest Service.
Ts he here?
Mr. PusnovicH : Yes. sir.
Congressman. MCCARTHY : T would like to say for the record, which I just
said on tho radio station here, that T hnve boon very favorably impressed bv
Hie cooperation of tho Forest Service. I think that anybody who has any smattering of knowledge about this whole thing must realize that this is something'
transcending individual agencies out in the field, that we are td^lg here about
national policy, and what is done out in the field really is a result of decisions
made at a much higher level,, and to try to focus responsibility on a field unit
I think is really to carry this too far. I've been most impressed with your
cooperation, and that of your colleagues, Mr. Pierovich, and I want you to
know that we appreciate it very much, and our report will so indicate.
I understand you would like to elaborate on the statements you made yesterday. PIEHOVICH. Thank you, Mr. Congressman, for your kind comments, and
Mr.
also for the way you've conducted this hearing. I think the Forest Service is
pleased with the way the hearing has gone. There are some significant elements of Forest Service concern that I felt should be made a part of the
record 'this morning, and I'll read essentially from that statement.
First of all, the Forest, Service has used phenoxy herbicides, but not since
the nationwide controversy broke last fall. In fact, the last use of herbicides
on the Kellner Russell project was June 11, 1969, and to the best of my knowledge, the last use of any herbicide by the Southwestern National Forest was
the August, 1909, on the Gila National Forest in New Mexico.
Second, it's apparent there are several persons in this area who believe
there are unknown, or suspected characteristics of these herbicides which may
have caused them damage, and this is of concern to us.
Three, it's apparent we must continue our efforts to ascertain the extent of
drift levels of herbicide residues, and the definite relationships between herbicides over environmental factors and the responses of plants and animals in
this area.
These studies are to be made public when they're completed.
Lastly, the extent of continued deferment of herbicide use in the Clmpnrrnl
program is dependent upon the outcome of our studies and of the Department's investigation of these matters.
Congressman MCCARTHY : Thank you very much. I wonder if you could for
the record, repeat what you told me yesterday relative to the drift of tho herbicide over streams, and into adjacent private property, and what steps, should
this be resumed, assuming that it can be shown to be safe, what steps would
be needed to correct that?
Mr. PIEROVICH. At this point, this will be my own opinion, but I first mentioned to you yesterday that our instructions to the applicator pilot were to
interrupt his spray application when he crosses streams, we had definite plans
for the project here to call for application away from the open water, nnrt
main stream courses. I do believe there was some drift into this stream course
as evidenced by some top kill on the Sycamores on the stream bottom. There
has been drift from the project area onto private property which we have
established so far as the visual effects are concerned, and from this I'm certain that we will be developing new guidelines to both assure that the herbicides that we might apply in the future are confined to the project area, and
to assure the safety of the public.
One definite indicator in this is that it would be desirable to use a much
more restrictive windspeed in application.
Does that answer your question, sir?
Congressman MCCARTHY : Yes, but what wind velocity do you think would be
safe? PIEUOVICH : I wouldn't want to speculate at this time, but we do have a
Mr.
general rule of 5 miles per hour, and we know that herbicides .were applied
here to 10 miles per hour, and we see new development in the herbicide application field, the use of inverts has become more and more popular, niul with
some corrective work recently done is this area I feel this will help us a pvont
deal.
Congressman MCCARTHY: Another point that I definitely sympathize with
you about is difficulty you have of getting information. I think the fact that
you weren't apprised of the Bionetics Kesearch Laboratory finding on teratogonicity until late last year suggests a problem in communications here, and if
you have any suggestions for new legislation I'd be grateful. Do you feel you
get enough information from Washington on such subjects?
Mr. Pircnovicn, I fool that in all of our—the exchange of information Is a
very complex thing today. We do make ourselves available to conferences, with'
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pie in these fields. Our technicians in herbicide work attend meetings
,y on this matter. We are expected to keep ourselves informed. The ll_
•
ture has been quite full of the controversies on 2,4,5-T, and we have been
aware of the developing controversies.
The most healthy thing that could happen in this area would be a definite
summary of literature that our technicians could refer to. There are abstracts
available now, but the combination of inputs from the universities and from
the various departments of government in one abstract bulletin would be helpfill to us.
Congressman MCCARTHY. Do you have anything to add, Mr, Pierovich?
Mr. I'IEHOVICH. No, I don't, sir.
Congressman MCCARTHY. Thank you very much, we appreciate it.
Mr. PIEROVICH. Thank you.
Congressman MCCARTHY. Our next witness is Dr. Paul Martin from the University of Arizona.
Dr. Martin, I understand you are accompanied by Dr. Russell?
Dr. MARTIN. That's right.
Congressman MCCARTHY. Would you like him to sit with you?
Dr. MARTIN. Yes.
Congressman MCCARTHY. Dr. Russell, would you care to join Dr. Martin?
Dr, Martin, we appreciate your being here. I wonder if you would identify
yourself and Dr. Russell for the record, your background and your particular
interest in this?
Dr. MAUTIN. I'm Paul S. Martin, University of Arizona, Department of Geology. I had training as a professional ecologist, and with me is Dr. Stephen
Russell who is a zoologist in the biology department in the University of Arizona. His special interest is in birds.
Congressman MOCABTHY. Thank you. Dr. Martin, I wonder before the record
if you would tell us about your involvement with the spraying project, and
any conclusions that you reached, based upon your analyses.
Dr. MARTIN. Well, I'm not involved in the spraying project, and I'm not a
herbicide expert. I .have no research experience with herbicides. I do hcive
interest in the vegetation of Arizona. I've spent years studying its fossil pollen
records, but the interest I had in Globe was in first seeing if indeed there was
any effect on vegetation as a result of herbicide treatment that had been
called to my attention, I have come up on four separate trips to visit the area
that was sprayed, and see what little I could of the community.
Congressman MCCARTHY. How long did you spend on these trips?
Dr. MARTIN. These were 1-day visits.
Congressman MCCARTHY. How many did you make?
Dr. MAHTIN. Four. As a result of seeing the area, and talking to some of the
people in the area, I was curious to see if just what degree the community
might have been affected by this. I wasn't prepared to believe that people, or
animals could be affected by herbicide sprays because the little I heard indicated that those who work with herbicides stand underneath the spray plane
and are occasionally drenched by the chemicals, and don't suffer ill effects. , , , ,
So it seems incredible that people in this community could be complaining of
such an effect, but they were.
Indeed as a result it seemed to me that it was important to listen to them
and try to understand what they were saying, and try to come to terms with
the only observers who witnessed an event that wasn't supposed to have happened.
It also seemed to me that some of the people involved in the work with herbicides were unprepared for this sort of experience, they weren't even listening
to the complaints. So I presumed to do that.
Congressman MCCARTHY, And what did you find in the course of your four
trips?
Dr. MARTIN. There is one other person that's involved in what I'm going to
say next, I don't know if she's here or not.
Within the last month a student from Massachusetts by the name of Miss
Adolnide Friclc and she was willing to go on a door-to-door basis, nnd interview
people in the community apart from the ones that I talked to.
. Congressman MCCARTHY. Excuse me, is Miss Frick present?
Dr. MARTIN. I have the results, a summary of her door-to^^r investigation
in the area, the purpose was to see if there complaints coini^^rom any other
source other than the individuals that I talked to. The trips^Klt I'd made up
here and the design was to on a door-to-door basis talk to approximately 50
people in the canyons close to the sprayed area, and to another 50 over in
Crestwood, which I believe is east of Globe at a further—at a point further
remote from the area that was sprayed.
So what Miss Frick did was then conduct a door-to-door interview with
people close to the sprayed area, and another group of 50 further away from it.
Congressman MCCARTHY, What did she find, do you have the report? We
would like to have that for the record.
Dr. MARTIN. I'd be glad to give you a copy.
Congressman MCCARTHY. Would you care to summarize it?
Dr. MABTIN, I'll simply read about a paragraph from the report that summarized it, and of course, the individuals are not identified in this report, and the
complete questionnaire is not represented here, simply the highlights of it.
There are three key questions, two that have to do with personal health,
and one that has to do with livestock. It turned out that few people do have
livestock in either—neither the spray area, or in Crestwood, but quite a
number have pets. This is what she found.
Regarding pets, 13 cases in which animals were effected, and one must presume some relationship to spraying although in no individual case perhaps
could this be directly proved.
This is the experiences of people living in this community who know the
nature of the community, and then feel that something has happened that's a
little bit out of the ordinary.
Thirteen cases in which animals acted, three kittens lost; two dogs lost;
infertile eggs, one; rabbits not breeding, two; chickens not laying, one; burro
lost, one; sick dogs, three reports.
Now, as far as people are concerned near the spray area, 23 of 56 indicated
illness over the past 2 years which may be spray associated. Some people had
absolutely nothing wrong with them, or were not concerned. They thought that
those that were complaining were imagining it happened, an event that had no
bearing in the real world, that it was in the minds of the people reporting.
Other reported, and we're quite convinced that their experiences were
related to the events of last June, or earlier when herbicide spraying had Imped.
Of the 23 reporting illness, 21 were reporting breathing difficulties. Many of
these are attributed to the times of spraying. Some are attributed to smelter
smoke, there's no avoiding the fact that this area that experiences a good deal
of smelter smoke. Some of these people may be reporting an effect that is
indeed caused by smoke, I don't know.
There were five reports of serious diarrhea, including one entire family.
Four reports of chest pains, including one false heart attack, one report of
coughing of blood, one report of subnormal temperature: Two reports of numb
pain in arms; two reports of hemorrhaging; two reports of irregular periods;
one report of miscarriage; two others by hearsay.
Fifty-six people interviewed, 42 mentioned some damage to plants, although
the purpose of this questionnaire was not to consider plant damage.
Now, in Crestwood at a great distance from the
Congressman MCCARTHY. Was the interviewer able to determine if such complaints were prevalent before the spraying began?
Dr. MARTIN. I don't know how one would do that. In fairness to the people
in the Forest Service who have worked with this project, one simply can't conduct a scientific experiment at this point in time. All we can do is talk to the
people who were the observers, or ones—or residents in the area, and while
their memories are still hopefully fresh, recover some information, just having
to take them at their word.
Congressman MCCARTHY. Let me jtist clarify. Is the interviewer ascribing
these conditions to the spraying based on the interviews with the people? Do
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they say that these phenomena results were the results of the spraying, or
"|ou't they know?
Dr. MAUTIN. Yes, some of them would rather not say. The question
effect, "Have you experienced any sickness which might be related to herbicide
spraying of this area,"
It's a leading question in part. It's not a question that denies any ignorance
of the fact that herbicide spraying had taken place in the area.
I am sure there are many faults of a questionnaire of this sort that a professional psychologist would recognize.
Congressman MCCAHTHY, Let me say as a point of information, we will
shortly have put into the record a scientific data of the results on human
beings of 2,4,fi-T, which I think you will find bear a similarity to phenomena
you've just described.
I wonder if you would go beyond Miss Trick's survey to give us benefit of
your own observations of what you saw, and if you were able to reach any
conclusions about the effects of the spraying,on either humans, vegetation, or
animals?
Dr. MARTIN, Well, the effects on vegetation impressed me as ones that have
to bo watched over a period of time. Again, this problem of who's to make the
investigation, and how it's to be conducted are important. The incident is over,
and iu the minds of some local people, hopefully will never occur again.
The problem is, what really happened? I was up on four separate trips, or 4
separate days, I saw some things that I have not seen in Arizona vegetation
before. Such as the presence on Century plants of flowering way out of season,
and immature new plants going on the old stocks of old ones without normal
seed being set,
I understand that this particular species of Century plant is known to do
that, and other botanists have seen such a feature.
The area that was sprayed, not all plants are dead in it, Some species like
Mnnzanita are remarkably resistant up to this point.
The effectiveness of the treatment is doubtful. The areas of spray aren't
(lend. The effects of spray on the outside areas on different plants have to be
watched over a period of time to fully appreciate the change in phenology, the
changes of flowerng time, the change of time when the leaves appear, and
when they fall, the way the tradition of plants may be as far as overall
growth is concerned, and if one wants to demonstrate the herbicide-caused
effect on vegetation. It's also necessary to take into consideration all the other
environmental variations that aren't under control either, such as rainfall and
temperature.
Congressman MCCARTHY. But, you did find evidences of drift outside the
project urea?
Dr. MARTIN. Yes.
Congressman MCCARTHY. Did you find evidences of 2,4,5-T in any of the
adjacent streams, or did you seek to find it?
Dr. MARTIN. No, I collected samples only from within the project area, soil
samples and water samples.
Congressman MCCARTHY. You found evidences of 2,4,5-T in the water you've
collected within the project area?
Dr. MARTIN. The samples that I collected and submitted to a laboratory in
California came back with a report of the presence of 2,4-D, and smaller
amounts'-.of 2,4,5-T.
Congressman MCCARTHY. In the water?
Dr. MARTIN. Thorn was a trace in the water, there was up to one part per
m i l l i o n in the soil of 2,4-D.
Congressman MCCARTHY. Is there anything that you or your colleague could
add which would he pertinent to our inquiry?
Dr. MARTIN. I would make one recommendation, and then if Steve Russell
1ms anything he would care to add.
The recommendation would simply be that hospital records, doctors' records,
t.ho veterinary records of those doctors and veterinarians in the Globe area be
Bone over very carefully by proper professional people.
Congressman MCCARTHY. At that point I think we should pnl into the
record a memo of conversation with Mr. Peter Kiddleberger of my staff, and
149
Dr. Grantville Knight, M.D., 2901 Wilsbire Boulevard, Sigto 3'I5, Santa
Monica, Calif.
^P
This conversation took place on February 6, 1970.
^^
Dr. Knight informed Mr. Iliddleberger that he has two patients under his
TOIre from Globe, Ariz. While his examination is not complete, he is of the
"(Union that their malady is associated with the recent spraying of Silvex containing 2,4,5-T by the U.S. Forest Servicei Dr. Knight is of the opinion that an
Investigation is warranted, and offered to submit a statement of his findings
upon completion of his examination subject to the approval of his patients.
Miss Frick is here now, and I wonder if she could sit next to Dr. Martin
mid Doctor, if you would be good enough to reread that portion alluded to?
Dr. MARTIN. This simply summarizes the interviews that Miss Frick conducted in the canyons that is Kellner Canyon, Russell, Sixsbooter, and Ice•Uouso. Fifty-six interviews in that particular area, and some people who had
serious complaints to make were not considered in this interview.
What I found just in tabulating what her questionnaire revealed was that
23 of 56 individuals indicated illness over the past 2 years, which may be
spray associated, 21 individuals reported breathing difficulties, many of these
nre attributed to the times of spraying, but not all. Some were attributed to
smelter smoke.
There were five reports of serious diarrhea, including one entire family.
Miss FRICK. Yes.
Dr. MARTIN. Four reports of chest pain, including one false heart attack;
one report of coughing of blood ; one report of subnormal temperature.
Two reports of pains, or numbness in arms; three reports of uterine hemorrhaging; one report of a miscarriage.
There were two others that I thought were hearsay, hut I wasn't sure had
really occurred in family that you interviewed, and then finally all the questionnaires wasn't directed to plant damage, there were 42 people interviewed
who mentioned at least some damage to their plants in that area.
Now, the Crestwood account shows much less effect, and this is what one
might expect because of the distance further away from the area of spray.
Congressman MCCARTHY. Doctor Russell, is there anything that you would
udd to the record here that would be helpful?
Dr. RXTSSELL. I don't think I would add to the record, but I'm in agreement
with Dr. Martin's statement.
Congressman MCCARTHY. You are, you've studied the information he lias
available?
Dr. RUSSELL. I have seen much of the general information, but I've conducted no investigation of my own into it.
Congressman MCCARTHY. Thank you, Gentlemen, and Miss Frick, very much,
I'd like to now recall I'rof. Galstori.
Doctor, as we discussed here I understand you have some scientific data on
the effects on human beings of 2,4,5-T. I wonder if you would cite the source
of this information, and the findings?
Dr. GALSTON. Mr. Congressman, I'm very happy to present this information
because in the course of my wanderings around on this day I have found that
certain individuals tend instinctively to disapprove any allegations of direct
damage to human beings or animals.
Now, as I hoped I made clear yesterday, very small doses of 2,4,5-T can
cause birth abnormalities in laboratory animals, and that is now actively
under investigation, and we've discussed to see whether it might be due to this
Impurity called dioxin, or whether it was due in fact to the chemical.
But now, the question is, can we actually produce an effect on mature individuals, let us say male individuals, totally apart from pregnant females bearing embryos in uteri, and I should say that there is a fairly sizable respectable scientific literature on this, and if one looks in a variety of sources,
including the sort of encyclopedia of clinical toxicology by Gleason and CougliHn, and can find citations to many articles, and I have reference to a few
here.
Now, 2,4-D can produce, if it's administered in very massive quantities, it
•can produce death in the small animals, and there are even a few cases
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151
recorded oC its having produced very severe symptoms in man. ' ^^
sidered more
however, comes from 2,4,5-T, and I would like to read to you a 1W account!• Mnnznnita and Oak, and the desired persistence of what t
—
^.-,1^^
of an article published in 1959 by T. Flint entitled "Dermatitis and Kidne!|;; a^airnble plants such as gerardia.
.: Now, I suppose a question could be raised as some of the local residents
Damage Ascribed to Weed Killer 2,4,5-T."
' huve been raising undesirable, and desirable, according to whose criteria, and
Flint relates an episode involving two sisters, age 4 and 0 years, who
played for several hours in a yard which had been sprayed heavily a slioi* bf what judgmental values. Manzanita and Oak do live on these hillsides, they
time'-before with the Ortho brand of 2,4,5-T, brush killer. This was used fo: tlo transpire to water, and I suppose their killing is desirable m the contention
..f wanting to avoid the evaporation of water. Whether after you are all
the control of poison oak.
through with the operation and plant to grass, which is the stated objective or
This spray contained 15.4 percent of the isopro ester of 2,4,5-T in an ol'
base.
(hi» clearing observation, you are going to save very much water, I m not sure,
«ml whether, in fact, the esthetics of the environment will be improved
Now, I should mention parenthetically, I don't have the exact data at Imni
another stated objective of this operation is also I'd say open to question, I
but Kuron contains much more than that, I believe in excess of 60 percent of
would think it would be a very useful operation for those groups charged with
this same ester.
iimkttiK policy to hold some public hearings at which citizens could come with
The next day both girls exhibited generalized erythema—reddening of tta
their points of view. I think a lot of this fracas is due to poor interchange of
skin—and edematus swelling of the oral and vaginal mucous membranes.
information between official agencies, and the citizens. If there had been open
The pulse rate and body temperature were not elevated, but both childrn,
were described as appearing slightly toxic. The limbs and eyelids were sliglitl! • lion rings, and announcements, this is what we intend to do, this is why we are
swollen as the mucous membranes of the mouth were inflamed. On the 3d dwEj' doing it, and this is how we are going to do it, and have objections recorded
there were signs of kidney damage. Albumen was noticed in the urine. Tlietfj|p *t the time, a lot of the acrimony that's built up here might have been
' - , , . . i *.
was no evidence of liver injury, the urinary abnormalities persisted for about^5" avoided.
Now, so far as the damage of plants around homes, there is no doubt about
2 weeks, but 2 months later the urine specimens for both patients
normal.
It, it has occurred. I have seen it, and as a plant physiologist, I could testify
Now, there are other reports in which 2,4-D, and 2,4,5-T are alleged to hnrep. Hint this is typical damage due to herbicide drift. I think that this points up
caused toxic effects on the nervous system as measured by the electroen-j|; n lesson when you discharge herbicides from the nozzles of spray on a helicopter, you are getting an assortment of droplet sizes, the big drops are going to
cephalogram. That is after ingestion, there was a desynchronization of the electrical activities of the nervous system, I bring these points up only to rein- _ fall quickly, the small drops are going to be carried for longer distances. I
force the fact that no chemical is completely innocuous. Some individuals are pi, think until the technology is improved, the so-called invert sprays is one possimore sensitive than others, and some may require a big dose, and some a pi- bility here, and new types of booms for spraying are another, it seems to me
that it's very unwise to spray in areas where homes are so intimately assocismall dose to have these abnormal effects produced, but I share with Dr.
ated with the forest and woodland, that you are trying to control. You cannot
Martin the view that when people appear and say that they have been
pinpoint the spray, you cannot, keep it out of the water, and you cannot preadversely affected by these chemicals, immediate and adequate attention
vent inadvertent spray damage to the nearby residences, and I would say that
should be given to the possibility that these reports will furnish yet additional
there are certainly many sprays in the country where the application of aerodata to supplement the rather large amount of scientific data already existing.
sol sprays is a highly beneficial practice.
Congressman MCCARTHY. Thank you, Dr. Galston. I wonder if you couldFrom my cursory look here that I would say the intervening of house and
give us your observations after your inspection of the sprayed area, and tlio
area where it drifted.
the canyons in which spraying is desired, is so intricate that the slightest miscalculation, the slightest air movement, the slightest malfunctions of the spray
Is there anything that you at this point care to have in the record ?
equipment would lead to damage to the property, and I don't know how that
Dr. GALSTON. Well, I'll say a few words. I want to make it perfectly clear
could be' worked out technically, and I would want assurance that those probthat after 24 hours in Globe, Ariz., I don't want to pose as an expert either OD
lems are looked into.
. ,
the program, or the effects on vegetation, or on people, but as a biologist workI think the people whose plants have been damaged ought to be compensated
ing in this area, there is some conclusions I think I can make which point out
In some way because the damage has been considerable around some homes,
the need for still further investigation, and everything I sny should be held In
that, light.
nnd I think it's unfair to expect these people to bear the brunt of this kind of
Inadvertent drift operation.
What did I see on my brief trip yesterday? Well, I would classify them in
several categories.
Now, I did see damaged animals, and I talked with humans who alleged
that they were adversely affected.
Number .1, at the helispot, overlooking the picnic area, I observed and
All I can say here is the damage is there, and spray operations did occur,
smelled residues, there was no doubt that you could smell residual diesel oil
but I know of absolutely no scientific evidence which would link the spray
which was primarily the carrier for the herbicide which had been splashed
operation to the damage, and I think the people who showed me the damaged
during the loading operation onto the helicopter.
animals showed it to me in the spirit that this could be a consequence of
Now, if you could smell it, there was a good deal aronnd, and that would
spray operations, but they weren't sure, and certainly I'm not sure, but unlike
indicate that there are definitely residues in certain selected areas, how niucli
some people I would not immediately offhand say this is ridiculous. It could be
there was I can't, say, how much there might be in the soil, or in the water, 1
us I have shown from my previous reading from this scientific compendium,
cannot say, but it seems to me that I could smell evidences at various points
nnd I could document further a lot of the symptoms that people are reporting
in my trip. So that there probably are residues here and there, and those
here have been reported for massive doses of 2,4-D. So we should not leave the
could serve as a continuous supply of leaching, I suppose, into the waters of
possibility that this did occur, but a much more scientific information is
the area, one should not discount that possibility.
required.
The second category was definite plant damage, and the plant damage was
My overall view after one day of looking around is one of puzzlement. I
both the desired plant damage in the canyon, and undesired plant damage in
wonder why it's desired to initiate this kind of an operation in this kind of an
the vicinity of homes, which was due to the drifting, I assume, the herbicide.
environment The stated objective is to improve water runoff, and water runoff
In the canyons we could see, and these were pointed out to me by some of
will benefit, I presume, the citizens of a nearby urban area, Phoenix, which is
our Forestry friends who were with me, the desired killing of such plants «s
growing rapidly, and which has a lot of water requirements, and their water
�152
153
requirements will grow as the years go by. We know this is an arid
way, not being an Arizona resident, and not being a politician, I peThaps
could say some things here which a lot of people were thinking, but haven't
brought forth.
Truly, water is going to be wilting in this area for others. So far as I can
see unless nuclear technicology makes it available on a massive scale, Which I
don't foresee, if you take water from this area to give to another area, you
are, in fact, robbing Peter to pay Paul. If you are robbing water from here,
you are going to partially change the kind of vegetation, perhaps you are
going to denude some of the areas in order to increase the runoff, this involves
a comparative set of rules. Whose object is going to be gored here, whose
interests are paramount? Well, clearly cities are not going to be able to grow
indefinitely, we are going to have to put some limit on them, we know, for
example, that the city of Los Angeles got into a lot of trouble with smoke
because there are just too many people there. In the same way cities in the
Southwest may have to limit their size ultimately based on the number of
people they can support on the amount of water resources there are. The
trying to take every amount of water out of the Country brings a possibility
of a very serious question.
Now that President Nixon among others is calling for a campaign to restore
the environment, it might be that, we would want to look at this whole project
in the context of what we are doing to the entire State, and to the entire
countryside.
Finally, I would like to merely renew my suggestions that the people who
formulated this policy, who set up this whole spray program should identify
themselves, and should request the contributions of the citizenry as an input
to this whole program.
I think that policy should not be made without question. This is a democratic society in which citizens have responsibijity to interest themselves in
the making of policy, and—my faith in the American people, and in their
desire to run (heir own country has been to a certain extent reinforced by
seeing a group of aroused citizens here out to protect their rights.
Thank you very much.
Congressman MCCARTHY. Thank you, Doctor Galston. I think the points you
make are valid. One that I would just enlarge on a bit is that I am presently
working on legislation to be established to support a National Growth Policy, I
think growth has to be commensurate with the resources and of course, in this
•case, water is a critical resource.
I would conclude these hearings now with a couple of observations. I think
it's important to know that 2,4,5-T was developed fit the Army's chief Genii
Warfare Research Center at Fort Detrick, Md. My experiences in investigating
the Army's chemical and biological warfare programs, and policies, has not
encouraged me about some of the actions' that have been taken, without taking
into consideration some of the unforeseen consequences. For instance, when
they wanted to dispose of waste from nerve gas production at the Rocky
Mountain arsenal near Denver, they first dumped this material into ponds on
the arsenal's property. They didn't expect that it would find its way out. They
thought it would be just absorped in the water on the pond. It wasn't, it was
carried out into adjacent streams, and the neighboring countryside, and killed
among other things livestock and 6 square miles of sugar beets.
They then dug a deep well and figured the best way to dispose of it was by
dumping it deep into the earth. That set off 1,500 earthquakes in' the> Denver
area, some of them up to six on the Richter scale, and caused great alarm in
the community. They finally had to pull out this material, and of course the
earthquakes stopped.
Then, they thought they should ship it across the entire United States. They
thought this would be safe. Scientists later said it would risk the lives of
thousands of people, the plans also called for dumping this large quantity of
nerve gas and other materials into the Atlantic Ocean. They thought that
would be safe.
Scientists later said it could destroy all marine life in 600 cubic miles of tile
Atlantic Ocean, with a cataclysmic effect on ocean's production cycle.
Now, I cite these instances not in reproaching the Arms^fc: the C.B.W.
establishment, but I think that this particular program ha^p questionable
record.
We find 2,4,5-T developed by the Army's Germ and Gas Warfare establishment, 25 years ago to this date. We do not know for sure whether it will produce birth defects in human beings, I find it unwise to say the least to use
such a substance 'without being sure that it is safe. For some reason the
burden of proof seems to be on me and my colleagues in the sense that the
uttitude is, "we'll keep using it until you can prove it unsafe." Well, I quarrel
with the basic assumption, I think that it should be just the reverse, I don't
think that any toxic substance whether herbicide, pesticide, drug, whatever,
should be used, sold ill the United States until it can be shown that it is not
harmful to human beings, that it doesn't produce cancer, or birth defects, or
genetic effects.
One would think that we have learned from the Thalidomide experience, but
apparently we haven't.
I also find it incredible that the Dow Chemical Corp. could have succeeded.
In helping reverse an order from The White House.
Now, I read this section from the statement of October 29 wherein the President's science adviser said that certain agencies of Government, the Department of Defense, the Department of Interior, the Department of Agriculture
would do certain things, will inaugurate a new policy. Now we have the letter
received today from The White House addressed to me, advising me that The
White House is backing off from this directive, and is saying that the statutory responsibility resides with the individual agencies.
.1 find it personally unconscionable that in light of the Bionetics findings,
und the scientific data cited by Doctor Galston this morning about the proven
effects of 2,4,5-T on females, that this substance would be continued to be used
on wide scale in the United States, and for that matter in Vietnam where
even larger quantities are used.
I welcome the U.S. Senate Subcommittee On Investigation into this. I will
prepare a full report which will appear in the public documents that will be
developed, as a consequence of our trip will be made available to not only the
Semite Commerce Committee, but appropriate other committees of the Congress, as well as to the study of the American Association for the Advancement of Science under the directorship of Professor Messelson of Harvard.
We finally conclude by thanking the officials who have been most helpful,
and to the residents of Globe who have been most hospitable, and I would
hope that this experience here might have effects far more reaching than the
small area of Globe, Ariz., and that perhaps as a result at least in part of
what we have discovered here, that we will stop using 2,4,6-T around Hie
world until we can run a series of tests that show that it is not harmful to
tills generation, and to the next generation.
Thank you very much.
Appendix 6
ALBUQUERQUE, N. MBX., February 26,
Hon. RICHARD D. MCCARTHY,
House of Representatives,
Washington, D.O.
DEAR MB. MCCARTHY: Thank you for yonr letter of February 16 and for the
opportunity to furnish additional documents or statements for the record of'
your hearing in Globe.
FOB THE RECORD REGARDING WINDS
In my testimony I promised to furnish you with additional data on windspeeds during the 1969 spray project. While windspeed was measured l(y tli<>
1' reject Air Officer who used a pocket anemometer, no record of observations
was made. He did, however, maintain a record of application flight times
�JL«J'±
phich shows when the work was shut down due to winds exceeding 10 miles
V hour. The following table summarizes these important times £
'cord;
Data
Time
June 8,1969...
J u n e S , 1969...
JuneS, 1969...
June 9, 19G9...
Remarks
1505 Shutdown (wind exceeds 10 m.p.h.).
.
1703 Resume operations (wind below maximum).
1935 End operation for day.
1018 Shutdown (wind exceeds 10 m.p.h.).
End operation (or day.
1115
Do.
1250
Do.
June 10,1969..
June 11,1969..
Because allegations.ol! "gale winds" during application have been made, it is
of interest to compare the above shut-down times with winds recorded at the
Globe Fire Weather Station, The Globe Station records are for observations
made only once daily at 1300 hours, but do not indicate the presence of "gale
winds" on any day of the project. These 1300 hours observations are as follows:
Date
June 9
June 10
June 11
Direction
--
-
- ..
J
SW
, SW
W
SW
Also on your field tour, there seemed to be some misunderstanding regarding
application of herbicide to the live stream in Kellner C^ki. While the
stream was flowing when you were in the area, it was not a !^Pstream at the
point visited at the time of application. We do not deny thaTsome herbicide
may have drifted to live streams, as evidenced by some tip damage to trees in
the Kellner Recreation Area where there was a live stream, but that drift
actually reached the water has not been established.
While the Interdepartmental Panel of Scientists headed by Dr. Fred H
isehirley arrived following your hearing, their findings are of sufficient importance to the matter under consideration, that we desire to have the enclosed
press release issued by them inserted in the record.
It was a pleasure working with you and Mr. Riddleberger during your visit
ir the lorest Service can be of any further assistance, please let us know AVe
will appreciate receiving three copies of the hearing record when available
Sincerely,
JOHN M. PIEBOVICH,
Assistant Regional Forester.
Speed (m.p.h.)
5
16
14
16
As can be seen from the two tables, the only day on which applications
extended beyond 1300 hours was June 8, when the 1300 hours observation was
only 5 miles per hour. The June 11 shut-down time of 1250 hours would tend
to infer that winds did possibly exceed 10 miles per hour when compared with
the 1300 hours observation of 16 miles per hour. Banger Moehn has stated
that winds did not exceed 10 miles per hour in the area of the spray application, and this is quite possible since spray work was high up in Russell Gulch,
in the lee of sheltering mountains to the Southwest, on that date.
OTHEB ITEMS FOB THE EECOKD
Additional copies of the Forest Service Interim Position Statement and of
the map showing the limit of infrared detection of dead and distressed vegetation (as of October 1009) are enclosed for the record.
As I recall, Professor Galston asked for additional information on the 3-Bar
research studies related to water yield. Since the Interim Position Statement
digests these, I suggest that the Statement will serve for the record, but would
be glad to arrange for you or for Dr. Galston to receive a copy of the rough
draft of the manuscript referenced in the Statement.
Since the herbicide container converted to a trash barrel, and found in Kellnor Canyon during your field tour, became a matter of importance to the
press, the following additional information may serve as a useful insertion for
the record: (1) The Dow Chemical Company label does not specify that the
container be destroyed (copy of specimen label enclosed) ; (2) As a matter of
good practice, we prefer that all pesticide containers not be reused, and when
it was found that trash barrels were being made of the containers by the
Globe District, the Kegional Forester directed by memorandum on January 29
that all Southwestern Region Ranger Districts discontinue such uses; (3)
Ranger Moehn, in response to the Regional Forester's direction, had all such
trash barrels picked up earlier in the week of your visit; (4) presence of the
container in the creek at the Kellner recreation area cannot be explained by
District personnel who were in the area and had not seen it prior to your field
tour; (5) the container had been washed with water and detergent prior to
painting for use as a trash barrel.
45-382 O - 70 - 11
�167
156
FOREST SSRVICiO INTERIM POSITION: K3IMSR CANYOWRUSSSU, GULCH HERBICIDE SPRAY PROJECT AND THE SOUTHWESTERN
REGION CHAPARRAL PROGRAM, February 9, 1970
INTRODUCTION
.
•.-..'
•
.-Background on Kellner Canyon-Russell Gulch Project
The Kellner Canyon-Russell Gulch -Project is a part of the Chaparral
Management Program of the Tonto National Forost. The primary objectiTa
of this project is to improve water yield, out other program objectives .
and resulting benefits are inttnied tp be. met as veil. Improved
water yield and other Chaparral Program objectives are discussed bel'ow.
This project was initiated in 1965 following extensive local discussions
and a press release which appeared in the local paper. Rather than
the usual practice of applying prescribed fire as the initial treatment, herbicides were used. This was because of the known tendency
for streams in this area'to produce flash floods; herbicide treatment was considered to be unlikely to contribute to flooding, ,wher.eas M
large -area.s treated by fire could.
Chemicals used in this project are listed by year of use in Table 1,
which is appended. These.are all Federally Registered Compounds
and were applied in keeping with the laws and label instructions
governing their safe use,
Following the 1969 Application of Herbicide, Tonto Forest Supervisor
Robert Courtney received a complaint in the form of a petition
bearing 15't signatures of people in and near Globe, Arizona. Following
the initial complaint, Courtney requested a team of qualified individuals
to visit the area for a general assessment of alleged herbicide
damage. This team reported some limited damage to vegetation on
certain private properties.
Chaparral Management Objectives •
Objectives of managing chaparral'on the Southwestern National Forests
are to:
1. Improve water quality and yield through reductions of the
potential for sedimentation following wildfire and through
reductions in evapo-transpiration losses where modification
of existing vegetation is proper.
2. To enhance the scenic value of the' Chaparral zone through
development of varied patterns resembling the natural variety
sometimes found in unprotected chaparral; these patterns
range from savanah-like grass and forb areas to newly regrowint;
chaparral, to relic,stands of mature chaparral.
3. To improve wildlife habitat through creation of "additional
f aodi
edge effect and through maintenance of vigor and new growtli
in desirable species.
H. To reduce the high .costs of protecting chaparral from wildfires '
through the establishment of breaks in heavy fuel continuity,
making it more possible to avert fires of conflagration proportions.
5. To increase forar;c production for wildlife and livestock
through the release of native grasses and the establishment •
of new grass stands.
6. To improve access for both the observer of wildlife and the
hunter through a system of.near-primitive roads to strategic
fire control locations and through the openings- that will
result in treated areas.
It is intended that each of the above objectives will be met through
Multiple Use Coordination Procedures. These require that regardless
of the primary purpose of any project, proper consideration be given
to other forest uses and values. Because of the intense interest in
improving Southwestern, water quality and yield, both Federal watershed
management arid cooperator funds have been made available • for this work
as a primary purpoje. Each of .the objectives of chaparral management
is fairly well understood by the interested public except for this one
of improvement in water yield. Even some experts have, until recently,
discounted the potential for augmenting water supplies through alteration
of shrub cover in the chaparral type.
Much of the research leading to improved understanding of the potential
for additional water has been done on the 3-Bar Experimental Watersheds
near Roosevelt Dam on the Tonto National Forest, Work there was begun
in 1956. Two reports from this work are of particular interest.
Pase, C.P., and P.A. Ingebo, 1965, "Burned chaparral to grass:
early effects on water and sediment yields from two granitic
soil watersheds in Arizona," Proceedings ninth Annual Arizona
Watershed Symposium, U pp illus.
Hibbert, Alden R., Unpublished 1970 Manuscript on file with
Rocky Mountain Forest and Range Experinent Station: "Increases
in streamflow vary with rainfall after converting brush to grass."
The latter report is cited because it contains data not previously
available which are regarded as more reliable (due to additional years'
of streamflow measurement) and which indicate greater pro-dice of
improved water yields than previously expected. Increases due to
watershed treatment have varied froiu 1.5 area inches to lH.0 area
inches. The two tost watersheds averaged an increase in water yield,
for the period 1959 through 1969, of from h to 6 area inches.
�159
Progress and Direction of Studios—The Kollner Canyon-Russell Gulch Project
northeast corner of the project. This corner was selected
as the best to test the hypothesis that soil residues from
drift might be found, since prevailing winds are from the
Southwest.
Task_ Forcen Ho. 1 and No. ?. (Completed Work)
The first two teams to examine the area were concerned with visually
detectable effects of the 1969 herbicide application. Due to the
similarity of some insect and desease symptoms to symptoms of herbicide
effects, the second teatn included specialists in cntovnolo^/ and plant
patholo;;y. It was on the basis of this team's findings that many
plants alleged to be damaged from herbicide drift were determined to
be affected by other causes.
Initial laboratory analysis reports have indicated low
concentrations of Silvex and 2, U-D at some locations
(maximum detected concentration off the project to date is
0,16 p.p.m. Silvex). Especially at these low levels
of concentration, it is possible that other sources of
contamination may induce "background" which could lead to
erroneous conclusions. For this reason, we are proceeding
to cross-check analysis procedures while, at the same time,
widespread sampling north of the project is scheduled.
It should be noted that while all complainants have been advised of
Forest Service claim-for-damage procedures, only one formal claim has
been filed. This claim was not for properties identified as damaged
in the Task Force Ho. 2 Report, and has thus been disallowed.
Infrared Photography and Interpretation for
Distressed Vegabation ('.-fork In Progress)"
While the second Task Force reported that some visually detectable
herbicide drift had occurred from the 1969 spray project, extending
approximately one-fourth mile north of the project, their assessment
did not include previous years' effects, nor was it concerned with
delineation of the sprayed area as a whole.
In order to more accurately define the limits of-herbicide effect on
plants from all years of spraying, aerial infrared photography has
been employed. Interpretation of these aerial photo/jraphs has made
possible a preliminary delineation of the exterior boundary of destressed
and dead vegetation. Both the visually detected drift line reported
by Task Force No. 2 and the External Limit of Infrared-detected
din-tressed and dead vegetation are shovm on the appended PRELIMINARY
map, It is important to note that internal exclusions have not been
delineated and that field verifications are not yet completed for
the infrared interpretation.
Environmental Effects (Work in Progress)
Work is underway in this study to assess the total effect of the
Kellner Canyon-Russell Gulch Project on the environment. Some of
the key considerations included in this study are listed below.
1. Possible further evidence of drift of herbicide sprays
through such herbicide residues aa arc detected in soil
samples north ol'. the pro.ject area. Initial soil sampling
was within the project and on two transects toward the
It would be premature to reach any conclusion regarding
drift at this time.
2. Herbicide levels in water samples. Water sampling and
analyses have been underway for some time. Project methods
• called for interruption of application at all stream channel
crossings, and as far as we have been able to-determine,
no herbicide was applied directly to water. Some soilleaching and runoff is to be expected. All samples we have
taken, or taken by private individuals and brought to our
attention, are less than the Federal water quality criterion
of 0.1 p.p.m. I/
3. Effect of Treatment on Esthetics. While it is evident the
dead vegetation over this area is not pleasing, our concern
here is with the next needed steps to actually provide
enhancement of the scenic resource. It is sometimes necessary
to tolerate temporary degradation of the appearance of an
area as a cost of .ultimate improvement. This study is
intended to better define tolerable limits, explore alternatives, and recommend treatments to completion. Concurrently,
we are assessing the past, present, and projected fire
hazard in order to build conflagration control concepts
into the landscape design.
It.
Effects on Animals and Plants. Initial observations by
wildlife experts have shown no marked effect upon wildlife.
I/ Surface water criteria for public water supplies table appearing in:
Water Quality Criteria issued as a report to the Secretary of
Interior, April 1, 1963, and published by the Federal Water
Pollution Control Administration.
�On the other hand, repeated claims have attributed varied
maladies of humans and animals to the project's herbicide
sprays. Lacking private medical histories or other solid
bases for evaluation, we believe it more sound to rely on
published results of laboratory tests. These are to be
used in determining expected effects on animals for rates
of application used. Yet to be published laboratory
results are needed to complete this study as it relates to
animals.
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A further consideration of this study is that for proper
perspective, all of the environmental influences on the area
must be weighed. Two examples help to brine this need to
focus, One is the frequent presence of smoke from nearby
sweltering operations, especially when an inversion and
northwest winds combine to produce a thick accumulation in
the basin north of the Final Mountains. The other is
household and industrial uses of herbicides which may
have induced additional residues Into the affected area.
It is our further position that it would be unwise to base decisions
on the future use of the herbicides employed, solely upon alleged or
suspected effects in the vicinity of the Kellner Canyon-Russell Gulch
Project. There are many environmental influences operating in this
aroa which must be better understood. Also, many of the questions
raised about t'ness chemical compounds can be resolved only through
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FOREST SSRVICS POSITION.
It is our position that the studies we have underway, as well as
the outcome of public meetings concerned with herbicides and with
the overall conduct of the chaparral program, must determine the
ultimate decision on deferment* For this reason, we believe it
would be premature to state at this time either when the deferment
may be lifted, or what new Guidelines will be followed.
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area nor the possible contamination by other herbicide uses
are known, their importance as suspect environmental effects
cannot be discounted.
V/e share deeply the concern of the people in this area with their
environment. The Forest Service has no intention of persuing a course
which will adversely affect the health and safety of its National
Forest neighbors, nor which will permanently detract from the scenic
or other qualities of the Forests.
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PRESS RELKASE - February 20, 1970
Government Interdepartmental Panel of Scientists
The panel ,i» carefully examining the evidence collected during
its visit. The study vill continue and will include analyses of
the numerous samples of blood, soil, vater, fruit and plants for
the herbicides, a possible contaminant (dioxin), as veil as various
agents producing disease in man, animals and plants. However, to date,
ve can summarize a few of our findings as follows:
1. The application of herbicides in the Final Mountains near.
Globe, Arizona vas made by the Tonto National Forest starting in
1965i The most recent application of the herbicide was made by
helicopter on June 8, 9, 10 and 11, 1969.
2. The materials used in the treatments in 196$, 1966, 1968 and
1969 included 2,1*-D, 2,U,5-T, and silvex. These chemicals came
from different sources. In 1969, 30 gallons of 2,1»,5-T produced by
the Hercules Chemical Company and 935 gallons of silvex produced by
the Dow Chemical Company were used. The silvex is reported by Dow
Chemical Company to contain less than 1 ppm of the dioxin. Analyses
will be made of eilvex and the other herbicides for dioxin and the
active herbicide ingredients,
R.16E.
�165
164
7.
There vaa evidence of woody plant mortality from root rot,
3. There are reports of the aircraft flying over private propertiei
and also visible damage to certain yard trees from several kinds
but not spraying; and other reports of the herbicide being applied
of insects and woodpeckers or sapsuckers.
Other plant injuries
Just outside the project area. There is clear evidence of drift of
ware observed that appeared to be caused by low soil moisture,
the herbicides on a number of plants on some of the nearby properties.
k. Human illnesses have been reported by several residents in the
air pollution and unusual soil properties.
8.
Globe region.
The phenoxy herbicides following normal use dd not usually
Many of the residents vith complaints vere interviewed
persist for more than 8 months in soil and vater. Additional
by a medical member of the panel. These are complaints that commonly
analyses ar« in progress to determine the presence or absence of
occur in the normal population; the eye irritation in one individual
may be related to the spraying. Nine doctors serving the area of
Globe vere interviewed and there vas general agreement that there
had been no significant increa'se in human illness related to the
spraying. However, blood samples vere obtained and additional
studies are planned to verify or rule out this possibility.
5. Reports from the wildlife specialists indicate no significant
effects on birds, deer, and other wildlife.
There are reports of
reductions of birds on a fev properties but there are other reports
that bird and other wildlife populations in and near the project area
are normal.1
6. Information obtained from ovners of livestock and observations
of animals did not indicate any illnesses that do not commonly occur
in other regions. It is doubtful that the spraying of the herbicides
or dioxin caused the afflictions in the goat and duck because the
goat vas born before the treatment and the duck was hatched about l»
niles away from the treated area.
herbicides. '•
Senator HART. We are adjourned to resume on the 15t3i of this
month in this room.
(Whereupon, at 5 :15 p.m., the Subcommittee was adjourned, to
resume on April 15,1970.)
�EFFECTS OF 2,4,5-T ON MAN AND THE ENVIRONMENT
WEDNESDAY, APRIL 15, 1970
U.S. SENATE,
COMMITTEE ON COMMERCE,
SUBCOMMITTEE ON ENERGY, NATURAL RESOURCES AND THE
ENVIRONMENT,
Washington, D.G.
The Subcommittee met, pursuant to adjournment,, at 10 a.m., in
room 1318, New Senate Office Building, Hon. Philip A. Hart, presiding.
Present: Senators Hart and Baker.
Senator HART. The Committee will be in order.
Our first and distinguished witness is the Surgon General, Dr.
Jesse Steinf eld.
f;,
STATEMENT OF DR. JESSE STEINFELfy SURGEON GENERAL, DEPARTMENT Or HEALTH, EDUCATION, AND WELFARE: ACCOMPANIED BY DR. DAVID GAYLOR, DR. DIANE COURTNEY, AND DR.
DALE LINDSAY
Dr. STEINJTELD, Thank you, Senator Hart.
Accompanying me are Dr. Diane Courtney, on my right, of the
Pharmacology and Toxicology Branch of the National Institute of
Environmental Health Sciences, Dr. Dale Lindsay, associate commissioner for science (FDA) and Dr. David Gaylor, chief of the Biometry Branch of the National Institute of Environmental Health Sciences.
I have a prepared statement.
Senator HART. Yes. I suggest you read it and if, there is any
footnoting or extension that you want to make as you go along, feel
free to do it.
Dr. STEINFELD. Thank you, sir.
I am pleased to appear before you today to discuss the herbicide
known as 2,4,5-T, our efforts to determine its hazard to health, and
subsequent action to protect human health.
The production of 2,4,5-T (2,4,5-trichlorophenoxyacetic acid) in
the United States increased from 8 to 40 million pounds per year in
the last decade. In the United States, 2,4,5-T is principally used as a
weedkiller in clearing range and pasturelands, roadsides and rightsof-way, in suppressing aquatic weeds, and in eliminating weeds in
croplands. It is also used to reduce weeds in turf. The use of 2,4,5-T
and its salts and esters on food crops has been registered by the
(167)
�168
f.S, Department of Agriculture on the basis of no residues i
marketed food.
To insure that the foods reaching markets are free of residues, the
FDA has monitored the food supply in selected cities. About 5,800
food samples were analyzed for 2,4,5-T and other pesticides in the
last 4 years. Residues of 2,4,5-T, at trace levels (less than 0.1 part
per million), were found in 25 of these samples. In 1965, one sample
contained 0.19 parts per'million; in 1966, another sample contained
0.29 parts per million. It is my opinion that the results of the monitoring program justified the registered use of 2,4,5-T on selected
food crops, in the absence of any known toxicity of 2,4,5-T.
The development of a balanced public policy which considers benefits and risks associated with the use of a compound such as 2,4,5-T
is an exceptionally difficult matter. Great public fear of the possible
implications for man has followed reports of harm in laboratory
animal tests. And yet frequently it is not known with certainty what
laboratory animal tests may mean for man. We are obligated to
make decisions of great health and economic importance on the basis
of very limited evidence of potential hazard; prudence allows no
other course. We are aware that both good and bad consequences
may result from our actions.
The enormous strides taken in achieving the prosperous and
healthy life we now enjoy in an industrial age has created problems
and uncertainties which are not easily overcome. The resolution of
these uncertainties and solution of these problems will require
national commitment and broad public education and understanding.
At this point, I would now like to read the joint announcement of
Secretaries Hardin, Finch, and Hickel, prepared in accord' with the,,
Tnteragency Agreement for Protection of the Public Health and the
Quality of the Environment in Relation to Pesticides. This is the
first public release of this announcement.
Agriculture Secretary Clifford . M. Karelin, Interior Secretary Walter ,T,
Hickel, and HEW Secretary Robert H. Finch today announced the immediate
suspension by Agriculture of the registrations of liquid formulations of the
weed killer, 2,4,5-T for use around the home and for registered uses on lakes,
ponds, and ditch banks.
Those actions are being taken pursuant to the Interagency Agreement for
Protection of the Public Health and the Quality of the Environment in Relation to Pesticides among the three Departments.
The three Cabinet Officers also announced that the Department of Agriculture intends to cancel registered uses of non-liquid formulations of 2,4,5-T
iirouwl the home and on all food crops for human consumption (apples, blueberries, barley, corn, oats, rye, rice and sugar cane) for which it is presently
registered.
The suspension actions were based on the opinion of the Department of
Health, Education and Welfare that contamination resulting from uses ol
2,4,5-X around the home and in water areas could constitute a hazard to
human health.
New information reported to HEW on Monday, April 13, 1970, indicates that
2,4,5-T as well as its contaminant dioxins, may produce abnormal development
in unborn animals. Nearly pure 2,4,5-T was reported to cause birth defects
when injected at high doses into experimental pregnant mice, but not in rats.
No data on humans are available.
These actions do not eliminate registered use of 2,4,5-T for control of weeds
and brush on range, pasture, forest, rights of way and other non-agricultural
land.
Users are cautioned that 2,4,5-T should not be used near homes or recreation
areas. Registered uses are being reviewed by the three Departments to make
169
certain that they include adequate precautions against graziM^treated areas
until long enough after treatment by 2,4,5-T so that no contc^Mited meat or
milk results from animals grazing the treated area.
^^
Wliile residues of 2,4,5-T in meat and milk are very rare, such residues are
Illegal and render contaminated products subject to seizure. There is no tolerance for 2,4,5-T on meat, milk or any other feed or food.
USDA will issue guidelines for disposal of household products containing
2,4,5-T. The chemical is biologically decomposed in a moist environment.
BACKGBOUND INFORMATION
Secretary Finch's Commission on Pesticides, which reported its findings in
November and December 1960, extressed concern that research conducted at
Hionetics Research Laboratories, under the direction of the National Cancer
Institute, indicated that 2,4,5-T had produced a number of birth defects when
fed or injected into certain strains of mice and rats. Because the test material
contained substantial concentrations of chemical impurities (dioxins), the birth
abnormalities could not be attributed with certainty either to 2,4,5-T, or to the
impurities known to be present.
Representatives of the chemical industry pointed to evidence of extreme
potency of the impurities as toxic agents. They demonstrated that 2,4,5-T now
being marketed Js of a greater purity than that which had been tested in the
IHonetics experiments and urged that further testing be undertaken to clarify
the questions raised.
Responding to this suggestion and utilizing materials supplied by one of the
major producers of 2,4,5-T, scientists at the National Institute of Environmental Health Sciences promptly initiated studies to determine whether 2,4,5-T
itself, its impurities or a combination of both had caused the earlier findings,
and whether the 2,4,5-T now being marketed produces birth abnormalities in
mice and rats.
The experiments were completed last week and the statistical analyses performed over the weekend. On Monday and Tuesday of this week the analyses
of the data were presented to the regulatory agencies of the Federal Government and to the members of the Cabinet.
The dioxin impurities and the 2,4,5-T as it is now manufactured, separately
produced birth abnormalities in the experimental mice.
Because absolutely pure 2,4,5-T was not available for testing, it is possible
only to infer from certain of the observations that the pure 2,4,5-T probably
would be found to be teratogenic if it were tested. But, since pure 2,4,5-T is
not marketed and could not be produced in commercial quantities, this is not n
practical issue for consideration.
In exercising its responsibility to safeguard public health and safety, the regulatory agencies of the Federal Government will move immediately to minimize human exposure to 2,4,5-T and its impurities. The measures being taken
are designed to provide maximum protection to women in the childbearing
years by eliminating liquid formulation of 2,4,5-T use in household, aquatic
nnd recreational areas. Its use on food crops will be cancelled, and its use on
range and pastureland will be controlled. Maximum surveillance of water supplies and marketed foods will be maintained as a measure of the effectiveness
of these controls. These measures will be announced more specifically in the
Federal Register shortly.
While the restriction to be imposed upon the use of this herbicide may cause
some economic hardship, we must all cooperate to protect human health from
potential hazards of 2,4,5-T, other pesticides and the dioxins.
The three Secretaries commended the chemical industry for its
prompt and willing cooperation with the National Institute of Environmental Health Sciences in the studies to clarify questions raised
by the initial studies of this herbicide and for working closely with
the FDA in the other studies still underway. They urged the full
support of industry, agriculture and the home gardner in insuring
the safe use of 2,4,5-T and other pesticides which contribute in
important ways to the welfare of the Nation.
�170
171
Fhat is the end of the press release and I would add that it is _
understanding that Secretary Packard of the Department'
Defense sent a memorandum to the Joint Chiefs of Staff saying the
Department will suspend the use of 2,4,5-T in all operations pending
evaluation of the data.
I will return to the prepared testimony.
At this point, we would like to provide for the record a summary
description of the results of these latest studies of the National
Institute of Environmental Health Sciences,1 completed this past
week. I shall be pleased to respond to questions about these data but
suggest that the Committee not be burdened by a detailed oral presentation of the findings which have been stated briefly in the foregoing announcement.
This leads me to brief mention of the.studies which will be presented next by Dr. Verrett. Commencing in the fall of 1969, Dr.
Verrett reinstituted tests of the embryotoxicity and teratogenicity of
2,4,5-T, its contaminating dioxins, and related chemicals.
Dr. Verrett is to be commended for promptly attacking these
problems and for going to the very considerable trouble of purifying the 2,4,5-T by repeated recrystallization. However, I must
express concern about the degree of reliance which has been placed
upon chick embryo studies. While the studies in chick embryos arc
in general agreement with those in studies of rodents at the NIEHS,
it is to be emphasized that they do not clarify the uncertainties as to
significance for man.
I believe that it is imperative that everyone involved in the development of a national policy for dealing with the many questions
posed by 2,4,5-T and other pesticides be aware of the complexity as
well as the importance of the issues, together with the limitations of
our ability to estimate potential hazards to human health posed by
these substances.
It is essential that we strive to respond wisely to the discoveries
which have been made in this field, and resist the temptation to
resort to measures which may be more extreme than the evidence
warrants. For example, 2,4,5-T is probably the most effective means
of controling poison ivy, poison oak, and other noxious weeds to
which a substantial portion of the population react badly. It has
been estimated that 60 percent of the American population is sensitive to either poison ivy or poison oak, and that from 5 to 10 percent of Americans suffer a reaction to the poisons from these weeds
each year. Some of these individuals become quite ill and incapaci- tated by their reaction to these poisons.
By contrast, we are not aware of any reliable evidence that 2,4,5-T,
indeed any of the pesticidal chemicals, has resulted in human birth
abnormalities. These remarks should not be interpreted as evidence of
indifference to what may be a potential hazard to health. The record
clearly reveals a series of responsible actions by the Administration
to the results of recent laboratory tests. Prudence has characterized
these decisions and actions and will continue to guide the Department in these matters.
In keeping with the pattern established with the naming of the
Secretary's Commission on Pesticides, the thorough s(B^ of pesticide problems by the Commission, and the Administraum's prompt
action to implement the recommendations of the Commission, we
HOW commit ourselves to the following actions:
We shall strive to develop better means for predicting in laboratory animal systems the potential hazard posed for man by chemical
pesticides.
We are aware of a great need for a centralized clearinghouse for
information of all types on pesticides. We plan to have such a clearinghouse established jointly by the National Library of Medicine
and the FDA in the very near future. Other agencies having similar
interests and needs will be invited to participate in this undertaking.
The need to continue certain closely restricted uses of 2,4,5-T will
require a high level of surveillance activity to insvire protection of
the human population from exposure through water sources. This
.will be done.
The Food and Drug Administration will continue to examine a
variety of foods for the possible presence of residues of pesticides,
and will take appropriate action through the interdepartmental
agreement to protect the public health.
This completes my prepared statement, Senator Hart.
My colleagues and I will be pleased to answer any questions.
Senator HART. Thank you Doctor.
Just as you began, we were joined by the able Senator from Tennessee, Senator Baker.
I understand that the announcement you just read us relates to
both powdered and liquid forms of 2,4,5-T shipped in interstate
commerce.
But what about the 2,4,5-T which is now on the shelf? What do
we do about that?
Dr. STEINFELD. You mean on the shelves in the homes and the
shelves in the stores?
Senator HART. Yes, the places for retail sale.
Dr. STEINFELI). I think there is a distinction between the suspension of the registration and the cancelling for registration and I
would like to call on Dr. Lindsay to describe in more detail, the procedures involved.
Dr. LINDSAY. The suspension is a little more drastic than the cancellation, because it is a final action until some other action is taken,
whereas the cancellation permits hearings and has the statutory procedure for appeal during which time the pesticide may be used while
it is being reviewed.
Senator HART. Well, the suspension, the more drastic remedy, was
directed at the liquid form.
Do I read that correctly ?
Dr. STEINFEI,D. Yes.
The suspension by Agriculture of the registrations of liquid formulations of the weed killer for use around the home and for registered use on lakes, ponds, and ditch banks.
We reviewed the concentration of 2,4,5-T in a number'of formulations and found the concentrated form is present in liquids and
could present a hazard.
1
See p. 08.
P:'
,
45-382 O—70
12
�«
'he amount of 2,4,5-T in some of the solid fertilizer-type mates was much less and therefore, the more drastic action was^
taken regarding those compounds.
^^
Senator HART. As you read that suspension sentence I did not
hear a suspension extended to the use of 2,4,5-T on food crops.
Dr. STEINFELD. The three Cabinet officers announced they, intended
to cancel the registered use of nonliquid formulation around the
home and on all food crops for human consumption, so that all of
these registered uses will be cancelled.
' ' "
Senator HART; But the use of liquid formulations on food crops,
as I understand the announcement, was not.
Dr. LINDSAY. As far as I know all of the use on food crops is
from the liquid application.
Senator HART. So there would be no application to food crops
under this order, as you understand it ?
Dr. LINDSAY. As I understand it.
I am not aware of any dry material used on food crops.
Senator HART. Well, let me get back to my point of departure.
You have suspended for certain applications 2,4,5-T in liquid form.
As Dr. Lindsay said, that is the more drastic sanction.
Now, with respect to that 2,4,5-T in liquid form, the order today
has what effect on the marketing and use on shelves or in homes ?
Dr. STEINFELD. Well, I don't know exactly what the Department
of Agriculture will do. This is not an FDA activity. I am certain
they will move quickly and appropriately. I think a significant
statement is on page 2 of the release, which says the "U.S. Department of Agriculture will issue guidelines for disposal of household
products containing 2,4,5-T. The chemical is biologically decomposed
in a moist environment".
The intent is to get rid of all 2,4,5-T around the household. 1
assume it would not be available for use in households where pregnant women would have access to it. I don't have the details of those
actions.
Senator HART. I see we don't have anybody on the witness list this
morning for the Department of Agriculture, but would you agree it
would be very inappropriate for the Department of Agriculture to
permit continued vending of liquid 2,4,5-T for any of the purposes
for which you have suspended it, even though it is now in retail distribution ?
Dr. STEINFELD. I think this announcement will have dramatic
impact. Our meetings with the Department of Agriculture on
Monday and Tuesday would lead me to believe they are going to
take appropriate and vigorous action.
Senator HART. Would you describe as appropriate, walking into a
store and seeing the thing on the shelf and saying, take it off ? That
seems appropriate to me.
Dr. STEINFELD. Idon't know the mechanisms which they have to
insure compliance.
Senator HART. If they have it and don't do it, don't you think it
would be inappropriate and if they don't have it, don't you think
Congress should give it to them ?
\<;
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Dr. STEINFELD. Certainly they should have the authority to do
what is required to protect the public health, and I itijk. they do
have this.
' ^P
Senator HAHT. Well, we will find out.
Dr. STEINFKLD. I am sorry, I don't know.
Senator HART. You are talking to another nonexpert, so don't feel
bad.
Mr. Bickwit has greater expertise than I, so we will let him deal
further with the problem.
But there is one passage in your announcement that particularly
interests me. In the press statement which you read, there is a paragraph which states: "The regulatory agencies of the Federal Government will move immediately to minimize human exposure to
'2,4,5-T and its impurities. The measures being taken are designed to
provide maximum protection to women in childbearing years by
eliminating formulation of 2,4,5-T use in household, aquatic and recreational areas. Its use on food crops will be canceled and its use on
range and pasture land will be controlled,"
You say on food crops its use will be "canceled."
But is 'it not a very technical definition only of that term that permits you to say it will be canceled on food crops, because in liquid
form'l take it, 'it may still be used, or am I wrong about that?
Dr. STEINFELD. When the use is canceled, such a notice is published in the Federal Eegister, I believe.
And then there is a 80-day period for comments, is that not correct, Dr. Lindsay ?
Dr. LINDSAY. Yes.
Dr. STEINFELD. After which appropriate action is taken.
Senator HART. I think what I am more concerned about is my
desire to understand precisely what may or may not be done with
this formulation in application to food crops.
In liquid form may it continue to be used ?
Dr. STEINFELD. You mean during the 30-day period while the—I
am afraid I don't understand.
Senator HART. It has been suggested to me that there would continue to be no restrictions with respect to the use in liquid form on
food products.
Now, is my information correct on that?
Dr. STEINFELD. No, sir, the use on all food crops will be eliminated as promptly as the law permits through cancelation of the
registration, whether in dry or liquid form or any form. There will
be no vise on food crops, Senator Hart.
Senator HART. All right. I think this is a desirable clarification,
since there were some who had felt otherwise.
You say it will be eliminated as promptly as is possible under the
law. .It could be eliminated more promptly by a suspension than a
cancelation?
Dr. STEINFELD. Yes.
Dr. LINDSAY. Yes, I am not aware of what the Department of
Agriculture's intent is with regard to carrying this on.
The main idea was to get it into effect at the earliest possible time
where it would be likely to come in contact with women of childbearing age.
�174
enator HART. I am trying to ask why the different treatml
Why with respect to certain forms and use is it merely canceled ?
Although that sounds very dramatic, it means if you want to use
it, go ahead and use it until somebody resolves differences which
may arise over the action. Why handle some uses on a cancelaticm
basis and some by suspension ?
Is it because those uses and forms that you suspended more intimately or directly come in contact with women of childbearing age?
Dr. STEINFEUX Yes, I believe that is the reason.
Right now there is a zero tolerance on foods, and any foods that
had any measurable toxicity would be subject to seizure. I believe
the intent was to move as quickly as possible, but we wanted to alert
women who may have liquid formulations around the home, who
may be spraying it, that it may present a hazard. We will take
appropriate steps to try to warn the female population, particularly
of childbearing age.
That is the reason for the more dramatic action in the one
instance, and the less dramatic but, I believe nonetheless complete,
action, however, nonetheless in others.
I guess I have here a legal phrase; I think for suspension one
must show an imminent hazard to health, and this, perhaps, is the
reason.
Senator HART. I don't envy you that business of interbalancing.
You describe the judgement that you seek to arrive at as a product of weighing the imminence of danger against the values that are
identified as following from the use of the pesticide. As a layman,
probably we would tend to oversimplify it.
Now, having admitted this may be an oversimplified impression,
why isn't it a more prudent balancing act to say, well, there is
danger here because we can't establish that there is no danger and
we are not going to get hung up on the degree of imminence of the
danger. We are just going to say, to be sure there isn't any danger,
we are going to suspend this.
Why aren't you tempted to resolve this balancing operation in
that manner ? .
Dr. STEINFELD. I am not sure I am the one who makes all these
decisions of balancing, Senator Hart. My role of course, is concerned with public health and safety. But we are always balancing
things,
Certainly in medicine, in picking drugs to use for diseases, sometimes the treatment is worse than the disease. If it should turn out
that these materials can be safely used on range and pastureland,
that there is a.period in which there is biodegradability during
which the materials will effectively disappear, and yet permit the
person who raises his cattle or dairy cows to have a better—I don't
really know the name, I am a city boy, a small-toAvn boy, not a
farmer—but better able to have better cows, more milk, better meat,
then there are appropriate reasons for using this chemical.
I think the real problem, Senator Hart, is that we do not have an
effective, adequate substitute for certain uses. I think this is the key
issue.
The other good chemical which kills poison ivy and poison oak is
a carcinogen in some animals and not proven for many, but it is a
very potent chemical that will destroy poison ivy and poison oak. So
there is another balance that one must weigh.
A
Senator HART. But into that formula you have to t»
of economic possibility that if this were suspended, if it ]ust
permitted to be marketed for this purpose, and if there is a neec tor
a cure for the ill that this thing treats, maybe there would be a
renewed effort to find a third alternative.
,
Dr. STEINFELD. I believe the action which has been taken today
will lead to more intensive research to find an alternative to 2,4,0-1
to destroy the particular kind of herbs it is capable of destroying.
Senator HART. Mr. Bickwit,
. ,
Mr. BICKWIT. I am sorry to go over the matter of use on food
crops again, but I do want to clear this up so that we know piecisely what the situation is. It says in the first paragraph^of yout
press release that liquid formulations of the weed killer 2,4,5-T foi
use around the home, for registered use on lakes, P™ds and d itch
banks will be suspended. Do you intend to include within that list ol
the wording for food crops is otherN
wise. It would be canceled rather than suspended.
Mr. BICKWIT. I am talking about liquid formulation.
Dr. STEIN™. As I read the actions taken there will be a. cancelation of registered use of nonliquid formulations around the home
r
™
is clear, but what I want to know is ^
notion is proposed with respect to the use of liquid formulations on
r ™ N F E L D . My interpretation of this
lawyer but I now see what you are driving at. I think this
have been worded, and we will have to check into it, 1 quid and
nonliquid formulations around food crops." The intent is not to use
liquid and nonliquid formulations on
™
speak for the three Cabinet officers. It is
my understanding that the intent is not to permit use on any loocl
crop for human consumption.
'.
„„„!«, y
Mr BICKWIT. Well, you will permit use on it pending appeals <
Dr.' STEINFELD. Pending the legal activities.
Dr. LINDSAY. But there is no permitted residue of 2,4,5-T on any
food. It would be subject to seizure.
Mr BK-KWIT. Now, I would like to deal with your statement that
an imminent hazard needs to be present before suspension can take
place. Is that to say that there is no imminent hazard from the use
.
studies which have been done flja market
basket sampling and the measurement of foods for 2,4,5-1, as 1
mentioned, it is a very rare instance where these things are found,
and in sugar cane the herbicide is probably destroyed in the processing by heat. We do not really know. The action we are taking is
based on teratogenicity in mice and the fact that dioxms also cause
teratogenicity in rats and perhaps in hamsters. It- is a possible
hazard.
�176
Mr. BICKWIT. Is that what you need to cancel as opposed
iend-a possible hazard ?
Dr. STEINFELD. I do not know the law that well. I really do not
know the exact wording of the law, do you, Dr. Lindsay ?
Dr. LINDSAY. No. I am sorry. This is Agriculture's bag, and I do
not know it.
Senator HART. Let us order printed in the record at the conclusion
of your testimony the appropriate sections of the Federal Insecticide
Fungicide and Rodenticide Act.
Dr. STEINFELD. Fine.
Mr. BICKWIT, liave you any information derived from your tests
on the degradability of dioxin ?
Dr. STEINFELD. Dr. Courtney is a pharmacologist.
Dr. COURTNEY. We have no information on that.
Mr. BICKWIT. In other words, then, it is possible that dioxin is
both persistent and accumulative in human beings ?
Dr. COURTNEY. That is possible. It is also possible that it can be
metabolized.
Dr. STEINFELD. I would like to volunteer something, that is, that
the dioxin which produced the results that we will submit for the
record is a very potent teratogen for mice in 10,000 to 30,000 times
smaller a dosage than 2,4,5-T as we could obtain to pinpoint which
chemicals were the villains. And I think it raises another issue, that
is, where else in man's environment could these chemicals be found?
We have not shown that these chemicals are teratogenic for man,
but we may want to take action. The Food and Drug Administration and Agriculture are presently studying a number of other pesticides in the mamifacture of which poly-chlorinated phenols are subjected to heavy temperatures and may produce dioxin. So I think we
are having an important study carried out there.
Mr. BICKWIT, Are you looking outside the herbicide area as well?
Dr. STEINFELD. We must look wherever polychlorinatecl phenols
are subjected to high temperatures. We must look for the presence
of dioxin and if we find them we shall have to take appropriate
action.
Mr. BICKWIT. But the appropriate action is not to find that an
imminent hazard exists ?
Dr. STEINFELD. I do not know what the appropriate action is. I
know we are going ahead with this activity.
Mr. BICKWIT. I take it you do know what the data are with
respect to 2,4,5-T and you do know dioxin is present and you do
know it is very potent and yet you have concluded it is not an imminent hazard. If it were you would have suspended rather than canceled use.
:
Dr. STEINFELD. You mean suspended all use everywhere? Is this
what you mean?
Mr. BICKWIT. Yes.
' Dr. STEINFELD. I think the question of imminent hazard would
relate to pregnant women, but we do not know it is teratogenic for
man. TJse out in rangelands and forests and so forth, I do not see as
a hazard to pregnant women.
Mr. BICKWIT. Clearly you have no evidence that it is not.
177
Dr. STEINFELD. No, I have no evidence that it is i^fnor that it
is, actually. It is a potential.
Mr. BICKWIT. And when you have no evidence either way you conclude that it is not an imminent hazard?
Dr. STEINFELD. I am tempted to make an analogy, but I probably
should not. It is difficult to state that there is no evidence that a
number of things are not a hazard to health. I think we are in a
never-never land, and where we can, we should try to get as much
good hard data as we can and act accordingly.
Mr. BICKWIT. Is there any evidence either way on the accumulativoness of dioxin ?
Dr. STEINFELD. I do not think there is any evidence on dioxin.
This is a new area which has opened up which we will have to study
intensively.
Mr. BICKWIT. Thank you.
Senator HART. I am not sure this will come out as an effective
analogy, but think for the moment of the general attitude on pot—
marijuana the prevailing view appears to be that since we cannot be
sure it is not harmful, it ought not to be used. Is it not correct now
that there is at least disagreement as to whether it is harmful or
not?
Dr. STEINFELD. I think most physicians, and I am the father of
teenagers, feel that pot is harmful.
Senator HART. Yon cannot be sure it is not harmful. Is not that
your parental attitude ?
Dr. STEINFELD. I feel it is harmful because it represents an
attempt to escape from reality at a time when children must adjust
to the outside world and become independent. So I find it harmful
as a crutch which particularly the teenagers and those growing up
must not use.
Senator HART. Well, you have destroyed my analogy. I was going
to pursue it on the assumption that you would agree you cannot be
sure it is not harmful. You say you are darn sure it is harmful?
Dr. STEINFELD. Yes, as far as teenage use, I think psychologically
it is harmful. I do not think we can be sure of enzyme changes or
long-term liver effects, this sort of thing. I do not think is is possible to be sure, but I would say it is harmful.
Senator HART. What if you were unsure, then would you say let
us go ahead, although I am not sure? Or would you say do not use
it? You say with respect to the pesticides, you balance it and say
since we are not sure it is harmful, go ahead?
Dr. STEINFELD. I think we have some evidence in animals that
2,4,5-T is a teratogen and dioxins are present, and while we cannot
be certain that women, mankind, behave similary to the mouse, yet
pregnant women should not be exposed to this. This is a prudent
action.
, •
Mr. BICKWIT. Do you know the date on which the National Cancer Institute received the first progress report raising the possible
teratogenic nature of 2,4,5-T in mice ?
Dr. STEINFELD. I have with me a chronology regarding 2,4,5-T. It
is a few pages, but it is triple spaced. If you would like I could read
it to you.
�178
Senator HART. Was that a part of the insert that you presented!^
Dr. STEINFELD. We can provide it to you, and if you would like I
can read it into the record.
Mr. BICKWIT. We would like it for the record.
Dr. STEINFELD. Maybe it would be useful to go through the chronology. With your permission, I will.
Senator HART. Please.
Dr. STEINFELD. In presenting the following chronology I should
take a moment of the Committee's time to commend Dr. Kotin and
Dr. Falk for their foresight and initiative in undertaking the studies
which were conducted under their guidance by Bionetics Research
Laboratories. This commendation extends also to the scientists .in,
the National Cancer Institute who assumed responsibility for successful completion of the study after Drs. Kotin and Falk transferred to the National Institute of Environmental Health Sciences.
It consumed large amounts of their time and energy without assurance that the investment would be rewarded. The total cost of this
study approximated $3.5 million, and approximately 20,000 animals
were studied.
Summer 1963; The National Cancer Institute (National Institutes
of Health) awarded a contract to the Bionetics Research Laboratories (Falls Church, Va.) to perform studies of the toxicology, carcinogenicity, teratogenicity, and mutagenicity of pesticides and
industrial chemicals which were to be selected by scientists of the
National Cancer Institute, according to protocols to be devised by
the scientists of the Institute.
During the fall, 1963, the chemistry and toxicology of the chemical compounds to be studied were examined and planning of the
large-scale carcinogenicity screening operations was initiated.
Fall and winter 1964: Large-scale screening activities in carcinogenicity were initiated and plans for teratology studies were drawn up.
June 1966: First indication of possible teratogenicity of 2,4,5-T.
At a dose of 113 mg/kg of body weight, 2,4,5-T, now recognized as
containing substantial concentrations of dioxin impurities, produced
an elevated incidence of cystic kidneys in one strain of mice. The
2,4,5-T had been administered by injection.
At that point we did not know whether the results produced by
injection were significant. The 2,4,5-T had not been fed.
November of 1966: 2,4,5-T of a similar grade of purity administered by injection at a close of 133v./kg. body weight was found to be
teratogenic in another strain of mice.
The results obtained in June and November 1966, in the absence
of information about rates of clearance of injected 2,4,5-T from the
blood stream, were regarded as of uncertain significance. This route
differs from human exposure and possible differences in metabolism
could be very important.
January 1968: Oral administration of 2,4,5-T of similar purity
was initiated in mice. The data produced in this study indicated teratogenicity (cystic kidneys and cleft palate).
May J968: Oral administration of 2,4,5-T of similar purity at a
dose of 113 mg./kg. of body weight produced cleft palate in another
strain of mice,
itV
September 1968: First draft of the final report of thedata on carcinogenicity arid teratogenicity was delivered to the N^fcial Cancer
Institute by the Bionetics Research Laboratories. IK should be
emphasized that these carcinogenicity data were in an incompletely
analyzed state and required scrutiny for possible errors, plus numerous statistical analyses. The first evidence of teratogenicity obtained
in rats fed 2,4,5-T was reported.
October &£, 1968: The draft report of the "raw" data mentioned
immediately above was provided to Dr. Fitzhugh in the Food and
Drug Administration.
October-November-December 1968: Scrutiny of the carcinogenicity data was undertaken by the National Cancer Institute scientists
and report writing begun.
January 30, 1969: At a meeting of scientists from the National
Institutes of Health with representatives of the regulatory agencies,
Consumer Protection and Environmental Health. Services, the
National Academy of Sciences, and the chemical industry, attended
also by Drs. Philippe Shubik and Samuel Epstein, the first two volumes of the final report of data on carcinogenicity, submitted by
Bionetics Research Laboratories were made available. In addition a
special preliminary report on the teratogenicity of 2,4,5-T, exclusive
of data pertaining to the other teratogenicity studies, was provided
to all participants in the meeting.
The analyses of the carcinogenicity data had been given priority
because of its volume and the apparent potential significance, based
upon the indications of the raw data. It had been intended to completely analyze the teratogenicity data immediately following completion of the analysis of the carcinogenicity data.
At the meeting of January 30 a number of uncertainties in the
analyses of the carcinogenesis data were pointed up by Drs. Epstein
and Shubik and one of the senior scientists in the National Cancer
Institute. On this basis, it was decided to withhold publication of
the data and findings until additional animal specimens had been
examined and certain features of the study design had been reanalyzed. For the same reason, it was decided that a presentation
planned for the March 1969 meeting of the Society of Toxicology
would be withdrawn from the program.
January-September 1969: Extensive statistical analyses of the
teratology data were performed by the National Institute of Environmental Health Sciences.
March 1969: In the course of the appropriations hearings, Dr.
Kndicott promised to provide the results of the carcinogenicity studies to the Congressional Record just as soon as the analyses could be
completed. This was accomplished in the last week of April or the
first week of May 1969.
June 1969: The preliminary report of the carcinogenicity findings
was made in the Journal of the National Cancer Institute.
June 1969: The Technical Panel on Carcinogenicity for the Secretary's Commission on Pesticides was appointed and included scientists from the National Cancer Institute and the National Institute
of Environmental Health Sciences.
June 1969: The intent to name a teratology panel to the Secretary's Commission on Pesticides was made known to the National
�180
•^incer Institute liaison member of the Commission. The s
^roous offer by the Institute's liaison member of the commisslo?
supply the Bionetics data on teratology was declined by a member
of the staff of the Commission.
July-September, 1969: Members of the staff of the National
Cancer Institute and the National Institute of Environmental
Health Sciences actively engaged in the work of the technical panels
on carcinogenicity and terabology. Further analyses of the teratogenicity data were performed.
August 15, 1969: Request made by the Teratology Panel for the
Bionetics data on teratogenicity.
/September 11, 1969: Data on teratogenicity provided to the Teratology Panel. Delay in part related to procedure involved in clearing
permission for the data and in part related to putting the data into
a condition suitable for examination by those who had not participated in their development.
Fall 1969; FDA studies on embryotoxicity, and teratogenicity of
2,4,5-T and dioxins reinstituted, as described in Dr. Verrett's testiNovember 25, 1969: Meeting of Natipnal Institutes of Health scientists with those from FDA and DOAV Chemical Co. to plan further
studies to clarify roles of 2,4,5-T and dioxin impurities in the production of teratological abnormalities.
November and December 1969; Secretary's Commission reports
published.
January 1970: New teratological 'studies initiated at National
Institute of Environmental Health Sciences using materials provided especially for the purpose by Dow Chemical Co.
April JO, 1970: Above teratological studies completed.
April 12,1970: Analysis of the above data completed.
April 13 and 14, 1970: Interpretations of the above-mentioned
findings by representatives of the regulatory agencies and parties to
the interagency agreement for protection of the public health and
the quality of the environment in relation to pesticides, and presen-tation of conclusions and proposed actions to members of the Cabinet.
That is a long chronology. I am sorry. I thought it would be
shorter.
Senator HART. You have taken the words from me, it is a"lorig"
time after that first bell was sounded before we got this morning's
action. I am sure it is always easier to play it from the 20-20 vision
of the grandstand up here than from the vantage point of the
summer of 1966 when the first bell rang. But that is still a long
time.
Dr. STEINFELD. The studies were initiated at a time when this sort
of thing was not ordinarily done. As we have more and more chemicals and materials put into our environment we must be more and
more careful about the effects they produce.
Senator HART. How can we compress the period between June of
1966 and April 15, 1970, in the future? What mechanism do you
now visualize which will avoid this sort of lag from recurring?
Dr. STEINFELD. If the procedures for registration of materials for
use on food crops required teratogenicity studies as well as other
181
long term chronic toxicity studies, as it is my undergfciding that
they now do, we may be able to avoid this in the future. ^P
The idea would be to prevent the introduction rather than react
some years later, after the material was used, not only ubiquitously
but in large quantities. I think this is the direction we must go, to
prevent the introduction of materials rather than to react after they
are used.
Senator HART. Wouldn't this require the burden of proof to be on
those who want a market ?
Dr. STEINFELD. Yes.
Senator HART. To make the affirmative case that it is not dangerous. That is correct, isn't it?
Dr. STEINFELD. Yes, I think the thing we really need are good
predicting systems for man. I think it would be ideal if we had
some in vitro systems which would tell whether a compound is going
to be toxic. This is what we need, a lot more research and correlation of animal data with human epidemiologic data. I hope we never
do experiments on man but we can collect data in retrospect epklemiologically in individuals who may have been exposed to chemicals
or certain diseases and so forth.
Senator HART. Mr. Bickwit?
Mr. BICICWIT. You obviously have done some thinking about how
to patch up the system and I don't want to cry unduly over spilt
milk, but do you have any idea why, when NCI received this first
progress report, that it did not immediately pressure Bionetics to go
into an all out effort to acquire further data quickly instead of
allowing them approximately 21/2 years to complete their tests ?
Dr. COURTNEY. The first statement NCI made was "Repeat the
study and make sure it is right," and that is just what we did. We
•went to a different strain of mouse, then we went to a rat. By the
time we did all of these studies, it took a bit of time.
Dr. STEINFELD. We were also studying similar chemical pesticide
structures, so we could see if it was a larger problem than just this
one, This was all going on at the same time.
Mr. BICKWIT. Did the other pesticides that you were -studying
exhibit the same kind of alarming data ?
Dr. COURTNEY. I don't know how you describe it as alarming.
Mr. BICICWIT. Would you not describe it as alarming?
Dr. COURTNEY. Yes. We had some other pesticides that we were
concerned with at the time and, of course, without repeated studies
wo could not make a judgment. So some pesticides were not as alarming and some were more and as we repeated the tests we got our
results. This pesticide seemed to give us a positive response every
time we studied it.
Dr. STEINFEU>. I would say we are not particularly pleased with
the fact that it took so long to get all the data out. The first time
around in one of these situations always takes longer and hopefully
in the future we will be able to move much more rapidly.
Senator HART. I was just thinking of all the things that have happened since that first alarm bell. We have elected two-thirds of the
Senate, a new President, gotten further into Vietnam.
Mr. BICICWIT. According to your chronology, if I read it correctly,
the data from Bionetics were first made available to FDA on October 24-, 1968?
�182
183
appeared to me it was the dioxin that was the lik|^ villain in
Dr. S'fEiNFELD. Yes, the draft of the raw data was
this piece, not the 2,4,5-T; the particular batch of the ^Jp-T used in
>. Fitzluigh on October 24-, 1968.
Hie
Mr. BICKWIT. Do you believe FDA, one of the government agon- | experiments was heavily contaminated with dioxin. Our goal
was to pin down the fact that it was dioxin and probably not 2,4,5-T
eies responsible for the regulation of pesticides, should have known
which was the teratogen and get rid of dioxins wherever they are
about these preliminary indications prior to a time more than 2
found.
years after the data first became available?
So I think last week I would have said the same thing, Senator
Dr. STEINFELP. I think in retrospect we could look at this and
Hart. The data over the weekend have changed the picture comspeed everything up and inform everyone very quickly. I can't give
pletely
you the reasons why, (a), the information was not rapidly dissemiSenator HART. Yes; that will be made part of the record.
nated as soon as it was confirmed and, (b) why things didn't move
Well, then we all wind up saying it is a darn shame this past
much more rapidly and on a larger scale. But I would point out
weekend had to be the first time when you got the solid information,
that the material used was heavily contaminated with dioxins. In
which information was a result of an alarm bell that rang in June
this interval we have identified the dioxins, and we are moving, 1
of 1966.
think, on a broad scale to try to find out where else dioxins may be
We all agree on that.
found. I am not trying to look for a silver lining in a dark cloud
Do you anticipate that the centralized clearinghouse which you
but I do think we have a lot better data and a lot more information
made reference to in your prepared testimony can assure that this
as to just what did the job; it probably was the concentration of the
kind of timelag no longer will occur ?
dioxins used in the Bionetics experiments which was responsible for
Dr. STEINFELD. I hope that that will help. Our other attempts at
the teratogenicity.
coordinating activities with regard to pesticides will also help. The
Mr. BICKWIT. Then you do regard this as a dark cloud ?
Secretary has a special commission: we have an interagency group
Dr. STEINFELD. I Avould say the darkest part is that, whatever the
of Agriculture, Interior, and HEW; we have Dr. Kussell Train,
rules were, we permitted the utilization of the matei-ial without testEnvironmental Quality Council; I hope all of these will help us
ing for what may be a significant hazard to man, teratogenicity.
avoid problems such as AVO are-facing today.
Senator HART. Doctor, I commented earlier on the fact that no
Senator HART. 1 would ask our staff to obtain for the record the
witnesses are scheduled today from the Department of Agriculture.
announcement Avhich you anticipate the Department of Defense is
My interest at that time bore on the action, if any, that would be
about to make. You did indicate that they Avere
taken to remove from retail channels and from shelves at home, perDr. STEINFELD. Idon't know if they Avill make an announcement
haps, this product as a result of the announcement that you gave us
If is my understanding that this is an action that Deputy Defense
today.
Secretary Packard has initiated this morning.
Th'e Secretary of Agriculture participated with Secretary Finch
Senator HART. If there is any announcement in connection with
and Secretary liickel in this announcement suspending or canceling
this, let it be a part of the record. I understand there is a big
2,4,5-T. I am' reminded and I must confess my own memory of this
departmental request outstanding for a major purchase order for
testimony is not clear, but it has been suggested to me that when
2,4,5-T. I Avould like to find out whether that contract request IIOAV
witnesses speaking for the Deparment of Agriculture testified before
Avill be withdraAvn in light of Deputy Defense Secretary Packard's
this subcommittee last week, they took the position that the evidence
position. I would assume it would. But let us make it a matter of
did not warrant an action such as is taken today.
record.
I won't say that they promoted or advocated its use, but—Mr.
Is there anything any of you would care to add, given the
Bickwit, have you found any passage that bears on this?
exchange we have had this morning ?
Mr. BICKAVIT. Yes.
Dr. STEINFELD, I Avould add one final statement. We used inbred
Senator HART. From the transcript this sentence is cited. This is
strains of animals and large doses of compounds in order to try to
from a Department of Agriculture witness who addressed us on the
find a particular phenomenon. The problem is that man is notseventh of this month.
inbred; Ave don't breed brothers and sisters and so Aye can't predict.
In view of all the information now available, we have not found that regisWe have a tremendous variation among people in this country; some
tered use of 2,4,5-T without a finite tolerance on food crops warrants a suspenpeople may have missing enzymes of a particular type that may
sion or cancellation of such registered use.
make a chemical extremely hazardous at a very IOAV dose.
NOAV, that testimony is April 7. You say that on April 13 the
We have taken actions because we muct act prudently. We don't
analysis which had been completed 2 days before were presented for
want to alarm the public, but we do want to react prudently and
proposed action. Whatever else you can say about it, it points up
protect the public health.
again the fact that on April 7, notwithstanding the patterns beginSenator HART. Amen.
ning in June of 1966, indicating possible serious danger, this one
Thank you very much, gentlemen.
Department was still telling us, on the record, what I just read you.
Dr. STEINFELD. Thank you.
Dr. STEINFELO. I would have agreed with that position last week.
(The information referred to earlier folloAvs:)
I was surprised to see the data that developed over the weekend. It
�184
185
necei
The Secretary, whenever he deems it neTessary for
effective administration of this Act, may require
th« submission of the complete formula .of the economic
poison. If it appears to the Secretary that the comREGISTRATION
position of the article id such as to warrant the proposed claims for it and if the article and its labeling
Sec. U.a. Every economic poison which is distriand other material required to be submitted comply with
buted, sold, or offered for sale in any Territory or
the requirements of section 3 of this Act, he shall
the District of Columbia, or which is shipped or
delivered for shipment frofli any State, Territory, or a register it.
the District of Columbia to any other State, Territory,] •> c. If it does not appear to the Secretary that the
or the District of Columbia, pr which is received frctt article is such as to warrant the proposed, claims for
any foreign country shall be registered with, the . ,. : it or if ,the article and its labeling and other
Secretary: Provided, That products which have the
material required to be submitted do not comply with
same formula, are manufactured by the same person,
the provisions of this Act, he shall notify the applithe labeling of which contains the same claims, and
cant for registration of the manner in which the
the labels of which bear a designation identifying
article, labeling or other material required to be
the product as the same economic poison may be regissubmitted fail to comply with the Act so as to afford
tered as a single economic poison; and additional
the applicant for registration an opportunity to make
names and labels shall be added by supplement statethe corrections necessary. If, upon receipt of such
ments; the applicant for registration shall file with
notice, the applicant for registration does not make
the Secretary a statement including—•
the corrections, the Secretary shall refuse to register
the article. The Secretary, in accordance with the
( ) the name and address of the registrant and the
1
procedures specified herein, may suspend or cancel
name and address of the person whose name will appear
the registration of an economic poison whenever it
on the label, if other than the registrant;
does not appear that the article or its labeling or
(2) the name of the economic poison;
other material required to be submitted complies with
(3) a complete copy of the labeling accompanying
the provisions of this Act. Whenever, the Secretary
the economic poison and a statement of all claims
refuses registration of an economic poison or -deterto be made for it, including the directions for use;
mines that registration of an economic poison should
and
be cancelled, he shall notify the applicant for regis(!»•) if requested by the Secretary, a full descriptration or the registrant of his action and the reasons
tion of the tests made and the results thereof upon
therefor. Whenever an application for registration
which the claims are based
is refused, the applicant, within thirty days after
service of notice of such refusal, may file a petition
requesting that the matter be referred to an advisory
committee or file objections and request a public
hearing in accordance with this section. A cancellation of registration shall be effective thirty days
after service of the foregoing notice unless within
such time the registrant (l) makes the necessary
corrections; (2) files a petition requesting that the
. matter be referred to an advisory committee; or (3),
�186
187
files objections and requests a public hearing. Each
advisory committee shall be. composed of experts, qualified in the subject matter and of adequately diversified
professional background selected by the National Academy
of Sciences and shall include one or more representatives from land-grant colleges. The size of the committee shall be determined by the Secretary. Members
of an advisory committee shalj. receive as compensation
for their services a reasonable per diem, which the
Secretary shall by rules and regulations prescribe, for
time actually spent in the work of the committee, and
shall in addition be reimbursed for their necessary
traveling and subsistence expenses while so serving
away from their places of residence, all of which costs
may be assessed against the petitioner, unless the committee shall recommend in favor of the petitioner or
unless the matter was referred to the advisory committee by the Secretary. The members shall not be
subject to any other provisions of law regarding the
appointment and compensation of employees of the
United States. The Secretary shall furnish the committee with adequate clerical and other assistance, and
shall by rules and regulations prescribe the procedures
to be followed by the committee. The Secretary shall
forthwith submit to such committee the application for
registration of the article and all relevant data before
him. The petitioner, as well as representatives of the
United States Department of Agriculture, shall have the
right to consult with the advisory committee. As soon
as practicable after any such submission, but not later
than sixty days thereafter, unless extended by the
Secretary for an additional sixty days, the committee
shall, after independent study of the data submitted
by the Secretary and all other pertinent information
available to it, submit a report and recommendation to
the Secretary as to the registration of the article,
together with all underlying data and a statement of
the reasons or basis for the recommendations. After
due consideration of the views of the committee and all
other data before him, the Secretary shall, within
ninety days after receipt of the report and recommendations of the advisory committee, make his determination
and issue an order, with findings of fact, with respect
' to registration of the article and notify tn^applicant
for registration or registrant. The applicant for reg;> istration, or registrant, may, within sixty days from
' the date of the order of the Secretary, file objections '
'._ thereto and request a public hearing thereon. In the
event a hearing is requested, the Secretary shall, after
V due notice; hold such public hearing for the purpose of
receiving evidence relevant and material to the issues
raised by such objections. Any report, recommendations,
underlying data, and reasons certified to the Secretary
by an advisory committee shell be made a part of the
record of the hearing, if relevant and material, subject
to the provisions of section ?(c) of the Administrative
Procedure Act (5 U.S.C. 1 0 ( ) . The National Academy
06c)
of Sciences shall designate a member of the advisory
committee to appear and testify at any such hearing with
respect to the report and recommendations of such com: aittee upon request of the Secretary, the petitioner, or
the officer conducting the hearing: Provided, That this
shall not preclude any other member of the advisory
committee from appearing and testifying at such hearing.
; As soon as practicable after completion of the hearing,
;• but not later than ninety days, the Secretary shall
• evaluate the data and reports before him, act upon such
;.- objections and issue an order granting, denying, or
cancelling the registration or reqxiiring modification
of the claims or the labeling. Such order shall be
; based only on substantial evidence of/record of such
hearing, including any report, recommendations, underlying data, and reason certified to the Secretary by
an advisory committee, and shall set forth detailed
findings of fact upon which the order is based. In
connection with consideration of any registration or
application for registration under this section, the
Secretary may consult with any other Federal agency
or with an advisory committee appointed as herein provided. Notwithstanding the provisions of section 3.c.
(k), information relative to formulas of products
acquired by authority of this section may be revealed,
when necessary under this section, to an advisory committee, or to any Federal agency consulted, or at a
public hearing, or in findings of fact issued'by the
Secretary. All data submitted to an advisory committee
45-362 O - 70 - 13
�188
V
V
189
support of a petition under this section shall ^Fca»j «««idered on the record as a whole, includi^lany report
be •%
recommendation of an adiri^ory committee. If applisidered confidential by such advisory committee: Pro
is made to the court for leave to adduce addivided, That this provision shall not be construed ju ,
evidence, the court 1may order such additional
hibiting the use of such data by the committee in con
to be taken before the Secretary, and to be
nection with its consultation with the petitioner or •;
i;'*Wuced upon the hearing in, such manner and upon such
representatives of the United States Department of Agri«|
®a and conditions as to the court may seem proper,
culture, as provided for herein, and in connection with;
such evidence is material and there were reasonable
its report and recommendation? to the Secretary. Not- >
withstanding any other provision of this section, the ••>: ^.gprounds for failure to adduce such evidence in the
Secretary may, when he finds that such, action is neces- If"proceedings below. The Secretary may modify his findplifina as to the facts and order by reason of the addisary to prevent an imminent hazard to the public, by
order, suspend the registration of an economic poison *•' ilional evidence so taken, and shall file with the court
immediately. In such case, he shall give the registrant pfnach modified findings and order. The judgment of the
prompt notice of such action and afford the registrant ; |;.wwrt affirming or setting ;aside, in whole or in part,
order under this section shall be final, subject to
the opportunity to have the matter submitted to an
;
^
ew by the Supreme Court of the United States upon
advisory committee 'and for an expedited hearing under
|«*rtiorari or certification as provided in section 125^
this section. Final orders of the Secretary under this
section shall be subject to judicial review, in accord- : |.of title 18 of the United States Code. The commenceance with the provisions of subsection d. In no event , |«*nt of proceedings under this section shall not, un||leos specifically ordered by the court to the contrary,
shall registration of an article be construed as a
^operate as a stay of an order. The court shall advance
defense for the commission of any offense prohibited
;0n the docket and expedite the disposition of all causes
.inder section 3 of this Act,
'filed therein pursuant to this section.
d. In a case of actual qontroversy as to the validity
of any order under this section, any person who will be v e. Notwithstanding any other provision of this Act,
^registration is not required in the case of an economic
adversely affected by such order may obtain judicial
poison shipped from one plant to another plant operated
review by filing in the United States court of appeals
fj oy the same person and used sc-lely at such plant as a
for the circuit wherein such person resides or has his
| constituent part to make an economic poison which is
principal place of business, or in the United States
Court of Appeals for the District of Columbia Circuit, ^registered under this Act,
within sixty days after the entry of such order, a
f. The Secretary is authorized to cancel the regispetition praying that the order be set aside in whole
tration of any economic poisofl at the end of a period
or in part. A copy of the petition shall be forthwith
transmitted by the clerk of the court to the Secretary, ji-; of five years following the registration of such
or any officer designated by him for that purpose, and ti economic poison or at the end of any five-year period .
^thereafter, unless the registrant, prior to the expirathereupon the Secretary shall file in the court the
: tion of each such five-year period, requests in accordrecord of the proceedings on- which he based his order,
as provided in section 2112 of title 28, United States • ance with regulations issued by the Secretary that such
registration be continued in effect.
Code. Upon the filing of such petition the court shall
have exclusive jurisdiction to affirm or set aside the
order complained of in whole or in part. The findings
of 'the Secretary with respect to questions of- fact shall
be sustained if supported by substantial evidence when
�190
Senator P!ART. Our next witness, as the Surgeon
ready indicated, is Dr. Jacqueline Verrett.
191
. |>,,.'
losses occurred after 3 weeks, and death ^fci weeks. The
f>#y observed was congestion of the lungs anc^mottled livers.
F fed 10 percent toxic fat in their rations lost hair on their
STATEMENT OP DR. JACQUELINE VERRETT, POOD AND
nnd shoulders (alopecia), and there was poor reproduction
ADMINISTRATION, DEPARTMENT OP HEW
lactation performance. Whelped pups were either dead or weak,,
,™ H»e mothers seemed to have an insufficient milk supply. Pups'
Dr. VERRETT. Thank you, Mr. Chairman, for this opportunity w
*•""—ed before weaning and fed a normal ration showed an immediscuss our investigations of the relationships between chlorinairf
und dramatic increase in growth. Other litters maintained on
phenoxy herbicides, chlorinated clibenzo-p-dioxins, and the chid
„ ll« toxic fat ration postweaning demonstrated inferior growth peredema factors.
'fennance.
p:•• Monkeys have demonstrated considerable sensitivity to toxic fat
Chick edema disease was first recognized in 1957, when large mmr
Materials. In one study nine monkeys received a toxic triolein at a
bers of broiler flocks in the United States suffered what appeared
level of 25 percent in their diets. One monkey died at 1 month, and
to be an epidemic disease. The affected birds appeared droopy, will
>four died at 3 months. At the 3-month period, corn oil was substiruffled feathers, and had difficulty breathing. In many flocks, more
than 50 percent of the birds died as a result of the disease. Of tl*jg tuted for the toxic triolein, but the other four monkeys died from
millions of birds affected, those autopsied consistently displayed* 9 Weeks to 5 weeks later, in spite of this substitution. Of the nine
hydropcricardium (accumulation of fluid in the pericardial sac),'f iWHtlikeys fed the toxic triolein, eight were autopsied and showed
irijrns of jaundice, pancreatic atrophy, and fibrosis, hemosiderosis,
accumulation of fluid in the abdominal cavity, subcutaneous edeinfcj
and additionally liver and kidney damage.
\
•fatty liver with necrosis, bile duct proliferation, and gross hemorIn 1958 the investigations of a number of .laboratories indicated i , rJwgo in the intestinal tract. No such pathology was seen in the
that the causal agent was contained in fats, and specifically in tin j i control -monkeys in this study.
A second study with 36 monkeys given a toxic fat at levels from
unsaponifiable fraction of fats in the commercial poultry rations. In; ^
laying hens the toxic fat caused a rapid drop in egg production.; fe 0.125 to 10 percent of their diet, demonstrated an inverse relationPullets receiving toxic fat during the full growing period did uol i |V' hip between the concentration of the toxic fat in the diet and their
come into production, and mortality was very high. Hydropericar- *-•"•• 'mean survival time. Those given the highest level (10 percent) had
ft mean survival time of only 91 days, while those given the lowest
dium, the most common lesion found in young birds, was not found
in birds of laying age.
level (0.125 percent) had a mean survival time of 445 days. It has
been estimated that the highest level provided approximately 728
The chick edema factor found in the toxic fat in the 1957 outbreak
micrpgrams total chick edema factors, while the lowest level diet
was presumed to have arisen as a byproduct of industrial production
provided approximately 100 micrograms total intake. The toxic fat
of stearic and oleic acids, since the unsaponifiable materials from this
was lethal at all levels studied, and the animals were sacrificed when
process were the components of fat in the poultry ration. Subpossible just before death. During the last 30 days of life, all monsequently, the toxic substance was found to be present in several
Koys developed alopecia, generalized subcutaneous edema, accumuladifferent types of fats. It was demonstrated to be present in samples
tion of fluid in the abdominal and thoracic cavities, and hydroperiof commercially produced oleic acids and triolein, in acidulated
cardium. There were decreases in red and white blood cell counts,
vegetable oils, and in inedible animal tallows. The demonstration of
total serum protein values, and altered serum-protein ratios. There
the presence of the chick edema factor in commercial fats led to the
was also cardiac dilatation and myocardial hypertrophy and edema.
ruling by the Food and Drug Administration in 1961 that higher
Finally, the experimental monkeys had reduced hematopoiesis and
fatty acids intended for food additive use must be free of the chick
spennatogenesis, degeneration of the blood vessels, focal necrosis
edema factors. The presence of the factor was to be ascertained by
of the liver and gastric ulcers.
a chick bioassay based on the volume of pericardial fluid in birds fed
the fat under investigation.
Limited experimentation with mice, pigeons, and turkeys, indicated that toxic fat in the diet led to reduction in growth without
Beginning in 1958, fat that had first been proved to be toxic to
hydropericardiiim or accumulation of abdominal fluid. Similarly,
chicks was used by various investigators in experiments with other
swine, fed toxic fat at a level of 9 percent of their ration, showed
species, and was demonstrated also to produce deleterious effects in
poor weight gain, but one pig sacrificed 6 weeks after the start of
rats, mice, turkeys, pigeons, guinea pigs, swine, dogs, and monkeys.
the study showed jno gross or microscopic lesions attributable to the
Early investigations of feeding toxic fats to rats indicated that
ration.
they are more resistant than chicks in short term feedings, but when
One important finding in the studies with chickens, is the apparent
fed in sufficient dosage, extracts of the toxic fat produced definite
storage of the chick edema factors in chick tissues. The unsaponideleterious effects as shown by growth depression, enlarged and fatty
fiable fraction of carcasses (exclusive of intestines, head, and feet)
livers, marked involution of the thymus, and enlarged adrenals.
of chickens fed the toxic fat was very potent in producing hydroGuinea pigs fed 2^ percent toxic fat stopped growing at 6 weeks,
pericardium in other birds when incorporated in their rations. Other
and death losses occurred at 8 weeks. At a level of 4% percent the
investigations of the distribution of the chick edema factors in the
lias b Hfg-I;,
•lis
�chick tissues indicated significant levels in bone, heart,
'kidney, liver, and skin. The liver contained more than 80 jj
the total detected. A similar determination of the distnlmion it
rats indicated the presence of chick edema factors only in liver
in the feces,
During the years that the previously described toxicity investigations were taking place, the toxic fats were undergoing intensiw
chemical analyses to concentrate, purify, and finally determine tfrt
nature of the compounds responsible for chick edema disease. At a!i
steps of these procedures, the path of the toxic material was cpfi'
firmed by assay in young chicks. This proved to be a time-consuniinf
and difficult job because of the complexity of the fatty materials, A
major breakthrough in this effort came when it was found thai t
highly purified crystalline material possessing the properties of chick
edema factor contained chlorine. This indicated that it was not i
natural component of the fat in which it occurred.
Work in several laboratories obtained similar results, and examii*
tion of the purified material by a variety of analytical techniques
suggested that chick edema factors could be highly substituted
(chlorinated) derivatives of naphthalene, biphenyl, anthracene, or
even structures common to the chlorinated pesticides of the DDT
family. These latter compounds were ruled out when tested in the
chick feeding assay, but some derivatives of the former classes of
compounds were tested and found in some instances to be toxic, and
indeed produce similar lesions to those observed with authentic toxic
fat. PIpwever, none of these compounds demonstrated the high order
of toxicity, or the complete chick edema syndrome when so tested,
Finally, by means of single crystal X-ray crystallography, it was
demonstrated that a pure compound isolated from a toxic fat wns
a hexachloro-dibenzo-p-dioxin, This structure was Aerified by infrared, ultraviolet, and mass spectrometry data. Final confirmation
came when this particular compound was synthesized and found to
produce the same lesions in chicks as the compound isolated from
the toxic fat.
The finding that a chlorinated dibenzo-p-dioxin was a chick edema
factor explained why different investigators had isolated materials
- similar-in their capacity to elicit chick edema disease, but yet in their
purest forms, had slightly different chemical properties. The large
number of isomers possible (more than 60) in this family ranging
from mono- to octa-chloro-dibenzo-p-dioxins illustrates the complexity of the problem. It then became a problem of determining
whether some or all of these compounds are, in fact chick edema factors, and what their relative capacities in this regard might be.
The chlorinated dibenzo-p-dioxin structures have been known in
organic chemistry many years, and became particularly noteworthy,
when in manufacturing processes with chlorophenols, their formation as byproducts posed serious occupational hazards., The most,
potent in this regard seems to have been the symmetrical tetrachlorop-dioxin which was formed in the manufacture of 2,4,5-trichlorophenol. These chlorinated compounds were found to cause a serious
and persistent disease referred to as chloracne. This disease was first
described in, 1899. Associations of this disease with chlorinated dibenzo-p-dioxins were made by the Germans, who had several out-
ittftka of this disease in their factories. There have also been similar
^ «mim<iices in the Netherlands and in this country, in f^juries man«*f*eturing chlorophenol compounds. It should also bc^pinted out,
i:;tis*l other compounds, such as the chlorinated naphthalenes, anthra].-*Bii«a, biphenyls and dibenzofurans, are known to be acnegenic, but
t:»t»m the case of the toxic response in chicks, these materials are less
'•^patent than the chlorinated dibenzo-p-clioxins. In the case of the
', «&k»racne associated with dioxin, the human symptomatology extends
l» other mucous membrane irritation, porphyria cutanea tarda, hirwtiRin, hyperpigmentation, increased skin fragility, severe damage
t» the, internal organs, particularly hepatotoxicity, and central nerv*ta system disorders, as indicated by neuromuscular symptoms and
j (wychologic alterations, and other systemic symptoms. Most of these
' wuputional exposures in Germany occurred in the 1950's, and foltowup examination of these affected workers in recent years indicate
Sltat tho recovery period is lengthy, with many workers still having
iiemonstrable adverse effects from prior exposure. Similar observa! lions have been made on exposed workers in the United States of
f-. America. The tetrachlorodibenzo-p-dioxin was demonstrated to pro;>';, clticc the chloracne in humans after the application of only 20 micro| groins. The rabbit ear is especially sensitive, with concentration of
;
g 0.001 percent to 0.005 percent producing severe reactions after local
T application. This assay using the rabbit ear is apparently used as.an
.'-• indicator in some plants of the content of this particular dioxin in
*:• tho manufacturing process. Hence, the serious health significance of
'•-•- those compounds for humans has, inadvertently, been clearly docu-
; mented.
Research in Germany and Japan indicated that the magnitude of
this problem was indeed large, since the formation of the chlorinated
di-benzo-p-dioxins would be facilitated in the saponification procedures used in various processes involving chlorophenols. A further
'•>'. complication is that a given chlorophenol preparation is generally
\ contaminated with other isomers, increasing the possibility of formation of a wide spectrum of chlorinated di-benzo-p-dioxins beyond
;' those to be expected from the predominant component. Evidence that
; this does occur will be discussed shortly in connection with the
chicken embryo studies of these materials.
During the time the previously described investigations of the
chick edema factors were underway, many of which were carried out
by FDA investigators, methodology was developed for detecting the
chick edema factors using sensitive gas-liquid-chromatographic
(GLC) techniques. It became apparent that authentic toxic fats
consistently gave peaks with specific retention times, and these peaks
were used as an indication of chick edema factor in a suspect sample.
Confirmation of this was obtained using the chick feeding assay. In
the light of recent knowledge of the chlorodioxins as chick edema
factors, it has been possible to establish that the materials being detected were hexa-, hepta-, and octa-chlorodibenzo-p-dioxins. Although toxic fat samples did indeed contain peaks corresponding
to dioxins of lesser chlorine content, i.e., di-, tri-, tetra-, penta-,
these are not detectable ,with this particular analytical procedure
because their particular peaks are obscured by other components,
�A test
However, in early 1969 there was another large outbreak of the
including pesticide residues present in the samples. Other
iliscaso in North Carolina and the toxic factor was tra^^to the fat
}cedures are being developed to detect these latter dioxins.
