1 15 20 https://www.nal.usda.gov/exhibits/speccoll/files/original/0bd7eefae477111446ce3989d7aafa1f.pdf 0f2840d2e9d4ceed9d9dcf177f8dfb71 PDF Text Text Item ID Number: Author 00092 Blackman, Geoffrey E. Corporate Author National Academy of Sciences. National Research Council Report/ArtlClO TltlB The Effects of Herbicides in South Vietnam: Part B, Working Papers, February 1974: Persistence and Disappearance of Herbicides in Tropical Soils Journal/Book Title Year 1974 Month/Day February Color Numbor of Images 60 DeSCPlptOn NOteS P 4 missing Friday. December 08, 2000 Page 92 of 106 �. > £s-T~ '—\4' (O \ M 5(7/1 The Effects of Herbicides in South Vietnam PART B: WORKING PAPERS FEBRUARY 1974 Persistence and Disappearance of Herbicides in Tropical Soils GEOFFREY E. BLACKMAN, JOHN D. FRYER. ANTON LANG, and MICHAEL NEWTON NATIONAL ACADEMY OF SCIENCES �THE EFFECTS OF HERBICIDES IN SOUTH VIETNAM * "PART B: WORKING PAPERS FEBRUARY Persistence and Disappearance of Herbicides in Tropical Soils GEOFFREY E. BLACKMAN, JOHN D. FRYER, ANTON LAJJG, AND MICHAEL NEWTON NATIONAL ACADEMY OF SCIENCES - NATIONAL RESEARCH COUNCIL WASHINGTON, D.C. 20Ul8 �Persistence and Disappearance of Herbicides in Tropical Soils GEOFFREY E. BLACKMAN, JOHN D. FRYER, ANTON LANG, AND MICHAEL NEWTONa SCOPE OF WORK A very obvious and legitimate question poses itself when an extraneous material has been introduced into the environment: how long will this material persist, or how fast will'it disappear? Disappearance in this case means loss of its characteristic activity either in its original form or in the form of derivative(s). For the military herbicide operations in South Vietnam (SVN) this question is particularly obvious and urgent. Herbicides have been used in those operations at levels roughly five to ten times higher than in normal agricultural practice, and some areas were sprayed twice and more often, sometimes within a relatively short time. Where effects of defoliation persist, as in the mangrove forests, is it because the herbicides are still active, or because of other changes induced by the herbicides, even though the latter themselves may have disappeared? In addition, claims have been made—in articles on the effects of the use of herbicides in the Vietnam war, in news reports, and * Professor Blackman, a member of the Committee on the Effects of Herbicides in Vietnam, is Professor Emeritus at the Department of Forestry, Oxford University, Oxford, England 0X1 3RB. Mr. Fryer, a member of the Committee on the Effects of Herbicides in Vietnam, is Director of the Weed Research Organization, Agricultural Research Council, Begbroke Hill, Sandy *~ Lane, Yarnton, Oxford, England 0X5 1PF. Dr. Lang, Chairman of the Committee on the Effects of Herbicides in Vietnam, is Director of the MSU/AEC Plant Research Laboratory, Michigan State University, East Lansing, Michigan U882U. Dr. Newton, a consultant to the Committee on the Effects of Herbicides in Vietnam, is Associate Professor of Forest Ecology, School of Forestry, Oregon State University, Corvallis, Oregon 97331- �elsewhere—that these compounds have "poisoned" the soil, rendering large areas incapable of supporting either indigenous or crop vegetation. Some of these reports convey the impression that vast parts of SVN are barren and will remain so for unknown but extemsive periods of time. The term "ecocide," i.e., destruction of the plant and animal community on a large scale and in an irreversible manner, has.been used. The Committee therefore considered soil studies on persistence i and disappearance of herbicides among its essential tasks. This task was approached in two ways. First, on our forays into different parts of the country we collected soil samples in areas that had been sprayed operationally with herbicides during the Vietnam war. As far as possible, emphasis was placed on sites that had received high total doses of herbicides, since it seemed that the prospects of finding measurable residues would be highest at such locations. However, this collecting activity was greatly limited by security problems. Thus, only one inland forest site could be sampled, and it had received only two sprays. The total number of samples from operationally-sprayed areas was U8. Most of the samples were subdivided into two or three parts and almost all of them were analyzed for two different herbicides, and some for three; hence, the total number of analyses was about 200. The Committee also collected some water samples from a river in the heavilysprayed Rung-Sat Special Zone mangrove area. Second, the Committee conducted a number of experiments in which the soil surface was sprayed with herbicides at a known rate equivalent to that used in a single operational spray mission. The �disappearance of the compound was then followed by sequential tests over a period of about 150 to 2f>0 days. El The reason for doing these experiments was that vhen the Committee started its work, one and a half years had elapsed since the termination of major herbicide operations. ,• It was thus too late to observe the early stages of herbicide behavior in the soil. However, a knowledge of these early stages is very important if valid conclusions are to be reached about the course of the disappearance of the herbicides. In addition, it appears that where crops had been destroyed by herbicides, farmers were sometimes hesitant to replant, being afraid that the new crops would also be killed or damaged. The Committee was on several occasions asked when replanting would be safe. About 1000 soil samples were analyzed for herbicide content during this phase of the Committee's work. In addition, a series of biological tests to evaluate the rate of disappearance of herbicides was undertaken. All herbicide experiments in SVW were conducted with the authorization of the GVN and, where appropriate, of tlu: proper local authorities. The Committee also conducted an experiment in which two strips of mangrove, each UOO ft (100 m) long by :2 ft (30 m) wide and totalling .0 less than 2 acres, were sprayed by helicopter with Agent Orange and Agent White, respectively. The objectives were to study the early responses of the plants, to determine differences in sensitivity, and to investigate the behavior of the herbicides in tl-.e soil under conditions comparable to those of wartime herbicide operations (or, more exactly, to follow up the consequences of the first herbicide application to vegetation). However, this experiment failed; the sprayed vegetation exhibited only minimal symptoms of damage and th .se had almost completely disappeared seven months later. The reasons are not entirely clear. The herbicide batches used were the same as those f.iiat proved very effective when sprayed by hand on soil. Most probably, :.hc equipment, which had not been in use for quit<_- a long time, was not functioning properly and did not deliver the specified amount of herbicide. No analyses for herbicides were done from these plots. �work. Biological determination was used primarily in the experiments with agricultural crops and soils because here the advantage of this method, as just mentioned, is particularly evident. If a plant does not show measurable effects six months after application of a herbicide to the soil, it can be concluded that this particular plant, if resown, will remain undamaged in future plantings, provided cultural and environmental conditions are not appreciably changed.- , The test plants that were chosen for the Committee's experiments were rice and other crop plants of considerable present or potential value in SVN and other tropical countries. Biological measurements of herbicide effects on mangrove seedlings were also carried out. Serial plantings were made in order to determine how long herbicides have direct adverse effects on mangrove seedlings, and also to obtain some information on conditions for reestablishment of the mangrove. The first chemical determinations were carried out by the Weed Research Organization, Yarnton, Oxford, England. Subsequently, the analytical work was conducted under a subcontract with the Huntingdon Research Centre, Huntingdon, U.K., an organization with considerable experience and an excellent reputation for this kind of work. The analyses were made mainly for 2,4,5-T, one of tha compounds in Agent Orange, and for picloram, one of the compounds in Agent White, because all experience indicates that these are more stable than 2,^-D (which is present in both Orange and White). 2,U-H was determined only in selected samples, usually those with a high content of either of the other two compounds. The analyses were carried out by means of electron capture gas chromatography. The methods used were the most up-to-date standard �residue procedures available and have beer proved in several different laboratories. For picloram a basic extraction followed by esterification with diazomethane (McKone and Cotterill, unpublished) was used; for 2,U,5-T the residues were extracted into an organic solvent and then the n-butyl ester ,« (McKone and Hance 1972) was the preferred derivative. Following routine procedure in herbicide work, all results are given as acid equivalents, disregarding the moiety of the molecule that forms the ester.' The results for soils are expressed as Ib/acre of soil surface, i.e.., as if all herbicide in a sample (or subsample) was deposited on the surface of the soil. Water samples are reported in parts per million (ppm). Values preceded by a < mean that no herbicide could be detected at this level of sensitivity. It will be seen from the results that the limit of detection varied to some extent from one batch of samples to another. This was because of slight changes in the sensitivity of the electron capture detector over a period of time, and in a few cases also because of varying amounts of material available, (if these amounts drop below a certain limit, the sensitivity of the analytical methods decreases.) The accuracy of the results was ensured, however, by the inclusion of some analytical standards with each set of samples. To check the reliability of the determinations, a random selection of samples with high, medium, and low herbicide levels was analyzed by the Gulf South Research Institute, New Iberia, Louisiana: the comparative results are shown in Table I. It can be seen that at low concentrations, near the detection limit, the agreement between the two determinations was generally good; at higher concentrations, the values found for the two forest soils by GSII were considerably lower than those of HRC. In our evaluations we used the HRC data, since their results for control tests (samples taken immediately after spraying) were— �1 I Table I. 1 Comparative determinations of 2,4,5-T and picloram in soil samples by Huntingdor Research Center and Gulf South Research Institute. •T , Data in ppm. < • below this detection limit. ster ND - not detected. 2,4,5-T Picloram e e (or ported HRC GSRI 1 2.99 3.293 0.44 0.260 1.28 1.114 0.342 0.102 0.59 ' 0.510 0.047 0.032 0.26 0.096 0.04 0.012 0.06 0.049 0.004 0.003 0.03 .0.016 <0.004 ND 1 0.03 0.005 <0.002 ND 2 ' Mangrove (Vung-Tau.SVN) GSRI 3 4 5 6 gardlng ' •' HRC 2 Soil . Sample No. 0.013* 0.005 <0.002 ND 0.005 <0.001 ND 0.005 <0.001 ND •.e Mangrove (Rung Sat) results !S to 3 <0.02 a 4 r rtain 1 2 3.16 0.412 0.402 0.115 0.58 0.245 0.060 0.041 0.02 0.016 0.003 ND 1 . 8.09 0.332 1.265 0.132 1.13 0.079 0.03 0.003 a Forest (Bnn-Me-Thuot,SVU) <().005 3 sctron 0.02 0.011 0.005 T 1 i Forest (Los Banos, Philippines) t of ulf are Section it higher insidersince 5) were — •Analyzed by Weed Research Organization Note: The samples for 2,4,5-T and for picloram are not always identical ND �with a single exception—very close to the theoretical values. (The GSR! analyses did not include such control tests.) Soil Sampling Soil sampling was done in two ways. Surface samples were collected mostly with the aid of metal (iron) cans, 5 in. (12.5 cm) high and of the same diameter, which were pushed into the soi;: and then extracted with the aid of a spade. This procedure was mainly used with mangrove soils, since they are soft enough to permit complete insertion of the can. Deeper samples, which will be called cores, were collected with soil samplers specially constructed by the Weed Research Organization. Two different but similar samplers were used, both consisting of a metal tube with sharpened bottom edges, either 2 in. (5 cm) or 1.75 in. (k.k cm) wide and either 36 in. (90 cm) or 30 in. (75 cm) long. Both samplers were provided with strong, interchangeable metal caps: one plastic-covered to minimize damage when driving the sampler into the soil, the other provided with handles to extract it again. There were also removable split inside liners for extracting the cores, but these proved to be of limited value and the cores were usually pushed out of the sampler tube with a metal rod with a disk at one end. When muddy soil (mangrove) was collected, an adjustable air vent at the upper end of the sampler tube was left open while inserting the sampler into the soil, to permit escape of air. The vent was closed, or the upper opening of the tube simply plugged with a rubber mallet, when the sampler was extracted, thus generating a vacuum that helped keep the soil in the sampler tube. Sometimes, a metal tube with a metal rod inside was pushed into the mud next to the sampler and 8 �the rod then withdrawn; this permitted air to penetrate to the end of the sampler, relieving a vacuum and facilitating the extraction of the sampler. In many cases, both with hard forest soils and with soft man- grove mud, the sampling proved an arduous and time-consuming task, and . * despite all precautions the contained cores were often considerably shorter than the length of the sampler because part of the core slipped out of the sampler and/or because the friction between the soil and the wall of the sampler tube either caused the core to compact or prevented the soil from entering the tube beyond a certain point. Wherever soil samples were collected, some control samples were taken from areas that had not been exposed to herbicides. These controls served to ascertain whether the soil contained natural materials interfering with the determination of the herbicides. If this was so, the analytical procedures were modified so as to exclude such sources of error. Criteria used for choosing the control sites were: information on herbicide missions in the area (printouts from the HERBS tape and, where available, information from local, military personnel); condition of the vegetation; and information from the local population (village and district officials, and farmers or woodcutters). In the mangrove, where sprayed areas exhibit pronounced damage and often complete kill of the mangrove trees, identification of control sites was not difficult. Good local information faciliated location of the forest sites at Pran Burl, Thailand, and Cau Muoi-Mot, SVN, as well. In other locations, the identification was a good deal less reliable, and it appearr, that some of the control sites had been exposed to herbicide, too. �The surface samples were stored in the cans, which were closed •with their lids. The cores were in most cases divided into two or three parts and placed either into double polyethylene bags or, more commonly, into plastic jars with airtight screw caps. In the case of mangrove soils,.j/hich are highly anaerobic (i.e., lacking in air and particularly in oxygen), care was taken to fill cans and jars to the rim and to tape the lids of the metal cans with masking tape, thus reducing exposure to air, which might cause changes in the soil and thus also affect herbicides still present in it. The samples from SVN and the Philippines were stored in a deep freezer and shipped for analysis in frozen condition. All effort was made to complete these safety procedures as rapidly as possible, and in most cases the samples were placed in the deep freezer on the evening of the day of sampling or on the nextday. However, particularly in the earlier part of the Committee's work, this was not always possible and samples occasionally had to remain unfrozen for as long as one week. In some cases, it was possible to keep them part of this time in a refrigerator, Water samples were kept in plastic containers in the dark to prevent breakdown of herbicides by light. Some formalin was also added to prevent decompostion by microorgejiisms. HERBICIDE DETERMINATIONS IN SOILS AND WATER FROM DEFOLIATED SITES Sites The sites that have been subjected to herbicide sprays in connection with the Vietnam war and from which the Committee took soil samples are listed in Table II. Two were in an area in Thailand that had been 10 �Table II. Location, herbicide spraying history, and soil sampling information for land sites that had received herbicides during military operation. Location - (SVN, unless otherwise noted) Date Forest, Pran Buri, Thailand: June 1965 herbicide spray test site. Plot 28 Spray history Agent Orange + picloram Amount Date No. & type of samples 9.1 Ib/acre 0.5 " Sep 30,1971 8 cores (2 ea. from 4 sites) ca. 16-26 in., divided in upper organic part (ca. 6 in.) and remainder Oct 1,1971 9B6 Ib/acre Purple 497 '" Pink 310 n Orange 128 " Dicamba Cacodylic acid (aBlus) 57 " Picloram 20 " = total of ca. 840 Ib 2,4-D; ca. 960 Ib 2,4,5-T; 128 Lb dicamba; 57 Ib cacodylic acid, 20 Ib picloram per acre Calibration Grid, Pran Buri. Thailand 1964-65 Crop land: dump site near Chieu-Lieu Hamlet TanDong -Hiep Village. Di-An Distr., Bien-Hoa Prov. Dec 1, 1968 1000 gal. Orange dumped on circular path from 1800 ft. forest: Cau Muoi-Mot strongpoint, ca.8 mi. (12 tan! north Dong-Xoai Village, Don-Luan Distr., Phuoc— Long Prov. Dec 1, 1968 White Apr 12,1969 Orange Oct 8, 1971 6 cores, ca. 26-32 in... 3 ea. from a bare (Nos. 1-3) and from a grass-covered (Nos. 4-6) area and 2 from an area supposedly outside the grid (Nn. 7.8): all divided into 3 equal sections (T-*top, M=middle. B^botton) 4 cores., ca. 28-35 in. divided into 3 approx. equal sections (T,M,B) 3 gal/acre Oct 16, 1971 4 cores, ca. 33-34 in., 3 " divided into 3 equal sections (T,M,B) �Table II, continued Location - SVN (Unless otherwise noted) Spray history Agent Date a ' Amount Date Mangrove, Rung Sat Special Zone Site tl No. s type of samples r 1965 1966 1967 1968 Aug 4, 1968 Aug 7 1968 ftug 3, 1968 ftug 9,1968 Aug 18, 1968 Mar 1, Jan 6, Nov 11, Aug 3, Orange Orange Orange White Orange Blue Orange White 3 gal/acre n 3 n 3 n 3 ii 3 n 3 ii 3 ii 3 H 3 Oct 9, 1971 3 surface samples. depth not determined, = RS-1 through -3 Mar 9, 1972 2 cores, 36 in., divided into 3 equal parts (T,M,B) = RS-4 and -5 Aug 31,1972 6 surface samples (5 in,) and 6 cores (ca. 30 in.) from same sampling sites divided into 2 equal sections (T,B) = RS-6 through -11 Site tS Aug 31,1972 6 samples same as Site #1 31 Aug 72 -- RS-12 through -17 S,T,B Site #6 Site §7 Site 18 Aug 31,1972 Orange a minimum of 86 Ib 2,4-D; 79 Ib 2,4,5-T; • 3 Ib piclcrara; 9 Ib cacodylic acid per acres compare text. One sample each, same as Site 11, Mar 9, 1972 (T,M,B) -. RS-18 through -20 �Table II, continued. Location - SVR (Unless otherwise noted) Date Mangrove, Ca-Mau Peninsula: Ca. 9-45 yd both sides of airstrip at Nam-Can village, Kan-Can Distr., AnXuyen Prov.' Ca. 3 mi. north-northeast of Nam-Can Naval Base, west bank of Kinh-Ngang canal Spray history Agent July 1968 Orange Sep 20-21, 1962 Purple Mar 11, 1970 White Apr 8, 1970 Orange Apr 21,1970 White Site appears to be on boundary of White and Orange sprays Amount Date No. & Type of Samples Oct 1 , 1971 3 samples each consisting of several cores, ca. 36 in., which had been divided into 3 parts;" 6 in.-6 in.remainder (T-M-B)j plus one sample 12 in. only: T and M 3 gal/acre 3 3 3 Oct 12, 1971 3 samples as in Ca-Mau Peninsula, above The figures are the depth of the hole. The length of the core was often considerably less, due to compacting and/or friction preventing the soil from entering the sampler. This was particularly marked with many mangrove samples. Agent Pink c - 60% 2,4,5=T-n=butyl ester 40% 2,4,5-T-isobutyl ester Dicamba =3,6 dichloro-o-anisic acid (controls certain phenoxy tolerant broadleaf weeds and brush species) �used for extensive trials with different herbicides in 1964-65, prior to large-scale use in SVN. No herbicide symptoms were seen on native plants, crops, or weeds. Samples were collected from one of the few trial plots that could still be definitely located (Plot No. 28), and from the center of the so-called Calibration Grid. The latter is an old rifle range at the Pran Buri Training Center, and all treatments that were used in the individual trials had been sprayed over this site. The direction of the sorties was at varying angles according to wind, but all intersected in a central area that, at the time of the Committee's visit, still stood out clearly because of several large patches of completely bare soil or very restricted vegetation, mostly grasses (imperata and others). This site had received truly formidable quantities of some herbicides, e.g., 70 times more 2,4,5-T and almost as much more 2,U-D as delivered on one regular herbicide mission with Agent Orange (or lUo times as much 2,4-D as on one Agent White mission). In SVN, the Committee was able to take soil samples in a so-called dump area, i.e., an area where, because of engine or other trouble, a plane delivering herbicides had released (dumped) the entire load over a relatively small area. The sampling site, which was in an area presently under crops (at sampling time, mainly peanuts), was originally located from the report of the pilot of the dumping plane and was subsequently verified by interviews with district and village officials and with villagers. It was definitely within the perimenter of the dump, although probably nearer its edge than its center. At the time of the Committee's visits, many dead trees—mostly fruit trees—could be seen, either still standing or, more frequently, felled and cut for firewood. �Some fruit trets had dead branches that may have been the result of herbicide damage, although the trees had produced new growth and were bearing some fruit. Some tall, unidentified tress looked, quite normal, however, suggesting that the dose of herbicide had not been excessively high as compared to regular herbicide missions. (The dump was made from a height of 1800 ft or 5^0 m, as compared to the '150 ft or 6(3 m of the regular defoliation mission.) , The one inland forest site that could be sampled in SVN had been subjected to one Orange and one White mission. The area had been heavily disturbed by long-term human activity; only a thin stand of large trees and much old bamboo were present, and the trees were either dead or exhibited clear herbicide damage.. In contrast, a relatively substantial number of samples were collected on several trips to the central part of the Rung-Sat Special Zone, in a mangrove area that has received more herbicide sprays than any other region in SVW. One site, our #1, had been within the recorded flight lines of nine missions and received six times as much 2,U,5-T and seven times as much 2,H-D as a single Agent Orange mission, or about 1^4times the amount of 2,U-D as a single Agent White mission. Moreover, these are minimum values; no less than 24 other missions had passed near the site between January 1966 and September 1966, and the site had almost certainly been reached by additional herbicide:--if not directly, then by drift. The level of herbicide exposure of the other, sampling sites in the Rung-Sat mangrove was undoubtedly similar. In the mangrove of the Ja-Mau Peninsula, the southernmost tip of the country, samples could be taken from two sites. One of these, situated close to the Nam-Can 15 �Naval Base, bad been hand-sprayed with Agent Orange. Datc(s) and amounts are not known, but since some of the mangrove plants showed regeneration, the level was probably not higher and perhaps somewhat lower than from an aircraft spray. The second site, on the Kinh-Ngang Canal about 3 miles (k.8 km) northeast from the first, had received a spray with Agent Purple, a precursor of Orange, in 1962, and two White and one Orange sprays in March-April 1970. The sampled area was entirely devoid of live mangrove trees but was covered in parts by a creeping grass (Paspalum sp.). As far as possible, sampling was done according to some pattern aimed at a uniform distribution over the accessible area that also included different parts of it. For example, in some of the mangrove sites completely bare areas were examined along with areas covered with grass (mai Faspalum vaginatum) and bearing some mangrove seedlings. Sometimes, however, e.g., in the Cau Muoi-Mot forest, the accessible area was so small that sampling had to be done at random. Water samples were taken on two occasions in the lower part of the main shipping channel to Saigon that runs through the Rung-Sat Special Zone, beginning at our land site RS #1 and going south on the Dang-Xay, Mu-Na , and Dong-Tranh Rivers.. Some control samples were collected in a small channel near our experimental sites in the mangrove of Chi-Linh near Vung-Tau (see below). The sites of the second Rung-Sat samplings, made on August 31, 1972, are shown in Figure IV C-5, Section IV, Mangrove Forests, Part A of the Report on the Effects of Herbicides in South Vietnam. No precise record was kept of the first sampling occasion in the Rung-Sat, but the area was similar to that of the second occasion. All samples were 16 �taken at outgoing tide and near the water surface. Results; Soil Samples Results of the soil analyses are summarized in Table III. Substantial levels of picloram and 2,4,5-T, sufficient to prevent growth or . 4 to cause serious malformations in many broadleaf plants, were present six years after application in some of the soil samples from the Calibration t Grid in Pran Burl, Thailand. Picloram was found throughout the length of the cores, but except in one (out of six) the concentrations in the bottom third (20 to 30 in. or 50 to 75 cm) were low. • 2,^,5-T was found in the top and middle thirds (top, 0 to 10 in. or 0 to 25 cm; middle, 10 to 20 in. or 25 to 50 cm) in one sample, and in the top third in three samples; none was found in the bottom thirds. Residues of both 2,U,5-T and piclorara were also found in some soil samples from the Rung-Sat mangrove. Sample Site #5 (samples RS #12-1?) seemed to contain 2,U,5-T consistently, particularly in the subsurface (below about 15 in. l3& cm] and to about 30 In. 175 cml). No detectable amounts of picloram were found at this site to the depth that could be reached with the soil samplers. Some samples at Site #1 (samples RS #1-11) contained 2,4,5-T or picloram, and picloram but no detectable 2,U,5-T was present in the single samples from .Sites #6, #7, and #Q (samples RS #18-20). In Sites #1 and #2, presence or absence of vegetation--including some mangrove seedlings—at the sampling sites seemingly was not linked with the variation of herbicide content .in the so:ll. All herbicide levels found in the Rung-Sat mangrove soil are below those that can be expected 17 �Table III. Herbicide residues in th^ soil samples of Table II, in Ib/acre. not sampled or analyze 1. = below detection lirut. S/T = surface samples or top portions of cores, B = bolton portions of cores. ?,4-D Site S/T Forest, Eran 'Purl, Plot 28 1-8 Calibration Orid, Pran liuri 1 2 3 5 6 7 8 Bump Site, Di-An Distr. 1-4 Bulked S/T' ' D B - Sample No. - f. 0.07 --0.03 .<;0.06 <ro.o6 -.'0.07 ^ 0.08 V, ^ 0.04b - £0.005a -^0.005a -; 0.06 -0.03 1.35 C.96 0.03 0.23 0.06 ''0.03 <0.o6 0.09 <0.04 C •'0.005 ^0.005 - <0.005d - " A A a ^0.006 o.oi 0.004 _ - <o.ooi <: o.ooi < 0.001^ < 0.001^ £ 0 . 001 <0.008 v. 0.035 <" 0.036 0.079 v.0.031 0.032 0.032 0.002 <0.006 v' 0.004 < 0.004 *0.004 < 0.004 •* 0.004 < 0.004 <: o.oo4 < o.oo4 C 0.004 < 0.004 <o.oo6 *' 0.004 ^0.004 * 0.00k ^ 0.004 <0.004 < 0.004 0.003 < 0.004 < 0.004 < 0.004 < 0.004 < 0.004 c 0.004 0.011 --0.007 0.007 0.003 0.179 0.023 0.057 0.109 0.1146 0.037 <.o.oo4 < o.oo4 0.006 f* . A < 0.005 ' <o.oo5 -•0.03-. OU0.03 <0.02fl 0.003 <ro.oo3 0.008 1.19 0.24 :0.03 . <:o.ooia <o.ooia 1.09 -.'0.03 -- 0.06 < 0.06 -'.0.06 Picloram S/T B 1.03 0.43 0.72 0.60 ^0.03 <' 0.06 <LC,03 Forest, Cau-Muoi-Mot 1-4 _ < 0.006 _ 0.010^ Mangrove, Site #1 RS-1 Rung-Sat RS-2 0.013° - <0.005d _ RS-3 «. 0.007 RS-4 - < 0.006 £0.007 RS-5 *0.04 <0.04 <:0.04 _ RS-6 0.09 RS-7 '.0.04 -10.04 <0.02 RS-8 0.19 RS-9 £ 0.0k 0.21 RS-10 •:0.04 - <0.03 RS-11 >• 0.04 0.015 Site- #2 RS-12 ^0.04 - <: o.oi RS-13 j' Q 0^.- 0.04 RS-14 0.24 _ 0.02 RS-15 j' O Qij <o.oi RS-16 ^ 0.04 - 0.015 RS-17 Site #6 RS-18 - 0.007 <. 0.007 ^0.007 Site #7 RS-19 - 0.007 ••^0.007 <0.004 - < 0.006 Site #8 RS-?0 / 0.007 Mangrove, Nam-Can Airstrip 1-3 Canal KinhHeane 1-3 | A CO. 02 - <o.oor <ro.ooiQ rt A ^Values in ppm since surface area of sample not known and therefore computation of Ib/acre rate not possible e 4 samples Only part of samples samples, bulied 4 samples, bulJced b 3 C 16 �to cause damage to crops in normal agricultural practice. No herbicide residues could be detected in the samples from Pran Buri Site #28-, the dump site in Di-An District, the forest near Cau Muoi Mot, and the two mangrove sites in the Ca-Mau Peninsula. The failure to find detectable herbicide in the second Ca-Mau site (at the KLnhNgang Canal) is interesting insofar as this site had been sprayed relatively late in the war, one and a half years before our sampling. Results; Water Samples Water samples from the lower part of the main shipping channel to Saigon were analyzed for picloram, the most persistent of the herbicides used for military purposes in SVN. Suspended sediment—mostly soil—was separated from the water by filtration, and the two fractions were analyzed separately. As Table IV shows, no herbicide was found in the filtered water, but the sediment of four out of eight samples contained amounts ranging from about 0.07 to 0.03 parts per billion (ppb) of water and from about 2.2 to 0.8 ppm of dry weight of sediment. If all the herbicide in the sediment were to become available in the water, the levels would, be far below the concentrations known to affect even the most sensitive species, but if only the sediments are considered, the levels are somewhat higher than those found in the Rung-Sat soil (maximum 0.01 Ib/acre = 0.05 ppm). Herbicide in. water is usually associated with suspended material if such is present, and turbid water may contain more herbicide than clear wa+.er, but the relatively high pic3oran content in the Rung-Sat sediment is somewhat unexpected. �Table IV. Analyses for plcloram in water samples from the Rung Sat. Values In ppra. ND = not determined Location and water sampling aite no. Concentration in filtered water Concentration in sediment Computed for water Computed for sediment (dry weight Vung-Taui No. 2 ND <0. 00002 <0.11 4 5 6 7 6 9 10 ND <0. 00003 <0. 00002 <0.00002 0.000043 0.000036 0.000066 0.000029 <0.17 <0.24 <0.50 1.26 1.06 2.24 0.77 Rung Sat, No. No. No. No. No. No. No. ND ND <0,0001 <0.0001 <0.0001 <0.0001 20 �EARLY STAGES OF HERBICIDE BEHAVIOR IN SOIL 'UNDER TROPICAL CONDITIONS This section summarizes the Committee's own experimentation on persistence and disappearance of herbicides fn certain tropical soils. Agricultural Sites -* In all experiments with agricultural lands, the soil, previously cleared of any vegetation, was sprayed with either Agent Orange or Agent White at the rates of ( ) three gal/acre (the rate used on most military l spray missions, (2) one gal/acre, and (3) one-third gal/acre. The agents, as manufactured, are much too concentrated to permit accurate dosage for the relatively very small plots treated in all our experiments, and therefore they were diluted. In the case of Agent Orange, which is insoluble in water, an aqueous 'emulsion was made with the aid of special emulsifiers. Agent White is water soluble, and the necessary additional amounts of water were added directly to the conmercial preparation. The application was by means of a carefully calibrated backpack sprayer and a spray boom designed to ensure a uniform pattern of distribution (constructed by the Weed Research Organization). All spraying was carried out by one and the same person (except in the mangrove microplot experiment). Comparable plots were left unsprayed as controls. None of the plots had been treated with herbicides before our experiments. Beginning some weeks after the herbicide application and at regular intervals thereafter, selected crops were sown or planted and their responses observed about one month later (in the case of paddy rice, additional data on total yield were taken after the plants had matured). were controlled during and between plantings by hand, and in the 2J �Philippines experiments also by application of a different herbicide: paraquat. Fertilizer was applied according to locally-established needs; during plantings, light surface cultivation was applied to the soil. The main characteristics recorded were number of surviving plants; % height and wet weight either of all plants on a plot or of a number of plants selected at randan; number of leaves and, for paddy rice, of tillers (side shoots emerging near ,the ground); plant color; and symptoms of herbicidal injury. Estimates of general vigor (on a scale of 0 to 10) and of ground coverage (i.e., the portion of a plot covered by the foliage of the crop, in percent) were also made for some plantings. In general, the most useful measurements--since they provided the clearest indication of herbicide effects or their disappearance—were survival., plant weight, and degree of herbicidal damage). In the case of paddy rice, the plants were grown to maturity and the yields of air-dried grain determined. The results are expressed as the time, in weeks, from the_ date of the herbicide application tp_ the date of that planting in which no effects on the growth of the plants, as determined by survival and weight, were noted, and if these times are different, also the time from herbicide application tp_ the date of that planting in_ which any herbicide symptoms had disappeared. This procedure is illustrated by Table V., which contains data for two. species that are relatively susceptible or resistant to the herbicidal agents, and by Figure 1, which is derived from these data. Two experiments were carried out, one in the Philippines and one in SVN. The Philippines experiment was conducted on the experimental farm of Tropical Agri-Search, Makati, Rizal Province, Philippines at 22 �Table V. Survival, growth and herbicide symptoms of naice and peanut* grown in soil trcatad with 3—1—1/3 gal/aera of Agents Orange and White at different tioee after application. (Experiment at Alabang, Third Series.) Survival and growth (fresh weight after 4 weeks) are expressed as percent of controls (plants grown on non-treated plots), symptoms on a scale from ++++ (very heavy) to 0 (absent), Interval between spraying the soil and planting Agent Orange Agent White Herbicide Symptom* Surviving Plants Plant Weight 1/3 1 3 (Gallons per acre) 1/3 1 3 (Gallons per acre) Surviving Plants 1/3 1 3 1/3 1 3 (Gallons per acre) (Gallons ptr acre) Plant Weight Herbicide SyeptoM 1/3 1 3 (Gallons per acre) (Gallons per acre) 1/3 1 3 ro Mais* 4 weeks 96 84 89 96 126 138 0 0 0 109 129 64 60 83 15 weeks 102 112 115 123 148 143 0 0 0 67 01 89 69 « 83 30* 36* 51* 0 0 0 96 0 0 0 + + + + • » Peanut 4 weeks 102 100 15 weeks 148 102 40* 128 89 101 99 + ++ *+* 124 115 128 0 0 0 22 weeks • Difference froa control statistically significant. 0* 58* 53* + + + 108 133 133 95 117 112 0 + + 78 112 77 99 87 104 0 0 0 �CROP AGENT AND DOSE (gal/acre) OR 1/3 1 3 WH ill 1/3 1 3 N "^ OR ro z 11 1 n. WH 1/3 1 3 1/3 1 3 8 8 1Q 12 14 15 18 20 22 TIME AFTER HERBICIDE APPLICATION TO SOIL (weeks) FIG. 1. Persistence of herbicide effects in naize and peanut (graphic representation of the results in Table V). The solid bars show the time between herbicide application to the soil and that planting in which effects of the herbicides on survival and Growth (as fresh veight) were no longer present. The broken bars show that tine after which no herbicide synptoiT.s were evident. lack of a broken bar means that herbicide symptoms were not present at all, or that they disappeared at the sane time (i.e., in the same planting) as effects on survival and growth. Thus, the effects of Agent Orange (OR) on maize were no longer present in the planting made k weeks after herbicide application; those of Agent White (WH) at 1/3 and 1 gal/acre were not present in the same (It-week) planting and those at 3 gal/acre in that planting after 15 weeks. (Maize, as well as other cereals and grasses, exhibits few if any of the herbicide symptoms found in broadleaf plants, such as twisting of stems'and leaves, reduction of the leaf blade, curling of leaf margins.) Effects of Agent Orange on survival and/or growth of peanuts on soil treated with 1/3 and 1 gal/acre were no longer present in the U-week planting, but herbicide syuptoms remained present until the 15-week planting, while on soil treated with 3 gal/acre, effects on survival (and herbicide symptoms) were present until the 15-week planting, but both had disappeared In the 22-week planting. In peanuts grown on Agent White-treated soil, effects on survival and growth were no longer present in the 15-week planting; and herbicide (plcloram) symptoms on soil treated with 1 or 3 gal/acre disappeared In toe 22-week planting. �Alabang, near Manila, under a subcontract with the International Rice ""search Institute, Los Banos, the Philippines. The experiment i3 "* jf -onslsted of two parts; one for paddy rice and one for "dry crops" t'.-.aize, sorghum, sweet potato, mung bean, peanut). The rice was planted in 82 by 66 ft (25 by 20 m) paddies, three : nldies were used for*each of the two agents (Orange and White), and each 1 i B*c *» w a* !^**8W - : o.ioy was subdivided by bunds into four sections for the three dose :ovols (3, 1, and 1/3 gal/acre) and an untreated control. Thus, each I *> 0 Jj JC i -rt £ P.** ''TI S.fc5 l |fa'* I gd-3 § 8 i! ^ *g 3 5% Tcntnent was replicated three times. Flooding was arranged in such a ..av that water could not move from one plot to another. The dry crops at Alabang were arranged in three different series. :.:i one series, the herbicide application was made on February 2, 1972, l-iring the dry season, and the first planting occurred on February 25, i'72. Plantings were repeated at six-week intervals; all plantings were Irrigated by soaker hoses and hand watering as long as the dry season t> *T8 Vested. In the second series, the herbicide was applied at the same • :.no as in the first (February 2, 1972), but the plot was allowed to !3 «d r---nain fallow until the onset of the wet season. Finally, in the • ::ird series, herbicide application was delayed until the wet season ii:: i was carried out on May 3, 1972. The first plantings in the second 'i:.l third series were then made on May 30, 1972, simultaneously with the •;ilrl planting of the first series (which had to be delayed one week o o TI a -J •••••ause of heavy rains). The objective of this threefold setup was to »': *ain some information on the persistence of the herbicides in the alst;nce of rain. One planting (July 16-17, 1972) was partly lost in a h'.-avy typhoon on July 19-20 and was therefore repeated on August 12-lU, 25 �resulting in a disruption of the schedule. The heavy rains caused losses of topsoil and may have thus caused some loss of herbicide as veil. However, a careful inspection of the pattern of herbicide symptoms in the most sensitive crop of this experiment—peanuts—gave no indication of cross contamination of the different agents and dosages. The number of replicates in each of the three series was three for the controls (no herbicide) and two for each of the three herbicide levels. Reduction to two replicates was necessary because of the restricted area available. The experiment in SVN was conducted at the Ea-Kmat Agricultural Research Station at Ban-Me-Thuot, Earlac Province, with the authorization of the GVN and the Director of the Agricultural Research Institute of the Ministry of Agriculture of the RVN, Dr. Thai-Cong-Tung. It was most capably supervised by the Manager of the Station, Ing. Nguyen-Van-Thoi, and his staff, in particular Ing. Truong-Duc-Bao. This experiment was restricted to crops capable of growth when receiving only natural rainfalJ The individual herbicidal treatments and the controls consisted of three replicates. The crops were dry (upland) rice, maize, sorghum, sweet potato, nrung bean, and peanut. Because of poor germination and growth of sorghum during the first two plantings, and because herbicide effects had mostly disappeared by that time, sorghum was replaced by soybean afte. these plantings. Rice, mung bean, and soybean suffered heavily from insect attack and the data obtained were less complete than for the other crops; nevertheless, comparisons between plants on herbicide treated and untreated plots were possible in most cases. The herbicide applications were made on March 2U, 1972, at the normal starting time of the rainy season. The first planting was on April 22, and subsequent 26 �plantings were made at Intervals of six to seven weeks, with the last one (for Agent White-treated plots only) on October 25, 1972. As the rainy season In 1972 began unusually late and was relatively short, and as no Irrigation system was available, the finst and last plantings • * suffered from drought. The growth of the firs- two plantings was impaired by a heavy growth of weeds. The soil at Ban-Me-Thuot is representative of the "red soils" of SVN. The Committee would have liked to conduct similar work for alluvial soils, since they represent the soils of the main agricultural region of the country, the Mekong Delta. However, with the time and manpower available this proved beyond our capacities. It would also have been desirable to repeat all experiments for a second year, in order to gain some insight about annual variations, but this was not possible either, because of the time limitation under which we were working. It should be realized, however, that little critical research involving quantitative studies of the persistence of herbicides in the tropics has been done. Thus, the information pertinent to the situation in SVN collected by the Committee represents, despite its limitations, a significant contribution of new knowledge to this general problem. The results of the Philippines experiment are illustrated in Figure 2, those of the Ban-Me-Thuot experiment in Figure 3. For the Philippines experiment, only the data for the third series (herbicide Application and first planting at the start of the rainy season) are shown. In the first series (herbicide application and first plantings In the dry season, with artificial irrigation) the time course of herbicide disappearance was quite similar to the pattern of the third, 27 �n ii n CD <C UJ I $ r>_n - C—« i !?—n Q.-M .££ 3DNVUO C-o JJ—n C.-o £2-n C.-PJ C — PI J Si—pi £2~-r I I 1 31IHM FIG. Z. The COUTM of disappearance of the effect* el herbicide* on selected crop* grown on •oil treated with 3( 1, and 1/3 gal/acre of Agenta Orange or White. Experiment at Alabang, Fhlllpplnea. Bar* • time In weeks from herbicide application to 'the aoll to that aequentlal aowlng or planting where effect* of herbicide* on eurvlvul and gro.'th or yield (eolid bare) and herbicide uynptoma (broken bars) vure no longer obiervabl*. (Lack of a broken bar • herbicide symptoms either not evident, or disappearing «t aanc time aa herbicide effect* on •urvlval, etc.) For further explanation, see text, Table V, and Figure 1. Faddy rice (variety HI-ZO, one of the "miracle varieties" produced by the International Rice Research Institute) wa* planted aa 3-week old nursery seedlings; sweet potato (local variety) a* cutting*: maize (meet corn, varletlei IV 601 and UFCA Synthetic #1 and #2), oorghum (variety Coaor jfe), nung bean (variety CES flk) and peanut (unknown varletle*) •• aeed*. Where vurietie* are known, the material was supplied by IRRI. Criteria for yield, survival aiid groulh: rice - grain yield; meet potato - *urvlval and length of longeat (hoot) other crop* -•survival and plant uclgkt (fresh) about k wetiki; after planting. �(JO*. I Itf . I/J 1/3 I 1/3 Sorghum The firat planting of sweet potato failed because of lack of rain, so no information is available until later sequential plantings. The second sowing of aung beans on the Orange plot, including the control (nontreated soil), was lost because of very poor germination. The effects of this herbicide at the 1 and 3 gal/acre levels may therefore have disappeared one sowing earlier than shown, i.e., 10 weeks after soil treatnent. c Sorghum was sown only twice, k and 10 weeks after herbicide application. In the White 3 gal/acre plots, socte effects on growth Bay still have been present in the second sowir.i;. It is probable that they would have disappeared by the next planting late, 17.S weeks after treatment. Soybeans were included or.ly beginning with the third sowing, 17.5 weeks after soil treatment. At this tine, Orange at all three levels and White at 1/3 gal/acre no longer produced any observable effects or symptoms on the plants. 'Peanuts still showed some plcloram symptoms in the fifth sowing; see text. 1 3 SMI POIMD 1/3 1 3 (a) 1/3 Mung torn Pcmt 1/3 1 3 1/3 1 3 Sorghum Potato 1/3 1 3 to) 1/3 1 3 '.1^3 <•> 1/3 Soybean Punut 1 3 1/3 1 3 10 12 14 16 IB 20 22 24 26 28 30 32 34 TIME AFTER HERBICIDE APPLICATION TO SOIL (weeks) FIO. 3. The course of disappearance of herbicide effects on selected crops grown on soil treated with 3, 1, and 1/3 gal/acre of Agents Orange or White. Experiment at Ban-Me-Thuot, SVN. Bars • time in weeks from herbicide application to the soil to that sequential sowing or planting where effects of herbicides on survival and growth or yield (solid bars) and herbicide symptoms (broken bars) were no longer observable. (Lack of a broken bar • herbicide symptoms either not evident, or disappearing at sane time as herbicide effects on survival, etc.) For further explanation, see text, Table V, and Figure 1. Sweet potato was planted as cuttings, the other crops as seeds. All crops were local varieties of unknown origin. Main criteria used to assess herbicide effects: sweet potato - number of surviving plants and length of longest shooti other crops - number of survivors and weight of plants (fresh), in all cases U-5 weeks after planting. �which means, of course, that the herbicide effects disappeared at correspondingly earlier calendar dates. In the second series (herbicide application in the dry season, but planting delayed for four months until the start of the wet season) the effects of the herbicides disappeared in general at the same time as in the third plantings. However, as long as symptoms were discernible they were somewhat more pronounced in the second than in the third series^ indicating a somewhat greater herbicide persistence. The reason for this is not clear; one possibility is that because of lack of competition by water the herbicide molecules were more effectively adsorbed to the soil particles and therefore somewhat less accessible to later loss and degradation. From Figures 2 and 3> the following conclusions can be drawn: 1. The herbicidal effects persisted in different crops for different lengths of time; i.e., the crops differed in their herbicide sensitivity. The three cereals (rice, maize, sorghum) were less sensitive than the broadleaf crops (mung bean, soybean, peanut, sweet potato) because the recorded deleterious effects stopped sooner. 2. The effects of Agent White lasted longer than those of Agent Orange. The main difference between the two agents is that White contains picloram while Orange contains 2,U,5-T; the observed effects can be attributed to the greater persistence of picloram. 3. In the Ban-Me-Thuot experiment, the persistence of the two agents, and particularly of .Agent White, was greater than in the Riilippines experiment. Symptoms of picloram injury were still evident in peanuts in our last planting at Ban-Me-Thuot, made about 31 weeks after herbicide application to the soil. AB this planting was completed at 30 �the onset of the dry season, and as the Riilippines experiment, second series (herbicide application to soil during the dry season, plantings Jelayed until the wet season) showed -hat the herbicides persist throughout a dry season, it is possible that some picloram symptoms might have been found if peanuts*had been planted again after the end of the dry season. The reasons for the difference in herbicide persistence between the two experiments may be due to any one or more of the following factors: (a) use of different varieties; (b) different soils; (c) lower temperature and less precipitation at Ban-Me-Thuot as compared to Alabang: rainfall during the period of the experiments at Ban-Me-Thuot was about 6k in., at Alabang about 90 in. including one typhoon with 7.25 in. in one day, and another'with 23.5 in. in two days, The results of our experiments, in their entirety, are in full agreement with past experience on the characteristics of 2,U-D, 2,U,5-T, and picloram. Specific and varietal differences in herbicide sensitivity are very well known. Indeed, the difference in the response of grasses and of many broadleaf plants to these compounds are the basis of most of their agricultural uses. Greater persistence of picloram, as compared to 2,U-D and 2,U,5-T, is also well established, and so is the influence of soil and climatic conditions on the disappearance of herbicides. More important than these variations is the fact, clearly brought out in both experiments, that even though the soil received the massive dose of 3 gal/acre, the herbicides did not affect growth, even of highly sensitive crops like the legumes, for more than about 30 weeks. Even if peanut plantings at Ban-Me-Thuot should show some picloram symptoms one year after soil treatment, it should .not be overlooked that effects on 31. �Growth and survival had disappeared after 17.5 weeks. It should also be appreciated that our results are overestimates in two respects. First, although the highest dose of herbicides used in our experiments, 3 gal/acre, was that used on the military herbicide missions, it is considerably in excess of what would in most cases reach the soil at .» least on a first mission over a particular region, when a major part of the herbicide would be intercepted by the vegetation and never reach * the soil in an active form. Second, if herbicide effects were present in a planting that was made four weeks after the soil treatment and observed for another four weeks, but were absent in the next planting, made 15 weeks after the soil treatment (e.g., the effects of Agent White on survival and growth of peanuts in the Philippines experiment), this result is shown as "no effects after 15 weeks." However, the herbicide may in fact have dropped below the effective level for this crop any time from 8 to 15 weeks after application to the soil. Forest Sites Experiments with forest soils were conducted on a cleared forest site on Mount Makiling near Los Banos, the Philippines, kindly provided by the Philippine Department of Forestry, and on a site presently used as a plantation, about 2? years old, of Hopea odorata, a dipterocarp, at the Ea-Knat Station at Ban-Me-Thuot. The former site consisted of two plots, 100 by 50 ft (30 by 15 m) in size, the latter of two narrower strips about 90 and 96 ft ( ? and 29 m) long and 6 ft (1.8 m) wide. The 2 plots were separated by buffer zones to exclude cross contamination of the two agents. One plot was sprayed with Agent Orange and the other wit; 32 �Agent White; the dos*.1 was 3 gal/acre, i.e., the rate used, by military spray missions; and the ground was cleared of vegetation as in the experiments with agricultural soils. At Los Baiios, both 10-in. (25 cm) .mrface samples and 30-in. (75 cm) cores were taken. At Ban-Me-Thuot, hocause of the dry conditions, the soil was very hard and only surface comples could be taken except on the last sampling occasion. Residues were determined by chemical methods alone; the results were, however, in agreement with observations on natural revegetation of the sprayed plots. The results of the Los Banos experiment are shown in Figure b, those of the Ban-Me-Thuot experiments in Figure 5- It can be seen that both 2,**,5-T and picloram disappeared from the soil quite rapidly, but picloram faded more slowly (it must be remembered that the initial dose of picloram was only 25 percent that of 2,U,5-T). The disappearance was greatest in the period immediately after herbicide application and then became relatively slower. It should be noted that the ordinate axes in Figure k and 5 (and likewise in Figures 6 and 7) are on a logarithmic scale; the: absolute drop iln the early period is thus much larger than may be apparent. For example, the picloram content in the top 10 in. of the Los Banos soil dropped from the theoretically-applied •lose of 1.6 Ib/acre to O.U9 Ib/acre, in 13 days to 0.29 Ib/acre, and in 31 more days to less than 0.02 Ib/acre. By the end of the experiment, ItJ} days after application, it had dropped relatively much more slowly, to 0.008 Ib/acre. The disappearance of 2,U,5-r was more rapid than that of picloram, considering the almost 10 times higher initial dose, but the difference was not striking, and by the end of the observations (189 days 33 �2.0 < 10 1.0 - < Los Banos Forest Soil 2.4,5-T 0 0-10 in. (0-25 cm) » 0-30 in. (0-75 cm) 1.0 Los Banos Forest Soil Picloram 0 0-10 in. (0-25 cm) x 0-30 in. (0-75 cm) 0.1 I I 0.1 0.01 0.01 0.001 30 60 90 120 Days 30 210 B 60 90 120 150 180 210 Days FIG. U. Disappearance of herbicides (A, 2,U,5-T; B, picloram) from a forest soil on Mount Makiling near Los Banos, the Philippines. The applied quantities were 13 Ib/acre 2,U,5-T and 1.6 Ib/acre picloran. Samples were 10 and 30 in. (25 and 75 cm, respectively) deep. Abscissa - days after herbicide application, ordinate - herbicide level in spil, as Ib/acre, on logarithnic scale. Vertical bars represent the 5 percent confidence limits. �2.0 10 - Ban Me Thuot Forest Soil Picloram (Surface samples) Ban Me Thuot Forest Soil 2.4.5-T (Surface samples) 1.0 U) VJ1 0.1 I I 0.1 0.01 0.01 0 30 60 90 120 150 Days i i 1,1 i 210 240 270 0.001 0 B 30 60 90 120 150 Days 180 210 240 270 FIG. 5. Disappearance of herbicides (A, 2,U,5-T; B, plcloram) from a forest soil near Ban-Me-Thuot, SVK. The applied quantities were 13 lb/acre,2,U,5-T and 1.6 Ib/acre picloram. Surface samples (5 in. = 12.5 cm) only. Abscissa - days after herbicide application, ordinate - herbicide level in soil, as Ib/acre, on logarithmic scale. Vertical bars represent the 5 percent confidence limits. �at Los Banos, 249 days- at Ban-Me-Thuot) the levels had dropped either to slightly above or to below the detection Limit. Disappearance of both herbicides in the surface and deep samples of the Los Banos experiment (0-10 in. and 0-30 in., respectively) proceeded in a similar manner (the discrepancy on the 105th day is most probably due to variations in the samples). As already mentioned, results of observations on revegetation of i the treated plots agree with the chemical data, indicating rapid disappearance of both 2,U,5-T and picloram (and of 2,4-D). At Mount Makiling, 3-5 months after the herbicide applications, both the Orangeand the White-treated plots had been fully revegetated, with a major component of broadleaf herbaceous plants including wild tomatoes. The latter—a species highly sensitive to all three herbicides used—had produced ripe fruits and must have started growth two months If not earlier after herbicide application. Two months later, i.e., 5-5 months after herbicide application, the plots were covered mainly with grasses that had largely replaced the broadleaf species, and numerous (unidentified) tree seedlings were present in the grass. The only possible herbicide effect still noted was an off-color individual of Philodendron sp. in the White plot. Otherwise, the treated plots were indistinguishable from the surrounding vegetation on nontreated soil. At Ban-Me-Thuot, the Orange-treated plot was at the last observation ( ^ days after 29 treatment) fully revegetated with the same species that occurred in the untreated surrounding areas; the White-treated plot was still sparsely vegetated, but was being actively recolordzed from the untreated areas. 36 �"-irvrovo The experiments with mangrove soils wore performed in a mangrove ftfa at Chi-Linh near the city of Vung-Tau. This area was within the p-rineter of the National Popular Forces Training Center; permission :'or the experiments was granted by the Commander of the C3nter and the City Council of Vung-Tau. The exact elevation of the experimental plots la not known, but they appear to be qu.ite frequently flooded at high tide. On all days we visited the area we observed either flooding or evidence of recent flooding. For the Initial experiment, an area 17'+ by 96 ft (about 50 by JO m) was cleared of all vegetation and divided into three parts, each '6 by 1*8 ft (about 30 by 15 m), separated by 15 ft (U.5 m) buffer strips. , Ono of the outer parts was sprayed with 3 gal/acre of Agent Orange, the oilier with the same amount of Agent White; the center part remained untreated as a control. Spraying was carried out at low tide, when the soil was dry. Soil surface samples were taken for the first time one -lay (two tides) after the spraying and sampling of both surface samples and cores was repeated five times: 20, 40, 72, 119, and 201 days after spraying. Seedlings of two major mangrove species, Rhizophora aplculata and Ceriopg tagal, were planted in groups of 100 at the time of the second, third, fourth, and fifth soil sampling and were observed about 2)» 35, and 50 weeks after the date of spraying. The number of groups was originally six per treatment, but in the Orange plot some groups were on the margin of the sprayed area and were discounted, reducing the c—b*r of some plantings to four. The results of this experiment, as "Mangrove Cleared," are shown in Figures 6 and 7. 37 �2.0 10 1.0 Vung Tau Mangrove Cleared Vung Tau Mangrove Cleared Picloram 0 Surface samples x Cores 2.4.5-T 1.0 0.1 © Surface samples * Cores I , - (i 0.1 - 0.01 I I - 1 1 © 0 O 0.01 I) , i i 30 i 1 60 j 1 1 1 1 120 90 Days 1 1 ISO 1 1 180 t 0.001 1 210 i 30 B 60 J- 90 120 Days i 150 i 180 210 FIG. 6.- Disappearance of 2,k,5-T (A) and picloram (B) from cleared mangrove soli. 2,U,5-T was applied at 13 Ib/acre, picloram at 1.6 Ib/acre. Surface samples were taken with 5-in. high metal cans, cores with a soil sampler 30 in. long, but the cores were usually shorter because of compacting and because of occasional loss of part of the core. Abscissa - days after herbicide application, ordinate - herbicide level in soil, as Ib/acre, on logarithmic scale. Vertical bars represent the 5 percent confidence limits. �w jJ! j; "»"7 1 t 1 to S <3 £ 3D .1 1 2 » ^^ ^ivrT ; |! 1 SK; Is 3 ;j : *f 1 ^•'j ^ ^ a i ' -rf^ "^ "7 $ *!•"-, _ I j. j. T'.u "j= 1^7 = •'it i ^ "• 5 t- "•P : i |<: « f 1 Y: 3I5:;- j!! 10 : !'! CONTROL • ; ' r? J ji: j rrl rfl rtf WHITE CONTROL F-nlir.jj V.iin Dam md THIMI Alln Sgr« '•••19 Ul vo 100 90 - .*L jiJ qr N IE I 1 i» o S i ,o < October 1971 -MWerki AflerS^rfving •I-*i ill :• i t; CONTROL ISNjMrnbnign -B .-.-edit Allffi Sfayng WHITE ' S • ;! i Hi< 1 9 : — " T ; " ;p i ; f JT S 1 ; • \ ORAM3E 1 MUCH 1973 -SOW— I n AfMi Soraying Tl" n A ORANGE n L-r-pP f-J ' WHITE « 73 ~ i] CONTROL ORANGE ».M il>h 4 & ^:p! £ € -1 L l i —1;5 1 I i t / i 1 (-i t j 1 CONTROL ••^ ~*''IC, : 'i i I n-n r-w-n , | F I !i:.5 I i [••] .';..J WHITE 1 I'^i L' TfIF 9 ORANGE \ ^9 . •::' j$ I fO.j ». -1 II I VUNG TAU MANGROVE CLEARED CEHIOPS w K'i II n r/i '• j ORWiat E 1 WHITE PUmmgi Wiih Odm •xl Taaii After Spiav'ng rrl ri i ^ B Am • CONTROL ORANGE 4 Ocnbw 1972 ""29 Wetki Alter Spraying WHITE CONTROL ORANGE WHITE 15 NMmbtr 1972 •*35 Wifef Afur Spr lying CONTROL ORANGE WHITE 1 Uvdt 1973 "^50 IVvcht Alltr Spriying FIG. 7. S-irvival of Rhizophora and Cerioiaa seedlings on cleared mangrove soil treated with Agent Orange (3 gal/acre) or Age-it White (3 gal/acre), or untreated (control). On the left, the situation at the start of the consecutive plantings ( 0 seedlings per group) is shown; going to the right, the survival at different 10 observation dates. At any one observation date, compare plantings of the same date, e.g., the April 6 control plantings with the plantings on Orange- and or. White-treated soil of the same date, and siailarly with the April 27, the May 33, and the July I1* plantings. �Figure 6 Illustrates the disappearance of 2,^,5-T and picloram from the soil. The trend is quite similar to that found in the forest soils (see Figures k and 5): an initial rapid rate of disappearance that falls off with time. The very marked drop one day after spraying, apparent especially in the case of picloram, may be in some part due to * washing out by the tide. Also, particularly in the case of picloram, the cores contain consistently higher levels of herbicide than the sur*i face samples, suggesting that the latter had penetrated into subsurface layers and possibly further. The levels of both herbicides 201 days after spraying were either near the lowest limit of detection, or below. Figure 7 summarizes the results of the planting experiments. Two features stand out. First, survival was low. Twenty-nine weeks after spraying, only between about 3 and 20 percent of the Rhizophora seedlings were still alive, the later plantings doing relatively better than the earlier ones. Most of the seedlings of the earliest Ceriops planting were dead, but this was mainly because they were transplants from a prepared bed; they had already formed roots and did not survive the shock of transplanting. In the later plantings, seedlings were collected from neighboring trees and survival was better. The decline in survivor numbers continued, although at a slower pace, to the last observation. One reason for seedling death is most probably Injury by crabs; seedlings with bitten roots were found partially pulled into the soil. However, we are in no position to say whether crab damage is a major or minor cause of the seedling loss observed. Second, even in the earliest planting (made three weeks after �, -«.-a and when the herbicide content of the soil was still 0.42 to *, . »j «-"O C '•»'• lb/acre of 2,U,5-T and O.OlU to 0 0 9 Ib/acre picloram) there was .2 •*•> ilff'.-rence in survival between the seedlings on the Orange, the White, »r.t the control plots. For example, on the October k observation date, • >•.«• survival in the April 6 planting of Rhizophora on the control plot „»« . 7 percent, on the Orange plot k.2 percent, and on the White plot ^ . ; percent; for the July Ik planting the values were 1 . , 18.5, and v 87 2l.'f percent, respectively. By the last observation date, the values Jr. the April 6 planting had dropped to 3-0, 3-0, and 1.6 percent, respectively, and in the July lU planting to 10.4, 6.0, and 10.7 percent, respectively. But in no case were there large differences between the rontrol, Orange, and White plots within plantings of one and the same 4ntr«; that is, there was no effect of herbicide residues on establishfr.t of Rhizophora and Ceriops seedlings. Because of this result, which was somewhat surprising in view of th* sensitivity of mangrove communities to aerial application of herbiclle, and because of the poor seedling survival, the experiment was rrfeated; however, it was modified to simulate conditions in an intact aar.grove that had not been cut or sprayed with herbicides. For this 2 purpose, 27 microplots of one square m (1.2 yd ) were cleared of vegetation. Eighteen were hand-sprayed, nine with Agent Orange and nine with A*?ent White at the same rates as in the "Mangrove Cleared" experiment; the last nine remained as untreated controls. Soil surface samples and cores were taken 26 or 28, 45, 82, and 138 days after spraying; Rhizophora •ri Ceriops seedlings were planted 4, 6.5, and about 12 weeks after *;r*yir.g, on each occasion 40 seedlings per plot, three plots each for �Orange, White, and control. Figure 8 shows that disappearance of the herbicides (2,U,5-T and picloram) proceeded much as in the "Mangrove Cleared" experiment. Perhaps it occurred a little more rapidly, since 2,U,5-T had dropped below the detection limit by 138 days in both surface samples and cores (in the former, it was undetectable even at 82 days) while in the "Mangrove Cleared" experiment traces were still detectable in core samples after 201 days. It should be noted that the soil of the microplots site contained more sand than that of the "Mangrove Cleared" experiment; tidal flooding may also have been somewhat less frequent. Figure 9 illustrates the behavior of seedlings. Survival was much better than in the "Mangrove Cleared" experiment--and in Rhizophora better than in Ceriops. Compare particularly the November 15, 1972 ' observation date in Figure 7 and the March 1, 1973 date in Figure $; both represent a similar period after spraying (about 3k and 35 weeks, respectively). It appears that the situation in a largely undisturbed mangrove can be a good deal more favorable for the establishment of mangrove seedlings than in a relatively large, cleared area in the mangrove. With respect to the herbicide effects, however, the microplot experiment gave results quite similar to those of the "Mangrove Cleared" experiment. Although at the time of the first planting the soil still contained substantial amounts of the herbicides, and although some seedlings of this planting on the Agent White-treated plots exhibited characteristic picloram symptoms (pale leaf color, ro3J.ed-up leaf margins), the numbers of survivors In this as well as in subsequent plantings were comparable on control, Orange-treated, and White-treated plots. In other words, both k2 �70 10 Vung Tau Mangrove Microplob Piclorwn 0 Surface samples x Cores Vung Tau Mangrove Microplots 2.4.5-T © Surface samples x Cores 0.1 1.0 I I 0.01 0.1 aoi 0.001 30 60 120 Days 'eiSO 30 B 60 90 Days 120 150 FIG. 8. Disappearance of 2,4,5-T (A) and picloram (a) from mangrove soil in small cleared plots within undisturbed mangrove. 