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                  <text>&lt;p style="margin-top: -1em; line-height: 1.2em;"&gt;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.&lt;/p&gt;&#13;
&lt;p&gt;For more about this collection, &lt;a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a"&gt;view the Agent Orange Exhibit.&lt;/a&gt;&lt;/p&gt;</text>
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                <text>Cupello, J. M.</text>
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                <text>Weed Science</text>
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                <text>1977-07-01</text>
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                <text>A Method for Simulating Subsurface Disposal of Herbicides</text>
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                    <text>item D Number

°3896

Author

Thalken, C. E.

D fj0t scanned

Department of Life and Behavioral Science, United Stat

RBpOrt/ArtlClB TitlB Absence of TCDD Toxicity to a Rodent Population
Following Massive Field Applications of 2,4,5 -T
Herbicide

Journal/Book Title
Year

1975

Month/Day

Ju|

Color

v

n

Number of Images

41

DeSCriptOU NOtBS

Submitted July 1975 for publication in the Journal of the
American Veterinary Medical Association

Friday, January 04, 2002

Page 3896 of 3927

�ABSENCE OF TCDD TOXICITY TO A RODENT POPULATION
FOLLOWING MASSIVE FIELD APPLICATIONS OF
2,4,5-T HERBICIDE

THALKEN, C. E., YOUNG, A. L., and W. E. WARD,
DEPARTMENT OF LIFE AND BEHAVIORAL SCIENCES,
UNITED STATES AIR FORCE ACADEMY, COLORADO

80840

SUBMITTED JULY 1975 FOR PUBLICATION IN THE JOURNAL OF THE
AMERICAN VETERINARY MEDICAL ASSOCIATION.

�Absence of TCDD Toxicity to a Rodent Population
Following Massive Field Applications of
2,4,5-T Herbicide
Charles E. Thai ken DVM, MS; Alvin L. Young, PhD;
William E. Ward, PhD
SUMMARY
Field studies were conducted on populations of beach mice, Peromyscus
polionotus, from a unique 92 acre military test site that received 87,186
pounds of active ingredient 2,4,5-trichlorophenoxyacetic acid herbicide
(2,4,5-T). Significant levels (10-710 parts per trillion - ppt) of the
contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were found within
the top six inches of test site soils. Liver tissue from rodents inhabiting
the test site contained 540-1,300 ppt TCDD. However, no gross or histological
evidence of teratogenesis or toxicity was found in 106 adults and 67 fetuses.
An analysis of variance of liver and spleen weights indicated significant
differences between control and TCDD-exposed animals. Analysis of plant
seeds revealed no detectable levels of TCDD (minimum detection limit of 1 ppt
TCDD). TCDD accumulation in liver tissue was thought to be associated with
pelt contamination from burrowing and subsequent ingestion of soil particles
via grooming.

�From the Department of the Air Force, Department of Life and Behavioral
Sciences, United States Air Force Academy CO 80840.
The authors thank Lieutenant Colonel Harold W. Casey, Chief, Veterinary
Pathology Division, Armed Forces Institute of Pathology, Washington, D. C.
20306, and his staff for histopathological contributions to this study.
The animals in this study were handled in accordance with the "Guide for
the Care and Use of Laboratory Animals", 4th Ed. 1972, Institute of Laboratory
Animal Resources (NAS-NRC), 2101 Constitution Avenue, N.W., Washington, D. C.
20418.
Presented before the Section on Public Health, 112th Annual AVMA Meeting
July 14-17, 1975, Anaheim CA.

�Concern over the level of contamination of 2,4,5-trichlorophenoxyacetic
acid (2,4,5-T) herbicide by the teratogen 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD)3'"'6'10'11'13 has prompted discussion on the safety of using 2,4,5-T
in forest and rangeland environments.12 Although numerous reports have
recently appeared in the scientific literature, most of these deal with
effects of 2,4,5-T and TCDD in laboratory systems.5,7,8,9,14,15,16
In general the effects and mode of action of TCDD on laboratory animals
can be characterized by a relatively small number of clinical signs. It is
reported that a single oral dose (25 yg TCDD/kg) caused an actual weight loss
for one week in young female rats, and young male rats receiving the same
dose had significantly decreased weight gain over a two week period.8 Slight
thymic atrophy, related to TCDD dose levels, was a common finding in young
mice receiving a single oral dose (50 yg TCDD/kg),7 while severe thymic
atrophy in young mice receiving a single oral dose of TCDD (150 yg TCDD/kg)
or four separate oral doses (25 yg TCDD/kg x 4) was reported.5 A single oral
dose of TCDD (50 yg TCDD/kg) in young adult rats and (3 yg TCDD/kg) in young
guinea pigs caused severe thymic atrophy.7 At these same dose levels slight
to severe centrilobular liver necrosis and degeneration of parenchyma! cells
in mice,5'7'8 rats8 and guinea pigs,8 together with ceroid pigment deposits
and hepatic porphyria in mice given four oral doses of TCDD (25 yg TCDD/kg)
at weekly intervals was seen.5 Acute death in guinea pigs has occurred

�following a single (3 yg TCDD/kg) oral dose of TCDD.8 A recent report indicated that four doses of TCDD (25 yg TCDD/kg) given at weekly intervals to
young mice induced the production of 6-aminolevinic acid (ALA) synthetase
and hepatic porphyria.5
TCDD was not converted or metabolized by the three liver microsome NADPH
systems in mice,ltf but rather, a major portion remained in the unmetabolized
form in the liver, partially concentrated in the microsomal fraction of the
cells.14 It appeared that the unmetabolized compound, rather than a metabolite,
was responsible for the toxic effect of TCDD and the endoplasmic reticulum of
the liver was the possible site of toxic effect.114 The fact that TCDD is a
very potent porphyrogenic chemical is evidenced by the 2,000 fold increase in
uroporphyrins following four oral doses of 25 yg TCDD/kg given at weekly
intervals to young mice.5 It may be through the combined direct toxic effect
of TCDD on the liver endoplasmic reticulum plus the indirect toxic effect of
TCDD on the ALA synthetase system and consequent production of toxic levels of
porphyrins that significant liver damage is produced in mice.
Laboratory data for rodents strongly suggest a correlation between histological lesions in the liver and lymphatic system and the amount of TCDD
ingested. Unfortunately, data relating to any actual effects on wild populations in their natural habitat are lacking. The problem of finding a field
site where a wild population of rodents has been exposed to significant quan-

�titles of TCDD is improbable because of (1) low levels of TCDD «0.1 ppm)
found 1n cur ently produced phenoxy herbicide, and (2) low rates of 2,4,G-T
•''''

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applied for brush control on .range!ands or for reforestation (1 to 2 pounds per

; acre). This report, however, documents the effects of residual TCDD on a
rodent population irihabitating a unique test site: a site previously treated
with massive quantities of 2,4,5-T herbicide and located on the Eglin ,Air
Force Base Reservation, Florida.

.

'

The Eglin Reservation has served various military uses, one of them having
been development and testing of aerial dissemination equipment in support of
Of military defoliation operations in Southeast Asia. It was necessary for , ;
. this equipment to be tested under controlled situations that would simulate
actual use conditions as near as possible. For this purpose an elaborate
'

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^testing installation, designed to measure deposition parameters, .was established on'tho Eglin Reservation with the place of direct aerial application/
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"restricted to.an area of approximately one mile square within Test Area C-BHA
';••.• in the southoastern part'of the reservation. Massive quantities of herbicidQs
( used in the testing of aerial defoliation spray equipment from 1962 through

:

; :

19/0,. wore released and fell within the'•• Instrumented test area, .'the; uniqueness.
: of the nrea lias promoted continued ecological surveys since 1967. As f» pesuU,
,'low ecosystems have been/su wo'll .studied and documented,

';'.

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•..,.

�MATERIALS AND METHODS

Description of Field.---Test Area C-52A (TA C-52A) covers an area of
approximately three square miles and is a grassy plain surrounded by a forest
stand that is dominated by longleaf pine (Pinus palustris), sand pine (Pinus
clausa), and turkey oak (Quercus laevis). The actual area for test operations
occupies an area of approximately one square mile and is a cleared area occupied mainly by broomsedge (Andropogon virginicus), switchgrass (Panicum
virgatum), wooly panicum (Panicum lanuginosum) and low growing grasses and
herbs. Much of the center of the range was established prior to 1960, but the
open range as it presently exists was developed in 1961 and 1962. The test
grid is approximately 93 feet above sea level with a water table of six to ten
feet. The major portion of this test area is drained by five small creeks
whose flow rates are influenced by an average rainfall of 61 inches. The mean

me

annual temperature for the test area is-6fr6 F while the mean annual relative
humidity is 70.8%. For the most part, the soil of the test grid is a fine
white sand on the surface, changing to yellow beneath. The soils of the range
are predominantly well drained, acid sands of the Lakeland Association with 0
to 3% slope. A typical three-foot soil core contained approximately 92% sand,
3.8% silt, and 4.2% clay with an organic matter content of 0.17%, an average
pH of 5.6, and a cation exchange capacity of 0.8.
Although the actual area for testing aerial dissemination equipment was

�approximately one square mile, the area of interest in this study was located
in the southern portion of the testing area and consisted of a 92-acre
instrumented grid. This was the first sampling grid and was in operation in
June, 1962.

It consisted of four intersecting straight lines in a circular

pattern, each being at a 45° angle from those adjacent to it. Although this
grid was discontinued after two years it received the most intense testing
program.

From 1962 to 1964, this grid (called Grid I) received 87,186 pounds

of 2,4-dichlorophenoxyacetic acid (2,4-D) and 87,186 pounds of 2,4,5-T. The
herbicide was disseminated as the water insoluble n-butyl and iso-butyl esters
(their military code names were Orange and Purple). Despite excellent records
as to the number of missions and quantity of herbicide per mission, there was
no way to determine the exact quantity of herbicide deposited at any point on
the instrumented grid. The first extensive soil sampling for residues of herbicides on Grid I was initiated in 1969 (five years after the last mission).17
At that time traces (parts per billion) of 2,4,5-T were detected.17 Analyses for
TCDD were initiated in 1972.

By midsummer 1973 analysis of soil samples indi| S fc^f-TV

cated that TCDD was detected only in the top s4x-inches of soil (e.g., analysis
ItsTfe^A

^0^^

of soil cores at"6 i-nek increments to a depth of ttueee-^eet indicated no
detectable TCDD in increments below six •inetie-s).17 Therefore, six sites on
i£«/^\
Grid I were sampled for TCDD in the top six-inch increment. One of the sites
was also subsampled at increments of 0-1, 1-2, 2-4, and 4-6 inches. Analysis

�of soil samples for TCDD was accomplished by a commercial laboratory.3
Animals.—The beach mouse, Peromyscus polionotus, is also referred to as
the old field mouse. It is a small mouse weighing about 13 g, approximately
120 mm in length, with brown (adult) or dark gray (juvenile) fur on the back,
and pale gray to white fur on the ventral region and legs.1

It may be found

in old field habitats and in areas of 5% to 60% vegetative cover, but prefers
sandy areas.2
Burrow and Diet.—The structure of fifteen mouse burrows was determined
by either preparing plaster casts of the tunnels and nests or by careful
excavation of the burrow complex. When plaster of Paris was used it was
poured into the hole, allowed to dry for 24 hours, and then the cast was
freed by removing the soil around it. Data on the diet of the beach mouse
were obtained by examination of litter both outside and within the tunnel and
nest. In addition, an analysis of stomach content was performed on ten mice.
Four 20 g samples of seed, composites from mature plants adjacent to burrows,
were sent to a commercial laboratory for TCDD analysis.3 The samples were
taken from four areas on the test site.