In the early 1960's the chicken embryo was being used in toxico- i| component of the feed. The toxicity was confirmed byWie chicken
logical evaluations of a wide variety of materials. It was hoped to ]h embryo test with fractions of the hexa-, hepta-, and octa-chlorodevelop a rapid and sensitive screening system to pinpoint coin- j k dibenzp-p-dioxins from the crude fat.
An investigation of the processing plant in which the toxic vegepounds with significant toxic and teratogenic effects for further ?p
table oil products were produced revealed a proximate operation for
study. In view of the demonstrated sensitivity of the chicken to chick ,Kedema factors, the chicken embryo was used to assay toxic fnt fe',. the manufacture of chlorophenol formulations. However, it is still
samples, and found to present the same syndrome as observed in the j|?, Hot possible to conclude that this accounted solely for the presence
chick feeding assay. A high mortality was observed with toxic fnt |j: of dioxins in the fat, or whether they were at least to some extent in
crude oil from a prior contamination.
extracts, and additionally, hydropericardium, generalized and masSince that time FDA has initiated an investigation of the oils of
sive edema, eye, beak, and leg defects, and necrotic livers were
other manufacturers and processors and in a few cases GLC analysis
apparent on gross observation. No microscopic studies have been
lias indicated the presence of chloro-dioxins. These have not as yet
conducted on embryos or hatched chicks in these investigations.
been confirmed by chicken embryo bioassay; however, it is noteIn parallel with other investigations, the chicken embryo was used
to test the toxicity of the chick edema factors isolated from toxic | worthy that in the case of these subject samples there is no known
adjacent manufacturing operation that would give rise to direct
fats. It was also found that the chlorinated biphenyls, naphthalenes,
; chloro-dioxin or chlorophenol contamination, so that entry of the
anthracenes, and other compounds did indeed elicit a toxic response,
chloro-dioxins from other sources must be considered.
and in some instances, the chick edema syndrome was present. But
This 1969 outbreak of chick edema disease, coupled with the quesin no case were any of these materials as potent as the toxic compotion of contamination of the herbicide 2,4,5-T by chlorinated dioxins
nents isolated from toxic fats, and were generally less potent by a
led us to renew this investigation. In the past 6 months the herbicides
few orders of magnitude.
2,4-D and 2,4,5-T, as well as the particular tetrachlorodioxin purAfter the identification in 1966 of a hexachloro dibenzo-p-dioxiii
ported to be the teratogenic agent responsible for the effects in the
as a chick edema factor, studies were initiated in which various
Bionetics study of 2,4,5-T have been under study.
isomers of chlorinated dibcnzo-p-dioxin were prepared and tested in
In an effort to assess the edema-producing capacity, the teratogenic
the embryos. Although the investigation was not extensive or comactivity, and the acute toxicity of these materials, samples of the
plete, it illustrated that isomers prepared by pyrolyzing selected
original Bionetics 2,4,5-T, from Diamond-Alkali, were obtained for
chlorophenols did give chloro-dibenzo-p-dioxins with GLC retention
comparison with a sample representative of the current manufacture
times duplicating those in the authentic toxic fats, and likewise, proof Dow Chemical Co.
duced the chick edema syndrome in the treated embryos. It was also
The Bionetics 2,4,5-T is reported to contain 27 plus or minus 8
apparent from this study that the various isomers .(that is, those
parts per million of the 2,3,6,7-tetrachloro dibenzo-p-dioxin, with the
with different chlorine content, and those with identical chlorine
content of other dioxins unknown. The current production of Dow
content, but with chlorine atoms positioned differently on the mole2,4,5-T has 0.5 parts per million of this dioxin, with no analysis for
cule) varied in their toxicity, although in all cases only microgram
higher chloro-dioxins reported, but does contain almost 5 percent of
or less quantities were required to elicit the toxic response. It is not
other impurities, mostly isomers of 2,4,-D 2,4,5-T, and chlorophenols
possible to give exact figures for the toxicities obtained in this study,
and chlorophenoxy compounds of undetermined structure.
since most of the individual dioxins were contaminated with traces
_ All investigations using the chicken embryo involved administraof others. Nevertheless, it was apparent that the symmetrical chlorotion of the compounds by injection through the air cell of the egg,
dioxin prepared from 2,4,5-trichlorophenol (2,3J6,7-tetrachloro dieither preincubation or at the 4th day of incubation.
benzo-p-dioxin) was more potent than any of the others tested, even
A comparison of the Bionetics 2,4,5-T with the Dow 2,4,5-T indirecognizing its lack of purity.
cates that with respect to the ability of the materials to produce
During this investigation, samples of the chlorophenols, both techembryonic mortality, the Bionetics 2,4,5-T is more potent. The.
nical and reagent grades, were examined by GLC to determine if
Bionetics 2,4,5-T has an LDM—that is, kills 50 percent of the treated
preformed chloro-dioxins were present. The presence of chloroembryos—of approximately 25 micrograms per egg, 0.5 parts per
dioxins was demonstrated by GLC, and these materials, which can
million, while the Dow 2,4,5-T LDGO is approximately 100 microbe_ removed by appropriate techniques, were then tested in the
grams per egg, 2 parts per million.
chicken embryo system and did indeed produce chick edema. A curWith respect to teratogenic effects, both samples produce chick
rent study of similarly contaminated chlorophenols, containing from
edema syndrome in the nonviable embryos, and hatched chicks, in18 ppb to 95 ppm of chloro-dioxins with six or more chlorine atoms
cluding eye defects, beak defects—predominantly cleft palate—short
are currently under test.
and twisted feet—the result of tendon slippage—and diffuse and
This investigation was not pursued further in view of the fact that
localized edema in various parts of the body.
there had been no known occurrences of chick edema disease since
the late 1950s and, hence, such research had a low order of priority.
�196
With both of these samples of 2,4,5-T these teratogenic o™
'observed at levels inducing no significant embryonic mortality.
The Dow 2,4,5-T still produces the chick edema syndrome at 50
micrograms, one part per million, a level where only 12 percent
mortality is observed, while the Bionetics'sample has similar effect*
as low as 6.25 micrograms per egg, 0.125 parts per million, a leve!
inducing only 16 percent mortality. Both of these mortalities a«
close to that induced by the solvent alone.
It should also be emphasized that the chick edema syndrome is
not observed in the embryos treated with the solvents only, at nny
level, or in the control flock.
A sample of 2,4,5-T from a chemical supply company was subjected to three recrystallizations before test. With the present GLC
techniques no chlorodioxins are detectable in this purified sample.
When tested in embryos it produced chick edema syndrome at 5, 10.
and 25 parts per million, all levels which induced no more than 15
percent mortality in the embryos.
This same sample was subjected to an additional purification by
seven extractions to remove dioxins that might have been present,
but were below the current detection levels.
This repurified sample is still clearly teratogenic in the embryos
since when tested at a level of 2.5 parts per million it produced 20
percent incidence of the malformations previously described, though
no significant edema was seen grossly. The mortality induced was
24 percent, which is higher than that of the sample prior to the
extensive extraction procedure.
It is also noteworthy that the embryonic mortality occurred soon
after treatment, and the hatched chicks had bleached down, indicative of an aberration in the normal pigment formation.
With respect to the 2,3,6,7-tetrachlorodibenzo-p-dioxin, early investigations of this compound in a preparation containing some
2,3,7-trichlorodibenzo-p-dioxin indicate a high order of toxicity and
teratogenicity.
Whether prepared by pyrolysis of 2,4,5-trichlorophcnol, or direct
chlorination of dibenzo-p-dioxin, the test preparations, which contained approximately 50 to 55 percent of the tetrachlorodioxin and
20 to 25 percent of the trichlorodioxin, produced significant mortality, that is greater than 20 percent, and chick edema syndrome in
more than 40 percent of the treated embryos at levels of five tenmillionth^ of a milligram per egg, or 10 parts per trillion.
More recent investigations, with two samples of the tetrachlorodioxin, both of purity greater than 95 percent, indicate edema and
terata at 20 parts per trillion. These samples have only become available within the past month and additional testing is underway at
lower and higher levels.
It should also be mentioned that the herbicide 2,4-D as a commercially available sample, and a purified sample, a mixture of the
K-butyl esters of 2,4-D and 2,4,5-T, and a sample of silvex, a related
herbicide, have been tested.
Terata and chick edema syndrome have been observed with all
of these materials at levels of 10 parts per million and above. Lower
levels are under investigation and the levels of dioxins in these
samples are also being determined.
197
.Studies have recently been initiated in the FDA tuaag pregnant
golden hamsters, intubated on day 6 through 10 of^|anogenesis
with the test compounds.
The Dow 2,4,5-T, 0.5 parts per million tetrachlorodioxin, tested at
1W m./k. yielded about 80 percent fetal deaths and those pups born
alive had gastrointestinal hemorrhages.
The thrice-rccrystallized 2,4,5-T sample referred to earlier, with
no detectable chlorodioxins, when tested at a level of 100 m./k. protlucd an average fetal mortality of 55 percent.
Among 38 live pups, three abnormals were found: One with a
deformed hind limb and two with inadequate fusion of the skull.
At lower doses the fetal mortality was less, but still higher than
that observed in control hamsters.
When the extensively repurified sample of 2,4,5-T was tested in
hamsters no gross terata were observed at 100 m./k., but the number
of early fetal deaths was 70 percent, indicating a definite embryotoxic effect and corroborating the observation of increased mortality
in the chick embryo studies. Additional tests with this compound
arc underway.
A dioxin preparation containing approximately 51 percent 2,3,6,7tetrachloro- and 21 percent 2,3,6 trichlorodibenzo-p-dioxin yielded
08 percent fetal deaths at 9.1 micrograms per kilogram. Gastrointestinal hemorrhages and eye anomalies—absence of lid—were
present in many of the pups.
• •
Tests with the purer tetrachlorodibenzo-p-dioxins are underway.
The numbers of animals in the hamster tests are too small to be
considered statistically valid, but there are definite indications that
alterations in fetal viability and gastrointestinal hemorrhages do
occur at the levels tested.
In summary, the chick embryo studies and additionally the preliminary hamster data indicate that the current production 2,4,5-T
containing 0.5 parts per million of the 2,3,6,7-tetrachlorodibenzo-pdioxin is teratogenic and embryotoxic in these test systems.
Further, an extensively purified 2,4,5-T sample, with no chlorodioxins detectable with the present techniques, has indicated significant embryotoxicity in the hamster and chick embryo, and addi-"
tionally produced gross terata in the chicken embryos, making it
impossible at this point in time to exonerate it of teratogenic or
other adverse effects on the embryos that may have some health
significance.
The data for 2,4-D in chick embryos likewise demonstrate these
effects in current production materials.
These studies have in no way assessed another and perhaps more
complicated aspect of this problem, and that is the interactions of
the various chlorodioxin isomers with each other in the many combinations in which they are likely to occur, or the possible interactions, including potentiation or synergism, between the chlorodioxins
and the chlorophenols, herbicides and other materials in which they
are found.
At this point, with your permission, Senator Hart, I would like to
insert in the record the documents containing the data from which
this testimony was derived.
Thank you. That is the end of my prepared statement.
�198
Senator HART. Yes, they will be received and placed in the rewr
j
after your oral testimony
Dr. VERRETT. I realize I presented a rather lengthy and complicated piece. I will be happy to answer questions and elaborate il
you wish,
I also have some samples of embryos, if you would care to s«
them, or chicks.
Senator HART. Yes, I heard about them.
The statement is not an easy one for one not trained in your
discipline.
I take it, Doctor, these are some of the chicks that have the deformities that you are talking about?
Dr. VERRETT. That is right. These are chicks that have been
treated with the tetrachlorodibenzo-dioxin. I would like to put them
out on the table but it is not safe.
As you can see, they cannot stand up. These chicks hatched early
this week. They have noticeable edema. This is really the ankle, if
you wish, of the chicken and this is really a result of the malformation,
These are normal chicks; as you can see, they have no difficulty
standing and walking.
These are chicks that have been treated with the ethanol, the solvent alone or have been untreated. We always run some at the same
time that have had no treatment and I think the comparison between
that one and this one, if you wish, is quite obvious.
Senator HART. Your last statement referred to a series of compounds.
Dr. VERRETT. Yes.
Senator HART. Exactly what are these ?
Dr. VERRETT. It is a family of compounds which I may say very
briefly is a series of phenols having a varying chlorine content. They
go from dichlorophenol, which is the precursor or the compound
from which 2,4-D is made, all the way up to pentachlorophenol.
All of these materials are very widely used as herbicides. They also
have a very broad use in industry and, in other words, are capable
of being in the environment and if they are contaminated with
dioxins, of also putting dioxins in the environment.
What I was trying to say is that we have looked at these chloro•phenols,' apart from just the herbicides which are derived from them,
and we know from past studjr, and there is a paper in the material
that I have included in the record which demonstrates, that these
materials are already contaminated with dioxin. The amount of the
contamination at this point is
Senator HART. It is very difficult to hear the witness. I do not
know what the distraction is, but please be patient.
Dr. VERRETT. We do know, at least by GLO (gas-liquid-chromatography) just chemical techniques—I will distinguish between chemical and biological techniques—by chemical analysis, we know these
materials are contaminated with chlorodioxins.
In addition to that, we know there are many' of these chlorinated'
dioxins that are involved and not simply the single one which has
been the subject of the 2,4,5-T. There are approximately 60 or more.
199
are
We are aware from our previous work that these
the point
trendy in chlorophenols as they are being used. This''
I was trying to make.
Senator HART'. The point I was trying to make by raising it is the
nm>8sity that there be no more delay with respect to establishing the
dangers, the hazards, the potentials for harm in this whole variety,
this whole family of products, given the kind of damage that you
|wvo so dramatically demonstrated here from one single element of
ilw, environment.
Dr. VERRETT. Yes. I might say—of course I could not bring very
many—these are chicks treated with the tetradioxin in question,
which is the most pertinent to the hearing. There are some here
which have been treated with 2,4-D. Thes are 2,4,5-T treated chicks.
\\'o do have underway an investigation of all the other dioxins. It
in a question of having to synthesize these materials and test them in
I>iiro form. But by inference at least, in our experience with the chick
wlc-ma problem in the past, we know all of these dioxins are in fact
toxic. We do know that. We have that information.
As I said, at least in clucks, we know there is a storage and possible transmission from chickens that have been fed these materials
to the human. The possibility does exist.
Senator HART. By handling, as you just did?
Dr. VERRETT. No, I hope not. The tetradioxin is potent. I do not
know whether handling this bird will harm you, but chloracne is a
very serious disease and persistent disease and there is no known
cure.
We are not aware that the other dioxins are as potent as that in
this regard, but they have yet to be evaluated.
To our knowledge the herbicides we have been discussing have not
actually been tested to see if any other of the dioxins are also
present.
The concern, apparently, has revolved around the particular
dioxin because it is extremely potent; even if the others are less
potent, of course, they may still be important.
Senator HART. I understood your explanation with the rats and the
monkeys to indicate that this—that the dioxin in'2,4,5-T has an accumulative effect.
Dr. VERRETT. The effects that are seen would indicate that. I would
lie unable to say that has been proven, but the fact that, for example,
in the one monkey study, when they were fed for 3 months and then
the animals that did not die during that course were put on a ration
free of it, still died in subsequent months, and that would indicate
that either the damage had been done by the initial exposure or
there was storage such that it finally did have its effect.
But it was fatal in all cases. So the animal data would indicate—
again we cannot apply that with certainty about the human—storage
or persistence, if you wish to use that word.
Senator HART. Then your attitude, which you say is not conclusive
and certainly does not relate directly to humans, points to the persistence,'the cumulative character of the dioxin, rather'than in the
other direction, that it is not cumulative?
Dr. VERRETT. I would say it indicates it is probably cumulative.
�200
Senator HAW. Do you know any experiments that point _ Ife
direction that it is probably not cumulative ?
"^
Dr. VERRETT. I am not aware of any, no. The human data I citw
with respect to occupational exposure would also indicate that it tf
probably cumulative in the human, of course.
Senator HAKT. You used a word here which I take it means burning.
Dr. VERKETT. Pyrolysis. Not exactly burning. In the sense I used
it, it means reacting under conditions of elevated temperature, bill
not the actual burning of the material itself. It indicates a high temperature reaction, perhaps, heat applied to the material in order to
make the reaction take place, but not in the sense of actually igniting it. _
But it does indicate that heat, in other words, facilitates the formation of these compounds (dioxins), if that is what you are arriving at.
Senator HART. What if some of this material is just put in the city
dump and burned? Could that burning inadvertently produce
dioxin ?
Dr. VERRETT. I could only say that the likelihood is there; yes.
Again, lack of actual experimentation does not give us any evidence or proof of this, but the fact is that these materials are formed
when chlorophenols are subjected to heat and that would indicate
to me that that is definitely a possibility.
Senator HART. What common products, or what common articles
contain chlorophenol ?
Dr. VERRETT. There are so many that I wouldn't be able to name
all. But, every piece of newspaper or paper of any kind probably
has chlorophenol used in the manufacture of paper. I am sorry I am
not in an area which would enable me to give you total usage figures, but they are considerable.
This is washed out to some extent, but there probably are some
chlorophenol residues, and paper would be one item and one which
we can say is very widely used.
Another example—well, leather is cured by using chlorophonols in
the tanning process. So leather materials contain it. There would be
any number of other eAreryday items that would possibly have it,
Senator HART. Well, just as I am reluctant to have pictures taken,
I am reluctant to make these contrary statement and yet this one is
not inappropriate.
If the materials that we customarily—and for generations, centuries I guess—have been throwing into a fire contain chlorophenol,
when you burn them, it is your opinion that dioxins can result—Dr. VERRETT. Could possibly result; yes.
Senator HART. Is it possible that some of the birth defects for
which there has been no medical explanation to date are the result
of this kind of thing, where we have always done it and it has never
seemed to hurt us 1
Dr. VERRETT. I would say it is a possibility. It would be for others
to assess this situation, but I think it is a distinct possibility because
of the fact that these materials (chlorophenols) are ubiquitous, and
if, in fact, chlorodioxins are formed in the environment, this is a
possibility.
201
•'•* I should point out the studies clone in mammals ha^fcbeen done
;l'.^y feeding, while the largest exposxire may come from^Hialation or
; «f«rmdl contact. That was the source of exposure occupationally to
]; lU» tetrachlorodioxin. So here we have to be concerned not only
tbrnit eating, but inhalation and perhaps contact exposure.
This brings up the subject of other materials. For example, we are
: »l«o investigating
,.'• Senator HART. I was just going to say that it is hard to visualize
' * jwbstitute for paper or leather, but can one have leather and paper
without this material ?
Dr. VERKETT. I would think so. I should point out some of the
materials (chlorophenols) are washed out in the processing. The
total amount used in the processing does not always remain in the
products. I did not mean to imply that. That brings up another
( Duration: Are they washed out into the rivers et cetera ?
:.• Nevertheless, I am not really sufficiently knowledgeable of the
h technology to say whether something else could be substituted or not.
</;•' Senator HART. Mr. Bickwit?
:1 Mr. BICKWIT. I have heard several people criticize these chick
;• embryo studies as being overly sensitive. I am not sure of this, but
•?, I believe the Secretary of HEW has criticized them.
:•'. Can you respond to that criticism?
Dr. VERRETT. Well, I have not as yet. As I mentioned in the testimony very briefly, this technique was started with just that idea, of
finding a sensitive technique, if at all possible, for assessing toxic
and also teratogenic response.
One of the great difficulties in all of the toxicology animal work,
using mammals and primates, even, is the difficulty of relating, of
course, to the human. There is perhaps another generation gap, if
you can use that word, between the chicken or an avian species.
I would say without hesitation that these studies, for example,
proved that this material is not for the birds, if I may phrase it that
way. I would certainly not say that you can conclusively state that
there is a human hazard from the results with the chick embryo.
However, inferentially we have evidence of that, and we have the
inadvertent tie-in because of the chlorodioxin toxicity already known
in primates and humans. I would certainly hope the human is less
sensitive than the chick, but I feel what we really need, and I think
that is pointed out very much by these hearings, is a very sensitive
test, and then it remains to show this is not the case in the species
more closely related to man.
I would rather demonstrate that something has an adverse effect
in a sensitive system and then, by appropriate study, find out
whether it will be relevant to man, than miss it altogether in an
insensitive test. Although we do not try to make direct correlations,
we feel certain anything seen in this system is worthy of further
study, and I should also like to add we are trying to keep the study
in the proper perspective.
That is, we use levels (doses) when possible that are relevant to
the human exposure or 'other animal exposure and not simply try to
produce effects with excessive amounts of material.
�Mr. BICKWIT. You state on page 4 that the most potent
he production of serious occupational hazards, seems to
he tetradioxin.
What actual experimental evidence is there that suggests or confirms that tetra is the most potent of the dioxins?
Dr. VERRETT. You mean in animal work ?
Mr. BICKWIT. Yes.
Dr. VERRETT. Of course the tests referred to earlier by Dr. Steinfeld show in that system that the tetradioxin is extremely potent in
rats. I am not aware that the other dioxins have ever been studied.
Mr. BICKWIT. Plave the other dioxins ever been compared in potency in chick analysis ?
Dr. VERRETT. Yes. As far as I know only in the chicle embryos.
Mr, BICKWIT. So, unless we can rely on-the chick studies, we may
be in very serious trouble, even more serious than it now appears on
the basis of available evidence.
Dr. VERRETT. That is right.
Mr. BICKWIT. Are there any data available, either in mammalian
•studies or chick studies on the relative toxicity of dioxins compared
to thalidomide?
Dr. VERRETT. If you used the chick as an exhibit or even the
mammalian studies with the tetradioxine, which is the only one
being studied at the moment, you would have to say this material
is some 100,000 to a million times more potent in these particular
species.
Now, I should add that the abnormal effects (terata) that we arc
seeing are not die same as we see with thalidomide, but the potential
for producing abnormalties that we do find, it is of that order;
Senator HART. Doctor, thank you very much.
(Thematerial referred to follows:)
'
BIBLIOGRAPHY
1. 'The Chick Edema Factor': Anon., Nutrition Reviews 26, 28 (1968).
2. 'Studies of the Chicken Edema Disease Factor': Friedman, L., Firestone,
D., Horwitz, W., Banes, D., Anstead, M., and Shuo, G., JAOAO J,%, 129 (1959).
3. 'The Occurrence of the Chick Pericardial Edema Factor in Some Oleic
Acids and Products Therefrom': Ames, S. R., Swanson, W. J., Ludwig, M. I.,
and Brokaw, O. Y., J. Am. Oil Chemists Soc. fSI, 10 (1900).
4. 'Studies of the Chick Edema Factor. II Isolation of a Toxic Substance':
Yarteoff, A., Firestone, D., Banes, D., florwitz, W., Friedman, L., and Neslieim,
S., J. Am. Oil Chemists Soc. 38, 60 (1961).
5. 'Collaborative Bioassay for Chick Edema Factor': Douglass, C. D., and
Flick, D. F., JAOAC 44, 3 (1961).
6. 'Progress in the Chick Edema Problem': Friedman, L,, Feedstuffs, March
17, 1962.
7. Occupational Intoxication Occurring in the Production of Ghlorophenol
Compounds': Bauer, H., Schulz, H., and Spiegelberg, TJ., Archiv fur Gewerbepathologic and Gewerbehygiene 18, 538 (1961).
8. 'A Technic for Testing Acnegenic Potency in Rabbits Applied to the
Potent Acnegen, 2,3,7,8-Tetrachlorodibenzo-p-dioxin': Jones, B. L., and Krizek,
II, J. Invest. Bermatol. 39, 511 (1962).
9. 'Studies of the Chick Edema Disease. 2. Preparation nnd Biological
Effects of a Crystalline Chick Edema Factor Concentrate': Flick, D. F., Firestone, D., and Marliac, .T. P., Poultry Science 44, 1214 (1965).
10. 'Chick Edema Factor: Application of Microcoulometric Gas, Chromatography to Detection of Chick Edema Factor in Fats or Fatty Acids': Firestone,
!>., Ibrahim, W., and Horwitz, W., JAOAC 46, 384 (1963)j^OAC (1965)
xwtions 20.087—26.006.
(•
11. 'The Injection of Chemicals into the Fertile Eggs Prior Wrncubation ns
« Toxicity Test': Mclaughlin, J., Marliac, J. P., Verrett, M. J., Mutchler, M.
K., and Fitzhugh, O. G., Tox, Appl. Pharm., S, 760 (1063).
12. 'Use of the Chicken Embryo In the Assay of Aflatoxin Toxicity': Verrett,
M. J., Marliac, J. P., and McLaughlin, J., JAOAC 47, 1003 (1964).
13. 'The Role of Toxic Fat in the Production of Hydropercardium nnd
Ascites in Chickens', Allen, J. R., Am. J. Vet. Res. 25, 1210 (1964).
14. 'Industrially Acquired Porphyria': Blieberg, J., AVallen, M., Brodkin, R.,
ami Appelbaum, I., Arch. Derm. SO, 793 (1964).
15. 'Electron Microscopic Alterations in the Liver of Chickens Fed Toxic
Fat': Allen, J. R., and Carstens, TJ. A., Lab. Inves. 15, 970 (1966).
10. 'Chick Edema Factor: Some Tissue Distribution Data and Toxicologic
Meets in the Rat and Chick': Campbell, T. C., and Friedman, L., Proc. Soc.
Kxp. Biol. Med. 1%1, 1283 (1966).
17. 'Studies on the Metabolism of Chick Edema Factor: Distribution in
Chick Tissue': Firestone, D., Higginbotham, G. R., Flick, D. F., and Ress, J.,
fc'DA Internal Preliminary Report, October 1966.
18. 'Light and Electron Microscopic Observations^in Macaoa mulatto, Monkeys
Vcd Toxic Fat': Allen, J, R., and Carstens, L. A., Am. J. Vet. Res. 28, 1513
(1967).
19. 'Note on an Improved Cleanup Method for the Detection of Chick Edema
Factor in Fats and Fatty Acids by Electron Capture Gas Chromatography':
Neal, P., JAOAC 50, 1338 (1967).
20. 'Oils. Fats, and Waxes': Neal, P., JAOAC 51, 489 (1968).
21. 'Chemical and Toxicological Evaluations of Isolated and Synthetic
Oliloro Derivatives of Dibenzo-p-dioxin': Higginbotham, G. R., Huang, A.,
Firestone, D., Verrett, J., Ress, J., and Campbell, A. D., Nature 220, 702 (1968).
22. 'Analysis of Commercial Chlorophenols for Trace Amounts of Their Condensation and Polymerization Products': Higginbotham, G. R., and Ress, ,T.,
FDA Internal Preliminary Report, November 1968.
23. 'The Identification and Crystal Structure of a Hydropercardium Producing Factor: 1,2,3,7,8,9-Hexachlorodibenzo-p-dioxin': Cantrell, J. S., Webb,
N. C., and Mabis, A. J., Ohem. Eng. News 45, No. 5, 10 (1967), and Acta Cryst.
U 25, 150 (1909).
24. ,'Clinical Picture and Etiology of Chloracne': Schulz, K. H., Arbeitsmedizln-Sozialmedizin-Arbeitshygiene 3 (2) : 25 (1968).
25. 'Report on Methodology for Chlorinated Aromatics in Fats, Oils, and
Fatty Acids': Ress, J., Higginbotham, G. R., and Firestone, D., JAOAC, in
press (1970).
26. Federal Register December 9, 1961, 26 F. R. 11828, 121.224; also Code
of Federal Regulations, Title 21, sec. 121.1070. further amended Federal Register August 25, 1966, 31 F. R. 11215.
27. Table: Embryotoxicity of Chlorophenols, Dibenzo-p-dioxins (Chick
Edema) FDA Preliminary Data, J. Verrett, 3/30/70. (Chick Embryos).
28. Table: Preliminary Report on Teratology Studies with Dioxin Using
Golden Hamsters: FDA Preliminary Data, T. F. X. Collins, \V. H. Hansen,
and C. H. Williams.
29. Table: Preliminary Report on Teratology Studies with 2,4,5-T Samples
Using Golden Hamsters: FDA Preliminary Data, T. F. X. Collins, AV. H.
Hansen, and C. H. Williams,
30. Letter: 1C D. Courtney, NIEHS to J. McLaughlin, FDA, of 12/4/09 re
Bionetics sample of 2,4,5,-T.
31. Letter: G. E. Lynn, Dow Chem. Co., to M. J. Verrett, FDA, of 2/9/70
re composition of Dow 2,4,5,-T sample.
32. Memo: FDA Internal from D. Firestone to A. D. Campbell, of 3/2/70 re
composition of chlorinated Dibenzo-p-dioxin Standard.
33. Memo: FDA Internal from D. Firestone to A. D. Campbell, of 3/20/70
re samples for Chicken Embryo Testing.
34. Memo: FDA Internal from J. Ress to A. D. Campbell, of 3/26/70 re
chlorophenol samples for Chicken Embryo Testing.
45-362 O - 70 - 14
�ir vitamin E)
nor"it-Ionium had any cur^B effect.
Sensitivity of different species to the
toxic factor in these fats varies consideri t'lrie
!•»»
ably. \Vhen monkeys were fed a sample of
ii- #ul^taiu'e was jircscnt in a triolpin that produced hydropericarduim in
v e ? of fats. These indndcd a
. . . ., ,-...
i.(,Vi, ,ii ( , f i within three
contained a toxic subnmahlv was present in
of
coiuair.i'd 25 per cent triolcin. The monkeys
showed signs of jaundice, pancreatic atrophy and libro.-is, bemosiderosis, f n t t y liver
f«Mrll>w;i« inriii|iuiuvv.
with necrosis, and gross hemorrhage in the
t * Vvcl oi 15 per cent, the, birds showed
w'yniptoms of hydropcricnrdium. f4im- intestinal tract. composed of equal parts
Pigs fed a ration
jynploms were produced when chicks. of a highly toxic chick ration and a normal
*«»? iVd rations containing distilhilcs or i-w'nip ration gained weight and appeared to
%«*!'if! (ecured in commercial production show no abnormalities 'Sanger et n'.. !<>o.
A fstiy ncid?. The toxic subs-lance (si was n'M. However, when a toxic fat was in•>•< frnmd in inedible animal tallows, aeicl- corporated into a swine ration at a level of
<«',•>.! vegetable oils, and several com- 9 per cent, the. live shoats gained 0.72
»»T').'.!'y produced olcic acid* and irioU-in pounds per clay while the controls gained
L •'Y'Mone, W, HorwitK, ]''ricdman, and G. 2 pounds (I.. C. Scott, J. Am. Vet. Med.
M. flme, J. Am. Oil Chc.m. Soc. 38. .',1S .Issn. 137. ?.W U.'WO)). Despite the pom•)W\>. A report from another laboratory weight gain-, the one pig sacrificed about
i«*f::mcil the presence of the. chick edema six weeks after ihc start of the <-Uidy showed
f'nnr in various industrial fats (J. O. no gross or microscopic lesions attributable
A'-xaiwk-r, R. J. Young, C. M. Burnett, and
the ration.
II. I'l. Hnthaway, Poultry Sri. 41. S3 to The fat from •the latter pig was rendered
f^fjlt
illlHI.t^vo
"*.~
n-rt i|u,ihty" (A. YiirUoff ft til.. ./. A in.
«*i,T'i. $,ie. 38. CO ( 1 / W l i i . When this
THE CHICK EDEMA FACTOR
i
A toxic factor in tunic, teed fjmt't? tot* and /«//// uciilt product'* li;ttl<'uiifticara'iuin
and asdics in'yo\\ng chicks u'hen 9 fig. per kilogram body weight ore. ft'.d per day.
There are a manlier oj these toxic conii*ounds. containing large amounts aj cltlftrine.
One has been diaraetcritctl and synthesized.
In 1957, large numbers of chickens suf- '
'fercd from what appeared to be an epidemic
disease. Losses a t t r i b u t a b l e to this epidemic
have been estimated in the millions of
dollars. The affected birds appeared droopy,
showed ruffled feathers, and had difficulty
breathing' (L. Friedman, l'\-cilf:tiiljfi. March
17, 1062 \.
In some flocks, over 50 per cent of the
birds died with typical symptom* of the
disease (V. L. Sanger i t til,, J. . t m , Vel.
Med. Assn. 133, 172 (JOSS)). Whi-n autopsied, the birds had pale hearts w i t h hydroperieardium, and livers that were pale,
mottled, and had an irregular granular surface. In, the advanced stages, the abdomens
were distended, and contained 100 to 500
ml. of clear, straw-colored fluid. The pcrieardial cavity is most, susceptible to fluid
accumulation, w i t h the abdominal cavity
next, and then subcutaneous tissue l\">. F.
Flick, C. D. Douglass, and L. Gallo, Paul-*
try Sc>. 42,.SflJ (IMS')). The kidneys were
pale and swollen; the f a t t y tissue of the
gizzard was edematons; and the duodenum
was swollen and soft (Sanger ct al, loc.
n't,). Ilydropericardium was not as prominent in the older birds as in the broilers
(Friedman, loc. cit.),
No acceptable explanation is available
for the abnormalities associated with feeding the chick edema factor. Despite ac-
cumulation of fluid in the pcncardial Ma
abdominal cavities of (he affected birds,tl*
hematocrit, blood volume, and moisUlnr
content of the heart, skeletal muscle, skin,
and kidney were normal. However, the total
body water content of the birds was significantly increased. On the basis of these oil*
servations, it, was suggested that (.he clink
edema factor increaMid permeability of th(
cardiac vascular bed (Flick, Douglass, mid
Gallo, loc, oil.). A d d i t i o n a l support l'ov thi'
hypolhwis came from Ihe rinding that the
toxic factor produced no appreciable clump 1
ii; the proportion or level of plasma prokin 1
(Flick, D, Firestone, and .1. 1*. Marline,
Poultry Kd. -14, 121.1, (IfiBi'i)).
The type of diel had a marked effect on
the rate at which the chicks' bodies accumulated water. Chicks fed a natural
grain ration containing 4 per eent toxic f;it
showed an increased body moisture content
(7S versus 72 per cent for the control')
only at the end of the third week. Birds
fed the same level of toxiu fat in a semipurified diet showed a marked increase in
body water content a f t e r seven days (70
versus 73 per eenl) (Flick, Douglass, anil
Gallo, he. cit.).
During 1958, a number of laboratories
traced the disorder to the presence of a
toxic substance in the unsaponifiablc fraction of fats added to commercial poultry
I'M presence of the chick edema factor
•i roivniK-rcinl fats led to the ruling by the live' ration containing-the rendered lard der<v,,l ;,i,d Drug Administration that methyl' veloped toxicity signs or symptoms. Tin?
•*n:r« of higher f a t t y acids intended for use finding suggested t h a t the pig did not store
-•k:t food additive must be free of the chick appreciable amounts of the IONIC substance
• i-i,ia factor (Federal Register Dec. 0,
its
;'•;(. K F.n. 1183$; J5J.M4). The presence in The adipose tissue.
apparent absence of Iho. toxic factor
••'. tin: factor was to be ascertained by ^ in pork fat is in contrast to chick*' fat,
1
•I.K'k hion-nay based on the volume of peri- where storage appears to occur. The uni..»Ji-d fiuid in birds fed the fat under in-' saponifiable fraction of carcasses of chick'•'••ti'.'iiMon.
ens fed the toxic fat was very potent in
Timf. Milt was necessary in the chicks' producing hydropericardiutn in other birds
fii'J'ih for the development of hydroperi- (Friedman el. al., J. Assn. Official Ayr.
fiii'lmiii \vhcn toxic fats were fed was suglSf>
V'H-'l by Alexander and co-worUevs. T\\ey r/ifin. 42, recent<l!).->9)). of the response of
A more
study
•iVurved typical symptoms of the diMurb- different species, to the chick edema faclor
:mri' only when the ration contained sodium utilized the unsaponifinblc fraction of a
• 'nl'iriric; extra ^ilt accentuated the con- to'xic f a t . This was fed to young chicks and
'liiinn. Alihougli this syndrome had many nils iT. C. Campbell and Friedman. Prnc.
'it I!IP •.•amiiirk-- of a vitamin K dplicien"y,
�206
Soc. K.rp. n\ol hied. .121, 1283 (HIGH)).
The w e a n l i n g rats fed I ho u n s a p o n i f i a b l e
f r a c t i o n nt a level of 9 //g. per k i l o g r a m body
weight per day of the chick edema f a e i o r
showed a 3.7 per cent loss in weight over the
six day feeding t r i a l . No gross t i s s u e alterations were apparent on autopsy. The livers,
however, were 21 per cent heavier t h a n
(hose of the controls when expressed on a
body weight basis, while t h e adrenals were
50 per cent heavier. The heart, kidney, and
spleen were of normal sue.
Chicks fed a c o m p a r a b l e a m o u n t of conc e n t r a t e developed h y d r o p c r i e n r d i u n i in six
days. The livers of these birds were 15 per
cent heavier than those of the c o u t r o U . The
statement was made t h a t " t h e increase in
l i v e r weight in the chicks was not due to
m o i s t u r e or fat." Such an observation
should have been d o c u m e n t e d and cheeked'
by h i s t o l o g i c a l studies.
, •'
A few years ago, H. I1',, l l a r m a n and collaborators (J.- Am, Client. Soc. 82. 207S
(IflGO)) reported i s o l a t i o n of a c r y s t i d l i n e
substance from a feed-grade t a l l o w . This
compound produced h y d r o p c n c a r d i u m in
chicks when incorporated i n t o a commercial
ration at a level of 0.1 p.p.in, A report
(Yartzoff ct al., loc. tit.) indicated t h a t the
crystalline substance c o n t a i n e d 47 per cent
chlorine.
Apparently, there are a n u m b e r of compounds t h a t behave l i k e (he c h i c k edema
f a c t o r . These compounds move closely w i t h
t h e (o.\ie factor d u r i n g m o l r c i i l a r d i s t i l l a t i o n , on thin layer e h r p n i a t o g r a p h y , and
show s i m i l a r peaks on gas chromato.!iraphy>
Xot all these compounds are toxic, and
l u x i c i t y appears to vary in those t h a t arc.
I ' a r t o f these d i f f i c u l t i e s may he n.vnlu,!
\ v i t l i c h a r a c t e r i s a t i o n and synthesis uf om
i'f t h e loxic. substances. By means of -in).'1'
cry.-tal x-ruy c r y s t a l l o g r a p h y , .1. S. Cnln i
X. C. U'fbb, and A. ,1. M a i n s rqm i '
(Abtlrficls,
Aiif. ('ryxlnllorirajiltin .Iw
Meelinii. 71. 37 (7.9671) t h a t the rnnipiiwi.)
isolated in I heir laboratories was I .2,1! 7 .
S . 9 - h c x a e h l o r o - d i b e n x o - p - d i o \ i n . This \\ w
was supported by infra-red, ultra-violil,
and mass spectromclry p r i n c i p a l l y by ,1 C
\Voolon and W. 11. Conrchene (,/. Ayr. I'uo'l
Chnn. .12, 94 (19G.1,)). This struct urn < i
verified by synthesizing the compound. Tin
l a t t e r produced the same, lesions, in chicki
a* (he compound isolated from llie loxic l i t
S i g n i l i e a n t progress has. been made i
i n d e i i t i f y i n g one of the l o x i c s u h s t a n f
present in some batches of f a t s i n t e n d e d iw
a n i m a l foods. There are s t i l l u immbci 1 ol
unanswered questions, the most impni'l iiil
of these being: What is tin; possible si«n li
cam1!1 of these compounds, t o h u m a n h e a l t h ?
•Since substances w i t h physiological prnjicrties s i m i l a r to the chick edema f a c t o r i n
be stored in the adipose (issue of chid <
can t h e y also accumulate in h u m a n time
when i n i n j m a l amounts arc i n a d v e r t e n t l y
ingested?
. A n o t h e r i m p o r t a n t q u e s t i o n is: What i'
t h e source of these compounds? A p p a r e n t l y ,
the chick edema f a c t o r has been associated
only w i t h f a t s and f a t t y acids subjected to
a considerable a m o u n t of heat d u r i n g tli i
processing. I f the source of t h e c h l o r i n e in
I lie l o x i c compound could In? i d e n t i f i e d , il
might bo possible; |.o devise methods for it
removal or e l i m i n a t i o n before m a r k e t i n g
the f a t .
207
Reprinted .from
Journal of the Association of Official Agricultural ChcmiS
February, 1!10'9
Studies of the Chicken Edcifya Disease Factor*
By I,. FRIEDMAN, D. FIRESTONE, W. UOIOTITZ, D. BANES, M. ANSTEAD, nnd
G.8HUE (Food and Drug Administration, Washington 25, D.C.)
we successfully produced the chniactcristic
symptoms of the "chick edema disease'' inWhen the problem of "X" or "Edema" volved n sample of fat that had been colin poultry was brought to our attention in lected by one of Our Food and Drug inDecember of 3057, a considerable amount spectors at a feed manufacturing plunt and
of work had already been done. It was es- which had been known to have produced
tablished that drugs added to tho feed, as the disease, and also a sample, of a "larry"
well as contiiniinnlion by drugs or heavy by-product from the manufacture of olcic
metals such as lead or arsenic, was not rc- nnd stearic acid, which presumably had been
n|Kinsible. Tho disease was not cruised by mixed into this fat sample to ihe extent of
kiclcrial, viral, or parasitic infection, and about 40%. In this experiment HID v i t a m i n
evidence was accumulating that tho only feed I?) hypothesis was tesletl. Also, tlio Miitabilingredient associated with all the various ity of the A.O.A.C. ration for this invc'-tig.'ioutbreaks was the fat. The symptoms have tion was checked against n feed. resembling
been described in detail in the preliminary more closely a pructical commercial r a l i n n .
report of Schmittlc, et. al. (J. Am. Yd. Mcd.
Basal Nation and the Vitamin K JliipolhAsm., 132, 210 (1058)), which also summarized sonic of the work leading to the esis,—Table .1 shous: the composition of the
iiicrimination of the fat added to tho ration. A.O.A.C. basal ration and the special basal
ration. The major differences arc (hn subThe characteristic symptoms of this dis- stitution of Drackctt (an isolated soybean
ease lire the presence of excessive, fluid in protein) for the casein of the A.O.A.C. diet,
tho pcricardial sac, in the abdominal cavity nnrl the inclusion of alfalfa leaf meal ami
(water belly), and less often subcutancous.ly, linseed oil meals. From the ri'.;alls shown
Accompanied by high mortality beginning in Table 2, it is clear that: (/) the f a t t y
approximately in the t h i r d week. The strik- field (F.A.) by-product is much more effecing resemblance of the "edema disease" tive in producing the disease thnn the. ^amsymptoms to those recorded for the exuda- ple of fat (INV. Fat), that had actually been
tive diathesis syndrome of v i t a m i n 1C de- used by feed manufacturers;- (2) the special
ficiency in chicks made a t t r a c t i v e the basal ration is to be preferred to the.
hypothesis that the. disease outbreaks were A.O.A.C. diet, tincc. the severity of thn edema
the result of an induced vitamin TO deficiency symptoms is incicascd, growth porfonnimco
caused by the use of poor quality fals. .
is improved, and gizzard erosion it elimiIt was convenient, because of other work nated; (;!) the feeding of vitamin K in bn;c
Introduction
in progress in our laboratory at the time, to
use the A.O.A.C. vitamin D3 chic): test ration and (.'ingle comb white. Leghorn chicks
in an attempt to produce the condition.
T u h l u l . UHSU] chick
r;il i n n s
AOAC
Whole yellow corn. uroinul
,r>R
2:>
Wluvit lluur imiidli i>i;g
1
12
Cnidc pi'ccipit'ik'd njifcin
Our first feeding trial with a sample of Calcium pho.^phnlc (piecipi- 2
tatiai)
feed collected from one of the "disease areas" .lodiiU'l Pall,
2
was a false start, since a f t e r six weeks on Noii.irru'li.".tc(! yc:i Kt
2
0 . 02
the ration the ibidej were normal nnd showed MnSO,. 111,0
,1
Coil I'lVLT oil
no unuciuil symptoms «po:i poslmodem ex- ]3i;l>j<Jm(i'.| n l f n l f n l< l ;if monl
.^.
a m i n a t i o n . The first experiment in which Linr'-'-il oil rni'iil
IJi'McUi.'lt protein
—
• I'n'M'lilnl III HIP Fi'wntv-srHiMil AIHI'M! M < i - l i n r "I
III" A - c i M i i l i < i n i.t (llli.'iiil ArnrMlluml Utiiinimi), Ocl.
13 IS, IflJS, ill U'lifliiiip.liin, U.C.
102.02
£|.rciltl
15
Id
2.H
2.01
H.2
0.112
1
1
10.11
1C,
1(10. i)ll
�209
208
^^
Table 2. Ell'ccl of Mispcet i'ul mid by-product fat on chirks
Av,
(llOIIJ)
1
2
3
4
5
20-Day
Wt, ((Jnims)
Dentil
Diet
Added Fat
H7
0
A.O.A.C. 0% lurd
0
• IS!)
A.O.A.C. ('.% 1,\V. fat
103
S|icdiil
G% INV. fat
i? 0
f
Special /3.0% lard +
12.4% )''.A. by-product
147
GO ''
[3.0% lard +
Special |2.4% F.A. by-product +
0
[vitamin li •
M3
No. of (.:hi.-.l<H Show mi
Hderini Syiuploinn
Mnrkrd
AliM
•
—
10
—
7
7
9
S
C
2
—
9
Chemical FJnr/inj/s.—Part of the chemicnl
data on these, samples is shown in Table 3.
Findings of particular significance were that
these "fats." (F.A. by-product) were actually
Tnblc 3. Comparison ol* >
mlh lard
{••
f:its
I.anl
lnv. fat
V. A. byproduct
0.35
3.8
HI. 7
llurrlsan)
rusiiivo*
Dil'Uoiiiii
lYcnpilrih)" 1
% Of UllMlp.
X'ntnp.
% of U l \ M I | i ,
23.0
70.11
02. ft
21.7
7.0
2.3
\
VOLATILE,IOO U C
FATTY ACIDS
1.3%
88%
I3grn.
880 gmUrea
Fractionation
i
1
UNSAP
10.7%
' I07gm.
5+
" JO mji r/f-alpliii incnpliiM'nl per edick per diiy by month,
Av. Miivivitl time, 29.2 days.
u
dotes docs not prevent the occurrence' of
the, disease, but docs seem to decrease, the
severity of the symptoms. However, in an
independent study by 1'. C. Underwood and
C. 0. Diirbin at Ilie Bcltsville Laboratories
of our Veterinary Medical Branch, w i t h
Hhodo Island Reds, the oral administration
of 1 ing per clay per chick of (W-alpha.
locophcrol acetate had DO effect, the symptoms being slightly more pronounced in the
supplemented group. The conclusion that
some material associated with the "fatty
acid by-product' 1 is responsible for the disease, and not n pimple v i t a m i n E deficiency,
was confirmed in a subsequent test, when the
fatty by-product was fed at 7% of the diet
and all chicks showed marked to severe
symptoms at autopsy; only 2 of 19 chicks
survived the. twenty-third day, and the average survival of 17 chicks was 19.4 days, In
n similar group fed 10 mg of rW-alpha tocopherol per chick per day, the average survival was 20.5 days for 15 chicks; 4 chicks
survived to the twenty-third day. All showed
marked to severe symptoms.
BY-PRODUCT
(lOOOom)
f )«•
Saponificalion
No. NoriuM
20
12
4
free 1'alty acids c o n t a i n i n g nn unusunlly
large amount of unsapouifiiible material.
Furthermore, tho unsaponifiablc fraction is
cumulatively different from t h a t ' f o u n d in
fresh animal fat such as lard, by virtue of
tho large percentage that reacts iii tho
Licbcrmann-T'iurchai'd test, compared to the
much smaller proportion that reacts with
(ligitnnin. In the lard the proportion if
almost one to one.
Ure,a Fractionalion.—Tho f a t t y acids from
the fatty by-product, nfter paponincation,
were fractionated (J'ig. 1) with uvca into
two portions: the, normally occurring f a t l y
acids that form urea adducf, and modified
or abnormal f a t t y acids that do not form
urea adducts. About 70% of, tho f a t t y acids
form urea adducts; the remainder ('<&%}
comprise, the, urea, filtrate.
K/Jcct of Urea Fractions on fiats wit .
Chicks.—The urea, f i l t r a t e f.'ilty acid fraction was fatal to weanling rats (40-00 R 1 )''
upon oral administration. T\\o successive
daily dopes of 0.4 nil resulted in marked loss
of weight and death by tho fourth day. Two
doses of O.'J nil caused a marked weight Joss
from which recovery bepnn at the fourth
day. This effect is similar to that observed
in our laboratory w i t h the i.-tlty acids tli.nl
do not, form urea adducts which were derived from healed cottonseed o i l , and to the
observations recorded by pivmipton, ct at,
(J. Nutrition, 14, ]77 (10.')!)) with heated
linseed oil. Chicks react, similarly, although
they RCfiin to be more resistant to tho l e t h a l
effects of direct feeding than (he rats. However, not enough observations were made to
establish the relative susceptibility of the
two specie,". All polymon; .ind hydrop;ciin(ion products (sen Fie;, J) w c i o toxic.
ADDUCT
76% (669gm.)
M.W. Me. Esters:
.
288
NON-ADDUCT
24% (2ll«jm)
M.W. Me. Esters:
323
Molecular
Disi II lotion
l30°C.,7ju
L....,_
POLYMERS/
1% (2cjm.)
,,.,
..
MONOMERS " .1
99% 209qrn.)
I. V.
i V R?
52 ' i
Hydrocicnation
10% P d - C .
Fig. J—Fmctionalion a\ fatly odd by-product. (Numbers fallowed by H- si<7» me le-xkil-v
tee lootnate c, Table C.)
Tho urea .ndrUict, fraction of the fatty acids of the liver markedly increase if., which was
produced no unusual effects cither in mts or accounted for only in part by au increase in
liver fat. No other signified)!!, differences in
in chicks.
the relative weights of organs (kidneys,
K/Jcci oj Vnsaponifiitble I1'radian on Rats. thyroids, hearts, adrenals, etc.) were noted,
—The significant, finding of feeding the un- although small differences caniwl be demonsaponifinbln fraction of the fatty ncid by- strated by such small groups.
product to r a t s are conn in Table 4. One
Although deleterious effects wi re produced
ml of the un.'iapomfiublo fraction was fed to
in rats both by the urea fihriU nnct by the
each of f o u r rats every day for 22 days.
unsaponifiablo portion, symptom.-: simihr to
Control animals received no supplement to
those of the "edema, disease" syndrome were
I IIP. basal r a t i o n . The experimental group
i.'fintainrd two inalo.s from different litter*, not observed in this species.
Effect on C/ii'c/.-s.—When add;- ! to (he diet
with lit lor mates in the control p.roup. The
females of both the test and control groups of chicks at a level equivalent K> '<% "f the
were, all from one litter. The growth rate original f a t l y by-product., the "ren l i l l i a l e
was pRniMcmilly deputed— females were produced a fisznificant growth ilivvcvsion b u t ,
more drastically .ifi'cclcd t h a n males; (lie as Table 5 shows, very few eiT••tun difeasn
H/C of thn tliyiruis decreased; and the t'm sym])tom?. Tho small amount of a c t i v i t y
�Tiiblo 4. Kvniilts of feeding uiitmponifinhlfg from "fully nolil ]>y-i\iro<luc
to weanling nils by stomach tub*! (1.0 ml/riil/dity)
Mn !<•»•
l-'oninlo.s11
Control
l-'od
Conlrol'
Toil
22-day wt gnin, urnms
KiS
Liver wt, g/100 g body-wt
% Fat in dry liver
Tiiymus, B/iOO g body wt
1J2
0.00
5.77
12.0
0 . 220
0.258
111 .
!)-»
0.05
10.03
H.S
-
114
lir.
5. OS
O.Oflfl
0.1(12
• MR!CM |);urod Ijy l i l t o r ; 2 Iill
b
FcimiK'3 all from ono liUur.
0,:!i
10.0
o.2;u
O.Jf/.'i
49
•IS
10.00
9.08
14.1)
0.08-1
0.073
Tnble 5. Effect of by-product fnt anil its frnctious on
t>|>t.
2
2
2
2
2
4
•t
4
4
indicated in tho urea, filtrate was (shown, in
later experiments to reside in tho, unsaponifiable residue that Imd not been eomplelely
extra clod. It is clear that tho edema disease-producing material is associated with
tho nnsaponifiablo portion of tho fatty byproduct. In Table 5 nro also shown the variations in severity of the disease produced by
a, series of graded doses. Although such a
t a b u l a t i o n as shown here cannot give a
complete picture of the range of observations
encountered, it indicates how the pattern of
symptoms seen in any one group depends
upon the close of active material fed. In
these ftudies the dose was usnnlly high
enough to produce severe symptoms, and
as we became more fnmiliar with the disease
we became more confident of the result a obtainable with smaller groups. Whenever possible we now use five chicks per gioup;
25%ETjO IN
PETR ETHER
ETHYL ETHER
FRIT
FRJH
9.1%
CO-tOH
7.6%
CO
la '
54%
t
lib
16%
0.1
FRACTION NO.
t>l 11
/•'if/. f—Ahiminn rJirmiKitaprn
abltf /racliun jrtim toxic latin
occasionally we have useJ ,'ts few as im>,
when (he amount.of sampfc was limited.
Coiicenlrnlion of Twxlc Factor
Alumina Chromaiography.—'Sl^ first, afp
m our attempts to isolate- the factor thai
caused (he "edema disease" from the tinsapomfiablo portion of the fatty by-produc'
was chromatography on ahunina "(1:0). A
chromalogram . (Kig. 2 ) was obtained by
plotting the weight of m.-Uorial eluted by
increments of solvent, 1'cUoleum ether was
ufed until the elution curve seemed to have
reached a minimum, then was followed by
25% ethyl ether in petrol'mm ether. The
elution was completed v.i;h ]00% ethyl
ether to give three distinct fractions of incnyiiig polarity, j,, „,„...,,,,_ j^,^ j
could be characterized as In diwarbon (I1C)
Fraction If as containing carbonyl conpounds, and Fraction III as. c o n t a i n i n g slcros. I ho ultraviolet absurj,!,-™ e])en(ra slltw
(hat- I-rrtchou I has characlf ristio absorpfioi)
peaks at 223, 227, 234, and M 3 m/,, suggestnig (he presence of significant amounts of
cholestadiene, cither the 2,4 or 3,5 isomer or
both.
In anticipation of the possibility that the
toxic.ty «-ould rondo in Traction I (S3"(i
oM-hc u n s i i K H i i f i i i b l o i n a l L . r i t l ) a sample of
d,u-choles[adie)ie was jirep/ued by \\ },
H a l in tune, to bo i n c l u / . d in tho third
feeding trial. The result, of (hi, experiment
indicated clearly that there was no response
to choteiadicne but t h a t ,'-]| the activity
responsible for (he chick e>i,,,,a disease was
»> Fraction H, which c o n . ' i l u t e d apjiro.xiHialely &% of (he imsapor-.ifiable or about
O.Su% of (he original
of FfMlinx
/'/.—Three differ-
Saranle
% >" Uio'
7
F.A. bj'-product
F.A. by-product +
7
• vitamin K b4.8
Urea adduct
1.6
Urea filtrate
0.70
TJnsnp.
0.50
Unsap.
0.25
Urisnr).
0.125
Unsnp.
O.OC2
Unsap.
No. Chicks pliowing
Kdomn Symptoms
Sovere Moderate
M"a
Dentil1
17(19.4)
2
15(20.5)
2
15 (20.2)
3 (20.3)
—
0
0
\
—
2
—
i
5
—
—
4
1
3
2
No. NorTon. Sroic*
mol
—
on
/I)
14
3
in
1U
4.9
4.7
0.2
/
4 . -~
4.0
2.4
2.0
• Flcuren in pnrcnUicscs indicate aycrniic! durvlvnl timo in dnys.
i tnlHilot«l na tli<- loxirity scoro.
«nt approaches were made simultaneously to
Iho problem of further purifying Fraction II:
( / ) counter current distribution (iso-octane:
mcthauol solvent system with 500 transfers 1 ), (%) chemical separation of carbonyl
ronlnining compounds by the Girard Re*gent T, and (5) re-ohromatography on
Alumina with a higher ratio of adsorbent to
(ample and more gradual elution.
potent, showing as much activity at a level
of 4.2 mg per 100 grams of feed as had been
observed with 5 grams of original product.
It is of interest that the toxic fractions 5
and 6 did not react with digitonin and did
not show the L.B. reaction.
In preparing material for the chemical
separation (Fig. f>) Fraction II was cut into
two approximately equal portions; a charFor the counter-current distribution ex- acteristic yellow band was used aa the laudperiment 8.4 grams of Fraction II were mark for the separation. Each of the fracloaded into the first 10 tubes of a 500 tube tions (IIA. and IIB) was treated identically.
Craig all-glass counter-current apparatus. First, after evaporation of the ether (he maAfter 500 transfers, the contents of every 10 terial was heated with methanol, (hen cooled.
consecutive tubes were combined, and after When the hot methanol solution cooled,
evaporation of the solvents the weight of white crystals were obtained. The infrared
tolids was determined in each of the 50 spectrum for these crystals indicated a carfractions obtained, The resulting distribu- bonyl compound with a long aliph.ilic chain.
tion curve, i.e., solute vs. tube numbers, is A synthetic compound, dipalmilcme (prepared by Jonas Carol), had a very similar
fhnwn in Fig. 3.
infrared spectrum.
The 50 fractions wave, combined accordApproximately 16% of Fraction IIA was
ing to the peaks indicated by the distribution curve into 10 fractions for biological insoluble in inelhanol. To the meth.inoltesting (Fig. 3). Each fraction was added soluble portion was added twice the weight
to the test diet at a level equivalent to 7% of Girard Keagcnt T and to the mixture
of the original fatty by-product. Figure 4 was added 10% its volume of glacial acetic
chows the separation procedure diagrammati- acid. The mixture was allowed to stand for
caliy, and indicates (he amount of material two hours at room temperature before the
recovered in each fraction. Tho activity water-soluble derivatives were separated
fcemcd to be equally distributed between from the unreactcd material ("non-kctonic").
Fraction 5 (tubes 240-320 containing 2.230 Tho "non-kotonic" portion was dissolved in
grams) and Fraction 0 (tubes 321-3-1.3 con- hot acetic acid and heated with (wo times
taining 0.000 grams), as indicated by the its weight of Girard Reagent T on the strain
lom-itv scores. Fraction 0 was the mo.?f bath for 10 minutes, and the unrcacfcd material was separated from tho valer-t-oluWc
1
Wo K m l r f u l l y nrkiuwli-'lr!'- 1 ll
«">' 'I''1'' <0 1'roclw &
derivative.-'. Approximately 23% of sample
Camilla Markers for fups«'tli'B
« folvont syslfiii.
�UNSAP
10.7% 107 gm.
Chromatography (1:6)
I
88.2 gm.
100
200
300
Tube No.
.
400
500
Fig, S—Counter-current partition ol Fraction II. Methyl alcohol ;iso-octane s-ystem, BOO lulia,
SB hours.
1
Ea
4.2gm.
Eb '
3.8cjm.
MeOH
Soluble
3.53gm.
MeOH
Insoluble
0.67 gm.
Girord -T
Reagent
(1:2)
FATTY
BY-PRODUCT
lOOOgm.
COLD
KETONIC
0.8! gm.
Saponification
HOT
KETONIC
0.28 gm.
(UN)
NON KETONIC
2.44 gm.
4.4*
Fig. S—Further fractionaiion of Fraction II,
COUNTERCURRENT DISTRIBUTION
500 Tubes
MeOH
~ Iso octane
I
.01
5
2 3 . 4
.03
.26
67
8
.49
2.57
.69
.55
0.1+
3.9*
3.8+
1.13
9
10
1.97 -82gm,
O.I*
Fifl, //—Pfirtitinn o/ Fraction II wi/h methyl alaohol:iao-nctone coiinlar-eiirreni fyslcm. (Numbers foU'iwifd by + sign urn toxicity score.!,' sco footnote c, Table. 6.)
utes. After the water-soluble derivatives
had reacted in the cold and 8% more with were separated from the unrcaeted nonheat; 69% did not voxel under these con- ketonic material, two-thirds of the 11X
ditions and was classified «" "non-ketomc
fraction proved to have reacted. Tim non(UN). The various fractions derived from ketonic residue was again treated with exFraction IIA in Fig. 5, as well as ft similar cess Girard reagent and heat, and this time
ret derived from Fraction 1IB, were also fed S0% of the "non-ketonic" material reacted.
at a 7% equivalent level in the diet. 'Jhe When the original material from the Girard
pynthetid dip.'dmitone was fed at a level of derivatives was regenerated and the reaction
10 mg per 100 grams. As indicated, there was repeated with excess Girard reagent,
WHS no discose-prodxicing activity in any of again only 1 67% reacted to form ketonethe fractions obtained from IIB and only the ' Girard derivatives: It was clear that our
"non-keloiiic" Frnclion UN was very active. active "non-kctonic" Fraction !1N consisted
Siirpris'inKly, however, the infrared spectrum largely of compounds containinp; "hindered"
of -Fraction UN definitely indicated the carbonyl prroiips that would react with Giranl
presence of rarbonyl groups. This co-called reagent v/ith great reluctance. Of additional
"noii-ketoiiitt" material was again trealC'l interest is the fact that th'>.e "hinrlci'-l
with Girard Hengent T, this time with five ketone" fractions had characteristic xdli; 1 times the weight of reagent instead of two
violel spectra willi a major ali-'orplion w::\times, and was heated at SO0 C for If. min-
�214
215
UN
NON KETONI c
2.44gm.
4.4+
Iso octane
0.41 gin.
2.2+
(3mg.%
80% EiOH
0.22gm.
4,2+
(2ma,%)
AUO-3, Chromatoqraphy (l'.40)
c. o
1
2% Ether in
2% Ether in
4% Ether
Iso octane
Iso octane
0.27gm.
I.l2qm.
0.63gm.
4.0 +
Partition Chromatography
I3mg.%)
Silane-Treated Celite
80% EtOH
0.24 gm.
5+
1
90% EtOH
0.47 gm.
1
CHCI3
O.I9gm.
-1
1
t2mg.%) 1
fig. G—Chromatography of Fraction UN. (Toxicily scores at the levels jcd appear below boxot.l
iinum at .233 m /4 and a secondary maximum
at 200 m/j,.
The disease-producing activity of Fraction
11M \vas furthcv concentrated (Fig. 6) by
chromatography, first on alumina. A ratio
of one part sample to 40 parts alumina was
used, and the column was chited successively
with iso-octane, 2% ethyl ether in ifo-ootune,
and 4% ethyl ether in iso-ocfane. The fractions were monitored by ultraviolet spoctrophotometry as they ware eluled and then
were combined for feeding on the basis of
spectral characteristics. The first part of
(he material eluted with 2% ethyl ether in
i.so-octane had a definite phenanthrene type
of ultraviolet spectrum with characteristic
maxima at 259 m/i and 300 m/i, and with no
peak at 234 rry. The fraction immediately
preceding, eluted with 100% iso-octane, did
not show a phenanthrenc type of spectrum
but did have a major absorption peak at
234 nifi and a smaller one at 200 m//, The
phenanthrone spectrum disappeared upon
further ohition with 2% ethyl ether in isooctane. Only the iso-octane and the first 2%
ether fractions Khmvcd toxieity at a level of
3 mg per ]00 grams of feed. The fraction
characterised by the phcnnnthrene ultraviolet spectrum' produced the more severe
edema symptoms.
Next, this "phcnaiithrcnc" material was
further concentrated by partition chromatogrnphy on a column consisting of 5 prams
of siUine-trcaled Celito plus 4 ml of isooclano. Mobile solvents were 80% ethyl
alcohol saturated with iso-octane, followed
by 90% ethyl alcohol saturated with isooolane. The first and second &()% alcolinl
fractions were both very toxic at a level o(
2 mg per ]00 grams of feed. These two
steps (Fig. 6) represent approximately nn
S-fold concentration of the toxic material
of Fraction UN. The first 80% alcohol cut
had a typical phenaiithrcnc ultraviolet spectrum itnd no peak at 234 m/i, whereas tlio
second SQ% alcohol fraction shows a major
absorption maximum at 234 ny, m'additidn
to the characteristic phenanthrene peaks al
250 and 300 m^.
Simultaneously with the work just discussed, another.study of chroraatography on
alumina was made with a higher ratio of
adsorbent to sample (1001,)) snid move gradual clution (Fig. 7). We had learned thai
Fraction II. obtained from the unonponififiblc
fraction could be cut directly from the firrt
alumina column into two parts (using (bo
characteristic yellow band as a landmark)
iiul that only the, first cut contained edema
disease-producing material (Fig. 2). This
Fraction IIA was clutcd from the second
column with largo volumes of petroleum
"I her (llircc arbitrary cuts), followed in succession by large volumes of 0.2%, 1%, '2%,
"'id 5% and 25% ethyl ether in petroleum
cilier. The seven fractions indicated in Fig. 7
were tested at a level of 3 mg per 100 grams
of feed. Only Fractions 2 and 3 showed
Activity. The methyl alcohol-insoluble portion from Fraction 3 showed activity. This
finding did not agree with earlier experience,
but njay bo explained by the larger quantities
of mntorial involved in r,hese particular operations as compared to the earlier experiment.
Apparently the active mater'ml has a more
limited solubility in mcthanol than first supItosed. In one of our most recent experiments, the mcthanol-sohible portion of Fraction 3 from the second alumina column was
subjected to partition chromatography on
«!nno-treated Celite as previously described
drig. 8). This time only 80% elhanol "was
used as the mobile phase and the eluate was
partitioned into forerun, tailings, and two
major fractions, 82A and 82B, according to
4.4+
3.0 nig %
their ultraviolet spectra.. Fraction S2A conFract
sisted mainly of material with a plienanthvenc spectrum, and 8211 showed the major
peak at 23-1 m/i. Fraction S2A was rcehromatographcd on the siliinc column, using
75% elhanol as the mobile solvtml, and again
cut according to the ultraviolet spectra into
a forerun, a fraction with a plicnnnlhvcne
spectrum, an overlap fraction showing approximately equal absorption peaks at 2r>9
and 234 iii/i, and a fraction with a mnjor
peak at 23-1- mp,, approximately 0 times as
intense as at 259 m/i. All of the fractions
were fed, and each of the major fractions
was found capable of producing the chick
edema disease when fed at a level of 0.5 mg
per 100 grains' of diet with a severity approximately equal to that observed in chicks
fed 5% of original fatty by-product. This
is the. first instance where fractions with
distinctly different absorption characteristics
had approximately the same biological activity.
Another recent experiment involves the
reduction of one of our potent fractions with
sodium borohydride. After (ha borohydride
was separated, tbc treated material was
3.0mg%
Fig, ?- Chrumalogrtiphy and clulion aj Pracl'wn IIA (1:100 column).
�MeOH
Soluble
0.220 qm.
Partition Chromalography
on S i l a n e - t r e a t e d Ceiite
( 8 0 % EtOH}
4.4*
828
Tailings
24mg. 2.0 mg% 147mg.
82A
48mg.
1 mg,
Partition Chromotoqraphy
on Silane-treated C'eliie
(75% EtOH)
1.2+
0.25mg%
A
6mg.
0.5mg%
0.5~mg~%
p = phenonthrene Spectrum (U.V.)
A=Absorplion Maximum a! 234mjj.
-Keversc phase partition ch'ninalography <i] JtlcOII-nolnble porlinn. nj Fnclion '3,
ohromntographod on alumina into three approximately equal (30:30:40) fractions of increasing polarity. The infrared spectra indicated that the first fraction was largely
hydrocarbon, the .second fraction had the
Sample
ASaSc
VIOLET
BLUEWHITE
a
9
WHITE
PALE
BLUE
1C. 0. JTaenni h;is beer": s t u d y i n g I lie duoivscenoe spectra, both activation and cniissinn, of each f r a c t i o n , and \ \ i l l report the
result when the data h.vvc been ;inal.vxcfl.
BLUE WHITE
BLUE
WHITE
carbanyl group present, indicating that biwhydride also reacts reluctantly and incompletely with the hindered ketones, mid tl*
t h i r d fraction contained hydroxyl groujw.
There wns very little disease-producing <f
tivity left, all of it; limited to the first fi if
(ion. This may mean (has either the activity
is lo.-t upon reduction hu* that nut all of \\v
inatcrinl reacts unclor tb<3 conditions used,
or that the reduced co:u pound retains only
part, of the original activity. In cilhc.r ci f,
these data arc additional 1 evidence lli.it I he
active mnleriiil contains a earbonyl com)Kinnd.
Each' of the fructions '•Jiscu.votl here, .1111!
many others havn been studied by ultraviolet and infrared spe&trophotomctry itnd
the pertinent observations have been mentioncd.
300
300
260
220
l''iy. fl—Paper chroniator/mphy, and uliravfaliit
mi iirx nj fluorrtcfnt spats obtained Jiotn v
hii/lily luxic jfnr.linn.
In iiddiiion, us (ho po'eney of Uio disease
prodiiciiin- factors lias bf' j n concentrated, \vc
hnve. tested the purity of each fraction by
pa PIT diroinatogr.'iphy. We arc usinc; SX
Wi Wlmtman No, 31 paper, washed with
swili.-niol nnd then coaled with mineral oil
iM has been pro-washed with eulfuric acid
sfi'l nieihanul. The papers! are developed
Wli inethanol in nn uscending system for
t*riods varying from 2 to 20 hours. For the
toiler periods the solvent, is permitted to
rvspornlo from the edge of the paper through
•tip flottcd glass cover. After the papers
*'e dried, they are examined under ultraviolet light and the fluorescent spots tiro
traced. 1'igurc 9 shows a typical paper
tbromatofimm. The spots arc cut out and
fluted with inethanol, and the ultraviolet
spectra of these solutions are determined.
Figure fl represents a fraction of high purity
that has been tested by a chick feeding
npflriment; it is of considerable interest, to
nolo that (lie ultraviolet spectra of thes.c
•pols are of both types discussed earlier,
namely, the, phenanthrene type with an abwrption maximum at 259 niju, and the type
wilh a maximum at 23-1 m^. The ultraviolet
curve of the original solution was of the
)>licn:inthreiie type,
If we were to speculate about the chemical
nature of tho disease-producing material
from the evidence available at this point,
we would visualize a pair or a series of
closely related compounds, with a 2-ring
naphthalene structure, or more likely a 3:
ring nucleus, similar to phenanthrcne; a
c.'irbonyl group in a long aliphatic side chain;
and moleeulnv weights of 500-COO.
Appearance of Poison in Flesh of Chiokcns
Once it had been 'established that thn
nhickcn edema disease was caused by a toxic
principle,' tho possibility that edible chicken
flesh might contain this toxic material had
to be considered. As soon as we discovered
tiie unusual characteristics of the unsaponifi-
able malcrial from the
fat and fatty
by-product, we compared tho unsuponifiablc
fraction of tho carcasses of chickens that had
been fed these products with tho uiisapomfiable fraction from the carcasses of normal
control chickens. About 25% more untaponifiable material per unit of fresh chicken
weight was obtained from the tai, chickens
than from the control group. When the unraponifiable material was -chromatographed
on alumina, about 18 times more hydrocarbon material was obtained from the test
group. The lest group hydrocarbon fraction
gave a strong positive reaction in the Licbermarm-Burchard Test whereas comparable
material from the control group was negative. Furthermore, upon comparing the
hydrocarbon fractions by differential ultraviolet spectrophotometry, definite absorption
peaks appeared which were similar to those
observed from Fraction I of the upsuponifiable portion of the fatty by-producl.
This evidence indicated beyond doubt that
some of the material from tho diseaseproducing fat was deposited in the flesh of
chickens when they received it in ihcir diet.
This, of course, did not prove that the
chicken meat contained tho toxic material,
especially • after it was learned (Iwt the
disease-producing activity was in Fraction II
and not in Fraction I. In order (o settle
. this point, the unsaponifiable portion obtained from freshly, ground chicken carcasses, not including intestines, head, or feet,
was fed to chicks at levels of 1.0, 0.5, 0.25,
0.12C, 0.05, and 0.025% in the test ration.
As can be seen in Tfiblc 0, symptoms of
cdcma-diseaso were observed at all levels fed.
Tho unsaponifiable material from normal
control chickens fed at 0.5% of the diet
produced no abnormal symptoms of any
kind.
Tul>lc d. Results of feeding mi$upoiiifiiililo extract of chickens llml
hncl boon fed fat. or fatty iicid by-product
I'.xpt. No.
3
4
it
(i
' J in Diet
1.0
0.5
0.2",
0.125
' o.o:>o
0.02^
»cnth-
2(13.5)5(10.0)
5010.0)
4 (21 .0)
-—
No. Oiick* Ph<uvinK Kflrma Symptoms
Mild
ff,vor(i
Moilenttti
— •
1
—
-_
iii t < n r r n l l i r w « ilinw nvri'.w wirviml lime In <l:iy*
.
•
—
_
—
'
3
1
'
• —•
—
—
,
2
1
No. Normal
—
—
—
1
a
�218
THE OCCURRENCE OF THE CHICK PERICABDIAI, EDEMA FACTOR IN SOME OLEIC
ACIDS AND PRODUCTS DERIVED THEREFROM * •^^
We have examined samples of various
types of fills and fatty acid products in a
survey that is still continuing. The edema
(liscrisc-produoinu product is a characteristic not confined to the production of a
single manufacturer.
Summary
In summary, from Hie studios co far, it
may bo concluded that:
(1) The chicken edema disease is caused
by a toxic factor in a fatly by-product of
stcaric and 'oleic acid manufacturing operations.
(2) The disease-producing factor has boon
concent rated approximately J.0,000 times by
saponification, chromntogrtiphy .of the unfapom'fiablc portion on alumina, and reverse
phase partition chromatography on silanetreated Oolite,
(3) The evidence presented indicates (a)
the presence of compounds with a two-ring
nucleus similar to naphthalene or a, threering nucleus similar to phemmthrene, (b)
tho presence of a "hindered carbonyl" group,
probably in n side chain, Based on the relatively low intensity of tho carbonyl peak
at 5.S fi a compound of molecular weight
500-600 is suggested. However, the evidence
on the presence of carbonyl in the toxic
compounds is not conclusive and does not
completely rule out the possibility of a toxic
hydrocarbon.
(<1) Tho fatty ttcid fraction from this
f a t t y by-product contains significant quantities of non-urea adduct-forming compounds
that are. toxic, although they do not produce
edema disease.
(5) The edema disease-producing compounds are present in significant amounts
in the flesh of chickens on rations which
contain toxic material.
(fi) Tho cdcnia disease-producing material
is not confined to the production of a single
manufacturer.
Note
Since this paper v.ns presented, rcj-riatcd
c'hroinalo,iTaphic purifimlions of tho toxic nialenals iin a l u m i n a and on silanc-tn.\'itt!il Oolite
haw now permitted (ho isnlulion of tcvrriil
distinctive fractions. One of these frr
possessing tho ultraviolet absorption spcclumi
of substituted naphthalenes (X,,,ux. at 236, SSC,
290 n i / i j ehoulder at. 32;i ni;i; \ n ,pn. at 253 ni/ii
ASH '— M X Am), proved highly toxic. Tho infnired ppcctrum of this ninUirial showed lilllc
absorption in tho carhonyl reidon. It is concluded thai, the "naphthalene" fraction comprises a m i x t u r e of hydror.'irbons. Assuminc
t h a t alkyl-.mbsUtuted n a p h t h a l e n e is the only
chromoplioro present, the average molecular
weight, calculated from llu; absorptivity at 23G
m/i (E, \%, 1 cm gs 3200) is in the vicinity
of 250.
Acknowledgment
The extensive work outlined in this paper
was made possible by the active cooperation
of many olher individual.?, both within nnd 1
outside (ho Food and Drug Administration.
We are indebted to \\. C. A u l t and I). II.
Saunders of tho Eastern Utilization Research
and Development Division of the U.S. Department of Agriculture, who prepared 1,330
KivimR of misriponifiablo material from (lit1
f a t t y by-product, and to fbcsc members of
tho Food nnd Drug Administration: Stanley
Neshcim, Division of Food, who prepared
nn equal amount of the unsnponifiable material; Jerome Eisner, Division of Food, who
helped prepare the charts; ,1. H. Jones, John
Winnifjer, and Meyer Dolni~);y, Division of
Cosmetics, who assisted in the countercurrent distribution s t u d y ; Jonas Cnvol,
Division of Pharmaceutical Chemistry, who
helped to interpret the i n f r a r e d curves and
to synthesize a sample of dipalmitonu; P. C.
Underwood and C. G. Durbin, Bureau o(
Medicine, who provided market-size chickens
that had been fed the toxic material, for
use in tho carcass residue studies; W. L.
Hall, Division of N u t r i t i o n , who synthesized
a sample of 3,5-cholcstadieno; and the numerous inspectors of t h e Food and Drug
Administration who provided essential information without which this work would not
have progressed.
Wo are also indebted to the other individuals and laboratories, who are simultaneously iiivoslipialinj: this problem, for discussions l.lmt provided information, helpful in
guiding \\t iu our studies. Among these arc
L'.'ilslon J ' u r i n a Company, the Procter &
Gamble Company, nnd Hjilw and Hunter.
Stanley B. Ames, William J. Swanson, Marion I. Ludwl^and
George Y. Brokaw, Research Laboratories, Distillation Products Industries,
Division of Eastman Kodak Company, Rochester, New York
A material causing the chick edema syndrome has been reported (1, 2, 3, 4)
to occur in specific lots of feed-grade animal fats. These fats were reported to
I'untain the edema-producing factor as a trace impurity produced during certain fat-processing operations (2, 3). Birds fed diets containing this unidentified factor develop an edematous condition, characterized by pericardial edema
(dlstention of the pericardial sac with fluid, also termed "hydropericardium")
«IK\ in more severe cases ascites and gross liver and kidney damage. In the
-"-'- 1 —-js symptoms are characterized by abdominal ilisg. Manifestations of the pericardial edema factor
""The ^ ^ S ^ ^ t factor has not yet been isolated but
.•crtain cha^Stlcs of the pericardial edema factor have been.reported by
Brew et al (2) as follows: a) presumably a hydrocarbon derivative possibly
of cholesterol, b) a molecular weight of about 360 c) assoc.a ed fractions
whieh give the Liebermann-Burchard test for sterol residues, d) "n be
concentrated by molecular distillation of fats containing the, toeto . r^ edoma
Hyndrome is not produced by fat per so but appears to be related to impurities
In certain lots of fats subjected to special fat-processing oiierafaons.
F W Hill of Cornell University reported to us that high-level feeding to
chicks of a sample of a glycerol ester of oleic anid resulted in some mortality
wmi gross svSns resembling pericardial edema Our studies have confirmed
his observations and furthermore have shown that the pericardial ederaaiirodupine fiotor is present in many samples of commercially-available oleic
Jidd as well a^ in glycerol esters made therefrom. Molecular_ distillation ,£ an
active oleic acid or of a glycerol mono-ester of an active oleic acid was found
to concentrate the pericardial edema-producing factor.
TECHNIQUE
In this investigation a rapid, sensitive bioassay procedure, developed in our
laboratories, was employed (5). The diet used is a modification of that: leseribed by Brew ef a,l. (2) and consists principally of purified casein, gelatin,
Klucose, and 36% test fat. Day-old chicks are fed this diet al H6. immediately
on arrival. Subgroups are sacrificed at 10, 14, and 21 days. In general, a higher
Incidence of pericardial edema is observed in the subgroups sacrificed at 10
and 14 days than in the group sacrificed at 21 days. At autopsy, chicks are
examined for the appearance of pericardial edema, ascitos, and the appearance
o£ complications, such as liver and kidney damage or labored breathing. Bioassay groups consist of 10 or 15 chicks for each sample of fat. Using a weightIns procedure described in Table I, a pericardial edema-activity score is calculated. This makes possible a semi-quantitative comparison between the relative
activities of various fat samples.
OCCURRENCE
Oleic acid samples (TJ.S.P.) from four manufacturers were assayed biologically and chemically (acid value and unsaponifiable content). As indicated
in Table I, several of the oleic acid samples were strongly active, but others
showed little or no activity. The pericardial edema activity scores showed that
no correspondence existed between the presence of the pericardial edemaproducing factor and the acid value or the content of unsaponifiables.
Tests for the pericardial edema-producing factor in certain commercial •
samples of glycerol esters (monoglycerides and monodiglycerides) of oleic acid
are summarized in Table II. Seven different manufacturers of glycerol esters
are represented. The majority of these ester samples were active. Again, there
is no correlation between unsaponifiable content and pericardial edema activity.
In four instances, samples of both the oleic acid and the glycerol estor pro'Communication No. 205 from the Research Laboratories of Distillation Product*
Industries, Division of Eastman. Kodak Company, Rochester, N. Y.
45-862 0—TO
15
�PERICAROIAL EDEMA ACTIVITY
Bioassay Results
Sampl e
No.
Cut.
Pereent
Anal
*
Pericaridal edema
sis
Unsap.
percent
Acid
value
Plus
death
Plus complications1
Plus
ascites
Uncomplicated
Other
deaths*"
Negative
Activity
ratio=
pos./tota!
Activity
score,^
percent
TABLE 1.— OLEIC AC10S AND THEIR PE (PERICAROiAL EDEMA) ACTIVITY
1
2
3
4
5..
0.45
0.68
0 77
0 52
0.18
0.28 .
0.51
0.50
0 77
0.52
0.20 .
201 0
202 5
201 2
202 S
203 0
202. 5
201 3
6.
7
8.
9
10
ll
204.9
204 4
203 9
202. 0
2
1
1 ..
1
2 .
1 ....
1
4
3
21
7
8
8
6
4
10
8
3
1
3 .
4
10
10
0/21
3/21
1/10
2/10
3/10
7/11
0/10
1/9
10/10
0/10
0/10
0
110
33
60
133
200
0
22
300
0
0
6/6
6/10
320
130
230
0
14
55
0
160
200
33
33
50
TABLE II.— OLEIC ACID ESTERS AND THEIR PE (PERICAROIAL EDEMA) ACTIVITY
I..
2...
3
2.2
16
17
2.2
5
6.
7 ..
8
9 .
11
12...
_
..
0 12
0.13
0 26
0 5
0 3
2.8
4 7
7.1
1.2
55
.
3
0 32
0 63
0.5
0.49
0 35
0.46
1.2
2
5 .
13
1
4
3
4
1
2
1
2
4
3
1
3
2
1
0
4
6
11
13
9
9
2
4
8
6
9
3
3
24/30
0/11
1/14
2/11
0/9
5/10
6/10
1/9
0/9
2/14
•
.
Seeff^f^^^''^^'^^
TABLE IV.-MOLECULAR DISTILLATION OF AN OLEIC ACID (SAMPLE 6,TABLE I)
Charge:
F 1
F 2
F-3
F-4.
F-5
F-6
4
11.9
10.3
4
10
l-l
--.--..
-
1/9
45
8/10
290
4
1
6/10
9/10
130
350
6
5 i:::::::::::
::::::::::::
200
0
2
3
7/11
0/10
8
_.
-—
1
—-
29.11
29.6/
...
2/9
67
1.2
TABLE V.-MOLECULAR DISTILLATION OF A GLYCEROL MONO-ESTER OF OLEIC ACID (SAMPLE 2, TABLE II)
Charge
F-2
F-3
F-4
F 5.
io ::::....
—
9.4-
30.61
31. 5f
-
3
—-
-
-—
-
1
4
;1
—
11.6
1.0
—
2
—-
5
— -,-1
;
*
----- -
—
—-
.
4/io
"
120
—
• Complications include gross liver and kidney damage or labored breathing.
Early deaths in excess of control groups, PE not observed.
k
I Tta •3re!WWS* ™SSSSl°iS« 5fa,'"^rily of the activity. The products of the number of chicks in each category times its weighting factor are summated and divided by the tota.
number of chicks as follows:
5x(PE+death)-p4X(PE-r-compl.)+3X(PE+ascites)+2X(PE)+lX(other deaths)
PE activity score=100 X
.. , . . .
Total chicks in test group
~~
fc/
^
C
�StTMMAKY
duced therefrom were bioassayed as summarized in Table III. The
L esters showed activity only when the corresponding oleic acid was acti^
f inactive oleic acids or those with low activity the corresponding glycer slfr
did not have increased activity. Thus the edema factor was not formed during
the manufacture of the glycerol esters.
A number of samples of commercially-prepared (TJ.S.P.)j^pc acids and
Klycorol esters of oleic acids were shown to contain a materiar^liich produces
t-crlcardial edema in chicles. The active material was concentrated from either
ihp oleic acid or the glycerol mono-ester by molecular distillation.
I'"our of the 11 samples of oleic acid were inactive; this indicates that the
jn-rleardiaL edema is not caused by the fatty acid itself but by an incidental
material in the fatty acid.
CONCENTRATION STUDIES
Molecular Distillation of an Oleio Acid, A sample of oleic acid known 10
contain a moderate amount of the pericardial edema-producing factor wn»
fractionated in a 14-in. molecular still into six fractions as indicated in Tahlf
IV. The first and last fractions and a composite of the middle fractions wt'f
bioassayed as described above. The first 12% strip cut was entirely free of HIP
pericardial edema-producing factor. The middle cuts, representing approximately 60% of the input fat, were only slightly active. The last 7% fraction
showed a concentration of the edema factor with an increased activity over
the input oleic acid. Thus the pericardial edema-producing factor was concentrated in the last fraction after the bulk of the oleic acid was removed l)f
molecular distillation.
Distillation of a Q-lyaerol Mono-ester of an Olclc Add. A sample of a gl.vcerol mono-ester of oleic acid, prepared from an oleic, acid known to contain
the pericardial edema-producing factor was fractionated into six fractions in
a 14-in, molecular still, as described in Table V, The first 10% cut, the Incl
3% fraction, and an intermediate fraction representing approximately 60%
of the input material were bioassayed as described above. An increased concentration of the pericardial edema-producing factor was observed in the first
10% cut. Some pericardial edema-producing factor was present in the other
fractions examined.
DISCUSSION
ACKNOWLEDGMENT
Appreciation is expressed to Louis .T. Lee and Richard E. Ardell for conducting the molecular distillations, to Willam 0. Lyman and Gary Brooks for
•wrelation of samples, to Herbert W. Rawlings of the Products Control
I-nboratory for chemical analyses and to David O. Herting, Philip L. Harris,
Slid Norris D. Bmbree for advice.
REFERENCES
"Potter, G. C., Brow, W. B., Patterson, R. L., and Sipos, B., J. Am, Oil Chemists'
Hoc., SC>, 214-217 (1050).
'Drew, W B.. Dore, ,T. B., Benedict, J. H., Potter, G, C., and .Slpos, E., J. Assoc.
Off, Aur. Cliem., 42, 120-128 (1050).
„,
_ T
' Kricdinan, L., Firestone, D., Horwitn, W., Banes, D., Anstead, M., and Shue, G., J.
"w'ooten, j!'C.,leand Alexander, J. a, J. Assoc. Off. Apr. Chem., 1,2, 141-148 (1059).
'Amos, S R,, Swansea, W. J., and Harris. P. L., ''Studies on a Factor Causing
I'crlcardlal Krtema in Chicks and Its Occurrence in Some Oleic Acids," Federation Proc.,
It. 323 (1000).
STUDIES OF THE CHICK EDEMA FACTOK. II. ISOLATION OF A Toxic STJBSTANCE?
The material which produces pericardial edema in chicks has been reported
previously only in certain lots of fats subjected to special fat-processing operations (1, 2, 3). Therefore the finding that some U.S.P. oleic acids possessed a
high degree of activity was unexpected. Feed -grade fats that contained tlio
edema factor characteristically had high unsaponifiable levels ranging above
0% (2). However, in the present study, samples of the more active oloic acids
had unsaponifiable levels ranging from 0.2 to 0.7%. This indicates a 10- to
30-fold concentration of the edema factor in the. unsaponifiaMe fraction and
stiggests that the unsaponifiable fraction of an active oleic acid might be a
starting material for isolation of the factor.
Molecular distillation of a fat which contains the factor concentrated this
factor in the more volatile fractions (2). Molecular distillation of an active
glycerol mono-ester of oleic acid also concentrated the edema factor in the
more volatile fractions, but separation from the monoglyceride fraction was
not as complete as it was from the higher-distilling triglycerides. Molecular
TABLE III.-COMPARISON OF PE (PERICARDIAL EDEMA) ACTIVITY OF OLEIC ACIDS AND THEIR CORRESPONDING
GLYCEROL ESTERS
!
Oleic Acid
(from Table 1)
5
1
3
(75%)
PE activity
ratio
pos.ltotal
3/10
0/10
0/21
1/10
PE activity
score
Sample No.
(from Table II)
percent
133)
0
133
0
33
PE activity
ratio
pos./loM
2
25/31
!
percent
',
6/10
3
4
5
PE activity
score
0/11
1/14
230
,!
distillation of an active oleic acid concentrated the edema factor in the les«
volatile fraction. Since fatty acids, such as oleic acid, distill at temperatures
lower than the corresponding monoglycerides, the distillation range of tiic
ortcma factor appears to lie above that of oleic acid and below that of the
glycerol mono-ester of oleic acid.
Andrew Yartzoff David Firestone, Daniel Banes, William Horwitz, Leo
Friedman, and Stanley Nesheim, Bureau of Biological and Physical Sciences,
Food and Drug Administration, Department of Health, Education and Welfare
Washington, District of Columbia
A crystalline halogen containing material producing chick edema symptoms
at 0.1 part per million in the diet has been isolated from a sample of triolein
which was toxic to monkeys. This material is similar to that reported by
Harman et al. (4) but differs somewhat in iiltraviolet spectral properties.
In a symposium on the chick edema disease in October, 1958, several laboratories (1,2,3) presented reports on their progress toward the isolation and
elucidation of the toxic factor responsible for the occurrence of this unusual
syndrome. It was established that the disease is caused by a toxic factor in
the unsaponifiable fraction of a fatty by-product of industrial stearic and
oleic acid manufacturing operations, and it was further suggested that the
factor might posses a polynuclear or steroidal structure. A note, later published
as an addendum to the contribution from this laboratory (2), reported that
the toxic factor was associated with eVuates from alumina and "silane-treated
Celite" chromatographic columns which exhibited the ultraviolet absorption
spectra of polysubstituted naphthalenes (Xiuax at 236 mp, secondary XM<UC at
286 and 296 m/i). Neighboring cuts from these chromatograins showed the
characteristic spectra of phenanthrene derivatives (Xmax at 259, 282, 292, 300
m/ii.) and of simpler naphthalene derivatives (XMax 228-233 mji, secondary
XM«* 270-280 m/i).
Subsequent purification of substances that had an absorption peak at 230
in/* demonstrated that they were not the most toxic fractions in our materials.
Furthermore our computations showed that the toxic factor must be potent
when present in the diet at levels of a fraction of one part per million, Recently Harman et al. (4) have reported the isolation of the chick edema
factor in crystalline form from a feed-grade tallow. Their substance was
toxic to chickens at 0.1 p.p.m. in the diet and had an ultraviolet absorption
spectrum with a major peak at 244 m/i, a lesser peak at 312 m^, and a shoulder
at 238 m/i. A private communication from Tishler of the same laboratory (5)
disclosed that the crystalline substance contains chlorine to the extent of about
47%.
Ames et al. (6) have observed the presence of the toxic factor in Some
commercial oleic acids. We have studied a sample of triolein which had been an
�225
ingredient in a series of dietary treatments involving changes in the^feL
and types of fats to which a group of Cebus monkeys had been subjecB^J
' e following summary of experimental results relative to these inonK?r*
received from 0. W. Portman and S. B. Andrus of tbe Department t4
Nutrition, Harvard School of Public Health.
Of a group of nine monkeys that received this triolein in their diets .fit »
level of 25% by weight, one died at one month and four at three months. After
three months on the triolein diet corn oil was substituted for the triolein. Tli*
other four monkeys died from three weeks to five months later even tlioupli
triolein had been discontinued and replaced by corn oil. Of 14 monkeys in thf
colony that did not receive troilein but were supplied other fats and oils «l
25% of the diet by weight, there was only one spontaneous death. Eight cf
the nine monkeys fed triolein were autopsied and showed the following findings: jaundice (4.S?) ; pancreatic atrophy and fibrosis ( 0 ) ; hemosiderosi<
(0) ; fatty liver (5) ; bile duct proliferation (3) ; oxtramedullary erythropoiosi*
( 3 ) ; necrosis of liver ( 2 ) ; gross hemorrhage in gastrointestinal tract ( 2 ) ;
and erythrocytophagoc.vtosis (1). Several features including marked anemia
in several instances suggested the possibility of a hemolytic process. Pancreatic changes were most pronounced in the two monkeys that survived longest (seven to nine months from the beginning of triolein feeding). The severity
of the lesions in the pancreas was unrelated to that of the hepatic changes.
With the exception of fatty changes in the liver, the above findings have not
been reproduced in rats. These observations are from an experiment not
designed to study a toxic principle, and it would be unwise to draw firm
conclusions with respect to the toxicity of the triolein from these limited datn.
The fact that, in our laboratory, marked symptoms of chick edema disease
were produced by this sample of triolein suggests the possibility that.tlic,
chick edema factor may have been responsible for the toxic effects noted in tlio
triolein-fed monkeys.
We now wish to report tbe ioslation of a highly toxic crystalline substance
from this triolein and to describe its properties.
EXPERIMENTAL
The triolein sample was of excellent quality with an unsaponifiable content
of 0,87% and a steroidal hydrocarbon (1) absorbance of 0.07. Only 0.01% of
oxirane oxygen (epoxide) was detected. Examination of the fatty acids ns
the ethyl esters by gas chromatography showed that oleic acid constituted
70.9% of the total acids with 3.6% linoleic, 0.3% linolenic, 13% palmitolcic,
i).4% palmitic and shorter-chain fatty acids, and 1.8% of a On fatty acid
containing one double bond (by inference from relative retention time). Urea
filtrate fatty acids were present to the extent of 4.73% (6a).
When fed to chicks at a level of 15% in the diet, the trioloin produced
the symptoms of chick edema disease with a severity approximately equivalent
to that observed with a diet containing the toxic fatty product used in our
original studies (2) at the 5% level. The unsaponifiable fraction of the triolein was to us.
available proportionately richer in the toxic factor than any other materials
The toxic factor in 17.6 kg. of triolein was concentrated by molecular distillation under pressures of 10-20 microns at temperatures up to 200°0. Tbe
distillate (289 g.), which contained all of the toxic factor, was saponified, and
166 g. of unsaponifiable material were recovered. The portion soluble in petroleum ether (155 g.), was chromatographed on 3 kg. of Fisher Alumina A
5'JO in a 4-ft. x 3%-in. column, using petroleum ether as eluent. Two-liter
fractions were collected, and substances with the absorption spectra of naphthalene and pbennnthrene derivatives were eluted in fractions 6-15. The
bulk of tbe material containing cholestadiene and related hydrocarbons (.12!)
g.) was eluted in the first three days, and the remainder was recovered by the
uso of more polar test.
in tbe chick edema solvents. These foreruns and tailings were devoid of potency
Fractions 6 to 15 were combined (668 ing.) and chromatographod on 2,500
g. of the more retentive Merck Alumina No. 71707. No material was eluted with
petroleum ether. Foreruns were eluted with 1-2% ethyl. ether in petroleum
ether, and fractions exhibiting the spectra of naphthalene and phenanthrene
derivatives (239 mg.) were obtained when 5% ethyl ether in petroleum other
was employed as eluent. This material was again chromatographed on Merck
»iiiinlim, using 250 g. in a 42x4.5-cm. tube. Several fractions^BWch eluted
*IMi fi% ethyl ether in petroleum ether exhibited an absorption i^Bmum near
^•'8 nip as well as the peaks previously observed at 234-236 m/i^nd 255-260
<»K. There fractions were combined, and the eluted material was partitioned on
*' K. of silane-treated Celite, by reverse-phase chromatography, using 4 ml. of
i«>-*wtnne in the immobile phase, and 80% alcohol saturated with iso-octane
** the mobile solvent.
Vructions exhibiting maximum absorption at 245 m/i, but not at 235 mp or
2-Yi/i, were combined for two further chromatographic purifications on alumina.
Merck alumnia was deactivated with 3% its weight of water, and tbe ratios
•>f iilumina to sample and column size 2,000:1 in a 25 x 1-cm tube and 25,000:1
in a 25 x 2-cm. tube, respecively. Iso-octane, redistilled and chromatographed
<»"or silica gel, was employed as eluent, and fractions were again monitored
I')' ultraviolet spectral absorbance fractions devoid of inflection
and 255
n>lt were combined and evaporated to dryness. Tbe solid residue was dissolved
In a small volume of boiling iso-octane and stored over-night in the refrigerator. White crystals weighing 2.64 mg. were obtained.
W)
Milwtance sublimed on tbe hot stage about 239° 0. The infrared absorption
Wctrmn of tbe substance, incorporated into a potassium bromide disc, showed
evidence of aliphatic and aromatic linkages but no bands characteristic of
oxygen or nitrogen functions.
The reported presence of chlorine in the toxic substance isolated by the
Merck group (5) prompted a Beilstein test, which showed the presence of
Inilogen., The presence of halogen in the crystalline material being reported
here was confirmed by examination in a microcoulometric gas chromatoKmph. 1 (In this instrument the sample is fractionated by gas-liquid chromatography, and, as they' are eluted from the column, the compounds are pyrol.vzed at 800° O. under oxidizing conditions. The halogen acid formed from
halogenated materials is titrated in a microcoulometer.)
In the chick biossay the substance, when fed at ca. 1 p.p.m. in the diet,
produced severe hydropericardium, hydroperitoneum, and liver damage, with
(loath occurring within 12 days. At 0.1 p.p.m. in the diet marked hydroperieardium was evident at autopsy after a three-week feeding period.
The isolation of the toxic substance had been complicated by the presence
of another material with an absorption maximum at 248 m/t. This second substance was isolated by the same techniques of chromatography, monitored
by ultraviolet spectrophotometry, and a yield of white crystals was obtained.
The ultraviolet spectrum of this material was identical with that of the toxic
substance except that it was shifted 3 HIM so that the major absorption peak
was at 218 m/». It behaved similarly to the toxic substance in the microcoulometric gas chromatograph, showing a similar retention time and a similar
halogen content. However, it was completely inactive in the chick edema test
when fed at 1 p.p.m. in the diet.
In order to obtain additional crystalline material the portion remaining
from nondestructive testing, fortified with the adjacent fractions from the
final chromatography (of high potency, judging from the ultraviolet spectrum),
was further chromatographed according to the previous isolation scheme.
Small quantities of phenanthrene derivatives were separated with a consequent diminution in the ultraviolet absorption in the 250-300 m/i region.
Furthermore, although there was no indication of nonhomogeneity by paper
chromatography, tbe ratio of absorbances of the shoulder at 240 m/j to the
peak at 245 varied from fraction to fraction, suggestion the presence of an
additional component.
The extinction values reported above may be in error because of inaccuracies
In weighing on account of the difficulties in handling the small amount of
material involved. There is no doubt however of the validity of tbe relative
absorbances at various wave-lengths. The spectrum of the substance reported
here differs somewhat from that reported by Harman ct al. (4), particularly
in the ratio of the absorbance at the maximum (244 or 245 m/i).to the absorbance at the inflection at 238 or 240 m/n.
1
Dohrmann Manufacturing Company, Palo Alto, Calif,
�«
DISCUSSION
^blf^
^^^^•R.v
^^H1R
ho toxic substance which we have isolated from triolein reseznbles tn^m
iverod by Harman ot al. (4) from animal feed tallows. However the
divergences in their properties suggest either that we are dealing with two
different but closely related compounds, or that one or both of the preparations is still a mixture of related compounds despite the fact that only «
single spot could be obtained on paper chromatography in a number of solvent
systems. In view of the manifest difficulties involved in isolating a pure compound in minute quantities from a myriad of substances with similar properties it would be hazardous, as Harman warns, to infer chemical structure*
from spectral data. Nevertheless it should be pointed out that the spectra
obtained by us and by Harman et al. are strongly reminiscent of those exhibited by highly substituted naphthalenes (7). Furthermore the toxic factor occurs in association with a bewildering array of aromatic naphthalene anil
phenanthrene derivatives, as we have previously noted (2). The detection of
chlorine in large proportions in a toxic preparation, and the ultraA'iolet
spectrum observed, suggest a possible relationship with chlorinated naphthalenes. Pontachloronaphthalene possesses an an absorption maximum at 243 m/i
and a secondary maximum at 312 m/j (8), and it has been shown to cause
hyperkeratosis in cattle (9) and several other species of animals including
chickens (.10). Other chlorinated naphthalenes also are toxic (11).
The possibility that the chick edema factor is a chlorinated napthalene
derivative cannot be ignored. Samples of tetrachloronaphthalene and hexnchloronaputhalene, kindly provided by Bngel and BelU of the Virginia Polytechnic Institute, who had demonstrated that these compounds could produce
hypcrkeratosis in cattle, were without effect in the chick edema test. Furthermore these compounds, despite the similarity of their ultraviolet spectra
and their chromatographic behavior to the toxic substance, showed considerable difference in the microcoulometric gas chromatograph. For instance,
chlorinated pesticides, aldrin and heptachlor, showed retention times of 10 min.,
tetrachloronaphthalene 9 min., and hexachloronaphthalene 14 min., whereas
the toxic substance, as well as its inactive analogue with the absorption maximum at 248 ia.it, had retention times of 37-38 min. It is tempting to speculate
that the greater retention-time of the toxic material is related to a greater
molecular weight or to a substituent conferring different solubility and polarity
properties.
We are continuing our studies toward the isolation of the toxic factor. It
is necessary that the chemical nature of this substance be elucidated to make
possible a rapid chemical test for its detection, to clarify its origin, to verify
the suggestion of its severe toxicity to primates, and to study its action in
other species.
ACKNOWLEDGMENT
Numerous individuals in addition to those mentioned in the text have contributed directly or indirectly to this project. We are particularly grateful to
O. TJ. Kline for his sustained interest, encouragement, and helpfulness in the
course of these investigations; to Benjamin Webb, who conducted the extensive molecular distillations; to Donald F. Flick and Linda Gallo for the
chick bioassays; and to Raymond .T. Gajan for the microcoulometric gas
chromatography. The cooperation of the various commercial laboratories that
were also working on this problem, in discussing their work with us, is also
greatly appreciated.
REFBIIENCES
1
Apr.Brow, W.B.,J,%, 120-128 (1959), J.H., Porter, G.C,, and Slpas, B., .T. Assoc. Offlc,
Chemists, Dore, J.B., Benedict,
- Friedman, L., Firestone, D., Horwltz, W., Banes, D,, Anstead, M,, and Shue, G., Mel.,
120-140.
3
Wooten, J.C., and Alexander, ,T.C., ibid., 141-148.
•' ITarman, R.E., Davis, G.B., Ott, W.H., Brink, N.Q., and Kuehl, F.A., ,T. Am.' Chem.
Soo., S3, 2078-2079 (1960).
'
5
0 Tlshler, M., Merck and Company, private communication, July 19, 1900.
Ames, S.K.,
4(10) (1900). Swanson, W.J., Ludwig, M.I., and Brokaw, G.Y., J. Am. Oil Chem. SI,
' Abadlr, Hoc.., 7S, 3348-3349 (1053).
Am. Clicm. B..T., Cook, .T.W., and Gibson, D.T., J. Cliem. Soc., 19B3, 8 ; Mosby, W.L., J.
"DllckenntafP, U.T., and Callen, J.K., Anal. Chem., 2S, 1080-1589 (1954).
"Slkcs, D., and Bridges, M.10., Science, US. GOO-507 (1052).
"Kohler, H., Arclilv. Experimentelle Votcrlnarmedizln, 8, 163-198 (1954).
"Bell, W.B., Vet. Med., J,S, 135-140 (1953).
Reproduced by the U. S. DEPARTMENT*OF HEALTH, EDUCATION, AND
Food and Drug AdminUttatlon
'ARE
Collaborative Bioassay for Chick Edema Factor*
By CARL D. DOUGLASS and DONALD P. FLICK (Division of. Nutrition, Food and
Drug Administration, Washington 25, D.C.)
A substance contained in certain processed
fats and fatty products used in poultry feeds
has' been implicated in the outbreak of the
condition called "chick edema disease" which
* Presented ns the report of the Associate
Referee on Bionssay of Chick Edema Factor,
Carl D. Douglass, at the Seventy-fourth Annual Meeting of the Association of Official
Agricultural Chemists, Oct. 10-12, I960, at
Washington, D.C.
occurred in 1957 (1). A number of laboratories are actively working to isolate and
identify the agent responsible for the disease (2-5) and each has developed its own
bioassay, which in each case has admirably
served its specific purpose.
Ames and co-workers (6), early in 1960,
reported this factor in oleic acid -samples
destined for human consumption. The pres-
�229
228
ence of this highly poisonous substance in
human food products immediately resulted
in a regulation from the Food and Drag Administration specifying that all such produc.ts must be, free of "chick edema factor"
in order to be incorporated in food. From
the point of view of the regulated industry
and the regulatory agency, tho need to develop an assay method of adequate specificity
and sensitivity, the results of which are
comparable from laboratory to laboratory,
can hardly be overemphasized. Since the
chemical characterization of the substance
has not proceeded far enough to provide the
basis for a chemical or physical assay, it is
necessary to use the bioassay. This method
lias successfully proved itself capable of
providing for the detection and semiquantitative estimation of small amounts of this
factor.
When the need for the standardization of
a method became acute, investigators in eight
laboratories that are currently engaged in
some phase of work on the chick edema factor were invited to participate with us in a
collaborative study and to submit suggested
procedures. All graciously responded, and
from the procedures which they forwarded,
tho authors selected what they considered
the most desirable features of each and com-
Coll.
2
2
3
3
4
4
2
^
1
2
2
3
0
O
4
4
3
15
14
9
8.S*
7.3
4.86
0.42
0.4
0.2
0.02
1.2 mg
1
1
2
2
3
3
4
4
^
I
AMOUNT, a
Folio acid (1.0% triturated in
powd. glucose)
10
Biotin (0.1% triturated in
powd. glucose)
7.5
Vitamin B,, (0.1% triturated
in powd. glucose)
2.5
Niacin
2.0
Ca pantothennte
0.50
Thiomine
0.50
Riboflavin
0.375
. Pyridoxine-HCl
0.200
Menndione
0.025
Celluflour (Alphacel), to make 500
2
3
3
4
(c) Fat-soluble vitamin mixture.AMOUNT/HO MIXT,
AMOUNT/K0 DIET
900,000
100,000
2.00
1.00
9,000
1,000
20
10
USP Units
1C Units
g
kg
USP Units
1C Units
mg
g
12
12
12
12
12
12
12
12
14
21
14
21
14
21
14
21
6
5
7
5
6
5
7
5
1
0
1
14
21
14
21
14
21
14
21
12
12
12
12
12
12
12
12
0
0
14
21
14
21
14
21
12
12
12
12
12
12
12
12
14
21
4
7
14
21
1
1
2
2
0
o
14
21
14
21
14
12
12
3
4
4
g
o
14
21
14
21
14
21
1
2
0/60 a MIXT.
Vitamin A acetate, cryst.
Vitamin Dj, cryst.
o-a-Tocopheryl acetate
Corn oil, to make
No.
oE
No
Early
of'
Pe^lS
Diet <Day«) Chlolu Deathg
i
A
1
i
(b) Vitamin mixture.—
CaCO.,
Caa(PO.).
K.HPO,
NaCl
Nii.HPO,
MgS0..7H,0
MnSO,,4H,0
Fe nitrate
ZnCO,
CuSO,.5H,0
Edema Incidence
Average Hydroperioardium
METHOD
Reagent*
(a) Salt mixture.
factor
Table 1. Summary data of tho collaborative aasay for chick
bined them into the procedure presented
here.