2,U,5-T was applied at 13 Ib/acre, picloram at 1.6 Ib/acre. Surface samples were taken with 5-in. high metal cans, cores with a soil sampler 30 in. long, but the cores were usually shorter because of compacting and because of occasional loss of part of the core. Abscissa - days after herbicide application, ordinate - herbicide level in soil, as Ib/acre, on logarithmic scale. Vertical bars represent the 5 percent confidence limits. �VUNG TAU MANGROVE MldtOPLOTS RHIZOmOHA 120 100 w •*. Lai -1 3ij I i -I I I *•:; * s ! t-S 43 i 20 CONTROL ORANGE WHITE CONTROL ORANGE CONTROL ORANGE WHITE WHITE ISNonrnlicr 1972 Pllnlinqt WilhOmitndTliMI AtUl Sprtying CONTROL ORANGE WHITE 1 Mtrch 1973 - 34 WMkt AIW Sprlying VUNG TALI MANGROVE MICROH.OTS cfmon 110 • 100 . I A "• VJ S n S m •}" a ? CONTROL ORANGE WHITE nm.nji WuhDnnndTinM Ar«rStK-lY«l CONTROL ORANGE WHITE 4O«ob«1972 ~t2WMki Aim Spravmt CONTROL ORANGE WHITE ISNonmlar 1972 ~19WHki AlnrSmiiiiiv CONTROL ORANGE WHITE I March 1973 -MV*r«ii AllcrS FIj. 9. Survival of Rhizophora and Cerlo-pa seedlings la small cleared plots within undisturbed mangrove treated with Agant Orange (3 gal/«re), Agent White (3 gal/acre), or untreated (control). See legend, Figure 7. �experiments show that three to four weeks after application to soil at the rates of 13 Ib/acre 2,U,5-T and 12 ILb/acre 2,^-D, or 1.6 Ib/acre picloram plus 9-3 Ib/acre 2,U-D, none of these herbicides Impaired the establishment of mangrove (Rhizophora aplculata and Ceriops tagal) seedlings. Nor was the seedling growth, as expressed by height, different • .» in both experiments on herbicide-treated and control plots (Table VI). i GENERAL CONSIDERATIONS OF THE DISAPPEARANCE OF HERBICIDES APPLIED TO THE SOIL IN VIETNAM AND SIMILAR ENVIRONMENTS Comparative Patterns of Persistence in Different Soils , The disappearance pattern of phytotoxic residues from all the soils examined in the present study is similar to that found in the more extensive investigations undertaken in temperate regions. In whole core samples (Figures ^, 6, and 8), the percentage of the theoretical amount initially applied that was left in the soil by the final sampling occasion (up to 2^0 days after application) ranged from 0.15 to 0.23 for 2,U,5-T and from 0.12 to 0.5 for picloram. Examination of the residual phytotoxicity to selected crops (Figures 2 and 3) indicates that, for the highest rate of application, the interval required before symptoms of injury were no longer observed varied. For Agent Orange the time ranged from U weeks for a resistant crop such as maize to 18 weeks for a susceptible species such as peanuts. The data for Agent White were ^ weeks for maize and 31 weeks for mung bean. For peanut, at the highest doses slight symptoms of phytotoxicity were still observable at the final assessment. Comparable data from other parts of the tropics are meager �Table VIC. Average height (in.) of shoot above ground level of mangrove seedlings planted on herbicide treated and on untreated (control) plots. (Date of observation March 1, 1973) Rhizophora Planting Date Control Orange Ceriops White Control Orange White "Mangrove Cleared" Experimenta j May 28, 1972 July 14 , 1972 16.2 14.8 15.5 14.8 16.4 16.6 11.3 10.9 10.2 H.o 10.5 11.5 10.1 9.2 9.1 9.1 Microplot Experiment August 5, 1972 October 10, 1972 14.4 16.5 13.4 15.8 a 14.1 15.9 9.4 '. 98 "Mangrove Cleared" Experiment: Herbicide application March 17, 1972. Number of plants per treatment measured between 7 and 58. Number of plants in two earlier plantings (April 6 and 27) too sma.11 for valid observations. Microplot Experiment: Herbicide application July 159 1972. Number of plants per treatment measured 32 to 4$. Only first and last plantings shown. 46 �and none exist for mangrove soils. The results of some relevant experiments have been collated in Table VII. .In the Puerto Rico field study on two susceptible species the time for disappearance of phytotoxic effects was 8 weeks for a mixture of 2,U-D and 2,^,5-T and 12 to 53 weeks for a mixture of 2,U-D,A 2,U,5-T, and picloram. These intervals are in the same range as those found in the present investigation. In the Puerto Rican forest soil experiment, the 0.7 percent picloram residue at the end I of one year is not out of line with the range of 0.3 to 0.5 percent in Figures ^B and 5B, considering both the much higher dose (9 compared to 1.6 Ib/acre) and the much longer interval before the terminal sampling occasion. For the other three sources quoted, comparisons with the data from Southeast Asia can only be tentative. If it is postulated that the shapes of the degradation curves against uime are relatively independent of the initial dos^s, then on the basis of the data for the two forest soils (Figures ^ and 5) interpolation would predict a 90 percent loss of both 2,U,5-T and picloram within 15 days, whereas the observed intervals in Texas were 1.5 months for 2,U,5-T and 2.3 months for picloran. Similarly, the intervals recorded for a 90 percent loss under temperate conditions in Table VII again exceed those predicted for forest soils in SVN and the Philippines. The processes by which a herbicide is lost from the soil and that hence determine its persistence can be divided into those that remove the chemical unchanged from the system, and those responsible for its decomposition within the system. The first group consists of volatilization, leaching, runoff, and uptake by plants. The second involves chemical breakdown by biological and nonbiolo;_Tical processes. The data �Table VII. Disappearance of 2,lt-D, 2,4,5-T and picloram from soil in warmer climates. Herbicide and dosage Observation Field Study in Puerto Riqo (Bovey et al. 1 6 ) 98 Time (in months) for no symptoms on: Soybean Cotton Picloram, 6 Ib/acre 6.5 3 2,4-D + 2,U,5-T, 12 + 12 Ib/acre 2 2 2,U-D + 2,4,5-T + picloram, 6 + 6 + 3 Ib/acre 6.5 3 Forest in Puerto Rico " (Dowler et al. 1969) Amount left in top hQ in. after one year (in percent) Picloram, 9 Ib/acre Picloram, 27 Ib/acre 0.7 5.5 Field Studies in Texas (Bovey et al. 1969, Bovey and Baur 1972) 2,4,5-T, 0.5 and 1 Ib/acre Picloram, 1 oz/acre Picloram, 1 Ib/acre Time for 90 percent or more degradation (months) Less than 1.5 0.7 2.3 For comparison, Temperate Climate Conditions (average figures) Time for about 90 ( 5 1 0 7-0) percent degradation (months) 2,U,5-T 3-6 ("Report on 2,U,5-T," 1971, Kearney 1 7 ) 90 Picloram, 1 oz to 1 Ib/acre (Hamaker et al. 1962, Herr et al. 1$>66, Scifres et al. 1971, Bovey et al. 1 6 ) 99 3-16 �reported in this chapter must be interpreted in the light of (1) what is known from previous investigations of the importance of these factors on the behavior of 2,U-D, 2,^,5-T, and picloram in soil, and (2) the characteristics of soil and climate in SVN that may affect herbicide persistence. tt Volatilization and Photodecopposition The n-butyl esters of 2,^-D and 2,U,5-T, the constituents of ™ t i Agent Orange, are moderately volatile (see Section II C, I&rt A of the Report on the Effects of Herbicides in South Vietnam) and might be expected to vaporize quickly on contact with soil or plant surfaces under tropical conditions. The ability of plants to hydrolyze esters of 2,^-D to the nonvolatile acid is well known, but some loss as a vapor from leaf surfaces before entry is a possibility. In soil, rapid hydrolysis occurs even at low moisture levels (Smith 1972); this could reduce or eliminate losses in a vapor form. Moreover, under very dry conditions adsorption of volatile herbicides by the soil surfaces is well known to reduce such losses. The salt forms of 2,^-D and picloram used in Agent White are not appreciably volatile, and significant losses by this route can be discounted. It is probable, therefore, that volatilization from soil was not of major importance. In laboratory experiments the acids can be decomposed by the action of light, but under field conditions it is generally unlikely that such losses would be appreciable; this expectation agrees with the observations already recorded at Alabang. Here the residual toxicity of Agent Orange applied in the dry season (February 2), with the soil surface left undisturbed until crop planting on May 30 was similar to the residual �toxicity in plots sprayed on May 3 and also planted on Kuy 30. Thus losses from soil due either to photodeccmposition or volatilir.ation are likely on theoretical grounds to have been small. Moreover, the lack of phytotoxic symptoms in susceptible crops planted in our control plots . * immediately adjacent to plots sprayed with Agents orange and White suggested that volatilization was of little consequence. Runoff Any material applied to the soil is liable to be carried from the site of action when rain causes surface runoff and water erosion; the degree depends on the intensity of the precipitation, the soil characteristics, the nature of the surface, and topoprapliical features. The amount of herbicide removed in the surface wash will also be dependent on the interval since application. In the present experiments the only evidence for runoff after heavy rain caim* from (l) the phytotoxic symptoms observed below the treated plots (which were situated on a slope in the forest soil experiment in the Philippines) and (2) from picloram symptoms on untreated plots of very susceptible crops observed after the heavy typhoon at Alabang (noted above),, It has also been mentioned that in the mangrove experiment on the cleared site the hcrbicidal contents of the surface samples taken two tides after application r.ay in part have been affected by tidal waters carrying away some of the surface particles. Leaching The proportion of a herbicide that is physically bound within the soil matrix is dependent on its physicochmical characteristics and 50 �the nature of the soil. A combination of these properties determines the amount that is transported downwards through the soil by water percolating from the surface. This leaching action is dependent on two interacting sets of conditions. The downward movement of the soil solution will occur only when on average the amount of incoming rain exceeds the amount of water lost by evaporation from the soil and transpiration from the vegetation. The amount of herbicide that moves downward is dependent on both the freedom with which the soil solution can pass through the soil pores, and the concentration of herbicide in solution (which will be in equilibrium with the amount retained on the surfaces of the soil particles). . Thus losses of herbicides will be greatest when rainfall markedly exceeds evapotranspiration, the soil is free draining, and the retentive power of the surfaces is low. Losses will be minimal ( ) in the dry season, (2) when the soil is relatively l impermeable, and (3) if the capacity for adsorption is high. For individual herbicides there is the further consideration that the greater the degree of binding on any one soil type the less the liability of leaching. Against this background it would be expected that the capacity for retention would be less for the forest soils at Los Banbs and Ban-Me-Thuot than for the heavy clay soil at Alabang ajid the estuarinc muds of the mangrove experiments. It would also be expected that leaching would be least at Ban-Me-Thuot, which had the lowest rainfall over the experimental period. Since no basic data are available for the conditions of mangrove soils, the effects of the fluctuations ir.the water table and intermittent flooding of the surface are not predictable, but it is likely—at least during the initial phase—that each time the water table recedes from the 51 �surface layer there will be some transfer of the herbicide to a lower depth. There is some suggestion of this in Figure 6A and B, since the cores tend to have a higher content than the surface samples. It is very difficult to disentemgle the losses caused by leaching from the losses related to the activitieis of microorganisms. But at least in some experiments it would seem i;hat the component of leaching was small. For example, it is apparent that orr the forest soil experiment at Ban-Me-Thuot (Figure 5A and B) the progressive disappearance of both compounds took place under conditions where, subsequent to the initial spraying, lack of rain led to steadily drier soil over the first three months; hence, there cannot have been appreciable leaching from the surface layers. Moreover, if leaching is a major component it would not be expected that in the Pran Buri Calibration Grid picloram would be still present after eight years, with the residues largely present in the top layers of the soil. These findings are in general agreement with the literature and the expected behavior of 2,1<-D and 2,U,5-T where they appear as the parent acids, which have a low solubility in water. The extent of the leaching of highly soluble piclorara is dependent on the soil type; it is seemingly most marked in sandy soils (Bovey et al. 1 6 ) Leaching is known to occur to considerable depths, and those who 99. have looked at depths greater than 3-^ ft have generally found traces, although higher concentrations occur near the surface. On the basis of picloram1s greater mobility compared with 2,4,5-T (Helling 1 7 ) it is 91, puzzling to note from Table VIII that in the first Uk days the proportion of the remaining residue of picloram found in the top layr is much larger than that of 2,U,5-T. 52 �Table VIII. Changes in the distribution of herbicides in forest soil, Los Baffos, the Philippines. (Figures are percent of total found at each depth) Days Depth (in./cm) * 13 44 105 2,4,5»T j •Sop (0-10/0-25) Middle (10-20/25-50) Bottom (20-30/50-75) Ib/acre (average) 68.7 17.9 13.4 44.9 31.1 24.0 1.37 0.16 l 59.8 35.7 4.5 0.09 Picloram Top (0-10/0-25) Middle (10-20/25-50) Bottom (20-20/50-75) Ib/acre (average) 98.3 1.1 0.6 90.9 5.5 3.6 0.27 0.03 53 22.6 43.B 33.5 0.03 63.1 23.4 13.5 0.01 �Degradation It is well established that 2,U-D and 2,4,5-T are degraded by soil microorganisms~2,4-D very rapidly, 2,4,5-T more slowly. There is also firm evidence that repeated treatment with these herbicides accel• ,* crates the rate of breakdown. Factors that favor the growth of microorganisms, such as high temperatures, moist conditions, and a ready > availability of substrates, also enhance the level of decomposition. Such studies, including the chemical pathways of degradation, have been extensively reviewed by Loos in Kearney and Kaufman ( 9 9 • 1&) The course of degradation of 2,U-D and 2,4,5-T by soil microorganisms is well documented. At first little breakdown takes place (lag phase), then the rate builds up rapidly to a high level, which is followed by a third phase when the rate declines. The data of Figures IfA, 5A, and 6A share these common characteristics:: the amount of herbicide present in the soil at first falls rapidly, but later the rate of disappearance slows down. Thus, on the basis of the similarities between these field studies and the laboratory studies of metabolic degradation, it can be postulated that both in the mangrove and forest soils microorganisms played a major role. Since, however, there was no prior information on the capacity of the microorganisms present in these types of tropical soils to decompose the more resistant 2,4,5-T, an experiment was carried out at the Weed Research Organization (Hance, personal communication) 14 in which radioactive 2,U,5-T n-butyl ester ( C-la.beled carboxyl group) was applied to both mangrove and upland soils from SVN. The results confirmed that these soils were capable of degrading 2,4,5-T to carbon dioxide in the laboratory. When the output of radioactive CC2 was plotted against �time, the curves showed the typical lag phase associated with microbial degradation (Audus 196*0 • The soil samples used for this experiment were taken from the area of the dump site in the Di-An District, the KLnhNgang Canal in the Ca-Mau Peninsula, and from Site #1 in the Rung-Sat Special Zone. The last two were mangrove soils. Reference has already been made to the possibility that 2,U-D and 2,U,5-T may be decomposed by the action of light, but there are other nonbiologi<Jal processes that participate such as hydrolysis, oxidation, and reduction. The surfaces of soil components may or may not catalyze these reactions; this matter requires further study. The pathways of disappearance of pidoram are not adequately understood. The available evidence suggests that photo-decomposition, leaching, and microorganisms are all involved, with the latter probably playing a major role (Upchurch 1 7 ) Thus there is supporting evidence 93. that the losses of picloram with time shown in Figures ^B, 5B, and 6B are associated with the activities of microorganisms. To conclude, the data collected by the Committee from experiments and soil samples in SVN, the Rulippines, and Thailand all indicate that the general disappearance pattern of the three herbicides is in line with that well known for temperate regions. Moreover, all information the Committee was able to gather locally from farmers, village and district officials, and agricultural advisers indicated that crops could be grown again with no reductions in yield or other ill. effects the year following one or more herbicide missions. If crops were not grown it was either because of lack of security, or apprehension that the herbicide treatment might make the produce unfit for consumption. 55 �GENERAL CONCLUEIONS • Several deficiencies in our studies have been mentioned before. We were able to collect soil samples in only one forest area in SVN that had been sprayed during the war; we were unable to make collections in ,* forest areas that had received the relatively heaviest sprayings. We were also unable to repeat our own experiments for a second year, and to i conduct them on the other major soil type of SVIT, the alluvial soils. However, these deficiencies are counterbalanced by two important considerations. First, our evaluation of persistence included different agricultural and forest soils (in SVN and the Philippines) as well as mangrove soils. The sites were selected because local conditions happened to be favorable and, except for trying to take samples in heavily sprayed areas, and to include soils from vegetation types most extensively subjected to military herbicide sprays, no attempt was made to give preferential coverage to a particular soil type or any other factor. Second, our findings, at least those on the fate of herbicides in soils, are in excellent agreement with general experience on this problem. Thus viewed, our data possess considerable internal consistency and, in our opinion, permit a number o:f general conclusions, namely: 1. The behavior of herbicides in the soils of Vietnam and the Philippines is similar to that reported for soils elsewhere. 2. Only where herbicides (2,4»D, 2,^,5--T, picloram) were applied in very massive doses (the former two in the magnitude of 1000 Ib/acre, picloram at 20 Ib/acre: Fran Buri Calibration Grid; see Table U , are ) they still in part present in concentrations that are above the threshold �likely to induce phytotoxic symptoms in some species. 3. Where applied to mangroves at total doses approaching 100 lb/ acre of 2,U-D and of 2,U,5-T, or 3 or more Ib/acre of picloram, the herbicides may still be present at low levels. Although the amounts present varied between sampling sites, the levels were such that the likelihood of damage to crops that could be grown -under these conditions can be discounted. They were far below the levels, that, in our own experiments, had no effect on the establishment of seedlings of Rnizophora apiculata and Ceriops tagal. Moreover, seedlings and young plants of mangrove species that we observed in heavily sprayed areas of the BungSat, where 2,^,5-T and/or picloram were still detectable by chemical methods, did not exhibit any herbicide symptoms. k. In areas subject to one or two herbicide missions 1.5 years before sampling, no phytotoxic residues could in general be detected. 5. Our results indicate that after a single application of Agent Orange, even where conditions are such that all the spray reaches the soil, crops sensitive to 2,U-D or 2,4,,5-T may be safely sown after four to six months of wet weather; after an application of Agent White under the same conditions, resistant plants like rice and maize can also be safely planted four to six months after application. In this connection, it is appropriate to point out once more that the dosage used in our experiments, i.e., about 6 or 12 Ib/acre of 2,4-D and 2,^,5-T, and 1.5 Ib/acre picloram, applied to the bare soil, was considerably higher than the dosage that would have reached the soil when the forest or mangrove sites were sprayed for the first time because of interception of the spray droplets by the canopy. 57 �6. Claims that the herbicides as they were used during the war have rendered the soil "sterile," permanently or at least for prolonged periods, are without any foundation. It should be noted that these claims were contrary to all existing information for the herbicides in question. . * REFERENCES * * Audus, L.J., ed. 196U. The physiology and biochemistry of herbicides. Academic Press, London and New York. 555 pp. Bovey, R.W. and J.R. Baur. 1972. Persistence of 2,^,5-T in grasslands of Texas. Bull. Environ. Contarn. Toxlcol. 8:229-33. , F.R. Miller, and J. Diaz-Colon. 1968. Growth of crops in soils following herbicidal brush, control in the tropics. Agron. J. 60:678-9. , S.K. Lehman, H.L.Morton, and J.R. Baur. 1969. Control of live oak in South Texas. J. Range Manage. 22:315-8. Dowler, C.C., W. Porestier, and F.H. Tschirley. 1969. Effect and persistence of herbicides applied to soil in Puerto Rlcan forests. Weed Sci. 16:^5-50. Executive Office of the President, Office of Science and Technology. March 1971. Report on 2,U,5-T: a report of the panel on herbicides of the President's Science Advisory Committee. U.S. Government Printing Office, Washington, B.C. 68 pp. Hamaker, J.W., C.R. Youngson, and C.A.I. Goring. 1962. Prediction of the persistence and activity of tordon herbicide in. soils under field conditions. Down to Earth 23:30-36. Helling, C.S. 1971* Pesticide mobility in soils. II. Application of soil thin-layer chromatography. Proc. Soil Sci. Soc. Am. 35s737-^3. Herr, D.E., E.W. Stroube, and D.A. Ray.. 1966. The movement and persistence of picloram in soil. Weeds 1^:2^8-50. Kearney, P.C. 1 7 . Herbicides in the environment. Agricultural 90 Research Service, U.S.D.A., Beltsville, Md. WC/70/WP/26. _ and D.D. Kaufman, eds. 1969. Degradation of herbicides. Marcel Dekker, Inc., New York. �McKone, C.E. and J.R. Hance. 1972.. Determination of residues of 2,U,5-trichlorophenoxyacetic in soil by gas chromatography of n-butyl ester. J. Chromatog. 69:204-6. Selfres, C.J., R.H. Hahn, and M.G. Merkle. 1971. Dissipation of picloram from vegetation of semiarid rangelands. Weed Sci. 19:329-32. Smith, A.E. 1972. The hydrolysis of 2,4-dichlorophenoxyacetate esters to 2,U-dichlorophenoxyacetic acid in Saskatchewan soils. Weed Res. 12:364-72. Upchurch, R.P. 1973. Herbicides in plant growth regulators. In Organic chemicals in the soil environment, Vol. 2. C.A.I. Goring and J.W. Hamaker, eds. Marcel Dekker, Ire,, New York. 968 pp. 59 � 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. 010 Folder The folder containing the original item. 0092 Series The series number of the original item. Series 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 Blackman, Geoffrey E. John D Fryer Anton Lang Michael Newton Description An account of the resource <strong>Corporate Author: </strong>National Academy of Sciences, National Research Council Date A point or period of time associated with an event in the lifecycle of the resource 1974-02-01 Title A name given to the resource The Effects of Herbicides in South Vietnam: Part B, Working Papers, February 1974: Persistence and Disappearance of Herbicides in Tropical Soils Subject The topic of the resource soil survey biodegradation ecological recovery Southeast Asia https://www.nal.usda.gov/exhibits/speccoll/files/original/8dd45d44d672ef1c545bc2019d64ceab.pdf dbb36089c3a06149ea2ff579fb93a3a3 PDF Text Text Item ID Number: Author Corporate Author 00054 Boush, G.M. University of Wisconsin, Department of Entomology, Madison, Wisconsin Pesticide Degradation By Marine Algae Journal/Book Title Year 1975 Month/Day A ril Color W Number of linages 23 DeSCrlptOU Notes Contract N00014-67-A-01 28-0023, Task No. NR 306-061 P ' Friday, November 17, 2000 Page 54 of 57 �Boush,G.M., et al 1975 Pesticides Degradation by Marine Algae .AD A 008 275 AD-A008 275 PESTICIDE DEGRADATION BY M A R I N E ALGAE G. M. Boush, et al Wisconsin University P r e p a r e df o r : Office of Naval Research 1 April 1975 DISTRIBUTED BY: Hiflimi TttfciHtil hififiiititi ftftfrt V. 1 BEMRTMENT if C8MMERCE �118104 10 Off lev of Haval Research Contract H0001U-67-A-0128-0023 00 o o Task Ho. HB 306-061 PHIAL REPORT Pesticide Degradation by Marine Algae by G. N. Bousb and F. Matsumura University of Wisconsin Department of Entomology Madison, Wisconsin 53706 April 1, 1975 Reproduction in whole or in part is permitted for any purpose of the United States Government Approved for public release: distribution unlimited This research was supported in part by the Office of Haval Research, Haval Biology Program, under Contract Ho. H0001U-67-A-0128-OO23, HR 306-061. Reproduced by NATIONAL TECHNICAL INFORMATION SERVICE US (topulnmt ol Comn«« Sprl.gfM<l. VA. 2JI51 �DOCUMENT CONTROL DATA - R I D l*ll> i <im«l w *nl*n4 wh*r< lh» •rmtmll ttffrl /« «•/««»<«•«) I*. RIPORT f C C U R I T V CLASSIFICATION I. ORIGINATING A C T I V I T Y Department of Entomology University of Wlseouln Madison, Wisconsin 93706 1. HtPMT TITL« Pesticide Degradation by Marine Algae 4. DC«CRIPTIV« NOTCt (Tyf» 01 np*r« and Final report »- »0 TMOMMI rFinf HMwTiiMffi Mail. lm»l MM) George M. Boush and Fumio Matsumura • - RCPOMT OATC If. TOTAL NO. OF FAOU 7». NO. Of KCFI April 1, 197? 0 M. eONTHACT ON CHANT NO ». PHOJ.CT NO. H B HOOO1H-OY-A"0120-0023 M. OMIOINATOITl KCPOHT NUMBCHI*) 306-061 M>. OTHCM HKPOMT NOIII (Any oHm Itwmbfn timt muy b* •• 10. DISTMIC JTION STATKMCNT Standard Distribution List II. SUPPlCMCMTAItV NOTt* 12. SPONSORING M I L I T A R Y A C T I V I T Y Office of Naval Research Rone I AStTRACT Various algae species are tested for their susceptibilities towards chlorinated hydrocarbon insecticides. Deildrin, which is the most frequently found pesticidal contasdnant in the 1)8, and its analogs were found to inhibit the growth of certain of algae species. Anacystis nidulans in particular showed narked susceptibility to endrin dieldrin, ketoendrln sad photodieldrin. This species was also susceptible towards dieldrin metabolites such as metabolite F and 0. Among DDT metabolites DDD (TDE) was found to be the most toxic material) followed by DDE, DDT and FW-152. It has not been mown that DDT should be more toxic to algae. In terms of acute toxiclty phenylmercuri acetate was by far the most alglcidal agent among all pesticidal chemicals tested. This pesticide is toxic to both A. nidulans and A. quadruplicatum at the concentration of 1 ppb. Algae, along with other plankton, are known to bioaccumulate pesticides and thereby play a vital role in the process of food-chain accumulation of these micropollutants Oar studies indicate that the rates of pick-up of pesticides are very rapid. To study the feasibility of constructing a model ecosystem we used algae as a key food chain organism. By this way we could demonstrate that TCDD, the most toxic contaminant of 2, >-T does not really accumulate in the aquatic organisms as compared to DDT. Algae as a whole are not very active in degrading pesticidal chemicals in vivo. They were found to play, however, a key role in the process of environmental alteration of pesticidal residues. The way they participate in such processes was found to be through aynerglstlc actions on photochemical reactions. Algal products, when tested in the form of aqueous extract from dead algal cells, were found to be excellent photosensitizers for DDT and mexacarbate degradation by the sun-light f«ini»-i>t.»rt «»n i«miO 00.^.1473 S/N 0102^)14^600 (PAGE t ) PLATE NO. 21BJ6 Security CUmlTic«tion PWCB SUBJECT TO CHANGE �Ill LINN A • HIV ••no* •OLB *T •»t oblarlmtrt hydrocarbon* ioMMllua quadrupliotiai •icrcfcial tegxmtetion DDT up-tak* t>ceuul»tlon food elmini teniflAl realdue* TCDD •otel •eocystmu i X i • DD (PAGE 2) < BACK » LIMN e LIMN- • N«Ll Security CU»ific«tlofi tt«LI •T �,UA«* /v/ TABLE CT COITOrTS Page or RESEARCH ACCOMPLISHED - r - i I. - Effect* of pesticide* on plankton - — . . — . — . 1 . II. - Effect* of degradation products . . — . — . . 2 . . .. III. > Effects of pesticide •icro-contaainaats -— - 6 nm or -nccraiic/u REPORTS -....17 or ALL PUHLICATICHS ..................i? MUOR ACCCMPLISBffiHTS - - 16 DOCUMENT caiTROiL DATA - R & D 19 KEY WORDS . - - - . _ . . . 20 �-1- SttMAHY OF RESEARCH ACCOMPLISHED I. Studies relating to the affects of pesticides on plankton. It has been suggested that the varying resistance of marine phytoplankton to effects of chlorinated hydrocarbons could have far-reaching effects in terms of phytoplankton population balance. Studies in this laboratory have also shown varied growth inhibition of planktonic bluegreen algae by chlorinated hydrocarbons. TABLE 1 shows the effects of aldrin, dieldrin, and endrin on growth rates (generations per 2U hr.) of Anacystis nidulans (freshwater species) and Agme'nellum quadrupllcatum (marine species).It is noticeable that generally the marine Isolate is more tolerant than the freshwater isolate. This may be due to the influence of the growth medium on the insecticide. The toxiclty of a pesticide in aquatic environments may vary according to the physical characteristics of water. Although much variation is noted in the data, the general trend indicates both algae are tolerant to these insecticides except at concentration* higher than reported in natural waters. Also notable is the sensitivity of A. nidulana to dieldrin, an isomer of TABLE 1 Growth Response of Agmenellum quadruplicate* and Anacystis nidulans to Aldrin*, Dieldrin, and """^ ppb Aldrin A. q u a d r u - A . 0 pllcatum*5 nidulans 950 U75 95 19 0.2 Control 6.2 * 0.7 7.1 * 0.5 6.6 * 1.2 6.8 * 0.3 6.U * i.o 6.6 * 0.5 Dieldrin Endrin A. q u a d r u - A ^ A. q u a d r u - A T plicatum nidulans plicatum nidulans 6.U * O.U 5.8 i 0.9 6.7 * 0.2 6.8 * 0.5 7.1 * O.U 7.2 i 0.3 6.S * O.U 6.0 - 0.8 6.0 - 1.0 6.5 - 0.7 5.3 * 1.2 6.2 i 0.9 3.2*0.8 3.9*1.5 6.9*0.6 7.2*0.9 6.7*1.3 . 6906 .*. 3.5 * 0 9 . U.8 * 1.5 U.9 * 2.2 5.6 * 1.3 *0.3 6.6 * 0.5 2.2 * 0.7 3.2 * 1.0 6.3 * 0.3 6.6 * O.U 7.0 * 0.5 6.6 * O.U Concentrations for aldrin 9 0 U55, 91, 18 and 0.2 ppb. 1, Values reported as number of generations per 2U hours, represents mean of 3 to 5 replicate cultures Aajaenellum quadrupllcatum (strain FR-6), Anacystic nidulans (strain TX20) In preliminary experiments the growth response of these two algae was also tested against phenylaercuric acetate (FHA), an algicide and fungicide once used extensively in 'Industry. The results are summarized in TABLE 2. �-2TABIE 2 Susceptibility of Two Species of Blue-Green Algae Against Fhenylmercuric Acetate 0.10 A. nidulans A. quadruplicatum . 109 Phenylmercuric acetate PPb) 0.75 0.25 0.50 1L.OO 100 109 112to 10 0 8° 7 6* 8 112 1.0 00 0 0 Expressed in % relative growth against controls as 100. Only 3 of U replicates grew during the experiment. *? Only 2 of k replicates grew. Only 3 of 6 replicates grew. Thus results showed A. nldulans to be affected by as little an 0.50 ppb PMA. At this and higher concentrations growth was irregular and vts preceded by lag phases. In view of mercury contamination reported in oceanic environments it was of interest to also consider the toxicity of FHA to A. quadruplieatum. Duplicate cultures in two experiments yielded the growth values as compared to controls (TABLE! 2). Thus it is evident that A. quadruplicatum is more tolerant to FMA than A. nldulans; however, neither organism showed any growth at 10 ppb IMA. ~~ Much research has shown that in addition to growth, beneficial activities of microorganisms can be affected by pesticides as well. Bacteria in soil which convert organic matter to ammonia, and several herbicides have been seen to influence soil nitrification. II. Effect of degradation products. Pesticides, as they may adversely affect microorganisms, involve not only the parent compound, but the intermediate and terminal residues of these compounds as well. Recent investigations have pointed out the potential of certain "terminal" residues to be as Ijxic as the original pesticides. Data from this laboratory alco support this obeservation. Anacystis nidulans and Agmenellum quadruplieatum were grown in media containing microbial degradation products of aldrin, dieldrln, and endrin. The data in TABIE 3 show that A. nldulans continues to be sensitive to photcaldrln and ketoendrin, two metabolites of aldrin and endrln, respectively. Agmenellum quadruplicatum appears resistant to both compounds. However, both organisms show of dieldrin, as shown in TABI£ U. formed microbially, photodieldrin by the action of UV or sunlight. continued sensitivity to metabolites While metabolites P and G are only is also known to form on plant surfaces Hence, it was of interest to assay the �-3- Qrowth Response* of Agmenellum quadruplicatum and Anaeystis nidulans to effects of Metabolites of Aldrin and Kndrin, toB-i»ll Photoaldrtn A. quadruplicatum A. nidulans ppb 950 *75 95 19 0.2 Control Ketoendrin A. quadrupllcatun A. nidulans 6.2 6.U 6.5 5.9 6.0 6.6 7.U - O.U 6.8 ± 0.2 7.3 * 0.3 7.1 * 0.8 7.3 * 0.6 6.6 i 0.5 * 1.0 * O.U * 0.7 ;0.7 * 0.6 t 0.5 5.3 -0.8 6.3 * 0.7 6.U 1 0.7 7.0 i 0.6 7.0 i 0.6 6.8 ± O.U fc.5 * 1.1 3.5 - 0.1 6.0 - 0.8 6.6 ;* 0.2 6.3 - 0.1 6.8 t O.U Conditions as in 1AHUE 1. TABLE Growth Response of Agmenellum quadruplicatum and Anaeystis nidulans to Metabolites of Dieldrin Metabolite F A. A. quadrunidulans ppb 950 475 95 19 02 . Control 5.* I 0.7 5.9 ±0.9 6.1» - 1.2 6 8 - 0.8 . 6.2 ±0.9 3.5 U.8 6.5 7.2 6.7 6.9 *Q.k ±0.3 ± 0.5 ±0.5 ±0.3 ±0.6 Metabolite 0 A. quadruA. plicatum nidulans Photodieldrin A. quadruA. plicatum nldulans M - 1.0 k.Z - l.U 6.U ±0.8 5.9 * 0.6 6.U * 0.9 6.7 * 0.1 6.U i 1.2 6.7 * 0.5 6.5 ± 1.1 6.U ± 0.1 6.2 ±0.9 6.9 * 0.6 5.6 - 1.0 6.9 ±0.5 6.6 ±O.U 6.U ±0.6 7 1 ± 0.2 . 6.2 ± 0.9 u.o Jo.u 5.3 7.2 7.1 71 . 6.9 ±0.8 ± 0.1 ±0.9 ±0.8 ± 0.6 response of other algal species to photodieldrin. TABUS 5 shows that of the algae tested, Hostoc sp. and the green alga Chlorella spporensis appeared only slightly affected by photodieldrin at high concentration, while A. nidulans was most inhibited. Continued growth of A. nidulans in successive cultures in medium plus various levels of photodieldrin did not improve initial growth rates. Other attempts to improve the organisms' tolerance by varying growth conditions and dark incubation intervals were unsuccessful. �ft. TABLE 5 Growth Response of Several Blue-Green Algae* to Photodieldrin*»c Alga* I II III V VI* VII3 Control 2.6 * 0.1 5 0.2 3.5 * 0.2 7.0 * 0.7 - v.i(,\ 6.2 * 0.6}? 3.3 * 0.3* ' 6 5 - I°- 2 /l! 3.6 * 0.5<6> 0.2 ppb 2.7 * 3.6* 6.7* 6.9* 3 3 7.1 * "-I""/^ 3.7 * 0.7<6> - ; 19ppb >..& * 0.2J3J - 950 ppb 95 PPb tf\ 2.7* 3.6* 7.1 - w.«/_\ SilitjSl I:!!?:!?! 3.5 * 0.5<6> k'.O * 2.9 * 0. 3.3 * 0. 5i2 3.2 7.1 3.fc * 0. * 0. * 0. * 0. * I • Anabaena variabilia; II » Hostoc «p. ; III » Anacyitig nidulana; IV Chlorella • _^___^ V * Agpenellum quadruplicatua (strain BS-l); VI » Agaenellua quadruplicatum' ( train PR-6J; VH Coceochloris elabans. a All algae tested here are blue-green species, except C soproensis, a gre* * b Values reported as number generations per 2k hours. ~ e Rubbers in parentheses indicate replicate cultures. d Indicates marine isolate; others are freshwater isolates. �-V ' S"*-^ •'' ,;;,•'*:tBT*1S, ". '!.-:i,"i £"«?£ i«*3. ':r E'-f».V< ;<*--;---;; ^m •BHI |J£J2*g|nlB£gB!|»Dxg ^Sf^^i^HKi^s^i^SiSfff^iHSH9^tMfUffS^HniSSo^3i^^SISKSS^SSS3^S^SSSESS3^Si^ Vjjpirfhi'^H -5ThHl£ 6 Growth Response of Agnenellum quadrupllc '.turn and Anacystis nidulans to DDT and its Metabolite** »» ppb DDT A. quadruplicatua 885 442 88 18 0.2 (13) 12) »0 0.0 5.3 5."» G.2 6.2 6.7 6.3 * 1.0 6 * 0.9 5 * 0.4 3 ± 0.3 ( 4 * 0.4 ( 4 * 0.6 ( 8 791 395 79 8 0.8 0.0 ODD I 1.1 J 1.3 * 0.6 ± 0.4 * 0.6 ± 0.7 791 395 79 8 0.8 0.0 DDE 5.1 5.* 6.3 6.5 6.1 6.1 3.4 5.3 6.7 6.8 7.0 6.k * 0.0 ( * 0.8 ( i 0.1 ( i 0.3 [ * Q.k * 0.7 1[ 6.9 * 0.2 ( 6.7 - 0.0 ( 7.k * 0.2 [ 7.1 * 0.0 7.k * 0.2 6.9 * 0.2 700 350 70 7 0.7 DBA ' 0.0 DBF 6.5 * 0.3 720 6.9 6.6 6.8 6.6 6.9 360 72 7 0.7 0.0 FW 152 ( 4) ( 4) (16) • 0.3 - 0.1 * Q.k * 0.1 * 0.2 8 0.8 0.0 5.8 - l.l 5.8 * 0.8 6.5 * 0.6 <f M "gUSjTaiJl-i-liai 2)c 2c k 2 2) 2) 6) 4.2 * 0.3 5.3 * 0.3 6.2 t 0.3 6.8 ± 0.3 6.4 * 0.3 7.0 4 0.3 ( j^ C 2) 2) 2) 6.2 * 0.1 2 5.9 * 0.2 2) 6.5 * 0.4 2 2) 3) 6.8 ± 0.0 6.0 ± 0.3 2 2 2 2 2 3) 6.4 t o.l 6.2 i 0.0 6.8 * 0.2 7.1 £ 0.»» 6.6 ± 0.1 6.0 ± 0.3 ( 2) 6.8 * O.I* ( 3) 6.6 * 0.3 ( »») •AM «t^«%v^ srn^t**^ •••••ilia •• 5 6.2 * 0.2 4 6.8 J 0.7 4 6.8 t 0.5 16) 5.4 - 2.0 6.5 * 1.5 U 6.5 * 0.4 ( 2) 7.2 * 0.0 ( 2 6.8 * 0.5 ( 7 6.6 i 0.6 i. 6) o o 7) 2 2 3 2 2 2 2 2 3) 6.3 i 0.4 2) 7.2 t 0.2 2) 7.4 * 0.2 ( 2) 7.2 ± 0.1 ( 2) 7.«» * 0.2 ( 2 7.0 * 0.3 ( 3 • Values expressed as number of generations/24 hours. • 16 4.6 * o.O 6.1 - 0.1 4) 6.8 * 0.3 3) 6.6 * 0.* 3) 6.3 * 0.* 4) 821 410 82 A. nidulans *«w* «MMt1 4 t^+^m Growth occurred in only one or two of several replicates. �-6TABUE 6 Incorporates data of several growth-response experiments of A. quadruplicatum and A. nidulans to DDT and five DDT-analogs. Again, although much variation occurred, the data show little growth-rate depression of either alga at concentrations below 100 ppb insecticide. Both species show continued, perhaps greater, sensitivity to the two analogs DDE and ODD, as well as DDT. However, the more polar compounds DDA, DBF, and FW 152 apparently have little effect as seen in these experiments. III. Microbial uptake and accumulation. Toxicants, one taken up by primary producers such as marine algae, can be passed up the food chain to higher trophic levels. In addition, many toxicants can, depending upon the environmental conditions and species involved, be accumulated within the cell to levels many-fold higher than ambient. From the few studies available, accumulation appears to be primarily by inactive surface adsorption. However, Glooschenko found that dividing marine diatom cells in light accumulated 2 °3Hg longer than did non-dividing cells, thus indicating the possibility of some active uptake mechanism. The exotic and demanding minor-element nutritional requirements of many organisms would tend to support active uptake in some instances. We have found that yeast cells of Rhodotorula gracilis rapidly accumulated yii> of the DDT in a 2-ppm aqueous solution! Likewise, another yeast, Torulopsis utilis, took up 9^6 of the DDT in 3 minutes. In TABLE 7, showing the percent radioactivity of the ^C-DDT in the cellular fraction, the control values showed a random distribution of DDT between the medium and the cellular fractions, ranging from 21 to 56£. These results were obtained by centrifuging, decanting, and filtering the aqueous medium, and the distribution of i^C-DDT between the medium and cellular fractions was determined by liquid scintillation counting. In contrast to the erratic control values, the cellular fractions accumulated DDT at a constant rate over the <X)£ level after 3 minutes. An extract of R. gracilis was prepared by sonicating the cells before the additior~of the ^C-DDT. The sonicated pellet was found to accumulate an average of 96£ of the DDT. We also attempted to correlate cellular lipid content with the uptake of DDT. Rhodotorula gracilis when grown on a medium rich in carbohydrates and deficient in nitrogen and phosphorus will produce approximately 6o£ lipids, whereas when not deprived of N and P, lipid production is reduced to approximately UOjt. When cultures were grown under both circumstances, no differences in DDT pick-up were noted. It is apparent that the complexities of pesticidal-microbial in- �-7- terrelationahips warrants continued study. It has been amply demonstrated that members of the microbial world vary widely in their response to pesticides and that several factors may influence the toxicity of pesticides. Likewise, the microbial tolerance of pesticides may be affected by growth conditions, physiological condition of the cells, and various stress factors which might exist in natural populations (e.g., temperature, limited nutrients, competition). For example, growth experiments with A. nidulans established separate tolerances of 1% NaCl and 800 ppb DDT (Batterton, Boush and Matsumura, 1972). However, growth of this alga is severely inhibited in meul^u containing both 1% NaCl and 800 ppb DDT. Figure 1 illustrates relative growth of A. nidulans in various concentrations of DDT r.nd NaCl. The resulting growth pattern indicates the combined stresses of NaCl and DDT significantly changes the tolerance of A. nidulans to either substance. However, in similar experiments with test-tube cultures, growth inhibition was contraindicated when the calcium concentration of the growth medium was increased fivefold. It is particularly interesting to note the similarities in nearly all c the uptake-accumulation studies. First, pick-vp of the toxicant is extremely rapid—varying from a matter of seconds to a few minutes. And secondly, removal of the toxicant from the medium is quite high— usually more than 90jt of the total being removed by the cells (dead or alive), even when ambient levels were many-fold higher than those usually encountered in nature., However, one factor should not be ignored. Few, if any, studies have included competitive adsorptive substrates. Might not DDT, for example, readily aifsorb to organic matter, silica, etc., if available? The apolarity, affinity for lipids, and low water solubility of virtually all of the persistent insecticides make studies in aqueous substrates difficult. Of even greater importance, cau we extrapolate from our work, even to a United degree, to conjecture as to what occurs in nature? It is doubtful if we can overstress the important of microbial accumulation. After more than 25 years of world-wide use and study, the real threat from persistent pesticides is in their unfortunate ability to concentrate with food chains. This would not occur were the toxicants not picked up from low background levels, concentrated in the cell, and finally, stable for considerable periods of time. The results shown in Figure 2 indicate the general susceptibilities of brine shrimp, Artemia salina, to various terminal residues and analogs of DDT. It can be seen that these analogs, though many of them have been regarded as non-insecticidal, are indeed toxic to this species. IV. Effects of chlorinated insecticides on NaCl-tolerance mechanisms. Since Na+, K -ATPases have been known to serve as the enzyme re- �-8sponaible for Na+ and K+ exchanging across many biological membranes, we have decided to study first the effect of DDT on the salinity regulatory mechanism of a blue-green algae (Batterton et al., 1972). The initial experimental results indicate that the susceptibility of a blue-green alga, Anacystis nidulans, against DDT varies greatly under different salt concentrations. At high NaCl concentrations the bluegreen alga becomes extremely sensitive to DDT. It is clear from the result that this fresh water species loses its NaCl-tolerance capability in the presence of a low level of DDT: the level normally would not affect the specie. . A spearate experiment in vitro showed also that DDT was indeed an inhibitor of Na+, X+-ATPases of A. nidulans. blocking all ouabain sensitive ATPase activities. The most important indication that the ATPase is related to the Nad-tolerance mechanism comes from the in vivo finding that Ca , when added externally to the medium, can antagonize the effects of DDT. TABLE 7 Percent Radioactivity of lJ*C-DDT in Yeast Cells Time (minutes ) 17.5 32.5 Culture 2.5 7.5 12.5 Control Torulopsis utilis Rhodotorula gracilis Extract-R. gracilis Protein producing medium-R. gracilis Lipid producing medium-R., gracilis 21 92 97 98 U2 96 98 56 91 97 38 95 98 Average 39 96 97 96 97 96 97 95 98 . 97 In another set of experiments, the brine shrimp, A. salina, was subjected to various chlorinated hydrocarbon insecticides under different salt concentrations (Figure 10). The results clearly indicate that the effects of these chlorinated hydrocarbons are strongest at either extremely low or high salt concentrations. The brine shrimp is noted for its great capabilities of tolerance on different salt concentrations. It is often found in abundance in inland salt lakes (e.g., salt ponds and lakes in Utah) where the salt concentration is so high that no other organism can survive. The loss of salt tolerance mechanisms for this species by the presence of these insecticides is, therefore, quite a surprising phenomenon. The examples illustrate only one aspect of pesticidal pollution. It is important to note, however, that such a finding comes from fundamental knowledge of the chemical interactions with biological materials. �-9AN'ACYST.'S _N!PULANS FIGURE 1. Relative growth of Anacystis nidulane in response to varied DDT and NaCl concentrations. Liquid culture (15 ml) in 60 mm petri dishes inoculated ( 2 , 0 cells/ml) and incubated 72 hr. under 1000 200 ft-c fluorescent lamps at 37° C. After correction for evaporation growth was measured as optical density at 660 run. All O.D. values normalized to 1.0. «* 0.4 a* u> CONCENTRATION xo IN PPM FIGURE 2. Differential toxicities of DDT analogs and metabolites on brine shrimp, Artemia salina; 2k hours exposure at 2l*° C. �" •• ' • > , • • - . -10- It is aJ.so necessary to stress that those stable terminal residues and contaminants would not have been detected from the environments if not for the specific knowledge accumulated through basic researches in the laboratory as to their chemical characteristics and behavior. Factors involved in the interactions of pesticides with variouii ecosystems are numerous and complicated, but it certainly is hoped that there are a number of rate-limiting, key factors that can be analyzed through controlled laboratory experiments. V. Effects of pesticide micro-contaminants: Model ecosystem study. While the problem of pesticidal contamination of the environment is far from beirg solved, considerable useful information has emerged from the research efforts made by many scientists in recent years. First, we now know by experience that the chemicals that cause environmental problems are the ones which are extremely persistent in nature, biologically active, and easily concentrated in biological systems. Compounds which lack any of the above qualifications usually do not play any significant role in pesoicidal pollution no matter how acutely toxic they are. The above analysis becomes more important, when one insiders other aspects of pesticidal pollution. For instance, we are concerned about only biological effects in considering pollution, with particular emphasis on the effects on non-target organisms. In the case of polychlorinated dibenzo-p-dioxins, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the question of bioactivity is indisputable, as it is one of the most toxic compounds known to occur as a pesticidal impurity. Its chemical stability is also questionable. Thus the central question of its hazard to the environment must be studied from the viewpoint of bioconcentration in various ecosystems. Published data on environmental fate of chlorodibenzo-p-dioxins are scarce at present. For instance, residues of dioxins were not found in several aquatic animals at detection limits of 0.01-0.01* pg/g. In the study reported herein we have made efforts to measure the degree of bioaccumulatior. of TCDD in relation to well established pesticides by using several model ecosystems. The data are still preliminary, In that several model ecosystems are still being compared for their relative merits in assessing the actual impact of pesticides in nature. 'The data obtained have been, however, useful in assessing the relative tendency of a pesticide in comparison with other pesticides. Materials and Methods—Approximately 100 microbial strains which have previously shown the ability to degrade persistent pesticides were screened for their ability to degrade TCDD. Screening was carried out and the metabolic products were examined by thin-layer chromatography (TLC) by the method of Matsumura and Boush. The pesticides (0.1 umole each) were deposited on 1 g of clean sea sand, which was placed on a *7* TS '*" ''' Urn *" '1 �-11column of sandy loam type soil. Water was then slowly dropped onto the surface of the sand at a rate of approximately 2 rnl/hr. The water and sections of soil were extracted with chloroform. Three groups of invertebrates were used for the pesticide accumulation study: Qatracoda species, Artemis, salina, and Aedes aegypti larvae, and one fish species northern brook silverside, Laludesthes sicculus sicculus. Pour pesticides were selected from representative groups of important compounds: dioxin (TCDD), DDT, /-3HC, and zectran. All compounds were l^-labeled in the benzene rings. Three model ecosystems were used to study bioaccumulation. In model I, the pesticides (5 and 10 pmole) dissolved in a solvent were added directly to water along with the primary food organism, such as algae and yeast, and this mixture was then added to the aquarium containing the invertebrate test organisms. In model II, the pesticides (20 pmole) were deposited on the inner surface of the glass container by evaporating the solvent to form a thin film. The primary food organism were grown in the container for 2U hr. and then transferred along with the culture media to the aquarium contairihg the test invertebrate organism. In model III, the pesticides (5 and 10 praole) were deposited on 1 g of sand and the solvent evaporated to form a thin film on the surface of the sand particles. The sand was added to the test aquarium containing invertebrates and/or fish. In all cases the test organisms were maintained in the aquarium at room temperature (2U° C), except for the fish cultures which were maintained at 12° C. Test organisms were either homogenized in counting solution or carbonized (Model 300 Packard Tri-Carb Oxidizer), and the amount of ^C02 measured. Measurements of the amount of labeled material in the water, primary food organism, and on sand and glass surfaces were made by extracting with chloroform. All studies were short-term (k-7 days), in small volume containers (200 ml). As shown in Table 8, the extent of translocation of TCDD from the sand to the organic soil layer is extremely small. Virtually no TCDD was found to leach out from the column. The mobility of TCDD in soil, therefore, must be considered much less than that of DDT. Thus, the mode of translocation of TCDD in the environment would be limited to movement of soil particles or dust-carried dispersion and biological transfer (but not plant-mediated transfer), particularly in aquatic environments. As for the microbially mediated degradation of TCDD, our current survey indicates that such capabilities are rather rare in nature. Approximately 100 microbial strains in which the ability to degrade persistent pesticides has been previously demonstrated were screened for this purpose. Among them, only five strains showed some ability to de- �-12- grade this compound. We ha e not been able to manipulate cultural conditions to increase the rate of degradation of TCDD in any of the microorganisms so far. In studying the extent of biological transfer of TCDD, three different model systems were devised. In model system I, pesticides in acetone were introduced directly into water along with the primary food organisms. In model system II, pesticides were applied to the inner surface of a glass container, and the primary food organisms were grown in the container for 2k hr. and were transferred to the aquarium. In model III, pesticide-coated sands were placed directly in the aquarium contairAig the test organisms. In the model I experiment (Table 9), DDT behaved quite differently from other pesticides, showing high degrees of affinity to each test organism, in close agreement with the phenomenon actually observed in nature. Although this model system is simple and appears to offer a quick straight-forward answer to the general tendency of pesticidal accumulation by biological systems, it has one weakness, i.e., that one is forced to work above the limit of water solubility of some of the compounds. TCDD for instance was measured at a level 100 times its water solubility. Also the extent of direct pick-up due to partitioning and food intake is uncertain. In the model II experiment, where only the portion of pesticide picked up by the primary food organisms and the media were introduced into the test aquarium, the levels of total pickup were further reduced in the case of TCDD (but not DDT) (Table 10). To circumvent the problem of solubility, the model III system was devised. In this way, only that portion of pesticide that is soluble should be present in water at any time. The results shown in Table 11 indicate that the rate of TCDD pick-up is extremely low in brine shrimp and fish under the experimental conditions. Mosquito larvae, which are bottom feeders, showed a surprising rate of TCDD pick up. The reaction is not at its maximal rate, since further increase in the level of the pesticide apparently increases the pick up by the larvae. Also noted is the difference between the bioconcentration pattern in fish as compared to other invertebrates. y-HHC, in particular, shows high degree of concentration in fish. To study the effects of food consumption, the same test was repeated in the presence or mosquito larvae. As expected, the level of TCDD (Table 12) in the fish increased in the presence of mosquito larvae, which are the best concentrators of TCDD among the organisms tested. On the other hand, the levels of other pesticides did not significantly change, indicating that the route through ingestion of mosquito larvae does not represent the major source of uptake in these pesticides. It is apparent from these data that the reaction of biological concentration is greatly influenced by the external conditions and the design of the experiment, the physical and biological nature of the �,. ,3t;i,,-*,i[*-i*>»^ ',.', "$&•:•" "'' *?• ., *uf. ; '.Tjr • .,!«,. . u. -13organisms, and by chemical characteristics of the pesticides. To facilitate understanding of the role of chemical nature of pesticides in determining the rate of bioconcentration, a comprehensive list has been prepared to illustrate their Important properties (Table 13). It can be seen here that general tendencies of bloaccumulation in invertebrate species follow closely the trend of the partition coefficients. In model II experiments, however, the valuos for TCDD come much lower than expected from this rule. Thus it is likely that water solubility (and solvent solubility) must play an important role where the initial pick-up is the rate-limiting factor. It is apparent that species-specific factors play a much more important role than once suspected. For instance, the pattern of bioaccumulation and concentration in fish is quite different from those in other organisms studies, in that both /"-BUG and zectran snow higher degrees of affinity than DDT and TCDD, respectively. Although the data are not sufficient to permit a definite conclusion, they suggest the possibility that water-soluble pesticides tend to accumulate in fish. TABLE 8 Vertical translocation of pesticides from sand to organic soil.a Pa s t ic ide c on tent, * DDT13 Top sand 0-0.5 cm 0.5-1.0 cm 1.0-1.5 cm 1.5-2.0 cm 2.0-2.5 cm Water 1st 2nd 3rd eluatec 50 ml 50 ml 50 ml Zectran" 90. Ul 7.32 1.0U 0.50 0.26 0.18 65.01 30.75 3.51 0.55 0.26 0.19 0.07 0.06 0.08 0.05 0.06 0.06 0.12 0.08 0.06 0.0*1. 0.02 1*9. U 17.6 29.1 0.09 10 x 1.5 cm glass column Pesticide introduced: 0.1 jamole each (33-8 for DDT, and 22.2 ;ig for Zectran) Water eluted per day, 50 ml for dioxin, 35.5 ;ig �-1UTABLE 9 Bloaccutnulation of pesticides by aquatic invertebrates for model I (pesticides introduced directly into ambient water with the primary food organisms). Test organisms (primary Pesticide food) Original concentration in water, ppb Final concentration found in test organisms, Concentration ppb factor Paphnia (algae) Dioxin DDT Zectran 32. ** 35.8 22.2 1,592 Ijl»,l6>» 1,969 '•9 123U 89 Ostracod .Tfalgae ) Dioxin DDT Zectran 32. k 7,069 50,771 7,265 218 1U18 327 Brine shrimp (yeast) Dioxin DDT /•-BHC Zectran 35.8 22.2 16.2 If 956 12,336 2,688 17.9 1U.7 11.1 121 689 183 1U 155 TABIE 10 BJ©concentration of pesticides by aquatic invertebrates for model II (primary food organisms allowed to pick up pesticide from glass surface and then given to the test organisms). Test organism (primary food) Pestici-ie Original amount, jig (theoretical concentration, ppb) Daphnia (algae) Dioxin DDT Zectran 6.U8 (162) 3.58 (179) 2.22 (111) Ostra :od (algae) Dioxin DDT Zectran 6.U8 (162) 3.58 (179) 2.22 (111) of the test. Final concentration found, ppb Water Test aquarium organisms O.U 22.9 15.1 2.6 50.8 H:.<, 879 U3.123 37,l»99 279 36,391 6,177 Concentration factor* 2,198 1,883 2,U88 107 716 1U2 �TABI£ 11 Bioconcentration of pesticides by aquatic organisms for model III (pesticides introduced into system in the form of rp-iuues on sand). Amount of pesticide Concentration found, ppb Test Water (including food) organisms Test organism Pesticide Brine shrimp Dioxin DDT V-BHC Zectran 1.62 1.79 1.47 1.11 0.1 0.5 5.2 5-0 Dioxin 1.62 O.U5 2.1+0 0.85 1.40 Mosquito larvae PC DDT /-3HC Zectran 3.24 1.79 3.58 1.47 2.9k 1.11 2.22 Fish (silverside) Dioxin DDT y^-SHC Zectran 1.62 1.79 1.47 1.11 6.6 13.1 5.45 10.8 0 2.1 1.8 4.7 157 Concentration factor 1,570 3,092 495 89 6,184 95 18 " 4,150 0 9,222 5,000 16,765 21,571 220 221 0 89 8 2 458 2,904 *?• 218 12,000 14,250 30,200 1,450 2,900 213 —. 1,613 U5 w* �-16'CABLK 12 Two-step bioconcentrution of pesticide by mosquito larvae, and northern brook silverside (model III). Pesticide 1.62 Dioxin DDT /•-BHC Zectran Water (including Hood) 1.3 1.1 1.79 l.U? 1.11 1.9 5 Concentration factor Mosquito Fish larvae 3,700 17,900 690 0 Amount of pesticide jig Concentration found, ppb Mosquito Fish larvae 2.8U6 16,273 708 337 1080 76 383 0 5^ 306 600 15 TABIB 13 Physiocochemical characteristics of dioxin in comparison with other insecticides. Water solubility Solvent solubility Water solubility 0.2 1.2 100 10 ioio 1 Dioxin DDT Zectran /•-BHC 5 ppb ppb ppm ppm Partition coefficient (vs. hexane) 106 10 * 105 100 ,0* 1000 0,0 100* 1,700 Benzene solubility, g/10Q g O.OU7 80 80 - Estimates The data indicate that TCDD is not likely to accumuhte in as many biological systems as DDT. This is likely because of TCDD's low solubility in water and lipids as well as its low partition coefficient in lipids. Since microbial degradation is not expected to be a rcajor factor, the predominant mode of elimination of this compound in the environment is photodecopposition by sunlight. VI. Degradation of pesticides by algae. Three salt water algae, Porphyridium sp., lAinaliella tertiolecta, ard Coccochloris elabans strain Di, were selected and studied for their ability to degrade a number of environmentally important pesticides (2,U-D, 2,U,5-T, mexacarbate, and DDT) and a pesticide contaminant �-17(tetrachlorodihcnzo d i n x i n ) . Pure algae cultures were grown on a dci'initlve raedla under laboratory c o n d i t i o n s . The compounds were studied under the following conditions: (l) growing al|,ao undor 2't hour light, (2) heat killed algae under 2'* hour light, (3) growing algae undor total dark (standard mrdia amended w i t h glucose) and ('0 controls (the medium alone but no algae) under ?.h hour light. The above studies were conducted for 7 days. Three co-npomids, 2,U-D, 2,U,5-T and TODD were reslstent to breakdown under those conditions. Mexn.carba.te, and DDT were readily broken down. Although inexaoarbatc was defended in the presence of light alone, in the presence of algae ( l i v i n g and d e a d ) , over '(0$ of the compound was converted to water soluble materials not fo^nd in controls. These compounds became solvent exl.ractable only after acid hydrolysis. A chloroform soluble metabolite was also dotcc'-ed v/hich was not found in c ntro.ls. This material had an Rf (ethyl ether, hexane, othanol; Y7:?0:3) belween methyl fonaami.do and formamldo mexacarbate derivatives. Tlie degradation of DDT under the above light conditions seem to give a small amount of DDA. In the presence of algae (living and dead), under light conditions, two other compounds were formed. One compound has tentatively been identified as DDE. The other compound using three TT£ systems has been identified as DDOH. Due to the fact that dead algae also forms the metabolites of mexacarbate and DDT it is postulated that a compound is formed by the algae v/hich causes photo-decomposition. The identification of the metabolites and the nature of the photosensitizers are proposed for future study. INDEX OF TECHNICAL REPORTS 1. No Technical Reports have been issued. BIBLIOGRAPHY OF ALL PUBLICATIONS 1. Batterton, J. C . , G. M. Boush and F. Matsuraura. 1972. DDT: Inhibition of sodium chloride tolerance by the blue-green algae Anaeystis nidulans. Science 176: llUl-llU3. 2. I'atsumura, F. and H. J. Benezet. 1973• Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzop-dloxin. Environ. Health Persp. 5: 253-253. 3. Boush, G. M. . Effects of pesticide terminal metabolites on algae and brine shrimp. (In preparation). �Mt~ *J -18M.JOR Varioi'-. alijue species are tested for i.hclr :iii;;<:opUb1 lities towards chlorinated hydrocarbon Insecticides. D i o l d r l n , which in the rnout frequently found pestlcidal contaminant in the Uf5, and Its analogs were found to Inhibit the growth of certain of algae :;pccles. Anacjfstis nidulans In pai-ticular showed marked susceptibility to endrin, die.l.drin, ketixrulrin and p h o t o d i e l d r i n . This species was also susceptible towards d l o l d r i n metabolites r.uch as metabolite F and (}. Among DDT me tab o I. i to s ODD (TDK) was found to be the most toxic material; followed by DDK, DDT and KW-l'p3. It has not been known that ODD should be more toxic to algae. In terms of acute toxicity phenylmercuric acetate was by far the most algicidal agent among all pesticldal chemi c a l s tested. This pesticide is toxic to both A_. nidulans^ and A. !n,a-lnipj l-.ratvrn at the concentrition of 1 ppb. Algae, along w i t h other plankton, are known to bioaccumulate pesticides and thereby play a vital role in the process of food-chain aoetnaulattnn of these mieruponutants. Our studies indicate that the rates of pick-up of p o s t i c i i l o s are very rapid. To study the feasibility of constructing a model censysl<.3i we u;:ed algae as a key food chain or(7xnism. By this way we could dc-nonntrate that 'WJDD, the most toxic contaminant of 2,'^,5-T does not really accumulate in the aquatic organisms as compared to DDT. Algae as a whole are not very active in degrading pesticldal chemicals iji vivo. They were found to play, however, a key role in the process of environmental alteration of pesticidal residues. The way they participate in such processes was found to be through synerfjistic actions on photochemical reactions. Algal products, when tested in the form of aqueous extract from dead algal cells, were fouiid to be excellent photosensitlsers for DDT and mexacarbate degradation by the sun-light (simulated sun lamp). � 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. 005 Folder The folder containing the original item. 0054 Series The series number of the original item. Series 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 Boush, G.M. F. Matsumura Description An account of the resource <strong>Corporate Author: </strong>University of Wisconsin, Department of Entomology, Madison, Wisconsin Date A point or period of time associated with an event in the lifecycle of the resource April 1 1975 Title A name given to the resource Pesticide Degradation By Marine Algae Subject The topic of the resource biodegradation dioxin pesticide testing 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. 092 Folder The folder containing the original item. 2303 Series The series number of the original item. Series IV Subseries IV 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 Givaudan, L. Title A name given to the resource Memorandum: Proposal for the Natural Degradation of TCDD, from L. Givaudan to President of the Regione Lombardia, March 10, 1977 Subject The topic of the resource incineration biodegradation dioxin https://www.nal.usda.gov/exhibits/speccoll/files/original/b29b54a15edda14ac6493b0c79bcf958.pdf 7aeb403cf849e31e37aa9c0d15379e01 PDF Text Text Item ID Number: AllthOT Corporate Author 00103 Harrison, Don D. Environics Office, Air Force Armament Laboratory, Eglin AFB, Florida Report/Article Tltlfl Residual Levels of 2,3,7,8-Tctrachlorodibenzo-p-Dioxin (TCDD) Near Herbicide Storage and Loading Areas at Eglin AFB, Florida Journal/Book Title Year wn Month/Day February Color Number of Images 33 DeSCrlptOD NOtOS Report No. AFATL-TR-79-20; PE: 62602F; JON: 06CD0101; 3 partial duplicateserrata for document. Full document has already had errata pages replaced. Friday, December 08. 2000 Page 103 of 106 �AF ATL-TR- 79-2O Residual Levels of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD)Near Herbicide Storage and Loading Areas at Eglin AFB, Florida MMJ. HARRISON RICHARD C. CREWS FEBRUARY 1979 FINAL REPORT FOR PERIOD JANUARY 1976-DECEMBER 1978 Approved for public release; distribution unlimited Air Force Armament Laboratory AIR FORCE SYSTEMS COMMAND*UNITED STATES AIR FORCE+EGLIN AIR FORCE BASE, FLORIDA �UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 1. REPORT NUMBER 7. OOVT ACCESSION NO 3. RECIPIENT'S C A T A L O G NUMBER AFATL-TR-79-20 4. TITLE (md Subtitle) 5. TYPE OF REPORT ft PERIOD COVERED RESIDUAL LEVELS OF 2,3,7,8-TETRACHLORODIBENZO-pDIOXIN (TCDD) NEAR HERBICIDE STORAGE AND LOADING AREAS AT EGLIN AFB. FLORIDA Final Report: December 1978 January 1976 6. PERFORMING 0=»O. REPORT NUMBER e.