Interpretive Analytical Services, Dow Chemical, U.S.A., Midland, Michigan,
48640. An 1KB 9000S computerized gas chromatographic-mass spectrometer
combination was employed.

�Traps—Live traps, sizes 0 and 1, for small mammals were used to
obtain the rodents.

These traps were baited with peanut butter and oatmeal.

Some traps were randomly placed on the test grid where 20 to 80% vegetative
coverage was present, while others were placed near openings to mouse burrows.
Still other traps were placed in rectangular patterns of five rows of traps,
each row located 20 paces apart, and containing five traps per row, at 15 pace
intervals. Four areas approximately 200 to 1,000 yards off the grid were
designated as control areas, and were trapped in the same manner as on the
test grid. Traps were checked daily and were moved to other locations after
four days failure to catch an animal. Captured mice were taken to the
laboratory for histopathologic examination and chemical analysis of the tissues.
Tissue Preparation—All animals were prepared for examination using a
cervical dislocation procedure to accomplish humane euthanasia. Euthanatized
animals were photographed, weighed, measured, skinned, and systematically
examined for developmental defects such as cleft palate, cleft lip, polydactyly
and microophthalmia. All internal organs were examined for gross lesions, and
individually weighed. Representative sections of each tissue were placed in
neutral 10% buffered formalin and processed for microscopic study.0

Havahart Traps, Department 1, P.O. Box 551, Ossining, M.Y. 10562.
G

Veterinary Pathology Division, Armed Forces Institute of Pathology, Washington,

D. C. ,20305.

�All remaining liver tissues and the pelts from mice captured in the test and
control areas were pooled according to sex ans maturity, placed in glass jars,
frozen, and submitted for TCDD analysis.
RESULTS
Soil Analysis—Analysis of 6-inch soil cores for TCDD taken at six
locations on the 92-acre area (Grid I) indicated wide fluctuations in TCDD
concentrations.

The results for the uniformly mixed top 6-inch increments

were 10, 25, 70, 70, 110, and 710 parts per trillion (ppt) TCDD.
*

Further

analysis of a duplicate core, obtained from the site having 110 ppt TCDD
concentration, indicated that TCDD was stratified within the top six inches
of soil. The analysis for depths of 0-1, 1-2, 2-4, and 4-6 inches resulted in
detectable levels of 150, 160, 700, and 44 ppts TCDD, respectively. These
data are shown in Table I and are placed in perspective with hypothetical
concentrations of TCDD which might occur with currently produced 2,4,5-T
herbicide formulations containing 0.1 parts per million (ppm) TCDD and applied
at normal rangeland or reforestation rates for brush control.
Trapping Data—In the eight weeks of trapping beach mice during the
summer (June-July) of 1973 and six weeks during the summer (June-July) of
1974, 106 specimens were collected from either Grid I or a portion of a grid
immediately north and slightly overlapping Grid I, and a control site. Since
many of the females were pregnant at the time of collection, 67 fetuses were

�recovered.

This brought the total number of beach mice collected and examined

over a period of two years to 173 (Table 2).
Burrows and Diet—From an examination of the burrows it was apparent
that no two burrows were identical. However, most were characterized by a
small mound of soil on the surface with a 2-inch diameter tunnel near the
center, leading down and away from the surface at a 45 angle. A plug of
soil was usually found within the first 10-12 inches of the tunnel entrance.
Eight to ten inches beyond the plug, the tunnel leveled and continued horizontally for another 18 inches. There it expanded into a spherical chamber
with a diameter of about 6 inches in which a nest was usually found: this
placed the nest approximately 12 inches below the soil surface.

Frequently,

beyond the nest, the burrow turned upward and to one side while at the same
time narrowing to its original 2-inch diameter.

This "escape tunnel" normally

extended to within 2-6 inches of the soil surface.
The nests were contructed of dried grasses (stems, blades, and inflorescences). The dominant grasses used for bedding were broomsedge and low
panicum. The litter within the nests consisted of caryopsis hulls, leaf fragments, twigs, insect exoskeletons, insect wings, and snake scales. From a
detailed examination of the caryopsis hulls, six species comprised the bulk
of the vegetative portion of the diet: rough button weed, Diodia teres;
spotted spurge, Euphorbia maculata; bitter polygala, Polygala polygama; common

�polypremum, Polypremum procumbens, and low panicum and switchgrass. Examination of insect parts indicated insects of the Orders Coleoptera (Beetles),
Hemiptera (True Bugs), and Homoptera (Cicadas and Leaf-Hoppers, etc.). Based
upon number of insect parts and seed hulls and upon each of their estimated
weights, it was assumed that about 90% of the beach mouse diet was seeds, and
the remaining 10% was made up of insects.
The results from the TCDD analysis of the four composite seed samples
indicated that TCDD was not detectable in any sample (minimum detection limit
of 1 ppt TCDD). The insects were not analyzed for TCDD.
Liver and Pelt Analysis—Results of liver and pelt analysis for TCDD are
shown in Table 3. Samples of liver, as well as pelts, of mice taken from
Grid I in which significant soil levels of TCDD were found, exhibited positive
evidence of accumulation of TCDD. TCDD was also found in the pooled liver
samples of both male and female control animals, although no TCDD was detected
on their pelts.
Histopathology—A series of histological examinations were performed on
the heart, lungs, trachea, salivary glands, thymus, liver, kidneys, stomach,
pancreas, adrenals, large and small intestine, spleen, genital organs, bone,
bone marrow, skin, and brain. Initially, the tissues were examined on a
random basis without the knowledge of whether the mouse was from a control or
test area. All microscopic changes, including those interpreted as minor or

�insignificant, were recorded. For example the following types of lesions were
interpreted as not significant or of a common finding when large populations
of wild animals are surveyed histologically: variation of nuclear size (poikilotosis) and of cytoplasmic staining in liver tissue from both control
and TCDD-exposed animals; subacute, focal myocarditis and sialitis in two
separate mice captured from a control area; poikilotosis of acinar cell nuclei
in a mucous salivary gland and poikilotosis of nuclei from the adrenal cortex
of two separate TCDD-exposed mice; and sarcosporidiosis of skeletal muscle in
one TCDD-exposed mouse.

Following the recording of all microscopic findings,

the tissues were re-examined on a control and test basis. Results of both
studies determined that the test and control mice could not be distinguished
on a histopathologic basis. Significant lesions were only found in two mice,
one from a control area (Fig 1) and one from a test area (Fig 2). Both had a
moderately severe, multifocal, necrotizing hepatitis. Sections from the liver
of these animals were stained with a variety of stains in attempts to identify
an etiologic agent.

Neither bacterial nor fungal organisms nor eeroid pig-

ments were demonstrated and the lesions were considered to be virus induced,
as they resembled the lesions seen in viral hepatitis of laboratory mice.
The gross lesions observed in the kidney of one other beach mouse proved to
be severe ectasia of the renal veins (Figs 3, 4, 5, 6, 7). Microscopically,
the vascular dilation was interpreted as being of little functional

�significance. All other lesions observed in both control and test mice were
minor and insignificant and of the type normally observed when a large group
of animals are examined at the microscopic level.
•

Due to the small size of the beach mouse and since both mature and immature specimens were collected, it was most difficult to grossly dissect the
thymus gland from salivary glands, fat, and regional lympth tissue. Upon
histological examination it was found that the tissue samples contained one
or more of the four tissues. Nevertheless, in those specimens that contained
thymus tissue, no histological evidence of thymus atrophy was noted in either
control or TCDD-exposed animals. Data on the gross weight of thymus tissue
were not statistically analyzed because of the uncertainty as to what other
tissues might be in the specimen.
Statistical Analysis—Tables 4 through 10 present statistical data for
physical parameters on beach mice collected from a control site and the TCDDexposed field site on Test Area C-52A. Pregnant females and sexually immature animals were excluded from statistical comparisons because of the widely
varying differences in both body and organ weights. Although microscopic
examination for ovarian follicles or sperm production confirmed sexual
maturity, animals with total body weights of 10 grams or greater were
considered mature and were included in statistical evaluations. Fourteen
of 46 females were pregnant. Since their body weights ranged from 11.48

�to 18.68 grams, inclusion of these animals into the population used for
statistical comparisons would have distorted the data for body and organ
weights. Table 4 presents the actual number (57) of beach mice used in the
statistical analyses. Unfortunately, all females from the 1973 control site
were either pregnant or immature.
Table 5 gives the mean total body weight by sex for mature beach mice
captured in 1973 and 1974 from control and test sites. A matrix of F-values
from the analysis of variance for total body weight for all possible combinations of treatment (location), sex, and year are given in Table 6. Statistically significant differences (95% probability level) existed only for sex,
or a sex-year interaction. No treatment differences were noted.

In general,

female beach mice are heavier than male beach mice.
Table 7 presents the mean values for liver weight by sex for mature beach
mice captured in 1973 and 1974 from control and test sites. The matrix of
F-values from the analysis of variance for all possible combinations of
treatment, sex and year are given in Table 8. Statistically significant differences were found for liver weights between sex (e.g., comparing control
females collected in 1974, comparison II and III) and with the sex year
interaction (e.g., comparing control 1973 males with control 1974 females).
However, the comparison of 1973 or 1974 control males with 1973 or 1974 Grid
I males (I with IV and II with V, respectively) yielded no significant

�differences in liver weights. The comparison of liver weights for females
collected from a control site in 1974 (III) with females collected in 1974
from the TCDD-exposed site (VII) indicated statistical significance at the 99%
confidence level. Note from Table 7 that the mean values for liver weights
would have suggested the results of the above comparisons.
Table 9 presents mean values for heart, lung, kidney and spleen weights
collected in 1974 from beach mice inhabiting control and TCDD-exposed field
sites. Statistical analyses of the weights for heart, lung, or kidney indicated no significant differences in the weights of these organs between sex
or treatment.