Three collaborative test samples were prepared by thoroughly mixing a fat, known
to be toxic, with cottonseed oil in ««ch proportions that the amount of toxic fat in the
samples was in the ratio of 1:2:4.
Each of the collaborators was sent the
procedure given below as well as four collaborative test samples identified by number.
Sample 1 was USP cottonseed oil; Samples
2, 3, and 4 contained, respectively, 1, 2, and
4 grams of toxic fat in 16 grams of sample.
Report sheets suitable for recording the following specific information were also sent:
chick number, initial body weight, final body
weight, weight gain, heart fluid volume,
presence or absence of peritoneal and subcutaneous fluid, and dates of early deaths.
Noteworthy features of the assay procedure are the use of white leghorn cockerels
as the assay animal; a semi-synthetic ration
in which the test samples are contained at
16%; and a 21 day feeding period. We included a request that groups be assigned to
provide for observations to be made at the
14th day as well. The collaborators were
asked to score the results visually, by use
of a suggested scale of values, as well as by
measurement of the pericardia! fluid volume.
21
14
21
12
12
12
12
12
12
1
1
2
2
14
21
14
21
12
12
. 12
12
2
1
1
0
2
3
5
o
o
1
3
4
Average
Weight Gain
Vitual
Score
Total («)
39.5 db 3.2
73.0 ± 4.3
Fluid Volume
(ml)
Score
From
Volume
0.054 ± ,004
0
1 + 0.12 d= .025
0
1+
± .009 0
db .039 9 +
3+
' ± .09
db .29 21 +
db.25 15 +
± .72 22+
Sub-
Aicitea
CUtRneoue
Hydropericardium*
0/12 0/12
0/12 1/12
0/12 0/12
0/12 7/12
1/12
1/12
2/12 10/12
2/12 6/12
6/12 3/12 10/12
0/12
1/12
0/12
0/12
1/12
3/12
1/12
52.8
78.2
47,0
09.4
20.2
39.4
±
d=
±
±
±
±
4.3
4.9
3.2
4.6
3,3
5.1
0
12+
7+
20 +
20 +
27 +
0.11
0.26
0.23
0.92
0.71
1.58
26.2
59.6
32.7
70.0
39.8
63.2
39.3
57.0
db 6.7
± 12.6
d= 9.2
± 4.3
± 2,9
db 4.7
db 7.6
± 9.6
1+
0
9+
10 +
7+
15 +
20 +
15 +
0.028 ± .013 0
0.072 ± .013 0
0.095 ± .021 0
7+
0.59 ± .24
0.155 dz .079 2 +
1.43 ± .49 14 +
2.05 ± .78 16 +
1.80 ± .66 15 +
0/5
0/5
0/8
0/5
1/6
2/5
5/7
4/5
0/5
0/5
0/6
0/5
1/6
1/5
6/7
3/5
0/5
0/5
0/0
4/5
1/0
5/5
5/7
5/5
0/12
'0/12
0/12
0/12
6/12
8/12
4/12
9/12
0/12
0/12
1/12
3/12
4/12
5/12
6/12
9/12
46.1 ±
104.1 ±.
51.4 ±
118,2 ±
48.7 ±
89.5 ±
45.4 db
86.1 db
2.0
3.3
2.5
6.3
2.6
4.9
3.3
4.4
0
0
4+
3+
12 +
7+
14 +
23 +
0.023 db .001 0
0.085 ± ,016 0
0.101 ± .018 1 +
0.145 ± .036 4 +
9+
0.39 ± .13
7+.
0.28 ± .12
0.54 ± .21 14+
1.46 ± .45 25 +
0/12
0/12
0/12
0/12
2/12
1/12
3/12
8/12
0
2
0
4
0
8
42.3 rb
91.3 d=
43.5 ±
41.8 db
28.1 ±
35.0 db
22.3 ±
32.3 d=
1.6
8.9
2.9
5.1
2.1
1.4
1.8
6.1
0
0
0
10 +
1+
16 +
11 +
19 +
0.059 d= .008 0
0.081 ± .034 0
0.127 ± .006 3 +
0.306 ± .052 10+
0.102 ± .021 2 +
0.66 ± .29 17 +
9+
0.37 ± .17
0.80 ± .25 20 +
0/12
0/12
0/12
1/12
0/12
10/12
1/12
8/12
0/12
0/12
0/12 0/12
0/12 3/12
1/12 8/12
2/12 2/12
8/12 11/12
5/12 6/12
9/12 9/12
0
0
0
1
2
2
1
9
22.6 ± 5.9
33.4 ± 10.0
11.2 ± 6.3
16.2 ± 7.2
25.4 ± 5.2
35.9 ± 10,3
12.4 ± 7.2
22.0 ± 15.6
0
0
0
8+
4+
10 +
10 +
6+
0.05 ± 0
0.054 ± .004
0.058 ± .005
0.196 db .093
0.166 ± .078
0.427 ± .203
0.391 dr .14
0.204 ± .078
0
0
0
3+
2+
9+
9+
6+
'0/12
0/12
0/12
1/12
1/12
1/12
3/12
3/12
0/12
0/12
0/12
4/12
2/12
2/12
5/12
6/12
0/12
0/12
0/12
1/12
1/12
4/12
5/12
5/12
0
0
0
1
42.9
100.8
54.5
78.1
0
3+
9+
15 +
0.061 ±
0.093 ±
0.125 ±
0.183 ±
0
0
2+
4+
0/12 0/12
0/12 0/12
0/12 0/12
0/12 2/12
0/12
0/12
2/12
4/12
o
0
0
1
o
1
0
o
±
db
±
±
3.8
7.3
3.2
4.7
.015
.013
.019
.023
(Confirmed)
�23i
230
' Attay Ration,—
Sucrose, commercial
Corn starch, commercial
Casein (vitamin-free)
Fat (USP cottonseed oil or
asay fat)
Gelatin
Salt mixt., .(a)
Vitamin mixt., (b)
Salt, iodized
Fat-sol, vitamin mixt., (c)
Chou'ne chloride, 25% aq. soln
203
203
20
16
13
6
2
12
1
OJ2
Treatment of Experimental Animali
Use" day-old white leghorn, single comb
cockerels. On day of receipt of chicks, tag
individually, record body weights, and place
in brooder cages equipped with heater. Use
room with controlled temp, and humidity.
Offer control ration contg 16% cottonseed oil
ns fat and HiO ad libitum. After 48 lir, weigh
chicks and do not use any chick which is outside limit of mean body wt by ±5 g. Place 12
chicks in each brooder cage and record body
wt and date of beginning feeding regimen.
Anay Period
Continue 1 group on control ration contg
10% cottonseed oil and substitute test fats for
all or part of the 16% cottonseed oil in assay
rations. , Check chicks daily for fatalities and
for presence of adequate food and H:0. Record all deaths and cause of deaths. For each
death due to other than accidental cause,
autopsy and record presence of hydropericardium, hydi'operitoneum, subcutaneous edema,
and amount of heart fluid to 0.01 ml. Autopsy
remaining chicks on 21st day and record findings as above.
Postmortem Examination for
Quantity of Heart Fluid
(a) Sacrifice of chicks.— Sacrifice by means
of cervical dislocation and proceed as follows:
(b) Exposure of heart.—With dissecting scissors make small transverse cut in skin over
full diam. of abdomen. Peel skin toward head
and lay skin fold over head. This cutaneous
incision permits wide field exposure of subcutaneous area over thoracic, and abdominal
cavities for examination for subcutaneous edema. Skin may be reflected caudally to afford
wider field of vision. Record (+ or — ) evidence of subcutaneous edema.
Insert blunt tip of scissors thru body wall,
and make transverse incision of musmlatu
to lower rim of rib cage, avoiding cutting into
organs ol peritoneal cavity.
Lift breastbone with fingers, insert blunt tip
of scissors, and carefully enlarge incision by
cutting on each side of chest cavity thru rib
joints up to clavicles. (Do not. cut into subclavian vessels.) With sufficiently wide cut,
fingers may be used to protract incised cheel
cavity and thus permit clear observation of
substernal attachment of pericardium. Firmly
clasp pericardial attachment with fingers and
reflect flap of sternum so as to expose heart
with pericardium intact.
Estimate visually and record severity of
pericardial edema according to following table:
Pericardial Edema
Absent
Slight
Moderate
•
Severe
Very severe
Collaborator 1.
Collaborator 2.
Collaborator 5.
,
14 Days
21 Days
14 Days
(c) Withdrawal of heart fluid.—(1) 'For vols
estimated as </.0 ml.—Firmly clasp apex of
pericardium with small forceps and make small
incision in heart sac. Insert small spatula into
incision of pericardium and push heart to one
side. Carefully aspirate1 fluid into 1.0 ml tuberculin syringe. Blunt-end 18 gauge needle permits more complete aspiration of small vols.
To reduce formation of air bubbles during aspiration, prerinse needle and syringe with
n-butyl alcohol. Do not include vol. of n-butyl
alcohol in measurement of fluid.
(#) For vols estimated as >7.0 ml.—Insert
sharp hypodermic needle on 10 ml syringe
into intact pericardium. Aspirate as much as
possible of the heart fluid (n-butyl alcohol
rinse is unnecessary). Collect and measure
remainder with blunt-end tuberculin syringe.
Record total vol. heart fluid.
(d) Other observations.—Observe and record other obvious changes such as peritoneal
edema (ascites), liver changes, kidney changes,
etc.
chicks of the age and w
bioassay.
From the observations reported by the
collaborators it may be generalized that of
the three anatomical sites in which fluid may
accumulate, hydropericardium occurs with
the greatest frequency, followed in order by
peritoneal and subcutaneous accumulation.
This is in agreement with earlier observations.
The appended charts (Fig. 1) show graphically the response of the volume of peri-
21 Days
14 Days
Score
0
+
++
14 Days
21 Days
Collaborator 7.
•
Collaborator &.
14 Days
Collaborator J,
!
.
21 Days
14 Days
21 Days
Collaborator 9.
1
14 Days
21 Days
21 Days
*Svsn
i
_
Seven of the nine cooperating laboratories
submitted results in time for inclusion in this
report. Their observations are compiled in
Table 1
It is seen that mortality due to the toxic
factor does not become appreciable until the
third week on the diet containing the highest
level of the toxic fat. Note that three of the
in the
Uw value /alls at (be high end of the normal
ttnge of the volumes of pericardial fluid for
•
_
ft
•
, t . rfa R&3 bfel
1
2
3
—*-i K$fl B88
4
1
Diets
fig.
1-
2
3
1liVi'
A-
1
2
3
4
1
2
3
4
Diets
•Volume of pericardial fluid (— S,E. of the mean) versus level of toxic fat for 14 and 21 day
feeding periods.
�232
—*—•••
Coll,
No.
Away No.
of
Period
of
Early
Diet (Day.) Chick. Deathg
3
3
4
4
9
14
21
14
21
12
12
12
12
0
2
1
5
1
1
2
2
3
3
4
4
14
21
14
21
14
21
14
21
12
12
12
12
12
12
12
12
0
1
0
0
0
1
0
5
233
Table 1. (Continued)
.."i
i
Average
Weight Gain
Average Hydroperioaj-dium
Edema Incidence
Total (g)
BS ""WJ-" £
Sub- Hydracuta- peHwA«oite« neous diua-
45.1 ±
72.5 ±
48.3 ±
75.1 ±
2.8 13 + 0.203 ±
8.3 28 + 0.878 ±
5.4 28 + 0.903 ±
6.4 26 + 3.44 ±
54.6 ± 3.3
104.4 ± 9.6
57.3 ± 5.6
108.0 ± 7,1
63.3 ± 3.6
104.0 ± 11.8
60.4 ± 4.889.6 ± 6 . 7
0
0
1+
|10 +
4+
36 +
21 +
35 +
0.09
0.10
0.15
0.29
0.19
2.86
0.84
4.52
.086 6 +
.339 17+
.303 20 +
1.40 19 +
± .01 ±.015
±.07
± ,05
± .045
± .55
± .24
±.86
0
sJ
10 +
5+
42 +
22 +
34 +
1/12
4/12
5/12
6/7
0/12
0/12
0/12
0/12
0/12
9/12
6/12
11/11
1/12
7/12
8/12
6/7
4/15
8/1 J
8/13
7/7
0/12 0/12
0/12 0/12
0/12 1/12
0/12
0/12
0/12 4/12
5/12 12/12
2/12 11/12
9/11 11/11
• Hydroperioardium incidence: baaed on measurement of pericardia! fluid.
collaborators reported deaths of four birds
on the control diet. Since these death's could
not have been due to the factor, this and the
foregoing observation indicate that mortality
measurement' alone is not a reliable index of
the presence of the chick edema factor. It has
been found that signs of chick edema disease
aro tlie development of hydropericardium,
hydropcritoncum, and subcutaneous edema,
in that order, as the-dose of toxic material is
increased. At higher dosage levels, death
will occur from the 10th day. Birds dying
of chick edema disease invariably exhibit
these signs. Deaths unaccompanied by any
of these signs cannot be attributed to the
toxic agent.
Weight gains of the chicks were somewhat
depressed at the higher levels of toxic fat.
In the control groups, the average weight
gains at the end of the assay periods were
quite variable'among the laboratories. This
variability may be due to borderline nutritional inadequacies of the basal diet that
possibly wore aggravated by hereditary or
environmental factors or a combination of
both, as may have happened in the case of
Collaborator 7's group. Perhaps it would
have been wise to follow the suggestion of
certain of the collaborators and to have an
antibiotic incoiporated in the test ration.
Visual subjective scoring of the 'degree of
hydropericardium according to the instruc-tions given in the collaborative procedure is
seen to agree fairly well with ft score calculated from the actual measurement of pericardia! fluid as shown in Table 2. The
figures given in Table 1 represent the sum
of the plus scores for individual chicks within
the group. It appears that there was greater
accuracy in visual scoring of large toxicity
responses than in the smaller responses.
While visual scoring may be adequate in
judging the presence of a large amount of
toxic material, it is inadequate in those borderline cases where controversy is most likely
to develop.
We have arbitrarily adopted 0.2 ml as the
upper limit of normal heart-sac fluid volume.
This choice is justified on the basis of the
work of Shue and Gallo (7), who report that
Table 2. Scoring by Referee of
pericardia! eclcrna
<0.20
0.21-0.40
0.41-1,00
1.01-2.00
>2.01
0
+
++
++ +
+ ++ +
(Hud to the level of toxic fat in the
<***• It is seen to vary directly with this
fc«l- II is likewise seen that an appreciably
t»**lfr response to the dose is obtained
*/'*f ft three-week feeding period than after
» Cwo-weck period. Although there is a high
evidence of negative responses in a group
«f cliicks on a low level of toxic fat, the
ivrage response of the group as measured
by the volume-of pericardia! fluid is proporiKmnl to the dose level within the dose limits
o( this experiment.
Comments of Collaborators
Most of the comments were directed at
tlw nutritional adequacy of the basal ration.
One investigator has suggested that dry,
itnhilizod vitamins A, D, and E be used
wthcr than the crystalline products used in
the procedure, This suggestion appears to be
meritorious and has been incorporated in
the recommended procedure as optional. Another objected to the' mode of addition of
rholine to the diet and suggested a 25% dry,
free-flowing preparation which is commercially available. For the sake of convenience,
this has likewise been'incorporated as optional. Objection was made by another eollnlx>rator to adding the water-soluble vitamins on Cclluflour as the carrier. Since many
laboratories routinely use other of the dietary
ingredients as carriers for the vitamin mixture, \ve see no reason why any of the major
components cannot be used for this purpose.
Weight gains obtained on the diet are not
optimal. This, however, is not critica-1 from
the standpoint of the assay except, as one
collaborator points out, that it is easier to
carry out the procedure of withdrawing the
heart-sac fluid from a larger bird than from
ft smaller one. Sinco i hr diet has given excellent pericardia! -liu'id responses, major
changes cannot be justified at this time.
Other collaborators have suggested that
an antibiotic be incorporated in the ration,
that crude casein be substituted for the vitamin-free casein, and that commercially available salt and vitamin mixtures be used. These
appear to be desirable from the standpoints
of economy and convenience and will form
the basis of a modification of the diet to be
used in further collaborative work. The level
of sodium chloride was li^He the subject
of question on the basis that 2% of salt is
too high and subjects the chick to extra
"stress." It has been found in our laboratory
that this level favors the development of
the edema. Selye and Stone (8) have found
that extra sodium chloride in the diet of
chicks accentuates the development of edema
produced by certain steroids. Blester and
Schwartz (9) state that 6-8 grama of sodium
chloride per day is not harmful to 9 week-old
chicks. We cannot see that a change here is
justified.
One collaborator reported that the ration
was unpalatable to his chicks. The weight
gains reported by him were much lower than
in any other laboratory. We have no explanation for this effect. It is suggested that this
laboratory may have some peculiar problem
and that the performance of these chicks
is not typical.
Several of the collaborators preferred to
use heavy breeds of chicks rather than leghorns. We have retained leghorns for the
reasons of uniformity of response, widespread availability, convenience, and the
demonstrated sensitivity of this breed.
Summary and Recommendation*
A successful collaborative study of a procedure for the detection and assay of the
chick edema factor in fats and fatty mate. rials has been carried out. The results indicate that the method as studied is satisfactory for the intended purpose. Although it
is demonstrated here that the method studied
is capable of differentiating contaminated
from uncontaminated fats, it would be desirable to carry out additional collaborative
studies on samples of lower potencies than
those used here.
It is recommended1—
(1) That the method for bioassay of chick
edema factor, presented in this report, be
adopted as first action.
(2) That collaborative studies be continued.
REFERENCES
(1) Editor's Note, This Journal, 42, 120 (1959).
1
These repommedations were approved by
tile General Kcfercc and by Subcommittee C,
and were adopted by the Association. See This
Journal, 44, 70 (1BG1).
�235
>
(3)
(4)
(5)
(6)
(7)
(8)
(9)
PKOGBESS IN THK CHICK EDEMA PROBLEM
Brew, W. B., Core, J, B., Benedict, J. H.,
Potter, G. C., and Sipos, E., ibid.,- 42, 120
(1950).
Friedman, L., Firestone, D., Horwitz, W.,
Banes, D,, Anstead, M., and Shue, G., ibid,,
42, 129 (1959).
Wooten, J. C., and Alexander, J. C., ibid,,
42, 141 (1959).
Harman, R. E., Davis, G. E., Ott, W. H,,
Brink, N. G., and Kuehl, F. A., J, Am.
Chem.Soc., 82,2078 (1960).
Ames, S. R., Swanson, W. J., Ludwig, M. I.,
and Bookaw, G. Y., J. Am, Oil Chemists'
Soc., 37, 10 (1960).
Shue, G. M., and Gallo, L., This Journal,
44, 456 (1961).
Selye, H., and Stone, H., Proc. Soc. Exptl,
Biol. and Med., 52, 190 (1943).
Blester, H. E., and Schwartz, L. H., Diseases of Poultry, 4th Ed., The Iowa State
University Press, Ames, Iowa, p. 112.
Collaborators
By Dr. Leo Friedman, Food and Drug Administnmon
;^TO>I
J. C. Alexander, The Procter & Gamble
Co,, Research Division, Cincinnati 39r Ohio
Stanley It. Ames, Biochemistry Department, Distillation Products Industries, Rochester 3, N.Y,
Carl D. Douglass, Food and Drug Administration, Washington 25, D.C.
0. F. Hixon, Laboratory of Vitamin Technology, Inc., 7737 S, Chicago Ave., Chicago
19, 111.
Walter H. Ott, Merck Institute for Therapeutic Research, Runway, N.J.
C. E. Poling, Swift & Co., Union Stock
Yards, Chicago 9, 111.
H. C. Sehaefer, General Research & Control Laboratories, Ralston Purina Co., St.
Louis 2, Mo.
It 1ms been almost four years since I first became aware of the problem
that.we know today as "chick edema disease." Because of our activity on this
problem, my colleagues and I have had the opportunity to become acquainted
with Many scientific groups and individuals working in the same area with
whom we have enjoyed a fruitful cooperation and pleasurable association. We
hope that they feel as kindly toward us as we do to them, but sometimes, I
known, they wish as we do that they had never heard of chick edema disease.
This problem has been most difficult and progress frustratingly slow. Despite
tlie meager amount of new information that can be added at this time, especially since the most recent advances were reported l>y Dr. Artman recently,
it is nevertheless worthwhile to revie-\y the several aspects of this problem
and see its present status in full perspective.
As you recall, during 1957 an epidemic disease caused millions of dollars in
losses among broiler flocks throughout a large part of the U.S. After elimination in succession of all other possibilities, attention was focused on the fat
ingredient of the feed as the etiologic agent. A series of reports in 1958 from
several laboratories described the manifestations of the disease and definitely
implicated a toxic fat or a toxic substance in fat as the cause. The characteristic symptoms were droopiness, ruffled feathers, labored breathing and high
morbidity and mortality. Autopsy findings revealed hydropericardium, abdominal ascites (water belly), subcutaneous edema, swollen liver, swollen and paie
kidneys, etc. In laying hens the toxic fat caused a rapid drop in egg production.
Pullets receiving toxic fat during the full growing period did not come into
production, and mortality was very high. Hydropericardium, the most common
lesion found in young birds, was not found in birds of laying age.
SYMPTOMS MFFEBENT
These differences in susceptibility and symptoms in different age groups
of the same species should be noted. The feeding of toxic fat to other species
has not produced such striking results as with young chicks, with the exception possibly of monkeys. However, every species that has been tested has
shown evidence of deleterious effects. Very little work has been done with rats.
Our very limited experience indicates that they are much more resistant than
chicks in short-term feedings, but that when fed in sufficient dosage, extracts
of the toxic fat produce definite deleterious effects as shown by growth depression, enlarged and fatty livers, and marked involution of the thyinus.
I recall few reports of the effects of toxic fat. on swine, but again, in our
own limited experience we have seen depressed growth . . . and have demonstrated the presence of toxic factors in the meat of .hogs that had been fed
toxic fat.
I am indebted to Dr. AVilcke of Ralston Purina for reports of studies on
guinea pigs and dogs. Guinea pigs fed 2i/£% toxic fat stopped growing after
six weeks, and death losses occurred after eight weeks. At a level of 4%%
toxic fat weight Josses occurred after three weeks and deaths after four weeks.
Control groups receiving non-toxic fats did not show weight loss or deaths.
The only observed pathology at the conclusion of the experiment was congestion of the lungs and mottled livers.
In experiments with three different breeds of dogs, using Purina Dog Chow
in which 10% of toxic fat was substituted for the usual normal fat, there was
poor reproduction and lactation performance. The females on the toxic fat
ration whelped pups that were dead or weak, and, furthermore, the mothers
seemed to have an insufficient milk supply. When the pups were removed before
weaning and fed a normal ration the increase in growth was immediate and
dramatic. Also, the females on the toxic fat ration tended to lose hair on their
backs and shoulders. With the ration containing toxic fat, post-weaning growth
tests (6-18 weeks) with five litters of pups demonstrated inferior growth performance using either weight gain or increases in body length as the criterion.
WORK WITH OTHER SPECIES
In these experiments with other species, fat that had first been proved- to
be toxic to chicks had been used. In the case of monkeys, a sample of trioleln
45-362 0—70-
-10
�236
had produced irreversible toxic symptoms for no apparent reason 1; _
was proved toxic to chicks and was the source from which we isolated a highly
purified crystalline "chick edema" factor. At the present time, then, "toxic
fat." that produces "chick edema disease" lias been demonstrated also to !>«>•
duco deleterious effects in rats, guinea pigs, swine, dogs and monkeys. It should
be emphasized that the toxic fat undoubtedly contains many other substance*
that may have effects in these other species. Definitive information as to tlie
effect of "chick edema factor" (OBF) in other species must await tests vritli
purified CEF. Triolcin-fed monkeys probably received the "purest" source of
OEF. However, purified OBF should be administered to monkeys to verify tlic
implication that the severe toxic symptoms observed with triolein were due
to CEF.
Relatively little lias been done to throw light on the mechanism of the toxic
effect. Flick and Gallo in our laboratories have reported that in young chick."
showing symptoms of the disease, the intra-cellular water was not, changed.
Neither were the total blood and plasma volumes altered so that the observed
edema was primarily interstitial. Hemoglobin and hematocrit levels were low
and Wood glucose levels were decreased in advanced stages of the disease.
Plasma sodium, potassium and chloride were not nffected. In the liver, neutral
fat was decreased and phospholipid increased. Preliminary experiments by
Flick give some indication of increased membrane permeability, but these must
be repeated under much more rigorous conditions.
DISSERTATION ABSTRACT
A most interesting study in chicks has been reported by .T. It. Alien, Jr. Part
of the results were presented at the federation meetings in March, 1001, and
the complete study is available as the dissertation of ,T. It. Allen, Jr., University
of Wisconsin, 1961. I will quote portions from the dissertation abstract [Dissertation Abstracts 22. [2], 645 (Aug., 1901)] :
"Experiments conducted to determine the effects of 'toxic fat' on mice,
pigeons and turkeys demonstrated a reduction in growth without hydropericardium or ascites."
In chicks, "Microscopic examination of the tissues of the test animals
revealed lymphocytic foci in the epicardium and myocardium. Jfldomatous fluid
separated the myocardial fibers. Edema of the lungs was a frequent observe-'
tion in the experimental birds.
". . . Blood pressure indicated the test birds had an elevated average mean
pressure in the right ventricle of 6 cm. water and 2 cm. water in the vena
«iva. Electron micrographs of the myocardium revealed shrunken, vacuolated
mitochondria in the test animals.
" 'Toxic fat' produces a reduction in growth rate of experimental animals.
Tliis reduction depends on the age of the animal and the level of 'toxic fat'
added to the diet. Hydropericardium and ascites are a frequent lesion in the
animals receiving from 1.0 to 5.0% 'toxic fat." When this level was reduced
to 0.25% in the diet, reduced testicular development was a more sensitive
criterion than hydropericardium, ascites or weight gain for evaluating chronic
toxicity.
"The mechanism by which 'toxic fat' induces hydropericardium and ascites
appears to be associated with degeneration and edema of the niyocnrdial fibers.
These data would tend to eliminate the kidneys, liver and endocrinos as the
primary cause of edema. The early development of hydropericardium, increased
venous pressure, enlarged hearts, mitochondrial changes in the myocardium and
generalized edema suggest that the myocardium may be directly inhibited;
however, altered capillary permeability lias not been excluded. It is believed
that cardiac decompensation and increased capillary permeability act together
in producing the excessive extravascular fluid collection- and the demise of the
animal."
The wide range of susceptibility within and among species and the variety
of toxic effects that have already been noted would make it appear logical
that some primary unit of structure and function such as the mitochondrion,
inny bo the target of Hie toxic factor and that the observed differences may be
explained by factors such as absorption, specific binding, transport, detoxication, etc,, that determine the local concentration of any substance in n specific
site.
237
Another possible mechanism that had suggested itself quite earlj^Bthat the
toxic factor interferes with the normal regulation of electrolyte^mcl water
tifUaiice. Selye and Stone, back in 1043, described the production of edema
symptoms in chicks by certain steroids and the accentuation of these symptoms
I)}' increasing the salt intake of the chicks. Alexander has shown that it is
l"isnible to produce hydropericardium in chicks by increasing the NaCt in the
ration and to prevent its occurrence even with OEF by eliminating NaCl from
t)m diet. AVork in our own and other laboratories has illustrated beautifully
flu> interaction of nutritional factors on the susceptibility of the chicks to a
1'ixic agent. For example, the present AOAO bioassay diet for "chick edema
factor" is probably four times more sensitive than the assay diet we used
originally, although both have approximately the same NaOl content.
A point to remember is that hydropericardium as a symptom of toxicity in
clucks is not new. In addition to NaCl and certain steroid hormones, chapter
'10 in Blester and'Schwarte on Poisons and Toxins indicates that:
(1) Zinc phosphide, used as a rodenticide produces "various degrees of
congestion with the accumulation of some serous fluid in the pericardial sac
as well as in the abdominal cavity in some cases."
(2) Alpha naphthyl thiourea, the rodenticide ANTU, shows in poisoned
chicks evidence of lung edema and excessive quantity of fluid in the pericardial
BUC. Sodium monofiuoroacetate, compound 1080, another very effective roden(3)
ticlde, produces in chicks distention of the pericardial sac with clear strawcolored fluid, in addition to other marked pathological changes on the heart
and lungs.
(4) Chlordane. "The primary lesions found in all fatal cases were in the
heart. Excessive quantities of fluid were found in the pericardial sacs. . . ."
The weed "corn cockle" and several species of Crotolaria produce seeds which
are toxic, and in chicks the toxic symptoms include hydropericardium.
However, in each case other characteristic pathology is usually present, and
In no case is the purified toxic principle of the same high order of activity as
the toxic substances that have been isolated from toxic fats.
It is well known now that chicks can efficiently utilize large amounts of
fat in properly balanced rations. The use of fat as a standard ingredient of
poultry feeds grew as the price of fat calories dropped and became competitive
with calories derived from corn. The chick edema disease epidemic of 1957 was
a totally unexpected consequence of this growing practice. Many of you are
familiar with the story of how the toxicity was associated with a fatty byproduct of stearic and oleic acid manufacture that had been blended with
feed grade fats.
Very large quantities of fatty acids are used industrially in the manufacture
of lubricants, rubber, paints, asphalts, roofing, chemicals, and to a much
smaller extent in foods. Relatively low grades of fat are split into fatty acids
and glycerol at high temperatures and pressures, sometimes with the aid of
catalysts. The glycerol is recovered and the fatty acids are distilled under
vacuum. The first distillate may be used directly as the highest grade of mixed
fatty acids or it may be. separated by a low temperature crystallization process
into stearic (saturated) and oleic (unsaturated) fractions. The residue from
this distillation is resplit and redistilled. The second distillate yields a lower
grade of fatty acids. The residue from the second distillation is usually suitable
for use on highways or in rubber manufacturing, hut occasionally it is again
recycled to obtain a third distillate and another residue. It was this third
residue that bad been blended with feed grade fat for use in feeds.
Every sample of residue of this type from several manufacturers of fatty
acids proved to be rich in chick edema toxicity. Our first impression, therefore, was that the toxic factor was produced during the splitting and distillation steps and that it was concentrated in the residue along with other
non-volatile unsaponifiable substances. Closer study of the various stages of
fatty acid production soon revealed that the toxic factor was distillable and
was present to some extent in the first distillates which were used for the
production of the best grades of fatty acids, and those intended for food purposes. This discovery was made independently and reported by Ames, et al.,
who had found several samples of oleic acid and a monoglyceride made from
such an oleic acid to be contaminated.
All tills happened just after the passage of the Food Additives Amendment.
The fatty acid manufacturing firms were very cooperative in providing us
�«
238
i information and samples. However, they sincerely believed that tb
e not part of the food business, that the bulk of their production wentf
non-food industrial purposes. In our visits and talks with their technical
we advised them to study the application of the new legislation to their industry. They realized that a substantial proportion of their highest grade of
materials did enter food channels when their customers started asking for
guarantees that the fatty acids met the requirements of the Federal Food and
Drug I/aw, At that point the technical committee of the Fatty Acid Producers
Council became actively engaged in a study of this problem, and we have
enjoyed the whole-hearted cooperation of the fatty acid industry. While they
made studies to determine what part of their processes were responsible for
the production of the toxic material, studies were continuing on the isolation
and chemical characterization of the active substances. Three years ago at the
AOAC meeting a symposium on chick edema was held at which reports from
the Purina laboratories, the Quaker Oats laboratory, the Procter & Gamble
research laboratories and the Food and Drug laboratories described the
progress made up to that time. The Merck group had recently entered the
problem, and, although they did not report, they also had traveled a similar
road.
Every step and experiment had to be followed by the chick bioassay. However, during the first year all the groups bad made steady progress at about
the same rate. First it was demonstrated that the "chick edema" toxicity was
entirely in the unsaponiflable portion of the fat. Then, in our work, the unsaponifiable was separated by cbromatography on alumina into three fractions
of increasing polarity by elution with petroleum ether alone, then with a
mixture of ethyl and petroleum ether and finally with 100% ethyl ether. The
first, or hydrocarbon, fraction contained 83% of the unsaponiflable, and its
ultra-violet absorption spectrum indicated the presence of cholestadiene. The
second fraction was characterized as ketonic and infrared absorption spectra
suggested the presence of dipalmitone. The third fraction consisted of sterols
and oxidized materials. Simultaneous bioassay of these three fractions and
synthetically prepared samples of cholestadiene and dipalmitone showed that
only fraction 2, but not the dipalmitone, was toxic.
Several approaches were tried simultaneously to purify fraction 2. I will not
take the time to describe our experiments with a 500 tube counter-current
distribution between iso-octane and mothanol, or the separation of carbonyls
with Girard-T reagent or the consecutive rechromatography on alumina and
Rilane treated Celite. Suffice it to say that as our fractions became more
potent we relied more and more on ultra-violet spectrophotometry as an indication of concentration and purity. Two distinct types of fractions were obtained, one with the ultra-violet spectra characteristic of naphthalene compounds and the other of phenanthrene compounds. Both fractions contained
the toxic factor. However, the sum of their toxicities was not comparable'to
that of the starting material in the final purification steps despite the fact
that practically all of it was accounted for by weight in the eluates. Since
only the fractions that had a significant weight had been tested biologically,
the insignificant, hardly visible residue of less than 0.5 mg. in the practically
"empty" beaker that represented the cut between.the two major fractions was
rinsed into a chick diet and to our surprise was very active at approximately
0.1 ppm., representing the most potent material we bad obtained. At this stage
we had practically exhausted our raw material, and we had to start from
the beginning once again.
During the course of these studies we had learned that Drs. Portman and
Andrus iiv the department of nutrition at the Harvard School of Public Health
had lost a number of monkeys in a nutritional study. They had used a synthetic
triolein as the source of fat and had to abandon the experiment. At a Gordon
Research Conference I had the opportunity to discuss this experience with Dr.
Portman. Fortunately about 40 Ib, of the triolein was still available. It proved
to be toxic in the chick edema assay. The triolein was of excellent quality,
containing only 0.9% of unsaponiflable material. This unsaponiflable proved
to be the richest source of the toxic material we had ever examined.
The. toxic fraction was separated from the triglyceride by molecular distillation. The distillate from 17.0 Kg. of triolein was saponified, and the
unsaponiflable was chromatographed on alumina to remove the cholostadiene
fraction. The naphthalene and phenanthrene-containing cuts were collected,
yielding 670 rag. of material. This fraction was rechronmtogrriphed on more
retentive alumina with 5% ether in petroleum ether, and 240 mg. of naphthalone-phenanthrene material was separated. This was rechroinatojjaphed in the
name system and three distinct types of U.V. spectra began tc^Bprge in the
fractions: Naphthalene (235 m/j,), phenanthrene (260 m/j) andlPnew peak at
245 m^i. These materials were combined and chromatographed on a silnne
treated Celite-iso-octane cohimn, with 80% alcohol saturated with iso-octane
as the mobile phase. The cuts with maximum absorbence at 245 m/i were
further purified by two more chroma tojrrnphic treatments on alumina at a
very high sample: adsorbent ratio (1:2,009 and 1:25,000). The final fraction
was completely free of absorption peaks near 235 and 200 m/i. This fraction
was evaporated to dryness, dissolved in a small volume of boiling iso-octane
und stored overnight in the refrigerator. White crystals were obtained, weighing 2.6 mg. and representing a concentration over the original triolein of three
million-fold. This material at 1 ppm. in. the diet killed the chicks in 12 days
and produced typical hydropericardium when fed at .05 ppm. in a 21-day test.
During the last stages of this work the Merck group [Harmon, et a).,
,TACS 82, 2078 (1060)], announced the isolation of a crystalline chick edema
factor from a toxic fat. On July 19, 1960. the Merck group informed us that
they had found 47% chlorine in their crystalline material. The presence of
chlorine in our material was quickly confirmed by the use of the Dohrmann
microcoulometric gas chromatograph.
In this test, a few micrograms of sample is injected into a gas chromatograph and the components as they emerge are pyrolyzed at 800° under oxidizing conditions. The halogen acid formed from halogenated materials is titrated
automatically in a microcoulometer.
Of interest also is the isolation of a crystalline material that had an ultraviolet absorption spectrum identical with that of the toxic material but shifted
8 mju so that the major peak was at 248 mp. instead of 245 m/t. It behaved like
the toxic substance, in the microcoulometric gas chromatograph, showing a
similar retention time and halogen content. However, it was completely inactive
in the chick edema test when fed at 1 ppm. in the diet.
The finding of chlorine in the toxic substance was a major breakthrough.
It was no longer necessary to think in terms of a naturally occurring material
that bad changed chemically under the conditions of industrial fatty acid
production. The possibility of contamination with one of many familiar
chlorinated hydrocarbons was obvious. Samples of tetracbloronaphthalene and
hexachloronaphtbalene, kindly provided by Rngel and Bell of Virginia Polytechnic Institute, who had demonstrated that these compounds produced liyperkeratosis in cattle, were without effect in the chick edema test. We have tested
a long list of chlorinated compounds including aldrin, dieldrin, lindane, DDT,
DDE, BHC, chlordane, toxapliene, methoxychlor, and a series of Halowaxes,
without any definitely positive indication. Furthermore these have all been
heated with oleic acid at 250° C for long periods and then fed to chicks, with
negative results. Also, on the theory that tallow may sometimes be bleached
with active chlorine materials which may chlorinate a sterol nucleus, we have
chlorinated cholesterol, squalene, estone and equilenin with negative results.
To test the theory that the toxic substance is a metabolite of a chlorinated
insecticide we have fed large doses of chlordane, methoxychlor, beptachlor,
aldrin and dieldrin to rats for a month and are feeding the unsaponiflable
extract of the rat carcass in the chick test. The results to date show that
chlordane and methoxychlor produce no response.
INDUSTRIAL EXPERIMENTS
Similar experiments have been carried out and are still in progress by at
least two industrial laboratories under conditions of fatty acid production.
The results so far have been largely negative or at best only suggestive but
not clear cut. Dr. Artman has reported that chlorination of naphthalene and
phenanthrene by substitution reactions has produced CISF active products. The
chlorinated phenanthrene is particularly promising, since preliminary fractionation experiments indicated the possibility that a highly toxic compound
was produced.
Dr. Boyd O'Dell and colleagues at the University of Missouri observed
hydropericardium and other symptoms of chick edema disease in chicks
housed in freshly painted cages. They traced the responsible agent down to
one of the paint ingredients, a chlorinated biphenyl sold under the name
�241
240
BIBLIOGBAPHY
ft
•Llol•!i of different chlorine content. Some were not toxic; others prod
.isease but only at relatively high feeding levels, e.g., 200 ppm. It may
that an impurity in these compounds is the toxic agent, or that they
Innately toxic at the high levels fed. Of interest is the use of one of tlu>«
Arochlors in some insecticide formulations. At the present time there has been
no real evidence developed to implicate any product or compound. However,
the circumstantial evidence is stimulating considerable speculation and activity.
The feed industry has managed by careful control of ingredients to nvoW
a recurrence of the 1957 epidemic. The color test developed by Brew, qt al., «l
Purina has been very useful in screening out toxic fats. This test is not specific J
but is useful since the presence of large amount of steroidal compounds thflt
give this test usually indicates still residue that may be toxic. It is useless1, J
however, for fatty acids and other products such as triolein, monogylceride*,
etc., that give no response in this test, but which sometimes are quite toxic.
GEORGIA OUTBMCAK
Last year, at this time, an outbreak of the chick edema disease occurred In
Georgia. Although considerable chlordane residues were found in the feed, vrp
do not believe they were responsible for the symptoms observed. Furthermore,
the best information we have indicates that only rendered fat was used In
this feed and no product of the fatty acid industry was involved. With cadi
now development the scope of the problem increases. First we were concerned
only with still residues, then also with fatty acid distillates and their derivatives. For a long time it was felt that only the fat derived from animals was
involved. Recent evidence from sources in the fatty acid industry and our
own studies indicates that some Vegetable fat sources may yield fatty acids
contaminated with CEF.
Still another development has occurred to further complicate the picture.
ICarly this year in the course of our regulatory activities, we examined n
sample of oleic acid. All the test chicks died by the end of the second week,
with symptoms of severely stunted growth, ascites, jaundice and pathology of
the liver and other organs, but with no hydropericardium. This sample lifl'l
been tested by the manufacturer last year before the adoption of the present
AOAO test procedure. The testing laboratory had used the procedure we
ourselves had used in our earlier work, and had found the sample negative for
chick edema. Repeat of the test in both laboratories by both procedures confinned both findings, The sample is free of chick edema disease factor when
tested on a diet of natural ingredients, and the chicks survive in apparent
good health. On the casein-sucrose diet of the AOAC chick edema test, tlie
chicks fail to grow and die early with the described symptoms but no hydropericardium. Furthermore, the dose response curve for this effect is very steep,
since the ratio of the dose that gives a maximum effect to the dose that produces a minimum effect is less than two, as compared to a ratio of four to five
Cor chick edema factor. Evidence from preliminary fractionation studies also
indicates that this is an entirely different substance. There is no other information as to its characteristics at this time. There is evidence that this toxic
contamination has occurred in different places from time to time and in a
variety of fatty acid samples and may occasionally occur together with C1W.
Here again we must anticipate that this material may occur elsewhere independently of the fatty acid industry.
For whatever comfort we may derive, it should be noted that chick edema
disease has been observed in England. In a letter to the editor of the Veterinary Record of June 10, 1961, C. C. Wannop of the Houghton Poultry Research
Station draws attention to a condition apparently identical with that reported
by Sangcr, et al., and Schmittle, et al., in 1058, that has appeared in several
broiler flocks. In a personal note dated Sept. 29, he says that the condition
has disappeared for the time in his country,
At the present time fatty acids can be used in the manufacture of foods or
food ingredients only if they are free from CEF. This requirement made necessary the development of a bioassay which has been accomplished by collaborative work and is now being adopted ns official by the AOAC.
I have tried to review the history of this troublesome problem, what little
is known of its toxicology and its physiological aspects, the speculations as to
the origin of the contamination, and attempts to track down its sources, 1
have sketched quickly our own attempts at isolation and identification of the
toxic factor and have alluded to the most recent developments along this line
that were reported at the AOAC Section of Fats and Oils by Dr. Artman.
Chick "Edema" Disease
1. Sangor, V. I et ai. •
., 133, 172 (1058), "
^
Sci., 37, 1300 (1958), "Effect of a tox-c sub-
, "Effect of certain toxic
performance, embryon.c
in fat on
r
development and health of djlckcji*
Disease Besearch
. r e m , s
.
chicken edema disease factor .
^^
Center, Athens,
42, 14i
(1959), Some
"tot status
Manufacturers Ass,.
d
iRHues special report on edema hi chicken ^
in animal
Asys^^'^s^ -*
Meat Insti tute
******
toFoundation
a
tease."
r 3 et al._pouury Science, 38, 579 (1959), "Relationship of
oxlLMegVatton to . toxicityin ce^}n JateJ ^ (1M9)i ,.studies on toxic
1Q
T^iiilflllOO
"VV. &-t Gt ^'
J, ULUH.J
- ^
fat in rations of laying hens and pullet^
IOiacn , N.V,
Science A
Au^usf f^Tne S
V AprU, 1000
^
products derived therefrom/'
2Q78 (1960).
"The isolation
, Brink, N. O., and Kuehl, F.
.
-
.
.
e
Ohom. Soc., 38, 418-422 (1001).
20. Douglass, C., and KlicK, u. L
: SS»ifciS!J
Horwlte, W.,
—"<»'
offlc _ Agri.
chemists, 44 (3),
, C. D, and Friedman, , ,
-
A
e
s Rex, ^^
24. Woot'ton, ,T. C. Alexander, J O ,
2
fed a chlorinated hydrocarbon
a n d Friedman, I*-
Qffic
Iderstine, A.—Poultry Sci, 40,
the edema producing factor m tox,
fat."
COMPOUNDS BY H.
MAY'20, 1901
INTRODUCTION, QENEBAIa
�242
an chemical works, the acne persisting over a long period and being ass
with other effects on the health.
Diseases in a group of 17 workers from a company in North Rhine/Westphalia were reported by BAADI3R and BAUER, as well as by BRINKMANN In
3O.TO/S.l. The workers in that company were engaged in the production of
pentachlorophenol. Apart from comedoue acne with various degrees of secondary pustular infection and boils, most of the workers, whilst still in the
flrst stages of the skin diseases, also experienced pain and weakness in tlic
lower limbs, mild paraesthesia, heart complaints and indeterminate psychovegetative disturbances. Subsequent examination of the records of 17 cases'
revealed the following findings :
All 17 were suffering from an acno, 4 of these being very severe, 8 fairly
severe, and 5 moderately severe to mild. In almost all cases there were extensive pustular infections and boils, 4 with bursitis on the elbow.
Other disturbances amongst the workers included 11 cases of bronchitis, 5
of myocardinc damage, 2 of cirrhosis of the liver (one of which proved fatal),
0 of neuritis symptoms (severe pains in the lower extremities in 7 patients,
sensibility disturbances in 4 cases, mild paresis without atrophy in 2 patients,
and 2 cases of weakening of the Achilles' reflex). Seven \yorkers complained of
physical conditions such as continuous fatigue, depression, lack of vitality,
nervousness, slight headaches, disturbed sleep, and decrease in libido ami
potency.
A larger number (about 60 cases, Prof. Hergt) of similar conditions occurred in two Mid-Rhenish companies amongst workers who had been engaged
for long periods, generally several years, in the production of trichlorophenol
(snponiflcation of 1,2,4,5,-tetrachlorobenzene to 2,4,5-trichlorophenol by treatment with methanolic caustic soda solution). These trichlorophenol workers,
like those in a third group of affected persons from the Hamburg region who
will subsequently be dealt with in more detail, suffered from further disturbances to health, these often not occurring until a fairly long time after occupational exposure had ceased. In the course of a discussion on a paper by
SP1EGELBERG, who referred briefly to our Hamburg cases in a lecture at
the 1960 North-AVest German Neurologists' and Psychiatrists' Congress in
Luueberg on psychopathological delayed and chronic damage following occupational intoxication. Janzarik described largely identical disturbances amongst
workers from the Mid-Rhenish companies.
The third group comprised 31 workers in a Hamburg company. Those of this
group who were affected were engaged in the trichlorophenol department of
the company, in which the herbicide 2,4,5,-trichlorophenoxyacetic acid was
manufactured from technical 2,4,5-trichlorophenol by heating trichlorophenol
together with caustic soda solution and monochloroacetic .acid in autoclaves.
After completion of this esteriflcation process, the end product was purified by
double recrystallization. The task of the workers consisted first of all in
charging the autoclaves, for which purpose the trichlorophenol in flake form
had to be removed by shovel from open barrels. In this operation, a fine dust
formed and dispersed throughout the room.
Other operations were concerned with filling and controlling centrifuges and
regulating feed and outlet pipes. Since it was the workers most exposed to
contact with trichlorophenol who suffered from the severest skin conditions,
it was logical from the outset to suspect the causal noxa to be present in the
trichlorophenol. The extent to which this assumption was valid is discussed
later in this paper in connexion with etilogy.
SOME CLINICAL OBSERVATIONS
Of the 31 workers of this Hamburg company, 9 are still receiving medical
attention 5 years after the termination of occupational exposure, this being
due to residues of their acne, chronic neuromuscular weakness of the leg
musculation, vaso-vegetative lability and, most especially, marked psychopathological disturbances. Details of the established complaints and damage
to health are given in the table. The development of the skin conditions in
the patients followed, by and large, a uniform pattern. Numerous comedones
formed, first on the face, especially on the cheeks above the malar bones, fore1
Unr t h a n k n arc duo to the BcrufnuonosHcnsehaft dor Chcmlschen Industrie for
jiliirliiK tlx-lr record* ntul other documents at our disposal, and also for their underxlniulhiii In the »omctlmc« lengthy clinical examinations.
243
head, temples, chin and ears, after which folliculitis, pustules, boiisJ^Bl retention cysts occurred as a result of secondary infections. As the dnHlse processed, these symptoms spread in the majority of patients, especially to the
"UK's of the neck, back of the neck, upper half of the back, chest, forearms,
Kcnitala and thighs. Numerous boils formed, particularly on the back of the
neck and on the back. The efflorescences were generally located so closely
together that scarcely any follicles remained unchanged,
In certain workers who had apparently been more strongly exposed, the
development of these acne-like symptoms preceded a dermatitis associated
with erythema and swelling, this extending to the region of the eyes, the cheeks
find the forehead. At about the same time, blepharoconjunctivitis occurred in
several patients, this, like the skin symptoms, becoming chronic in some cases.
As the table of findings shows, spots or, in certain cases, patches of pigmentation occurred in the faces of some patients, these giving the skin a dirty,
Kreyish-brown appearance.
The overall clinical picture was identical to the symptoms occurring after
working with chlorinated naphthalenes, diphenylenes and other aromatics as
flrst described by HERXHEIMER (1899) and subsequently by several other
authors (BETTMANN, HOLTZMANN, TELEKY, HERZBERG, BRAUN,
GRIMMER, etc.). (For further details, see W. BRAUN and A. RISSESUNDKRMANN (I960)). Although not a completely exact description, the
designations "chloracne" and "perna disease" have become the most popular
for these forms of occupational intoxication.
TABLE OF FINDINGS
Skin and mucous»membranes: Dermatitis of the face in initial stage;
comedones, retention cysts, nodules, pustules, boils; patches of pigmentation;
blepharoconjunctivitis.
Internal organs: Loss of appetite; abdominal complaints; loss of weight;
reduction in general condition; altered acidity of the gastric juice; gastritis;
damage to liver; pulmonary emphysema, dyspnea; myocardiac damage; blood
pressure; edema; pathological urine finding (renal damage).
Nervous system.—Neurological: Muscular pains; weakness in legs; (general)
fatigue; increased sleep requirements; parestbesia; headaches; attacks of
giddiness; orthostatic collapse tendency; paresis (implicit) ; coordination disturbances ; hypaesthesia; reflex irregularities; vegetative hyperexcitability;
EEG findingJ; EMG finding.8
Nervous system.—Psychopathological: Decrease in initiative and interests;
hyperaesthetic-emotional traits; pronounced fluctuations in intensity; disturbances in memory and concentration; disturbances in libido and potency;
alcohol intolerance; depressions; decrease in impulsion; affective disturbances
In the restricted sense of the term; experimental weakness in mental capacity;
organic Rohrschach psychogram; individual neurotic traits. ,
n. = normal, aim. = abnormal, p. = pathological, n.s.p. = not definitely
pathological, v.mU. = premature fatigue in the electromyographic series stimulus test.
The course of the dermatological manifestations proved to be extremely
obstinate in our cases. The therapeutic measures employed (drainage of the
comedones, external keratolytic and antibacterial measures, as well as the
Internal administration of antibiotics in severe cases) could not prevent the
reformation of comedones, retention cysts and boils in the first year, or two,
although there was no further contact with the causal noxae. Only after a
long time did the tendency to relapses cease. A residual condition now to be
found, particularly amongst the serious cases, is closely arranged pitted scars
which have a disfiguring effect, especially where localization occurs in the face
(pseudo-atrophodermia vermiculata).
All affected workers reported pronounced fatigue and weakness in the legs,
often with pain, especially in the region of the proximal leg musculation.
These conditions were marked, even in the early stage of the disease, and
in some cases even before the development of skin changes Paraesthesia was
reported in the records or in spontaneous information in only 2 of'9 cases.
2
Our thanks nre due to Do/,. Dr. BOCHNIK and Dr. BTTSCHART for conducting theolcctro-encephalographlc investigations, and to Dr. PUFF and Dr. RUEDAS for the
clectro-niyographic Investigations.
�-,
ft;
Implicit paresis or atrophy, weakening of the reflexes, or absence
| expansion reflexes as a sign of toxic polyneuropathy was not esta~
any of the cases. Two of the patients examined indicated a decrease
bility with isolated epicritical disturbances in the lower limbs. No deilnlt*
signs of neurogenic damage that could have been expected with peripheral
nerve lesions were established electro-myographically; premature fntlpt*
which was recorded in the series stimulus test, requires further confirmntN
regarding both the method and the raised findings. As these findings shnu,
the neuromuscular disturbances do not fit in with the typical picture of toxk
polyneuritis or polyneuropathy.
The electroencephalographic examination produced an abnormal electroencephalogram in 6 cases, with frequency lability and dysrhythmie groups of *
partly asymmetrical character. In one of the patients examined, aceentimtc)
dysrhythmia and raised cerebral excitability were revealed after photostlinnli 1 •
tion, The electroencephalographic changes found were uncharacteristic (tini
afforded no diagnostic viewpoints of any real consequence.
Some of the workers examined complained of headaches, attacks of gidclinw*
and orthostatic collapse tendency. In 5 of the 9 patients examined, there were
distinct signs of vegetative hyperexcitability, fine tremor of the hands, increased perspiration on the hands and legs, axillary perspiration, raised dermagraphism and suggestions of Chvostek's sign. The blood pressure value menuured during out-patient check-ups were all in the normal region, though «'
the lower limit, of the norm in 5 of the 9 patients examined. Orthostatic collapse tendency was not established either during out-patient visits or diii'lnK
in-patient observation by an internist. In 2 cases, myocardiac damage wfl*
suspected.
Abdominal complaints such as a feeling of fullness, pressure in the stomach
and liver region, and slight pain, were reported by 5 of the 9 patients. There
were 4 reports of disturbances in the gastric secretions, 3 of subacidity, one
of hyperexcitability and one radiographic finding of gastritis.
Very thorough investigations were conducted as part of repeated out-patient
examinations and in-patient observation to ascertain any liver damage.'1 Whilst
the liability reactions were uncharacteristic in all cases, the bromphthalein
test indicated slight delay in the dyestuff excretion in 2 instances. In 3 eases,
the liver biopsy produced pathological findings, these comprising 2 cases of
slight perihepatitic changes and in one case a fatty liver with inflammatory
symptoms and slight fibrosis of the liver. Owing to the clinical and histologic.nl
findings, it was suspected that a condition following virus hepatitis existed
in this instance. Deposits of ferrous and non-ferrous yellow-brown pigment
were established in this case, although these did not correspond to the grey,
non-ferrous pigment discovered by KALK and WI1VDHIRT in chlorophenol
intoxication, The excretion of erythrocytes in the urine of one worker whose
renal findings wore otherwise normal remained unaccounted for.
The psychopathological changes in the chlorophenol workers who were nil
psychiatrically and psychopathologically examined were especially remarkable.
In 6 cases the course could be observed over a 2-year period and there \vns
an opportunity for objective anamnesis investigation and experimental psychological examinations. 4 With a very large degree of agreement, n subjective
syndrome of complaints was reported by the patients under investigation, this
syndrome extending from the psychoneuropathic complaints in the region of
the extremities, cardiovascular and abdominal symptoms to the mental/
spiritual sphere, especially in the modes of behaviour associated with the vital
forces (BiiRG'ER-PRINZ). Considered in detail, there were reports of disturbances in the vital senses such as general .sense of weakness, feeling of
fatigue, indisposition, sense of insecurity, inner restlessness and a feeling of
illness. The basic mental mood was reported to be deteriorated and lowered
towards behaviour characterized by dissatisfaction or sullenness and irritation.
Not infrequently, a mood component of fear and unease was present. Changes
in affectivity in the restricted sense of the term were reported by the patients
.in the form of increased emotional reactions, irritability, tendency to fits of
temper and also a certain hebetude.
•Trot Dr. HORNBOSTEL and Dr. SCHONFELDEH, I. Mod. Univ.-KHnlk. HamburgKppcmlorf.
4
Our thanks arc dun nt this point to Dip], Psychologist W. von SCHUBERT, for conducting the tests (Rorschnch poychogram and Hamburg-Wcclislcr intelligence tent for
nclulta).
General loss of strength and reduced inner vitality and impulsion were
*?ni[itoms noted in each of the cases observed. The probands d^feU)ed reduc6!<m in initiative and interests, weak willpower, reduced efficiei^^and more
Njilil exhaustion in physical and mental/spiritual matters.
The subjective and objective anamnestic psychopathological picture is
farther complicated by a number of additional symptoms that are present with
* greater or lesser degree of regularity. Disturbances of the instincts occurred
In practically all cases. Thus, probands suffered from a sharp reduction in
{Money, and most also from decreased libido. The appetite had deteriorated
*ft'l occasionally there were substantial fluctuations in weight. The sleep and
*'<H'|I requirements of most probands were distributed in that there was an
iserwise in disturbances to sleep itself and concurrently, but less often, to
Hie process of falling asleep.
In certain cases, a decrease in mental capacity, especially disturbances of
Hw memory and perception, were mentioned. In the majority of instances,
alcohol intolerance was noted. Finally, mention should be made of hyper•I'Stlietic traits with hypersensitivity to light and noise. Marked fluctuations
the psychopathological syndromes described were reported in nearly all
ISON.
Cbanges were described in the intensity of the symptoms, . daily patterns
«>f fluctuation occurring in favour of early morning, late morning or evening
liuurs. in many probands, there were intervals of some days or weeks in
which they were practically free from complaints;
For completeness' sake, a further two less common phenomena are described. Two probands stated that the general symptoms and the polyneuroimthic symptoms were relieved for a varying length of time by the use of
colrt media (cold showers and washing with cold water). One proband reiwted abnormal, constantly changing eating habits, such as a wish for nothing
tint black bread, milk soup, or three litres of milk daily, there being no desire
tor food of other types at these times.
Compared with the multifarious polysymptomatic sxibjective pictures, the
objective psychopathological signs can be recorded at less length. In exploratory conversations, the majority of the probands displayed a distinct, slightly
depressed and subdued mood, which could be brightened only slightly or not
nt all. So far as impulsion was concerned, the patients examined gave an
impression of lif elessness ; their psychomotivity was feeble and fatigued. The
Impression was rather one of slight cerebral organic impulsion reduction than
»f inhibition. The affective modes of behaviour were occasionally notable for
their reduced reactivity and oscillation capacity, though also because of lability
nml decompensability. In 2 oases, pronounced hypochondria and, in one case,
slight but distinct alienation of the total personality were recorded.
The psychological tests are significant for the discovery of finer intellectual
performance shortcomings and psycho-organic disturbances in affectivity. In
Hie majority of the probands tested by the Hamburg-Wechsler intelligence
tost for adults (HAW1E), there was a significantly raised percentage of deKcneration, this providing a certain indication of an acquired decrease in
mental capacity. In the Rorschach psychogram, coartation of the experiential
typo, signs of weakened emotional reactivity, poor concentration, reduction
In tempo, sluggishness of the mental processes and a tendency to perservation
point to cerebro-organically governed changes.
DISCUSSION
On the basis of the findings in three independent groups of chlorophenol
workers, which together included more than 100 affected by diseases, a
characteristic clinical picture is provided, the most important features of this
being the following disturbances :
1. Following initial dermatitis of the face and symptoms of irritability on
the part of the conjunctiva ; often together with gradually developing acne
primarily in the region of the face, then the back of the neck, shoulders and
upper trunk, and in severe cases on the entire body, with comedones, pustules,
boils and patches of pigmentation. In several cases with severe irritation of
the mucous membranes of the face and the upper respiratory tract ; sometimes
with continuing blepharoconjnnctivitis,
2. In several cases, disturbances connected with the internal organs,
especially damage to the liver, with deposits of a nonferrous pigment as a
�246
.1 characteristic biopsy finding. In some cases, chronic bronchitis
instances of inyocardiac' damage.
3. Ill all cases, general fatigue and weakness principally affecting the pro*
iraal muscles of the lower limbs, often with pain in the muscnlation and IB
some cases paraesthesia and slight hypaesthesia. In isolated cases only, more
pronounced disturbances of sensitivity, slight paresis (implicit) and weakenim
of reflexes.
4. A psychovegetative syndrome with the following disturbances: Subjective:
Disturbances of the vital senses, disturbances in the basic mental mood nwl
affectivity, disturbance in impulsion, weakness of memory and concentration,
hyperaesthetic traits, vegetative dysregulation, tendency to orthostasis, slwp
disturbances, much increased sleep requirements, disturbances of the instind
sphere, reduction in libido and potency, and alcohol intolerance.
Objective,—psychopathological: Reduction in impulsion, subdepressive trail'
of u type characterized by genuine vital moments of depression, i disturbance*
in affectivity in the sense of a certain levelling-out, increased' excitability:
occasionally hebetude, hypochondria and personality alienation.
Experimental psychological, HAWIE: Increased degeneration percentage;
Rorschach psychogram: Coartation of the experiential type, signs of weakened
emotional reactivity, weakness of concentration, reduction in tempo, sluggishness of mental processes, tendency to perseveration.
The dermatological picture of the chlorophenol intoxication described shown
extensive agreement with the disease caused i>y chlorinated aromatic hydrocarbons as first described by HERXHEIMER and later by several authors
(see BRAUN, RISSE-SUNDERMANN). On the basis of the observation*
that chlorinated naphthalenes were principally responsible, \VAUER, ami
later TBLEKY, suggested the designation "perna disease" (.WJTZchlorinatcd
A r 4phthalene). TELEKY pointed out that the chloracne already described by
HERXflEIMER in 1890 was produced not by pure chlorine but by chlorinated
hydrocarbons or the simultaneous action of chlorine and tar. Further observations on perna disease made by MITTELSTADT, FLINN and .TARW1K,
DRINKER and collaborators, and GREENBURG and collaborators, indicated
that not only the skin symptoms but also fatigue, loss of appetite, giddiness,
and severe liver damage with acute yellow atrophy of the liver leading to
death can result from work with chlorinated naphthalenes. BAADER mentions epidemics at American shipyards during the Second World War. Jn
his description of the cases occurring in America and Great Britain, sometimes with a fatal outcome, TELKKY refers to the report of BROWN, President of the Halowax Co., New York (1937), that only the manufacture of
the higher stages of chlorination and the combination with cb.lorinn.tod
diphenyls and other substances led to severe damage to the health and in
some cases to fatal acute yellow atrophy of the liver. TEIjEKY also refers
to the animal experiments by 0.. K. DRINKER and collaborators to support
the view that only the higher chlorinated diphenylamines produce serious
damage.
The general symptoms in occupational chlorophenol intoxications are apparently more pronounced than those occurring with the lower chlorinated
naphthalenes employed earlier. This fact was also observed by TRUHATJT
and collaborators amongst workers who had been using pentachlorophenol
for wood preservation, as well as KUBOTA in Japan, who mentions multifarious disturbances of the autonomous nervous system and who observed
several fatal cases. In all three German groups of chlorophenol intoxication,
liver damage was established, the damage that was most pronounced and
studied most intensely being that found amongst the cases of disease occurring in two Mid-Rhenish companies (HERGT, KALK and WILDHIRT). I"
all the groups, several cases of chronic emphysema bronchitis and inyocardiac
damage were found, although these disturbances did not occur yearly so regularly as the pronounced fatigue and neuromuscular weakness, which we
observed in all our patients. The psychosyndrome described was equally
regular, this being found not only by us but also by .TANZARIK and RTCHERT
to a completely identical degree amongst the Mid-Rhenish workers.
The psychopathological syndrome could be distinguished with a sufficient
degree of certainty by differential diagnostics from endogenic psychosis,
especially mild cyclothymia diseases, neurotic personality developments and
organic psychosyndroines of different etiology, and somewhat presenlle or
247
Wclirosclerotic processes of degeneration. Phenomonologicall^Mhe relationi'ilpn to the pseudoneurasthenic syndrome, which is describecffn connexion
with a large number of occupational intoxications such as those caused by
toid, carbon monoxide, manganese, thallium, arsenic, carbon disulphide, tri'liloroethylene, etc. (for relevant articles, see BORBELY, von HATTINGSWKKU MEGGENDORFER, MOESCHUN, PBNTSOHEW, TELEKY)—and
«^|K'cially the relationships to particular endogenic mood conditions—are
"livlous. Lowering of the vital level, moments of depression, vegetative symptoms, and, not least, fluctuations in intensity can be observed predominantly
in endogenic-depressive conditions. On the other hand, alcohol intolerance,
bypnraesthetlcally excitable and polyneuropathic traits influence the differential
diagnostic aspect more in the direction of an apparently exogenic condition.
Tin1 somewhat older psychiatric literature should be borne in mind in this
fwnexion (MEGGENDORFER, STBRTZ), this placing the neurasthenic
M'wlrome quite definitely in the pattern of exogenic symptom complexes. Not
li'iist, reference should be made to the phenomenological relationship of our
'^nervations to the (exogenic) hyperaesthetic-emotional conditions of weakness of BONHOFFBR, which, from the psychopathological aspect, have significantly been designated by BWALD as no longer heteronomous but homonomous in the sense propounded by KLEIST.
Despite the phenomenological relationships discussed, the psychopathological
delayed syndrome of the chlorophenol workers scarcely corresponds completely
with any of the known clinical pictures. In any case, the question of a special
I'N.vchic-vegetative delayed intoxication syndrome, which was discussed by
Nl'IlOGELBERG in connexion with observations on persons suffering chronic
occupational damage from military poison gas, also demands consideration in
view of the observations mentioned in this paper.
It has been possible to rule out psychogenic-neurotic moments so far as our
subjects are concerned, provided that individual neurotic conditions, i.e.
tlmracterogenic and experiential situative data are involved. Two of the
nine probands exhibited considerable psychopathic or neurotic structural
elements. However, it was easily possible to separate these two probands from
Hie other completely or largely non-neurotic cases. Certain "collective-neurotic"
factors have, in our opinion, to be taken into account as an unfortunate but
practically unavoidable fact in all group investigations but especially those
Involvirig etiological evidence (SPIECELBERG). Reactions of this type have
also been observed in our cases in the sense of a superimposed psychogenic
accessory with, as it were, "physiological" but not inadequate, individualneurotic (complex-determined) idemnification wishes. The psychopathological
analysis of the individual case and the comparison of the findings in each
Instance with such independent collectives of the same etiology afford sufficient protection from authoritative and scientific false assessments. Despite
the long course, the prognosis of the psychopathological intoxication results
appears favourable. Although technical aspects of the pension situation have
not yet been finally clarified, there was, on the whole, a certain subjective
Improvement in the symptoms, or else they remained static. We have not
observed any objective deteriorations, except for the momentary intensity
fluctuations. The experience of the Mainz Nerve Clinic (RICHER) and the
impressions of works medical staff (KNECHT) suggest a benign course of
acute and chronic intoxications, provided no toxic parenchyma damage, as
such, influences the prognosis unfavourably.
It seemed appropriate to attribute the toxic action to the high-chlorinated
chlorophenols, this view being supported by animal experiments conducted
I'.v MACHINE and THOMAS, H. KITZMILLER, a series of other investigators
(KEHOR, DE10HMANN, GRUEBLER, BOYD, McGAVACK, TERRANOVA,
1'ICCIONE cited ace. to von OETTINGEN) and also our own animal experiments. KTMM1G and SCHUL55 were, however, able to show that the use of
non-industrial, analytically pure, high-chlorinated chlorophenols (trichlorophpnols, pentachlorophenols) does not lead to the characteristic symptoms of
chlorophenol intoxication.
Animal experiments were carried out with a view to discovering the noxae
causing the symptoms. The rabbit's oar proved to be a suitable test object
since it is possible to produce the changes on this with the substances causjng
chloracne, these changes closely resembling those of human chloracne
(HOFMANN and NEUMANN, BRAUN, LANDES, etc.). Brushing with a
�248
substance which is active in tills respect leads at first to patches of
in conjunction with reddening, swelling and flaking; then, some dlPrlntfT,
hyperkcratosis linked with the follicles and also small cysts occur, the*
being easy to record histologically, as well. In addition to the brushing exi*1''ments, tests were carried out on rabbits to determine the general toxicit;.
whilst cats, too, were used for testing a number of substances. In these tod*,
it was found, in corroboration of findings derived by OETT1QL and also IIOFMANN from similar cases of intoxication in a large chemical works !i
southern Germany, that the substances producing chloracne possess inarM
liver toxicity in rabbits. It was possible to trace effectively the damage t*
the parenchyma of the liver intra vitam with the micro-modification of U*'
bromsulphthalein test given by HOITMANN and OETTKL. In autopsies, dlf 1
fuse steatoses and extensive necrosis of the parenchyma of the liver vfin
found. The investigations, which have already been reported (SCHULZ 1950;
KIMMIG and SCHULZ 1957) led to the following results:
The effective substances must have occurred in the alkaline hydrolysis of
1,2,4,5-tetrachlorobenzene to 2,4,5-tricblorophenol, this having been carried
out technically under pressure at about 180°C in the presence of metlmnol
and caustic soda solution. However, it was not the tricblorophenol itself Imt
the by-products that formed in small quantities in the course of the pressurized
phenol process that were regarded as the causal noxae; for it was not possible
to produce any of the above-named changes on the rabbit's ear with pure,
repeatedly distilled 2,4,5-trichlorophenol or with 1,2,4,5-tetrachlorobenzene,
although they did occur with the tricblorophenol used technically.
Since the isolation of defined compounds from the residue occurring in the
distillation of technical tricblorophenol was not possible at first, compound*
were synthesized by chemical means and given to us for testing on animiil*
where there was a certain likelihood that these substances may occur as byproducts in the saponification of tetrachlorobenzene to trichlorophenol. The
substances initially available were various chlorination products of tlie
diphenyl ether and the dibenzofuran (diphenylene oxide).
Although the diphenyl ether and its IX to 4X chlorinated derivatives, and
also dibenzofnran and monochlorodibenzofuran were ineffective in experiments
on animals, 3X and 4X chlorinated dibenzofnrans, even in concentrations as
• low as '0.05%, produced the symptoms mentioned on the rabbit's ear1. Single
closes of 0.5 to .1 mg/kg administered orally produced severe liver damage in
rabbits, this leading to the death of the animals in most instances.
The clinical observation of a laboratory assistant engaged elsewhere, wlio
fell ill with severe chloracne after exposure to tetrachlorodibenzodioxine,
indicated the chlorine derivatives of the dibenzodioxine.
Tetrachlorinated dibenzodioxines, especially 2,3,6,7-tetrachlorodibenzodioxine,
were highly effective on the rabbit's ear, even in low concentrations. Tlivoe
brushed applications with 0.01-0.005% solutions (in polyglycol) were sufficient
to cause severe areas of inflammation and follicularly arranged hyperkeratosis.
When administered orally, single doses of 0.05-0.1 mg/kg body weight led to
severe liver damage and generally the death of the animals.
The assumption that 2,3,0,7-tetrachlorodibenzodioxinc is actually of considerable importance in causing the chloracne diseases'occurring in the chemical'
works received further substantial support from the chemical angle. It wns
possible to prove that this compound is formed from two molecules of sodium
trichlorophenolate in association with the cleavage of NaCl under the pressure
and temperature conditions prevailing in the autoclave.
It was, 'moreover, possible to isolate the named tetrachlorodibenzodioxine
from the by-product occurring in the technical pressurized phenol process
(alkaline sanonification from tetrachlorobenzene to trichlorophenol). To
prove that 2,3,6,7-tetrachlorodibenzodioxine is capable of producing alterations
in the form of chloracne not only on the rabbit's ear but also on human skin,
one of us (SCHULZ) carried out a test on his own body. Two brushed applications of a 0.01% solution on a circumscribed skin area of the forearm led
within two days to a mild dermatitis, then some days later to a follicular
hyperkeratosis and comedones, these also being easy to record histologically.
The etiological significance of this substance for the diseases described here
seems to us to be sufficiently evidenced by this experiment. However, it is not
impossible that other chlorinated aromatic compounds with highly toxic
characteristics may occur in this technical process, these possibly not having
been so far identified or tested in experiments on animals.
249
The experimental, and clinical findings are an impressive ^•fcmp of the
antities us
that, in works pathology, substances occurring in smt^Bua
'ItrixhictR in chemical processes can be of importance. I n^Sper e n t s to
nperim
<il*oover the origin of frequency occurring occupational intoxications, this
'Iwimiiit should not be ignored. If it is possible to prove that impurities
•'Inched to the main product should be regarded as the causal noxae of an
r«vu)>ational disease, this constitutes an important prerequisite for successful
'iaxy.
tn our own special case, it was possible, by changing the plant chemistry
»*|i(>ct of the manufacturing process, to prevent the formation of the highly
l»*k', multi-chlorinated dibenzodioxines and dibenzofurans. Since than, 2,4,5trlehloropbenol is again being manufactured in this works and processed into
Hit1 herbicide 2,4,5-trichlorophenoxyacetic acid, without symptoms of intoxication of any kind occurring amongst the workers.
A TECHNIC FOB TESTING ACNEQENIO POTENCY IN BABBITS, APPLIED TO THE
POTENT AONEGEN, 2,3,7,8-TETBACHLOHODiBENZO-p-DioxiN*
B. Linn Jones, M.D. and Helen Krizek, Ph.D.
Pollicular hyperkeratosis is an important feature of the occupational disease
known as chloracne, which is characterized by the appearance of papules,
comedones and cysts after exposure to industrial materials containing highly
chlorinated diphenyls, highly-chlorinated naphthalenes, and other chlorinated
aromatic compounds. A characteristic epithelial byperplasia and hyperkeratosis
can be produced on the inner surface of the rabbit ear by such compounds
(1, 2), and a difference in intensity of response has been noted and suggested
ns a basis for comparative tests (2). Experimental studies heretofore (1-7)
have been, however, directed chiefly to the ability or the failure of various
materials to produce this effect in experimental animals and in man, and in
delineating its gross and its histological features; these studies have used
cither material of unstated origin, or else mixtures (e.g. Halowax, 1014). We
Imve attempted -to study the phenomenon of acnegen-induced hyperkeratinIzation on the rabbit ear in a quantitative fashion by recovering and weighing
the keratin formed after applying known amounts of a single, well-characterized chemical compound under controlled conditions. To recover keratin a new
Icchnic was developed based on the resistance of this material to digestion by
pepsin. As test compound we have chosen 2,3,7,8-tetracluorodibenzo-p-dioxin,
which has been reported to be so potent that painting the rabbit ear three times
with a 0.05%-0.001% solution was sufficient to produce the acneform response
(7). With a compound of such potency, the expected effects could be produced
without the necessity of applying the material in an ointment or as a crust,
circumstances which would have made very uncertain the quanity actually in
contact with the skin.
EXPERIMENTAL
Preliminary gross and histologic observation indicated that 0.3 inierograms
npplied to the rabbit's ear gave, by gross observation, a minimal follicular
plugging whereas 0.02 micrograms caused no observable effect. Accordingly, 0.3
inierograms was chosen as the lowest dose; in addition dose levels of 1.0, 3.0
and 10.0 micrograms were studied.
Seven mature, white, male rabbits were used for each dose level, except in
the first studied (0.3 micrograms) for which only six were used. Seven days
after wax epilntion t, of the inner surface of the ears, 1 ml. acetone solution of
the compound was applied to one ear of each of the rabbits, and 1 ml. acetone
to the other. Special effort was made to distribute the liquid uniformly over
* From tho Section of Dermatology, Department of Medicine, University of Chicago,
ChicaRO 37, Illinois.
_
, ..
This research was supported by the Research and Development Division. Office of the
Surgeon General. Department of the Army, under Contract No. DA-40-007-MD-III
and by United States Public Health Service Medical Training Grant No'. 2A-ti20,S(Cl).
Presented at the Twenty-third-A-imnar-Meeilng of The Society for Investigative Dermatology, Inc., Chicago.. HlrTTune 26, 1962.
t With "Improved Zip."
k.
�251
250
the entire inner surface, and to aid in securing uniformity the |
The complete removal of all non-keratinized tissue from the bJM^y by pepsin
:-*te«tUoii was confirmed by histological section of a keratin disc.^H. 10).
divided into three applications, made on successive days. The righriif »t»
•;'• In Kcneral, the keratin discs recovered from the treated amHils were less
used as control for some rabbits in each group, and the left for others. K«'
;(irag)li> and thicker than the controls. At lower dosages the follicular keratin
teen days after the first application, three biopsy samples, extending tlireisS
;..•*»* umially observed as tree-like forms representing casts of the multiolobated
the cartilage, were taken under procaine anesthesia, with a 9 mm. pnncli. <**
• *rfi»Cf>ous glands. (Figs. 5A, 6, and 7). These follicular projections were not
sample was taken from the middle, one from the posterior and one from (1*,
in the controls. (Fig. 5B). At higher doses the keratin in the follicles
anterior area of the ear, at a level about 15 mm. distal to the notch of <w
as a larger structure of smooth, oval shape typical of comedones.
ear. (Fig. 1) The discs were washed free of blood without delay. Under « &*
secting microscope at OX magnification, the moist samples were plucked tnvf
«. 8).
Wi'lghts of keratin recovered from the biopsy samples are given in Tables
any adhering hairs and clots; and the cartilage was removed with sharp #S»
t * ml 2. In Table 1 are listed average weights for the three biopsy specimens
Bard-Parker scalpel. A thin coating of white petrolatum was applied In »S*
token from each ear. In subsequent experiments biopsy specimens taken from
epidermis. Each disc was floated in a 50-mm. petri dish containing 10 ml. Ol'i
•he anterior, the middle and the posterior portion were distinguished, in an
pepsin (Worthington Biochemicals "2X Crystallized" product) in 0.24 K lift,
*ffort to assess the importance of the site of biopsy removal with respect to
and incubated 4 hours at 37°0., at the end of which period the keratin din"
was gently lifted out, free of any undigested dermis by inverting and (.'pni'f
Mie weight of keratin recovered.
DISCUSSION
irrigating it, and resuspended in 10 ml. 1:1 V/V ethanol; diethyl ether inixtuf*1
A light aluminum-foil cup 0 mm. in diameter, with perforated bottom, I"?
for each rabbit there was calculated a value for the relative increase in
viously washed with ether and weighed to 0.02 mg., was brought close toll*;
. fcvlKht, (T — G)/G, where T = average weight for the three biopsy samples
disc, which was then gently transferred, with follicular projections up, wllk';
• from the treated ear and G — the corresponding average' for the control.
the aid of a scalpel handle, into the cup. After four hours at room temiwr*ture in the covered petri dish, the solvent was aspirated. A similar lenelilaf ||;;>* Thews values and their average for each dose level are plotted (Fig. 0) against
On1 dose. The averages vary approximately linearly with the logarithm of the
with 10 ml. ether was made, after which the cup in the covered petri dish w»*
'lose, but individual (T — 0)/0 values at each dose deviate widely. The deviadried
mg. in vacuo overnight. The cup and sample were weighed to the nearest <M&
Hon may be due, in part, at least, to failure to secure uniform spreading of
thi« aenegen, in spite of the precautions taken. Loss of keratin during manipuAdditional biopsy samples were taken on about half the animals and examlation of the samples may be another, source of deviation; however, such an
ined histologically after routine homatoxylin and eosin staining.
error would be expected to be most important for smallest amounts of keratin
The following method was found convenient for preparing a small quantify
handled (i.e. the controls). An inspection of the control weights does not
of 2,3,7,8-tetrachlorodibenKO-p-daoxin.1 The sodium salt of 2,4,5-trichloroplieiKi!
reveal a corresponding spread of values. Further, the probability of losing kerwas prepared by dissolving 1.6 g. metallic sodium in 25 ml. absolute ethanol Is
atin was greater for the rabbits showing greatest response, because these
a 100-mI. round-bottomed flask, adding 20 g. of 2,4,5-trichlorophenoI (Eastmai
tonded to have some loosely adhering scales and large comedones which might
"Practical Grade," recrystallized from petroleum ether) and distilling off ttrf
IIP expressed and lost in the course of tissue removal and subsequent processethanol. The salt was cautiouly heated until the copious evolution of nci'5
Ing; actually, therefore, the spread of response may be larger than our results
fumes which took place at 200-250° C. subsided, after which the flask wn*
Indicate, In addition, gross observation indicated that some rabbits gave a
kept at 350-400° C. for 30 hours. Two zones were found in the distilling headweaker response than others at the same dose level. We feel, therefore, that
The lower of these, a dense, compact mass, had a melting point of 230-300° C
the values reflect a real individual variation. Inspection of Tables 1 and 2
and was only slightly soluble in chloroform. This was the crude acnegen. Tit*
upper zone of coarse crystals soluble in chloroform was probably a tetracliloroTABLE I.-RECOVERY OF KERATIN FROM EARS OF RABBITS AFTER APPLICATION OF 0.3 MICROGRAMS 2,3,7,8benzene formed in a competing reaction. Two batches of the crude acncgon
TETRACHLORODIBENZO-P-DIOXIN
were combined and recrystallized twice from anisole to yield 0.25 g. product.
Its synthesis is represented in Fig. 2.
Milligrams of keratin per 9-mm
Analysis. Calculated for CioI-LO.CU : C, 44.76% ; II, 1.25% ; 01, 44.05%.
biopsy sample >
Found: O, 44.31%; H, 1.40%; 01, 44.18%," The melting point wan
Control
Treated ear
205-300° C.; literature values for 2,3,7,8-tetrachlorodibenz;o*?)-dioxin prepared
Rabbit
by chlorination of dibensso-p-dioxin: 205° C. (8), 320-325° C. (9). The infrared
0.97
spectrum showed a doublet at 1310-1322 cm."1 in the range reported for n
I
0.98
.1.07
series of clibenzo-p-clioxin derivatives (10). The ultraviolet absorption spectrum
<9
..
1 31
of the compound in absolute ethanol had a maximum at 233.m/t find one at 307
1.18
1.88
66
1 20
m/i; the respective molecular extinction coefficients were 46,500 and 4,250.
. ...
1.13
1.31
RESULTS
1
Histological sections showed a characteristic hyperkeratosis of the folliculnr
epithelium and a marked hyperplasia of the surface epidermis. At low dose
level some sebaceous cells were present;. at high dose level, only a few were
seen, and the follicle was filled with a keratinous mass. (Figs. 3 and 4). There
was marked thickening of the epidermal keratin, although this was only rarely
observed on the slides, presumably because it was lost in the cutting and processing.
1
Following the practice of Chemical Abstracts we describe our command no 2,3,7,
8-tetraehlorodibeno-;)-dloxin. From a comparison of its preparation with the mode of
oridin in the Industrial process discussed by Klmmig and Sehulz (7) we conclude that
it IB the same as their 2,t),6,7-tetra-chlorodibenzo-p-dioxin.
" Analyses by Micro-Tech Laboratories, Skokie, 111.
Average of three samples from, respectively, anterior, middle, posterior.
reveals some tendency for greater response to occur in animals for which control values were high, but this tendency is by no means clear-cut. We found no
correlation between intensity of response and weight of the animal.
There appears to be no consistent difference in the three positions, anterior,
middle and1 posterior, along the line of section, either for the control or for the
treated eaz , with respect to weight of keratin recovered. Average value for keratin recovered from the controls was remarkably constant: 1.11 ± 0.10, 0.85 ±
0.18, 1.02 ± 0.26 and 1.02 ± 0.16 mg. for the rabbits treated with, respectively,
0.3, 1.0, 3.0 and 10.0 micrograms.
45-362 0—70
17
�253
h
of acnegonicity of various m^e^Nu'thi11?3'*mlsibst be used for *
*^
and 2,3,7,8-tetTachlorodibenzolfhovin tl H
™ «nce is applied ,_- „,.
avoid the compltottoKff^^^totoe^auch
a procednTUfc,
number of animals necessary Control ' " l d u a l , fences, and redllc(, lSrt,
from untreated animals Three b i o n w . n i C°U d be secured taaepcnden*.
ysis^of the data showed"S vS Sfflr^ot^..BO«««7?««|-»':
ie (the nikl(!i+s
on the nvcnvt'
tlons. Thulaff O^an^n^n^"^^^^1- but ««re were"smne
as showing folllcular dilatat
of keratin. At 3.0 micr0g^7^T^^^ r.°.ur sh,owed increase in wolfM
loiiIenHy 0.3 micrograms in acetone, died at the end of JMays, and two
receiving, respectively, 30 and 2 micrograms died witl^^. week. How,' *»*r, tliere were undoubtedly other contributing factors, because in all animals
«mi<Hl subsequently no toxic symptoms were observed.
r
SUMMAHY
I. HyperkeratimiKation induced on the rabbit ear by the aenegen 2,3,7,8i»trnclilorodibenzo-p-dioxin is studied by n new technic based on weighing
i*>i'i)tlf] recovered after careful pepsin digestion.
, 'A When applied in acetone solution to the rabbit ear, 2,3,7,8-tetrachloro*f>vnzo-;>-(iioxin is effective at micrograin levels. Effect of dose and individual
ittffi'rnnces in response are discussed.
:. 'A. The new technic, using 2,3,7,8-tetrachlorodibenzo-p-dioxin, is suggested for
, *Mni>nring acnegenicity of various substances.
BEFKBENCE8
Milligrams of keratin per 9-mrn. biopsy sample
1. ADAMS, E. M., IEISH, D. D., SPENCEK, H. 0. AND HOWE, V. K.: The response
of rabbit skin to compounds reported to have caused acneform dermatitis.
Inc. Med., Ind. Hyg, Section, 2:1, 1941.
2. HOFMANN, H. T. AND NEUMANN, AV.: Eine Methode zur tierexperiruentellen
' Pi-titling der Hautwirkung chlorierter Naphthaline. Zbl. Arbeitsmed., 2:
169, 1952.
.1. BBAUN, W.: Ohlorakne. Editio Cantor, Aulendorf i. Wurtt, 1955.
4. HAMBEICK, G. W., JK. AND BLANK, H.: A microanatonical study of the
response of the pilosebaceous apparatus of the rabbit's ear canal. J.
Invest. Derm., 26 : 185, 1956.
5. HAMBRIOK, G. W., JB. : The effect of substituted naphthalenes on the 1 pilosebaceous apparatus of rabbit and man. ,T. Invest. Derm., 28: 89,1957 .
(1. SHELLEY, W. B. AND KLIOMAN, A. M.: The experimental production of acne
by pentand hexachloronaphthalenes. A.M.A. Arch. Derm., 75: 689, 1957.
7, KIMMIO, J. AND SCHULTZ, K. H.: Berufliche-Akne (sog. clilorakne) durcli
chlorierte aromatisclie zyklische Ather. Dermatologica, 115: 540, 1957.
8. TOMITA, M., UEDA, S. AND NABISADA, M.: Dibenzo-p-dioxin derivatives.
XXVI Synthesis of polyhalo-p-dioxin. Yakugaku Zasslii, 79:186, 1969,
Ohem. Abstr., 53 : 13152, 1959
0. SANDEBMANN, W., STOCKMANN, H. AND OASTEN, R.: Tiber die Pyrolyse des
Pentachlorphenols. Ohem. Ber., 00: 600,1957.
10. NARISADA, M.: Infrared absorption spectra of aromatic ether compounds.
II. Characteristic bands of dibenzo-p-dioxin derivatives. Yakugaku
Zasshi, 79:183,1959; Chem. Abstr., 53: 10967,1959.
DISCUSSION
other, comedones were observed; but the response in terms of weight recovered
was less than for two which gave the gross impression of follicular papules.
Attention should bo called to the great toxicity of 2,3,7,8-tetrachlorodibenMp-dioxin. Kimmig and Schulz (7) reported that 0.5-1.0 mg. per kg. orally was
lethal to most of their rabbits. In our preliminary experiments a rabbit receiv-
DK. PETEH IfLESCH, Philadelphia, Pa.: Since the criterion of acnegenic activity appears to be the conversion of the sebaceous cells into keratin-forming
cells, I would like to ask, what did you see in the histologic sections?
Dn. E. LINN JONES, (in closing) : In the paper we will have histologic sections of treated glands and the digested keratin disc.
In regard to histology there is varying response, depending on the amount
applied. With lower doses there is conversion of the cells in the follicle to kerfitinizing squamous cells, with occasional remnants of selbaceous cells in pockets here and there. With larger doses no sebaceous cells can be found. The
entire follicle is converted into a large keratin-filled papule.
�STUDIES OF THE CHICK EDEMA DISEASE
ljy' 2. PREPARATION AXD BIOLOGICAL EFFECTS OF A CRYSTALLINE
CHICK EDEMA FACTOR CONCENTRATE
BY
U. F. FLICK, D. FIRESTONE AND J. P. MARLIAC
Reprinted (rom I'our/niY SriKM'R: Vol. XL1V, No. 5
September, 1965
(255)
�256
m
WH, .
257
phcnanthrene derivatives were collected,
combined and concentrated. The concentrate was chromatographed on columns of.
Cclite:H 5 SO,:fuming II-SO, (1:1:1) and
cluted with CCI4. The foregoing procedure
was a modification of AOAC method 24.111
(a) (Horwilz, 1.960). The CC1, eluates
were re-chromalographett on Merck Alumina, and individual fractions were collected and checked by ultraviolet speclrophotomctry. Fractions eluting with 10%
dielhyl ether with absorption spectra of
naphthalenes (absorption maxima in the
range of 240-250 my..) were combined and
:,,.,«./.
re-chromatographed on Merck Alumina
I?' The purpose of this paper is to report
with isooctane as the ekiant. Individual
fcW recent studies on the purification and
fractions were collected and bioassayed,
,|Wo1ogical' effects of a concentrate of the
and a highly toxic crystalline fraction
|chick edema factor (CEF) isolated from a
(CEF concentrate) weighing 79 mg. was
|J crude toxic fatty material (TFM) known
obtained.
'f to produce the chick edema disease (Flick
The CEF concentrate was examined by
Itl al., 1962, 1963). Preliminary data are
microcoulometnc and electron capture gas
:-included on the effects of CEF-containing
.; material on egg halchability and develop- chromatography. A microcoulometric gas
chromatograph (Dohrmann Manufacturing
ment of the chick embryo.
Company, Palo Alto, California) was used
at a column, temperature of 250°C. with a
MATERIALS AND METHODS
l
Preparation of CEF Concentrate. Eigh- 6-foot X /\ inch (i.d.) aluminum colStudies of the Chick Edema Disease
teen pounds of unsapom'fiable material was umn packed with 20% Dow-Corning High
2. PREPARATION" AND BIOLOGICAL'EFFECTS OF A CRYSTALLINE
Mated from 180 pounds of toxic fatty Vacuum Grease on ackl-%Yashed ChromoCHICK EDEMA FACTOR CONCENTRATE
material (TFM) by saponificatioi^and ex- sorb W. Details of this technique were detraction- of the unsaponifiable fraction with scribed previously by Firestone ct al.
D. F. FUCK, D. FIRESTONK AND J, P. MAUI.I/U:
petroleum etheridiethyl ether (1:1, v./v.). (1963). For electron capture detection, an
Divisions of Ki/lrilioii, Food Chemistry, anil Taxicologlcal Kvaluatiow, Footl and Driip. Administration,
The petroleum ether was redistilled and Aerograph Hy Fi (Model CiOOft) gas chroWashington, D.C. 2020J
:
the solvent boiling between 40-60°C. was matograph (Wilkins Instrument and Re(Rercirwl for puMirolioil February 23, 19fiS)
collected and used. The .unsaponifiable search Inc., Walnut Creek, California) was
INTRODUCTION'
1959; Wool (on and Alexander, 19S9; Ames | fraction was chromalographed on Fisher used. This instrument was equipped with a
M)LLO\V)NG (he early report's of the ct- al., 19(50; Harinan ct at,, 1960; Yarlzoff |
; Alumina (Cat. No. A 540) essentially as tritium source electron capture detector, A
chick edema disease (CED), Snngcr f.l al, 1961; Wool (on rf til., 1962; and
described by Yartzoff ct al. (1961). Frac- stainless steel column, 5 feet X ?4 inch,
<V til. (1058), Puller ct a!. (1059), Allen Wootlon and'Comx-hene, 1964).
tions with ultraviolet absorption spectra packed with 5% SE-i2 silioone gum rub(1961) ami Allen and Lalich (1962) rc)!rew c.l al. (1959) reported that broiler
that matched those of naphthalene and ber on 60-80 mesh Chromosorb W, was
porlcd toxicologlcal studies which cMnb- chicks developed .(lie diswtsc when fed a
pcnanthrenc derivatives were combined and used at. a column temperature of 202°C.
chromatosraphed on Merck Alumina (Cat. and a nitrogen (carrier gas) flow rate ol
lished iha.l fb<! disease differed /coin known fraction purified ,3200-fo!<J I earn (he origipoultry diVea.U'r-. A number of invcsti^alors nal starting material. A fraction purified
No. 71707). Foreruns were eluled with 50 ml./minute.
reported on the purification, isolation and 10,0'00-fold which elicited CED was reportpetroleum ether. Additional fractions that
Feeding Procedure and Biochemical Mrtltpartial chemical chnracleri/.iilion of toxic ed by Friedman ft al. (l'J59). Yarlxoff ft
clutcd with 5% dielhyl ether and that exods. Day-old Single Comb While Leghorn
factor (Urew ct til., 1950; Kricdrmm ft al., al. (1961) reported that a fraction purified
' hibited the spectra of naphthalene and
>,000-(old from a low potency commertrlolcin produced hydropericardium
) when fed to day-old chicks at 50
». in the diet, and resulted in death at 1
:rr,n. WootlOQ and Courchcne (1964)
.band one fraction (designated a 3.02)
5*Mch was highly toxic for the chick. These
}J»wkcrs estimated that ingestion of 5 \>.g.
£it the « 3.02 fraction was enough to kill
ISM chick. Firestone et al. (1963) reported
|$*l signs of the disease were elicited by
|«W fraction fed at 0.1 p.p.m., which subthe report by Yartzoff et al.
I
�zrja
Corn meal, yellow
Wheat flour middlings
' Alfalfa meal
Linseed meal
Vcas(
NaCI, iodized1
Cod liver oil
Cottonseed oil
% (W/VV)
36
15
1
8
. 1(1
3
3
1
I
16
1
Tor composition of A.O.A.C. salt mixture, sec
report by Flick ct al. (1903).
1
This addition gave a total of 1.99% NaCI in the
diet,
Hotel/ability Study. For this pn
study, commercially available
Leghorn fertile eggs were injected
test materials into the yoik sac pri*
mcubation according to the lechniqst
cnbed by McLaughlin ct al. (1963).' '
trol eggs were injected with 0.1 ml.
oil. Experimental eggs were injected
10, 20 or 50 ,*!. of the unsaponWable
tion of crude TFM from which (he a
line CEF concentrate was prepared.
"•ere candled each day after the 5th 6tf«
incubation. Dead embryos were rcnwwj
and examined for gross malformations. Aft*'
incubation, unhatched eggs were opened to!
gross observation. Chicks which halcM
were observed for 3 days.
cockerels were fed ad libitum and provided
with fresh water at all times, They were
handled and maintained as reported previously (Flick el al., 1963).
RESULTS AND DISCUSSION
The composition of the basal diet used is GLC Analysis ofCBF Concentrate. »
G»l
shown in Table 1. The crystalline CEF chromatographic separations of crystal!*'
concentrate, dissolved in chloroform:ether, Ui* concentrate were obtained by
"
was added to the cottonseed oil, and the oil both the microcoulometric and elo
was warmed with stirring to remove sol- capture techniques. A typical electron ap• vent. The CEF concentrate was fed at lev- ture chromatogram of our purified prepaid
els of SO and 200 p.p.b. for three weeks.
Bodj' weights and feed intake were determined and at weekly intervals chicks burS'' P
, "' Prcsb>'tc™" HospUal, KC..
were anesthetized with diethyl ether, vol- burgh, Pennsylvania for detection of COHW.
?CUy lnl ° lhc *>»< ha. bm
ume of hydropericardium (HP) was mea- stud j
CggS (pcraon
on rom Dr. McLaughlin) wi.l, " com~
sured according to the procedure outlined
.on I'" T 1?'°°°
approximUcl,
00 chcmic,,s, lK,.a , nk anf)
"
by Douglass and Flick (1961), and postC njU cti
mortem observations were recorded. Blood
d
l'" , ° " °" «'lth several differ!
samples were withdrawn from the right ven- stud es. Reefed materfals were found to conMrt«ntly rema.n m the yolk nnd no great diflku'l'f.
. I ride with needles and syringes moistened ^encountered which were ^ ^^
with heparin, and the following tests were
performed: microhematocrit (Natelson, of
fraction is a liquid residue
fatty material which is a sli'J l»|.
1961), whole blood glucose (Somogyi, of
1952) and total plasma proteins and plasma ionoT
protein fractions (Gornall el al., 1949). oT of ,
a(
°;;H, , •
™
Other blood samples were allowed to clot, tamed hydrocarbon residue nn,| fattj- ac
and copper determinations' were made on < a , o n products m addiMon ,o ( he 'chi
the sera,
' Samples of scrum were colledcd in acidrinscd tubes, frozen under carbon dioxide and sent
,. ^., *.,*. uipiijjoim.ianic. material .contained <0.5% (v./v) of prcdpitable material.
Sec Brew et al. (1959), Friedman ct al. (1950),
and Woolton and Alexander (1959).
MINUTES
P- Fw. 1. Gas chromatogran. of (a) toxic concentrate, (b) Procter & Gamble a 3.0*• »»* J> P££r
K'Cunhte a 3.1J. See text for details on instrumentation. The numbers adjacent to chrom.logi.phic pea
lit retention times relative to aldrin.
lion is shown in Figure l(a). The chromatogram revealed that within a 70 minute
nin at least eight components were present
in the preparation. Woollen ct al. (1962)
Isolated two components, which were designated o. 3.02 and a" 3.17 (gas chromatographic retention lime relative to methyl
arachidate). Each of ihese two component*
contained a high-melting fraction that produced edema and a low melting fraction
that did not produce edema. Our toxic concentrate was chromatographically compared to the low melting fractions of Woollon
ct al.3 The peaks of the. latter two coinpounds, obtained with the electron capture
technique, arc shown in Figure l(b) and
1
Kindly supplied by Dr. N. It. Artman, Procter S; Gamble Company, Cincinnati, Ohi:>.
l(c). The chromatogram of our CEF concentrate shows a. peak (retention time of
10.S relative to aldrin) having the same
retention time as the a3.02 inactive isovner.
The relative peak area (r/c of total area
of the chromaiographic peaks) of Ihe 10.5
component was estimated by using the retention X peak height method of Carroll
. (1961). By this method the 10.5 peak obtained in the microcoulometric chromatogram represented about 20^ ot total components.
Chick Response. Results obtained on
weekly weight gain, food consumption,
feed/gain ratio and calculated consumption
of the CEF are tabulated in Table 2. The
weighl gains among control bird? increased
at a fairly constanl rate each week. Weight
�260
261
TA
T/\fflWr,—Weekly weig/il gain, feed eoiisimip/io>i, feed/gain ratio and iiiecslion of crystalline CEF c<m«»*i*l
CEI'
level
No. of
chicks
Menu
WClRllt
1
gnin
g-
0
50
200
52
47
47
39
36
38
23
20
22
58.4 + 2.1
27.5±3.0
'•10.6±2.7
1
Menu val ucs./cnce
. . . , , , *,J
f8-inrliM'c/»...
Feed/gain
CEF concentrate conBi«i|
ratio
''•I
1
. Week!
38.5
42.0
42.7
Week Z
S2.6
80.5
72 1
36.7±1,6
29.3±2.0
39,0±2.2
0
50
200
No. Of
deaths
_
£.
27.4±1.1«
29.8±1.4
2.8.0±1.4
0
50
200
Diet
consumed'
*—
..i.^,
0
0
0
1.41
1.41
1 53
0
2.1
0
0
1*
2.25
2.75
1.85
0
4.0
14.4
2.39
4.07
3.29
0
5.6
9rt 7
Week 3
139.3
0
112.0
0
133.7
3'
I- C £
UIC l l t U l l l l .
-
,.
Total iig.
.
xg./lM t, %
B.W. y|
o
4
3.2
.1
"•'
?,
;!
-j
14.4
•';
0
4.4
A
1
- —*
.
—
1
This chick died on l l t h day with edema.
' These chirks were severely cclcmaious.
gains among birds fed 200 p.p.b. CEF were
similar lo those of controls for the first two ond week, and at SO p.p.b. led (o a dccreM
weeks but were less than those of controls m diet consumption during the third week,
for Ihe third week. The birds fed SO p.p.b. ..The feed/gain ratio was elevated durCEF grew less during the second and third ing the second week among birds fed 50
weeks thnn the controls or the birds fed p.p.b. CEF and was further increased dur200 p.p.b, Weight gains, were equivalent ing the third week. The feed/gain ratio for
the birds fed 200 p.p.b. CEF concentrate
among nil groups during (he first week.
was clevaled during the third welt but it
There were a few deaths, all of which
were associated with feeding CEF concen- was lower than the ratio fqr the birds fed SO
trate (Table 2). The chicks that died had p.p.b. From the data,'however, it is apparedema. Birds with severe edema always ent that feeding of the-concentrate was achad gross organ changes. These pathologi- companied by a moderate decrease in feed
utilization.
cal changes were not so severe as those obFrom determination of feed intake and
served among chicks fed the crude TFM
(unpublished observations). This observa- knowledge of the amount of crystalline
tion may bo an indication that the purified CE1< concentrate incorporated into each
CEF preparation has been at least, par- diet, the amount of puriml material intially separated from substances in the gested was calculated. These ca'culations
crude /at that not only enhance edema for- are given in. Table 2. The total v-,. CEF
mation but also may lend to extensive and concentrate consumed was ]•)./ (j.g./diick
for those fed p.p.b. and 49.6 w?./chick for
more severe pathological changes.
those fed 200 p.p.b. The amount of CEF
From (he data in Table 1 on diet consumption, i( is apparent that the CEF con- concentrate ingested in ,,.g./)oo g. of body
centrate did not alter (he amount of diet weight/chick is shown also in Table 2. The
ingested during the firsl week. CEF con- total amount of purified concentrate incentral!.! at 200 p.p.b. caused a moderate de- geslcd (19.6 ,,.g.) ,VM about ninefold greatcrease in t/iet consumption during the sec- er than the amounl (S w;.) ,;,.-,, \yooit<in
''•'•at. (10M) calculated could kill one
iiecnani
Flick ct al. (1963) that the mecTianism of
CfiF toxicity resulting in edema formation
may be the effect of CEF or. capillary perTolal
A/O
ffT
meability, and perhaps mor« specifically,
Gluliulin
Albumin
relln
,
Ji rolr'iti
(ml
~S~%~~
on causing chemical alterations in cndolheir. %
g. %
]}il V-ci'(1
lial intercellular cenifnt substance, as pos]. 1V± .OS" 1.7H- .13 I.27:t 12 J,, 9 S ± ..10
Wuk /
tulated by Allen (1961). Even though
•2. 03 ± .07 I.2S* .HO 1.66-t .\1 3. 311.,01
1.,19± .09 l , 3 8 r .12 1.47.1; .10 3. 37± .07
there was no appreciable, depression of cir1 1,96i .13 1.23* .09 l.5'):t .15 3.,231',.08
n'ccjt 2
culating albumin, at least sufficient to re2 .21 J- .11 1 . 0 7 + .01 I. (18 '• .11 3 .31*. .11
2 .SIJ. .05 o.vs± .Of. a.O.IJ; .11) 3 .M>± .10
sult in hypoalbuminemic edema (15cst and
.21 3 .08± .22
2 .12:1: . 1 7 0.')5± ,08 2.2'l±
H'ret 3
Taylon, 1955; and Smith and Jones,
2 .20± .11 \.3<>x .11 1.70-' .20 3 .H8i .11
1 .'»* .20 I . S 7 ± ,.M 1.17± .18 3 .88t .37
1961), it may be that even slight decreases
1 .%± .16 ;.15± .12 1 . 7 7 ± .20 3 .15 + ,20
in albumin could enhance fluid transfer
1
S.K. of the menu.
across endothelial membrane?'damaged by
[thick. The difference between these values the chick edema factor.
The degree of development of HP in re:< way be clue to a number of reasons. First,
tWootton el. at. (1962) did not repnrt the sponse to the inrre.st.ion of the. CEF concenWiount of food ingested by ihcir birds fe:l trate is shown in Table 4, The (.oncentrate
; Ihe a 3.02 fraction. Second, they vised 'a fed at the 200 p.p.b. level prod need a mar1
heavy breed of chick, while we have used ked increase in fluid which accumulated
» light breed. Third, though both diets were within the pericardia! sac. 'Hie magnitude
*j'n(helic, they were not identical. Fourth, of this response is of the samrt order as retheir « 3.02 fraction was essentially homo- ported previously by Flick <.* al. (1963),
geneous, while our prcparalion contained using 4% crude TFM in a similar diet.
perhaps as many as eight components The HP group scores show (has. the SO p.p.b.
(compare a, b and c, Figure 1). Never- level of CEF did not result in abnormal
theless, the prediction of Brew ct al. volumes of pericardia! fluid according to
(1959) that the chick edema factor may the ranges of volumes-reported by Shue
be detectable when fed to chicks at levels and Gallo '(1961). The HP incidence
of 2,0 p.p.m, or less has been confirmed shows that all the birds fed 200 p.p.b. CEF
by our studies and by those of Woolton had abnormally high levels of fluid in the
pericardinl sac. Also shown in Table 4 are
rial. (1962),
The results obtained for plasma albu- values for hcmatocrit, serum copper and
min, globulins, A/G ratio and total'pro- whole, blood glucose. The' packed cell volteins are presented in Table 3. In general, umes were decreased by the t\vo levels of
there were no appreciable differences in ei- CEF concentrate fed. These decreased liether the plasma protein fractions or the matoc.rits were not. so low as those reported
total plasma proteins when considered on previously by Flick ct at. (1963) from
r
Uie group basis. Some chicks with severe feeding 5 /o crude TFM with a practical
edema had marked hypoallnimincmia and type of ration.
Scrum copper levels (Table 4) were elehypoproteinem'm, in confirmation of the
observations of Alexander c.l al. (1962). vated at the SO p.p.b. level but no statistiFrom the data it seems unlikely that the cally significant change from normal ocmassive edema formed was the result of curred at the 200 p.p.b. level of CfiF concensuch modest alterations in circulating pro- trate. Since it had been observed that the
teins. These data support the hypothesis ol liver, heart and kidney were adversely in. .—\\'telily Ictds of l>!a
5. C. Wliile !.Fgl,tmt cockerel* fa! CKI*
�262
263
TAD IE 4.—Ilydropmcardiiim (111'), hcmattiml, .tenini cn/ificr and whole lilana i'/wotc values o/S.C, i
Willie Leghorn cockerels fed CKl{ concentrate for 3 weeks
CEF
' level
P.P.».
0
50
200
•
-
•
Hydropericardium
:
Mean volume
Score'
ml.
0.0<JO±0.012(S)>
0.1J8iO.OI8(4)«
4.63 ±1.64(6)
0
o
10+
Hematocrit
Observations
Scrum
Copper
Glucose"'
Untnp.
bjetlcd
nig. %
Incidence
'• 'Hi.
i ill $& si'isl
°/°
27.1 ± 1 , 4 (8)
.j 1±
g(5)
No. eggs
injected-
217±IO<» ij
'L °'
_„
.
Hatch
Embryo
%
93'
40
20
0
20
20
20,
. 5
- 10 '
,. M
50
m, n
;
.
•
(Malformations of right
{cerebral hemispheres, legs
and beaks; small embryos
Chick
Normal
[Weight retardation,
{sparse and deformed
feathers.
•—
m ' Controls were injected with 100 ,,1. of corn oil.
| ' Normal expected % hMch (Mclaughlin el al, 1963).
t
. u, uiii ini-iin imimncr ol observations),
sample of HP fluid lost from this group.
volvcd in thn chick edema disease (.Allen,
1961; Alexander et al., 1962; and Flick ct vanced stages of the disease (see footnote^
2, Table 4).