~"C*GNTRACT OR <3RANT"NUMBER(«J 7. AUTHORS Don D. Harrison Charles I. Miller Richard C. Crews 9. PERFORMING O R G A N I Z A T I O N NAME AND ADDRESS 10. PROGRAM ELEMENT, PROJECT. T A S K AREA ft WORK UNIT NUMBERS Environics Office Air Force Armament Laboratory Eglin Air Force Base, Florida 32542 PE: 62602F JON: 06CD0101 12. REPORT DATE II. C O N T R O L L I N G OFFICE N A M E AND ADDRESS Air Force Armament Laboratory Armament Development and Test Center Eglin Air Force Base, Florida 32542 February 1979 13. NUMBER OF PAGES 32 14. MONITORING AGENCY NAME fi ADDRESSfl/ dlllerent from Contr<-l!tng Ottice) 15. SECURITY CLASS, (ol this report) UNCLASSIFIED I5«. DECLASSIFI CATION''DOWN GRADING SCHEDULE 16. DISTRIBUTION STATEMENT fof ffif* Report) Approved for public release; distribution unlimited. 17. DISTRIBUTION STATEMENT (ol lha abstract entered In Block 30, II different from Report) IB. SUPPLEMENTARY NOTES 19. KEY WORDS (Continue an reverse aide It necessary and Identity by black number) Hards band 7 2,4-dlchlorophenoxyacet1c add (2,4-D) 2,4,5-tr1chlorophenoxyacet1c add (2.4,5-T) 2,3,7,8-tetrachlorodibenzo-p-d1ox1n (TCDD) Herbicide Orange »0 (Agent Orange) Herbicide Blue Herbicide White Herbicide Purple D1ox1n A B S T R A C T fC'onllriuv nr- reverse side II necessary mud Identify by blorh number) A study was made of the residual levels of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) in the areas surrounding three hardstands on Eglin AFB, Florida, that had been previously used for storing end loading military herbicides. The study deals only with areas in the immediate vicinity of these hardstands and their associated drainage systems. Massive quantities of herbicides, including Herbicide Orange, were loaded at these hardstands between 1962 and 1970. Only one of the three storage and loading areas was found to be contarninatec wich TCDD. Hardstand 8 and the East End of Taxiway 9 were relatively free of FORM I JAN 73 1473 EDITION OF I NOV 65 IS OBSOLETE UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When Data Entered) �UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGErH'hsrt Data Entered) 19. CONCLUDED Chlorinated phenols Environmental Monitoring Phenoxy Herbicides Defoliant Environment Contamination 20. CONCLUDED TCDD residues in the soil. Soil from around Hardstand 7, the most intensively used hardstand, still has concentrations of TCDD as high as 275 parts per b i l l i o n (ppb), however. Concentrations of one-third that amount were present to a depth of one meter at two sampling sites. Soil contamination around Hardstand 7 is confined to a small area immediately surrounding the concrete pad. A map of TCDD soil concentrations is presented. "•"CDD was found to be present in biological organisms in the immediate vicinity of Hardstand 7 and the Hardstand Pond. However, no TCDD was found in any of the environmental samples collected in a bayou immediately downstream from the Hardstand 7 area. UNCLASSIFIED ',t< . i K i r y Ci • ','iiFH.ArioN O' '• "• r>>"'-' " ' " ' ' " »'" " " �PREPACK This report is the result of research conducted by the Air Force Armament Laboratory, ADTC from January "1976 to December 1978 under Air Force Exploratory Development Project 06CD0101. The Brehm Laboratory and Department of Chemistry at Wright State University (WSU), Dayton, Ohio, performed all tetrachlorodibenzo-p-dioxin (TCDD) analyses in this study except where noted. Most soil samples analyzed under this study employed Gas Chromatography-Quadrupole Mass Spcctrometry (GC-QMS) at the 100 - 1000 picogram/gram (parts per trillion) detection limit. Some soil samples and all biological sajnples analyzed by WSU were analyzed employing Gas Chroma tography - High Resolution Mass Spcctrometry (GC-HRMS) at the 30 picograrns/gram detection limit. The primary reason for the predominant use of the low resolution method was the lower cost of analysis. The reduced cost per sample permitted more samples to be taken. For the purposes of this study, a more intensive sampling effort was considered more important than a low detection limit. This report has been reviewed by the Information Officer (01) and is re leasable to the National Technical Information Service (NTIS). At NTTS it will be available to the general public, including foreign nations. This technical report has been reviewed and is approved for publication, FOR THE COMMANDER A. FARMER Thief, Environics Office (The reverse of this page is blank) ��TABLE OF CONTENTS Section Title Page I INTRODUCTION 1 II BACKGROUND 2 1. Description of Monitoring Areas 2 a. East End of Taxi way Number 9 2 b. Hardstand 8 4 c. Hardstand 7 5 2. Herbicides Used on Eglin 9 a. Herbicide Orange 9 b. Herbicide White 10 c. Herbicide Blue 10 d. Herbicide Orange II 11 e. Herbicide Purple 11 3. Chemical Properties and Effects of TCDD. . 12 III TCDD Analysis 14 1. East End of Taxiway Number 9 14 2. Hardstand 8 14 3. Hardstand 7 17 a. Sediment Samples b. Soil Samples 17 c. Biological Samples IV 17 21 CONCLUSIONS 25 REFERENCES 26 �LIST OF FIGURES Figure ' Title Page 1 East End of Taxiway Number 9, Eqlin AFB, Florida 2 2 Eglin AFB Herbicide Storage and Loading Sites with Associated Aquatic Drainage Areas 3 3 Hardstand 8, Eglin AFB, Florida 4 4 Aerial View of Hardstand 7, Eglin AFB, Florida 5 5 Hardstand Pond Located Behind Hardstand 7 6 6 Beaver Pond Located Downstream From Hardstand 7 6 7 Typical Storage of Herbicide on Hardstand 7 7 8 Locations of Known Herbicide Storage Sites and Disposal ?it on Hardstand 7 8 Total Herbicide Application on Test. Area C-52A, Eglin AFB, Florida, 1962 Through 1970 9 9 10 Soil Sampling Sites at East End of Taxiway Number 9, Eglin AFB, Florida 15 11 Soil Sampling Sites at Hardstand 8.. Eglin AFB, Florida . . . 16 12 Sampling Sites at Hardstand 7, Eglin AFB, Florida 19 13 Tom's Pond, Eglin AFB, Florida 22 14 Snapping Turtle (Male) Collected 1978 from Hardstand Pond, Eglin AFB, Florida 24 IV �LIST OF TAULES Table Title Page 1 Approximate Total Volume of Herbicides Applied to Test Area C-52A, Eglin AFB Reservation, Florida, 1962 Through 1970 11 Total Pounds of Active Ingredients of Herbicides Disseminated on Test Area C-52A, Eglin AFB Reservation, Florida, 1962 Through 1970 12 Results of TCDD Determinations in Soil Samples Collected January 1976, from East End of Taxi way Number 9, Eglin AFB, Florida 14 Results of TCDD Determinations in Soil Samples Collected January 1976, from Hardstand 8, Eglin AFB, Florida 17 Results of TCDD Determinations in Sediment and Soil Samples Collected for TCDD Analysis in 1977 18 Results of TCDD Determinations in Soil Samples Collected January 1978 from Hardstand 7, Eglin AFB, Florida 20 TCDD Analysis of Organisms Collected on Eg'lin AFB, Florida 23 2 3 4 5 6 7 (The reverse of this page is blank) ��SECTION I INTRODUCTION Reported here are the results of a study made of the residual levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the areas surrounding three hardstands that had been previously used for storing and loading military herbicides at Eglin Air Force 3ase, Florida. TCDD is an extremely toxic material that has been reported to be mutagenic, teratogenic, and carcinogenic in some organisms, although those effects are unconfirmed in man (References 1, 2, and 3). It was shown to be a contaminant in several herbicide formulations which were used extensively on Cglin test ranges from 1962 through 1970 (Reference 4). The objective of this effort was to determine concentrations and distribution of contamination detected during work reported in AFATL-TR75-49 (Reference 5). The study was designed so that data obtained from it could be used to map TCDD soil concentrations in the immediate vicinity of three hardstands and to determine range boundaries of the contamination emanating frorr the hardstands. The hardstand areas were subjected to massive amounts of herbicides due to spills, leaking drums, purging of aircraft spray systems, and malfunctions of aircraft spray nozzles. For lack of exact information on the time, amount, and type of herbicide released to the environment at each specific site, precise degradation rates for the TCDD could not be calculated at the completion of this study. As more samples are analyzed in the future, using data in this report as a baseline, valuable conclusions on degradation rates may be established. Because of the dynamic nature of the areas studied for this report and the many factors that can influence movement and degradation of any contaminant, caution should be used in applying these data to other situations. �SECTION II BACKGROUND 1. DESCRIPTION OF MONITORING AREAS a. East End of Taxiway Number 9 This area is the end of a taxiway that was used briefly during the beginning of the defoliation test program at Eglin (Figure 1). The quantity of herbicide previously stored or loaded at this location is unknown but is suspected to be small. However, at the time that this location was used, the herbicides involved were predominantly Purple and Orange, both of which contained TCDD as a contaminant.' The concrete runway area is bordered by an asphalt strip and is approximately 55 feet (17 meters) above sea level. The soil surrounding the area is sandy with excellent drainage potential. Excess water is drained by a storm sewer into a bayhead to the east of the runway. This bayhead forms a stream which empties into Tom's Bayou approximately 1000 meters downstream {Figure 2). Figure 1. East End of Taxiway Number 9, Eglin AFB, Florida �Figure 2. Eglin AFB Herbicide Storage and Loading Sites with Associated Aquatic Drainage Areas �b. Hardstand 8 Hardstand 8 (Figure 3) is an asphalt and concrete aircraft parking area located west of the north-south runway on the main Eglin airdrome (Figure 2), approximately 65 feet (20 meters) above sea level. The hardstand is connected to the airdrome by an asphalt taxiway. This hardstand was used for limited herbicide storage and loading during the latter portion of the spray program. The area surrounding Hardstand 8 is level with very little runoff occurring. The soil of this area is sandy with excellent drainage potential. Figure 3. Hardstand 8, Eglin AFB, Florida �c. Hardstand 7 Hardstand 7 (Figure 4) is -ar asphalt and concrete aircraft parking area located west of the north-south runway on the main Eglin airdrome, approximately 65 feet (20 meters) above sea level. The hardstand is connected to the airdrome by an asphalt taxiway. This hardstand was the most extensively used site for herbicide storage and loading during the 1962 through 1970 spray test program. The soil of this area is sandy with good drainage properties. Directly behind the hardstand is a ravine that drops off approximately 15 meters to a small pond (Figure 5). Because of the packing caused by vehicular traffic and the water-repellent nature of the oil-based herbicide contamination, runoff of excess water caused an erosion problem in some spcts which led to the frequent use of fill dirt. Eventually, a dike covered with asphalt was constructed on the rim of the ravine for soil stabilization. A storm drain was alse installed to help control erosion. The pond behind Hardstand 7 drains into a small stream which flows north until it enters a man-made reservoir named Beaver Pond (Figure 6). The drainage system eventually flows into Tom's Bayou and Choctawhatchee Bav. " *'*•* , •''• '*''•."?«. " Figure 4. Aerial View of Hardstand 7, Eglin AFB, Florida 5 i *', ;b * �.1' Figure 5. Hardstand Pond Located Behind Hardstand 7 Figure 6. Beaver Pond Located Downstream From Hardstand 7 �Several hundred 55-gallon drums of various types of herbicide were stored around Hardstand 7 for later transfer of their contents into tanks aboard spray aircraft (Figure 7). Known storage locations are shown in Figure 8. Much of the area immediately surrounding this hardstand was contaminated with herbicide due to accidental spills during loading operations and transfer procedures, leaking drums, and purging of spray systems before and after missions. A pit was dug in 1969 (according to the best available information) to the southwest of the hardstand as a temporary means of preventing the excess herbicides from entering the stream back of the hardstand (Figure 8). After several months of use, the pit was filled with soil. —r . ™—™: - "•" •* •.' ' ' Figure 7. Typical Storage of Herbicide on Hardstand 7 �' FIVE FOOT A S P H A L T COVERED DIRT MOUND W \ ASPHALT N \ Pit DIRT MOUND HERBICIDE DRUMS igure R. Locations of Known Herbicide Storage Sites and Disposal Pit on Hardstand 7 �2. HERBICIDES USED ON. EGLIN Several herbicides and/or mixtures of herbicides were tested at Eg!in during the period 1962 through 1970. These herbicides were loaded into aircraft spray tanks at the previously described storage areas. Spray equipment and planes were also washed down at bhese sites after the spray missions. Characteristics of these herbicides are listed below (Reference 6), Although other formulations of 2,4,5-T were also used (e.g., Pink, Green), the formulations listed below were the predominant ones used in Eglin test programs. As seen in Tables 1 and 2 and Figure 9, massive quantities of these herbicides were stored and disseminated on Eglin test ranges during the period 1962 through 1970 (Reference 7). 5 ^ PURPLE 55 C ORANGE ^ H WHITE a z > I } BLUF z 62 63 64 65 66 67 68 f VEAR OF APPLICATION 70 Figure 9. Total Herbicide Application on Test Area C-52A, Eglin AFB, Florida, ]962 Through 1970 The names Orange, White, Blue, and Purple used below were code names and unrelated to the color of the materials. a. 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 contained about 4.21 pounds of the active ingredient of 2,4-dichlorophenoxyacetic acid (2,4-D) and about 4.41 pounds of the active ingredient of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). Orange was formulated to contain a 50:50 mixture 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 49.49 free acid of 2,4-D 0.13 n-but;yl ester of 2,4,5-T free acid of 2,4,5-T 48.75 1.00 inert ingredients (e.g., butyl alcohol and ester moieties) 0.63 b. Herbicide White White was a dark brown, viscous liquid that was soluble in water bit insoluble in organic solvents and diesel fuel. One gallon of White contained 0.54 pound 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 "!:4 mixture of the triisopropanolamine salts of picloram and 2,4-D. The percentages of the formulation were: triisopropanolamine salt of picloram 10.2 triisopropanolamine salt of 2,4-D inert ingredient (primarily the solvent triisopropanolamine) c. 39.6 50.2 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 hydroxydimethlarsine oxide (cacodylic acid). Blue was formulated to contain both cacodylic acid (as the free acid) and the sodium salt of cadodylic acid (sodium cacodylate). The percentages cf the formulation were: cacodylic acid 4.7 sodium cacodylate 26.4 surfactant 3.4 sodium chloride / water 5.5 59.5 antifoam agent 0.5 10 �It should be noted that cacodylic acid and sodium cacodylate contained arsenic in the form of the pentavalent, organic arsenical. This form of arsenic has a low mammaliam toxicity. Of the total formulation, 15.4 percent was arsenic in the organic form, and only trace quantities were present in the inorganic form. The term Herbicide Blue was applied to powdered cacodylic acid in 1961 through 1964. This herbicide contained 65 percent active ingredient cacodylic acid and 30 percent sodium chloride and was mixed in the field with water. d. Herbicide Orange II Orange II was the military designation of a formulation similar to Orange with the difference being ths substitution of the isooctyl ester of 2,4,5-T for the n-butyl ester of 2,4,5-T. The physical, chemical, ana toxicological properties of Orange II were similar to those of Orange. e. Herbicide Purple The first record of the use of Purple in large quantities was in the Camp Drum, New York defoliation test in 1959. The formulation was a brown liquid, soluble in diesel fuel aid organic solvents but insoluble in water. One gallon of Purple contained 8.6 pounds of the active ingredients 2,4-D and 2,4,5-T. The percentages of the formulation were: r-butyl 2,4-D 50 n-butyl 2,4,5-T 30 iso-butyl 2,4,5-T 20 The physical, chemical, and toxicological properties of Purple were similar to those described for Orange. TABLE 1. APPROXIMATE TOTAL VOLUME OF HERBICIDES APPLIED TO TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, 1962 THROUGH 1970 Herbicide Gallons Disseminated Orange 19,807 Purple 16,164 White 4,172 Blue 4,395 11 �TABLE 2. TOTAL POUNDS OF ACTIVE INGREDIENTS OF HERBICIDES DISSEMINATED ON TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA, 1962 THROUGH 1970 Pounds Active Ingredient Chemical 2,4-D 169,292 • 2,4,5-T 160,948 Piclorain 2,253 - Cacodylic Acid and Sodium Cacodylate 3. 13,624 CHEMICAL PROPERTIES AND EFFECTS OF TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is molecule that has received a great deal of public since 1970 because of its highly toxic properties its being a contaminant in the environment by the from trichlorophenols. a heterocyclic organic and scientific attention and the possibility of use of products made TCDC is a synthetic, chlorinated hydrocarbon produced from trichlorophenol at high reaction temperatures. Trichlorophenols are used in the production of several chemicals. Since trichlorophenol is a precursor of 2,4,5-T, TCDC was present in varying quantities as an impurity in herbicides that contained 2,4,5-T (e.g., Orange and Purple). TCDD is a solid which is very insoluble in water (0.2 parts per billion (ppb) at 25° C); very slightly soluble in fats (44 parts per million (ppm) in lard oil); slightly soluble in hydrocarbons (570 ppm in benzene); and somewhat more soluble, but still not very highly so, in chlorinated organic solvents (1400 ppm in ortho-dichlorobenzene)., Its solubility in Orange is 580 ppm. Like other chlorodioxins, TCDD is relatively stable when subjected to heat, acid, and alkali. For thermal decomposition, a temperature of about 800° C is required (Reference 3). 12 �iCDD is an extremely toxic material that is aloo reported to cause hirth defects and embryo mortality (Reference 2). It is an extremely stable compound which has a relatively long half-life (References 8 and 9). U(!(,dir;r! of it.1, insolubility in water, TCDD is considered to be relatively immobile in the environment. It is impossible to state the exact concentrations of TCDD in herbicides Orange and Purple that were used on Eglin, but analyses of samples from herbicide Orange left over from ~;he Vietnam conflict can be used to estimate the probable concentrations. The mean TCDD concentration range in Herbicide Orange was <0.02 to 15 ppm with an overall mean of 1.98 ppm. The mean TCDD concentration range in Herbicide Purple was 17 to 47 ppm with an overall mean of 32.8 ppm (Reference 6). Herbicides used at Eglin from 1962 throuph 1970 probably contained concentrations of TCDD similar to those analyzed from Johnston Island and Gulfport, Mississippi. 13 �SECTION III TCDD ANALYSIS 1. EAST END OF TAX IWAY NUMBER 9 In January 1976, five soil samples were taken from the east end of Taxiway Number 9 and one sediment sample from the exit of the storm sewer drain (Figure 10). The soil samples were taken from the top 10 cm at sites considered to be likely areas for herbicide contamination. No TCDD was found in any of the samples (Table 3). TABLE 3. RESULTS OF TCDD DETERMINATIONS IN SOIL SAMPLES COLLECTED JANUARY 1976, FROM EAST END OF TAXIWAY NUMBER 9, EGLIN AFB, FLORIDA TCDD Concentration Sample TCDD (ppb) Detection Limit (ppb) 6 0.045 7 NDa 0.030 8 NDa 0.030 9 NDa 0.030 10 NDa 0.032 11 a NDa NDa 0.040 Not Detected 2. HARDSTAND-8 Five soil samples from the top 10 cm were also collected in January 1976, from Hardstand 8 (Figure 11). Site Number 5, with 0.034 ppb, was the only sampling point around this hardstand that contained a detectable quantity of TCDD (Table 4). 14 �Stream 6 concrete 10 5Om Figure 10. Soil Sampling Sites at East End of Taxiway Number 9, Eglin AFB, Florida 15 �Concrete N i 15m I igure 11. Soil Sampling Sites at, Hardstand 8, Eglin AFB, Florida 15 �TABLE 4. RESULTS OF TCDD DETERMINATIONS IN SOIL SAMPLES COLLECTED JANUARY 1976, FROM HARDSTAND 8, EGLIN AFB, FLORIDA TCDD Concentration Sample TCDD (ppb) Detection Limit (ppb) 1 0.030 2 NDa 0.030 3 NDa 0.030 4 NDa 5 a NDa 0.034 0.030 _b Not Detected Not Applicable 3. MAROSTAND 7 Because of the potential for TCDD contamination of the aquatic system draining Hardstand 7 and the previous data obtained from that area (Reference 5), sediment and soil samples, as well as biological organisms, were collected from several locations at this area in February 1977. a. Sediment Samples. Two sediment samples were collected from the hardstand pond directly behind Hardstand 7. Silt sample 1 consisted of organic dctritis, and silt sample 2 was taken from below that layer and was predominantly sand. Silt sample 3 was from Tom's Pond taken downstream from the man-made Beaver Pond dam (Figjre 2), which in turn was approximately 15 meters below a natural beaver dam. Silt sample 4 was taken from the head of Tom's Bayou at the creek entrance. This sample was taken from the surface to a depth of 15 cm and consisted almost entirely of detritis. Results from these samples are listed in Table 5. b. Soil Samples. In addition to bhe sediment samples, soil samples were taken ir February 1977from two locations at Hardstand 7 where herbicide con Lamination was known to exist (Figure 12). These samples were designated sample 5 and sample 6 and were taken from 0 through 10 cm and 40 throuch 50 cm, rcspecLively, ?. meters northeast of the concrete edge of the hardstand. Samples 7 and 8 were taken from 0 through 10 cm arid 40 throuch 50 cm, respectively, 2 meters southwest of the concrete edge of the hardstand. Results from these soil samples are given in Table 5. 17 �TABLE 5. RESULTS OF TCDD DETERMINATIONS IN SEDIMENT AND SOIL SAMPLES COLLECTED FOR TCDD ANALYSIS IN 1977 TCDD Concentration TCDD Date Collected (ppb) Sample Location Collected Detection Limit (ppb) 1 Hardstand 7 Pond (Detritis) Feb 77 NDa 0.370 2 Hardstand 7 Pond (Sand) Feb 77 NDa 0.037 3 Tom's Pond May 77 4 Tom's Bayou Mar 77 5 2 Meters N of Concrete (0-10cm) Feb 77 6 2 Meters N of Concrete (40-50 cm) Feb 77 7 2 Meters S of Concrete (0-10 cm) Feb 77 8 2 Meters S of Concrete (40-50 cm) Feb 77 a 0.65 NDa 37.8 0.69 275 37.1 -b 0.031 -_b _b _b _b Not Detected Not Applicable Since relatively high levels of TCDD were found in soils around Hardstand 7, it was decided to conduct a more thorough monitoring effort at this hardstend. A grid system was established around Hardstand 7 (Figure 12) in order to facilitate the monitoring effort. Beginning at the center of the hardstand pad, 14 radians were established at 22.5 degree intervals, designated A through N, with A being the south southeast radian and proceeding clockwise. Sampling points on each radian were established at 20, 25, 30, am1 40 meters from the center point. These sampling points were designated by the numbers 1, 2, 3, or 4, respectively, following the letter designating the radian. The third designation for each sample identification indicates the depth at which the sample was taken: 1 = 0 through 10 cm, 2 = 20 through 30 cm, 3 = 55 through 70 cm, and 4 = 95 through 110 cm. Soil samples were collected using this grid system during January 1978. All designated sairpling sites were not actually sampled since asphalt covered some areas, and other areas had been covered with overfill to the extent that the sanple would have been meaningless. Those sites used and the TCDD concentrations found at those sites are presented in Table 6 and Figure 12. 18 �FIVE FOOT A S P H A L T COVERED DIRT MOUND N D4 DIRT MOUND C4. 20m Note: Soil concentrations of TCDD in ppb are given at each site by depth from top to bottom. Depths sampled were 0-10 cm, 20-30 cm, 55-70 cm, and 95-110 cm. £11 depths were not sampled at each site, however. Figure 12. Sampling Sites at Handstand 7, Eglin AFB, Florida 19 �TABLE 6. RESULTS OF TCDD DETERMINATIONS IN SOIL SAMPLES COLLECTED JANUARY 1978 FROM HARDSTAND 7, EGLIN AFB, FLORIDA TCDD Concentration Sample TCDD (ppb) TCDD Concentration Detection Limit {ppb) Sample b A21 1.5 A22 NDa NDS A31 4.2 NDa A41 NDa Bll 198.9* B12 53.6 B13 20.4 ND d 1 .0 F41 1.0 b A32 Detection Limit (ppb) F33 1.0 A23 TCDD (ppb) NDa 1 .0 G31 NDa 1.0 632 NDa 1.0 1 .0 G33 NDa 1.0 b H31 0.2 1.0 b H32 ND3 1.0 H33 NDa 131 0.5 1.0 b b _b B21 NDd 1.0 132 NDa 1.0 B22 NDa 1.0 133 ND3 823 NDa 1.0 021 0.8 1.0 b B31 NDa 1.0 J22 NDa 1.0 B41 NDa ND3 cn 3.0 1.0 _b 023 J31 0.5 1.0 b C12 1.0* J32 NDa i.n 03 5.9* 041 NDa C14 0.7* 1.0 b b b b Kll 70.2* C21 NO 3 1 .0 K12 20.2* C22 NDS 1.0 K13 16.6* C23 NDd 1.0 K14 19.0 C31 a ND 1.0 K21 3.9 C32 N0a 1.0 K22 0.8 C33 a ND 1.0 K23 0.030** C41 NO3 1.0 b K31 0.4 Dll D12 D13 D14 77.7* K32 127.4 97.1* 4.5 L12 0.133** LI 3 b _b NDa 111 b 0.5* K41 _b 121.4 HDa b b b b b b b b 1.0 b b D21 0.079** L21 0.4 023 NDa 1.0 L22 NDd 1.0 D31 NDa 1.0 L23 NDa 1.0 032 N0a 1.0 L31 ND8 1.0 033 NDS 1.0 L41 ND3 D41 NO3 Mil 0.8 1.5* 1.0 b 1.0 b Ell Ml 2 NO3 1.0 E12 NO3 1.0 M21 NDa 1 .0 E13 NDa ND3 1 .0 Q.I 1.0 b M22 E14 H31 NDa 1.0 E21 0.5 M41 NDa E22 NDa 1.0 Nil 1.0 b E23 NDa 1.0 N12 N0a 1.0 E31 NO 3 1.0 N13 NDa 1 .0 E32 NDa 1.0 N21 NDa 1.0 E33 a ND 1.0 N22 NDa 1.0 E41 NDa 1 .0 N23 NDa F31 NDa 1.0 N31 3.0 1.0 t> F32 a 0.7 D22 NDa 1.0 N41 NDa b _b 18.9 1.0 _b 1.0 Not Detected riot Appl icable 'samples contained varyi ig quantities of red dye. Red dye was used as an indicator for test purposes during the 1962 through 1970 spray m i s s i o n s . A slightly different extraction technique was used for these samples. "samples were subjected to high resolution mass spectrometry (GC-HRMS). 20 �The highest concentration of TCDD found in the soil surrounding Hardstand 7 from this set of samples was 198.9 ppb. This was found in the surface sample at site 81. Several other sampling sites nearest the concrete pad were also heavily contamirated. This was expected, however, since drainage of spills from the concrete pad woulc migrate to those areas and also because of leakage from drums that had been stored at those areas. The presence of TCDD at depths down to 100 cm at site Dl was also expected because of the pit which had been dug in that area to restrain herbicide runoff. The high concentrations in the deeper samples at sites Bl and Kl, however, are not as easily explained. Probably several factors are involved. The most obvious explanation is the probable saturation of the soil with herbicide to a considerable depth when the hardstand was being used for loading operations. TCDD could have been transported to those depths via the herbicide solvent. After the degradation of the herbicide, the TCDD remained at the lower depths. The vertical movement of TCDD in the soil probably decreased greatly at that time, however. Although it is recognized that TCDD has little vertical mobility in soil, some vertical movement probably has occurred around Hardstand 7 because of the high concentrations. It should be pointed out that the detection limit for the samples from Hardstand 7 was 1 ppb except where noted in Table 6, Therefore, TCDD in concentrations less than 1 ppb could have been present in samples reported as "not detected" in this study. An important finding in this study was that soils containing high levels of TCDD contamination around Hardstand 7 were in a very confined area. TCDD contamination above 1 ppb was found predominantly in an area 3 to 4 meters wide surrounding the perimeter of the concrete pad. Soils outside this area were largely below the detection limit of 1 ppb. Although soil levels of TCDD below 1 ppb cannot be dismissed as insignificant, the magnitude of concern is obviously different from soils with much higher levels. c. Biological Samples. Biological organisms were collected from Hardstand 7, the Hardstand Pond, Beaver Pond, Tom's Pond (Figure 13) and Tom's Bayou (Table 7). No TCDD was detected in any of the samples collected from Tom's Bayou., Tom's Pond, or Beaver Pond. However a large snapping turtle (Figure 14) collected from the Hardstand Pond contained 1.5 ppb TCDD in fat tissue. TCDD was not detected in muscle, liver, or testes of the turtle, although it should be remembered that the detection limit in the liver tissue was high (0.260 ppb) due to high levels of interfering compounds. The turtle was not subjected to pathological examination, but no gross abnormal pathological findings were noted during visual observations. It is interesting and perhaps of some significance to note that the turtle was collected immediately after having been observed in the act of copulation. The fact that TCDD was found in fat tissue yet absent 21 �(at the admittedly high detection limit) in the liver might point toward species dependence not only for biological effects, but also for sites of bioaccumulation. Snapping turtles are near the top of the food chain in the aquatic system draining the contaminated hardstand. Therefore, the potential for TCDD accumulation from both the environment and contaminated food existed. Because of this, the TCDD concentration found in this specimen may be high compared to other organisms existing at this area. This specimen was old enough to have been living during the 1962 through 1970 spray orogram, although it cannot be determined if he actually lived in the Hardstand Pond/Beaver Pond area all that time. Figure 13. Tom's Pond, Eglin AFB, Florida 22 �TABLE 7. TCDD ANALYSIS OF ORGANISMS COLLECTED ON EGLIN AFB, FLORIDA TCDD Concentration Location Collected Date Collected Snails (NerUina _recl_avala) Tom's Bayou Mar 77 Alcwlfc (Ppnnlohus pseudoharongus) livor Tom' 1 ) Bayou Mar 77 TCDD (ppb) Detection Limit (ppb) NDa (l.(!ll NDa ND Sample 0.015 0.109 ND3 0.0l r i NDd 0.026 0.030 d muscle Cldins (4) (Pnlymp.soda carol irn ana and Rangia cuneata) Tom's Bayou Mar 77 Crab (4) ( C a l l incctus sapidus) muscle Tom's Bayou Mar 77 viscera ND° Bass (Micrpptprus salinoirles! 1 1 ve'i Tom1'. Pond May 77 ND3 NDfl *Turtle (Cnelydr.) serpent ina) fat llardstcind Pond 0.017 0.010 1.5 IIIUSClL' _b Mar 78 3 livor ND iiusi.le NDa 0.009 lestes NDa 0.011 *Beach Mice ('!) (Poromyscus polionotus) 1 i vcr lldrdstand 7 Mar 70 0.5SO 0.053 skin 0 f) *Sunfish (Lc'.P ."!J:'. l ' - ) muscle Beaver Pond _b o.oon NDa 0.007 Nof Delected b _b Nl)° viscera a Mar 78 0.2f>() Not A|)pliu)blc Samples were analyzed by !.ne Dcparlmcnt of Chemistry, University of Nebraska, under contract from the Department of Chc-ir.isr.ry and Biological Sciences, 1,'SAFA, Colorado (Contract *F056117RC0063). Funds provided by ACLC.'LO in support of studies on the Environmental rate of Herbicide Orange. 23 �Figure 14. Snapping Turtle (Male) Collected 1978 from Hardstand Pond, Eglin AFB, Florida A pooled sample of one female arid two male beach mice collected from Hardstand 7 contained 0.550 and 0.053 ppb TCDD in liver and skin samples, respectively. Weights of the mice were 6.96, 12.24, and 13.18 grams with liver weights of 0.44, 0.77, and 0.76 grams, respectively. Total weight of the three skins was 4.25 grams. Liver tissue was examined grossly and histologically for congenital and teratogenic defects by the Armed Forces Institute of Pathology (AFIP), Washington, DC. Microscopic examination of tissue showed no abnormal characteristics. 24 �SECTION IV CONCLUSIONS Analyses from this study revealed that only one of the three storage and loading areas included in the monitoring effort was appreciably contaminated with TCDD. Samples from near the surface around Hardstand 7 had concentrations of TCDD as high as 275 ppb with one-third that amount to a depth of 1 meter. However, this contamination was largely confined to an area approximately 3 meters wide around the perimeter of the concrete pad (a total area of about 350 square meters). At Hardstand 8, only one sample was found to be contaminated, and it contained only 0.034 ppb TCDD. No TCDD was found at the east end of Taxiway 9. The minimal contamination at Hardstand 8 and its absence at the east end of Taxiway. 9 was not surprising since these two hardstands were not used nearly as much as was Hardstand 7. TCDD has apparently migrated from Hardstand 7 as far downstream as Tom's Pond. The presence of TCDD downstream, however, does not necessarily mean that the material is spreading at this time. Much of the downstream TCDD contamination probably occurred during the actual loading operations and before the dike was built in back of the hardstand. Soil erosion and water runoff were obvious during that time but are well controlled currently. TCDD has been picked up by biological organisms in some contaminated areas, but the paucity of analyses makes it impossible to draw any conclusions concerning the extent of bioaccumulation at this time. The absence of TCDD in the fish collected from Beaver Pond during this sampling program could indicate that TCDD is degrading in that ecosystem. Whole body samples of sunfish from Beaver Pond in 1974 contained 0.014 ppb TCDD although muscle and liver tissue were free of contamination. Additional sampling of biological organisms is required in Beaver Pond and Tom's Pond to further define the extent of contamination in the biota. The final conclusion from this monitoring effort is drawn from the fact that no TCDD could be detected in either the silt or a wide range of biological organisms collected from Tom's Bayou. That no contamination occurs in Tom's Bayou is significant because it is the first place contamination would occur off the Eglin AFB Reservation. Therefore, data obtained from this study indicate that TCDD migration from Hardstand 7 is minimal and currently limited to a small area on Eglin AFB. 25 �REFERENCES 1. Courtney, K. D., D. W. Gaylor, M. D. Hogan, J. L. Falk, R. R. Bates, and I. Mitchell". Teratogenic evaluation of 2,4,5,T. Science 168: 864866, 1970. 2. Schwetz, B. A., J. M. Norris, G. L. Sparschu, V. K. Rowe, P. J. Gehring, J. L. Emerson, and C. G. Gerbig. Toxicology of chlorinated dibenzo-p-dioxins, Environ. Health Perspectives Experimental Issue No. 5: 87-100, September 1973. 3. Lang, Anton, et. al. The Effects of Herbicides in South Vietnam. National Academy of Sciences, Washington, D.C. 1974. 4. Biever, Helen. Defoliant History of Test Area C-52A, Working Papers, Vitro Corporation of America and Armament Development and Test Center, Eglin AFB, Florida, December 1969. 5. Bartleson, Fred D., Jr., Don D. Harrison and John D. Morgan. Field Studies of Wildlife Exposed to TCDD Contaminated Soils. AFATL-TR-75-49. Air Force Armament Laboratory, Eglin AFB, Florida, 1975. 6. Young, Alvin L., John A. Calcagni, Charles E. Thalken, and James W. Tremblay. The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange and Its Associated Dioxin. OEHL TR-78-92. USAF Occupational and Environmental Health Laboratory, AMD, Brooks AFB, Texas, 1978. 7. Young, Alvin 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 AFB, Florida, 1974. 8. Young, Alvin L., Charles E. Thalken, Eugene L. Arnold, James C. Cupcllo, and Lorris G. Cockerham. Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the Environment: Summary and Decontamination Recommendations. USAFA-TR76-18. United States Air Force Academy, Colorado, 1976. 9. the the Air Young, Alvip L., Charles E. Thalken and Milliam E. Ward. Studies of Ecological Impact of Repetitive Aerial Applications oF Herbicides on Ecosystem of Test Area C-52A, Eglin AFB, Florida. AFATL-TR-75-142. Force Armament Laboratory, Eglin AFB, Florida, 1975. 26 �INITIAL DISTRIBUTION AUL/LSE 71-249 ASD/ENFEA IK) USAF/SAMI AIIVINT IKJ lAC/DRA FAG/1 NAT ASU/ENESII/S. Johns US Army TRADOC ATTN: ATAA-SL 1 1 1 1 1 1 1 (Tech Lib) 1 HQ USAFE/DOQ HQ PACAF/DOOFQ ASD/XRP COMIPAC/I-232 AFATL/DLODL DDR&E (Tech L'-b) USAFA/DFCBS AFLC/MMNO SAAMA/SFQT HQ USAF/RDP AFSC/DEV 1 3 1 1 2 1 1 1 1 1 1 DDR&E (Env & Life Sciences) Chemical Systems Lab DRDAR-CLJ-L (Tech Lib) USAF (OEHL/Capt Young) NWC Env Eng (Mr Ouimette) 1 1 10 1 AMD/RD AMRL/THE (Dr London) AMRL/THT (Dr Back) ADTC/CSV ADTC/SGPE AFESC/EC AFLC/DC ADTC/DEN US Army Natick Lab AFATL/DLV 1 1 1 1 1 1 1 1 1 25 Accessions Div DDC-DDA Deseret Test Center/Tech Lib Naval Weapons Ctr/Tech Lib US EPA (Dr Edwin L. Johnson) Dow Chemical Company US EPA (Dr Jack Moore) The Brehm Lab & Dept of Chemistry 12 1 1 AFCEC/EO AFATL/DL 1 1 USDA/ARS IJSDA Forest Insect & Disease Mgt US EPA (Office of Research & Monitoring) EPA (Dr Ralph Ross) Naval Const Battalion Center 2 1 1 1 1 27 (The reverse of this page is blank) 1 2 1 2 ��AD EGLIN AFB. FLA. 32542 OFFICIAL BUSINESS PENALTY FOR PRIVATE USE S3OO THIRD CLASS � 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. 012 Folder The folder containing the original item. 0103 Series The series number of the original item. Series 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 Harrison, Don D. Charles I Miller Richard C. Crews Description An account of the resource <strong>Corporate Author: </strong>Environics Office, Air Force Armament Laboratory, Eglin AFB, Florida Date A point or period of time associated with an event in the lifecycle of the resource 1979-02-01 Title A name given to the resource Residual Levels of 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) Near Herbicide Storage and Loading Areas at Eglin AFB, Florida Subject The topic of the resource biodegradation herbicide blue herbicide white herbicide purple 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. 035 Folder The folder containing the original item. 0742 Series The series number of the original item. Series III 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 Hay, Alastair Source A related resource from which the described resource is derived Nature Date A point or period of time associated with an event in the lifecycle of the resource November 18 1976 Title A name given to the resource Seveso: The Problems Deepen Subject The topic of the resource Italy biodegradation soil decontamination congenital birth defects ao_seriesIII 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. 038 Folder The folder containing the original item. 0827 Series The series number of the original item. Series III 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 Klopffer, W. G. Rippen R. Frische Source A related resource from which the described resource is derived Ecotoxicology and Environmental Safety Date A point or period of time associated with an event in the lifecycle of the resource 1982 Title A name given to the resource Physiochemical Properties as Useful Tools for Predicting the Environmental Fate of Organic Chemicals Subject The topic of the resource biodegradation ao_seriesIII 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. 049 Folder The folder containing the original item. 1264 Series The series number of the original item. Series III 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 Matsumura, Fumio Herman J. Benezet Source A related resource from which the described resource is derived Environmental Health Perspectives Date A point or period of time associated with an event in the lifecycle of the resource 1973-09-01 Title A name given to the resource Studies on the Bioaccumulation and Microbial Degradation of 2,3,7,8-Tetrachlorodibenzo-p-dioxin Subject The topic of the resource dioxin biodegradation ecological fate ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/9346e6068481f593b699f8853bda813b.pdf 553b9c533e1e82c5b45c3ef79964b2ae PDF Text Text Item ID Number Author CorpOratB Author 00184 Patrick, Michael A. Environics and Human Factors Office, Air Force Armament Laboratory, Armament Development and Test Center, Eglin AFB, Florida Toxicological and Recalcitrant Properties of a Proposed Propellant Ingredient, Triaminoguanidine Nitrate (TAGN). I. Microbiological Study Journal/Book Title Year 1976 November Wnr Number of Images Project No. 5066; Task No. 01 ; Work Unit No. 001 Friday, January 05, 2001 Page 184 of 194 �AFATL-TR-76-139 TOXtCOLOGICAL AND RECALCITRANT PROPERTIES OF A PROPOSED PROPELLANT INGREDIENT, TRIAMINOGUANIDINE NITRATE (TAGN) I. MICROBIOLOGICAL STUDY ENVIRONICS AND HUMAN FACTORS OFFICE NOVEMBER 1976 FINAL REPORT: APRIL - NOVEMBER 1976 Approved for public release; distribution unlimited AIR FORCE ARMAMENT LABORATORY AIR F O R C E S Y S T E M S COMMAND • UNITED S T A T E S AIR F O R C E EGLIN AIR F O R C E B A S E , F L O R I D A �UNCLASSIFIED SECURITY CLASS'FICATION OF THIS =>AG€ (When Rate Entered) READ INSTRUCTIONS BEFORE COMPLETING FORM 2. GOVT ACCESSION NO, 3. RECI^'FWT'S CATALOG NUMBER REPORT DOCUMENTATION PAGE 1, REPORT NUMDER AFATL-TR-76-139 4. TITLE (and Subtitle) 5. TVFE OP REPORT * PERIOD COVERED Final Report - April to November 1976 TOXICOLOGICAL AND RECALCITRANT PROPERTIES OF A PROPOSED PROPELLANT INGREDIENT, TRIAMINOGUANIDINE NITRATE (TAGN). I. MICROBIOLOGICAL STUDY 6. PERFORMING ORG. REPORT NUMBER 7. AUTHORfs) 8. CONTRACT OR GRANT NUMBERfa) Michael A. Patrick, Lt, USAF 10. PROGRAM ELEMENT. PROJECT, TASK APE* ft WORK UNIT NUMBERS 9. PERFORMING ORGANIZATION NAME AND ADDRESS Environics and Human Factors Office Air Force Armament Laboratory Eglin Air Force Base, Florida 32S42 Project No. 5066 Task No. 01 Work Unit No. 001 tl. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE Air Force Armament Laboratory Armament Development and Test Center Eglin Air Force Base, Florida 32542 November 1976 14. MONITORING AGENCY NAME ft AODRESSfff different from Controlling Office) 15. SECURITY CLASS, (at thlt report) 13. NUMBER OF PAGES 72 UNCLASSIFIED 15«. OECLASSIFtC ATI ON/ DOWNGRADING SCHEDULE 16. DISTRIBUTION STATEMENT (of this Report) Approved for public release; distribution unlimited. 17. DISTRIBUTION STATEMENT (of th» abttroct entered in Block 20, If different tram Report) 18. S U P P L E M E N T A R Y NOTES Available in DDC. 9. K E Y WORDS (Continue on reverse side It necessary and identify by block number) Toxicological Properties Recalcitrant Properties Triaminoguanidine Nitrate Microbial Populations 20. ABSTRACT (Continue on reverse mid* If necessary end Identity by block number) The toxicological and recalcitrant properties of a proposed propellant ingredient, triaminoguanidine nitrate (TAGN), were investigated. Pure cultures of microorganisms isolated from Eglin Air Force Base, Florida, as well as cultures obtained from US Army Natick Laboratories, Natick, Massachusetts were exposed to TAGN and evaluated. During the course of this investigation, it was determined that microbial populations were not adversely affected by short-term exposure to TAGN. The following parameters were not significantly DD 1473 EDITION OF 1 NOV 65 IS OBSOLETE UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGE (When Dmtu Entm-nS) �UNCLASSIFIED SCCURITY CLASSIFICATION OF THIS FAG£fiMj«n Da** Entered) (Item 20 concluded) altered by TAGN concentrations up to 50 ppm: ' growth rate, respiratory activity, and viability. At concentrations greater than 100 ppm, TAGN was bacteriostatic but not bacteriocidal. Of the two bacteria tested, Pseudoaonas aerugi.nosa and Escherichia coli, both were capable of removing (degrading) TAGN from aqueous solution. UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGEfflfmn D*>* �PREFACE This technical report is the result of research conducted by the Air Force Armament Laboratory, Armament Development and Test Center, Eglin Air Force Base, Florida, from April 1976 to November 1976 under Air Force Exploratory Development Project 50660101. Reference to specific manufacturers or suppliers of scientific equipment used in this study is for the sole purpose of identification and does not constitute endorsement of these products by the United States Air Force. The assistance of Cadet Ron Alford, USAF Academy, in the bacteriocidal portion of this study is gratefully acknowledged. This report has been reviewed by the Information Office (01) and is releasable to the National Technical Information Service (NTIS). At NTIS, it will be available to the general public, including foreign nations. This technical report has been reviewed and is approved for publication. FOR THE COMMANDER: A. FARMER Chief, Environics and Human Factors Office i (The reverse of this page is blank) ��TABLE OF CONTENTS Section I II III IV Title Page 1 INTRODUCTION MATERIALS AND METHODS, . . . Cultures . . . . . Inhibitory Studies . . Bacteriocidal Effects Oxygen Uptake. . . . . . . . TAGN Degradation . . . . . . . . ..... ... RESULTS AND DISCUSSION CONCLUSIONS. . . . . . . . ill 3 3 3 3 3 4 5 . . 7 �LIST OF FIGURES Figure 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Title Page Standard Growth Curves of (A) Pseudoaonas aeruginosa QMB 1468 and (B) Bacillus megaterium QMB 1605 Exposed to TAGN . . 8 Standard Growth Curves of (A) Staphyj.ococcus aureus QMB 1458 and TO Bacillus cereus QMB 1597 Exposed to TAGN. . 10 Standard Growth Curves of (A) Escherichia. coli QMB 1557 and (B) SR 401 Exposed to TAGN , 12 Standard Growth Curves of (A) SR 403 and (B) SR 404 Exposed to TAGN , 14 Standard Growth Curves of (A) SR 405 and (B) SR 407 Exposed to TAGN 16 Standard Growth Curves of (A) SR 408 and (B) SR 409 Exposed to TAGN . 18 Standard Growth Curves of (A) C 1 and (B) C 4 Exposed to TAGN . . . . . . . . . . . . . . . . . . . . 20 Standard Growth Curves of (A) T 6 and (B) Arthrobacter sp. QMB 1631 Exposed to TAGN. 22 Standard Growth Curves of (A) Bacillus subtilis QMB 1611 and (B) Serratia marcescens QMB 1466 Exposed to TAGN 24 Standard Growth Curves of (A) SR 402 and (B) SR 406 Exposed to TAGN . . . . . . . . . . . . . . . . 26 Standard Growth Curves of (A) T 4 and (B) T 100 Exposed to TAGN ............ 28 Endogenous (A) and Exogenous (B) Oxygen Uptake by Pseudomonas aeruginpsa QMB 1468 Exposed to 500 ppm TAGN . 30 Endogenous (A) and Exogenous (B) Oxygen Uptake by Bacillus megaterium QMB 1605 Exposed to 500 ppm TAGN 32 Endogenous (A) and Exogenous (B) Oxygen Uptake by Staphylococcus aureus QMB 1458 Exposed to 500 ppm TAGN. . . . . . 34 Endogenous (A) and Exogenous (B) Oxygen Uptake by Serratia_ marcescens QMB 1466 Exposed to 500 ppm TAGN 36 Endogenous (A) and Exogenous (B) Oxygen Uptake by Escherichia coli QMB 1557 Exposed to 500 ppm TAGN . . . . . . . . 38 Endogenous (A) and Exogenous (B) Oxygen Uptake by Arthrobacter sp. QMB 1631 Exposed to 500 ppm TAGN . . . . . . . . 40 Endogenous (A) and Exogenous (B) Oxygen Uptake by Bacillus cereus QMB 1597 Exposed to 500 ppin TAGN 42 IV �LIST OF FIGURES (CONCLUDED) Figure 19 20 Title Page Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 402 Exposed to 500 ppm TAGN 44 Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 404 Exposed to 500 ppm TAGN 21 22 23 24 46 Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 406 Exposed to 500 ppm TAGN . 48 Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 407 Exposed to 500 ppm TAGN 50 Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 408 Exposed to 500 ppm TAGN. 52 Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 410 Exposed to 500 ppm TAGN 25 54 Endogenous (A) and Exogenous (B) Oxygen Uptake by C 4 Exposed to 500 ppm TAGN 26 27 56 Disappearance of TAGN from Cultures of (A) Pseudomonas aeruginosa QMB 1468 and (B) Escherichia coli QMB 1557 as a Function of Cell Density (O.D.) Thin-Layer Chromatogram Depicting the Disappearance of TAGN from the Cell-Free Supernatant of a Growing Culture of Pseudomonas aeruginosa as a Function of Time 58 . 60 LIST OF TABLES Table 1 2 Title Page Exposure of Bacterial Cultures Obtained from US Array Natick Laboratories to TAGN 61 Exposure of Bacterial Cultures Indigenous to Eglin AFB, Florida, to TAGN. . 63 v (The reverse of this page is blank) ��SECTION I INTRODUCTION An experimental propellant consisting of approximately 45 percent triaminoguanidine nitrate (TAGN), 19 percent nitrocellulose (NC), 30 percent cyclotetramethylene tetranitramine (HMX), 5 percent isodecyl pelargonate, and I percent resorcinol is being considered by the Air Force for use in gun ammunition employing high-density, armor-piercing penetrators. It is a matter of environmental policy to determine toxicity and evaluate methods for disposal of new propellant constituents before inventory acquisitions. Prior to this study, no information was available concerning the biodegradation or toxicity of the major component, TAGN. The objectives of this initial study were to investigate whether TAGN is degradable by microorganisms and to determine if TAGN adversely affects microbial populations indigenous to soil and water habitats where appreciable amounts of TAGN may accumulate during propellant testing and disposal. 1 (The reverse of this page is blank) ��SECTION II MATERIALS AND METHODS CULTURES Bacterial strains used in this study were obtained either from US Army Natick Laboratories, Natick, Massachusetts, or isolated from soil or water samples collected at Eglin Air Force Base, Florida. Original cultures were preserved under liquid nitrogen. Subcultures were maintained on Trypticase soy agar (TSA) at 4°C and were transferred monthly. INHIBITORY STUDIES Starter cultures were grown overnight in Trypticase soy broth (TSB) with agitation at 20°C and were diluted 1:10 in 15-mM phosphate buffer (pH 6.6) prior to use. Experiments were initiated by inoculating 0,1-aJt cells into S.Q-m£ filter-sterilized (0.45-um Millipore filters) TSB containing TAGN at concentrations of 500, 100, 50, or 10 ppm. Control samples contained no TAGN. All experiments were performed in triplicate at 20°C with agitation on a gyratory shaker (120 rpm). Growth was monitored periodically by recording the optical density (O.D.) of each sample with a Bausch and Lomb Spectronic 20 Spectrophotometer at 520 nm.1 All sanples were corrected for O.D. discrepancies due to tube variations and TAGNinduced absorption. BACTERIOCIDAL EFFECTS Cultures were grown overnight in 50-mH TSB at 25°C with agitation and were harvested at late log or stationary phase by centrifugation at 6,000 rpm for 15 minutes in an IEC/B20 refrigerated centrifuge. Following resuspension in sterile phosphate buffer, the cells were diluted to give a final O.D. reading of 0.2 at 520 nm. Diluted samples (5.0 mi) were added in duplicate to 5.0-m£ phosphate buffer containing TAGN at final concentrations of 0, 500, and 2,000 ppm. Samples were shaken in sterile 15-m£ centrifuge tubes at 25°C for 1-hour or 5-hour time periods. Afterwards, the exposed cells were centrifuged at 6,000 rpm for 15 minutes to remove TAGN and were resuspended in equal amounts of phosphate buffer. Samples were subsequently diluted with buffer to a final titer of 3.0 x 10a 3.0 x 10s colony-forming units/mi (CFU/m£) and were spread plated in triplicate. Following incubation overnight at 34°C, the plates were counted with the aid of a Quebec Colony Counter. OXYGEN UPTAKE Cultures were grown at 25°C, harvested, and resuspended in phosphate buffer as previously described. Endogenous preparations contained 1.5-ntfc �cells, 1.5-ml phosphate buffer, and a final TAGN concentration of 500 ppm, Exogenous preparations contained 1.5-mi cells, 0.5-mH phosphate buffer, 1.0-m£ TSB, and a final TAGN concentration of 500 ppm. Exogenous samples were pre-incubated 30 minutes at 30°C prior to data collection. All control samples were identically prepared but contained no TAGN, Oxygen uptake measurements were performed with a YSI 5331 Oxygen Probe at 30°C with airsaturated solutions. TAGN DEGRADATION Twelve liter fermentors (Virtis Research Equipment, Gardiner, New York) containing 10 £ of a mineral salts medium consisting of 1,79 g/Ji KH2POi», 1.65 g/£ Na2HPO% • 7H20, 0.12 g/£ MgSQK, 0,03 g/£ CaCl2, and 5.0 g/£ glucose were inoculated with 25 m£ of an actively growing culture of either Pseudomonas aerujinosa or Escherichia coli.2 Immediately following inoculation, filter-sterilized TAGN was added to give a final concentration of 61 ppm for the P. aeruginosa culture and 75 ppm for the E_, coli culture. NaNOs (0.7 g/£) was added to each fermentor at a designated time following inoculation. Periodically, samples were aseptically withdrawn, centrifuged at 6,000 rpm for 15 minutes, and analyzed for TAGN by a modification of the ninhydrin assay.3 Freshly prepared ninhydrin reagent (3.0 m£3 was added to l.Q-mJl sample and heated for 5 minutes in a boiling water bath. The sample was cooled, and the resulting optical density was determined at 570 nm against a blank containing no TAGN. Culture densities were aeasured prior to centrifugation at 520 nm. In order to rule out the effects of other ninhydrin reacting substances, 50 u£ supernatant samples were spotted on silica gel GF thin-layer plates (Analtech, Inc., Pittsburgh, Pennsylvania) and were developed against a solvent system of methanol-water-methyl sulfoxide (40:30:30). Plates were sprayed with 0.2 percent ninhydrin in watersaturated butanol. �SECTION III RESULTS AND DISCUSSION Growth of the majority of the bacterial isolets examined was not adversely affected by TAGN concentrations up to 50 ppm. However, at 100 ppm and above, 16 of the 22 bacterial cultures tested were markedly inhibited (Figures 1 to 8). Since continued incubation of these cultures for up to seven days did not result in further growth initiations, it must be assumed that the inhibitory effects of TAGN were absolute and not merely the result of greatly extended lag periods. Of the 6 remaining bacterial cultures capable of growth in the presence of 100 ppm TAGN (Figures 9 to 11), all exhibited prolonged lag phases prior to logarithmic growth. Although the overall growth rates of these cultures were considerably retarded, the ultimate cell densities attained, when compared to controls, were not significantly affected by exposure to TAGN. At the present time, inadequate evidence is available to explain the inhibitory actions of TAGN on bacterial cultures. However, it is clear that the inhibitory effects were not due to TAGN-induced pH changes, since the addition of this substance had no pronounced influence on initial hydrogen ion concentration of the media. To determine whether TAGN was bacteriocidal, cells were suspended in buffered solutions of TAGN at concentrations of 500 or 2,000 ppm for up to 5 hours. These concentrations were high enough to prevent cell proliferation in a suitable medium such as TSB, but as shown in Tables 1 and 2, viability of the cultures was unaffected. In every case the bacteria were capable of renewed growth following TAGN removal by centrifugation. Therefore, while TAGN was bacteriostatic under the specified conditions of this test, it was neither bacteriocidal nor significantly toxic to the microbial cultures examined . Oxygen uptake, a method of evaluating cellular oxidative capabilities, was investigated at a constant TAGN concentration of 500 ppm (Figures 12 to 25). In several instances oxygen uptake was markedly depressed by 500 ppm TAGN, as in the case of the common soil inhabitants Arthrobacter sp. (Figure 17) and Bacillus cereus (Figure 18). But the majority of the bacteria tested were not significantly influenced by exposure to TAGN. In most cases, the bacteria assimilated oxygen at nearly identical rates to those determined for the controls. A few isolets, such as StaphylocQccus^ aureus (Figure 14) and SR 406 (Figure 21), were even stimulated by exposure to TAGN. Both Pseudomonas aeruginosa and Escherichia coli were examined for their ability to degrade TAGN under batch fermenter conditions (Figure 26), In each case the bacteria significantly reduced the quantity of TAGN available in solution, persumably through degradation, although no methods were �available to determine active bioaccumulation or adsorption. Thin-layer chromatography of the resulting cell-free supernatants of the Pseudpmonas aeruginosa culture (Figure 27) failed to show the presence of any soluble TAGN byproducts, but did provide an additional method to verify the reduction of TAGN in aqueous solutions under these culture conditions. �SECTION IV CONCLUSIONS The results of this study indicate that, while TAGN was generally bacteriostatic at concentrations of 100 ppm and above, the bacteria tested were not otherwise adversely affected by concentrations as high as 2,000 ppm for contact periods up to 5 hours. Under the experimental conditions of this study, TAGN was neither bacteriocidal nor did it appreciably affect the respiratory activity of the cells. Following exposure and subsequent removal of TAGN from solution, all bacteria tested were capable of normal growth resumption. Moreover, some bacteria were capable of degrading or at least removing TAGN from solution, thereby effectively reducing the aqueous concentration of this compound as might result from testing and disposal of this proposed propellant. �1.6 1 (A) PseydoMonas aeruginosa 1,4 X 1.2 •M •H in (3 4> O 1.0 <D u o O.f <M 1/5 (fl c 0.6 (U Q en u p. o 0.4 0.2 10 Time (hours) 12 �1.6 -.1 (B) Bacillus megaterlum 1.4 , . X *J 1.2 •H t/> Q 2 1.0 SC O 0.8 - - X •M •H tfi g 0.6 Q Q •H 0.4 0.2 -- 8 10 12 14 16 18 20 Time (hours) Figure 1. Standard Growth Curves of (A) Pseudoiaonas aerugino sa QMB 1468 and (B) Bacillus megaterium QMB 1605 Exposed to TAGN �1.6 -r 2 (A) Staphylococcus aureus 1.4 - o 10 n Time (hours) 14 16 20 �l.fi-r 2 (B) Bacillus cereus 1,4- - X 1.2 • H tn c i.o- - O C. S to X g C.6 Q o • r4 10 12 14 16 18 Time (hours) Figure 2. Standard Growth Curves of (A) Staphylococcus aureus QMB 1458 and (B) Bacillus cereus QMB 1597 Exposed to TAGN �1.6 — 3 (A) Escherlchia coll 1.4- X 1.2. tn (3 O Q 1.0 - fl> U 3E C 0.8 - - CM LTS X +-> •H w C (U Q 0.6 - - tfl o •r-l *J 0.4 . . 0.2 . . Time (hours) �1.6 T 3 (B) SR 401 1.4 - - X1.2-- c Q -« 1.0 - i—i u 35 o o.e + LO X +J g 0-6 + Q ,0,4 - - 0.2 - - 50 ppm 100 ppm 500 ppm I Time (hours) Figure 3. Standard Growth Curves of (A) Escherichia coli QMB 1557 and (B) SR 401 Exposed to TAGN �4 (A) SR 403 1.4. . X 1.2. _ w C tt» o l.C o CJ s: O 0.8-- LD X 4-1 •—! £ 0.6. - ctt o .-4 a. 0.4 . _ o 0.2 - - Time (hours) 20 �1.6 -r Time (hours) Figure 4. Standard Growth Curves of (A) SR 403 and (B) SR 404 Exposed to TAGN �1.6 _ 1.4 - X 1.2 to 1.0 4» U s^/ as O 0.8 CM tn X 4-1 Pi 4> 0.6 Q O 0.2 -- 10 Time (hours) 12 14 16 18 20 �1,6-*5 (B) SR 407 1.4-- 1.2-0> o 1.0- 0) u O 0.8- CM LO W g 0.6' O n) U •H a, 0.4 O 0.2 -- 10 12 14 16 Time (hours) Figure S. Standard Growth Curves of (A) SR 405 and (B) SR 407 Exposed to TAGN 18 20 �1.6 10 Time (hours) 12 14 16 18 20 �<£> 10 Time (hours) 12 u Figure 6. Standard Growth Curves of (A) SR 408 and (B) SR 409 Exposed to TAGN 18 �7 (A) C 1 K) o Time (hours) �1.6 —. 8 10 12 14 U Time (hours) Figure 7. Standard Growth Curves of (A) C 1 and (B) C 4 Exposed to TAGN �N) K> 10 Time (hours) 12 14 16 18 �8 (B) Arthrobacter sp. 1.4. . X 1.2. . o> cs 2 i.o4- o 0.84- CN U^ •Jl g 0.6-JQ td o •H cL 0.4- . 0.2 - - 10 12 14 Time (hours) Figure 8. Standard Growth Curves of (A) T 6 and (B) Arthrobacter sp. QMB 1631 Exposed to TAGN 16 18 20 �1.6 — 9 (A) Bacillus subtilis 1.4. . X 1.2 •H c OJ a 1-0 tt» u 2 0 0.8 r-4 tn X g 0.6 o 03 O 0.2 20 Time fhours) �9 (B) Serratia marcescens a> Q •-< 1.0 - - u v_/ z O 0.8 r-j NJ cn tn g 0.6- - a 0.4-- 0.2- - 10 12 16 18 Time (hours) Figure 9. Standard Growth Curves of (A) Bacillus subtilis QMB 1611 and Serratia marcescens QMB 1466 Exposed to TAGN �1.6 -_. 20 Time (hours) �1.6 10 (B) SR 406 1.4 - - X 1.2 - . <D Q 1.0 - 0) u 3E O 0.8 fM LA tn C 0.6 o Q 0.2. . 16 Time (hours) Figure 10. Standard Growth Curves of (A) SR 402 and (B) SR 406 Exposed to TAGH IB 20 �1.6 ro 00 8 W Time (hours) 12 14 16 18 �1.6 -r 11 (B) T 100 Time (hours) Figure 11. Standard Growth Curves of (A) T 4 and (B) T 100 Exposed to TAGN �100 T 12 (A) PseudoiBonas aeruginosa (Endogenous) ( N O fti O CO 80- - o (U o TAGN (U O. Relative Rates: .Control (100 %) .500 ppm TAGN (86"r) 70- - 60 i I 10 Time (min") 12 14 16 18 �100 T 12 (B) Pseudoroonas aeruginosa (Exogenous) ^ Control \ D . 40 J_ Relative Rates -Control (100?) 500 ppm TAGN 20 10 14 16 Time (min) Figure 12. Endogenous (A) and Exogenous (B) Oxygen Uptake by Pseudomonas aeruginosa (^ffi 1468 Exposed to 500 ppm TAGN 18 20 �100 T 13 (A) Bacillus rnegaterium (Endogenous) Control 70 - - Relative Rates: V .Control (1002) .500 ppm TAGN (lOOt) I 60 10 Time (min) 12 14 I 16 18 20 �13 (B) Bacillus megateriurn (Exogenous) Relative Rates: Control (IOCS) 500 ppm TAGN (114%) 20 8 10 12 14 16 Time (rain) Figure 13. Endogenous (A) and Exogenous (B) Oxygen Uptake by Bacillus megaterium QMB 1605 Exposed to 500 ppm TAGN 18 �100 - . 14 (A) Staphilococcus aureus (Endogenous) 70 Relative Rates: .Control (100%) ,500 ppm TAGN (142%) 60 10 Time (min) 12 14 16 18 20 �100 T 14 (B) Staphylococcus aureus {Exogenous) Control 90 . _ o 3 r—i O 80. . D U SH 0> \ 500 ppm \ TAGM V \ \ N a. 70 . _ Relative Rates: .Control (100$) ,500 ppm TAGN (200%) f 60 S 10 12 a 14 16 Time (min) Figure 14. Endogenous (A) and Exogenous (B) Oxygen Uptake by Staphylococcus aureus QMB 1458 Exposed to 500 ppm TAGN 18 20 �100 . 15 (A) Serratla marcescens (Endogenous) 90 - - 500 ppm TARN <M o CD *—I o 80 - - c d> u o> ^ a. 70 . _ Relative Rates: .Control (100°*) ,500 ppm TAGN (112%) 60 10 Time 12 14 +— 16 f 18 20 �100 15 (B) Serratla inarcescens (Exogenous) 90 - - CM O ppm TAGN JO 3 80 - - c 4) o H 4) Q. 70 - - Relative Rates: .Control (100%) — -.-.—500 ppm TAGN (93%) 60 10 iz 14 Time (min) Figure 15. Endogenous (A) and Exogenous (B) Oxygen Uptake by Serratia inarcescens QMB 1466 Exposed to 500 ppm TAGN IB �100 -r- 16 (A) EscheH.chla colj (Endogenous) 90 - - . Control ( M O 0 ) 500 ppm TAGN •s r-t i—i O in 80 - CM 00 4) u 70 . . Relative Rates: .Control (100X) .500 ppm TASN (103%) 60 10 Time (min) 12 14 16 is 20 �100 16 (B) Escherichia coll (Exogenous) to OJ ( X 40 4- Relative Rates: .Control (100%) .500 ppm TAGN (118S) 20 10 12 14 16 Time (min) Figure 16, Endogenous (A) and Exogenous (B) Oxygen Uptake by Escherichia cpli QMB 1557 Exposed to 500 ppm TAGN 18 20 �100 .*17 (A) Arthrotuctar sp. (Endofenous) 500 ppir, ' v. TAGN CM O Control 3 i—i O 80. *-> 4> U f-i 70 Relative Rates: .Control (100%) ,500 ppm TA6N (56%) 60 10 Time (min) 12 14 16 18 20 �100 TT 17 (B) Arthrobacter sp. (Exogenous) 80 4- (M O 4> ! >. 500 ppm v TAGN V—* . 0 O CO 60-4- -(-I Q> CJ ^H 0) a. 404Relative Rates: .Control (1005:) .500 ppm TAGN (90?.) 20 10 Time (rain") Figure 17. Endogenous ( ) and Exogenous (B) Oxygen Uptake by A Arthrobacter sp. QMB 1631 Exposed to 500 ppm TAGN �100 T 18 (A) BacjVTus cereus (Endogenous) O C <£> O (H (U a, Relative Rates: .Control (10OT) _ _ _ _ _ _ 500 ppm TA6N (103'* 80 10 Time (min) 12 14 16 18 �100-t- 18 (B) Bacillus cereus (Exogenous) 500 ppm TAGN 90- - Control O 0> rH £3 •—t O CO 80- . c <u u CD ^ D, 70. - Relative Rates: Control (1005) 500 ppm TAGN (69°",) 60 10 14 16 Time (rain) Figure 18. Endogenous (A) and Exogenous (B) Oxygen Uptake by Bacillus cereus QMB 1597 Exposed to 500 ppm TAGN 18 20 �100 19 (A) SR 402 (Endogenous) 500 ppm TAGN 90- - Control o 0> i-H • § t-H O !) / 80- - <o o f-l 0) .. 0 70. _ Relative Rates: .Control (100%) ,500 ppm TAGN (72%) 60 10 Time (min) 1.2 14 16 18 20 �100 __- 19 (B) SR 402 (Exogenous) 90- 500 ppm TAGN (M o 3 i—t O 80 _ _ O> o !-. 03 O. 70 _ . Relative Rates: .Control (100%) 500 ppm TAGN (99*) 60 10 12 14 16 Time (min) Figure 19, Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 402 Exposed to 500 ppm TAGN 18 20 �100 _ 20 (A) SR 404 (Endogenous) 90- - O tu r-1 , 0 3 c 0 U 500 ppm TAGN Control 70- . Re1atl¥e Rates: , Control (100*) 500 ppm TAGN (84%) 60 10 Time (rain) 12 34 16 18 20 �100 _ 20 (B) SR 404 (Exogenous) 80- - CM o , 0 <—I O CO 60- - c <o u ft Relative Rates: 40- - .Control (100?) 500 ppm TAGN (1125) 20 H \ 8 10 12 16 Time (min) Figure 20. Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 404 Exposed to 500 ppm TAGN 18 20 �100, 21 (A) SR 406 (Endogenous) 4:1. oo 70. . Relative Rates: Control (100%) 500 ppm TASN (140%) 60 10 Time (min) 14 16 18 20 �100--. 21 (B) SR 406 (Exogenous) 90 - - o 0> I—I 3 O r—I 80-- -ft10 0) o (H 0) a. Control 70 Relative Rates: ..Control (100*) — _ — _. 500 ppm TAGN (87% 60 8 Figure 21, ^ 10 Time (rain) 12 14 16 Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 406 Exposed to 500 ppm TAGN 18 �100 T 22 (ft,) SR 407 (Endogenous) 90- - CM O <u 1—I , 0 3 80- VI O 4J CD O N S> £L, V 500 ppm TAGN S Control 70. Relative Rates: .Control (100*) .500 ppm TAGN (91%) 60 I I 10 Time (min) 12 14 16 18 20 �1CW T 22 (B) SR 407 (Exogenous) 80 . . r-j O 3 i-H O 60 - - O !~i 0> a. 500 ppm\ TAGN \ 40 - - Relative Rates: Control (100%) 500 ppm TAGN (134%) 20 10 14 16 Time (min) Figure 22. Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 407 Exposed to 500 ppm TAGN 18 20 �100-_ 23 (A) SR 408 (Endogenous) in r-o 70 Relative Rates: .Control (lOOt) 500 ppm TASN (107%) 60 10 Time (min) 12 14 16 18 20 �90-_23 (B) SR 408 (Exogenous) 70. - CN o O en 50- - 500 ppra TAGN o UJ F-t <u Cu 30. Relative Rates: .Control (100%) 500 ppm TAGN (77%) 8 10 12 14 16 Time (min) Figure 23, Endogenous (A) and Exogenous (B) Oxygen Uptake by SR 408 Exposed to 500 ppm TAGN 18 �100 T 24 (A) SR 410 (Endogenous) 90 . > o w TAGN GJ F-H •§ o I—I t/3 80 . _ Control C CO u f-l 70 _ . Relative Rates: .Control (10021) .500 ppm TAGN (102%) 60 10 Time (min) 12 16 18 �100 T 24 (B) SR 410 (Exogenous) CM O 0) f—t O 60 •M C 0) u 40 - - Relative Rates: .Control (1001) ,500 ppm TAGN (77° 20 8 10 12 14 16 Time (roin) Figure 24. Endogenous (A) and Exogenous (B) Oxygen Uptake by SR410 Exposed to 500 ppm TAGN 18 20 �100 25 (A) C 4 (Endogenous) 90 CM O 4) O to 80 c <u O 0) CL, 500 ppm TAGN 70- - Relative Rates: .Control (100%) __ _ _ _ 500 ppm TAGN (94%) 60 1*4 Time (min) re �100 T 25 (B) C 4 (Exogenous) \ 90 . . \ \ \ \ Cvl O t-t \ f> t-t O 01 500 ppm TAGN 80 __ 0) u \ N 0) o. 70 __ — Relative Rates: .Control (100S) 500 ppm TAGN (92%) 60 8 10 12 16 Time (min) Figure 25. Endogenous (A) and Exogenous (B) Oxygen Uptake by C 4 Exposed to 500 ppm TAGN �—.0,16 80 __ 26 (A) Pseudowonas aeruglnosa 70. . & O, ui z 'J DO Culture Density (O.D.; pp« TAGN 80 100 Time (hour) 120 140 160 180 �0.16 26 (B) Escherich^ia coil * en 20 40 60 80 100 1?0 140 160 Time (hour) Figure 26. Disappearance of TAGN from Cultures of (A) Pseudomonas aeruginosa QMS 1468 and Escherichia coli QMB 1557 as a Function of Cell Density ( . . 