However, significant differences were noted for spleen weights

between control and test site animals. A matrix of the F-values from the
analysis of variance for spleen weights is given in Table 10. Note that the
comparison of sex within the same treatment is not significant; e.g., I with
II or III with IV. Differences are noted for all treatment comparisons; e.g.,
I with 5, I with IV, or II withft II with IV.
DISCUSSION
Soil Analysis—The application of massive quantities of 2,4,5-T herbicide
to Test Area C-52A has created a unique field site in which to assess not only
the ecological impact of the herbicide (2,4,5-T) but also the toxic contaminant TCDD. The method of application; i.e., by aerial dissemination,
resulted in unequal distribution of the herbicide. Three major flight paths

�intersected the 92-acre instrumented grid. If a soil sample wet»e obtained
from an area not under one of the flight paths or if it w$re obtained near
the intersection of all three flight paths then the residue levels would be
expected to vary significantly.
The data suggest that TCDD may persist for long periods of time in the
environment. However, caution must be exercised in making such a statement.
As noted in Table 1, it was probable that Grid I received highly contaminated
herbicide. The herbicide was most likely produced in the 1950s or early
1960s and thus was subjected to preparation treatment different from those
controlled procedures subsequently used. A conservative estimate for TCDD
contamination may be 8 ppm in the formulation. Using the 8 ppm figure for
4600 pounds of butyl esters of 2,4-D and 2,4,5-T applied per acre (equivalent
to 947 pounds of 2,4,5-T acid) in the years from 1962-1964, the amount of
TCDD applied was 0.0368 pounds per acre. This is 12,267 ppt TCDD in the top
six inches of soil.

At the least, this has declined to 710 ppt in about 8

years. This is a loss of about 95 percent. Thus, the apparent high residue
is probably due to the massive quantities applied rather than to the resistance of TCDD to biological and/or physical degradation.
d

The value of 0.0368 pounds per acre = 4600 x 8 x 10"6. 0.0368 pounds of TCDD

in 3 x 106 pounds per acre-foot of soil = 0.0368 x 1012/3 x 106 = 0.0368 x
106/3 = 36,800/3 = 12,267 ppt.

�Liver and Pelt Analysis—The presence of TCDD in the liver samples of both
male and female mice collected from the control site in 1974 may have been
due to high levels in one or more specimens in the pool of samples. Mice
from the test area could have migrated to the periphery of the grid and wandered into the area designated as control. The closest point from the control
site to the test area was 200 yards. A previous trapping study on this test
site17 reported that the mean random travel distance (or average habitat
radius) for the beach mouse was 65 yards. The distance traveled on the
longest radius observed was over 1000 yards, but this unusual observation
was regarded as a freak occurence.

However, it emphasizes that a mouse (or

mice) could have been contaminated in this way, and thus have contaminated
pooled samples analyzed for TCDD. Nevertheless, the use of these data as
truly control data must be viewed with caution.
The levels of TCDD in the livers of beach mice collected from Grid I
substantiated bioaccumulation of TCDD; i.e., an accumulation of TCDD TJT_ an
organism from its environment. In general, levels of TCDD in the livers were
no greater than the most concentrated zones of TCDD in the soil. There are
no data from this study to support biomagnification of TCDD; i.e., an increase
in concentration of TCDD in successive organisms ascending the trophic food
chain.

�Although the concentration of TCDD on the pelts of beach mice from the
test area was only 10-15% of that in their livers, it was apparent that the
mice continually contaminated themselves by repeated movement in an out of
their burrows. The soil data substantiated the presence of a zone of TCDD
within the top six inches of the surface (Table 1). Thus, whenever an animal
burrowed into the soil it was exposed to TCDD. Moreover, the sand plug located
within a short distance of the tunnel entrance suggested that recurrent
burrowing activity occurred even in established burrows.

Likewise, the loca-

tion of the escape tunnel suggested that even the nest itself may contain
detectable levels of TCDD.
Histopathology—The only significant lesions seen on histopathologic
examination of 173 adult and fetal beach mice were two instances of moderately
severe multifocal, necrotizing, hepatitis and a single mouse with severe
venous ectasia of the renal veins in one kidney. All other lesions were of
the minor or insignificant type, normally observed in microscopic surveys of
large numbers of field animals. The absence of liver lesions (necrosis and
porphyria) in animals that had liver levels of TCDD from &lt;20 ppt to 1,300 ppt
(Table 3) is most significant in view of the massive quantities of both
2,4,5-T and TCDD that must have been applied to the test site. Moreover, a
previous study17 of this area, which terminated in the summer of 1970, indicated that a significant population of beach mice were inhabiting the test site.

�The average life-span of a related species, Peromyscus maniculatus, has
been recorded to be less than five months and only a few mice lived the full
potential of three or more years.2 A single female beach mouse is capable of
producing eighty or more young under laboratory conditions with litters being
born at approximately 26 day intervals.1 It is further reported that beach
mice on Santa Rosa Island, Florida (within 20 miles of Test Area C-52A), may
have produced 10 generations per year.1 At this frequency the animals collected in 1974 on Grid I may be 40 generations removed from the population
first noted in 1970.

However, a more conservative estimate would be 6 gener-

ations per year (giving a female 60 days to reach sexual maturity), for a
total of 24 generations.
It must be stressed that the populations of beach mice noted in 1970 were
probably subjected to much greater levels of residual TCDD in the soil than
those animals collected in 1974.

The absence of pathological signs in mice

collected in 1974 indicated that TCDD was neither mutagenic (somatic or germinal) nor carcinogenic in the field at the concentrations noted in Table 1.
Since none of the 34 fetuses examined from animals captured on the test grid
showed teratogenic defects it must also be concluded the levels of TCDD
encountered failed to induce observable developmental defects.
As animals mature, the thymus gland undergoes gradual regression until in
the adult it is often found only as a rudimentary structure.

Because of the

�age differences in animals captured from the field, it was impossible to
obtain mean thymus weights.

In the literature where thymic atrophy is

reported following single oral doses of TCDD, young animals of a similar age
were used for the laboratory study.7 Microscopic examination of all thymus
gland tissue from both control and TCDD exposed animals further substantiated
the lack of thymic lesions in the field situation where animals were exposed,
via their burrowing and grooming habits, to soil levels of TCDD as seen in
Table 1.
Statistical Analysis—Although 32 control and 74 test animals were
collected from the field, the population selected for statistical analyses
was necessarily smaller (19 and 48, respectively). Removal of all immature
mice and pregnant females from the population reduced the range of variability
between individuals. However, it also eliminated the data on control females
collected in 1973.

This is important to note since the only remaining com-

parison of liver weights between females were those collected in 1974 from the
control site and Grid I and which were significant at the 99% confidence level.
Since the population numbers were low for this comparison (4 versus 4), caution must be used in the interpretation of the results (histological examination did not support differences in liver tissue between control and test
animals).

�Data on spleen weights between the control and Grid I mice were statistically significant (Table 10).

It has been reported6 that rats given 10.0

pg/kg became moribund or died between 17 and 31 daily treatments.

Remarkable

changes were consistently observed in the spleen and lymph nodes. These
changes consisted of a relative depletion of lymphoid cells and pyknosis of
the nuclei and degenerative change in the multinucleated megakaryotic type
giant cells of the spleen. In the present study, an increase in spleen weight
was found in those animals (male and female) collected from the TCDD-exposed
field site. However, as with the liver data, histological examination (gross
and microscopic) of the spleens did not support differences between the control and test animals. It is interesting to note the magnitude of the standard deviations between spleen weights from the two locations. The magnitude
of the differences in the standard deviation may reflect the fluctuations in
soil levels of TCDD throughout Grid I. Thus, not all animals from the test
site received the same exposure levels. Because the population numbers were
relatively large for this comparison (19 versus 48), and hence a measure of
their reliability, the data suggest that the spleen may be the most sensitive
organ by which to assess field exposure to TCDD.
This report has reaffirmed that results from laboratory experiments and
field studies present widely varying differences in biological organisms'
responses to chemicals found in the environment.

It should further serve as

a ceveat regarding conclusions reached from laboratory experiments alone.

�List of Figures
Fig 1—Hepatitis in a beach mouse captured from a control area.
tory cells can be seen in the sinusoids and periportal areas.

Inflamma-

H&amp;E stain;

x 130. AFIP Negative No. 74-14883.
Fig 2---Acute necrosis and inflammation in the liver of a beach mouse
captured from the test area.

H&amp;E stain; x 130. AFIP Negative No. 74-14874.

Fig 3---Gross illustration showing ectasia of renal veins on the left with
the normal contralateral kidney on the right. Beach mouse was collected from
test area. AFIP Negative No. 74-86541.
Fig 4—Close-up view of veins illustrated in Fig 3. AFIP Negative No.
74-86542.
Fig 5—Close-up view of normal contralateral kidney illustrated in Fig 3.
AFIP Negative No. 74-86543.
Fig 6—Microscopic appearance of venous ectasia in the kidney of the beach
mouse shown in gross illustration Fig 3. H&amp;E stain; x 90. AFIP Negative
No. 74-14876.
Fig 7—Microscopic appearance of a normal kidney from a beach mouse captured
on the test area. Compare this figure with Fig 6. H&amp;E stain; x 90. AFIP
Negative No. 74-14881.

�Fig 1—Hepatitis in a beach mouse captured from a control area. Inflammatory cells can be seen in the sinusoids and periportal areas. H&amp;E stain;
x 130. AFIP Negative No. 74-14883.

•'-VW
'•V&amp;
,&amp;'&lt;™m

*li*"l.te'&lt;4 '« «•''*
:'*'

' V».^M

Fig 2-—Acute necrosis and inflammation in the liver of a beach mouse
captured from the test area. H&amp;E stain; x 130. AFIP Negative No. 74-14874.

�Fig 3—Gross illustration showing ectasia of renal veins on the left with
the normal contralateral kidney on the right. Beach mouse was collected from
test area. AFIP Negative No. 74-86541.

Fig 4-—Close-up view of veins illustrated in Fig 3. AFIP Negative No.
74-86542.

�Fig 5—-Close-up view of normal contralateral kidney illustrated in Fig 3.
AFIP Negative No. 74-86543.

Fig 6—-Microscopic appearance of venous ectasia in the kidney of the beach
mouse shown in gross illustration Fig 3. H&amp;E stain; x 90. AFIP Negative
No. 74-14876.

�Fig 7—Microscopic appearance of a normal kidney from a beach mouse captured
on the test area. Compare this figure with Fig 6. H&amp;E stain; x 90. AFIP
Negative No. 74-14881.

�TABLE 1. Comparison of 2,4,5-T/TCDD Application Rates to Rangelands (Normal Use)
Versus Rates Applied to Grid I, Test Area C-52A, Eg!in AFB, Florida (Military Aerial
Spray Equipment Test Program)
SUBJECT

NORMAL RANGELAND USE

Pounds 2,4,5-T Active Ingredient
Per Acre

2

947

184

Total For 92 Acre Area
TCDD Concentration of 2,4,5-T
Formulation

GRID I APPLICATIONS
(1962-1964)

87,186

&lt; 0.1 ppm (Current Production
Standards)

&lt; 0.1 - 47 ppm*

Concentration of TCDD in Soil Profile
(parts per trillion):
0-1 inch

0.8 ppt**

150 ppt***

1-2 inches

Not Detectable

160 ppt

2-4 inches

Not Detectable

700 ppt

4-6 inches

Not Detectable

44 ppt

Below

Not Detectable

Not Detectable

Range of TCDD Contamination in Herbicide Stocks Returned from Southeast Asia in
1971 and stored on Johnston Island, Pacific Ocean. (In Disposition of Orange
Herbicide by Incineration, November 1974, Department of the Air Force Final
Environmental Statement.)
** Assuming no TCDD degradation and the application of 2,4,5-T to bare soil. If two
pounds 2,4,5-T, containing 0.1 ppm TCDD, are applied and uniformly mixed into top
one inch of an acre of soil, then 2 x 0.1 x TO"6 pound TCDD per 6 acre in one inch
of soil weighing 3 x 106 pound per acre-foot or about 0.25 x 10 pound per inch
acre equals 0.2 x 10~6/0.25 x 106 or 0.8 x 10"12.
*** Soil profile samples collected in 1974.