.^
a/., 1963), and thai changes in these organs
From what is known to date, the chick I
had been involved in the mechanism of
edema formation (Smith and Jones, 1961), edema factor elicits a number of signs of I
it was thought that determination of the intoxication which not only accompany tk I
levels of serum copper in the birds with the feeding of crude TFM but are more severe f
disease might be of some diagnostic value. when the crude material is fed. The finding -1
Scheinberg and Sternlieb (1963) reported (hat . our CEF concentrate contained 8 '
that human patients with Wilson's disease peaks by electron capture gas chromatoghad severe liver disease, and many patients raphy (Figure 1) indicates that at least S
had serum copper dyscrasias caused by ex- compounds were present in the purified
cessive urinary excretion, , malabsorption preparation fed in these studies. It may be
from the intestine, decreased protein syn- that only one, or' a few, of these comthesis (particularly in severe malnutrition) pounds possess the necessary molecular
and severe hepatic dysfunction. I t may be configuration to produce signs of the disthat the elevated serum copper among the • ease equivalent to the estimated potency of
chicks fed SO p.p.b. CEP'' concentrate was the « 3.02 fraction.
associated with either decreased liver utilization of copper or decreased renal excre- Untcfiabilily St,,dy. The preliminary retion. The adverse effect on serum copper of sults obtained from injection of White
CEF concentrate fed at SO p.p.b. is not Leghorn eggs with the unsaponifiablc fracclear, but may be indicative that CEF tion of TFM,-which conlained CEF, arc
toxic activity is oligodynamic and more shown in Table S. The percent hatch of
specifically oligof.oxic (more toxic at low control eggs was within the expected range
reported by McLaughlin ct al. (1963)
levels than at higher levels).
Whole blood glucose levels were not ap- Chicks which hatched appeared to be norpreciably altered by feeding the concen- mal i" size and development by gross obtrate (Table 4), Crude TFM, however, fed servation. When fertile CW!S ,,ere ,fc.jn.
in a purified ration, frequently elicited a
marked hypoglycrtnia among chicks in ad-
jectcd with 10 ,,1. of the undiluted unsapon"liable fraction containing CEF, (he hatch
was 40^; injection of 20 v.]. resulted in
blood glucose; injection of CEF-containing
unsaponifiable fraction and determination
of its effects on embryonic development
and on egg halchability.
The following observations were made:
1) presence of approximately eight components (GLC separation) in crystalline material, 2) moderate growth depression at
SO p.p.b. level of CEF concentrate, 3) essentially normal feed consumption, 4) moderately increased feed/gain ratio, S) increased
HP at SO p.p.b. level o( CEF concentrate, 6) .severe HP at 200 p.p.b. level of
CEF concentrate, 7) essentially no group
changes in .plasma proteins, 8) moderate
decrease in 'hematocrit, 9) no change or
moderate elevation of' serum copper (SO
p.p.b'. CEF concentrate), and 10) normal
blood glucose levels.
A preliminary hatchability study revealed
that a CEF-containing material led to a
SUMMARY
decreased hatch of injected eggs and to deThe following studies were.- performed: velopment of embryonic deformities.
gas-liquid chromatographic (GLC) separaACKNOWLEDGMENT
tion of a purified crystalline concentrate
• The isolation of CEF concentrate was
containing the chick edema factor (CEF);
largely carried out by Mr. ^dr.ej&.&'jirt*
feeding of the concentrate at SO or 200
v
phy was perparts per billion (p.p.b.) in a scmi-synthel- $~-~
ic d i e t . l o day-old S. C. White Leghorn 2™j£
. F T C K S S I s u y , lood and
cockerels for three weeks and determinaDrug Administration,
tion of growth, feed intake, 'feed/gain
REFERENCES.
ratio, mortality, total intakte of CEF concentrate, plasma proteins, hydropcricardi- Alexander, J. C., R. J. Young, C. :M. Burnett
um (HP), hematocrit, serum copper, whole - ami II. D. HaUiaway, 1962. Hydropericard.um'
hatch and SO p.l. completely inhibted hatching. Embryos which failed lo
tolcli exhibited one or more of the followlng developmental anomalies: malformed
<beak, lack of development of the right
mcsencephalon, eye defects, growth retardation or leg deformities. The deformities
^hscrved were not studied further. The
deformities found were common to the embryopathies \vhich occurred and were not reis lated lo level of CEF-containing fraclion injected. Embryos which hatched afler injection with 10 or 20 y.1. of unsaponifiables
exhibited'sparse and defective, feathering
(down) and were small compared to the
controls. This study revealed that CEFcontaining unsaponifiables are capable of
interfering with normal embryonic development a'nd halchability.
�264
assay and safety of fats and fatly ncid products. Poultry Sci. .11: 22-32.
Allen, J. K., Jr., 1961, The role of "toxic fat"
in Uic production of hydro|)c;-icardium and
asdics, Dnclornl Thesis, X.'nivmity of Wisconsin.
Allen, J. K., and J. L. Lalich, 1062. Response of
chickens to prolonged feeding of crude "toxic
fat." IVoc. Soc. Rxptl. Uiol. Mod. 109: 48-51.
Ames, S. K., \V. J. Swansnn, M. I, J.udwig and
G. Y. Brokaw, I960. Tlic occurrence of the
chick pcrirnrdi.il edema factor in some olcic
acids and products derived therefrom. J. Am.
Oil. Chemists Soc. tl: 10-11.
Best, C. If, nnrl N. B. Taylor, 1955. The Physiological Basis of Medical Practice, 6th Ed., The
Williams & Wilkins Company, Baltimore, Md.,
37-10.
Brow, W. B., J. B. Dorc, J. II. Benedict, G. C.
Potter and 15. Sipos, 1959. Characterization of
a type of unidentified compound producing
edema in chicks. J. Assoc. Offtc. Agr. Chemists,
4 2 : 120-128. '
,
Carroll, K. K., 1961. Quantitative estimation of
peak areas in gas-liquid chromalography, Nature, 191: 377-.17R.
Douglass, C. D., and D. F. Flick, 1961. Collaborative bioassay for chick edema factor. J.
Assoc. Offic. Apr. Chemists, -14: -H9-156.
Firestone, D., W. Ibrahim and \V. Ilorwitz, 1063.
Chick edema factor. Ill Application of microcoulomelric gas chroinnlogrnphy to detection of chick edema factor in fals or fully
acids. J. Assnr. Offic. Agr. Chemists. 4 7 : 3S4-396.
Flick, D. F., L, Gallo, J. Wiiilm-h, C. D. Douglass
and L. Friedman, 1962. Bioassay of the chick
edema factor: 1061 collaborative study. J.
Assoc. Offic. Agr. Chemists, 45: 231-239.
Flick, D. F., C..D. Douglass and L. Gallo, 1963..
Studies of the chick edema disease. 1. Body
water distribution and effect o f , diet. Poultry
Sci. 4 2 : 8SS-S62.
Friedman, L., 1). Firestone, W. HonviU, I). Banes.
M. Anstead and G, Shuc, 19S9. Studios of the
chicken edema disease (actor. J. Assoc. Offic.
Agr. Chemists, 42: 129-140.
Gornall, A. C,., C, J. Unrdawill and M. M. David,
1949, Determination of serum proteins by
means of the biurel reaction! J. Kiol. Clicm.
177: 751-766.
Ilarman, R. K, 0. B, Davis, W. II. Ott, N. G.
Brink and F. A. Kuchl, Jr., 1960. The isolation and characterization of the chick edema
factor. J. Am, Chcm. Soc. 82: 2078-2079.
Horwitz, W., Editor, 1960, Official Methods of
Analysis, Association of Official Agricultural
Chemists, Washington, D. C., 345.
McLaughlin, J., Jr., J. P. Marliac, M. J.
Verrctt, M. K, Mulchlcr and 0. G. Filzhugh,
1963. The injection of chemicals into the yolk
sac of fertile eggs prior to incubation as fl
loxic.ity test. Toxicol. Appl. Pharmacol. S:
760-771.
Natelson, S., 1961. Microtechniques of Clinical
Chemistry, 2nd Ed., Charles C Thomas, Springfield, III., 76-79.
Potter, G. C,, W. B. Brew, R. L. Patterson and
E, Sipos, 1959. Current status of the toxic
principle causing chick edema syndrome. J.
Am'. Oil Chemists Soc. 36: 214-217.
Sanger, V. L., L. Scott, A. Hamdy, C. Gale and
W. D. Poundcn, 1958. Alimentary toxemia in
.chickens. J. Am. Vet. Mod. Assoc. 133: 172176.
Schcinber«, I, H., and I. Sternlicb, 1963. Wilson's
disease and the concentration o f . ceruloplasmin
in serum. The Lancet, June 20, 1420-1421.
Shue, G., and L. Gallo, 1961. A study of the
normal variation in chick pericardia! fluid. J.
Assoc. Offic. Agr. Chemists, 4-1: 456-459.
Smith, H. A. and T. C. Jones, 1961. Veterinary
Pathology, 2nd Ed., Lea &• Febiger, Philadelphia, Pa., 121-122.
Sumogyi, M., 1952.< Notes on sugar determination. J. liifll. Chcm. 195: 19-23.
Woottnn, J. C.i and J. C. Alexander, 1059, Some
chemical characteristics of the chicken edema
disease factor, J. Assoc. Offic. Agr, Chemists,
42: 141-148.
Woollon, J. C.,. N. R. Artir.an and J. C. Alexander, 1962. Isolation of three hyilropcricardium-prodncing factors from a toxic fat. J.
Assoc. Offic. Agr. Chemists, 45: 739-746,
\Vootton, J. C., and \V. I>. Courcheno, J964. A
contribution to the knowledge of the structure
of two hydropcricaidium-producing factors from
a toxic fat. J. Agr. Food Chcm. J-2: 94-98.
Yartwff, A., D, Firestone, 11. liancs, \V. HonviU,
L. Friedman and S. Nosneiin, 1961. Studies of
the chick edema factor. II, Isolation of a toxic
substance. J. Am. Oil. Chemists Soc. JSi 6062.
265
Reproduced by the
U. S. DEPARTMENT OF HEALTH, EDUCATION, AND WKLFARH
Food and Drug Administration
NUTRITIONAL ADJUNCTS
Chick Edema Factor. III. Application of MicrocouEomc.fric
Gas Chromalography to Detection of Chick. Edema .Factor in
Fats or Fatty Acids
By DAVID FIHKSTOXE, WALID JBJiAHIM, and WILLIAM HCfflYvTfZ (Division of
lrood, Food nnd Drug Administration, Washington 25, D.C.)
A rapid screening test for detecting
ohick edema factor in ,falfi consists of
adsorption cliromaiopraphy of extracted uiisapoiiiliablcK on alumina,
followed liy HHMlysis of specific fraclions by a inicrocoulomctric gas chromnlogi'iiph M'liioli is ftr.imilivc only to
)ifilogr.iiH. Tltls rliroinsitogrophic nirlhod
nppears to be more stMisitive limn tlic
cliick bioHssay.
Toxic f a t yielded pas cliromaloRrapliic peaks vilii retention times relative to aldrin of 5 or more. AH samples
which failed lo reveal those fliromato(jraphie peaks have been shown lo be
nontoxie in the ohick bio;.iBsny,
Tlin \vii|p.--)irfiui iicciin'oiice of chick c'dcin:i
(lisciisc in 111")? rwnllcd in t h e ilcalhs of millions n f young chickens. The lox'u1 iiKilcrials
(his disease have bron found to be
clilorinatod tiroinnlic hyclrociuljons, occnrrinR
in loNio fats in association with n lurpe iniinbpr of relatively nontoxic nromntic materials
\vilh similur chemical ami physical propertics, This paper describes a screening procedure for detection of such toxic fals.
Specific frjiclions of misnpoiiifiablr 1 m a t t e r
isolaled from tlic fats arc examined by using
a niimicotilmnclnc mii= rhromalouraph, ah
mslniment. which can dclei-l siibmimiprum
amuunls of lialogcn. r)'hc prosencc of f-lowcltiiin); snbslances is an indication of (lie
chick cilein:i factor in the f.-i!.
Tlic precise stniclnrc n! tlic Mih.-tance*
CiiMsinu chick edema disc.-pc have yet to
be determined. IVlimin.'iry work on the
detection, isolation, and cli;ir:iclciiy:i(ion of
the luxic aficnls \va-t rcjiorird in l!i.">!i by
se\cral lalioraloriiv (I-.'!). ^idiM'iiiicntly,
�267
266
Hnrmon and co-workers (4) isolated a toxic
subst:mcc in crystalline form • from .1 feed
grade (allow. A private coinnumicnlion from
Tishlc.r of the same laboratory (5) disclosed
• that HIP. crystalline substance contained
about >17% chlorine.
\
YarUoff and co-workers (C) isolated a,
crystallino halogen containing material that
produced chick edema symptoms at 0.1 ppm
in (ho diet from a sample of triolein. This
triolo.in was toxic to monkeys, producing
changes in the liver, kidney, pancreas, and
other organs. More recently, Wootton and
co-workers (7) isolated three compounds
from a, toxic fat which produced chick edema
disease. IVfiw spectra of two of the, compounds indicated a molecule which has a
molecular weight of 3JU and contains six
chlorine atoms. Ultraviolet spectra wore
consistent with the concept that these materials arc highly chlorinated aromatic compounds.
Ames and co-workers (S) and Firestone
and co-workers (9) reported (he occurrence
of chick edema disease factor in oleic acid
samples destined for human consumption.
A food additive regulation 1 -of the Food and
Drug A d m i n i s t r a t i o n now requires that food
grade f a t t y acids he "free of chick edema or
other toxic factor." At present, the detection
and assay of chick edema factor in fats is
carried out by n bioassay procedure (10-12).
t h a t requires 21 days to complete.
We observed t h a t unsaponifiable matter
from toxic f a t s contained a number of chlorinated 'components which had greater retention 'limes t h a n chlorinated pesticides
when examined in a. microeoulometric gas
chromalograph;' 1 our observation prompted
this investigation of the use of microcoulometric gas elu'om.ilogniphy for detecting the
presence of chick edema factor in fats. Chick
edema factor is presumed to be present if
one or more gas chromatographic peaks with
r e t e n t i o n times relative to aldrin of !> to 20
are f o u n d ; its absence is presumed if analysis of the. equivalent of 100 g of a fat or
f a t t y acid fails to reveal the presence of
these gas chromalograpin'c peaks.
METHOD
Kxlrnciion o] wisapnnifiablc matter (modification of AOAC method 26.06,4(5). — Reflux
111 g sample w i t h 270 ml alcohol ami 55 ml
fiO% (wAv) KOI! 'for 1 hour. -Transfer mixture to 2 L separator, rinsing flnsk with 325
ml U = 0, mid add rinsings to separator. Add
300 ml petroleum ether, A.C.S. (redistilled, retaining cut w i t h b.p. •iO-CO°C), and shake vigorously. Let layers separate, breaking emulsions
that may have formed by adding 10 ml alcohol and swirling gently. Draw off lower layer,
and transfer upper layer to Another separator.
Repeat extraction 3 times with 300 ml portions
of petroleum ether and combine extracts.
Wash extracts twice with 60 ml portions
of H:0 by swirling gently. .Wash petroleum elhcr extracts first w i t h 60 ml H.,0 and
tlien with CO ml of an alkaline dilute alcohol
soln (dissolve 28 g anhydrous KiCOi in 600
ml H,0 and then add 400 ml alcohol), and
repeat washings in same order. WnHi extracts
with 60 ml portions of H.-O until neutral to
phenolphthulein. Transfer extract to a 2 L
crlen'moyer and dry by adding 20 g tuihydroua
NtisSOi, swirling vigorously, and letting the
solution stand a half hour. Decant solution
through a'glass funnel, containing u pledget of
cotton in the neck and holding 20 g nnhydrouti
Na:SO.. into another 2 J, crlenmeyer. Wash
first erlcnmeyer 'and funnel with lined 10 ml
portions of petroleum ether, transferring washings from the erlenmcycr through (lie funnel
and into the filtered solution. Evaponito most
of solvent on steam bath, imd transfer extract
to 100 ml tared fat flask containing several
boiling chips. Evaporate solvent on ."Loam bath
and complete drying under a gentle current of
air, or by evacuating flask to 0.5 cm of mercury while swirling on steam bath, Determine
weight of unsaponifiablc matter.
PraclioiiHlinn uj Miuttpmiifiablc matter by
alumina cliromalograpliy. — To a chromntographic column, 25 mm o.d. X 300 mm long,
fitted lit the bottom with a coarn; porosity
fritted glass disk and Teflon stopcock, ndd rcdislillod petroleum ether, dried prior to use
with anhydrous Nit:SOi, u n t i l column is %
full. Weigh 50 g aluminum oxide (Merck reagent, No. 71707), and transfer lo column.
Store tlin alumina in tightly closed I'otllu, nml
' Cmli' of I'Vdi'riil !(cK"liifimiH, Tillo 21, ficc- How boll IP «s «>on us possible after weighing
li.m 121.1070.
out portions for chromalography. Let alumina
1
1 inliriiwnu M a n u f a c t u r i n g Company, I'alo settle, and when air bubbles slop riv.ng lo the
All.i. < ' n l i f .
jig Aldrin = (peak area, in.3) X [recorder
sensitivity (min./in.)(mv/in.)] X (35.5g/eq.)
X (OOuoB/min.) X (10' /»g/g)(10-'v/mv)
(10)/(scnsilivity range, ohms) X (% chlorine
in compound) X (96,500 coulombs/eq.).
For n 0.1 mv/in. recorder sensitivity, 2 min/
in. chart speed, and 12.8 oiims sensitivity range
resistance, the equation alxove reduces to:
jig Aldrin = (area X 34J>)/% chlorine.
surface of the solvent, place a disk of coarse
filler paper on top of the alumina.. Cover the
disk with 20 g nnhydroua NojSOi. Drain the
excess petroleum other so that it is level with
the upper surface of the NoiSOi.
Transfer unsaponifiable matter to the chroirmlographic column, using u total of 20 ml
petroleum ether. Allow liquid level to fall so
that it is just below the top of the NaiSO,.
151ute sample with 400 ml portions of each of
the following solvents (dried prior to use by
shaking with anhydrous NiuSO.): Petroleum
ether (fraction 1), 5% ethyl ether in petroleum
clhi.'r (fraction 2), and 25% ethyl ether in petroleum ether (fraction 3). Collect eluatcs in
GOO ml erlcnmoyer flasks, add several boiling
chips, and evaporate, lo small volume on steam
bath. Transfer residues to tared fat extraction
flasks, evaporate solvent, and weigh. Transfer
to 2 g short stylo vials having screw cap with
tin liner, and evaporate solvent.
'Aficrocaulomclric 0os c/tronm/or/rnp/ij/.—Dissolve 4 g silicon'; grcnno (Dcnv Corning High
Vacuum Grcuw) or Dow Corning DC 200
silicouo fluid (12,.r>00 cunlislokcs) in 200 ml
chloroform on sloani bath. Add 10 g acidwashed Chromosorb W (Jolms-Manville Co.),
and stir coutiuiiotisly until most of solvent
evaporates (about half an hour). Let stand
on steam bath 1 hour, and place in vacuum
oven at 60°C overnight to remove residual
solvent.
Pack the coated Chromosorb W into a 3'
length of 0.25" o.d. aluminum tubing plugged
at one end with glass wool, using a Burgess
Vibratool. (Two 3' columns may be prepared
from 20 g coaled Chromosorb W.) Add a plug
of glass wool to the open end of the column
and bend it into a light spiral, using a 3"
diameter mandril. Condition the column at
275°C for 48-72 hours, passing nitrogen
through at 20 ml per minute.
Prepare a 1.00 X 10-'% solution (10 mg/L)
of aldrin in hexane or benzene and chromatoRraph 100 /d portions in n Dohnnann microeoulometric gas chrnmatograph at 2<16-2-18°C,
»"ing a nitrogen flow rain of 60-100 ml per
minute so that aldrin elutcs in 2.3-3 minutes.
Use the 128 ohm range setting. a Determine
area of aldrin peak by (riangulution, or with
a disc chart or electronic integrator installed
en Die strip chart, recorder, nnd calculate recovery of aldrin using the following equation
(applicable to chlorinated compounds):
The number of strokes of a disc chart integrator coupled to a chroronlograpliy recorder 4
equivalent to each square inch of urea can be
determined as follows: (a) Remove the fuse
from (he. strip chart amplifier; (6) move the
pen upscale on the strip chart a known distance from the baseline; fc) run the chart a
known distance; and (rf) divide the calculated
area (height X distance traversed by the pen)
by the number of strokes obtained.
By using the formula, cafrculalcd as described
above, n recovery of at least 70% of the aldrin
injected nliriiild bo obtaiuwl.
Dissolve fractions 2 un<l 3 from alumina
chromatography separately in benscnc to give
100 fil solution, and ehroinsitograph each solution in the Dohrmann in.tlrmncnt. (For analysis of more than about 60 tag of each fraction,
approximately 50% benzene solutions of up to
250 fi\ volume should bo prepared and injected.
Do not, inject more than 125 mg material into
column). First ehromatograph Via of the fraction,, and if no chromatographic peaks with
H\ = 6 or greater are observed, ehromatograph
the. remaining %o of the fraction (equivalent
to'100 g starling sample). Chromatograph a
portion of aldrin before each sample, and calculate. /?A value (relcntiofi time relative to
aldrin) of each peak in tlv rumple chromalogram, using a millimeter rule to measure retention times. Record Jt*. of gas chroinntographic peaks in the rangR K>. = 6-20. Peaks
in this rang<! are indicative! of the presence of
chick edema factor. The presence of broad
bands with no definite peaks is not indicative
of the presence of chick eitema factor..
(Note: Types of sample* which are found
from experience to be generally free of componenls characteristic of toxic fats may be
examined as described at»ovo in 100 g portions, the sample saponified by refluxing with
2JO ml alcohol and 60 ml 50% (w/w) KOH,
and all of each of Ihe polnr alumina fractions
gas chromalographcd.)
' This Hotting w i l l hnvo a rcKiKtiuico of 12.S
oluns wlicu Hie clH'<mw(ogr;u>lt is used with a
I niv slrip chart retonler,
' MiMaeapolis-ITnnu.vwcIl Model T ]fi3X
(Mimn'npnliK-lIom'ywcll Kcf-ulalor C»., I'hiladl'lpliin, I'll.), or equivalent.
*'
48-362 O - 70 - 18
�268
Kciiulu and Dincusaions
at 250"C on silicone columns. When a !<>«••
Relative Retention Times ol Chlorinated melting inactive isomer" having the win*
Pesticides awl Chlorinated Materials from retention time (/JA = C.O) as one of (I*
Toxic, Fats.—A number of chlorinated pesti- toxic factors was chromatographed in ll*
cides iincl several chlorinated materials iso- Dohrmann instrument, the following Rf, vallated from toxin fats were chromatographcd ues were obtained at 2-10°, 248°, and 250*
in the Dohrmnnn instrument at 24S°C with respectively: 0.0, 0.4, and 0.2.
the 3' column. Retention times 'relative to
Preliminary Analysis of a Group oj Totk
aldrin (#A) ^re shown in Table 1. The and Nontoxic Fats. M icrocoulomctric Andpesticides arc representative, of the whole ysis of Uivsaponifiable Matter Without Prioi
range of retention, times displayed by chlo- Fractionalion on- Alumina.—A group of 7
rinated pesticides. A toxic factor isolated toxic, and 7 nonloxic fats were examined
from triolein (G), an inactive analogue, and initially. The fats are described in Tables J
a concentrate prepared from a, toxic fat, all
yielded chrqmatogrnms with peaks of R.\'~
Tnble 2. Data on toxic fate
5 or greater whereas the pesticide peaksOrmnio
were all less than ]{A = 4. The toxic factor
Clln
Un««from triolein as well as the toxic fat concen% Unsn- ponidjvbl'
trate produced chick edema when fed to
Manu- ponilmbln Mailer,
Component
facturer
Alattcr
ppra
young chicks at a level of 0.1 ppm in the
diet.
1. Tullow acids,
1
18.3
10
still distillate
2. Tallow acids,
Table ]. Relative retention limes of
. 1
14.8
47
chlorinated pesticides und materials
still distillate
isolated from toxic fats
3. Tallow ncidtt,
2
U
10.1
(3 foot, }4 in. diameter column, 20% silicone
still distillate
grouse, 80% Chromosorb W) carrier gas flow 4. Tallow acids,
3
2.0
25
"
" i•
'
'
still distillate
rate, about CO ml/min.; column temperature,
248°C; injection block temperature, 270°C)
5, Tallow acids,
1
4.5
2000
still residue
Retention
6. Tallow
2
5.1
39
Time r«. Aldrin
7, Fat from broiler
Sample
4
2.5
8000
feed
Clilordano
1.0
Ileptachlor
0.0
and 3, respectively. Presence of chick edema
Kcpone
2.2
disease wns determined by bioassay (2) using
Mircx
3.0
a special basal ration. Organic halogen in
Strobnnc
'
0.6-3.5
Tcdioii
the unsaponifiable matter (assumed to be
3.4
Toxnplicnc
chlorine) was determined by microcoulomc0.0-3.8
Toxic factor from triolciii
5.0
trlc gas ohromatographic analysis of 50 ing
Innctivc analogue from triolein
9.0
portions of unsaponifiable mailer without
Concentrate from u toxic fnt
2.3, 3.G, 5.4
prior fractionation by alumina chromatograpliy. Both toxic and nonloxic fats conThe chick edema-producing /actors iso- tained widely variable amounts of unsaponlated by Wool ton and co-workers (7) had ifiable matter and organic chlorine. Sources
retention times relative to methyl arachi- refer to individual manufacturers. Gas chrodatc of 1.17, 3.02, awl 3.17 when chromato- matograms of unsaponifiablo matter from
graphed at 2f>0°C on a 20% silicone column. two of the toxic fats (Nos, 1 and 2) show
Since aldrin chiles (wire as fast as methyl peaks with R\ values of 14. Gas chromatoarachidate under these condition.1', it would grams of Unsaponifiable m a t l e r from the
be expected .that these toxic factors would
* Ptipjilicd by Dr. N,
have Rf. values of about 2.4, (5.0, and 0.3 Sainfoin Co,, Cincinnati, K, ArUnnii, T'roctcr nml
Ohio,
269
Tnlilo 3. Hula <m nonloxic Cuts
*
Component
1. Cottoniiced oil
(OSO)
J. Onttonsnncl oil
J. ORO foots,
still residue
1 090 fntty acida
8. Vegetable, oil
footfl
6. Tullow fatty
ucidn, still
residue
7. Corn nil
Mnnufn.,|il"'r
% UlWlHjmliabln
Multnr
Orcnme
Cl in
IJnan*
inniiinhln
MnUcr,
lirnii
8
0.0
80
jj
ft
0.5
13
12.1
5
31 ,
2
5
0.4
2.2
: 28
0
2.2
7
7
O.C
1,10
oilier toxic fats and from nontoxio fats- obtained without prior fraction i i i o n ' by uhimini ohf<m:itn|»ruiihy wer- fiimilar; most of
the organic linlogen cluk'd in 2-4 minutes,
nml tlierc were im P'-alrs with « A values
greater lhaii S. Since fuinponcnts with JYA
values of ,r> or mnre urn usually present in
the unsaponifiable matter of toxic fats at
very low levels, n concent ml ion stop by
alumina chrimmtn|'.rai>hy ix iici;r-s:«ir.v prior
to microcoiildiiictric analysis.
Frnctionaliun oj llnsapnitifiMc
Matler
on Alumina- Prinr ta .\firroriiiihntrtrk Annli/.tis.—-Tho "I toxie and 7'nonloxic fats were
Ihoii iiiiiilyzi-d liy procedures essentially a*
described'nl'ove. A t fust, 0-fuoi, and later,
d-foot cliriiinatuKi'iipliii' col'iiMii." won: uwil
in the. miruicoulomclric gap ohromatograph.
With shorter columns, f.'isler I'ltiliou permitted analysis of 12 wimples cadi working
day, and results were comparable to those
oblained with (he ctmvpntion.il 0-foot columns. Jieciiise 10-10(1 mj: portions of sample were rcpratrdly injeetrd, all romponents
of Hie imrrocoiilomrfric gas chromatugr.iph
were rlcatn.il every 2-4 weeks as required.
C.Jis chromaloftrapliic columns were replaced
each 1-2 months. I n CIIEI-K wheru samples
conluined large nmounls of unsaponifinblc
matter, larger alumina column? were used
for the column chromrifoKrapliy TO Hint the
ratio of alumina lo miwipnmfuililc mailer
was at Icfisl 20 to 1, l u thwe eaww appro-
elutmg solvents
priately largor nmounts o T e h
were iilw> nuwl.
Fractions.' obtained by admrptinn rhromatogrnphy on alumiiiii of uiisiipnnifmhle matter were, .'maJymMl liv itiirrocoulinnetrir gas
ehromnliifti.ipliy. (<ns ••liromntogmms of
polar Irai'Unns from toxic, fats (cluted with
5% and 2.1% ethyl ether in petroleum clbcr)
(ill showed peaks with KA values greater
t h a n 5. No peaks with /?A values greater
than 5 vow found in piae ehromatograms of
these frnctions from tlto nonloxie fats.
Portions of alumina fractions 2 and 3
equivalent to only abc/ut .V) g of the nontoxic
still rct'iiluws (Table. 3, .samples 3 and 6)
we.re chrrjiniitogrnphcd iit the Dolirmann instrument l)"'!»nne of the presence o( it large
amount ol crystalline 1 material in these fractions. The infrared spectrum of this material (i.-i.Ini.ed by recryslaHizing from petrolpuni-clhci'1 resembled th.-i' of dipalmitone.
An nildi'i'innl clenniip pvof.'dui'e must be developed for routine nnalysi* of 100 p samplesof such still residues. Additional examination of utiNiponifiablos from 2000 g portions
of three nontoxic vegetable oils (Table 3, samples 1, 2, and 7) failed to reveal chromatogniphic j«!!ik.s with }tA values greater than 3.
Taljlc 4, Slow-clutitiR peulis in inicro-
cnulonicti'ic KHS chromntnitrnmg from
nuulsis nf taxic fnt«»
Mnnu-
Nn.
1
2
3
4
5
G
Toxic. Vat
Tn'loH' acids.
Still distillate
Tallow acids,
slil1 residue
Tullow acids
Tallow
Tullow acids,
still distillate
Tallow acids
lar!-yer
HA SB
j
0, 9, 12,21
. ]
6, 9, 10, 14
>
2
2
0, 9, 10, 18
6, 9, 12
6, 9, 10, 18
3
6, 10
Table 4 lists the slow-eluiing peaks found
in gas chromatograms from 0 of the 7 toxic
fills. Figure 1 shows chroimilogram.s of alumina fruction 3 from three still distillates,
each of which was obtained from a different
n i a n n f a e l u r e r of commercial f a t t y ncids. A
similar p.'iUern of slow-ehuing peaks SUR-
�270
gests that, a common complex contaminant
may bo Vesponsible for the presence of chick
edema factor in fats. Each peak probably
represents a complex mixtures of closely related compounds. In fact, when polar alumina fractions from several toxic fats were
further fractionated by additional column
cliromnlogrnphy on alumina, such purification often resulted in partial resolution of
the,corresponding peaks into at least 2 components.
The fat from a chick edema-producing
sample of broiler feed (toxic sample 7) contained over 400 ppm chlordnne, and this
sample required special treatment because
largo quantities of chlordanc olutcd in alumina fractions 2 and 3. Although the R A
of chlordnne = 1, the large amounts present
produced overloaded chromatograms which
interfered with gas chromatograjihic detection of other components. A portion of combined fractions 2 and 3 was molecularly
distilled in a "cold finger" pot still for 2
hours at 85°C and-50 p. pressure. The. cblordane was volatile under these conditions and
271
was eliminated from the residue! which was
analyzed in the Dohrmann 'instrument.
Peaks with flA values greater than 5 were
then found in the chromatograms.
These results indicated thaJt alumina chromatography of unsaponifiaMe matter followed by rnicrocoulometric fffis chromatogrnphy of appropriate fractions might be
used as a screening procedure to detect
chick edema factor in fats and fatty acids.
Additional work on cleanup procedures is
required before this technique can be applied
routinely to examination of 100 g samples
of low-grade fate, such as still residues containing large amounts of material that elute
in alumina fractions 2 and 3.
Effect of Alumina Activity and Column
Dimensions on Adsorption Chromatography.
—Alumina activity was found to affect (lie
rate of elution of substances from toxic fnts
which are responsible for the1 gas chromalographic peaks of jRA = 5 anct greater which
are characteristics of toxic samples. Various
batches of Merck .alumina used for this
work were found to vary in activity from
tc)
R
A=6
R
B|
A °
(a)
10
20
minutes
Fty. I—Mitrocoulomntric goi chromatorjrami of alumina fraction 3 liolntod from toxie bertch jlill d'utillnlol
obtained Irom 3 monufaclurors of commercial fatly acldi. The fraclioni wer» liolaled from (a) 5 g.
(b} 5 g, and (c) 20 a of lot.
Brockrmmn activity I to activity II. Activities were determined by observing the rate
of travel of solutions of specific pairs of azo
dyes (13, 14). Using activity I alumina, unwiponifiablo matter from toxic fat or from
toxic fat added to USP cottonseed oil was
cluted from the columns so that the characteristic slow-cluting peaks were found in gas
chromat'OKrams of alumina fraction 3. With
activity II alumina, these peaks were, found
in chromatograms of alumina fraction 2. .
Generally, the alumina used in this work
wus not standardized; it. required gas chromatographic analysis of alumina fractions 2
and 3. Standardization of the alumina
should permit elution of the slow-cluting
compounds in one fraction, reducing the
number of samples required for gas chroma. tography. For example, Merrk alumina
'heated 48 hours at 200°C had a Brockmann
&el\yity I, and all the slow-eluting peaks
from several toxic fats examined were found
in chromatograms of fraction 3. Work is
continuing on a procedure for standardizing
alumina in !i simple and reproducible manner.
Column dimensions also were found to
affect the elution of characteristic substances
from toxic fats. When activity I alumina
was used, these substances were eluted in
fraction 3 from a 25 X 300 mm column,
whereas they eluted in fraction 2 from a
30 X 300 mm column.
B/Jcct o/ Column Tcmperalwe and Flow
Hate on Gas Chromatogra.phy of Klow-duting
Components o/ Toxic fats.—Studying the
gas chromatographic behavior of chlorinated
pesticides, Burke and Johnson (15) found
that varying the column temperature and/or
the carrier gas flow rate resulted in variations of relative retention times of the pesticides. Similar variations in relative retention
times were observed with the slow-eluting
components of toxic fats. fiA values, however, were affected more by variations in
temperature than in flow rate. A toxic substance isolated from triolein (6) had the
following «A values at 246', 250°, and
252°C: 5.0, <!.!), and 4.7. An inactive analogue isolated from the sample had the following # A values at these temperatures: 9.7,
8.0, and 7.8.
Because of the design of the
metric gas chromatograph used for this work,
whereby oven temperatures are controlled
only by a variable transformer, line voltage
fluctuations result in continuous variation of
column temperature. A variation of d: 1°C
within a 1-2 hour period is the best stability
to be expected. Even with these variations,
however, the instrument is suitable for detection of .slow-eluting materials in toxic
fats bp'-ause of the large difference in retention times between these slow-eluting
materials and the chlorinated pesticides and
other fast-eluting chlorinated materials
found in all fats examined,
Analysis o/ Bioassay Collaborative Samples.—A recent collaborative study of the
AOAC bioassay method for detection of
chick edema disease (12) indicated that the
lower lir.iit of sensitivity was obtained with
a'test cample containing 1.56% 'of a toxic
fat in DSP cottonseed oil. One hundred g
portions of this sample, the toxic fat, and
the original cottonseed oil were analyzed.
Extracted unsaponifiables were chroma tographed on 50 g alumina as described above,
and the 5% and 25% ethyl ether cluates
(fractions 2 and 3) were gas ehromatographcd. Chromatograms of fraction .2 from
the cottonseed oil without and with added
toxic fat are1 shown in Figs. 2 and 3, respectively. Peaks with RA values greater than
.1.5, including the 7JA = 6 and ftA = 10
peaks, are due to the toxic fat, A chromatogram of alumina fraction 2 from 100 g of
test sample containing 0.78% toxic fat in
Table 5. Annlysid of DSP cottonseed oil
containing various levels of nddcd toxic fat(
microcoulometrlc RUB chromutogrnphy of
. alumina (ruction 2
Dlao Integrator nenponse
(No. of Pen Strokes)
Toxic, Put Added, %
Rt - 5
11* -10
0.00
0.78
1.6G
3.12
4.08
0
0
2
G
13
12
28
0.24
6
14
32
32
47
�272
273
the USI' cottonseed oil is shown in Fig. 4.
Tlur 7r\ t= 0 and A'A — 10 peaks cull still
bn definitely detected nt this! level of toxic
f:it, which was luilf of that, found to be nt
the lower limit' of detection of the AOAC
bioassay.
Samples of I'tM' cut (unseed oil containing
various levels of adilwl toxic fnl up to G.24%
were analyzed. The disc integrator response
of the KA = 6 and li\ — 10 peaks are.shown
in Table 5. The integrator response (number of pen strokes) is approximately proportional to the level of added toxic fat in the
cottonseed oil. Each stroke is equivalent to
about 0.05 ;.ig of organic halogen.
Analysis oj Coinnmreuit Olvic and Ktcarir
Acids and /Jcri'i-atii't-s.-'-Twclvo food grade
oleic acids were examined liy this ehronuilo.graphic method. The examination of I) of
these samples for chick edema toxicity was
reported earlier (0). Can duMmatograms of
uluinina fractions from a nontoxic mid n toxic
acid are shown in Figs. 5 and 0, respectively,
Chromatographic jieaks of H\ — 0 and
greater from the toxic sample are .uhowu in
Fig. 6. These ]wakH were, present, in ahimina
fractions 2 and 3. The "peak" with /?.A = G
R A -I.25
(b)
8
10
minutes
Fig. 2— Microcoulometric 'fl
ehromatogromj of (a) oldrin itandord (1 )ifl), <""*
USP cotlomeed oil without added toxic fat.
alumina fraction 2 from
in the ehronuitograin from a
alumina fraction 1 i« believed to be an artifact due to
overloading of the coulomcter.
Results of analysis of the 12 oleic ncids,
compared w i t h the chick bioa.'*ay using a
special Iwwil ration (2), arc shown in Table
ti. llydroperk'ardmm activity, the primary
index 'of the presence of chick edema factor, was estimated as described by Firestone, and co-workers (!)). Six samples were
positive, by both the biossay and ehromatograpliii: methods. Otic sample (No. 0) was
negative by the regular bioassay when fed
at the usual level of 10% in the diet. However, a positive response-was obtained when
extracted un.sapouifiaMe matter was fed at
a. level equit~ale.nl to ('» limes that present, in
Die normal tost diet.
Sample 10 was negative by the chick
bioassay, but gave a weak positive response,
by the chromatographic method. A small
chromatoRraphic peak with R,\ = 10 was
obtained from alumina fraction 2. In coinpaving bionssay activity and peak areas of
plow-ehiting components of the other toxic
oleic acids, it would be expected that the
level of chick edema factor in sample 10
40
minutes
Fig. 3—Microcoulometrit 901 ehfomalogram of alumina fraclion 2 from USP cotlorueed oil containing 1.56%
toxic fal.
;":
Hf.
-Microeoulometrit (jos chroma tog ranu of (o) oldrin ilandard (1 //g), apd (b) USP coltonieed oil
containing 0.78% toxic fal.' Alto iliown are disc intcgrolor pen itroKci. *•
�274
275
Tahlc 6. Anulvhim of commercial oloic acids
to)
CIlronultoKraji'tlo Analysis, Holalivn I'tnk Area {Integrator S'.roki'ip)
Sample No.
llyilrmiciicimlium
Activity (0)
+0.2
+3
K^ 1
E'i 2
(b)
(o)
3
4
S
0
7
0
10
11
12
.
+0.3
+0.1
+0.2
Kt - 6-0
74
340
08
US
40
150
100
RA - 1S-18
KA - 0-10
105
0,0
0
8
104
9
14
00
196
150
88
—
—
+0.1'
—
—
—
25
• Kftch Bti-uki) in equivalent to lihout 0.05 tig organic h;il"Hvi',.
1
Unsaponifiable mutter fed ftt a Invel etiuivalent to 0 tinn.-tt that present in the normal tent diet.
10
20
30
40
minutes
fig, 5—Microcoulometric gas diromalogram) of (o) alumina fraction 1, (b) alumina fraction 2, and (c) alumina fraction 3 from a non-toxic commercial oleic acid,
(c)
R =10
A
(o)
10
20
30
40
minutes
Fig, 6—Microcoulomelric gal cJiromaloBrom! of (ct) olumina frodion I, (b) alumina (raclion 1, ond (c) alu
rnlna traction 3 from a toxic commercial of«ic acid.
iniglil bo below that detectable; l>y the bionssay.
In comparing hydropericardiuni activity
and gas eliromatographie response (relative
peak area), no parallel relationship was
found between the bioassay response of the'
toxic oloic acids and the eliromatographie
response. It should be emphasized, however,
Hint the sltivv'-eluting compounds in toxic
samples represent doth toxic and relatively
iiontoxic materials, and most of the gas
chromalogr'aphic "response is probably due
(o relatively nonloxii! substances. Nevertheless, microooiilonietrie pas ehromatographic
detection of slow-fluting compounds appeal*
(o be an c.fiVotivo scriieninf; tool for segregating (jiidiilionnble sampk;s and diverting them
to noitudiblc. uses or for lurlliur testing by
bioassay.
The R«S chronia(ogr/ij)liic response of the
/?A = 9 peak present at widely different
levels in 2 o!eic nrids was checked by analyfis of individual samples on diHermit days,
using different gas eliromatographie columns
for each run. Results are shown in*Tnblo 7.
Sample I contained a low level of material
responsible for (he Rf, = 9 peak. The differences in integrnlnr resp(jn>e are due both to
variation in coulomcter response and to in(iccurac'ies of disc integrator ri'spoiise at low
halogen levels. The larger a m u u n l s of maU'rial in sample 2 responsible for I he J?A — (1
peak were distributed in alumina fractions
2 nnd 'A to varying extents depending upon
the alumina activity. The total integrator
response from both alumina fractions was
fairly constant, however, varying from 136
to 169 integrator pen strokes.
In addition to the oloic acids, a number
of derivatives of oleio arid (triolein, glycerol
monooleate, etc.) were analyzed. Nine of
ten samples were negative by both the bioassay and chromatographic analysis. One
toxic sample, a triolein, gave gas chromatograms'with peaks of 7?A = 0, 10, and 19.
Ten steitric acids and derivatives examined
were negative by the bion.isny and chromatogrtiphic mxfliod,
Anuliisit of Animal find Vegetable Fats
ami (,'uin'i»vri;iul Yi'gelalilii Oil Falty Acids.—
Fifti'i'ii animal and vegetable fats were examined. Three lo,\ic animal tallows were
positive by the chroniiitographic method.
Table. 7. KcsponRc" of KA. •= 9 peak
from 2 oleio uculs
Bmt
Simple 2
Hun
No.
Frnolion 2
Fraction 3
Fraction 2
I
0.2
O.S
0.0
5.0
0
20
0
0
0
H(i
2
3
4
10S
100
Fraction 3
Hfl
48
0.5
SO ',.
« J)is<j inbwiilur rc.i|H)ii«r in munhi'r of ilisc i
Total
13G
144
ion
150
�276
277
(b)
to)
RA« 8.4
minutes
Fig, 7—Mlcrocoulomelric go* chromatogroms of (a) alumina fraction 2, and (b) alumina fraction 3 from o
growth-dopreuinQ coHotuecd oil which did not produce chick edoma disoaio,
Ten vegetable fats (including cottonseed oil,
corn oil, peanut oil, sii/flowcr oil, and soybean oil) were examined, and were negative by the bioassay and chromatographic
method. Chromatograms from several of the
vegetable oils showed broad bunds with no
definite peaks. Chroraatograms of alumina
fractions 2 and 3 from one of those samples,
a USP cottonseed oil, arc shown lit Fig. 7.
The brond band in chromalogram (a) is
typical of that found in (ho other oils. This
sample depressed the growth of young chickens, but did not produce symptoms of chick
edema disease. The broad band in chromatogram (a) with a maximum at 8.4 is not
characteristic of a toxic f a t . No additional
work has been do'iie to identify the substances causing these broad bands.
Twelve commercial vegetable oil fatty
acids, nonloxic by the chick edema bioassay,
were examined. These samples -included
f.itty acids from coconut, cottonseed, corn,
palm, soybean, and (nil oils. Eleven of the
samples wore negative by the chromatographie method, but one of the samples (a
tall oil fatty acid) gave a small chromatographic peak with fiA = 5, iw.licative of »
toxic sample.
Acknowledgments
The authors wish to express Oheir appreciation to Andrew Yartzoff who ."suggested 111"
initiation'of this work; to Gleiu Shuo, Donald Flick,, and Linda Gallo wl'i/o conducted
the bioassays; and to Benjamin AVebb, Alvin
Frccland, and Peter LeNard wiiw carried out
most of the analytical work.
RBI'BHBNCES
(1) Brow, W. B., Dora, J. B., IJprandict, J. H,
I'ollor, G, C,, and Si]ios, E., This Journal,
• 42, 120 (1959).
(2) Friedman, L,, Firestone, D., itonvitz, W,
Banes, D., Anstead, M., amcl Slme, G.(
ibid., 42, 129 (1959).
(3) Wootlon, J. C., nnd Alexnindor, J. C.,
ibid., 42, 141 (1959).
(4) Harmon, 11. E., Davis, G. K.,. Oil, W. II.,
Brink, N. G., nnd Kuehl, F.. A., J. Am.
Chem.Kne., 82,2078 (1900).
(5) Tislilcr, M., Merck nnd Conipumy, privnlo
coiniminicivtion, July 19, 1900.
(fl) YHrlxuft, A., Firestone, 1)., Bunes, D., (12) Flick. D. F., Giillo, L., "^^1^1, J., Doughiss, C. D., and Fricdnnm, L., ibid., 45,
Hcirwilz, \V,, Friedman, I.., and Noslmim,
2U1 (1SI02).
S., ;. Am. Oil Chi'miats Hoc.., 38, 00
.(13) Ih'flmiuin, K, Clmnnnlogrophv, RcinhoUl
(1901). •
1'ublisliiiiK Corp., New York, 1061, p. 34.
(?) \Voolloii, J. C., Aiiiuiin, N. H., ami Alexmulct, J. C., Thi* Journal, 45, 739 (1902). (14) Bvockmtum, 11., mid Sclioddcr, II., Her.,
74, 73 (HMD.
(8) Ames, S. It., Swimson, M'. J., UulwiR, M.
I., imd lirnkaw, 0. Y., J. Am. Oil Chem- (15) Burke, J., and Johnson, 1'j., 7Vn'j Journal,
45, 318 (10G2).
i*l»' Stir.., 3 7 , 4 (10) (1900).
(0) Fii'cslour, D., IlonviU, W., Friedman, I/.,
and Slum, G.M., ibid., 38, 418 (1901).
KulimitU'd to Wit's Journal for publication
<10) Uoiigluss, 0. D., anil Flick, D. F., 1'his Novi-mlier L'O, 10(12.
'J'liis i>n|n-i' w:\K pvownlml :il tin- RovcntyJuiirniil, 4 4 , 4 4 0 (1901).
u i x l l i Auniiiil Mrolitij; of Hie Ast-oci:iUn» of Olii(11) "Cliiiiigcs in Mi'lliods," ibiil., 44, 146 ciiil ApriciilUirnl Clicinists, Oct. lfl-37, 3062,
at Wnsliint-ton, !).(.'•.
(1901).
�278
279
Reprinted from TOXICOLOGY AND APPUKD PHARMACOLOGY, Volume 5, Number 6, November 1963
Copyright © 1M3 by Academic Preti Inc.
Printed in V. S, A.
TOXICOLOGY AND API-LIED PHARMACOLOGY 5, 760-771 (1963)
The Injection of Chemicals into the Yolk Sac of
Fertile Eggs prior to Incubation as a
Toxicity Test
JOSEPH MCLAUGHLIN, JR., JEAN-PIERRE MARLIAC, M. JACQUELINE
VERRETT, MARY K. MUTCHLER, AND O. GARTH FITZHUGH
Division of Pharmacology., Food and Drug Administration, Department of
Health, Education, and Welfare, Washington 25, D. C.
Received January 24, 1963
The increasingly large number of food additive chemicals introduced
into the market each year has necessitated the development of rapid and
reliable methods for the evaluation of their toxicity. Toxicologic studies
of all these chemicals by the usual methods using animals are very difficult, and such studies sometimes give inconclusive results.
The toxicity of some chemicals, and especially of food additives, may
be determined by injection of the chemical into the yolk sac of fertile eggs
prior to incubation and subsequent observation of the effects of the chemical on the embryonic development of the chick, This appears to be a
promising method in that it may be carried out much more economically
in terms of money and space than would be possible with larger animals.
Hundreds of chicken embryos .may be observed in a minimum of space,
and over a comparatively short period of time: The feasibility of using
such large numbers is valuable also in the statistical evaluation of toxicity
data.
A review of the literature shows how little work has been done in this
field except in a fragmentary way on isolated cases. Most of the reports
refer to injections of chemicals made after the fourth or eighth day of
incubation and examination of the embryos killed before they hatch.
The earliest work that we have found in the literature was by Fere
(1893). During ten years after this date he published about sixty-seven
papers; a review article (Fere, 1899) contains a summary of many of his
studies. His work consisted mainly of injection before incubation, but the
eggs were usually opened on the third day of incubation. His interest was
mainly in the teratogenic effect of chemicals.
contain a wealth of information on the avian embryo.
Preliminary reports of this investigation have been presented by Marhac
(1962) and McLaughlin and Mutchler (1962).
EXPERIMENTAL
'
7 « of *e y«ar. No important variation »as detected^
a
atles 95% A further restriction is based on the waght of the eggs
aU t?ose weeing less than 52 g or more than 63 g are reacted. After
1 Truslow Eggs, Chestertown, Maryland.
�|fc needle (hypodermic linen long ei^r n, « ^^«J
Ilk viscosity of the liquid to be injected) i m s
withdrawHV
Car
The initial experiment with a given chemical is for range-finding
IH A cell into the yolk (Fig. \
^^ ^ce such damage could
, to avoid damaging the vi elhne^^^rane sm
^ ^ ^^
performed at two or more concentrations of the chemical with 10 e&o'r^t ~
the yolk to spread ou m the>to™^ The needle shouW be
level. On the basis of this information, 20 or more eggs are injected l |
l|
ifc
yolk on it, the injection is not satlst™
the appropriate amount of the chemical.
As soon ^ the egg
a sterile gauze pad ^^^^ a small piece of
If the chemical proves to be nontoxic, the experiment is repeated -^
andling, the eggs are randomized in order to avoid series of infertile
i any one experiment.
the minimum number of eggs that will give a reliable and reprodutilft|
value for the hatchability. In the case of a toxic chemical, additional
are injected to determine the specific effects of the chemical. The .—u
number of eggs used for a chemical depends upon the data obtained initu%;,
and upon the kind of information desired. Hence, data for some chemkAl
are based on less than one hundred eggs, whereas data for others are b*8*l-f
on several hundred eggs.
"|
Technique of injection. The injections of pure chemicals, chemical wit"!
tions, or suspensions are made at volumes up to 0.10 ml. When necessary!
dilutions are made with solvents such as water, propylene glycol, corn
peanut oil, or other nontoxic solvents.
,
In order to avoid contamination, the injections are carried out in MM
Isolator Box2 with a sterile atmosphere created by using formaldehyde j
vapors (produced by mixing 2 g of potassium permanganate and SO mi ij
of 37% formalin). During the period of a year, more than fifteen hundred;^
noninjected eggs were exposed to formaldehyde vapors; no toxic effect was !i
noticed. After exposure to these vapors for 30 minutes, the eggs are ready
for injection.
\.
the entire air cell.
GERMINAL DISC
V1TELLINE MEMBRANE
ALBUMEN
CHALAZA
SHELL MEMBRANE
FIG. 1, Diagram of egg and position for injection.
Incubation and hatching. The injected eggs are put into the incubator
The large end of the egg is wiped with a sterile gauze pad moistened |
trays with the large end up; the trays are placed in the incubator,4 which
with a 70% alcohol solution, and a hole is drilled in the shell in the center J
automatically rotates hourly and is maintained at an optimum temperature
of the surface over the air cell (Fig, 1). Care must be taken not to daraag* |
of 38°C and a relative humidity of 60%. The eggs are candled on the fifth
8
the shell membrane with the point of the drill ; this is to avoid, if possible,
day of incubation and every day thereafter. Clear eggs and dead embryos
contact of the air with the egg membrane. Fine particles of shell are reare removed for examination. On the seventeenth day of, incubation the
moved with an aspirator to prevent the needle from carrying them into
fertile eggs are transferred to the hatcher5 and kept at a temperature of
the yolk.
37°C until they hatch.
Immediately before the injection, each egg is shaken with a quick twist
EVALUATION OF DATA
of the wrist. Since the germinal disc occasionally sticks to the air cell and
The injection of the chemical into the egg may produce one of four
it is possible to damage it with the needle, this movement will allow the
disc to float free in the egg.
possible results'. (1) the chemical is highly toxic at the level injected, and
2
8
Kewaunee Scientific Company, Adrian, Michigan.
Burgess Vibrocrafters Inc., Grayslake, Illinois.
* Humidaire Incubator Co., New Madison, Ohio; model no. SO, capacity 4SO eggs.
5
Brower Manufacturing Co., Quincy, Illinois.
�283
i
. all ^Jembryos are killed during the first 20 hours of incubation (before Qj
emmra
*Hn, and this coagulation may decrease the availability of the chemlPFas
the two-somite stage); (2) the chemical is toxic but allows a numberet,jj
**!! as some yolk nutrients, and thereby alter the response of the embryo.
embryos to develop only up to a certain point, and some possibly even W. |
If the pH is highly acidic or basic, a pH effect differing from the true
hatch; (3) the chemical has little effect on the hatch; and (4) the chct8-||
toxic effect of the chemical may be obtained due to interference with the
ical has no effect on the hatch or on the posthatch development of the chid |
|
|
normal acid-base equilibrium in the egg.
("no effect" level).
^
The ionic concentration is also important for a similar reason. The obIf the chemical appears to be highly toxic and all the embryos are killed,;
lervation of increasing toxicity with increasing concentration of a chemical
the experiment is repeated with smaller doses of the chemical until sow
should be interpreted cautiously, since highly concentrated solutions may
hatch is obtained. If the chemical is toxic, but allows the embryos to deUpset the physical equilibrium of the yolk by causing osmotic effects.
velop for a longer period of time, dead embryos are examined pathologically
and the chicks that do hatch are examined for eye damage, color of the
Finally, the introduction of a chemical into the yolk may cause a special
feathers, weight, length of the legs, form of the beak and of the rump,
type of toxicity because it destroys, alters, or combines with essential
hematologic changes, and condition of the internal organs (liver, kidneys,
nutrients such as vitamins and minerals.
heart, gall bladder, and spleen).
EXPERIMENTAL DATA
It is advisable in all cases to observe the chicks for a period of a few
Twenty-five thousand eggs have been used in our laboratory during
weeks in order to detect any delayed effects. Since as much as 30°/o of the
the past three years to test more than 100 chemicals with the following
yolk remains at the time of hatching and is absorbed during the first 7
days thereafter, effects of a toxic chemical may first be observed at this
TABLE 1
time. There may be weight retardation, death during the first week, or the
NONTOXIC CHEMICALS
appearance of nerve damage occurring as late as 2-6 weeks after hatching.
Solution injected
The toxicity of a chemical is evaluated mainly from the percentage
Per cent
Quantity
hatch
of hatch at varying dosages of the chemical as compared to noninjected
(ml)
Concentration
Chemical
(control) eggs, from a study of the embryonic development of the eggs
95,
0.05
Water (boiled)
r\t
—
95
that fail to hatch, and from a study of the appearance and development
0.05
Undiluted
Propylene glycol
90
of the chicks that do hatch. However, several other factors must also be
O.OS
Undiluted
Corn oil
90
considered in this evaluation: these are specific gravity, solubility, coagulat0.05
Undiluted
Peanut oil
90
ing effect, pH, and the ionic concentration of the chemical tested.
0.05
0,9% in water
Sodium chloride
70
0.05
If the chemical has a high specific gravity, there is the possibility of its
5.0% in water
30
0.10
5.0% in water
settling out in the bottom of the egg and thereby giving a value of ap20
0.05
10.0% in water
parently low toxicity.
90
0,05
5.0% in water
Dextrose
The solubility is quite important since the availability of the chemical
for utilization in the chick embryo is partially dependent on its solubility
results: (1) nontoxic chemicals injected at an appropriate level allowed
in the egg. However, since egg yolk is an emulsion, solubility problems are
the embryo to develop and to hatch as did the controls; (2) toxic chemicals
somewhat minimized. In the case of insoluble chemicals that are injected
produced effects at dose levels which may be compared to those producas suspensions, it is also necessary to consider particle size in the evaluation
ing effects in feeding experiments using animals; and (3) this technique
of toxicity data.
often provided toxicologic information which had not been shown by conIn order to have a toxic effect, the chemical must come in contact with
ventional methods.
the embryo either directly or indirectly through the bloodstream. Chemicals
Table 1 lists the results obtained with some chemicals in common use
such as the lower aliphatic alcohols have a coagulating effect on the proin food and shows the dosages used and the percentages of hatched chicks.
, 49-362 O - 70 - 19
�285
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use in food. However, it must be pointed out
effec t
.
d at a sufficiently high concentration, may have some
Isince our data and those reported in the literature indicate
I phase of the study was not carried beyond a prehrmnary e,
&n that nontoxicity also was shown for * - b byjh h*k
- ^
tmbryo technique. This is of theoretical as well as practical i
ffor the evaluation of any new technique to be used m tox.cologic
Bc»k deformity (»hor!
. i*Wnwt, »Mwtll»*<<
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Beak deformity
• 1i
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Most of the developin;g embryos died
on the 15th day o f incubation
Hatched chicks showed growth re-
284
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Lead acetate
in no
at a
of 1 mg per
Autopsy
of the dead embryos showed extensive brain damage, as has beeri reported
; ;by deFranciscis and Bocalatte (1962) and by Karnofsky and Ridgway
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• chloride showed no hatch even at a level of 0 5 mg per egg.
.mourea is a known carcinogen with a basic effect ^^^
f The hatch time was delayed with increasing amounts of this chemical At
| IteTof™egg no chicks hatched and the embryo require 35 days
""" to develop to the stage normally attained at 20 days. At 2 5 m g _ p e r e g g
some chicks hatched, but most of them had to be helped out of the shell.
This effect has been reported also by Yushok (1950)
Cavanagh (1954) reported that triorthocresyl phosphate (TOLF). a
weUkno'n plasticizer fo'r nonfood use, causes paralysis when.fed£> aduU
chickens. We observed this paralysis in some chicks which hatched from
eggs injected with 10 mg of the undiluted chemical.
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questions as to the safety to the consumers of any food contaminated with
C
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bending of the tibia and a general shortening of the bones of the l
egg,
Sodium selenite proved to be highly toxic. At a level of 0.1 mg
no embryos developed to more than the 5-day stage.
Drbutyl-tin-dilaurate showed no hatch at a level of 10 mg per egg The
majorhy of the embryos, which did not live more than 15 days at th,s
�286
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level, had a short and/or flexed mandible; in addition, some embryos
^,
/-*' showed subcutaneous edema.
i
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Aroclor 1242 gave no hatch at a level of 25 mg per egg. At a level of
o
it 10 mg per egg, one chick hatched out of 20 injected eggs, but died 2 days
S
iji
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pO
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''t later. Some embryos, which were examined after they died, showed beak
in 13
bfl 13
deformities (often a short upper beak), edema, and growth retardation.
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Table 3 lists preliminary data on the toxicity of some chemicals which
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have been shown to be toxic in some degree to animals, and which may be
w£
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1* found in some processed foods. Included in this group are various solvents,
plasticizers, and insecticides. Some of the chemicals on this list require
/—*—.
further study, including observation of the chicks until they reach ma55 ^o o^ **•• • *o So fi TH o o g 3 Si
turity, before they may be classified as to low or high toxicity.
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The injection of chemicals into the yolk of fertile eggs prior to incubation is a method which can be advantageously used as an element in the
evaluation of the safety of food additive chemicals and drugs, and which
^
could be used to screen new products-and eventually to correlate their
toxicity with that of similar products already tested.
If one considers that a chemical injected into the yolk may be comi pared to a substance which has the power to cross the placental barrier,
¥
this technique, in addition to being an embryonic feeding study, assumes
further importance in that it is also a reproduction study. The unfortunate
experiences recently suffered with chemicals that have teratogenic effects
|"4 in humans, and the failure of conventional testing methods to produce
this effect in animals, emphasize the urgent necessity for new methods of
analysis. Preliminary work that we have done in this area has given satisfactory results (Verrett and McLaughlin, 1963).
f
Since this represents a system in which the chemical is in direct contact
with the embryo throughout development, it is more than likely that any
1
toxic or teratogenic effects would be readily observed. However, there is
i
always the possibility that the chicken will not be a species susceptible to a
i?
particular compound, just as it has been shown that the other commonly
i
used species of animals do not respond to all chemicals in a similar manner.
1
It is also possible for the chicken to be more sensitive to a chemical than
other species.
f
Finally, this technique may be applied also to the study of the synergistic effects of chemicals. Results of experiments in our laboratory on
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DISCUSSION
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�288
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the potentiation of a few pesticides have been very encouraging (Marfi«|
and Mutchler, 1963).
SUMMARY
An evaluation of toxicity by injection of the chemical into the yolk sac of
eggs prior to incubation gave the following results:
Water, propylene glycol, corn oil, peanut oil, isotonic saline solution, and isotoaic '|5|i
glucose solution showed no toxicity or a very low order of toxicity.
";|if
Mercuric chloride, lead acetate, selenium, triorthocresyl phosphate, £,#'-diam!<
diphenylmethane, thiourea, Aroclor 1242, and dibutyl-tin-dilaurate showed a W
order of toxicity and/or teratogenic effects at certain levels.
Acetone, methanol, ethanol, n-butanol, diethylene glycol, ethylene glycol, ii* '
propanol, di-2-ethylhexyl phthalate, hydrochloric acid, carbon tetrachloride, etijf \
acetate, malathion, heptachlor, and styrene showed an intermediate order of toxidiy,
REFERENCES
CAVANACH, J. B, (3954). The toxic effects of tri-ortho-cresyl phosphate on li*
nervous system: an experimental study in hens. /. Neural. Neurosurg. Psychial. il,
163-172.
DE FRANCISCIS, P., and BOCALATTE, F. (1962). Lead acetate and develqpment of If*
chick embryo. Nature 193, 989-990.
FERE, C. (1893). Note sur 1'influence, sur 1'incubation de 1'oeuf de poule, d'injectiom
pre'alables dans 1'albumen, de solutions de sel, de glucose, de glycerine. Compl. Rend,
Soc. Biol, 45, 831.
FERE, C. (1899). Teratogenie experimentale et pathologic gene>ale. Cinquanlenoin
de la SocUte de Biologic, Vol. jubilaire, pp. 360-369.
GOERTTLER, K. (1962). Der 'teratologische Grundversuch1 am bebruteten Hiihnchenkeim seine Mo'glichkeiten und Grenzen. Klin, Wochschr. 40, 809.
HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the
development of the chick embryo. /. Morphol. 88, 49-92.
KARNOFSKY, D. A., and RIDCWAY, L. P. (1952). Production of injury to the central
nervous system of the chick embryo by lead salts. J. Pharmacol. Exptl. Therap. 104,
176-186.
KEMPER, F. (1962). Thalidomid und Entwicklung von Huhnerembryonen. Arzneimittel-Forsch. 1 2 , 640.
. , ' ,
, . , , . ,
MCLAUGHLIN, J., JR., and MUTCHLER, M. K: (1962). Toxicity of some chemicals
measured by injection into chicken eggs. Federation Proc. 21, 450.
MARLIAC, J. P. (1962). Injection of chemicals into chicken eggs as a toxicity test.
Federation Proc. 21, 450.
MARLIAC, J. P., and MUTCHLER, M. K. (1963). Use of the chick embryo technique
for detecting potentiating effects of chemicals. Federation Proc. 22, 188.
PLATT, B. S., STEWART, R. J. C., and GUPTA, S. R. (1962), The chick embryo as a
test organism for toxic substances in food. Proc. Nutr, Soc. (Engl, Scot.) 21, XXX.
RIDGWAY, L. P., and KASNOFSKY, D. A. (1952). The effects of metals on the chick
embryo: toxicity and production of abnormalities in development. Ann. N.Y. Acad.
Sci. 66, 203-215.
289
A. L.
(I960). The Avian Enoryo: Structural and Factional Develop-
mtnt, 1st ed. Macmillan, New York.
, A. L., and ROMANO^ A. J. (1949). The Awn ff
M. J., and MCLAUGHLIN, J., JR. (1963). use 01 me t«
iff compounds on the rat liver.
IMi.
York
*
^
/
�290
291
injected at two or more levels when there
was sufficient material available. More than
8,000 eggs have been used in these studies to
date.
The injections into the eggs were made
by one of two routes: into the yolk, or into
the air cell. The technique for injection into
the yolk has been described previously (2).
The volume injected into the yolk was 0.05
ml or less in all cases. For injections into
the air cell, n hole of about 5 mm diameter
was drilled in the shell over the air cell.
The solution was then deposited on the egg
membrane by a syringe, and the hole was
scaled with adhesive cellophane tape. The
eggs were allowed to remain undisturbed in
a vertical position (air cell up) for about
an hour to let the material disperse. The
volume injected into the air cell was rer^., The sensitivity of the chicken embryo to stricted to 0.04 ml or less.
The solvent used for all injections was
JS tflstoxins was reported in 1962 by Platt.-et
IF' «/. (1) who observed that preparations of propylene glycol, which was known, from
$': "groundnut toxin" injected into the yolk of previous investigations (3), to be nontoxic in
6-day old chicken embryos caused deaths, the eggs at the levels used. However, eggs
and'that the quantities required were about were periodically injected with this solvent
l/200th of those required .for a positive re- in appropriate amounts and incubated with
the aflatoxin-injected eggs. Noninjected conwit in the day-old duckling.
An investigation of the feasibility of using trols and drilled-only controls were also inthe chicken embryo for a bioassay of afla- cluded in the experiments to provide the
loxin toxicity is currently underway in our necessary data on the background mortality.
The eggs were incubated (2) and candled
Inboratories. The preliminary results reported here consider only the general sys- daily from the fourth inc.ubation day on, at
temic toxicity of the aflntoxins lo the chicken which times all nonviablc embryos were reembryo. At the present time, the studies moved and examined grossly.
fef »«« resulted in a toxic response that
ft*f«*rr*latod well with that obtained by
fjkjedion of pure aflaloxin B, solutions
"$JH llic same dose levels, nnd in most
i the chemical analysis was conThe presence of aflaloxins G,,
,,-„ ond G,, had no apparent effect on
1*0 loxicily due to aflatoxin B,, at the
* Itvels nl which they occurred in the
particular samples tested.
The separation of aflaloxin B, from
ftmlnmiiuitcd extracts by thin-layer
(bromnlograpliy, and its subsequent
tlulion from the plates and injection
Into the eggs, confirmed lhat the toxicily
«f these extracts was due primarily to
Uielr aflatoxin Bj content.
Use of the Chicken Embryo in the Assay of Aflatoxtn Toxicilj'
By M. JACQUELINE VERRETT, JEAN-PIERRE MARLIAC, and JOSEI'lI McLAUGHLIN, JR. (Division of Toxicological Evaluation, Food and Drug Administration, Washington, D.C. 20204)
The possibility of using the chicken
of aflaloxiii-producing mold cultures inembryo as a test organism for the assay
dicated that the chicken embryo mi
of fiflaloxin toxicity lias been investisensitive to these compounds. A dosegnled and found to he feasible. The
injections of test solutions were made response was exhibited in that the toxicity of the samples was related to the
before incubation, in fertile White Leg- mortality at Uie j
t m e of hatching.
horn eggs, by either of iwo routes:
Extracts of nflatoxin-free pcnnul
yolk or air cell. The development of
products were found to Le nontoxie lo
the embryos was observed for the full
the chicken embryo. The addition of
21 day incubation period. The vehicle
aflatoxin B, to such ui.coiiiuminatcd
for all injections was propylene glycol.
The injection of solutions of pure extracts produced the expected toxicily
nflatoxiiis B l nnd G |t and of extracts in the embryos. The i.ijeclion of extracts from contaminated peanut prod-
are not sufficiently complete to verify
whether the aflatoxins produce any specific
pathological lesion in the embryos.
Experimental
The sensitivity of the chicken embryo to
aflatoxins was. studied by injecting the following: ()) solutions of pure aflatoxin "S>1 and
pure aflatoxin G t ; (2) extracts of aflatoxinproduciug mold cultures; (3) extracts of raw
peanuts, roasted peanuts, peanut meal, and
peanut butter; and finnlly (4) aflatoxin B,
obtained from crude extracts by clution from
(lun-laycr chroinfttographic. plates.
The solutions were injected into fertile
While Leghorn eggs before incubation.
Groups of at least 20 eggs were used at each
dose level of a sample, nnd each sample was
Results and Discussion
1'nrificd Aflatoxin Solutions and 1(1 old Culture Extracts.—The toxicity of solutions of
crystalline aflatoxin B, and aflatoxin G, 'to
the chicken embryo was first determined.
The toxicity of aflatoxin B, to the chicken •
embryo was greater when injected via the
air cell route than when injected into the
yolk. Figure 1 contains plots of the mortality at 21 days due to the injection of
several dose levels of nflaloxin B, by both
the yolk and the air cell routes. The LD5ns
obtained were 0.048 nnd 0.025 /ig for the
yolk and air cell routes, respectively.
Allatoxin (!,, which was injected inlo the
yulk only, showed a lower loxicily to the
chicken embryo than that obtained "with
�292
aflaloxin 33,. The injection of 1.0 us of nflatoxin 0, produced ft mortality of 00% (nt
21 days), while 2.0 //.g wns. required to produce a mortality of 00%.
fig- ?—Toxicity of aflatoxin Bi in Ihe chicken embryo; mortality at 21 clays, LOsoi yolk, 0.048 /ig; air
cell, 0.025 fig. Open squares: yolk injection, Closed
squares: air cell injection.
Examination of nonsurviving embryos from
eggs injected with nflatoxin B, by either
route revealed a severe growth retardation
in most cases, In addition, edema, hemorrhage, undordevfilopmciit of the moseneephnlon (in embryos that died before the seventh
day), mottled and granular liver surface,
short logs, and slight, clubbing of the down
were also observed in many of those embryos.
Extracts of several cultures of nflntoxinproduciug molds were used to determine the
sensitivity of the chicken embryo to combinations of nflntoxing B n G,, B,,, and G.,. The
concentrations of these four constituents in
(he exfracts were known from prior chemical
analysis. The extracts were injected into
eggs by both routes, in amounts designed
to contain .specific levels of aflaloxin B,, to
compare their loxicities to I how of solutions of (ho pure nflnloxin B, at the same
dose levels.
293
and air cell injections were made in embryos
route, and tlmt the toxicity was not
The toxicilics of these extracts to ll*
from 1 to 18 days old.
iiliy different from that obtained
bryos showed a good correlation wi&
These experiments revealed that, with both
jj*i* UK* solution of pure adatqxin U t of the
standard solution of nflatoxin B,. Tl*ff
injection routes, the embryonic, sensitivity to
M* concentration.
no apparent alteration in the toxicity «< (A*
the aflatoxin decreased rapidly as the emAfntoxin-conlaminated Peanut Products.
toxin Bj due to the presence of nftiWHW?
bryo age increased, and the maximum toxic
A c l s ' from raw or roasted peanuts,
B,, G,, and G 2 at the levels at wliiefc tf
effect was obtained with pre-inc.uba.tion inmeal, and peanut butter, which were
occurred in these extracts.
n to contain aflatoxins by chemical amil- jections.
Aflatoxin-free Extracts of I'camil
Evaluation uj Sumjilc. Toxicily.—In the
were injected into eggs by both routes.
nets.—The usefulness of the chicken
course of these studies it was also observed
amount of extract to be injected in the
in a bioassay of aflatoxins in peanut
that the toxicity of aflatoxin B, injected at
was calculated on the basis of the nflanets depends on whether the corisfiltr
the higher dose levels was apparent early in
teun Jl, content determined chemically, irreaflaloxin-freo peanut product extract*
the incubation period, since most of the om'livo of the amounts of aflatoxins U 5 , G,,
inherently toxic to the embryos.
• bryos did not survive beyond the eighth to
. present. In most instances the. results
To determine the toxicily of these u»i*<
tenth day. With lower dose levels', it is necflwitolxmited the chemical analysis, since the
rials, extracts of raw • peanuts, roasted p»j
essary to continue observations for the duraftaieity was comparable to that obtained
nuts, peanut meal, and peitnut butter, »fc«ii
tion of the 21 day period, since many emWlh the injection of equivalent amounts of
were found to be free of afiatoxins by diw*
bryos survive longer than the tenth dny but
UK pure aflatoxin B, solution.
eal analysis, were injected into eggs 1>) Wfe
fail to hatch. In these instances an evaluaAfatoxin B, Obtained by Thin-Layer
the yolk and air coll routes. In most of tin*
tion of the toxicity of a sample on the basis
Cknmatoornphy (TLC).-ln order to conexperiments the equivalent of original pac,
of survivors at 8 or 10 days would be preInn Hint the toxicily of the contaminated
nut product injected ranged from 1 l o J f
mature, and the true toxicity of a sample
fcrtrncts wns primarily due to their aflatoxin
per egg. The toxicity was low for nil rf
might be underestimated.
B, content, separate portions of some cxthese extracts; in general, it was equal I*
IrtcU were subjected to TLC and the roor only slightly higher limn that of Ifcr
Acknowledgments
wlliiiK aflatoxin B, spots were removed from
background, which ranged from 0 to SC^
We wish to thank Mary 1C. Mutchler and
U»e plates, eluted, dissolved in propylene
mortality.
William F. Scott for their technical assistance
fljcol, and injected into eggs.
One experiment was carried out with w
Separate experiments were performed to in this work.
extract of aflatoxin-frco raw peanuts, *•
The Division of Kood Chfinittry, Food and
confirm that no toxic materials were derived
jccted by both the yolk and air cell roiih
Drug Administration, supplied the pure, aflafrom the TLC process itself. TLC "blanks"
in quantities corresponding to a raw pe.im*
toxin B, and G t and the extracts of mold
injected into eggs in a similar manner had
equivalent of 1, 2, 4, and S g per egg. Tbt
cultures and peanut products, nnd per* very low toxicity and .were comparable to
toxicity observed for the S g level wns «*
formed the chemical analyses of these exbackground.
significantly higher than dial observed f(*
The toxic.it.ies of these eluted aflatoxin D, tracts.
the ] g level pr that of the background,
•pots from more than 20 extracts of a variIn the same experiment, known qnantilM
HRFUBKNCKS
ety of peanut products correlated very well
of the pure aflatoxin B, were added to lira
(1) Pln.lt, B, S., Stewnrt, U. J. C., nnd Gupta,
with that of standard nflatoxin B! solution
raw peanut extract, and injected at the same
I?.., Proc. Niilr. ,Soc., 30, 21 (t%2).
injected at the same dose level, and verified (2) Mclaughlin, J., Jr., Marline, J. P., Vcrlevels mid by both routes. This was done l»
(hat the toxicity of these extracts was, .in
vctt, M. Jacqueline, Mulchler, Mary K.,
determine whether the nfinUixin B, toMnty
am! 1'iUhMRh, 0. G., Toxicol. Appl. Phur(act, primarily due to aflatoxin B,.
could be masked or enhanced by the presEmbryo Age and Aflatoxin Toxicity.—The
, macol, 5, (CO (1903).
ence of increasing amounts of peanut innlftoxicity'of aflatoxin I}, to the chicken em- (3) Mclaughlin, J., Jr., Marline, J. P., Vervetl,
rial. Separate groups of eggs were injected
M. Jncr|iK'line, Mutclilcr, Mnry K., nnd
bryo n't various stngcs of incubation was also
with corresponding amounts of the st.iwl.inl
• FilzlniRh, 0. G., Am. Ind. llyg. Aasnc. J.,
investigated. Single injections into the yolk
aflatoxin B, solution for comparison. Tltc
25,282 (UIQ-i).
wore made up to the fourth inco.ba.lion day,
results of this experiment, indicated that ni
1
little as- 2. ) pph of aflatoxin B, in the original raw peanuts could be oasily detected by
Reprinted from the Journnl of the Association of Official Agricultural ChenM,, Vol. 47,
administration of the extract by either inDecember 1964.
. Tills impel- wns prcwnhil nt tliu "sw7n'lyijitflilh Aimiiiil Mwliiii; nf llic Asmrinlinn nf
Ollicml Agrii'iiltm-iil ClirmiKts Oct. 10 -"2 111(11
nt Wn'shiiiKlnii, U.C..
�294
295
THE BOLE OF "Toxic FAT" IN THE PRODUCTION OP HYDBOPEBICAKDIXJM
AND ASOITES IN CHICKENS
ie renu
Mood counts were conducted on a portion of the sample, and the remainder was
allowed to clot. After centrifugation the serum was saved for total protein,'
7
electrolyte concentration, and nonprotein nitrogen determinations' and electrophoretic studies.21
In experiment 1, surviving birds were used for vascular perfusion studies.
Immediately after they were killed, the thoracic aorta was perfused with 5.0%
dextrose to remove blood, and 1.0% silver nitrate was injected to outline the
"cement substance" between the endothelial cells of the mesenteric capillaries.
Slides were prepared of the mesenteric vessels and examined for alteration of
Interendothelial silver precipitate in birds consuming toxic fat.
Five control and 5 test birds given (he ration containing 1.0% toxic fat were
"elected for hemodynamic studies after 150 days on trial. Venous pressures
were determined by making an incision through the skin over the jugular vein.
The vein was dissected away from the surrounding tissue, and 2 ligatures
were placed around the vessel. The superior legature was made secure, and a
small incision was made into the vessel. A No. 5 cardiac catheter was placed
In the vein and made secure by the inferior ligature. The birds were placed
under a fluoroscope and the exact position of the catheter determined. The
catheter was attached to a 3-mrn, water manometer filled with physiologic
saline solution, thus enabling pressures to be determined in the venae cavae
flnd right ventricles.
At the termination of experiment 2, tissues were obtained for electron microscopy by severing the cervical spinal cord and exposing the heart by removal
of the sternum. Sections were taken from the left ventricle and cut into blocks
of approximately 1 mm. with a razor blade. The tissues were immediately
fixed in 1.0% osmium tetroxide, dehydrated, and embedded as outlined by
Palade.15 Thin sections2 were cut on a cicrotome.1 Tissues were examined with
an electron microscope opera/ting at 50 kv.
James E. Allen, D.V.M., Ph.D.
SUMMARY
When "toxic fat" was added to the diet of experimental birds at concentrations from 0.25 to 5,0% for 35 to 150 days, edema of the myocardium, skeletal
musculature, and lungs; hydropericardium; ascites; and foci of lymphoytes in
the myocardium and epicardium were observed. Appreciable changes were not
observed in the total serum protein levels, albumin: globulin ratio, electrolyte
balance, or in the nonprotein nitrogen levels of the blood. There was dilation,
edema, and lymphocytic infiltration of the heart. The myocardial mitochondria
were vacuolated and shrunken. An increase in venous pressure was also
noticed. The fluid imbalance observed in birds that consumed toxic fat' did not
result from a decrease in total blood proteins or from an alteration in the
albumin: globulin ratio, but was associated with cardiac decompensation and
increased capillary permeability.
Schmittle et al.™ were the first to incriminate some fats as responsible for
the production of hydropericardium and ascites in young chickens. Subsequent
studies 3i" have demonstrated a reduction in growth rate, retarded sexual
development, and increased mortality in pullets that have consumed toxic fat
A marked reduction was observed in the hatchability of eggs from hens fed
toxic fat." Turkeys and ducks appeared to be4 less susceptible than chickens to
the detrimental effects of this fat in the diet. Fat-soluble tissue extracts from
chickens fed toxic fat were capable of producing hydropericardium and ascites
when uadded to the diet of other birds." Associated with the transudate, Sanger
at al. observed necrosis of the liver, subepicardial hemorrhage, and lymphocytic infiltration of muscle fibers in chickens consuming toxic fat. In addition to
these changes, Simpson et al.™ also noticed bile duct hyperplasia and proliferation of the endothelium in the parenchymal tissues. The unsaponifiable portion
of some batches of fat was found responsible for the toxicity.2'18'17'2* Repeated
passages of this fraction through alumina and silica gel columns led to the isolation of a crystalline factor of unknown structure which was able to produce
anasarca.8
The primary aim of this study was to investigate the mechanism by which
toxic fat produces anasarca. Preliminary studies suggested that a vascular or
cardiac injury may be responsible for the transudation of fluid into tissues. In
the hope that some clarification of the mechanism might be obtained, arterial
perfusions, recordings of hemodynamic changes, and ultrastructural myocardial
studies were undertaken.
MATERIALS AND METHODS
In experiment 1 (acute), 100 White Leghorn cockerels, 1 day old, were separated into 5 groups of 20, placed in heated batteries,- and fed rations containing 0, 0.5, 1.0, 3.0, and 5.0% toxic fat* for 35 days. Because the exact chemical
nature of toxic fat is not known, the amount of the toxic material undoubtedly varies in fats from different sources. The fat used in these experiments
was from the same source, identical shipments, and had the samd LDso when
f e d t o day-old chickens.
'
.
.
.
.
,
In experiment 2 (chronic), forty-eight 4-week-old cockerels of comparable
weight were given, for 150 days, a ration** containing 0, 0.25, and 1.0% toxic
fat The birds were fed and watered daily. Throughout the trial period Hie
general appearance, food consumptions, and mortality were spleen, pancreas,
kidneys, adrenal glands, skeletal muscle, brain, and bone marrow were fixed in
10.0% neutral formalin, embedded in paraffin, sectioned at 7 p, and stained
with hematoxylin and eosin. Frozen sections were prepared from the livers
and kidneys and stained with Sudan IV to demonstrate neutral fats. At the
termination of the trials, blood was obtained from the cephalic vein, complete
Received for publication .Tan. 8, 1084.
Krom the Depnrtment of Pathology, University of Wisconsin Mecllcnl School, Madison.
This Investigation was supported in part by Public Henlth Service research grant
nF-898!) from the Nntionnl Heart Institute, Public Health Service.
The author Is Indebted to Mr. Homer Montague for the photography.
• Emery Industries, Cincinnati. Ohio.
•• McMlllen Feed Mills, Fort Wayne, Ind.
RESULTS
i
In experiment 1, birds given rations containing 0 to 1,0% toxic fat survived,
whereas those consuming rations with 3.0 and 5.0% toxic fat had a 25.0 to
G5.0% mortality, respectively (Table 1). As the level of toxic fat in the diet
TABLE 1.—EFFECTS OF TOXIC FAT CONSUMPTION ON CHICKENS
No. birds
0
0.5
20
20.
20..
20
20
Toxic fat In
diet (percent)
:;
12
12..
12..
12...
1.0
3.0
5.0
0
0,25
0.5
1.0
Days on trial
No. died
35
35
35
35
35
150
150
150
150
0
0
0
2
11
0
1
4
6
Ascites
Hydropericardium
L
.
20
20
0
1
0
2
20
20
0
4
8
11
was increased, there was a corresponding decrease in growth rate. The control
birds averaged 348 Gm., whereas those given rations with toxic fat averaged
176 Gm., at the end of 35 days. Hydropericardium and ascites were not
observed in the chickens fed rations containing 0 and 0.5% toxic fat. Perionrdial fluid volumes ranged from 0.5 to 5.0 ml. in the birds given rations with
1.0 to 0.5% fat. Ascites was also observed in these groups, but the volume was
not determined due to the partial coagulation of the transudate.
In experiment 2, 6 birds fed 1.0%, 4 fed 0.5%, and 1 fed 0.25%.toxic fat in
the ration died. The mean survival time for the groups was 80 ± 20, 100 ±
14, and 135 days, respectively. The control group gained an average of 10.5
Gm., whereas the survivors of the group fed the ration containing 1.0% toxic
fat gained 14.9 Gm. per day. The weight gain of the surviving birds of .the
1
1
Porter-Blum m)cro f ome, Ivan Sorvall. Inc., No-rwalk. Conn.
RCA, BMU-3G, Radio Corporation of America, Cnmden, N.J.
�296
297
fed rations containing 0.5 and 0.25% toxic fat was almost
to that of the controls. Hydropericardium was observed in 11 and ascites
birds fed rations containing 1.0% toxic fat. Eight of 12 birds on the 0,5%
toxic fat ration had hydropericardium but were free of ascites. The average
volume of pericardial transudate in the test birds was 13.5 ml. No appreciable
difference was noticed in the organ weights of the test and control birds, With
the exception of the testes and hearts of the birds in experiment 2. The average weight per testis of birds in the control group was 14.9 Gm., whereas that
of the 0.25% group was 6.6 Gm., the 0.5% group was 4.1 Gm., and the 1.0%
group was 3.9 Gm. The hearts of birds of the control group averaged 15.0 Gm.,
and those of birds in the test groups averaged 23.0 Gm. and were markedly
dilated in most cases (Fig. 8).
Tissue sections of the various organs were examined microscopically. Tlie
birds with hydropericardium had fibrinous deposits and foci of lymphocytes on
the visseral pericardium. The muscle fibers were separated by edematous fluid.
A number of the small myocardial artieries were surrounded by lymphocytes.
There were also foci of lymphocytes between the myocardial fibers (Fig. 12).
The lungs were congested and had a moderate amount of peribronchial lymphoid hyperplasia. Pulmonary edema was found in those birds which died during
the experiments. Livers from birds .with marked ascites frequently had thickened capsules. In many cases there was coagulation of the transudate on the
convex surface of the liver which formed a false capsule (Fig. 3). There was
moderate fatty infiltration of the hepatic cells in birds given toxic fat in
experiments 1 and 2 (Fig. 4). Extensive lymphoid hyperplasia around the perlportal areas was found consistently (Fig. 5). Five of the test birds in experiment 2 also had myeloid hyperplasia scattered throughout the parenchyma!
tissue of the liver.
Microscopic examination of the testes of the birds given toxic fat in experiment 2 revealed a reduction in size of the seminiferous tubules. The Sertoll
cells and spermatogonia appeared normal, but there was a reduction of the
primary and secondary spermatocytes. These maturing cells were reduced to
the point where no spermatids and spermatozoa were observed. Cells in tlie
testes of the test birds appeared normal. The major difference between testes
and 143 mEq./llter in the control birds. The potassium levate were 5.3
raEq./liter in the test birds and 5.5 mEq./liter in the control birds. ^A
When 1.0% silver nitrate solution was perfused in the thorac^aorta and
the mesenteric capillaries examined microscopically, a difference was found in
the cement substance located between the endothelial cells of the test and control birds in experiment 1. The control group had uniform diamond-shaped
bands of silver precipitate between the capillary endothelial cells, whereas the
bands in the test birds were irregular and indistinct (Fig. 9,10).
Catheterization of the heart revealed tlie mean right ventricular pressure of
the control birds to have an average value of 15.3 Cm. of water, whereas the
test birds "had an average of 21.3 Cm. of water. The pressures in the inferior
vena cava averaged 5.5 cm. of water in the control birds and 7.1 cm. of water
in the test birds (Table 3).
TABLE 2.—SERUM PROTEIN STUDIES ON CHICKENS FED TOXIC FAT
Toxic fat in diet
(percent)
0 ..
0.5...
1.0...
3,0..
5. 0...
Serum protein
(Gm./lOO cc.)
3.36
3,24 .
3.14 .
3.08 .
3.06
Albumin
(Gm./lOO cc.)
Globulin
A/G
(Gm./lOO cc.)
Nonprotein
nitrogen
(mg./100cc.)
Sodium
(mEq./liter)
Potassium
(mEq./lilsr)
1. 06
2. 30
46
21.7
143
5.5
0.94
2.12
44
20.7
146
5.3
of birds in test and control groups was a lack of spermatogenesis and n
marked reduction in tubular size (Fig. 6,7) in testes of test birds.
Total blood proteins were determined by the micro-Kjeldahl method. Tlie
control group of experiment 1 had an average value of 3.3C Gm./lOO cc. The
0.5, 1,0, 3.0 and 5,0% groups had values of 3.24, 3.14, 3.08, and 3.00
Gm./lOO cc. of protein, respectively (Table 2). In experiment 2, it was found
that the groups given rations containing 1.0% toxic fat had an average of 4.1
Gm./lOO cc., the 0.25% group averaged 4,5 Gm./lOO cc., and the controls had
an average value of 4.8 Gm./lOO cc. of serum protein. The protein level of the
ascitic fluid of the experimental birds was 1.7 Gm./lOO cc., with no appreciable
difference in the levels of the various groups. The albumin: globulin ratio was
determined by paper electrophoresis, and little difference was found in the protein ratio of the various groups.
Sodium, potassium, and nonprotein nitrogen levels were determined on serum
Rumples from 6 birds given rations containing 5.0% toxic fat and 6 control
birds. No appreciable difference was found in the nonprotein nitrogen levels of
the 2 groups, with the controls averaging 21.5 mg./lOO cc. and the test birds
20.7 mg./lOO cc. The sodium values were 146 mEq./liter in the experimental
TABLE 3,-HEMODYNAMIC ALTERATIONS DUE TO TOXIC FAT CONSUMPTION IN CHICKENS
Mean right
ventricular
pressure
0
. .
.
o
0
0
0
5,0...
5.0
5.0
5.0
5.0
„
"
". ."
Mean
inferior
vena cava
pressure
(cm. HjO)
Toxic fat in diet (percent)
Mean
superior
vena cava
pressure
(cm. HsO)
(cm. HsO)
5.6
5.4
5.2
5.8
5.4
7.4
7.0
7.2
6.5
6.8
5.2
5.3
5.6
5.8
5.7
7.0
7.1
6.9
7.2
7.4
15.4
15.8
15.2
15.0
15.0
22.2
20.0
21.0
25.5
20.4
Preliminary data obtained from electron micographs of heart muscle from 10
test and 10 control birds of experiment 2 indicted that the major changes
occurred in the mitochondria of the cardiac muscle cells. In some cases the
mitochondria were markedly shrunken and vacuolated with indistinct cristae.
In the birds with more advanced cases, many of the mitochondria had disappeared, leaving large areas devoid of any organelle.
DISCUSSION
There was normal development of all organs with the exception of the testes
of birds in experiment 2, -which were markedly reduced in size. Kumaran and
Turner10 outlined the tubular and spermatogenic development in chickens at
various states of maturity. It would appear that the consumption of toxic fat
In these experiments retarded testicular development by approximately 2
mouths in the 6-month-old roosters. Despite the reduction in testicular size,
secondary sex characteristics such as comb size and body conformation were
unaffected.
In earlier studies,1 young birds were killed every other day for 3 weeks to
determine when hydropericardium or ascites developed. Hydropericardium was
usually noticed about the 16th day after feeding toxic fat in the ration was
Initiated, without ascites or pulmonary edema. Fluid accumulation in the lungs
and peritoneal cavity invariably developed later. When the concentration of .
toxic fat in experiment 2 was reduced to a low level, the birds failed to
develop ascites, but hydropericardium was a consistent observation.
The most prominent lesions at necropsy were hydropericardium, ascites, and
pulmonary edema. A number of procedures were conducted to resolve what
caused the anasarca. Birds with ascites and hydropericardium in many cases
had higher serum protein levels than those in the control group. The albumin:
globulin ratios of experimental and control birds were approximately comparable. The nonprotein nitrogen and electrolyte levels of the blood were within
normal range in the test groups. There was a reduction in the cement substance in the experimental birds with hydropericardium and ascites. Since a
large quantityu of fluid escapes between the endothelial cells rather than
through them any alteration in the cementum would affect the permeability
o£ the vessels." Whether this alteration in the cement substance of the capll-
�299
298
larics wa$ associated with liypoxia of the endothelial cells resulting from cardiac decompensation or from a direct effect of toxic fat on the capillary mem- •
brane remains to be clarified.
Hemodynamic studies revealed an increase in right ventricular and vent
cava pressures in the experimental birds. Associated with these pressure
changes were dilatation and hypertrophy of the right side of the heart of
birds given toxic fat. Pulmonary edema, cardiac dilatation, and increased
venous pressure are indicative of cardiac decompensation.
What do these data reveal in regard to the causes for the excessive extravascular fluids? The lack of any appreciable change ia the total blood protein
and albumin: globulin ratio would indicate that the liver was producing
adequate amounts of albumin. There were no marked alterations in kidney
function, because the serum albumin and nonprotein nitrogen levels were comparable in the 2 groups, which suggests that the kidney tubules and adrenal
cortex were not affected by toxic fat ingestion. The microscopic observations
fortify the biochemical data, because the alterations in the liver, kidneys, and
adrenal glands of test birds did not appear of sufficient magnitude to account
for the anasarca.
Since myocardial fibers are very active, there is a constant demand on tlie
respiratory enzymes located in the numerous mitochondria between the
myoflbrils. A rather high metabolic demand is implied, - because the ratio of
mitochondria in cardiac muscle to skeletal muscle is approximately 500:1."
Any significant alteration in the mitochondria would affect, the .respiratory
enzymes and sooner or later lead to cardiac failure. Certainly this would
appear to be a logical explanation for what occurs in birds consuming toxic
fat. As the heart becomes less efficient, there will be an increase in hydrostatic
pressure in the veins and capillaries, with a predisposition for the extravasation of fluid into the tissues and body cavities.
REFERENCES
1. Allen, J. R.: The Role of "Toxic Fat" in the Production of Hydropericardium and Ascites. Ph.D. Dissertation, University of Wisconsin, Madison, June,
1001. Dissertation Abst, 22, (1961) : 545.
2. Brew, W. B., Dare, ,T. B., Benedict, J. H., Potter, G. C., and Sipos, E.:
Characterization of a Type of Unidentified Compound Producing Edema in
Chickens. ,T. Assoc. Off. Agric. Chem., 42, (1959) : 120-128.
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The Isolation and Characterization of the Chicken Edema Factor. J. Am.
Chem. Soc., 82, (1960) : 2078-2079.
9. Kingsley, G. R.: The Direct Biuret Method for the Determination of
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10. Kumaran, J. D. S., and Turner, 0.: The Normal Development of Testes
in Plymouth Rocks. Poult, Sci., 28, (1949) : 511-520,
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Failure. ,T. Chron. Dis., 9, (1959) : 424-441.
12. Machlin, L. .T., Gordon, B. S., Meisky, K. A., and Maddy, K. H.: Relationship of Exudative Degradation to Toxicity in Certain Fats. Poult. Sci., 38,
(1959) : 579-585.
13. Marches!, V. T.: The Passage of Colloidal Carbon Through Inflamed
Endothelium. Proc. Roy. Soc. Biol., 156, (1962) : 550-552.
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'el14. Naber, E. C., Bletner, J. K,, and Touchburn, S. P.: Effect of
Toxic Fats and Their Derivatives on Growth, Reproduction, Embryoni>
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15. Palade, G. E.: Study of Fixation for Electron Microscopy. J. Exptl. Med.,
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Physiol. Rev., G. C., Brew, W. B., Patterson, P. L., and Sipos, B.: Current
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Status of the Toxic Fat Principle Causing the Chick Edema Syndrome. J. Am.
Oil18. Sanger, V. L.,(195ft) : L., Hamdy, A., Gale C., and Pounden, W. D.: AliChem. Soc., 36, Scott, 214-217.
mentary Toxemia in Chickens. J.A.V.M.A., 133, (Aug. 1,1958) : 172-176.
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Probably Due to a Toxic Feed—Preliminary Report. J.A.V.M.A., 132, (March 1,
1958) : 216-219. C. F., Pritchard, W. R., and Harms, R. H.: An Endotheliosls in
20. Simpson,
Chickens and Turkeys Caused by an Unidentified Dietary Factor. J.A.V.M.A.,
134, (May 1, 1959) G., Pickels, E. G., and Curren, E. L.: Improved Hanging-strip
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SUMMARIO IN INTERLINQUA
Le Rolo de "Grassia Toxic" in le Production de
Hydropericardio e Ascites in Gallinas
Quandro "grassia toxic" esseva addite al dieta de aves experimental a concentrationes de 0,25 a 6,0 pro cento durante 35 a 150 dies, le sequente alterationes esseva observate: (1) Edema del myocardio, del musculatura skeletic, e
delpulmones; (2) hydropericardio; (3) ascites; e (4) focos de lymphocytes in
le myocardio e le epicardio. Appreciabile alterationes non esseva observate in
le nivellos de proteina total in le sero, in le proportion albumina a globulina,
in le balancia electrolytic, e in le nivellos de nitrogeno non ligate a proteina in
le sanguine. Esseva notate dilatation, edema, e infiltration lyrnphocytic del
corde. Le mitochondrios myocardial esseva vacuolate e contrahite. Un augmento del tension venose esseva etiam notate. Le imbalancia de Hquido observate in aves que consumeva grassia toxic non resultava ab un decline in total
protelnas de sanguine o ab un alteration in le proportion albumina a globuina
sed esseva associate con dlscompensation cardiac e un augmento del permeabilitate capillar.
PORPHYRIA
INDUSTRIALLY
INDUSTRIAL!* Ai;^uin^ *
Twenty-nine patients working in a chemical factory engaged in the manufacture of 2,4-dichlorophenol (2,4-D) and 2,4,5-trichlorophenol (2,4,5-T) exhibiting
features of chloracne were studied for the presence of porphyria cutanen
tarda. In 11 cases urinary uroporphyrins were elevated.
Two of these patients who shpwed evidence of acquired porphyria with
chloracne were hospitalized. The features of chloracne as well as the clinical
and laboratory features of acquired porphyria have been discussed. There
appeared to be an etiologic but not quantitative relationship between the chloracne in workers 'engaged in the manufacture of 2,4-D and 2,4,5-T and porphyria cutanea tarda of the acquired type. It is our feeling that either the
finished chemicals or some intermediate are responsible for both diseases.
Since Waldenstrom first implied that porphyria cutanea tarda might be
acquired, a growing number of chemials have been implicated in the pathogenesis of this disease. These chemicals have included alcohol, sedatives, fungicides, etc.1"1' While treating a severe outbreak of chloracne in a factory which
manufactures 2,4-D and 2,4,5-T, a number of workers were noted to have
hyperpigmentation, hirsutlsm, fragility of the skin and vesiculobullous eruptions on exposed areas of skin, together with cutaneous findings of chloracne.
Investigation revealed evidence of porphyria cutanea tarda of varying degrees
of severity in 11 out of 29 workers investigated. Porphyria cutanea tarda has
never before been described as related to chloracne, nor has it been ascribed to
4B-362 0—70
20
�301
300
iwsltesj;
PI |
industrial exposure in the United States. This outbreak is therefore of int*H*:Sjj iSSlinotelns, albumin-globulin
in adding more evidence to the growing concept that porphyria cutanea Unfaj ^twculiitlon, and thymol
may be an acquired disease occurring after various insults to the liver, TWwtl "Igwer, as well as serological
cases were studied in detail.
' -j$%
From the Departments of Dermatology and Medicine, Newark Beth »-—*•''•'
Hospital.
'
,,„,
Chief of Dermatology, Newark Beth Israel Hospital (Dr. Bleiberg) ; SenWJst
.
r :
»sident Physician in Merlini'tip. Nuwnrlr TSoth Tc,.ool Hospital /T-IResident PllVBirfnn in Medicine, Newark Beth Israel 7T/u,nUni (Dr. Woll»n»«- .«
Assistant in Dermatology, Newark Beth Israel Hospital (Dr. Brodkin);
tor of Medical Education and Consultant in Medicine, Newark Beth In»tli!
Hospital (Dr. Applebaum).
(jK^fft =«•£ SStSS " SHs
S
$«*r u.8 wood'. mm. I'sj?4jssStie «»mum;ioi',oi^i;;troi""i"8Mn<ss.
fe^",si°. xri»KJ^i»«!*,rs^Sinrf ^«»»l^^x^^^^^s^^^^A.
REPORT OF CASES
CASE 1.—A 48-year-old white male who was employed at the factory f«>ii
three years as a chemical operator. His work brought him into intimate cosvj
tact with the suspected chemicals. His past history included two attacks of bO- .'
iury colic prior to 1953. He was never a heavy user of alcohol. A diagnosis ot'$
cholecystitis had been made and a cholecystectomy was performed early i»'*|
1953. After this he came to work at the factory in question. In 1956 he bcgaa ||
to notice some darkening of his skin and suffered right upper quadrant pala. '
A diagnosis of common duct obstruction was made, and this patient was opcr-'.
ated on again in January of 1956. An unsuccessful attempt was made to prol* .
the common duct, and no further operative procedure was done. During the
postoperative course, this man received 2 gm of barbiturates. The patient
stated that his urine had turned "the color of Coca-Cola" at least one year
prior to the second operation. That spring, an eruption of bullae appeared on
the face, ears, and hands. These lesions could be produced either by exposure
to the sun or by pressure. In addition to the vesicular eruption, the patient
noted progressive darkening of his skin and marked hirsutism, especially over
the temples. Inspection of the urine revealed a Coca-Cola coloration and, under
the Wood's Light, a brilliant red fluorescence. The exact laboratory data on
this patient are no longer available except for the presence of quantitatively
markedly increased excretion of urinary porphyrins including uroporphyrins,
coproporphyrins, and porphobilinogen.
This man is now alive and well and apparently is suffering minimal if any
symptoms of porphyrim cutanea tarda. His present job does not entail the use
of any chemicals. He has failed to present himself for further testing.
CASE 2.—This is a 60-year-old white male who has been employed in the factory for seven years as a welder. In the course of his work, which consisted of
welding tanks and pipes, lie was brought into frequent and prolonged contact
with chemicals. He was admitted to the Newark Beth Israel Hospital for
investigation. He stated that three months prior to admission, he had noted an
increased darkening of the skin, thickening of the eyebrows, and a darkening
and reddening of his urine. His family history and his past medical history
were unrevealing, except for moderately heavy alcohol intake for some years.
Physical examination revealed numerous comedones and small epidermoid
cysts and furuncles the face, chest, and shoulders. There was intense grayishbrown Jiyperpig-mentation with a purplish tint on the exposed surfaces of the
face, neck, chest, and hands and moderate hypertrichosis of the temples. The
scalp hair showed a lusterless, dull silver color change. The liver edge was
palpable about 3 cm below the right costal margin and was smooth and nontender. The remainder of the physical examination was within normal limits.
A casual urine specimen revealed a strong tea color with a deep fluorescence,
reddish, under the Wood's light.
Laboratory studies revealed increased urinary uroporpliyin, coproporphyrin,'
and urobilinogen excretion. There was no demonstrable porphobilinogen. The
foc.es showed increased uroporphyrins and coproporphyrins. All porphyrin
determinations were qualitative and done by the Watson-Schwartz method.
Other significant findings included an elevated serum glutamic oxaloacetic
transaminase ranging between 41 and 51 units on five different days. Serum
glutamic pyruvic transaminase on corresponding days ranged between 53 and
04 units. The sulfobromophthalein retention was 6% in 30 minutes. The erythrocyte sedimentation rate (Westergren) was 94 mm In the first hour. All
other Htudies, which included complete blood count, bleeding and clotting time,
urirmlysis, glucose tolerance test, serum bilirubin, blood urea nitrogen, total
tlfeUy. About four days aftei the^onse^ ^ ^^
" " i eruption on
i stopped, but
finer i t
.in tu« ov.«**" ~
appeared. ^g^ tlme
-..
. : o,«»» - ——-^i^^l
I
—
htlent
:t
J
Chloracne'
Mild —
12..
13..
H..
15..
. Severe
Mild
do
. Severe
. Mild
do
;
do
Severe
. Mild
.. Moderate
.. Severe
: None
do
do
Moderate
17.
18.
19.
20.
21.
22
23
24.
25
26
. Mild
... Moderate
Severe
"„ Miid
None
" .. .do
" Mild
. None
. . ..do
."..."...do
• «V."
s
t
I
I
lo'."."
11
is:.
;
:.
"
.
mffiion
mentation
do
Hirsutism
••••-
Urine
Utoporphyrins
pos
None
do
.do
. Pos
None
. Pos
None
^j^
Contact
Moderate
Skin FwgllHl
P°s-
Do.
None...
None
-.do
Mild
:
Do.
"."Mild..:
...do
do
Mo'd'efaYe-— - Nes
None
Do.
"None
Moderate
.do
Do.
"
do
do
Mild
Do.
1
..do
Moderate—. "Severe
..do
Do.
" Mild
Severe
. Mild
..do —
Do.
Moderate— .
• Moderate—
"". Moderate
do
Do.
..do
Severe
' ...do
do— .
Do.
None
'
do
" None
Do.
".".".'.-"-do—
..do
Moderate
do
Po
... Pos
do
do
"" do
Do.
None
""Moderate—
. Severe
N
" Mild
.do
Ne
8
Moderate... " Marked
..do— "" Mild
'00.
None
....do
" Mild
Do.
"""" ..do— "" ....do
Marked
ModerateDo.
""
Pos
None
""
do
Marked-- " Moderate—
Do.
Pos
"•"• . . .do
Mild
Do.
None
-, " None..... . None
. .do
Do.
"""
—do—
...do....
.do
..do—.
..do—
Do.
.do
"....do
"" ..do....
.do—
Do.
..do
ido
..""—do...,
Do.
""""Pos
—do —
""".""..do
do—
"",".". -do— ",. Pos— —
:--:&--><»•
i Brief period of employment.
OH
S3SSBS3SS5S3H**
�303
302
hirsutism which involved the temples. The dull silvery tint of the taSf
ible. He stated that in the past he had had episodes of blistering of IS*
exposed skin. He also had noticed that his urine was dark on voiding. IW
family history was noncontributory. The physical examination revealed t*
intense hyperpigmentation of the face, neck, and hands. There was severe U*1
sutism involving the eyelids, eyebrows, and lateral aspects of the forelini
(Fig 2). Oomdones and small epidermoid cysts were very prominent, and tliw
were numerous furuncles scattered over the entire body. The remainder of tfe*
physical examination was within normal limits except for prolapsed hemW"
rhoids. The following laboratory studies were within normal limits: complete
blood cell count, urinalysis, bleeding and clotting time, prothrombin (lux. .
Mood glucose tolerance test, urea nitrogen, cholestrol, bilirubin, alkaline pl«*
phntase, total protein and albumin-globulin ratio, ceplialin flocculation, tliyidd
turbidity, serum electrolytes including sodium potassium, chlorides, COi com*'
bining power, calcium, and phosphorus. The serum glutamic oxaloacetic trans*minase on five successive days ranged between 39 and 56 units while II*
serum glutamic pyruvic transaminase on corresponding days ranged betweffl i
47 and 72 units. The sulfobromophthalein retention was 8% in 30 minutes. Ttif
electrocardiogram was normal. The chest x-ray revealed a diffuse nodular ;
infiltration of both lungs due to pneumoconiosis. This was consistent with th»
patient's history of having worked a number of years as a coal miner. T!«,
plain film of the abdomen was negative. The urine revealed a negative
Watson-Schwartz test The urine failed to fluoresce under the Wood's light Th*
erythrocyte sedimentation rate (Westergren) was 24 mm in the first hour.
.
A liver biopsy was performed and the specimen immersed in saline. Under
the Wood's light the specimen and saline in which it was immersed Buoreswd
faintly. On microscopic examination, the liver biopsy showed evidence of liver
cell regeneration and hemofuscin deposition. A skin biopsy showed brown granular pigmentation as the basal margin of the epidermis. There was a mild
chronic inflammatory infiltrate scattered through the dermis. No sebaceous
glands were visible in the sections.
Since the man's chloracne has-been so severe, he had been removed from
contact with chemicals two years prior to his admission to the hospital. Till*
probably was responsible for the failure to prove qualitative chemical evidence
of tarda is in the urine. It also may indicate that acquired porphyria cuteneaporphyrinsreversible.
SCREENING TESTS
Twenty-six additional men working at this chemical factory were studied on
on ambulatory basis. In addition to routine urinalysis, each urine specimen
was tested for uroporphyrin by the Watson-Schwartz method. Bight out of tlie
26 manifested significantly increased excretion of urinary uroporphyrius by tlie
Watson-Schwartz method. If the three cases described in the case reports
above are added, this is a total of 11 cases of porphyria cutanea tarda of
varying degrees of severity out of 29 patients tested, or 37+% (Table).