0D) �Solvent Front R f 0.11 Origin 0 hr 48 hr Mobile Phase: 72 hr 96 hr 120 hr 140 hr Standard TAGn Methane1 - Water - Dimethyl Sulfoxide (40:30:30) Figure 27. Thin-Layer Chroaatogram Depicting the Disappearance of TAGN from the Cell-Free Supernatant of a Growing Culture of Pseudomonas aeruginosa as a Function of Time 60 �TABLE 1. EXPOSURE OF BACTERIAL CULTURES OBTAINED FROM US ARMY NATICK LABORATORIES TO TAGN (N is the number of replicate samples; a is the observed significance level) Sample Incubation (hr) TAGN (ppm) N Standard Deviation Mean a 1 0 6 2.2xlO? 0.6xl07 .. . 1 500 5 l.SxlO7 0.9x10 0.114 1 2000 6 1.4x10 5 Pseudomonas aeruginosa 0 5 6.3x10 7 7 7 0.4x10 7 1.1x10 7 5 500 6 3.8x10 5 2000 4 3.2x10 1 0 6 11x10 7 1.0x10 7 7 0.2x10 6 Bacillus megaterium 6 1 500 6 5.8xl0 2.7xl0 1 2000 6 6 6.7x10 1.2x10 5 0 6 2.0x10 6 — 0.004 0.002 6 2.0x10 6 0.011 6 — 0.003 0.002 6 0.7x10 5 5 l.OxlO 0.3xl0 5 Bacillus cereus 500 — 0.007 2000 6 2.8xl06 0.6xl06 0.022 1 0 6 2.1x10 0.6x10 1 500 6 2.6xl06 2.0xl06 1 2000 6 2.9x10 5 0 6 2.7x10 5 500 6 1.2x10 5 2000 6 2.6x10 61 6 6 6 6 6 6 2.0x10 5 6 6 0.147 0.103 6 0.7x10 6 0.5x10 1.4x10 6 0.002 >0.2 �TABLE 1. EXPOSURE OF BACTERIAL CULTURES OBTAINED FROM US ARMY NATICK LABORATORIES TO TAGN (CONCLUDED) /v (N is the number of replicate samples; a is the observed significance level) Sample Incubation (hr) TAGN (ppm) N Mean Standard Deviation A- a 6 7,3xl07 I.SxlO7 500 6 7.9xlO? I.SxlO7 0.13 2000 6 6.6xlO? 1.9xl07 0.128 5 0 6 7.7xi07 O.SxlO7 5 500 6 S.lxlO7 2.1xl07 0.169 5 2000 6 7.9xlO? 1.2xl07 0.182 1 0 6 7.0xlO? 0.4xlO? 1 500 6 8.2xl07 3.3xlO? 0.109 1 2000 6 llxlO7 2,0xl07 0.002 5 0 6 8.2xlO? 2.0xl07 5 500 6 7.2xlO? 1.4xl07 5 Eseherichia coli 0 1 Serratia marcescens 1 1 Staphylococcus aureus 2000 6 8.2xl07 I.SxlO7 1 0 6 l,3xlO? 0.7xlO? 1 500 6 1.6xlO? 0.9xl07 0.138 1 2000 6 i.SxlO7 O.SxlO7 0.002 5 0 3 6,9xl07 3.1xl07 5 500 6 2.8xlO? I.SxlO7 0.042 5 2000 6 2,lxlO? 0.7xlO? 0.031 62 0.097 �TABLE 2. EXPOSURE OF BACTERIAL CULTURES INDIGENOUS TO EGLIN AFB, FLORIDA, TO TAGN A (N is the number of replicate samples; a is the observed significance level) N Mean Standard Deviation 0 6 l.SxlO7 0.2xlO? 1 500 5 l.SxlO7 0.3xl07 1 2000 6 l.SxlO7 5.6xlO? 5 0 4 2.8xl07 6.3xlO? 7 7 Sample Incubation (hr) SR 409 1 TAGN (ppm) 5 6 l.SxlO O.lxlO 5 SR 404 500 2000 6 2.3xl07 O.SxlO7 1 0 5 O.SxlO6 0.4x10 6 f\ a ___ 0.033 >0.2 — 0.194 >0.2 1 500 6 l.SxlO O.lxlO — 0.013 1 2000 5 l.lxlO6 0.2xl06 0.100 5 0 4 7.0xl06 0.6xl06 6 6 6 — <0. 00025 5 6 3.9xl0 l.lxlO 5 SR 406 500 2000 4 J.OxlO6 2.4xl06 1 0 6 5.6xl07 l.OxlO7 _ — 1 500 6 5.7xlO? 0.9xlO? >0.20 1 2000 6 S.OxlO7 0.6xlO? 5 0 5 8.7xl07 l.OxlO7 7 7 — 0.001 5 500 5 8.4xl0 l.SxlO — 0.191 5 2000 6 7.6xl07 1.9xlO? 0.076 63 �TABLE 2. EXPOSURE OF BACTERIAL CULTURES INDIGENOUS TO EGLIN AFB, FLORIDA, TO TAGN (CONTINUED) (N is the number of replicate samples; a is the observed significance level) Sample Incubation (hr) SR 402 1 TAGN (pprn) Standard Deviation a 0 6 4.3xl07 l.OxlO7 — «.— 500 6 4.8xl07 2.0xl07 0.154 1 2000 6 5.8xl07 l.SxlO7 0.024 5 0 6 5.9xl07 0.9xlO? --- 5 500 6 lOxlO7 l.OxlO7 <0. 00025 5 2000 6 S.SxlO7 0.9x10 0.002 1 0 6 4.8x10 1.9xlO? 1 500 6 4.1xl07 O.SxlO7 0.114 I 2000 6 7.4xl07 l.lxlO7 0.009 S 0 6 3.7xlQ7 l.SxlO7 -__ 5 500 6 5.2xl07 2.1xl07 0.061 S SR 408 Mean 1 SR 407 N 2000 6 2.9X107 O.SxlO7 0,092 1 0 6 2.4xl07 0.6xlO? 1 500 6 1.9xlO? 0.2xl07 0.028 1 2000 6 S.lxlO7 0.6xlO? 0.029 5 0 6 3.2xl07 0.7xlO? 7 7 A. _ __ 5 500 6 2.8xl0 0.4xl0 — 0.083 5 2000 6 3.0xl07 O.SxlO7 0.150 64 �TABLE 2. EXPOSURE OF BACTERIAL CULTURES INDIGENOUS TO EGLIN AFB, FLORIDA, TO TAGN (CONCLUDED) (N is the number of replicate samples; a is the observed significance level) Sample Incubation (hr) SR 405 1 TAGN (ppm) Standard Deviation N Mean 0 5 1.0x10 1 500 6 1 2000 6 5 0 6 5 500 6 1.2xlO? 7 1.0x10 7 1.6x10 7 1.0x10 0.4xlO? 7 0.2x10 7 0.3x10 7 0.4x10 5 2000 6 0.3xlO? 0.2xl07 7 0.1x10 65 (The reverse of this page is blank) /•>• a 7 — 0.080 — — 0.009 <0. 00025 ��REFERENCES 1. Oster, G., and A.W. Pollister, (eds.). Physical Techniques in Biological Research, New York: Academic Press, 1955, Vol I, pp. 51-76. 2. Norris, J.R., and D.W. Ribbons, (eds.), Methods in Microbiology, New York: Academic Press, 1969, Vol I, pp. 473-504. 3. Housewright, R.D., and C.B. Thorne, "Synthesis of Glutamic Acid and Glutamyl Polypeptide by Bacillus anthracis; I. Formation of Glutamic Acid by Transamination," Journal of Bacteriology, 1955, 60:89. 67 (The reverse of this page is blank) ��INITIAL DISTRIBUTION DDC 12 AUL (AUL/LSE-70-239) 1 ASD/ENFEA USAF (AF/SAMI) 1 1 Ogden ALC/MMWM AFIS/INTA Veg Con Div (SAREA-CL-V) DDR§E (Tech Lib) 2 1 1 1 USAFA/DFCBS 1 AFLC (DS) Deseret Test Cen (Tech Lib} 1 1 AFLC/MMNO SAAMA/SFOT 1 1 NWC (Tech Lib) NWL (Tech Lib) USDA/Pesticide Coordinator USDA/Agr Env Qual Inst AFSC/SDW DDR§E (Env § Life Sci) Edgewood Arsenal (SAMUEA-SA) AFSC/DEV AEDC/DEE Edgewood Arsenal (SAREA-TS-L) 1 1 1 1 1 1 1 1 1 1 Edgewood Arsenal (SAREA-CL-V) 1 CINCPAC(JSAl) USAF Env Health Lab NASA Miss Test Facility NWC Env Eng AMD (RD) 1 1 1 1 1 USA Natick Lab 1 AMRL/THE AFCEC/EQ AMRL/THT Eglin AFB: ADTC/DEN ADTC/SGPE TAWC/TRADOCLO 1 1 1 1 1 1 AFATL/DL 1 AFATL/DLOSL 9 AFATL/DLV 10 ADTC/CSV 1 69 (The reverse of this page is blank) �� 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. 017 Folder The folder containing the original item. 0184 Series The series number of the original item. Series 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 Patrick, Michael A. Description An account of the resource <strong>Corporate Author: </strong>Environics and Human Factors Office, Air Force Armament Laboratory, Armament Development and Test Center, Eglin AFB, Florida Date A point or period of time associated with an event in the lifecycle of the resource 1976-11-01 Title A name given to the resource Toxicological and Recalcitrant Properties of a Proposed Propellant Ingredient, Triaminoguanidine Nitrate (TAGN). I. Microbiological Study Subject The topic of the resource microbial populations biodegradation https://www.nal.usda.gov/exhibits/speccoll/files/original/9aa3c7403d775662daeff3c3b2db666c.pdf b9edb2dc9b63d6208296861a49a4d9d4 PDF Text Text Item ID Number: Author CorpOratB Author 00102 Rosenberg, Arthur Department of Agronomy, Cornell University, Ithica, New York Roport/Artldo TltlO Microbial Degradation of Pesticides Journal/Book Title Year 197S Month/Day November 17 Color Number of Images 30 DOSCrlptOn Notes Contract No. N00014-78-C-0044; Task No. NR 205-032: Annual Report Friday, December 08, 2000 Page 102 of 106 �Annual Report October 1, 1977-Sept.30,'78 Micrcbial Degradation of Pesticides • . MMOMMINO OH«. Nft»O«t N T CONTRACT OH «NMT MOU«IIV«j N00014-78-C-0044 . ^.^ Arthur toaenberg aixi Martin Alexander ORGANISATION NAMI AND -X 00 of Agronomy Cornell tluvwrsity , Now York 14853 II NR 205-032 II. MPOMT O*r« CONTMOLLINO OFPtCl NAMI AND ADDMIII 17 NowtMr 1978 Office of Naval Research Department of the Navy, Code 200 ITONlNQ AOCNCV NAMI 4 A I* •If" «lff*r«nr ta* ConN«IIHi« OHIcij OtltmtuTlON ITATCMlNT f*f Ml* Jt*»o/O Distribution of this report is unlimited., »; DEC 7 IT CMITMIIUTION ITATIMKNT (•/ Hi* »78 *l*o* >0. 8 NOTII U. ». KIV WOIIOI fCoiltaM «n rtrcrM «M* </ n«o««MTr MM (4wi(lf|> k? M*«k i.iaikw) Kbcrcblal degradation, Herbicides, Pesticides, Pollutants, 2,4-D, 2,4,5-T M AMTMACT fCmlfniM «n «•»•»•• •«• If Ma«M«r •»* an next page n •WTJOH OP I NOV •• IB OMIOLITI t/H 0101-014-IWI i lifted ••nu II �{. \. HBSTWSCT ^ ^ Fitt:y-two bactati.<i isolated frxin twiwaqt?, tai^>ci.itr soil, and var Tropical aoils were tested foi their «bility to attack 2,4-D and 2,4, V'r F Mirteen caused the disappearance of JO to 100% of the 2,4-0, and nine bixxight about the deatmction of 20 to 100% of the 2,4,5-T. None of t.h»orqaaiww oould uae 2,4-D or 2,4,5-T aa a sole source of carhun. Degradation of 2,4-D and phanoxyacetic acid in nonaterile aowaqo and a tropical soil was greatly <u>hanoed by pretreatincj the sewage and soil with these coipounda, suggesting the selection for organisms capable of attacking 2,4-D and phenoxyaoetic acid. Cell yields of the three most active 2,4,5-T degradera in a madi.um with glucose, glycerol, and sodium succinate and in a banzoate-supplement. medium with and without 2,4,5-T did not differ, suggesting oometabolic attack. Resting dell suspensions of nine of the isolates cleaved chlorine from the 2,4,5-T molecule while metabolizing more than 40% of the 2,4,5-T, suggesting ring cleavage of the herbicide. Eight isolates produced chlorinated phenol from 2,4,5-T. Studies of the respiratory activity of three isolates also suggested ring cleavage of 2,4,5-T, By use of (^Q-ring-UL) 2,4,5-T, it was found that the herbicide was readily metabolized in a tropical son. MCUWTV ci.Mwric*rieM OP AOK(lNll|BJ 197 �•f NAVAL RESEARCH Task No. NR 205-032 A;I.HIM, AU'JKT JJICRCBIAL gBGRADATION OF J>ESnCIDEB Ay'Stoaonborg 4£ M./Alexander Cornell University Department of Agronomy Ithaca, New York 14853 7 /^ 7 3tf Reproduction in whole or in part is permitted for any purpose of the United States Government Thin document has been approved for public release; its distrilution is unlimited ±>2ij �WriUUUCTlGN herbicides such as 2,4-D and 2,4,'j-T ^ro .imnny the mnet ocrnnrmly usecl horbicidaa foi selective weed control and for defoliation. The metabolic fates of 2,4-0 and 2,4,5-T are of obvious concern because of the potential toxicity of the herbicides and their metabolites to nontarget organisms. Although considerable work has been done on the persistence of 2,4-D in the soils characteristic of agricultural lands of continental United States, little attention has been given to the persistence and fate of this herbicide or 2,4,5-T in tropical soils of the Pacific Ocean area. The research in this report was designed to determine the persistence and fate of 2,4-D and the persistence, fate, ard role of ccmetabolism in the biodegradation of 2,4,5-T in such soils. MATERIALS AND METHODS »^ Materials. 2,4-Dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyaoetic acid (2,4,5-T) were obtained from Dow Chemical Co., Midland, Mich.; phenoxyacetic acid (PA.), 2,4-dichlorophenol (OCP), 2,4,5-trichlorophenol [TCP), and catechol fron Eastman Organic Chemicals, Rochester, N.Y.; and phenol and sodium benzoate fron Mallinckrodt Chemical Works, New York, N.Y. Uniformly ring-labeled (14C] 2,4,5-T (sp act 1.61 nCi/ranol) was purchased from California Bionuclear Corp., Sun Valley, Calif. The purity of the C~labeled compound was 98% as determined by thin-layer chrcmatography. Unlabeled 2,4,5-T and 2,4-D were purified by recrystallizing them twice in benzene. The purity of the oonpounds was greater than 99% as determined by thin-layer chronatography and melting point determinations. The compounds were prepared at 10,000 ppm in either 95% ethanol (Mallinckrodt) or as the ilium salt in distilled water. �(jJMWware. Glassware was cltMied by .1 24-h immersion in 20» (vol/vol) Nitric acid was removed by multiple washings in tap water followed by distilled water. Isolation of sewage and soil microorganisms. Bacteria capable of degrading 2.4-D and 2,4,5-T were isolated by the enrichment culture technique usinq the following sewage and soils at A concentration of 10% as initial sources of inocula: sewage collected at the primary effluent of the Ithaca, N.Y. sewage treatment facility (used within 30 min after collection), a temperate-zone soils mixture, Philippine soil (pll 6.8), Puerto Rico soil (pH 5.8), Nigerian soil (pH 5.9), and Trinidad soil (pll 6.1). Ttw; enrichment medium was an inorganic salts medium (3) and contained (per liter): (NH.)-SO., 0.5 g; NCI, 0.2 g; NaCl, 0.1 g; CaCl0.2H20, 50 mg; MgS04-7H2O, 0.2 g; PeCl.j.6H2O, 20 mg; and bufr fered with 12 mM potassium phosphate buffer, pH 7.2. When used as the source of carbon, the compounds were added to final concentrations of 250 and 1000 ppn. When used as a substrate for cometabolism, the compounds were added to a final concentration of 100 ppm in the inorganic salts medium containing 0.3 g/liuu: each of glucose, glycerol, and sodium succinate (basal medium). The medium was sterilized by filtration through sterile 0.2 urn membrane filters (Millipore Corp., Bedford, Mass.). The enrichment cultural (10 ml total volume) were incubated statically in screw-cap tubes at 29°C. 2,4-D, 2,4,5-T and PA disappearance were determined by UV absorbance. Once significant loss and visible turbidity occurred, 1.0 ml of the enrichment culture was transferred to fresh medium. After two successive transfers, the enrichments were streaked on plates containing either the inorganic salts medium with 15 g/liter agar (Difco) and amended with 250 or 1000 ppm of the compound if used as carbon source or basal medium with 15 g/liter agar and amended wii.: 100 ppn of the test compound. Isolates able to attack 2,4-D and 2,4,5-T were subsequently recognized by their ability to degrade the compound in liquid medium. Growth �4 . curvei and kinetics of 2,4,5-T disappoarancx' were mudted by tjrowinq selected ^ organisms ui basal median amended with r>0 ppm 2,4, VT or mineral uclu medium with 300 ppm 2,4,5-T at 29°C and 150 rpm. Periodically, portions won; remtjved, and either- the optical density determined or the samples were oenlrifuged at 10,000 X a at 4°C for 15 mui and the supernatant fluid assayed for 2,4,5-T disappearance. Nonbiological disappearance of 2,4,5-T was assessed using sterile incubation medium. Resting cell preparations. To prepare resting cells, cultures were grown in 1-liter Erlenmeyer flasks containing 500 ml of basal median amended and uriamended with 25 ppm 2,4,5-T and incubated at 29°C and 150 rpm for 36 h. The cells were harvested by centrifugation for 15 nvin at 10,000 X ^ at 4°C and washed three times with and resuspended in 10 ml of 10 nM phosphate buffer, pH 7.2 to an optical density of 1.5 at *20 nm. lt> 10 ml of the resting cell sus.„_, pens.Lon was added 25 ppm 2,4,5-T, and the suspensions were incubated for 24 h at 29°C and 150 rpm. The reaction mixtures were centrifuged, and the supernatant fluid was used for the analytical and chemical procedures. Nonbiological degradation of 2,4,5-T was assessed with sterile medium. Manometry. Standard manonetcic procedures were used (20). Each flask received either 0.33 ml of a solution with 1.1 pinoles of substrate as 2,4,5-T (sodium salt) in distilled water or inorganic salts medium with glucose or sodium benzoate as sole carbon source. The endogenous flask contained 0.33 ml of 10 nM phosphate buffer, pH 7.2, in the side arm. The main compartment contained 2.67 ml of cell suspension in the phosphate buffer, and the center well received 0.2 ml of 20% KGH. The cells were grown in the sane median as used for respiration studies. Degradation of (14C-ring-UL) 2,4,5-T. To determine the persistence anil ^_ degradation of 2,4,5-T in soil, 25 and 75 vq 2,4,5-T (14Oring-UL)/g aoil wire add<2d to 5.0 g of Philippine soil (pH 6.8, organic matter about 3%) in 50 ml �Erlanneyeu flaska. The screw C»UH weio mxlititxl n> aouomnudare \.atlun-ailieone discs (Pierce Chemical Co., Rxrkford, Til.). The herbicide was dissolved in 95% ethanol, And the uolvent was allowed to evaporate before mixing with the soil. The soil was wetted to 70% of field capacity with dist.'lled water. One flaak with each chemical concentration contained a sanple of soil irradiated with a tot*I dosage of 6 megarads; this dosage was sufficient to totally inactivate the soil microflora. Periodically, the soil was acidified with) 10 N HjSO., and air was passed into the soil for 90 min. The air passing out of each soil was bubbled through filter sticks (Ace Glass, Inc., Vineland, N.J.,) into disposable scintillation vials (Kimble, Toledo, Ohio) containing 2.0 ml of carbon dioxide-trapping agent ( 0 mMet, Amer sham/Searle Corp., Arlington 0. "oights, 111.) and 13 ml of aqueous counting scintillant (ACS, Amarsham/Searle ^^rp.). Anadytical methods. Turbidity was measured at 420 ran in a Bausch and Lamb spectrojihotaneter, model Electronic 20. 2,4,5-T, 2,4-D, and PA disappearance were monitored by ultraviolet (UV) absorbanoe measurements at 292, 280, and 268 ran, rea{iective.Lyf in 1-on quartz cuvcittes in a Beckman grating spectrophotaneter, model DO-G. Chloride release was determined by the technique of Bergmann and SaniK () 6. Ph<anol production was determined by the method of Chrastil (10). Catecnol production was determined by the method of Arnow ( ) 4. Carbon dioxide-14C activity in the scintillation vials was determined by counting in a Beckman liquid scintillation counter, model LS-100C. All counts corrected for quenching and background. �RKSU1.TS Isolationofj»jjjj>Jja. Km ichmont oultuu\>; Abl<- to dogr.ide 2,4-D 2,4,5-T ware isolated I" run se*«*ie and soils. IXii irxj the course of the onrichnwnt studies, the par*1 is tenet? of 2,4-0, 2,4,5-T and phannxyacet ic acid (PA) was dotenr.med for sewage and Philippine soil. Sewatjo .ind soil was amended with 100 ppm of the compound, and the degradation was followed by a decrease in UV absorbancy. vtian cxn|Mred to au toe laved sowaqe (cxintnil), qi-oater than 90% of the 2,4-D and PA disappeared after 7 and 12 days, respectively; however, 2,4,5-T was not attacked after 60 days (Pig. 1). Subsequent additions of 2,4-D and PA to thct sewage showed greater than 75% disappearance after 2 and .1 days, respectively, suggesting the selection for organisms capable of attacking the ccn|xxmds. Similar results wore obtained with Philippine soil, except that the time needed 'to obfwrve 90% disappearance of 2,4-D and PA was 14 and 16 days, respectively,, while 3 and 4 days were required for 75% disappearance of subsequently added 2,4-D and PA, respectively (Pig. 2}. 'To determine the number of isolates f ran each inoculum t' at could degrade 2,4-D and 2,4,5-T, the bacteria were grown in basal medium amended with 50 ppm 2,4-D or 2,4,5-T. Disappearance of the compounds was monitored periodically by recording the UV absorbancy of the culture's supernatant fluid, and these data were ocnpared to the disappearance of the confounds in sterile medium (control). A sximnary of the study to show the number of isolates from each inoculum capable of degrading 2,4-D and 2,4,5-T is given in Table I. Sewage and Philippine soil provided the most 2,4-D-metabo.lizing isolates, whereas the numbnr of 2,4,5-T-metabolizlng isolates, although highest from sewage, were fairly evenly distributed among the various soils. All enrichments yielded organisms capable of natabolizing 2,4-D and 2,4,S-T. Phenoxyacetic acid, Malt, �245-T 100 SE 2,4-D ADDITION COND PA DlTiON 75 UJ !5 (T 25 8 12 16 60 DAYS Fig. 1. Disappearance of 2,4-D, 2,4,5-T, and phenoxyaoetic acid in sewage amended with 100 ugAnl of each compound. �SECOND (SECOND PA ADDITION 20 60 Fig. 2. Disappearance of 2,4-D, 2,4,5-T, and phenoxyaoetic acia in Philippine soil amended w. th 100 Mg/ftg of each ccnpound. �1. Nuifcar of 2,4-D and 2,4,5-T •tihnUttnrj bactaria Uolatad fro* ',4-0 taldwnt atdjau-ataS- Soil Sewage Tenpera*a Philippina Puerto Rloo Nigeria and •oil 2,4,W Trinidad Soil SCMMje Tenperate Philippine Puerto Rico Nigeria Trinidad 2.4-Dfe 25 1 3 1 0 0 1 1 1 1 0 0 2,4-D£ 0 0 0 0 0 0 0 0 0 0 0 0 2,4,5^lfe 4 1 2 0 0 0 2 1 1 1 1 1 2,4,W£ 0 0 0 0 0 0 0 0 0 0 0 0 P)£ 23 0 3 2 2 0 2 1 1 1 * n£ 2 2 1 1 0 1 0 1 3 3 1 1 1 0 2 0 - N*£ 2 2 1 0 0 1 1 1 1 1 ••£ 1 1 1 1 1 0 i. 1 0 1 Ph»£ 3 0 2 1 0 1 2 1 1 0 1 PhamlS 1 0 1 1 0 0 1 1 0 0 1 1 1 KP* 2 1 1 0 1 1 0 0 0 0 0 0 DCT£ 0 0 0 0 0 0 0 0 0 0 0 0 *P^yB» 1 1 1 1 0 0 1 1 1 0 1 1 H^pCL 0 0 0 0 0 0 0 0 0 0 0 0 MA. 4tt»ri•tiona 2,4-D, ! • • t A. *• * * -* i . *m t A <_<•.•< for 2,4, 5-T *- * e>.*tic acid; PA, pharocyacatic acid; NaB. oodiia txruB- �8 p- .and phenol were used as a carbon source unci as a substrate for crme'-abolism, whereas 2,4-D, 2,4,5-T, DCP, and TCP were used as a substrate for cometabol ion only. The isolates were studied further to ascertain their morphological and biochemical characteristics. The bacteria exhibited the following diversity: 88* 'nere Grant-negative, 78% wore .xd-shaped, 58% were motile, 32% were pigmenbad and/or fluorescent, 68% were oxidase positive, and 94% were catalase positive (liable 2) . A breakdown of the characteristics of the isolates by inoculum indicated that the inocula contained a high percentage of Gram-negative, rod-shaped, catalase-positive bacteria, while the remaining characteristics fluctuated from a low of 5% pigmented and/or fluorescent bacteria fron the temperate soil to 88% oxidase-positive bacteria from the Philippine soil. Metabolism of 2,4-D and 2,4,5-T. Nineteen isolates were capable of metabolizing 2,4-D and 2,4,5-T. Resting-oeli suspensions of these bacteria were prepared, and 2,4,5-T disappearance, phenol and catechol production, and chloride release were determined. When compared to sterile controls, eight isolates caused the disappearance of greater than 50% of the 2,4,5-T. Extensive metabolism occurred with isolates 2A3 (88%), 4C3 (88%), and 5DJ (92%) (Table 3). Twelve isolates released chloride! in the medium, with isolates 2A3, 4C3, and. 5D3 liberating 90% or more. Eight isolates produced phenol from 2,4,5-T. to catochol was detected in the medium after a 36-h incubation period. The loss of tJV abaorbancy, release of chloride, and absence of phenol and catechol indicated that certain isolates were metabolizing 2,4,5-T by destroying the aromatic ring. The production of phenol with and without chloride release suggested that some of the isolates were converting 2,4,5-T to 2,4,5-trichlorophonol and mono- and dichlorophenol. �9 • ^ TABTE 2. Morphological and biochemical characteristics of bacteria isolated fran sewage and soil Percentage of isolates showing characteristic Gram Rod Pigmantad/ Qxidaae Catalase negative shaped Mobility fluorescent positive positive Source Sewage 90 95 69 50 87 100 Taiperate soil 92 60 60 5 40 97 Philippine soil 88 86 71 43 88 91 Puerto Rico soil 73 76 49 28 67 100 Nigeria soil 91 75 50 29 57 85 Trinidad soil 94 76 49 37 69 91 _ 88 78 58 32 68 94 Mean �10 tf K 3. M^tahnLiant of ^,4, VI', lolwnHo of i-Jiloi idu, and pmducHon of i>hejv by reatmi-ooll Phunol 2.4.S-T ,£> ,» (as'*) LAI 0 24 60 2AI 8 0 60 2AJ 0 40 •JO 2AJ 88 «M 0 4AJ 0 0 35 4A5 44 24 0 -, % 0 0 itr; 44 40 0 6B2 24 0 10 2C1 52 0 0 2C2 0 0 75 2C4 64 30 0 4C1 44 50 0 4C3 88 90 0 4C5 8 0 30 403 4 24 25 503 92 90 0 3E3 72 40 0 5F4 68 50 0 -Initial concentration: 25 of chlcvldft in 25 ugAil 2,4,5-Tj 10.5 �•^ " Tho throe most active J^.VT-iloii.uli!*) iHol.iti»H (2A\, 4C1, .uvl MM) wutt- grown in basal medium .inundud w i t h '>() \\>\\ ot .>,4,'>-T 01 iimnjamr H . i l t s int-tlium unanded with JOO ppm 2,4 f r >-T. cubatod v/ith the isolate. I'onlrol t Links (.xaUaiiumj bnaal mud tun weir in- C.rowth. monsumi by *iptical density, W«H with 2,4,T>-T disappearancx: rtH uiLvisured L~v lk>8S ol tlV nbsorlxuK^. of the presence 01 Absence ot 2,4,S-T in the bns-il medun\, isolate 2A.J .1 sigmoidal qruwth cutvt^ with nvotinwl qniwtJi .iftor JO h (Fiq. J) . iance ot" 2,4,5-T at^irttxl dbout 8 h after inoculation, .\iid .tfter 90 h, greater than 90* of the herbicide had disappeared. Sterile controls had leas than 2* disappearajice after 90 h. No qrc*rt± occurred in the inoixjanic salts medixm after 90 h. The inability of 2,4,5-T to serve as sole carbon source, *he disappearance of the ccropound in the presence of an external carbon source, ^^J the lack of significant differences in growth between amonded and unamended bas.il medium indicated that the loss of the herbicide resulted from cumetabolism. Conetabcilism ot" 2,4,5-T was also indicated for isolates 4CJ (Kig. 4) and 5D3 (Fiq. 5). The rate and extent of O uptake was measured with resting-cell suspensions ot 2A3, 4C3, and 5D3 prepared from cultures grown on glucose-uxarganic salts madium with and without 2,4,5-T (30 ppm) or inorganic salts medium with sodium benzoate as sole carbon source. The oxyqen o.>iisumption values are the means of two replicates corrected for endogenous respiration (less than 3 nmol of O- in 1 h) and are determiivad for sodium benzoate or glucose as sole carbon source and 2,4,5-T in the glucose-inorganic salts medium. The oxidation of 2,4,5-T, glucose and sodium bonzoate is presented in Fig. 6. Oxyqen consumption was high for sodium benzonto (5 »imol of O ' -nol of Jium benzoate). Glucose was completely oxidized (6 iinol of O_/nnol of qlucoae) by 2A3. Although O2 consumption for 2,4,5-T was negligible by 2A3 when grown �IOC) PTICAL DENSITY o- Q75 75 e> gO.50 ti 50 in « csf 0.25 - 25 2,4,5-T 30 60 HOURS 90 Pig. 3. Growth of isolate 2A3 in the presence (closed circles) and absence (open circles) of 50 ug 2,4,5-T/tal and 2,4,J-T disappearance. �TICAL DENSITY 100 8" 75 in cvf ^ 25 30 60 HOURS 90 Ficj. 4. Growth of isolate 4C3 in the presence (closed circles) and absence (open circles) of 50 ug 2,4,5-T/tal arid 2,4,5-T disappearance. �100 .OPTICAL DENSITY '8- 75 50 I in + CM* .4.5-T 30 60 HOURS 90 Fig. 5. Growth of isolate 5D3 in the presence (closed circles) and (open circles) of 50 ug 2,4,5-T/taU, and 2,4,5-T disappearance. �6- 120 160 MINUTES Fig. 6. Metaboliaro of glucoM, •odiun bwwoat*, and 2,4,5^ by cell* of 2*3 grown in 0.5% gluooM (cloMd oirclw) or 0.5% auditm cirolM) inorganic Mlts broth. iting �12 with glucose, the cells consumed «l»xit 1.7 iimul of oy'iimul of 2,4,'i-T whan grown with sodium benzoate, suggesting a subatrate-anahxj at initiation of respiration. The amount of 0^ ixjnsumed was about 2b» of the theoretical amount needed to oxidize the 2,4,5-T completely. Resting cells of 4C3 were prepared frun cultures qrown in qluooae-inorganicsalts medium with aivi without 2,4,5-T. The cells cutpletely oxidized qlucosc (Fig. 7) . Oxygen consumption in the presence of 2,4,5-T by cells not grown in the presence of 2,4,5-T was 2 umol of OVumol of substrate. However, cells exposed to 2,4,5-T in the growth medium consumed nearly 4 umol of OVionol of 2,4,5-T, suggesting that the presence of 2,4,5-T stimulated metabolism and ring cleavage of the herbicide. Resting cells of isolate 5D3 were prepared similar to 4C3, and these cells o oxidized all the glucose (Fig. 8). Cells not exposed to 2,4,5-T in the •v ^ growth medium consumed nearly 2 umol of O^umol of 2,4,5-T while "pre-exposed" cells consumed 3 umol of 0_/umol of substrate. The enhanced 0_ consumption (about 40% of the theoretical amount for complete oxidation of 2,4,5-T) suggests, similar to 4C3, that the presence of the herbicide during growth stimulated subsequent 2,4,5-T oxidation. To determine further the persistence and degradation of 2,4,5-T in soil, the Philippine soil was amended with 5 and 15 ppm of 2,4,5-T ( V-ring-UL). The CO2 evolved was measured periodically by acidifying the soil and trapping the CD- i"a trapping solution. Compared to gamma-irradiated controls, CD- evolution was detected in the soil with 5 ppm 2,4,5-T (Table 4). The amount of 1 4 (X>2 detected was 16% of the initial radioactivity added after 2 wceka and 23% after 3 weeks. No significant CO- evolution occurred in the soil with 15 ppm 1,'>T-14C in a 3-week period. Evolution of mf ditional evidence of ring cleavage. GC*2 from the soil provided ad- �60 120 180 MIMJTES Fig. 7. itetAbolism of glucose and 2,4f5-T by resting cells of 4C3 grown in 0.5% glucose-inoxqanic salts broth (cloned circles) and broth amended with 30 u<-j 2,4,5-T/ml (open circles). �GLUCOSE cr CO CD CO I 3 O _l O 2,4,5-T 120 180 MINUTES Fig. 8. Metabolism of glucose and 2,4,5-T by resting cells of 5D3 grown 60 in 0.5% glucose-inorganic sa.lts broth (closed circles) and broth amended with 30 ug 2,4,5-T/ml (open circles). �13 TABLE 4. Degradation of 2,4,5-T( C-ring-lJL) by Philippine 9011 Degradation (*)Vteeks 5 ptm 15 pptn 1 0 0 2 16.3 < 1.0 3 22.6 1.8 ^Measured as % of 2,4,5-TC C-ring-UL) evolved as CO,. �IHHIUSMCN Enrichment-culture techniques using 2,4-1), 2,4,0-T, «nd irv»loquen indicated that the inocula fran natural ocosystuns contained many bacteria cai**M'.of destroying 2,4-D and 2,4,5-T but only by crmetabol ism. Studies with resting cell suspensions of the bacteria showed that 74% of the isolates deatioyad ficm 8 to 92% of the 2,4,5-T in the medium, 63% released chloride from 2,4,5-T, arid 42% produced phenol. In addition, 64% of the bacteria that degraded 2,4,5-T also released chloride. The liberation of chloride with loss of UV absorbancy indicated ring cleavage of the herbicide. Further, the data from manometric studies suggested ring cleavage of 2,4,5-T by two "induced" bacterial isolates, 4C3 .and 503. The cleavage of the herbicide agrees with the findings of Ou and Sikka (17), who found extensive degradation of the aromatic ring of a structurally similar molecule, 2-(2,4,5-trichlorophenoxy)propionic acid by aquatic bacteria. In contrast, other workers (2,9) have reported that phenoxy herbicides with a chlorine in the meta position of the aromatic ring were resistant to microbial degradation in soil. The failure to show microbial degradation of these chemicals may have resulted from soil inocula in which the appropriate microorganisms were- either absent or not present in sufficient numbers to produce degradation of the compound in the time of the experiment. In addition to organisms degrading 2,4,5-T, bacteria were also isolated that could produce a phenolic compound from the herbicide. In some cases, phenol production was accompanied by chloride release, suggesting the conversion of 2,4,5-T to the mono- or dichlorophenol. In other instances, phenol was produced without a loss of UV absorbancy and chloride release, suggesting the production of 2,4,5-trichlorophenol. Sharpee (18) showed also the production of 2,4,5-trichlorophenol from 2,4,5-T. The appearance of the chlorinated phenols in the culture medium suggests that 2,4,5-T degradation proceeds via the cleavage; �of the acetate moiety as described for 2,4-1) doqr.idcit ion ( 1 , 7.8, \'>, I')) . If the trxohloroiphenoj. is dehaloyenaturt, then the molecule rr»y Ix? decomposed l»y »hu pathways described for 2,4-D degradation (18,19) . The pathway of deqrdd.it;on of the trichlorophenol, however, remains unknown (1). Alternately, 2,4,5-Tmay be converted to a hydroxylated, dehaloguvated organic product such as 3,rj-dichlorocatechol as described by Horvath (12) . If so, then the catechol may be deoorvosod as described for 2,4-D since the catechol as also produced during the bacterial degradation of this herbicide (14,18,19) . The data frxm the enrichment cultures indicated that 100 ppm of 2,4,5-T persisted in sewage and Philippine soil for 60 days. However, a subsequent experiment using a larger sample of Philippine soil amended with only 5 14 2,4,5-T ( C-ring-UL) indicated ring cleavage and evolution of 140 , within 0, <eeks. Inasmuch as 2,4,5-T does indeed disappear fron soil (1,11) and evidence exists that microorganisms are involved (12) , one might expect that microorganisms could be obtained which use it as a source of carbon and energy. However, such an organism had yet to be found. In this stud", the three most active bacteria attacked 2,4,5-T by cometabolism. Come tabol ism is the metabolism by a microorganism of a conpound that will not supply that organism with energy or an essential nutrient. The species thus does not replicate at the expense of the compound; hence, should the initial cell number be small, there will be no increase in cells with the requisite enzymes so that the rate of decorposition will ranain low and also will not show the typical increase with time that is characteristic of substrates supporting growth (13) . This long persistence coinciding with an apparent microbial transformation (5) is typical of the behavior of 2,4,5-T in soil. The persistence, however, of 2,4-0 ard 1,5-1 is dependent on many factors, not the least of which is abundance of ^v •* 2,4-D aixJ 2,4,5-T-metabolizing bacteria (1,14). Thus, a small soil sample may �1ft not ix>nt -tin i 'v Ixsciei la in sutl iciei.l number to dogiticlu ^,4-L; .ind (\specMlJy 2,4,5-T w i t h i n U>e teat, per ml. ^ dependency of 2,4-D and 2,4,'i-T metiib'jlian on size >n ; v > i l sarples has be«ui shtjwn (16,21) . research w i l l be di.rect.ed t o (^; determining the fdte <irvd pcraisteiv?e ot 2,4,5-T in Pacific Island soils utilizing gas-liquid chrcmat'xjraphy and 14 C-tagged herbicide, (b) determining tho role of cunetabolian in the bioof 2,4,5-T and 3eeking means to enhance the process and (c) assessing the effect of selected pesticides 01 the function of microbial cotitunities of sewage. CREDIT Ti.i.s research was supported in part by the Office of Naval Research, Microbiology Program, Naval Biology Project, under contract N00014-78-C-O044. �J/ MIHMOCHAI'IfY 1. Aii'-xandei, M. 1974. Miciubuil format ion <>l (jnvironmcntal i»)l lutanta. Advan. Appl. Microbiol. 18:1-71. 2. Alexander, M. and M. r. II. Akwni. L961. Effect of chunical structure an microbial decunposition ci miTHtic herbicides. J. Agric. Food. Chein. 9:44-47. .}. Alexander, M. .and B. K. Lust iqnvm. 1966. Effect jf chemical structure on microbial degradation of substituted benzenes. J. Agric. Food Chem. 14:410-413. 4. Arnow, L. E. 1937. Colorimetric determiiiation of the ccnvjnents of 3,4diJiydroxyphenylalaniije-tyrosine mixtvres. J. Biol. Chan. 11Q-531-537. 5. Audus, L. J. 1951. The biological detoxication of hormone herbicides in soil. Plant Soil 3:170-192. 6. Bergman, J. G., and J. Sanik. 1957. Determination of trace amounts of chlorine in naphtha. Anal. Chem. 29:241-245. 7. Bollag, J.-M., C. S. Helling, and M. Alexander. 1968a. 2,4-D metabolism: enzymatic hydroxylation of chlorinated phenols. J. Agric. Food Chem. 16:826-828. 8. Bollag, J.-M., G. G. Briggs, J. E. Dawson, and M. Alexander. 1968b. 2,4-D matabolism: enzymatic degradation of chlorocatechols. J. Agric. Food Chem. 16:829-833. 9. Burger, K., I. C. MacRae, and M. Alexander. 1962. Decomposition of pnenoxyalkyl carboxylic acids. Soil Sci. Soc. An. Proc. 26:243-246. ].0. Chraatil, J. 1975. Colorimetric estimation of phenols and tyrosine. Anal. Chan. 47:2293-2296. 11. De Rose, H. R., and A. S. Newnan. 1947. The comparison of the persistence of certain plant growth regulators when applied to soil. Soil Sci. Soc. Am. Proc. 12:222-226. �IB 12T"Horv»tli, R. S. 1970. Microbial acnDtabol ism of 2,4,5-LrichloroiJJ»noxyaoetic acid. Bull. Environ. Content. Tcwicol. 5:537-541. 13. Hotvath, R. S. 1972. MicrobiaL oan*>tsLoIism and the degradation of organic cun»unds in nature. Bacteriol. Rev. 36:146-155. 14. Loos, M. A. 1975. Phanoxyalkanoic acids, p. 1-128. In P. C. Kearney and 0. D. Kaufman (od.), Herbicides: Chemistry, degradation, and mode of action, vol. 1, 2nd ed. Marcel Dekker, New York. 15. Loos, M. A., R. N. Roberts, and M. Alexander. 1967. Formation of 2,4-dichlorophenol and 2,4-dichloroanisole from 2,4-dichlorophenoxyacetate by an Arthrobacter sp. Can. J. Microbiol. 13:691-699. 16. Ou, L.-T., D. F. Rothwell, W. B. Wheeler, and J. M. Davidson. 1978. The effect of high 2,4-D concentrations on degradation and carbon dioxide evolution in soil. J. Environ. Qual. 9:241-246. iTTcu, L.-T., and H. C. Sikka. 1977. Extensive degradation of silvex by synergistic action of aquatic microorganisms. J. Agric. Food Chem. 25:1336-1339. 18. Sharpee, K. W. 1973. Microbial degradation of phenoxy herbicides in culture, soil, and aquatic ecosystems. Ph.D. Dissertation. Cornell Univ., Ithaca, N.Y., 94 pp. 19. Tied'ie, J. rt., and M. Alexander. 1969. Enzymatic cleavage of the ether bond of 2,4-dichlorophenoxyacetate. J. Agric. Food Chen. 17:1080-1084. 20. Umbreit, W. W., R. H. Burris, and J. F. Stauffer. 1964. Manonetric techniques, 4th ed. Burgess, Minneapolis. 305 pp. 21. Yoshida, T., and T. F. Castro. 1975. Degradation of 2,4-D, 2,4,5-T, and picloram in two Philippine soils. Soil Sci. Plant Nutr. 4:397-404. �OFT ICE OF NAVAL RtSEAWlt M.ICROBIQLO(.Y PHOC^RAM STANDARD DISTRIBUTION LIST Administrator, Defense IXxTmentation Center Cameron Station Alexandria, VA 22314 ( 6) Director, Naval Hesearch I.aboratory Attention: Technical Information Division Code 2027 Washington, DC 20390 ( <i) Code 1021P (ONRI, DOC) Office of Naval Research 800 N. Quincy Street Arlington, VA 22217 ( 3) Office of Naval Research Department of the Navy Code 443 Arlington, VA 22217 ( 2) Commanding Officer (Code 00) Naval Medical Research & Development Command National Naval Medical Center Bethesda, MD 20014 ( 2) Technical Reference Library Naval Medical Research Institute National Naval Medical Center Betheada, MD 20014 ( 2) Office of Naval Research Department of the Navy Code 200 Arlington, VA 22217 ( 1) Office of Naval Research Branch Officer 495 Summer Street Boston, MA 02100 ( 1) Office of Naval Research Branch Office 536 South Clark Street Chicago, IL 60605 ( 1) Office of Naval Research Branch Office 1030 East Green Street Pasadena, CA 91101 < 1) Conrand Officer U. S. Haval Medical Research Unit 12 Box 14 APO, "an Franciso 96263 �10 STANDARD DISTRIBUTION LIST—Cont inuocl of Copies ( 1) Commandinq Officer U. S. Naval Medical Research Unit #3 FPO, New York 09527 ( 1) Coimanding Officer U. S. Naval Ntedical Research Unit #5 APO, New York 09319 ( 1) Officer in Charge Submarine Medical Research Laboratory U. S. Naval Subnarine Base, New London Groton, CT 06342 ( 1) Scientific Library Naval Bioscienoes Laboratory Naval Supply Center Oakland, CA 94625 ( 1) Scientific Library Naval Aerospace Medical Research Institute Naval Aerospace Medical Center Pensacola, FL 32512 ( 1) Commanding Officer U. S. Naval Air Development Center ATTN: Aerospace Medical Research Department Johnsville, Warminster, PA 18974 ( 1) Commanding General U. S. Army Medical Research and Development Command Porrestal Building Washington, DC 20314 ATTN: MEDDH-Sr ( 1) Director of Life Sciences Air Force Office of Scientific Research Boiling Air Force Base Washington, DC 20032 ( 1) STIC-22 4301 Suitland Road Washington, DC 20390 ( l) Director Walter Reed Army Institute of Research Walter Reed Army Medical Center Washington, DC 20012 � 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. 012 Folder The folder containing the original item. 0102 Series The series number of the original item. Series 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 Rosenberg, Arthur Martin Alexander Description An account of the resource <strong>Corporate Author: </strong>Department of Agronomy, Cornell University, Ithica, New York Date A point or period of time associated with an event in the lifecycle of the resource November 17 1978 Title A name given to the resource Microbial Degradation of Pesticides Subject The topic of the resource biodegradation soil decontamination 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. 055 Folder The folder containing the original item. 1447 Series The series number of the original item. Series III 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 Travis, Curtis C. Holly A Hattemer-Frey Source A related resource from which the described resource is derived Chemosphere Date A point or period of time associated with an event in the lifecycle of the resource 1978 Title A name given to the resource Human Exposure to 2,3,7,8-TCDD Subject The topic of the resource dioxin biodegradation ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/373c0ef0b1de1f948ab7cf276942002a.pdf 7bdc005fa5bab510c2b0a3ddc943e2df PDF Text Text Wilson, James G. Advisory Committee on 2,4,5-T Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency Journal/Hook Titlo Year 1971 Month/Day Ma Color ' v' ! 79 Alvin L. Young filed this item under the category "Human Exposure to Phenoxy Herbicides and TCDD" Thursday, April 05, 2001 Pago 1152 of 1180 �CHILDREN'S V<.-*$VJ HOSPITAL MEDICAL CENTER Y"ha Children's Hospital Research Foundation titling* & Bethesda Ave. 'Cincinnati, Ohio 45e?£?9 Department of Pediatrics College of Medicine University of Cincinnati May 7, 1971 Mr. William D. Ruckelshaus Administrator Environmental Protection Agency Washington, D.C. 20460 Dear Mr. Ruckelshaus: On behalf of the membership of the Advisory Committee on 2,4,5-T, I am pleased to submit the attached .Report. We earnestly hope the Report will prove helpful to you and your staff in establishing policy and procedures relative to future regulation of the use of the herbicide 2,4,5-T. I shall endeavor to be of further service in this connection if needed. Sincerely yours, James G. Wilson, Chairman Advisory Committee on 2,4,5-T JGW:bh ADOLESCENT CUNl'O • CHILDREN'S DENTAL CARE FOUNDATION • THE CHILDREN'S HQSP'TAL THE CHILDREN'S HOSPITAL RESEARCH FOUNDATION • CHILDREN'S NEURPMUSCLJL.AR DiAGNOE'?IC!Cl'.IIMIC _ CONVALESCENT HOSPITAL FOR CHILDREN • HAMILTON COUNTY DIAGNOSTIC CLINIC FOH THEi MENTALLY 7>kT/*PQK;'J. '' LINITGD CEREBRAL PALSY OF CINCINNATI. INC. AFFILIATED WITH THE UNIVERSITY OF CINCiN r: - �R E P O R T of the A D V I S O R Y C O M M I T T E E ON 2,4,5-T to THE A D M I N I S T R A T O R of the E N V I R O N M E N T A L P R O T E C T I O N A G E N C Y Submitted, May 7, 1971 �CONTENTS Page Membership of the Advisory Committee 1 Introduction 3 I. Factors Influencing Exposure to Man A. Patterns of use of 2,4,5-T B. Fate in soil, air, water and plants Fate of 2,4,5-T Fate of TCDD References cited in Section I A and B C. Fate in animals Fate of 2,4,5-T Fate of TCDD References cited in Section 1 C II. Toxicity of 2,4,5-T and TCDD in Animals and Man A. Nonteratogenic toxicity of 2,4,5-T of TCDD References cited in Section II A B. Teratogenic Potential of 2,4,5-T 1. Scope of embryotoxicity 2. Data from laboratory animals 2,4,5-T in rats TCDD in rats 2,4,5-T in mice TCDD in mice 2,4,5-T in hamsters TCDD in hamsters 2,.4,5-T in rabbits 2,4,5-T in sheeps 2,4,5-T in rhesus monkeys Summary of data on laboratory animals 3. Human exposure during pregnancy Vietnam Summary of Vietnam data on Human Embryotoxicity Globe, Arizona Swedish Lapland References cited in Section II B 8 8 9 9 14 18 23 23 25 26 28 28 28 33 34 37 37 39 41 45 46 47 48 49 49 49 49 50 51 51 57 58 59 60 �III. General Conclusions IV. Recommendations V. 64 66 Statement of views of Dr. Theodor D. Sterling entitled, "Objections to and Modifications of the Final Report and Recommendations of the 2,4,5-T Advisory Committee". 68 VI. List of Persons conferring with the Committee 76 �-1MEMBERSHIP of the ADVISORY COMMITTEE James G. Wilson, Ph.D., Chairman Professor of Research Pediatrics and Anatomy, Children's Hospital Research Foundation and College of Medicine, University of Cincinnati, Ohio Roswell K. Boutwell, Ph.D. Professor of Oncology, McArdle Laboratory for Cancer Research, University of Wisconsin Medical Center Madison, Wisconsin Donald E. Davis, Ph.D. Alumni Professor, Department of Botany and Microbiology, Auburn University, Auburn, Alabama Frank N. Dost, DVM Associate Professor of Veterinary Medicine, Science Research Institute, and Environmental Health Sciences Center, Oregon State University, Corvallis Wayland J. Hayes, Jr., M.D.,Ph.D. ^Professor of Biochemistry, Department of Vt Biochemistry, University School of Medicine, Nashville, Tennessee Harold Kalter, Ph.D. Professor of Research Pediatrics, Children's Hospital Research Foundation and College of Medicine, University of Cincinnati, Ohio �-2- Ted A. Loomis, M.D., Ph.D. Professor Pharmacology and State Toxicologist, Department of Pharmacology, University of Washington School of Medicine, Seattle Arthur Schulert, Ph.D. President, Environmental Science and Engineering Corp. and Associate Professor of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee Theodor D. Sterling* Ph.D. Professor, Dept. of Applied Mathematics and Computer Science, Washington University, St. Louis, Missouri David L. Bowen Secretariat to Advisory Committee Environmental Protection Agency �-3- INTRODUCTION On October 29, 1969, the President's Science Advisor, Dr. Lee A/. s DuBridge, announced that a series of coordinated actions was being taken by several governmental agencies to restrict the use of the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). This was precipitated by release a few days earlier of the findings of a large-scale screening study of a number of pesticides and industrial chemicals conducted by Bionetics Research Laboratories in which it was found that mice and rats treated during early pregnancy with large doses of 2,4,5-T gave birth to defective offspring. The announcement, together with reports of an increased occurrence of birth defects by South Vietnamese newspapers during June and July 1969, elicited far-reaching reactions from governmental agencies, segments of the scientific community, various lay groups concerned with environmental problems, and from the public communications media. Government-sponsored panels of experts, special commissions set up by scientific organizations, hearings before subcommittees of the U.S. Senate, and conferences attended by representatives.from industry,, government, and universities examined available data and heard expert opinions. None of these groups, however, was able to provide a generally acceptable answer' to the central question of whether 2,4,5-T, as currently produced and'usad, constituted a risk for human pregnancy. At least one reason for failure to reach a>satisfactory resolution of the issue was the paucity of reliable, scientific evidence. Additional animal experiments performed early in 1970 confirmed that the purest available sample of 2,4,5-T, given in large doses to �-4- pregnant mice, did indeed result in the delivery of some malformed offspring. The question then becomes one of whether, or to what extent, such animal data could be extrapolated to man. On April 14, 1970, an attitude of caution was expressed by the Secretary of Health, Education and Welfare, who advised the Secretary of Agriculture that: "In spite of these uncertainties, the Surgeon General feels that a prudent course of action must be based on the decision that exposure to this herbicide may present an imminent hazard to women of child-bearing age." Accordingly, on the following day the Secretaries of Agriculture, of Health, Education, and Welfare and of Interior jointly announced the suspension of the registration of 2,4,5-T for: "I. All uses in lakes, ponds or on ditch banks. II. Liquid formulations for use around the home, recreation areas and similar sites." (USDA-PRD PR 70-1, 20 Apr. 1970) A notice for cancellation of registration was issued on May 1 for: "I. All granular 2,4,5-T formulations for use around the home, recreation areas and similar sites. II. All 2,4,5-T uses on crops intended for human consumption." (USDA-PRD PR 70-13, 1 May 1970) All registrants of 2,4,5-T were advised of these actions, and two of the registrants, Dow Chemical Company and Hercules Incorporated, exercised their right under Section 4.c. of the Federal Insecticide, Fungicide and Rodenticide Act (7 U.S.C. 135 gt seq.) to petition for referral of the matter to an Advisory Committee. The National Academy of Sciences supplied a list from which was selected a nine-member Advisory Committee of scientists with appropriate qualifications from universities and research institutes over the country. At its first meeting on February 1 and 2, 1971, the Advisory Committee was given a charge which in substance asked that it: 1) consider all �—5— relevant facts, 2) submit a report and recommendations regarding registration for certain uses of 2,4,5-T, and 3) state the reasons or bases for these recommendations. It was the concensus of the Committee that the central issue was whether use of the herbicide does in fact constitute an imminent health hazard, especially with respect to human reproduction. Accordingly, the Committee has undertaken to examine all available information and to evaluate its relevance to the potential hazard of human exposure during pregnancy. During the intervening months since restrictions were placed on the use of 2,4,5-T a number of additional studies have been carried out on several animal species and a few reports on human exposure during pregnancy have been further evaluated. Although the new data have not answered all of the questions that have been or could be raised, they have undoubtedly provided a more substantial basis for making a scientific judgment about possible effects of this herbicide on prenatal development than previously existed. In undertaking such judgment the Committee has taken into account certain considerations that seem appropriate to the issue, as follows: 1) As is frequently the case, available data are insufficient for a definitive statement of conditions under which a specified risk might occur, assuming that freedom from risk is ever attainable. 2) Since most chemicals under suitable laboratory conditions could probably be demonstrated to have teratogenic effects, and certainly all could be shown to produce some toxic effects if dosage were raised high enough, it would not be reasonable to consider the demonstration of toxic effects under conditions of greatly elevated dosage sufficient grounds for prohibiting further use of a particular chemical. 3) Benefits are to be expected from the continued use of �-6- 2,4,5-T. The necessity of making a value judgment of benefit vs. risk, therefore, must be accepted, not only for this herbicide, but for numerous valuable drugs, some natural nutrients, and many other chemicals, some of which are known to be teratogenic in laboratory animals. The risk vs. benefit judgment for a particular herbicide or drug can be evaded only if it can be shown that another compound is equally as efficient and involves less risk. This presupposes that the risk potential of a substitute herb- icide is at least as well known as that of the original (in this case 2,4,5-T), a fact that may be difficult or impossible to ascertain. The substitution of a relatively unknown pesticide for an older one with known adverse effects is not a step to be taken lightly. Even with steadily improving methods for safety evaluation of new chemicals it is impossible to anticipate all of the conditions and permutations of use that could result in undesirable effects. The task of making a judgment about the central question of hazard to human pregnancy is complicated by still other considerations. Although herbicides are of economic benefit to man, their use is not without possible hazard to the environment and to other aspects of human welfare. In various connections questions have been raised about: 1) damage to non-target plants caused by spray drift or by movement in water, 2) damage to subsequently planted sensitive crops owing to herbicide persistence in the soil, and 3) acute or chronic toxicity to man or other animals aside from that related to pregnancy. In addition, there is some concern that traces of the chemical or its contaminants in food may cause unsuspected effects in man or that minute amounts in the environment >may -adversely affect untested species in the ecosystem. �-7- It is scientifically impossible to prove that a chemical is without hazard. Pesticide regulations now require that new agents be tested for acute and chronic toxicity, mutagenicity and carcinogenicity. These tests may involve the use of two or more species of animals taken through several generations and the examination of thousands of individuals. Since it is necessary to extrapolate from effects in test animals to man and since species are known to differ in sensitivity to chemicals, the permissible residue levels in food must always be many-fold below the minimal effect level for the species tested. Concern that some unexpected detrimental occurrence may outweigh the benefit of a pesticide has doubtless been heightened by the finding that DDT residues in the environment have adversely affected reproduction in certain predatory avian species that are at the top of their food chains and, as a consequence, ingest large accumulations of this persistent compound. With these considerations in mind the Advisory Committee has examined all available information relating to factors that may influence human exposure to 2,4,5-T and the toxic reactions, nonteratogenic as well as teratogenic, such exposure of man and other animal species may entail. �-8- I. FACTORS INFLUENCING EXPOSURE TO MAN, Human exposure to an environmental chemical such as 2,4,5-T depends on 1) pattern of usage, i.e., how widely and frequently applied and in what amounts, and 2) its fate in the environment, i.e., does it accumulate or is it degraded as fast as applied. A. Patterns of use. The chloro-phenoxy herbicides 2,4-D and 2,4,5-T have been widely used to control broad-leaved weeds for over 20 years. Because 2,4,5-T is more expensive than 2,4-D (2,4-dichlorophenoxyacetic acid) it has been primarily used to control woody plants and a few herbaceous species against which it is more effective than 2,4-D, and because of the cost difference, commercial formulations containing 2,4,5-T are usually mixtures of the two herbicides. In 1964 the uses of 2,4,5-T were: rights-of-way - 49%, non-farm forests - 10%, hay, pasture, and rangelands - 7%, all other farm uses - 12%, lawns and turf - 7%, federal agencies - 6%, and other miscellaneous uses - 9%.—' The tocal domestic use at that time was about 9 million pounds and incomplete information indicates that this value may also be approximately correct for 1969.-/ Most of the 2,4,5-T used is applied as a spray to foliage. Lesser amounts are sprayed on the trunks and branches of dormant trees, injected into the bases of trees, poured or sprayed into frills around the trunks of trees, or sprayed or painted on newly cut stumps of trees. Amine salts of 2,4,5-T dissolved in water are most often used when the herbicide is applied to foliage and esters dissolved in oil are most �—9— often used when it is applied to bark. The spray concentrations usually vary between 0.1 and 2.5% and the rates of application are usually between 0.5 and 8 Ib per acre, depending on the size, and sensitivity of the plants being treated. Higher rates and concentrations have been used in Vietnam for military purposes. On domestic rice, 0.50 to 1.25 Ib per acre of 2,4,5-T is used in 3/ 5 to 7 gal of water—' , applied from the air when the rice is 7 to 9 weeks old, has emerged from the water, and is'standing erect. Directions for the use of 2,4,5-T warn against allowing it to drift onto susceptible plant species and require that the herbicide not be allowed to contaminate water used for irrigation or domestic purposes. B. Fate in soil, air, water and plants. When 2i4,5-T is applied as a spray, the great bulk of the herbicide and any contaminant it may contain, e.g., 2,3,7,8-tetrachlorodibenzo-paradioxin (TCDD), are deposited on the foliage of the plants, on the ground in the immediate vicinity or, in the case of rice, on the impounded water. Much smaller amounts may be inserted into the air or settle on streams of water and by either means be carried many miles from the site of appl'ication. After 2,4,5-T and TCDD are applied, however, each moves through the biosphere and accumulates or degrades according to its own chemical and physical properties. The fate of 2,4,5-T has been more extensively studied than that of TCDD. Fate of 2,4,5-T. Once the herbicide reaches the soil it is immediately subjected to physical and chemical actions that continually reduce the amount remaining at the site of application. These actions include degradation by soil microorganisms, leaching and surface �-10- movement in water, volatilization, movement by wind, and photochemical decomposition. The persistence of 2,4,5-T is influenced by its rate of application and by various climatic and edaphic factors, and occurs most rapidly under conditions that are optimal for the growth of soil microorganisms.— At least two bacterial isolates, Mycoplano sp. and Achro- mobacter sp.— — — — and one actinomycete, Streptomyces viridochromogenes— from soil are known to metabolize 2,4,5-T. Br.evibacterium sp. has been shown to cometabolize 2,4,5-T to a product tentatively identified as 3,5dichlorocatechol— . Morris— found that 2,4,5-T was decarboxylated in the litter of the forest floor and had a half-life of approximately 40 days. 12/ Loos— has thoroughly reviewed degradation of phenoxyalkanoic acids, including 2,4,5-T. Loss of all phytotoxicity of 2,4,5-T applied to the soil 13/ was reported to occur 3 to 6 months after application.— No chemically detectable amounts of 2,4,5-T were found in the soil 1 year after an application of 2 Ib per acre and only very small amounts were found 3 to 7 months after application.— Although the rate of disappearance varies, there have been no reports of carry-over of 2,4,5-T from one year to the next, indicating that no build-up in the soil would result from recommended rates of treatment repeated annually. When phenoxy herbicides were first introduced, highly volatile esters were available and farmers were inexperienced in their use, there were several instances in which the drift through air produced severe injury to sensitive crops, usually in adjoining fields or more rarely at some distance from the point of application. Owing to accumulated experience and the institution of regulations regarding the conditions under which applications can be made, as well as the removal of the volatile esters from the market, �-11injury to crops is now unusual. The elimination of drift sufficient to injure most crop plants, however, does not eliminate the possibility of drift that can be detected chemically. Some of the 2,4,5-T applied as spray can be transported in the atmosphere as droplets of spray, as the gaseous phase of 2,4,5-T, or adsorbed on dust or other particulate matter in the air. In a survey in the State of Washington, 2,4,5-T was detected 9 days out of 99 at Pullman, o in average concentration in positive samples of 0.045 yg/m . At Kennewick it was found 14 days out of 102 at average concentration in positive samples 3 147 of 0.012 yg/m — . In Cincinnati, Ohio, 0.04 ppm was found adsorbed on dust in a trace of rain—' presumably from applications in Texas. Photo- chemical degradation would be expected to occur in the air, particularly at high altitudes and in dry climates where ultraviolet radiation is highest. Kearney et al.—' report that exposure of 5 and 10 ppm water solutions of 2,4,5-T to ultraviolet light from a 450 watt Hanovia lamp greatly reduced the 2,4,5-T present within 5 minutes. It is not possible to extrapolate accurately from these data to the rate of decomposition in sunlight, but it is obvious that photochemical degradation could play a significant role. Probably most of the 2,4,5-T that gets into the air very soon either settles out or is washed out by rain and thereby is returned to soil and water. There is no evidence to suggest that 2,4,5-T remains in the air for more than a few weeks after insertion. Measurable quantities of 2,4,5-T could enter water iL'n several ways, e.g., by inadvertent direct spraying, from surface leaching of treated soils and plants, or in rain that falls through air containing 2,4,5-T; but undoubtedly most of it is washed from treated plants and soil. �-12- Apparently the amounts are usually quite small since only 28 of 322 water samples taken in western states, where 2,4,5-T is widely used for brush control, were shown to contain 2,4,5-T— in concentrations ranging from 0.01 to 0.07 ppb. In closely controlled watershed studies in Waynesville, North Carolina, no 2,4,5-T was found in any sample of run-off from an area one-fourth of which was treated with 2 Ib of 2,4,5-T per acre in 1968 or 18/ 1969— . When one-fourth the area was treated with 4 Ib per acre some herbicide was found in the water after the first and second rain storms, but the highest concentration found was 0.048 ppm in run-off water during a storm that occurred 8 days after application of the herbicide. No 2,4,5-T was found in the last sample collected that day or in those collected on subsequent days. Few data are available on the rate of disappearance of 2,4,5-T from water. It would be expected to be adsorbed on clay particles or adsorbed by aquatic species within a few days. The concentration of picloram, a considerably more persistent herbicide than 2,4,5-T in most situations, decreased from 0.965 ppm to 0.129 ppm in 3 weeks in a test in which it was applied at 19/ the rate of 4 Ib per acre to a pond— . All available data suggest that the amount of 2,4,5-T entering water is quite low and that it does not remain in the water very long. Absorption, translocation, and metabolism of 2,4-D and 2,4,5-T by plants have been extensively investigated. Most investigations have dealt with 2,4-D, but numerous studies have involved both and it is apparent that their behavior in plants is similar. Ready absorption of 2,4,5-T by leaves, , , 20/21/22/23/24/25/ n f stems, and roots of plants is known to occur. Once absorbed 2,4,5-T may either move upward in the xylem or bidirectionally in �-la- the phloem. Some 2,4,5-T is absorbed by the leaves, transported down the 22/ stem and into the roots, and excreted by the roots into soil solution— . Decarboxylation of 2,4,5-T has been demonstrated in a number of plant 127 species— . The varied sensitivity of different species to 2,4,5-T may be attributable in part to different rates of metabolism. 25/ Slife et al.—• found only traces of unidentified metabolites 8 days after applying C- carboxyl-labeled 2,4,5-T to wild or cultivated cucumber plants, both species susceptible to 2,4,5-T. Easier and associates—'—' applied labeled 2,4,5-T to excised blackjack oak leaves. C-carboxyl- They found no decarboxyl- ation but did find that an average of 59% of the 2,4,5-T was broken down into three major unidentified metabolites in 24 hours. 28/ Morton—' reported that 80% of 2,4,5-T applied to mesquite was metabolized in 24 hours. Fitz- 297 gerald et al.— identified 2,4,5-trichlorophenol as a common metabolic product of 2,4,5-T in sweetgum and southern red oak but found that no 2,4,503 / trichloroanisole was formed. Morton et al.— have studied the metabolism of various formulations of 2,4,5-T by beardgrass, little bluestem, and sideoats gramma and observed only moderate effect of formulation and species on the rate of metabolism. to 2.9 weeks. Half-life values in green tissues ranged from 1.6 In one experiment using radioactive 2,4,5-T ester and silver beardgrass, little bluestem, and dallis grass, alcohol extracts of green tissue taken 1 week after application contained no 2,4,5-T ester, 50% 2,4,5-T acid, and 50% unknown radioactive metabolite. Authorization to use 2,4,5-T on food crops depends on demonstrating that no residue exists in the edible product at harvest. The following studies illustrate the amounts of 2,4,5-T that may persist in food crops at various intervals after treatment. When 2,4,5-T was applied to apples as a �-14- spray concentration of 40 ppm, residue in the fruit had fallen to 0.004 ppm in 22 days.—' The application of 2,4,5-T to blueberries at 1 Ib per acre resulted in a concentration in the fruit of 0.05 to 0.33 ppm 44 days after 31 application although none was found 733 days after application.— / No detect- able 2,4,5-T (sensitivity =0.01 ppm) was found in rough rice 50 days after •JO/ applying 2.25 Ib per acre of 2,4,5-T.—' The rice straw contained 0.18 to 1.04 ppm 2,4,5-T 50 days after but none 84 days after application. Further evidence that very little 2,4,5-T gets into food is seen in results of assays of raw agricultural products and in the Market Basket Survey samples. From about 10,000 food and feed samples examined from 1964 through 1969 only 25 contained trace amounts of 2,4,5-T (less than 0.1 ppm) and only two contained measurable amounts, 0.19 ppm in a sample of milk in 33/ 1965 and 0.29 ppm in a sample of sugar beets in 1966.—' Furthermore, of the 134 total diet samples involving 1600 food composites (Market Basket Survey) analyzed from 1964 through April 1969, only 3 contained 2,4,5-T. Two were dairy products containing 8 to 13% fat with 0.008 and 0.19 ppm in the fat. A single meat, fish and poultry composite from Boston consisting 33/ of 17 to 23% of fat was found to contain 0.003 ppm 2,4,5-T on a fat basis.—' 34/3S/ It is concluded from the foregoing that: 1) The herbicide 2,4,5-T does not accumulate in any compartment of the biosphere. 2) The risk of human exposure to 2,4,5-T in food, air or water is negligible. Fate of TCDD. Under present conditions of manufacture this con- taminant is usually present in 2,4,5-T at less than 1 ppm, thus insuring that very little TCDD is inadvertently applied with 2,4,5-T. Like 2,4,5-T any contaminating TCDD would be deposited on the leaves of treated vegetation �-15- or on soil and water in the vicinity, although as indicated for 2,4,5-T, smaller amounts could enter air or water and be carried some distance from the site of application. Water transport, however, is sharply limited by 36 / the fact that the solubility of TCDD in water is only 0.2 ppb.— As a consequence it would tend to remain on the surface of plants and soil at the site of application. Photochemical decomposition of TCDD has been studied at Dow Chemical 37/ og/ Co.— and the United States Department of Agriculture.— Exposure of 1.02 mg of TCDD in 100 ml of water-saturated chloroform to ultraviolet light at 35°C caused 50 to 100% degradation in 2.5 hours. The Department of Agriculture, using a sunlamp with a peak emission at 310 nm, irradiated TCDD dissolved in methanol and found it to have a half-life of 3.5 hours. Rapid decomposition was also reported in natural sunlight when approximately 5 ml of a 24-ppm methanol solution of TCDD was sealed in glass tubes and exposed to 7,000 to 9,000 footcandles of sunlight, with a half-life of approximately 5 hours, virtual disappearance in 48 hours, and none detectable after 72 hours. Similar rates of decomposition, however, were not observed when the TCDD was placed on the surface of dry soil where irradiation for 96 hours with a sunlamp (maximum energy at 310 nm) did not cause any significant loss by either photodecomposition or volatilization. The same was true for wet soil irradiated for 6 hours. Interactions of TCDD with soil have also been studied by the United 39 States Department of Agriculture.