�TABLE 2—Numbers of Beach Mice Collected During the Summer 1973
and 1974 Studies of Control and TCDD-Exposed Field Sites
1973

1974

CONTROL
MALE

5

12

17

FEMALE

5

10

15

FETUSES

12

11

33

SUBTOTAL = 65
TEST
MALE

26

17

43

FEMALE

18

13

31

FETUSES

25

9

34

SUBTOTAL = 108
TOTAL

= 173

�TABLE 3—Concentration (parts per trillion) of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) in Liver and Pelt Samples From Beach Mice, Peromyscus polionotus
Collected From Control and TCDD-Exposed Field Sites, 1973 and 1974.
TREATMENT

YEAR

SEX

LIVER

PELT

CONTROL

1973

MALE AND FEMALE

&lt; 20*

N.D.**

CONTROL

1974

MALE

51

&lt; 40*

FEMALE

83

&lt; 40*

GRID I

1973

MALE AND FEMALE

GRID I

1974

MALE
FEMALE

* Minimum level of detection.
** Not determined.

540

N.D.**

1,300

130

960

140

�TABLE 4—-Number of Peromyscus polionotus Used in Statistical
Comparisons of Populations Collected From Control Site and TCDDExposed Field Site. Pregnant Females and Immatures Excluded
LOCATION

SEX

1973

1974

MALE

4

11

FEMALE

0*

4

CONTROL

MALE

12

14

8

4

TREATMENT
FEMALE

* All females were either pregnant or immature (See text)^

�TABLE 5—Mean Values For Total Body Weight (Grams) of Peromyscus
pollonotus Collected From Control Site and TCDD-Exposed Field Site.
Pregnant Females and Immatures Excluded.
LOCATION

SEX
MALE

1973
11.88+1.03

1974
12.02+1.21

CONTROL
FEMALE

—*

11.77+1.13

MALE

12.17+1.13

11.49+0.93

FEMALE

13.28+2.27

14.05+2.21

TREATMENT

* All females were either pregnant or immature.

�TABLE 6—Matrix of F-Values From Analysis of Variance of Total Body Weight for Peromyscus
polionotus Collected From Control Site and TCDD-Exposed Field Site.

MATRIX NUMBER

MATRIX
NUMBER

LOCATION

SEX

YEAR

I

CONTROL

MALE

1973

II

CONTROL

MALE

1974

III

CONTROL

FEMALE

1974

IV

GRID I

MALE

1973

GRID I

MALE

GRID I

FEMALE

GRID I

F=1.37
ns*

III

F. = 1.19

IV

F = 1.20

VI

VII

ns*

ns

F=1.24
ns

F = 1.15
ns

F = 1.14
ns

F = 1.70
ns

F = 3.50
P .05

F = 3.34
P .10

F = 1.01
ns

F = 1.47
ns

F = 4.04
ns

F = 3.85
ns

F = 1.48
ns

F = 4.01
P .025

F = 3.82
P .05

F = 5.94
P .005

F = 5.67
P .025

1973

VII

IJ

1974

VI

I

FEMALE . 1974

*Not significant at a probability of less than .10 (i.e., 90% confidence level)

F.= 4.80 F.= 4.58
P .10
ns

F = 1.05
ns

�TABLE 7—Mean Values For Liver Height (Milligrams) of Peromyscus
pollonotus Collected From Control Site and TCDD-Exposed Field Site.
Pregnant Females and Immatures Excluded.
LOCATION

SEX ,

MALE

1973

707.50+143.61

1974

611.00+111.34

CONTROL
FEMALE

—

*

678.25 + 26.29

MALE

861.11+263.36

664.79+150.54

FEMALE

1115.00 + 355.65

860.25 + 151.90

TREATMENT

* All females were either pregnant or immature.

�TABLE 8—Matrix of F-Values From Analysis of Variance of Liver Weight For Peromyscus
polionotus Collected From Control Site and TCDD-Exposed Site.
MATRIX NUMBER

MATRIX
NUMBER

LOCATION

SEX

YEAR

I

CONTROL

MALE

1973

II

CONTROL

MALE

1974

III

CONTROL

FEMALE

1974

IV

GRID I

MALE

1973

V

GRID I

MALE

GRID I

FEMALE

GRID I

FEMALE

F = 1 .66
ns*
—

III

IV

V

.VI

VII

F = 29.85
P .01

F = 3.36
ns

F=1.10
ns

F = 6.13
P .10

F = 17.94
P .025

F = 5.59
P .01

F=1.83
ns

F = 10.20 F = 1.86
P .005
ns

—

F = 100.39 F = 32.80
P .005
P .01
—

F = 3.06
P .05

1973

VII

—

II

1974

VI

I

1974

*Not significant at a probability less than .10 (i.e., 90% confidence level).

—

F = 1.12
ns

F = 183.07 F = 33.39
P .005
P .01
F = 1 .82
ns

ns

F = 5.58
.005

F = 1.02
ns

F = 3.01

F = 5.48
P .10
—

-

�TABLE 9—

Mean Values for Heart, Lung, Kidney and Spleen Weights (Milligrams) for Peromyscus polionotus

Collected in 1974 from Control Site and TCDD-Exposed Field Site. Pregnant Females and Immatures Excluded.
'

ORGAN
KIDNEY

SEX

HEART

LUNG

CONTROL

MALE

100.55 + 12.98

98.64 + 13.82

191.27 + 20.12

16.82 +_ 6.42

CONTROL

FEMALE

91.75 + 19.82

114.25 +_ 33.98

197.25 +_ 30.90

21.75 +4.65

GRID I

MALE

93.93 + 20.75

99.93 +_ 21.04

193.50 +_ 20.33

23.07 + 14.66

GRID I

FEMALE

104.50 + 13.23

96.00 + 17.15

226.50 + 24.77

24.75 + 20.34

LOCATION

SPLEEN
*

�TABLE 10—Matrix of F-Values from Analyses of Variance of Spleen Weight
for Peromyscus polionotus Collected in 1974 from Control Site and TCDDExposed Field Site.
MATRIX
NUMBER

I

LOCATION

SEX

CONTROL

MALE

I

MATRIX NUMBER
II
III

IV

CONTROL

FEMALE

III

GRID I

MALE

F = 5.22

F = 10.05

ns*

II

F = 1.91

P .01

P .005

F = 9.96
P .05

F = 19.16
P .025
F = 1.92

ns
___
IV

GRID I

FEMALE

* Not significant at a probability of less than .10 (i.e., 90%
confidence level).

�REFERENCES

1. Bowen, W. W.: Variation and Evolution of Gulf Coast Populations of
Beach Mice, Peromyscus polionotus, Bulletin of the Florida State Museum,
University of Florida, Gainesville, Florida, 12:1, (1968): 1-19.
2. Blair, W. F.: Population Structure, Social Behavior, and Environmental Relations in Natural Populations of the Beach Mouse (Peromyscus
polipnotus 1eucocephalus). Contribution from the Laboratory of Vertebrate
Biology, University of Michigan, Ann Arbor, Michigan, Number 48, (Jun, 1951):

1-47.
3. Buu-Hoi, N. P., Pham-Huu Chanh, Sesque, G., Azum-Gelade, M. C., and
Saint-Ruf, G.: (1) Enzymatic Functions as Targets of the Toxicity of "dioxin"
(2,3,7,8-Tetrachlorodibenzo-p-dioxin). (II) Organs as Targets of "dioxin"
(2,3,7,8-Tetrachlorodibenzo-p-dioxin) Intoxication. Naturwiss., 59, (1972):
173-175.
4. Cunningham, H. M., and Williams, D. T.: Effect of Tetrachlorodibenzop-dioxin on Growth Rate and Synthesis of Lipids and Proteins in Rats. Bull.
Environ. Contam., 7:1, (1972): 45-51.
5. Goldstein, J. A., Hickman, P., Bergman, H., and Vos, J. G.: Hepatic
Porphyria Induced by 2,3,7,8-Tetrachlorodibenzo-p-dioxin in the Mouse. Res.
Commun. Chem. Pathol. Pharmacol., 6:3 (Nov, 1973): 919-928.

�6, Greig, J, B.: Effect of 2,3,7,8-Tetrachlorodibenzo-p-dioxin on Drug
v Metabolism in the Rat. Biochem. Pharmacol., 21, (1972): 3196-3198.
7..,' Gupta, B. M., Vos, J. G,, Moore, J. A., Zinkl, J..G., and Bullock*
; 0. C,.*' Pathologic Effects of 2,3,7,8-Tetraehlorodibenzo~p-dioxin in Laboratory. Animals.. Environ. Health Persp., 5, (1973); 125-140.

,

,

;

8. •'Harris, M. W., Moore,.J; A., Vos, J. G.s and Gupta, B. N.: General
\ Biological Effects of TCDD in Laboratory Animals. .. Environ.. Health Persp., B,
y (1973): 101-109.
9.
',

i

' ' ' • ' '

;' :',/.:.,..V '; ' i

• '••.

'''• '' ' '' '^^ • ' ! ' • ' • . ' •

'-" .' '

Joiifjs, 6., and Butler, W. H,':, A Morphological Study of the Liver
'

'

.

'

.

'

I

1

'

,

.

"

',

i

!

'

lasion Induced by ^O^jS-Tetrachlorodlbenzo-p-diaxin in Rats,
'."'112:2,.''(1974): 93-97.:''

•' • . ' • • ' ; ; • ' •

' ' • ' , ,i, . ,' ,": ' ; '

'

•'',

/•

J. Pathol.,
;

•

,'

.. ;.; : . .,;• ' "' ;

10..: Kiiiibrough,'R, D., Llndor, R, E., and Gaines, T. B.: Morphological

;

Changes in Livers of Rats Fed Polychlorinated Biphcriyls. Arch. Environ1
:
"

'

,'

Healtiii',25, (1972); 3EJ4-361.

• '

.. .

,

;

'•

'

&gt;

-

,

.1 . i . ' . ' ! • ' ' • " '

,'" '

:

;

,
.'/'

' . ' - : V,',.'.':•;•'.'