COMMENT
Hyperpigmentation in these workers was limited to the sun-exposed I areas
of the head, neck, and hands. It was more frequently observed in the Negro
patients involved. The degree of hyperpigmentation was roughly proportional
to the severity of the chloracne. The hyperpigmentation varies from mild redness in extremely fair individuals to dark gray intense dusky bronzing of the
skin. The degree of hirsutism was also proportional to the severity of the
chloracne. This too was quite variable in degree but always involved the temples between the lateral half of the eyebrow and the temporal hair of the
scalp. The hirsutism in a few cases, notably case 3, extended beyond this and
involved both the upper and lower eyelids. The hair was of approximately tlie
same texture and density as that of the eyebrows.
The occupational environment of these men consists of a group of basic
chemicals including acetic acid, phenol, monochloracetic acid and sodium
hydroxide, plus the finished products 2,4-D and 2,4,5-T as well as many
unknown intermediary products. It is known that one of the intermediaries ia
a highly volatile chlorinated phenolic ether which contains six chlorine atoms.
Thin particular compound, because of its volatility, in strongly suspected-of
being a possible causal agent. Porphyria has been described in many cases as a
defect
of ingestion of
laiysis 01 uie mmc. "- -— -p,,^,,-,.,, of chloracne does not usually. w™?^"" "ioT2 ~13,T6). — IS^micals (patients 1, 8, 11, 10 or patients 2 ^8'^r^<Jf'cheillcai exposure
!$•••« prophyria does not usually correspond to tUeaegre
of ^^
p-fLUenta 7, 20, 25, 26 or patients 2, 8, 4, 10,12,U, W
tg ±> 4> g,
fej ,wne does not usually correj*n^ to ™° Prft would appear that there is some
mil. or patients 5, 20, 25, 26). T1^refoie^ wom^TO
fl,
thflt .
H*'1'. Individual susceptibility to these disease. It lias been oose
_ _ _ _ * „ , „_,„„
4... » , _ , . , _ _ _ i _ iii, ,,/iniacuiaiir. time tend to
.»«. < » — >.
* «annot be overlooked.
.,
h
, offered by Dr. DonaldMC
P 3>
"
, 40 Union Ave, Irvington, NX
BEFEBENCES
- Dirin (I8tanbul)
to
-W.M~.tro.,
j,
A . : UUOC4 vu^u»u
^.-
.-
v
.: The Porphyrias, Advances In Internal Medicine, Chicago: Year Book
"warnes, JEI. u.. iuM,..j..^ In Bantu Races on the Wltwatersrand, S Afr Med J
7
20:781-784, U., and Schwartz, S.: Experimental Porphyria: III. Hepatic Type Produced
Schmla, 1955.
by Sedormld, Proc Soc Exp Blol Med 81:665-G89, 1952.
* Goldberg, A., and Rlmlngton, G.: Experimentally Produced Porphyria In Animals,
Proc Roy Soc A., and 143 :257-279, C.: Diseases of Porphyrln Metabolism: American Lec• Goldberg, (Blol) Rlmlngton, 1955.
ture Series, Springfield, 111.: Charles C Thomas, Publisher, 19R2, p 194.
10
Solomon, H. M., and Klgge, IT. H. J.: Disturbance In Porphyrin Metabolism Caused
i... Woo/iiv,^ niotiivi l.4r-Dlhydro-2,4,6"Trlmethypyrldlne-3,5-Decarboxylate, Proc Soc Exp
I.
3. E.; and Rimlngton, C.: Nervous and Blochemlci
Intoxication, Nature (London) 191:363-_..,
•JIIB ri-uutcui -^ Porphyria—Some Facts and Questions, New
5, 1960.
il.: Acquired Porphyria From Liver Tumor, Clln Scl 16 :517-527, 1957.
ELECTRON MICHOSCOPIC ALTERATIONS IN THE LIVER OF CHICKENS ,
FED Toxic FAT*
Toxic fat is the name applied to certain fats that produce hydropericardium,
hydrothorax, and ascites when added to the diet of chickens.'1 "•" It has been
demonstrated that the toxic fraction fe associated with the unsaponiflable portion of the fat.10-" The toxicity can he increased by repeated passages
through silica and alumina gel columns."' * Crystalline preparations of the
toxic fraction have been prepared, but the chemical nature of the compound
remains to be identified."
Accepted Department o£ Pathology and
From the for publication March 21, IflOfl. the Regional Primate Research Center, University of Wisconsin, Madison, Wlnconsln.
,'
* This research -was supported in part by grants HB-08081 and FR-0167 from the
National Institutes of Health.
�304
1
^, -ae following alterations that resulted from the consumption of toxic .-;!,
10
have been reported, ganger, Scott, Hamdy, Gale, and Pounden observed f<K«S|;
necrosis of the liver, epicardial hemorrhage, and lymphoeytic infiltration at ttej|
cardiac muscle. Simpson, Pritchard, and Harms" reported bile duct hyper|ii**l|
sia and proliferation of the endothelial lining of the smaller blood vesoetufi'
Allen and Lalich' observed testicular hypoplasia. Marked dilation and edwt*;;
of the myocardium and altered right ventricular and vena cava pressures liinM
been demonstrated.8
.f
The present experiment was initiated to determine the morphologic effect rf;;
toxic fat upon the liver of chickens and to correlate these findings with 11*
development of anasarca.
;
I
305
A
|{[-*ml a shift in the albumin-to-globulin ratio of the treated chicsPI. Similar
| |ricctrophoreticpatterns were obtained on the ascitic and pericardial fluids,
;
Ij although the total protein of these fluids was only 1.8 gm. per 100 ml. Blood
gi.riectrolytes were not appreciably altered.
P^
<i>f&t
1
GROSS AND MICROSCOPIC
OBSERVATIONS
jUj- A large quantity of colorless, semiclotted fluid accumulated in the subcutaft; neons tissue and the pectoral, thigh, and lower leg muscles were pale and edeIjjmatous. The abdominal cavity of each experimental chicken contained approxil^vWntely 40 ml. of ascitic fluid. The liver was somewhat mottled with rounded
W < margins and a thick gelatinous material resembling coagulated plasma was
EXPERIMENTAL PROCEDURE
|, Brmly attached to Jthe capsule in 25 per cent of the chickens that had received
|
One hundred sixty-eight 1-day-old White Leghorn-New Hampshire chicken* ; '^toxlc fat. The kidneys were pale and swollen. All of the organs within the
were divided into groups of 48 and 120 chickens. One-half of the chickens frota ,
i abdominal cavity appeared edematous. There was marked distention of the
each group were given a commercial diet containing 3.0 per cent toxic M
jjlpericardial sac with a clear, slightly yellow fluid. Five to 10 ml. of pericardial
(Emery Industries, Inc., Cincinnati, Ohio), while the remaining chicken*-, flktfluld were obtained from each chicken. There was a noticeable dilation and
received a comparable diet which contained 3.0 per cent corn oil. In tlie group ; 1;: i hypertrophy of the right side of the heart and the myocardium was pale, with
of 48 chickens, three control and three experimental chickens were killed every
P; no gross lesions evident. The lungs were extremely edematous and the tracheoother day for 16 clays and sections of the liver were obtained for electron , |f .bronchial tree was filled with blood-tinged, foamy fluid. The brain was edemamicroscopic evaluation.
i
i|' ,tous but free of other gross lesions.
The group of 120 chickens remained on the toxic fat diet until the LDw wa» j p/ The major microscopic alterations were observed in the liver. Its capsular
established. From the survivors, blood was obtained for hematocrit," henioglo- ,; {§;' surface was covered by a thick layer of homogeneous, eosinophilic material
bin,8 total serum protein,13 serum electrophoretic pattern,23 and electrolyte studies." ' |i .containing numerous fibrin strands. Although the general architectxire of the
The ascitic fluid was also collected for total protein and electrophoretic pattern
|u; Hver parenchyma was maintained, numerous foci of necrosis were observed
determinations. All chickens that died during the course of the experiment n» :$ J > ! (Fig. I), These lesions varied in size, from only two or three cells to a major
well as those sacrificed at its completion were necropsied. Portions of the liver
5'ji portion of the lobule. There was a fairly sharp line of demarcation between
were fixed in 10 per cent buffered formalin for 24 hours, dehydrated, embedded
5;:, the viable arid dead cells. Immediately adjacent to these necrotic foci, the cytoin paraffin, sectioned and stained with hematoxylin and eosin. Other portions
jj: plasm of the parenchymal cells was quite vacuolated, but otherwise not
of tlie liver were sectioned on a cryostat and stained with Sudan IV for neu;S~ remarkable.
•
tral fats. Additional paraffin-embedded tissues .were stained with aniline blue
THIN SECTIONS FOB LIGHT MICROSCOPY
and phosphotungstic acid-hematoxylin.5
Better visualization of the histologic alterations was obtained from tissues
Small sections of liver from the 48 chickens of the first group plus 20 conembedded in the Araldite-Epon mixture than was possible using the conventrol and 20 experimental chickens of the second group were 7 obtained at tlie
tional paraffin embedded preparations. The parenchymal liver cells of tlie contime of death, cut in small cubes, and fixed in Oaulfield's and Millonig's
trol chickens stained uniformly with toluidine blue. Their nuclei were dark
fixatives.10 These tissues were dehydrated through a graded series of ethanol
blue and the cytoplasm was much lighter and granular. The degenerating paand embedded in an Araldite-Epon mixture." Thin sections of approximately
renchymal liver cells of the toxic fat chickens were of two types on the basis of
0,5 ft were cut on an 3ultramicrotome for light microscopy and stained by tlie
their affinity for toluidine blue. One cell type had a decided affinity for the dye
toluidine blue method. " Ultrathin sections were placed directly on 400-mesli,
so that both the cytoplasm and nucleus were very dark (Fig. 2). Many of
uncoated copper grids, stained with urany! acetate, and examined with an
these cells were shrunken, distorted and contained large morphologic features
RCA BMTJ-3G electron microscope.
of the organelles were fairly well maintained in the cells obtained during the
first 5 days of the experiment. However, the organelles were in very close
TABLE l.-ALTERATIONS IN THE PERIPHERAL BLOOD OF CHICKENS CONSUMING TOXIC FAT
apposition as a result of what appeared to be shrinkage of the cells and loss
of the cytoplasmic matrix. Although the external mitocliondrial membranes
were quite irregular, the shape and distribution of the cristae remained unaltered. The membranes of the endoplasmic retieulum were in close apposition
and had abundant ribosomes along the outer surface. Free ribosomes were dismEq.lliter mEg./liter ffm.periOO
persed throughout the cytoplasm. In the dark cells, the Golgi complex, lysosomes, and microbodies were usually sparse and only vaguely discernible. Only
isolated areas within the cytoplasm contained glycogen granules. Numerous
myelin-like structures appeared to arise from the cytoplasmic membranous systems of these cells. The nuclei of these dark cells had approximately the same
electron density as the cytoplasm. The paired nuclear membranes appeared as
one wide, irregular dense band containing few discernible pores. Except for the
When 3.0 per cent toxic fat was added to the diet of young chickens 50 per
extreme density of the nucleoplasm, the chromatin material and nucleoll
cent died within 15 days. Approximately 24 hours prior to death, the chickens
became listless and moved only when agitated. Because of the marked abdomiresembled those in the control cells.
From the 7th through the 15th day of the experiment, the dark parenchymal
nal distention the chickens assumed a ducklike gait when forced to walk.
liver cells became increasingly electron-dense, the intercellular spaces wider
Moist rales were present in the lungs and considerable amounts of clear to
and myelin-like bodies more abundant along the plasmalemniae. The cytoblood-tinged fluid were
ens at the time of death. observed in the oral cavity of the experimental chickplasmic organelles became more difficult to visualize. Numerous oval to oblong
clear vacuoles of variable size developed in the cytoplasm. Myelin-like bodies,
On the 15th day, blood studies were made on the surviving chickens. Those
identical to those along the plamnalemma, were present throughout the cytofed toxic fat showed a reduction In several of the blood components (Table 3).
plasm. At this time it was difficult to visualize the existence of a distinct
Hemoglobin levels decreased from 10.0 gin. per 100 ml, to 0,0 gm. per 300 ml.,
nuclear and cytoplasmic separation because of the electron density and the
and hematocrits wore reduced from 31.0 per cent to 18.0 per cent. There was a
decrease In total serum protein from 3.4 gm. per 100 ml. to 2.3 gm. per 100 ml.
t
�306
^
307
absence of any distinct nuclear membrane. Eventually these cells d*
into dark, shrunken, fairly homogeneous masses which exhibited little i
WM also be related to altered «*«»»* ^ toxic effect of this fat upon
logic resemblance to liver parenchymal cells (Fig. 6).
The second type of parenchymal cell in the degenerative areas was i«n« »•»»,{ ipMota confirm a previous report regarui 6
very electron-lucent. An isolated cell of this variety was seen most freqwWfrij ISfWiKmlature of chickens.
BimMAKY
within a group of the previously described dark cells. The plastnaleuiiwf'rf|
these cells were in close apposition to the adjacent dark cells. The mlewWjl
16
projecting into the space of Disse were shorter and less abundant than tUfty
the chickens died
rent
of the dark cells (Fig. 7). Many of the organelles were markedly dWwfct]
Small segments of the endoplasmic reticulum were scattered throughout <l»j
edema, ascites, anu BUU^
cytoplasm. The majority of these membranes contained ribosomes along iMfe »-?|Mtla o£ these chickens, M.IIC 6*»™ —--outer borders. Numerous free ribosomes were also scattered throughout &*|
cytoplasm. The mitochondria were large and disorted. Their exlurart'l
membranes were quite irregular and formed many bulblike projection ft1*;';
the surface of the mitochondria. Small vesicles were apparent between tW,'J
external and internal, mitochondrial membranes. The enlarged cristne pr**^
cally filled the interior of the mitochondria (Fig. 6). Large, clear vacuolwuwjj
cytoplasmic degenerative bodies were abundant throughout the cytoplasm wlfflfe ^Wto of the chickens^a^consumed^fa predisposed to^a^orUo^the
glycogen granules were very sparse in the light cells. The Golgi complex Wra it
sisted of collapsed vesicular sacs dispersed in small groups throughout ti*J
cytoplasm, The mitochondria were large and distorted. Their extffWfl?
chromatin material quite evenly dispersed throughout the nucleoplasm.
;'Jj
Acknowledgments. The,.a£r,°rBf "supplying the toxic
During the terminal portion of the experiment, the large electron-lucent cdfc^
dustries, Inc., Cincinnati, Ohio, for suppiyi B
developed openings in their plasinalemmae. Their cytoplasmic organelles wat.y
BBFEBENCES
observed in the sinusoids, space of Disse, and in the intercellular spaces.
j
The endothelial cells which line the sinusoids showed similar change* to i
those -previously described in the dark parenchymal cells. The endotlidttM
changes were most frequently observed adjacent to a large focus of dark cril*oi
The long, slender cytoplasmic processes of the endothelial cells becftn*':
extremely dark with the cytoplasmic and nuclear structures becoming indlrtta- •;
guishable. Large fragments of these endothelial cells were frequently seen It •;•
the sinusoidal spaces. Similar changes were observed in the bile duct eplllx* i
Hum. It was not uncommon to see two or three extremely electron-dense celt* .
which epithelial cells (Fig. 8).
normal had lost all their internal structure adjacent to a number of seemingly '.
DISCUSSION
,.•;•;
The gross lesions produced in chickens fed toxic fat have been well doco- |
mented, yet the mechanism by which this fat produces ascites, hydroperlear- ';
dium, hydrothorax, and edema has not been clarified. Vascular lesions and ear- V
diac decompensation have been mentioned as possible causes of the massive 7
accumulation of extravascular fluid in chickens that have eaten toxic fat.1 In :J
this experiment the liver was investigated to determine its association with j
the accumulation of extravascular fluid in chickens whose diets contained toxic -i
fat, '
";;•
There was a decided reduction in the total blood protein of the toxic fat ;';
chickens. The electrophoretic patterns of their sera showed the drop in protein ;•
resulted from a decrease in the serum albumin. When the liver tissues were '•'.
examined microscopically the explanation for the decrease in serum albumin ;
***At, . .
became apparent. There were numerous areas of focal necrosis throughout the
liver of the experimental chickens.
There is no clear explanation of why some parenchymal liver cells shrink nncl ]
become extremely electron-dense, while others appear to swell and become very
electron-lucent. Undoubtedly each of these changes is a degenerative process
which eventually terminates in the death of the cell. One could postulate that
these two cell types perform different functions and because of this functional
W. D.
difference, the reactions of these cells to this toxic material were manifested
anger, V. L., ScottJ^ Hamdy,
^ 1958
nB
bnbly
differently. Such changes in size and lucency,' however, are not unique for
toxic fat but resemble those observed in hepatic lesions produced by monocrotaline and x-irradiation." Further studies will be necessary to clarify the difand turUyn <*»M|1 by an unitlennnao
^ fltalnlng epolj
fering responses of various parenchymal cells to this toxic substance.
The accumulation of large quantities of extravascular fluid in the tissues
and body cavities of chickens that have consumed toxic fat can be attributed
at least in part to an alteration in the permeability of the vascular bed. The
high level of protein in the ascitic and pericardial fluid and the electrophoretic
.
8
-SrfvSftS^S--^"^^*
"SS& - -»•;;
s
5
u;;wrivV*t!%. .!!iS ,r „». PWS.;;'?. a sss:» «=,';,
«crr^^jBsar?ii?£H%"BS::
�309
308
g»s* niu-3U on ANAKBOM ^/J, -„,-, rip(.p(.tor 200"U, nasu iietuci. --« ~ > =.
CHICK EDEMA FACTOR : SOME TISSUE DISTRIBUTION DATA AND
§li««»tui«, carrier ga., 60W-O. M f«*<°L"efl«ne purge PM „„»„„„ «
180 cc/min.
TOXICOLOGIO EFFECTS IN THE BAT AND CHICK.* (31020)
*l».. Mlum column oven cc/mm«rgo»,MA o][iiii|iWy
fc
The chick edema factor (CEF), responsible for a large number of deattattjj!
the broiler industry in the fall of 1957, was traced to the unsaponlftri&tjl P
r
matter (unsap) of the fat used in the broiler rations. It since has been cij*r^j
tallized (1,2,3) and its structure proposed as that of a hexachlorohexalij<tB|>;|
phenathrene (2). It is known that in the toxic fat, a mixture of related w*;;^
pounds can be found, some toxic and some relatively nontoxic.
Y1*
To lay the groundwork for a study of the specific physiological effect* **; If«k8 1 through 8 as shown. Peak
pure CEF compounds, a few short studies have been completed to learn IS* li?'>wparation.§
rr n i>io TT
distribution of the toxic material in the body and which organs were priniarfil W( Results and discussion. -LaufL.e „ fnr the rats. The results j.ui «.^
--»_
^.
• -d-feedconsumptionjrnthenr|^
affected.
Eatperimcntal. Adult rats and day-old White-Rock chicks were used. Bccaittf
the pure material was not available in sufficient quantity, we used, from tin*
toxic fat, the unsap which represented 38% of the original toxic fat and wtt.
estimated to contain at least 10 ppm CEF. The unsap was force fed because tb*
animals' food intake was drastically curtailed when it was mixed in the dlA;
AH animals were offered water and commercial feed ad libitum.
.
Table I shows the experimental plans and dosage levels employed for nil I
TABLE ll.-GROSS EFFECTS IN RATS
Trials. Feed consumption and fecal and urinary excretion were measured fw
the rats. Body weights were recorded in all experiments. All animals, upca
sacrifice, were examined grossly for pathology and selected organs were
weighed and frozen. Hydropericardial fluid (HPF) volume was measured I*
the chicks.
In addition to the examination for gross effects, the presence of CEF mate-'/J
rial in various tissue was determined. Adrenals, kidneys, and livers wew'J;
assayed in the rats; livers only have been analyzed in the chicks. To obtain »
picture of the amount of material being absorbed and perhaps excreted, analyses of the feces and urine of rats and of the combined chick excreta wew
made.
£.,RL°o',tt«r.-S,,"K. szsfgSSSZS**. «-
ftsy^raTSMt?rei? s^sxx H
-'. (s>
e
ferr»n?x s.™~n — - * ^jrs-,
TABLE 1.—EXPERIMENTAL PLAN OF TRIALS
L
_
Dosage (per kg)
Trial
1. H days
Species
Rat
animals
Group
High
!
High
Control '
III. 6 days
Chick
High
! Low
Control'
6
1
r
M
TABLE III.-ORGAN WEIGHTS EXPRESSED
-'
Unsap
CEF (Mil'.-•;
(ml/day) nated (ntlttfl v
2.0
1.0
2.0
1.0
0
5.4
1.1
0
•H
H ;
0
41
. 10
0
Control animals were not intubated.
The assay method for CEF is being reported in detail elsewhere.t In short,
the sample is homogenized with water and saponified with alcoholic KOH. The
unsaponifiable portion is extracted with petroleum ether and chromatographed
first on an alumina column. The eluate obtained with 25% ethyl ether in
petroleum ether, following prior elution with petroleum ether and 5% ethyl
ether in petroleum ether, is concentrated and chromatographed on 500 fi thin
layer silica gel plates with 3% ethyl ether in petroleum ether. The silica gel in
the area of Kr 0.80-1.00 is removed and eluted with ethyl ether, the solvent
removed, and the residue is redissolved in isooctane for gas chromatography
We used an F & M Model 400 gas chromatograph with an electron capture
detector and a U-tube column, 3 ft X 6 mm (o.d.) X 4 mm (i.d.), packed with
* Supported by TJSPHS Grant EF 00305. Contribution 858 from Dept. of Nutrition
and Food Science. Mansnchiiflfttts Inst. of Technology.
tChick Edema Factor."L. Friedman In preen, J. AOAC, "Chemlcnl Assay and Isolation
of T. C, Campbell nn<J
1
i
1
HPF—hydropericardial fluid (ml),
'Significant statistically at 1 percent probability level.
1
»At 55percent probability level ,(Hogben L testXS),
At percent prouaunuj .o.u .,..-„—
(0 /ig/kg/day) showed an increase over controls of 21% while the chicks (10
Mg/kg/day) showed an increase of 15%. (The increase in liver weight in the
chicks was Table III, neither heart nor spleen weights in either species are sigAlso, in not due to moisture or fat.)
nificantly affected. In rats, there appears to be a slight increase in kidney
weights, though not statistically significant, and a highly significant increase
o£ 50% in adrenal weights for animals on the high level. Whether this adrenal
weight increase is simply a non-specific stress effect from intubation is not
known, although it would seem that this cannot be entirely responsible, since
the high level group showed an increase of nearly twice that of the low level
group. other observations which have been made on previously studied birds
Some
In this laboratory are of interest. Hematocrit values are depressed. Of a total
of 43 birds, we have observed that, with an average of 0.08 ml HPF in control
§ The chromatograin for the Firestone preparation IB presented elsewhere.
�311
310
U;
V
FIGURE 1.—Toxic CEF components found in unsap used in this study.
FIGURE 2.—Rat liver extract showing 2 CEF peaks.
FIGURE 3.—Rat feces extract showing CEF peaks, with altered Nos. 4a and 7
FIGURE 4.—Chick liver extract showing 2 CBJF peaks. (Large peak just before
No. 4 found to be contaminant leached from liner of sample vial.)
birds (considered normal), there was a packed cell volume of 33.0%, while for
diseased birds having 0.73 ml HPF, the packed cells volume was 27.9%.
Control animals not receiving OEF did not show these peaks. These chromatoKrams show that only peaks 4 (or 4a?) and 7 were present in the liver. In the
fecal extracts all peaks were found, with the exception that instead of peaks
4ft and 7 showing their original retention times, these were slightly increased
in each case and have been designated 4a' and 7'.
These retention times increases in the fecal components were measured by
noting their retention times in relation to their neighbors, as shown in Table
IV. Whether the 2 components of the liver are the products of components 4
nnd 7 or the original unaltered substances cannot be accurately determined
from these chromatograms, since the rest of the OBF chromatographic pattern
Is missing. It may be concluded, however, that there is a selective absorption of
Itoiks 4 (or 4a) and 7, with metabolism by the liver and excretion into the
Intestine.
It appears, on the basis of the tissues analyzed, that the liver is the target
organ. Furthermore, the pattern of OEF chromatographic peaks presented
here, Nos. 4 and 7 are the key components. We have succeeded in partially
separating the CEF components such that 4a and 7 crystallized together, indicating a certain chemical as well as physiological similarity.^ There is no way
of differentiating 4 from 4a with regard to its absorption and metabolism on
the basis of the available evidence.
Summary. "Unsaponiflable matter isolated from a toxic fat and containing an
estimated 10 ppm chick edema factor (OEF) was force-fed daily to adult rats
nt levels of 2.0 cc and 1,0 cc/kg body weight/day in 2 studies of 14 and 6
days, respectively. Feed consumption, body weight, and digestibility were
depressed. Heart and spleen weights were unaffected, kidney weights seemed to
be slightly increased, and adrenal and liver weights were significantly
Increased. In the chick, typical hydropericardium, ascites, and subcutaneous
edema were observed. There were no significant changes in heart or spleen
weights. Liver weights were significantly increased. The rat was as sensitive as
the chick to CEF according to increase in liver weight. Of the 8 or 9 CEF
components, shown to be in this unsap, 2 (Nos. 4 and 7) were found to be
absorbed and located in the liver, while the other components were not
TABLE IV.-CHANGES IN CHROMATOGRftM RETENTION TIMES OF CEF PEAK NOS. 4a AND 7
Relative retention times
"
Unsap '
1
1 Unsaponifiables administered to animals in this study.
Includes feces of 11 individual rats and the composite
1
No. of runs
R;:«
Ru:i
31
Material analyzed
«1.10±.01
l.lldbOl
1.14±.03
4a not found
1.22±.01
2
feces of 2 groups of 6 chicks each.
The deviation Includes the total range of values.
detected. In place of Nos. 4 and 7, there were 2 new peaks in the feces with
slightly increased retention times. This suggests that the 2 active CEF components are metabolized in the liver and excreted into the intestine via the bile,
both in the chick and the rat. No CEF-like material was found 'in kidneys,
adrenals, or urine.
FIGURE 6—Chick feces extract showing OEF peaks, with altered Nos. 4a and 7.
Also, we observed that whereas control birds will show disappearance of n
given amount of an I.V. injected dose of T-1824 dye (Evans Blue) from their
vascular system of approximately 1%/min, poisoned birds will show a disappearance of approximately 2%/min, supporting the observation of Allen (4),
that the permeability of the vascular wall appears to be increased.
Distribution of the OEF In animal body,—Of the rat tissues and samples
examined, CEF was detected only in the liver and feces, Fig. 2 through 5 show
typical chromatograms of purified extracts of feces and liver of both species.
STUDIES ON THE METABOLISM OF CHICK EDEMA FACTOR :
DISTRIBUTION IN CHICK TISSUES
D. Firestone, G-. R. Higginbotham, D. F. Flick and J. Ress
The distribution of chick edema factor in chick tissues following consumption of rations containing toxic fat has been of considerable interest. Chick
1
Harman, R. E., Davis, G. H., Ott, W. H., Brink, N. Q., Kuehl, F. A., J. Am. Oil
Chom. Soc., innO, v82, 2078.
1
Wootton, J. C.. Courclicix!. W. I/,, J. Afrrlc. Food Chom., 1964, v!2, 04.
" Ynrt?,oft, A.. Firestone, D., Banes, D., Horwltz, W., Friedman, L., Neshelm, 8., J.
Am. Oil Chem. Soc., 10(11, v38, no.
4
Allen, ,T. R., Ph. D. Thesis, 1981, Univ. of Wisconsin.
' Hogben, C, A. M,, J. Lab. Clln. Med., 1904, v64, 815.
* Recelverl January 13, 1906. P.S.B.B.M., 1966, v!21.
�313
312
liver, and skin showed peaks characteristic of the presence IHchick edema
factor. No characteristic peaks were found in the adrenals, brain, skeletal
muscle, and testes; perhaps the concentration was too low to be detected in
these tissues.
The toxic fat as well as the toxic unsaponiflables used for this study exhibited 4 characteristic GLO peaks of Ba = 10.6, 12.4, 18.6, and 21.6 (See Table
2), The intestine chromatograms exhibited greatly diminished 10.6, 18.6 and
21.6 peaks, and a peak appeared at 12.0 in place of the 12.4 peak of the toxic
fat and toxic unsaponiflables. The 10.6 and 18.6 peaks were not evident in
chromatograms from the other tissues exhibiting characteristic peaks, and the
12.0 peak was the major characteristic peak. In addition, the bone, heart,
kidney and skin extracts from the chicks fed toxic fat (Group A) exhibited a
12.4 peak which occurred as a shoulder on the major 12.0 peak.
These results suggest a selective absorption of the chlorinated components of
the toxic fat; this is most clearly indicated by the diminished 10.6 peak in the
intestine extract which is completely absent in the other tissue extracts. The
appearance of peaks at 12.0 or 12.0 and 12.4 in place of the 12.4 peaks of the
toxic fat can also be explained by selective absorption and deposition of individual components if we recognize that the GLC peaks observed each represent
more than a single component. Campbell and Friedman (2) have also observed
selective absorption of chick edema factor components in rats as well as in
chicks. However, these authors only detected chick edema factor in the liver
and feces.
edema factor was detected in chicken tissues by electron capture gas chrora
tography, and an estimation of the concentration in various tissues was made
by evaluation of gas chroniatographic response.
Thirty samples consisting of homogenized tissues and parts from three
groups of chickens, submitted by the Division of Nutrition, were examined for
chick edema factor by a recently developed method.1 One group had been fed ft
ration containing 3% of a reference toxic fat. Another group had been fed a
ration containing unsaponifiables equivalent to 3% of the toxic reference fat.
The last group received a ration free of toxic fat. The weights of tissue samples ranged from 0.4 to 93.4 grams. The samples were received in glass stoppered Erlenmeyer flasks, in ethanol.
DETERMINATION
(1) Extraction of unsaponiflable matter from animal tissue (Modification of
AOAO Official Method 26.071).
Quantitatively transfer an alcoholic solution of the homogenized tissues to fl
24/40 round bottom flask, add ethyl alcohol to give a final volume of 4
ml/gram tissue, but not less than 50 ml. Add 2 ml KOH solution (3 + 2) per
gram of tissue.
Saponify by boiling with occasional swirling on a steam bath for one hour
under reflux air condenser. Transfer alcohol soap solution while still warm to
separator using water (equivalent to twice the volume of ethyl alcohol). Kinsc
saponiflcation flask with the same volume of ethyl ether and transfer to separator. Shake vigorously, let layers separate and clarify, breaking any emulsion
by adding up to 1/20 volumes of alcohol and swirling gently. Drain lower layer
and pour ether layer through top into a second separator containing water (2
ml/g tissue), but not less than 20 ml. Make two more extractions of soap solution with ethyl ether (8 ml/g tissue). Binse pouring edge with ethyl ether and
add rinsings to second separator.
Botate combined ether extracts gently with the HaO (violent shaking at this
stage may cause troublesome emulsions). Let layers separate and drain
aqueous layer. Wash with two additional portions of ILO (2 ml/g tissue),
shaking vigorously. Then wash ether solution two times witti alternate por- ,
tions (2 ml/g tissue) of KsCOa and HjO. If emulsion forms during washiug,
drain as much of aqueous layer as possible, leaving emulsion in separator with
ether layer and proceed with next washing, "Wash final solution with HiO
until washings are neutral to phenothalein.
Transfer ether solution to erlenmeyer, rinsing separator and its pouring edge
with ether, adding rinsings to main solution. Dry ether solution by adding •
anhydrous NaaSO« (1 g/g tissue) and swirling vigorously ca 1 min. Let solution stand 10 min. Decant ether solution through glass funnel containing
pledget of pre-rinsed cotton in neck and holding 26 gr anhydrous NasSOi into
another erlenmeyer containing boiling chips. Wash first erlenmeyer with ether
and transfer to second erlenmeyer.
Evaporate most of solvent on steam bath and transfer to 100 ml (preweighed) extraction flask. Evaporate residual solvent on steam bath under N.
Dry flask to constant weight and obtain weight ,of unsaponifiable matter.
Proceed.with fractlonation of unsaponiflable matter on alumina and cleanup
of alumina fraction 3 as directed in method (1), analyzing the residue by electron capture gas chromatography as directed.
RESULTS AND DISCUSSION
All residues except for liver samples were initially taken up in 100 ul of
iso-octane and 5 ul injected. The liver samples were taken up in 250 ul of isooctane and 1 ul injected. Examination of the chromatograms indicated the
presence of a small contaminant (Ra 11.1) in the reagents used in the cleanup
of the negative control samples.
Six out of 10 samples were "toxic" from each group of chicks fed toxic fnttr
material. The liver sample in each group was the most "toxic", containing
roughly 83% of the total "toxicity" as indicated in Table /. The terms "toxic"
or "toxicity" as used here to describe results of gas chromatographic analyw*
refers to the amount of material in an Individual tissue which produces characteristic peaks at Ba 12 and 22 resulting from feeding the toxic fat. Of the
pooled adrenals, bone, brain, heart, intestine, kidney, liver, skeletal muflcle,
skin, and testes that were. examined, only the bone, heart, intestine, kidney,
BEFEBENCES
\
1
Hlgginbotliam, G. 11., Ress, J., and Firestone, D., JAOAC, in press.
'Campbell, T. C., ana Friedman, L,., JAOAC, t9 824-828 (1966); Proc. Soc. Bxp.
Biol. Med. Ml, 1283-1287 (1966).
TABLE I.-RELATIVE % OF CHICK EDEMA FACTOR IN POSITIVE TISSUESi
Group A
(3 percent
toxic fat)
Tissue
„
7.1
0.6
2.4
2.4
82.8
4.7
Heart.
J
liver.
Skin
*
Group B
(unsap.~
3 percent
toxic (at)
""
4.8
1.2
2.4
1.8
83.6
7.2
1
The relative percent of CEF in each positive tissue was estimated by adjusting the volume of each sample so that <
5 ul injection of each would yield a chromatogram exhibiting the major peak (Ra 12) with a height of ca 6 cm. The following
formula was used to calculate percentages:
% CEF=
volume of sample
xlOO
\Total volume of all samples in group/
TABLE II.-RETENTION TIMES (HA)' OF CHARACTERISTIC OF CHICK EDEMA FACTOR COMPONENTS IN TOXIC FAT
ADDED TO DIET AND ISOLATED TISSUES
§
rubber
(Barber Colman Model 5360 gas chromatography; 7 foot 1/4 Id glass column packed with 2^%SE52 silicone gum rubbei
on 60-80 mesh Gas Chrom Q; 3 x 10-8 amperes full scale; detector voltage, 30; injector temp., 240° C; column temp.
. . . . .
-.^j5 -.
200" C; detector temp., 210 C)
Sample
Toxicfat
Unsaponifiables (from toxic fat)
Adrenals
Bone
Brain
Heart
Intestine
Kidney
Liver
Skeletal Muscle
Skin
T«te»..,:
Ra'Retention time relative to aldrin.
Group A (Fed 3% toxic fat)
10.6,12.4,18.6,21.6..
None
12.0,12.4,21.6
None
12.0,12.4,21.6
10.6,12.0,18.6,21.6.
12.0,12.4,21.6
12.0,21.6
:.
None
12.0,12.4,21.6
None
Group EJ (fed unsap. s (3%
toxic fat)
"".".""."..". 10.6", 12.4,18.6,21.6
None.
12.0,21.6
None
12.0
10.6,12.0,21.6
12.0,21:6
12.0,21.6'
None
12.0,21.6
None.
�314
315
LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS IN Macaco, mulatto,
MONKEYS FED Toxio FAT
J. E. Allen, D.V.M., Ph.D., and L. A. Carstens, B.S.
SUMMARY
Thirty-six Macaco, mulatto, monkeys were given a diet that contained 0125
to 10.0% of a fat capable of producing hydropericardium, ascites, and deatli In
chickens. There was an inverse relationship between the concentration of toxic
fat in the diet and the survival time of the monkeys. The monkeys given tlie
greatest level of toxic fat had the mean survival time of 91 days, and the
monkeys given the lowest level and the mean survival time of 445 days,
During the last 30 days of life, the monkeys developed generalized subcutnneons edema, ascites, hydrothorax, and hydropericardium. There were
decreases in erythrocytes, leukocytes, total serum protein values, and altered
albumin :globulin ratios. There was also cardiac dilatation and myocardlal
hypertrophy and edema. Experimental monkeys had reduced hematopoiesis and
spermatogenesis, degeneration of the blood vessels, focal necrosis of the liver,
and gastric ulcers. It was proposed that toxic fat exerted its injurious effects
upon the parenchymal cells of the liver, endothelium, and myocardium with
subsequent development of generalized anasarca.
Fats from plant and animal sources have been used to increase the caloric
level of diets for animals. As a result of increased demands by feed manufacturers for low-cost fats, almost every available source of these products has
been utilized. Certain fats were found to be extremely toxic to poultry, and
hundreds of thousands of chickens died or were killed after they were fed
diets containing these fats. Results of experiments indicated that young chickens developed hydrothorax, pulmonary edema, ascites, and subcutaneous edema
in 1 or 2 weeks when there was, toxic fat in their diet.1'8'15"17 The accumulation of large quantities of extra'vascular fluid in chickens seemed to result
from altered permeability of the vascular bed, cardiac decompensation, and
liver necrosis'.1'2
Chemical studies on toxic fat indicated that the toxic fraction was located
in the unsaponifiable portion.8'20 By repeated passages through alumina and
silica gel columns, crystalline preparations of the toxic fraction were prepared";
however, the chemical composition of the compound was not determined.
Before the cause of this intoxication of poultry was established, many chickens that had been fed toxic fat were processed for human consumption. Since
that time, the clinical, histologic, and electron microscopic 15
changes that
occurred in the intoxicated chickens have been enumerated.1''' '" Data are
not available, however, concerning the effects of toxic fat on primates. Since
the chemical composition of this fat is unknown, the possibility exists that it
may once again adulterate various edible fats. This is a report on experiments
undertaken to determine the effect of toxic fat on lower primates. The results
of the experiments may be helpful in postulating the effects that toxic fat
might have in man.
MATERIALS AND METHODS
Since the chemical nature of toxic fat was unknown and chemical procedures were not available to determine the toxicity, a biological assay was performed on the fat used in the experiments. When the diet of 1-day-old chickens contained 3.0% toxic fat, 50.0% died within 15 days.
In the initial experiment, 16 Macaca mulatta monkeys (av. weight, 4.2 kg.)
were allotted to 4 groups and fed diets containing 0 (control), 1.0, 5.0, and
10.0%, respectively, of toxic f a l i . T n the 2nd experiment, 20 M. mulatta monkeys (av. weight, 6.0 kg.) were allotted to 4 groups and fed diets containing 0
(control), 0.125, 0.25, and 0.5%, respectively, of toxic fat. The toxic fat was
combined with corn oil to obtain similar fat intake levels in all groups of monRocelvod for publication .Tune 23, 1966.
From the Department of Pathology and the Regional Primate Research Center, University of Wisconsin, Madison, Win. 53706.
This Institutes of supported In part by grants HE-08681 and FR-0167 from the
Nationalresearch w»n Health.
The authors thank Mlns Karen Welke and Mrs. Adricnno Cuppas for technical
aiiHistance,
«l
1
•>
f
,
keys. The following data were obtained at181-month intervals: total serum protein value,' serum electrophoretic10 pattern, complete blood count," prothrombln time,13 serum bilirubin value, cholesterol level,' serum electrolytes,8 blood
urea nitrogen value,11 and body weight Observations were made each day on
the general appearance and amount eaten. Needle biopsies of the liver were
performed each month throughout the experiment to study sequential changes
that occurred in hepatic tissues.
All monkeys were necropsied immediately after death. When possible, the
monkeys were killed immediately prior to their anticipated death to ensure
procurement of fresh tissue for light and electron microscopy. Tissue sections
from heart, lung, liver, spleen, mesenteric lymph node, sternal bone marrow,
skeletal muscle, testis, gastrointestinal tract (3 zones of stomach and every 10
tin. of intestinal tract), skin, adrenal gland, pancreas, kidney, cerebrum, cerebellum, pituitary gland, thyroid gland, parathyroid gland, and urinary bladder
were fixed in 10.0% neutral formalin, dehydrated, embedded in paraffin, sectioned at 6 p., and stained with hematoxylin and eosin stain. Frozen sections
from liver and heart were stained with Sudan IV for neutral fats.
Sections of liver and heart were taken from each monkey, cut into small
cubes, and fixed in Millonig's11 and Caulfield's" fixatives. These tissues were
subsequently dehydrated through a graded series of ethanol and embedded in a
plastic resin mixture." Sections of the tissues were cut on an ultramicrotome,
TABLE l.—TERMINAL HEMATOLOGIC CHANGES IN MONKEYS FED DIETS CONTAINING TOXIC FAT
Group
Controls
F«d toxic fat
<
•
;
.
Total serum
No. of
protein
monkeys (Gm./lOO ml.-)
9
27
7.5
5.4
Serum
albumin (%)
Packed cell
volume (%)
White blood
count
xlO»/cmm.
Red blood
count
xlO«/cmm.
61
35
41
16
6.8
3.0
6.5
2.5
placed on 400-mesh uncoated copper grids stained with uranyl acetate, and
examined with an electron microscope.*
RESULTS
In the initial experiment, monkeys in the groups fed diets containing 5.0 and
10.0% of toxic fat had the mean survival time of 01 days. The monkeys in the
group fed the diet with 1,0% toxic fat had the mean survival time of 100
days.
In the 2nd experiment, the monkeys survived for a much longer period. The
monkeys in the groups fed 0.5, 0.25, and 0.125% toxic fat' had mean survival
times of 202, 274, and 445 days, respectively.
There were considerable differences in survival times of monkeys in the various groups; however, the major clinical and pathologic changes were similar
and occurred during the terminal 30 days regardless of whether the monkey
survived for less than 4 months or longer than 1 year. Therefore, the data
from the experimental monkeys will be presented collectively.
HEMATOLOGIO EVALUATIONS
Total serum protein values of the monkeys were reduced approximately 2.0
Gm./lOO ml. during the experiment (Table 1)'. The mean percentage of serum
albumin in the monkeys fed toxic fat on the last determination prior to death
was 35%, whereas that of the control monkeys averaged 61%. There was a
gradual decrease in the cellular elements of the blood during the experiments.
Packed cell volumes were reduced from 32% to 16% during the last 30 days of
life. These findings were substantiated by the total red blood cell counts which
averaged 2.5 million/cmm. of blood immediately before the monkey died. The
hemoglobin values followed a similar course, with the mean value of 6.0
Gm./lOO ml. of blood being obtained in the last 30 days of life. Comparable
observations were recorded for white blood cell counts; these averaged 3,000
white cells/cmm. of blood on the last hematologic evaluation of the experimen* RCA KMU-3G Electron Microscope, Hadlo Corporation of America, Camden, N.J.
45-362 0—70
21
�316
Jtl monkeys. Prothrombin times, serum bilirubin values, serum electrdl^K :1
|
mood urea nitrogen values, and cholesterol levels of serum were not chanpiYfj
appreciably during the experiment.
• •
317
tnyelold and erythroid series (Fig. 3). Most of the bone marrow i^Pcomposed
of fatty tissue and proteinaceous fluid. The blood vessels and sinuses contained
only a limited number of cells.
Skeletal Musculature.—The skeletal muscle was pale and edematous. MicroCLINICAL OBSERVATIONS
•coplcally, the muscle bundles and fibers were widely separated by fluid, but
otherwise seemed normal.
The major changes in the monkeys were the development of generalized nlsTcstes.—Grossly, the testis seemed normal; however, when examined micropccia and subcutaneous edema 1 to 2 months before death, Edema, first notiwd , ^
scopically, active spermatogenesis was not found. The seminiferous tubules had
around the lips and eyelids, progressed to the remainder of the face and even- • -j
abundant spermatogonia and Sertoli cells, but only a limited number of pritually involved the subcutaneous tissue of the trunk and extremities. Es|*-^|
mary spermatocytes. There were no spermatids or mature spermatozoa. Intercially obvious was the marked edema of the scrotum and sheath wltlcfc "fKtltial tissue was moderately edematous, but the Leydig cells did not seem
developed during the last few weeks of life and, in some monkeys, partially :|
affected.
obstructed the flow of urine. During the last month of life there was decreased j|
Gastrointestinal Tract.—In 18 of the 27 experimental monkeys, marked
food consumption and the subsequent loss of body weight was frequently u-.ty
hypertrophy of the gastric mucosa occurred in the fundic and pyloric regions.
much as 1 kg. Diarrhea developed in 75% of the experimental monkeys during fti
mucosal layer.
the last few days of life. Results of bacteriologic cultural examinations of |In the same areas, small gastric ulcers penetrated the tract were seen.(Fig. 4).
In 6 monkeys, inflammatory changes in the intestinal
Results
feces were negative for pathogenic enteric organisms,
of bacteriologic evaluations of these lesions indicated no pathogenic organism
that could have been associated with the gastrointestinal disturbance. MicroGROSS AND MICROSCOPIC FINDINGS
.',;:
scopically, the hyperplastic gastric mucosa was seen to form many large, inter(Hgitating folds. Adjacent to these proliferative areas, the mucosal lining was
The findings at necropsy substantiated the presence of extensive subcuU- :;
eroded, and the underlying tissue was necrotic. Large numbers of polymorneous edema in over 75% of the monkeys fed toxic fat.
5
phonuclear leukocytes were in the necrotic tissue and underlying musculature.
Heart.—Dilatation of the heart was especially obvious on the right side- ii
Blood vessels of tjie edematous submucosal and muscular layers of the stomThis was further clarified when the circumference of the valves was deter- f|
ach adjacent to the ulcerated areas were free of obstruction.
mined. The mean tricuspid and mitral valve circumference of the experimental i|
Enteritis in 6 monkeys had caused moderate denudation of the intestinal
monkey hearts was 55 and 45 mm., respectively; in contrast, the tricuspid and :
|
mucosa and considerable hemorrhage Into the lumen. The mucosal lining near
mitral valves of the control monkey hearts averaged 40 to 36 mm., respec- ,-|
the base of the crypts was intact, and the underlying musculature was normal.
tively. Hypertrophy of the cardiac muscle was also apparent in the experimen- J;
Skin.—There was marked edema .of the dermal layer of the skin, causing
tal monkeys. The hearts of the experimental monkeys were 0.56% of the body -i
weight, whereas hearts of the control monkeys were 0.30%. Microscopically, .| disarray of the collagen fibers. The epidermal layer was comparable in the
|
control and experimental monkeys. There was an absence of any detectable
the muscle fibers were distinctly separated by fluid. Individual muscle cells
change in the hair follicles of the monkeys given toxic fat.
were hypertrophic, and their nuclei were enlarged, distorted, and hyperchromlc
The adrenal gland, pancreas, kidneys, cerebrum, cerebellum, pituitary gland,
(Fig. 1). There were no distinct valvular lesions in hearts of the experimental
thyroid gland, and 'urinary bladder of the experimental and control monkeys
monkeys.
.;fM
Lungs.—Lungs of experimental monkeys were not altered appreciably. Iso- ;|§ were comparable grossly and microscopically.
lated areas of atelectasis, congestion, edema, and flbrosis were observed. The /
ELECTRON MICROSCOPIC CHANGES
proliferation of fibrous connective tissue was associated with the presence of j
lung mites (Pnewnonysis simicoli).
,
,
.
,
, ,.|
The extent of electron microscopic change in the liver correlated well with
Liver.—Livers of experimental monkeys were small, firm, and moderately .the level of toxic fat in the diet and the duration of toxic fat consumption. An
yellow. On microscopic examination, moderate distortion of the architecture ,
early change in the parenchymal cells was the disruption of the orderly
was found. Many parenchymal cells were enlarged, multinucleated, and had '
arrangement of the granular endoplasmic reticulum. The cisternal spaces were
only moderate affinity for stain, whereas other cells were small and markedly
dilated, and the loss of ribosomes from the outer surfaces of the cisternae
hyperchromic. There was also focal necrosis of the parenchymal cells in the
resulted in an apparent increase in the smooth endoplasmic reticulum. As a
centrilobular zone (Fig. 2). Many parenchymal cells contained vacuoles in
result of mitochondrial swelling, the cristae seemed shorter and less abundant
their cytoplasm which stained positively for neutral fat when frozen sections
than those of the control monkey hepatic cells (Fig. 5 and 6).. Cytosomes of
were prepared. Small areas of fibrous connective tissue occurred in the peri- "
variable content and size were moderately prevalent in the cytoplasm. Small
portal area; however, they did not alter the architecture appreciably.
vesicles and flattened lamellae comprised the relatively small Golgi complex.
Spleen.—Spleens of experimental monkeys averaged only 0,074% of the body
ITat vacuoles were abundant throughout the cytoplasm (Fig. 6). The nuclei
weight, and those of the control monkeys were 0.13%. Microscopically, the gercontained distinct nucleoli and abundant chromatin dispersed throughout the
minal centers were surrounded by only a narrow zone of lymphocytes, the
nucleoplasm.
blood sinuses were practically devoid of cells, and the trabeculae were espe- \
Results of hepatic biopsies made a few weeks before death, and experimencially prominent.
tal tissues obtained at the time of death, indicated distinct alterations. Many
Mesenterio Lymph Nodes.—Lymph nodes were light tan and edematous.
parenchymal cells were shrunken and electron dense (Fig. 7). These dark cells
Microscopically, the germinal centers were surrounded by a narrow band of
were seen in various stages of degeneration. In some cells, the cytoplastnic
lymphocytes, The medullary cords were indistinct, and the sinuses were filled
organelles were still visible despite the extremely dense matrix. Vacuoles were
with proteinaceous fluid.
dispersed between the cellular organelles. Myelin bodies were abundant in the
Sternal Bone Marrow.—Grossly, the bone marrow resembled coagulated
cytoplasm, along the plasmalemma, and in the intercellular spaces. The nuclei
plasma. Microscopically, only a small number of hematopoietic cells were seen .
were also electron dense, and the nuclear envelope was only vaguely discerniin the marrow, and those were approximately equally divided between the
ble. Other dark cells had lost all resemblance to normal parenchymal cells,
�319
318
-
along the plasmalemma adjacent to
...„ ,„„*,<= yiuuess. u.ue cytoplasmic organelles were quite
i abundant matrix, There was marked disruption of the endopla
.--.I, with only short fragments being scattered throughout the
Abundant free ribosomes were quite evenly dispersed between the urpuwtn*
The external contours of the mitochondria were frequently irregiilnr, »s* m
matrix was moderately electron dense. Occasionally, bulblike projection* «w*f
formed by the external mitochondrial membrane. The plasmalemma? tmfc
irregular, and the microvilli were short and sparse. Occasionally, thcr* «w*C.
myelin bodies along the plasmalemmal surface. In some instances, the K04
cell plasmalemmae
extracellular space. had ruptured, and organelles were dispersed througliWiiSt
ons have been reported in
PSfe by which toxic fat exerts its im,^^,, -research wiu be
IfeMilOKenoRls has not been determined, and
fmttwl to elucidate these points.
monkeys and the development
relationship of toxic fat consumption'^^^^ ioA i C My, under
$»*mlerate to extensive alopecia has not "f^^ monkeys. However, only in
%*Ml conditions, there is a loss £!£« ^^Ha that of the monkeys
fWW«l Instances has alopecia become as extensive
ppm Interesting and not well-understood lesion was the development of gastric
^Btnnd lesion was
ftl*a toxic fat.
microscopic
$*»« in more than 66% °f the monkey fedJo^cja^ ^ ^
MjtNKwrniice of the ulcers was similar toi tnat; oob«
equaUy evasive. The
|tS* and determination of the etiologk factors ™s ^ ^ vasculature
^gS^SXSZSfi^^™
«*the ulcers were
JBtsi^ra^^ff ^^—ra ^vS
Bile duct epithelium was affected markedly in monkeys fed diets conti
'
toxic fat. Many of the epithelial cells were so electron dense that tlic
plasmic organelles were difficult to visualize, and their nuclear ineinbB*48 gpwUtlon of large quantities of fluia.^. t1ileB_ decrease in total serum protein
de
e l
were irregular and extremely dense. The interlocking plicae of adjacent <*8*|| Bftkcnthe blood protein values were tested a ™ fo und (Tabie i). The
fpri a reversal in the albumin :tfojuj* ratio were lou ^ ^ attrlbuted
were widely separated. As a result of the shrunken condition of the epHWtitr''
cells, microvilli on the luminal surface of the plasmalemmae seemed thta i:
Mfaj foftcraiBe in serum protein values, J^0™?"J/tacrease in bi00d urea nitrogen
elongated.
'|| gto altered renal function ^^^"^^a^tton for the decrease in
Endothelial cells in some areas of the liver had a distinct resemblance to fltf fe# albumin in would be ^ated ?st.^og":1fn1Tir£ observed in the liver. ^^ _
the urine. The Jm to the chan^bserveci i
^ Focal
{
dark parenchymal and bile duct epithelial cells. The dark endothelfal «W; liltrum protein
ob
degenerative
were shrunken, and their internal structures were distorted. There were *W* |l*m»8 of necrosis and degeneration -were^ ^rvea m
o£ the
ened fenestrations between the endothelial cells. Changes in the cytoplawftfj I/awntnl monkeys. Examination of electron nucrogru^
organelles were comparable with those observed in the parenchymnl wB*: ^.*wu, substantiated the light .mi«.o«°P£ ^Symal lells, there was a
*H«ed functional status of the liepatic pa«^ »•
^^ pressure
Occasionally, large cytoplasmic sequestra from the endothelial cells wwf$
observed in the lumen of the vessels.
''^f
Crease in albumin production. This wouia, in mru^
extravascular spaces.
Heart.—The main differences between hearts of control and experlment*i'j| ;«t the blood sufficiently to produce ^"^"le^ect of toxic fat unon the
dark) shrunken,
monkeys were the dilatation of the intercellular spaces and the wide dlsix?r«i;r! r Another important aspect of the pH)ble.n vw the^£
.'wsculature. Many of the hepatic »^ mj ocara^
these vasc ular
of the myofibrils in the latter. Between the widely separated groups of m)'«&fi
Qed ^tu tue Hght
brils (Fig. 9), there were mitochondria, occasionally a segment of sarcopltt«ml* fj il«l seemed to have undergone degenerative cnanb
thnnges were not detected when the tissues• ™™ fl b d nad undergone
reticulum, and abundant matrix. Usually, only the Z lines could be readily Ti*-:.:,|
ualized. Gytosomes were much more abundant in the muscle cells of expcrf-,;"! Microscope, it is likely that other•vessel^f^rg^ ^y^tth the vascular
w
c
1
mental monkeys and were usually found near the nucleus (Fig. 10). In mow,'v* i-rimllar changes. These observations °"^u °gd toxic fat. As a result of cel:Hmnges recorded in chickens *at ^™ c^umrf t«u at ^^ ^ &
than 75% of the hearts of the experimental monkeys there was a dlstlnrt '|
would eventually
swelling of the mitochondria. The cristae were widely separated, and the nilto-1 s lular shrinkage, the intercellular BparoB were wia
chondrial matrix was abundant (Fig. 11). Large myelin figures were observed••%
iqucnt increase in the l'0108}*^ ?* ^uJfSd predispose to the accumula ion
fend to an increase in vessel peimeability ana^ p
P hypertrophy in all of
within and surrounding many mitochondria. Myocardial nuclei, components fit ••;{
"of extravascular fluid. There was ^/^^^he^yocardium was examined
the transverse tubular system, as well as elements of the sarcoplasmic retlc«-';"|
n
m
: the monkeys that consumed toxic fat Whe ™e y°cft
^ and
lum, seemed unaffected. There was a noticeable separation of intercalnted %
microscopically, there was hyper rophj' oi'.to^n™Uc£mmonly reported in
disks in many of the experimental monkeys (Fig. 12). The various bnwlt ; |
the
a
marked interstitial edema. Both of . **"" ? Jecame less efficient, there was
formed by the tissue.
tous fluid in the myofibrils were often masked due to the abundance of edema-"'-'',
.;
patients with cardiac insufficiency. As \he h«art^ame less ^^ ^
Many of the endothelial cells appeared electron dense and shrunken, with ,'.'
likely a gradual increase in ^°^ep^Se spaces. An increase in right
distortion of the nuclei and cytoplasmic organelles (Fig. 13). In most ',
instances, the organelles were in close apposition as the result of the cell ';
,: ^^^^*~^^^°» tUat P°StUlated £OT '
monkeys has been reported in chickens fed toxic^at
permeability, and
ma
shrinkage. There were cytoplasmic myelin bodies in many of the cells. Occa- .1
sionally, intercellular stromal cells had changes similar to those in the endo- .
Reduced osmotic pressure of themblood,jd
^^"-TYhe causes for development of
thelial cells.
cardiac insufficiency have been ^ «iaiJ d]^cStC *"etermine at this time
anasarca in monkeys fed toxic fat It ^ a™cg^\ikel expianation is that all
DISCUSSION
'""'
"""
'
entity at a particular
Toxic fat consumption had a decided effect upon hematopoiesis and spennn-togenesis. Myeloid, erythroid, and lymphoid elements of the peripheral blood
stage of the disease.
were markedly reduced. Germinal centers in the lymph nodes and spleen and
the islands of hematopoietic cells in the marrow were extremely sparse. Inhibited spermatogenesis was also observed in the monkeys fed toxic fat. Similar
�321
320
REFERENCES
1
Allen, J. R.: The Role of Toxic Fat In the Production of Hydroperlcardium
Ascltes in Chickens. Am. J. Vet. Kes., 25, (July, 1964) : 1210-1219.
3
Allen, J. K., and Carstens, L. A.: Electron Microscopic Observations in the
Chickens Fed Toxic Fat. Lab. Invest., 15, (1900) : 970-979.
s
Allen, J. R., and Lallch, J. J.: Response of Chickens to Prolonged Feeding of
Toxic Fat. Proc. Soc. Exptl. Biol. & Med., 109, (1902) : 48-51.
„ ., ™
« Bowman, R. E., and Wolf, R. C.: A Rapid and Specific Ultramlcromethod for T«W ;'|;3
Serum Cholesterol. Clin. Chem., 8, (1902): 302-309.
' Gornall, A. G., Bardawlll, C. J., and David, M, M.: Determination of Serum Prolriw
by 8 Means of the Bluret Reaction. J. Biol. Chem., 177, (1949) : 751-766.
,
--,
Hald, P, M.: The Flame Photometer for the Measurement of Sodium and Potas»!«» 'ij,|
in Biological Material. J. Blol. Chem., 187, (1947) : 499-511.
-.'.'4
"Harman, R, E., Davis, G. E., Ott, W. H., Brink, N. G., and Kuehl, F. A.: Tw ,,-M
Isolation and Characterization of the Chicken Edema Factor. J. Am. Chem. Soc., f* /.-f
(1960) : 2078-2079.
.. •'.'
10
Mallory, H. T., and Evelyn, K. A.: The Determination of Bilirubln with tw
Photoelectric Colorimeter, J. Btol. Chem., 119, (1937) : 481-490.
„,»
11
Mlllonig, G.: Further Observations on a Phosphate Buffer for Osmium Solution* I* :'i
Fixation. Vol. 2. Proc. 5th Internatl. Cong. Electron Microscopy. Academic Press, pr* ,'gjj
York (1902) : 8.
13
Mollenhauer, H. H.: Plastic Embedding Mixtures for Use In Electron Microscopy.
Stfiln Tech., 39, (1904) : 111-114.
" Quick, A. J,: Hemorrlmglc Disease. Lea & Febiger, Philadelphia, Pa., 1957.
"4!
11
Rosenthal, H. L. : Determination of Urea in Blood and Urine with Diacetyl MonoiImc. Analyt. Chem. 27, (1955) : 1980-1982.
irt
" Sanger, V. L., Scott, L., Hamdy, A., Gale, C., and Pounden, W. D.: AllmenUrj.«
Toxemia in Chickens .T.A.V.M.A., 133, (Aug 1, 1958) : 172-170.
, *
ia
Schmlttle, S. C., Edwards, H. M., and Morris, D.: A Disorder of Chickens ProliaMj ;
Due to a Toxic Feed — Preliminary Report. J.A.V.M.A., 132, (March 1, 1958) : 216-219.
" Simpson, C. F., Prltchard, W. R., and Harms, R. H.: An Endothelosls In Chicken* -,,
and Turkeys Caused by an Unidentified Dietary Factor. J.A.V.M.A., 134, (May 1, 1059)!
410-416.
18
Williams, F. G., Jr., Pickets, E, G., and Durrum, E. L.: Improved Hanging Strif ,
Paper-Electrophoresls Technique. Science, 121. (1955) : 829-S32.
10
Wintrobe, M. M.: Clinical Hematology. 5th ed. Lea & Febiger, Philadelphia, Ft.
20
Wootton, J. C., and Alexander, J. C.: Some Chemical Characteristics of the Chltl
Edema Disease Factor. J. Assoc. Off. Agrlc. Chem., 42, (1959) : 141-148.
CALCULATED DIETARY INTAKES OF CHICK EDEMA FACTOR(CEF) FROM DATA PUBLISH ED BY ALLEN AND CARATONJ
(AM. J. VET. RES, 28, 1513-26 (1967))
Moan
survival
time
(days)
Dietary level of toxic fat
(Percent)
CEF intake
per animal
per day
(ug.)
ToW
Ctf
Inlikl
(ut)
MONKEYS (MACACA MULATTA)'
.
0.125
0.25
0.50
1.0
5.0
10,0.
445
• 274
202
169
90
0.225
0.45
0.90
1.80
8.0
1(0
123
182
ZM
721
l/HH;iv 1.^1*. * i » « -• "-" "
t. Capture Gas Chroinalograpby
CHICKS (DAY-OLD)»
3.O..
15
0.36
5.4
( w.nrl riiomi«trv Food and Drug Administration,
liy PAUL NKAL (Division of Food Uioinisir>,
I"
Washington, D.C. 20201)
, f
Tho elytron cupUirc GLO scmen.ng test
for rhi.'k cduma factor 0, « lias Lie-on
1
Calculations based on average daily food consumption of 225 gm. for a 5.0-kg. monkey (F. Sperling, personal, communication, June 12,1969).
' Calculations based on average dally food consumption of day-old chicks (personal studies, see Flick, et al., Poultry
Science, 45,630-36 (1966)).
i
|I
nf
HIP wtrolcum ellier extract from Die s\il° J™^11^,,,,,^ on „„ alumina column,
,' ' f • ftH( | H(.,M1,,p „/ ll,e tl.iul «l'»»;'«l
•! - w/, ,.c.lnclion in simple plonnvip i""pm i l s a f i O / r ic'Midio"
' , , „ (....-imcnt
Tlie inodifiofl procedure i m n n f ! " I.IL.IUIII,
v1
""
•
f in 2i ire indicative of the
vw « llln " "' 10
,
vn,.p o[ chick c drma fnflor.
�322
323
Table 1. Comparison of sulfitric acid cleanup with saponifieation for detection of chick edema
factor by electron capture gas chromatojjraphy (ECGLC)
ECGLC Analysts*
Sample"
HjSO< Cleanup
Saponlflcation
10.1 (18), 11.8 (14), 17, 6 (40),
20.4 (34)
Low positive reference fat (1.0% toxic
fat in USP Cottonseed Oil)
10.1 (45), 11.8(48), 17.6(65),
20.4(60)
Toxic animal tallow
10.2(74), 11.9(82), 18.8(94),
20.G(98)
Toxic ololc acid
10.1 (147), 11.8(23), 17.8
(>500), 20.4 (200)
10.1 (98), 11.8 (10), 17.8 (>500),
20.4 (154)
Toxic glyceryl monooleate
10.1(50), 11.8(23), 17.8(200),
20.4(82)
10.1 (48), 11.8(14), 17.8(258),
20.4(98)
Vegetable oil soapstock (nontoxic)
10.4 (trace), 13. 5 (trace)
10.4 (trace), 13. 5 (trace)
Oleicacid (nontoxic)
10.4 (trace), 13.5 (trace)
10, 4 (trace), 13. 5 (trace)
Cottonseed oil (nontoxic)
10.4(23), 13.5(22)
10. 4 (trace)
Blank
10. 4 (trace), 13. 5 (trace)
10.4 (trace), 13 .5 (trace)
.
10.2(56), 11.9(4?), 18.8(85),
20.6 (90)
" Toxic and nontoxic refer to results of AOAC chick bioassay (AOAC Official Motlmcls of Analysis, 10th Ed.,
1965, 26.087-26,091).
6
The first values (without parentheses) refer to retention time of peaks at 200°C vs. aldrin; the values In
parentheses rafer to peak area which is equal to retention time- (cm) X peak height (cm).
Method
IteaKcnIs uittl -Apparatus
'Hinsc all glassware with appropriate solvent
before use. Do not use polyethylene containers
to store solvents (6).
(a) Petroleum ether.—Reagent grade; redistill in glass between 30" and 60°C (available from Burdick. and Jackson Laboratories,
Muskngon, Mich.).
(b) Carbon telrachhride.—Distillcd-in-glass,
(c) Celile,—Johns-Mansville #645, acidwnshed. Wash well with petroleum ether and
dry.
(d) filler paper.— #5-19 S&S Blue Ribbon,
or equivalent,
/)«rermiiintj«iii
Std/uri'c acid cleanup.—Dissolve 2.5 g fat in
10 ml CGI., in 100 ml beaker (heat, if
necessary). Add 10 ml concentrated H 2 80j
and thc-n 20 g Oolite; mix with heavy glass
stirring rod during additions and stir until
homogeneous mixture is obtained. Adrl 125 ml
petroleum ether, mix well, let solids settle, and
ftltr>r the supernatant liquid through filter
paper in 00 mm conical funnel. Repeat with
additional 125 ml porlion of petroleum ether.
I'lVttporalo combined petroleum ether filtrate
to 5 ml for alumina column frni-tioiia.tic.in.
Complete dclerminalion as outlined in the
method of lligginbothani f t al. (1).
Kcsull* iitid Discussion
GLC retention limes and prmk areas for
negative, positive, and blank samples were
compared for both the sulfnric acid and tlic
saponificalion methods; sec Tab'.c 1. Results
are comparable as indicators of toxic material.
Gas chromatographic peak heights were lower
in some ea^es with the sulfuric acid cleanup;
however, Hie presence of toxic factor win
clearly indicated in the low positive reference
material, The nontoxic cottonseed oil samples
would have been judged toxic by the saponipnu
ll
ficalion mollieid because of Hie relatively largo
GLC peak of R,, 13.5, ft ponk detected at low
levels in gas chroinalograms from blanks and
other nonloxic samples; see Tnblo 1. Small
peaks of It,, 10-25 wore observer! in both procedures in the blank mil] nonloxic samples.
However, they did not interfere with identification of the toxic fats and differentiation of
toxic from nonloxic samples. Gas ehromalograms of blank and low positive reference
samples aflor saponificatiou and preliminary
culturie acid treatment are shown in Figs. 1
and 2.
Acknowledgments
The author expresses sincere thanks to David
Firestone for guidance and encouragement
throughout the development of this project
and to Richard Staaf who performed many of
the analyses. •
REFERENCES
(1) Higginbolhnm, G. R., Firestone, D,,
vez, Linda, and Campbell, A, D,,
Journal 50, 874-879 (1907).
(2) Higginbotham, G. R., Ross, J., and
stone, D., ibid. -SO, 884-885 (1607).
ChaThis
Fire-
Ftg. 2—Gas chromatograms of (a) blank and (b)
low positive reference fat after cleanup with sulfurlc acid treatment. See Fig. i for GLC condition!.
(3) Davidow, B., tbirf. 33, 130-132 (1MO).
(4) Dingle, J. H. P., Analyst 90, G38 (IOCS).
(5) Burke, J., , and Giufl'rida, Laura, This
Journal 47, 326-342 (1964).
•
LOW POSIWf KerfRENCC
""V"~'J -—;,-—• 4- •-/.,-" •# • jj, - '
fl«
W»WTI1
fig, J—Gas chromatograms of (a) blank and (b)
low positive reference fat after cleanup with
saponifieation.
GLC conditions: T X 4 mm i.d. glass column
packed with 2.5% StT-52 on GO-SO mesh GaJ
Chrom Q at 205°C. Amount injected: 1/SOth of
alumina column fraction 3,
Reprinted from the Journal oj the Association of Official Analytical Chemists, Vol. 50,
December 1967.
-
�325
26. OILS, FATS, AND WAXES
[) The official, first.action GLO-microcoulometric method for chick edeS
factor, 26.092-26.096, was changed by adding the following to 26.092:
(g) Ethyl ether for alumina chromatography.—Ether (not >2% alcohol) or
absolute ether (not >0.01% alcohol) (available from Burdick and Jackson
Laboratories).
(2) The official, first action electron capture method for detection of chick
edema factor, This Journal 50, 216-218(1967) was changed by addition of the
following to (b) in the Determination section:
After ". . . 26.094" in line 6 add "(using ether specified in 26.092(g)" (item
(1) above).
(3) The following rapid screening method for detection of chick edema
factor was adopted as official, first action :
PRINCIPLE
Samples are subjected to preliminary HaSO( cleanup and extd with petr.
ether. Ext. is purified on A12O3 column and examined by electron capture GI/C,
after addnl H2SO« cleanup. Gas chromatographic peaks with retention time
relative to aldrin of 10-25 are indicative of chick edema factor.
REAGENTS AND APPARATUS
(a) Petroleum ether.—Eedistd in glass, b.p. ,30-60° (available from Burdick
and Jackson Laboratories, 1953 S. Harvey St., Muskegon, Mich. 49442).
(b) Ethyl ether for alumina chromatography.—Ether (not >2% alcohol) or
absolute ether (not >0.01% alcohol) (available from' Burdick and Jackson
Laboratories).
(c) Carbon tetrachloride,—Bedistd in glass (available from Burdick and
Jackson Laboratories).
(d) Celite.—No. 545, acid-washed. Wash well with petr. ether and dry at
room temp.
(e) Alilrin standard soln.—0.1 jug/ml. See Reagents and, Apparatus, section
(a), JAOAC 50, 216(1967).
(f) Chick edema -factor low positive reference sample.—1.5% reference toxic
fat in TJSP cottonseed oil. (Available from Division of Food Standards ami
Additives, Food and Drug Administration, Washington, D.O. 20204).
(g) Activated alumina.—See Reagents and Apparatus, revised 26.092(1)),
JAOAC 50, 216(1967).
(h) Alumina chromatographic column.—To dry chromatographic tube, 17
mm o.d. (14.6 mm i.d.) X 250 mm long, fitted at bottom with coarse porosit}'
fritted glass disk and Teflon stopcock (tube without fritted disk but holding
glass wool plug in bottom may be used), add redisd petr. ether, dried before
use with anhyd. Na2SO<, until column is % full. Weigh 15 g A1203 and transfer to column in small portions, tapping tube as AUOa settles. When last portion of AlaOa settles and air bubbles stop rising to surface, add 5 g anhyd.
Na3SOi. Drain excess petr. ether so that it is just above upper surface at
Na2SO(.
(i) Oas chromatoffraphic column.—Glass, 5-7' X %" i-d., packed with 2.5%
SE-52 silicone gum rubber on 60-80 mesh Gas Chrom-Q (Applied Science Laboratories, State College, Pa. 16801). Coat support with substrate as follows:
Weigh 2.5 g silicone gum rubber stationary phase and dissolve in 300 nil
CHsCls-toluene (1 + 1), heating to dissolve. Add 97.5 g Gas Chrora Q and let
stand 10 mm with occasional gentle stirring. Dry in rotary evaporator held in
50° bath. Pack coated material into chromatographic column by adding small
amts while vibrating column at packing level with Vibro-graver tool (Fisher
Scientific Co., Pittsburgh, Pa, 15219). Fill to within I" on exit side and 3" on
entrance side, and fill remaining space with silanized glass wool. Condition
column at operating pressure 2-5 days at 250'.
(j) Gas chromatoyraph with electron capture detector.—See Rear/cuts anil
Apparatus, (.e), JAOAC 50, 216(1967).
DETERMINATION
(a) Preliminary sufurie acid cleanun.—Dissolve 2.5 g fat in 10 ml CCt< in 400
ml boaker; mix with heavy glass stirring rod while adding 10 ml H»SO<. Add
G g anhyd. Na2SOi and stir well while adding 20 g Celite, until homogeneous
uiixt is obtained. Add 125 ml petr. ether, mix well, let solids settle, and filter
supernatant thru paper in 90 mm conical funnel. Repeat with^idnl 125 ml
petr. ether. Evap. combined petr. ether filtrate to 5 ml for AliC^Humn fractlonation,
(b) Fractionation of petroleum ether filtrate T>y alumina ehromatography.—
Dry solvents prior to use by shaking with anhyd. Na8SO». Transfer petr. ether
nitrate from (a) to A1803 chromatographic column, using total of 15 ml petr.
ether. Let liquid level fall to just above top of NaaSO(. Elute sample with 100
ml petr: ether (fraction 1), 50 ml 5% ether in petr. ether (fraction 2), and 100
ml 25% ether'in petr. ether (fraction 3). Relatively fast flow rates of ca 8-0
ml/min give satisfactory results. Keep liquid level above top of NaaS04 at all
times. Discard fractions 1 and 2, and collect fraction 3 in 125 ml erlenmeyer.
Add several boiling chips and evap. to dryness on steam bath. Transfer residue
with petr. ether to 10 ml g-s. graduate and evap. petr. ether soln to 3 ml.
(c) Sulfurie acid cleanup of alumina fraction 8.—See Determination, section
(c), JAOAC 50, 217(1967).
(d) Electron capture gas chromatographif of petroleum ether extract.—See
Determination, section (d), JAOAC 50, 217(1967).
(4) The official, first action method for methyl esters of fatty acids,
26.055-26.059, with changes in This Journal 49, 231-232 (1966), was revised
ns follows;
(A) 26.058(c), revised third paragraph, TMs Journal 49, 232(1966), after
". . . to obtain calibration factor." insert "Reference mixts simulating most fats
and oils may be obtained from Applied Science Laboratories, Box 440, State
College, Pa. 16801; Supelco, Box 581, Bellefonte, Pa. 16823; and Lipids Preparation Laboratory, Hormel Institute, Austin, Minn. 55912)."
(B) 26.059, change to read as follows: "Two single detns of major components (>6%) performed in 1 laboratory shall not differ by >1.0 percentage
unit, Two single detns performed in different laboratories shall not differ by
>3.0 percentage units."
(5) The official, final action lead-salt ether method for determination of saturated and unsaturated fatty acids, 26.040, was changed as follows:
(A) Change first paragraph, first sentence to read: "Accurately weigh 10
(for plant fats used in common household cooking oils) or 20 g sample into
200
"
(B) Add the sentence "Reserve ether filtrate (contains ether-sol. Pb soaps)."
to the end of the second paragraph.
(C) Fifth paragraph, line 8, after ". . . HCl-free", insert "(no ppt with
AgN0 3 )."
(D) Revise the sixth sentence of the fifth paragraph to read: "Distill ether,
avoiding any loss of fatty acids, and heat, over steam bath to constant wt
under controlled flow of N to prevent oxidation of fatty acids. Cover steam
batli with towel to prevent splashing 11,0 into erlenmeyer."
(E) Paragraph 6, line 1, insert "reserved" after "Transfer". Line 6, add
"Repeat HC1 hydrolysis until no more PbCU is pptd." after ". . . into beaker."
(F) Change paragraph 7, lines 2 and 3 to read, ". . . until HC1 is removed
(no ppt in wash H2O with AgNOa). Dehydrate ether with ca 2 g anhyd.
Na2SOi and transfer ether soln . . . ."
(G) Change paragraph 8 to read: "Det. in duplicate I numbers of 0.2-0.3 g
oil from unsatd fatty acid fraction, and from entire satd fatty acid fraction.
(I number of satd acid fraction is due to presence of some unsatd acid.)"
(6) The following gas-liquid chromatographic method for butylated hydroxyanisole (BHA) (121006) and butylated hydroxytoluene (BHT) (128370) in
corn and rice breakfast cereals was adopted as official, first action.
APPARATUS
(a) Gas ehromatoffraph.—Barber-Colman Model 5000, or equiv., with H
flame ionization detector and strip chart recorder. Establish following operating conditions: temps—column 160°, detector 210°, flash heater 200°; N flow
rate, sufficient to elute BHT in 3-4 min from QF-1 column and elute BHA in
3-4 min from Apiezon column; H flow rate, ca 40 ml/min for Apiezon L and
ca 25 ml/min for^QF-1; air flow rate, ca 340 ml/min; electrometer sensitivity;
500 X (5 X 10 lo) amp full scale deflection) with 5 mv recorder. Adjust H and
air flow rates, if necessary.
Adjust electrometer sensitivity so 0.1 /ig BHA gives ca 50% deflection.
Repeat injections until constant peak heights are obtained on successive injections of identical vol. of std mixt.
Order of appearance on Apiezon column (4 f ) : BHA, BHT, di-BHA. Order of
appearance on QF-1 column (6') : BHT, BHA, di-BHA.
�327
326
Iho reference loxic fa' \vcio eacli fraelionntcd by pruparaMvo O1,C (lliermnl c'liidurtivity detection), \wh\p n
U font x .( uini column at 2fiO" C packrd w i t h 10 per cent
'/JC'-'Jinf mi IJU/Sti ntesli '(Inn C'hrom Q'. fK-ilnt.cd components from the 2.:t.-|,(l-lelnU'lilnruplieiinl pyrolynutc and
from the e b l m i n n l e d dilieui',o-ji : di"\in were examined by
electron capture (!!/' itiul iufrnreil (Spectrn^eupy. and were
iilsn nnal\ p sed by mass Kpeetmmctry 1o dett-nniiin mnlec.uInr wei^hl find numlier nl' chlorine atoms per miitecutc,
Uenults nre rccfi'd'-d in 'I'able 2. The comjionciils isnlaled
from the reference tnxie fat Imvo iwi yef been rxnutimvl
l from Nature, Vol. 220, Wo. S16S, />/>. 7ft2-^OJ, W<nvi»b«r 1(5, J96S)
. . . ..... _
2,fl,4,(1'TKTIlACIIl,()KOPIlHN''I. I'Vli
m/'pcnk
tf,nl
Xfi.
2nd* f
Mixture
dt ,m><'mi)-ji-ill"*l
n-o
17'4
20-0
Chemical and Toxicologicai Evaluations
of Isolated and Synthetic Chloro
Derivatives of Dibenzo-p-dioxin
ONK of tlm toxic compounds Unown ns hydropericnrrlimn
fuclor (clticU ocdcmin factor) its l,2,3,7,(i,0-hoxflchlorodiboirAo-ji-dioxin1. llccftufio it is highly improbablo th'it '
theso \ - ory toxic compfjunds nro nutnrnlly occurring compononls of tho fats frnm which they have U«m isoluted, •
tho qtifHtifin oi' l,)icir origin baa aroused Bpi'Diilation.
A clnn to the possiblo origiu of hydroporicartlUim factor
is found in a report by Tomiin c( tiL* of tho ayntlicftin of
polyh»loclibenxo-j>-(lioxins: chlorophcnols nnd their miltR,
when healed, nnrlcryo condensation reactions imd form
chlorinated dorivptiveH of (lib(iii70-;;-dioxin. BOCEIUHO of
tliis obsoi'vntion, \\~f.\ studied tho pyrolysis of n number of
coinniorciftlly avnilnblu ohloroplienols which nro widely
used in agriculture HIK! industry, nnd present vesulu of
HOITUI pi'olinu'nai'y pxp^riininitB \\hicJi tuip^est Umt hydropcrirnrdinni fnctor could arise fi'om cerlnin ohlorophenols.
Tlio commeiTiul clilnrophennU u.scd in this work \veve
pyrolyfefl in neconlnneo w i t h tins gcnci'al proccclurcH
described3. Heuwno extracts of thu reaction mixture*
wero fiiH'tionnted by passing tbeni through an alumina
column. Residues of the beii'/.euc effluents wore oxl.rne.tcd
with pctroloum el her at room tempcnUviro, and tho
poll-oleum el her \\nft then removed. The resultini; products wni'o sufticienily pure in most CHRCK for uxnininnlion
by electron capluru gns--lic|iiid chvomutonra])liy ( t J l » ( 3 )
and liinlogieftl ie»linjr hy (ho chicken embryo nwsny*.
Tablo 1 hhoxvrt retention limes of Iho Ol.C peaks on a
7 font, a-5 per emit W;.52' column at S'OO* C nml iho
resuUHof lljccliickei) emhryo ansny. I'lHlividimlly isolated
hydnmericni'ilium fnc!|.oi's of known chemical utruduiva
wiicu not lu'ailidilo for Ut*n as Klandnn.!:!; cnnKetjuent ly.
a cimcenlrale IViim tho nininponifialil" fmcl i"ii of a certain
eiHumerc.ia) li»xic f a t l y ncid mati'rinl u m uned as a (!!.(.'
reference. The unsapnniliablu fraction of Ihe f a t t y ucid
nuilorial \vt\» known to contain trneo (imouiUs of the
hydnipei'iciiRliiim factor. Thu toxic fatty ncid innlcriiil,
a hy.pnuluot obtniitcd from tlio mannfucturn of oleie and
utcaric acids, has bcon u«ed as a reference in nil our prc
viou« chemical and biologicnl work1.
Tnlilo 1. RXAMINATIOH OF CIUOKOI'MBNI
3niii|)tR pyrntyfed*
2,.|'))tclil»r<iplicnol (K)
/ r a n t snir c of com
|ionciHi| In pyrolynfl
1-0
2Xo-Trli'lilurmik>no1 (111
,.|.0-Trlrlilui'(tiilu>nol ( H I
?•»
,!M,0-Tc(riu>!|Wan1wnol (T) fl'0,10-0, 11-2.17'4,
ao o. nfl'O
I'rulfH'lilortiplU'iiol (K)
SJHt
Clilfirlnnl-'rt illlii'ii/n.ji-rtioxln
1 -0,1 'ft, 3'5,
fl-G
llcrcrcncii toxic fnl comuoiiPiiti
1-0. 1-8, 3-It, 0-fi, 8-0,
10-0, 11-2, 1 7 - 4 ,
G-U
PII
li'O
0'05
100
60
100
27
1 j|... r r l r n f l o i i Untr» nl'i^Un Hr.llvn tn Uio rAttnllon time of hWrln. .
1 1 /in -20 n.ii.h, In tin; i-fin, Stinutle InjTlcil Into (he n\t cell nf frr*h
ferUln uffl* Iwroro IncuimiloH. " Solvents usod: ctlmiiol, nccUnm and cliloroS Embryonic niorlnlllv iU 21 riny
ftolvrnt-lit^'Cind controls Viiu 10-ln
The morlnllly of non-lnjcclPd and
rent.
Tho chromntO[;rnm of components from tho toxio fntty
acid nirttci'ial conUiinvd ft nnmbor of pea UK of widely
varying retention limes; four of Ihoflo pcutt« had H»
vahiee grcaler than H). According to n current cJcetrcm
capturo OLO lest4, the presonco of ono or more GLC
pcatiH with /in values of 10 or more indicates that hydro*
periciii'dinm factor is jimse-nt in a fat. Tlio jjyi'oiysftlfl
from Luclim'eal grado S.^.-J^-lctmchlorophcnol was llto
only product Mmt showed u peak pattern iiidiciiling tlie
presenco of hydrupcrii-ardium factor; tho peak pntlcrn
rtiHcmhlud that UiKplnyod by the long rolentinii time compoiiLMils of th<* refereiino toxic, fat. \Vhen tested l)y tlw.'
ollicinl cbifk hioasfiny* for hydrojH-ricardinm factor, llic
mixture produced I be rjiso-fisu a(. a tfteltuy lord of 0-1 p.pJiiIn addition, dilK'n/.o./)-din\iu was chlorinated directly
in order In prepan- y,;t,V 1 R-li'lni(ihluKnUl>eM/.o-/)-dioNin l .
Thin malurinl jirovr-d to he cxtrenvly Uixie in llrti chic.ltcit
cml.M-jo anrtny (Tfthle 1). and provided attnuivn for i^lofioii
of u t'riehloi'o Hf well a-i tlio tuirnchlonxlibeiv/.o./j.diitx-iii.
The tee.hniual ^rad-: a.H.'l^l-tulrachlorophriiol pyrolyti*
ale, ehlorinated dil»',ii/,o-/'-di'ixin, nnd c!i>mpoui?ntfi fVoia
Infrared spectra of individual components exhibited
characteristic absorption bnndu in the return of J,3301.280 cm p l attributed to asymmetric titrotehin^ vibration
of C-*-0---CJ in the flibeu/o-;j-(lioxiu rinf/. The- two principal compouiulH isolated from the cihlorinntcd dibcu/.o-;)dioxtn were '2,3,7-trichloi-odibcnx.o-/).dioxiii ami 2,11,7,8toLmohltiifxlibettv.o-jj-dioxm. The infrared Kpeelrum of
tbn trichloro compound luw briuds f\t 870 em- 1 and 805
cm-1 (1,2,-1-Hubstiluted brii/.cne). and Sfid cm-1 (1,2,4,,'}.
Huhslituted hen/cuu), Tho infrared t*}i:*e|rum t>f tho
tdtrne.hloro compound has a strong band at 8fiS cm-'
(l,2,4,fi-Hul'9titule(l bmxcnn). Tho infrared fipectrum of
tln % letrachloro compound matched (ho fcpcutrum of
2,.'l,7,S.t[itracblorotlibcir/,o-/)-dioxiii published by 'J'omitn
fit (tl,*. Chicken cmljryo assay showed ilitit the tetrnchlovo
compound was more toxic t h a n the trichloro conipoxitid,
The fir«t four pcnka in t!ie chromnloft-rajii from tho
telrachlorophetio! pyrolysalo were caused by positional
isomei'fl, each containing six cblorino atomR,; tho fifth
and tiixth peaka were caused by positional isomcrs, o«ch
contnining sovcn chlorine atoms. A seventh ponk in the
olij-omntogriim, observed when Ihe snmpl'.i injection wns
overlonded, had a retention time icleuficul to (hat of a
anmplo of ochtuhlnrodtbenxo-;}-r]io\in prepared by pyrolysis of pentacililorophonol. The presence of pentaolilfrophcniil in tcclnu'cul ^I'iido 2,3,4,0-tctrachlgrophenol
nccountu for Ihn formntion of tho two hrptacliloro ifiomera
nnd tho oclnchlorodibew.o-p-dinxin.
A fourth hoxaehloi'o ifiumer, in addition to the ihreo
oxpcctcel from pure 2,3,4.li-tetrachlorophenol, is prohnbly
cnufiecl by the iiresenco of ft tctrnohlorophpiiol other than
the 2,3,'1,0-iaomrr in the otortinK material. Infnu-ed
npcotrn of peaks Ii and 4 from tho 2,ii.4,(i-teinH'hloruphenot
pyrolysato M'ore very Hiniilnr (o pubd'sln/cl K|jcctrn- of two
liydroperieardium factors iKuhvlcf! from a contamitifited
fnl«.
Tho conmonenlff isnlated by preparative C!IX: from the
refercnci! tuxit 1 f a t . tbe ehloriutited dihen/O'/t-dioxin. and
the 2,3,-1,0-leirachlnrf.pheiiol pyrol.vsnle Mere mialy.si.-il by
electron e i i p t u r e Of.C,' at 2<m" (Tand by the ehiekcn'etnbryri
HHqay. These resulls an- shown iti Table It. The q u i m t u y
o f i n r t l e n t i l m each fraetKMi from the reference (tixic nit
wa« csl ii mi led on ihe Imam of tbe \\t-ijihl of naiuple.
iuj'-cted m l ' t the prepnrntivo gas chromalnjrnipli niul the
uuntber uf fractions collected," The immunls of material
ifinliilr-d frniu tbe elilnriiuited dibeni'.n-;)-dioxiii and 2.1),'!.(!telraehloni|)lieiiiii pynil\vnle were rou{;h esltmnti-n based
on enmpari^dii of (il,C! peak areatt of isolated eumpnnenl?*
find tin.- sluriiuf,' mixiutv^. A m o u n t s of individual componenlfl in the mixtures were estimated tV<nn normalised
paak area ealeulntinnfi. The d a l u Kttc^eht t h a t one- hexa
fl-n
10-0
It-2
* H was ritt|imttl!fll1iiilnliniltSO/t K <)rtii»U>ilnlw > niililji>r.ti-innli>cnclir|lir.
t SI-R rooliioitB3nnO 4,Tithlc t .
t ComiKineiilsrroincWi.flimM ill\«>n/o-j)-(lltfxlii.
ond ono lieptn-i^oniei 1 ure less toxic than the ollmr iKOincrs.
Work IB now under wny tu jJi-epin'O indivjdun) eomponndH
of high purity in sufficient tjiiBuHticB Tor ndditionnl
e.lioimcul nnd bioloRicul Icsling.
Refcrenco toxic fa I components with low /?• vnlucn
(less timn ffa 8-U) cmilcl !«• Huo (o oJiloro-orgnnir pesliRides nnd/or lower cliliiro(lilwn/o-;).tiioxina.
Additional work is required to identify iho conijiovmds remwins)blo for the penkH with low Ite vnlnpB. Tho pof\U» with
high }{„ vnlnc-s .(tlioso cqunl to nr ^renter than (Ml) correspond to timso of the liexneliloro nnd heptixchloro
positionnl ismners ifjolnleil from ihc pymlj-Mite of 2,3,4,0letmehlnropbeiiol. Tlie overall ro«ulln of thin work
Rupf-L-Bt tbut chtnmphcnoU conUt lie precursor;* of hydropci'iRwrdinm ft\rlor. Comnicicinl chloroplirnols nnj widely
nnf'd for such divo.rsc npplienfions as contiiet lierbiciclcs.
defoliant s nnd lermitc control njieiUfi, n« Mi'U us for
control of miorobinl nttnek in (tin jmmufacfurt' of n
number of products. There me ninny opportunities for
fata nnd fatty acids, the only products in which hydropericardium lac tor bns bt-on found , to bccumo contaminated. "When crude ftxts and tflt[o\\P nrc subjected
f.o heating operation!* (hyilrojyms, (listiltntion) in tho
production <jC ctunmcrcial f n t t y nnHs, residues of coinmercial cblorophennla preKCiil in Ilip fnt ini(dit be converted in part to ebip.U oudemn faclorH. Add it tonal work
is r*'(|tiired tt) deloi'mhu.1 whether liydtt'periciiitliuin
ftictors arc formed from residual chlorinnted i>l»piiola
during production of connnemul f a t t y acids. This laboratory i^ developing methods ftir detecting cblomphenola in
fats and fnlty neifln.
\Vo thank Dr Jo-Vun Chen, Dr Donald P. Flick, Mr
Robert Barrcm, Mr Jamca K)ilum, Dr 1C, K. llcynnldo,
ML- William V. Scott niul Mra Mnry K. Mutchlcr for tccbnicnl npsistanpc,
G. H. HKiGINHOTIMM
1
ANITA HUASG
DAVID FIRKSTONK
jArqUKi.iKK VKUIIBTT
JOJIN KKSS
A. U. CATilVnKM.
]Jnrt*ftti of Science,
Food and Drug Arbninisti-alion,
Deprtrlnicnt of Dealt)*. Kdueutiun
and \\'elf«re..
Wellington. I'M.'.
,! AiiRiitt b; r
ri 27. 1
1
C/JI-IH, Km], ,V<ic»,45, So. 5, 1H (1007).
•Tomlta, M., 1'i-itn, S., nn.l N.uh:i.1n, >(., Ynl,i,,nla Zattfti. 79, IfO (1050):
''
'
iiin[<-l.iiiGl.liri,J.,.;. X'i"^ (I
• V i T n - t l . M .K.Miiil
^7, KHIII (iflrtt).
1
Xi-til. )'., ./. A***, OJlic. Anal. i'l<rn,!*l<, BO. \ : « (10i17)
' Offi''i"l ,Vi(/J(v/» nf Aiinliiti*. l l l l l i r.l., »i-i'«. 'Jfl.nftT-Sfl.oni. (
Oli'i.-lnl Acrlrnli nr.it rlictnlfitp, WiiRlilimUii), ]).(•.. l<wi;,).
•WooMon. J. C., \ilnmn, NT. U., m»^ Afc-faii'li-f. .t. f'.. ,/. «M
-
nird in. Oreal dnlHJn by I'Wk'i, Kn!»hl A Co , Lid . St. Atb*n»
�328
INTERNAL PRELIMINARY REPORT ANALYSIS OF COMMERCIAL CHLOROPHENOLS
FOR TRACE AMOUNTS OF THEIR CONDENSATION AND
PLYMERIZATION PRODUCTS
By G. R. Higginbotham and John Ress
Laelair (1) has reported an ultraviolet absorption study of the components
of technical pentachlorophenol, separated by vacuum sublimation, and disclosed
only pentachlorophenol (POP), 2.3.4.6-tetrachlorophenol, and an unidentified,
dark brown, high melting, "chlorophenol" containing 58.3% chlorine. The unidentified material was presumed to be a polymerization product produced
during process of manufacture. Tomita et al. (2) have demonstrated that
chlorphenols and their salts, when heated, undergo condensation reactions and
form chlorinated derivatives of dibenzo-p-dioxin (chick edema factors, CEF).
Recently, we examined the unsaponifiable fraction of a number of commercial chlorophenols and obtained results which suggest that the impurities of
chlorophenols consists of chloro derivatives of dibenzo-p-dioxin in addition to
other components which have not been identified.
Thirteen chlorophenol samples were analyzed for chick edema factor. The
samples were carried thru the 2.5 gram saponification procedure and examined
by electron capture GLC after AlaO3 column chromatography and HaSOi
extraction [JAOAC, SO, 884 (1067)1. GLO peaks indicative of chick edema
factor were observed in most of the chromatograrns from the thirteen samples.
Six of the thirteen samples were selected for further study. The unsaponiflables from each sample were extracted and chromatographed on an AUOi
column, according to A.O.A.C. Method 26.092-26.094. The unsaponifiable fractions were submitted to the egg embryo biassay. Two components (Ra 6.3 and
Ra 11.6) were isolated by preparative GLC from the unsaponiflable fraction of
Eastman's technical grade 2,3,4,6-tetrachlorophenol. The components were
examined by mass spectrometry and submitted to the egg embryo bioassay.
Results are tabulated below.
Table I shows the amount of uusaponifiables isolated from 100 g samples of
six. representative commercial chlorophenols. In every case the amount of CEF
in the total sample is estimated to be less than 0.3%. If a lipid sample is contaminated with pre-formed chick edema factor,* high levels of poly chlorophenols would also be expected to be present in the sample.
Results from the chick embryo assay are recorded in Table II. Table III
summarizes the results obtained on two components isolated from
2,3,4,6-tetrachlorophenol. The molecular weight and the number of chlorines for
eacli component are not consistent with a chloro derivative of dibenzo-p-dioxin.
The Ra values of these components are not consistent with known chick
edema factors. The compounds may be photodecomposition products of technical grade 2,3,4,6-tetrachlorophenol,
Further work will be required to characterize the unsaponiflables from commercial chlorophenols. The overall results of this preliminary study emphasize
the need for a rapid method for polychlorophenols in fats and fatty acids. The
unsaponiflable and CEE1 (hexa-, hepta- and octachlorodibenzo-p-dioxin) content
of a number of reagent and technical grade commercial chlorophenols"are
shown in Table 4. OEF content was determined by GLO using a synthetic CEF
mixture as a reference material. CEIT was found in all the chlorophenols
examined, varying from a trace (ca 0.001 /ig/g) to 205 /
* Pre-formed chicle edema factor IB defined here as a group of chloro derivatives of
dibenzo-p-dioxin initially present in a sample before it is subject to any type of heat
treatment.
329
REFERENCES
1
Laclair, J. B., Anal. Chern. 2S, 1760 (1951).
'Chern. Abstr. 5S, 1315d (1959).
TABLE I.-EXAMINATION OF CHLOROPHENOLS FOR PRE-FORMED CHICK EDEMA FACTORS
Wt. Unsap.,
mg.
Sample (100 i.)
» 126. 5
223.7
323.8
2, 3, 4, 6-Totrachlorophenol (Eastman)
2, 4, 5-Trichlorophenol (Pract.) ..
Pentachlorophenol (tech.) .
2, 4, 6-Trichlorophenol (Reagent)
2, 3, 4, 6-Tetrachlorophenol (Baker)
2, 4-Dichlorophenol (Baker)
. .
2.0
»135.8
12.2
.. .
Percent
Wt. AWi
Fr.3
CEF'
109.2
11.9
228.5
0,9
120.2
0.3 . .
0.11
0.012
0.23
0.001
0.12
' Estimate based on weight of AljOj ft. 3 and assuming that the fraction consists only of CEF.
' A second extraction of the unsaponifiables (after acidification) of the soaps afforded 5.0 mg of material.
1
A second extraction of unsaponifiables gave 9.8 mg of material.
TABLE 2.-CHICK EMBRYO ASSAY
pg/egg
Sample (unsaponifiables)
10.0
,
,
2,4,6-Trichlorophenol (R)
2, 4-Dichlorophenol (T)
100
0,55
10.0
0.60
10.0
0.6
4.8
50.0
15.0
2,4,5-Trlchlorophenol (T)
2,3,4,6-Tetrachlorophenol (Baker)
Pentachlorophenol (T)
Percent
mortality
30
100
15
100
55
70
40
13
TABLE 3,-EXAMINATION OF TWO COMPONENTS FROM THE UNSAPONIFIABLE FRACTION OF TECHNICAL 2,3,4,6TETRACHLOROPHENOL
Chicken embryo assay
pi/egg
R« of GLC component
Mol. wt.
408
442
6,23
11.8
Percent
mortality
2.0
1.0
0
0
Cl atoms
7
8
TABLE 4.-EXAMINATION OF COMMERCIAL CHLOROPHENOLS FOR CHICK EDEMA FACTORS (HEXA-, HEPTA- AND
OCTACHLORODIBENZO-P-DIOXINS)
mg. Unsap.
No. Compound"
1
2
3
4
5
6
7
8
9
10
11
12
13
14
2,4-DichloroplienoKR)....4
2,4-Dichlorophenol(T)
,.
2,5-Dichlorophenol (R)
2,4,5-Trich!orophenol(T)
2,4,6-Trichlorophenol (T)
2,4,6-Trichorophenol(R)
2,3,4,6-Tetracrilorophenol (T)
2,4,5-Trichlorophenol (R)
2,3,4,6-Tetrachlorophenol (T)
Pentachlorophenol (T)
Pentachlorophenol (R)
Dowclde B (sodium 2,4,5-trichlorophenate (T)
OowcideG (sodium pentachlorophenate (T)
Reference toxic fat
• R«=reag«nt grade,
^technical grade.
° Hexa-, hepta- and ocUchlorodibonzo-p-dloxins.
,
,
iig/g. of CEFb
2.0
2.4
0.8
0.018
0.219
0.008
trace
trace
0.013
205
0.021
32.5
12.3
1.9
22.5
3.0
,
35.0
67.1
1.4
1.8
25.8
223
96.5
121
0.167
trace
47.0
3.0
�331
330
Reprinted from Acta Cryaallngraflilca, Vol. 1125, I'art I, January 1969
I'lUNim IN OKNMAHK
Ada Cryn. (1969). 1125, 150
The Identification and Crystal Structure of a Hydroperlcnrclium-Prodncing
Factor: l,2,3,7,8,9-Hcxaclilorodibenzo-/)-dioxin
BY J.S.CANTRELL,* N.C.Wrinn AND A.J.MAtiist
The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, Ohio 45239, U.S.A.
(Received 4 December 1967)
A crystalline material, isolated from a contaminated animal feed fat, and capable of producing hydropericnrdium in chicks, was shown by solution of its crystal structure to be 1,2,3,7,8,9-hcxachlorodibeiwo-B-dioxin (CizOjH'CU). The triclinic unit cell has the dimensions o»7-952±0-005, />.= 9-379
+ 0-01, <: = 9-433+ 0-0.1 A, « = 92-35°±0-20°, 0=92-39°±0-20", >>= 10!l-92°±0-30°, The calculated
density is 1-958 s.cm-» for Z<=1, compared with 2-01 B.cm-> measured for (lie bulk material. A statistical
treatment of the 1158 measured reflections indicated a center of symmetry; the space group was therefore assumed to be /*!. The structure was solved by the symbolic addition method of Karle & Kane.
The nearly planar molecules arc almost parallel to the (Oil) crystallogrnphic planes. No unusual bond
lengths or angles were found. The structure was rclincd to R= 10-5%.
cc-3-17 material, docs indeed produce the hydropcricardium condition in chickens.*
Experimental
Two types of crystals were isolated from a warm
bcnzcne-hcxanc solution of the K-3-17 material. The
bulk of the crystalline material appeared to differ in
phase from the material used for this study. No crystals
of the bulk phase were found to be satisfactory for
single-crystal studies, and only two crystals of the
studied phase were isolated. Measured r/-spacings of
X-ray powder patterns taken of the bulk phase material did not match rf-spacings calculated from the unit
cell of l,2,3,7,8,9-hexachlorodibcnzo-/)-dioxin. However, when the bulk phase was heated to just below
the melting point (230"C) n phase change occurred.
Measured rf-spacings from X-ray powder patterns of
the transformed bulk phase match the calculated r/spacings of l,2,3,7,8,9-hcxachlorodibcnzo-/;-dioxin reasonably well. Therefore, it was assumed that the material used for this crystal structure determination was
a high temperature phase of the bulk crystalline material known as ot-3-17 H1TF.
The single crystals used were diamond shaped and
had the approximate dimensions 0-18 xO-10x0-08 mm
(a x b x c).
The uni [-cell parameters were determi ned from singlecrystal data using a General Electric single-crystal
orienter and Mi-filtered Cu Ka radiation (A= 1-5418 A).
The parameters of the triclinic cell chosen according
to Dirichlct (Balashov & Ursell, 1957) are as follows:
ff=7-952 ±0-005 A «= 92-35±0-20° es = l-958g.cm-3
6 = 9-379 i 0-01
/!= 92-39 + 0-20 e» = 2-0l g.cnv'
c = 9-433+ 0-01 • v'= 109-92 ±0-30 Z = 2
1'=662-8 A»
where t>0 was measured for the bulk phase by flotation.
Two-dimensionally integrated equi-iuclinatiori Wcissenberg data were collected for the ir-axis /ones, 0/c/4W, and for the /j-axis zones /tO/-/i5/ using the multiplefilm technique (one pack each of four films, Eastman.
* The composition for the structure reported here, namely
CuOzHzClg, agrees well with unpublished niicrochemical
analyses performed by Professor Wolfgaiiu J. Kirslen, University of Uppsala, Uppsala, Sweden, at a very curly stage of
tills structure work.
Quantity
(1964). One of the active fractions of material isolated
was that called a-3-17, where this nomenclature refer*
The isolation, chemical analyses, and spcctroscopic to the vapor phase chromalographic behavior as dedata on the hydropcricnrditim toxic factor (1IPTF) scribed by Wootton cl til. (1962). Wootton and his colmaterial have been described by Wootton, Artman & leagues proposed that lll'TF was a chlorinated teaAlexander (19(0), and by Wootlon & Courehcnc hydrophciwnthrcne with the empirical formnli
C M H|oCI<j. l-'ollowin;. the molecular identification
• IVcscnt luklrcss-. M i a m i Univcrsity.Dcparlincntdf Cliem- herein reported, Woolton (1966) showed that a synl<lry, Oxford, Ohio. U.S.A.
thetic hcxachlorinated ihbcn70-/wlio\in, whose ph)>1 Itcprinl rcquc.sK sluniltl beflcUla-sscdto this author lit the
ical properties arc remarkably similar to the isolated
Procter & Gimihle address.
Introduction
45-362 O - 70 - 22
Noil-centrosymmetric
0-886
0-736
1-000
Kodak No-Sctcciv). Intensity data were recorded for
both crystals, reduced separately, then compared,
edited, and averaged. Absorption corrections were
made separately for each crystal using Busing & Levy's
general absoiption correction program as modified by
Jeffrey (1964).
Owing to the very tiny ciyslals, and in part to the
. integration, very long exposures of approximately 1150
hours were required to obtain satisfactory multiplefilm data. The entire Wcis;.enbcrg camera was placed
inside a plastic bag and a helium atmosphere was provided to reduce background due to air scattering. Of
the 3030 possible reflections, 115S (38%) were recorded; 397 of these reflections had intensities less
than a minimum threshold value and were classified
as 'Icss-thans'. The intensities of most of the reflections
were measured by a Joyce .Locbl microdcnsitometcr
scanning at right angles to Ihc longer integration direction. The weakest reflections were estimated visually.
A standard intensity strip wns prepared and used for
the visually estimated intensities. To ensure that both
types of intensity data were on the same scale, a sufficient number of medium intensities were measured
both visually and by the dcnsitometer. Radiation damage effects were found to be negligible by retaking data
for earlier crystal settings.
Statistical treatment of the intensity data by K.tmachandran & Srinivasan's (1959) modification of the
method ofHowclls, Phillip & Rogers (1950) indicated
a center of symmetry. The s.pace group was assumed,
therefore, to be I' I (Cj) and this assumption was confirmed during the direct method calculations.
Solution anil refinement of the structure
Initially we knew the weight of the molecule and the
number of chlorine atoms per molecule, and we knew
that the molecule possessed some aromatic character.
Attempts to solve the structure from the three-dimensional Patterson map were not successful. The symbolic
addition method of Karlc & Karle (1963, 1966) was
then employed.
The phases were determined for the 251 most intense
reflections in terms of four algebraic quantities, n, b, <•,#.
A summary of the calculation of the unitary structure
factors or £-valucs used for tlus determination is compared with theoretical Values and is as follows:
Ccn (rosy in metric
0-798
0-968
1-000
0-3%
5-0%
32-0%
Kartert n/. (1964)
. J-lndolj-la-cctic acid
0-772
0-769
0-970
0-934
1-000
1-031
0-J%
02%
4-SV.
3-3%
36-1%
30-8%
1-158 rclleclions
1-28-9 reflections
761 non-zero
8W non-7cro
397 unobserved
424 zero
Clcss-than')
C^HjOiCltf
�333
332
e overa temperature actor was ' 4
The os'cnill tempcnilure rnctor wns 2-24.
A summary of the sixteen cases that resulted from
thc sign permutation of Ihc four algebraic assignments
is as follows: •
No. rcD.
ycneialcd
No. incortcct signs
% \viong
sijin
Objections
lo choice
Ctisc 1
a
H.4.
1}
c
—
c
•t-
—
—
119
132
117
46-6
v/
2
-14+
3
•1•1-
4
+
5
.).
—
—
•i142
109
smnc changes made in carbon and chlorine assignsome c a n g e s mae n c a r o n an c o r n e assgn
l.r._
I i 1 -i tl n r. , ,
j
mcnls resulting :. ideally, a p.1. n a r 1,2,.1,7,8,9-tara,j_
in, - I . ii.. - l a
chloroanthraccne with mm symmetry, This change iill
the structure dropped the R value to 35%. The plan1C
125
4—
-f
153
98
132
52-6
49-8
52'6
J
'<
10
II
I?.
13
14
15
121
130
128
115
136
•I125
12S
121
130
122
129
6]
16
128
123
120
53-0
•1
8
•/
—
183
68
132
—
•1114
137
133
129
118
133
13D
251
0
129
121
130
121
H-
122
129
111
/ Klcctron density peaks were found nl ccnlcrs or symmciry wlicn llic special scclionfi .v«0 nnd ,v« 1 \vcrc compuled.
1 ll is very unlikely llial nil 251 most inlense rcHcclions would be positive,
* This correct cusc was selected after examining fhrcc-dimensioiiiil electron <lc^sily maps for cas,cs 9 lo 1^.'