— / When TCDD was placed on the surface of five very different soils and subjected to leaching with water, the TCDD did not move into any of the soils, probably because of its very low water solubility. Similarly, in leaching experiments using soil thin- �-16- layer chromatographic technique, no TCDD moved from the spot of origin in OQ / either a Hagerstown silty clay loam or a Norfolk sandy loam.— It would thus appear that most of f-ho chemical falling on the soil surface would remain there. The fate of TCDD mechanically incorporated into.soil has 39/ been investigated by the Department of Agriculture.— Radiolabeled TCDD was mixed into soil at the rates of 1, 10, and 100 ppm and soil extracts radio-assayed 20, 40, 80, and 160 days after application. The amount of radioactive material (probably TCDD) in the soil decreased 15 to 20% in 160 days, indicating that this compound was very slowly degraded in the soil and could persist for more than a year. The possibility that TCDD incorporated in the soil might be absorbed 39 by plants has been studied.— / Soybean and oat plants x^ere grown on Lake- land sand containing 0.06 ppm radiolabeled TCDD, which is 40,000 times the amount that would appear in soils treated with 2 Ib per acre of 2,4,5-T containing 1 ppm TCDD. Less than 0.2% of the available TCDD was absorbed by either type of plant, with radioactivity reaching a peak at 10 days at which time it measured about 0.12 ppm in oats and 0.05 ppm in soybeans on a dry weight basis, then it declined to an insignificant level at 40 days. The most likely source of plant contamination by the TCDD present as a contaminant in commercial 2,4,5-T is by way of foliar application. 39/ When radioactive TCDD was applied to the surface of leaves— no material was translocated from the site of application on the plant; but about 40 percent of the applied TCDD could be leached from the surface of the leaves by water, probably because the TCDD was added with a surfactant. This suggests that surface contamination could be the source of a very small TCDD residue in leafy food plants, but 2,4,5-T is not used on such �-17- plants. Even if 2,4,5-T containing 1 ppm TCDD were applied to such plants at the rate of 2 Ib per acre, the resultant TCDD contamination would be at 2 the extremely low level of 0.224 yg per m on the exposed surface. It is concluded from the foregoing that: 1) There is no indication that TCDD accumulates in air, water or plants, although it fyught accumulate in soils after heavy application of a highly contaminated sample of 2,4,5-T. 2) Direct application of 2,4,5-T containing TCDD could result in minute quantities of the latter remaining on the surface of foliage. than 0.2% of TCDD in soil is known to be absorbed into plants. 3) Less �-18- References Cited in Section I A and B 1. USDA, 1970. and 1966. Report and tables on domestic use of 2,4,5-T, 1964 Supplied by Production Resources Branch, Farm Production Economics Division, April, 1970. 2. Byerly, T.C., 1970. Use of 2,4,5-T in the United States. Letter from Dr. Byerly to Senators Magnuson and Hart. 3. Dow, 1970. 245. Petition, Part III, Item 3. Specimen labels for VEON The Dow Chemical Co. 4. De Rose, H.R. and A.S. Newman, 1947. The comparison of the persistence of certain plant growth-regulators when applied to soil. Soil Sci. Soc. Amer. Proc., 12;222-226. 5. Bell, G.R., 1957. Some morphological and biochemical characteristics of a soil bacterium which decomposes 2,4-dichlorophenoxyacetic acid. Canad. J. Microbiol., 3_:82l-8^0. 6. Bell, G.R., 1960. Studies on a soil Achromobjicter which degrades 2,4-dichlorophenoxyacetic acid. Canad, J. Microbiol., j$:325-337. 7. Steenson, T.I. and M. Walker, 1958. Adaptive patterns in the bacterial oxidation of 2,4-dichloro- and 4-chloro-2-methylphenoxyacetic acid. J. Gen. Microbiol., 18;692r-697. 8. Walker, R.L. and A.S. Newman, 1956. Microbial decomposition of 2,4-dichlorophenoxyacetic acid. Appl. Microbiol., 4_:201-206. 9. Bounds, H.C. and A.R. Colmer, 1965. Detoxification of some herbicides by Stre^tomyces. Weed Sci., 13:249-252. 10. Horvath, R.S., 1971. Microbial cometabolism of 2,4,5-trichloro- phenoxyacetic acid. Bulletin of Environmental Contamination and Toxicology, _5:537-541. �-1911. Norrls, L.A., 1970. Degradation of herbicides in the forest floor, p. 397-411. In; Youngberg, C.T. and C.B. Davey. Tree Growth and Forest Soils. Oregon State Univ. Press, Corvallis. 12. Loos, M.A., 1969. Phenoxyalkanoic acids, p. 1-49. Kearney and D.D Kaufman. Dekker, Inc., New York. 527 p. In; P.C. Degradation of Herbicides. Marcel 394 p. 13. Kearney, P.C., R»G. Nash and A.R. Isensee, 1969. pesticide residues in soils, p. 54-67. In; Persistence of Chemical Fallout; Current Research in Persistent Pesticides, ed. M.W. Miller and George G. Berg, Chas. C. Thomas, Ft. Lauderdale, Florida. 14. Bamesberger, W.L. and D.R. Adams, 1966. the environment. Organic pesticides in Adv. Chem. Ser., 60. ACS Publ. Wash., D.C. 15. Weibel, S.R., R.B. Weidner, J.M Cohen and A.G. Christiansen, 1966. Pesticides and other contaminants in rainfall and runoff. J. Amer. Water Works Assn., 58;1075-1084. 16. Kearney, P.C., E.A. Woolson, J.R.. Plimmer and A.R. Isensee, 1969. Decontamination of pesticides in soils. Residue Reviews, 29;137-149. Edited by F. Gunther. Springer-Verlag, New York. 17. Manigold, D.B. and J.A. Schulze, 1969. Pesticides in Water - Pesticides in Selected Western Streams. A Progress Report. Pesticide Monit. J., _3:124-135. 18. Sheets, T.J., 1970. picloram. Watershed studies with 2,4-D, 2,4,5-T and Communication from George Irving, Jr., to T.C. Byerly, April 3, 1970. (ARS Contract 12-14-100-893 ( 4 ) 3). 19. Hoffman, G.O., E.D. Robinson and M.G. Merkle, 1969. Loss of picloram into surface and ground waters. Weed Science Soc. of Amer., Abstract 75. Supplemented by personal communcation from M.G. Merkle. �-20- 20. Easier, E., 1962. Penetration, movement, and behavior of herbicides in plants. Proc. Southern Weed Conf., 15;8-15. 21. Fisher, C.E., C.H. Headers and R. Behrens, 1956. Some factors that influence the effectiveness of trichlorophenoxyacetic acid in killing mesquite. Weed Sci., JK139-147. 22. Hurtt, W., W.A. Wells and C.P.P. Reid, 1970. Foliar uptake and root exudation of picloram and 2,4,5-T by selected woody species. Weed Sci. Soc. Amer., Abstract 145. 23. Morton, H.L., E.D. Robinson and R.E. Meyer, 1967. Persistence of 2,4-D, 2,4,5-T and dicamba in range forage grasses. 24. Perry, P.W. and R.P. Upchurch, 1968. Weeds 15;268-271. Growth analysis of red maple and white ash seedlings treated with eight herbicides. Weed Sci., 16:32-37. 25. Slife, F.W., J.L. Key, S. Yamaguchi and A.S. Crafts, 1962. Penetration, translocation and metabolism of 2,4-D and 2,4,5-T in wild and cultivated cucumber plants. 26. Easier, E., 1964. Weed Sci., 10:29-35. The decarboxylation of phenoxyacetic acid herbicides by excised leaves of woody plants. Weed Sci., 12:14-16. 27. Easier, E., C.C. King, A.A. Badiei, and P.W. Santelmann, 1964. breakdown of phenoxy herbicides in blackjack oak. The Proc, Southern Weed Conf., 1J:351-355. 28. Morton, H.L., 1966. Influence of temperature and humidity on foliar absorption, translocation, and metabolism of 2,4,5-T by mesquite seedlings. Weed Sci., 14:136-141. �-2129. Fitzgerald, C.H., C.L. Brown and E.G. Beck, 1967. Degradation of 2,4,5-trichlorophenoxyacetic acid in woody plants. Plant Physiol. j42:459-460. 30. Edgerton, L.J. and D.J. Lesk, 1963. Determination of residues of 2,4,5-trichlorophenoxyacetic acid in apples by radioisotopes and gas chromatographic methods. Proc. Am. Soc. Hort. Sci., 83;120-125. 31. Trevett, M.F., 1964. A request for approval of a contact method of applying 2,4-D and 2,4,5-T for control of woody weeds in Maine lowbush blueberry fields. Unpublished data. Cited in Dow communication dated Jan. 19, 1971. 32. Syracuse University Research Corporation, 1970. 2,4,5-T residues in rough rice and straw. Unpublished data. Cited in Dow communication dated January 19, 1971. 33. Duggan., R.E., 1971. Memorandum to Way land J. Hayes. Unpublished. March 12, 1971. 34. Corneliussen, P.E., 1969. Pesticide residues in total diet samples. Pesticide Monit. J., .2:140-152. 35. Duggan, R.E., H.C. Barry, and L.Y. Johnson, 1967. Pesticide residues in total diet samples. Pesticide Monit. J., JL:2-12. 35a. Martin, P.J. and R.E. Duggan, 1968. Pesticide residues in total diet samples. Pestiicde Monit. J., JL:11-20. 36. Dow, 1970. Petitions, Part III, Item 20. Solubilities of 2,4,7,8tetrachlorodibenzo-p-dioxin. The Dow Chemical Co., December 8, 1964 (Revised April 1970). 37. Dow, 1970. Petitions, Part III, Item 19. The degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin by ultraviolet light. The Dow Chemical Co., May 1970. �-22- 37a. Lynn, G.E., 1971. p-dioxin. Photodegradation of 2,3,7,8-tetrachlorodibenzo- Unpublished work by Dow Chemical. Letter dated February 1, 1971. 38. USDA, 1970. Progress report on dioxin research. IV. Unpublished report dated March 25, 1970. 39. Kearney, Philip C., 1970. Chlorinated dioxin research. Presented before a Joint Meeting on Pesticides, United Kingdom, Canada, United States. November 5. �-23- C. Fate in Animals Fate of 2,4,5-T. Information on absorption, distribution, and metabolism of 2,4,5-T is not extensive. The most thorough studies are 1/2/ those reported by Erne — — who demonstrated that the triethanolamine and alkaline salts of 2,4,5-T and 2,4-D were readily absorbed, distributed and eliminated from the body. Rats and pigs given single doses of 100 mg/kg of the amine salt showed plasma half-life values of 3 and 10 hours respectively. Residues in kidney, liver, lungs and spleen sometimes exceeded plasma levels, but there was little indication of penetration into brain or adipose tissues. via the kidney. The compounds were excreted mainly With repeated administration plasma levels decreased and excretion rates increased. Up to 20% of the material in blood was in erythrocytes. As with single doses, little was found in adipose tissue or in the central nervous system. Placental transfer was found to be rapid in swine. Tissue half-times ranged from 5 to 30 hours and,were lowest in rats. There was no apparent retention of either 2,4-D or of; 2,4,5-T after repeated administration. 3/ According to St. John et al.,— when a cow was given 450 mg of 2,4,5-T acid divided among four daily doses, all of the administered material was excreted in the urine as the salt within 6 days. Zlelinski and Fishbein- found that a dose of 100 mg/kg of 2,4,5-T in mice was lost from the body more slowly than were several other herbicides, with disappearance rates ranging between 1 and 4% of the original dose per hour. The rate of excretion of 2,4,5-T in man is unknown, but it seems to be slower than that of 2,4-D. A man who committed suicide with a mixture of the two compounds had substantial concentrations of 2,4,5-T in all organs analyzed but no 2,4-D in any organ?-'. �-24- Using massive doses it is possible experimentally to exceed the ability of domestic animals to eliminate 2,4,5-T and thereby to produce measurable residues in their tissues. Four or more 250 mg doses of 2,4,5-T given to sheep produced levels of 33 to 113 ppm in fat and 40 to 100 ppm in muscle. The residues were 99% or more in the acid form regardless of whether the acid form or an ester was fed SJ. The Chemical form of the agent, however, may influence its deposition. Oral administration of the propylene glycol or butyl ether esters of 2,4,5-T to yearling cattle for 32 weeks at rates of 0.15 and 0.75 mg/kg/day produced no residues greater than those occasionally found in untreated controls. Subsequently Clark et al., — fed 2,4,5-T, 2,4-D and Silvex (2,4,5-trichlorophenoxy propionic acid) to sheep and cattle at several dietary concentrations for 28 days. Sheep receiving 2000 ppm 2,4,5-T in the diet were found to have muscle tissue residues of 1.0 ppm when treatment was terminated, and no detectable residue o/ 7 days later. Newton and Norris —' analyzed tissues from deer that had ranged over reforested land treated with 2,4,5-T and found essentially no residues. Several factors limit the intake of 2,4,5-T by domestic animals and man following recommended use of the compound, namely, low rate of application and breakdown by plants, animals, photochemical degradation and soil microorganisms. Owing to both the limited nature of prescribed use and the decomposition that occurs in the environment, 2,4,5-T almost never reaches a detectable level in human drinking water or food (see Section I B, pp. 11 and 14). Examination of approximately 11,600 samples of food offered for retail sale in the United States revealed only five �-25- with measurable residues, the highest concentration being 0.29 ppm. The highest level in potable water was 0.00007 ppm . 12 / Fate of TCDD. Piper and Rose —' have reported a preliminary study of the tissue distribution and disposition of C-labeled TCDD administered as a single oral dose of 0.05 mg/kg to male rats. The biological half-time for this dose was approximately 20 days, and fecal excretion accounted for the greater part of the TCDD removal. Three days after administration 3.1% of the total dose per gram was recovered from liver and 3.0% of the total dose per gram was contained in fat. The residual radioactivity in these tissues was not identified and therefore cannot be assumed to be TCDD. The fact that 8.3% of the dose was recovered from 14C02 in expired air indicated that some of the TCDD was completely metabolized. The Dow Chemical Co. has recently provided comparative solubility data on TCDD and p.p'DDT at 24°C, as follows: corn oil lard oil water TCDD 28 44 PPM of Solvent p.p'DDT 86,000 86,000 0.0002 0.001 Although suggesting a petitioning toward fat, these data clearly indicate that, unlike DDT, TCDD is so insoluble in fat that it would not be expected to accumulate in body fat depots in appreciable amounts. It is concluded from the foregoing that: 1) 2,4,5-T is rapidly excreted in all animals studied using doses in the range of those likely to be encountered in the environment; 2) 2,4,5-T is not known to be accumulated in any animal tissues or product used for human food; �-26- 3) 2,4,5-T has been detected in animal tissues or products used for human food very infrequently and then only in minute quantities; 4) limited data indicate that TCDD is also eliminated, at least some by metabolic breakdown, with a half-life of 20 days; and 5) the solubility of TCDD in fat is limited which would preclude appreciable accumulation in body fat. References Cited in Section I C 1. Erne, K., 1966. Distribution and elimination of chlorinated phenoxyacetic acids in animals. 2. Erne, K., 1966. Acta. Vet. Scand., 7:240-256. Animal metabolism of phenoxyaeetic herbicides. Acta. Vet. Scand., _7:264-271. 3. St. John, L.E., D.G. Wagner and D.J. Lisk, 1964. Kuron, Silvex and 2,4,5-T in the dairy cow. Fate of atrazine, J. Dairy Sci., j47:1267-1270. 4. Zielinski, W.L., Jr. and L. Fishbein, 1967. Gas chromatographic measurement of disappearance rates of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice. J. Agr. Food Chem., 15;841-844. 5. Curley, A., 1971. Personal communication to Wayland J. Hayes. 6. Clark, D.E., J.S. Palmer and C.H. Ayala, 1970. Residual and toxicological aspects of 2,4,5-T and an ester in sheep and cattle. Presented at the meeting of the Pesticide Division, American Chemical Society and Chemical Institute of Canada, Toronto, May 28, 1970. 7. Clark, D.E., H.R. Crookshank, R.D. Radeleff and J.S. Palmer, 1971, Tissue residues of chlorophenoxy acid herbicides in cattle and sheep. Am. Chem. Soc. Meeting, April 2, 1971. �-278. Newton, M. and L.A. Norris, 1968. Herbicide residues in blacktail deer from forests treated with 2,4,5-T and atrazine. Western Soc.. Weed Sci., Proceedings pp. 32-34. 9. Corneliussen, P.E., 1969. Pesticide residues in total diet samples. Pest. Monit. J., j2:140-152. (Mar.) 10. Duggan, R.E., H.C. Barry and L.Y. Johnson, 1967. Pesticide residues in total diet samples. Pest. Monit. J., JL:2-12. (Sept.) 11. Martin, R,J. and R.E. Duggan, 1968. Pesticide residues in total die-t samples. Pest. Monit. J.,. JL:11-20. (Mar.) 12. Piper, W.N. and J.Q. Rose, 1971. The excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Unpublished report from Dow Chemical Co. dated March 18, 1971. �-28- II. TOXICITY OF 2,4,5-T AND TCDD IN ANIMALS AND MAN Currently available commercial preparations of 2,4,5-T can be characterized as having at least 95% 2,4,5-T with less than 0.5 ppm TCDD and no other toxicologically significant compound. Many earlier studies on the adverse effects of 2,4,5-T employed preparations containing considerably greater concentrations of TCDD than this, and others used 2,4,5-T samples of unspecified purity. Toxicological studies utilizing 2,4,5-T preparations which were not known to conform to the standards suggested above, nevertheless, have some value because any error attributable to larger amounts of TCDD would have been toward the conservative side, that is, would have suggested greater toxicity than if a purer 2,4,5-T had been used. A. Nonteratogenic Toxicity. Of 2,4,5-T. Among the earlier reports of 2,4,5-T toxicity that did not fully identify the composition of the product under investigation were the studies of Drill and Hiratzka— in which oral LD5Q for dogs was estimated to be in excess of 100 rag/kg, and of Rowe 2/ and Hymas— in which the oral LD,-n to various rodents was found to be greater than 350 mg/kg. Drill and Hiratzka found no adverse effects in dogs which were fed 2,4,5-T five times a week for 90 days at dosage levels of 2.5 and 10 mg/kg. Four dogs were treated at a level of 20 mg/kg 2,4,5-T and died at 11, 49, 59 and 75 days after the first dose, Rowe and Hymas reviewed the toxicologic information available on 2,4,5-T at that time, and concluded that the acute lethal oral toxicity, in terms �-29- of LDcQ and-19/20 confidence limits, of 2,4,5-T was for male rats 500 (391-640) mg/kg; for male mice, 389 (245-619); guinea pigs, male and female, 381 (307-472); chicks, male and female, 310, (211-456). The latter authors also used various commercial formulations of the butyl, isopropyl and amyl esters of 2,4,5-T in single oral-dose animal-feeding experiments in rats, chickens and guinea pigs and reported LDc0 levels with 19/20 confidence limits which were all greater than those listed above. They concluded that oral administration of 2,4,5-T could be tolerated without adverse effects in doses only slightly smaller than those which caused toxic effects and stated that this fact demonstrates o/ that 2,4,5-T has a low degree of chronicity. Palmer and Radeleff—' found that the propyl glycol butyl ether ester of 2,4,5-T was lethal to one sheep after 369 daily oral doses of 100 mg/kg and to another sheep after seven doses of 250 mg/kg. A single cow also succumbed to the latter dose. The triethylamine salt of 2,4,5-T caused no observed effect (sheep) after 481 doses of 100 mg/kg/day. The propionic acid butyl ether ester of 2,4,5-T was lethal to a sheep after 11 daily doses of 100 mg/kg orally and lethal to a cow after 29 such doses. Five daily 250 mg/kg oral doses also killed a cow. Fifty mg/kg/day orally had no effect after 73 days. In 1969 an investigation of the carcinogenicity in mice of 120 pesticides and herbicides— ; 2,4,5-T was among the compounds tested which did not cause significant increase in tumors after oral administration. The purity or dioxin content of the sample was not described. This represents the only report of long-term treatment with 2,4,5-T other than the few farm animals mentioned above. The dose was the maximum �-30- tolerated dose (zero mortality) determined with single doses, 6 daily doses, and finally 19 daily doses. The dosage was given by stomach tube at 21.5 rag/kg from the end of the first through the fourth weeks and thereafter it was mixed in the diet at 60 ppm of food and continued until 18 months of age. It is presumed that all animals survived the 18 month test period although this was not stated in the publication. Johnson— has reported acute oral, single dose toxicity studies on commercial 2,4,5-T in which the LD5Q for 2,4,5-T was 500 mg/kg in the rat and 380 mg/kg in the guinea pig. Ninety-day feeding studies with 2,4,5-T containing 0,5 ppm TCDD were recently reported by McCollister and Kociba.— The acid form was administered to groups of 10 male and 10 female rats at 100, 30, 103 and 0 mg/kg/day. No significant adverse effects were observed in the groups receiving doses at or below 30 mg/kg/day, but those receiving 100 mg/kg/day showed a depression of body weight gain, a decrease in food intake and elevated alkaline phosphatase levels. The males showed slightly increased serum .glutamic-pyruvic transaminase levels and slight decreases in red blood cell counts and hemoglobin levels. Histological evidence of toxicity were minor and inconsistant. In an earlier experiment at Dow Chemical Co.—', the mono-, di-, and tripropylene glycol butyl ether esters of 2,4,5-T were administered orally to rats over a similar 90-day period at doses as high as 186 mg of 2,4,5-T acid equivalent per kg per day. At the highest dose and at 62 mg/kg/day of acid equivalent various evidences of toxicity developed, but no adverse manifestations attributable to the agent were detected at dosages of 18.6 or 6.2 mg/kg/day. Q / The Dow Chemical Co.— has prepared an extensive health inventory of 126 manufacturing personnel in an effort to identify adverse effects �-31- of inhaled 2,4,5-T. The inhalation rate of the agent was estimated to be 1.6 to 8.1 rag/day per worker, depending on the work assignment, for periods of up to three years and at total career exposures in excess of 10,000 mg. The survey indicates that no illness was associated with 2,4,5-T intake. Specifically there was no increase in skin ailments or of alkaline phosphatase or SGPT levels as compared with controls having no exposure to 2,4,5-T. The result was entirely different in a plant where the 2,4,5-T produced contained a high proportion of dioxin. The latter plant was 9/ studied by Bleiberg— in 1964 and again six years later by Poland et al.— who also reviewed earlier studies in factories in other countries where TCDD had been a problem. Poland and associates reported on 73 employees whose health was found to be improved compared to that of workers in the plant six years earlier. Eighteen percent of the men had suffered moderate to severe chloracne, the intensity of which correlated significantly with the presence of residual hyperpigmentation, hirsutism, and eye irritation and with a high score on a test indicating a manic reaction. The chloracne did not correlate with job location or duration of employment at the plant or with coproporphrin excretion. One of the men had uroporphyrinuria but, unlike the situation six years earlier, no porphyria could be found. Systemic illness such as may be produced by TCDD was markedly less than that reported in previous studies of 2,4,5-T plants and probably no greater than expected in unexposed men of the same age. Dogs and rats tolerate oral intake of 2,4,5-T at a rate of 10 mg/kg/day or higher without detectable clinical, biochemical, or pathological change. The tolerance limit of people is not known but �-32- no injury was detected in workers with the highest recorded, prolonged exposure in a factory making low-dioxin 2,4,5-T, i.e., 8.1 mg/man/day or about 0.11 mg/kg/day°/. In view of the small and highly infrequent occurrence of residues of 2,4,5-T in human food (see Section IB), it is clear that exposure from this source is too small to measure accurately. Thus, although it is impossible to estimate how much greater the 2,4,5-T exposure of workers is than the exposure of the general population, it is clearly much greater than the corresponding one for DDT—. In fact, exposure to 2,4,5-T is trivial even for persons who daily eat unpolished rice. The very small number of cases in which human irigestion of 2,4,5-T led to clinical illness offer no information on the minimal dosage of the compound that is toxic to man. In animals, however, the toxicity of 2,4,5-T is similar to that of 2,4-D, consequently some information on 2,4-D is of interest. When 2,4-D was investigated as a possible treatment for disseminated coccidiodomycosis, the patient had no sideeffects from 18 intravenous doses during 33 days; each of the last 12 doses in this series was 800 mg (about 15 mg/kg) or more, the last being 2000 mg (about 37 mg/kg). A 19th and final dose of 3600 mg (67 mg/kg) produced mild symptoms—/. Suicidal ingestion of a quantity of 2,4-D as a single dose known to be greater than 6500 mg 13/ (in excess of 90 mg/kg) was fatal— • Butler —' —' has reviewed Fish and Wildlife Service studies of pesticide and herbicide effects on marine organisms. Several 2,4,5-T derivatives were examined (TCDD content was not known). A 96-hour exposure of oysters to the polyglycol butyl ether esters of 2,4,5-T at a concentration of 0.14 ppm in the water caused a 50% decrease in shell �-33- growth rate, with recovery in one week. The 24-hour LD^Q of this ester to juvenile esta"3<jine fish was 0.32 ppm. A concentration of 2,4,5-T acid at 2 ppm caused no decrease in growth after 96 hours. A level of 50 ppm 2,4,5-T was not lethal to juvenile mullet and killifish in 48 hours, and 1 ppm was without effect on shrimp in 48 hours. It is thus apparent that aquatic species tolerate higher concentrations of 2,4,5-T than have been reported in water samples taken from heavily sprayed areas (see Section I B, pp. 11-12). It is concluded from the foregoing that: 1) most species tested can survive a single oral dose in excess of 100 mg/kg and several, excepting the dog, can survive daily treatment for a number of days at this level or higher; 2) dogs die after 11 to 75 doses at the rate of 20 mg/kg/day and rats show toxic signs at repeated daily doses of 100 mg/kg; both species tolerate 10 mg/kg/day without detectable effect; 3) no proven instance of toxicity associated with 2,4,5-T intake in man has been found in industrial or agricultural workers known to have had repeated, relatively high levels of exposure to 2,4,5-T of low dioxin content; and 4) the safety factor for the general population is estimated to be several orders of magnitude greater than that of 2,4,5-T factory workers. Of TCDD. TCDD has been recognized as a contaminant of commercial preparations of 2,4,5-T for several years; however, there has been no extensive study of its toxicity. According to JohnsonM' the acute LDtjQ for TCDD is 0.022-0.045 mg/kg in the rat and 0.0006 mg/kg in the guinea pig. Because of the high potency of this compound in the guinea pig, these experiments were repeated and confirmed by �-34- Dow Chemical Co. Some information on the toxicity of TCDD is available from a study of TCDD teratogenic effect in the rat (see Section II B, p. 44). No evidence of clinical effect on the dams was found at doses of 0.0005 mg/kg/day, although embryotoxicity appeared in litters of females given 0.000125 mg/kg/day. Some vaginal hemorrhage was caused by 0.002 mg/kg/day and 0.008 mg/kg/day caused pallor and debilitation. As far as occupational exposure is concerned, it is clear that any danger of 2,4,5-T formulations resides in their TCDD content. The primary manifestation of industrial TCDD intoxication is chloracne, an easily detected, in fact highly disfiguring, dermatitis. It is significant that this condition has not been a problem in factories producing 2,4,5-T with a low content of TCDD, nor among persons who apply the herbicide as a part of their regular occupation. It is therefore highly unlikely that exposure to traces of TCDD will have any effect on persons who use 2,4,5-T formulations occasionally or who merely encounter possible traces of it in the environment. Data are too limited for a firm conclusion but there is no evidence to suggest that TCDD as a contaminant in 2,4,5-T is likely to be encountered by animal or man in. sufficient dosage to cause toxic reactions. References Cited in Section II A 1. Drill, V. A. and T. Hiratzka, 1953. Toxicity of 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid. A Report on Their Acute and Chronic Toxicity in Dogs. AMA Arch. Indust. Hyg. Occup. Med., ^:61-67. �-35- 2. Rowe, V,.K. and T.A. Hymas, 1954. Summary of toxicological Information on 2,4~D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. 3. Palmer, J.S. and R.D. Radeleff, 1964. Am. ,J. Vet. Res., 15:622-629. The toxicologic effects of certain fungicides and herbicides on sheep and cattle. Ann. N.'Y. Acad. Sci., 111:729-736. 4. Innes, J.R.M., B.M. Ulland, M.G. Valeric, L. Petrucelli, L. Fishbein, E.R. Hart, A.J. Pallotta, R.R. Bates, H.L. Falk, J.J. Gart, M. Klein, I. Mitchell and J. Peters, 1969. Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note., J. Nat'l Cancer Inst., .42:1101-1114. 5. McCollister, Susan, B. and R.J. Kociba, Sept. 18, 1970. Results of 90-day dietary feeding study on 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) in rats. Dow Chemical Co. Internal report. 6. Johnson, J.E. Paper presented at Symposium of A«*,issaucan Institute of Biological Sciences, Bloomington, Indiana, August 26, 1970. 7. Dow Chemical Co., Internal communication, Nov. 27, 1961. Results of 90-day dietary feeding studies on Dowanol 97B ester 2,4,5-T in rats. 8. Dow Chemical Co., letter with attachment from C.G. Kramer to J.E. Johnson, April 7, 1970 (see Dow Petition). 9. Bleiberg, J., M. Wallen, R. Brodkin and I. Applebaum, 1964. Industrially acquired porphyria. Arch. Derm., 89:793-797. 10. Poland, A.P., D. Smith, G. Metter and P. Fossick, 1971. A health survey of workers in 2,4-D and 2,4,5-T plant, with special attention to chloracne, porphyria cutanea tarda, and psychologic parameters. Arch, Environ. Health, 22:317-327. �-36- 11. Laws, E.R., Jr., A, Curley and F.J. Biros, 1967. Men with intensive occupational exposure to DDT. Arch. Environ. Health, 15:766-775. 12. Seabury, J.H., 1963. Toxicity of 2,4-dichlorophenoxyacetic acid for man and dog. Arch. Environ. Health, 2.:202-2°9- 13. Nielsen, K., B. Kaempe and J. Jensen-Holm, 1965. Fatal poisoning in man by 2,4-dichlorophenoxyacetic acid (2,4-D): of the agent in forensic materials. Determination Acta Pharmacol. Toxicol., j22:224-234. 14. Johnson J.E., 1970. Symposium on Possible Public Health Implications of Widespread use of Herbicides, AIBS Meeting, August 26, 1970. 15. Butler, P.A., 1963. Pesticide-Wildlife studies; A review of fish and wildlife service investigations; Circular 167. Commercial Fisheries Investigations. 16. Butler, P.A., 1964. Pesticide-wildlife studies, 1963; A review of Fish and Wildlife Service investigations during the calendar year, circular 199. Commercial Fisheries Investigations. �-37- B. The Teratogenic Potential of 2,4,5-T 1. Scope of embryotoxicity. Teratology is the science dealing with the causes, mechanisms, and manifestations of deviant structural or functional development. Such deviation can be the result of mutation in which case the defect may be transmitted by heredity, or it may be induced by unfavorable environmental conditons during the developmental period: usually during the formative stages of the embryo, less often during functional maturation of the fetus, and possibly even during the final stages of development postnatally. Many types of adverse factors in the environment have been shown to initiate abnormal development when applied during pregnancy in laboratory animals, including: certain dietary deficiencies (mostly of vitamins); many classes of chemicals, including some drugs; various physical factors such as ionizing radiation, drastic temperature changes, and alterations in atmospheric gases; a few viral infections; some maternal endocrine and metabolic imbalances; and undoubtedly some combinations of these. Relatively few of these experimentally demonstrated teratogenic agents have been shown to be effective in man. High doses of ionizing radiation such as are used in therapy for cancer or emanate from nuclear explosions are well known to be teratogenic when applied during early human pregnancy. Two infectious agents, rubella and cytomegalic viruses, have been clearly implicated. Three types of drugs - thalidomide, folic acid antagonists, and androgenic hormones - have been established as causes of malformations in man and a few others are suspected but are not at this time proven to be teratogenic. Maternal metabolic diseases �-38- such as endemic cretinism, diabetes, phenylketonuria, and adrenal hyperplasia account for a small percentage of human developmental disease. One environmental pollutant, methylmercury, proved to be teratogenic for man when it reacned high concentrations in certain watet in Japan from which fish were eaten as a large part of the diet. Adverse effects on development are difficult to evaluate because they vary greatly in degree and type. Collectively these effects can be designated as embryotoxic because they most often have their inception in the embryo and include such manifestations of toxicity as lethality, teratogenicity, prenatal growth retardation, and postnatal functional deficiencies. Few attempts are made to evaluate functional deficiency except as it may be reflected in postnatal survival data. On the other hand embryolethality, teratogenicity, and growth retardation can under laboratory conditions be readily detected and quantitated. Difficulty is often encountered, however, when all three toxic manifestations are simultaneously evaluated, since the phenomena involved are rarely affected to the same degree by a given embryotoxic agent. Although most chemical substances probably could be shown to be teratogenic under suitable experimental conditions and all could be shown to have some toxic effects when dosage is sufficiently high, some would be more strongly teratogenic, others would be predominantly embryolethal, whereas still others would tend mainly to cause intrauterine growth retardation. While these toxic manifestations vary directly with dosage, they may not show parallel dose-response effects. Any one of the three may begin to appear at a somewhat lower dose than either of the others. Lethality is probably the most variable from one agent to another, sometimes appearing at low doses and increasing slowly as dosage is increased, sometimes �-39- ''appearing abruptly at doses already causing considerable teratogenicity and growth retardation. Teratogenicity is probably the most predictable of the three in' that it usually has an easily demonstrable no-effect range of dosage and a steep dose-response curve once teratogenicity begins. These variations in embryotoxic manifestations are particularly troublesome when it is necessary to establish the highest no-detectableeffect or the lowest effect level of dosage. A level that has no detec- table teratogenic effect may already be in the effect range for lethality or growth retardation. The solution to this dilemma requires either that one form of embryotoxicity be selected as the criterion of interest or that the one showing the lowest effect level arbitrarily be accepted in setting tolerance limits. Such complications have been encountered in attempting to evaluate 2,4,5-T results, particularly in those experiments in which data on all embryotoxic manifestations were not reported. In some experiments only results pertaining to teratogenicity were given and in these cases dose-response evaluation had to be limited accordingly. For others, the Advisory Committee has tried to extrapolate the data in such a way as to approximate a no-effect level, i.e.,, the largest dose at which no increased lethality, teratogenicity, or growth retardation occurred. 2. Data from laboratory animals. In 1964, the National Cancer Institute contracted with Bionetics Research Laboratories to perform screening studies for carcinogenicity and teratogenicity on a number of pesticides and industrial chemicals. The results, released in October 1969, indicated that of the 53 compounds examined, 2,4,5-T in particular showed embryotoxicity in two stocks of mice at a dose of 113 mg/kg/day �-40- when given for several days during organogenesis. Cleft palate, cystic kidneys, intestinal hemorrhage and fetal mortality occurred in higher percentages of treated than of control animals although a clear doseresponse relation was not evident at lower doses. The results have been reviewed elsewhere =J±J and published in summary form _' and therefore require no extensive discussion here. Certain inconsistencies in the data —/—- likewise need no comment because the sample 2,4,5-T used in the Bionetics study is known to have been contaminated with 27 * 8 ppm of TCDD and the results can no longer be considered a valid indication of the teratogenicity of the herbicide. This contaminant itself has since been shown to have teratogenic and embryolethal properties, as will be discussed later. Despite the limitations of the original Bionetics study, it served two useful functions, in: 1) highlighting the possibility that herbicides may cause previously unknown adverse effects on nontarget organisms, including mammals, and 2) emphasizing the need for more thorough safety evaluation of such compounds before they are approved for widespread use. The discovery that the contaminant TCDD was present in the herbicide used in the Bionetics study made it necessary to determine whether the reported teratogenicity was caused by 2,4,5~T or TCDD. Additional studies relating to this question have been completed at the Dow Chemical Company, the Food and Drug Administration, the National Institute for Dental Research, the National Institute of Environmental Health Sciences, the Department of Agriculture Animal Disease and Parasite Research Division, the Food and Drug Directorate of Canada, Bionetics Research Laboratories, and �-41- the Children's Hospital Research Foundation of Cincinnati, on rats, mice, hamsters, rabbits, sheep and rhesus monkeys using samples of 2,4,5-T containing known concentrations of TCDD as well as relatively pure samples of TCDD, These studies are summarized below, species by species and separately for 2,4,5-T and TCDD. Insofar as the original reports permits, data are summarized on maternal toxicity, e.g., death or failure to show normal weight gain during pregnancy; as well as on embryotoxicity and fetal toxicity, e.g., prenatal death teratogenesis, intrauterine growth retardation, and perinatal signs of other toxicity. It is recognized that fetal death, either individual or as whole litters may also reflect maternal toxicity, and therefore may be difficult to interpret. 2,4,5-T in rats. Sprague-Dawley rats at Dow Chemical Co. were fed 1, 3, 6, 12, or 24 mg/kg/day of 2,4,5-T containing 0.5 ppm of TCDD on days 6 through 15 of pregnancy —'—'U . No maternal death or reduced maternal weight gain during pregnancy was noted. There was also no increase in prenatal mortality, only slight impairment of fetal growth in a few cases at the 24 mg/kg dosage, and no malformations. The poor ossification of the 5th sternebra noted in some cases was probably a sign of mild transient retardation of skeletal development and of no known significance. Pregnant rats of the same stock were fed 50 or 100 mg/kg/day of "commercial production grade" 2,4,5-T containing 0.5 ppm TCDD on days 6 through 15 of gestation, or 100 mg/kg/day on days 6 through 10 _/OL/, The only effects observed after the lower dose were intestinal hemorrhage in one of 203 offspring and a slight increase in �-42- frequency of delayed ossification of skull bones. The larger dose produced 83% maternal death and early death (resorption) of the entire litters in most of the surviving pregnant animals. Surviving offspring were reduced in size but had no anomalies except delayed ossification of skull bones, and this retardation was overcome within three weeks after birth, Sprague-Dawley rats at the National Institute of Dental Research —' —' were given orally 60, 80, 100, or 120 mg/kg/day of 2,4,5-T containing 0.4 ppm TCDD over various periods of consecutive days during the middle third of gestation. Maternal toxicity data were not reported. No treatment greatly increased the intrauterine mortality rate and many had no effect. growth retardation was not mentioned, Prenatal Apparently the offspring were examined only for external malformations and those of the oral cavity. Very few with such defects were found (7 of 1500 at susceptible periods). of cleft palate. from females treated A mixture of 2,4,5-T and 2,4-D produced one case To rule out the possibility that the chemical might not be reaching the fetus, millipore filters soaked with 0.05, 0.1, 0.11, or 0.125 mg 2,4,5-T were applied to amniotic sacs on day 12, 13, 14, 15, or 16 of gestation. Of 68 fetuses surviving to the time of examination two had cleft palate, one had a tail defect, four had limb defects, and others were small or edematous. A second study of this type yeilded one possible limb defect and four possible tail defects in 68 survivors. Rats of the FW-49 stock in Germany recently were given 25, 50, 100, or 150 mg/kg/day of 2,4,5-T (containing <C0.02 ppm TCDD) on days 6 through 15 of gestation (cited by Tschirley 12/ —• ) . Macro-and Microscopic examination revealed no signs of teratogenicity even with the highest �-43- dosages. There was an increase in the prenatal mortality beginning at the 50 mg/kg dose and a reduction in the mean fetal weight beginning at the 100 mg/kg dose. Complete details of the study were not given. Charles River rats at the National Institute of Environmental Health 13/14/15/ Sciences — — — received samples of 2,4,5-T CO.5 and 30 ppm TCDD) at the rate of 10 to 80 mg/kg/day orally or subcutaneously on days 6 through 15 of gestation, or 2,4,5-T C^ 0.05 ppm TCDD) at the rate of 150 mg/kg/day subcutaneously on days 14 and 15 of gestation. The 80 mg/kg dose was stated to be the maternal LD, dose but data were not presented. The 80 and 150 mg/kg doses caused a reduction in maternal weight gain and increased prenatal mortality, but fetal weight was unaffected. A low incidence of fetal kidney anomalies was noted, but could not be attributed with .confidence to the treatment. In addition, pregnant females were fed 50 mg/kg of 2,4,5-T (< 0.05 ppm TCDD) and allowed to deliver. The offspring examined periodically for 3 weeks postnatally during which time mortality, weight gain, and general development did not differ from those of control animals, Wistar rats at the Food and Drug Directorate of Canada ~ were fed dosages of 25, 50, 100, and 150 mg/kg/day of 2,4,5-T acid or of 2,4,5-T butyl ester containing <0.5 ppm TCDD on days 6 through 15 of gestation. No apparent adverse effects on pregnant females were noted, but fetal weight was reduced. At the largest dosage there was an apparent increase in the frequency of "spontaneously occurring" skeletal anomalies. The largest dose of the acid form killed 3 of 8 pregnant females and reduced maternal weight gain but lower do'ses were without maternal toxicity. Intrauterine death was increased at 50 mg/kg and was pronounced at larger dosages, at which levels weight of surviving �-44- fetuses was reduced. At the higher doses there was also increased skeletal variations some of which did not occur spontaneously in controls. Postnatal survival of young was not adversely affected by maternal dosage with 100 rag/kg. The 2,4,5-T butyl ester was without effect. The foregoing rat experiments all involved repeated daily treatment of pregnant females with 2,4,5-T. The possibility exists, that owing to maternal homeostatic mechanisms, e.g., inducation or inhibition of metabolic enzymes, the most sensitive teratological test would be one involving a single treatment during early organogenesis. To test this possibility rat experiments were carried out at the Institute of Developmental Research, Children's Hospital Research Foundation of Cincinnati ^-'. Using a sample of 2,4,5-T containing 0.5 ppm of TCDD, groups of pregnant Wistar rats were treated by gavage on day 9 of gestation with doses of 100, 200 or 400 mg/kg in 0.2% carboxymethylcellulose. Day 9 is generally regarded as the time at which the rat embryo is teratogenically most susceptible. Dose Days of mg/kg treatment Control Control 20 100 200 400 none 9 7-13 9 9 9 Whole litters res orbed 0/40 0/45 0/11 0/11 0/11 2/10 Litters continuing to day 20 Total % dead or mean wt.of % survivors implants resorbed survivors malformed 509 558 170 170 156 122 5.4 7.2 8.8 9.0 11.5 25.4 3 . 7 gin 3.8 3.7 3.7 3.8 3.2 1.9 0.8 0.7 1.9 5.1 11.0 Cumulative untreated control over past 4 years. Cumulative vehicle treated control (per gavage) over past 4 years. ^Types of malformations; anophthalmia, microphthalmia, curly or short tail, hydronephrosis, ectopic testes, agnathia. �-45- The data in the accompanying table indicate that a single dose of 100 rag/kg at a highly sensitive time in rat emhryogenesis did not cause an increase in abnormal development or a decrease in intrauterine growth but did cause a slight increase in intrauterine death. This effect was accentuated at higher doses and a moderate increase in malformations above control levels was also noted. Intrauterine growth was affected only at 400 mg/kg, a dose sufficient to cause severe embryotoxicity as evidenced by complete resorption of 2 of the 10 whole lititers. In summary, it appears that rat strains vary considerably in their susceptibility to the embryotoxic effects of 2,4,5-T. A low level of teratogenicity may appear in some strains when repeated dosage exceeds 100 mg/kg/day, or single dosage on day 9 is at 200 to 400 mg/kg of maternal weight. Some increase in intrauterine death and decrease in intrauterine growth, as well as maternal toxicity, was sometimes noted at lower daily dosage, e.g., 50 mg/kg. TCDD in rats. Rats have been treated during pregnancy with TCDD in 18/ appreciable dosage in only two laboratories. At the Dow Chemical Co. ~ pregnant Sprague-Dawley rats received 0.00003, 0.000125, 0.0005, 0.002, or 0.008 mg/kg/day of dioxin (91% TCDD) orally on days 6 through 15 of gestation. Only one maternal death occurred and maternal weight gain was depressed only by the largest doses. Prenatal mortality was greatly increased at the 0.002 mg/kg dosage and all fetuses were killed at the 0.008 mg/kg level. Fetal weight was greatly reduced at 0.002 mg/kg and somewhat reduced at lower doses. tions of the tail and limbs. Only two offspring had possible malforma- Edema and intestinal hemorrhage were observed in some offspring of females treated with 0.000125, 0.0005, or 0.002 mg/kg. 9/ In a second Dow study — pregnant rats of a stock of unstated origin received �-46- by an unstated route 50 mg/kg/day of "pure" 2,4,5-T Cprobably containing 0.05 ppm TCDD) to which was added 0.00001, 0,00003, 0.00006, 0.000125, 0,0005, or 0.001 mg/kg/day of TCDD on days 6 through 15 of gestation. Cleft palate occurred in ten litters, mostly in those receiving the 2,4,5-T plus 0.0005 or 0.001 mg TCDD. The frequency of offspring with cleft palate, as well as procedural details and toxicity were not described. Charles River rats at the National Institute of Environmental Health Sciences — received TCDD subcutaneously 0.0005 mg/kg/day on days 6 through 10 of gestation, or 0.002 mg/kg/day on days 9 and 10 or 13 and 14 of gestation. No malformations or excessive fetal mortality were noted; but various possible kidney anomalies and several instances of intestinal hemorrhage occurred. Thus, TCDD given to pregnant rats caused embryolethality and occasional teratogenicity at doses below the maternal toxic level. 2,4,5-T in mice. Mice of GDI, C57BL/6J and DBA/2J strains received, subscutaneously, 50, 100, 113, 125, or 150 mg/kg/day of 2,4,5-T containing <L, 0.5, or<0.05 ppm TCDD on days 6 through 15 of pregnancy at the 14/ National Institute of Environmental Health Sciences — . No maternal death occurred and maternal weight was depressed only in C57BL mice at the 100 mg/kg dosage. Fetal mortality was increased only in GDI mice at the 150 mg/kg level. Fetal weight was reduced in all three lines of mice at dosages of 100 mg/kg and greater. Cleft palate occurred in low but consistent frequencies in all three lines of mice at doses of 100 mg/kg and more with all three samples of 2,4,5-T. Where sufficient data were available, a dose-response relation for fetal mortality and growth retardation was noted. Paradoxically the frequency of kidney �-47- anomalies, types unspecified, was increased above the low level of background occurrence by the 2,4,5-T containing 0,05 ppm TCDD, but not by the 2,4,5-T containing 0.5 ppm TCDD. In general, these mice showed a low level of teratogenicity at 100 mg/kg/day during embryogenesis, and some embryolethality and decreased fetal weight at lesser doses. Moore 197 found no appreciable difference in teratogenic and embryolethal —' potential between 2,4,5-T as free acid and its butyl, isooctyl and butyl ether esters at approximately molar equivalent dosage in...jnice. Mice of the NIH all-purpose albino stock were given subcutaneously 113 mg/kg/day of 2,4,5-T containing 0.4 ppm TCDD or 113 mg/kg/day of a mixture containing 50% 2,4,5-T and 40% 2,4-D ("Orange") usually on days 6 through 14 of gestation at the National Institute of Dental Research —• . Cleft palate occurred in 9 of 141 offspring, but no data regarding maternal toxicity and other fetal effects were reported. In a recent study made by the Bionetics Research Laboratories, commissioned by Hercules Incorporated, GDI mice were injected subcutaneously on days 6 through 15 of gestation with 100 mg/kg/day of 2,4,5-T supplied by the Dow Chemical Co. and Hercules Inc. No maternal death seems to have occurred and maternal weight gain was unaffected. Intra- uterine mortality was not increased but mean fetal weight was slightly reduced. The only malformation that occurred was cleft palate and its frequency was 11.1% (27/243) with the Dow sample and 1.3% (3/235) with the Hercules sample. Both products had<0.5 ppm TCDD. TCDD in mice. TCDD given to mice at 0.001 or 0.003 mg/kg/day subcutaneously on days 6 through 15 of gestation did not affect fetal mortality, maternal weight gain, or fetal weight, but did produce low frequencies of cleft palate in the three mouse lines used in the �-48- National Institute of Environmental Health Sciences study In one experiment 2,4,5-T (100 rag/kg) and TCDD ( . 0 mg/kg) given 001 together apparently produced no greater frequency of cleft palate than when each was given alone. TCDD greatly increased the background rate S'f. kidney anomalies, especially in C57BL mice. Thus the limited data indicate that TCDD has some teratogenic potential in mice at doses even lower than those causing appreciable intrauterine death. 2,4,5-T in hamsters. Golden hamsters of a commercially obtained stock were treated orally on days 6 through 10 of gestation with 20-100 mg/kg/day of 2,4,5-T from seven sources, at the Food and Drug Administration 20/21/ Four of the samples contained 45, 2.9, 0.5 and 0.1 ppm TCDD, respectively, and three contained no detectable TCDD. on maternal toxicity was not given. Information Fetal mortality was greatly increased by the TCDD-containing 2,4,5-T samples and its frequency was usually directly related to both 2,4,5-T dosage and dioxin content; but it was also moderately high and dose-related after 2,4,5-T containing no detectable dioxin. The mean weight of surviving fetuses was unaffected or only mildly so for the different samples. A low to moderate incidence of gastrointestinal hemorrhage was observed, but this was probably not developmental in origin. Malformations were noted in offspring exposed to 2,4,5-T containing TCDD or not, but their frequency was usually higher after 2,4,5-T containing dioxin than after 2,4,5-T that did not. No malformations were produced by 2,4,5-T alone below the 100 mg/kg dose, whereas all dosages of dioxin-containing 2,4,5-T produced malformations. Very few malformations (cleft palate, 2 cases, and ectopic heart, 1 case) resulted from use of dioxin-containing 2,4,5-T, and only at 100 mg/kg. �-49- The most frequent defects, poorly characterized as "bulging eyes" and "poor head fusion", occurred in low percentage (15.8% and 11.4%, respectively) after 2,4,5-T with or without TCDD. Some apparent discrepancies were present in the calculations of the malformation rates. In addition 150 mg/kg/day of a recrystalized and extracted 2,4,5-T was used 22J and produced a high fetal mortality rate but no malformations. TCDD in hamsters. Hamsters were given dioxin (21% tri, 53% tetra CDD) orally at 0.00013, 0.002 or 0.0091 mg/kg/day on days 6 oo/ through 10 of gestation at the Food and Drug Administration — . Maternal toxicity was not mentioned. Mean fetal weight was reduced only at the two highest dosages. Eye anomalies and prenatal mortality were most frequent at the highest dose. Gastrointestinal hemorrhage was noted at the 0.0005 and 0.002 mg/kg doses. 2,4,5-T in rabbits. New Zealand white rabbits were treated orally with 10, 20, or 40 mg/kg/day of 2,4,5-T containing 1 ppm TCDD on days 6 through 18 of gestation at Dow Chemical Co. — —'• No deaths of pregnant females occurred, and maternal weight gain and fetal mortality and weight were unaffected. No congenital malformations were noted and developmental variations were not increased in frequency. 2,4,5-T in sheep. Sheep were fed 100 mg/kg/day of Dow production 2,4,5-T or of Dow production 2,4,5-T propyleneglycolbutylether ester on days 14 through 36 of pregnancy at the Department of Agriculture Animal 9/ Disease and Parasite Research Division ~ . Two of 19 ewes died on days 35 and 36 of pregnancy, but their fetuses were normal. The other 17 delivered normal offspring at term. 2,4,5-T in rhesus monkeys_. No further details were provided. The Poisons and Pesticides Board of �-50- 23/ Sweden has commissioned a study in pregnant rhesus monkeys — a t doses of 5, 10, 20 and 40 tng/kg given three times per week for 4 weeks between days 20 and 48 of gestation. The sample of 2,4,5-T contained 0.5 ppm of TCDD. Twelve fetuses removed by hysterotomy at 100 days of gestation from females treated with one of the three lower doses (4 pregnancies each dose), were developmentally normal and fell within the range, of weight for untreated fetuses of this age. Two of 4 pregnant females treated with the highest dose yielded normal fetuses and the other 2 have not been hysterotorn!zed at this writing but are still pregnant. One- f emajL treated at this level aborted on day 61 of gestation and the conceptus was too macerated for examination. Abortion rate among untreated females in this colony is about 7% prior to day 100 of gestation. Summarizing available data on exposure of pregnant laboratory animals, it is notable that rats were used most often and under the widest variety of conditions. Pregnant females of several stocks received orally administered low-dioxin-content 2,4,5-T in doses up to 400 mg/kg for durations varying from single treatment to periods including much of embryonic development. In the studies in which dosage was kept below the toxic level for the pregnant females malformed offspring rarely occurred, and at higher dosages only a low teratogenic potential was revealed. Results of rat studies with TCDD were variable. In two of three experiments very few malformed young occurred, but in the third an appreciable incidence of cleft palate was reported. Mice proved to be more susceptible than the other species to the embryotoxic effects of both 2,4,5-T and TCDD. Both compounds produced low to moderate frequencies of cleft palate in all stocks tested but �-51- they did not appear to be more teratogenic when given together than when given separately. Hamster studies with large doses of 2,4,5-T containing no detectable or low concentrations of dioxin (0.1 and 0.5 ppm) produced significant fetal mortality but relatively few instances (14/760 = 1.8%) of maldevelopment. Trials with high-dioxin-content 2,4,5-T (2.9 and 45 ppm) also caused appreciable fetal mortality but moderate frequencies of anomalies (16/209 = 7.6%). Based on these data jL_t can be concluded that: 1) doses of low- dioxin-content 2,4,5-T and of TCDD below the level producing maternal toxlcity were without significant effect on prenatal development, producing little or no embryotoxicity in, rats, rabbits, hamsters, sheep, and rhesus monkeys, and 2) these chemicals were more embryotoxic in mice, producing a low to moderate frequency of a specific malformation, cleft palate. The significance of the finding that TCDD in mice increased certain anomalies of the kidney, which occurred in low frequency in controls, can only be resolved by further investigation. 3. Human exposure during pregnancy. Reports have appeared in the news media that the use of 2,4,5-T was associated with an increased occurrence of congenital malformations and/or stillbirths in human beings in Vietnam; Globe, Arizona; and Sweden. Vietnam. Because of the increased use of several defoliating chemicals by the United States Military in South Vietnam during the past several years, particular concern was aroused by the reports in Vietnamese newspapers between June 26 and July 5, 1969 of human birth defects attributed to these chemicals. Two surveys have been undertaken to evaluate the situation. One was conducted by Dr. R. T. Cutting, U. S. �-52- Army Medical Research Team (Walter Reed Army Institute of Research), Dr. Tran Hun Phuoc, Ministry of Health, Government of the Republic of Vietnam, and three collaborators from the Military Assistance Command, 24/ Vietnam. A report was issued in December 1970 — and will be referred to here as the Army report. A second survey was recently made by the Herbicide Assessment Commission (HAG) of the American Association for the Advancement of Science, consisting of Drs. M. S. Meselson, A. H. O c IO£ / Lesting and J. D. Constable The Army study surveyed obstetrical records mostly for the years 1960-69 of 22 provincial, district, and maternity hospitals in 18 cities and other areas in various geographical localities. In most hospitals the records consisted of daily summary ledgers, prepared by the chief midwives, and contained such relevant information as the age and parity of the mothers and the sex, weight, and general condition at birth of the babies. Space was provided for additional remarks concerning maternal or infant complications. In three hospitals such ledgers were not kept but instead individual records were available. In the hospital yielding the largest number of births, the Tu-Du Maternity Hospital in Saigon, as system of automatic data processing existed, which provided for separate recording of numerous categories of malformations. Almost half a million births were included in cumulative records, and the overall recorded stillbirth and congenital malformation rates for the entire period were found to be 33.7 and 4.9 per 1000 livebirths, respectively. Attempts were made to analyze the information by geograph- ical area, by year, and by intensity of herbicide spraying. can be summarized as follows. The findings (1) In four geographical regions - capital, �-53- coastal, interior, and delta - the rates per 1000 livebirths of stillbirth and congenital malformation were 32.5 and 5.8, respectively, in the capital area, and 36.7 and 2.9 in the three remaining areas. The differences in these rates may be attributable to better maternal and neonatal care, or to more competent or thorough examination for congenital malformations in the capital area. (2) The rates for stillbirths de- clined and for congenital malformations remained unchanged during this 10-year period. (3) The only differences in these rates between the years 1960-65 and 1966-69, periods of relatively light and heavy defoliant spraying, respectively, was a downward trend (from 36.1 to 32.0 for stillbirths, and from 5.5 to 4.5 for congenital malformations). (4) There were no consistent differences between heavily and lightly defoliantsprayed areas. For the most part, however, the possibility of meaningful interpretations of the results of the Army study were precluded by their several limitations. First, it is obvious that only a fraction of the total births that occurred during these years were included in the records examined by the survey team. The report states that "RVN [Republic of Vietnam] officials estimate that currently only 70% of all births are reported to the MOH [Ministry of Health]" (emphasis added). The«New York Times Encyclopedic Almanac for 1971 (p. 877) gives the estimated population of South Vietnam for 1970 at 18 million and the birth rate as 3542 per 1000 population, which would yield between 630,000 and 750,000 27/ births in 1970 — . The last complete year for which records were examined by the Army survey, 1969, yielded a total of 87,153 births. Also the births that were included in the Army survey were far �-54- from evenly distributed throughout the country, the three capital-area hospitals contributing over 67% of the total. In addition, the Chinese, 211 who comprise a significant fraction of the population (over one million)—- , were probably completely omitted since, as was stated by the Army report, they did not attend the hospitals surveyed. Finally, any hope of relating the results to variations in the degree or geographical region of herbicide spraying was frustrated by a number of factors. For example, changes in the local practices of referring difficult obstetrical cases to provincial hospitals, determined by availability of trained personnel in the centers, and increasing with gradual improvement in transportation and security, probably greatly influenced stillbirth and malformation rates in •specific hospitals. Probably most significant was the fact that populations most heavily exposed to 2,4,5-T were those most likely to be underrepresented in the Army survey or to be inadequately dealt with when recorded. Thus, as the HAG report stated, the bulk of 2,4,5-T used in Vietnam was sprayed in relatively remote and sparsely populated areas; the population directly exposed to 2,4,5-T probably did not exceed 5% and may have been 1% or less of the total population of Vietnam; and very likely a significant proportion of the exposed population consisted of Montagnard people, whose births usually did not occur in hospitals and rarely were included in medical records or statistics. It is equally probable that other remote and lightly populated areas were similarly underrepresented and incompletely recorded. The Army survey noted that during 1960-69 there was a countrywide downward trend in the stillbirth rate. But, as was pointed out in the �-55- HAC report, this was heavily influenced by data from the capital area, in which 67.8% of all the surveyed livebirths occurred, and which generally experienced little or no exposure to 2,4,5-T. Deducting the capital area data and considering only data from the other parts of the country apparently reverses the trend, giving stillbirth and malformation rates for 1960-65 (years of no or light spraying) and 1966-69 (years of heavy spraying) of 31.9 and 2.3, and 38.4 and 3.1, respectively. This would seem to indicate that in the remoter areas, where exposure could have been intense, stillbirth and malformation rates increased during years when spraying was heavy. A possible explanation for these apparent differences was provided by the HAC report, in noting that more complete recording and increased referral of difficult pregnancies from the countryside to the provincial hospitals occurred in these years. A more likely explanation, however, is that in recent years, as a larger and larger proportion of births was registered (e.g., number recorded in noncapital areas in 1960-65 was 37,951; in 1966-69, 113,358) a larger proportion of stillbirths was ascertained and a more complete examination for and/or recording of congenital malformations was made. Particular attention was directed by the HAC reports &o the records for the Tay Ninh Provincial Hospital, because although "the total number of directly exposed Vietnamese to 2,4,5-T is* probably low, the northern portion of Tay Ninh has been heavily defoliated and the rivers draining the areas of defoliation run through the remainder of the province and are a source of fish for some of the population." Examining records that were apparently not available to the. Army survey, the HAC found that �-56- in 1968-69 the stillbirth rate recorded at the Tay Ninh City Provincial Hospital was 68.5, which they believed to be a higher rate than that found anywhere else by the Army survey. Although this is true, it should be noted that in two provincial hospitals, Qui Nhon, for which records only for 1966-69 were available, and Da Lat (nonpaying patients) during 1960-65, the stillbirth rates were not far below this, being 62.7 and 61.4, respectively. The HAG also discovered how unreliable the records were regarding congenital malformations, since they noted that not a single malformation was recorded for the 2551 births in 1969 in the Tay Ninh Provincial Hospital, and on questioning the midwives it was learned that although a fair number of deformities had been seen none were reported. Another hospital, at Vung Tau, during most of 1968-70 reported no congenital malformations in 6198 births and a much lower stillbirth rate than did the Tay Ninh Hospital, yet it closely bordered and included in its referral area a zone of intense defoliation. A further point needing critical scrutiny is the finding by the HAG of an apparent increased prevalence of children with spina bifida and isolated cleft palate among admissions to the Saigon Children's Hospital, the former increasing from 0.7% in 1959-66 to 2.1% in 1967-68, and the latter from 0.5% in 1959-65 to 2.6% in 1966-68. It should be emphasized that the figures do not pertain to- incidence at birth, but to the percentage of malformed children admitted to this Hospital some time after birth for operative care. It should also be stated that at least 77.5% of all the admissions in 1959-69 came from Saigon or nearby areas in which defoliation was not practiced. Again the most lilcely explanation �-57- of the apparent sudden rise in prevalence of these two malformations is more thorough examination and increased referral for surgical repair. Supporting this probability is the fact, noted by the Army Report, that the frequency of congenital malformations recorded in the capital area hospitals was much higher (although it varied greatly among the three hospitals) than in the remainder of the country, a fact in turn attributable to availability of more complete and competent medical services and personnel in the former than in the latter. • Summarizing the Vietnam data on human embryotoxicity, it can be said that (1) the sample of births surveyed was from year to year a variable but usually very small fraction of the total number, (2) it was quite unrepresentative of the geographic and ethnic distributions, (3) the heavily sprayed and otherwise exposed areas were greatly underrepresented, and (4) the birth records were not trustworthy and, therefore, the rates of stillbirth, and especially of congenital malformation, derived from them were equally unreliable. For example, the overall congenital malformation rate found in South Vietnam, 4.91 per 1000 livebirths, is about half of what was reported in other studies in various 24/ parts of Asia — , and possibly a quarter of what might actually exist at term. A further indication that the newborn children were not carefully examined is tlie absence of Down's syndrome in the list of specific malformations compiled by the Army survey, despite the fact- that some Oriental populations have been reported to have an incidence of this condition not 28/ unlike that in Western populations — . Finally there is, and can be, no precise knowledge or reasonable approximation of the exposure to 2,4,5-T experienced by pregnant �-58- Vietnaraese women, Including what amounts they ingested or absorbed and when this may have occurred during pregnancey. Thus, any attempt to relate birth defects or stillbirths to herbicide exposure is predestined to failure. It can only be concluded that the birth records that have been surveyed, and probably any that will be surveyed in the future, for South Vietnam for the period 1960-1970 cannot answer positively the questions about possible adverse prenatal effects following human exposure to 2,4,5-T. It must be emphasized, however, that the searches that have been made almost certainly would have revealed any marked increase in the incidence of birth defects or the introduction of a striking defect such as that produced by thalidomide. In spite of considerable effort, no such occurrences were found. 