11. Poland, A. P., Smith, D., Matter* G./et_ a±.: A Health Survey of
Workers In, a 2,4«D Plarit and 2,4,5-T Plant, Arch. Environ, Health, 22» (1971)
, 316-327,';'- ••'.'.':•', ; /&gt;•' ^V';'..."^ :'.;'''".;"' ; '
;••.';.,'•:••

•'.' ' • ; • • •' ''•' : '.'? .i^' I ••,•'.; '•

12, Report on 294,5-T, A Report of the Panel on Herbicides of:The ' •:;;

, Prosidont's Science Advisory Committee&gt; Executive Office of'.The1 President,.
Office of Science and Technology, March, 1971&gt;

'\

&gt; '••','•

�13. Schwetz, B. A., Norn's, J. M., Sparschu, G. L. et al_.: Toxicology
of Chlorinated Dibenzo-p-dioxins. Environ. Health Persp., 5, (1973): 87-99.
14. Vinopal, J. H., and Casida, J. E.: Metabolic Stability of 2,3,7,8Tetrachlorodibenzo-p-dioxih in Mammalian Liver Microsomal Systems and in
Living Mice. Arch. Environ. Contain. Toxicol., 1:2, (1973): 122-131.
15. .Vos, J. G., Moore, J. A., and Zinkl, J. G.: Effect of 2,3,7,8Tetrachloridibenzo-p-dioxin on the Immune System of Laboratory Animals.
Environ. Health Persp., 5, (1973): 149-162.
16. Woods, J. S.: Studies of the Effects of 2,3,7,8-Tetrachlorodibenzop-dioxin on Mammalian Hepatic &lt;$-Aminolevulinic Acid Synthetase. Environ.
Health Persp., 5, (1973): 221-225.
17. Young, A. L.: Ecological Studies on a Herbicide Equipment Test Area
(TA C-52A), Eglin AFB Reservation, Florida. AFATL-TR-74-12, Air Force Armament Laboratory, Eglin Air Force Base, Florida, January, 1974. Unclassified,
distribution unlimited; available from Defense Documentation Center, Defense
Supply Agency, Cameron Station, Alexandria, Virginia

22314.

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                  <text>&lt;p style="margin-top: -1em; line-height: 1.2em;"&gt;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.&lt;/p&gt;&#13;
&lt;p&gt;For more about this collection, &lt;a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a"&gt;view the Agent Orange Exhibit.&lt;/a&gt;&lt;/p&gt;</text>
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                  <text>&lt;p style="margin-top: -1em; line-height: 1.2em;"&gt;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.&lt;/p&gt;&#13;
&lt;p&gt;For more about this collection, &lt;a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a"&gt;view the Agent Orange Exhibit.&lt;/a&gt;&lt;/p&gt;</text>
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                <text>Cockerham, Lorris G.</text>
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                <text>Environmental Toxicology and Chemistry</text>
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                <text>The Absence of Hepatic Cellular Anomalies in TCDD-Exposed Beach Mice - A Field Study</text>
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                  <text>&lt;p style="margin-top: -1em; line-height: 1.2em;"&gt;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.&lt;/p&gt;&#13;
&lt;p&gt;For more about this collection, &lt;a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a"&gt;view the Agent Orange Exhibit.&lt;/a&gt;&lt;/p&gt;</text>
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                <text>Hamme, N. A.</text>
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                    <text>Item D Number

°3833

Author

Arnold, E. L.

D N0t Scanned

Department of Life and Behavioral Sciences, USAF Aca

KOpOrt/ArtlClB Title Typescript: A Rapid Method for the Determination of
Several Phenoxyalkanoic Acid Herbicides in Soil
Samples

Journal/Book Title
Year

1974

Month/Day

February

Color

D

Number of Images

16

DOSCrlptOn NOtBS

Includes two memorandums clearing the typescript for
publication

Friday, January 04, 2002

Page 3833 of 3927

�DEPARTMENT OF THE AIR FORCE
HEADQUARTERS UNITED STATES AIR FORCE ACADEMY
USAF A C A D E M Y , COLORADO 80840
REPLY TO

ATTN OF: OIPM (Capt Boyle/4050)
SUBJECT:

2 0 FEB 1974

Review of Manuscript

TO: DFLS (Capt Young)

The Department of Defense has reviewed your manuscript, "A Rapid Method for
the Determination of Several Phenoxyalkanoic Acid Herbicides in Soil Samples,"
co-authored by Maj. E.L. Arnold. It is cleared, with no changes, for open
publication.

WILLIAM F. FRENSLEY, Major, USA|
Deputy Director of Information

Atch
Manuscript (2)
Cy to: DFSS

"MAN'S FLIGHT THROUGH LIFE IS SUSTAINED BY THE POWER OF HIS KNOWLEDGE'

�DEPARTMENT OF THE AIR FORCE
HEADQUARTERS UNITED STATES AIR FORCE
WASHINGTON, D.C.

SAF/OIS (74-103)

15 February 1974

Paper, "A Rapid Method for the Determination of Several
Phenoxyalkanoic Acid Herbicides in Soil Samples"
AFA/OI
The Department of Defense has reviewed subject paper and
has no objection to open presentation.
FOR THE CHIEF OF STAFF

JOHN J. PELSZYNSKI
Colonel,iUSAF

1 Atch
Subj as above

Chief, Office for Security Review
Office of Information

Underwrite Tour Country's Might - Buy U.S. Savings Bonds

�A RAPID METHOD FOR THE DETERMINATION OF SEVERAL PHENOXYALKANOIC
ACID HERBICIDES IN SOIL SAMPLES

f0R

20

E. L. ARNOLD AND A.L. YOUNG
DEPARTMENT OF LIFE AND BEHAVIORAL SCIENCES
USAF ACADEMY, COLORADO 80840

740x03

�A rapid gas chromatographic method for the measurement of several
phenoxyalkanoic acid herbicides in soil samples is described. Average
recovery of herbicides added to soil ranged from 99.7% to 87.5% (±4.9%)

�INTRODUCTION:

,

;

;

•

, &gt;-;?^

The problems associated with the ecologically safe disposal of contamin^CU^&gt;
ated or excess pesticides chirr received increased attention in recent years.
Among those compounds requiring a safe method for disposal are the halogenated
phenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D),
2,4,5 trichlorophenoxyacetic acid -(-£,3,5-?), and their esters. One of the""

r

mei&gt;ns of disposal of these herbicides which has been suggested is soil biodegradation, whereby the herbicides are incorporated in soil and degraded by
soil microorganisms. (1).

In order to evaluate the effectiveness of this

technique, analytical procedures are necessary for the measurement over a
wide range of concentrations of both the n-but.yl esters and the sodium salts
of 2,4,-D and 2,4,5-T. Since evaluation of the biodegradation technique will
require the analysis of large numbers of soil samples taken from relatively
large field plots, an analytical procedure which is not only accurate and
precise but also rapid and simple to perform is required. A number of different
technioues have been devised for this type of analysis (2-4), however, many of
these procedures tend to be complex and time consuming since they were designed
for samples where herbicide concentrations were low and many interfering substances were present. This paper will describe a simple, rapid and accurate
.procedure for the simultaneous determination of the n-butyl esters of 2,4-D
arid 2,4,5-T and their correspond ing hydrolysis products in soil samples.

•

�EXPERIMENTAL:

,., ;^H&lt;M ,,. „

^

'
'' ) i •
APPARATUS: A Beckman Gas Chromatograph, Model GC-45 equipped with a

*

hydrogen flame ionization detector was employed. Columns were 6 ft x 2 mm
i.d. glass u-tubes packed with 80/100 mesh chromasorb W coated with 10%
DC-200 silicone gum as the stationary phase. All columns used were sugglied...^,^
'

by the instrument manufacturer.

'

:

'

' '

-

|v

• ' •

Conditions for the chromatography-werie-:--"-**-"-'-™--"-

temperatures; inlet, 245°C; column, 220°C; detector, 250°C; air flow:;

=,V

carrier (helium), 60 cc/min; detector make up (helium), 60 cc/min; hydrogen, Tfw;:
'

'

.

'

,

,

'

•

&gt;

'

' • • ' - ; • ' &gt; ( ;(.i'

50cc/min; air, 250 cc/min. One micro!iter samples were injected directly on

'

column.
REAGENTS: Methanol, chloroform, acetylchloride and anthracene were
~

f :•;••• ':
;

ACS reagent grade chemicals (J.T. Baker) and were used without additional
purification. The sodium salts of 2,4 dichlorophenoxyacetic acid (2,4-D) ;&gt;,and 2,4,5 trichlorophenoxyacetic acid (2,4,5-T) were practical grade (Dow
,'•-.'

;

':• '';".'.',".:

. -

-

;
;

•-

, • ' ' . * ' . ' ' '--.. : •«' '•'"•
j

,Chemicals:Co.):,C The:• n-bii.tyl esters of 2,4-D and 2,4,5-T were analyzed to be y;" ? ;;Cv
greater than 99% purity (Hercules, Inc.): 3% solution of anhyrdous hydrogen,,
chloride in methanol was prepared by reacting 5 ml of acetyl chloride with ",^0
100 ml ; 0 f methanol. (5). • ' • - ' •

,,

,

,.,

•••:-^'*,-;y'ffi\£$i

. • . " • . ..-\

• ^PROCEDURES: Standard solutions of the n-butyl esters and the sodium - T V ;";
salts of 2,4-D and 2,4,5-T were prepared by dissolving weighed amounts of
each compound in either chloroform (n-butyl esters) or methanol (sodium salts)&gt; /
.

'

•

-

•

'

.

"

•'

. . . . . • . . • - - ' y '-..
'

After serial dilutions were made, the sodium salt solutions were reacted With
:

•" . . - . . • • ' •

.

-"';i"-f

•

a .3% (w/w) solution of anhydrous HC1 in methanol (5 ml herbicide salt standard**'','!
'

'

,

,

.

'

I

.. ,

,

•

'

'

,

'

'

'

^'''

ir' '

solution plus 1.5 ml 3% HCl": methanol•) in order to form methyl esters suitable ';
' ' ' ' , ' '

• . . ; . , .

,

^

.

•

.

-

'

'

'

'

for chromatography. After heating for 15 minutes at 55°C, a sufficient volume
of a chloroform solution of anthracene internal standard was "added ta obtain a^:,*
'

'''

�final internal standard concentration of 10 yg/ml, 50 yg/ml or 250 yg/ml
depending upon the herbicide concentration of the standard solution. '
Sufficient internal standard solution was also added to the n-butyl ester
solutions to obtain the same range of internal standard concentrations as
used with the sodium salts. All standard solution volumes were adjusted to
10 ml with chloroform prior to chromatography.
V: /
;

1

Standard curves for each herbicide were prepared by the chromatography •

* of standard solutions of varying herbicide concentration, calculating the
ratios of the individual herbicide GC peak areas to the GC peak area of
the internal standard and plotting these ratios against the actual herbicide
concentrations in the standard solutions. Concentration curves were prepared
, for each of the four herbicides at three different concentration ranges,
,
&lt;

:

(0-10 yg/ml, 10-100 yg/ml, 100-1000 yg/ml).
Soil samples used for evaluating the procedures were prepared by
dissolving weighed amounts of each of the four herbicides in anhydrous
methane! and adding this solution to representative control soil samples
contained in 1 liter round bottom flasks. Solvent was then removed from the

;&gt; samples using a rotary flash evaporator. Control soil samples had been
'';-, previously analyzed and shown to be free from herbicide residues.
Soil samples were analyzed in the following manner. Five grams of
treated soil were placed in 15 nil glass screw top culture tubes, and the
herbicides extracted with 10 ml anhydrous methanol by heating the sealed
tubes at 55°C in a water bath. Samples were shaken periodically during the
heating period. After cooling, a .'•) ml aliquot of the methanol extract was
.//transferred to a clean culture tube and mixed with 1.5 ml of 3% HC1rmethanol.