'
Case 13 was choscu over the other seven iiCccpliiblc
choices because of the appearance of a chemicnlly reasonable strucliirc in the t'leciron density plot in ;m
(0^4) plane, H had already bct-i\ dctennincd t h a t the
molecule had to be oriented approximately in alternate
(O'f'l) planes, based on (rt) the early analysis of Ihc Patterson map, (h) the very high intensity of 032, (c) electron densities calculated from models based on the
Patterson vectors, and (//) p.'icking considerations, A
nearly planar hcxachlorinated plienanlhrcnc slructurc
wns initially Titled lo the y^-nia]1) peaks-located near an
(034) plane.. An R value of 50% was obtained for the
initial trial coordinates and a three-dimensional electron density map suggested t h a t the phenanthrcnc
skeleton should be changed lo on anthracene one with
of the molecule was originally in alternate (034) planes
but required some lilting and when these changes were
made the tt vjihic dropped to 24%. One cycle of least
squares reduced the residua) lo 19%. Al this point the
two bridging atoms were ix-co^ni/cd as oxygens, since
their relative electron densities were 30% higher than
those for Ihc carbon atoms and the individual temperature factors for these mom? had gone negative.
Substituting oxyp.cn atoms for these carbon atoms in
the proposed model resulted hi an R value of 16%.
There was a careful editing of the data, especially of
those reflections classified as 'less-Hums', at the /< =
16% stage of refinement. This editing consisted of deleting a few doubtful reflections recorded near the edge
of a film, correcting transcription errors, and rcclas-
4
The fractional coordiniites have hr-cn multiplied by 10 and the teinpcraliirc factors by 10-; the via mlard deviations arc in parcntlicses. The antsolroriic tenipcralurc' factors of Ihc chlorine atoms me m the form
Cl(l)
0(2)
CIO)
CK4)
CHS)
CK6)
C( 1 )
C(2)
C(3)
CX-1)
0(6)
C(7)
C'(8)
C'(9)
C'( 1 1 )
C(IZ)
C(I3)
C'(|4)
0(5)
0(10)
X
-0099 (6)
-0145 (S)
3314(7)
9-19.1 (6)
6188(6)
2919(6)
1762(24)
H I S (21)
3286(25)
475? (M)
77 I J (23)
7706(20)
6219 (27)
4754 (21)
3292 (23)
4776 (24)
6726(21)
4752 (30)
6312(15)
3266(15)
j,
3158(6) '
0579 (61
-0257 (6)
7375 (6)
8434 (6)
6936 (6)
2601 (2?)
1502(20)
11 IS (32)
1920(23)
5516(21)
6572(19)
7051 (2-1)
6238 (19)
3471 ( 2 1 )
29'MC!?i
4 8 1 1 (191
5271 (19)
3742(1.1)
4591 (14)
7.
5185(6)
2931 (5)
2621 (5)
10547(5)
10752(5)
8629 (51
4879(20)
3979(18)
3853 (20)
4673(21)
8394 (19)
9388 <!(>)
9503 (22)
8539(17)
57V8(I9)
5629 (19)
7461 ( I K )
7591 (17)
6496(13)
671.1(13)
Kor «,,
58V (20)
362 (23)
487 (26)
372(23)
395 (24)
282(21)
354(41)
262 (35)
3S6M3I
370 (-12)
321 (39)
219 (3-D
427 (45)
249 (35)
325 (39)
.160 (421
240 (3-D
213 (32)
355 (27)
334 (27)
«J!
376 (291
378 (30)
411 (30)
457 (3d)
375 (29)
421 (29)
B»"
487 (26)
417(7.71
3Vfi (26)
347 (24)
309 (23)
473 (27)
fln
flu
Ba
'149 (21)
025 (22)
163 (24)
17-1 (23)
016 (22)
148(21)
-035(17)
-162(19)
-056 (21)
-018(18)
034 (18)
028(18)
-OS8 (22)
-043 (22)
-084 (24)
-105(22)
-024 (21)
-102(23)
sifylng sonic of the 'Icss-Uians' to observed reflections
on the basis of visutil estimations or the weakest reflect ions. ,
The It value droppccPto 13% during the next leastsquares refinement. Fixing the chlorine atoms and refining only the carbon skeleton lesnlted in an R value
of 11-8% at which time (he anisolropic temperature
refinement ou the chlorine atoms was undertaken and
gave (lie final R of 10-5%. Of the 397 'Icss-tliun' reflections only 42 calculated were larger than their threshold
values and none by more than 3d%. A weighting function similar to that given by Hughes (1941) was chosen
so as to have l i t t l e dependence on /v, it was taken as
)'«• = *•/(/-„+ 53-4) for !•,> I-,,, nnd \'w=-KI(l'm + tt-4)
for F, < Tin, where Fm- 21 '4, and the quantity minimised
was i'liVo-Fr)3.'Less-Hum' reflections having l"c> lr,
(thrcshnid) contributed like an ordinary reflection, but
those having /•"„</•',, did not influence the refinement
at a l l . A full-matrix least-squares procedure was used
throughout the refinement, and in the last cycle all
parameter,shifts were less than \<j. In a difference map
calculated at the /^ = I 1-8% stage, the residual electron
density ranged from -0-8 to + 0-G c.A->, and the
maxima and minima did not show any chemically or
structurally significant features.
The final position and thermal parameters are given
in Table 1. The observed and calculated structure factors are listed in Table 2. In this work, the scattering
factors.arc taken from International Tables for X-ray
Crystallography (196?.); those for carbon arc by FreeTable 3. Distances of dlants from Icftst-squares planes
Distances for atoms not defining Ihc plane arc maikccj with an
asterisk; s.d. is the standard deviation of the atoms defining
the plane. Under the (044) heading are listed lite deviations
from the (034) plane containing the molecule. The plnnc* are
defined in direct space by equations /'.vH-Qy f Rz^S,
am
Cl(2)
Cl(3)
Cl(4)
C1(S)
Cl(S)
CO)
C(2)
C(3)
C(4)
C(6)
C(7)
C(8)
CX9)
C(ll)
C(I2)
C(13)
CO 4)
0(5)
0(10)
Origin
s.d.
f
Q
li.
S
All
-0-10
-0-11
O'OO
-0-16
-O'OS
0^13
-0'02
-0-06
-0'02
0-OS
O'OO
-O'OS
-O'OI
-0-01
006
0-05
005
0-06
0-OS
O'OO
1'57«
0-07
-1-1589
-5-1655
6-8500
1-5675
C+0
-0-10*
-0-12*
-0-03'
-0-1S*
'-0-06*
0-13*
-0-03
-0-08
-O'OS
0'03
-0-03
-0-07
-0-03
-0-02
005
0-03
0-02
0-05
0-05
0-08
1-55*
0-05
-I-IOS6
-6-0031
6-M04
1-5497
(05(4)
0-33«
0-10'
-0-25*
-0-45*
0-12*
0-54'
0-15'
0-02'
-0-16*
-0-17*
-0-25*
-0-21*
0-03*
0-13*
0-13*
-0-09'
-0-10'
0-12*
-0-17'
0-25* •
1-67*
0-00
O'OOOO
-6-6618
6-6618
1-6655
man (1959); those for oxygen by tlcrghuis, I laanappcl,
I'otlers, Uiopslra, MacGillavry & Vtcncndaal (IV55);
and those for chlorine by Uawson (1960).
Discussion
The molecule is nearly planar and the molecular plane
is tilted 8° to Ihc (Oil) plane. Deviations of atoms from
the (034) plane essentially containing the molecule and
Fig. I. Molecular packing in the (03-1) plnnc conlp.inmp the
molecule. Large shaded ciiclcs aie Cl, solid ciicles me C, atul
open circles Rrc O.
Cl(3')
Fig. 2. Projection onto Y7. (ij.iii
�334
from the least-squares planes of t h e entire molecule
nml of t h e carbon-oxygen skeleton are given in Table 3.
The molecule' appears to he s l i g h t l y bowed in the
middle :ind s l i g h t l y twisted about a line from Cl(3) to
Cl(5). The packing arran.ccmcnt of chlorines 4, 5, and 6
appears to be more crowded t h a n t h a i for chlorines
I , 2, and 3. This packing dillcrcnee could account for
the slight twist of the molecule.
Fig. I pictures the molecular packing in the (O^T4)
plane c o n t a i n i n g the molecule, and Fig.2 gives a projected view of two adjacent molecules related by the
center at (1, !,•?)• Intermolecular distances in this (034)
plane of less t h a n 4-0 A are shown in Fig.3. Between
ccnlrosymmetriciilly related molecules there are a number of CI(»}-Cf(w') and equivalent CI(m)-CI(V) distances of 4'0 A of less. I-'rom t h e parent molecule to
the one related by the center at QvM) the distances
are CI(2)-CI(4')-3'S5 A, CI(?)-CI(5')"3-S6 A, and
CI(3)-CI{(/) = 3 ; K 3 A ; by the center at (0,0,.J)-CI(1)CI(3') = 3-84 A; by the center at (0,-J,-J)-C!(l)-CI(r) =
3'39 A nnd CI(2)-CI(6') = 3'98 A; and by the center at
\\
/
\
V"
»/\
A'
,W o V» A,\
,
V -''Z..U,_,.J\..--CCI(T|
vii"~5~w~~"''w>™
^-v :
.cit4
335
(1,J,1)-CI(4)-CI(6') = 4'00 A. The least-squares planes
of the two adjacent molecules related by the center at
(J,-J,-J) arc 3 ' J 3 A a p a r t ; between these two molecules
the shortest, interatomic disumcc is 3-30 A from a C(I2)
to an O(IO).
Fig.3 indicates the bond distances and angles. The
mean standard deviations arc as follows: (TC-C~
O025A, o r .oi'<H>19 A, n-c o = 0'022A; for angles
n~2'0°. The bond distances are not significantly different from those found by Davydova & Slruchkov
(1962) and Gafncr & Hertistcin (1962) foi 1,4,5,8letrachloronaphthalenc where molecular over-crowding results front the presence of m a n y chlorine atoms
s u b s t i t u t e d on adjacent aromatic positions. This compound belongs in group (I) according to the classification d u e lo l l a r n i k , Mcrbslcin, Schmidt & Hirshfcld
(1954) for compounds that are affected by molecular
over-crowding.
An electron density map shotted in the (034) plane
containing the molecule is shown in Fig.4.
The authors wish to acknowledge their appreciation
to Dr J, M.Stewart of the University of Maryland, who
furnished the X-ray 63 computer program and provided much information on its use. In addition, we
wish to thank Dr Lyle Jensen for a number of helpful
discussions on the use of the X-ray (53 computing system and on approaches to the solution of the structure
in general.
We wish lo express our appreciation to Drs Jerome
and Isabella Karlewho provnkd assistance in applying
the direct method for determining the phases of a
number of the most intense reflections.
Thanks are.due to Mr Robert Gloss who obtained
part of (he data and provided the computer programs
used in generating the relations between reflections
necessary for applying the direct method.
References
BAUSIIOV, V. & UnsiiLL, M. D. (1957). Ada Crysl. 10, 582.
BEftOlllilS, J., HAANAI'Pin., U, M,, 1'OTTUtS, M., LOOPSTRA,
MR..I. C") Inieraloiviie ctiM;im:e<;. IVimcil atoms .ire on ni-iyhboring molecules in ihc same pl,ino, ffo-c**0-025 A;
ffr-t.i ---O'OI'J A ; ou-ii'--0-022 A. View corresponds to l;'ig. j .
B. O., MA'CCILLAVUY, C. H. & VECNKNHAAL, A. L. (1955).
Ada Cryst. 8, 478.
DAVVDOVA, M, A. & STRUCIIKOV, Yu, T, (1962). Zli,
Slrukl. Khlmil,\ 184,
DAWSON, 11. (I960). Ada Crysl. I.I, 403.
Fltl.niAN, A. J. (1959). Aria Crytt. 12, 261.
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10SI.
G A I X I R , G. A HhRii.siF.iN, F. II. <1963). Nnlm-c, Lontl. 200,
130.
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,
..
Hucillt.s ]•;, W. (19(1). J. Amcr. Client, Sue., 61, 1737.'
Inlcrnalhtiml Ttihlwfar X-ray Cry>>l<i!ltw(ii<liy, (1962). Vol.
111. Hirminpjiam: Kynoch Press.
JEHIUV, G. A. (1964). I'rivalc ccvnmunication.
1
'1
fig.4. Electron density in the
projection*! 01
3
;.A"S starting at e.A-'. The m
4) plane contnining Ihc molecule. Contours arc at I e.A"sstarting at 22e.A~ . The xx marks arc
om the electron density maxima, which in most cases are a short distance, from (044).
KAHU!,I.L.,BRiTTS,K.&auM,P.(1964)./<<•/« Co*', 17,496.
KARLU, I.,L.'& KARI.E, 3. (1963). Ada Crysl. 16, 969.
KAM.E, J. & KARLE, I. L. (1966). Ada Cryst. 21, 849.
RAMACHANDHAN, G. N. & SKINJVASAN, R. (1959). Ada
Crysl. 12, 410.
WOOTTON, J. C., ARTMAN, N. & ALEXANDER, J. C. (1962).
/. Assoc. Offic. /tgr. ChemisH, 45, 739.
WOOTTON, 1. C, & COURCHCNE, W. L. (1964). 'J. Agrlc.
Food Cliem. 12, 94.
WOOTTON, J. C. (1966). Unpublished results.
�337
336
S T S INCOUPORATED,
SCIENTIFIC TRANSLATION SEBVICE
Ann Arbor, Midi.
CLINICAL PICTUBE AND ETIOLOGY OP CHLOHACNK
By K. H. Scultz, University Dermatology Clinic, Hamburg-Eppendorf
Chloracne is the name for forms of occupational acne which develop as •
result of intoxication with certain chlorinated aromatic compounds. The name
dates back to Herxheiraer who described the first case in 1899 and fltlll
assumed that in analogy to bromine and iodine, the acneiform eruptions are
the result of free chlorine as the etiological toxin. This view proved to !'<•
incorrect. The proposed designation "perna disease" of Wauer, Teleky ami
others based on the finding that this clinical picture occurred more frequently
under the influence of perchlorinated naphthalenes also does not go to tlte
heart of the matter, since other chlorinated aromatics in addition to chlorinated naphthalenes are also etiologically important.
The clinical picture of symptoms primarily affects the skin. Beyond tills,
internal organs may be affected and nervous system and emotional disorders
may appear.
The skin symptoms are in the regions of the follicles. Comedones, resulting
from a follicular hyperkeratosis, predominate and frequently are so numeroin
that hardly a single follicle remains untouched and the affected region of the
skin obtains a dirty-gray appearance. In addition, at the peak of the disease,
fairly large sebaceous cysts, inflammatory nodules, pustules and furuncles
appear and in some of the patients, large spots or patches of pigmentation
appear in regions exposed to light. Preferential sites are the face as well as
the exposed areas of the throat and neck. Frequently, the external ear, especially, the ear lobes, are involved where small cysts can be easily palpated. In
more pronounced forms, changes can also be found on the back, chest and
extremities and in males, on the genitalia. Hands and feet usually are not
involved. It is not rare that the symptoms of acne are preceded by a dermatitis with erythema and edema. In this phase of the condition, photosensitivity
frequently exists which evidently contributes to the development of dermatitis
and the mentioned pigmentations (S. Braun, Grimmer).
Generally, a differential diagnosis is not particularly difficult. The primary
problem is to define the condition compared to other forms of occupational
acne and acne vulgaris, which is generally possible with consideration of the
clinical aspect, localization and especially the patient's history. Acneiform derma toses caused by tars, pitch and mineral oils are found primarily on the
extremities and trunk, while the face is more rarely involved. The predominance of inflammatory changes, such as folliculitis and furuncles in oil and tar
acne arid of comedones in chloracne are other characteristic features. Drugcaused acneiform exanthemas due to iodine, bromine or cortisone also have a
picture differing from chloracne.
The course is eminently chronic. In spite of intensive local and general therapy, recidivism may occur even years after the elimination of the causal
toxins. Healing frequently takes place with extensive pitted, permanently disfiguring cicatrization (Schmidt and Boslet).
The skin is not the only indicative region of intoxication with chloracnecausing substances. Damage of internal organs is not rare, with the liver being
in the foreground. Several authors have reported on grave damage of hepatic
parenchyma accompanied by icterus and functional disorders, including a
number of fatal cases of acute atrophy of the liver (see reviews of W. Braun
arid A. Risse-Sundermann). The pronounced liver-toxocity of chloracne-ca using
substances was also confirmed in animal experiments (Bennett, Drinker and
Warren ; Hofmann, Oettel ; Schulz).
In addition to liver damage, changes in the kidneys, pancreas, gastrointestinal tract and myocardium can also be observed, although much more rarely.
Nervous system and psychological disorders were found primarily among
workers occupied in the production and processing of chlorinated phenols
(Trubant et al. ). General fatigue, weakness of the legs, headache, attacks of
vertigo, paresthesias, muscle pain, tendency to orthostatic collapse, local
and disturbed nonHihllity, anomalies in reflexes as well as an autonomic
Mvnrll'nirlf wlHl Imvnror'l (Irlvn
flonr^«Hlnri
rnrlnpfvl Tmwor nf rrvnll nntl
trillion, disturbed sleep, Irritability, loss of appetite, reduced libido and impotPiice have been reported as the most frequent neurological and psychopathological symptoms of intoxication which become manifest often only several
months after it occurs (Bauer, Schulz and Spiegelberg). With regard to the
question of distinguishing the latter from psychoneurotic obsessions (wish for
compensation), reference is made to the discussions of Spiegelberg.
Ktlology.—When we review the literature, we find that periods of greater
Incidence of chloracne have existed in the last 60 years, which can be correlated with industrial development, W. Braun has described these relationships
In Themonograph. were observed near the turn of the century when chlorine
his first cases
nnd hydrochloric acid began to be produced by the electrolytic route. At that
time, the, condition occurs primarily among workers having the assignment to
clean the so-called hydrochloric acid towers, but only when tar was used as
the protective coating of the walls. Although the causal toxin could not be
determined at the time, it can be assumed on the basis of our present knowledge that the reaction products of chlorine and aromatic components of the tar
must be considered as etiological factors of this condition.
The next period of increased chloracne frqucy coincides with the introduction of so-called halogenated waxes. These are mixtures of highly chlorinated
naphthalenes and diphenyls with a waxy consistency and a number of valuable
properties. They are water-repellent, nonflammable, resistant to acids, are a
good dielectric and are not pest-promoting. The halogenated waxes developed
(luring the first world war at that time were used primarily for the manufacture of gas masks. Numerous cases of chloracne occurred in the manufacturing
plants. middle twenties, these halogenated waxes were used in the mining
In the
Industry as a water-repellent and nonflammable insulation for detonators. The
high incidence of diseases observed in detonator manufacturing plants has
been' described by Teleky.
The next massive occurrence is related with the rise of the electrical and
radio industry. Chlorinated naphthalenes and diphenyls were in more widespread use for the insulation of wires and condensers at the start of the thirties. Several hundred cases including one fatality with liver atrophy became
known especially in the United States.
With the entry of the United States in the second world war, the field of
application of these materials expanded also into ship building; this had the
following reason: It was found that the halogenated waxes were in the position to insulate ships from the dangerous weapon of German magnetic mines.
Consequently, large quantities of these materials were used in American shipyards for the impregnation of ship hulls. Mass incidences of chloracne with
several fatalities were the results.
In spite of all negative experiences, the use of chlorinated naphthalene
waxes did not stop after the last war. Chloracne cases of greater or lesser frequency were repeatedly observed in the electrotechnical and cable industry
(Braun, Grimmer, Risse-Sundermann). From the pathogenic aspect, it is of
interest to note an observation of Herzberg of 7 patients who developed intestinal symptoms and acneiform dermatoses following the use of industrial
chlorinated greases for frying.
The question of the relationships between chemical structure and acneproducing effects of chlorinated naphthalenes is the subject of several experimental studies. Teleky as well as Drinker and Warren arrived at the conclnsiou
about thirty years ago that the toxieity of the molecules increases with an
increasing number of chlorine atoms on the ring. Later experiments conducted
by Sehley and Kligman with human subjects and with the use of several
chlorinated naphthalenes showed that penta- and nexachloronaphthalenes produced the strongest effects; compounds with 1 to 3 as well as 7 and 8 chlorine
atoms were far less toxic of inactive. We confirmed this finding in animal
experiments using rabbit ears (Schulz 1965).
In the last 10-15 years, halogenated waxes have become less important as
etiological factors of chloracne. Evidently, this is related witli the fact that
they are no longer as important industrially and have been replaced by synthetics of the most diverse nature in most fields of application.
In the fifties, the incidence of chloracne was observed in entirely different
sectors of industry i.e. in the production and processing of chlorinated phenols.
Reports of group involvements have been published from at least three
�338
339
industrial plants in Western Germany. Baader and Bauer as well as Brinkman
described 17 workers of a plant in Nordrhein-Westfalen who developed the
typical skin symptoms as well as damage of the internal organs and central
nervous system disorders in the production of pentachlorophenol.
A larger number (about 60 cases) of similar disorders were recorded several
years later in the region of southwest Germany among workers occupied in the
production of of trichlorophenol (Hergt, Oettel, Hofmann), Approximately at
the same time, 31 workers in a Hamburg plant became ill after working with
industrial 2,4,5-trichlorophenol, an intermediate of the synthensis of trichlorophenoxyacetic acid, a weed killer.
Siliar high' incidences of the disease occurred a few years ago in chemical
plants of the Netherlands and the U.S. during analogous production processes.
The high frequency of cases in Hamburg led to studies of the etiology. They
were conducted by us together with Dr. Sorge, the former manager of the
chlorophenol plant.
Rabbit ears were used as the biological substrate on which symptoms corresponding to human chloracne can be produced by local painting as demonstrated by Hofmann and Neumann with chloronaphthalenes. The results, which
have been reported earlier (Schulz 1957; Kimmig and Schulz 1957), can be
briefly summarized as follows:
First, it was found that it was not possible to produce changes in the rabbit
ear in the form of chloracne with the use of the chemically pure compound in
contrast to the technical grade of 2,4,5-trichlorophenol used in the plant. Pure
1,2,4,5-tetrachlorobenzene also was inactive. The toxic factor therefore must
have formed as a byproduct during the alkaline hydrolysis • of tetrachloro-,
benzene into trichlorophenol.
Under the conditions of a salt fusion in a solvent-free state^2 molecules of
sodium trichlorophenolate form 1 molecule 2,3,6,7-tetrachlorodibenzodioxine
with the elimination of 2 molecules of NaCL. Dr. Sorge synthesized the compound and in addition, isolated it from the distillation residue of industrial
trichlorophenol.
Animal experiments conducted with
tetrachlorodibenzofuran
and
2,3,6,7-tetrachlorodibenzodioxine showed an extremely high toxicity of these
compounds. Even concentrations of 0.001-0.005% of tetrachlorodibenzodioxine
led to severe reactions on the rabbit ear after local application. On human
skin in a self-experiment, two applications of 10 y of the substance produced
the symptoms characteristic of chloracne. On the rabbit ear, tetrachlorodibenzofuran showed an activity which was about 10 to 20 times less pronounced. Moreover, the unexpectedly high hepatotoxic action is worthy of note,
particularly after tetrachlorodibeuzodioxine. Single oral doses of 20-50 y/kg
body weight regularly produced lethal liver necrosis, while doses of 10 y/kg
were lethal for about 50% of the rabbits.
On the basis of these chemical and toxicological findings,, it is justified to
conclude that 2,3,6,7-tetrachlorodibenzodioxine played an important role in the
etiology of the cases of chloracne which occurred during the industrial production of trichlorophenol. It cannot be ruled out, however, that other, as yet
unknown chlorinated aromatics of highly toxic properties may form during the
industrial process under certain conditions. The results of the study are an
example that materials which form only in small amounts as byproducts of
large-scale syntheses can be of importance in occupational medicine. If such
toxic byproducts can be uncovered and their mechanism of formation can be
elucidated,' this will create an important prerequisite for successful prophylaxis. In our special case, the plant succeeded in avoiding the formation of
highly toxic byproducts by modifying the production process.
Our animal experiments were extended to other chlorinated aromatics to
which other authors ascribed a chloracne-causing action on the basis of clinical observations. (Reviews of these compounds in the monograph of
W. Braun.) Neither benzenes and phenols with 1 to 6 chlorine atoms nor chlorinated diphenylethers produced an effect in animal experiments It seems indicated to assume, therefore, that neglected toxic byproducts were of decisive
etiological importance in these cases rather than the main products.
In connection with the acne produced by chloronaphthalenes, the question
arose whether toxic byproducts rather than the chloronaphthalenes themselves
might not be considered as the true toxins (Oettel). In the production of
industrial naphthalene by fractional distillation of tar, the presence of other
aromatic compounds deriving from the tar apparently cannot be ruled out. In
the following chlorination process, such substances might then also undergo
CL-substitution. These question prompted us to carry out animal experiments
on rabbit ears using chemically pure chloronaphthalenes of different degrees of
chlorination specially synthesized for this purpose.*
In agreement with the findings of Shelley and Kligman, we found that naphthalenes containing 5-6 chlorine atoms have a chloracne-producing effect. The
necessary concentrations, however, were about 100 times higher than those of
tetrachlorodibenzofuran (diphenylene oxide) about about 1000 times higher
than for 2,3,6,7-tetrachlorodibenzodioxine (tetrachlorodiphenylene dioxide).
On the basis of the present state of the art, therefore, the chloracne-producing activity of the following compounds appears to be sufficiently demonstrated
or at least highly probable:
OH
4- NaOH
+ CH30H
1,2,4-tetrachlorobenzene
2,4.5-trichlorophenol
Alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene into 2,4,5-trichlorophenol.
Since the isolation of well-defined compounds from the distillation residue of
trichlorophenol was unsuccessful, a number of especially synthesized substances were investigated which might have formed as a byproduct of the
cited saponification process on the basis of theoretical considerations. The
majority of investigated compounds proved to be inactive. Only dibenzofurans
with 3 and 4 chlorine atoms (diphenylene oxides) and 2,3,6,7-tetrachloro-•
dibenzodioxine (tetrachlorodiphenylene dioxide) led to the characteristic
changes on the rabbit ear already in low concentrations. Moreover, it was demonstrated that 2,3,6,7-tetrachlorodibenzodioxine had formed by the following
reaction route in the industrial process of alkaline hydrolysis of 1,2,4,5-tetraciilorodibenzene.
Cl-X\-ONa
Cl-ljJ-Cl
Cl
31-/VC1
NaO-L
NaO-
J-C1
Sodium salt of 2,4,5-trichlorophenol
Naphthalenes containing 5-6 chlorine atoms:
2NaCl
2,3,6,7-tetracb.lorodibenzodioxine
(2,3,6,7-totrachlorodiphenylone dioxide)
A
�341
340
Dibenzofurans (diphenylene oxides) with higher degrees of chlorination:
XX
01-
01
Trubaut, B., G. Vitte and E. Broiissomart: Research on the toxicology of
pentachlorophenol. Arch. Mai. Prof. 13, 561 (1952).
AVauer: Occupational diseases due to chlorinated hydrocarbons'. Zbl. f.
Gcwerb-Hyg. 6, 100 (1918).
Address of the author: Prof. Dr. K. H. Schulz, University Dermatology
Clinic, Martinistr. 52, 2 Hamburg 20.
*I am indebted to Prof. Dr. Zeile, Fa. 0. H. Boehringer Sohn, Ingelheim, for
the synthesis of the compounds.
2,3,6,7,-tetrachlorodibenzodioxine:
In conclusion, it should be noted that the toxicodermatosis represented I'T
chloracne results from intoxication with certain chlorinated aromatics. Tlie
causally responsible compounds partly involve highly toxic substances which
can cause damage in various internal organs, especially the liver and nerrtna
system, in addition to the skin. Since the skin does not always represent the
only manifestation site, it' is recommended that thorough internal and neurological as well as psychiatric examinations be made in cases of suspected chloracne.
LITERATURE
Baader, E. W. and H. J. Bauer: Industrial intoxication due to pentachlorophenol. Ind. Med. Surg. W, 286 (1951).
Bauer, H., K. H. Schulz and TJ. Splegelberg: Occupational intoxication
during the production of chlorophenol compounds. Arch, f. Gewerbepath. und
Gewerbehyg. 18, 538 (1961).
Bennett, G. A., C. K. Drinker and M. F, Warren: Morphological changes In
the livers of rats resulting from exposure to certain chlorinated hydrocarbons.
,T. Industr. Hyg. a. Toxicol. 20, 97 (1938).
Braun, W.: Chloracne. Monograph Supplements to the journal Berufsdermatosen, Vol. 1, Editio Cantor, Aulendorf/Wurtt
Drinker, C., M. F. Warren and G. A. Bennett: The problem of possible systemic effects from certain chlorinated hydrocarbons. J. of industr. Hyg. and
Toxicol. 19, 283 (1937).
Grimmer, H.: Occupational acne by chlorinated aromatic hydrocarbons
(chloracne, perna disease). Zbl. f. Arbeitsmed. 5, 70 (1955).
Hergt, W.: Comment in discussion Occupational Physicians' Conference, Bnd
Durkheim, 1955.
Herzberg, J, J,: Chloracne following consumption of chlorinated paraffin.
Dermat Wschr. 119, 425 (1947).
Herxheimer, K.: On chloracne. Munch, med. Wschr. 278 (1899).
. Hofmann, H. Th.: Paper before the Occupational Physicians' Conference,
Bad Durkheim, 1955.
Hofmann, H. Th. and W. Neumann: A method for the animal-experimental
study of the dermatological effect of chlorinated naphthalenes. Zbl. Arbeitsmed.
2, 169 (1952).
Kimmig, J. and K. H. Schulz: Chlorinated aromatic cyclic ethers as a cause
of so-called chloracne. Naturwiss. 44i 337 (1957).
Kimmig, J. and K. H. Schulz: Occupational acne (so-called chloracne) by
chlorinated aromatic cyclic ethers. Dermatologica (Basel) 115, 540 (1957).
Oettei, H.: Clinical and animal-experimental experiences with highly toxic
chlorinated hydrocarbons; a contribution to the perna problem. Paper before
the Occupational Physicians Conference, Bad Durkheim, 1955.
Risse-Sundermann, A.: Intoxications by chlorinated aromatics. Monograph,
Cologne University. Dissertation, Cologne, 1959.
Schulz, K. H.: Clinical and experimental studies on the • etiology of.chlQr-,,
acne. Arch. Klin, exper. Dermat WC>, 589 (1957).
Schulz, K. H.: Unpublished experiments, 1965.
Schmidt, W. and W. Boslet: Contribution to a knowledge of permanent skin
changes in chloracne patients with demonstrable insurance claims. Berufsdermatosen //, 109 (1956).
Shelley, W. B. and A. M. Kligman: The experimental production of acne by
penta-und hexachloronaphthalenes. Arch, of Dermat. (Chicago) 75, 689 (1957).
Spiegelberg, U.: On the question of delayed and permanent psychopathological damage following occupational intoxications. Med. Klinik 56, 436 (1961).
Teleky, L.: Perna disease (chloracne). Klin. Wschr. 845 (1927); Klin.
Wschr. 897 (1927) ; Klin. Wschr. 214 (1928).
REPORT ON METHODOLOGY FOB CHLORINATED AEOMATICS IN FATS,
OILS, AND FATTY ACIDS
By JOHN RBSS, G. R. HIGGINBOTHAM, and DAVID FIRESTONE, (Division of Food Chemistry and Technology, Bureau of Science, Food and Drug
Administration, Consumer Protection and Environmental Health Service, Public
Health Service, Department of Health, Education, and Welfare, Washington,
D. C. 20004)
ABSTBACT
The official, first action electron capture GLC (EC-GLC) method for chick
wleina factor (polychlorodibenzo-p-dioxins) have been reviewed. This general
procedure, which underwent collaborative study in 1967, has undergone several
minor modifications which result in better recoveries of polychlorodibenzo-pdioxins and increased specificity in interpretation of the gas chromatographic
results. The EC-GLC method can be used as a screening test, or where a typical pattern of GLC peaks is obtained as a preliminary test, but confirmatory
tests are needed to demonstrate structure and toxicity of polychlorodibenzo-p(lioxins. Preliminary work with combined GLC-Mass Spectrometry indicated
that this technique might provide a suitable test, if adequate sample cleanup
can be accomplished. A chicken embryo assay has been developed to the point
where toxicity can be observed in three to five days after injection of eggs.
A preliminary procedure has been developed for isolation and gas chromatography of chlorophenols in fats and fatty acids. Polychlorophenols have been
found to be precursors of chlorodibenzo-p-dioxins. The use of a non-specific
microbiological test for chlorophenols employing the Bacillus megaterhtm was
evaluated. Chlorophenols were found to produce uniformly graded growth inhibition of the test organism in the range 1-100 /tg.
The widespread use of toxic organochlorine compounds in agricxilture and
industry requires development of sensitive methods for their detection in a
wide variety of commodities. In addition, it is equally important that methods
be developed to^detect toxic breakdown or conversion products of organochlorine compounds. One of the most urgent needs in the fat and oil industry is for
a rapid and specific method for polychlorodibenzo-p-dioxins (chick edema factors) in fats, oils, and fatty acids. The official, first action, microcoulometric
and electron capture methods for chick edema factors (CEF) are essentially
screening procedures (1,2,3), Both methods, at present, require a rather time
consuming three-week chick bioassay (4) for confirmation.
The purpose of this report .is to review the current status of chemical and
biological methods for chlorophenols and chlorinated dibenzo-p-dioxins in fats,
oils, and fatty acids.
CEF consists of a mixture of chlorinated dibenzo-p-dioxius which occur occasionally as ,1 trace contaminant in fats. Recently, a communication (5) from
this laboratory reported the results of a preliminary study which demonstrated
the possibility tiiat CBF could arise from residues of pentachlorophenol and
2,3,4,6-tetrachlorophenol in fats and fatty acids. Chlorophenols and their salts,
when heated, undergo condensation reactions and form chlorinated derivatives
of dibenzo-p-dioxin. The following equation illustrates this condensation reaction.
Technical grades of pentachlorophenol contain ca 10% of 2,3,4,6-tetrachlorophenol which also undergoes thermal condensation reactions and forms hexachloro derivatives of dibenzo-p-dioxins. The condensation of 2,3,4,6tetrachlorophenol with pentachlorophenol forms two heptachloro derivatives
of dibenzo-p-dioxin.
An electron capture GLC method has been developed for pentachlorophenol
and 2,3,4,6-tetrachlorophenol in fats, oils, and fatty acids (6). However, recov-
�342
343
orios of the two volatile polychlorophenols were low and varied over a wic
range; nevertheless, the method appears to be satisfactory for qualitative
measurements at the 0.5 ppm level, Several samples of oleic acid known to
contain (JjiJF were analyzed and were found to be contaminated with residues
of pentachlorophenol. The method requires further study.
The official electron capture and microcoulometric methods for CEF were
developed before their chemical structures were known. The methods are
screening procedures and are based on the observation that toxic fats contain
a number of chlorinated components (now known to be polychlorpdibenzo-pclioxins) which have greater retention times than chlorinated pesticides. The
electron capture method has received wide acceptance. It is approximately
2000 times as sensitive and requires less sample than the microcoulometric
method. In addition, electron capture gas chromatographic equipment is simpler and is in general use in many laboratories.
Recently, it has come to our attention that a number of laboratories that
routinely use the electron capture method for control work are not aware of
improvements (3) that have been made in the original procedure (2). Kecent
changes which have not been published include a minor modification of the
HaSOi cleanup step and a slight modification of the procedure for packing the
GLO column. The modified method, which includes these changes, replaces all
existing GLC methods for the chemical assay of CEF.
stiHr
solvent, add 5 g anhydrous NaaSOj. Drain excess petroleum etc iso that it is
just above upper surface of NaaSOj.
(11) Gas chromatographic column.—Glass, 0-7' long x %" i.d., packed with
21/2% SE 52 silicone gum rubber on 60/80 mesh Gas Chrom Q (Applied Science
Laboratories, State College, Pa. 16801). Coat the support with substrate as follows : Weigh 2.5 g of the silicone gum rubber stationary phase and dissolve in
300 ml of 1:1 methylene chloride-toluene, heating to dissolve. Add 97.5 g of
support material to liquid mixture and let stand 10 minutes with occasional
gentle stirring. Dry in rotary evaporator. Apply vacuum to the chromatography column, and pack the coated material into the column by adding small
amounts while tapping the column at the packing level after each addition.
Fill to within 1" on the exit side and 3" on the entrance side, and fill the
remaining space with silanized glass wool. Condition the column at operating
pressure at 250° C for 2-5 days.
(12) Gas chromatograph with electron capture detector.—A tritium source
concentric type detector is recommended. Operate instrument in accordance
with instructions of manufacturer, and obtain a stable baseline before carrying
out analyses. Choose an operating voltage (ca 50-80 volts) that will cause
between 0.6 and full scale deflection for 0.1 mg of aldrin (2 ftl of standard
aldrin solution) at a sensitivity setting of 1 x 10"° AFS. Keep the column temperature at 200° ±1°C, and adjust nitrogen flow rate so that aldrin elutes in
1-1.5 min. (3-4 min. per in. chart speed). Inject 2 jA. of the standard aldrin
solution before injection of each reference or test sample.
METHOD
Reagents and Apparatus
Rinse all glassware with appropriate solvents before use. Do not use polythylene containers to store solvents.
(1) Concentrated HaSOi.—Reagent grade.
(2) Petroleum Ether.—Reagent grade, redistilled in glass between 30 and
60°C (Available from Burdick and Jackson Laboratories, 1953 S, Harvey St,
Muskegon, Mich. 49442).
(3) CCU—Distilled in glassl (Available from Burdick and Jackson Laboratories, Muskegon, Mich.)
(4) Anhydrous Na2SOi.—Analytical reagent grade.
(5) Ethyl ether.—Analytical reagent grade (not >2% alcohol) or absolute
ether (not >0,01% alcohol).
(6) Iso-octane.—Distilled in glass (Available from Burdick and Jackson
Laboratories, Muskegon, Mich.)
(7) Standard aldrin solution.—Dissolve aldrin in iso-octane to make 0.05
/ig/ml solution.
(8) Chick edema factor low positive reference sample.—1.5% reference toxic
fat in USP cottonseen oil or other suitable vegetable oil. (Prepare from reference toxic fat available from the Division of Pesticides, Bureau of Science,
Food and Drug Administration, Washington, D.C. 20204).
(9) Activated AUOa (Fisher No. A-540, do not substitute).—Activate 100 S
portions by heating 4 hours at 260° C. Transfer without cooling to dry container and close tightly. Check activity of AlsO,i by analysis of the low positive
reference sample, examining A1203 fractions 2 and 3. With sufficiently activated AlaOa, chick edema factor elutes predominantly or entirely in AlsOa fraction 3 as indicated by the gas chromatograms. (Chromatogram should show n
series of GLO peaks with Ra values between ca 8 and 45).
(10) AlaOu chromatographic column.—To a dry chromatographic column, 17
mm o.d. x 2BO mm long, fitted at bottom with coarse porosity fritted glass disk
and Teflon stopcock (a column without the fritted disk but holding a glass
wool plug in the bottom may be used), add redistilled petroleum ether, (fried
prior to use with anhydrous NazSO*, until column is % full. Weigh 15 g AUOi
and transfer to column in small portions, tapping the column as AlaOa settles.
When last portion of AlaOa settles and air bubbles stop rising to surface of
DETERMINATION
(a) Analysis of 1.5% reference toxic fat in USP cottonseed oil.—Dissolve 2.5
g of the 1.0% reference toxic fat in 10 ml of C01< in a 500 ml glass stoppered
Erlenmeyer flask. Proceed with determination as described below in sections
( b ) , (c) and ( d ) . Take up residue from (d) in 250 /tl iso-octane and inject 5
microliters of reference solution (equivalent to 50 mg of the original sample)
into calibrated gas chromatograph. The resulting gas chromatogram should
exhibit a series of GLO peaks with Ra ca 8-45, depending on operating conditions. Peaks at Ra S-13 are due to hexachlorodibenzo-p-dioxin isomers, 2
peaks at Ra 17-22 are due to the 2 heptachlorodibenzo-p-dioxin isomers, and a
peak at Ra 35-45 is due to octachlorodibenzo-p-dioxin.
(6) Preliminary sulfuric acid cleanup.—Dissolve 2.5 gm of fat in 10 nil CC1(
In 600 ml glass stoppered Erlenmeyer. Add 10 ml cone. HzSOi, stopper, and
shake for 30 sec. Add 125 ml of petroleum ether, stopper and shake vigorously
for ca one minute. Allow layers to separate and decant supernatant liquid into
a 500 ml erlenmeyer, avoiding transfer of lower layer. Repeat extraction with
additional 125 ml portion of petroleum ether. Evaporate the combined petroleum ether extracts to 5 ml for AljOa column fractionation.
(a) FractionaUon of Petroleum ether filtrate 'by alumina chromatography.—
Solvents must be dried prior to use by shaking with anhydrous NaaSO4. Transfer petroleum ether filtrate from (b) to AlaOa chromatographic column using
total of 15 ml petroleum ether. Let liquid level fall to just above top of
NaaSOv Keeping liquid level above top of NaaSOj at all times, elute sample
with 100 ml of petroleum ether (fraction 1), 50 ml of 5% ethyl ether in petroleum ether (fraction 2), and 100 ml of 25% ethyl ether in petroleum ether
(fraction 3). Relatively fast flow rates of ca 8-9 ml/min give-satisfactory
results. Ground glass stoppered separatory funnels (250 ml) are satisfactory
reserves for eluting solvents. Discard fractions 1 and 2, and collect fraction 3
eluate in 125 ml Erlenmeyer. Add several boiling chips and evaporate solvent
to dryness on steam hath. Transfer residue with petroleum ether to 10 ml
graduate cylinder equipped with a ground glass stopper, and evaporate petroleum ether solution to 3 ml.
�344
(A] Sulfurio acid cleanup of alumina fraction 3.—Add 2 ml of concentrated
HsSO< to graduated cylinder containing 3 ml petroleum ether solution from
(c), stopper and shake vigorously for 30 sec. Allow layers to separate nnd
decant petroleum ether layer into 10 ml beaker avoiding transfer of H»SOi
layer. Add 2 nil petroleum ether to cylinder, swirl vigorously, allow layers to
separate, and decant petroleum ether layer into beaker. Add ca 0.5 g of solid
naHOOii to beaker and stir ca % min. Let stand five minutes and decant
petroleum ether layer into clean 2 or 4 dram vial. Wash NaHCOa with 2 ml
petroleum ether and decant washing into vial. Evaporate solvent under N.
(c) Electron capture gas chromatography of petroleum ether extract.—Take
up residue in 250 pi iso-octane (redistilled in glass), stopper vial tightly and
rotate so that solvent wets sides of vial. Inject 1 microliter of sample solution
(equivalent to 10 mg fat) into calibrated gas chroma tograph. Gas chromatographic peaks with Ea S-45 are indicative of the presence of chick edema
factor. Compare Ka values of sample peaks with Ka values of peaks from reference toxic fat. See (a) for identification of peaks. If peaks indicative of
chick edema factor are not observed, inject 5 <ul of sample solution (equivalent
to 50 mg fat) into gas chromatograph. Check reagents for possible interferences by running a blank with each set of samples. The chromatogram from
the blank should show a smooth low baseline from Ba 8—Ra 45. (Types of samples found by experience to be generally free of components characteristic of
toxic fats may be examined by initial injection of 6 pi of solution.)
DISCUSSION
Analysis of the low positive reference sample serves as an overall check on
instrument performance and sample cleanup. The chromatogram from the low
positive reference fat should show a distinct peak pattern as illustrated in
Figure 1. The lower chromatogram (B) represents an injection equivalent to
50 mg of the original low positive reference fat.' Chromatogram (A) represents a mixture of synthetic polychlorodibenzo-p-dioxins prepared by pyrolysis
of 2,3,4,0-tetrachlorophenol and pentachlorophenol. As stated previously, peaks
1 through 4 are due to four positional isomers of hexachlorodibenzo-p-dioxlns.
The isomer associated with the small shoulder (peak 3) is probably caused by
the presence of a tetrachlorophenol other than the 2,3,4,6-isomer in the starting
material. Peaks 5 and 6 are due to two positional isomers of heptachlorodibenzo-p-dioxin. Peak 7 is due to octachlorodibenzo-p-dioxin.
Aldrin is used to calibrate the instrument sensitivity for chick edema factor
analyses. The similarity of detector response vs. applied voltage for aldrin and
an extract from the 1.5% reference toxic fat (low positive reference sample)
is illustrated in Figure 2.
CHEMICAL AND BIOLOGICAL CONFIRMATORY METHODS
The need for rapid chemical and biological confirmatory tests has led to
investigation of mass spectrometry 'as well as two biological toxicity assays.
Preliminary work has suggested that rapid EC-GLC screening for chick edema
factors can be carried out initially; if the presence of chick edema factors is
indicated, then larger portions of sample would be fractionated and cleaned up
for chemical and biological confirmation.
MASS SPECTROMETBY
Results of a preliminary investigation of combined GLC-mass spectrometry
(GLC-MS) indicated that the use of this technique might be suitable if adequate sample cleanup can be accomplished. A 7-foot coiled glass column packed
with 2.5% SE-52 on 60-80 mesh Gas Chrom Q was used with an Atlas CH-4
mass spectrometer and single-stage Llewellyn (silicone membrane) separator to
345
examine standards and an extract from a 2.5 g sample (posra^e for chick
(posflf^e
edema factor by EC-GLC). The GLC oven temperature was 220°C with a
helium flow at 75 ml/minute. Injection temperature was 235°C and silicone
membrane temperature was about 160° O. MS sensitivity setting was 32 x at 40
PA.
GLC retention times as well as molecular weight and number of chlorine
atoms in the molecule were determined for a standard mixture and an extract
from the test sample. Two /j\ of 10/tl solution from the test sample was
injected; it was estimated that 2 pi test sample contained 0.4 ps of hexa-,
liepta- and octachlorodibenzo-p-dioxins in addition to other unidentified constituents. A summary of results are in Table 1. Comparison of fragmentation
pattern and relative abundance of the ions from standards and sample might
afford additional specificity; impurities in the test sample prevented such evaluation at this time.
CHICKEN EMBBYO ASSAY
Extracts from a reference toxic fat and several test samples were subjected
to the chicken embryo assay (7) ; 111 g samples of fat were fractionated
according to AOAC (1965) 26.093-26.094, and alumina fraction 3, and cleaned
up with sulfurlc acid (JAOAC (Changes in Methods) 50, page 217, section (c)
(1967)). Small portions of each extract was retained for EC-GLG analysis and
the remainder, in chloroform solution, was subjected to the chicken embryo
assay (10-15 eggs per sample were tested by injection of portions of the
sample extract in the air cell) at three levels equivalent to ca 40, 30 and 10 g
starting sample. Assay results are shown in Table 2. These results indicate
that the chicken embryo test can provide a sensitive indication of toxicity as
well as a measure of specificity due to observations of localized and genralized
edema. In many instances evidence of toxicity can be observed (by periodic
candling) in 3-5 days.
B. megaterium TOXICITY TEST
The use of a non-specific biological test employing the Bacillus megaterium
was evaluated (8). This test involves observation of inhibition of a seeded
petri dish holding a B. megaterium spore suspension in agar medium. Filter
paper discs of sample extracts are placed on the surface of the agar plates
and inhibition zones are observed after 18 hour incubation at 37° C.
Five samples (111 g each) were fractionated according to the general procedure of AOAC (1965) 26.093-26.094. These test samples consisted of two
highly toxic fats, one low toxic reference material (1.5% TEF in TJSP cottonseed oil), one nontoxic oil, and a reagent blank. In addition to the three alumina fractions obtained with petroleum ether, 6% ethyl ether in petroleum
ether and 25% ethyl ether in petroleum ether, a fourth fraction eluted with
400 ml of 100% ethyl ether was obtained. The four alumina fractions were
cleaned up twice with sulfuric acid according to JAOAO (Changes in Methods)
50, 0.217 Sect. C (1967). The residues of fractions 2,3, and 4 were spotted on
filter discs (7.5 mm diameter) at two levels equivalent to 2.B and 54 g starting
sample. EC-GLC analysis of alumina extracts indicated that chick edema factors (polychlorodibenzo-p-dioxins are predominantly concentrated in alumina
fraction 3. In addition, solvent blanks, a synthetic reference standard consisting of hexa-, liepta- and octachlorodibenzo-p-dioxin (CDPD), and a sample of
technical grade 2,3,4,6-tetrachlorophenol (2,3,4,6-TCP) were spotted at the following concentrations : 0.01 jug, 0.1 pg, 0.5 pg, 1.0 pg, 10.0 pg, and 100 pg.
After incubation, inhibition was observed in six cases as shown in Table 3.
The sensitivity of the B. megaterium test for CEF appears to be limited to 100
ppm and even then only a very small zone of inhibition was noticed. It
appears, however, that a rapid confirmation test for chlorophenols can be
developed at levels of ca 2-3 ppm or less. These are the low levels at which
aflatoxin Bi shows inhibition of B, megaterium.
�347
346
ACKNOWLEDGMENT
The authors wish to express their appreciation to Mr. Joseph Barandy,
Drew Chemical Company, Boonton, N.J. and Dr. B. N. Gerhardt, Emery Industries, Inc., Cincinnati, Ohio for suggesting modifications of the H2S04 cleanup
procedure which have resulted in improved recoveries of cliick edema factor.
The contribution of the following members of the Food and Drug Administration are gratefully acknowledged: to Dr. M. J. Verrett for the chicken
embryo assays; to Joseph N. Damico and Bobert P. Barron for the mass spectrometric analyses: to Robert M. Eppley for the B. megaterium tests; ami
Thomas J. Dols for his helpful discussions concerning GLC operating parameters.
a
1
KBFEBENCES
1
Official Methods of Analysis, 10th ed., Association of Official Agricultural Chemist",
Washington, D.C., 1065, sees. 26.002-26.006.
' "Changes in Methods. 26. Oils, Fats, and Waxes," ibid. SO, 217-218 (1067).
"Neal, P., Tills Journal SO, 1338 (1067); "Changes in Methods. 20. Oils, Fats, and
Waxes," Mil. Si, 489-400 (1968).
•Official Methods of Analysis, 10th ed., Association of Official Agricultural Chemists,
Washington, D.C., 1965, sees. 26.087-26.091.
5
Iligginbothani, G. E., Huang, A., Firestone, D., Verrett, J., Ress, J., and Campbell,
A. IX, Nature M0, 702 (1968).
" Iligginbotham, G. R., Ress, J., and Rocke, A., JAOAC, In pross.
1
Verrett, M. J., Marline, J. P., and McLaughlin, J. ibid. J,t, 1003 (1964).
'Clements, N. L., ibid. 61, 611 (1968).
TABLE 1.—MASS SPECTROMETRIC ANALYSIS OF ISOLATED COMPONENTS
GLC
peak no.
Standard mixture
Identity
1 Kexachlorodibenzo-p-dioxfn ...
2
.do
3 Heptachlorodibenzo-p-dioxin...
1 Hexachlorodibenzo-p-dioxin ..
3 Heptachlorodibenzo-p-dioxin
- .
Test sample
No. ol
Chlorine
atoms
indicated
Molecular
weight
found
ca
6
6
7
6
7
388
388
422
388
422
TABLE 2.—CHICKEN EMBRYO ASSAY OF EXTRACTS
EC-GLC analysis
for chick edema
factor
Sample
Reference toxic fat
Tost fat No. 1
Test fat No. 2
Tost fat No. 3
Reagent blank
Chl6roform solvent
Control eggs
. .
positive
do
do
do
negative.... ,
...do
. .do
Estimated level
Percent
of hexa-, heptaand octachloromortality
dibenio-p-dioxchicken
ins in fat, in ppm embryo assay
'
2 4
0.1
0.7
0.6
Assay observations
100
73
100
93
30
20
7
Do.
Do
Do.
TABLE 3.-RESULTS FROM B. MEGATERIUM TEST
Sample
Nontoxic USP cottonseed oil Fr. 4 (2.5 g extract)
TEF-F797,- Fr. 2, (2.5 g extract)
100 «g synthetic (CDPD) reference standard
1«2,3,4,6-TCP
10/jg2,3,4,6-TCP
100^g2,3,4,S-TCP
Observation
Barley visible around disc.
Do.
Do.
Do.
16 mm inhibition zone.
36 mm inhibition zone.
45-382 O - 70 - 23
�349
348
SECTION 121.1070
FATTY ACIDS
The food additive fatty acids may be safely used in food and in the manu' facture of food components in accordance with the following prescribed conditions :
(a) The food additive consists of one or any mixture of the following
straight-chain monobasic carboxylic acids and their associated fatty acids manufactured from fats and oils derived from edible sources: Capric acid, caprylic
acid, lauric acid, myristic acid, oleic acid, palmitic acid, and stearic acid.
(6) The food additive meets the following specifications:
(1) Unsaponiflable matter does not exceed 2 percent.
(2) It is free of chick-edema factor or other factors toxic to chicks, as evidenced during the bioassay method for determining the chick-edema factor as
prescribed in paragraph (c) (2) of this section.
(c) For the purposes of this section :
(1) Unsaponiflable matter shall be determined by the method described in
section 26.049 of the Official Methods of Analysis of the Association of Official
Agricultural Chemists, Ninth Edition (1960).
(2) Chick-edema factor shall be determined by the bioassay method
described in the Journal of the Association of Official Agricultural Chemists,
Volume 44, page 146 (1961). The presence of chick-edema factor shall be determined by a comparison between the mean log of the pericardial fluid volumes
of a test group and of a concurrent negative control group. The significance of
the difference in pericardial fluid volumes between the test group and the negative control group is determined by calculating a t value according to the formula : The test sample is judged to contain chick-edema factor if the calcu-
100
1.1.1
co
7'
O
n.
CO
1.1 j
a:
GO
where:
$ i and £ c are the means of the log of the pericardial fluid volumes of the test
and control groups, respectively;
rij and n,2 are the number of chicks in the respective groups;
s(' and sc are the variances of the test and control groups, respectively.
The variances are calculated as follows:
sa=
n(n-l)
where:
Xx is the sum of the logs of the perioardial fluid volumes;
2x8 is the sum of the squares of the log of the pericardial fluid volumes for
either the test t or control c group date.
20
co •
VOLTS
i
100
lated t exceeds 1.3 and the mean log of the pericardial fluid volume obtained
from the negative control group multiplied by 100 is less than 1.1461.
(3) "Other factors toxic to chicks" referred to in paragraph (b) (2) of this
section shall be determined during the course of the bioassay test described in
subparagraph (2) of this paragraph on the basis of chick deaths or other
abnormalities not attributable to chick-edema factor or to the experimental
conditions of the test.
(d) It is used or intended for use as follows:
(1) In foods as a lubricant binder and as a defoaming agent in accordance
with good manufacturing practice.
(2) As a component in the manufacture of other food-grade additives.
(0) To assure safe use of the additive the label and labeling of the additive
and any premix thereof shall bear in addition to the other information
required by the act the following:
(1) The common or usual name of the acid or acids contained therein.
(2) The words "food grade," in juxtaposition with and equally as prominent
as the name of the acid.
�350
SECTION 121.1071
351
SALTS OF FATTY ACIDS
The food additive salts of fatty acids may be safely used in food and in the
manufacture of food components in accordance with the following prescribed
conditions:
(a) The additive consists of one or any mixture of two or more of the aluminum, calcium, magnesium, potassium, and sodium salts of the fatty acids
conforming to § 121.1070.
(&) The food additive is used or intended for use as a binder, emulsifler,
and anticaking agent in food in accordance with good manufacturing practice.
(c) To assure safe use of the additive, the label and labeling of the additive
and any premix thereof shall bear in addition to the other information
required by the act the following:
(1) The common or usual name of the fatty acid salt or salts contained
therein.
(2) The words "food grade," in juxtaposition with and equally as prominent
as the name of the salt.
Title 21—FOOD AND DRUGS
Chapter 1—Food and Drug Administration, Department of Health, Education,
and Welfare
SUBCIIAPTER B—FOOD AND FOOD PRODUCTS
PART 121—FOOD ADDITIVES
Subpart D—Food Additives Permitted in Food for Human Consumption
FATTY ACIDS
The Commissioner of Food and Drugs has received a petition (FAP GA2003)
from Fatty Acid Producers' Council. Division of the Soap and Detergent Association, 295 Madison Avenue, New York, N.Y. 10017, proposing that § 121.1070,
the food additive regulation providing for safe use of fatty acids in food and
in the manufacture of food components, be amended:
A. To provide for the use of a screening method for 'determining the presence of chick-edema factor in the fatty acids that, within certain conditions,
may be used in lieu of the hioassay method prescribed by paragraph (c) (2),
and
B. To delete references to "other factors toxic to chicks" from the section.
From the available information it can be concluded that the anomalies presently identified as due to other toxic factors, which may be evidenced during
the bioassay method for determining chick-edema factor, are directly associated with the same conditions or substances producing chick-edema factor, and
the proposed physicochemical method is adequate as a screening test for
detecting the chicle-edema factor complex of toxicants.
Based on the information sbumitted in the petition, and other relevant material, the Commissioner has concluded that the regulation should be amended as
petitioned. In addition, the references identifying the chick-edema bioassay
procedure was updated to refer to the Official Methods of Analysis of the
Association of Official Agricultural Chemists.
Therefore, pursuant to the provisions of the Federal Food, Drug, and Cosmetic Act (sec. 409(c) (1), 72 Stat. 178G; 21 U.S.C. 348(c) (1)), and under
the authority delegated to the Commissioner by the Secretary of Health, Education, and Welfare (21 CFR 2.120; 31 F.R. 3008), § 121.1070 (b) (2) and (c)
(2) and (3) are amended to read as follows:
SECTION 121.1070
(b)
FATTY ACIDS
(2) It is free of chick-edema factor:
(1) As evidenced during the bioassay method for determining the chickedema factor as prescribed in paragraph (c) (2) of this section; or
(ii) As evidenced by the absence of chromatographic peaks with a retention
time relative to aldrin (RA) of five or more using the gas chromatographicmlcrocoulometric method prescribed in paragraph (c) (3) of this section. If
chrouiatographic peaks are found with RA values of five or more, it shall meet
the requirements of the bioassay method prescribed in paragraph (c) (2) of
this section for determining chick-edema factor.
(c) * * *
(2) Chick-edema factor shall he determined by the bioassay method
described in Official Methods of Analysis of the Association of Official Agricultural Chemists, 10th Edition (1965), sections 26.087 through 26.091.
(3) The gas chromatographic-microcoulometric method for testing fatty
acids for chick-edema shall be the method described in Official Methods of
Analysis of the Association of Official Agriciiltural Chemists, 10th Edition
(1965), sections 26.092 through 26.096, except that the following procedure is
substituted for that described in section 26.092(b) :
Activated alumina.—(Fisher No. A540 or equivalent.) Activate 250-gram portions by heating 4 hours at 260" C. Transfer without cooling to dry container
and close tightly. Use within 1 week after preparation. Check activated A1,O»
by analysis of a reference standard by examining fractions 2 and 3. Chickedema factor should elute in Ala03 fraction 3 as indicated by the gas chroma togram. (A sample of the reference standard may be obtained on request from
the Bureau of Science, Food and Drug Administration, Washington, D.C.
20204.)
*
*
*
*
-»
Any person who will be adversely affected by the foregoing order may at
any time within 30 days from the date of its publication in the FEDERAL REGIBTEH file with the Hearing Clerk, Department of Health, Education, and Welfare, Room 5440, 330 Independence Avenue SW., Washington, D.C. 20201, written objections thereto, preferably in quintuplicate. Objections shall show
wherein the person filing will be adversely affected by the order and specify
with particularity the provisions of the order deemed objectionable and the
grounds for the objections. If a hearing is requested, the objections must state
the issues for the hearing. A hearing will be granted if the objections are supported by grounds legally sufficient to justify the relief sought. Objections may
be accompanied by a memorandum or brief in support thereof.
Effective date. This order shall become effective on the .date of its publication in the FEDERAL REGISTER.
(Sec. 409(c) (1), 72 Stat. 1786; 21 U.S.C. 348(c) (1))
Dated : August 18, 1966.
J. K. KIBK,
Acting Commissioner of
Food and Drugs.
[F.R. Doc. 66-9263; Filed, Aug. 24,1966; 8:47 a.m.]
�£icbrvoto:-:icitv of Chlorophgr.ols, Olber.go-p-dio:-:ir.s (Chick edera)
Table 12
Estimated Dose Levels to Produce Indicated Mortality (Percent)
1.5
0.05
0.2
.1.0
2,3,4,5- Tc trachlorooaer.ol 2.5
?sr.caehlorop>.inol
0.4
0.01
1.0
2,4,5-Trichio-cr.har.ol
2 ,4, o-Tfichloroohar.ol
.0.7
1.5
2,4-Dicr.lcroph.enoxyacetic
Unsaponifiable*
Fraction (Dose in rig)
100
50
0
Pyrolysis*
Products (Doss in ing;
100
50"
0
Phenols
(Doss in ag)
>0.5
2.5xlO~ 5
0.25
5x13"°
0.01
2.5xiO~6
0.005
2.5x10-3
2x10-*
l.OxiO' 4
0.05
IxlO"3
0.2
0.4
2
_
2.5xlo'
i.o ^o.i
-
'
>-, 5x10-3
l.SxlO"2
SxlO'1-
2Kl3~3
-
.05
6xKr3
"
1.2xlO"3
6xlO~4
2
3
ixlfi-3
.->lxlO"
SxlO"
-
r
2 , 4 , 5- Tr ich lorophar.oxyacetic acid
Eior-etlcs
Dow £123449
0.25
1.0
1.0
co
en
bO
< .05
—0.5
Polvchlorodiben3o-p-3ioxins *
0.5
Diberv^o-p-dioxin
Chlorinated dibonzo-^-dioxin
2.5:-:10"D
IxlO-5
Tetrachloro diber.so-p-dioxln
2xlO"4
5xlO"5
5xUT4
- :"
1.25xlO"4
2xlO~3
Ho::achlcro iscniers (4)
z.. Xcst toxic
Hcr^tachl^ro iso:r.ers(2)
a. Xost toxic
h. Least toxic
Uapublls-.-ad data of J. Varrctt
* >'aziy of c'aesa ara z;i:cturas
Code:
- =3 " 2O pp=
1 jit - 20 jpb
3
1.25x.lO-
2.5xlO'
2.5x10 <>
-
4
•
-
IxlO"3
~
1.25X10'3
Air- Call Iroactions
Pra-incubation
PS,~LBii:-liSY RHFCST CH Tr5AXOI.CC? SVUDIT.S ma FICXIN USISG GOI2a? ^.I-lSrEF.S
Oocpoun'J
.Amou.:it Intubateil
try..-, f, - 10
Ccatrol
0
C'hlovod Lbcr.ro-p9.1 .'g/!-.g/<lay
t.io;;ia
<21'.% trichloro,
2.0 2/KS/<3ay
53» tctracblorc,
2j-nthcii=cd ia FDA)
0.5 <:£/hc/^y
0.13 .-s/^/^y
?c-i i
titter
'Iota! i1
of Fo.ti
*•• Bcra
Alive
Av£. :.:t.
Tcsr.ta
^ £,<?.<•.£
a.s
43
41
Ecus
2 - 1 ED, 1 ID'
1.55
10.3
62
1
1
eye arsffl^slics
5 1 - 4 ME, 47 ID
1.33
10.8
43
41
Srtr,tro-
2 - 2 2S
1.47
10.5
63
62
1 > 1 O<
1.6S
11.6
58
55
2 - 2 CD
1.53
hosr.orruags
Sac-troLntGiti'-'-al
iicraorrhaga
Hons
H) - Uatly r.-.ac, U5 - Lctc Coae .
75Z2K:
t-r. ".?.£. Collins, I5f. W.H. Haas en, 3>r. C.E. Uillie=3
Kictcua ™ox:".color>,y Srcnch
r=:vi;lca o£ ?^r.-:i.cidc ',tic;-ictry •!- T
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co
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�354
355
«H
*
I"
,'
4.)
|j
DEPARTMENT Off HEALTH, EDUCATION, AND WELFAR|
PUBLIC HEALTH SERVICE,
Research Triangle Park, N.G,, December 4, 1969.
P
!?? I J. MCLAUGHLIN,
F)H.
i
" *if! '!-'- Food and Drug Administration,
^ *• '"lip :T- '
1 ml
Washington, D.O.
tj
"» fji'^1 |>;
DEAH DR. MCLAUGHLIN : I tun sending you under separate cover a sample of
1
k?
'<W,t>'T (2,4,5-trichlorophenoxy acetic acid) from the supply that was used at
fci
Blonetics Research Labs, Inc. for the Teratogen Screening Study for NCI.
Sincerely,
« &**1I fy "'
^*
u S^'tf?
K. DIANE COURTNEY, Ph.D,
Pharmacology and Toxicology Branch,
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THE DOW CHEMICAL COMPANY,
Midland, Mich., February 9,1310.
•'). M. JACQUELINE VEHRETT, Bureau of Science, Division of Toxicology, Department of Health, Education, and Welfare, Food and Drug Administration,
Washington, D.C.
A,"i''*« j
DEAR DR. VEBBETT :The complete assay on the sample of 2,4,5-trichloro^*t ^
I'lionoxyacetic acid (Dow production batch 120449) which we recently sent to
'•
™ *
ion is as follows:
!M <> " a, i
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2,3,7,8-tetrachlorodibenzo- p-dioxin = (0.5 ppm)
2,0-dichlorophenoxyacetic acid = <0.02
2,5-dichlorophenoxyacetic acid = '0.42
2,4-dichloi'ophenoxyacetic acid = 0.05
2,3,6-trichlorophenoxyacetic acid = 0.55
2,4,6-tichlorophenoxyacetic acid = <0.1
Bis-(2,4,5-trichlorophenoxy)acetic acid = 0.4
3 isomers of dichloromethoxy phenoxyaeetic acid = 2.9
2,4,5-trichlorophenol = 0.23 (Max.)
sodium chloride = 0.035
2,4,5-trichlorophenoxyacetic acid = Balance
Freezing Point = 152.9" 0
Total assay by acid titration = 100%
Sincerely,
GEORGE B, LYNN
Government Regulatory Relations,
Dow Life Sciences.
�357
356
MARCH 2,1971
CHLORINATED DIBENZO-P-DIOXIN STANDARD (F768)
1. We have supplied Dr. C. Billiams with a mixture of tri- and titrncliloi*-HJIJJ
dibenzo-p-dioxins (F768) prepared by direct chlorination'of dibenzo-p-dloxln »( ;-^lf
room temperature for two and one-half hours (see memo of Ress to CuuipWJ,/{%
1/30/70 and memo of Firestone to Campbell 2/5/70).
f !|
|(
2. This mixture, previously analyzed by EC-GLC and found to contain aboul J\jif|
f50% tetrachlorodibenzo-p-dioxin (GLC peak area), was reexamined by GUJ:pj,|||
with flame ionization detection (6 foot glass column; 200°C; 3% C
'*
Packard GO model 871), which gives a more accurate determination of
sition. By this latter analysis, it was determined that the chlorination mixturt~';t;|j|
F768 consists of two components as follows:
ff'
(a) 38% 2,3,7-trichlorodibenzo-p-dioxin
*'•"<
( 6 ) 62% 2,3,7,8-tetrachlorodibenzo-p-dioxin
,C;
•,
.
MABCH 26, 1970. ;,ii
SAMPLES FOU CHICKEN EMBRYO TESTING
'•''.'•';.
1. The following samples were delivered to Dr. Verrett on 3/17/70 for chicken ;
embryo testing:
'.;.j
Our. No.
Identification
F871
2,3,7,8-tetrachlorodibenzo-p-dioxin, Dow, pure (ca. 95% by GLC)
F877
Remarks
„ 0.0878ms in 10 ml
1
of acetone.
0.3806 mgin 10ml
of acetone.
.-.; 2,3,7,8-totrachlorodibe(izo-p-dlo)iin, FDA, prepared by Dr. Pohland (99.5% 0.1735m«in 10ml
pure by GLC).
of acetone.
2,4,5-tricdlorophenoxyacetic acid; Dr. Williams' purified sample; further ex- 1.712 grams.
tracted 3 x with petr. ether and 4 x with 1+i petr. ether—diethyl ether.
SylvexiDr. Williams'purified sample; further extracted 3x with petr. ether and 2.519 grams. .
4xwkh 1+1 petr. ether—diethyl ether.
2,7-dichlorodibenzo-p-dioxin, Dow, pure(ca, 99% by GLC)
F883.....
F881-A-1
F881-B
D. FIEESTONE, Head,
Fats and Oils Section.
'' '
CHLOROPHBNOL SAMPLES
'
MARCH 26, 1970.
' '•'
1. About 3 grams each of the following chlorophenols were delivered on 3/23/70
to Dr. Verrett (at her request) for chicken embryo testing.
Our No.
F888-sub A
F888-sub B
F888-subC
F888-subD
F888-sub E
F888-SUD F
Identification
EC-GLCanil
PPB'CEF
Pentachlorophenol (purified) Aldrich Chemical Co
2,3,4,6-Tetrachloropnenol (technical) Eastman Chemicals
2,4-Dichlorochlorophenol (purified) Eastman Chemicals
Ortho-chlorophenol (purified) Fisher Scientific
2,4,5-Trichlorophenol (technical) Eastman Chemicals
2,4,5-Trichlorophenol (reagent) Dow Chemical Co
167.
96,500.
18.
No analysis.
Trace.
Trace.
•Hexa-, hepta-, and octachlorodibenzo-p-dioxine.
J. RESS,
Fats and Oils Section.
I. Cleft Palate. 21-day Embryo. 0.02 micrograms 2,3,6,7-Tetrachloro-dibenzo-pdioxin.
j
�358
II. Abnormal (incomplete) Development of Eyelid. 21-day Embryo. 0.002i ;'^
mierograms 2,3,6,7-Tetrachloro-dibenzo-p-dioxin.
;' : i
359
III. Edematous Cyst Covering Rump. 21-day Embryo. 0.02 ^ (micrograms),
2,3,6,7-Tetrachloro-dibenzo-p-dioxm.
�361
360
Senator P!ART. The hour being 12:25, I suggest we recess until
2:15.
(Whereupon, at 12:25 p.m., the hearing was recessed, to reconvene
at 2:15 p.m. this same day.)
AFTERNOON SESSION
Senator HART. We resume this afternoon to hear first from Dr.
Julius Johnson of the Dow Chemical Co. If a corporate entity can
have a spirit attached to it, the comparing is a distinguished corporate constituent of mine.
STATEMENT OF DR. JULIUS E. JOHNSON, VICE PRESIDENT AND DIRECTOR OP RESEARCH, THE DOW CHEMICAL CO.; ACCOMPANIED
BY ETCYL BLAIR, DIRECTOR OP DOW AGRICULTURAL CHEMICAL
RESEARCH; V. K. ROWE, DIRECTOR OP THE DOW TOXICOLOGICAL
LABORATORY; AND 'GEORGE LYNN, DIRECTOR OP GOVERNMENT
REGULATORY RELATIONS OP THE DOW CHEMICAL CO.
Dr. JOHNSON. Thank you, Senator Hart.
I have with me Dr. Blair, director of Dow Agricultural Chemical
Research, Mr. Howe, director of our Toxicological Laboratory, and
Mr. George Lynn, director of our Government Regulatory Relations.
Senator HART. Thank you. You are all welcome.
Doctor, you have given us a statement. We will order it printed in
full in the record and as you go along, if there is any extension or
summation you care to make, the record will contain the full statement in any event.
Dr. JOHNSON. Senator Hart, the policy decision has already been
made and announced this morning. I will, however, with your permission, read my testimony, all except the last part which deals with
some historical matters that already appears in the record,
Then, if you would permit, 'I would like to make some additional
comments which may be appropriate to the process of shortening the
time interval between the discovery of a suspected toxic phenomena
and taking appropriate action.
I would_ also like to refer back to some earlier work done under
Senator Ribicoff's guidance in his committee and quote at least one
passage from that work published in 1966, which I think is appropriate to the issue, if I may do so.
Senator HART. By all means.
Dr. JOHNSON. Thank you.
Mr. Chairman, I am Julius E. Johnson, vice president and director
of Research and Development of the Dow Chemical Co., Midland,
Mich. I also served as a member of the Secretary's Commission on
Pesticides and Their Relationship to Environmental Health, May 8,
1969, to November 7, 1969, chairman, Emil M. Mrak.
I have with me George Lynn, director of Government Regulatory
Relations of the Dow Chemical Co. V. K. Rowe, director of the Dow
Toxicological Laboratory and Etcyl Blair, director of Dow Agricultural _ Chemical Research, are also present to assist if necessary.
This statement is concerned with the herbicide 2,4,5-trichlorophenoxyacetic acid, which has often been referred to as 2,4,5-T and
I
1
•4
the chemical "intermediate 2,4,5-trichlorophenol used in the manufacture of 2,4,5-T.
An announcement was issued October 29, 1969, by Dr. Lee Dubridge of the Office of Science and Technology which referred to
birth defects observed in tests by the Bionetics Laboratories using
2,4,5-T in various dosage ranges in mice and rats.
This announcement preceded the final report of the Panel on
Teratology of the Mrak Commission appointed by Secretary Finch
which, since May 8,1969, had been reviewing the effects of pesticides
upon health and the quality of environment. At the time, October
29, 1969, members of the Mrak Commission had not seen the Bionetics report on teratology.
Following the announcement by the Office of Science and Technology, I became particularly concerned because Dow is a manufacturer of this herbicide. Consequently, I made a diligent effort to
trace the source of samples used and learned that the 2,4,5-T sample
came from the Diamond Alkali Co. (which no longer makes 2,4,5-T).
Moreover it was learned that '2,4,5-trichlorophenol also tested by
the Bionetics Laboratory came from Coleman-Mathison-Bell who
had obtained the sample from McKesson-Robbins who in turn had
procured it from the Dow Chemical Co.
2,4,5-trichlorophenol is used as an intermediate in the manufacture
of 2,4,5-T. Hence, the quality of 2,4,5-T is related to the quality of
its intermediate 2,4,5-trichlorophenol. The chemical process used by
Dow for manufacture is as follows:
1,2,4,5-tetrachlorobenzene is hydrolyzed in a solution of methanol
and sodium hydroxide in water to form sodium 2,4,5-trichlorophenate. This is in turn reacted with sodium monochloroacetate to
form sodium 2,4,5-trichlorophenoxyacetate. The solution is acidified
to precipitate and recover the 2,4,5-trichlorophenoxyacetic acid.
Since 1950 we have been keenly aware of the possibility of a highly
toxic impurity being formed in 2,4,5-trichlorophenol as a side reaction under conditions • of elevated processing temperatures. The
most sensitive toxic reaction observed in humans to this impurity
was manifested by a condition known as chloracne, a skin disorder
mostly prevalent on the face, neck, and back.
It is similar in appearance to severe acne often suffered by teenagers. We also knew that if the impurity was present in the 2,4,5trichlorophenol it could be carried forward to the end product,
2,4,5-T. It is not formed during the manufacture of Dow 2,4,5-T
from the 2,4,5-trichlorophenol, nor does it form on storage even at
high temperatures. To avoid the impurity in 2,4,5-T it is necessary
to keep it out of the 2,4,5-trichlorophenol.
Our early control test was a bioassay. This consists of applying a
solution of the material to the inner surface of a rabbit's ear and
observing for the typical skin response described in a paper published in 1941 by Dow scientists. I wish to insert in the record at
this point the paper entitled "The Response of Rabbit Skin to Compounds Reported to have cause Acneform Dermatitis," by E. M.
Adams, D. D. Irish, H. C. Spencer, and V. K. Rowe, published .in
Industrial Medicine, January 1941.
Senator HART. It will be printed.
(The information follows:)
�363
362
The Response of Rabbit Skin to Compounds Reported to
Have Caused Acneform Dermatitis
E. M, ADAMS, D. D. Inisii, H. C. SPENCER.
and V. K. ROWE,
Biochemical Research Laboratory,
The Dow Chemical Company
Midland, Michigan
HOSE of us acquainted with the industrial lion of some hundreds of test substances arc
field have recognized the need of an experi- easily arranged according to type.
Certain of the strongest irritants produce a
mental method lor studying skin irritation.
We would profit greatly by knowing the potential rapid destruction of the tissue (necrosis), without
skin hazards of a substance before it is put into the skin having an opportunity to show an active
use; we would be able to take proper precautions response. Irritants with milder and slower actions
In the cheapest and most satisfactory manner and lhan this have some effect upon the tissues, as •
result of which we see certain responses on tinmany undesirable incidences could be avoided.
In the literature there arc many instances of part of the tissue. Most irritants have resulted
irritation tests upon the skin of animals, but ap- ir responses in the rabbits' skin which tend l">
parently there has not been a comprehensive develop rapidly and to subside in a short time.
which
study. In an attempt to develop an experimental • This relatively rapid response, reaction, we have
may inmethod, we began about six years ago to study the termed a simple irritation or
the severity, any of the
responses of rabbits' skin to various types of sub- clude, depending upon congestion, inflammation,
following: hyperemia,
stances. We considered the possibility that if • exfoliation, edema, blistering, sloughing, exudaenough were known of these responses to different tion, crustation, necrosis, induration, hair loss.
types of compounds, particularly to those with
may
which there has been considerable human experi- Microscopically one edema,see hyperemia, congesblistering, leucocytic
'ence, then these responses could be organized to tion, hemorrhage,
infiltration, sloughing, and various degenerative
form the basis of an experimental method.
Acneform dermatitis, characterized by such changes.
One type
has been
lesions as folliculitis, comedones, nodules, papules, ever, which of response somewhat observed, howa
longer interval
pustules, and inflammatory changes, has been re- in which to requires apparent, and which has D
become
ported arising from exposure, to quite varied sub- much more prolonged course. This latent reaction
stances including petroleum oils and greases, is a proliferative response which may possibly
shale oil, paraffin, zinc oxide, chlorine, tars, occur in any of the structures of the skin, but that
pitches, chlorinated diphenyls, chlorinated naph- about which we are particularly concerned now
thalenes, and crude chlorinated phenols.'1 '• '• '• '• '•
is epithelial hypcrplasia, with its resultant thickI. 8. 10. 11. IS, H. 15. Jfl. IT. IS. 10, 20, U. 13, It. IT. !l. 10, 90. 33. K. !H.
ening of the skin, follicle enlargement and si1™ The recent occurrence in this country of such
l a 1 8 t UB
nil acnct'orm eruption, '• "• '
sometimes called quellae.
Naturally responses vary to some extent, anil
"chloracne," lias attracted particular interest, and we have observed various combinations of these
iv c included in our animal studies five types of reactions, depending upon the substances applied
substances known to cause the reaction. Today to the skin and the intensity of action.
we wish to describe the unusual response of the
For purposes of classification we have arhitarily
rabbits' skin to these materials and to consider its divided the prolifcrativo response into the followpossible significance,
ing five groups according to intensity;
1. Least detectable.
Expevimcntnl Part
2. Very slight. .
,
,
N OUR experiments, materials have been ap3. Slight.
plied to the inner surface of the ear of albino
4. Moderate.
rabbits and to the shaven belly. The undiluted
5. Severe.
materials have been used as well as solutions of
While there are naturally no sharp breaks bevarious concentrations in olive oil, paraffin oil
U. S. P., propylenc glycol, ethanol, and water. tween these, and some overlapping occurs, dibrcn very
Liberal applications were made on the ear with- vision was rather easy and hashi/jiorplasia: useful.
Least detectable epithelial
This
out any covering. The applications on the ab.degree of response is manifest as an increased
domen were made in a small cotton pad which prominence of the hair follicles on the inside nf
was covered by a large bandage of filter cloth
held in place by adhesive tape. Applications were the ear. The little dots t h a t one sees on the insirip
made mice a day. live days a week, for four of the car simply become slightly larger. After
exposures arc ended this enlargement regresses
weeks or u n t i l a marked reaction resulted.
The responses obtained following the applica- in a short time, leaving the skin apparently normal.
T
I
lium cracks and lifts off in la^^fiioci-s like porThis degree of response is commonly seen as part tions of a cast. Often beneath these is a soft,
of a mild simple irritation which is maintained cheesy, foul-smelling material, which soon drcs
by repeated exposures. Thus far we have been and comes off revealing a markedly exfoliating
unable to attach a particular significance to this skin beneath.
The exfoliation often has a granular consistency
intensity of reaction.
Venj slight epithelial hyperplasia: This reaction at first, which later becomes flaky. There is a
appears on the ear as a slight enlargement of the complete hair loss.
hair follicles, which protrude and become hard,
This proliferation of the epithelium seems to
causing the ear to feel rough. The thickness of progress only to a certain extent, even with rethe ear may be increased. A very slight scaly peated applications of thi1 provoking agent. The
exfoliation may accompany this degree of re- slowness and persistence 'Of this latent reacli-in
sponse, but seldom is there any detectable is to be emphasized. Thr maximum of a severe
hyperemia or hair loss. On the abdomen one sel- hyperplasia usually has occurred in the neighdom sees any gross evidence of hyperplasia.
borhood of two weeks, the largest amount of exSlight epithelial hypcrplasia; In this reaction foliation around four weeks, and a scaly exfoliation
the ear increases in thickness to about twice and hyperemia have persisted for months.
normal and feels slightly stiffened and "leatherAlthough wo make exposures upon both ear
like." There is some hyperemia, scaly exfoliation, and belly, the skin of the car appears to respond
and hair loss. The hair follicles become slightly in the most satisfactory manner. There the mildest
enlarged, raised and hard. On the abdomen there reactions are more apparent and the enlarged
may br a slight thickening ol' the skin and an ex- follicles are more easily seen. As a rule the abfoliation, but enlargment of the follicles is not dominal skin shows a more marked simple irapparent.
ritation.
Moderate epithelial hyperplasia: This reaction
consists of a thickening of the ear to 3 to 4 times Histology
normal as a result of which it is quite stiff and
ICROSCOPIC examinations were made usleathery. The follicles on the ear become modering 10% formalin as fixative, paraffin for
ately enlarged, raised and hard, causing the sur- imbedding, and hematoxylin-eosin as stain.
face of the ear to feel like the coarsest of sand
The slightest hyperplastic response is shown by
paper. After a time the protruding hard masses a very slight increase in thickness of the epithelium
can be easily expressed by the finger-nail or by and the development of small projections (like
bending the ear. At limes the enlarged follicles papillae) of but a few cells in size. The early
are not apparent until after considerable exfolia- stages of more severe responses show increasing
tion has occurred. A moderate hyperplasia is degrees of thickening of the surface and follicular
usually accompanied by a slight to moderate epithelium. Numerous projections reach downhyperomia. Exfoliation of a granular or scaly type ward from the surface epithelium, nearly to the
is of modenili' intensity and hair loss is nearly cartilage of the ear. The follicular epithelium
complolc. After a number oi weeks the oar is spreads outward and downward, often completely
completely deuuilod ol' hair, slightly pitied, with engulfing hair follicle and sebaceous glands. Apa slight or moili-nitc- hy|,ori'mia and possibly some parently there is also a hyperplasia in the corium.
I'Nt'olialion. Tlu> abdominal skin may show a Accompanying this hyperplasia, one may see conUR'iilrv ximplr irritation than docs, the car; hy- gestion, even occasional h .-morrhagos, edema, and
peromia, edema, mid even sloughing and exuda- leucocytic infiltration.
tion have occurred, llypcrcmia is usually mainLater the rate of proliferation apparently lestained during the course ol' thickening. The ab- sens and those changes resulting in keratinization
dominal skin finally becomes hard and stiff, fol- become more evident. As those changes leading
lowed by a marked scaly and granular exfoliation, to keratinization progress from the lowermost
layer of the epithelium, which constitutes a basal
which persists for weeks.
Severe hyperplaftta:
This reaction is usually layer markedly displaced from the original, largepreceded by a marked simple irritation, including masses of material are thrown off. Thus in one
even necrosis; however, there may be only section of abdominal skin we see a thick layer
hyperemia and edema. As a severe hyperplasia of partly keratinized and degenerate tissue being
progresses, a 1 marked hyperemia is evident until thrown off above a flat, normal-appearing stratum
obscured by the thickened epithelium. The thick- vorneum. At the hair follicles most of the tissue
ness of the ear is increased to many times normal, undergoes complete keratinization, forming the
ears at least 1 cm. thick having been formed. As hard plugs that may be expressed. Completely
a result they become very stiiV, hard, and heavy. engulfed follicles and glands are destroyed as the
Fixfoliation at first has a granular consistency, hyperplastic epithelium is keratinized and thrown
later flaky, and persists for months. The enlarged
hair follicles are buried under the thickened epi- off.
The sebaceous glands have seemed to bo inthelium and become apparent only after con- active. One sees them, apparently normal, being
siderable exfoliation has occurred. From them engulfed by proliferating epithelium. Some glands,
large masses of keratin may be expressed leav- of normal size and appearance, arc scon opening
ing pits that may reach 2 to 3mm. in width.
into the pits or cysts; others arc seen with their
On the abdomen the hardened mass of epithc-
M
43-362 O - 70 - 24
�364
rhicts extending through large masses of keratinized epithelium.
Sections taken at a late stage show an ntropliic
or Very .slightly thickened surface epithelium and
luimorous large pits surrounded by slightly hyprrpliislic epithelium. The curium may still be
thicker than normal.
U l t i m a t e l y there is a tendency tor the pits to
broaden nut and become shallower, mill one sees
n very irregular atrophic epithelium.
Discussion
'"pHERE are certain points which indicate a
JL relationship between this reaction observed
in the rabbit and the acneform dermatitis of man.
First, the reaction in the rabbit was produced by
5 types of substances known to cause an acneform
dermatitis in man. They were chlorinated diphenyls, chlorinated naphthalenes, chlorinated
diphenyloxides, crude chlorinated phenols, and
petroleum oils. A few other types of substances
have produced the epithelial hyperplasia,but there
has been no exposure of these on man. Wacker
and Sohmincke'-' reported the experimental production of epithelial hyperplasia with various oils,
fats, and paraffin, Sachs,2'' and others, apparently,
have produced I he identical epithelial hyperplasia
in rabbits with a number of dyes. In his review
of the pertinent literature, Sachs states that the
most common dermatosis arising from exposure to
aniline and coal tar dyes is eczema; however,
warty growths and acneform dermatitis have also
occurred. Thus it appears probable that the devclopment of an outstanding hyperplastic response
of the rabbits' skin is specific for those substances
capable of causing an acneform dermatitis in man,
and possibly, the related papular and warty erupSecondly, by gross and microscopic examination, the enlarged follicles produced in the rabbit
•escmble the comedones, nodules, and cysts of the
lormatitis in man. In both cases there is a relaively large pit or cysl whose walls are composed
if e p i i h e l i u m and which contains varying ainnunls
if kcralini/.cd epithelium, and at times hair, hair
follicles, nnd debris.
In both the rabbit and in man there is hyperplasia of the epithelium. Proliferative changes
have not been stressed in descriptions of the
h u m a n reaction and probably have not been seen
to a greater extent because tissues were taken
at relatively late stages of the reaction. There are
reports of increased numbers of mitoses and of
thickening of the rete Malpighii. "• '«• »>• H " Prosser White 3 * describes acanthosis in the "primary
papule" and considers one important factor in the
production of oil folliculitis to be the chemical
irritant causing auxetic cell growth. The ability
of tar to cause active mitosis is well known. 1J
Bornemann's 3 first case, examined nt a late stage,
ornemanns rst case, e x a m n e d nt a late stage,
showed more mitoses than normal and slight
thickening; but in his second case, examined at an
earlier stage, the thickening of the epithelium was
much more marked.
Although menlion is often made of sebaceous
cysts in descriptions of the ucnoform ilorm»liH;
only two instances were found of the specific n**
lion of sebaceous glands in doscriplinnti "I ft;
microscopic picture. Jones nnd Alden" rrj»«W#
slight cdemato.iK elianges in n few glands UMf
saw; Curgil and Acton" snid that Hie srlwivw'
glands were unaffected. Bornemann 3 fell that Ikcysts were of sebaceous origin hut udmilliil (l»<
d i f f i c u l t y of proof, and his description shows (I**
to be essentially epithelial structures.
These facts, together w i l h our experimental itsuits, indicate Hint the so-called "sebaceous cj-><«'
of the acneform dermatitis in man are dlricO)?
the result of an epithelial hypcrplasia. Their tjustent of sebaceous-like material is probably tat
to the occurrence of inflammatory and dcgoncr»tive changes in the mass of epithelial tissue. W
course, the retention of sebum may also occur,
and influence the picture to some extent, but Iba
appears to be a secondary reaction. Bacterial infection may be a factor influencing the imtuif
of the reaction.
Conclusions
,rr,n-n
'PHERE have been a number of hypotheses con*~ cel'ni"S 'he formal ion of this iicneform cru|>tlon
W(1 fed lhi
'" man '
" evidence shows llm
acllefo
™ dermatitis to be :he visible response »<
the skm to a
" '"•'la"! acting upon it from UKexterior, and that I his response takes the form i)(
first
epithelial hyperplasia, second inflammalorjr
and
degenerative changes, and finally regencralive processes.
And in conclusion, it is possible that this aj>parently unusual response of the rabbits' skin offers us an experimental method which will indicate the ability of substances to produce an
acneform dermatitis in man.
Bibliography:
1. BEVTMANN: "Chtor-alu.e.' 1 oinc bcKoiidpir Form von
pi-o/essinriollE'r Itnulcrknmkunij, Dc'lif, mrd. Wwhlixflir.
27, 437-440 (1001).
2. BI.ASCHKO: DcrtaaM. Xi'il. In, 70-72 (1011).
3. BOIINEMANN. W.: Ucbor <lio Ilislnloiilc dcr Chlorncne, Arrli. /. Di'rni. n Smth. 02. 75-80 (1002).
4. BUTTl.EH. M. G,: Acnrfon.) IJcrnuilnsis Produced by
Ortho-(3-Oil»ro|iln'i.yl) Phoiml Kndluni and TVIrncl.lorphenol Sodium. Arch. limn, nilrt S|//.lil/. 35, 251-254
(11)37),
5. Coi'irrois-SiimT, TOUHAINE KT MKimiKl.: Eludr
•.ur rint.ixic'ntion prof.'ssioiin.'llo pnr IP Irirhlont-nfiphIhnlene, Attualn de nit'd. h'f/. de crhttln. 14, 422-427
(1934).
6. CVBGII,, D, F., nnd H. W. ACTON: Jute Dermntllis,
lildion J. Med. Res. 12, 257-201) (1924-5),
7. DimoN, W. F\: Petroleum Dennntitis, M«dirnl Record, 140, 650-552 (1834).
8. DUVOIB, M.I ApropuH des d'^'mnloses protesslnnnelles par le triclikirniinpMhiili'Mr. Aintuli'x ik> tnpd. Itg.
de rrlnilii, H, 539-544 (11134).
9. FUI.TON, W. H.. nnrl J L. MATTIKWH: A Prcliminury
Report ot Iho DormnliiinKkiiV ii'nri "sysinnic 'rcM"e'd's""af
Exposure to Hc.vHchlom-miplifhjiicne nnd o.lordiphenyl,
D
Pennn. Di'pl, '........".....' H u l l..... 43 (1D30V
°"-" ""•" I-nbnr, Sprrinl " , No, ~
10. HrnxiJLiMKi?. K.: Urhpr C'hloi'nUnt'. A/iiiirrln'ii. Mcif.
Woelniiichr. 46, 27fl (1890).
H; JAMIJON: Trcniinc-ni rif rOcxcmn with ('mil Tnr, Ann,
rtc dernt. cl de suph,, .Inn,. 1900, pngo 22. Quoti-d by R.
365
The Dormnlcrg.ises. H. K. Lewis iiWVu., Ltd., I/ondon,
1034.
^5. SACHS, (V: KliiiiKcho und oNiM-rlmentelle Unlcrsuchunt,'.'ii iK-bci- die EU.wirkunfl vtm AnilinfnrbsttjETen nut die
r.inisrliliclii 1 und Tieiisi'lle Hunt, ^rclt. /. Dvrtn. u. Si/ph.
I in. 555-684 (1913).
26. SroTT. A.: The Occupuliunal Dcrmaloses of the
I'urnltiii Workers (if the Scottish Shnle Oil Industry, Brit.
Med. J. 2, 381-3B5 (1922),
27. SKZAHY, P. VAI.I.KBV-HAIKIT, and BENOIST: Mclanose
' ("MACKENZIE, S.: A Case ol Tnr Eruption, Brit. J. de Riehl, bullion d'huile, hypcrkdrntose folllculnlre chez
un ouvrler tuumeuv f:ur mutiuix, Bull. aoc. ftdnf. dp.
tttim. 10, 417 (1898).
, 18. MAVEBS, M. R., and M. G, SiLViBBF.no: Skin Condi-, rtcnunt. el d.' »|/nh. 34. 139-143 (1927).
Won, Resulting from Exposure to Certain Chlorinated
211. SULZBEBGER. M. B., A. ROSTENBERCJ, J R . , i.nd J. J.
Hydrocarbons, J. Indiut. H|/». * To.t. 20, 244-258 (1938). RFIEH: Acneform Eruptions, N. Y State J. Met. 34, 899-908
17. N.COI.AS and J. LACASSAONE: Un^eas d ««"* ?hlOT"
(19341.
29. TEI.EKY: Die Pernnkmnkhcit, Klin. Wrhnichr. 6.
<fu, Bull. sac. Jrtnic. tlcrmot, el Sl/pll. 36, 223 <!»'»>•
IB. NOC.UEH-MORE, S., and M. C,BA«-BAHBraA| ContriJ'u- 845-8411 (1927)! 6. 897-901 (1827); 7, 214 (1928).
30. TOTSUKA, R.: Study o( the So-cnlled "Oclkrntzo,"
Ikm ft I'ctude des polkllodermles t. propos do trow CM OL
Pi-oduL-rd by IhQ Induslvinl Use of Mineral Oil and Turpcnlh.e, Jn|i. Xeil. f. Dorm. ... l/rol. 17, 395 (1917). Ab19 OOSTON, O.: On the Local EtVocls ot Crude Paraflin, stnii'lod in Brit. J, Dormnlol. 29, 227-228 (1917).
31. TounAlNE, SOI.ENVB. MKNr.TRB... nnd AUBRUN: Clqu, Idhi M.'d J 17. 544-547 (1B71-2).
%
O rEN,i,IM, M.: Aftcctinns ot '*« Skin Cjiujed * iintp-quatiT ens d. di-rmntilis pjir Irtchloronnpl.Oiulrne,
Hmipnllon nnd P.-ofosaion, The Mcdlcnl ]Vw» 169 (N1I8>, fli.H. tar. tram: de derm, el A? svph. 41, 265-208 (1934).
32. TUHNEII, ,T. A.: An O::cupntionul Dermntoco..iosis
"il" iC" C «., nnd L. n. Bmhndl: Oil Folllc-ulllis, J. iimimK Zinc Oxide Workers, U. S. Public Hcnlth Reports
:16. 2727-2732 (1921).
33. WABBE. W.: A Cose of Oil Folllculitls, Bril. J. Dcrinntiil. 12,212-213 (1900).
34. WHITE. R. PBORSEH: Tbe Dermatcriioses, H. K. Lewis
nnd Co., Ltd., Ixjndon, 1934, pp. 195-241.
' 35. Occupation nnd Henlth, lnlcrnation.nl Labour Office,
, H. C., nnd J. W. O,or««: The Problom o£ the . Genevn. 1930. Sec Cl.lorlne, Con'. Tars. Paraffin. PetrolOwwoi-ks PUch Industries and Cancer. The J. H. Me- ' cum Oils. Pitch, Shale Oil Industry.
Fedden Researches, 1913. Quoted toy R. PBOSBEH WHITE,
IWcr Wliitc, The Dcrrmitoi-BOsoB, H, K. Lewis nnd Co.,
IM, Inrndon, 1034.
_,
IJ JONES, J. W.. and H. S. AU.EN: A n .^;»j'"7" ° trm •IrrjMls, Arch. Derm, nncl Suph. 33. 1022-1034 (1036 .
13 I.EWIN. b.: UcbC'i- nllgomelno und HmitvcrMitlun*
*nch I'cti-oloum, Virclwiu's Arch. 1. Palliol., Annlonur u.
f»»,. 112, 35-119 (1888).
H. MCEWEN: Acne due to Tiir, J, Cut. DH. 35, 1153
�367
366
Dr. JOHNSON. Thank you.
-:;m
THE DOW CHEMICAL COMPANY
MIDLAND, MICHIGAN
48B4O
~~. — r^ulJCU uiuoracne and our bionssav ¥
"*rs&?fflri^r^&?rtf
by incineration S
ANALYTICAL
.June 22, 1965
METHOD
MLW.65.11
THE DETERMINATION OP 2,2,7,8-TETRACHLORODIBENZO-p-DIOXIN
IN 2,4,5-TRICHLOROPHENOXYACETIC ACID BY
GAS-LIQUID CHROMATOGRAFHY
. We confirmed
to the point where
method for the
P.p.n, in
Dow Chemical Co.
Soaator HAHT. It will be printed<
( I he information follows :)
'
1.
by early' 1905 |Scope
as chromatographic
This 'method i's applicable to the determination of 2,3,7,8-
tetrachlorodibenzo-p-dioxin in 2,4,5-trichlorophenoxyacetic acid.
The dioxin can be detected at the one ppm level with a lower
limit of 0.5 ppm possible at optimum operation conditions.
_, 2,4,5-tri- .,w
Ohromatography," by the ,si
fl
'-B
Principle
The 2,3,7,8-tetrachlorodlbenzo-p-dioxin is separated from
the 2,4,5-trichlorophenoxyacetic acid by means of an extraction
with chloroform. The chloroform extract is concentrated and then
chromatographed. The 2,3,7,8-tetrachlorodibenzo-p-dioxin in the
sample is measured and compared to a known standard.
3. Safety Precautions
2j3j7,8-Tetrachlorodibenzo-p-dioxin is capable of causinp a
severe .delayed skin response (chloracne) upon minimal contact.
Samples suspected of containing any of this compound should IK-handled so aa to prevent all skin contact and inhalation. Wealimpervious gloves (rubber, polyvinyl chloride, etc.) at nil times
when contact is a possibility. Clean all equipment with acetone
followed by a chloroform wash. Dispose in such-a manner as to
prevent all skin contact, any potentially contaminated equipment
or materials which are not readily cleaned with chloroform, i.e.,
towels, gloves, etc.
ty. Apparatus
(a) Gas chromatograph, Aerograph A-600-D with flame
ionization detector, Wilkins Instrument and Research, Inc.,
Walnut Creek, California, or equivilent.
(b) Recorder, -0.05 to +1.05 millivolt, full span, one-second
full response time.
(c) Syringe, Hamilton microliter. No. 701N, or equivalent.
(d) Syringe, Multifit 5 cc, Becton, Dickinson and Company,
or equivalent.
(e) Syringe, Yale 1/i) cc, Becton, Dickinson and Company,
or equivalent.
�368
369
(f) Centrifuge
•: •}:
(g) Injector 'Insert, Pyrex glass for A-600-D. Available
•••-•• —
."3
vo/
from Wllkins Instrument and Research, Inc., Walnut Creek,
' ,j
California (Note lla).
\v;
(h) Column, 1/8-inch O.D., 0.081-inch I.D., stainless steel .-*,?
;
tubing, five feet in length .packed with reagent 5(c).
;y
5. Reagents
(e) Add 25 ml. of I N sodium hydroxide to the chloroform
txtract and shake for 15 minutes (Note lie) .
(f) Centrifuge for five minutes. ••
.
(g) Using a five-milliliter syringe, draw off as much of the
Bottom chloroform layer as is possible into a small vial. Note
Mils volume.
. •
(h) Evaporate to dryness in a hood.
(i) Take up with chloroform to 5-0$ of the volume noted in step
ig) . This final solution represents ten grams of sample per ml,
of chloroform.
(j) Inject 1.0 microliter into the chromatograph and measure
the response of the 2, 3,7,8-tetraohlorodibenzo-p-dioxin . Figure II
•hows a representative chromatogram.
fa) Solid support, Chromosorb W, 60/80 mesh, Johns-Manville. .:;
-:•
(Note (b) Partitioning agent, SE-30, Silicone gum rubber-methyl
lib).
(c) Column packing, five percent by weight of SE-30 on 60/80 ';;
mesh Chromosorb W. Available from Wilklns Instrument and Research*]'
Inc., Walnut Creek, California.
•-}?.
d) Carrier gas, nitrogen, commercial grade.
. .
%; 9. Calculations
e) Chloroform, ACS grade,
' ,|
f j 2,3 J 7,8-Tetrachlorodibenzo~p-dioxln, available from
+':/!
Let:
•• •
The Dow Chemical Company, Midland, Michigan.
.;-•:'
A = The area of the 2, 3,7,8-tetrachlorodibenzo-p-dioxin in
(g) Sodium hydroxide, 1 N solution. Dissolve 40 grams of
\\i
reagent grade sodium hydroxide in one liter of water.
'•*
the sample.
6. Chromatographic Conditions
B - The attenuation of the chromatograph for the sample.
Oven temperature, 225°C.
Inlet temperature, 260°C.
( c j Carrier gas flow rate, 75 ml. per minute as determined
by the moving soap bubble technique,
(d) Attenuation, such that a response of at least 50$ of
scale is obtained from a 1.0 microliter sample of a standard
containing 100 micrograms of 2,3.>7,8-tetrachlorodibenzo-p-dioxin
in one milliliter of chloroform.
7.
C = The .micrograms per milliliter of the 2, 3,7,8-tetrachlorodibenzo-p-dioxin In the standard.
D = The area of the response from the 2,3,7,3-tetrachlorodibenzo-p-dioxin in the standard.
E = The attenuation of the chromatograph for the standard.
Preparation of Standard
(a) "Again read Section 3(b) Weigh, using a micro-balance, one milligram of 2,3,7,8tetrachlorodibenzo-p-dioxin into a ten ml. volumetric flask.
(c) Dilute to the mark with chloroform.
(d) Inject a 1.0 microliter sample Into the chromatograph,
See Figure I for a typical chromatogram.
8. Procedure
'a Weigh 10.0 grams of the sample into a four-ounce bottle,
b Add 20.0 mllliliters of chloroform and shake for one hour.
c Place the solution in a centrifuge tube and, with
proper balancing, centrifuge for five minutes.,
......
(d) Using an eye-dropper, draw off as much of. the clear
chloroform layer as possible into a two-ounce bottle.
Thon.
li pin of.' 2",3,7,8-tetrachlorodibenzo-p-dioxin = nA X, X,?B x,?
\J • c.' X
Xv.'
m
10, Accuracy
The accuracy of this method is - 5$, or less, relative,
11,
1,'otes
(a) Glass inlet liners have been found "o be necessary to
provide reproducible results.
�371
370
FIGURE I
(b) Silicone FS-1265 ( f l u r o ) has been f o u n d - t o work well as
a stationary phase. It also is available from Wilkins Instrument
and Research, I n c . , Walnut Creek, California.
(c) Any 2,^.,5-trichlorophenoxyacetic acid which has dissolved
in the chloroform extract must be removed as it will interfere with
the chromatographic analysis of the 2,3,7,8-tetrachlorodibenzo-pdioxin.
Attenuation 1 x 1
2,3,7,8-Tetrachlorodlbenzo-p-dioxin
114.4 mierograma/ml.
# * # * * # # # # * * *
The analytical procedures given herein have been adapted from
literature sources or developed upon the basis of experimental
data which are believed to be reliable. In the hands of a
qualified analyst they are expected to yield results of sufficient
accuracy for their intended purposes. However, The Dow Chemical
Company makes no representation or warranty whatsoever concerning
the procedures or results to be obtained and assumes no liability
in connection with their use. Users are cautioned to confirm the
suitability of the methods by appropriate tests. Anyone wishing
to reproduce or publish the material in whole or in part should
request written permission from The Dow Chemical Company.
0
Start
4
6
8
Retention Time (minutes)
�372
373
Dr, JOHNSON. Thank you.
Attenuation 32 x 10
FIGURE II
Attenuation 1 x 1
2,3>7 ( 8-Tetraohlorodibenzo-p~dloxin
2.5 ppm
v
0
Start
Attenuation
32 x 1
2
Retention Time (minutea)
Senator Hart, the 2,4,5-trichlorophenol plant was redesigned to
insure, insofar as possible, the production of a product containing a
minimum of the tetrachlorodibenzo-p-dioxin. By so doing we were
able to control the quality of Dow 2,4,5-T.
By May 1965 we had the technology to establish a manufacturing
specification of no detectable 2,3,7,8-tetrachlorodibenzo-p-dioxin in
2,4,5-trichlorophenol and 2,4,5-T, using an analytical method sensitive to 1 p.m.m. While the plant was being rebuilt, we purchased
2,4,5-trichlorophenol and 2,4,5-T on the basis of this specification.
The now plant came on stream in 1966 and since that time Dow
2,4,5-trichlorophenol and 2,4,5-T have met this specification, and
most has contained less than 0.5 p.p.m. of the 2,3,7,8-tetrachlorodibenzo-p-dioxin.
• I apologize for repeating these long chemical names but the position and number of chlorines is important.
Senator HAUT. The day will come when I can pronounce them,
even if I can't understand them. I can't do either yet.
Dr. JOHNSON. When the difficulty was encountered in 1964 we
notified the Michigan Department of Health, the Institute of Industrial Health, University of Michigan, and various other health
oriented individuals in private medicine and industry.
In addition we called a meeting which was held in March 1965 to
notify other manufacturers of 2,4,5-T of the difficulties encountered.
We described to them the nature of the health hazard and shared
our test procedures and analytical standards.
With this background—and firsthand experience—it was only
natural that my associates and I would inquire about the identity
of the sample used for the Bionetics tests. The 2,4,5-trichlorophenol
tested was Dow material and the 2,4,5-T was a Diamond Alkali
sample.
It is important to emphasize that 2,4,5-trichlorophenol was reported to show no significant increase of anomalies by the Bionetics
Laboratory, but the sample of 2,4,5-T did display a significant increase of anomalies.
This prompted examining our past records of tests run in 1964.
The records of analytical determinations of different supplies showed
that samples of Diamond Alkali 2,4,5-T in fact did contain tetrachlorodibenzo-p-dioxin up to levels of 16 ppm. It should be emphasized at this point that Diamond Alkali has since stopped manufacturing 2,4,5-T.
I presented the essence of the above information to the Mrak Commission November 7, 1969, and showed pictures of the chloracne
observed in humans and pictures illustrating the rabbit ear test.
Moreover, I stated that the Bionetics test with 2,4,5-T may have been
complicated by an impurity in the 2,4,5-T. •
I further emphasized the importance of tests using procedures
recognized among experts as being valid and meaningful; the importance of representative materials which could be better obtained
by consultation witli industry; and the importance of knowledge.of
composition and purity of the materials tested. These points were
made in the course of writing the final draft or recommendations of
the Mrak Commission.
�374
In view of ,
2,4,5-T and the «.«,
defects in cattle or &u
we found it difficult to UBUBV
the registered uses of 2,4,5-T.
375
I'vtAformation has been supplied primarily through Dr. Burger of the
'*
rfiii'fi
°f rreased incidenco
rangelands s ra ed
P y
On December 11, 1969, Dr. V. B. Robinson and V. K. Rowe met
itli Drs. Falk, Courtney, and Gaylor at the Research Triangle and
'^tscussed with them the design of teratological study to be conducted
T+ i
•
jT
' '" ""ra :
6
,i) *tt Dow regular production 2,4,5-T. Agreement on the design of the
as
iSjBperiment was easily achieved and was followed in our studies.
tested
Bionetics
taminated
|if At this meeting Dr. Courtney of the NIEHS Laboratory provided
if
the
If 4 two gram sample of the 2,4,5-T used by the Bionetics Laboratory.
BThis sample was examined at Dow with the following results:
I?' L Rabbit ear tests showed a positive reaction characteristic of the
|4' contaminant.
if" 2. Analysis by gas liquid chromatography indicated the presence
for tests which would be z,c™r,tM« f theirB sclenfcisdisc"ss protocol I Of 27 plus or minus 8 p.p.m. of 2,3,7,8-tetrachlorodibenzo-p-dioxin.
A • < v v . to
asked Dr. McLauo-hlin of S?M
ts- Dr. LimMj |K In late December Dr. Burger of the OST requested a review of the
^bera, l^lr^^t^nSg1^^ ^ ™ ^1 i-dicmistry of 2,4,5-T production to be presented to Dr. BaldeschDrD J.^SSSiS^Jft^J?^ of a? fational Cancer Instill |weiler, a consultant of the agency. The information for this report
k'Wiis organized by Dr. Blair of Dow and presented at a meeting with
lister;
flio OST on December 29, 1969 in Washington.