29/ Globe, Arizona was another site of human exposure — . The herb- icides used in the Kellner Canyon-Russell Gulch spray project near Globe were, in 1965 and 1966 the isooctyl esters of 2,4-D and 2,4,5-T, in 1968 an ester of Silvex (2,4,5-TP), and in 1969 about 97% Silvex (3680 Ib) and almost 3% 2,4,5-T esters (Hercules Co., 30 gal.). The reports of harmful effects to animals and people from the spraying began during and after the 1969 spraying. Those concerning possible reproductive and embryonic effects consisted of two miscarriages by a woman, one in April and the other in December 1969; a number of stillbirths of kids; and one miscarriage in a goat. Two other alleged cases consisted of a deformed goat approximately 5 years old, and therefore born before any herbicide spraying in the area (incidentally the defects were not of developmental origin), and a chicken with a slipped tendon which was incubated 4 miles from the sprayed area and after the spraying occurred in 1969. In all likelihood none of these reported effects was due to the sprayings. Competent �-59- medical and agricultural experts have been unable to find evidence of adverse effects on either human or animal reproduction that could be attributed to the defoliants applied during the Kellner Canyon-Russell Gulch spray project. Swedish Lapland. Swedish government defoliation projects to improve the quality of forests in Lapland have been associated in the public press with the occurrence of human malformations and abortion among reindeer. The chlorophenoxy acids 2,4-D and 2,4,5-T were used in routine fashion for a number of years without reports of untoward effects until the spring of 1970 when several instances of unexplained death and abortion among reindeer were attributed to use of these compounds. A group of scientific experts has investigated these claims 30 / for the National Poisons and Pesticides Board — and has failed to find a substantial basis for relating the toxic manifestations in these animals to ingestion of herbicides. Subsequently two instances of con- genital malformations in human infants have been attributed to alleged exposure of pregnant women during application of the herbicides. Highly competent medical scientists at the Institute of Hygiene and the Teratological Laboratories of the Karolinska Institute of Stockholm and at the Institute of Human Genetics at Mllnster, Germany have beeh unable to find temporal or clinical evidence to suggest that the occurrence of these human birth defects was more than coincidentally related to defoliating operations in Sweden. �-60- References Cited in Section II B 1. Report of the Secretary's Commission on Pesticides and their Relationship to Environmental Health. U.S. Department of Health, Education, and Welfare, Washington, D.C. 2. 1969. Report on 2,4,5-T of the Panel on Herbicides of the Office of Science and Technology, April 1971. 3. Courtney, K.D., D.W. Gaylor, M.D. Hogan, H.L. Falk, R. R. Bates, and I. Mitchell, 1970. Teratogenic evaluation of 2,4,5-T. Science 168;864-866. 4. Klingman letter to DuBridge, December 22, 1969. 2,4,5-T Advisory Committee Exhibit 5. 5. Emerson, J.L., D.J. Thompson, C.G. Gerbig and V.B« Robinson, 1970. Teratogenic study of 2,4,5-trichlorophenoxyacetic acid in the rat. Toxic. Appl. Pharmacol., JL7:317 (abstract). 6. Emerson, J.L., D.J, Thompson, R.J. Strebing, C.G. Gerbig and V.B. Robinson, 1971. Teratogenic studies of 2,4,5-trichloro- phenoxyacetic acid in the rat and rabbit. Food Cosmet. Toxicol., in press. 7. Thompson, D.J., J.L. Emerson and G.L. Sparchu, 1971. Study of the effects of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on rat and rabbit fetal development. Teratology, in press. 8. Sparschu, G.L., F.L. Dunn, R.W. Lisowe and V.K. Rowe, 1971. Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) on rat fetal development. Unpublished study. �-619. Johnson, J. E., 1970. The public health implications of widespread use of the phenoxy herbicides and picloram. Presented at the Symposium on Possible Public Health Implications of Widespread Use of Pesticides, American Institute of Biological Sciences, Bloomington, Indiana, August 26, 1970. 10. King, C. T. G., 1971. Teratogenicity studies of 2,4,5-T and 2,4-D. Unpublished report, February 25, 1971. 11. King, C. T. G., E. A. Horigan, and A. L. Wilk, 1971. Screening of the herbicides 2,4,5-T and 2,4-D for cleft palate production. Teratology, in press. 12. Tschirley, F. H., 1971. 2,4,5-T. Report on status of knowledge regarding Submitted by the USDA to the EPA, March 5, 1971. 2,4,5-T Advisory Committee AE-20. 13. 2,4,5-T Advisory Committee Exhibits I-13a, 1-14, and 1-15. 14. Courtney, K. D. and J. A. Moore, 1971. 2,4,5-T and tetrachlorodioxin. Teratology studies with Submitted to Toxic. Appl. Pharmacol. 15. Moore, J. A. and K. D. Courtney, 1971. Teratology studies with the trichlorophenoxyacid herbicides 2,4,5-T and Silvex. Teratology, in press. 16. Khera, K. S., B. L. Huston and W. P. McKinley, 1971. Pre- and postnatal studies on 2,4,5-T, 2,4-D, and derivatives in Wistar rats. Toxic. Appl. Pharmacol., in press. 17. Wilson, J. G., 1971. Unpublished data. 18. Sparschu, G. L., F. L. Dunn and V. K. Rowe, 1970. Teratogenic study of 2,3,7,8~tetrachlorodibenzo-p~dioxin in the rat. Toxic. Appl. Pharmacol., 17:317 (abst.). �-62- 19. Moore, J. A., 1971. Personal communication to 2,4,5-T Advisory Committee. 20. Collins, T. F. X., and C. H. Williams, 1971. with 2,4,5-T and 2,4-D in the hamster. Teratology, in press. 21. Collins, T. F. X., and C. H. Williams, 1971. with 2,4,5-T and 2,4-D in the hamster. Teratogenic studies Teratogenic studies Unpublished studies. 2,4,5-T Advisory Committee AE-16. 22. Effects of 2,4,5-T on Man and the Environment. Hearings before the Subcommittee on Energy, National Resources, and the Environment of the Committee on Commerce, U. S. Senate, April 7 and 15, 1970. Serial 91-60, p. 354. 23. Wilson, J. G. Preliminary report submitted to Swedish Poisons and Pesticides Board, April 19, 1971. 24. Cutting, R. T., T. H. Phuoc, J. M. Ballo, M. W. Benenson, and C. H. Evans. 1970. Congenital malformations, hydatidiform moles, and stillbirths in the Republic of Vietnam 1960-1969. Govt. Printing Office, Washington, D. C. 25. Meselson, M. S., A. H. Westing and J. D. Constable, 1970. Background Material Relevant to Presentations at the 1970 Annual Meeting of the AAAS. Herbicide Assessment Commission of the American Association for the Advancement of Science. Revised January 14, 1971. 26. Summary of presentations by the Herbicide Assessment Commission of the American Association for the Advancement of Science, Chicago, Illinois, December 29, 1970. 27. Foster, L., and M. Harth, eds., 1971. The New York Times Encyclo- pedic Almanac 1971. New York Times, New York. �-63- 28. Kikuchi, Y., H. Oishi, A. Tonomura, K. Yamada, Y. Tanaka, T. Jurita and E. Matsunaga, 1969. Translocation Down's syndrome in Japan: age. 29. its frequency, mutation rate of translocations and parental Jap. J. Human Genet., 14:93-106. Binns, W., C. Cueto, B. C. Eliason, H. E. Heggestad, G. H. Hepting, P. F. Sand, R. F. Stephes, and F. H. Tschirley, 1970. of Spray Project near Globe, Arizona. February 1970. Investigation Investigation Conducted 2,4,5-T Advisory Committee AE-15. 30. Rapport frlm en expertgrupp, 1971. Fenoxisyror, granskuing av aktuell information, Giftnamnden, Stockholm. �-64- GENERAL CONCLUSIONS The Advisory Committee on 2,4,5-T has accepted as its primary objective the evaluation of hazards to human reproduction of continued use, under appropriate regulations, of the herbicide 2,4,5-T. Toward this end it has examined all available information pertinent to a scientific consideration of the subject. The level of human exposure depends on rate of application of the herbicide, balanced against the rate at which it is removed from the environment. Current patterns of usage of 2,4,5-T and its known fate in various compartments of the environment, including the plant and animal foods of man, are such that any accumulation that might constitute a hazard to any aspect of human health is highly unlikely. Special note has been taken of the toxic contaminant TCDD. .The limited data now available indicate that this dioxin is not as rapidly degraded in the environment as is 2,4,5-T, but modern methods for the manufacture of the herbicide are capable of routinely producing a product with such a low level of contamination as to eliminate the likelihood of human toxicity from exposure to TCDD. Manufacturing standards must, however, be subject to continued monitoring. Much of the general toxicity attributed to 2,4,5~T in the past now appears to have been caused by the contaminant TCDD. The herbicide when essentially free of this contaminant, e.g. 1 ppm, has relatively low toxicity for all animal forms in which it has been tested. Particular attention was given to the teratogenic potential of �-65- both 2,4,5-T and TCDD. Acceptable data are now available on the embryotoxicity of 2,4,5-T in 6 mammalian species, mouse, rat, hamster, rabbit, sheep and rhesus monkey. None of these showed adverse effects at dosage of 40 mg/kg/day of maternal weight. The mouse appears to be more sensitive than the other forms studied in that it shows a low level of teratogenicity (cleft palate) at 100 mg/kg/day given throughout organogenesis, whereas hamster and rat required higher dosage to obtain comparable effects. It is likely that all species could be caused to show some embryotoxicity if 2,4,5-T dosage were raised high enough, a fact already well known for many prevalent environmental chemicals such as aspirin, caffein, nicotine and organic mercury. The dioxin contaminant TCDD also has been shown to have a low teratogenic potential at doses in excess of 0.001 mg/kg, but this dosage level is virtually impossible with currently produced 2,4,5-T. No evidence has been found of significant potentiative interaction between 2,4,5-T and TCDD. No evidence has been found of adverse effects on human reproduction in three separate locations, namely Vietnam; Globe, Arizona; and Sweden, where pregnant women have allegedly been exposed to high levels of 2,4,5-T. On the basis of these observations, it is concluded that, as presently produced and as applied according to regulations in force prior to April 1970, 2,4,5-T represents no hazard to human reproduction. �-66RECOMMENDATIONS The Advisory Committee on 2,4,5-T after careful consideration of available information on potential hazards to man, -particularly as regards reproductive functions, of continued, regulated use of 2,4,5-T, recommends the following: 1. That registration for use of 2,4,5-trichlorophenoxyacetic acid and its esters be restored to the status existing prior to April 1970, with the following 2. exceptions. That certain specific limitations and qualifications be added to the previously existing registration, as follows: a. A permissible residue or not more than 0.1 ppm of 2,4,5-T on the edible parts of food products and in potable water for human consumption be accepted. It is recognized that very few foods tested to date have contained this level of residue, but it is probable that some of the reports of no residue in the past were due to limited sensitivity of the analytical method. In view of recent and future advances in methodology, which tend to make zero residues of anything increasingly unlikely, a more realistic policy would be the setting of safe tolerance limits at this time. b. A limit of 0.5 ppm of contamination with 2,3,7,8- tetrachlorodibenzo-p-dioxin be set for existing inventories of 2,4,5-T, except as specified in item c. below, and a limit of 0.1 ppm of contamination with this dioxin be established in all future production of 2,4,5-T. Surveillance should be maintained by requiring that a manufacturer submit a reference sample and a certified analysis of each future production lot to the Environmental Protection Agency. �•67- c. All formulations to be used around the home and in recreational areas as of present date should be limited to 0.1 ppm of the dioxin, TCDD, and also should bear a conspicuous warning, e.g., "This compound may be dangerous to pregnant women and animals and its use must be such as to reduce the possibility of exposure to an absolute minimum". 3. That existing deficiencies in information relative to possible accumulation in the soil and possible magnification in the food chain of the dioxin TCDD be rectified by specific research directed to this end, with these questions to be subjected to scientific review within three years of the present date and yearly thereafter until these questions are resolved. 4. That additional post-registration monitoring for adverse effects of agricultural chemicals be established, to include both surveillance for such effects in man and domestic and wild animals, as well as consideration of the applicability of new methodology that may be evolved for specialized testing, e.g., for carcinogenesis, mutagenesis or teratogenesis. Date: May 7, 1971 Respectfully / submitted, James G. Wilson, Ph.D. �-68- Objections to and Modification of the Final Report and Recommendations of the 2,^,5-T Advisory Committee The report by the 2,U,5-T Advisory Committee is basically an accurate statement of the present state of information; however, it falls short of being completely fair in its evaluation of the evidence on which conclusions are based. It is true that considerable uncertainty exists about the tera- togenic potential of 2,^,5-T (and of its impurities) especially for the small doses at which man may make effective contact with this suostance. On the other hand, the report is overoptimistic in assessing the implications of data so that it may well underestimate what dangers may lurk in the unrestricted use of 2,H,5-T. Particularly: The data do not necessarily justify a conclusion that there is a level at which TCDD is not teratogenic. There is an unjustified certainty that Vietnam birth records do not show teratogenic effects. The report fails to consider the consequences of the (admitted) uncertainty about the fate of TCDD in the food chain and in tissue. The report does not weigh risk vs. benefits, as it was charged to do. The report presumes to lecture the scientific community on the wisdom of instituting a "permissible residue" of substances thought to be teratogenic. The report is overoptimistic in believing that recommendations for needed research will be followed by industry or by public agencies once a decision has been rendered to restore 2,lt,5-T to unrestricted use. �-69We can only conclude that the Surgeon General was justified in feeling that a prudent course of action must be based on the decision that exposure to this herbicide may present an imminent hazard to women of childbearing age. Hence, we can only recommend that the registration of 2,l|,5-T be suspended and/or cancelled for use around the home, recreation areas, and similar sites and on all crops intended for human consumption. However, the use of 2,U,5-T may be permitted under certain conditions for uses in forestration and rights-of-way providing: 1. That the limit be set of 0.1 ppm of contamination with 2,3,7,8tetrachlorodibenzo-p-dioxin for all future production of 2,lj,5-T. (However, the use of present inventories may be permitted until used up providing the amount of contaminant in them does not exceed .5 ppm of the dioxin TCDD.) 2. That 2,^,5-T be applied no more often than once a year at any one site. 3. That 2,lj,5-T be applied with proper caution so that it will not contaminate other areas where it may come into human contact. We also recommend that this action be reviewed again when the existing deficiencies in information relative to possible accumulation in the soil and possible magnification in the food chain of the dioxin TCDD have been rectified by specific research directed toward that end. It is always difficult to make decisions in the face of uncertainty. insufficient data makes the work of the committee very difficult. The The fact that ours is the view of the minority ought to strengthen the impression that the committee labored honestly and conscientiously to deduce the best recommendations from a confused aggregate of observations. c- /) sv us-~ 5/5/71 l^s i—? i Theodor D. Sterling �-70Additional Comments It Is- Hot _Quite Certain .at What Dpje TCDD Has Mo Effect The report gives the impression that 2,^,5-T shows a teratogerxic effect uniformly at high doses only. One reason for that impression is that most investigations concentrate on doses of 100 mg/kg or more so that data are lacking to a large extent on how much of teratogenic effects could show up at smaller doses. Yet, there are a number of studies that do show definite effects for doses of less than 100 mg/kg of weight.* Also, the dose effect of 2,H,5-T depends largely on its impurities, especially on TCDD. The experiments which provide the basic animal data and the analysis of these data unfortunately were not done with the sophistication necessary to throw light on the effect of 2,H,5-T and TCDD at very low doses. Many of the reports presented no more than tables of group means, and some even presented pages and pages of undigested numbers on individual observations. It is difficult to draw any final and firm conclusion from data such as these. Nevertheless, there are sufficient instances where teratogenic * For example, on rabbits increased resorption and diminished fetal weight reported by Emerson, J. L., Thompson, D. J,, Gerbig, C. G., and Robinson, V. B.: Teratogenic Study of 2,^,5-Trichlorpphenpxyacetie Acid in the Rabbit, The Dow Chemical Co., Zionsville, Indiana. A number of instances are cited by Epstein, S. S., of the Children's Cancer Research Foundation, Inc. and Harvard Medical School, Boston, Mass., lt/lV70, Subject: Teratogenic effects of 2,^,5-T formulations. Another example is the study on hamsters by Courtney, K. D. , Moore, J. A., Gaylor, D. W., Hogen, M, D., Falk, H. L.: Summary Teratogen Study NIEHS. �-71properties have been observed at lower doses than 100 rag/kg so that the question whether there is a zero effect for some dose is not easily answered. A case in point is the data included in this report by the chairman of the committee. The experiment provides for doses of 100, 200, and ^00 mg/kg but only tests at one single low dose, that of 20 mg/kg, although the effect of a dose at this low level is of utmost importance. Also, there are no control animals. Cumulative experience with untreated controls over the past four years and cumulative vehicle treated controls (per gavage) over the past four years are used as controls. Given oxir knowledge of variation in experiments, it is difficult to understand why this important experiment was performed without a concurrent control. Yet, per cent dead or resorbed fetuses show a trend toward lower dose which is still detectable at 20 mg/kg. (Whether or not we think of this effect as large or small will depend on whether or not we are willing to accept Control 1. or Control 2. of that Study.) Taking these factors into consideration, it becomes difficult to see how the report can conclude that "doses of low-dioxin-content 2,H,5-T and of TCDD below the level producing maternal toxicity were without significant effect on prenatal development producing little or no embryo-toxicity in rats, rabbits, hamsters, sheep, and rhesus monkeys" (page 50, Final Draft). If anything, the conclusion ought to have read that, despite the small amount of data present, some of it does point to teratogenicity at lower doses. The Uncertainty About the Vietnam Human Data Does Mot Mean These Data Show No Effects pf___2,U',5-T on Stillbirth and Malformation 2,^,5-T was used extensively in Vietnam for defoliation. Unfortunately, birth records show a confused and confusing picture of what happened to malformation in Vietnam during the years in which defoliation efforts increased �-72- and how stillbirth rates compare between regions that are heavily defoliated and regions that are not. The Army survey notes that during 1966 to 1969 there was a countrywide downward trend in the stillbirth rate. But the HAC report points out that this conclusion was heavily influenced by data from the capitol area in which 67.8 per cent of all live births surveyed occurred and which, in addition, generally experienced little or no exposure to 2,1*,5-T. Deducting the capitol area data and considering only that from other parts of the country, reverses the trend and results in lower stillbirth and malformation rates for 1960-65 (years of no or light spray) than for 1966-69 (years of heavy spraying). Also, HAC found that the 1968-69 stillbirth rates recorded at Tay Ninh City Provincial Hospital, a hospital which was in a region heavily defoliated and through which rivers draining areas of defoliation run, had a recorded stillbirth rate of 68.5, which they believe to be higher than that found anywhere else. The 2,U,5-T Advisory Committee report goes into the unreliability of all the human data that comes from Vietnam in great detail. It is true that the instances cited might easily have created a spurious impression of an increased stillbirth rate for the defoliated regions or periods. the opposite might be just as true. However, Factors could just as easily have worked to hide a large stillbirth rate than to spuriously create one. If we already speculate, we might just as easily speculate the other way. Thus, the best we can say is that these data do not definitely show an effect of defoliation. However, they do show an effect which has to be explained away. It is up to the committee to register our doubts, but it is unseemly to spend page after page denying the reality of the Vietnam observation in the face of the careful report by a select committee of the American Association for the Advancement of Science. �_73~ The Report Fails to Consider the Uncertainty About the Fate of TGDP It is not possible to produce 2,H,5-T without impurities, especially the dioxin TCDD. These impurities have been shown to be toxic and teratogenic in the extreme. (In fact, the recommendations of the report to restrict the permissible level of TCDD in 2,lj,5-T and to monitor this restriction is in recognition of its hazard.) While 2,if,5-T is quickly eliminated from the soil by biological and other actions, the same cannot be said of TCDD. The report notes that TCDD might accumulate in the soil from one year to the next. There is also evidence that a small amount of the soil's TCDD is absorbed by plants. But there is no information on the concentration of TCDD in the food chain at this level, nor is there information as to what extent TCDD may be stored by animal tissues. Again, the report speculates that TCDD is not stored to a significant extent because it is not easily soluble in oils. However, may it not be stored by other tissues besides fat, and, in fact, may it not be stored more by animal fatty cells than in oils? (There is some evidence that TCDD, when digested, finds its way to every tissue in the body.) There is no question but that some doubts exist in the minds of the committee on this point. The recommendations acknowledge these doubts and ask that the deficiency in information relative to possible accumulation in the soil and possible magnification in the food chain of the dioxin TCDD be rectified by specific research directed to this end, with this question to be subjected to scientific review within three years of the present date and yearly thereafter until these questions are resolved (Section 3 of Final Draft Recommendations). �-74We find it difficult to go along with this reasoning. After recent experience with DDT and with mercury, it would be reckless to leave such questions in abeyance while approving the unrestricted use of 2,U,5-T. Nothing would be lost by waiting two or three years while this question can be settled. On the other hand, a great deal of damage may be created if the committee restores 2,H,5-T to its normal use while hoping that further research will justify our confidence in having made a correct guess. Moreover, the restriction of 2,^,5-T from regular use would work as a powerful motive, spurring industry and concerned government agencies to seek to settle this important question by initiating appropriate studies. Yet, at the same time, the use of 2,lf,5-T could be maintained in all those instances where contamination of food or people would be unlikely. Considering the Risk/Benefit Equation A curious situation emerges in evaluating the benefit of 2,^,5-T. It is apparently of major value in its nonfarm uses, especially for forestry and road clearance, where it can be applied sparingly and with a great deal of expertise. Also, in forestration 2,U,5-T may be reapplied only every few years. The other uses in or near food crops and around the home represent less frequent areas of application but may expose very large numbers of individuals to 2,^,5-T, to its impurities, and to its residues. Thus, it is not true that all uses of 2,lt,5-T are equivalently beneficial. Many of the nonfarm uses of 2,1*,5~T clearly have national benefit. On the other hand, the use of 2,H,5-T for the growing of crops, especially rice, is basically of benefit to a few farm industries because it increases the yield per acre of cultivated ground. Since there are available many acres that are not now cultivated, there does not appear to be a national �-75urgency to support that limited use of 2,U,5-T until important questions about buildup of TCDD have been settled. Similarly, the use of 2,U,5-T for grazing land is of relatively small benefit for a small number of people. Finally, although the use of 2,lj,5-T around the house may be thought of as of national importance, adequate alternate means for home gardening do exist, so that it may be best to avoid for the moment the possible risk of direct contact with man. Theodor D. Sterling 5/5/71 �-76- LIST OF PERSONS CONFERRING WITH THE COMMITTEE Mr. Harold G. Alford, PRO Dr. Theodore Byerly, USDA Mr. Robert L. Caswell, PRB Dr. Cipriano Cueto, PRD Dr. K. D. Courtney, NIEIIS Mr. Charles Dunn, Hercules Incorporated Mr. Elaine Fielding, OGC Dr. John Frawley, Hercules Incorporated Dr. Dan Gaylor, NIEHS Dr. R. E. Johnson, EPA, OP Mrs. Joan Katz, Attorney representing Harrison Wellford, et al. Dr. Albert Kolbye, FDA Mr. John Kuniholm, Hercules Incorporated Mr. D. D. McCollister, Dow Chemical Company Dr. I. A. Mitchell, Office of Surgeon General Dr. J. A. Moore, NIEHS Dr. David P. Rail, National Cancer Institute Mr. George Robertson, OGC Dr. Virgil Robinson, Dow Chemical Company Mr. V. K. Rowe, Dow Chemical Company Dr. Jessie Steinfeld, Surgeon General Mr. Paul Whiteaker, PRD � 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. 046 Folder The folder containing the original item. 1152 Series The series number of the original item. Series III 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 Wilson, James G. Description An account of the resource <strong>Corporate Author: </strong>Advisory Committee on 2,4,5-T Date A point or period of time associated with an event in the lifecycle of the resource May 7 1971 Title A name given to the resource Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency Subject The topic of the resource dioxin herbicide toxicology animal testing biodegradation ao_seriesIII https://www.nal.usda.gov/exhibits/speccoll/files/original/dd4280761c30ba23ac1c8e04d3d1a0a7.pdf 72b4a89c20932245745139584e7e6195 PDF Text Text 00187 Young, Alvin L. USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas ReOOrt/ArtJCiB Tlth Herbicide Orange Site Treatment and Environmental Monitoring: Summary Report and Recommendations for Naval Construction Battalion Center, Gulfport, MS 1979 November Color n 46 Friday, January 05, 2001 Page 187 of 194 �Report OiHL HERBICIDE ORANGE SITE TREATMENT AND ENVIRONMENTAL MONITORING SUMMARY REPORT AND RECOMMENDATIONS FOR NAVAL CONSTRUCTION BATTALION CENTER GULFPORT MISSISSIPPI November 1979 Approved for public release; distribution unlimited USAF Occupational and Environmental Health Laboratory Aerospace Medical Division (AFSC) Brooks Air Force Base, Texas 78235 �S11H TREATWHf MID ENVIRQHiffililAL MQHITORIMG AND FOR NAVAL CONSTRUCTION BAOTM.ION November 1979 . Prepared for AIR FORCE IiOGISTICS COMMAND WB OH ��- .. JECURlTV Ct A»riCATiON OF THi« SEAS INSTRUCTIONS BEFORE COMPt-ETlNG FORM REPORT DOCUMENTATION PAGE 2, OOVT ACCfSflON NO. «fHT*S CATALW tttttllf• OIHL-fR-79-169 S. TYPE OF REPORT * CEHlOO COVEHIO *. TITLE Herbicide Orange Site Treatment and Environmental final Monitoring* Sawary leport and !eeoiui»ndatlons for Naval Construction Battalion Center, i, PBMPemuita ens. RSPDMT Gulfport MS I CONIHACT 9» HAIT . Alvin L. Young, Major, USAF Charlea 1. fsalkan, Lieutenant Colonel, USA?,-VC Williaa J. Gainsay, Major, QSAF, BSC onaAiiiAfiON MMII *«§ USAP Occupational and Environmental Health laboratory iroofes Mr Force Base, Itoas 78235 ^ ^ i, g^/i,jimraMis' I, ItiPQllT BAT• I H, COttT«Ot.lilN« OF^tet NAMI ANO ABPflltll USAF Occupational and Environmental Health Laboratory Brooks Mr Force Base, Texas 78235 Moveaber 197! 36 Ti, »OllrTOlHS8 AOSNCY MAMI ft AODMBSSfJl dilftt^i Irom Ooatrenini Olllef) !•• MCUWTT CUASI, f*f Unclassified It. ITATIMtNT f*l » for public release *, distribution unlimited •>, DtlTNilUTION STATSM8NT (el tfe* *fe«if*«t fnitrti in Mae* M, I lUPPLEMIMTAIiy NOTIt , I. KEY WONBI CCenltniM MI tmmtm «)<*• // n»s««»«ry «nd id«n(//y by M«eft nu»b«o aquatic studies bioaeovmulation biod»fradation of herbicides biodegradation of TCDD chlorinated phenols ecological effects 2,4-dicnlorophenoxyacetic acid {2,4-D) environmental aonitoring herbicides Herbicide Orange dloxin Orange phenos^ herbicides PACER HO 0, ABfrftACT fCenthMM on «»««• *!4t It n*a***«y »nd ia»nlHy kr Moo* mambtr) Snviroiu»ntal surveys of the soils, plants and the aquatic system in and around a 12-acre Herbicide Orange storage area at Gulfport MS were conducted from 1970 through 1979. The major objectives of the surveys were to (1) determine the magnitude of Herbicide Orange contamination on the storage area| (2) determine the fate of the phenoxy herbicides 2,4-D and 2,4,5-T, their phenolic degradation products and fCDD in soils of the storage area?{3) monitor movements of residues from the storage area into adjacent water, sediments and biological organisms! and (4) recommend managerial techniques for niniaizing the impact EDITION Of 1 NOV ft IS OMOLETE Unclassified CtAStlfICATtOM Or THIS PAOC �HCURtTy CI.»SSIFIC*"nOH OF THJkf AqgftHJMQ D»t* soil microbial studies TCDD 2,3»7,8HMtr«ehlorodibenso-p-dloxin (TCDD) 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) 20. of the herMcldes and TCDD residues on the ecology and human populations adjacent or near the storage area. High levels"of TCDD (e.g., 100-200 parts per billion [ppb]) were associated with spill sites on the herbicide storage area. Sediment samples from the storage area contained 2.7 to 3.6 ppb TCDD and biological organisms closely associated with the sediment contained 0.14 to 7.2 ppb TCDD. Water staples collected in the same area were negative £or TCDD at a detection level of 0,02 ppb. Two of five off-base samples were positive for TCDD (ft crayfish and a sediment sample both contained 0.02 ppb TCDD). The primary recommendation is that the 12-acre Herbicide Orange storage area be left undisturbed permitting the continuation of "natural" degradation of the herbicides and TCDD. It is recommended that the area be restricted and that efforts be immediately undertaken to minimize future erosion of contaminated soil into the ditches. The prevention of soil and silt movement from the area may be accomplished by stabilizing the ditch banks, constructing silt catchments within the ditches and constructing a silt retaining pond prior to the stream leaving the NCBC. Unclassified SECURITY CLASSIFICATION OF THIS PAQEfffftwi Dm* �PURPOSE The report was prepared to present senior Mr Force leaders the latest available data In the continuing environmental monitoring studies of a 12-acre storage area on the Naval Construction Battalion Center (NCBC), Gulfport MS, ftie area had been used for the long-term storage of approximately 8 0 0 0 gallons of Herbicide Orange from mid-1968 to 4,0 mid-1977, SASIC HISTORY . Since 1970,, various Air Force and contract laboratories have been conducting environmental surveys and analyses of the soils, plants, and the aquatic system in and around the Herbicide Orange storage area. As some leaking became evident and as more information became available on the toxic contaminant 2,3,7,8-tetrachlorodibenzo'~p-dioitin (TCDD) contained in the herbicide, more extensive monitoring programs were conducted, fhe entire inventory was redrummed in 1972 and checked for leaks continuously thereafter. In the summer of 1977, the herbicide was transferred to a specially equipped ship and destroyed by at-sea incineration dwinf Project PACER HO. fhe Air Force Plan and the 1PA permits for the disposal of the herbicide committed the Air Force to a follow-on storage site reclamation and environmental monitoring program, fhe major objectives of this program were to (1) determine the magnitude of Herbicide Orange contamination in the storage area; *Updated to include data received 3 Dec 1979 subsequent to report preparation, �(2) determine the soil persistence of the pheonxy herbicides 2f4-dichJ.orophenoxyacetic acid (2,4-D) and 2,4,5-T, their phenolic degradation products and TCDD in soils of the storage area; (3) monitor for potential movement of residues from the storage area into adjacent water, sediments and biological organisms; and (4) recommend managerial techniques for minimizing any impact of the herbicides and TCDD residues on the ecology and human populations adjacent or near the storage area. STORAGE SITE CWTAMIHATIOti AND FATE The monitoring approach used to determine storage site contamination consisted of analyzing soil samples selected from 42 different sites within the storage area. Sampling points were selected in groups depending upon whether a spill of the herbicide had occurred in that area or not. Previous studies had shown that residue did not appreciably move within the acid soil or significantly penetrate the impervious concrete-stabilized hardpan located approximately six inches below the soil surface. Soil samples were also analyzed for microorganisms. The results indicated that approximately 15% of the 12-acre site is significantly contaminated with Herbicide Orange and TCDD. Levels of 2,4-D and 2,4,5-1" in the samples, which were greater than 100,000 parts per million (ppu) in July 1977, have decreased to one-third that level in IS months. Data from spill sites monitored for this same time period also suggested that TCDD levels are decreasing but at a slower rate. The soil penetration of the herbicides was low while penetration of TCDD was negligible. Sterilization of the soil did not occur; rather, certain microflora proliferated under high levels of herbicides. �RESIDUE MOTEMBI1 IMTO ADJACENT AlffiAS To monitor for potential movement of residue from the storage area, soil and biological samples were collected from the drainage ditch directly adjacent to the site. A Novenbar 1978 analysis of this nearby on-base drainage ditch found positive TCDD residues [o.14-3.6 parts per billion (ppb)]. She TCDD movement was presumably caused through soil erosion from the annual (Jan-June) heavy rain season {approximately 60 in). Drainage ditches carry heavy rain from the storage site and other parts of the bas« into Ixsng Beach Canal 11» approximately 9,000 feet from the site. The canal runs from the city of Long Beach through the base carrying municipal surface drainage, and until July 1978, carried treated sewage materials. The canal eventually runs into Turkey Creek approximately 12,000 feet from the storage site. Due to the November 1978 findings, further samples were collected at varying distances from the site in January, February, and June 1979. Following extensive and difficult analyses in contract laboratories, the results were received in September, November, and December 1979, The results confirmed the November 1978 data and indicated slightly higher levels (sediment levels of 1.7-3.6 ppb and biological levels of 0.14-7.2 ppb). Water samples collected in the same area were negative for TCDD at a detection level of 0.02 ppb. TCDD appears to move only as a part of soil sediment. Sediment and biological sauries taken downstream at 3,000, 7,000, 9,000 and 12,000-feet from the site indicated that some TCDD residue was now present but at very low levels. A crayfish collected at 9,000 feet and numerous fish collected at 12,000 feet were analyzed with .032 ppb the highest level detected. This figure of .032 ppb is three times lower than the Pood and Drug iii �Administration suggested maximum permissible level of 0.1 ppb. With present "state-of-the-art" detection limits, readings as low as these in biological samples have only been considered reliable in recent months. RECOMMENDATIONS To control the now verifiable but very low levels of residue, the report recommends the following actions: - Stabilize drainage ditch banks to prevent water erosion during heavy seasonal rainstorms. - Construct siltation traps in the drainage system allowing for greater silt catchment prior to drainage water leaving the base. - Leave the storage area in its present undisturbed state and continue to limit access so that the "natural" degradation of the herbicide and its TCDD continue to occur. - Allow the continued growth of native vegetation in the contaminated storage area and drainage ditches since this plant community inhibits water erosion. - Continue sampling to ensure that preventive actions do control contamination. - Develop follow-on reserach to determine possible methods for returning the storage area to full and beneficial use. iv �PREFACE This technical report represents the culmination of a two-fear environmental monitoring program of an area previously used for the long-term storage of Herbicide Orange at the Naval Construction Battalion Center. The study was conducted by personnel of the United States Mr Force Occupational and Environmental Health Laboratory, Brooks Mr Force Base, Texas and the United States Mr Force Academy, Department of Chemistry and Biological Science, USAF Academy, Colorado. Funds for this program were provided by Air Force Logistics Command through the San totonio Mr Logistics Center, Directorate of Fuels, Kelly Air Force Base, Texas, fhe report was prepared for the Mr Force Logistics Conaand, Wright-Patterson &FB, Ohio. �Acknowledgements Analyses of herbicides, phenols, and soil TCDD were perforaed by Dr B. Mason Hughes, Mr W.H. McClennen, Mr L.H. Wojcik and Mr F,D. Hilematn, Flaranability Research Center, the University of Utah, Salt lake City Uf 84108. The analyses of ethers and isooctyl esters of trichlorophenol and herbicides were conducted by Or E.L. Arnold, formerly of the Clinical Sciences Division, USAF School of Aerospace Medicine, Brooks AFB TX 78235. High resolution GG-MS analysis of TCDD in selected biological and sediment samples was performed by Dr M.L. Gross, Mass Spectrometry Laboratory, University of Nebraska, Lincoln NE 68588. The assistance of Mr Tom Murphy, Epidemiology Division, USAF School of Aerospace Medicine, in statistically analyzing herbicide data is gratefully acknowledged, The assistance of Mrs Joyce Kidd, Secretary to the Vice Commander, USAF Occupational and Environmental Health Laboratory, in typing and editing this report is gratefully acknowledged. WILWMI E. MABSOM, Colonel, USAF, BSC Commander VI �IMTBOOUCTIOII During the sooner'of 1977 the United States Air force (0SAF) disposed of 2.22 million gallons of Herbicide Orange by high temperature incineration at sea. This operation, Project PACER HO, was accomplished under the very stringent criteria set forth in an tF.S. Environmental Protection Agency (IPA) ocean dumping permit. Among the numerous conditions of thi« UPA-approved disposal operation was the requirement for the USAF to conduct extensive environmental and occupational monitoring of the land-transfer/loading operations, shipboard incineration operations and subsequent storage site reclamation and environmental monitoring. Details of the proposed site monitoring programs were documented in April 1977 by the Air Force Logistics Command (AFLC) in a programming plan for the disposal of Herbicide Orange ( ) In this plan, AFLC proposed that 1. soil samples from the storage sites at both the Naval Construction Battalion Center (NCBC), Gulfport MS, and Johnston Island (JI), Pacific Ocean, be Qollnated and analyzed for Herbicide Orange after the completion of transfer operations, These analyses were to aid in the establishment of a •chadule for future monitoring. The site monitoring program would be flexible to requirements generated by construction of any facility on the storage site and would be concluded upon mutual agreement of all agencies involved. In July 1977, following the completion of the PACER HO dedrunming and subsequent site clean-up operations at NCBC, the USAF Occupational and Environmental Health Laboratory (USAF OEHL) initiated an extensive site monitoring program, fhe objectives of this program were: 1. To determine the magnitude of Herbicide Orange contamination on th« storage site. •. �2. To determine the soil persistence of the two phenoxy herbicides contained in Herbicide Orange and a. dioxin contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). 3. To monitor for any movement of residues from the site into adjacent water, sediments and biological organisms. 4. To recommend techniques for managing the storage area with the ultimate goal of returning the area to full beneficial unrestricted use. HISTORICAL BACKGROUND (GENERAL) In April 1970, the Secretaries of Agriculture; Health, Education and Welfare; and the Interior, jointly announced the suspension of certain uses of the herbicide 2,4,5-trichlorophenoxyacetic acid ( , , - ) These 2457. suspensions resulted from published studies indicating that 2,4,5-T was a teratogen. Subsequent studies revealed that the teratogenic effects had resulted from a toxic contaminant in the 2,4,5-T, identified as 2,3,7,8-tetrachlorodibenzo-p-diojcin (TCDD). Subsequently, the Department of Defense suspended the use of Herbicide Orange [a mixture of 2,4,5-T and 2,4-diehlorophenoxyacetic acid C2,4-D)] in South Vietnam. At the time of the suspension, the Air Force had an inventory of 1.37 million gallons of Herbicide Orange in South Vietnam and 0.85 million gallons at the Haval Construction Battalion Center, Gulfport MS. In September 1971, the Department of Defense directed that the Herbicide Orange in South Vietnam be returned to the United States and that the entire 2.22 million gallons be disposed of in an environmentally safe and efficient manner. �The 1.37 million gallons were moved from South Vietnam to Johnston Island, Pacific Ocean, for storage in April 1972,' HISTORICAL BACKGROUND (NCBC) Craig (2), in a historical review of herbicides for Southeast Asia noted that the storage of Herbicide Orange became an item of significant importance with the temporary suspension placed on all uses of Herbicide Orange by the Assistant Secretary of Defense on 15 April 1970. Prior to 1970, shipments of herbicides into and out of the Mobile Outport and the Naval Construction Battalion Center were handled in a routine manner. As the herbicide inventory began to accumulate in Southeast Asia, the San Antonio Air Logistics Center, Directorate of Fuels (SA ALC/SF), Kelly AFB TX, discontinued shipments from the port of embarkation to Southeast Asia in 1963 to avoid exposing large quantities of herbicides * to possible damage by enemy action. The SA ALC then had to determine disposition of the product at the port and that scheduled for delivery. Bather than return the product to the manufacturer and suspend delivery to the port, SA ALC decided to arrange for the product to be temporarily placed in storage. Since the Mobile Outport, Mobile AL, was routinely used as the port of embarkation for herbicides, this was the logical place for the temporary storage. It was anticipated at that time that the storage period would be about six months. Herbicides were sent to the Mobile Detachment for storage between April and June 1968, and were removed from storage between September and December 1968. Except for �one shipment to Southeast Asia during September 1968, herbicides removed from this storage site were used only to fill equipment test requirements at Iflin AFB PL. On 26 June 1968 an Interservice Support Agreement was made by and between SA ALC and NCBC, to provide services related to receiving and storing approximately 50,000 18-gauge, 55-gallon drums of herbicide. The agreement was effective for the two-year period 1 July 1968 - 1 July 1970. It was to be reviewed annually by both parties. Input of herbicides to Gulfport began in July 1968. Additional Interservice Support Agreements were made in 1970 and 1972. Storage was considered a better alternative than the return to the manufacturer where storage charges would have been more expensive, lite NCBC agreed to receive and store the drums of herbicide and remove from storage quantities of drums as designated by SA ALC while SA ALC agreed to provide personnel in support of this operation. This was modified in July 1968 to reimburse NCBC for material and supervisory personnel salaries. The Gulfport outside storage area was about two miles from the docks, with convenient access to the railroads. It was fenced and isolated from public traffic. The NCBC provided surveillance personnel as well as a controlled access. It was planned and set up for long-term storage. To provide good drainage, 2 x 6-inch dunnage (creosoted lumber) was laid on a hard surface and drums, positioned horizontally with the bung closure pointing outward, were stacked in double rows, three high, in pyramidal fashion. The number of drums in each single row, bottom to top, was 55, 54, and 53. To allow inspection of the bungs, there was an 18-inch walking space between each double row. �HOC urns the only Continental Onited States (COOTS) storage facility used daring the last half of FT69 and through Pf70. The Mobile Outport intransit storage facility was not used after Deeeatoer 1968 when the last drums of herbicide were moved to NCBC. At the end of FY70 there were 833,855 gallons of Herbicide Orange in storage at NCBC. Except for a small quantity stored at iglin AFB FL for test purposes, Gulfport was the CONUS storage point. & few damaged drums were received at NCBC with leaks around the bung closures because the seals had vibrated loose. In such cases the producer was notified to supply new bung closures. NCBC personnel took the corrective action. Usually the leaks could be stopped by removing the cover and tightening the bung or replacing the bung gasket. When damaged leaking drums were spotted while in storage, they were redrumtad by the people on duty. It was discovered that a herbicide moistened area usually appeared on the drum two or three weeks before noticeable loss occurred, and the contents could be saved by transferring it to a new drum when the damp area was noted. In May 1971, during an inspection of the inventory, it was noted that deterioration of some of the drums had required HCBC personnel to redrum the product. As drums were removed from the stacks, indications of additional leaking drums became apparent. Previously, leaking had been attributed to breakdown of the bung seals used in the drum closures or an occasional seam leak. Now there were indications of leaks starting in the drum surfaces. During 1972, military personnel moved, inspected, and redrummed as required, the entire inventory of approximately 15,400 drums. Thereafter, an intensive drum surveillance program was initiated �in which all drums were routinely inspected and moved or redrummed as required. The drum surveillance program was continued until May 1977 when Project PACER HO began. The observations in 1971 and 1972 that drums were deteriorating prompted AFLC to task the USAF Environmental Health Laboratory (EHL/K) , Kelly MB TX and the Department of Chemistry and Biological Sciences (USAF/DFCBS) , USAFA CO, to undertake a cursory chemical and biological monitoring program of the storage site. & review of these efforts is provided in a subsequent section of this report. DESCRIPTION OF JHEBBICIEB Pour military herbicides were stored for various lengths of time at NCBC. These herbicides were code-named Herbicides Orange, Orange II, Blue and White. Herbicides Blue and White were intermittently stored at NCBC during 1968 and 1969. However, all stores of these materials were shipped to South Vietnam. Since these two herbicides (Blue and White) were only briefly stored at NCBC, site monitoring programs did not include these materials. The herbicide inventory that underwent long-term storage was comprised of primarily Herbicide Orange (approximately 13,855 drums) and a relatively small quantity of Orange II (1,545 drums) . Young, et al. (8) have described these herbicides. 1. Herbicide Orange Orange was a reddish-brown to tan colored liquid, soluble in die se 1 fuel and organic solvents, but insoluble in water. One gallon or Orange theoretically contained 4.21 pounds (Ib) of the active ingredient of 2,4-0 and 4.41 Ib of the active ingredient of 2,4,5-T. Orange was formulated to contain a 50s 50 mixture of the n-butyl esters of 2,4MJ and 2,4,5-T. The percentages of the formulation typically weres �n-twityl 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 49.49 0,13 48.75 1.00 in*rt ingredients {e.g., butyl 0.63 alcohol and ester moieties) 2. Herbicide Orange II Orange II was a formulation similar to Orange with the only difference being the substitution of the iaooctyl eater of 2,4,5-T for the n-butyl e«ter of 2,4,5-T. The physical, chemical, and toxicological properties of Orange II were similar to those of Orange. Orange IX was produced solely by one chemical company. A detailed analyses of the inventory of Herbicide Orange and Orange II stored at NCBC was prepared in 1975 by Hughes, et al. (4) and Fee, et al (3) A summary of manufacturers and TCDD contents is presented in Table 1. SUMMARY Of SAKL? EJiViaOHMENTAL MONITORING PROGRAMS As early as 1970 the Air Force was expressing its concern about the possible adverse environmental impact of the storage of Herbicide Orange at NCBC, Gulfport MS. Environmental scientists from Eglin AFB visited the storage site at the request of SA ALC/SP and conducted an environmental survey of the plant and aquatic animal community in and around the herbicide storage cite. No significant environmental problems were noted at that time. In 1972, members of the OSAP Environmental Health Laboratory, Kelly AFB TX (EHL/K), conducted an environmental survey of the storage area and also found no significant environmental problems. �TJBL! 1. Identification Data on Herbicide Orange Stocks Stored at the Naval Construction Battalion Center, Gulfport MSa Manufacturer Analysis Total Number Transportation b Seepienee of Drums *TCDDC Control No. (TCN) Mo. with Same TCN (ppm) Hercules Co 9 6 8156 0 0 44 01 Hercules Co 9464 8192 001 14 Diamond Co PY9461 7165 0001AA 18 60 14. 2e Diamond Co PY9461 8156 001AA 11 421 8.62f Thompson Hayward Co 9463 8155 X032 8 1 500 2,152 <0.05 n&a 1,546 0.32 0.12 Dow Chemical Co 9463 8155 X052 10 6,976 Thompson Co 9463 7184 X011 3 46 HA Thompson Co 9463 8155 X012 5 808 0.17 Monsanto Co FY9463 7163 X0001XX 4 563 NA Monsanto Co PY9463 8183 X002XX 6 2,185 15,257 a SOURCEs 7.62 Pee, et al. (3). Each separate purchase of herbicide was designated by a separate TCN G Tetrachlorodibenzo-p-dioxin (TCDD) content. Results reported in this column are the average of six samples collected from six different barrels of Herbicide Orange having the same TCN. d Not Analysed. ^Average value of five samples: 12, 17, 12, 15, 15. Other sample value was 0 0 with recheeJcs. .7 ^Average value of four samples: 8.0, 8.1, 8.7, and 9.7. Other two samples each averaged <0.05 with rechecks. *0n the bajis of 280 samples of Herbicide Orange taken from the Gulf port inventory, the weighted mean concentration of TCDD was 2.06 ppm. �In July 1974, members from the OSAF Academy Department of Chemistry and Biological Sciences conducted an extensive survey and ecological assessment of the herbicide storage area and collected soil, water, and biological samples. There was considerable evidence of herbicide contamination within th« storage area itself (i.e., visual evidence of leaks and •pill* on the soil)i however, there was no evidence that any of the material had been carried from the storage area by the surface drainage system. Soil samples collected between the stored drums, on the banks of the drainage system and silt deposits at various points in the drainage ditches had no detectable levels of herbicide at the 1 part per million (ppm) level, One soil sample was taken only six feet from the drums where prior leakage had been detected as evidenced by discoloration of the soil surface. Hater samples from the drainage ditches had no detectable levels of herbicide at the 50 parts per billion (ppb) level. One of the water samples did, however, contain hydrocarbon residues apparently from washing operations in the area. The presence o£ the fuel in the water gave the stream an oily appearance which may have lead some people to conclude that a herbicide residue was present. The biologicals (frogs, tadpoles, minnows) that were collected were not analyzed because there was no evidence that the aquatic drainage system was contaminated at that time. Upon gross examination no abnormalities were seen in any of these aquatic specimens. A complete survey of the flora surrounding the storage area was also completed during the July 1974 visit by the USAF Academy personnel. Plant damage of a herbicidal-nature (twisting and bending of leaves and stems) was noted on two plant species as far as 85 yards west (downwind) of the drum storage site. �In December of 1974 Dow Chemical Interpretive Analytical Services reported the first known TCDD positive soil sample frent between the rows of barrels on the storage site. Two soil samples were analysed. One sample had nondetectable levels at a detection limit of 4 parts per trillion (ppt) while the second soil sample was positive for TCDD at IS ppt. During the period of August 1974 to October 1976 representatives of the EHL/K made 11 trips to the Naval Construction Battalion Center to monitor pilot plant activities, drum rinse studies and conduct environmental monitoring including the collection of water samples from the herbicide storage area drainage ditches. Water sample values for 2,4-D had a range of average mean value of 0.15 ppb to 409.4 ppb; the 2,4,5-T range of average mean values for water was 0,3 ppb to 519.4 ppb and a 1976 TCDD positive sample that had an average mean value of 7.7 ppt. Sediment samples collected from the drainage area contained 2,4-D in a range of average mean values of 0.04 ppm to 0.24 ppm; the 2,4,5-T range of average mean values for sediment was 0.04 ppm to 0.42 ppm. All sediment samples for TCDD were negative} however, the analytical laboratory could not establish a level of detection for TCDD because of interferences. In the October 1976 report it was noted that of the 26 water samples analyzed, 13 were reported as containing more than 10 ppb herbicide. However, at the base discharge sample point leading off base, there were no water samples analysed that exceeded this lower detection limit of 10 ppb. Also, of the 23 water samples that were analyzed for TCDD, there was only one that had a positive reading and that sample was collected near the storage area. TCDD. Samples collected further downstream had no detectable The detection limit in these samples was 0.01 ppb. These results indicated that although some herbicide was entering the drainage system, 10 �it was not leaving the base and most likely was being held in the bottom sediments of the drainage ditch system. Visual observations of the drainage ditch system indicated that there. were no deleterious effects being exerted on the biotic community and that fish, frogs, snakes and other normal fauna and flora seemed to flourish. Only two of the sediment samples analyzed exceeded 1 ppm herbicide. These samples were collected near- the storage area. The sediment samples collected near the base discharge point never exceeded the 1 ppa herbicide leval and no fCDB was ever detected in any of these sediment samples. However, the analytical laboratory could not establish a level of detection for TCDD because of interferences. Soil sample data in October 1976 was not sufficient to make an interpretation as to the degree of severity of the herbicide contamination of the soil. Recommendations from the October 1976 EHIi/K report weres 1. The levels of Herbicide Orange (HO) in the ambient air were not high enough to create any concern about any on- or off-base exposure. Thi« was also borne out by the biomonitoring that had been performed during the Agent Chemical Inc (ACI) operation at NCBC. If the TCDD analytical result* were viewed as upper limits, as suggested by the analytical laboratory [Wright State University {WSU)], then there was no need for concern. 2. There was no indication of any off-base discharge of TCDD in the water or sediment samples. 3. Quarterly environmental monitoring surveys should be continued. 4. There is need for a comprehensive sailing program of the soil in the HO storage area to permit a better evaluation of the degree and extent of contamination by both HO and TCDD. 11 �In January 1976, members from the USAF Academy, Department of Chemistry and Biological Sciences,conducted an extensive aquatic and soil survey of the herbicide storage area. During this survey, many soil, sediment and biological samples were collected from throughout the storage area and the surface drainage system. These samples were frozen and archived as baseline samples should the need arise to evaluate similar types of samples during or after the dedrumming operation. Selected samples frost this collection were later analyzed in 1978. Data from these samples are incorporated into the Results and Discussion Section of this report. USAF OEHL SITE MONITORING PROTOCOL Four problem areas were apparent in the design of a study: 1. Over 25 individual chemical components in Herbicide Orange had been identified [Hughes, et al. ( ) . Should or could a monitoring 4] program include all of these components? The low percentage in content of most of these components combined with their known low toxicity and/or rapid biodegradability (e.g., butanol, toluene and xylene) suggested that only the principle herbicides (acid and ester formulations of 2,4-D and 2,4,5-T), their major breakdown products (di- and trichlorophenol) and TCDD should be followed. 2. What criteria should be used to determine the number and location of sampling sites on an area of approximately 12 acres? Spills, due to handling of the drums during dedrum operations (during and prior to PACER HO) or to leakage (prior to PACE! HO), could have occurred almost anywhere on the storage area over the eight-year period. Certainly, the persistence and fate of individual herbicides, phenols or dioxin might be determined if a technique could be used to determine old spills from new spills. 12 �3. What factors associated with'the actual storage Urea at NCBC will have influenced the penetration of herbicides/TCDD into the •oil profile? This problem would certainly influence the depth of sampling that would be required, 4. In an "ideal" monitoring program, some method would be required to determine a minimum level of residue that could be considered biologically and ecologicallf acceptable, i.e., a "no significant effect" residue level. Should this no effect level be based upon soil micro- organisms, surface vegetation or some other criterion? Previous environmental studies in 1974 and 1976 by Young, 19), and Ifoung, et al. (10), showed that movement of the herbicide components of Herbicide Orange and the TCDD contaminant was low, suggesting that both lateral movement and soil penetration of the water-insoluble Herbicide Orange and TCDD would be minimal. Thus, surface sampling, e.g., the top three inches (S cm) of soil, should constitute the primary sampling depth. As noted above, the depth of routine sampling was of major concern in designing the residue monitoring program. Young, et ai. ( 0 had shown that 1} neither the herbicide components of Orange nor the TCDD had appreciably moved in the soil during biodegradation studies at Eglin AFB PL or the APLC test lange Complex, Hill AFB OT. However, these studies had involved soils treated with herbicides by using a hand sprayer and at concentrations greatly below those encountered in spills. Certainly some of the spills that had occurred at NCBC were "old" spills and the effects of time (years) on these spills was essentially unknown. Another factor in sampling depth was that the soil in the outdoor storage areas of NCBC had been treated in the 1940s with cement and compacted ( ) This treatment had created a 6-12 inch (15-30 1. en) layer of hardened stabilized soil. This "hardpan" was relatively 13 �impervious to water and presumably herbicide; however, in 1977, the hardpan was 3 to 6 inches (8-15 cm) below surface due to the addition of soil and gravel during the intervening years. This upper layer of soil was primarily sandyloam in texture. Selected sites where heavy spills had apparently occurred had also been treated with a 2 inch (5 cm) layer of oyster shells. All of these factors influenced the decision to select only one depth as the primary sampling depth which was the top three inches (8 cm). In July 1977, a preliminary sampling study was initiated. This consisted of assessing the heterogenity of the soils on the sites and the heterogenity of the herbicide concentrations. Twelve sites were selected for sampling; six were in areas of obvious spills and six in areas that showed no spill. Not only were the spills discernible by sight but also by smell. Winston and Ritty (7) had previously found that the olfactory senses can detect a butyl enter formulation of 2,4,5-T at levels of 0.4 ppb. The results of this fir»t sampling after PACER HO are shown in Table 2. Significant concentrations of herbicides, phenols and TCDD were detected in soils from spill sites. The variation in concentrations and in the portion of acids to ester* suggested that the spills were from different time periods. Accordingly, a more extensive protocol was proposed for future sampling. 197§ fROTOCQE The sites selected within the storage area for monitoring of residue were determined by whether a spill had occurred or not occurred at that specific location. The basis for determining a spill was whether a herbicide stain was discernible (heavy, light, absent) and whether a herbicide odor was detectable (strong, mild, absent). Thus, within the Storage Area numerous location* were found that had a heavy stain ajid strong odor (labeled 8/H, presumably representing a recent spill)? a light stain and 14 �2. Concentration parts per Million, of total herbicides, total phenols, and TCDD in 12 soil samples collected July 1977 from the Herbicide Orange Storage Area, Naval Construction Battalion Center, Golfport MSa Location total Herbicides (ppm) Total Phenols13 (ppm) TCDD Spill Sitesc 1 51,600 132,400 3 5 8 10 11 37,350 34,840 117,060 Mean = 95,000 78,040 42,395 87 109 166 96 303 152 (5) 90 019 .00 0.6310 »008) (.049 0.1900 0.0185 MA. 0.2371(4) 4- 0.2718 Mo Spill Sitesd 2 4 6 7 9 12 •f 12.4 0.7 0.2 0.1 0.6 0.2 0.2 0.3 + 0.2 NA NA NA MA HA NA a Analysis by the Flasmability Research Center, The university of Utah, Salt Lake City OT. Air Force Contract No. 561178C0062. Report submitted 17 May 1 7 . 