�/*

Reaction was allowed to proceed at 55°C for 15 minutes. The samples were .««,..**
allowed to cool to room temperature and'^fcJHnl of a chloroform solution of""** """
internal standard added. The final concentration of internal standard ob&lt;?

7

T

tained was either 10 g/ml, 50 g/ml or 250 g/ml, depending upon the
expected range of herbicide present.

pif

' '"-

One microliter samples were analyzed

by gas chromatography. Soil concentrations were determined by multiplying
the concentrations in mg/ml obtained from the standard curves by a factor of
V

four to obtain the concentrations in micrograms of each herbicide per gram
;: of soil. Soil samples obtained from biodegradation test plots were analyzed
i;

in the same manner.

A'7-; RESULTS AND DISCUSSION:
•:,•• ;

Employing the chroma'tographic conditions outlined above, the four herbi-- . &lt;
f

, cides of interest could be separated from each other in less than ten minutes
, ,

as shown in Figure I. This figure also illustrates the freedom of the samples &gt;

'/^ ;;,;; from interfering substances which might be present in the soil. In the, : /
chromatogram, the herbicides which were present, in the soil as either salts
or free acids appear as methyl esters. The esterification reaction appeared

•;V

to produce a quantitative yield of methyl esters regardless of the equilibrium^h
form of the herbicide which was present.
Five replicate soil samples,for each of five different herbicide concentrations were analyzed. All samples were analyzed on the same day as they
• w e r e prepared in order to minimize hydrolysis of n-butyl esters due to
the prevailing alkalinity (ph 8.4) of the soil which was used. The results
of these analysis are given' in Table I. Three additional replicates at two
.concentration levels were stored at 4°C for 24 hrs then analyzed for the
purpose of evaluating the degree of hydrolysis. These data are presented

;

';
',

�TABLE I
RECOVERIES OF.HERBICIDES ADDED TO SOIL SAMPLES*

Amount added
mg/g soil'

Compound
2,4-D, Na
2,4, -D, n-butyl
2,4,5-T, Na
2,4,5-T, n-butyl

2.00
2.00
2.00
2.00

2.4-D. Ha
2,4-D 5 n-btityl
2,4,5-T, "3
2,4,5-T 5 n-Duty"i

2.19
1.85
2.08
1.84

-

2,4-D, n-butyl .
2 5 4,5-T, : Na - , ; 1 ^
2,4,5-T, ;n-b'uty I''' -' . -

-, 0.050
0.050
0,050
f:\ I 0.050

112
86
98
88

0.264
„, ,
,,
,0.212
':X:-:k &gt;f --;f 0.251 : ':'•• - '
: ;

0.250:
0.250
0.250 *..
:{
": 0.250

2,4-D, Na
2,4-D, n-butyl
2,4,5-T, Na
2,4,5-T, n-butyl

no

1.12
0.86
0.98
0.88

1.00
1 .00
1.00
1 .00

9 4_n &gt;HC
£,i*-L , v=

Percentage
Recovery

Average
Recovered**(mg/g )

; ';;; ;--.:'•• •;;- -^ 0.201

93
104
92

.

106
85
100
81

- -:

x

2,4-D, Na ;- :/~-yV
:: o.oio
2, 4-D,&gt; n-butyl - ; •--:- H f; 0.010
2,4,5-T, Na
v
; : 0.010
2,3,4-T, n-butyl
0.010
f

0.061

0.048
0.052
0.041

112
96
104
82

* 0.012
0.007
0.009
0.007

: -

,•

120
70
90
70

c

* Samples analyzed immediately following preparation.
**Mean value of 6 analyses.

o

- "if
?i ^~~*

�in Table II. Average recovery,of: total;added .herbicide ranged from 99&gt;7% ,;;v &gt; v
at soil concentration of 8 rng/g to 87.5% at soil concentrations of 0.04 mg/g.
The mean relative standard deviation for all four compounds of interest
was ±4.9%. It appears from the data contained in Table I that approximately
8% of the n-butyl esters of 2,4,5-T and 12% of the n-butyl esters of 2,4-D
contained in any sample are converted to methyl esters by the derivative
forming reaction. Since this rate of conversion is relatively constant at
all herbicide concentrations, a conversion factor may be added in order to
improve the accuracy of the analysis. The data presented in Table II indicates a relatively rapid hydrolysis of the n-butyl esters of 2,4-D and 2,4,5-T
when exposed to alkaline soil. In the 24 hr period of exposure, 9% of the
n-butyl ester of 2,4-D and 8% of the n-butyl ester of 2,,4,5-T were hydrolyzed.
' It should be noted that in this calculation it is presumed that some of the '
/hydrolysis of esters noted, is due to the derivative forming reaction. This
*• ' ,

presumption is based upon studies made with different concentrations of HCl.^
•.

-f

At catalyst concentrations lower than 3% (w/w), recovery of the n-butyl esters
from soil samples approached 100% while recovery of the herbicide salts

.

decreased rapidly if the samples were analyzed immediately after preparation.,;
These data illustrate the necessity of analyzing field soil samples as soon
after removal from the test plot's/is possible if a comparison of esters and
salts is necessary.
At the conclusion of the analysis of the prepared soil samples, a
number of samples were obtained from USAF soil biodccjradation test plots and
analyzed for herbicide residues. A herbicide formulation of the n-butyl
enters of 2,4-D and 2,4,5-T had been incorporated at several different
application rates Into the soil of these plots. Table III contains the data

�TABLE II

-

HYDROLYSIS OF HERBICIDE ESTERS ADD TO ALKALINE SOIL SAMPLES

Compound

Amount
added mg/g
soil

Time of
exposure
hrs

Average
amount
recovered

2,4-D, Na
2,4-D, Na

2.00 --.."
2.00

0
24

2.19
2.71

2,4-D, n-butyl
2,4-D* n-butyl

2.00

0
24

1.85
1.38

2,4,5-f, Na\

2.00
•2.00 .

0

24

2.08
2.40

2.00 .

0
24

1.84
1.58
1.12
1.32
.86

Percent increase,
decrease of 24 h'
exposure

+26.0
2.00

. '

-24.0
9 5 ~r 3 _- T 5
£ 4 £ i

*!a
; *-CJ
"'"

2,4,5-T, n-butyl .
2,4,5-T, n-butyl

-

+16.0
2.00

-13.0

? 4— n f?
2*4-?! Na .

1.00

1.00

0
24

2,4-D, n-butvl
2,4-D, n-butyl

] .00
1.00

0
24

.72

2,4,5-T, Na
2,4,5-T, Na

1.00
1.00

0
24

0.98

1.00

0 .
24

0.88
0.71

+20.0

-14.0

1.14

+16.0
2,4,5-T, n-butyl
2,4,5-T, n-butyl

'1.00

-19.0

�TABLE III
DETERMINATION OF HERBICIDE CONCENTRATIONS IN SOIL BIODEGRADATION TEST PLOTS
2,4-D Na
mg/g

2,4-D n-butyl
mg/g

2,4,5-T Na
rog/g

2,4,5-T n-buty'
mg/g

1-3
1-4
1-5

0.740
0.698
0.731
0.740
0.726

0.376
0.354
0.368
0.385
0.361

0.700
0.650
0.684
0.706
0.671

0.326
0.303
0.340
0.327
0.316

Mean ± a

0.727±.018

0.369±.012

0.682±.023

0.322±.014 j

2-5.

0.335
0.910
0.856
0.795
0.852

1.05
1.15
1.08
1.06
1.10

0.410
0.426
0.410
0.392
0.408

1.62
1.70
1.66
1.57
1.60

Mean ± a

0.850±.042

1.09±.040

0.409±.010

1.63±.043

3-5

0.125
0.130
0.117
0.115
0.132

0.015
0.015
0.013
0.011
0.014

0.098
0 . 1 00
0.110
0.095
0.112

0.032
0.042
0.035
0.040
0.038

Mean ± a

0.124±.015

0.013±.002

0.103+.008

0.037+.004

Sample. £

-

1-1 V
1-2 .."

:

Soil
Depth

- 0-6" . -

G-5 :

2-1
2-2
2-3
2-4

3-1
3-2,
3-3 '
3-4

6-12 i:
•

-- .

"

10

^
'-:-

:

.-

�obtained from five replicate:analyses from three different samples taken
1

'

'• '

t

.

"

from the test plots. As can be seen from the data in the table, the concentrations of the different herbicide residues in these plots varied greatly,
however, the mean relative standard deviation (± 6.1%) for all samples varied
only slightly from that determined when prepared samples were used. This
indicates that the analytical technique is applicable for the determination
of herbicide residues over the large concentration ranges found in actual field
samples. - . ' , ' • • • '

' .•

';/-'-.

In addition to the relatively high precision and accuracy of the analy- '.".•••'
tical procedure, one of its major advantages is the number of samples which
can be analyzed for four different components in a short period of time. The
time required for one individual to analyze 12 soil samples was 3-4 hours or
about 30 minutes per sample. Sensitivity of the procedure allows accurate ,
measurement of any of the four compounds at levels greater than 4 tnicrogram/
.' •

• -

v

.

-

gram soil. It was also noted during the investigation that,1 if desired, two
other ester formulations (octyl and iso-ocytl) of 2,4-D and 2,4,5-T could
be determined simultaneously with the analysis described previously.
ACKNOWLEDGEMENT:

.-''•-',

, '"i^ty

The authors gratefully acknowledge Anthony M. Wachinski for providing . -;;v r ;
, the soil samples from soil biod.egradation test plots used in this work. Further
- reproduction is authorized to satisfy the needs of the U.S. Government.

,

11

&lt;&lt; ^*,

�1. M.A. Loos, Degradation of Herbicides, P.C. Kearney and D.D. Kaufman,
Eds., Marcel Dekker, Inc., New York, 1969, pp 18-24.
2. D.E. Clark, J. Agr. Food Chem., 17:1168 (1969).
3. D.E. Gutni.ck and G. Zweig, J. Chroma tog. 13:319 (1964).
4. M.L. Taylor, R.L.C. Wu, E.L. Arnold, B.M. Hughes and T.O. Tiernan,
Abstracts; 21st Annual Conference of the American Society for Mass
»
Spectrometry, San Francisco, Calif., May 1973.
5. Applied Science Laboratories, Inc., State College, Pa., Catalog 16,

p 78, 1973.

12

�Figure 1: Gas chromatogram of the extract of a prepared soil sample containing
1 tng/g each 2,4-D and 2,3,5-T sodium salts and 1 nig/g each 2,4-D and 2,4,5-T
n-butyl esters. Salts were converted to methyl esters prior to chromatography. Chromatographic conditions as outlined in experimental section,
attenuation = TOOOx.