''". Dr. Blair is on my right.
;•••' By January 12, 1970, we had made enough progress in the teratological study in rats with Dow production grade 2,4,5-T to make a
would bo tli
,.-.' report to Dr. Egeberg, Assistant Secretary for Health and Scientific
that, for {lit if
Affairs, HEW. Copies were sent to other involved persons in DHEW
report showed that the
the kforlrL^ Sf^r? ?, fSory experiments m' r and USD A. Thisdid not cause birth defectsDow 2,4,5-T of regular
production grade
as determined.by gross
Health
in tlle
tories of a third'party ?iX^ ^ ^
>
labom-.^ examination of fetuses. The dosage levels used were selected in conG
nment or Dow o r
in the Dow laboraE o^ToS °J °r
) fl
Falk, Courtney, and
nel of the Department PIEW °bservatlon afc any time by perW"4 sultation with Drs. were able to report toGaylor of NIEHS. a pilot
Furthermore, we
Dr. Egeberg that
study with pregnant rabbits fed the same 2,4,5-T had not caused
it
0
h>
™
birth defects. Dr. H. L. Richardson, pathologist, FDA, observed the
results of both of these tests.
T -i
*>-***w ouuu.j' yieiueu. DOS
These preliminary observations were followed by the more time
consuming microscopic examinations of the tissues and detailed
ifc
id
necess-aryt'rmi feKesS *« Jffl ,™ ^^ ™' ^
skeletal examinations, This work confirmed the preliminary findings.
and on refined 2 4 5 T Ttl? #raded levels of the contaminant
The final report of the study was presented before the Society of
Dow wo Id
samples of 2,4,5-T and 2 S 7 s /f^ll ^
» P^vide
Toxicology in Atlanta, Ga., on March 17, 1970. I wish_to insert into
to
National Institute of^vSS^ff^Pfl°™
«'«
nmental Health
the record at this point an abstract of this report entitled "TeratoResearch Triangle N 0
Science laboratories at
genic Study of 2,4,5-trichlorophenoxyacetic Acid in the Rat" by J.
L. Emerson, D. J. Thompson, C. G. Gerbig, and V. B. Robinson, The
laboSTesT:?^rl^Sl^f^ conce ld ^ ^ ™HS
™
them
Dow Chemical Co.
the test methods to be used
™ing tlie details of
Senator HAKT. That will be received.
D rid and B k
(The information follows.)
h 5f
A study to determine the embryotoxicity or teratogenieity of 2,4,5-triicholorthe sample tested by Bionetics onr? P]°SS1?1lhfcynfo a contaminant in
ophenoxyacetic acid containing less than one part per million of 2,3,7,8Dow. At this meetijo- the samp ^ f the \ ™^^ known to
tetrachlorodihenzo-p-dioxin has been completed in Sprague-Dawley derived rats.
8
as
to the Mrak Commfeion Thl ^CfJTi^ ^
Panted"
Five treatment groups, each consisting of 25 females were administered 1, 3, 6,
for
cussed with Drs.1HCJjau hIm B nf
McLauo-hli^ 7?
additional testing as dis12 or 24 mg/kg/day of the compound via gavage in 0.25% METHOCEL® on
also presented.
S > ates, and Mitchell of DHEW was
days 6 through 15 of gestation. A single group of 50 females received the sus.
practical hazard existed
^offiTi^T ^?.^
of »Tt'•apS'tiroflS ?• ^ ? *• - %
6
nterested in M
for^atFo^lfdotltKd'^T-n'" i
- «^hen it was availabl,?^:?^^^^^
pending vehicle and served as controls. The following parameters were examined: clinical observations, maternal body weights (prebreedtng and day
20), number and position of fetuses and resorptions, number of corpora luten,
imp weight and aex, gross external examination of pups, and macroscopic
examination 'for intestinal hemorrhage in pups. Two-thirds of each litter were
fixed in Bouin's solution and one-third was prepared for alizarin red-S staining
�376
377
;
ff
and skeletal examination. Examination of Wilson sections under the clissortktB,
microscope or of alizarin stained skeletons of all fetuses from the 24 mg/kg/dWf'|
group and an erjual number of control fetuses was performed. KepresentfttW f
stained lustologic sections through the head, thorax, and abdomen of 10 conltsi
and 10 high level fetuses were studied for histopathologic changes.
No clinical or gross pathologic signs of adverse chemical effect were o
in treated dams during the period of treatment or gestation. Similarly
size, number of fetal resorptions, birth weights and sex ratio of pups tipixw
to be unaffected by chemical treatment. Skeletal and visceral examination *«
high level and control fetuses as well as histopathologic examination of certain H
fetuses failed to reveal any terntogenic or embryotoxic effects.
•!
The results of this study fail to substantiate the findings reported reecnll}••':j
(unpublished data: Bionetics Besearch Laboratories, Bethesda, Maryland) «f>>
serious effects in fetuses obtained from dams given comparable daily doses of
2,4,5-T containing approximately 27 parts per million of the contaminant'
2,3,7,8-tetrachlorodibenzo-p-dioxin.
• , '
'
".,
Dr. JOHNSON. Thank you.
'.'"
In accordance with the plan discussed with the DHEW in Decent
her, as soon as the preliminary results of the 2,4,5-T study on rat*
indicated no fetal anomalies, we proceeded to conduct a teratology'
study in rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dosages were
used which bracketed the levels of the contaminant which were given ;
inadvertently to the rats in the Bionetics study. The results of this
experiment indicated that a high of maternal and fetal toxicity WKS,
associated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Dr. H. L. Richardson of FDA and Dr. C. T. G. King, National Institute of Dental
Research, NIH, participated in the observations made on.these
animals at necropsy at the Dow Laboratories in Midland.
We concluded that the presence of the tetrachlorodibenzo-pdioxin in the sample tested in the Bionetics Laboratories could have
accounted for the observations reported and attributed 2,4,5-T. At
this point I wish to insert into the record an abstract of the report
as presented to the Society of Toxicology, March 17, 1970, Atlanta,
Ga., entitled "Teratogenic Study of 2,3,7,8-Tetrachlorodibenzo-pdioxin in the Bat"1 by G. L. Sparsc'hu, F. L. Dunn, and V. K. Howe,
The Dow Chemical Co.
Senator HART. That will be inserted.
Dr. JOHNSON. Thank you.
The detailed results of these tests were also presented by Dow
personnel to the scientists of FDA, NTH, NIEHS, in Washington
on February 24,1970.
In addition to our investigations with laboratory animals, we have
also utilized the medical records of Dow employees accumulated
throughout their Dow careers.
Our physicians have made an in-depth evaluation of the health of
male employees who have been exposed to 2,4,5-T in manufacturing
operations for from 6 months to approximately 20 years. From the
medical data available, over 50 clinical parameters were selected for
statistical evaluation. The control population for this evaluation consisted of 4,600 other individuals for whom similar data were available.
After careful study of this information, it was the conclusion of
our medical staff that there was no evidence that exposure to 2,4,5-T
had resulted in adverse effects.
1
Scop. 470.
It is our belief that the adverse effects reported by the Bionetics
•Laboratories were the resivit of a contaminant—2,3,7,8-tetrachloro<libenzo-p-dioxin—and were not caused by 2,4,5-T.
Moreover it is our belief that 2,4,5,-T produced under specifications
requiring less than 1 part per million of 2,3,7,8-tetrachlorodibenzo;. |Xlioxin present no practical hazard when used in accordance with
good agricultural practices,
; Now, from here on it is historical.
', If I may I would like to add some recent information and this
like the information presented this morning, was only obtained this
past weekend, as a matter of fact.
The Midland Labs extended the work by Dr. Robinson using the
Dow 2,4,5-T on rats. This is the production grade of Dow 2,4,5-T.
The dosage of 50 milligrams per kilogram, higher than that recom1
mended in the consultation at NIEHS, was administered. At this
dose we found one fetus in 203 viable fetuses with intestinal hemorrhage. At the 100 milligram per kilogram dose level; there was 75
percent mortality of the dams and high fetal mortality.
:
At this point I should point up that these levels of 2,4,5-T put
extreme stress on the physiology of the test animal.
' The next experiment involved drinking water supplied to rats.
This drinking water was saturated with 2,3,7,8-tetrachlorodibenzop-dioxin and in this case no effect was detected in 21 litters of 259
pups.
In a third experiment we studied combinations of pure 2,4,5-T
with 2,3,7,8-tetrachlorodibenzo-p-dioxin. In this experiment 2,4,5-T
was fed at 50 milligrams per kilogram and maintained at a base
level throughout all tests to which graded or incremental dosage of
additional 2,3,7,8-tetrachlorodibenzo-p-dioxin were added. Bear in
that this base amount of 2,4,5-T was administered orally in a single
dose each day.
I will read the list of results. When 50 milligrams per kilogram
of pure 2,4,5-T were administered with zero added 2,3,7,8-tetrachlorodibenzo-p-dioxin, no adverse effects were observed in rats.
When one-hundredth of a micrpgram per kilogram of the tetrachlorodibenzo-p-dioxin was combined with the 50 milligrams of
2,4,5-T, again no effect was observed. When three-hundredths of a
microgram per kilogram per day of the tetrachlorodibenzo-p-dioxin
was combined, again no effect was observed. When six-hundredths of
a microgram per kilogram of the tetrachlorodibenzo-p-dioxin was
combined with the 2,4,5-T, we observed 8 percent of gastrointestinal
hemorrhages and one cleft palate in 155 fetuses. When 0,125 micrograms of the tetrachlorodibenzo-p-dioxin was combined we had an
increase in the resorptions and some gastrointestinal hemorrhage.
We also saw subcutaneous edema in three of the 134 fetuses.
When five-tenths and 1.0 micrograms of the tetrachlorodibenzo-pdioxin were combined with the base level of 2,4,5-T, we observed an
increased incidence in the fetal mortality, in resorptions,' in gastrointestinal hemorrhages, and in subcutaneous edema, cleft palates
were observed in four out of 14 and five out of 15 litters respectively.
Again I should stress the base level of 2,4,5-T was extremely high,
approaching the toxic dose; moreover the base level of dioxin was
extremely high.
�«
379
378
Dr. JOHNSON. The simplest way to think of it is one^ath of the
here any further comment to be added on that, Mr. Rowe?
weight of a grain of sugar over a lifetime. Senator Ha^^T am not
ROWE. I think you covered it very well.
sure how you would like to proceed. I have some other remarks to
Dr. JOHNSON. These data were only available yesterday. The
make which get to tins question, perhaps in shortening the time inmicroscopic study of soft tissues and skeletal study have not been
terval. Would yoxi prefer for me to answer questions now or at a
done. These animals were stressed to the limit of 'their tolerance of •
2,4,5-T and, in addition, to a toxic stress of the tetrachlorodibenzo-p- 1 later time?
Senator HART. I have a few questions. Suppose we ask a few
dioxin.
questions here and if they do not raise items that you intended to
I personally feel it is important to consider the dosage related
discuss, we would welcome the additional reaction.
to the response with these materials and later I will get into a more
One very quick one has to do with the test that you report having
definitive discussion of that, if you desire.
made on 110 male employees. I am sorry, 130 with no evidence of
Based on the proposed finite tolerance of 2,4,5-T in food but less
adverse effect.
than the 1 part per million of tetrachlorodibenzo-p-dipxin in the
Has there been any evaluation made of the effect on female em2,4,5-T the safety factor for humans as derived from animal studies
ployees or were any exposed ?
in several thousandfold. This is well in excess of the safety factors
Dr. JOHNSON. None were exposed, Mr. Chairman.
judged adequate by toxicologists in some branches of government
Senator HART. Was that on purpose consciously ?
and many others in the scientific community.
Dr. JOHNSON. No, the manufacturing plant is not a desirable
I would like to make a few comments, because the word dioxin has
place for most women to put in their time.
become almost a cause celebre, in order that we get some conception
Senator HART. I am not sure you speak for all women. That is
of the amounts involved. If this rather simple illustration is okay, I
another subject entirely.
guess it really isn't very simple but I will try it, if it is all right.
Dr. JOHNSON. This has imraveled far enough.
Now, this is assuming what is not going to happen. It is assumed
Senator HART. I am sorry, I didn't realize there was a vote signal
that all food ingested by man contained 0.2 part per million of
and we must recess to permit me to get to it. So I will be back
2,4,5-T. This is not going to happen but I am just making that
shortly.
assumption for purposes of illustration.
(Recess.)
The total, 2,4,5-T ingested per day in the food of a person would •"I
Senator HART. Doctor, let us ask these few qxiestions and then if
be three-tenths of a milligram, that is equal to 300 micrograms, ft
they do not raise all of the items that you would like to make advery small amount. If the 2,4,5-T contained 1 part per million of the
ditional comment on, we would welcome your making a comment.
tetrachlorodibenzo-p-dioxin, the daily food would contain one one- ,*
When did you first have reason to believe that a dioxin conmillionth of this 300 micrograms or
taminant in 2,4,5-T could cause chloracne?
Senator HART. Doctor, back up.
Dr. JOHNSON. Just a moment, sir, and I will get the exact date.
If the two parts per million
Senator HART. Yes.
Dr. JOHNSON. Two-tenths.
Dr. JOHNSON. Senator Hart, in regard to the 2,4,5-T, .it was 1964
Senator HART. Had how much dioxin?
when we first developed an awareness of this possible buildup of the
Dr. JOHNSON. One part per million.
dioxin potential in the product—in October.
May I proceed?
Senator HART. There was no, as far as you were aware, earlier
Senator HART. Yes.
study here or in Europe that identified tetradioxin as causing
Dr. JOHNSON. If the two-tenths parts per million of 2,4,5-T in
chloracne ?
the food of a person contained one part per million of tetrachloroDr. JOHNSON. Not in 2,4,5-T.
dibenzo-p-dioxin, the food would contain one-millionth of the 300
Senator HART. Tetradioxin in some formula had been found to
micrograms or 300 picograms.
cause chloracne?
A picogram is one-trillionth of a gram and a gram is one-twentyDr. JOHNSON. Yes. In 1950 the Germans ran into difficulty with
eighths of an ounce.
chloracne and they isolated the dioxin. This was in the process of
If a person ingested this amount of dioxin each day for 100 years,
manufacture of 2,4,5-trichlorophenol. We were aware of it.
which is an optimistic period, the total amount ingested woiild be
Senator HART. When did you begin the manufacture of 2,4,5-T?
only 11 micrograms. A grain of sugar weighs about 120 micrograms.
Dr. JOHNSON. We began the manufacture of 2,4,5-T in 1948. I
So this 11 micrograms of tetrachlorodibenzo-p-dioxin would be no
should point out in addition, however, that we were monitoring our
more than one-tenth of a granule of sugar and that level affords
workers for chloracne since 1941. This was also when the rabbit ear
a 6,000-fold safety factor over the amounts, as we observed, to cause
test was developed by Dow.
no embryotoxic effects in rats.
Senator HART. If the German study indicated the tetradioxin as
I realize that this has nothing but illustrative value, but it is not
a chloracne cause in 1950, would that have suggested to you' any test
very much the tetrachlorodibenzo-p-dioxin we are talking about.
run on the potential presence or dangers of this contaminant in the
That is my main point, Senator.
2,4,5-T that you were introducing?
Senator HART. All right, some who are better equipped than I
Dr. JOHNSON. Yes, and we monitored products made with 2,4,5to handle the technical aspects may pursue it with you. I just contrichlorophenol knowing that the dioxin resided in the intermediate.
fess that I will have to inhale it if not ingest it.
It did not appear in the 2,4>5-T until 1964.
4B-302 0—70
2ti
�380
,
_Senator HART. Did those tests include any tests to determine W]
carcinogenicity of dipxin in the formula that you were producing!,?
Dr. JOHNSON. No, sir, they did not.
Senator HART. Then in June of 1964 you were concerned nboe$
the chloracne, and in your testimony you say I think that you nolified a number of people. You notified the Department of Heallfe
of Michigan and the Industrial Health Institute in Ann Arbor and
various other health oriented individuals in private medicine ana
industry, and you had the meeting in March of 1965 notifying other
manufacturers. Why not the FDA and the U.S. Department of
Agriculture?
Dr. JOHNSON. At that time, Senator Hart, we considered our
obligation discharged by removing the dioxin from our product, by
notifying health authorities in the State and we thought we hud
the problem solved.
In retrospect it would have been much preferred had we notified
the U.S. Department of Agriculture, the agency that has statutory
authority for the registration.
Senator HART. I would agree. It would seem to me, and as I sny,
it is easier second guessing, that it would have been more appropriate and foremost to notify the agency that registers the product.
But what about the 2,4,5-T that you learned in June of 1964 hnd
this contaminant? Is it a practice to make an effort to remove tlio
contaminated product from the shelves ? What about the product in
the back?
call houses and retail channels ? What retrieval effort is made ? What
Dr. JOHNSON. Senator Hart, according to the procedures we wero
using at the time we did not produce or sell contaminated 2,4,5-T
within the limits of sensitivity we had available for measure.
Senator HART. What was it you were notifying people about in
19(14?
Dr. JOHNSON. This was the chloracne problem.
May I add a comment ?
Senator HART. Yes.
Dr. JOHNSON. As indicated in the formal testimony, the manufacture of 2,4,5-T, when pushed by temperature or heat, will produce
this contaminant; it builds up as a caustic insoluble oil. This problem produced the chloracne which initiated the actions we took.
Senator HART. What percentage of dioxin was in the 2,4,5-T that
you produced prior to the correction made at this time?
Dr. JOHNSON. It was—now you are asking about the 2,4,5-T?
Senator HART. I beg your pardon ?
Dr. JOHNSON. You are asking about the 2,4,5-T. At the time there
was an undetectable amount using the rabbit ear test as an indicator.
It might be important to point out that chromatographic proce-dures for analysis were developed during the late 1950's and early
1960's and applied with increasing sensitivity. This is a changing background of analytical capability. At the time we were using the
rabbit ear test and we did not know that dioxin was present, if any.
Senator HART. As of now, do you believe that the earlier 2;4,5-T
that you were producing was safe or unsafe?
Dr. JOHNSON. Safe, because we were monitoring the intermediate
2,4,5-T-chlorophenol and similar products since 1941.
Senator HART. But you really do not know how much dioxin was
in it. How can you say that ?
381
;
Dr. JOHNSON. It was, according to our ability to determine dioxin,
at that time, Ave thought zero.
, Senator HART. But you know better now, don't you ?
Dr. JOHNSON. We know better now because we have more sensitive
methods.
Senator HART. How can you say it was safe earlier when we know
now it was not?
Dr. JOHNSON. In our firm, Mr. Chairman, the matter of safety is
considered to be related to the dosage. The product as we sold it and
monitored it; and, as it was used, according to the label, we are
convinced it was safe. There was not sufficient exposure to the
tetrachlorodibenzo-p-dioxin, even if it had been there in the amounts
known today, one part per million or a half part per million. We
are convinced it was safe.
- Senator HART. Did you feel any requirement at any time to engage
others in making the judgment which you just made about the earlier
formula?
Dr. JOHNSON. The answer is no.
Senator HART. Now, do you agree with the position that has been
taken, as announced this morning by the three Secretaries?
Dr. JOHNSON. In a matter of practical hazard, an an imminent
hazard to health, I do not agree. Under the climate of pressure today,
it was a wise decision.
Senator HART. That sounds like you are planning to run for
reelection, but you do not want to announce it.
I am reminded that the action taken today was to cancel, not to
suspend the nonliquid 2,4,5-T and that means, as I understood the
Surgeon General's testimony this morning, the Secretaries do not
regard that form of 2,4,5-T as imminently hazardous to health.
Do you want to rephrase your answer so as to respond specifically
to the finding on the nonliquid ?
Dr. JOHNSON. The nonliquid form I consider to be safe under
labeling registrations.
Senator HART. I think the Food and Drug Committee finds it has
potential hazard to health and therefore cancels rather than suspends
it. Do you agree with that ?
Dr. JOHNSON. I dp not agree that cancellation is necessary.
Senator HART. Is it your intention to appeal the action?
Dr. JOHNSON. This must be considered. I cannot answer at this
time. We have few, if any, products of our own that are nonliquid
formulations of 2,4,5-T.
Mr. BIOKWIT. Do you have any evidence on the degradeability of
2,4,5-T?
Dr. JOHNSON. Yes. Just one moment, please. The evidence is present in the literature published by the land grant colleges and the U.S.
Department of Agriculture, predominantly. A publication by Dr.
P. C. Kearney, E. A. Woolson, J. R. Plimmer, and A. R. Isensee,
reviews the subject of degradation in a chapter entitled "Decontamination of Pesticides in Soils."
Page 139 indicates the persistence of 2,4,5-T to last 5 months.
There are additional references and review articles that, if you like,
I could submit for the record. It would take quite a bit of time
to read these, but I could do so, Mr. Chairman, if you like.
Senator HART. They will be received.
�382
List of References on Degradation of
phenoxy herbicides, including 2,4,5-T
The Dow Chemical Company
April 15, 1970
Audus, L. J. (1960) Microbial Breakdown of Herbicides
in Soils, p. 1-19 in Herbicides and the Soil, edited by
E. K.Woodford and TTT R. Sagar, Blackwell Scientific
Publications, Oxford. (45 references)
Sheets, T, J. and L, L. Danielson (1960). Herbicides in
Soils, p. 170-181 in The Nature and Fate of Chemicals
Applied to Soils, 'Plants, and Animals, ARS 20-9, USDA
(62 references)
Tliiegs, B. J, (1962). Microbial Decomposition of Herbicides, Down to Earth, Fall 1962,
(31 references)
Freed, W-, H. and
of Herbicides in
Residue Reviews,
Inc., New York.
M. L. Montgomery (1963). The Metabolism
Plants and Soils, p. 1-18 _in Vol. 3 of
edited by F. Gunther, Springjer-Verlag,
(115 references).
Kearney, P. C. (1966). Metabolism'of Herbicides in Soils,
p. 250-262 in Organic Pesticides in the Environment,
Advances in~5hemistry Series 60. (42 references)
Kearney, P, C., E. A. Woolson, J. R. Plimmer, and
A. R. Isensee (1969). Decontamination of Pesticides in
Soils, p. 137-149 _in Vol. 29, Residue Reviews, edited by
F. Gunther, Springer-Verlag, New York.
Loos, M. A. (1969). Phenoxyalkanoic Acids, p. 1-50 _i£
Degradation of Herbicides, edited by P. C. Kearney and
D, D. Kaufman, Marcel Dekker, Inc., New York.
(166 references)
Midwest Research Institute (1967). Herbicide Residues
and Their Persistence, 'and Some Factors Determining the
Fate of Herbicides, p. 208-231 and 232-249 in Assessment
of Ecological Effects of Extensive or RepeaTed Use of
Herbicides, final report processed for Defense Documentation Center, Defense Supply Agency, AD 824 314, U. S. Dept.
of Commerce.
Alexander, M. and M. I. H. Aleem '(1961). Effect of
Chemical Structure, on Microbial Decomposition of Aromatic
Herbicides. J. Agr. Food Chem. 9_, 44.
Bell, G, R. (1960). Studies on a Soil Achromobacter which
Degrades 2,4-Dichlorophenoxyacetic AcidTCan. J. Microbial.
6, 325.
Bollag, J, -M., C. S. Helling, and M. Alexander (1968a).
Enzymatic Hydroxylation of Chlorinated Phenols. J. Agr.
Food Chem. 16, 826,
.m
,m
383
Bollag, J. -M., G. G. Briggs, J. E. Dawson, and .
(1968b). Enzymatic Degradation of Chlorocatechols.
Food Chem. 16, 829.
J. Agr.
Tiedje, J, M., J. M. Duxbury, M. Alexander, and J. E, Dawson
(1969). 2,4-D Metabolism: Pathway of Degradation of
Chlorocatechols by Arthrobacter sp. 'J, Agr. Food Chem. 17,
1021.
—
Duxbury, J. M., J, M. Tiedje, M. Alexander, and J. E. Dawson
(1970). 2,4-D Metabolism: Enzymatic Conversion of Chloronaleylacetic Acid to Succinic Acid. J. Agr. Food Chem. 18
(2), 199.
.
—
Rogoff, M. H. (1961). Oxidation of Aromatic Compounds by
Bacteria, p. 193 in Volume 3 of Advances in Applied Microbiology, edited by W. W. Umbreit, Academic Press, New York.
Aly, 0: M. and S. D. Faust (1964). Studies on the Fate of
2,4-D and Ester Derivatives in Natural Surface Waters.
J. Agr. Food Chem. _12, 541.
Crosby, D. G. and H. Tutass (1966). Photodecomposition of
2,4-Dichlorophenoxyacetic Acid. J. Agr. Food Chem. 14, 596.
Crosby, D. G. and M. -Y. Li (1969) . Herbicide Photodecomposition, p. 321-363 jln Degradation of Herbicides, edited by
P. C. Kearney and D. D. Kaufman, Marcel Dekker, Inc.,
New York. (102 references)
Brown, E. and Nishioka, Y. A. (1967).
Pesticides Monitoring J. 1(2), 38-46.
Pesticides in'Water.
Norris, L. A, (1968). Stream Contamination by Herbicides
after Fall Rains on Forest Land. Western Society of Weed
Science Research Progress Report, p. 33-34.
Montgomery, M. L. and L. A.- Norris ( 9 0 . A Preliminary
17)
Evaluation of the Hazards of 2,4,5-T in the Forest Environment, USDA Forest Service Research Note PNW-116.
Sheets, T. J. and J.. F. Lutz (1969). Movement of Herbicides
in Runoff Water. 'Presented at the Dec. 1969 meeting of the
American Society of Agricultural Engineers, Chicago, Illinois.
Bailey, G. W., J. D. Pope, Jr., andD. R. Cochrane (1968).
The Degradation, Kinetics, and Persistence of Silvex Under
Impound Conditions. Abstracts p. 43, WSSA Meeting, New Orleans,
Louisiana, February 1968.
�384
^aurfi L i I^Mu*..-.Iu,n..
^
PIUaM tkMOa I i^A'.A.^iSKi2«w».ii^iisii Ux2/ll'
Urea, triazine, and piclorarn herbicides
i'
7T^?!5!W:^^Pfip{|^>^i
^ - - .
Benzole acid and amide herbicides
' - ^ •, tv^i^ji ^'ti t....
Phenoxy, toluidine.and nitrile herbicides
u
RESIDUE
Carbamate and aliphatic acid herbicides
Decontamination of pesticides in soils
By
P. C. K EARNEY) * E. A. WooLsoN
18
Fig. 1. Persistence of pesticides in soils
J. R. pLIMMER>* and
I. Introduction
',
senfe one or more
<n ft. bar
pesticides. The organic herbicides
nonlh. depend^ on
orA gri cu, ture over ot h ers
P rsistcnt
°
major herbicides that persist for a month or longer are shown' in Figure
1. The phosphate insecticides do not persist for long periods in most
soils. A more detailed picture of organochlorine pesticide persistence
is shown in Figure 2, Chlordane and DDT usually persist for several
years while heptachlor and aldrin extend their activity through the
formation of their respective metabolites, i.e., heptachlor epoxide and
dieldrin.
Why are we concerned about pesticide residues in soils? Their
effects would appear to be very subtle and not directly related to man
or his environment. The need for pure water is obvious, for man
directly consumes processed water. Not so with soil, and therefore, why
is there concern over soil contamination?
There now exists unequivocal evidence that most plants can absorb
and translocate residual pesticides from contaminated soils (NASH
1968). Uptake and translocation have been demonstrated with radiolabeled pesticides incorporated directly into the soil and then seeded
with several agronomic crops. Many of the soil variables that influence
this uptake process have been studied. For example, increasing .the concentration of dieldrin and DDT in soil causes a corresponding increase
in the amount of insecticide recovered by the wheat plant. The plant
is apparently indiscriminate in its ability to absorb most substances
from soils. Therefore a link exists between residual pesticides in soils
and man's food chain. In addition, residual pesticides are potential
pollutants of water and air.
�387
386
?ll
Phosphate insecticides
Orgonochlorine insecticides
r",1";1"} "*,""", ' T"l '
TZ?77?&ZZ £252Z2i2i...•llAu^JU
Chlordone
Dicuinon
2t£-:?.";'-'i''-*'l
Di-Syston
DOT
BHC, Dieldrin
Heptachlor, Aldrin, Metabolites
I
I
I
i
I
1 '
2
i
Phorate
3
4
Years
5
6
,
21
Malathion, Parothion
l
1
1 .
I
)
2
4
6
8
Weeks
i
1
10
l
12
Benzoic acid and amide herbicides
Urea,Iriazine, and piclorom herbicides
.. ^ . -:'.i..' -.S • •'. l - tf. j
Propazine,
PHYSICAL ^BIpLDGlCKE; CHEMICAL
Photodecom- t/Rpot'up'tai(eSK~i Oxidation
position , Metabolism » , Hydrolysis
Volatility
•
,.
3,3,6-TBA
.
Simozine
Leaching
Bensulidc
*
~
• *^ •
.'.W^' . 'S'.-*!.- ''.'..VSV.-. •'' I* '.'.^.-> .'.' j'.^
Atra/ine, Monuron
Fig. 3, Processes that determine fate of a pesticide in soil
Diphenomide
Diuron
Linuron, Fenuron
Prometryne
I
I
I
0 3 4
CDAA, Dicambo
4
I . I
i
I
6
8
10
12
Months
L
8 10 iz 14 is re
Months
Phenoxy, foluidine.and nitrile herbicides
.i^ito" "f!(?./ffi')'.'I'i> :'J>1' ^.ii-v- '- [- J; '^.^"V'vjj
TCA
Trif lurolin
i:rtiiLKIiiiA&*i' i.?.'? i-.r
teii&.a"!Li:j^ii3
2, «*,*!-T
Dolopon.CIPC
^'iiki^ri^'X^'^ '••'; '^'1
CDEC
Oichlobenil
MCPA
IPCJEPTC
2,4-D
0
1
2
Corbarnofe and aliphatic acid herbicides
,_3
4 ,
5
6
Borban
l
3
2
! Months
1
4
1
6
Weeks
l
8
1
10
, 1.
12
Fig. 2. Persistence of individual pesticides in soils
Now that we have defined the problem and the need for decontamination in soils, what methods are available for removing persistent
pesticides? The fate of a pesticide in soils' is determined by a number
of processes which come under the general heading of physical,
biological, and chemical (Fig. 3). Under physical, they include photo-
decomposition, volatility, leaching, and adsorption. Under biological,
they include root uptake and microbial metabolism and under chemical, they include oxidation, reduction, and hydrolysis. Several of these
are responsible for decomposing pesticides. For example, soil enrichment techniques for the proliferation of specific microorganisms effective in metabolizing foreign substances have been a favorite method
for microbiologists. It is conceivable that "catch plants" or plants with
a high affinity for certain pesticides could be grown on contaminated
soils and then removed after taking up some part of the residual
pesticides. It is possible that a combination or several of these methods
could be employed to reduce pesticide concentrations in soils.
If each of these processes were active on a pesticide, then soil
residues would not exist. If soil microorganisms could be induced to
rapidly metabolize DDT to the level of carbon dioxide, then soils
would be an ideal medium for decontamination. They don't, and
therein lies the problem of reducing soil residues. The work to be
reported today deals with two approaches to reducing pesticide residues. The first concerns the use of light to decompose transported
pesticides in water and hence its application to irrigation waters and
soils. The second concerns the decontamination of field soils containing
DDT by flooding and inoculation with microorganisms.
II. Pesticide decontamination in water
A major source of environmental contamination is caused by the
movement of materials from their site of application. Pesticides move
primarily in the liquid or vapor phase. Pollution of water by organic
�388
389
rorn
impounds is undesirable, contamination by biologically active com-
pounds is potentially dangerous. Two particular situations in which
pesticides in water cause concern relate to the waste problem encountered in static or lagoon operations and to irrigation systems. The
danger of the latter situation is best illustrated with the water-soluble,
mobile herbicide picloram (4-amino-3,5,6-trich]oropicolinic acid). Minute amounts of this potent herbicide irrigated oa sensitive crops could
have disastrous results. Concentrations as little as 10 p.p.b. in soils
have a lethal effect on such sensitive crops as soybeans.
What methods are available for removing pesticides in water? The
use of energetic radiation (ultraviolet or gamma ray) has been suggested (MARCUS et al. 1962) for fragmentation or destruction of organics in water. This method should be effective on a large number of
pesticides especially in dilute aqueous solutions. Unfortunately, the
technology has developed little beyond die experimental stage. Large
scale ultraviolet and gamma irradiation techniques are in early technological stages and wider industrial application is the needed stimulus
for further development.
We have determined the periods of exposure required to destroy
the biological activity of a number of herbicide solutions in a smallscale ultraviolet irradiator. The method may be applicable as a pretreatment for waste waters or as a treatment for contaminated
irrigation systems.
The reaction system is a borosilicate glass vessel and holds 250 ml.
of the solution to be irradiated (Fig. 4). A quartz, water-cooled,
double-walled tube is fitted into this well and is immersed in the
solution. A 450 watt Hanovia lamp is suspended in the well. The
quartz well transmits a large part of the available energy down to the
shortest wavelengths emitted by the lamp.
Solutions of herbicides in water wore irradiated (250 ml. at a time)
for periods of 5, 10, and 15 minutes. Picloram,' 2,4,5-T, bromacil, "
diphenamid, and 2,3,6-TBA were die herbicides used in the initial
experiments. These compounds were chosen because their solubility .
and persistence are sufficiently high for them to be potential contaminants in irrigation water. Oats were used to bioassay picloram,
2,4,5-T, and diphenamid and cucumber was used for bromacil and
2,3,6-TBA. The treatments consisted of zero, 5, 10, or 15 minute irradiations of the solutions at 1, 5, or 10 p.p.m, concentrations and a
control in which no herbicide was added. The time required to destroy
the five herbicides is shown in Figure 5 (PUMMER 1968).
A five-minute irradiation greatly reduced the phytotoxicity of
picloram and 2,4.5-T at five and 10 p.p.m. and bromacil at one p.p.m.
Diphenamid and 2,3,6-TBA required a 10-mim;te exposure. These
initial results indicate that five minutes or less exposure to ultraviolet
irradiation of solutions in the range of one p.pm. would significantly
lower their phytotoxicity to plants. More pesticides, under conditions
Water
Lamp
Quartz
gloss well
Solution
irradiated"
Fig. 4, Photochemical reaction vessel
5
Picloram
2,4,5-T
5 and 10 p.p.m.
Sp.p.rn.
Bromacil
2,4,5-T
I p.p.m.
lOp.p.m.
Diphenamid
10 p.p.m.
Bromacil
2,3,6-TBA
10
lOp.prn.
lOp.p.m.
Fig. J5. Times required to destroy herbiddal activity by irradiation
15
�391
390
approaching large, volumes of water on flow systems need to be
investigated before wide application to pesticide decontamination is
attempted.
of f-ve percent SE 30
sorb W, Column temperature was 210
ml/minute, Detector temperature was 21& |j
mtamn
ined consider-
III. DDT decontamination
Turning our attention to field soils, one of the most serious residue
problems occurs with orgnnochlorine insecticides. As previously mentioned, DDT persists for several years in most agricultural soils. Complete removal of these residues may be impossible. However, lowering
existing residues below some arbitrary threshold level may result in
minimal plant residues. Our objective, then, was to find some agent in
nature that could attack the DDT molecule. This agent did not necessarily have to cause complete destruction of DDT, but perhaps alter it
to a more biodegradable or labile form. Obviously, organisms indigenous to most soils do not possess this agent. Intestinal microorganisms
in the rat, however, are able to alter DDT extensively.
Whole cells or cell-free extracts of Aerobacter aerogenes catalyze
the degradation of DDT in vitro to at least seven metabolites (WEDEMEYER 1966), previously reported from rats given DDT orally (PETERSON and ROBISON 1964). These reactions proceed by dechlorination,
elimination, oxidation, and finally decarboxylation to yield dichlorobenzophenone. Therefore, it occurred to us soils inoculated with A.
aerogenes may be capable of metabolizing residual DDT.
To test this hypothesis, three soil types were amended with zero, 5,
10, and 20 p.p.m, of DDT, The soils were Lakeland sandy loam,
Hagerstown silty clay loam, and Sharkey clay. Four-hundred g. of
soil were weighed into pots and DDT was applied in chloroform
solution. Since metabolism of DDT by A, aerogenes appears to occur
most rapidly in still cultures or under partially anaerobic conditions,
two-thirds of the soils were flooded to simulate partial anaerobiosis.
The water covered the soils to a depth of approximately one inch.
One-third of the DDT-treatcd soils was maintained at field capacity,
one-third was flooded, and one-third was flooded and inoculated with
A. aerogenes.
Cells of A. aerogenes from slants obtained from the American Type
Culture (ATC 13048) were mass cultured in three percent trypticase
soy broth at 36° C, for eight hours, The cells were harvested by highspeed cenlrifugation, washed, and resuspended in the original volume
of fresh broth solution, The cells were incubated for three days in still
cultures, harvested again, washed, and concentrated 10-fold in a one
"percent yeast-extract solution, Aliquots (10 ml.) were added to the
flooded soils and mixed into the surface layers. All soils were sampled
at weekly intervals. Residual DDT and products v/erc measured by
electron-capture ga,s cbromritography. Moist soil samples were extracted with a 3:1 mixture of hexaneiisopropanol and injected on to a
Flooded
Flooded- A. aerogenes
Fig. 6. DDT decomposition in three soils (see text)
Flooded - A. oerogeneS'
Flooded
Lakeland
Fig. 7. DDT decomposition in three soils (sec text)
disappeared more rapidly in the inoculated soils. Second, complete
loss occurred in the Sharkey clay, while lesser amounts were lost from
Lakeland and Hagerstown in both Hooded series. DDD (TDK) was
observed to occur in most soils, but its appearance did not parallel
�392
393
Tosses in-DDT. In other words, there was a net loss of DDT-DDD in.
this system and no other products were detected by gas chroma!*'
graphy. Somewhat the same picture is encountered at five p.p.in. of
DDT (Fig. 7), with complete loss in the Sharkey clay, more accelerated disappearance in the inoculated soils, and a general trend for
total loss of DDT-DDD with time. Recovery values for DDT during
the early sampling periods on Lakeland were high and explain the
values obtained above five p.p.m. in the flooded soils.
Additional studies were initiated to determine the fate of the DDT
in these systems. The experiment using Lakeland soil at five p.p.in.
plus DDT-1<JC was set up in a closed glass system for trapping ^COj.
Nitrogen was bubbled through the system, and any gaseous carbon
dioxide was trapped in base. Samples of the carbon dioxide trapping
solution were removed periodically and analyzed for radioactivity.
Less than one percent of the total added activity was released in
volatile components. This would be in general agreement with results
previously reported (GuKNZi and BEARD 1967).
Therefore, several other alternatives are available to explain the
disappearance of the DDT from these soils, A polar metabolite could
be present in the aqueous phase and not removed by the hexane:isopropanol extraction, or a metabolite is absorbed on some soil component and not recovered. It is also possible that u volatile metabolite
is formed which escapes from the system with time. The presence of
a polar metabolite can apparently be ruled out, since only 7 to 28
percent of the added DDT-^C could be detected in the aqueous
phase. Subsequent research with "C-DDT in a similar type of experiment indicated that up to 20 percent of the 1''C could not be extracted
from flooded soils after a four-week incubation period, The radioactivity is apparently tightly bound to soil particles in some form not
recoverable with hot- hexanciacetone, ethyl acetate, or ethanol
(KEARNEY and WOOLSON 1969). Therefore, we must conclude the loss is
real and reproducible, although the mechanism is not fully understood.
IV. Conclusions
Radiation as a method for preventing the spread of pesticides in
water systems deserves further consideration. Ultraviolet radiation for
sterilization processes is in commercial use. High energy radiation has
similar applications and its use in food processing has been studied.
Ultraviolet radiation has been used for the complete removal of organic materials from sea water samples on a laboratory scale (ABMSTRONG et at 1966). A pebble-bed type of reactor has been described,
but not further developed, which appears suitable for continuous
ultraviolet irradiation of solutions. A radioactive material is incorporated into impervious ceramic "pebbles" together with a suitable
phosphor which emits ultraviolet radiation. A flow system is envisaged
with the pesticide in dilute solution flowing over the ceramic pebbles.
In addition to thermal methods of destruction, we-suggest that radiation methods be further explored as a simple means of removing peslicicles in situations where applicable.
Removal of pesticides from soils is a far more complex process,
since the system is static and not conducive to flow-through operations,
Several methods have been suggested for reducing residues. The use
of calcium polyphosphate on the residual chloro-s-triazines has not
been successful under field conditions (HAIUUS et al. 1968). The use
of absorbents (charcoal) for removing toxic materials in replanting
certain nursery stock has been successful; extention of this technique
to field conditions for atrazine and organochlorine residues has been
attempted ( LICHTENSTEIN et al 1968). In these situations, however,
the cure may be worse than the sickness, since a new and far less
understood variable is now being introduced into the soil. Such may
be the case with microbial decontamination of DDT by A. aerogenes.
Many experimental variables would have to be studied before largescale field studies would be justified.
In the final analysis any decontamination method would have to be
economically feasible before it would be acceptable to the farmer. The
most promising and yet still unexplored method for reducing soil
pcsticjde residues lies at the molecular level. A thorough understanding of the electronic and steric factors that render a pesticide molecule
susceptible to the natural biological degradation pathways is still in
early developmental stages. This approach would appear to offer the
most challenging chemical method for reducing residues on a continuing basis.
••a
Tnble I. Common and chemical names of pesticides mentioned in text
Bromacil
5-bromo-3-scc-butyl-G-metliyluracil
DDD
1, l-dicbloro-2,2-bis (p-chlorophenyl) ethane
DDT
l,l,l-trichloro-2,2-bis(7J-chloroplienyl)ethane
Diphcnamid
N,N-dimel.hyI-2,2-diplienylacetamide
Picloram
4-amino-3,5,G-tn'cliloropicolinic acid
2,4,5-T
2.4.5-trichlorophenoxyacetic acid
2,3,6-TBA
2,3,6-trichlorobenzoic acid
Summary
A limited number of methods are available for decontaminating
soils and. water. Complete destruction of organic herbicides in water
may be effected by exposure to intensive high-energy radiation. Measurement of the rates of photodecomposition of picloram, diphenamid,
bromacil, 2,3,6-TBA and 2,4,5-T by bioassay techniques is in progress.
Experiments with model continuous flow cells indicate rapid deslruction of organic dyes. No picloram could be detected in a solution
of initial concentration of 1 p.p.m. after 30 minutes or less exposure
to high intensity UV irradiation. Where complete removal is not feasible, reduction of existing residues below a level in soils where the
�394
395
significance of plant uptake becomes minimal may be desirable.
Biological alteration of a persistent pesticide to a more degradable
form is another method of reducing residues. DDT residues in three
soils (Sharkey clay, Hagerstown silty clay loam, and Lakeland sandy
loam at rates of 5, 10, and 20 p.p.m,) were reduced in flooded soils
and flooded, enriched soils inoculated with Aerobacter aerogencs.
Losses were most rapid in the inoculated Sharkey and Hagerstown
soils receiving the lowest rate of DDT application during the first
week. Parallel experiments conducted with ring labeled DDT-^C
showed no 14CO2 evolved from the inoculated soils. Conventional
' chromatographic and radiometric techniques indicated a conversion of
DDT to DDD and a reduction of the total DDT-DDD residue in soils
with time.
Zusammcnfassung *
Resum6 *
Decontamination de pesticides dans les sols
On dispose d'un nombre limite de methodes decontamination des
sols' et des eaux. On pent effectuer une destruction des herbicides
organiques dans 1'eau par exposition h. des radiations intenses de haute
6nergie. La mesure des taux de photodecomposition du piclorame, du
diphenamide, du bromacile, du 2,3,6-TBA et du 2,4,5-T par bio-essais
est en progres.
" Des experiences a 1'aide de cellules a courant continu indiquent
une destruction rapide-des colorants organiques. Aucunc trace de
piclorame n'a pu etre clecelee dans une solution qui en contenait
initialement 1 p.p.m., apres 30 minutes ou moins d'exposition a une
irradiation UV de haute intensive. Dans les cas ou une elimination
complete n'est pas .possible, une reduction des residus presents en
dessous d'une certaine limite peut etre souhailable pour les sols ou
1'irnportancc de 1'absorption par les plantes devient minime, La transformation biologique d'un pesticide persistant en une forme plus
aise"ment decomposable est une autre methode de reduction des
residus. Des residus de DDT dans trois sols (urgile de-Sharkey, limon
argilcux alluvionnaire de Hagerstown et limon sableux de Lakeland
aux concentrations de 5, 10 et 15 p.p.m.) ont etc reduits dans des sols
inonde\s et des sols inondes cnrichis, inocules avec Aorobactcr aerogenes. Les pcrtes ont ete plus rapides dans les sols de Sharkey inocuMs
ct les sols de. Hagerstown ayant re9u la plus faible concentration en
DDT clurant la premiere semaine. Des experiences paralleles avec du
DDT marque "C n'ont rev61e aucun dcgagement de 14CO2 des sols
inocules. Les techniques chromatographiques et radiometriques conventionnellcs ont indique" une conversion du DDT en DDD et une
reduction des residus totaux de DDT-DDD dans Ics sols en fonction
du temps.
Dekontaraination von Pcstiziden im Bodcn
Eine begrenzte Zahl an Methoden steht zur VcrfiJgung, um Boden
und Wasser zu reinigcn. Vollstiindige Zerstorung von organischen
Herbiziden in Wasser kann durch Belichtung mil intensive!" Strahlung .
von boher Energie bewirkt wcrden. Messungen dcr Photoabbauraten
von Picloram, Diphenamid, Brornacil, 2,3,6-TBA und 2,4,5-T durch
Biotesttechniken sind im Fortschrciten begriffcn. Expcrimenle mit
Modelldurchflusszellen zeigen schnellc Zerstorung von organischen
Farben. In einer Losung mit einer Anfangskonzentration von ein p.p.m.
konnte nach 30 Minuten oder weniger Belichtung mit hoch intensivcr
ultravioletter Strahlung kein Picloram mehr nachgewiesen werden. Da,
wo vollstilndige Entfernung nicht moglich ist, wird die Reduzienmg
von vorhandenen Riickstanden in Boden unter eine Menge, wo die
Bedeutung fiir die Pflanzenaufnahme minimal wird, wunschenswert.
Biologische Veranderung eines persistenten Pcstizids zu einer abbaufiihigcrcn Form ist eine andere Methode, um Riickstande zu
reduzieren. DDT Ruckstande in Boden (Sharkey Ton, Hagerstown
sandiger Ton-Lehm und Lakeland sandiger Lchm mit Ralen von 5, 10
und 20 p.p.m.) wurden in uberfluteten Boden rcduziert und weggeschwcmmt und angercicherte Boden mit Aerobactcr acrogencs,
geimpft. Die Verluste waren am schnellsten in den Sharkey und
Hrgerstown'Bb'dcn, welchc wahrcnd der ersten Woche die niedrigsle
Rate von DDT Bchandlung erhallen batten. Parallele Untersuchungen,
welche mit 14C-ring-markiertcm DDT durchgefuhrt wurden, zcigten
keine 1"CO2 Entwicklung in den beimpften Bb'den. Konvenlionelle
chromatographische und radiomctrische Techniken dcuteten die Umwandlung von DDT zu DDD an und eine Reduktion des GesamtDDT-DDD-Ruckstandes in Boden mit der Zeit.
»
References
ARMSTRONG, F. A. J., P. M. WILLIAMS, and J. D. H. STRICKLAND: Removal of
organic matter from sea water by ultraviolet light. Nature 112, 481 (1966).
GUENZI, W. D., and W. E. BEARD: Anaerobic biodegradation of DDT to DDD in
soil. Science 156, 1116 (1967).
HAHRIS, C. I., D. D. KAUFMAN, T. J. SHEKTS, R. G. NASH, and P. C. KEAUNEY:
Behavior and fate of s-triazincs in soils. Adv. Pest Control Research S, 1
(1968).
KEAHNEY, P. C., and E, A. Woor.soN; Personal communication (19G9).
', R. G. NASH, and A. R. ISENSKE: Persistence of pesticide residues in soils.
In M. W, MII.LEH and G. G. Bisno, cd.: Chemical fallout: Current research
on persistent pesticides, Chapt. 3, pp. 54-67. Springfield, 111.; Charles C
Thomas (1969).
LTCHTENSTEIN, E. P., T. W. FUHKEMANN, and K. R. SCHULZ: Use of carbon to
* Ubcrsctzt von A. SCHUMANN,
8
Traduit par S. DOHMAL-VAN BEN BIUTEL,
45-362 O - 70 - 26
�''TM
396
! Industdal
^
647
o66).
Photochemistry. j
>d ondrin f rom
soil,
A gt
>
'•
by Acrobacter aerogcne, Science 1
*
Mr. BICKWIT. Do you have any evidence on the degradeability of
jj-wxm?
^^
; Dr. JOHNSON. In terms of photodegradation, we have somd^Bdence
j *"tl)is is not exhaustive—but nevertheless some evidence that would
^'indicate a half life of—excuse me, I will get that in just a moment.
"•• This is the type of experiment run in a laboratory in a solvent.
lHi& half life was 21/2 hours under a typical type of sun lamp. This
» only an indicator type of test. The prognosis is in the presence
,*f light, dioxin will degrade. Degradation in soil we do not have
information on. We are diligently, however, preparing carefully
totaled—radiochemically labeled—2,3,7,8-tetrachlordibenzo-p-dioxin,
mul supplying this to the U.S. Department of Agriculture for tests
M quickly as possible, because this is the fastest way to get the
answer.BICKWIT. Do the light conditions you are using exist in
Mr.
nature?JOHNSON. Primarily in ranges and pastures, yes. Because
Dr.
2,4,5-T is intercepted on the upper surfaces of weeds and brush more
deposits in exposed than in shaded conditions. The predominance
would be exposed to light conditions. Obviously some is going into
the shade. Under those conditions of lesser light intensities, I can't
reply. BICKWIT. Is it ultraviolet light you are using?
Mr.
Dr. JOHNSON. Yes, with a typical sunlamp.
Mr. BICKWIT. Do I summarize your findings correctly when I say
you believe 2,4,5-T is degradeable in a matter of months, and witli
respect to dioxin there is some evidence it is degradeable, but we
doDr. JOHNSON. Correct. I it is? again emphasize if specifications
not know whether or not should
are low, the minute amount presents an extremely small exposure.
Mr. BICKWIT. With regard to the calculations that you offered us
earlier, you have assumed that a person who ingests 2,4,5-T is
ingesting one part per million dioxin. Isn't it possible
Dr. JOHNSON. May I clarify the statement for the assumption?
Mr. JOHNSON. The 0.2 parts per million referred to a hypothetical
Dr. BICKWIT. Sure.
situation.
Dr. JOHNSON. People do not ingest that much, and that is the
total dietary intake, and moreover, 2,4,5-T has not been a residue
in food. It is, in effect, zero tolerance.
Mr. BICKWIT. I understood that. What I still understand to be
your assumption was that any amoxmt of 2,4,5-T, no matter how
small, that was ingested, would have one part per million dioxin in
it. That was the basis of your calculation.
Dr. JOHNSON. The specification we have set for our own product
is less than one part per million. Actually, it is 0.5. The assumption
was to make it easy mathematically, on the high side, and some
day I will learn not to draw these mathematical analogies. They
never quite makeWell, I hope you learn right now. What I was sugMr. BICKWIT. the mark.
gesting as a possibility is, that since 2,4,5-T is degradeable, and
since we do not know whether or not dioxin is degradeable, that
although your product, when sprayed, has 0.5 parts per million of
�"'
:' Dr. JOHNSON. I think Mr. Bickwit is talking about ^^possibility
*f the 2,4,5-T degrading in the food consumed, whereas the degradation question only applies to environment.
Mr. BICKWIT. No, I am not.
.Senator HART. Try it again.
Mr. BIGKWIT. You are assuming that the maximum amount that
'*o would ingest would be 0.2 parts per million in every amount of
iood, in every iota of food, that we eat. From that you conclude
- that the amount of dioxin in that food would be one-millionth of
! 0.5} parts a million, which I admit is an extremely small amount. I
am questioning your assumption of whether the amount of dioxin,
compared to the amount of 2,4,5-T that is ingested, would be onemillionth merely because dioxin is one-millionth of the 2,4,5-T
amount when it is sprayed ?
What I am suggesting is that the dioxin consumed may actually
l» more than the amount of 2,4,5-T consumed, even if it is only one
1
part per million when sprayed.
Dr. JOHNSON. Is the situation that you have in mind that the
2,4,5-T is sprayed?
Mr. BICKWIT. Yes.
Dr. JOHNSON. The 2,4,5-T is sprayed into the environment. This
contains one part per million. Then the 2,4,5-T degrades. The dioxin
does not. And over a period of time, is there is a buildup of dioxin ?
Is this the problem?
Mr. BICKWIT. I have another way of getting at this, perhaps.
If one gram of 2,4,5-T is sprayed on a blueberry—an unlikely
assumption—and the 2,4,5-T degrades so that it complies with the
tolerance of 0.2 parts per million, you may still have one microgram
of dioxin sitting on that blueberry, without any violation of the
tolerance of 2,4,5-T, And that one microgram may well be toxic.
Dr. JOHNSON. I would like to find a degradeable material like that.
Theoretically, obviously, if the one gram of degradeable material
contained the one microgram or one part per million of nondegradeable material, then the one gram degrades, that one microgram
would still bo sitting there.
Is that the point you are trying to make ?
Mr. BICKWIT. That's right.
Dr. JOHNSON. That's right,
Mr. BICKWIT. Well, how then does a 0.2 parts per million tolerance for 2,4,5-T protect us from dioxin ?
Dr. JOHNSON. Because the ratios of those two during the process
of growing and supplying the food are going to remain essentially
the same.
Mr. BICKWIT. You have no evidence for that statement.
Dr. JOHNSON. I know I don't, but you have no evidence for the
hypothetical question, either.
Mr. BICKWIT. I do have some evidence, by your own statement,
that dioxin is more likely to be nondegradeable than 2,4,5-T.
Dr. JOHNSON. But these are matters of relative rates. You are
suggesting an instantaneous degradation.
Mr. BIGKWIT. I am using a hypothetical situation, as you were. •
:
._. _,~^WJ.T. uertamly not.
, '>
Mr. BLAIR. So your question as phrased has, no meaning as,I triwj
to interpret it. Unless you mean that possibly as 2,4,5-T degrade*,-,
that it would degrade through dioxin, or into a dioxin product
Then, while up. percentage would go up, the exact concentration ;
has not gone the
Mr. BICKWIT. What I am saying is that your calculations nre
based on the view that the hypothetical view—that we would allow
a 0.5 parts per million tolerance for 2,4,5-T. You then conclude, if 0.2
parts per million of 2,4,5-T were ingested, that an extremely minute
amount of dioxin would be ingested.
What I am
that premise? questioning is whether the conclusion follows from
Dr. BLAIR. Yes, it would be an extremely small amount, and it
would not increase with time.
Mr. BICKWIT. What I am suggesting is that the 2,4,5-T could
break down so that it complies with the 0.2 parts per million tolerance. In fact, it might even disappear. Yet, we would be ingesting
a good deal of dioxin for which there would be no tolerance.
Dr. BLAIR. It is not possible.
Mr. BICKWIT. I don't understand why not.
of Dr. BLAIR. millioncould you ingest 2,4,5-T that contained a tenth
a part per How
Mr. BICKWIT. It contained that much when it was sprayed.
Dr. BLAIR. Yes, and with time the concentration of dioxin in that
environment has not increased one iota.
Mr. BICKWIT. That's right. The amount has not increased.
Dr. BLAIR. But is not possible to ingest more than what is there,
It doesn't make sense.
Dr. JOHNSON. Mr. Chairman, could I make a comment?
Senator HART, I wish I could be helpful. I would like to see if a
rephasing of
Mr, Bickwit. the question might not elicit the point that bothers
Dr. JOHNSON.
rephrased first? Do you want to comment first, or have the question
Senator HART. Make your comment.
399
�400
-Jr. JOHNSON. My purpose in bringing up what appears to ha*
been a rather foolish example was merely to give a feeling for the
magnitude of how much dioxin we are talking about.
Mr. BICKWIT. And my contention is that once the assumption on
which the hypothesis was based is removed, then it does not give
that feeling.
Dr. JOHNSON. I am sorry. I don't agree.
Senator HART. That is one of the fortunate features of having_a
reporter here. We can all grab the record in the morning to see if
we can count out an understanding and agreement of yesterday.
Mr. BICKWIT. You state in your statement that members of the
Mrak Commission had not seen the report prior to Dr. DuBridge's
October 29 announcement.
Last week Dr. Kotin of NIEHS told us as work was completed it
was promptly passed along to the Commission.
Do you mean to imply that the Bionetics report was not complete
until this time?
Dr. JOHNSON. I did not receive a copy of the report of the Panel
on Teratology until very late in the deliberations.
Mr. BICKWIT. I think that is unfortunate. I wonder if you have
knowledge as to why that was so ?
Dr. JOHNSON. No.
Mr. BICKWIT. We have have been told that whenever you burn a
polychlorinated phenol, dioxin prodiiction is possible, or even likely.
Could you enumerate a few of your products that contain such
phenols ?
Dr. JOHNSON. There's trichlorophenol, tetrachlorophenol and pentachlorophenol, and sodium salts thereof.
Mr. BICKWIT. What is a Dowicide product ?
Dr. JOHNSON. The products I just mentioned. Since you related it
to the phenols, I assume that is what you are talking about.
"Mr. BICKWIT. Yes. I wonder if you could furnish us with a list'of
all such products
Dr. JOHNSON. Mr. Howe clarified a point here that our Dowicide
trademark applies not only to chlorophenols, but to other antimicrobials.
Mr. BICKWIT. I wondered if we could have for our files a list of
the products which you produce which contain polychlorinated
phenols ?
Dr. JOHNSON. Yes. I prefer the term chlorophenols. Polychlorinated phenols would indicate a polymer. Chlorophenols.
Senator HART. Now I am getting into something that you don't
have to be anything to understand except efficient.'
'
Do you have anything to do with Lake St. Claire?
Dr. JOHNSON. I am not really prepared to discuss that in detail, I
don't have any direct responsibility for the Sarnia Plant.
Senator HART. I would be disciplined severely by my outdoor
friends if I didn't ask what you are going to do about the mercury
that has found its way into Lake St. Claire.
Dr. JOHNSON. I might say this, we intend to exercise responsible
action.
Senator HART. That is like the lawyer admitting his client is innosnt. But I won't push you if you are not prepared.
^^
cent. JOHNSON. I am not prepared to say any more.
Dr.
^J
Senator HART. Thank you very much.
Are there any further items that you have ?
Dr. JOHNSON. Yes, if I can take a few more minutes.
Senator HART. Time is not a problem.
Dr. JOHNSON. I would like to make a comment or two, again on
dioxin before getting into the last part of my testimony.
There have been questions raised about the decomposition of
2,4,5-T and dioxin. 2,4,5-T is unstable at elevated temperatures. We
were able to get degradation when 2,4,5-T or dioxin were burned on
paper. We were unable to detect any dioxin residue in the smoke.
Now, these are very early bits of information, strictly preliminary
but I thought I should mention it at this time.
This morning you asked a question about shortening the time
interval between the early indication of some possible difficulties and
the learning of sufficient truth about a situation so that we can take
appropriate action prior to an imminent hazard to health. Hopefully, if we can improve some of our combined procedures between
Government and industry, we can shorten that time.
I have a few remarks to make that apply to the scientific community as well as to this interface with Government. Screening tests,
as we have heard, (and I commented on some of my own,) I think
are of value. As early indicators. But screening tests alone, without
the consideration of quantitative data, can be quite misleading and
if necessary
Senator HART. Can be what 1
Dr. JOHNSON. Quite misleading and I think it is necessary to
develop understanding in meetings where open reports are published
early and where scientific information is submitted to the challenge
of other scientists; scientists of the universities; government; industry and professionals so these things can be considered in the caldron of open technical debate. quickly to shorten this gap if there is
I think we can operate more
more openness about reports. Moreover, I think another procedure
for shortening the time is better to understand test procedures. The
screening tests are simple but those tests which are substantive
enough to justify intelligent action require, the consideration of scientists outside of the generating laboratory, and the review of
appropriate methods.regulatory action are—or for clearance of new
Test methods for
products, in particular, require consideration. I don't mean we
sho.uld lock every clearance procedure into a lock step because the
development of techniques is a mutable, changing thing. But the confirmation of tests prior to official acceptance as this relates to public
policy is important. of Official Agricultural Chemists has established
The Association
a useful procedure. When these gentlemen and this association are
considering new tests, the topic is identified, the first scientists to
work on the test are identified, collaborators are found to_ work out
objective tests and these tests are run in separate laboratories so that
confirming methodology can be established.
�402
As a result the test must be reliable; that is, it miist give accifll<
Precise and reproducible results when used by qualified analysts^!*
must be practical; simple. It must be available to all analysts and !l
must be substantial; that is, supported by collaborative study.
Now, I will say these types of tests apply primarily to cleaninc*
procedures or to regulatory tests. I am not saying extreme tests are
not in order. But those which support the authority of regulatory
backup need this type of scientific consideration.
The American Standards Society also has procedures for standardizing tests. These are physical tests. They involve one or more
laboratories in the preliminary study and interlaboratory studio.1*,
maybe as many as 10, in the more refined studies, and this results in
a standard test method which can be used.
The Department of Commerce, National Bureau of Standards,
does an excellent job in developing standards. Moreover they have
an industrial associates program which permits the access of industry into these laboratories, for a period of time to work, so thai
industry representatives can better understand the problems of
standards and go back to their companies better informed.
Protocols for clearance of new and existing products, I think are
important for the Food and Drug Administration to consider seriously. Again I don't mean standard tests would remain forever, but
protocols. Industi'y needs to know where it stands, industry also
need to participate; appropriately and at arm's length, to be sure,
nevertheless; to participate with specific information.
The period of time between 1966 and now could have been shortened by sharing appropriate information, and I admit we could
have assisted by volunteering earlier. But there seems to be some
reverse of togetherness and there seems to be great concern that communication between governmental agencies and industry is suspect
and would be misused.
I would hope that one valuable result to come of this would be a
continuation of developing science fortified by interest in this important matter of communication,
On the point of developing protocols (and I frankly think the
whole subject of teratology requires the vigor of scientific debate
among professional peers involving industry, university and government) I would hope the National Academy of Sciences could provide such a forum, if not the National Academy perhaps the New
York Academy of Sciences and I intend to help encourage this
because it is important. It is important that these ideas are traded
openly and published.
I would mention that the World Health Organization, in a document entitled "Principles for the Testing of Drugs for Teratogenicity," the World Health Organization Technical Report, Serial No.
364 or 1967, has the following to say on test animals for teratology,
on the subject of the chick embryo, page 7.
The chick embryo contributes greatly to basic embryological knowledge.
Hownver, for the screening of drugs for teratogenicity, its use is not recommended. It is too sensitive to a wide range of agents and affords no parallel
with the anatomical and physiological relationship existing between pregnant
mammals and her conceptus.
403
.1
I will support the idea of the use of a chick embryo^P screening
tests, early indications, but from that point on additional protocols
and understanding, I think, are necessary.
Again in the line of helping to shorten this interval, I think strict
standards are necessary in defining matters of safety and public
health. No question about it. I don't object to tought regulations.
But industry needs to know where it stands and the dialogue is
important. The dialogue between agencies can help to shorten this
time and I am glad to see distinct improvement in communications
between the Department of Health, Education, and Welfare and the
Department of Agriculture.
Again I would like to say we are not talking about a locked-in
procedure that could be used as an excuse to avoid fxirther responsibility for progress.
Another point; millions have been spent on cancer' research. Yet
to this day, adequate experiments have not been devised or supported on an adequate scale by government to establish thresholds.
By that I mean levels below which no response occurs. Without that
type of information—there will be continuing public fear as analytical methods become more and more sensitive.
Senator HART. May I interrupt you there?
Dr. JOHNSON. May I finish the point and come back?
With teratogens, mutagens, radioactivity and teratology, this all
applies. In other words, if the dose is low enough can any of these
effects be avoided. This is the big question today. It is the question
which coupled with sensitive analytical methods makes the Delaney
clause a difficult thing to live with.
I would like to make the comment that vitamin A, an essential
nutrient, is a teratogen at high doses. So this matter of developing
test procedures and a big enough experiment with carcinogens,
mutagens, teratogens or low levels of radioactivity, needs to be done
on a national scale and supported so that some of this public fear
can be avoided and so we know where we stand.
Scientists should put limitations on their speculations and preconceptions and get the facts but also realize we have a job in serving
the public policy that does not exactly follow the scientific method.
I hope we can work out quicker procedures to go from early indications to reliable decisions by involving some of the procedures I
have talked about. After responding to your question, I would like
to make that final statement.
Senator HART, I am not sure I need to ask the question now. I
agree completely on the desirability of doing that which our best
minds can suggest to shorten the time.
The question I intended to interrupt you with I think you did
answer. I was going to ask to what extent in developing these
thresholds and having broader sampling and more reliable data, to
what extent is that the responsibility of the Federal Government?
Dr. JOHNSON. On the matters of carcinogenicity, teratology,
radiation, and mutagenesis, where such public concern is involved,
and since this involves a wide variety of suspected material, it is my
opinion that the Federal Government should play a major role,in
supporting this research, both in its own laboratories and in extra-
�404
4U5
iRRESTATEMENT OF DR. SAMUEL S. EPSTEIN, CHILDREN'S C
;OOL,
^•ural research done in universities and in industry to help g
; SEARCH FOUNDATION, INC., AND HARVARD MEDICO
^uversity of methods and the best thinking of the nation.
' ''flf
BOSTON, MASS.
Senator HART. All right, then to the extent that the story tlm( *<*||||
have heard today and heard last week reflects delay and a failure«*^if|
Dr, EPSTEIN. Mr. Chairman, it is a privilege to testify before you
communication, I would hope that the suggestions you have ma*!*'-" ' today. The subject of my presentation is teratogenic effects of 2,4,5-T
and the discussions that we have had all will insure that there wjR
be no delay, there will be no barrier to free communication, aiv4 ,, { formulations.
My professional background and experience, as stated in the
there will be no suspicion attached as agency and producer try tt»''' attached appendix 1, broadly relate to the study of hazards due to
drive through to the answer.
_
.'.',5 chemical pollutants in the human environment. Relevant to the presNow, you wanted to remind us of what the Ribicoff comrniUrt';4| ent testimony is my recent role as Cochairman of the Advisory Panel
said.
on Teratogenicity of Pesticides, and as Chairman of the Advisory
Dr. JOHNSON. Yes, if I may.
Panel on Mutagenicity of Pesticides, of the Secretary's Commission
This is read from Report No. 1379 of the 89th Congress, 2d fison Pesticides and Their Relationship to Environmental Health, HEW,
sion "Interagency Environmental Hazards Coordination, Pesticides
and Public Policy", a report of the Committee on Government Oper- SI GPO, December 1969.
The Panel on Teratogenicity was composed of outside consultants
ations United States Senate, made by its Subcommittee on Reorganiand representatives of HEW agencies—CPEHS, FDA, NCI,
zation and International Organizations, I will read from page 50.
NIEHS, and NIDR. The HEW Panel report on teratogenicity is
And this puts the scientists on the spot, including me.
included in the appendix (II).
The heading of the paragraph is "The Responsibilities of the SciA. Teratogenicity as a public health hazard: Potential hazards
entists" and we are talking about industry scientists, governmental,
posed by environmental pollutants and drugs include toxicity^ or poiand university.
soning, carcinogenicity or induction of cancer, mutagenicity or
"The committee asked the scientific witness for meaningful
induction of genetic damage, and teratogenicity or induction of
advice for the Congress, but much of the testimony was inhibited bv
developmental abnormalities in the growing embryo.
defense of past positions, employer loyalties, and lack of authority.
Although teratogenic effects of various agents have been recogScientists should do as thorough a job of preparing answers on
nized for several decades, it was only as a reaction to the thalidoaspects of research administration and planning as they do on the
mide episode of 1962 that a requirement for teratogenicity data
technical details of the work.
became established.
The maker of public policy must have alternatives from which to
Teratology is the study of congenital malformations. These are
choose. There are always strong vested interests which resist change.
generally denned as stmctural abnormalities which can be recogUnless, the technological situation (in this case, ecology) can be
nized at or shortly after birth and which can cause disability or
clearly explained and related to public policy issues, the decisiondeath. Less restrictedly, teratology, also includes, microscopical, biomaker is hardput to recommend any new course. This understanding
chemical and functional abnormalities of prenatal origin.
must be extended also to the citizen. No great social issues have ever
Congenital malformations pose incalculable personal, familial and
been decided until the needs were clear to the man in the street.
social stresses. The financial cost to society of one severely retarded
Scientists cannot assure that their knowledge will reach the decichild, computed on the basis of specialized training and custodial
sionmaker through the normal channels of publication and review in
care alone, approximates to $250,000 (Oberle, 1969), This figure
the scientific community. Without shortcutting the classical methods
excludes further costs to society due to deprivation of earnings.
of assessing the truth, there is still an obligation to interpret what is
In the absence of a comprehensive national surveillance system,
known and replace emotion, rumor, and misconception with a clear
the precise overall incidence of congenital malformations is
explanation of the facts.
unknown. This incidence has been variously estimated as ranging
The role of the scientist in relation to the legislator is limited to
between 3 and 4 percent of total live births.
an area somewhere short of the decisionmaking point. Proper use of
Three major categories of human teratogens have been identified
scientific advice requires considerable effort on both the part of the
—viral infections, for example German measles; irradiation, for
scientific community and the body politic.
example X-rays; and chemical agents, for example thalidomide and
This is the end of my statement.
mercury.
Senator HART. I am glad you reminded all of us of it.
B. Methods for teratogenicity testing: teratogenic effects of chemDoctor, thank you very much.
icals and other agents should of course be identified in experimental
Do your associates have anything to add in light of the statement ?
animals rather than in human beings following accidental or unreAll right, thank you very much.
cognized exposure. Test agents should be administered to pregnant
Dr. JOHNSON. Thank you.
animals during active organogenesis of their developing embryos.
Senator HART, Next we have Dr. Samuel Epstein, Children's
Shortly before anticipated birth embryos should be harvested by
Cancer Research Foundation.
caesarean section and examined.
Doctor, we welcome you.
�Parameters to be considered in test and concurrent control anil
^ The report of the advisory panel on teratogenicity states unambigsh^k include the incidence of abnormal litters, the incideiw*
;|»ously * * *
.
^^
abrrarmal fetuses per litter, the incidence of specific congeri*»{| h Pesticides should be tested at various concentrations including le^^rsubw
abnormalities, the incidence of fetal mortality, maternal *J|P*$j ,, *teiiUally higher than those to which the human population are likely to be
gains in pregnancy, and maternal and fetal organ/body wei^{|5 m
ratios.
."•'!$ |!;,«|K)sed,
; The,report also emphasizes the insensitivity of standard test sysAdditionally, some pregnant animals should be allowed to p»*r,
tems imposed by the relatively insufficient numbers of litters convenbirth in order to identify abnormalities that may otherwise mnnun* .;•
tionally tested.
only in the perinatal period.
'•'
The report further states * * *
Agents and their known metabolites should be administered to tw1 5:
Thus, compounds showing no increase (in birth defects) cannot be considor more mammalian species under various nutritional condition* '
ered nonteratogenic.
during active organogenesis and by a variety of routes reflecting.;
Epidemiological surveys of human populations may provide post
possible human exposure.
hoc information on geographical or temporal clusters of unusual
Of interest in this connection is the lack of data in the availabw
types or frequencies of malformations following exposure to undeliterature on teratogenicity testing by the respiratory route. Respiratected or untested teratogens in the environment. However, logistic
tory exposure is particularly important for pesticide aerosols uw
« considerations, quite apart from inadequate current surveillance sysvapors.
Agents should be tested at higher dose levels than might be untici- ;
tems, limit the utility of this approach.
It should be emphasized that no major known human teratogen
pated in humans following high-level accidental exposure, as well as <
;
, such as X-rays, German measles, mercury, or thalidomide, has been
following extensive low-level exposure. This is essential to attempt
identified by retrospective epidemiological analyses, even in industo reduce the insensitivity of conventional test systems based on very
;
trialized countries with highly evolved and sophisticated medical
small numbers of animals compared with the millions of humans al
presumptive risk.
facilities.
Prospective epidemiologic surveys on agents previously shown or
To illustrate this further, let us assume that at actual human
suspected to be teratogenic, by experimental studies or by retrospecexposure levels, a pesticide induces teratogenic effects or cancer in as
tive population surveys, are clearly inappropriate.
many as 1 out of 10,000 humans, then the chances of detecting this
C. Bionetics studies on teratogenicity of 2,4,5-T: Bionetics
in test groups of less than 50 rats or mice exposed at these actual
Research Laboratories, Inc., of Litton Industries, under a contract
levels would be very low. Indeed, many more than 10,000 rats or
from the National Cancer Institute, tested 48 pesticides, including
mice, depending on their spontaneous incidence of teratogenic effects
2,4-,5-T and related compounds, for teratogenic effects during
or cancer, would be required to demonstrate a statistically significant
effect, if we assumed that rats and humans have similar sensitivity
1965-68.
Although the bionetics studies were originally designed for purto the teratogen or carcinogen being studied.
poses of large-scale screening 2,4,5-T was tested more extensively
For some teratogens, humans may be less or may be more sensitive
than any other pesticide. Thus the data on 2,4,5-T may be regarded
than test animals. Meclizine—a drug used for morning sickness in
pregnancy—for example, is teratogenic in the rat, but not apparas more definitive.
The Bionetics Research Laboratory report is included in the
ently in a restricted number of humans studied (King, 1965;
appendix (III). A revised and more detailed statistical analysis of
Yerushalamy and Milhovich, 1965).
these data is summarized in tihe report of tihe Advisory Panel on TeraWith thaiidomide .conversely, the lowest effective human teratogenic dose is 0.5 mg. per kg. a day. Corresponding values for the
togenicity of Pesticides (appendix II).
2,4,5-T was tested on repeated occasions from 1965-68 in three
mouse, rat, dog, and hamster are 30, 50, 1.00, and 350 mg, per kg. a
day (Kalter, 1968).
strains of mice and in one strain of rats by subcutaneous and/or oral
administration over a dose range from 4.6 to 113 mg. per kg. The
Thus humans are 60 times more sensitive than mice, a hundred
total numbers of litters tested at each dose level, by each ro\\te in all
times more sensitive than rats, 200 times more sensitive than dogs,
700 times more sensitive than hamsters.
strains and species, excluding C3H mice in which only one litter was
Clearly, attempts to determine a safe level for thalidomide, based
tested, were as follows, and I list these in a table enclosed.
As can be seen, the bulk of the data was obtained with BL6 mice.
on animal teratogenicity data, would clearly expose humans to significant teratogenic hazards. Accordingly, it is routine practice to
Due to control variability, the BL6 data have been considered for
test for teratogenicity and carcinogenicity at a range of concentrathree time intervals—prior to September 1966, from September to
tions, including those higher than human exposure .levels,, and
November 1966, and from November 1966 to August 1968.
extending to maximally tolerated doses (MTD).
Data on AK mice were considered for two time intervals—prior to
Even at MTD levels administered to mice from day 7 of life until
November 1966, and from November 1966 to August 1968.
sacrifice at 18 months, less than 10 percent of the 1J/.0 pesticides
Data for BL6 mice, AK mice, and Sprague Dawley fats, as
tested in the recent Bionetics study were shown to be carcinogenic.
derived from the bionetics report, are as follows:
�408
JP3L6 mice: 2,4,5-T administered on days 6-14 or days 9-17, a
mice sacrified on day 18 of pregnancy.
BL6/AK/imce: 2,4,5-T administered on days 6-14, and mice sacrificed on day 18 of pregnancy.
AK mice: 2,4,5-T administered from days 6-15, and mice sacrificed on day ID of pregnancy.
Sprague Dawley rats: 2,4,5-T administered from days 10-15 and
rats sacrificed on day 20 of pregnancy.
In the 4 tables that follow dealing with a wide dose range, the
results are expressed as percentages of abnormal fetuses. I would
point out those occasions where statistically significant incidence of
results was noted.
Major abnormalitities in mice were cleft palates and cystic kidneys, and in rats, cystic kidneys and gastrointestinal hemorrhages.
Increased fetal mortality was generally concomitant with these
abnormalities. It is of particular interest that 39 percent abnormal
embryos with cystic kidneys were seen in rats even at the lowest
dose tested. Thus the no effect level was not reached even at 4,6
mg./kg.
_ _
_
Teratogenicity data on 2,4,5-T, as summarized in the bionetics
report (appendix III) are quoted in extenso below. Some critical
sentences are italicized:
This compound was given by the oral route to BL6 mice at dosages of 40.0
und 113 nig/kg and to AKR mice nt 113 mg/kg. It was given by subcutaneous
injection to BL6 mice nt dosages of 21.5 and 113 mg/kg and to AKR mice and
B6AK hybrids at 113 mg/kg. It was also given subcutaneously to 03H mice ut
215 mg/kg, but there were too few of these to merit inclusion in the discussion
which follows.
Administration was for eight days (6th through 14th) in most cases; for
nine days (6th through 15th) in some; and for five days (10th through 14th)
in one case—the details are indicated in the tabulated results. Subcutaneous
administration used DMSO as a vehicle; oral used 50 percent honey.
With the single exception of the lowest dosage used (21.5 mg/kg to BI/6 subcutaneously) all dosages, routes and strains resulted in increased incidence of
abnormal fetuses. The incidence of cleft palate was high at the 113 nig/Us
dosage, but not at lower levels. The incidence of cystic kidney was also high
except in the AKR strain and in the BL6 mice which received 46.4 mg/Jtg
orally. Fetal mortality was increased in all groups given 113 mg/kg for eight
or nine days, but not in mice (BL6) given this dosage for only five days nor
in the two groups of BL6 mice given lesser dosages (46.4 mg/kg orally and
21.5 mg/kg subcutaneously.
Most fetal and maternal measurements showed inconsistent changes from
which no conclusions can be drawn. In contrast, there was a highly consistent
decrease in maternal weight gain in BL6 mice given 113 mg/kg by either route.
Tjower dosages and the AKR strain showed either no change or a slight
increase. All dosages, strains, and routes showed an increase in the maternal
liver weight and this led to a further study discussed separately below.
These results imply a hazard of teratogenesis in the use of this compound.
The problems of extrapolation preclude definition of the hazard on the basis of
these studies, but Us existence seems clear.
The observed influence of 2,4,5-T on maternal liver weight as mentioned
above raised a question as to its effect on the fetal liver. This was answered
by a study carried out in Bt/6 mice using subcutaneous injections of DMSO
solutions at a dosage of 113 mg/kg only. The period of administration was
lengthened to cover the period from the 9th through 17th day of gestation.
Separate control groups were used concurrently. Except for the inclusion of
fetal liver weight, measurements were made as previously described.
The fetal livers of the 2,4,5-T treated mice weighed significantly more than
those of controls given DMSO only and the weights of the whole fetuses were
409
liver
nignificantly less. Correspondingly, there was an increase in
weight expressed as percent of body weight.
Other observations were consistent with those reported above. The incidence
o/ abnormal fetuses was unusually high as were those of cleft palate and
cystic Udney.
Because of the potential importance of the findings in mice, an additional
ntudy was carried out in rats of the Sprague-Dawley strain. Using dosages of
21.5 and 46.4 mg/kg suspended in 50 percent honey and given by the oral route
on the 6th through 15th days of gestation, we observed excessive fetal mortality (almost 80 percent) and a high incidence of abnormalities in the survivors.
When the beginning of administration was delayed until the 10th day, fetal
mortality was somewhat less, but still quite high even when dosage was
reduced to 4.6 mg/kg.
The incidence of abnormal fetuses was threefold that in controls even with
the smallest dosage and shortest period used. Fetal and maternal measurements showed only occasional instances of significant differences from controls
except in the case of maternal liver weight which was consistently increased
in all 2,4,5-T treated animals.
It seems inescapable that %,4,5-T is teratogenic in this strain-of rats when
(liven orally at the dosage schedules used here. These findings lend emphasis to
the haeard implied by the results of studies on mice.
I). Recent reanalysis of the Bionetic data on teratogenicity of
2,4,5-T. More refined and more appropriate additional statistical
analyses of these data were presented and discussed in the report of
the Advisory Panel on Teratogenicity of Pesticides (appendix II).
These are clearly confirmatory of the original conclusions of the
Bionetics report on the teratogenicity of 2,4,5-T. Some relevant portions of the HEW panel report are quoted in extenso below:
Tested more extensively than other pesticides, 2,4,5-T was clearly teratogenic
as evidenced by production of statistically increased proportions of litters
affected, and increased proportions of abnormal fetuses within litters in both
DMSO and honey for both C57BI//6 and AKR mice. In particular, cleft palate
and cystic kidneys were significantly more prevalent. In addition, a hybrid
strain resulting from a C57BI//6 female and AKR male showed significant
Increases in anomalies, in particular cystic kidney, when administered at 113
mg/kg of body weight in DMSA,
Additionally, 2,4,5-T was tested in Sprague-Dawley rats. AVhen given orally
at dosages of 4.6, 10.0, and 46.4 mg/kg on days 10 through 15 of gestation, an
excessive fetal mortality, up to 60 percent at the highest dose, and high incidence of abnormalities in the survivors was obtained. The incidence of fetuses
with kidney anomalies was threefold that of the controls, even with the smalleat dosage tested.
E. Recent studies on teratogenicity testing of relatively pure
2,4,5-T. In view of the fact that the Bionetics study was conducted
with a sample of 2,4,5-T which was subsequently shown to contain a
relatively high concentration, 27 ppm, of a tetrachloro dioxin contaminant, testing has been recently repeated with relatively pure
samples containing less than 1 ppm of this particular dioxin. *
The results of these studies were presented by the FDA and
NIEHS at a recent conference of February 24, 1970, at the FDA;
the Dow Chemical Co. data were presented at the 9th annual meeting of the Society of Toxicology, Atlanta, March 17,1970.
As can be seen from the data summarised ~below, purified tl^fi-T
is teratogenic in three species—rats, mice and hamsters. These data
should be regarded as preliminary. Confirmatory data on chick eggs
are not presented here.
1. Dow Chemical Co. studies (Emerson et aL, 1970). 2,4,5-T with
0.5 ppm dioxins, as a probable contaminant, was tested in pregnant
�A
411
410
rats by repeated oral administration at doses of 1, 3, 6, 12, and 24
mg./kg.; the maximal dose tested was 24 mg./kg. No embryo deaths
or weight losses were noted within the dose range tested. However,
at 24 mg./kg. there was a sevenfold increase in the incidence of
fetuses with defective ossification of the fifth sternebra; poor sternebral ossification was noted in four out, of 103 control fetuses, and in
• 29 out of 103 fetuses of 2,4,5-T treated groups.
Defective sternebral ossification has been described in the rat as an
expression of the teratogenic effects of drugs such as protamine zinc
insulin and tolbutamide (Lichtenstein et ah, 1951; Dawson, 1954).
2. NTEHS studies: Using the purest sample of 2,4,5-T, made
available by Dow Chemical Co., teratogenic effects were induced in
Swiss-Webster mice. Cleft palates were noted at dose levels of 150
mg./kg. and scattered abnormalities at 100 mg./kg.; the cleft palate
incidence in control mice was essentially zero.
3. FDA studies: Hamsters were injected with five doses of 100
mg./kg./day of various batches of purified 2,4,5-T between days 6-10
of pregnancy. In one of these studies, there was a 66-percent incidence of mortality in 50 fetuses. Of the surviving fetuses, 17 percent
had congenital abnormalities—crooked tail, missing limb, and defect
in skull fusion. No data was presented on possible effects induced by
doses less than 100 mg./kg.
Of additional interest was a report also presented at the same conference on purified 2,4-D, which produced a 22-percent incidence of
congenital abnormalities in hamsters at a dose level of 100
mg./kg./day.
>
.
.
,
,
1 should like now to address myself to the toxicity of dioxins.
Toxicity of dioxins: Rabbit ear skin is highly sensitive to dioxins,
repeated application of which can produce chloracne, as a cumulative manifestation of local toxicity. Approximately 0.3 micrograms
of the tetra isomer will produce a positive response; "more than 10
micrograms on a surface wipe sample indicates acute hazard" (to
man) (Silverstein, 1970).
The acute oral LD-50 dose of tetra dioxin in male guinea pigs is
0.5-1.0 micrograms/kg., and in male and female rats, 22.5 and 45
microgram/kg,, respectively. Feeding chicken edema factor diets,
containing dioxins, produced cumulative toxicity in monkeys (Allen
and Carstein, 1967). Storage of hexa, hepta and octa isomers, as
identified by GLC, has been reported in chickens and rats fed
chicken edema factor diets (FDA, unpublished). Chronic administration of 2,4,5-T or 2,4-D to dogs produces cumulative toxicity with
gastrointestinal haemorrhage, suggestive of cumulative dioxin effects
(Drill and Hiratzka, 1953).
TERATOGENICITY OF DIOXINS
1. FDA studies (FDA Conference, Feb. 24, 1970) : A mixture of
dioxins, 21 percent trichloro and 53 percent tetrachloro isomers, were
injected in hamsters between days 6-10 of pregnancy over a dose
range from 0.5 to 9.1 microgram/kg. per day. At the highest dose,
the incidence of fetal mortality was 82 percent and the incidence of
congenital abnormalities, 82 percent. At the 0.5 microgram/kg. dose,
there was a 5 percent incidence of abnormalities. The no-effect level
was thus not reached at 0.5 micrograms per kg.
,
f
2. Dow studies (Sparsclm et al., 1970) : The tetra dk^fc isomer
—
-
-
-
.
'
•
»
«
^
w
n ^^V
was fed to Sprague Dawley rats between days 6-15 of ^&t
over a dose range from 0.03 to 8.0 micrograms/kg. per day. There
was a marked increase in resorption sites at the 2 microgram level.
Gastro-intestinal hemorrhages occurred over a range from 0.125 to 8
micrograms, dose-dependently. Additionally, at the 0.125 microgram/kg, level there was a decrease in male fetal weights.
It should be emphasised that cystic kidneys were not seen at the
0.125 microgram/kg. dose of the tetra isomer or even higher levels.
In the Bionetics study, 2,4,5-T at 4-6 mg./kg., containing 25 ppm of
the tetra dioxin isomer equivalent to 0.124 microgram/kg., produced
a 39 percent incidence of congenital abnormalities with, cystic kidneys.
There is thus a clear discrepancy between the teratogenic effects of
2,4,5-T containing 25 ppm of dioxin, and the effects of the equivalent concentration of the same dioxin. It is, however, conceivable
that this discrepancy may reflect synergistic interactions between
dioxin and 2,4,5-T.
SOME UNRESOLVED PROBLEMS RELATING TO 2,4,5-T AND DIOXINS
1. Chemical composition of 2,4,5-T formulations: Currently used
2,4,5-T formulations contain about 5 percent of known impurities,
largely polychlorophenols. Analytic data on a sample of 2,4,5-T
(Dow data, on production batch 120449) in the following table substantiates the approximate 5 percent of polychlorophenol impurities
in 2,4,5-;T formulations as currently used.
There are no available data on the presence and concentration of
the more than 60 positional isomers of dioxin, other than the
2,3,7,8-tetrachloro dioxin isomer, in this batch of 2,4,5-T, or in other
batches produced for food crop or other purposes in the United
States and abroad.
In view of the relatively high concentration of polychlorophenol
impurities in 2,4,5-T, it is likely that a wide range of dioxins are
also present. 2,4-D and other phenoxy herbicides are similarly chemically uncharacterized.
. The higher positional dioxin isomers, hexa, hepta, and octa, have
been identified in 2,4-dichlorophenol, a precursor of 2,4-D. Apart
from the presence of dioxins in polychlorophenols, heating of polychlorophenols will produce additional and very high yields of
dioxin.
Illustratively, heating 5 g. of pentachlorophenol at 300° C. for 12
hours yielded 1.5 g. of the octa-dioxin isomer (Cowan, 1970). There
are no available data on the possible production of dioxins from
combustion of 2,4,5-T or 2,4-D. While improved production techniques may well reduce the levels of polychlorophenols and the
levels of the 2,'5,7,8-dioxin isomer, apart from other isomers, in
2,4,5-T and other phenoxy herbicides, the degree to which this is
practical does not yet appear to have been clearly defined.
2. Stability and persistence of dioxins: The extent of• usage of
2,4,5-T and other phenoxy herbicides on food crops and for other
purposes in the United States and abroad dictates the scale of
resulting environmental contamination with 2,3,7,8-dioxin and other
45-362 0—70
-27
�413
412
isomers. The following data are illustrative and (Agricultural Kco*
nomic Eeport No.-131, IJSDA, 1968).
These data reflect deliberate applications of phenoxy herbicides on
crops, and do not reflect unintentional crop contamination following
the more extensive application of herbicides for brush control or
.other purposes. There are no available data on the extent of such
unintentional contamination. It is, however, well known that phenoxy herbicide dusts may drift for miles, even on nonwindy days, following routine application (Federal Register, 1969). The concentration of phenoxy herbicides in the air in Washington in 1964 reached
a maximum of "' —--«~~«or>-, /™ 3, with n,n averaee of 0.046
maximum 01 3.4 microgram/m.a „»„
„
microgram/m3 (Bamesberger and Adams, 1966).
These figures probably underestimate the proportional concentration of atmospheric dioxins, in view of their high stability relative
to phenoxy herbicides.
I now present a table on calculated dioxin contamination of an
acre of soil, following 2,4,5-T application.
CALCULATED DIOXIN CONTAMINATION PER ACRE FOLLOWING 2,4,5-T APPLICATION
Based on 0.5
ppm Dioxin
Basod on
ppm "ioiin
0.57
5.70
2M
Application 2,4,5-T (Ibs/acre)
2.5 (domestic .use).
25.0(export use)...
n
Now, these calculations are based on the 2,3,7,8-dioxin isomer
alone, and ignore additional contamination due to other dioxin isomers. The figures for export use should be adjusted to reflect varying concentrations of 2,4,5-T in different formulations.
The high concentration of polychlorophenolic impurities in 2,4,5T, approximately 5 percent, apart from other sources of
polychlorophenols, may resiilt in extremely high yields of dioxins.
As mentioned previously, heating of 5 grams of pentachlorophenol
at 300° C. for 12 hours results in a yield of 1.5 grams of the octadioxin isomer. Combustion of shrub, brush, timber, or other materials exposed to phenoxy herbicides or other polychlorophenols, may
thus liberate high concentrations of dioxins in the atmosphere.
It is thus of interest to examine the data on stability and persistonce of dioxins in the environment. The 2,3,7,8-tetra isomer is known
to be heat stable up to 800° C. There are, however, no available data
on the heat stability of other dioxin isomers. There are also no
available data on the stability and persistence of the 2,3,7,8- and
other dioxin isomers in soil, water, crops, milk, and animal or
human tissues.
Most importantly, there are no available data on the possible accumulation and transmission of 2,3,7,8- and other dioxins in the food
chain—air, soil, and water—to plants, brush and crops—to fish,
birds and cattle—to man, with attendant accumulation in man.
The heat stability of the tetra isomer, the general lipid solubility
of the dioxins, and their cumulative toxicity in experimental' am1
smismals, all serve to enhance the possibility of food chain tr
sion of the various dioxin isomers.
'
3. Teratogenicity of relatively pure 2,4,5-T.
The relatively contaminated 2,4,5-T used in the Bionetic study,
containing about 27 p.p.m. of the tetra-dioxin contaminant, induced
congenital abnormalities in mice and rats, particularly cystic kidneys, that were not produced by the pure tetra dioxin. Illustratively,
the contaminated 2,4,5-T at 4.6 mg,/kg./day oral dose level in rats
produced a 39 percent incidence of congenital abnormalities. This
dose of 2,4,5-T is equivalent to 0.124 micrograms/kg./day of the
tetra-dioxin. as reported in the Dow studies, presented at the Society
However,
of Toxicology meeting in Atlanta on March 17, 1970,_ 0.125
micrograms/kg./day of the tetra dioxin did not produce cystic kidneys' in rats. This discrepancy may however conceivably reflect
synergistic interactions between 2,4,5-T and dioxins.
Additionally, as indicated above, relatively pure 2,4,5-T, containing one p.p.m. of the tetra-dioxin contaminant, was teratogenic
in preliminary studies with three species—mice, rats, and hamsters,
over a dose range of 24^-150 mg./kg. In some of these studies, for
example, in hamsters at 24 mg./kg./day, no effect levels were not
reached. recent studies on relatively pure 2,4,5-T clearly confirm its
Thus,
teratogenecity and lend further emphasis to the following conclusions of the report of the HEW Teratogenicity Panel: "The use of
currently registered pesticides to which humans are exposed and
which are found to be teratogenic by suitable test procedures in one
or more mammalian species should be immediately restricted to prevent risk of human exposure."
All these considerations confirm the following conclusion stated in
the Bionetics reports (App. Ill, p.19) : "These results imply a
hazard of teratogenesis in the use of this compoxind, 2,4,5-T. The
problem of extrapolation preclude definition of the hazard—but its
existence seems clear."
4. TOXICOLOGY Or DIOXINS
The toxicology of dioxins is of particular interest in view of the
previously reviewed data on the very high acute toxicity, embryotoxicity, cumulative chronic toxicity of 2,3,7,8-dioxin and related isomers, and also the stability, widespread environmental distribution,
and likelihood of accumulation and transmission of any dioxins in
the food chain. data, in the available literature on the carcinogenicity
There are no
or mutagenicity of positional isomers of dioxins. Recent studies on
the teratogenicity of dioxins have been largely restricted to the
2,3,7,8-isomer; there are no available experimental data on behavioral or psychopharmacological effects due to dioxins; this would be
of interest in view of the possible psychiatric effects described in
humans exposed to dioxins. There are no data in the available literature on any toxicological studies on any dioxin isomer following
acute or chronic administration by inhalation.
�414
The extreme inadequacy of toxicological data on dioxins dearly
precludes consideration of potential human hazards .due to, dioxin*
in air, food or water, and consideration of possible safety margini
following exposure to dioxins.
I would like to reemphasize the conclusions of the HEW panel on
teratogenicity:
"The use of currently registered pesticides to which humans are
exposed and which are found to be teratogenic by suitable test procedures in one or more mammalian species should be immediately
restricted to prevent risk of human exposure. Such pesticides, in
current use, include Gaptan; Garbaryl, the butyl, isopropyl, and
isooctyl esters of 2,^,-D Folpet; mercurials; PGNB; and H
The teratogenicity of 2^.,-D, the other salts and esters of both ,i
and 2,4,5-T, and that of IPO should be investigated further."
Finally and critically, available data on tlae toxicology of the dioxins, and more importantly on the lack of data on the toxicologyacute and chronic toxicity, carcinogenesis, mutagenesis, and teratogenesis—of the numerous positional isomers of dioxins, indicate an
urgent need for restriction of human exposure to dioxins. Similar
restrictions should extend to polychlorophenols, polychlorophenolic
containing formulations, and their combustion products.
I thank you, sir.
(A list of the references to the statement follow:)
BEFERENCES
Oberle, M. W. Science 165, 991-992, 1069.
King, 0 J. Pharm. JStsp. Therap. J.Jf7, 391, 1965.
Yerushalamy, J. and Milkovich, L. Am. </. O&s. Gynac., 93, 553,1965.
Kalter, H. Teratology of the central nervous system, University of Chicago
Press, 1968.
Emerson, J. L., Thompson, D. J., Gerbig, 0. G., Eobinson, V. B. (Dow Chemical Co.) Teratogenic study of 2,4,5-Trichlorophenoxy acetic acid in the rat.
Society of Toxicology, 9th Annual Meeting, Atlanta, Georgia 3/17/70.
Lichtenstein, H., Guest, G. M. and Warkany, J. Proc. Soc. Exp. Mol. d MeA.
78, 398-402, 1951.
Dawson, J. E. Diabetes 13, 527-531, 1964.
L. 0. Silverstein, Dow Chemical Co. Memo. February 7, 1970. Safe Handling of
Tetrachlorodibenzo-p-dioxin (TCBD) in the Laboratory.
Allen, J. R. and Carstein, L. A. Light and electron microsocopic observation in
Macaco, mulatto, monkeys fed toxic fat. Am. J. Vet. Res. 88, 1513-26,1967.
FDA. Unpublished data.
Drill, V. A. and Hirateka, T. Intl. Hygiene & Occupat. Med. I, 61-67, .1953.
Sparschu, G. L., Dunn, F. L., and Rowe, V. K. (Dow Chemical Co.) Teratogenic study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the rat. Society of Toxicology 9th Annual Meeting. Atlanta, Georgia 3/17/70.
Cowan, ,T. C. USDA Memo 2/12/70. Possible sources of polychlorodibenzodioxins
in fats.
Quantities of Pesticides used by Farmers in W6Jt. Agricultural Economic
Report No. 131. Economic Research Service, USDA, Washington, D.C. 1968.
Federal Register, May 21, 1969.
Bamesberger, W. L., and Adams, D. R. In, Organic Pesticides in the Environment Advan. Ohem. Ser. GO, (1966).
Senator HART. Thank you, Doctor.
In your summary you refer to 2,4-1) among other things, and earlier in your statement you said that 2,4-D produced birth deformi-
415
ties in the hamster. What is your opinion as to the teratogenicity of
2,4-D in comparison to 2,4,5-T ?
Dr.' EPSTEIN. The findings of the Bionetics study in relation to
2,4-D, were certainly not as conclusive as for 2,4,5-T. But, on the
other hand, in the wide range of tests performed in the Bionetics
study there were strong suggestions of teratogenic effects. That was
as far as one could go. For these reasons the HEW Panel on Teratogenicity clearly recommended that further work should be done on
2,4-D and that a high degree of suspicion should be attached to it.
Moreover, the recent FDA studies on hamsters would seem to confirm the earlier suspicions of the Bionetics studies in mice and
would indicate to say the least the need for a high degree of caution
and minimally, indicate the need for restriction of further human
exposure to 2,4-D pending clarification of these problems.
I think to await further definitive studies before taking action
would not be appropriate in the light of the available data.
Senator HART. That gets \is back, and we return here all the time,
to this balancing of principle.
On the point that Mr. Bickwit was developing with Dr. Johnson,
on the business of the cumulative effect of dioxin I take it you
regard them as cumulative; is that correct?
Dr. EPSTEIN. On the basis of the available data in the literature,
one could say there is a strong suggestion of cumulative effects. The
rabbit ear skin test depends on cumulative toxicity and the chick
edema effect is cumulative. One can demonstrate the pickup of dioxins in rat and chicken liver. So available data indicate they are persistent and cumulative, although I would regard these data as far
from definitive.
Senator HART. What is your feeling as to the adequacy of the evidence? What is the extent of the evidence on the degradability of
the dioxins in ultraviolet light ?
Dr. EPSTEIN. I would submit that data on strong sunlight and
shortwave TJV is probably irrelevant. If you expose dioxins to
strong sunlight you are exposing material to light which contains
short wave ultraviolet in a manner not closely related to normal conditions.
I would like to see studies on conditions of photo-degradability of
dioxins on conditions of shade and normal daylight. This is not
available.
Senator HART. Well, we always seem to run into the lack of available data in pursuing answers to these questions. Now let's get to the
availability of substitutes. What alternatives exist, or are you in
position to know, for 2,4,5-T and 2,4-D ?
Dr, EPSTEIN. T am obviously not an agricultural expert and we
are now talking about crop use, I presume. Does your question relate
to crop use of herbicides or total use ?
Senator HART. I guess I should ask it totally and if there are
areas where there is an alternative, identify that.
Dr. EPSTEIN. With the qualification that I am no expert in this
field and not qualified to comment on the relative merits of various
�417
416
herbicides, I would merely point out they fall into two categoric*. :.,
the pre-emergence and post. For the pre-emergency group, you have
amiben, trifluralin, and atraxine, which all seem to be appropriate , "
•for use under a wide range of conditions.
. ;
As far as post-emergence herbicides, 2,4-D and 2,4,5-T are herbi- •;;;;
cides with which we are familiar, additionally there are others e.g.» «;
endothal, atrazines and dinoseb. Atrazihes are pre-emergence and ^
post-emergence herbicides. There is thus a wide range of herbicides ,<;
for use on both pre- and post-emergence levels.
Senator HART. We have staff inquiry as to whether you have any ,
knowledge as to the cost differences, if any, as between 2,4-D and
2,4,5-T and some of these other items.
Dr. EPSTEIN, I am not competent to answer that.
Senator HART. What tests have been made on the dioxins for genetic damage or for cancer ?
Dr. EPSTEIN. There have been no tests made on any dioxin for
• genetic- damage. There have been no tests made on any dioxin for
carcinogenicity, either by oral administration or by injection or bv
inhalation. I must point out that we are talking about very small
levels of highly potent compounds and we must remember certain
carcinogens are active at less than a part-per-million level. We have
no ball park data on this at all. We just don't know whether we are
dealing with significant ranges or nonsignificant ranges.
Senator HART. Is the same true with respect to testing of _ the
exposure by inhalation or respiration of the herbicides or dioxins?
Early in your paper you remind us of this.
Dr. EPSTEIN. There are no data in the total literature of chronic
carcinogenicity or mutagenicity of any pesticide whatever by inhalation. This is a point brought out quite forcefully in the Mrak Panel
report. Inhalation is a very significant route of human exposure and
the experimental test systems do not reflect recognition of this exposure.
Senator HART. What possible dangers may result from either
2,4-,5-T or 2,4-D in noncrop use, and other herbicides in noncrop use?
Dr. EPSTEIN. Well, a wide range of possibilities exist and it is not
possible to define many of these.
First of all, you get drifts of the actual 2,4,5-T and 2,4-D on the
crop use and I have given data on the extent of this and the concentration of herbicides in Washington. Over and above that, material
which is sprayed like timber, pasture, and shrubs may well be
burned.
In fact it is quite common to burn shrubs after you spray and we
already know if you heat 5 grams of pentachlorophenol at 300
degrees you get 1.5 grams of the octadioxin isomer. So you are
putting into the environment material rich in polyphenols and
which are likely to be combusted. We have no idea how long these
dioxins will persist and these data are just not available.
But there are very real possibilities of formation wPsignificant
quantities of dioxins from phenoxy herbicide use. But I must
emphasize that similar considerations also obtain to a rather wide
range of polychlorophenols which we have not adequately discussed
here.
Senator .HART. If you had an opportunity to examine the announcement made and reported to us today, with respect to the suspension
or cancellation of 2,4,5-T, given the possible danger of the noncrop
_ use that you discussed, do you concur in the action taken? Do you
think it goes too far or not far enough?
Dr. EPSTEIN. Do I have to answer that?
Senator HART,. Sir ?
Dr. EPSTEIN. Do I have to answer that ?
Senator HART. Well, you don't have to, but it would be helpful. I
am sure it would be accepted as from a person whose qualifications
to make the judgment are excellent.
Dr. EPSTEIN. My view then, based on available data and also lack
' of data on the phenoxy herbicides, is that their use under conditions
where humans are exposed is clearly not warranted.
Senator HART. Does that include the application on grazAnglnnd?
Would you include that use when you say use to which humana
would be exposed I would submit that phenoxy herbicides should,
Dr, EPSTEIN. ?
under no circumstances, be used on the crops. I would submit there
is a strong presumption for suspension of their use under any circumstances in the environment.
Mr. BICKWIT. What possible dangers do you see resulting from use
of .Dr. EPSTEIN. ?Before responding to that, might I just list the cate• chlorophenol
gories of use of polychlorophenols? Again, this is an industrial area
in which I am not competent to go into any detail. But as far as I
can see polychlorophenols are used for a wide range of purposes.
First of all, for treatment of timber; then as slimicides and fungicides; for paints, varnishes, lacquer; laundry starch; for shampoos:
paper and paper coatings, including food wrappings; for curing 01
hides; rendering of fats; production of feeds for animals and for
man; and for manufacture of a wide range of pesticides.
Pentachlorophenol is used as a herbicide quite apart from being
used on sugarcane, so there is a wide range of use for this in the
environment. If you take 5 grams of pentachlorophenol and heat it
at 300 degrees centigrade, you form 1.5 grams of the octadioxin.
We are not now _ talking about a billionth or trillionth part per
gram; we are talking about 1.5 gram of the octadioxin isomer. I
cannot tell you the degree of resulting hazard because nobody has
tested it for carcinogenicity, mutagenicity, either by injection, skin
application, or inhalation.
�419.
418
Therefore, your question is unanswerable except to say wliat
information wo have on the dioxins indicate that these are hig
toxic. That is as far as one can go.
Senator HART. Well, the data on the risk side, then, in so many of
these areas, are a little skimpy, if not nil. So that when a^encw*
have to make this judgment balancing the benefits which are identifiable against the possible health risk, how can they make that kind
of judgment in the absence of adequate risk data?
Dr. EPSTEIN. I agree entirely. The concept of balancing risk
against benefit is predicated on two assumptions. We usually gut *
good idea what the benefits are, and secondly we should know as
exactly as possible what the risks are,
In this instance we do not know what the risks are, and, therefore,
this equation cannot be evaluated here. We cannot discuss benefit if
wo have no information whatsoever on carcinogenicity, mutagenicity.
and teratogenicity.
Senator HART. Well, without answering the hard question of
whether you take a hard line and not permit anything on the market
until you get the needed data or whether you use the lack of data as
tlie reason for permitting it to go out until the data are available,
the long and short of your testimony, and that of others we have
heard today, is that it is extremely important to develop and intensify our efforts to get that data. Is not that the obvious message of
today ?
Dr. EPSTEIN. This is one of the messages, yes. But 'we have hftd
information on dioxin for 20 years. We have known of the carcinogenicity of DDT since the late 1940's.
Senator HART. Well, if you are being critical, that is fair enough,
but wouldn't you agree that whether we are slow in reacting or not,
wo had better react now ?
Dr. EPSTEIN. Yes.
Senator HART. Doctor, thank you very much for this wonderful
statement. I perhaps have indicated my own feelings by my interruption. I have had the same reaction on this subject today as we
have on so many subjects. If we could stop the world for 7 days and
get one part of one set of problems fixed, then crank the world up
before stopping it again to move on the rest, in a lifetime we might
get most of the things fixed. But you do have a feeling of wondering
whether we will manage to move in time with sufficient intelligence
to respond to all the problems that confront us.
(The appendices follow:)
APPENDIX I
SAfVJH. S . r . I ' S I I U J
Person;) 1:
U.S.
Boston,
1947
1950
1952
IDS'1,
1958
1963
1950
1951
195?.
ii.Sc. (Physiology) London U n i v e r s i t y , E n g l a n d .
M . B . B . S . (Bachelor of M e d i c i n e , Bachelor cf Surgery)
(Double Honors) London U n i v e r s i t y , E n g l a n d .
D . T . M . H . (Diploma of Tropical Medicine and H y g i e n a ,
Bacteriology and Parositology) London U n i v a r s i t y .
D . P a t h . (Diploma Of Pathology) London U n i v e r s i t y .
M . U . (Doctorate of Medicine, Thesis in Pathology and
Bacteriology) London U n i v e r s i t y , E n g l a n d .
Dipoloniate, i n Public. Health and Medical Laboratory
Microbiology, of the American Board of Microbiology.
,nstrator Morbid Anatomy, Guy, H o s p i t a l , Undon.
uc,,....,,
Demo J .r8toi",; Morbid Anatomy, buy *. n ^ - . ^ — , _ .
House P h y s i c i a n , St. O o h n ' s H o s p i t a l , London.
Postgraduate Student in Tropical M e d i c i n e , Pathology
[
Bacteriology and Parasltolocy, Royal Army Medical College,
\
London.
S p e c i a l i s t in Pathology, Royal Army Medical Corps
Lecturer in Pathology and Bacteriology, Institute of
Laryngology and Otology, U n i v e r s i t y of London.