99 "Total herbicides refers to concentrations of acid and all esters detected of 2,4-D and 2,4,5-f. °Total phenols refers to concentrations of dichlorophenol and trichlorophenol. %he sample consisted of a cube (3x3x3 inches) of soil removed from the center of an area designated spill or no spill. 8 HA • Mot Analyzed. ( ) refers to number of samples included in obtaining the means and standard deviation. %D • Not Detected at the detection limit specified in parenthesis. 15 �mild odor (labeled L/L, presumably representing an older spill); and no stain and no odor (labeled O/O, presumably representing an uncontaminated area). Fourteen replications of each treatment were then randomly selected to represent the storage area (thus a total of 42 permanently marked sampling locations). Twelve of these locations had been tentatively located and marked on 28 July 1977 with the remaining 30 located and marked on 17 January 1 7 with sampling being conducted on these dates, as well 98 as 6 November 1978. In collecting the soil samples, a 3-inch square was marked, 6 inches away from the site marker pin. At each sampling tine, soil was taken from a different "point of the compass" with reference to the marker pin to insure a fresh and undisturbed profile. At the designated site, a 3x3x3-inch cube of soil was removed with a ceramic spatula which was rinsed with acetone between uses to prevent carryover of residue and microorganisms. Wherever possible, sediment samples were collected from the drainage ditches in a similar manner. CHEMICAL ANALYSES Each soil sample consisted of approximately 200 grans and was placed into new glass jars ( 0 ml) appropriately labeled and transported to the 40 laboratory where they were uniformly mixed and subsampled. The subsample used for chemical analysis was immediately frozen. The remaining sample was used for microbial studies (see Microbial Analyses). All soil samples collected from NCBC in July 1977, January 1978 or November 1978 were submitted for chemical analyses to the Flantmability Research Center, University of Utah, Salt Lake City UT. Each soil sample was analyzed for the esters and acids of 2,4-D and 2,4,5-T. In addition, each sample was analyzed for diand trichlorophenols (intermediate degradation products of 2,4-D and 16 �2,4,5-7) and selected samples analyzed for TCDD. & brief description of the netted employed in the analyses has been published ( ) 5. MICROSIAL ANALYSES SubBttRf>le8 of all soils were sent to the Department of Chemistry and Biological Sciences, USAF Academy CO for microbial analyses. Ml samples were analyzed for total populations of actinomycetes, fungi and bacteria. In addition, Jcey species presumably responding to the presence of herbicides were identified. The method employed in the microbial analyses has been previously described by Young ( ) It was hoped that quantitative and 9. qualitative studies of the microorganisms from each of the treatment classes used in association with residue data would permit an establishment of a no effect level. CTSULTO AMD DISCUSSIONS, Of HERBICIDl AMP MICBQBIAI* DATA A summary of the analytical results for the 42 sites sampled in January and November 1978 is shown in Table 3. A statistically significant decrease in the levels of total herbicides and total phenols was found to occur between the two dates. There was also a downward trend in TCDD levels, but it was not statistically different {P.05), This trend'in decreasing levels of TCDD (as well as in herbicides and phenols) is even more pronounced when the July 1977 data (Table 2) are compared to the 1978 data (Table 3). Unfortunately, because of differences in site delineation between 1977 and 1978, data for spills vs no spills between the two years cannot be "paired" and statistically analyzed. Nevertheless, the data suggest that TCDD may be degrading within the time period of this study (18 months). Data on the soil penetration of the herbicides, phenols, and TCDD are shown in Table 4. This site (site 17) was a site where a herbicide spill 17 �TABLE 3. Mean concentrations, parts per million, of total phenols and TCDD in soils collected in January and November 1 7 frost selected sites on the Herbicide 98 Orange Storage Area, Naval Construction Battalion Center, Gulfport MS* Location Number of Sites Sampled*3 Total Herbicides (ppit)c Total Phenols (ppm)a 14 14 32af 36* 3.5a 04 .0 TCDD (pp«) "Ho" Spills ( / )e 00 Jan 78 78 ND(4) "Old" Spills ( / ) LL Jan 78 Kov 78 14 14 1,2020. 4920 86a 230 14 14 51,2850 30,0050 437tt 2530 O.G3641(3) 003(} .483 "New" Spills (H/B) Jan 78 Nov 78 026(0a .041) 014(1a .441) a Samples analyzed by the Flammability Research Center, The University Of Utah, Salt Lake City OT. Mr Force Contract Mo. 561178C0062. Reports submitted 17 May 1979 and 7 November 1 7 . 99 Each soil sample consisted of a cube of soil (3x3x3 inches) removed adjacent to a designated marker. °fotal herbicides refers to the concentration of acid and all esters of both 2,4-D and 2,4,5-T. %otai phenols refers to total concentration of both dichlorophenol and tr ichlorophenol . e The coding O/O, L/L and H/H are described in the text. Means within columns within subtitles followed by the same letters are not significantly different at the 0.05 probability level. For the statistical analyses, the Wilcoxon Paired-Sample Test was used. A test for a one- tailed hypothesis with paired samples was used in the procedure for nonparametric data since it could not be assumed that the levels of residue detected were from a normal distribution and it was expected that the residues would decrease with time. See Reference 11. Detected? the number of samples analyzed is in parentheses. The detection limit was generally 0.0002 ppra ( 0 ppt) . 20 n NA-Mot Analyzed. %he number within parentheses refers to number of positive samples used in calculations of the means. In L/L sites, the other 11 samples were either ND or not analyzed; in H/H sites the remaining samples were HD. 18 �TABUS 4. Penetration of herbicides, phenols and TCDD in soil collected June 1979 from a site (Nuaber 17, H/H) where a herbicide spill occurred in 1977 on the Herbicide Oranfe Storage Area, Haval Construction lattalion Center, Gulf port MSa Description of Siteb Soil Depth (Inches) Total Total Herbicides (Pl»»}c Phenols (ppa)d KDD (ppm) Surface Layer 0-3 61,650 365 0.325 Above Hardpan 3-6 34,690 95 0,340 Within Hardpan 6-9 1,620 48 0.021 Within Hardpan 9-15' 322 " 11 MDe *Sanf»les analyzed by the Flaamability Research Center, fhe Uniwrsity of Utah, Salt lake City OT. Mr Force Contract No. 561178C0062. Report submitted 7 Marorober 1979. See text for description of flardpan. C fotal herbicides refers to concentration of acid and all esters of both 2-4D and 2,4,5-T. total phenols refers to total concentration of both dichlorophenol and trichlorophenol. e ttot Detected. The detection limit was 0.00048 pp» ( 8 ppt) for this 40 sample. 19 �had occurred during the PACER HO Operation in Jane 1977. The soil core was collected in June 1979; thus, a period of at least two years had elapsed from date of spill to date of sampling, A decrease in concentration of residue occurred with depth. The hardpan (soil stabilized with cement at least 30 years earlier) was relatively impervious to any residues, despite the high annual rainfall (60 inches) received in this geographic location. These data suggest that soil penetration of residue as a route for contamination of subsurface water will be negligible. Some additional observations of the residue data that may influence future monitoring programs concern the nature of the remaining residues. Although most of the sites, where high levels of residues have been found, have been associated with a spill of Herbicide Orange, two of the sites contain significant levels of the isooctyi esters of 2,4-D and 2,4,5-T. These data suggest that Orange II was spilled at these sites rather than Orange. Whereas the butyl esters of 2,4-D and 2,4,5-T have rapidly hydrolyzed in the soil, the data from Orange II sites show little or no degradation of the isooctyi esters over the two-year period, especially the isooctyi esters of 2,4,5-T. In addition, in these two sites detailed studies of the reiidue indicate the presence of an apparently very stable isooctyi ether of 2,4,5-trichlorophenol. Unpublished data by Arnold* of the studies on soils treated with Orange II in 1972 and collected six years later, have shown negligible degradation in the isooctyi ether of 2,4,5-trichlorophenol. The stability of this ether has permitted its use in confirming the actual concentration of herbicide in the soil at the time of treatment. It may be possible to use this "marker" ether to date selected spills at NCBC, *E.L. Arnold, August 1979. Analysis of Herbicide Orange Components in Selected Soil Samples. USAPSAM/NGP, Brooks APB TX. Report submitted to USAF OEHL. 20 �Data from the micrcbial analyses of soil samples collected from the storage area in July 1977 and January and November 1978 are shown in Tables 5 and 6. Although the biological activity was high in all three treatment areas ( / , L/L, and H/H) trends in populations were discernible. The 00 July 1977 data in fable 5 indicate the impact that activities associated with Project PACER HO may have had on the storage area. During PACER HO, not only did personnel and vehicular traffic disturb the entire site, but when the operation was complete, the site was leveled and a layer of oyster shells was placed in selected sites where spills of herbicide and fuel oil had occurred. The bacteria were especially affected} note that the July 1977 levels in either no spill or new spill sites were ituch lower than the other two dates. However, these data may also reflect both an effect of PACER HO and a lag-phase effect in the adaptation of the bacteria to herbicide. The highest levels of bacteria were found in highly herbicidecontaminated sites (January 1978). Of the several bacterial genera isolated and identified, Psuedoponas spp. predominated in samples with the highest levels of herbicides. Levels of fungi decreased both with time and herbicide concentration. Only 50 percent of the H/H sites in January or November 1978 had detectable levels of fungi, and then, as noted in Table 6, they were not always of genera found in O/O or control soils. Proliferation of certain organisms could indicate their ability to metabolize or co-metabolize herbicide or herbicide degradation products or it could indicate elimination or inhibition of natural competitors. Specific metabolic activity studies using the predominant organisms would be necessary to determine their exact role (if any) in biodegradation. 21 �TABLE 5. Microbial population levels (number of organisms per gram of soil) in soils collected in July 1977, January and November 1978 from selected sites on the Herbicide Orange Storage Area, Naval Construction Battalion Center, Gulfport MSa Fungi, xlO5 Number of Sites Bacteria, xlO7 "No" Spills ( / )b 00 Jul 77 Jan 78 Nov 78 6 14 14 29.7 45.6 40.2 29.6 Old Spills (L/L) Jan 78 Nov 78 14 14 41.8 36.3 1 .2 ( ) 0 8 4.2 ( ) 8 6 14 14 15.4 49.4 34.6 28.6 (5) 7.7 ( ) ? 6.1 (7) Location tsr 7.8 6.2 New Spills (K/H) Jul 77 Jan 78 SOV 78 Control*1 Jan 78 1 38 3.0 Sov 78 I 35 3.2 a Microbial analyses conducted by Department of Chemistry and Biological Sciences, USAF Academy CO. Final report received August 1979. **fhe codling 0/0, L/L and H/H are described in text. c The number within parentheses refers to number of samples where colonies could be counted. Fungi in soils contaminated with herbicide frequently showed no growth after 7 days or growth was random. Control taken in open grassy area one mile from Storage Area. 22 �TABLE 6. Fungal genera found in soils collected from selected sites in 197? and 1978 on and off the Herbicide Orange Storage Area, Naval Construction Battalion Center, Gulfport MSa Predominant Genera Off-Site Control On Site o/o VL Aspergillus spp. Panici Ilium spp. Gunninghamella spp. Zygorhynehus sp. Alternaria sp. Mycelial Molds Candida^ spp. Rhodotorula sp. Geotrichum sp. Triehoderma spp. Muoor. spp. Rhizopus sp • Absidia sp. X X X X X X X X X X X X X X X X X X X X X A X X X X X X A X Miorobial analyses conducted by Department of Chemistry and Biological Sciences, OSAF Academy CO. Final report received August 1979. ft» ceding 9/0, L/L and H/H refer to no spill ( / ) old spill 00, (L/L) and new spill (H/H) and are further described in text. 23 *Jf / f ^ t* I /H I X �AQUATIC SYSTEM MOIilTORIHG FOR TCDD RESIDUE, 1 7 - 9 9 9717 The extreme toxicity associated with 2,3,7,8-TCDB (Reference 8) and its occurrence as a contaminant in 2,4,5-T (and hence Herbicide Orange) dictated that it must be the focus of any residue monitoring study. The location of the NCBC in relation to the major population center of Gulfport MS and to the associated aquatic system is shown in Figure 1. Previous ecological studies on the environmental fate of TCDO by Young ( ) 9 and Young, et al. ( 0 suggested that aquatic drainage systems could be 1) contaminated by water erosion of soil particles containing TCDD. The herbicide storage area is drained by a series of snail ditches that connect into a single ditch immediately adjacent to the area. This larger ditch is fed by other small ditches as it transversea the property of the NCBC. Zn an effort to obtain baseline data on TCDD in this aquatic system, archived biological samples (collected in the immediate storage area and frozen in January 1976) were analyzed in November 1978 and found positive for TCDD residue. Thereafter, additional environmental samples were collected in January, February and June 1979 at varying distances downstream from the storage area. These designated Aquatic Sampling Sites are shown in Figure 2, Aquatic Site III was located at the NCBC perimeter. Aquatic Site IV was at a culvert discharge from the drainage ditch into Long Beach Canal Number 1. Aquatic Sampling Site V was at the confluence of the canal and Turkey Creek. The analytical results from some of these environmental samples were received in September and November 1 7 . 99 A summary of all available TCDD residue data for the aquatic system draining from the storage area is shown in Table 7. It should be again noted that TCDD data in Tables 2, 3 and 4 are presented as parts per million (ppm). Aquatic monitoring studies detected residue levels in 24 �4» O O -H 3 *J C (0 g« o «o ® r-i +J 15 111 > -H 4 O 3 0 CO C S ffi 5° •P C I I tie Is H 14 (D S +J e 9 V Il l rt i & 25 �ci4e M O 4) 41 « §« •H £8 re i-t -ft C 4J •H A (0 i) 31 •H «H 0) -P .si ^ «• D» 3 n. jjj S J3 io (0 rt o * "51 §• a> «g ««4 « O « • tt a 1 tie s H M \ \ V \ 26 M 10 �TABLE 7. Summary of results (parts per billion) for TCDD residue studies in water, sediments anil biological organisms associated with drainage from the Herbicide Orange storage area, Naval Construction Battalion Center, Gulfport IB* Aquatic Sampling Site Distance from Storage Area (Feet) Water Cppb) Maximum Concentration in Sediments (ppb) I Immediate Area ND 3.6 Biologicals (ppb) 0.14-3,5fc 1.6 -7.2 II III 3,000 NAd 7,000 NA 0.01 005 .4e IV 9,000 NA 0.02 0.02f V 12,000 NA ND ND 0.2-2.2 ND9 The analyses for TCDD were conducted by the University of Nebraska, Mass Spectroroetry Laboratory, Lincoln HE, under Mr Force Contract Ho. P0561178C0063 and the Oniversity of Utah, Salt Lake City Of, under Air Force Contract No. 5S1178C0062. Reports submitted 6 September 1979 from the University of Nebraska and 17 May 1979 and 7 November 1979 from the University of Utah. m » Not Detected. Detection limit varied with the sample. All water samples were analyzed by the university of Utah and the detection limit was 0.02 ppb. Sediment samples from Sites I, II and V were analyzed by the University of Utah by low resolution GC-MS where the detection limit was 0.5 ppb. Sediment sauries from Sites III and IV were analyzed by the Dniversity of Nebraska by hifh resolution GC-MS where the detection limit was O.OOS ppb. All biological samples were analyzed by the University of Nebraska and the detection limit ranged from approximately 0.05 to 0.005 ppb, °First sample set collected in January 1976 and analyzed and reported in January 1979; second sample set collected in January 1979 and reported in September 1979. NA - Not Analyzed. This value is an average for a single biological, a crayfish, which was analyzed twice. The mean detection limit was 0.01 ppb. This value was for a single biological, a crayfish, which was analyzed twice. The mean detection limit was 0.008 ppb. A single biological sample, a composite of mosquitofish, was analyzed three times. The sample was considered negative at a mean detection limit of 0.007 ppb. 27 �parts per billion (ppb) and pacts per trillion (ppt). Thus, the average mean level of TCDD in storage site soils (spills) in July 1977 was 237 ppb (0.237 'pftt, see Table 2) j 206 ppb in January 1978 and 144 ppb in November 1978 (see Table 3). Data in Table 7 in very low parts per billion are two orders of magnitude below levels in the storage area soils. Water Samples - Surface Drainage System Herbicide storage Area h total of 61 surface drainage system water samples were collected (Aquatic Sampling Site I) during the history of the project. One sample collected in 1976 was positive at an average mean value of 7.7 ppt TCDD. All remaining samples were negative for TCDD at detection limits ranging from 5-37 ppt. Water Samples - Potable Water System and Wells on the NCBC h total of 36 potable water system and well water samples taken during th« history of the project have contained no detectable levels of TCDD at detection levels as low as 10 ppt. Sediment Samples Two of eight sediment samples collected (Aquatic Sampling Site I) in the immediate surface drainage system of the herbicide storage area in June 1979 were positive for TCDD at levels of 2.7 ppb and 3.6 ppb. Of the remaining six samples, five contained no detectable TCDD at a detection limit of 2 ppb. The sixth sample contained no TCDD at a 37 ppb detection limit. The maximum positive value for this location is shown in Table 7. Two sediment samples have been collected from Aquatic Sampling Site ZZ. These samples were collected in June 1979 and were found negative for TCDD at a detection limit of 0.5 ppb. 28 �Two sediment samples have been collected from Aquatic Sampling Site III (located at the NCBC perimeter}, One of these samples was collected in February 1979,- the other in June 1979. The June sample (data reported in November 1979) was negative for TCDD at a, detection limit analysis of 0.5 ppb [low resolution Gas Chromatography-Mass Spectroraetry (GC-MS}], while the February sample (data reported in September 1979} was positive for TCDD at a level of 0.01 ppb (high resolution GC-MS analysis). the datum from the February sample is reported in Table 7. One sediment sample collected in February 1979 off-base, 9,000 feet from the herbicide storage area (Aquatic Sampling Site IV), in the drainage system leading away from the herbicide storage area and the NCBC, was positive for fCDD at 0.02 ppb with a lower detection limit of 0.01 ppb (report received September 1979). One additional sample collected from the same area (Aquatic Sampling Site IV), in June 1979 contained no detectable TCDD, when the detection limit was 0.5 ppb (report received November 1979). A single sediment sample was collected from Aquatic Sampling Site v. The sample was collected in June 1979 and analyzed by low resolution GC-MS. The sample was found negative for TCDD at 0.5 ppb. Biological Samples Aquatic biological samples (snails, fish, tadpoles, crayfish, and insects) collected over the past three years from the drainage ditch serving the immediate herbicide storage area (Aquatic Sampling Site I)» contained TCDD levels that ranged between 0.14 ppb and 7.2 ppb (Table 7). Aquatic biological samples (snails, tadpoles, fish and crayfish) collected over the past three years from the drainage ditch 3,000 feet 29 �downstream from the herbicide storage area (Aquatic Sampling Site II), contained TCDD levels that ranged between 0.2 ppb and 2.2 ppb. A large crayfish was collected in January 1979 and the muscle tissue and intestine trace separately analyzed. The intestine was found to contain 1.1 ppb TCDD, while the muscle tissue contained 0.0? ppb TCDD. A crayfish sample collected in February 1979, 7,000 feet downstream from the herbicide storage area (Aquatic Sampling site III), just before the drainage system exited the NCBC property, contained 0.045 ppb TCDD. A crayfish sample collected in February 1979, 9,000 feet downstream from the herbicide storage area (Aquatic Sampling Site IV), offbase in the drainage system serving NC1C was found to contain 0.02 ppb TCDD. A mosquitofish sample collected in February 1979, 13,000 feet downstream from the herbicide storage area (Aquatic Sampling Site V), in the off-base drainage system, contained no detectable TCDD at a detection limit of 10 ppt. 30 �Environmental studies of an area on the Naval Construction Battalion Center, previously used for the storage of Herbicide Orange from mid-1968 through mid-1977 were conducted during the period 1970 through 1 7 . The 99 following are conclusions from those studies: ' 1, .approximately 1-2 acres of the 12-acre area are contaminated with Herbicide Orange and its associated dioxin. 2. Levels of 2,4-0 and 2,4,5-T herbicides in selected saaples from the top three inches of soil profile were greater than 100,000 ppmdaean 78,040 ppn) in 1977, bat rapidly decreased to one-third that level in 18 months. 3. No accurate estimate of TCDD persistence is possible from these studies. However,, data from spill sites monitored for 18 months suggest that TCDD levels are decreasing. 4. Soil penetration of the herbicides was low while soil penetration of TCDD was very low but measurable. 5. Soil sterilization did not occur as a result of Herbicide Orange contamination. 6. Proliferation of certain microflora occurred under high levels of herbicide (specifically members of the fungal order Mucorales, white nonsporulating mutants, soil yeasts, and Pseudomonas spp.) 7. Yeast and Pseudomonas spp. predominate in samples with highest levels of herbicide, 8. Proliferation of certain organisms could indicate: a. ability to metabolize HO or degradation products. b. Ability to co-metabolize HO or degradation products. c. Elimination/inhibition of natural competitors. 31 �9. The low solubility of TCDD in water would suggest that its solubility in water alone could not account for the levels of TCDD found in the drainage ditch sediment. 10. The movement of TCDD from the storage sites is primarily through soil erosion, especially that caused by water. 11. Organisms that come into direct and intimate contact with TCDD-contaminated soil generally become contaminated themselves, (A wide variety of organisms have been examined.) 12. TCDD was found in a crayfish collected on base 3,000 feet downstream from the storage site. Levels in the intestine were 1.1 ppb, levels in muscle tissue were only 0.07 ppb. Movement of contaminated soil from the storage area downstream may have resulted in the contamination of crayfish. However, crayfish are highly mobile and nay have migrated from the storage area to the point of capture. 13. TCDD was found in two samples (1 sediment and 1 biological) collected off-base of NCBC. Although the levels of TCDD were extremely low (20 parts per trillion in each sample), it is apparent that some contamination from the storage area has occurred. Contamination from the storage area is not yet extensive and can be controlled. RECOMMENDATIONS The principle recommendation for management of the 12-acre area at the Naval Construction Battalion Center, formerly used as a storage area for Herbicide Orange, is that the area be left undisturbed permitting the continuation of "natural" degradation of the herbicides and TCDD, Specific recommendations to prevent further movement of contaminated soil from the area include: 32 �1. Limiting access to the storage area, and preventing motor vehicle traffic froa crossing the area and potentially "tracking" TCDDcontaminated soil particles to other parts of the installation. 2. Preventing water erosion wherever possible by stabilizing the drainage ditch banks with concrete or asphalt material. The ditch banks should be slightly elevated on the contour to allow pooling of water from the storage area prior to entering the ditch creating an initial siltation catchment. The ditches should be allowed to have plant growth in them to slow the movement of water and allow for more silt catchment, In several places along the ditch drainage system concrete dams should be constructed to slow water movement and provide a wide shallow overflow {in effect creating snail siltation ponds in the ditch drainage system). 3. Constructing one or two larger siltation ponds in the drainage system prior to the drainage water leaving the base. 4. Allowing native vegetation to invade the storage area and establish a plant coaanunity to help prevent both wind and water erosion, 5. Developing a research protocol to determine possible methods for returning the storage area to full beneficial use. This protocol might include techniques to: a. decontaminate TCDD-laden soils. b. increase TCDD degradation rates. c. characterize the distribution and effects of TCDD in the aquatic environment. 33 �LITERATURE CITED 1. Anonymous. 1977. Air Force Logistics Command Programming Plan 75-19 for the Disposal of Orange Herbicide. San Antonio Mr Logistics Center, Kelly AFB TX. Annex 8, pp 2-4. 2. Craig, D.A. 1975. Use of Herbicides in Southeast Asia. Historical Report. San Antonio Air Logistic Center, Directorate of Energy Management, Kelly AFB TX. 58 p. 3. Fee, D.C., B.M. Hughes, M.L. Taylor, T.O. Tiernan and C.E. Hill. 1975. Analytical Methodology for Herbicide Orange, vol lit Determination of Origin of OSAP Stocks. Technical Report ARL-TR-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB OB. 36 p. 4. Hughes, B.M., D.C. Fee, M.L. Taylor, T.O. Tiernan, C.E. Bill and R.L.C. Wu. 1975. Analytical Methodology for Herbicide Orange. Vol Xi Determination of Chemical Composition, Technical Report ARL-TR-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB OH. 365 p. 5. Httfh**, B.M., F.B. Hileaan, i,H. Wbjeik and W.H. MeClennen. 1979. A rapid method for the analysis of low levels of Herbicide Orange (butyl **t*rs of 2,4-D ami 2,4,5-T), 2,4-ST 2,4,5-T, dichlorophenol, trichlorophenol and tetrachlorodibenzo-p-dioxin (TCDD) in environmental samples. Division of Analytical Chemistry, American Chemical Society. Abstract, 177th ACS Rational Meeting, Honolulu HI. 6. Hummel, R.A. 1977. Clean-up Techniques for the Determination of Parts Per Trillion Residue Levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDB). Journal of Agricultural and Food Chemistry 25(5)s1049-1053. 7. Winston, A.W. and R.M. Ritty. 1971. What Happens to Phenoxy Herbicides When Applied to a Watershed Area. Industrial vegetation Management 4(1):12-14. 8. Young, A.L., J.A. Calcagni, C.E. Thalken and J.W. Tremblay. 1978. The Toxicology, Environmental Fate and Human Risk of Herbicide Orange and its Associated Dioxins. Technical Report OEHL-78-92. USAF Occupational and Environmental Health laboratory, Brooks AFB TX. 247 p. 9. Young, A.L. (Ed). 1974. Ecological studies on a herbicide-equipment test area (TA C-52A), Eglin AFB Reservation, Florida. Technical Report AFATL-TR-74-12. Air Force Armament Laboratory, Eglin AFB FL. 141 p~. 10. Young, A.L., C.E. Thalken, E.L. Arnold, J.M. Cupello and L.G. Cockerham. 1976. Fate of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the Environment: Summary and Decontamination Recommendations. Technical Report USAFA-TR-76-18. Department of Chemistry and Biological Sciences, USAF Academy CO. 41 p. 11. Zar, J.H. 1974. Biostatistical Analysis. I»renti0e-Hall Inc., Edgewood Cliffs NJ. pp 124-126. 34 �ADDENDUM Additional residue data from selected biological samples collected June 1979 were received 3 December 1 7 . These data are shown in Table A-l. 99 Vh«M data offer additional support of the previous conclusion, that TCDD from the Herbicide Orange storage area is present in selected biological samples obtained outside the boundary of the Naval Construction Battalion Center. 35 �TABLE A-l. Summary of results (parts'per billion) for TCDD residue in biological organisms collected June 1 7 fj?o» the 99 drainage system associated with the Herbicide Orange storage area, Naval Construction Battalion Center, Gulfport MS* Aquatic Sampling Distance iron Site Storage Area Mature of Sample Concentration Detection of Limit TCDD (ppb) (ppb) 11 3,000 Composite: Crayfish/Fish 015 .7° 0.035 III 7,000 Composite: Crayfish/Fish Turtle ( a ) Ft 0081 .8* HD® 0.010 0.035 IV 9,OOO Composite: Crayfish/Fish 001 .3f 0.017 V 12,000 Composite: Crayfish/Fish Frog (whole body) 000 .2 006 ,0 008 .0 O.OOS *fhe analyses for TCDD were conducted by the University of Nebraska, Mass Spectrometry Laboratory, Lincoln HE, under Mr Force Contract No. F056118C0063. Report submitted 3 December 1 7 . 99 This composite sample and subsequent composite samples in this table consisted of mosquitofish and snail crayfish. Q Average of three analyses. dAverage of two analyses. eND * not detected. Average of two analyses. 36 � 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. 017 Folder The folder containing the original item. 0187 Series The series number of the original item. Series 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. Charles E. Thalken William J. Cairney Description An account of the resource <strong>Corporate Author: </strong>USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas Date A point or period of time associated with an event in the lifecycle of the resource 1979-11-01 Title A name given to the resource Herbicide Orange Site Treatment and Environmental Monitoring: Summary Report and Recommendations for Naval Construction Battalion Center, Gulfport, MS Subject The topic of the resource biodegradation Mississippi dioxin Pacer Ho https://www.nal.usda.gov/exhibits/speccoll/files/original/3f4bef0956a64c1c127a1b2894821266.pdf 3181b7d44f6de47aef9776417196ede7 PDF Text Text Item ID Number 00193 Author Young, Alvin L. Corporate Author Department of Chemistry and Biological Sciences, USA Report/Article TitlO Fate of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCCD) in the Environment: Summary and Decontamination Recommendations Journal/Book Titlo Year Month/Day Color Number of Images October [J 49 Monday, January 22, 2001 Page 205 of 341 �USAFA-TR-76-18 FATE OF 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIQXlN (TCDD) IN THE ENVIRONMENT: SUMMARY AND DECONTAMINATION RECOMMENDATIONS CAPTAIN ALVIN L. YOUNG MAJOR CHARLES E. THALKEN LT COLONEL EUGENE L. ARNOLD CAPTAIN JAMES M. CUPELLQ MAJOR LORRIS G. COCKERHAM DEPARTMENT OF CHEMISTRY AND BIOLOGICAL SCIENCES USAF ACADEMY, COLORADO 80840 OCTOBER 1976 APPROVED FOR PUBLIC RELEASE; DISTRIBUTION UNLIMITED Prepared for: HEADQUARTERS AIR FORCE LOGISTICS COMMAND WRIGHT-PATTERSON AIR FORCE BASE, OHIO 4S433 DEAN OF THE FACULTY UNITED STATES AIR FORCE ACADEMY COLORADO 80840 �Editorial Review by Lt Colonel J. M. Shuttleworth Department of English and Fine Arts USAF Academy, Colorado 80840 This research report is presented as a competent treatment of the subject, worthy of publication. The United States Air Force Academy vouches for the quality of the research, without necessarily endorsing the opinions and conclusions of the author. This report has been cleared for open publication and/or public release by the appropriate Office of Information in accordance with AFR 190-17 and DODD 5230.9. There is no objection to unlimited distribution of this report to the public at large, or by DDC to the National Technical Information Service. This research report has been reviewed and is approved for publication. PHILIP J/QZRDLET Colonel, USAF Vice Deah of the Faculty Additional copies of this document are available through the National Technical Information Service, U. S. Department of Commerce, 5285 Port Royal Road, Springfield, VA 22151. �UNCLASSIFIED SECURITY CLASS'FICATION OF THIS »AGE (When Data Entered) READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 1. REPORT NUMDER 2. GOVT ACCESSION NO 3. RECIP'FN'T'S CATALOG NUMBER USAFA-TR-76-18 4. TITLE (and Subtitle) 5. TYFE OF REPORT ft PERIOD COVERED Fate of 2,3,7,8-TetracMortdibenzo-p--dioxin (TCED) in the Environment: Surtmary and Decontamination Recxxtmendations Summary Report 6. PERFORMING ORG. REPORT NUMBER 8. 7. AUTHOR?.) Alvin L. Young, Capt, USAF, PhD; Charles E. Thalken, Maj, USAF, VC, DVM, MS; Eugene L.Arnold, LtCpl, USAF, BSC, PhD; James M, Cupello, Capt, USAF, PhD; Lorris G. CocTcerham, Maj, USAF, MS CON1 RACT OR GRANT NUMBERf»> 10. PROGRAM F.LEMENT. PROJECT, TASK AiHTA ft WORK UNIT NUMBERS 9. PERFORMING ORGANIZATION N A M E AND ADDRESS Department of Chemistry and Biological Sciences DFCBS-R USAF Academy, Colorado 80840 t. CONTROLLING OFFICE NAME AND ADDRESS 12. REPORT DATE October 1976 Department of Chemistry and Biological Sciences DFCBS-R USAF Academy, Colorado 80840 13. NUMBER OF PAGES 14. MONITORING AGENCY NAME & ADDRESSf// different from Controlling Otlice) 15. SECURITY CLASS, (of thla report) 44 UNCLASSIFIED 15«. DECLASSIFICATION/DOWNGRADING SCHEDULE 6. DISTRIBUTION STATEMENT (ot thla Report) Approved for public release: distribution unlimited. 7. DISTRIBUTION STATEMENT (of the abstract entered In Block 20, II different from Report) 8. SUPPLEMENTARY NOTES 9. KEY WORDS (Continue on reverse aide It necessary and Identity by block number) Animal SUTVey; Aquatic Studies; Bic>accumulation; Biodegradation of Herbicides; Biodegradation of TCDD; Ecological Effects; 2,4-dichlorophenoxyacetic acid (2,4-D); Fish Studies; Herbicide; Histopathology; Insect Studies; Maranals; Necropsy; Orange; Reptile Study; Soil Microbial Studies; TCDD; Teratogenic; 2,3,7,8-tetxochlorodibenzo-pdioxin (TCDD); Test Area C-52A, Eglin AFB Reservation; 2,4,5-trichlorophenoxyanifl \rf f ^ * *rf__ jfr f T Vegetative Succession. • —• irtffrYftrr H-hM HiVfAM ^r^-VT^_ _* ^y VMV^ ^ * ^- - «''*^^ *' -. *"^ ' " — -..,..-— ———.... , —, 0. ABSTRACT (Continue on rovora* aide (/ ri «vt** «ry ant/ I ((entity by bjpck number) . . /msmr\\ i* U.^ Studies on the fate of 2,3,7,8-tetrachiorodibenzo-p-dioxin (TCDD) have been conducted on biodegradation plots and field test areas that have received massive quantities of Orange herbicide (a 50:50 mixture of the n-butyl esters of 2,4lichlorophenoxyacetic acid [ , 4 D and 2,4,5-trichlorophenoxyacetic acid 2'-] 2,4,5-T]). From the studies reviewed in this report, it is apparent that 1) TCDD may persist (in biotic and abiotic components) for long periods of time when initially present at extremely high concentrations on the soil surface, 2) TCDD will accumulate in the tissues of rodents, reptiles, birds, fish, and W DD , FORM73 1473 JAN M >l EDITION OF 1 NOV 65 IS OBSOLETE Mg F i rs UNCLASSIFIED �SECURITY CLASSIFICATION OF THIS PAGEfWhen Data Entered) 20. Abstract (Continued) insects when these organisms are exposed to TCDD contaminated soils (however, the levels of TCDD in the tissues apparently do not exceed the levels of TCDD found in the environment), (3) organisms tolerate, i.e., based on no observed deleterious effects, soil levels between 10-1,500 ppt TCDD, (4) TCDD is degraded by soil microorganisms, especially when in the presence of other chlorinated hydrocarbons, (5) TCDD is degraded in the presence of sunlight, (6) movement of TCDD in the abiotic portions of the environment can be by wind or water erosion of soil particles, but leaching by water alone does not appear to occur, and (7) TCDD is probably not readily released or degraded in the environment when bound to activated coconut charcoal. SECURITY CLASSIFICATION OF THIS PAGE(Wien Date Entered) �TABLE OF CONTENTS Title Page Introduction 1 Soil Incxjrporation/Biodegradation Studies 6 Fate of TCDD in an Ecosystem Geographical and Vegetative Features Sampling Grids and Herbicide Deposition Preliminary Ecological Studies Soil Studies of TCDD Residues Rodent Studies Trapping Data/Histopathology Liver and Pelt Analysis Burrow and Diet Studies TCDD Laboratory Uptake Experiment Hepatic Ultrastructural Study Reptile Studies TCDD in Aquatic Organisms TCDD in Birds of TA C-52A Vegetative Succession Studies on TA C-52A 17 ... 18 18 18 .21 23 23 25 25 26 27 29 30 31 32 Laboratory and Greenhouse Experiments with TCDD 34 Recormiendations 39 �LIST OF TABLES Number 1 2 3 Page Analyses of the Top 15-on Layer From Each of the Soil Biodegradation Sites 7 Descriptions of Three Biodegradation Studies Involving Use of Herbicide Orange 8 Concentrations of Herbicide Orange and TCDD in Plots Originally Treated with 4,480 kg/ha, AFLC Test Range Complex, Utah, at Various Sampling Dates After Application. (TCDD in parts per billion) 9 4 Concentrations of Herbicide Orange and TCDD in Plots Originally Treated with 4,480 kg/ha, Garden City, Kansas, at Various Sampling Dates After Application. (TCDD in parts per trillion) . 9 5 Concentrations of Herbicide Orange and TCDD in Plots Originally Treated at 4,480 kg/ha, Eglin AFB, Florida, at Various Sampling Dates After Application 10 6 Movement of Herbicide Orange and TCDD in a Soil Profile, Eglin AFB, Florida. (TCDD in parts per trillion) 12 7 Comparison of Herbicide Orange Degradation Rates in Plots at the Eglin AFB, Florida, Site, Receiving Either Herbicide, Herbicide Plus Soil Amendments, or Herbicide Plus Amendments and Charcoal .14 8 Approximate Amounts of 2,4-D and 2,4,5-T Herbicides Applied to Test Area C-52A, Eglin AFB Reservation, Florida 9 10 11 19 Concentration of TCDD in Soil Profile (1974) of Grid I, Test Area C-52A, Eglin AFB, Florida 22 Numbers of Beach Mice Collected During the 1973 and 1974 Studies of Test Area C-52A 22 Concentration (Parts Per Trillion) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Liver and Pelt Samples from Beach Mice, Peromyscus polionotus, Collected from Control and TCDD-Exposed Field Sites, 1973 and 1974 24 11 �UST OF TABLES (Continued) Nottogr 12 13 14 Page Concentration (Parts Per Trillion) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Liver and Pelt Samples from Beach Mice, Peronyscus polionotus, Dusted with Alumina Gel Containing No TCDD (Control) or 2.5 Parts Per Billion TCDD (Test) 28 Concentration (Parts Per Trillion) of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in Composite Samples of Viscera or Trunk from Six-Lined Racerunners, Cnemidophorus sexlineatus, Collected from Control and TCDD-Exposed Field Sites 28 Degradation of TCDD (Parts Per Trillion) in a Greenhouse Experiment, Eglin AFB, Florida 37 111 �INTRODUCTION The heterocyclic organic molecule 2,3,7,8-tetrachlorc-dibenzop-dioxin (TCDD) has received a great deal of attention in the last 6 years because of its highly toxic properties and the possibility of it being widespread in the environment by the use of products made from trichlorophenol, especially the herbicide 2,4,5trichlorophenoxyacetic acid (2,4,5-T). Although TCDD may occur as a contaminant in products made from trichlorophenol, the levels of TCDD found in any given lot of trichlorophenol is dependent upon the manufacturing process. TCDD may be produced as a by-product during an alkaline hydrolysis reaction when the temperature for making 2,4,5-trichlorophenol from tetrachlorobenzene exceeds 160°C. However, there is less likelihood of TCDD formation in the manufacturing process which starts with phenols and chlorinates them to form trichlorophenol since little or no heat is required in this reaction. Public interest in TCDD originated in 1970 when the herbicide 2,4,5-T was implicated as a potential teratogen in pregnant rats ( ) Later tests indicated that the teratogenesis may have been 1. caused by 27 ± 8 ppm of TCDD present as a contaminant in the 2,4,5-T. As more data have been obtained (2), it has become apparent Courtney, K.D., D.W. Gaylor, M.D. Hogan, J.L. Falk, R.R. Bates, and I. Mitchell. Teratogenic evaluation of 2,4,5-T. Science 168:864-866, 1970. 2 Schwetz, B.A., J.M. Norris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Oner son, and C.G. Gerbig. Toxicology of chlorinated dihenzo-pdioxins. Biviron. Hlth. Perspect., Experimental Issue No. 5:87-100, September 1973. �that TCDD is one of the most toxic chemicals known; the oral LD5Q for many animal species is in the range of micrograms per kilogram. Purthermore, the known effects of TCDD include anorexia, severe weight loss, hepatotoxicity, hepatoporphyria, vascular lesions, chloracne, gastric ulcers, and teratogenicity ( ) The 2. hazard posed by the presence of even a small amount of this substance in the environment has therefore been of concern. For a person or animal to be poisoned with TCDD, a rare set of circumstances would be required. Since present production methods are able to reduce the TCDD level to less than 0.1 ppm, it is unlikely that contaminated 2,4,5-T herbicide or even contaminated trichlorophenol would be implicated in such a poisoning. Nevertheless, two accidental poisoning episodes involving TCDD have been recently reported. In 1975, Carter et al. (3) identified TCDD as the apparent cause of an outbreak of poisoning in humans, horses, and other animals on a horse breeding farm in eastern •H .• , Missouri in 1971. Exposure to TCDD followed the spraying of contaminated industrial waste oil on riding arenas for dust control. An investigation concluded that a hexachlorophene (made from trichlorophenol) factory in southwestern Missouri had accumulated distillate residues containing 306 to 356 ppm TCDD. It was this distillate residue that was subsequently disposed of via a salvage oil company and sprayed on the horse arenas. 3 Carter, C.D., R.D. Kiiribrough, J.A. Liddle, R.E. Cline, M.M. Zack, Jr., W.F. Barthel, R.E. Koehler, and P.E. Phillips. Tetrachlorodibenzodioxin: an accidental poisoning episode in horse arenas. Science 188:738-740, 1975. �The second incident of TCDD poisoning occurred in July 1976 in Seveso, Italy ( ) The source of the TCDD was a chemical 4. factory that produced trichlorophenol through the alkaline hydrolysis of tetrachlorobenzene. When the temperature in a steamheated reaction vessel rapidly increased, a.safety disk ruptured sending a plume of trichlorophenol, TCDD anici other products 30 to 50 m high above the factory. The cloud apparently rose into the air, cooled, and came down over a cone-shaped area about 2 km long and 700 m wide. An area of 110 hectares (ha) was evacuated after hundreds of animals had died and many people had reported skin disorders. Several measurements of TCDD on vegetation in an area adjacent to the factory were in the 1 to 15 ppm range, with one reading as high as 51.3 ppm. An Italian government commission (5) recommended: "removal of topsoil to a depth of 10 cm in an area of 113 ha, the. dismantling of all buildings in the Seveso area, and the total disruption of all wildlife." The need for data on the fate of TCDD in the environment is not confined to solving problems associated with the above two incidents. During the latter portion of the last decade, a program of aerial application of herbicides was conducted in Southeast Asia by the United States Air Force. In 1969, at the conclusion of this program, considerable amounts of herbicide were left unused. 4 Rawls, R.L., and D.A. O'Sullivan. Italy seeks answers following toxic release. Chem. Engr. News 54(35):27-35, August 23, 1976. Itay, A. Toxic cloud over Sevesco. Nature 262(5570):636-638, August 19, 1976. �One of the herbicides used extensively in this project was a herbicide designated "Orange" which was formulated as a 50:50 mixture of the n-butyl esters of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-T. In 1970, approximately 2.3 million gallons of this material was placed in storage by the Air Force. An analysis of TCDD in the Orange herbicide stocks (6) indicated that the range in concentration was 0.1 to 47 ppm TCDD. The weighted average concentration of TCDD for the 42,015 55-gallon drums of herbicide was 1.859 ppm. Because of the TCDD concentration, the herbicide could not merely be declared surplus and disposed of on the agricultural markets. Many methods have been evaluated for disposing of this material. However, regardless of the final method selected for its disposition, the storage sites where the material is currently stored (Naval Construction Battalion Center, Gulfport, Mississippi, or Johnston Island, Pacific Ocean) will need to be decontaminated. At the request of Headquarters, Air Force Logistics Command, Wright-Patterson AFB, Ohio, in April 1972, the Department of Chemistry and Biological Sciences, United States Air Force Academy, initiated studies on herbicide Orange and TCDD. The objectives of these studies were: (1) to investigate soil incorporation/ biodegradation as a disposal method for herbicide Orange; (2) to investigate the ecological effects associated with past uses of Department of the Air Force. Disposition of orange herbicide by incineration. Final Environmental Statement, November 1974, pp. 36-37. �herbicide Orange; and (3) to investigate the soil persistence and food chain accumulation of TGDD. This report documents the available data on TCDD from these studies. Furtherxnore, using these data, recommendations for decontamination of an area exposed to TCDD are presented. �SOIL INCORPORATION/BIODEGRADATION STUDIES One potential method proposed for the disposal of herbicide Orange was subsurface injection or soil incorporation of the herbicide at massive concentration rates. The premise for such studies was that high concentrations of the herbicides and TCDD would be degraded to innocuous products by the combined action of soil microorganisms and soil hydrolysis. In order to field test this concept, biodegradation plots were established in three climatically different areas of the United States; Northwest Florida (Eglin AFB), Western Kansas (Garden City), and Northwestern Utah (Air Force Logistics Command Test Range Complex). A comparison of the soils of the three sites is given in Table 1. The Utah site had a mean annual rainfall of 15 on, while the Kansas and Florida sites had 40 and 150 cm, respectively. Table 2 describes the experimental protocol for the three sites to include when the plots were established, the method of herbicide incorporation, the experimental design and the initial calculated herbicide concentration, ppm, at the time the plots were established. Further details on the experimental protocol can be obtained from Young, Arnold and Wachinski ( ) 7. Tables 3, 4, and 5 compare the rate of disappearance of TCDD with that of Orange herbicide for selected plots at the Utah, Young, A.L., E.L. Arnold, and A.M. Wachinski. Field studies on the soil persistence and movement of 2,4-D, 2,4,5-T, and TCDD. Appendix G. Department of the Air Force. Disposition of orange herbicide by incineration. Final Environmental Statement, November 1974. �TABLE 1. ANALYSES OF THE TOP 15-CM LAYER FROM EACH OF THE SOIL BIODEGRADATION SITES ORGANIC MATTER (%) SAND (%) SILT (%) CLAY (%) 5.6 0.5 91.6 4.0 4.4 Garden City, KS^ 7.0 1.7 37 42 21 Silt loam AFLC Test Range Complex, UT0 7.8 1.4 27 53 20 Clay loam LOCATION pH Eglin AFB, FLa SOIL DESCRIPTION Sandy loam located on Test Area C-52A, Eglin AFB Reservation, Florida T>lots located on the Kansas Agricultural Experiment Station, Garden City, Kansas °Plots located 75 miles west of Salt Lake City, Utah �TABLE 2. LOCATION Eglin AFB, Florida CO Garden City, Kansas DESCRIPTIONS OF THREE BIODEGRADATION STUDIES INVOLVING USE OF HERBICIDE ORANGE DATE ESTABLISHED 2 Apr 1972 10 May 1972 AFLC Test 2 Oct 1972 Range Complex, Utah METHOD OF INCORPORATION TREATMENT CALCULATED INITIAL HERBICIDE CONCENTRATION (PPM)C 4,480 kg Herbicide/haa 4,480 kg Herbicide/ha, plus soil amendments^ 4,480 kg Herbicide/ha plus soil amendments and activated charcoal 5,000 5,000 Preplant Incorporate (Rototiller) 2,240 kg Herbicide/ha 4,480 kg Herbicide/ha 1,000 2,000 . Simulated Subsurface Injection (8 cm band width) 1,120 kg Herbicide/ha 2,240 kg Herbicide/ha 4,480 kg Herbicide/ha Simulated Subsurface Injection (30 cm band width) 5,000 10,000 20,000 40,000 of herbicide calculated as active ingredient. Herbicide injected at 10-15 cm level or preplant incorporated in the 0-15 cm level. All plots duplicated. xhe amendments included 4.5 kg lime, 13.5 kg organic matter, and 1.4 kg fertilizer (12:4:8 for N,P,K, respectively) uniformly mixed within the top 0-30 cm of soil in the plot. °Contained in the top 0-15 cm layer. �TABLE 3. CONCENTRATIONS OF HERBICIDE ORANGE AND TCDD IN PLOTS ORIGINALLY TREATED WITH 4,480 KG/HA, AFLC TEST RANGE COMPLEX, UTAH, AT VARIOUS SAMPLING DATES AFTER APPLICATION. (TCDD IN PARTS PER BILLION) DAYS AFTER APPLICATION TOTAL HERBICIDE3 (PPM) TCDD ., (PPMxlO ) 282 8,490 15.0 637 4,000 7.3 780 2,260 5.6 1,000 2,370 3.2 1,150 1,150 2.5 a Composite sample from replicated plots, 0-15 on increment TABLE 4. CONCENTRATIONS OF HERBICIDE ORANGE AND TCDD IN PLOTS ORIGINALLY TREATED WITH 4,480 KG/HA, GARDEN CITY, KANSAS, AT VARIOUS SAMPLING DATES AFTER APPLICATION. (TCDD IN PARTS PER TRILLION) DAYS AFTER APPLICATION TOTAL HERBICIDE3 (PPM) TCDD3 , (PPMxlO~°) 8 1,950 ~b 77 1,070 225 189 362 210 600 40 659 a 490 <:L —b —b —b 42 Composite sampling from replicated plots, 0-15 cm increment HSbt determined �TABLE 5. CONCENTRATIONS OF HERBICIDE ORANGE AND TCDD IN PLOTS ORIGINALLY TREATED AT 4,480 KG/HA, EGLIN AFB, FLORIDA, AT VARIOUS SAMPLING DATES AFTER APPLICATION DAYS AFTER APPLICATION TOTAL, HERBICIDE (PPM) TCDDa , (PPMxlO~b)C 5 4,897 375 414 1,866 250 513 824 75 707 508 46 834 438 -b 1,293 <10 b — Composite sample from the plot containing only herbicide (i.e., no lime, organic matter, or fertilizer added). Sample from the 0-15 cm increment. Analysis not completed. °TCDD in parts per trillion. 10 �Kansas, and Florida sites, respectively. Although the number of analyses have been limited, the data have indicated that TCDD (and phenoxy herbicide) degrade more rapidly in the Kansas soils (Ulysses Silt Loam) than in the Florida soils (Lakeland Sandy Loam), and least rapidly in the Utah desert soils (Lacustrine Clay Loam). The levels of TCDD and herbicide in a soil profile from one of the Bgl.in AFB, Florida, biodegradation plots are shown in Table 6. Initially (e.g., day 414), the data indicate that both the herbicide and the TCDD may be leaching down into the lower soil increments. However, note that the analysis for herbicide in a soil profile obtained on day 557 shows no leaching. The method of collecting soil samples, i.e., by the use of a soil auger contaminated the lower soil increments whereas the trenching technique showed no contamination. The analysis of soil profiles at all three locations for biodegradation indicated that neither the herbicide nor the TCDD appreciably penetrated below the 15-30 cm level. Thus, we believe that the disappearance of the herbicide and the TCDD can be attributed to the action of soil microorganisms, rather than leaching. Data for TCDD are not available at this time (analysis in progress) on the influence of soil amendments (e.g., lime, fertilizer, and organic matter) on the degradation of TCDD in the Eglin AFB, Florida, biodegradation study. However, preliminary indications are that the addition of these amendments in these Florida soils does appear to slightly enhance herbicide degradation. On the other hand, the presence of activated coconut charcoal in 11 �TABLE 6. MOVEMENT OF HERBICIDE ORANGE AND TCDD IN A SOIL PROFILE, EGLIN AFB, FLORIDA. (TCDD IN PARTS PER TRILLION) DAYS AFTER APPLICATION3 4l4b 557C HERBICIDE (PPM) DEPTH (CM) HERBICIDE (PPM) 0-15 1,866 250 824 15-30 263 50 11 30-45 290 <25d <10d 45-60 95 <25d <10d 60-75 160 <25d <10d 75-90 20 <25d <10d TCDD ,. (PPMxlO ) a Composite sample from the plot containing only herbicide r~ Increments obtained by use of a soil auger having cup dimensions of 7.6 x 15.2 on, diameter and length, respectively £i Increments obtained by use of porcelain spatula from the side of 90 cm deep trench < T)etection limit 12 �the Eglin plots, at the 12 on level, prevented degradation of the herbicide and probably also prevented degradation of the TCDD. These data are shown in Table 7. In no instance can it be shown that TCDD levels reached a non-detectable level (less than 10 parts per trillion) within the designated time periods (see Tables 3, 4, and 5). Although biodegradation appears to reduce the level of herbicide and TCDD, the data did not follow simple exponential decay curves. For the mixture 2,4-D and 2,4,5-T herbicides, disappearance was rapid initially, but slowed substantially in the later portions of the test period. With this type of decay kinetics, meaningful half lives are difficult to calculate; however, a reasonable estimate appears to be in the range of 150-210 days. The degradation of TCDD followed a similar decay pattern. However, it appears at this time that the decreased rate of degradation of TCDD as a function of time may be even more pronounced than for the herbicides. One might speculate that the enzymes responsible for herbicide metabolism are inducible and also are involved in TCDD breakdown. If this is the case, it is not surprising that TCDD metabolism slows or ceases when the initial massive concentrations of herbicide are removed. Microbial studies have been conducted in the biodegradation plots in both Utah and Florida ( , ) For the Utah plots, 89. Q Stark, H.E., J.K. McBride, and G.F. Orr. Soil incorporation/ biodegrada.tion of herbicide orange. Vol I. Microbial and baseline ecological study of the U.S. Air Force Logistics Command Test Range, Hill AFB, Utah. Document No. DPG-FR-C615F, US Army Dugway Proving Ground, Dugway, Utah 84022, February 1975. 13 �TABLE 7. COMPARISON OF HERBICIDE ORANGE DEGRADATION RATES IN PLOTS AT THE EGLIN AFB, FLORIDA, SITE, RECEIVING EITHER HERBICIDE, HERBICIDE PLUS SOIL AMENDMENTS, OR HERBICIDE PLUS AMENDMENTS AND CHARCOAL TREATMENT HERBICIDE PLUS HERBICIDE PLUS AMENDMENTS 3 HERBICIDE AND CHARCOAL" AMENDMENTS DEPTH (CM) DEPTH (CM) DEPTH (CM) DAYS AFTER 0-15 15-30 0-15 15-30 0-15 15-30 APPLICATION (PPM) (PPM) (PPM) (PPM) (PPM) (PPM) 5 4,897 302 5,703 232 3,074 134 98 4,280 580 5,422 <50 414 1,866 263 2,015 193 2,767 c <50 c 463 1,217 222 c 824 11 161 c c 557 1,796 c 707 508 <10 c c 2,660 c <50 c 834 438 <10 184 <10 c c 1,293 <10 <10 <10 <10 1,556 <10 amendments included 4.5 kg lime, 13.5 kg organic matter, and 1.4 kg fertilizer (12:4:8 for N,P,K, respectively) uniformly mixed within the top 0-30 cm of soil in the plot. A 1 cm layer of activated coconut charcoal was applied to the trench prior to application of the herbicide. ^ °Not determined. 14 �samples were taken three times throughout the year (summer, winter and spring, 1973-1974), and nacrobial species present (bacteria, actlncmycetes and fungi) were determined. Bacterial counts were higher for soils with greater concentrations of the herbicide and with greater moisture content, but the herbicide, in any concentration, had no significant effect on the mycoflora. For the Florida plots (9), soil samples were taken from all plots in June and August 1974, and in April 1975. Although bacterial and fungal levels were similar for control plots or plots receiving either herbicide or herbicide plus the soil amendments lime, fertilizer, and organic matter, the levels were significantly higher in the plots receiving the activated charcoal. Microorganisms tended to be concentrated in the level which contained the charcoal (0-15 cm), but greatly reduced in number at depths immediately below the charcoal. This effect of increasing the number of microorganisms may have been due to adsorption of growth promoting substances (e.g., nutrients and water) on the surface of the charcoal particles. Although the number of organisms were greater in these plots, the level of herbicide residue was also greatest (Table 7). Apparently, the binding of the herbicide by the charcoal prevented it from being degraded by the microorganisms. These two microbial studies have shown that the application of 2,4-D and 2,4,5-T at massive rates (5,000-40,000 ppm) not only Q Cairney, W.J. Determination of soil microorganism populations in the Eglin AFB, Florida, biodegradation plots. Department of Chemistry and Biological Sciences, United States Air Force Academy, CO, 1975, unpublished. 15 �does not sterilize the soil, but indeed stimulates the growth of certain rnicroflora. That these bacteria, actinomycetes and fungi are proliferating to such an extent indicates that they are probably using the herbicide and TCDD as a carbon source (the exception being the charcoal plots at Eglin), and, as such, are conjxibuting to their degradation. 16 �FATE OF TCDD IN AN ECOSYSTEM The biodegradation plots offered little opportunity to evaluate the ecological effects associated with the use of herbicide Orange or to investigate food chain acramulation of TCDD. Although studies were conducted on the microorganisrns, plants and dominant resident vertebrate and invertebrate animals on and adjacent to these plots (8,9), they were limited to studies of less than 0.5 ha. Therefore, concurrent with the biodegradation studies/ an investigation was initiated on the much larger ecosystem (terrestrial and aquatic) of a unique military test area in Northwest Florida. In support of programs testing aerial dissemination systems, Test Area (TA) C-52A, Eglin AFB Reservation, Florida, received massive quantities of military herbicides. The purpose of these test programs was to evaluate the capabilities of the equipment systems, not the biological effectiveness of the various herbicides. Nevertheless, after several applications, test personnel began to express concern over the potential ecological and environmental hazards that might be associated with continuance of the test program. This concern led to the establishment of a research program in the fall of 1967 to measure the ecological effects produced by the various herbicides on the plant ccnniunity of TA C-52A (10). Ward, D.B. Ecological records on Eglin AFB Reservation—the first year. AFATL-TR-67-157, Air Force Armament Laboratory, Eglin AFB, Florida, 1967. 17 �Geographical and Vegetative Features In 1962, the Armament Development and Test Center (ADTC), Eglin AFB, Florida, established an elaborate testing installation designed to measure deposition parameters of aerially applied herbicides on the Eglin Reservation. The direct aerial application 2 was restricted to an area approximately 2.6 km within TA C-52A in the southeastern part of the reservation. The entire test area 2 covers approximately 5 km and is a grassy plain surrounded by a forest stand that is dominated by long leaf pine (Pinus palustris), sand pine (Pinus clausa), and turkey oak (Quercus laevis). The actual area of test flight paths and herbicide application is in 4 a mechanically cleared area now occupied mainly by broomsedge (Andropogon virginicus), switchgrass (Panicum virgatutn), and other low growing herbaceous vegetation. Sampling Grids and Herbicide Deposition Four separate test grids were established on the 2.6 km2 test area during the 1962 through 1970 testing period. Table 8 indicates the approximate total amount of herbicides (active ingredients) applied on each test grid and the time periods of those applications. Preldmnary Ecological Studies The first in depth animal survey was initiated on the herbicide equipment test grids and surrounding area in 1970 ( 1 . This 1) T>ate, B.D., R.C. Voight, P.J. Lehn, and J.H. Hunter. Animal survey of test area C-52A, Eglin AFB Reservation, Florida. AFATLTR-72-72, Air Force Armament Laboratory, Eglin AFB, Florida, April 1972. 18 �TABLE 8. APPROXIMATE AMOUNTS OF 2,4-D AND 2,4,5-T HERBICIDES APPLIED TO TEST AREA C-52A, EGLIN AFB RESERVATION, FLORIDA TEST GRID GRID AREA (HECTARES) 1 37.25 39,540 (1962-1964)a 39,540 (1962-1964) 2 37.25 15,885 (9416) 16-96 15,885 (1964-1966) 3 37.25 4 97.0 HERBICIDES (KILOGRAMS ACTIVE INGREDIENT) 2,4-D 2,4,5-T 1,263 (97 16) 19,959 (9817) 16-90 19,959 (9817) 16-90 When the major portion of the herbicide was applied. 19 �survey was conducted during the time that aerial spray equipment was actively being tested. The purpose was to determine species variation and distribution patterns on the test grids and surround2 ing 28.5 km . Of the 86 species of vertebrate animals observed or collected, it was concluded that the beach mouse (Beromyscrus polionotus) and the six-lined racerunner (Cnemidophorus sexlineatus) were present in sufficient numbers to conduct population studies. In the spring of 1973, analyses of random soil samples from the test area indicated that low levels (e.g., parts per billion or parts per trillion) of TCDD were persisting in areas (i.e., the flight paths) that had received repetitive aerial applications of 2,4,5-T. Based on the beach mouse populations and the residue information a study was initiated in the summer of 1973 to obtain rodents for analysis of TCDD in body tissues and for examination of TCDD in body tissues and for examination of gross and microscopic evidence of teratogenic and mutagenic effects. A trapping survey was also conducted to study habitat preference of the beach mouse to determine if population distribution was related to vegetative cover. Data from these studies (12) indicated a correlation between the levels of TCDD in rodent liver and soil; however, there was no evidence of toxic histopathology in any rodent tissue. It was also found that indeed the population distribution was related to vegetative cover. *oung, 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 AFB, Florida, 1974. 20 �In the sunnier of 1974 a team of military and civilian scientists undertook a more extensive investigation of the numerous components of the ecosystem of TA C-52A. Using the information from previous studies, they obtained data on the fate of TCDD in soils, rodents, reptiles, aquatic organisms, birds, and vegetation. These results have been published by Young, Thalken and Ward (13), and are summarized in the following sections of this report. Soil Studies of TCDD Residues Soil samples (the top 0-15 cm increment) were collected from all four of the test grids on the test area and analyzed for TCDD. With the exception of Grid I, TCDD residues were in the range of <10 (minimum detection limit) to 30 parts per trillion (ppt, 1x10-12) Soil analysis of 20 separate samples from Grid I detected levels of TCDD in the range of <10 to 1,500 ppt. This wide fluctuation in TCDD concentrations was attributable to the locations of the actual flight paths on the test grid ( . . not all of the 37 ha ie, received the same amount of aerially applied herbicide). It was • also apparent that the massive amounts of herbicides applied to this area in 1962-1964 contained very high levels of the TCDD contaminant. Further analysis of a duplicate soil core, obtained from a site having 110 ppt TCDD, indicated that TCDD was stratified within this top 0-15 cm of soil (Table 9). Young, A.L., C.E. Thalken, and W.E. Ward. Studies of the ecological impact of repetitive aerial applications of herbicides on the ecosystem of test area C-52A, Eglin AFB, Florida. AFATLTR-75-142, Air Force Armament Laboratory, Eglin AFB, Florida, 1975. 