�INTERNAL STANDARD
1U I

2, 4-0, METHYL
.ESTER
Jf

C/3

2
O

' 0.

co

Ul

or
oc
LU
a
DC
o
O
uu
oc

2, 4, 5-T,
METHYU
ESTER

2,4-D, n-BUTYL ESTER

2, 4, 5-T, n-BUTYL ESTER

0

3

4

5

TIME (MINUTES)

8

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Author

Kotchman, G.S., Jr.
Non-Explosive Munitions Division, Air Force Armament
Laboratory, Air Force Systems Command, United
States Air Force, Eglin AFB, Florida
Typescript: Flushing Techniques For Defoliant Spray Tanks, June 1970

Journal/Book Titla
0000

(Month/Day
D
20

Technical Notes AFATL-TN-70-?; Typescript is followed by handwritten editing notes
and reference materials. Included in the typescript is a table describing the military
codes, trade names, common names and formulations of agents orange, white, and
blue.

Friday, January 05, 2001

Page 167 of 194

�AFATL-TN-7G-

FLUSHING TECHNIQUES FOR
DEFOLIANT SPRAY TANKS

by

G. S. KOTO-WAR, Jr., 1st Lt, USAF
A, L. YOUNG, 1st Lt,
N. A.

TECHNICAL

AFATL-TN-7Q-1

JUNE 1970

NON-EXPLOSIVE MUNITIONS DIVISION
AIR FOICE
LABORATORY
AIR FORCE SYSTEMS
UNITED STATES AIR FORCE
EGLIN AIR
BASE, FLORIDA

�ABSTRACT
In support of development of the F-4D/PAU-7B (modified TMU-28)
defoliant spray tank, flushing techniques were develop'fbr flushing mnd/or
washing defoliant agents Orange, White or Blue from spray tanks and/or
from aircraft surfaces in the event of contamination. In addition, a
solvent technique was developed for dissolving the precipitates formed
following the mixing of defoliant agents Orange or White with agent Blue.

�GENERAL COMMENTS

The use of more than one defoliant agent in Southeast Asia (SEA) has
resulted in compatibility problems between the agents.

Remaining quantities

of the defoliants Orange or White in the spray tank or in other parts of
the spray system, if not properly removed, will fern persistent precipitates
with agent Blue. The need for a solvent system, applicable for use in the
field, which will dissolve these precipitates, is of interest to defoliant
operations. In addition the development of the F-4D/PAU-7B (modified TMU-28)
defoliant spray tank for use with agents White and/or Blue has generated a
requirement for techniques in flushing and/or washing defoliant agents from
not only the spray tanks but also from the aircraft surface in the event of
contamination.
A description of the three agents used in defoliant operations in SEA
is given in Table 1. Further data on these agents can be obtained from a
technical note by Young and Wolverton ( ) or from a technical report by
5
Darrow, Irish, and Minarik ( ) Orange is readily soluble in fuel oils or
4.
&amp;eorganic solvents, but insoluble in water. Agents Blue and White are soluble in
water but insoluble in organic solvents and diesel fuel. None of the three
agents are compatible with each other, more especially agents Orange and White
with agent Blue. The liquid formulation of Blue was introduced to the SEA
operations in 1965 ( )
4.

In a technical manual on the use of herbicides (1),

dated 22 November 1966, agent White was first described for use in defoliant
operations. In a "CAUTION" statenent in this manual the compatibility of Blue

�TABLE 1. Descriptions of the three military defoliants used in Southeast Asia with their military
code, trade name, common name, and formulation.
Military Code

Trade Name

Common

Orange

Brush Killer

2,4-D, 2,4,5-T

50-50 mixture of the 80% n-butyl esters
of 2,4-D (2,4-dichlorophen.oxyacetic acid),
and 2,4,5-T (2,4,5-triehlorophenoxyaeetie
acid).

White

Tordon 101

2,4-D, picloram

10.2% of the triisopropanolamine salt of
picloram (4-aminQ-3,5f6-trichloropicolinic
acid), 39.6% of the triisopropanolamine
salt of 2,4-D, and 50.2% inert ingredients
(primarily triisopropanolamine).

Blue

Phytar 560 G

cacodylic acid,
sodium cacodylate

4.7% cacodylic acid (dimethylarsinic acid),
26.4i sodium cacodylate (sodium dimethylarsinic acid), 3.4% sufactant, 5.5% sodium
chloride, 0.5% antifoatn agent, and 59.5%
water.
/

Formulation

�(Phytar 560 G) with White (Tordon 101) was noted: "When Phytar 560 G and
Tordon 101 are mixed a precipitate will form that will clog the spray system.
Particular care will be exercised to remove residual Phytar by liberally
flushing with water prior to filling aircraft tank with'Tordon. Residual
Tordon will be removed by the same means prior to filling with Phytar."
No mention was made as to how to remove this precipitate if it occurred or of
the compatibility of Orange with Blue.
Compatibility of Tordon 101 and Phytar 560 G was discussed by Buerge
in a research memorandum of the Dow Chemical Company (3)«

Buerge noted that

a white precipitate formed when the two formulations were mixed in a ratio
of 1:19 or 19:1, Tordon 101 to Phytar 560 G, respectively. This precipitate
was recognized as the sodium salt of 2,4-D which is only 4-5% soluble in

7

water. Ingredients which were trjLed to prevent this precipitation from
0
**•
forming included kerosene, #2 fuel oil, heavy aromatic naphtha, @&gt;lyglycol P-400,
(Pblyglyeol P-26-2 and each of the Dowanols. However, none of these chemicals
prevented the precipitate from forming.

Thus, Buerge concluded that the only

way to prevent a precipitate^ from forming and to make the two formulations
compatible was to dilute the formulation remaining in the spray tank with
Jl5r^

water.

In considering the JJinfet A/OlSY-l J|efoliant jlispenser, Buerge stated:
O
"If a. minimum of 150 gallons of water is added to either formulation remaining
$ the tank and then the resulting mixture pumped or drained back down to 50
I
in
gallons, no precipitate will form when 950 gallons of the other formulation is
added. If it is not desirable to pump or drain the mixture of water and
*~\
formulation in the tank back down/to 50 gallons, then 150 gallons of water can
be added if Phytar 560 G is leftain the tank, and then Tordon 101 can be added
(to bring tsfae total volume in the tank) to 1000 gallons. If Tordom 101 is left

�in the tank, however, approximately 500 gallons of water would have to be added
before the Phytar 560 G is added to 1000 gallons to prevent a precipitate,
Even then a little precipitate will form in this mixture after standing
overnight." Furthermore, Buerge noted that to prevent the possibility of
any precipitate forming in the lines when spraying is started, the remaining
formulation in the tank should be diluted with water and pumped through the
lines to clean the concentrated formulation out before the tanks are filled with
a new formulation.
In a Dow Chemical Company research memorandum, Buerge (2) reported on
the compatibility of Tordon 101 and Orange. Mixing of these two defoliants did
not produce a precipitate. The only problem recognized with the mixing of
Tordon 101 and Orange was a separation of the two on standing (with Tordon 101
on top) when the ratio of Orange to Tordon 101 was high and sufficient agitation
was not present.
The need for solvent systems, applicable for use in the field, that will
flush the spray tanks and systems efficiently is obvious.

Improper flushing

requires a solvent system to remove the formation of any precipitate. The
1
efforts reported in this note were undertaken not to 'clearly identify the precipitates formed from the mixing of the defoliants, but rather to determine solvent
systeias and techniques which would meet the above requirements.

Considerations

were given to chemical availability in normal supply channels and to cost
effectiveness.

�LABORATORY

AND

The precipitate formed when White and Blue are mixed has been
identified as the sodium salt of 2,4-D,which is only 4-5% soluble in
water ( ) The precipitate fonued when Orange and Blue are mixed nay thus be . 3.
the sodium salt of the n-butyl ester of 2,4-D or 2,4,5-T or a nixture of the
two. The precipitates formed from mixing prange or White with Blue appeared
- 6
to be different as indicated by various solubility tests. To find a solvent
system which would dissolve these precipitates, many solvents were tried.
Solvents which were unsuccessful in dissolving both precipitates included
chloroform, hexane, ethanol, keorsene, benzene, petroleum ether, carbon
tetrachloride, toluene, butanol, methanol, and dilute hydrochloric acid.
The precipitates were partially soluble in acetone^ani mot water with
detergent broke up the precipitate fomed from Orange and Blue.
solvent system which will dissolve both precipitates is a 501

,

,j

/by volume) solution of N,N-dinethyl formanide (DMF) in water, pith
l— ~
sufficient amopnts of the solvent system in the tank (with scrubbing if possible),
A

the precipitate should be removed. If difficulties should still persist, a
higher concentration of DMF should be used. Purging the systen with water
will remove any remaining DMF. DMF has a boiling point of 153°C. (A*-with
the vapor can be harmful; thus, DMF should be
kept away from heat and open flame. Contact with skin and clothing should also
be minimized.
Solvent system recommendations for efficient flushing of individual
defoliants (Orange, White, and\Slue, respectively) were basej on a residual
content of 1 gallon in the F-4l^PAU-?B defoliant spray tank,
application- of the r«€OHieitd»ti0n-s--S'hould--r€aiove-any-precipitatioft-&gt;--prcJ!W"eiis.

�The recommended flushing agent techniques are as follows:
a. To flush Orange from tank prior to the addition of Blue:
(1) Add approximately 5 gallons alcohol (methanol, denatured alcohol,
s

or isopropyl alcohol). Mixy Flush as much as possible from tank. Note:
\
flushing twice with alcohol u^ing 3 ^ gallons each time would get better
s^
results if flushing can be done^ easily.
(2) Add approximately 10 gallons water and mix. Flush milky solution
froa tank. Again add 10 gallons water and mix. Flush slightly^ cloudy solution
n

froii tank.

If solution is still milky at this stage, repeat water flush

third time.
(3) Add 2 gallons alcohol to tank, nix, and flush. Repeat
adding 2 gallons alcohol, nix and flush.

A check can be made at this stage by

saving a snail amount of the solution flushed out and adding to it a small amount
of Blue. Mixture should be clear and tank should be ready for adding Blue.
b. To flush Blue froa tank prior to the addition of Orange:
(1} Add approximately 5 gallons alcohol to tank. Mix and flush
immediately so that residue does not settle out.
( ) Repeat adding S gallons alcohol, mix, and flush.
2
( ) Repeat adding 5 gallons alcohol, mix, and flush. Better
3
cleaning can be obtained by flushing five times with 2 to 3 gallons of alcohol.
Save small amount of final flush solution and add small amount of Orange. If
the mixture remains clear, the tank is satisfactory for filling with Orange.
If solution is cloudy, flush again until aixture of flush solution and Orange
remains clear.

7

�a. To flush White froa the tank prior to the addition of Orange or Blue:
(1) Flush system with 5 gallons of detergent and water,
(2) After flushing thoroughly with water, add approximately 5
gallons of alcohol to tank, mix and flush,
(3} Save a snail amount of flush solution and add mint snail
amount of Blue. If mixture remains clear, the tank is satisfactory for filling
with Orange or Blue. If solution is cloudy, flush again until mixture of flush
solution and Orange or Blue remains clear.
If a spray tank is only to be used with defoliants White and Blue, then
the flushing recommendations would be as follows:
a. Assuming 1 gallon of WHITE is in the tank, a Minimum of 10 gallons
of water should be added. Flush this from the tank saving a saall amount of
the flush solution. To the small amount of the flush solution add a small
amount of Blue, the defoliant to be used in the tank. Allow to set several
minutes.