B r i t i s h Eunire Cancer Campaign Research F e l l o w , in
195B - I960
conjunction w i t h the Chester Beatty Cancer P>esearch
' Institute and T u m o r ' P a t h o l o g i s t ' a t ' t h e H o s p i t a l for
Sick C h i l d r e n , Great Orrcond Street, London.
Consultant in Pathology, The Memori?! H o s p i t a l , Pc-terhorout;h,
1960
England,
Research Associate in Pathology and Microbiology, The
C h i l d r e n ' s Hospital Medical Center and the Chil'dren's
1961 to date
Cancer Research Foundation, I n c . , Doston.
C h i e f , laboratories of Carcinogenesis and Toxicology,
A p p l i e d Microbiology and H i s t o l o g y , the C h i l d r e n ' s
1961 to date
Ci>ncer Research Foundation, I n c . , liorton.
Senior Research Associate in Pathology, the C h i l d r e n ' s Cancer
Research Foundation, I n c . , Boston, and Rsr,etrch Associate
1962 to date
in Pathology, Harvard "cdical School, Boston.
1952 - 1955
1955 - 1958
�Awards:
1.
2.
Socle ty Mejnbarsh 1 ps:
Committees:
British Medical Association
Society of Clinical Pathologists
Society for Pathology and Bacteriology
Society for General Microbiology
Society of Protozoologists
Air Pollution Control AssoclAtion
American Association of Pathologists and Bacteriologists
American Society for Experimental Pathology
American Association for Cancer Research
American Board of Microbiology
Society of Toxicology
Environmental Hutagenesis Society
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h
(i
j
k)
Cong r_es r. i onal> Tes.tj ngny:
Military Awards in Royal Army Medical Corps, 1953
(a 'Montefiore Go'ld Medal in Tropical Medicine
b Montefiore Prize In Tropical Hygiene
c Ranald Martin Prize in Military Surgery
Society of Toxicology, 1969 Achievement Award
Member, Committee on the Relation of Protozoology
to Public Health. The Society of Protozoologists,
19S2--1969.
Chairman, Committee on Biological Effects of Air
Pollution, Air Pollution Control Association,
1963-1969.
Member, Technical Council of the Air Pollution
• Control Association,, 1963--1969.
Executive Secretary, Environmental Mutagenesis
Society, 1969.
Chairman, Committee on Cyclainates and Caffeine,
Environmental Mutagen Society, 1969.
Chairman, Committee on Liaison, Environmental
Mutagen Society, 1969.
Chairman, 1969, HEW Pa-nel, Hutagenicity of Pesticides "
Chairman, 1969, HEW Panel, Teratogenicity of Pesticides
Member, 1969, HEW Panel, Pesticide Interactions
Member, 1969, HEW Panel, Cardnogenicity of Pesticides
Chairman, NIMH Panel on Chronic Non-psychiatric Hazards
of Drugs of Abuse, 1969.
On "Cancer and Mutation-Producing Chemicals in Polluted
Urban A i r " , at Hearings before the Sificommittee on Air and
Hater Pollution of iho Committee on Publia Harks, July 2931, 1968, presided by Senator Edmund S, Muskie.
1. Epstein, S. S., and Winston, P.:
Intubation granuloma.
0. Laryntjol. and Otol., 7j_: 17-38, 1957.
2. Epstein, S. S., Winston, P., Friedmann, I., and Ormerod, F. C.:
The vocal cord polyp.
J. l.aryngol. and Otol., 7J_: 673-688, 1957.
3. Epstein, S.' S., and Shaw, H. J.:
Metastatic cancer of the larynx as a cause of carotid-sinus syndrome.
Cancer, 1_0_: 933-937, 1957.
4. Epstein, S. S.:
An intra-oral inoculation technique for the production of experimental
pneumonia in mice.
J. Hygiene, 56: 73-79, 1958.
5. Epstein, S. S., and Stratton, K.:
Further studies in the mouse Intra-oral inoculation technique.
J. Hygiene, 56: 81-83, 1958.
6. Epstein, S. S., and Shaw, H. J.:
Multiple malignant neoplasms in the air and upper food passages.
Cancer, Vl_: 326-333, 1958.
7.
Winston, P., and Epstein, S. S . :
Papilloma of the larynx: A clinico-patholoqical study.
....
J. Laryngol. and Otol. j 72: 45Z-464, 1958.'
8.
Epstein, S. S . , and Friedmann, I.:
j<]ebs_ij:l_]_a_ serotypes in infections of the ear and upper respiratory
tract".
0. Clin. Path., 2_: 359-362, 1958.
9.
Epstein, S. i.:
A "stripping" technique for the examination of the total epithelial
surface of the larynx.
J. Path, and ISact. , 7.5: 472-473, 1950.
10.
Fropnian, T., Wakefielcl, G. S., nnd Epstein, S, S . :
Platelet-agglutinating factor in glandular fever complicated by
jaundice and thromSocytopciria.
The Lancet, 383-385, October 25, 1058.
�422
423
Publ
PuM ications
11.
Epstein, S. S. , and Brcidboar, T. I.:
A case of primary diphUir-rti.io otil.fs media.
J. Laryiifiol. anJ Otnl., 72; 1001-1003, lflf.8.
21.
Epstein, S. S . , Weiss,, 0. I!., IHisli, P . , and Causey, I).:
Vit-iiiiii! B,/, tiiitl growth of Cug'lena G r a c i l l s .
Fed. Proc., _20_(l): 4bO, Mroxli "Te'MV
12.
Epstein, S. S.:
22.
Epstein, S. S . , ai:d Tii,v,,i1s, G. II.:
"Simplfl" Vit.fniiin B 1? antiire-Ubo'litos.
Proc. Amor. A s s o c . Cantor R e s . , 3 ( 3 ) : 223, 19C1.
23.
Epstr.-iti, S. S. , and Burrouijhs, M. :
Some factors Influencing the photodynai'ilc rusponse of Paj'anisc'li'iii
caudt.tuii! to 3,4-b2itxpyi'crifi.
Hnturo',"]_9.3..: 337-33(1, 1962.
24.
Epstein, S. S . , Burroughs, M., Small, M., and Verbrugghsn, M.:
The pho'todynamic toxi.dty of polycycllc hydrocarbons.
Proc. Aniar. Assoc. Cancer Res., 3_(4): 316, 1962.
25.
Epstein, S. S . , W e i s s , 0. B., Causeley, D., and Bush, P.:
Experimental K'lebsjella pneumonia in mice with particular reference
to peri arterial changes.
J. Path, and Bact., 78; 389-390, 1959.
13. Epstein, S. S.
The biochsmistry and antibiotic sensitivity of the Klobsi.ella.
J. Clin. Path., 1_2: 52-58., 1959.
14. Epstein, S. S., end Payne, P. M.:
The effect of some variables on experimental KlcbsJ.ella infections
in mice.
J. Hygiene, 57; 68-80, 19D9.
15.
Shav), H. J., and Epstein, S. S . :
Cancer of the epiglottis.
Cancer, 1?: 246-256, 1959.
16.
Epstein, S. S. , and Weiss, J. B . :
The extraction of pigmants from Euc|luna_ cjracjllis_.
'Biochem. J., 75: 217-250, 1959.
17.
Timmis, C. M., and Epstein, S. S . :
New antimetabolites of Vitamin B,,,.
Nature, 184: 1383-1384, 1959.
27.
18.
Epstein, S. S . , Payne, P. M., and Shaw, H. 0 . :
.
Multiple"primary malignarit'neoplKsms in the air and upper food passages.
Cancer, 13.: 461-463, 19GO.
28.
19.
Epstein, S. S . , and Weiss, 0. B.:
Measuring the size of isolated cells.
Mature, 187.: 461-463, 1900.
Epstein, S. S . , Burroughs, M., and Small, M.:
Tho photoclynamic effect of the carcinogon ; 3,4-ben?.pyrene on
Paramecumi cauclatuin.
C"a"nceir"Re~s".7"2S: K-44, 1963.
29.
Epstein, S . S . :
L'ffectsof some benzinvidazolc.'S on a Vitamin B^-requirinrj alga.
Nature., 188: 143-144, 1960
Epstein, S. S . , Small, M., Koplan, J., and Mantel, N.:
Photodynaiiiic bioassay of t>eiizo[a'lpyreno using Paijjmiecluin amdatuii).
J. Nat. Cancer Inst., 31; 163..168', 1S63.
"" "
30.
Epbioin, S. S . , Small, M., Jones, II., Koplan, 0. , and Ifankel , N . :
A photodynaiiiic bioassay of atmosphurlc pollulanls.
Proc. Amor. Assoc. Cancer lies., _4(1): 18, 19C3.
20.
Influence of Vitamin B,, on the size and growth of Euglena qracillis.
J. Protocol., 9_, 336-339, 1962.
"
26.
Epstein, S. S . , and Timmis, 6. M.:
Effect of Vitamin B,, antagonists and other compounds on the C1300
tumor.
Biochsm. Pharrnacol., 11: 743-746.. 19S2.
Epstein, S. S . , and Timmis, 6. M.:
Simple antinrotabolites of Vitamin B,,. •
l£
- .J. Protpzool., JO; 63-7.3,. 1953.
�425
424
Pub! ica'tions
j'ubVi.catipns
41.
31.
Acta Unlo internsi., i,um..«
32. Epstein, S. ~S., Small, M., Koplan, J., Mantel, H., Falk, It. 1., and Sawicki, V.
Photodytisuic bioassay of polycyclic. air pollutants.
A.M.A. Archives of Environmental Health, 7: 53'l-b37, 1SS3.
42. Small, M., Brickman, E,, and Epstein, S. S.:
Uptake of polycyclic compounds by phagotrophic protozoan.
Fed. Proc. , ?A_: 684.. 196b.
43.
. Small, H. , Jones, I!., and Epstein, S. S.:
I'hotodynavAic activity of polycyclic compounds.
Fed. Prpc., 22: 316, 19G3.
34. tpstein, S. S., Small, M.,betweenH. I., and Mantel, carcinogenic activities
On the association Falk, photodynamic and M.:
in polycyclic compounds,
Cancer Res., 24: 855-862, 1964.
E p s t e i n , S. S , , Saporoschetz, 1. I!., S m a l l , M . , Park, W . , and Mantel, f ! . :
A s i m p l e bioassay'for a n t i o x i d n n t s based on protection of Tetjjihyiiiei
p y r i f o r a i s from the photodynamic t o x i c i l y of b(inj:o[a](iyrone.
KaUir"e', 200: 655-C58', 1965".
33
Epstein, S. S.., Forsyth, J., and Bulon, I.:
A simple bioassay for antioxidtnts.
Fed. Proc., 24j ,623, 1965.
44. Epstein, S. S.:
Bioassay for polycyclic atmospheric pollutants and for antioxidants
based on photodynamic response of protozoa.
Abstract from Second International Conference of Protozoology, London,
August, 1965. Reprinted from Excerpta Msdlca International Congress,
Series 91.
35. Foley, G. E., and Epstein, S. S,; .
Coll culture and cancer chemotherapy.
in "Advancfts in Chemotherapy", ]_: 196-1, Academic Press, New York.
45.
36. Epstein, S. S., Bulon, I., Kaplan, 0., Small, H., and Mantel, N.:
Charge-transfer complex formation, carcinogenicity and photodynamic
acti.vlty in polycyclic compounds.
Nature, 2U4_: 750-754, 1964.
46. Epstein, S. S . , and J o s h i , S. R . :
Obstructive renal f a i l u r e in random-bred Swiss mice.
Fed; P r o c . , 25: 237, 1966.
37. Epstein, S. S,, Gulon, I., and Kaplan, J.: carcinogenicity and photodynamic
Charge transfer complex formation,
. - . activity in polycyclic-compounds.
Fed. Proc., 23_(2);' 287, 1964.
38.
Epstein, S. S.:'
Photoactlviition of polynuclear hydrocarbons.
A.M.A. Archives of Environmental Health, 1_0: 233-239, 1965.
39. Epstein, S. S., Small, M., Sawicki, E., and Falk, H. l.\
Photodynawic bioassay of polycyclic atmospheric pollutants.
J. Air Poll. Control Assoc., 15: 174-176, 1965.
, '
40.
Epstein, S. S..:
A simple
photodynamic assay for polycyclic atmospheric p o l l u t a n t s .
World Health Organization Report, UHO/BL/S1, 1965.
E p s t e i n , S, S. :
The l u n g as a transplant site for m a l i g n a n t tumorsin rodents.
Cancer, 19_: 454-157, 1966.
- 4.7,..Eps.tein, S. S . , Saporoschstz, I. B . , and M a n t e l , N , :
Interactions between antioxidant and photosensitizer in the
photodynamic bioassay for a n t i o x i d a n t s .
Life Sciences, 5; 783-793, 1966.
48. Epstein, S. S . , Forsyth, J . , Saporoschetz, I. li., and M a n t e l , N . :
An exploratory investigation on the i n h i b i t i o n of selected photosensitize
by agents of v a r y i n g a n t i o x i d a n t a c t i v i t y .
Rad. Research, £8: 322-335, 1966.
49.
E p s t e i n , S. S . , and Tabor, F. B, - .
P h o t o s e n s i t i z i n g conipounds in extracts of U . S . A . d r i n k i n g water.
Science, 1.5_4.(3746): 261-263, 1966.
50. E p s t e i n , S. S , :
Two s e n s i t i v e tests for carcinogens in the a i r .
J. Air P o l l . Control Assoc., 1 6 ( 1 0 ) : 546-546, 1966.
�427
426
Publications
51.
52.
53.
54.
55.
Publications
Small, A . , Mantel, N., and Epstein, S. S . :
The role of cell-uptake of polycyclic compounds in photodynanric
injury of Jetrajiy_mena_ pyrvformis.
ExperimentaT'Cel 1 'Research ,~Wr""206-217, 1967.
Epstein, S. S. , Joshi, S . , Andrea, J., Forsyth, J., and Mantel, N.:
The null effect of antioxidants on the carcinogenicity of 3,4,9,10dibcnzpyrene to mice.
Life Sciences, (5: 225-233, 1967.
Epstein, S. S., and Niskanen, E. E.:
Effects of Tween 60 on bsiuo[a]pyrene uptake by Tetrahymena £JZ-!!Pr"v'.s.
and by isolated rat liver mitochondria.
Experimental Cell research, 46: 211-234, 1967.
Epstein, S. S., Ooshi, S., Andrea,J., Clapp, P., Falk, II., and Mantel, N.:
The synergistic toxicity and carcinogenicity of Freons and
piperonyl butoxide.
Nature, 21_4: 526-528, 1957.
56. Epstein, S. S., Saporoschetz, I. B., and Hutner, S. H.:
• - - - . - - • Cytoxicity of antioxidants to Tetrahyaiena- pyrlforrr,is;
J. Protocol., J_4_: 238-244, 1957.
'"
57.
58.
59.
Nagata, C . , Fujii, K. , and Epstein, S. S . :
Pliotodynamic activity of 4-n1troC|Uinoline-l-oxide and related compounds,
Nature, 21_5: S72-973, 1967.
Epstein, S. S . :
Cjrcinogonici ty of organic extracts of atmospheric pollutants.
J. Air Pollution Control A s s o c . , 17, 7?fi-7!>(i, 1967.
61.
Epstein, S. S . , Andrea, J., Clapp, P., and Mackintosh, D . :
Enhanceii'iSnt by piperonyl butoxide of acute toxicity due to Freons
lien?.o(a)pyreno, and Grisoofulvin in infant mice.
Toxicology and Applied Pharmacology, VI, W-448, 19G7.
62.
McCarthy, R. E., and Epstein, S. S.:
CytocheiTiical and cytoofinetic effects of maleic hydrande on
cultured mammalian cells.
Life Sciences, 7_, 1-6, 1968.
63.
Epstein, S. S , , Mantel, N., and Stanley, T. W . :
Photodynaiivic assay of neutral sub-fractions of organic extracts
of particulate atmospheric pollutants.
Environmental Science and Technology, £, 132-141, 1968.
64.
Epstein, S. S . , and Mantel, N,:
Hepatocardnogem'city of maleic hydroxide following parenteral
administration to infant Swiss mice.
Intel-national Journal of Cancer, .3, 325-335, 1968.
65.
Rondia, D . , and Epstein, S, S . :
The effect of antioxidants on photodecomposition of benzo(a)pyrene.
Life Sciences, 7_, 513-518, 1968,
Epstein, S, S . :
Carcinogenicity of Tetraethyl lead,
Cxperlentia, 2_4_, 580, 1968.
"
'
!
"
67.
Epstein, S. S. and Shafner, H.:
the of mammals in a practical screening test for chemical mutagens in
the human environment.
Mature, 2_19, 385-387, 1968.
68.
Epstein, S. S., Andrea, J . , Joshi, S . , and Mantel, N.:
llepatocarcinoganicity of cjriseofulvin following parenteral
administration to infant mice.
Cancer Research, 27_: 1900-1906, 1967.
Epstein, S. S., Andrea, J., Mantel, II., and Fall;, II.:
Carcinogenicity of the herbicide uialeic hydrazido.
Nature, 215: 1308-1390. 1967.
60.
66.
Epstein, S. S,, Joshi, S., Andrea, J,, Mantel, H., Sawicki, (.., Stanley, T . ,
and Tabor, E. C , :
Carcinogenicity of organic parti cul ate pollutant;, in urban air after
administration of trace quantities to neonatal mice.
Nature, 212: 1305-1307, 1966.
Jaffe, J . , Fujii, K. , Sengupta, M., Cuerin, II., and Epstein, S. S . :
In yi_vo inhibition of mouse liver microsomal hydroxylating systcmr,
by wetiiyloii(:UioiVpl\fjiiyl insectitidal syneryists and related compounds.
Life Sciences, 7_, 1051-1052, 1908.
�428
429
Publications
69. Epstein, S. S.:
Cancer and mutation-producing chemicals in polluted urlmn air.
Air Pollution (Air Quality Criteria) Hearings before the Subcommittee on Air and Water Pollution of the Committee on Public ' .
Works.. U. S. Senate, 91st Congress. Washington, D. C., 1968.
Publications
78. Lpslcjin, S. S. :
Introduction to sympovia on toxicologic and epidemiologic bases for
air quality criteria.
Journal of Air Pollution Control Association, 19, 629-630, 1969.
11
70. Epstein. S. S.:
Irradiated roods
Science, 161_, 739, 1968.
71. Fujii, 1C.,'Jaffe, H. and Epstein, S. S.:
Factors influencing the haxobarbital sleeping time and zoxazolamine
paralysis time in mice.
Toxicol. Appl. Pharmacol., 13, 431-138, 1968.
72. Epstein, S.'S., and Saporoschetz, J.B.:
On the association between lysogeny and carcinogenicity in
nitroquinolines.
Experientia, 24_, 1245-48, 1968.
73. Jaffe, H., Fujii, K.. Sengupta, M., Guerin, H., and Epstein, S, S.:
The bl-modal effect of piperonyl butoxide on o- and p-hydroxylation
of biphenyl by mouse liver microsomes.
Biochem, Pharmacol., 18, 1045-1051, 1969,
74. Epstein, S. S.:
Chemical mutagens and the Environmental Mutagen Society
-"Current Opinion" editorial
Medical Tribune and Medical News,"10., pp IT-IS, June ?.', 1969.'
75. Pagnatto, L. [K , and Epstein, S. S.:
The effects of antioxidants on ozone toxicity in mice.
. Exporientia, 25, 703-704, 1969.
76. Epstein, S. S.:A oaioh-all toxicological screen.
Exporientia, £!j_, 617-618, 1969.
77.
Epstein, S. S., and St. Pierre, J. A . :
Mutagenicity in yeast of nitroi|uino1inos and related compounds.
Toxicol, Appl. Pharmacol., 15, 451-4GO, 19C9,
79. L'pstoin, S. S. :
' • Chcmicali Iwards in the human environment.
Ca-A Cancer Journal for Clincians, J_9, 277-281, 1969.
80. Epstein, S. S., Hollaonder, A., I.ederberg, J., Leoator, M., Richardson, II.,
and Wolff, A. H.:
Cyclainate Ran
Science, 166, 1575, 1969.
81. Lijinsky, W., and Epstein, S. S.:
liiti'osann'nes as environmental carcinogens,
Nature, 225, 21-23, 1970.
82. Epstein, S, S., Fujii, K., Andrea, J., and Mantel, H.:
Carcinogenicity testing of food additives and antioxidants by
parenteral administration to infant Swiss mice.
Toxicol. Appl. Pharmacol. 16, 321-334, 1970.
83. Fujii, K., Jaffe, H., Bishop, V., Arnold, E., Mackintosh, D., and
Epstein, S. S.:
Structure-activity relations for methylenedioxyphsnyl and related
compounds on hepatic microsomal enzyme function, as measured by
prolongation of hexobarbital narcosis and ?.oxazolai»1ne paralysis
in mice.
Toxicol. Appl. Pharmacol. 16., 482-494, 1970.
84. Epstein, S. S., Arnold, E,, Steinberg, K., Mackintosh, D., Shnfner, H.,
and Bishop, Y.:
Mutagenic and antifertility effects of TEPA and METEPA in mice.
Toxicol. Appl. Pharmacol,
85. Epstein, S.. S., Joshi, S. Pv,,_ Arnold, E., Page, E. C., and Bishop, Y.
Abnormal zygote development in mice after paternal exposure to a
chemica] mutagon.
Nature
86. Epstein, S. S., Csillag, R. G., Guerin, H., and Friedman, M. A.
In vitro effects of methylonedioxyphenyl insecticidal syncrgists
on hydroxylations of biphenyl by mouse liver microsomes.
Biochem. Pliannatol.
87. Epstein, S. S. , Bass, W., Arnold, F., and Bishop, Y.
The failure of caffeine to induce mutagenic effects or to synergizc
thfc offe.cts of known mutagens in mice.
Fd. Cosmot. Toxicol.
�430
431
APPENDIX II
Publications
•88.
Epstein, S. S., and Lederbcry, J.
Chronic non-psychiatric haxards of drugs of abuse
Science
89. Friedman, M., and Epstein, S. S.
Stability of piparonyl butoxido.
Nature
90. Epstein, S, S.s Bass, W., Arnold, E,, and Bishop, Y.'
The imitauenicHy of trimethyl phosphate in mice.
Science
91, Epstein, S. S. , arid Fujii, K.
Synorgism 1n carcfnogenesis with particular reference to synorgistlr.
effects of piperonyl butoxide and related insacticidal synergists
(Chsp coy*)
In, Chemical Tumor Problems, ed. Nakahara, W., Tokyo, 1970,
92. Epstein, S. S.
The failure of caffeine to induce mutagenic effects or to synerqize
the effects of known mutacjens in mice. (Chapter)
In, Chemical mutagens. ed. Vogel, F., Heidelberg, 1970.
93. Bateman, A . , and Epstein, S. S.
Dominant lethal mutations in mammals. (Chapter)
In, Environmental chemical mutagens. ed. Hollaender, A . , Plenum
Publishing C o . , New York, 1970.
94. Joshi, S. R., Paye, E. C., Arnold, E. , Bishop, Y . , and Epstein, S. S.
Fertilization and early embryonic development subsequent to mating
with TEPA-treated male mice.
. •
Genetics
95. Oaffe, II., Epstein, S. S., and Neumeyer, 0. L.
_•
,
Comparative effects of .piperonyl butoxide and N-(4-pentynyl)
phthalimido on mammalian microsamal enzyme functions.
.J. Mecl. Chem.
96. Epstein, S. S., and Rohrborn, G.
Recommended procedures for testing genetic hazards due to chemicals
based on the induction of dominant lethal mutations in mammals.
Nature
SUMMARY AND CONCLUSIOKS
Teratology deals with the etiology and development of congenital
malformations. Congenital malformations are generally denned as
gross structural abnormalities of prenatal origin, present at birth or
manifesting shortly after, which kill or disable. In a broader sense,
teratogenesis is considered to include histological, biochemical, and
functional abnormalities of prenatal origin.
Congenital malformations present obvious personal, medical, and
social stresses. Additionally, it has been recently estimated that the
costs to society of one severely malformed child, in terms of medical
and other care and deprivation of potential earnings, amount to several
hundred thousand dollars.
There are now well over 400 substances that, in various forms and
combinations, are currently used as pesticides. Pesticides may represent an important potential teratogenic hazard. Therefore any teratogenic pesticide to which the population is exposed should be promptly
identified so that appropriate precautions can be taken to prevent risk
of human exposure. It is feasible to test these substances for teratogenic
effects in test animals so that potential hazards to human health can
l>e evaluated.
For these and other reasons detailed in the report, we conclude that:
a. All currently used pesticides should be tested for tetratogenicity
in the near future in 2 or more mammalian species chosen on the basis
of the closest metabolic and pharmacologic similarity to human beings
possible. Pesticides should be tested at various concentrations including
levels substantially higher than those to which the human population
are likely to be exposed. Test procedures should also reflect routes
related to human exposure. Apart from the obvious route of ingestion,
attention should be directed to other routes of exposure, including
inhalation exposures from pesticide aerosols and vaporizing pesticide
strips used domestically and exposures from skin absorption. Parenteral administration is an appropriate test route for pesticides to which
humans are exposed by inhalation, or for pesticides which are systemically absorbed following ingestion.
b. The use of currently registered pesticides to which humans
are exposed and Avhich are found to be teratogenic by suitable test pcocedures in one or more mammalian species should be immediately
�432
433
restricted to prevent risk of human exposure. Such pesticides, in current use, include Captan; Carbaryl; the butyl, isopropyl, and
isooctyl esters of 2,4-D Folpet; mercurials; PCNB; and 2,4,5-T. The
teratogenicity of 2,4-D, the other salts and esters of both 2,4-D and
2,4,5-T, and that of IPO should be investigated further.
c. Pesticides found to be' inactive after appropriate testing can
be considered as provisionally safe, unless other evidence of teratogenicity develops.
d. No new pesticide should be registered until tested for teratogeuicity by suitable procedures. Any pesticide found to be teratogenic
should only be used in circumstances where risk of human exposure
is minimal.
e. Efforts should be made to improve and standardize procedures
for teratogenicity testing and population monitoring.
A scientific group or commission should be charged with responsibility for continued surveillance of the whole problem of pesticide '
teratogenesis.
ment is apparently undisturbed, and above which delM in utero
results.
Most agents are teratogenic only in the developmentally labile early
period of gestation, during which active organogenesis occurs. In
humans, this sensitive period extends aproximately from the end of the
first week of pregnancy to the 12th week. Other circumstances may
also influence the effectiveness of human teratogens, such as maternal
nutritional, demographic, socioeconomic, and cultural factors, physiological states, and temporal and seasonal situations. Thus a potential
teratogen may manifest its effect only when particular conditions
conjoin.
The relationship between human exposure to a teratogen and subsequent induction of congenital abnormalities is generally not obvious.
Any one teratogen may produce a multiplicity of effects and any specific effect may be produced by various teratogens. In test animals, the
teratogenetic response may differ from species to species. In humans,
differences in genetic, metabolic, and environmental influences may
contribute to a variety of specific effects from exposure to a particular
teratogenic agent. Induced and spontaneous effects may be difficult to
distinguish. The teratogenicity of thalidomide might have been missed
had it not produced malformations rarely encountered; additionally,
only a fraction of the pregnant women who took thalidomide had
defective children.
Consequently, further data on the possible teratogenic effects of pesticides in experimental animals are urgently needed to provide a basis
for evaluating potential hazards to human health.
Ancillary methods
Preliminary screening can be accomplished by the use of nonmammalian species, particularly the chick embryo. These tests may give
useful ancillary data prior to further testing in mammals. However,
negative results in these systems alone should not be considered proof
of safety.
METHODOLOGIES FOR TERATOGENICITY TESTING
Introduction
Prior to 1963, the Food and Drug Administration did not require
evaluation of teratogenicity. As a result of the thalidomide disaster,
the need for data on teratogenicity became evident. In 1963, the President's Science Advisory Committee on "Use of Pesticides" recommended that toxicity studies on pesticides include effects on reproduction through at least 2 generations in at least 2 species of warmblooded
animals. Observations to be included were effects on fertility, size and
weight of litters, fetal mortality, teratogenicity, and growth and development of sucklings and weanlings. Such toxicity studies including
the three-generation procedure were not designed primarily to detect
teratogenicity and thus may not be appropriate.
The potential teratogenicity of chemicals may be detected by two
complementary approaches. First, chemicals or other agents may be
administered to experimental animals to determine whether they
induce prenatal damage. Secondly, and on a post hoc basis, human populations may be epidemiologically surveyed to detect geographical or
temporal clusters of unusual types or frequencies of congenital malformations. Combinations of these approaches are likely to ensure early
detection and identification of teratogenic hazards.
Experimentally, a complex of factors are needed to elicit teratogenic
effects. These relate to gestation period, genotype of the pregnant animals, dosage, mode of administration and metabolic transformation of
teratogen. For example, teratogens may be effective only at a certain
dose range, whether high or low, narrow or wide, below which develop-
Use of lower mammalian species
a. Purity, composition, stability, and source of compounds under
test should be determined.
b. At least two mammalian species should be tested. These should
be chosen on the basis of metabolic and pharmacokinetic similarity to
humans. If possible, commercially available inbred strains should be
used; if not, intra-species variability must be recognized. Species commonly used include mice, rats, hamsters, rabbits, dogs, cats; sheep and
swine have also been used.
c. Preliminary mammalian experiments should determine--the
amounts of the compound and its appropriate metabolites necessary
�434
to produce serum levels comparable to ranges likely to be found in humans after high level accidental exposure as well as potential exposures assuming extensive use of that pesticide. Multiples of these dosages, up to the mammalian maternal LDBO should be administered to
determine the lowest dosage causing a significant increase in fetal
death, or resorption. Dosage in this critical range should be tested for
teratogenic effects .with care to distinguish these effects from other
embryotoxicity and to determine dose-response relationships.
d. Compounds should be administered, by appropriate routes,
within the critical dose range determined by preliminary tests. Parenteral administration is an appropriate test route for pesticides to which
humans are exposed by inhalation, or for pesticides which are systemically absorbed following ingestion. Compounds should first be
tested by single administrations of a range of doses at various times
during the phases of active organogenesis. The substance should be administered at discrete times throughout the period of organogenesis as
various organs are developing, since some substances have specific
effects on the development of particular organs. By this technique,
the possibility of inducing hepatic microsomal or other enzymes facilitating metabolic detoxification or activation of the substance is
also minimized. If no teratogenic effects are detected by this technique,
subsequent testing should be based on repeated administrations of the
substance at daily intervals or if feasible, intervals of less than 24 hours
during the entire period of organogenesis.
e. When appropriate, metabolites should also be tested for teratogenic effects.
f. Additional investigations should include—
i. Determination of appropriate plasma and fetal levels of
compounds;
ii. Determination of the biological half-life of the compound
in test animals;
iii. Metabolic studies to identify mechanisms of detoxification or
activation of compounds when appropriate; and
iv. Determination, when appropriate, of the possible potentiating effects of protein deprivation or concomitant exposure to
other pesticides or other environmental agents. '
'
g. All procedures, including those relating to animal.breeding,
housing, handling, feeding, husbandry, methods for examining fetuses
for congenital malformations, defining the onset of pregnancy, and
classifying congenital malformations should be rigorously standardized. Numbers of pregnant animals and offspring must be adequate
for statistical significance. All tests must be replicated on independent
occasions and with contemporaneous controls.
435
Nonhuman primates .
Results from lower mammalian species may warrant subsequent
testing in nonhuman primates The following considerations should be
noted:
a. Records of menstrual cycles are essential. Primates whose reproductive history is known and have previously delivered normal young
should be selected for testing. Timing of ovulation, and therefore gestation, should be accurately determined by allowing the males and
the females to be together for no more than 3 consecutive days. Vaginal
smearing, to determine the presence of spermatozoa should be avoided;
the use of Tullner's method for determining chronic gonadotropin
levels and rectal palpation is preferable.
b. Compounds should be carefully administered in controlled
dosages.
i
c. Pregnant animals should be handled only minimally.
d. Compounds should be administered during the various phases
of organogenesis. Embryos can be obtained by laparotomy any time
after the first 100 days of gestation; the mother may be subsequently
used for other experimental procedures. Additionally, some young
should be allowed to go to term to identify possible teratogenic effects
detectable only in the neonatal period.
Population monitoring
It has been shown (see Literature Review) that some pesticides induce congenital malformations in experimental animals providing a
critical dose, is appropriately administered at critical times. "When
animal experiments indicate that a pesticide is teratogenic, human
effects should be retrospectively evaluated, when possible, by study of
pregnancies during which the mothers were inadvertently exposed to
the pesticide, such as a result of farm work, accidental ingestion, or industrial exposure. Prospective epidemiologic approaches may involve
follow-up of large numbers of people over long periods of time, and be
slow, tedious, expensive, or difficult to implement. It is not appropriate
to conduct prospective epidemiological studies on human populations
with pesticides previously shown to 'be teratogenic by experimental
animal studies or retrospective human data. Human exposure to such
compounds must be minimized by appropriate regulatory preventive
action.
Prospective epidemiological approaches for pesticides in current
use may provide important information, however, it should be realized
that no major teratogen has yet been recognized in this way. The malformations induced by X-ray, German measles, thalidomkle, and
mercury—Minamata disease, Avere each recognized by an alert medical
�practitioner who observed a cluster of cases and then traced the cause
tojjs source.
m
^PLat can be done to enhance prompt recognition of such clusters
should they occur from previously unsuspected teratogens in the future? A variety of existing data resources can be used for this purpose. In each, the occurrence of congenital malformations in substantial segments of the population is being recorded in a standard fashion. The best of these resources are local, rather than statewide or national. The prepaid medical program of the Kaiser-Permanente Hospitals and Clinics in the San Francisco Bay Area are of particular interest. A detailed study there of the occurrence of malformations
among 16,000 births represents a good model for additional investigations. A similar study has been made by the Health Insurance Plan
of Greater New York, but its 30 or more cooperating clinics are less easily coordinated than the Kaiser system.
A citywide surveillance, known as the Metropolitan A'tlanta Congenital Defects Program (jointly directed by Emory University
School of Medicine, the Georgia Department of Public Health, and
the National Communica,ble Disease Center, USPHS), involves reports on all children with congenital malformations born to residents
of the five-county Atlanta area.1 As yet, no cluster of cases has suggested an environmental influence since the program began in October,
1967.
In a substantial number of States, birth certificates contain an item
concerning congenital malformations. The completeness and accuracy
of such reporting varies considerably and depends on the physician's
interest and diligence and on the conspicuousness of the abnormality.
Birth-certificate data on malformations in New York State are more
extensive than those of many other States and have been effectivel}'
used in several research studies. Nationally, however, no attempt has
been made to collect and evaluate all data on malformations that are
available on birth certificates.
A select committee convened by the National Center for Health
Statistics (NCHS), has recommended, in an excellent but little known
report, that efforts be made to improve and use information on congenital malformations recorded on birth certificates (Vital and Health
Statistics, Documents and Committee Reports, NCHS Series 4, Number 7, March 1968). Implementation of this recommendation would
bo of great value, for monitoring to detect the teratogenic effects of
newly introduced or geographically localized environmental chemicals
or other agents.
To enhance our ability to recognize significant changes in congenital malformation rates, a systematic collection of data from
concentration points should be established. Specifically, a surveil-
lance 'should be made of claims submitted to private, State, or local
agencies for the medical care of children with birth ded^ks. Because
the Children's Bureau, DHEW, has so much experien^r with these
agencies, its assistance should be sought in planning the surveillance
network.
Data from foreign countries should also be evaluated as part of
a national effort to study possible relationships between pesticides
and congenital malfunctions.
In studying the possible relationships between exposure to pesticides
and the occurrence of diseases, statistical associations, if present, will
provide important information. However, when possible it is important to secure additional information concerning the following:
a. Dose-response relationships.
b. Absence of alternative explanations.
c. Biological plausibility.
d. Consistency with other knowledge from clinical, laboratory,
and epidemiologic research.
e. Disappearance of the effect when the presumed cause is
removed.
In particular, as clusters of specific anomalies are recognized,
through whatever resources that presently exist or may be developed,
any possible relationships to pesticides would be clarified by the use
of laboratory techniques to measure the maternal, fetal, or neonatal
body burden of suspect chemicals.
There are national units engaged in teratologic research, but each
is following a set method. There is a critical and immediate need to
establish a national or international center to study congenital malformations in man not by a single method but by whatever techniques
are most appropriate for testing or generating hypotheses. The center should be diversified and fast moving, ready to use local, national,
or international resources in order to determine the significance of
laboratory or clinical data.
LITERATURE REVIEW
Animal studies
For convenience, detailed results of the Bionetics study are presented in a subsequent section.
Much of the total available literature and data reviewed by this
Panel were methodologically inadequate to support definitive conclusions. Additionally, the authors of many reports tended to confuse
or equate em'bryotoxicity and other adverse effects on reproduction
with teratogenicity. It is also apparent from the literature that insufficient attention has been directed towards problems of interactions
in testing for teratogenesis.
�The Panel considered the following information to be of
a. Captan and Folpet.—These pesticides have been shown to be
terntogenic in chicken embryos (Verrett et al., 1969). Captan was also
shown to be teratogcnic in rabbits (McLaughlin, 1969), although other
rabbit studies yielded negative results (Kennedy et al., 1968; Fabro
et al., 1965). The enhancement by protein deprivation of the acute
toxicity of captan to rats (Boyd, 1968), was noted with particular
interest. The teratogenicity of captan and Folpet in mice was demonstrated in Bionetics studies. Unpublished data on captan in monkeys were evaluated and found inadequate; in these studievS, the duration of organogenesis was not entirely covered mid controls were not
appropriate. However, the 3/7 abortions observed at the highest dosage
given, 25 mg./kg., may be indicative of an em'bryotoxic hazard due to
captan.
b. Carbaryl.—This was tested at 66.7 and 200 p.p.m. in the diet of
pregnant mice (FAO/WHO, 1967). In tivo litters at the 200 p.p.m.
level, a total of seven instances of skeletal malalignment, nonfusion,
incomplete ossification, and one case of cleft palate and gross facial
malformation were noted, as opposed to no malformations in the lowlevel group and-two cases of cleft palate in controls. Teratogenetic
findings for carbaryl are also reported in the Bionetics study. In a.
study in beagle dogs fed carbaryl during gestational periods at levels
of 50, 25, 12.5, 6.25, and 3.125 mg./kg. body weight daily, teratogenic
effects were found at all but the lowest dose level (Smalley, 1968).
c. Mercurials.—Organomercury compounds: Various mercury
containing pesticides were evaluated under, the heading "phenylmercury acetate (and other organomercury compounds)" by the 1966
Joint Meeting of the FAO Working Party and the WHO Expert Committee on Pesticide Eesidues (FAO/WHO, 1967). The results of additional experimental work have been reported in the 1967 Evaluations
of Some Pesticide Kesidues in Food. Additional information on
"Methylmercury" was published by the Ecological Research Committee, the Swedish Natural Science Research Council (1969) Bulletin
no. 4. by Goran Lofroth, where embryotoxic effects in mice (reported
by Fro'len and Ramel) were discussed along with other data. When
given subcutaneously, in doses of 0.11 mg. on day 7 of gestation,
phenylmercuric acetate was reported to cause fetal malformations in
mice. Eye,'tail, and central nervous system defects were noted (Murakami et al., 1956).
d. Organ&chlorine,—Embryotoxicity in rats and dogs has been
reported for organochlorines including dieldrin, chlordane, and
kepone. In the absence of convincing data, kelt'hane has been claimed
to be teratogenic in mice (An Der Lan, 1964); see also Bionetics
studies.
^^
e. Organophosphates.—The cholinesterase-inhibitn^p organophosphate insecticides, guthion, parathion, diazinon, Bidrin, Trithion,
and EPN, have been shown to be teratogenic when injected directly
in the yolk sac of chick embryos. The malformations were nonspecific or
common to all organophosphates (Fish, 1966). It was also claimed that
these compounds are teratogenic in mice. The data reported, however,
suggested that organophosphates, like the organochlorines, act by
reducing litter size and producing embryotoxicity rather than by
producing specific teratogenic effects. See also Bionetics studies.
/. Thiram.—Thirarn was reported to be teratogenic in hamsters
at 250 mg./kg. (Ilobens, 1969). In the Bionetics study it Avas not found
to be teratogenic. In a study of three generations of rats, no toxicological effects were observed at a dietary level of 48 p.p.m. (FAO/WHO,
1967). However, Thiram should be further investigated for possible
teratogenic effects.
g. Miscellaneous reproductive effects,—Placental transfer of
dieldrin and incidence of stillbirths have been studied in cows (Braund,
1968); increased stillbirth rates have been claimed in cows fed with
DDT (Labon, 1965). The estrogenic activity of o,p'DDT has been
related to reproductive effects in chicken, quail, and rats (Baseman,
1968, Wurster, 1968; Porter and Weimeyer, 1969). Diminished population size and reproductive failure have been produced in sparrow
hawks by DDT and dieldrin (Porter and Weimeyer, 1969). These
resulted from a decreased eggshell thickness, increased breakage of
eggs, and increased egg eating by parent birds. Other studies of
interest include the following: Finnegan, 1949; Tauber, 1950; Fisher,
1952; Narpozzi, 1956; Swann, 1958; Cottrell, 1959; Marliac, 1964;
Backstrom, 1965; Hathaway, 1967; Ware, 1967; Weike, 1967; Carlton,
1968; Keplinger, 1968; Khera, 1968; Verrett, 1969; Legator, 1969.
Bionetics animal studies
Bionetics Research Laboratories of Litton Industries, during
1965-68 under a contract for the National Cancer Institute (NCI
Contracts PH 43-64-57 and PH 43-67-735), tested various pesticides
and related compounds for teratogenic effects. These studies were designed as large-scale screening tests. The Bionetics data were reanalyzed statistically to account for litter effects. The results of this
statistical re-evaluation are presented in this section. More detailed
material on these pesticides will be published in the future.
a.. Summary of findings from Bionetic animal studies.—Tested
more extensively than other pesticides, 2,4,5-T was clearly teratogenic
as evidenced by production of statistically increased proportions of
�440
, and increased proportions of abnormal fetuses within
litters in both DMSO and Honey for both C57BL/6 and AKR mice.
In particular, cleft palate and cystic kidneys were significantly more
prevalent. In addition, a hybrid strain resulting from a C57BL/6
female and AKR male showed significant increases in anomalies, in
particular cystic kidney, when administered at 113 mg./kg. of body
weight in DMSO.
Additionally, 2,4,5-T was tested in Sprague-Dawley rats. When
given orally at dosages of 4.6, 10.0 and 46.4 mg./kg. on days 10
through 15 of gestation, an excessive fetal mortality, up to 60 percent
at the highest dose, and high incidence 6f abnormalities in the survivors was obtained. The incidence of fetuses with kidney anomalies
was three-fold that of the controls, even with the' smallest dosage
tested.
PCNB produced an increase in renal agenesis between litters,
and within litters, when administered orally from days 6-14 or days
6-10 of pregnancy. However, renal agenesis was not produced when
PCNB was administered only from days 10-14 of pregnancy. These
effects were produced in only the C57BL/6 strain of mice.
Other pesticides producing a statistically significant increase
in the proportion of litters containing abnormal fetuses and in the
increased incidence of abnormal fetuses within litters were: Captan,
Folpet, 2,4-D isooctyl ester, 2,4-D butyl ester, 2,4-D isopropyl ester,
carbaryl (Sevin), and IPC. These pesticides produced elevated incia.nd in one solvent only. The resuHs for carbaryl and for IPC were
less consistent than for other compounds. (The pesticides 2,4,5-T,
PCNB, captan, Folpet, carbaryl, IPC, and the butyl and isopropyl
esters of 2,4-D were statistically significant at the .01 level, for one or
more tests. This criterion is similar to that adopted by the Technical
Panel on Carcinogenesis, Chapter 5, to identify "positive" compounds.
The isooctyl ester of 2,4-D was significant at the 0.05 level.)
Compounds inducing only an increase in the proportion of abnormal fetuses within litters were: a-naphthol, and 2,4-D methyl
ester. The statistical significance of these results was relatively
weak; further study is required before any conclusions can be
reached. Similarly, 2,4-D produced only an increase in the proportion
of abnormal litters during 1965 in AKR mice. Due to the teratogenic
activity of certain of its esters, 2,4-D should be studied further.
Carbaryl plus piperonyl butoxide did not show an overall increase in nonspecific anomalies, but resulted in significantly more
cystic kidneys for doses above 10 mg./kg. carbaryl plus 100 ^,l./kg.
piperonyl butoxide.
It must be emphasized that failure to detect statistically significant increases of .anomalies may be due to insensitivity resulting
441
from experimental variation and small numbers of littesRested. In
addition, higher fetal mortality among some of the "negative" compounds may be selectively eliminating abnormal fetuses.
b. Methods.—Four strains of mice were used: C57BL/6, AKR,
C3H, and A/Ha. Most of the studies were performed with the
C57BL/6 strain. A hybrid fetus resulting from mating a C57BL/6
female with an AKR male was used to study a few compounds. More
restricted studies were also made on Sprague Dawley rats; results
of these with reference to 2,4,5-T a.re considered separately.
Most compounds were administered subcutaneously in 0.1 ml.
solutions of dimethylsulf oxide (DMSO). Water soluble compounds
were administered in saline, and some times also in DMSO. Compounds
administered orally were given by gavage in 0.1 ml. in a 50-percent
honey solution. Groups of positive controls and untreated controls
were included, as well as controls receiving only DMSO, saline, or
honey. While controls were run periodically throughout the duration
of the study, compounds and controls were not matched with respect
to either route or date of administration.
Virgin females were used in these studies. The onset of pregnancy
was determined by detection of vaginal plugs. Compounds were
administered daily from the sixth to the 14th day of pregnancy (15th
day for AKR mice). Mice were sacrificed on the 18th day (19th day
for AKR mice) of gestation. On sacrifice, fetuses were examined for
anomalies. Approximately two-thirds of the fetuses were then stored
in Bouin's solution until necropsy. Remaining fetuses were stained
with alizarin red S after proper processing. Numbers of resorption
sites and dead fetuses were also scored.
G. Statistical analysis,'—All analyses were performed on a per
litter basis rather than a per fetus basis, since initial investigations indicated that the occurrences of anomalies among fetuses within litters
were correlated. The large litter-to-litter variation may reflect some
maternal effect-, an indication of the effective dose level of the compound actually reaching the fetuses, experimental variation, or, as is
most likely, some combination of the three factors.
While there were no statistically significant time trends within
the various control groups in terms of the onset of fetal anomalies in
the C57BL/6 mice, the incidence of fetal mortality was certainly timedependent in this strain, with 1965 being characterized by a low incidence of prenatal deaths. Furthermore, there was a period of approximately 6 months, extending from the latter part of 1965 into
early. 1966, during which no control animals were tested. During this
period a change in .the substrain of C57BL/6 mice used in the study
took place. Finally, among abnormal litters, as defined by litters con-
�443
442
at least one abnormal fetus, there was some suggestion that'
the distribution of abnormal fetuses per litter was stochastically larger
in the DMSO controls than it was in the untreated controls. Thus, the
possibility exists of a time/strain/solvent interaction that is undetectaible in the controls because the level of background teratologic
activity is relatively low. This potential interaction effect could either
enhance or dissipate the effect of any given compound, depending on
the conditions under which it was administered. Thus, the data were
necessarily separated by both time period and solvent for the purposes
of analysis. Similarly, an increase in fetal anomalies in the DMSO
controls of the AKR mice was noted after November 1966. Thus, the
AKR data were analyzed separately in two time periods.
It should be noted that not all compounds were administered on
more than one occasion or in more than one solvent or strain. Thus, in
general the compounds in the study cannot be compared for teratogenic potential, since those that were tested extensively were more
likely to show some adverse effect and, perhaps, less likely to appear
consistent over time, solvent, and/or strain,
As noted, approximately two-thirds of the fetuses were stored in
Bouin's solution until necropsied; the remainder being stained with
alizarin red. However, in many instances the proportion of necropsied
fetuses was slightly higher for the compound under investigation than
for the corresponding controls. It is doubtful if this discrepancy could
have any appreciable effect on the conclusions since the incidence of
anomalies detectable only by necropsy among control animals was
relatively low. Furthermore, if all of the control and test mice had
been necropsied, the significance of the differences observed in this
study would be intensified. Thus, no effort was made to correct for inequalities in the necropsy/stain ratio in the present analysis. Additionally, no attempt was made to correct for differences in litter sizes
or sex-ratios within litters, since both of these factors may, at least in
part, reflect effects of the Compound under test.
(I, Itcvults.—Data for pesticides yielding a statistically increased
level of anomalies in C57BL/6 and AKR mice are listed in tables 1 and
•2, respectively. The proportion of abnormal litters gives the proportion of litters containing one or more abnormal fetuses, as a measure of
the prevalence of anomalies across litters. The proportion of abnormal
fetuses per litter gives a measure of the prevalence of anomalies within
litters. The proportion of abnormal fetuses per litter for litters containing abnormal fetuses gives a measure of the prevalence of anomalies within effected litters. A significant increase of dead fetuses and
resorptions is also listed. Some tests were conducted on only one par-
tioular day or on adjacent days as listed. Eye anomalies, j^fcfily microphthalmia and anophthalmia, accounted for approximate^St) percent
of the individual anomalies in C57BL/6 mice. To a large extent, results in table 1 reflect changes in the incidence of eye anomalies. Yet,
when the data were analyzed excluding fetuses with microphthalmia
only, there were no striking changes in the results. In the last column
of table 1, statistically significant increase in various types of anomalies other than eye anomalies are listed. The .positive controls, trypaii
blue and ethyleneimine, table 1, and 6-aminonicotinamide, table 2,
showed elevated levels of anomalies, although the latter control did
not yield consistent results over all dose levels.
Only those test conditions which resulted in statistically elevated
incidences of anomalies are listed in tables 1 and 2. Some compounds
gave no increase in anomalies (based on the overall incidence if tested
in both time periods) when tested in other solvents, strains, or dose
levels (table 3). It must be emphasized that failure to detect a statistically significant increase in anomalies may only be a reflection of
experimental insensitivity due to experimental and biological variaton and insufficient number of litters. Thus, compounds showing no
increases cannot be considered nonteratogenic. For example, trypan
blue in DMSO at the highest dose level tested, 3Y.5 mg./kg., did not
show an increase in anomalies, possibly due to higher fetal mortality.
Standard corrected 2 X 2 chi-square tests (1) were used to compare
the proportion of abnormal litters for the compound with the controls
in the same solvent. In the cases where tests were conducted in two
time periods, the results from the two chi-squares were combined (1).
The levels of statistical significance for the combined tests are listed
under the total column for proportion of abnormal litters.
The distribution of the proportion of abnormal fetuses per litter
(tables 1 and 2) for compounds were compared with the appropriate
control distribution by use of the nonparametric Mann-Whitney litest (#). This test requires that the proportion of abnormal fetuses per
litter is independent from litter to litter, but requires no assumption
about the frequency distribution of these proportions. Again, where
litters were run in both time periods, the significance level for the
combined tests is given under the total column. Bracketed data include
groups which were combined before statistical tests were conducted.
STATISTICAL
REFERENCES
( / ) SNEMicoH, G. W. AND CocHRAN, \V. G.: Statistical Methods, 6th ed. Iowa
State Univ. PresH, Ames, Iowa (1967),
(2) STEEL, K. G. D. AND TOBKIE, ,T. H.: Principles and Procedures of Statistics.
McGraw-Hill Book Co., Inc., New York (1960).
45-362 O - 70 - 29
�TABLE 1.—Tests which displayed significant increases of anomalies (C57BL/6 mice)
Dose per
Solvent kg of body
Negative controls:
Untreated
.
None
Controls
. ... DMSODo
. . Saline __ _
Do....
Honey
Positive controls:
Trypanblue. ..
DMSODo.
_.
DMSODo
DMSO..
Do
Saline...
Do.
...do
Do
...
.. do.
Proportion of abnormal
litters
Proportion of abnormal
fetuses per litter
1965
Compound
1966-68
Total
1965
1968-68
Total
1965
1966-68
Total
.39
.41
.37
.42
... .53
.52
.40
.46
.43
.08
.16
.13
.11
.12
.10
15
.10
.13
.11
15
.18
.33
.24
28
28
28
32
25
29
26
32
f
5-Omg
12. 5mg
37. 5 mg
5.0 mg
12. 5 mg
37. 5 mg
.60
.86
.60
LOO
.71
. 71
Proportion of abnormal
InTests No. of live
fetuses per litter in abnormal creased repeated
litters
litters
mortalover
.
•".60
... .86
.60
.LOO
.71
- .71
32
44***
.36
. 61***
.49" 33*
32
44*"
.36
.61***
.49**
33*
54
52"
1965 1966-68
26
70
31
46"
.49"*
49*** Yes
.61"
.69"*
"'"
69
112
46
32
5
7
54
Yes
52** Yes
60
Yes
61**
69*** Yes
46" Yes
.60
Increased
anomalies
other than
1
iHydro1 cephaly.
5
5
7
7
cephaly.
Ethyleneimine .
Experimental:
2,4,5-T..
2 4 5-T
...do
4. 64 ;sl
DMSO_. 113 mg
do
LOO*
1. 00*
. 79**
1 00*
1 00"
46. 4 mg
113 mg
Folpet
215 mg
464 mg
464 mg
100 rag 1 00*
48 A*!
130 pi
100 ftl
94^1
1. 00 *
100 mg
1 00 *
54
DMSO
DMSO
DMSO
DMSO
850 mg
10 mg
106 mg
1. 00 **
86
.43
49***
. 79**
-.56*"
37**
70***
LOO*
1.00"
.88*
.88*
67] '
67")
LOO]
1 OOJ
61
. 71***
77 ** .77 **
LOO *
67
.67
.75 ** .75 **
.70 ** .70 **
do
do
do
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
PCNB (days 5-14)
PCNB (days &- 14)
PCNB (days 6-10)
.49***
.56*"
37**
70***
.25**
25)
38)
27
.29 ***
381
35"*
.29 ***
.28 **
.25 ***
.26 ***
58***
37
70***
.25"
25l
r
71***
.28 **
.25 ***
.26 *** -
24
24
.58"
""24" "
71***
7
No
14
Yes
.37
.70*** Yes
.29
.29
38
38
.37
37
.49 ** Yes
44
38 * ' .38 *
24
.41 *
.41 *
.34 . ... i
.34
37 *
.37 *
-
No .
Cleft palate
cystic
kidney.
6 ..
9 ..
Cleft palate
cystic
kidney.
8 .12 ..
. 10 ..
No
No
6
IS
13
.1
,
J Renal
agenesis.
6 -.
15
20 Agnathia.
20
ester.
IPC
Butoxide.
Do
Significance level:
DMSO
*(.10).
83
50
100Ml
46 4mg4464 ;il
"(.05).
-50
"(.01).
71 **
.71 *
.86
83
50
.50
46 **'
16
26 **
.46 *
.46 ***
33 *
.09
.27 **
39 *
30 *
13
29
.37
6
11
7
7.
36
26
-.46 *
.38
.36
.26
.21
.21
.46 *
.38
30 *
13
.10
.10
Hydrocephaly, skeletal
7 ..
fi
No
'...
fi 1
[Cystic kidney
12
^
(^,
�446
447
TABLE 3.—Tests which showed no significant increase
(with particular doses, solvents, or test strains)
S.I
oo
Compound
s
a 2v >,
^SSS
a ss
s
.8
"3
e
.
es
fig
S8
8
S
88
•I
_:
s
I
"S.
I
°<
- .§
S8
>e
88
•
M
to
S
"Si
°Q
S I
S
o
to
ft
23
XO
2J ^
ss
«
R tt
|'?
i.
•v" 4"
I
5 ±M
^-S.
ce Le
11/66
With t
£
S 2
•3 -g
8
'Sig
fTh
Not
$
o
8
Strains
2,4,5-T
PCNB (days 10-14)...
PCNB
Captan
Do
Folpet.
Do
2,4-D Isooctyl ester
Do
Do
2,4-D Butyl Ester
Do
2,4-D Isopropyl Ester..
Do
Carbaryl
Do
Do ... .
IPC
IPC
2,4-D Methyl Ester
Do
o,p'-DDD...
Do
2,4-D
Do...
Do
Do
C57
CB7
AKR
C57
AKR
C57
AKR
C3H
A/Ha
AKR
C57
AKR
C57
AKR
C3H
C57XAKR
AKR
03H
AKR
AKR
C57XAKR
C57
AKR
057
057
C3H
057 X
AKR
057
AKR
C57
AKR
C3H
057
C3H
057
057
AKR
C3H
057
057
AKR
C3H
057
AKR
Zectran
Do
Thiram--Do
Ferbam
Do
Monuron
Do
Do
> .
Do
Diuron
Do
2,4-D Ethyl Ester
Do
Atrazine
Do
Do
Solvent
Doso per kg.
bodywt.
DMSO
Honey
Honey
Honey
DMSO
Honey
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Honey
DMSO
DMSO
10 mg.
10 mg.
10 mg.
115 mg.
4.64 mg.
4.64 mg.
215 mg
215 mg
; 215 mg
215 mg.
215 mg
215 mg
86 M!
86 M!
46.4 mg
46.4 mg
46.4 mg
Total
number
of litters
6
9
21.5 mg.
464 mg.
464 mg.
100 mg
100 mg
100 mg
100 mg.
48 M!24 jd.
130 M!.
46 M!.
100 /ul.
46 M!.
94 M!.
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Honey
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Increased
mortality
(G67BL/6)
100 mg
100 mg
464 mg.
850 mg
850 mg.
106 mg.
106 mg.
100 mg
100 mg.
100 mg
,
Yes
100 mg
100 mg
98 mg.
7
7
8
7
.:
-
9
12
13
5
13
6
5
8
6
10
6
6
8
6
13
11
13
7
5
13
12
16
12
6
11
.-
......
.a..
6
6
7
13
9
13
6
6
7
7
6
13
15
�449
448
TABLE 3.—Tests which showed no significant increase oj anomalitt
(with particular doses, solvents, or test strains)—Continued
Compound
Piperonyl Butoxicle
Do
Do.p,p'-DDD
„'
p,p'-DDT
Carbffryl + Nicotinamide
Nico Unamide
CIPC
Nabam
'.
Do
Do
Do
Do
Propazine. Dicryl
Perthane.'
Ovox
Tedion
Amitrol
Do
Do
-.
Strains
C3H
057
C57
C57
C57
C57
C57
C57
C3H
C57
057
AKR
AKR
C3H
C57
C57
AKR
AKR
C57
C57
AKR
Solvent
DMSO
DMSO
DMSO
, DMSO
' DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
Saline
DMSO
Saline
•DMSO
DMSO
DMSO
DMSO
DMSO
Saline
Honey
Saline
Dose per kg.
body wt.
Increased
mortality
. (C67BL/6)
0
6
6
1000 Ml
1000 M!
21.5 M!
46.4 mg.
46.4 mg.
100+61
mg.
61 mg.
1000 mg
21.5 mg.
46.4 mg
46.4 mg
46.4 mg
46.4 mg.
464 mg.
21.5 mg.
100 mg
185 mg
217 mg.
464 mg.
215 mg.
464 mg.
Total
number
o( llttera
—
0
Yes
-.
-'.
--
.-
6
0
10
'..
-
Yes
•.
e
6
6
14
5
14
6
6
6
7
6
13
8
14
Human studies
Epidemiologic data on possible effects of pesticides on human reproduction and teratology are grossly inadequate. Prospective studies
on this subject are difficult to design and almost nonexistent, except
for the community pesticide program of the Food and Drug
Administration.
Chlorinated hydrocarbons.—In a recent review (Khera and
Clegg, 1969), no adverse human reproductive effects were attributed
to DDT and other chlorinated hydrocarbons. Studies on 240 pregnant
women indicated that 21 percent had significant first trimester pesticide exposure, and that 52 percent were exposed during their entire
pregnancy. No statistical difference in numbers of patients with anomalies existed between these exposed groups (Nora et al., 1967), Low
values of DDT residues have been found in a small number of human
placentas (Rappolt et al., 1969). Sharply reduced tissue levels were also
found in 68 newborn infants (Zavon, 1969). Pesticide levels in human
milk have not shown any relation to perinatal toxicity (Laug et al.,
1951; Lofroth, 1969; Curley and Kimbrough, 1969). Studies on 152
mothers showed transplacental passage of DDT and DD^T (O'Leary,
1969). Low placental and high vernix levels were noted; ietal blood
levels were one-half maternal levels. In a similar study on premature
infants (O'Leary, 1969), high fetal levels were noted; no relationship
between maternal blood levels of DDE and DDT and the incidence
of first trimester spontaneous abortion were found, although the num-'
ber of pregnant women reported on was inadequate for firm conclusions.
Organophosphates.—Evidence of teratogenic potential of organophosphates in humans has been reviewed and found inconclusive
(Khera and Clegg, 1969).
Mercurials.—Consumption by Japanese pregnant women of fish
and shellfish contaminated by methylmercury produced a high incidence of infantile cerebral palsy (Matsumoto et al., 1965). This condition has been termed fetal Minamata disease.
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�450
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'
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451
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1967.
.
.
TAUBER, O. E., and HUGHES, A. B.: "Effects of DDT ingestion on total cholesterol
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VEBRETT, M. J., ET AL : "Teratogenic effect of captan and related compounds in the
the developing chicken embryo." Ann. N. Y. Acad. Sci. 160:334-43, 1969.
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WARE, G. W., and Goon, E. E.: "Effects of insecticides on reproduction in the
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WEIKE, JI.: "Effects of DDT on reproduction in hens." Acta. Pharmacol. 25:
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�452
Senator HART. Before adjourning, I should add for the record^P'6
nad been scheduled Dr. DuBridge of the Office of Science and Technology, 'but Mr. Bickwit advises that in the face of a 5:30 appointment,
he has asked not to be heard, but instead submits his statement for the
record.
(The statement follows:)
STATEMENT OF DR. LISE A. DUBBIDGE, DinECToa, OFFICE OF
SCIENCE AND TECHNOLOGY
SENATE COMMERCE COMMITTEE,
APBIL 15, 1970.
Mr. Chairman, Members of the Subcommittee, Let me say at the outset Unit
I am pleased to have an opportunity to discuss with this Subcommittee certain
aspects of the herbicide, 2,4,5-T. An examination of the subject illustrates a
number of important issues relating to the Federal Government's involvement
with pesticides. I believe that these deserve some discussion and I am glad to
have the privilege of exploring them with you.
The herbicide, 2,4,5-trichloropherioxyacetic acid is a member of a family of
pesticides which have served mankind very well for a long period of time.
This group of compounds, known as phenoxy or auxin herbicides, have been
vised since the late 1940's and resulted from research work performed during
the 1940's on herbicides and defoliants for military as well as civilian use.
The toxicity of 2,4,6-T was studied in line with the requirements for its registration by the Department of Agriculture. The toxicology required for this
registration of 2,4,5-T was aimed primarily at determining its acute toxicity.
In this regard, it is now quite clear that the experiments performed for this
purpose (almost all of which were done by or for the manufacturing industry
seeking the registration) revealed that 2,4,5-T was relatively non-toxic
This herbicide demonstrated a pWsistence in soil and water which was very
short (on the order of three months for total disappearance). It is true also,
ns you heard last week, that only rare instances of 2,4,5-T residues have been
rliscovered in the food surveys performed by the Department of Health, Education and Welfare.
As a result of these findings, plus its proved utility as an herbicide and as a
defoliant, 2,4,5-T was considered a very beneficial and safe herbicide and with
good reason. As evidence for this, it is plain that the demand for 2,4,5-T has
risen, especially in the last several years. The production of 2,4,5-T in the
United States increased from 7.0 million to 42.5 million pounds between 1960
and 1968. Domestically, it has proved its worth ns a valuable adjunct in the
clearing of range and pasture lands of brush, in the clearing of roadsides and
rights-of-way, in the suppression of aquatic weeds, in the limited use for control of weeds in croplands, • and for altering physiological responses of crops.
The increase in production apparently has reflected the demand for 2,4,5-T
both domestically and as a defoliant for military operations in southeast Asia.
In fact, the domestically used quantities actually decreased between 1964 and
1966.
In 1964, the National Cancer Institute of the National Institutes of Health
undertook on contract with the Bionetics Research Laboratories, Incorporated,
a screening study of a large number of economic poisons. As you have heard,
the general purpose of this study was to ascertain the potential for cancer, for
genetic alteration, and the potential of producing birth defects for this long
list of pesticides. All who have been concerned with this subject recognize the
value of the Bionetics study as a screening mechanism for these potential hazards. 2,4,5-T was among the list of materials screened. One of the results of
this study was that a particular lot of commercial grade 2,4,5-T provolced birth
453
defects in mice and rats if administered in sufficiently Iq^^doses at nn appropriate stage of pregnancy in these animals. These resUr were available in
1908 and they were subsequently further analyzed statistically by the National
Institute of Environmental Health Sciences.
No further action was taken on the findings of the Biouetics study after
August 1908 nor was the information on teratogenesis publicly available. However, copies of the study reports did find their way to members of Congress, to
journalists, and to some members of the scientific community. Coincidentally,
in May or June of 1969, a number of anecdotal articles appeared in the Vietnamese press which reported an unusual incidence of congenital abnormalities
and abnormalities of pregnancy in certain parts of Vietnam. In some cases,
these reports were linked to defoliation operations. The evidence for this is,
however, extremely doubtful.
It was with this background that I met in October of last year, in my
capacity of Executive Secretary of the Environmental Quality Council, with
representatives of the several Federal agencies which were most concerned
with the use of this herbicida It was the consensus of these representatives
that this research information from the Bionetics study warranted serious consideration including certain restrictions on the use of 2,4,5-T. The announcement of these intended actions occurred on October 29.
One of these actions was a limitation of defoliation operations in Vietnam.
The limitation, which did occur subsequently, took the form of restricting
defoliation to non-populated areas. Another announced action was aimed at the
Government's own use of 2,4,5-T domestically in programs of brush and weed
control. These programs were mainly pursued by the Department of Agriculture and the Department of the Interior. Here the Government did restrict the
application of this herbicide so as to reduce possible exposure to uinn. TinState Department, which to some extent had been a party to the use of 2,4,5-T
along our border with Canada, took steps to reduce human exposure here and
make available to foreign countries technical data about the subject.
The Department of Agriculture agreed to cancel the registration Of 2,4,5-T
for use on food crops by the first of the year unless the Food and Drug
Administration could, by that time, satisfy itself that it had enough evidence
to establish a negligible tolerance limit for human exposure. The food crop
uses of 2,4,5-T incidentally represented a minority of its total domestic use.
At the time of the announcement of concerted Government actions, I also
assembled a panel of experts within the Office of Science and Technology to
review all that is known about of 2,4,5-T. This Panel has prepared a report on
the subject which I expect to make available within a few weeks. During the
course of this review, it became known that an impurity of 2,4,5-T was of
potential importance. The impurity, a polychlorinated dioxin, was apparently
very toxic and had been identified in batches of 2,4,5-T as early as 1957. It
arose partly as an impurity of the chlorphenol starting material and partly as
a result of the temperatures and pressures of certain of the reactions in the
manufacturing process. It had provoked severe skin irritations among workers
in 2,4,5-T plants in Germany and in the United States. The discovery of this
industrial hazard had led one U.S. manufacturer to curtail his process until he
was able to reduce the dioxin content to less than 1.0 ppm in the 2,4,5-T product. This eliminated the skin irritation problem.
Within weeks after my announcement, some additional animal experiments
were begun in two laboratories simultaneously. These experiments were
directed towards confirming and extending the results of the Bionetics studies.
In addition, they were aimed at finding out whether the apparent teratogenic
agent was 2,4,5-T itself, or a potent impurity.
Fortunately, the experiments needed to test for teratogenesis are essentially
acute, short-term studies. With an expectation of meaningful results from
these experiments in a fairly short period of time and. in view of the potential
�454
of t l m o x i n impurity, the Department of Agriculture in consultation with
Interior, HEW, "and my office delayed any action toward cancellation of the
food crop registrations for 2,4, 5-T as you heard last week.
One of the sets of confirmatory studies was undertaken by the Government,
itself. The results of these experiments, pursued at the National Institute of
Environmental Health Sciences, were made known to you by Dr. Steinfeld this
morning. Essentially, these results implicated both 2,4,5-T (in the purest form
available) and its dioxin impurity as potential teratogens. This story, I think
you will agree, represents an example of some appropriate Government actions.
As you have heard and as Dr. Steinfeld pointed out, the results of these confirmatory studies were translated into immediate actions in the form of a series
of announced Government restrictions on uses of 2,4,5-T.
Let me now turn to what I believe are some important lessons to be learned
from tliis fascinating case study. First, let me review. We have here an example of a chemical substance intentionally placed into the environment by man
for the betterment of his welfare. Where the aim has been to exchange capital '
for labor in land and waterway management, there can be no doubt that this
herbicide has proved its worth.
In view of what we have now learned, I am persuaded that we must consider some changes in our procedures and we must be willing to submit our
regulatory systems for pesticides, as for other chemicals in the environment, to
examination. The very excellent report of the Secretary's Commission on Pesticides, headed by Dr. Kmil Mrak, will, of course, serve to point up this issue.
From a number of indications it is quite apparent that we, as a society,
have relatively recently begun to ask more sophisticated questions about
adverse effects on health of a variety of chemical substances. In part, this has
come about because of some additional scientific knowledge and investigative
tools. In part, it has arisen simply by virtue of some increased concern about
the safety of environmental chemicals. For example, the realization that environmental agents may be major contributors to the incidence of cancers in the
epidemiological sense is a fairly recent observation.
In brief, then, we have set our sights higher in terms of the questions we
would like the scientific community to ask about pesticides. As I reminded you
at the outset, the total amount of background toxicology performed on 2,4,5-T
had been limited to studies of acute toxicity— performed essentially by industry as directed by the Federal Government. No one had seriously suggested
that the hazards of birth defects, genetic change, or cancer be tested for in the
case, of 2,4,5-T, nor were there tools to screen for these diseases. The Bionetics
study represented a step up in degree of sophistication of research.
Certainly this evolutionary process is a highly commendable situation and is
one which is to be encouraged. There do exist some dilemmas, however. The
major dilemma accrues from the fact that there is no real end point to this
questioning process. The more research that is performed,1 the more new questions will be raised about the chemical under investigation . That is, it is quite
obvious that decisions virtually always will have to be made on admittedly
Incomplete information. Perhaps the goal we should seek is a sufficiently flexible system to allow us to change our minds (when confronted with new information), coupled with an explicit acknowledgement of the perpetually interim
state of our scientific knowledge. Again, the Bionetics study is illustrative. The
455
Bionetics' results were new and unexpected findings—albeit tABlve findings.
It is the nature t>f science that experimental results are alwayWrobject to further confirmation and refinement. The discovery of teratogenesis in experimental animals required confirmation and further investigation to make that
finding meaningful. Fortunately, the experiments to do this were begun almost
immediately, as you know, and the results have just now become available.
What these results have done is to sustain our earlier concern about this herbicide. At the same time, scientific logic would dictate that we should continue
to apply more research effort to better understand these
findings.
.
Then too, the more sophisticated the scientific investigations become, theV
more expensive they are. The cost of the Bionetics study was approximately \.
two and one-half million dollars. Remember, this was only a screening study.r__J
Much more extensive studies would surely be desirable.
A related question that is raised concerns the distribution of these costs. As
I have noted, in the case of pesticides, the tradition has been for the Government .to impose on industry the obligation of proving that a material is safe
and of performing the toxicology necessary for that proof. As the cost and the
time required for this background research rises, the manufacturers may be
less and less inclined to pursue the development of new products of limited or
uncertain marketability. Since we depend on the manufacturing industry for
this development, we may be discouraging innovative and improved products, i
Hence, I submit that additional public investment may have to be made in the \
future in background research relating to health and other effects of environ- 1
mental agents—including pesticides. The President's Science Advisory Commit-'
tee is studying these issues presently.
Let me touch now on the subject of the translation of research findings into
policy decisions and regulations. I have made the point that the heart of the
Federal Government's control over pesticides resides in the process of registration with the Department of Agriculture. This registration is based, in part, on
toxicological information supplied by the manufacturer. There has been relatively little thought given to the subject of how to incorporate new, unexpected information which is collected outside the registration process into the
regulatory process. This was clearly demonstrated after the Bionetics study.
Finally, let me raise the question of the latitude available for regulation of
pesticides. Under the existing Federal Insecticide, Fungicide 'niid Rodentlcide
Act (which law regulates pesticides), the burden of proof of safety resides
with the manufacturer. In the case of an existing registration, the options for
action available to the Government, however, are relatively few.- These are
cancellation or suspension of the registration. Both of these are relatively
drastic actions and are not supposed to be entered into capriciously. If a registration is cancelled (which was the uigge«*ion made for 2,4,ti-T), the decision
nitty Lie appealed by the manufacture r ami it then befalls the Government to
prove that a hazard exists, rather than the industry to establish its safety. In
short, there does not exist a mechanism whereby the Government may exercise
prudent and unequivocally effective restraint temporarily on the receipt of
new, unexpected information, and while awaiting more definitive results.
There are now under discussion a series of proposed amendments to the
Federal Insecticide, Fungicide, and Rodenticide Act. These matters are seriously being considered in these discussions before your Committee.
�457
456
Watershed Studies with 2,4-D, 2,4,5-1, and Picloram'
Description of Watersheds
The two experimental watersheds are located in the southern Appalachian
near Waynesvi1le, North Carolina. Watershed 1 contains 4.64 acres, and
watershed 2 contains 3.66 acres. The slopes of both watersheds averaged about
35 to 40 percent. The predominant soil is Halewood clay loam. The watersheds
are delineated and enclosed so that no surface or subsurface flow'can enter,
Each is equipped with a Weir i n s t a l l e d to bedrock, and total flow from the
watersheds Is measured. Three 0.05-acre plots with catchment devices for
surface runoff determination are superimposed on watershed 2. The vegetative
cover was a mixed grass sward containing discontinuous infestations of
herbaceous weeds and small woody plants.
Experimental Procedure
The map of watershed 1 (Figure 1) shows the nine 0.05-acre plots sprayed
In 1967 and the three large plots (1.16 acres) sprayed in 1968 and 1969.
There are three replications of three treatments in I9&7. Large plots A, C,
and D were sprayed w i t h the same herbicides in 1968 and 1969. The a p p l i c a t i o n
rate was 2 Ib/A. When large plots were used, there was one replication per
watershed. The map of watershed 2 (Figure 2) shows the small plots sprayed
In 1967 and 1968. The plots ware sprayed as shown in 1967. In 1968 herbicide
treatments ware rotated to so that each plot received a different herbicide. The
a p p l i c a t i o n rate was 2 Ib/A in 196? and 1968. In I9&9 the large plots were
sprayed at a rate of 4 Ib/A. The treatments were adjusted so that the herbicide
assigned to each surface runoff plot had not been applied to that plot in 1967
or 1968. There-were three replications of treatments when small plots were used.
Treatments on large plots were unrepllcated w i t h i n a watershed.
The chemical 'and common names of the herbicides were 3,6-dichloro-o-anisic
a c i d [dicamba], 2,4-dlchlorophenoxyacetic acid [2,4-0], 2,4,5-trIchlorophenoxyacetlc
acid [2,4,5-T], and 4-amino-3,5,6-trichloropicolinic acid [picloram].
The herbicides were applied in September, 1967.and in August, 1968 and 1969.
All applications were made with a Knap-sac sprayer. The herbicldal formulations
were as follows:
dicamba (dimethylamine salt)
2,4-D (alkanolamine salts)
picloram (potassium salt)
2,4,5-T (propylene glycol butyl ether ester in 1968)
2,4,5-T (trlethylamine salt In 1969).
•
•
,
,
A contribution of the North Carolina State University Agricultural Experiment
Station. This research was supported by the U, S. Department of
Agriculture under Contract No. 12-14-100-8938(3*0.
For several months after spraying, grab samples of water were collected at
the flumes during storms, and runoff samples were removed from the surface-runoff
tanks at the end of each rain storm. Water and soil samples were shipped to
R a l e i g h for analysis. Soil samples were frozen before shipment. Water samples
were shipped as soon as possible after collection and were stored at 4°C on
arrival in the laboratory. Usually, analysis of water samples began w i t h i n 3 to
4 days after collection.
An electron-capture gas chromatographtc method was developed for simultaneously
measuring residues of the four herbicides. Low l i m i t s of detection of 2,4-D in
water was 0.002 to 0.003 ppm; for picloram, 2,4,5-T, and dicamba the l i m i t was
0.0005 to 0.001 ppm.
Results
Water samples collected from flumes at the base of each watershed during end
after rain storms in 1967 contained 2,4-D, but concentrations of picloram and dicamba
were below the l i m i t s of detection. The highest concentration of 2,4-D (0.028 ppm)
occurred shortly after peak runoff of the first storm after application. The level
decreased with each subsequent storm and was below the l i m i t of detection in samples
taken between September 27, 1967 and June 17, 1968 when sampling was discontinued
u n t i l the 1968 application.
Although one-fourth of watershed 1 was sprayed with each of three herbicides
In 1968, neither 2,4,5-T nor picloram was detected in flume water, and only low
concentrations of apparent 2,4-D (0.003 to 0.005 ppm) occurred sporadically
(Table 1). A small interference peak with a retention time in the gas chromatograph
equal to that of 2,4-D raises some doubt about the authenticity of 2,4-D values In
the 0.002 to 0,004 ppm range (Tables 1 and 2).
Concentrations of the herbicides in flume water samples collected In 1969
from watershed U.were below the detection l i m i t i n . a l l cases (Table 3).
After the 1969 applications, 2,4,5-T was detected in water samples taken at
the base of watershed 2 during the first and second storms (Table 4). The highest
concentration was 0,048 ppm in a sample collected while runoff was increasing during
the second storm. The concentration was less In other samples and decreased to
less than 0.001 ppm when flow returned to normal. Low concentrations of picloram
were detected in flume samples during the second storm also. The maximum concentration was 0.003 ppm in a sample collected while flow rate was decreasing.
Picloram was detected at 0.002 ppm In the first base-flow sample taken after the
storm, but levels were less than 0.001 ppm in all samples thereafter.
Residues of 2,4-D, 2,4,5-T, and picloram in soil at several times after
application are shown in tables 5, 6, and 7, respectively. The 2;4-D disappeared
rapidly from soil. Although picloram persisted for several months, none was
detected 1 year after application. A very small amount of 2,4,5-T was present
In t;he 0 to 6-inch soil depth at 3 and 7 months after application, but none was
found 12 months after.
�458
Table
459
Concentrations (ppm) of 2,4-D, 2,4, 5-T, and picloram in water
from the flume of watershed 1 over a 4-month period after
a p p l i c a t i o n of 2 Ib/A of each herbicide to 25% of the watershed area on August 21, 1968.
Flow
condi t ion
2,4-D
(ppm)
2,4,5-T
Peak
Base
<0.002
<0.002
Peak 1
Table 2.
Concentrations (ppm) of 2,4-D, 2,4,5-T, and picloram in water
from the flume of watershed 2 over a 4-month period after
a p p l i c a t i o n of 2 Ib/A of each herbicide to 4% of the watershed area on August 20, 1968.
I
Flow
condi t ion
2,4-D
(ppm)
2,4,5-T
(ppm)
Picloram
(ppm)
Half-up
Peak
Ha If -down
<0.002
0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
8-31-68
Peak 1
<0.002
<0.0005
<0.0005
9- 1-68
Ha If -down
Peak 2
Peak 3
Base
<0.002
<0.002
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
9- 6-68
Base
<0.002
<0.0005
<0.0005
10- 3-68
Ha If -down
Base
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
10- 6-68
Ha If -down
Base
0.003
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
10-16-68
Base
<0.002
<o.ooos
<0.0005
10-24-68
Base
<0.002
<0.0005
<0.0005
11- 6-68
Base
0.003
<0.0005
<0.0005
12- 1-68
Half-up
<0.002
<0.0005
<0.0005
12-13-68
Peak 1
Peak 2
Peak 3
<0.002
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
Picloram
(PPm)
^
,
<0.0005
<0.0005
<0.0005
<0.0005
1
<0.002
<0.0005
<0.0005
Ha If -down
Peak 2
Peak 3
Base
<0.002
<0.002
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
9- 6-68
Base
<0.002
<0.0005
<0.0005
9-13-68
Base
<0.002
<0.0005
<0.0005
Ha If -down
Base
0.005
0.002
<0.0005
<0.0005
<o.ooos
10- 6-68
Ha If -down
Base
0.004
0.003
<0.0005
<0.0005
<0.0005
<o.ooos
10-16-68
Base
<0.002
<0.0005
<0.0005
10-24-68
Base
<0.002
<0.0005
<0.0005
11- 6-68
Base
0.002
<0.0005
Date
8-25-68
8-31-68
9- 1-68
10- 3-68
.
(ppm)
.
Date
8-25-68
i
<0.0005
:
•
12- 1-68
Peak
<0.002
<0.0005
<0.0005
12-13-68
Peak 1
Peak 2
Peak 3
<0.002
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
<0.0005
12-14-68
Base
<0.002
<0.0005
<0.0005
12-20-68
Base
<0.002
<0.0005
<0.0005
12-22-68
Peak 1
Peak 2
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
12-24-68
Ha If -down
<0.002
<0.0005
<0.0005
12-14-68
Base
<0.002
<0.0005
<0.0005
12-26-68
Base
<0.002
<0.0005
<0.0005
12-20-68
Base
<0.002
<0.0005
<0.0005
12-22-68
Peak 1
Peak 2
<0.002
<0.002
<0.0005
<0.0005
<0.0005
<0.0005
12-24-68
Ha If -down
<0.002
<0.0005
<0.0005
12-26-68
Base
<0.002
<0.0005
<0.0005
'
1
*
45-382 O - 70 - 30
�460
Table 3.
Concentrations
from the flume
application of
area on August
.461
(ppm) of 2,4-D, 2,*t,5-T, and picloram in water
of watershed 1 over a It-month period after
2 Ib/A of each herbicide to 25% of the watershed
13, 1969.
Flow
condi t ion
2,k-0
Ha If -down
Base
<0.003
<0.003
<0.001
<0.001
Peak
Ha If -down
Base
<0.003
<0.003
<0.003
8-29-69
Base
9- 5-69
Date
8-16-69
8-22-69
(ppm)
2,^,5-T
(ppm)
Table k.
snea area on nugust it, 1303.
•^
Picloram
(ppm)
9-19-69
2,^-D
(ppm)
2,4,5-T
(ppm)
Pi cloram
(ppm)
Ha If -down
Base
<0.003
<0.003
<0.001
0.019
<0.001
<0.001
Half-up
Peak 1
Peak 2
Hal f-down
Base
<0.003
<0.003
<0.003
<0.003
<0.003
O.OW
0.031
0.006
0.003
<0.001
8-29-69
Base
<0.003
<0.001
<0,001
9- 5-69
Ha If -down
Base
<0.003
<0.001
<0.001
<0.003
<0.001
<O.OOI
9-12-69
Base
<0.003
<0.001
<0.001
9-19-69
Peak
Half-down
Base
<0.003
<0.003
<0.003
<0.001
<0.001
<0.001
<0.001
<O.OOI
<0.001
9-23-69
Half-up
Peak 1
Ha If -down
Peak 2
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
Peak 3
<0.003
<0.003
<0.003
<0.003
<0.003
9-2^-69
Base
<0.003
<0.001
<0.001
10- 1-69
Base
<0.003
<0.001
<0.001
10- 2-69
Base
<0.003
<0.001
<0.00!
10-' 8-69
Base
<0.003
<0.001
<O.OOI
Flow
Date
condition
<0,001
<0.001
8-16-69
<0.00!
<0.001
<0.001
<0,001
8-22-69
<0.003
<O.OOI
<0.001
Ha If -down
Base
<0.003
<0.003
<0.001
<0.001
<0.001
;
<0.001
;
Base
<0.003
<0.001
<0.001
<0,001
<0.001
.;
;
9-12-69
Concentrations (ppm) of 2,^-D, 2,it,5-T, and picloram in water
from the flume of watershed 2 over a 2-month period after
a p p l i c a t i o n of k Ib/A of each herbicide to 25% of the water-
Ha If -down
Base
<0.003
<0.003
<0.001
<0.001
<o.ooi
Peak 1
Ha If -down
<0.003
<0.003
<0.003
<0.00l
<0.001
<0.001
<0.001
<0.001
<0.001
9-2^-69
Base
<0.003
<0.001
<0.001
10- 1-69
Base
<0.003
<0.001
<0.001
10- 2-69
Base
. <0.003
<0.001
<0.001
10- 8-69
Base
<0.003
<0.001
Base
<0.003
<0.001
Peak
Half-down
Base
<0.003
<0.003
<0.003
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
12-10-69
Peak
Ha If -down
Base
<0.003
<0.003
<0.003
<0.001
<0.001
<0.001
<0.001
<0.001
<0.001
12-17-69
Base
<0.'003
<o.'ooi
<0.001
12-29-69
Base
<0.'003
<o;ooi
<o;ooi
0.003
0.002
<0.001
12- 7-69
<0.001
<0.001
0.002
<0.001
1 1-28-69
:
.
9-23-69
Peak 2
<0.001
;
i
:
i
<0.001
�462
463
Table 5. Residues of 2,4-D (lb/A) in soil at 0 to 3 months
after a p p l i c a t i o n of 2 lb/A August 20, 1968 to
Watershed 2.
Table 6.
>
Residues of 2,4,5-T (lb/A) in soil at 0 to 12 months after
application of 2 lb/A August 20, 1968, to Watershed 2.
Soi 1
depth
(i nches)
0-3
_
,
Months after application
Soil
depth
(inches)
:
Months after a p p l i c a t i o n
0
1.5
1.14
0.15
3.0
0.03
0.02
3-6
<0.02
6-12
<0.04
<0.04
<0.04
0.04
<0.01
0.01
<0.01
<0.01
<0.01
0.04
<0.02
<0.02
<0.02
0.08
<0.02
-
18-24
0.0k
0.03
0.03
12-18
<0.04
18-24 '
12.0
'
6-12
<0.04
12-18
7.0
3.0
3-6
<0.02
0.02
<0.02
-
0.06
Total
0.79
1.5
0
0-3
.—_—_____
0.79
.
Total
1.14
0.30
0.03
0.04
<0.04
•
Table 7.
Residues of picloram (lb/A) in s o i l 0 to 12 months a f t e r
application of 2, lb/A August 20, 1968 to Watershed 2.
Months after application
Soi 1
depth
( i nches)
7.0
12.0
0.37
' 0.08
<0.02
0.11
0.08
0.06
<0.02
6-12
0.18
0.04
0.06-
<0,04
12-18 '
0.10
0.06
-
18-24
0.04
0.06
-
0.95
0.61
0
1.5
1.27
'0.52
3-6
0-3
1
Total-
1.27
3.0
'
0.20
<0.08
�WATERSHED
MOUNTAIN
I
RESEARCH
STATION
WAYNESV1LLE, N.C.
A
PICLORAM
B
DICAMBA
C
2,4-D
D
2,4,5-T
Fiqure 1.
a
Map of watershe-' I ihowinc t L l e r.ine O.C>-c-t_re p l o t s (sr-.all
p l o t s A, B, and C) sprayed in 1967 and ire I.l6-acre plots
(A, C, and 0) sprayed in 1553 and 1563.
FLUME
WATERSHED
MOUNTAIN
RESEARCH
'
2
STATION
WAYNESVILLE, N.C.
A
PICLORAM
B
DICAMBA
C
2,4-D
D
2,4,5-T
FLUME
! ~ 7 a r i IJ6^
Figure 2.
K3P of .jarer'-ed - =fi
-jnj :'••-- ' - a c r e -'-J'.s
�466
467
•e™ror
SeHlor HART. May I thank all who assisted in the development of
this very informative hearing. We stand adjourned.
(Whereupon, at 5:15 p.m., the subcommittee was adjourned.)
(The following material was subsequently received for the record:)
spoiled areas which remain in our industrialized country to l^freserved as
natural regions. I was dismayed to learn recently that a 20-foot wide strip
through the middle of one of our most beautiful national parks has been defoliated with another herbicide, picloram.
Mr. Chairman, it is incredible to me that Glacier-Waterton International
Peace Park should be subjected to defoliants for the creation of a "North American DM55" through its center. It is an unpleasant irony that a park which was
established to commemorate the peaceful relationship between two nations and
•to preserve a unique glacial wilderness must be divided in two by a senseless and
ugly defoliated strip. Moreover, even less is known about the possible teratogenic
and carcinogenic effects of this chemical picloram than about 2,4,5-T, the agent
under immediate suspicion.
Our lack of a ""national policy in the area of herbicides and pesticides reaches
its ultimate absurdity in this defoliated strip in Glacier Park. I urge the Subcommittee to consider the far-reaching measures which are necessary to control
all aspects of the use of pesticides. Only a comprehensive approach to the problem will enable us to avoid mistakes such as DDT and 2,4,5,-t.
STATEMENT OF HON. LEE METCALF, U.S. SENATOR FBOM MONTANA
Mr. Chairman, I appreciate the opportunity to outline briefly a few of the
special problems which arise from the use of herbicides such as 2,4,5,-T in
Montana.
I will not go into the potential health dangers involved with the widespread
use of 2,4,5,-T and other insufficiently tested herbicides. Others, far better qualified than I to assess these dangers, have already presented their professional
opinions to the Subcommittee. What I would like to bring to the Subcommittee's
attention, however, is the particular impact the use of herbicides has upon n
vast agricultural State such as Montana.
Montana, as you know, is in the unique situation of lying at the head of three
major international drainages which carry waters into the Gulf of Mexico and
the Pacific and Arctic Oceans. The river systems whose headwaters lie in Montana, the Columbia, the Missouri-Mississippi, and the Saskatchewan, pass
through over half of the land area of our nation and part of that of Canada.
Into these drainages enormous quantities of herbicides and pesticides are
sprayed each year. Forests, grazing lands, grain fields, power line rights of way,
and highway shoulders are sprayed with a wide variety of chemical poisons.
Herbicides are used by Federal agencies, State agencies, private companies,
farmers, and home gardeners. The State undergoes a veritable deluge every year.
I hardy need to point out the potentially dangerous effects of such chemicals, if
the.y are found to persist and collect in water supplies and in the food chain.
Kcologists have pointed out that the little understood processes of accumulation,
biological magnification, and synergism can bring about completely unexpected
results from commonplace chemicals.
One would assume because of the pervasive use of 2,4,5,-T and other herbicides
and because of the harmful side effects which have already been discovered to
result from some group of these cheimicals, that truly exhaustive tests would be
required to be run on any new candidate for herbicidal use. Unfortunately, this is
not the case. To our astonishment we have been told in effect that the burden of
proof is on the public to prove that a proposed substance is dangerous rather than
on the supplier to prove that it is not.
The result of this convoluted notion of regulation is that enormous amounts of
potentially dangerous herbicides are spread about our land, they eventually find
their way into water systems and merge with others as they travel their way to
the oceans. Once the Department of Agriculture places its seal of approval on a
chemical, the substance can be sprayed with impunity. Whatever detrimental
effects may result are thereafter extremely hard to ascertain. The unfortunate
results may in fact only become visible years from the time they are used.
The Montana State Legislature Is now considering a new Pesticide Commission which would provide much better regulatory machinery at the State
level. However, due to the problem of human and financial resources, such State
commissions will always have to take their cue from the Federal regulatory
bodies. And as it now stands, the Federal regulating process is a very confused
and weak one.
What is vitally needed, Mr. Chairman, is a clearly delineated Federal regulatory policy in the field of pesticides, herbicides, and defoliants which would:
1. Place the burden of proof of the safety of a product upon the manufacturer ;
2. Provide for thorough, independent testing in government laboratories;
3. Place the sole responsibility for the approval of the health dangers of
a product with the Secretary of the Department of Health, Education and
Welfare;
4. Substantially strengthen the enforcement machinery in the agencies
which control pesticides; and
fi. Create a national policy for the use of pesticides by Federal agencies.
Concerning the final point, I think it is extremely important that the use of
these chemicals in national parks be critically examined. Our national parks and
primitive areas were set up specifically in order to sector off the few small, un-
OFFICE OF THE SECRETARY OF DEFENSE,
Washington, D.O., April 21,1910.
Hon. PHILIP A. HART,
Chairman, Subcommittee on Energy, Natural Resources, and the .Environment,
Committee on Commerce, U.S. Senate, Washington, D.C.
DEAR MB. CHAIRMAN : The following information is provided for the record in
response to a staff request from your Subcommittee :
Deputy Secretary of Defense David Packard on April 15 ordered the immediate suspension oC the use of 2,4,5-T within the Defense establishment
pending a more thorough evaluation of the system.
Sincerely,
J. F. LAWRENCE,
Brigadier General, USAIC,
Deputy Assistant to the Secretary for Legislative Affairs.
DEPARTMENT OF AGRICULTURE,
OFFICE OF THE SECRETARY,
Washington, D.C., April 31,
Mr. LEONARD BICKWIT,
Staff Co/tnxcl, Subcommittee on Energy, Natural Resources, and the Environment, U.S. Senate, Washington, D.C.
DEAR MR. BICKWIT : The following comments on the April 15 joint U.S. Department of Agriculture, U.S. Department of Health, Education, and Welfare, and
U.S. Department of the Interior release "Home Uses of 2,4,5-T Suspended" are
offered for the record of the Hearings of the Committee.
The release is not clear on the cancellation of registered uses of 2,4,5-T ou
food crops. The action to be taken is the cancellation of all registered uses of
2,4,5-T on apples, blueberries, barley, corn, oats, rice, rye, and sugarcane.
The term "cancellation" as here used means that each registrant will be notified that his registered products subject to this action shall cease to move in
interstate commerce 30 days after notification of cancellation. During this 30-day
period, the registrant may correct the labels on his cancelled products to delete
the cancelled uses. The registrant may appeal the cancellation action and ask
for an advisory committee or public hearing. In such case, the registrant's cancelled products may move in interstate commerce until action on the appeal is
completed.
The term "suspension" as used in the release with reference to action to suspend all registered uses of liquid formulations around the home and all registered uses of 2,4,5-T on lakes, ponds, and ditch banks, means that such suspended
products shall cease to move in interstate commerce upon receipt of notice of
the order of suspension by the registrant. The registrant may appeal the suspension action but in such case the suspended products cannot move in interstate
commerce while action on the appeal is in process.
Sincerely,
T. 0. BYERLY,
Assistant Director, Science and Education.
�468
CENTER FOB STUDY OP RESPONSIVE LAW
Washington, D.G., April 80,
Senator PHILIP HABT,
U.S. Satiate,
Washington, D.G.
DEAB SENATOR HAM : May we commend yon and the Senate Commerce Committee for investigating so thoroughly the hazards presented by weed-killer
2,4,5-T. It, was encouraging to see USDA relent and prohibit certain uses of the
chemical. However, after this brief commendation, we would like to make clear'
our feeling that the bans on 2,4,5-T were inadequate, and the press release
announcing the ban misleading to the public.
According to the official press release, "In exercising its responsibility to safeguard public health and safety, the regulatory agencies of the Federal Government will move immediately to minimize human exposure to 2,4,5-T and Us
impurities. The measures being taken are designed to provide maximum protection to -women in the child-bearing years by eliminating formulation, of 2,4,5-T
use in household, aquatic and recreational areas (emphases added)." We contend that the measure taken reflect an utter disregard on the part of the USDA
for public health and safety and represent minimal rather than maximal
protection.
1. The use of Silvex (also called 2,4,5-TP ; 2-(2,4,5-trichlorophenoxy) propionic
acid) has not been suspended. Silvex is as closely related to 2,4,5-T as pancake
are to waffles. Because of this similarity one can predict with confidence that
Silvex will prove to be about as teratogenic as 2,4,5-T. Furthermore, the series
of chemical reactions by which Silvex and 2,4,5-T are made are almost identical.
These reactions lead unavoidably to the formation of the dreaded tetra-dioxin
contaminant. If any serious attempt were being made to "minimize human exposure" to hazardous weed-killers and the impurities contained therein, the
home use of Silvex would certainly be banned. Silvex, incidently, is a more
common ingredient in garden products than is 2,4,5-T.
2. "Minimizing human exposure to impurities in 2,4,5-T and its impurities"
would demand an immediate, total recall of 2,4,5-T and related compounds from
retail stores and homes (with the manufacturers reimbursing stores and consumers). Although the USDA has called for recall in a private communication
to manufacturers, no recall will be complete unless the announcement is published in the Federal Register and a list of products containing the dangerous
substances is published in newspapers so that consumers may return what they
had previously purchased.
3. "Minimizing human exposure" to impurities in 2,4,0-T would necessitate
suspending the use of 2,4-D (the most widely used weedkiller), 2,4-DP (found
in Scott's Turf Builder Plus 4), and pentachlorophenol (present in Ortho Triox
Liquid Vegetation Killer). The processes by which all three of these compounds
are made lead unavoidably to formation of dioxins.
4. "Minimizing exposure to 2,4,5-T and .its impurities" would necessitate suspending' all uses and formulations of all chlorophenoxy and chlorophenol weedkillers :
(a) The possible persistence of dioxins represents a very real danger, as
pointed out by Drs. Verrett and Epstein, Mr. Bickwit and ourselves at the
Commerce Committee hearings.
(6) Accidental exposure to the weed-killers or their contaminants due to
direct spraying, drift or residues on food is possible, indeed inevitable, when
these weed-killers are used on crops, ranges, rights-of-way, neighbors' yards,
etc, In testimony before the House Subcommittee on National Security and
Scientific Developments on December 2, 1069, Dr. Arthur Galston of Yale
University said, "I suggest that its (2,4,5-T) teratogenicity is such that even
its use in such apparently innocuous domestic manners as clearing brush
near powerlines is undesirable. Such chemicals could find their ways into
water supplies and could be Ingested in teratogenic doses."
If the regulatory agencies truly wish to exercise their responsibility to safe-.,
guard public health and safety, particularly that, of women of childbearlng age,
by minimizing human exposure to 2,4,5-T and it.H impurities they will certainly
have to go far beyond the recently announced minimal actions,
If possible we would like this letter made part of the hearing record.
Thank you once again for your interest in this important matter.
Sincerely yours
HAHBISON WEIXFOED.
JAMES TUBNEB.
469
STATEMENT OF R. L, CUSHINO ON BEHALF OF THE HAWAIIAN S^^ PLANTERS'
ASSOCIATION, HONOLULU, HAWAII
^J
The Hawaiian Sugar Planters' Association, an association representing all of
Hawaii's 24 sugar-producing companies, wishes to present its views <m the
proposed cancellation of registration lor the herbicide 2,4,5-T. By. implication,
the fate of other phenolic-derived pesticides, including the important herbicide
2,4-D which is chemically related to 2,4,1-T, is also at stake.
, 2,4,5-T and 2,4-D have been used widely in Hawaii since about 1948 for control of weeds in sugarcane fields. They are valuable for this purpose and there
are no'other equally effective herbicides registered by the U.S. Department of
Agriculture for use in sugarcane. Cancellation of registration of 2,4,5-T and
possibly that of other related pesticides, as originally proposed by Dr. Lee
DuBridge, the President's Science Adviser, on October 26, 1969, would cause
serious economic hardship to sugarcane growers. A letter explaining our position
was sent to Dr. DuBridge on February 5,1970, and he gave us a very thoughtful,
thorough response on March 3.
We believe adverse publicity about all pesticides, such as the unconfirmed
reports of birth defects in Vietnam attributed to 2,4,5-T, is. based largely on
emotion and on inadequate scientific investigation. At the same time we recognize and emphasize that massive aircraft treatments of crops, water sources,
jungle, and possibly villages in Vietnam differs almost totally from the moderate,
controlled agricultural spraying in the U.S.
We recognize the possible hazards from poorly controlled pesticides and the
need for adequate protection of the public. This is the purpose of the federal
regulatory statutes governing pesticides; we believe these laws and regulations
are sufficient to insure food crops free of harmful pesticide residues and to
protect the public from contact with the chemicals. In support of this statement
we quote from Dr. DuBridge's reply to our letter.
"Among 5,800 samples of foodstuffs tested for this material by the Food and
Drug Administration during the past five years, only two were found to contain
more than trace quantities of 2,4,5-T; only 25 contained detectable amounts.
Thus occurrence of 2,4,5-T in foodstuffs is indeed rare."
Laws of the State of Hawaii permit use of 2,4-D and 2,4,5-T herbicides when
applied in such a manner that detectable amounts of spray do not drift to sensitive crops, home gardens, or ornamental plants. Hawaii's semi-tropical climate
and year-round conditions favorable for plant growth make the control of all
weed species an imperative, constant necessity if crops are to be grown successfully and economically. Application of herbicides, such as 2,4-D and 2,4,5-T, and
other pesticides are moderate, as dictated by economic factors and by state and
federal laws. Moreover, sugarcane in Hawaii is a two-year crop. Control of weeds
by application of herbicides is necessary only in the first few months of growth.
Even then the amounts are small and the applications are made with care and
precision. Because of these circumstances, no residues of 2,4-D or of 2,4,5-T have
ever been found in the harvested crop. In the many years of using 2,4,5-T and
related herbicides there have been no known cases of illness or physical defects
either in the men who applied them, their families or the public. Neither is there
any record of injury or hazard to wildlife or recreation.
It now seems probable that the birth defects ascribed to 2,4,5-T in the Bionetics
Laboratories study were caused by a contaminant present in the single sample of
the herbicide apparently used in the investigation. We understand this fact was
pointed out to the Food and Drug Administration by the Dow Chemical Company
who had been aware of the contaminant for several years and had modified its
manufacturing plant so as to avoid producing it. It is difficult to understand how
a carefully controlled scientific study of such importance could be conducted
without first determining the purity of the 2,4,5-T used by Bionetics,
It is unfortunate that, formulated products from several commercial sources
were not included as well as highly purified 2,4,5-T. We feel the subsequent
publicity given the Bionetics work in the press, in Science magazine, and in the
Whiteside article in the New Yorker, was hasty, poorly informed, emotional, and
calculated to gain public attention at the sacrifice of scientific verity.
We believe the regulatory personnel of the U.S. Department of Agriculture and
the Food and .Drug Administration acted with discretion and sound Judgment, in
the face of considerable pressure, to suspend final action against 2,4,5-T until all
relevant data were evaluated from other studies to be completed this spring. It is
�470
471
our uiiTrerstandJng at this time that the "no residue" or zero tolerance registrau nTrers
tions for 2,4,5-T have been extended for the remainder of 1970 to establish whether
finite tolerances can be assigned. We concur in these decisions and believe much of
the credit for new, more accurate scientific data should go to the laboratories of
Dow Chemical Company and of the Food and Drug Administration.
Tt is not our intent to use pesticides whose application can be shown to be
detrimental to the field workers or to the public. Our main concern is the unfortunate consequence for ourselves and all agriculture if concellations of registrations of these and other useful pesticides can take place suddenly and without
adequate notice. It Is probable that pressure for more cancellations is likely to
come, as this one seems to have, from emotion rather than from careful consideration of sufficient scientific evidence.
The collection of information necessary to obtain registration may take many
years. There is now no satisfactory alternative for 2,4,5-T. One alternative is
known and field testing on it was first begun in Hawaii in 1963. However, field
evaluations, residue analyses, and soil- and water-movement studies in a two-year
crop to, provide data to meet registration requirements take so much time that the
chemical company has not yet been able to file for registration of this alternative
compound.
It is our hope that the investigation of the safety of 2,4,5-T and of other
phenolic-derived pesticides will continue, but that the results will be carefully
and scientifically evaluated. We fear that decisions basd on emotion instead of
on fact and reason could deprive American farmers of essential pesticides. We are
confident that your committee will provide such reasoned judgment.
(The following is referred to on p. 376.
nilfflai
tonnd to have a kinked tail and two of its feet were somewhat inilfflapen. Skeletal
examination, however, revealed no evidence of bone abnormalities.
The 8,0 /ig/kg/day dosage level proved to be toxic to the dams. There were no
viable fetuses in the dams which were examined on day 20 of gestation. All resorptions occurred early and no evidence of fetal tissue was found.
'Skeletal examinations revealed delayed ossification of some sternebrae and
skull bones. This occurred generally throughout the various groups, including
controls, and is not considered to be of practical significance.
The results of this study indicate ft high level of maternal and fetal toxicity to
l)e associated with 2,3,7,8-tetrachlorodibenzo-p-dioxiri. Its presence in the sample
tested in the Bionetics Laboratories study could well have 'accounted for the observations reported and attributed to 2,4,5-trichlorophenoxy acetic acid.
ABSTRACT
Toratogenic Study of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Rat G. L.
Sparschu, F. L. Dumi and V, K. Rowe, The Dow Chemical Company, Midland,
Michigan.
2,3,7,8-Tebrachlorodibens!O-p-dlioxin. has been found to occur in small amounts
as a contaminant in some commercially manufactured samples of 2,4,5-trichlorophenoxy acetic acid. The purpose of this study was to learn whether the presence
of this impurity possibly could awount for the fetal abnormalities in test animals
reported in a recent study (unpublished data: Bionetics Research Laboratorfiesi
Bethesda, Maryland).
The dioxin was administered by gavage in 9 :1 corn oil-acetone solution in doses
of 0 (control), 0.03, 0.125, 0.5, 2.0 and 8.0 micrograms per kilogram body weight
per day to groups of 24 (control) and 12 (treatment) pregnant female SpragueDawley derived rats on 'days 0 through 15 of gestation.
On day 20 of gestation, each dam was sacrificed and a cesarean section performed. The number of viable and dead fetuses and early and late 'resorptions was
recorded. Bach fetus was examined for any gross abnormalities. Two-thirds of
each litter were fixed in Bourn's solution, Wilson sections were examined under
the 'dissection microscope, and tissues were studied for histopathology. One-third
of each litter were fixed in alcohol and examined for skeletal abnormalities by
alizarin red-S staining.
Presented at the meeting of the Society of Toxicology, Atlanta, Georgia, March
17, 1970.
No differences were observed in the fetuses taken from dams treated at the
dosage of 0.03 /ig/kg/day and those taken from dams that received the solvent
vehifle only. At the 0.125 /ig/kg/day dosage, all parameters studies were within
normal limits except for a very slight decrease in average weight, and the occurrence of intestinal hemorrhage (18/127) and subcutaneous edema (22/80) in the
fetuses from dams that received this treatment. At the 0.5 /ig/kg/day level, the
number of fetuses was reduced and the number of resorptions and fetal deaths
was increased. The average weight of the viable fetuses was very slightly decreased. The incidence of intestinal hemorrhage (36/99) and subcutaneous edema
(31/65) was markedly increased over that seen in the 0,125 /ig/kg/day treatment.
A<t the 2.0 /ig/kg/day level, only 7 viable fetuses were obtained. These were
from 4 of the 11 litters examined. Resorptions were numerous, intestinal hemorrhage wan frequent (4/7), and subcutaneous edema was present in all of the 4
fetuses examined by Wilson section, One fetus from this treatment level was
O
�
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
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Alvin L. Young Collection on Agent Orange
Description
An account of the resource
<p style="margin-top: -1em; line-height: 1.2em;">The Alvin L. Young Collection on Agent Orange comprises 120 linear feet and spans the late 1800s to 2005; however, the bulk of the coverage is from the 1960s to the 1980s and there are many undated items. The collection was donated to Special Collections of the National Agricultural Library in 1985 by Dr. Alvin L. Young (1942- ). Dr. Young developed the collection as he conducted extensive research on the military defoliant Agent Orange. The collection is in good condition and includes letters, memoranda, books, reports, press releases, journal and newspaper clippings, field logs and notebooks, newsletters, maps, booklets and pamphlets, photographs, memorabilia, and audiotapes of an interview with Dr. Young.</p>
<p>For more about this collection, <a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a">view the Agent Orange Exhibit.</a></p>
Text
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Box
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176
Folder
The folder containing the original item.
5169
Series
The series number of the original item.
Series VIII Subseries I
Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Date
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1970-04-01
Title
A name given to the resource
Effects of 2,4,5-T on Man and the Environment: Hearings Before the Subcommittee on Energy, Natural Resources, and the Environment of the Committee on Commerce, United States Senate, Ninety-First Congress, Second Session, on Effects of 2,4,5-T on Man and
ao_seriesVIII