If no semi-solid particles form, the tank is satisfactory for

filling with Blue.

If a precipitate forms, add more water to the tank and

test a small amount of the flush solution with Blue to insure that the solution
remains clear,
b. Assuming 1 gallon of Blue is in the tank, a minimum of 10 gallons of
water should be added. Flush from the tank and test a small amount of the flush
solution by adding a small amount of White.

If no precipitate forms after

setting for several minutes, the tank is satisfactory for filling with White,
If a precipitate forms, repeat the flushing and test the flush solution until
the solution shows no precipitate.

�For all recommendations, the wash containing the diluted Orange, White, or
Blue should, if possible, be diverted into pits or settling basins for
incorporation into soil.

It should not be directed into a stream or river.

�LITERATURE CITED

1. Air Force, AFPS, SAMMA. 22 November 1966.
Herbicides. T.O. 42C-1-17.

Technical Manual-Use of

2. Buerge, T. £., 30 June 1966. Compatibility of Tordon 101 and Orange.
Research Memorandum, Dow Chemical Company, Midland, Michigan.
3. Buerge, T. E., 1 July 1966. Compatibility of Tordon 101 and Phytar 560 G.
Research Memorandum, Dow Chemical Company, Midland, Michigan.
4. Darrow, R. A., K. E. Irish, and C. E. Minarik. 1969. Herbicides used in
Southeast Asia. Technical Report SAOQ-TR-69-11078, Plant Sciences Laboratories,
Fort Detrick, Frederick, Maryland.
5. Young, A. L., and B, C. Wolverton. 1970. Military Herbicides and
Insecticides, Technical Notes AFATL-TN-70-1, Air Force Armament Laboratory,
Eglin Air Force Base, Florida.

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�1. Description of Defoliants

i
BLUE Is the code designation for a neutralized^ liquid formulation
_-4»ntaIniag--cacodylic acid ^diraethylarsinic-acid) arid its sodium salt.
The BLUE currently in use is a liquid formulation produced by Ansul
Company and is designated Phytar 560G, It contains';a minimum of 21%
sodium cacodylate with additional free cacodylic acid for a total
clime thy larslnic acid equivalent of not less than 26%; or 2.48 Ib/gal.
Phytar 560G contains 5% by volume of a surfactant plus 0.5% of an
antifoaia agent.
'
\
WHITE is the code-designation -for Tordon 101, a liquid formulation
containing picloram (4-amino-3,5,6-trichloropicolinic acid) and 2,4-D
in the form of triisopropanolamine salts. The composition of MUTE,
based on weight of salt, is: picloram, 10.2%; 2,4-D, 39.6%. Based on
.weight of-active-ingredient or acid, the composition is: picloram,
5.7% or 0,54 lu/gal; 2,4-D, 21.2% or 2.0 Ib/gal. The remainder
consists of water, wetting agent'j and other inert ingredients.
Picloram or Tordon is a proprietary item produced by Dow Chemical Company,
2. Physical, Chemical, and Biological Properties
a. Physical State
;

BLUE (Phytar 560G) is a liquid formulation of cacodylic acid
and its sodium salt. It is a reddish or brownish, free-flowing liquid.
WHITE (Tordon 101) is a dark brown, somewhat viscous liquid.
b. Solubility

BLUE is readily soluble in water and alcohol but insoluble in
diesel fuel and oils.
.~ *
WHITE is misclble in water but insoluble in diesel fuel and
other oils. "The" triisopropanolamine forms of 2,4-D and picloram in WHITE
are both readily soluble in water.
c. Volatility
BLUE is nonvolatile.
low vapor pressures.

Cacodylic acid and its salt have extremely

�WHITE is considered nonvolatile. Picloram has a vapor pressure
of 6.16 x 10~7 mm of Ilg at 35 C.
WHITE, because of.the low volatility of its active ingredients,
has been suggested as a substitute for ORANGE for defoliation in areas
adjacent to rubber plantations and sensitive broad-leaved crops. However,
because of the high activity of the picloram component, extremely small
amounts can damage sensitive species.
WHITE is stable and moderately resistant to ultraviolet radiation.
Despite its water base, the formulation WHITE is flammable with a flash
point of 35 C.
d. Corrosive Action
Corrosion tests with undiluted BLUE showed little or no effect
on brass, copper, aluminum, and tin after an explsure of 1 month. Steel
showed a rapid initial reaction and moderate corrosion with BLUE. Zinc
was the least resistant metal, exhibiting a galvanic reaction in the cacodylate solution.
NHITE is noncorrosive on common metals and materials used in
spray equipment.
e. Biological Properties
BLUE is a desiccant or contact herbicide and causes rapid browning
of foliage. It shows relatively little translocation or movement within
the plant. Foliage of broad-leaved vegetation affected by BLUE may abscise
or shrivel and remain on the plant. Grasses may show rapid browning and
death of top growth but regrowth of resistant perennial species may occur
within 1 to 2 months. Defoliation of woody vegetation by BLUE shows shortterm effect, with ~ maximum effect at 2 to 6 weeks after treatment.
. «
WHITE is a mixture of two systemic herbicides, picloram and
2,4-D, It is partially selective in its defoliant and herbicidal action
on an array of plant species and is effective, principally, on woody and
certain broad-leaved herbaceous plants. Both the picloram and 2,4-D
components are relatively ineffective on grasses, bamboos, and other
monocotyledonous plants. Forest areas defoliated with WHITE often show an
immediate increase in these resistant plants.
Both picloram and 2,4-D are readily absorbed by the foliage.
Picloram, apparently, is more easily absorbed by the root system than are
2,4-D and other phenoxy herbicides.

�The picloram component of WHITE is more persistent in soils as
compared with other chemicals such as ORANGE and BLUE. Microbial
decomposition of picloram is limited and losses from soils occur
principally by leaching and some photodecompositioii. At rates used for
defoliation it may persist in some soils for 1 year\or more.
i
3. Storage
\
Herbicides are delivered in 55-gallon steel drums marked with an
identifying color band. Drums may be stored in either a horizontal or
vertical position. Under prolonged storage, stockpiles should be checked
• periodically to determine the condition of the "containers and remove
leakers or damage drums. BLUE and WHITE are stable chemicals with a
storage life of several years. The chemicals may outlast the life of the
metal containers under prolonged storage exposure to tropical heat, rain,
and humidity.
4. Handling

\

BLUE may be handled safely with ordinary sanitary precautions in
avoiding prolonged contact with skin or clothing. Cacodylic acid is
readily absorbed through the skin; prolonged absorption may lead to a
distinct garlic odor in the breath. Spillage should be avoided and
=removed by liberal flushing and rinsing with clear water.
,

Only ordinary precautions are recommended for handling WHITE, as
for any common agricultural chemical. It may be mildly irritating to skin
and eyes on prolonged contact. Contaminated clothing should be washed
before reuse. Spillage on the skin should be rinsed with clear water.
BLUE should not be used in a spray system either following or
preceding WHITE, without thoroughly flushing the tank and system with
water. A mixture of these two agents results in a precipitate consisting
of the sodium salt*of 2,4-D (a component of WHITE}. If a change in agents
is to be made, the tanks or spray system should be filled at least half
full with clear water and the system exhausted of liquid before the new
agent (BLUE or WHITE) is added.
5. Methods of Disposal
Loading and storage areas subject to chemical spillage may be
partially decontaminated by repeated washing with ammonia water and
flushing with clear water. Runoff water from such flushing operations
should be diverted to settling basins or restricted areas not subject to
overflow on cropland. Picloram in WHITE is persistent in spray equipment
and containers or in soil, thus full decontamination of equipment or areas
subject to spillage is extremely difficult. A vigorous regimen of soap

�I

and water, ammonia water, and clear water rinses and flushings is necessary.
Equipment used for WHITE should not be used for other purposes such as
fertilizing or applying insecticides.
i
Equipment used for application of BLUE should b'e thoroughly cleaned
prior to storage or disuse. Several flushings of soapy or detergent water
to which ammonia has been added may be used, followed by a clear rinse.
For roost spray systems a final rinse with diesel fuel may serve to prevent
:
rust or sediment accumulation.
;
Excess spillage of BLUE in loading or storage areas should be removed
by thorough washing with clear water and/or dilute ammonia. Runoff or
wash water containing diluted BLUE should, if possible, be diverted into
pits or settling basins for incorporation into soil. The chemical is
rapidly adsorbed by soil and deactivated.
Used containers and residual chemical should be disposed of by burial
whenever possible.
\
\
6. Toxicology
Cacodylic acid or dimethylarsinic acid, the active component of BLUE,
contains arsenic in the innocuous pentavalent state rather than the toxic
;trivalent form. The acute oral toxicity (LDgg) of cacodylic acid in rats
i is 1,400 mg/kg for males and 1,280 mg/kg for females. For the formulation
Phytar 560G or BLUE containing the acid and its sodium salt, the acute
»
oral toxicity (UDgo) for rats is 2,600 mg/kg, a toxicity level lower than
that of aspirin (1,750 mg/kg).
Cattle fed 24.5 mg/kg of cacodylic acid daily in a 60-day feeding
test showed no arsenic in the milk and no storage of arsenic in the animal
body on a cumulative basis.
In tests with fish, Gambusia, shiner, and largemouth black bass were
able to withstand concentrations of cacodylic acid of at least 100 ppm
for 72 hours, a rate equivalent to 270 Ib.of a chemical per acre-foot of
water. It may, thus, be considered nontoxic to fish and animals.
When BLUE or cacodylic acid is applied directly to soil, the chemical
is rapidly adsorbed and deactivated by the soil colloids so that new crops
can be planted during the same growing season.
Toxicity data have been collected for the components 2,4-D and picloram
and for the mixture WHITE. Picloram is extremely low in toxicity; the LD50
for acute oral toxicity in rats is 8,200 mg/kg; for sheep and cattle it is
488 to 650 mg/kg.
For Tordon 101, or WHITE, the acute oral LD5Q for rats has been reported
as 3,080 mg/kg; for sheep 2,000; and for cattle 3,163.

�In a feeding test on cattle, 97.7% of the administered picloram was
recovered unchanged in the urine; no picloram was detected in milk.
Median tolerance limits of fish to Tordon 101 ranged from 64 ppm for
fathead minnow to 240 ppm for brook trout.
Toxicological studies conducted by Cdgewood Arsenal and Dow Chemical
Company indicate that a single direct exposure to a spray of WHITE, at
prescribed rates, would not constitute \a. hazard to the skin or a
systemic hazard by inhalation.
The chemical, picloram, is considered nontoxic and not hazardous to
humans, animals, and fish.

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&lt;p&gt;For more about this collection, &lt;a href="/exhibits/speccoll/exhibits/show/alvin-l--young-collection-on-a"&gt;view the Agent Orange Exhibit.&lt;/a&gt;&lt;/p&gt;</text>
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