21 �TABLE 9. CONCENTRATION OF TCDD IN SOIL PROFILE ( 9 4 17) OF GRID I, TEST AREA C-52A, EGLIN AFB, FLORIDA SOIL PROFILE GRID I APPLICATIONS OF HERBICIDES ( 9 2 1 6 ) 16-94 PARTS PER TRILLION ( P ) TCDD PT 0-2.5 on 150 2.5-5 on 160 5-10 on 700 10-15 on 44 Below (15-90 on) None detectable TABLE 10. NUMBERS OF BEACH MICE COLLECTED DURING THE 1973 AND 1974 STUDIES OF TEST AREA C-52A CONTROL 1973 1974 TOTAL Male 5 12 17 Female 5 10 15 12 11 33_ Fetuses Subtotal 65 TEST 1973 1974 TOTAL Male 26 17 43 Female 18 13 31 Fetuses 25 9 34 Subtotal 108 TOTAL 173 22 �Rodent Studies TRAPPING DATA/HISTOPATHOLOGY. In the 8 weeks of trapping beach mice during the summer of 1973 and 6 weeks during the summer of 1974, 106 specimens were collected from Grid I. Many of the females were pregnant at the time of collection, providing 67 fetuses for examination. Table 10 indicates the numbers of males, females and fetuses collected from Grid I during 1973 and 1974. The only significant lesions seen on histopathologic examinations of 173 adult and fetal beach mice were two instances of moderately severe, multifocal, necrotizing, hepatitis (one test, one control animal) and a single test 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 mature animals that had liver levels of TCDD from 20 ppt to 1,300 ppt (Table 11) 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. There was no evidence to indicate that TCDD was mutagenic nor carcinogenic in the field at the concentrations noted in Table 11. None of the 34 fetuses examined from animals captured on the test grid showed teratogenic defects. This leads one to the conclusion that the levels of TCDD encountered in the field failed to induce observable developmental defects. An analysis of the organ to body weight ratios of each of the control (males and females) and 23 �TABLE 11. COSfCENTRATiai (PARTS PER TRILLION) OF 2,3,7,8-TETr'RACHLORODIBENZOP-DTOXIN (TCDD) IN LIVER AND PELT SAMPLES FROM BEACH MICE, PEROMySCUS IE. IOUONOTLJS, COLLECTED FROM CONTROL AND TCDD-EXPOSED FIELD S T S , 1973 AND : PELT TREATMENT SEX Control 1973 Male and Female 20a ND13 Control 1974 Male 51 40a Control 1974 Female 83 40a Grid I to .fc- YEAR 1973 Male and Female 540 NDb Grid I 1974 Male Grid I 1974 Female a Minimum level of detection determined LIVER 1,300 960 130 140 �test (males and females) using the Wilcoxon Rank Sum Test indicated no statistical differences between field control males and field test males nor field control females and field test females (P^O.05). LIVER AND PELT ANALYSIS. The presence of TGDD 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 m. However, it is emphasized that a mouse (or mice) could have been contaminated in this way, and thus have contaminated pooled samples analyzed for TCDD. Therefore, the use of these data as truly control data must be viewed with caution. The levels of TCDD in the liver of beach mice collected from Grid I substantiated bimccumulation of TCDD; i.e., an accumulation of TCDD in 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 these studies to support biomagnification of TCDD; i.e., an increase in concentration of TCDD in successive organisms ascending the trophic food chain. BURROW AND DIET STUDIES. In all burrows that contained mice a consistant finding was a plug of soil pushed up into the tunnel within the first 2.5 to 25 on of the entrance. Frequently an "escape tunnel" would extend from the nest area to within 2.5 to 25 �15 on of the soil surface. Although the concentration of TCDD on the pelts of beach mice from the test area was only 10-15 percent of that In their livers, Table 11, it was apparent that the mice were continually contaminating themselves as they repeatedly moved in and out of their burrows. The soil data, Table 9, substantiated the presence of a zone of TCDD within the region of the tunnel entrance. Likewise, the location of the escape tunnel suggested that even the nest itself may contain detectable levels of TCDD. An examination of the plant and insect litter within the nests indicated that the beach mouse diet was made up of about 90 percent seeds (based on caryopsis hulls) and about 10 percent insects (based on insect exoskeletons and wings). Four composite seed samples were analyzed for TCDD with no TCDD being detected in any sample (at a minimum detection limit of 1 ppt TCDD). The insect remains are currently being analyzed for TCDD. TCDD LABORATORY UPTAKE EXPERIMENT. Twenty-two beach mice from the designated control area were brought into the laboratory and divided into a "control" group of 10 animals and dusted with 100 mg of alumina gel 10 times over a period of 28 days while the "test" group of 12 animals was dusted with 100 mg alumina gel containing 2.5 ppb TCDD 10 times over the 28 day period. Table 12 indicates control levels and test levels of TCDD in the composite liver samples and on the composite pelt samples of the alumina gel and alumina gel plus TCDD dusted animals. These animals were given complete histopathologic examinations at the completion of the experiment with no differences being 26 �seen between control and test animals. An analysis of the organ to body weight ratios of each of the control males to test males and control females to test females using the Wilcoxon Rank Sum Test indicated a statistical difference involving only the spleens of control male and test male animals at the 95 percent confidence level. This difference was not supported by either histopathological evidence nor by morphometric data as indicated in the Hepatic Ultrastructural Study section which follows. HEPATIC ULTRASTRUCTURAL STUDY. After the liver was removed from 30 beach mice (15 control and 15 from the test area) and weighed, a section was taken from the center of the median lobe. Representative electron micrographs were made from the liver tissue of each animal and the data obtained from each micrograph using a stereology technique. This method of quantitative analysis of the cell ultrastructure used morphometric procedures based on the techniques developed by Weibel et al. (14), as modified by Buchanan (15) With this method, a transparent grid of intersecting lines was placed at random over the micrographs and all the line intersections (points) which were over the required cell structures were counted. All of the points lying over the mitochondria, the damaged (swollen and ruptured) mitochondria, the granular vfeibel, E.R., G.S. Kistler, and W.F. Scherle. Practical stereological methods for morphometric cytology. J. Cell. Biol. 30: 23-38, 1966, Buchanan, G.M. Effect of high dietary molybodenum on rat adrenal cortejc. Unpublished thesis. University of Colorado, Boulder, CO, 1973. 27 �TABLE 12. CONCENTRATION (PARTS PER TRILLION) OF 2,3,7,8TETRACHLORPDIBENZO-P-DIOXIN (TCDD) IN LIVER AND PELT SAMPLES FROM BEACH MICE, PEROMYSCUS POLIONOTUS, DUSTED WITH ALUMINA GEL CONTAINING NO TCDD (CONTROL) OR 2.5 PARTS PER BILLION TCDD (TEST) TREATMENT SEX Alumina Gel Malea Female Alumina Gel + TCDD Male3 Femalea LIVER PELT NDb NDC NDb NDC 125 45 125 89 a Male and female livers composited for analysis Minimum detection level - 10 ppt detection level - 8 ppt TABLE 13. CONCENTRATION (PARTS PER TRILLION) OF 2,3,7,8TETRACHLORODIBENZO-P-DIOXIN (TCDD) IN COMPOSITE SAMPLES OF VISCERA OR TRUNK FROM SIX-LINED RACERUNNERS, CHEMIDOPHORUS SEXLINEATUS, COLLECTED FROM CONTROL AND TCDD-EXPOSED FIELD SITES LOCATION VISCERA Control Site NDa Test Site 360 TRUNK 370 a Minimum detection limit - 50 ppt Tiinimum detection limit - 40 ppt 28 �endoplastnic reticulum (RER) and the agranular endoplasmic reticulum (SER) were then counted. The total area of the cytoplasm was then measured in the same manner. The volume fraction of each structure was determined to be the ratio between the point count of that structure and the total point count of the cytoplasm. In this manner the ratio of mitochondria volume to cytoplasm volume of the hepatic parenchyma! cell was determined for each animal, as was the ratio of damaged mitochondria volume to total mitochondria volume, RER to cytoplasm, SER to cytoplasm, and RER to SER. Using these volume fractions or ratios as quantitative measurements of the structures in question, the hepatic parenchymal cells from treated animals were compared with those from control animals. Similar data were collected from 22 mice brought from the field into the laboratory and exposed to 30 days of external dusting with alumina gel (with or without 2.5 ppb TCDD) . Analysis of the morpheme-trie data using the Wilcoxon Rank Sum Test indicated no statistical differences between field control and field treatment animals, nor were there statistical differences between the control and treatment animals of the dusting study Reptile Studies ANALYSIS OF REPTILE TISSUE. Chemical analysis for TCDD in body parts of the six-lined racerunner indicated significant levels of TCDD in both the visceral mass and in the trunk, Table 13. 29 �Gross post-mortem examinations were performed on 19 racerunners collected from either a control site or from Grid I with no evidence of gross abnormalities seen in any of the specimens. TCDD In Young, Lehn and Mettee (16) conducted species diversities and food chain studies in two aquatic ecosystems draining TA C-52A. Erosion of soil occurred in to a pond on the test area and in to a stream irrmediately adjacent to the area. TCDD levels of 10-35 ppt were found in silt of the aquatic systems, but only at the point where eroded soil entered the water. Species diversity studies of the stream were conducted in 1969, 1970, 1973 and 1974. Insect larvae, snails, diving beetles, crayfish, tadpoles, and major fish species from both aquatic systems were analyzed for TCDD. Species diversity studies indicated no significant change in the composition of ichthyofauna between these dates or a control stream. Concentrations of TCDD (12 ppt) were found in only two species of fish from the stream, sailf in shiner (Nbtropis hypselopterus) and mosquitofish (Gambusia affinis) . The sample of mosquitofish consisted of bodies with heads and tails removed. Two samples of sailf in shiner were analyzed; one containing viscera only and the other bodies less heads, viscera, and caudal fins. Only the viscera contained TCDD. Samples of skin, muscle, gonads, and gut were obtained from spotted sunfish (Lepomis punctatus) Young, A.L., P.J. Lehn, and M.F. Mettee. Absence of TCDD toxicity in an aquatic ecosystem. Weed Sci. See. Amer. Abst. 107, p. 46, 1976. 30 �from the test grid pond. Levels of TCDD in those body parts were 4, 5, 18, and 85 ppt, respectively. Gross pathological observations of the sunfish revealed no significant lesions or abnormalities. TCDD In Birds of TA C-52A Bartleson, Harrison, and Morgan (17) have conducted an extensive survey of the birds of TA C-52A. Between March 1974 and February 1975, they visited study areas twice each week at various times of day and night, and observed a total of 77 species of birds. Of this number, 44 species were observed on Grid I, the area of greatest TCDD residue. The remaining birds were seen in the surrounding clearing and bayheads projecting into the clearning. A small collection of specimens was made for species identification and for TCDD analysis. Only three species could be classified as residents which nest on the test grids. These were southern meadowlark (Sturnella magna), morning dove (Zenaidura macroura), and bobwhite quail (Colinus virginianus). TCDD residues were found in meadowlark livers (100-1,020 ppt) and in the stomachs and stomach contents (46 ppt) of these same birds. An analysis of liver and fat tissue from doves indicated concentrations of 50 ppt. An analysis of seed in the crop of the doves showed no detectable residue of TCDD. Two routes of TCDD contamination Bartleson, F.D., D.D. Harrison, and J.D.-Morgan. Field studies of wildlife exposed to TCDD contaminated soils. AFATL-TR-75-49, Air Force Armament Laboratory, Bglin AFB, Florida, 1975. 31 �were proposed for these birds. The first was through the process of dusting and subsequent ingestion of contaminated soil while preening. A second possible route was through the ingestion of soil-borne insects from the test grid; an analysis of a single composite insect, sample indicated a concentration of 40 ppt TCDD. Vegetative Succession Studies on TA C-52A TCDD analysis of vegetation has been limited to seed samples collected in support of the rodent diet study. No TCDD was found in four samples of seeds collected from vegetation on Grids I or II. The minimum level of detection was 1 ppt. A vegetative succession study has been conducted by Young and Hunter (18) to document the re-vegetation of an area denuded first by mechanical means and then by hundreds of applications of phenoxy herbicides. Nine months (June 1971) after the last defoliant-equipment test mission, a detailed survey of the vegetation was initiated. The area was divided into a grid of 169 sections (each 122 by 122 m), and within each section the percentage vegetative coverage was visually ranked as Class 0, 0-5 percent; I, 5-20 percent; II, 2040 percent; III, 40-60 percent; IV, 60-80 percent; and V, 80-100 percent. Three sections within each class were selected at random and surveyed for dicotyledonous plants. An unsprayed area 0.32 km northwest of the test area was also surveyed. In June 1973, each Young, A.L., and J.H. Hunter. A long-term field study of vegetative succession following repetitive application of phenoxy herbicides. Weed Sci. Sec. Amer. Abst. 1977. 32 �of these areas was again surveyed, but in addition/ a square-foot 2 (0.093m ) analysis technique was performed in 15 additional sections. These sections were randomly selected and within each 2 section, nine areas, each 0.093m , ware analyzed for species composition and ground cover density. Both methods of vegetative survey were repeated in June 1976. The number of dicotyledonous species increased from 74 in 1971 to 107 in 1973, and to 123 in 1976. In 1971, 20 percent of the test area had less than 20 percent vegetative cover, while 26 percent of the test area had more than 60 percent vegetative cover. In 1976, no sections had less than 20 percent vegetative cover, but over 73 percent of the test area had a cover of more than 60 percent. The major grass species were Panicum yirgatum and Panicum lanuginosum. The major dicotyledon was Diodia tores in 1971, but was replaced by Chrysqpsis graminifolia in 1976. These data demonstrate the rapid invasion of dicotyledonous species despite the unusually heavy applications of phenoxy herbicides. As a concluding remark, Test Area C-52A, Eglin AFB, Florida, has offered a unique opportunity to examine the effects of longterm, low-level exposure of biological systems to TCDD. Perhaps when the herbicide 2,4,5-T (contaminated by TCDD) was first applied to the test area (1962-1964), the levels of TCDD that accumulated on the soil may have been sufficiently high to be toxic, although there is no mention of animal deaths in the records of test missions for this area. It is of interest to note that in the 33 �Italian TCDD episode, an estimated 0.9 to 4.5 kg TCDD were 2 disseminated on an area of 1.4 km . This is approximately equal to 6.5 to 32 g/ha. Grid I on Test Area C-52A probably received between 0.07 and 1.86 kg TCDD on an area of 37 ha, or approximately 2 to 50 g TCDD/ha over a 2-year period. This range of values was arrived at using the arithmetic mean and maximum concentration of TCDD contamination of the herbicide Orange presently in the United States Air Force inventory. 34 �LABORATORY AND GREENHOUSE EXPERIMENTS WITH TODD Two additional studies have been conducted in support of the previous investigations of TCDD in field ecosystems. One of these studies has been conducted by Cupello and Young (19) on the potential uptake from soil of 14C-TCDD by plants. In this study, 2,240 kg active ingredient Orange herbicide/ha, containing 14 ppm 14C-TCDD, was placed beneath the soil surface in specially designed growth boxes containing 100 plants of Sorghum (Sorghum vulgare) per box. The plants were grown under controlled environmental conditions for 9 weeks; 14-hour photoperiod, diurnal temperature of 35 ± 2°C and 15 ± 1°C, and a relative humidity of 60 and 85 percent, day and night, respectively. On day 64 the plants were cut at the soil surface, ground in a Wiley Mill, and extracted with hexane in a Soxhlet Extraction apparatus for 4 hours. The TCDD in the extract was then concentrated by using the Dow Chemical Company Analytical Method ML-AM 73-97. The "TCDD concentrate" was quantitatively transferred to a scintillation vial using benzene, and 15 cc of Aquasol added to it. Analysis of the counting data from a liquid scintillation counter indicated no significant uptake of hexane extractable 14C-TCDD activity in the plant material. An analysis was also performed on the plant tissue prior to hexane extraction, and after hexane extraction for 4 hours. These plant samples were combusted in a 19Cupello, J.M., and A.L. Young. Radiochemical bioassay of TCDD uptake in plant material. Department of Chemistry and Biological Sciences, United States Air Force Academy, CO, 1976, unpublished. 35 �Packard Model 306 sample oxidizer, the OCL collected in Packard Carbo-Sorb, this solution diluted in Packard Permafluor-V, and the filler counted in a liquid scintillation counter. These data indicated the presence of sufficient C activity in the unextracted plant material to be equivalent to approximately 430 ppt TCDD in the plant tissue. This activity was not significantly reduced by hexane extraction. This relatively high 14C activity in the plant tissue could be explained by one of at least four hypotheses. It could represent the presence of (1) bound (non-hexane soluble) TCDD, (2) a soil biodegradation product of TCDD that was taken up and bound by the plant, (3) a metabolic breakdown product of the TCDD that was formed after incorporation of the TCDD into the plant/ or (4) a contaminant in the original C TCDD stock solution that eventually found its way into the plant either as the original contaminant or as a metabolic of it. A second study has been conducted by Bartleson, Harrison, and Morgan (17) on the effect of tilling TCDD contaminated soil. One cubic meter of soil was collected from Grid I, TA C-52A, and removed to the laboratory. Four samples were taken from the uniformly mixed soil, analyzed and found to contain 1,100 ppt (2 samples) and 1,300 ppt (2 samples) TCDD. The contaminated soil was placed in two groups of four pots (20 cm deep and 20 cm in diameter). The four pots in each group were treated as follows: two were left outside and exposed to natural elements, and two were placed in a greenhouse and watered with a nutrient solution. 36 �One of the two containers in each location was left undisturbed, and the other was stirred (tilled) weekly with a spatula. This stirring was not complete, and soil in the bottom of the pots was relatively undisturbed. The soil in each of the pots was emptied into a clean tray and mixed thoroughly before samples were collected and analyzed for degradation of TCDD. The data shown in Table 14 suggest that sunlight, tilling and perhaps increased temperatures (associated with the greenhouse) may promote more rapid degradation of TCDD. There also may be an additive effect from use of nutrients. 37 �TABLE 14. DEGRADATION OF TCDD (PARTS PER TRILLION) IN A GREENHOUSE EXPERIMENT, EGLIN AFB, FLORIDA LENGTH OF EXPOSURE 0 (PPT) 9 weeks (PPT) 23 weeks (PPT) Tilled 1,200 1,100 520 Untilled 1,200 1,000 530 Tilled 1,200 640 460 Untilled 1,200 810 530 TREATMENT Full Sunlight3 Greenhouse Samples placed outside of greenhouse Samples watered with a nutrient solution 38 �RECOMiyENDATIONS FOR DECONTAMmTION OF TCDD EXPOSED FIELD SITES Although there are many potential options for the decontamination of an area exposed to TCDD (see reference 4), data provided in this report would suggest that one of the most feasible options would be soil incorporation/biodegradation. The data base used in selecting this option is as follows: 1. TCDD may persist (in biotic and abiotic components) for long periods of time when initially present at extremely high concentrations on the soil surface. 2. TCDD will accumulate in the tissues of rodents, reptiles/ birds/ fish/ and insects when these organisms are exposed to TCDD contaminated soils (however, the levels of TCDD in the tissues apparently do not exceed the levels of TCDD found in the environment). 3. Organisms tolerate/ i.e./ based on no observed deleterious effects, soil levels between 10-1,500 ppt TCDD. 4. TCDD is degraded by soil microorganisms, especially when in the presence of other chlorinated hydrocarbons. 5. TCDD is degraded in the presence of sunlight. 6. Movement of TCDD in the abiotic portions of the environment can be by wind or water erosion of soil particles, but leaching by water alone does not appear to occur. 7. TCDD is probably not readily released or degraded in the environment when bound to activated coconut charcoal. 39 �Specific Re<xirntrendations In locations where accidental TCDD contamination covers Significant geographical area, e.g., many hectares, an in situ biodegradation program may be most effective in reducing levels of TCDD residues. Incorporation of organic material, lime, and fertilizer to enhance microbial activity may be advantageous. The biodegradation site should be tilled frequently so as to expose residue to sunlight. Watering of the site is recommended to reduce wind movement of contaminated particles and to enhance biodegradation. In locations where a limited area has been exposed to accidental contamination of TCDD, the top 0-15 cm of soil should be removed and taken to an isolated area where biodegradation procedures can be conducted. Similar treatments should be applied to these plots as would be for an in situ program. Protective clothing should be worn by all site personnel. The contaminated clothing should be incinerated at an approved incinerator. Following use, all equipment should be rinsed with an organic solvent (e.g., diesel fuel) to remove TCDD residue. The solvent containing TCDD residue may be collected in activated coconut charcoal and either incinerated or placed in an approved sanitary landfill, although if a sufficiently isolated land area is available, biodegradation may be feasible. It should be noted that some TCDD residue will remain in a contaminated site. However, research on the ecosystem at Test Area C-52A, Eglin AFB, Florida, indicated that organisms do have 40 �a tolerance to low levels of TCDD. Therefore, in those areas having soil residues below 1 ppb, further efforts to decontaminate the area are not practical. These areas should, however, be fenced and posted to prevent livestock and human exposure. 41 � 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. 017 Folder The folder containing the original item. 0193 Series The series number of the original item. Series 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. Charles E. Thalken Eugene L. Arnold James M. Cupello Lorris G. Cockerham Description An account of the resource <strong>Corporate Author: </strong>Department of Chemistry and Biological Sciences, USAF Academy, Colorado Date A point or period of time associated with an event in the lifecycle of the resource 1976-10-01 Title A name given to the resource Fate of 2,3,7,8-Tetrachlorodibenzo-P-Dioxin (TCCD) in the Environment: Summary and Decontamination Recommendations Subject The topic of the resource biodegradation ecological fate soil decontamination Eglin AFB https://www.nal.usda.gov/exhibits/speccoll/files/original/5c56b76a52235883573a7e79bc378e2f.pdf 2abd017e641043200a441dac5938a007 PDF Text Text Item ID Number °1165 Author Young, Alvin L. United States Air Force Occupational and Environmental Report/Article Title The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange and its Associated Dioxin Journal/Book Title Year Month/Day Color Oct ber ° D Number of Images 263 DeSCrlptOU NOtBS A'v'n *-• Young filed this item under the category "Human Exposure to Phenoxy Herbicides and TCDD" Thursday, April 05, 2001 Page 1165 of 1180 �• 4 MTlftrO A-L. eVdL Report OEHLTR-78-92 r ^j-—-' j •••4>^ USAF OEHL TECHNICAL REPORT THE TOXICOLOGY, ENVIRONMENTAL FATE, AND HUMAN RISK OF HERBICIDE ORANGE AND ITS ASSOCIATED DIOXIN Alvin L. Young, Captain, USAF John A. Calcagni, Lieutenant Colonel, USAF, MC Charles E. Thalken, Lieutenant Colonel, USAF, VC James W. Tremblay, Major, USAF, BSC October 1978 Final Report I Approved for public release; distribution unlimited PREPARED FOR: The Surgeon General United States Air Force Washington, D.C. 20314 USAF Occupational and Environmental Health Laboratory Aerospace Medical Division (AFSC) Brooks Air Force Base, Texas 78235 �NOTICES This report has been released to the National Technical Information Service, 5285 Port Royal Road, Springfield, Virginia 22161, for sale to the general public. *** Qualified requestors may obtain copies of this report from Defense Documentation Center (DDC), Cameron Station, Alexandria, Virginia 22314. *** This technical report has been reviewed and is approved for publication. WILLIAM E. MABSON, Colonel, USAF, BSC Commander �UNCLASSIFIED S E C U R T t V ' t V A S S I F I C A T I O N OF T H I S P A G E (When Data Entered) READ INSTRUCTIONS BEFORE COMPLETING FORM REPORT DOCUMENTATION PAGE 2. GOVT ACCESSION NO 1. REPORT NUMBFR 3. RECIPIENT'S C A T A L O G NUMBER USAF OEHL - 7 8 - 9 2 i t " . ; ':" C-inrt Subtitle) 5. TYPE OF REPORT & PERIOD COVERED The Toxicology, Environmental Fate and Human Risk of. • i , Herbicide Orange and Its • Associated. Dioxin - • , Fin; I 6. PERFORMING ORG. REPORT NUMBER 3 ?. AUTHOR,,, /\yvin L. young, Captain, USAF John A. Calcagni, Lieutenant Colonel, USAF, MC Charles E. Thai ken, Lieutenant Colonel, USAF, VC : James U. Tremblay, Major. USAF, BSC 8. C O N T R A C T OR G R A N T NUMBERfa.) 9. P E R F O R M I N G O R G A N I Z A T I O N N A M E AND ADDRESS 10. PROGRAM ELEMENT, PROJECT, TASK A R c A & WORK UNIT NUMBERS US Air Force Occupational and Environmental Health Laboratpry Brooks AFB TX 78235 11. 12. CONTROLLING OFFICE NAME AND ADDRESS REPORT DATE October 1978 The Surgeon General US Air 'Force 13. NUMBER OF PAGES Washington, DC 20314 14 M O N I T O R I N G A G E N C Y N A M E 4 ADDRESS^/ different Iron Controlling Olficv) 15. S E C U R I T Y CLASS, (at this report) Unclassified 1Sa. DECLASSIFI CATION/ DOWN GRADING SCHEDULE 16. D I S T R I B U T I O N S T A T E M E N T (ol this Report) Approved for public release; distribution unlimited. 17. D I S T R I B U T I O N STATEMENT (ol the abstract entered In Block 30, It different 18. tram Report) S U P P L E M E N T A R Y NOTES Authors: A.L. Young, PhD J.A. CaVcagni, MD C.E. Thalken, DVM J.W. Tremblay, P.E. 19. KEY WORDS (Continue on reverse aide II nocessary and identity by block number) clorinated phenols 2,4-dichlorophenoxyacetic acid (2,4-D) dioxin environmental monitoring herbicides Herbicide Orange phenoxy herbicides Pacer HO Ranch Hand South Vietnam toxicity - animal toxicity - human 20. A B S T R A C T fContinue on reverse aide If necnssary and Identify by block number) The use of herbicides in South Vietnam between 1962 and 1971 was reviewed, including the nature and quantities of herbicides used, their handling and application.' Emphasis was placed on Herbicide Orange, a 50:50 mixture of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), with its associated contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The atrisK US military population in South Vietnam was defined to establish the potential for exposure in handling "and application of Herbicide Orange. The environmental fate of the phenoxy herbicides and TCDD was reviewed to evaluate E O . T I O M O F I - . ' 6 5 I S OBSOLETE U N C L A S S I F I FD SS.CURITY CLASSI f : » iT, r;) A G E (Wien Deta Entt-rr ' �UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAOBftWiwi £>•<« Entered) Item 19. Key Words (cont): 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) Item 20. Abstract (cont): the potential for human risk associated with exposure to areas previously treated with Herbicide Orange. The occupational and environmental aspects of the project to incinerate at sea 2.22 million gallons of Herbicide Orange durinc the summer of 1977 were summarized to assess the potential for human exposure in handling large quantities of the material. Scientific data were reviewed on incidents and episodes involving suspected poisoning of humans or animals by phenoxy herbicides or TCDD. Literature dealing with animal toxicology and the effects of human exposure to 2,4-D, 2,4,5-T and TCDD was reviewed to correlate exposures with symtomatology. iv UNCLASSIFIED SECURITY CLASSIFICATION OF THIS PAGEfHTion Data Entered) �PREFACE The use of herbicides in support of tactical military operations in South Vietnam from 1961 to 1971 has had (and continues to have) a negative impact on the use of pesticides by numerous facets of our society. fjrior to the Vietnam conflict, herbicides were considered invaluable to agriculture, innocuous to human life and of little environmental concern. Today, seven years after the last herbicide mission in Vietnam, these same herbicides are the center of scientific debate involving not only ecological but also medical, legal and political issues. The United States Environmental Protection Agency (EPA) has recently issued a Notice of Rebuttable Presumption Against Registration (RPAR) of pesticides containing one of these "Vietnam" herbicides, while at the same time some Veterans of the Vietnam Conflict have reported medical problems they claimed were the result of herbicide exposure while assigned to military duties in Vietnam. In April 1978, the Surgeon General of the United States Air Force (USAF) tasked personnel'of the USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas with updating previous scientific assessments of possible adverse effects to human health resulting from exposure to the herbicides, especially Herbicide Orange used in South Vietnam during the Vietnam Conflict. The present report was assembled using the latest available published scientific information, previously unpublished data, and observations from medical and scientific personnel intimately associated with the herbicides in question. The report reviews the use of phenoxy herbicides in Vietnam, their environmental fate, and pertinent animal and human toxicological studies. In addition, a description is given of the 1977 military operation for the disposal of Herbicide Orange emphasizing the facets of environmental monitoring and industrial hygiene. The document concludes with an assessment of the risk to human health following exposures to the phenoxy herbicides. Special emphasis was placed upon the chemistry, environmental fate and toxicology of the trichlorophenoxy herbicide contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin). The authors are indebted to Kenneth C. Back, PhD, Chief Toxicology Branch, Toxic Hazards Division, United States Air Force 6570th Aerospace Medical Research Laboratory, Wright-Patterson AFB Ohio for his critical review of-this document and the many recommendations he made to improve the qualify of the discussions on toxicology. Special thanks are expressed to Rodney W. Bovey, PhD, United States Department of Agriculture", Texas A&M University, College Station, Texas, for his assistance in providing copies of the foreign literature on the phenoxy herbicides. �The services of many staff members and consultants of the United States Air Force Occupational and Environmental Health Laboratory are acknowledged. Special acknowledgement is made to Mrs Joyce G. Kidd who served as the general editor, and to the following typists who willingly worked numerous overtime hours in preparing this manuscript: Ruth S. Bledsoe; Yolanda Carrisalez; Irma Ledesma; Lorraine M. Polonis; Nancy L. Ragan and Trina Roark. �CONTENTS Page PREFACE v CHAPTER I .THE USE OF HERBICIDES IN SOUTH VIETNAM I. INTRODUCTION 1-1 II. THE HERBICIDES USED IN SOUTH VIETNAM A. Historical B. Descriptions of the Herbicides Used in Operation RANCH HAND 1-1 1-1 1-3 C. Quantities of Herbicides Sprayed in South Vietnam D. Land Area Sprayed with Herbicides in South Vietnam III. THE AIRCRAFT, SPRAY SYSTEMS AND MISSION CONCEPT IN OPERATION RANCH HAND A. Historical B. Spray Systems and Characteristics of the RANCH HAND Aircraft C. Mission Concepts 1-8 1-11 I-11 1-11 1-14 1-15 IV. PERTINENT DEPLOYMENT AND BIOLOGICAL FACTORS OF THE HERBICIDES 1-18 A. Use Patterns of Individual Military Herbicides B. Canopy Penetration of Defoliants « V. 1-18 1-20 ESTIMATED QUANTITIES OF INDIVIDUAL CHEMICALS SPRAYED IN SOUTH VIETNAM A. Herbicide Orange and its Components 2,4-D, 2,4,5-T and TCDD B. Military Projects that Involved Handling Herbicides Orange, Purple, Pink or Green VI. SUMMARY 1-21 1-29 1-29 LITERATURE CITED 1-32 LIST OF TABLES 1. Selected physical, chemical and toxicological properties of the three major military herbicides used in South Vietnam, 1962 - 1971. VI 1 1-5 �2. Number of gallons of military herbicide procured by the U.S. Department of Defense and disseminated in South Vietnam during the period January 1962 - December 1964. 1-9 3. Estimated number of gal of military herbicide procured by the U.S. Department of Defense and disseminated in South Vietnam during the period January 1965 - February 1971. 1-10 4. Comparison of data from three sources of the estimated number of acres treated in South Vietnam during the period of January 1962 - February 1971. Data make noi allowance for multiple coverage. 1-12 5. The number of acres treated in South Vietnam, 1962 - 1971, with military herbicides within the three major vegetational categories. Data represent areas receiving single or multiple coverage and for 90 percent of all areas treated. 1-13 6. Concentration, ppm, of TCDD in samples of Herbicides Orange and Purple. 1-23 7. Composition, percent, of selected samples of Herbicide Orange in relation to military specifications. 1-27 8. Estimated quantities of herbicides and TCDD disseminated in South Vietnam from January 1962 - February 1971. 1-28 9. Data on the major military projects involved in the handling and/or spraying of Herbicides Orange, Purple, Pink or Green in support of military programs in South Vietnam. 1-30 LIST OF FIGURES 1. Chemical structure and nomenclature of the major herbicides used in South Vietnam, 1962-1971. Formulas A and B comprised Orange, C and D - White, and E was Blue. 1-6 2. Structure and physical/chemical characteristics of 2,3,7,8- tetrachlorodibenzo-p-dioxin, TCDD or dioxin. VII I 1-22 �CHAPTER II DISPOSAL OF HERBICIDE ORANGE Page I. INTRODUCTION 11-1 II. HISTORICAL BACKGROUND II-l III. DESCRIPTION OF LAND-BASED OPERATIONS A. NCBC, Gulfport MS B. Johnston Island I1-2 II-3 II-4 * IV.. LAND-BASED OPERATIONS MONITORING PROGRAMS A. Monitoring Equipment and Procedures B. Analytical Procedures and Methodologies V. LAND-BASED MONITORING RESULTS II-5 II-6 II-7 II-8 A. NCBC, Gulfport MS II-8 B. Johnston Island 11-10 VI. SUMMARY AND CONCLUSIONS 11-15 LITERATURE CITED , 11-19 LIST OF TABLES 1. Results of industrial hygiene air samples collected inside the dedrumming facility Project PACER HO NCBC, Gulfport MS, 24 May 10 June 1977. II-9 2. Results of ambient air samples collected at Gulfport MS, Project PACER HO, 24 May - 10 June 1977. 11-11 3. Results of industrial hygiene air samples collected inside the dedrumming facility, Project PACER HO Johnston Island, first loading 27 July - 5 August 1977 11-12 4. Results of industrial hyigiene air samples collected inside the dedrumming facility Project PACER HO, Johnston Island, second loading, 17-23 August 1977. 11-13 5. Results of industrial hygiene "breathing zone" samples collected inside the dedrumming facility Project PACER HO, Johnston Island. IX 11-14 �6. Results of downwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. 7. Results of upwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. 11-16 11-17 CHAPTER III ENVIRONMENTAL FATE OF 2,4-D, 2,4,5-T AND TCDD I. THE ENVIRONMENTAL FATE OF THE PHENOXY HERBICIDES III-l III-l IJ.I-4 III-6 THE ENVIRONMENTAL FATE OF TCDD 111-7 A. Analytical Limitations 111-7 B. Laboratory Studies of TCDD C. Field Studies of TCDD III-7 III-ll D. Environmental Production of TCDD E. Photodegradation of TCDD III. III-l A. Physical/Chemical Factors Influencing Disappearance of Herbicide B. Biological Degradation of the Phenoxy Herbicides C. Accumulation and Metabolism of Phenoxy Herbicides in Animals II. INTRODUCTION 111-20 111-21 IV. SUMMARY LITERATURE CITED II1-22 III-24 LIST OF TABLES 1. Concentrations of TCDD, parts per trillion, in the Herbicide Orange biodegradation plots, AFLC Test Range, Utah, four years after applications, 111-15 2. Concentration of TCDD in soil profile of Grid 1, Test Area C-52A, Eglin AFB, Florida. 111-17 LIST OF FIGURES 1. Semi-loaarithmic olot of soil concentrations (parts per million) of herbicide in Herbicide Orange biodegradation studies at Eqlin AFB, Florida, and Hill AFB, Utah. TIT-13 �2. Semi-logarithmic plot of soil concentrations (parts per trillion) of TCDD in Herbicide Orange biodegradation studies at Eglin AFB, Florida, and Hill AFB, Utah. 111-14 CHAPTER IV THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS I. II. INTRODUCTION IV-1 REVIEW OF 2,4-D TOXICITY IN ANIMALS . A. The Acute and Short-Term Toxicity Potentials of 2,4-D IV-3 IV-3 B. The Subacute and Chronic Toxicity Potentials of 2,4-D . . IV-5 C. Absorption, Distribution and Excretion of 2,4-D D. Embryotoxic, Fetotoxic and Teratogenic Potentials of 2,4-D IV-17 E. Carcinogenic and Tumorigenic Potentials of 2,4-D IV-20 F. Mutagenic and Cytogentic Potentials of 2,4-D in Animals IV-23 REVIEW OF 2,4,5-T TOXICITY IN ANIMALS IV-26 A. The Acute and Short-Term Toxicity Potentials of 2,4,5-T III. IV-16 IV-26 B. The Subacute and Chronic Toxicity Potentials of 2,4,5-T C. Absorption Distribution and Excretion of 2,4,5-T IV-26 IV-31 D. Embryotoxic, Fetoxic and Teratogenic Potentials of 2,4,5-T IV-36 E. Carcinogenic and Tumorigenic Potentials of 2,4,5-T IV-46 F. Mutagenic and Cytogenic Potentials of 2,4,5-T IV-47 IV. REVIEW OF TCDD TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of TCDD IV-50 IV-50 �B. The Subacute and Chronic Toxicity Potentials of TCDD IV-52 C. Absorption Distribution and Excretion of TCDD IV-56 D. Embryotoxic, Fetoxic and Teratogenic Potentials of TCDD IV-61 E. Carcinogenic and Tumorigenic Potentials of TCDD F. Mutagenic and Cytogenic Potentials of TCDD IV-71 SUMMARY OF THE LITERATURE REVIEW OF THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS IV-72 A. 2,4-D IV-72 B. 2,4,5-T IV-73 C. TCDD IV. IV-63 IV-74 • LITERATURE CITED IV-76 LIST OF TABLES 1. Summary of literature data on the no-effect, LD^Q and LD-iuu levels of the acute toxicity of 2,4-D in nn animals IV-6 2. Summary of literature data on the subacute and chronic toxicity of 2,4-D in animals IV-13 3. Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4-D in animals IV-21 4. Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4-D in animals IV-24 5. Summary of literature data on the no-effect LD50 and LD-inn levels of the acute toxicity of 2,4,5-T in animals IV-27 6. Summary of literature data on the subacute and chronic toxicity of 2,4,5-T in animals IV-32 7. Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T in animals IV-41 8. Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4,5-T in animals XII IV-48 �9. Summary of literature data on the no-effect, 1050 'and levels of the acute toxicity of TCDD for animals IV-53 10. Summary of literature data on the subar.ute and chronic toxicity of TCDD in animals IV-57 11. Summary of literature data on the embryotoxic, fetoxic and teratogenic potentials of TCDD in animals IV-64 12. Summary of literature data on the carcinogenic and tumorigenic potentials of TCDD in animals IV-69 CHAPTER V 2,4,5-T/TCDD EPISODES I. II. INTRODUCTION V-l INDUSTRIAL EXPERIENCES V-2 A. Industrial Processes B. Industrial Episodes ' V-2 . V-5 III. VIETNAM EPISODE IV. , EASTERN MISSOURI HORSE ARENA EPISODE V. THE SEVESO, ITALY EPISODE VI. V-12 V-17 V-19 GLOBE, ARIZONA EPISODE V-21 VII. THE SWEDISH LAPLAND EPISODE V-24 VIII. THE AWAMUTU, NEW ZEALAND EPISODE IX. DISCUSSION OF LITERATURE AND CONCLUSIONS X. SUMMARY V-26 V-28 V-32 LITERATURE CITED V-33 LIST OF TABLES 1. Total United States production and use of 2,4,5-T herbicide for the period 1961 through 1969. V-3 2. Industrial incidents associated with the manufacture of chlorinated phenols. V-7 XTM �3. Some clinical features observed in cases of chloracne associated with production of 2,4,5-T and other chlorinated phenols. V-10 LIST OF FIGURES 1. Synthesis scheme for production of the n-butyl ester 2,4,5-T (NBE 2,4,5-T) and site where formation of TCDD may occur. V-4 CHAPTER VI HUMAN EFFECTS OF HERBICIDE ORANGE I. II. INTRODUCTION . VI-1 PHARMACODYNAMICS VI-1 A. B. C. D. VI-1 VI-1 VI-2 VI-4 Percutaneous Entry of Phenoxy Herbicides Ingestion of Phenoxy Herbicides Tissue Analyses for the Phenoxy Herbicides Pharmacodynamics "of TCDD III. ADVERSE EFFECTS VI-4 A. Limitations of Referenced Studies B. Phenoxy Herbicides That Do Not Contain TCDD C. Trichlorophenol (TCP), 2,4,5-T and TCDD D. Cancer VI-12 VI-27 CONCLUSIONS VI-28 A. Pharmacodynamics B. Effects of the Herbicides VI-28 VI-29 C. Effects of TCDD IV. VI-4 VI-6 VI-30 V. SUMMARY VI-30 LITERATURE CITED VI-31 LIST OF TABLES 1. Levels (part per million) of phenoxy herbicides in human tissue or body fluid following ingestion of fetal dose. VI-3 2. TCDD levels in a human body. VI-3 3. Distribution of symptoms in 292 workers employed in the production of the amine salt and the butyl ester of 2,4-D VI-7 xiv �4. Distribution of adverse effects in case reports following the ingestion of non-TCDD containing phenoxy herbicides. VI-9 5. Distribution of reported adverse effects following exposure of field workers and applicators to 2,4-D. VI-11 6. Organ systems reported affected after occupational exposure to PCP, TCP, 2,4,5-T or TCDD. VI-13 7. Signs, symptoms, and disorders reported after occupational exposure to TCP, 2,4,5-T or TCDD. VI-14 8. Special clinical studies following occupational exposure to TCP, 2,4,5-T or TCDD. VI-15 9. Organ systems reported affected after exposure to TCP and TCDD following an industrial accident. VI-17 10. Signs, symptoms and disorders reported after exposure to TCP and TCDD following an industrial accident. VI-18 11. Special clinical studies after exposure to TCP and TCDD following an industrial accident. xv VI-19 �CHAPTER I THE USE OF HERBICIDES IN SOUTH VIETNAM I. INTRODUCTION The introduction of herbicides in 1962 into the armed conflict in Vietnam represented an application of a new technique for modern warfare. Their use in a defensive role was for defoliation. Their use in offensive roles was for food crop denial. The herbicides most widely employed were the phenoxyacetic acids. They were extensively used for almost a decade throughout the forested, semi-populated, regions of South Vietnam. Assessments -of their ecological impact in South Vietnam have been published (see Chapter V). An assessment of the effects of herbicides on the human indigenous populations of South Vietnam has also been conducted (11). No assessment has been made of potential adverse human effects of the phenoxy herbicides or the toxic contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on personnel of the U.S. Military forces. Adverse human effects in military personnel due to the herbicides or the contaminant would be predicated on the assumption that an exposure occurred. The presence of military spray aircraft or the observation that drums of herbicide were stored on a military installation, or even smelling the odor of "herbicides" in the air does not necessarily constitute an exposure to the herbicide per se. An exposure would have had to involve physical contact for a sufficient period of time to permit the chemical(s) to penetrate the body. This chapter examines those factors that would have influenced the likelihood of such exposures. They include: 1. the nature of the herbicides used in South Vietnam, 2. the nature of the herbicide applications, 3. the procedures employed in the handling of the herbi- cides, and 4. the quantities of individual chemicals sprayed in South Vietnam. Detailed examinations of these "parameters" are reported in the following sections. II. THE HERBICIDES USED IN SOUTH VIETNAM A. Historical The discovery and early history of the phenoxy herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) have been reviewed by Peterson (31). Peterson noted 1-1 �that the effectiveness of these plant growth regulators as "herbicides" was determined in mid-1944 field trails at Beltsville and Camp Detrick (now Fort Detrick), Maryland. The outstanding effectiveness of these two herbicides in controlling the growth of broad-leaved plants and weeds, coupled with their apparently low mammalian toxicity and low application rates, resulted in their rapid acceptance in world agriculture. Peterson (31) reported that the annual production of 2,4-D alone exceeded 14,000 pounds in 1950 and 36,000,000 pounds in 1960. Irish et al (26) and Darrow et al (14) have documented the early military use of the phenoxy herbicides. They reported that the earliest aerial spray trials (conducted in military aircraft) occurred in 1944 and 1945. Three different mixtures of 2,4-D were used in these early tests. Although herbicides were not used in tactical military operations in World War II, a small program for screening potential herbicides for military use continued after the War. By 1951, personnel at Fort Detrick had determined that the vegetationcontrol chemicals of choice were mixtures of the butyl esters of 2,4-D and 2,4,5-T. In 1959, the Crops Division, Fort Detrick, conducted the first large-scale military defoliation effort at Fort Drum, New York. This project involved the aerial application of the butyl esters of 2,4-D and 2,4,5-T to approximately four square miles of vegetation. Its success prompted the Office of the Secretary of Defense (OSD) in May 1961 to request that the Crops Division determine technical feasibility of defoliating jungle vegetation in the Republic of Vietnam. As part of a project to evaluate herbicides and defoliation techniques (Project AGILE) in Southeast Asia, Brown (7) conducted eighteen different aerial, spray tests (defoliation and anticrop) 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. Also, other factors had to be considered, such as availability in large quantity, costs and known or proven safety in regard to their toxicity to humans and animals" (7). The results of these tests were that significant defoliation and anticrop effects could be obtained with two different mixtures 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. Brown (7) reported that the first shipment of Herbicides Purple and Blue was received 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 1-2 �RANCH HAND. These were code-named Pink and Green and will be described in the subsequent section. By January 1965, two additional military herbicides had been evaluated and brought into the spray program. These were code-named Orange and White, and are also described below. Herbicide Orange replaced all uses of Purple, Pink, or Green and eventually became the most widely used military herbicide in South Vietnam. In April 1970, the Secretaries of Agriculture; Health, Education and Welfare, and the Interior jointly announced the suspension of certain uses of 2,4,5-T. These suspensions resulted from published studies indicating that 2,4,5-T was a teratogen. Subsequent studies revealed that the teratogenic effects had resulted from a toxic contaminant in the 2,4,5-T, identified as 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD). 'Subsequently, the Department of Defense suspended the use of Herbicide Orange (4). At the time of the suspension, the Air Force had an inventory of 1.37 million gallons of Herbicide Orange in South Vietnam and 0.85 million gallons at the Naval Construction Battalion Center (NCBC), Gulfport, Mississippi. In September 1971, the Department of Defense directed that the Herbicide Orange in South Vietnam be returned to the United States and that the entire 2.22 million gallons be disposed of in an environmentally safe and efficient manner. The 1.37 million gallons were moved from South Vietnam to Johnston Island, Pacific Ocean for storage in April 1972. B. Descriptions of the Herbicides Used in Operation RANCH HAND The following military herbicides were used in South Vietnam in Operation RANCH HAND. The first three were extensively used in both defoliation and anticrop programs. Only limited quantities of the herbicides Purple, Pink or Green were used in South Vietnam, and then primarily during the 1962-1964 time period. 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 (gal) of Orange theoretically contained 4.21 pounds (lb) of the active ingredient of 2,4-D and 4.41 lb of the active ingredient of 2,4,5-T. Orange was formulated to contain a 50:50 mixture 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) 1-3 49.49 0.13 48.75 1.00 0.62 �Some of the physical, chemical, and toxicological properties of Orange are listed in Table 1. The structures of the n-butyl esters of 2,4-D and 2,4,5-T are shown in Figure 1. 2. Herbicide White White was a dark brown viscous liquid that was soluble in water but insoluble in organic solvents and diesel fuel. One gal of White contained 0.54 Ib of the active ingredient of 4-amino-3,5,6trichloropicolinic acid (picloram) and 2.00 Ib of the active ingredient of 2,4-D. White was formulated to contain a 1:4 mixture of the triisopropanolamine 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 Some of the physical, chemical, and toxicological properties of White are listed in Table 1. The structures of the triisopropanolamine salts of 2,4-D and picloram are shown in Figure 1. 3. Herbicide Blue Blue was a clear yellowish-tan liquid that was soluble in water, but insoluble in organic solvents and diesel fuel. One gal of Blue contained 3.10 Ib of the active ingredient hydroxydimethyarsine oxide (cacodylic acid). Blue was formulated to contain both cacodylic acid (as the free acid) and the sodium salt of cacodylic acid (sodium cacodylate). The percentages of the formulation were: cacodylic acid 4.7 sodium cacodylate surfactant 26.4 3.4. sodium chloride water antifoam agent 5.5 59.5 0.5 Some of the physical, chemical, and toxicological properties of Blue are listed in Table 1. The structure of the sodium salt of cacodylic acid is shown in Figure 1. It should be noted that cacodylic acid and sodium cacodylate contained arsenic in the form of the pentavalent, organic arsenical. This form of arsenic was essentially nontoxic to animals as can be noted by the LD^Q value for white rats. Of the total formulation, 15.4 percent was arsenic in the organic form, only trace quantities were present in the inorganic form. The term Herbicide Blue was first applied to powdered cacodylic acid in 1961 through 1964. This first Herbicide Blue contained 65 percent active ingredient 1-4 �TABLE 1. Selected physical, chemical and toxicological properties of the three major military herbicides used in South Vietnam, 1962 - 1971.a- Herbicide Code Name Orange White Blue a Molecular Mass Specific Density, 25°C Viscosity, Centipose, 23°C Weight Total Acid Ester Equivalent Ib/gal Ib/gal Soluble in Water Specific Tpxicity for Mhite Rats mg/kgb. Relative Toxicity 589 1.28 43 10.7 8.62 No 1,173 1.12 125 9.4 2.54 Yes 3,080 Very Low 296 1.32 14 10.9 3.10 Yes 2,600 Very Low 566 Low Source: (35) Milligrams of the herbicide per kilogram of body weight of the test animal lethal to 50 percent of white rats. �n-butyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D) B. n-butyl ester of 2,4,5-trichlorophenoxyacetic acid (^,4,5-T) 0 0-CH0C-0~ +NH[C H ^ 3 6 03 Cl D. triisopropanolamine salt of 2,4-D - 0 -4- C-0 NH[C_H OHl 36 3 triisopropanolamine salt of 4-amino-3,5, 6-trichloropicolinic acid (picloram) CH - As - 0 Na' sodium salt of hydroxydimethylarsine oxide (cacodylic acid; FIGURE 1. ' Chemical structure and nomenclature of the major herbicides used in South Vietnam, 19621971. Formulas A and B comprised Orange, C and D - White, and E was Blue. 1-6 �cacodylic acid and 30 percent sodium chloride and was mixed in the field with water (7, 14). 4. Herbicide Orange II Orange II was the code-name of a formulation similar to Orange with the difference being the substitution of the issocytl ester of 2,4,5-T for the n-butyl ester of 2,4,5-T, The physical, chemical, and toxicological properties of Orange'II were similar to those of Orange. Orange II was produced solely by one chemical company. Approximately 950,000 gal of Oramge II were shipped to South Vietnam during 1968 and early 1969 (12). How much Orange II was returned to Johnston Island from South Vietnam in April 1972 was not determined. 5. Herbicide Purple Purple was first formulated in the mid-1950s time period. It was used in the Camp Drum, New York, defoliation test in 1959 (26). The formulation was a brown liquid soluble in diesel fuel and organic solvents but insoluble in water. One gal of Purple contained 8.6 Ib of the active ingredients 2,4-D and 2,4,5-T. The percentages of the formulation were: n-butyl 2,4-D 50 n-butyl 2,4,5-T 30 iso-butyl 2,4,5-T 20 The physical, chemical, and toxicological properties of Purple were similar to those described for Orange. 6. Herbicide Pink Pink was a formulation of 2,4,5-T used extensively in early RANCH HAND operations (7) and in the defoliation test program of 1963 and 1964 in Thailand (15). Pink was a mixture of the n-butyl and iso-butyl esters of 2,4,5-T. No data were available on the physical, chemical, or toxicological properties of Pink- However, Darrow et al (15) reported that it contained 8.16 Ib active ingredient per gal. The percentages of Pink formulation were: n-butyl 2,4,5-T 7. 60 iso-butyl 2,4,5-T 40 Herbicide Green Green was a single component formulation consisting of the n-butyl ester of 2,4,5-T. It was used in limited quantities in the 1962-1964 period (3). However, the only reported use of Green 1-7 �was in an evaluation program of herbicides for use against manioc and [(7), and correspondence between personnel of the Air Force Armament Laboratory, Eg!in AFB, Florida, and personnel of the Crops Division, Fort Detrick, Maryland, dated 5 Sep 63]. No data were available on physical, chemical, or toxicological properties of Green. Brown (7) reported that Green contained the same amount of active ingredient as Pink. 8. Other Herbicides Used in South Vietnam In addition to evaluating Herbicides Purple, Pink and Green, Brown (7) also evaluated Dinoxol, a mixture of 20 percent each of the butoxy ethanol esters of 2,4-D and 2,4,5-T; Trinoxol, 40 percent butoxy ethanol ester of 2,4,5-T; Diquat, 6,7-dihydrodipyridol (l,2-a:2'5 l'-C) pyrazidinium dibromide; and small quantities (grams) of 16 different chemicals. The latter chemicals were applied on native grasses and bamboo at the Saigon Navy Yard. Darrow et al (14) reported that small quantities of soil-applied herbicides were used on base camp perimeters, mine fields, ammunition storage areas, and other specialized sites requiring control of grasses and woody vegetation. The soil-applied herbicides evaluated for use in South Vietnam included Bromacil, 5-bromo-3-sec-butyl-methyluracil; Tandex, (3,3dimethyluneido) pheny1 -tert-butylcarbamate; Monuron, 3-(p-chlorophenyl)-!. 1-dimethylurea; Diuron, 3-(3,4-dichlorphenyl)-l, 1-dimethylurea; and Dalapon, 2,2-dichloropropionic acid. C. Quantities of Herbicides Sprayed in South Vietnam The estimated number of gal of the various military herbicides sprayed in South Vietnam from 1962 through 1971 have been obtained by examination of procurement and disposition records. The data obtained from these sources were compared to other sources when available; e.g., actual tactical mission records. Table 2 presents a summary of the available data on the number of gal of herbicides Blue, Green, Pink and Purple procured and disseminated in South Vietnam between January 1962 and December 1964. (3) Table 3 gives a comparison of the estimated number of gal procured and disseminated in South Vietnam between January 1965 and February 1971 as reported by the National Academy of Science (11), Westing (34), and Craig (12). The discrepancies in total herbicide quantity between the three sources occurred because Craig's data were from procurement records only, while the NAS and Westing data are based on both records and estimates. The latter two reports used different assumptions in calculating the total herbicide volume. These included such factors as spray line data (length and width of the spray swath), rate of application (1.5 or 3 gal/acre, and the amount of herbicide disseminated during a mission, 1-8 �TABLE 2. Number of gallons of military herbicide procured by the U.S. Department of Defense and disseminated in South Vietnam during the period January 1962 - December 1964.a Military Herbicide Gallons of Formulation Pounds Active Ingredient Blueb 5,200 10,000 Greenc 8,208 66,980 Pinkc 122,792 1,001,980 Purple 145,000 1,180,300 281 ,200 2,259,260 Total a Source document: Memorandum for Assistant Secretary of Defense from the Office of the Under Secretary, Department of the Air Force, Washington, D.C.; dated December 15, 1961. Subject: Summary of Current Status Project "RANCH HAND" Chemicals. (3) e was procured as a fine white hygroscopic powder which contained 65 percent cacodylic acid (active ingredient), 30 percent sodium chloride, 3 percent sul fates and 2 percent water. Approximately 290 1b of powder were mixed with 100 gallons of water (6). Thus, a total of 5,200 gal of Blue were probably disseminated in South Vietnam (primarily by the HIDAL Spray System). c d Pink and Green contained approximately 8.16 Ib active ingredient per gal (7). Purple contained approximately 8.14 Ib active ingredient per -gal (see Section V.A.3., Chapter I, p 1-26) 1-9 �TABLE 3. Estimated number of gal of military herbicide procured by the U. S. Department of Defense and disseminated in South Vietnam during the period January 1965 - February 1971. Military Herbicide Craig, 1974 ( 1 2 ) a NAS Report, 1974 (11 )b Westing, 1976 ( 3 4 ) c Orange 10,645,904 11,266,929 11 ,712,860 White 5,632,904 5,274,129 5,239,853 Blue 1,144,746 1,137,470 2,161 ,456 17,423,554 18,936,068 19,114,169 Total Data compiled from procurement and disposition records maintained by the San Antonio Air Logistics Center, Directorate of Energy Management, Kelly Air Force Base, Texas. The data for expenditures of Herbicide Orange, White, and Blue were based on procurement and delivery records for late FY 64 through FY 72, less those quantities of herbicides returned to Johnston Island in April 1972 or retained in the Continental United States. b See Table III C-l of the referenced National Academy of Science report (11) c See Table 3.3 of the referenced report. 1-10 �D. Land Area Sprayed with Herbicides in South Vietnam As noted, in Section C above, the estimate of acreage sprayed with herbicides was often based on spray line data and/or the quantity of herbicide expended. The National Academy of Science (11) discussed these parameters as they applied to the HERBS tape, the major source of all mission maps and tabulations of herbicide operations in South Vietnam for the period August 1965 through February 1971. Table 4 presents a comparison of data from three sources on the estimated number of acres treated in South Vietnam from January 1962 through February 1971. Included in these figures are the same areas of land counted more than once if they were sprayed more than once. Table 5 is a comparison of the data for acreage sprayed within the three major vegetational categories. These data have been corrected for multiple coverage. The National Academy of Science Report (11) concluded that herbicides were sprayed on 10.3 percent of the inland forests of South Vietnam, 36.1 percent of the mangrove forests, and 3 percent of the cultivated lands or approximately 8.6 percent of .the total land area in South Vietnam. Westing (34) estimated that approximately 10 percent of South Vietnam was sprayed. III. THE AIRCRAFT, SPRAY SYSTEMS AND MISSION CONCEPT IN OPERATION RANCH HAND Almost all herbicide used in South Vietnam was sprayed from aircraft. Irish et al (26) have described some ground delivery systems for herbicides, but noted these were used primarily for control of vegetation on minefields and perimeter defenses. U.S. military personnel were responsible for operating and maintaining the aircraft used in Operation RANCH HAND. The number and types of aircraft, their load capacity, ease of loading, and the spray system employed in them, all were important factors in determining the number of personnel required in performing the herbicide missions. Standard procedures were adopted in all herbicide handling phases of the operation. This section, then, reviews the aircraft factors where military personnel were likely to have physically contacted the herbicides. A. Historical The first aerial spray trials for herbicides were conducted by the military in 1944 and 1945 (14, 26). These early tests were accomplished using the U.S Army Chemical Corps M-10 smoke tanks hanged externally on a B-25 aircraft. By 1953, the U.S, Air Force had accomplished prove-out and acceptance testing of the large-capacity (1,000-gal) spray system known as the Hourglass or MC-1 Spray System. In 1960 and 1961, Air Force personnel assigned to the Special Aerial Spray Flight, La ITley AFB, Virginia, acquired two MC-1 Spray Systems 1-11 �TABLE 4. Comparison of data from three sources of the estimated number of acres treated in South Vietnam during the period of January 1962 February 1971. Data make n£ allowance for multiple coverage. ACRES TREATED YEAR NAS Report (11) Irish et ail. (26) Westing (34) MEAN 1962 NAa 5,68T 5,724 5,703 1963 NA 24,947 24,920 24,934 1964 NA 93,842 93,869 93,856 1965 75,50lb 221 ,559 221 ,552 221,555 1966 608,106 842 ,764 845,263 765,378 1967 1,570,114 1,707,758 1,707,784 1 ,661 ,885 1968 1,365,479 1 ,330,836 1 ,696,337 1,464,217 1969 1,365,754 NA T, 519,606 1,442,680 294,925 NA 252,989 273,982 1,259 NA 3,346 2,303 1970 1971 Total of Mean = 5,956,493 a Data not available (NA) ^Data for period August 65 through December 65. 1-12 �TABLES. The number of acres treated in South Vietnam, 1962 - 1971, with military herbicides within the three major veqetational categories. Data represent areas receiving single or multiple coverage and for 90 percent of all areas treated. ACRES TREATED Vegetational Category NAS Report, 1974 01 )a Westing, 1976 (34 )b Inland Forest 2,670,000 2,879,000 Mangrove Forest 318,000 746,000 Cultivated Crops 260,000 595,000 3,248,000 4,221,000 Total a See page II1-39 of the referenced report. b See Table 3.6 of the referenced report, Data for Inland Forest was woody subtotal, less acreage for mangrove forest. 1-13 �and modified them to spray insecticide and to interface with the newly acquired Fairchild-Hiller C-123 air transport. Irish et al (26) noted that in October 1961, six C-123 aircraft were made available to the USAF Tactical Air Command with a high-priority directive to install the MC-1 Spray System. Fabrication was accomplished-expeditiously and on 7 January 1962, three of the configured-aircraft arrived at Tan Son Nhut Air Base, Republic of Vietnam. During the early months of 1962, the C-123/MC-1 system and the HIDAL (Helicopter, Insecticide Dispersal Apparatus, Liquid) were evaluated for dissemination characteristics. The aircrews were members of the Special Aerial Spray Flight, Langley AFB, and were on temporary duty to South Vietnam as part of Operation RANCH HAND. Air Force,personnel engaged in the herbicide program did not receive permanent change of station assignments until 1964. In late 1962 and early 1963, an intensive RDT&E (Research Development, Testing and Evaluation) program was initiated between the Crops Division, Fort Detrick, and the Air Force Armament Laboratory, Eglin AFB, Florida, to provide improvements in spray system components in support of RANCH HAND (26). Concurrently, operational employment of the spray capability by the RANCH HAND units was intensified steadily with time and availability of resources. B. Spray Systems and Characteristics of the RANCH HAND Aircraft Tests and evaluations of aircraft and spray systems were conducted on the calibration grids on Test Area C-52A, Eglin AFB, Florida (6, 14, 22, 27, 35) and on the calibration grid at Pran Buri, Thailand (11, 13, 15). The C-123/MC-1 spray configuration was initially calibrated to spray 1 to 1.5 gal of herbicide per acre (gal/A) (14, 26). Thus, in 1962 and 1963 the herbicide missions conducted using this initial system resulted in the dissemination of herbicide at this lower rate. The numerous modifications and extensive evaluations of the equipment configurations at Eglin AFB and Pran Buri did not result in equipment changes for Operation RANCH HAND until 1964 (14). . Darrow et al (14) and Irish et al (26) have reported that in early 1964 the rate of 3 gal/A was obtained at first by making double passes with the aircraft but by late 1964 the modifications were complete and the system was capable of spraying 3 gal/A in a single pass. The modified 1,000-gal C-123/MC-1 spray system was capable of depositing 3 gal/A on swaths 240 feet wide when spraying at an airspeed of 130 knots at a 150 feet altitude. Two 20-hp pumps were needed to achieve the required flow rate of 430 gal/min of Purple (26). The HIDAL spray system was capable of deposits of 1.5 gal/A when flown inwind at 55 knots and at an altitude of 100 feet (26). The tank volume for this system was 200 gal. 1-14 �In early 1966, following its development, the A/A 45Y-1 Internal Defoliant Dispenser, replaced the MC-1 in all C-123 aircraft. However, completion of calibration tests and performance characteristics for this spray system did not occur until 1968 (16, 22, 27). The A/A 45Y-1 defoliant dispenser was a modular spray system for internal carriage in cargo aircraft. The module consisted of a 1,000 gal tank, pump, and engine (20 hp) mounted on a frame pallet. An operator's console was an integral part of the unit but was not mounted on the pallet. The C-123 aircraft had wing booms 1.5 inches in diameter and 22 feet long extending from the outboard engine nacelles toward the wing tips. A short tail boom 3 inches in diameter was positioned centrally near the aft cargo door. There were 16 nozzles on each wing boom and eight on the tail boom. The nozzles were check valve bodies with 3/8-inch orifices (no nozzle tips). The system was capable of spraying at the rate of 240 gal/min, which, when released at 150 feet altitude at 130 knots airspeed produced a swath 260120 feet wide with a mean deposit of 3 gal/A in a coarse spray having an MMD (mass median diameter) of 320 to 350 micron (p). Spraying time was approximately 3.5 to 4 minutes, which was adequate to dispense 950 gal of chemical on a spray line about 8.7 statute miles (14 km) in length. In order to achieve predictable deposits, it was recommended that the missions be conducted under inversion to neutral temperature situations and calm wind conditions. Craig (12) has reported that each aircraft had a crew of 3 men: the pilot, co-pilot (navigator), and flight engineer (console operator). However, observers (Vietnamese and American) frequently accompanied the aircrews on herbicide missions (32). C, Mission Concepts The objectives of the defoliation and anticrop programs in South Vietnam have been thoroughly reviewed by Huddle (23) and others (11, 14, 34). It is the objective of this section to elaborate only on the background and mechanics of a "typical" herbicide mission that would have influenced the degree of exposure to herbicides by aircrew and/or ground personnel. The following scenario of events or "standard operating procedures" has been compiled from reports by Craig (12), Darrow et al (14), Irish e't al (26) and the National Academy'of Science Report (11). 1. Each of the 11 different companies that manufactured military herbicides packed them in new ICC 17C 55-gal 18 gauge steel drums for shipment to Southeast Asia (12). Until 1967, lined drums were used only for shipment of Blue. However, because of the results of compatibility tests, lined drums were also used to ship White beginning in 1967. 1-15 �2. ^ach herbicide drum was marked with a three-inch color-coded band around the center to identify the specific military herbicide. This marking was initially a 12-inch band, but was changed to a 3-inch band in March 1966. 3. Shipping time from the arrival of the herbicide at a U.S. port until it arrived in South Vietnam varied from 47 to 52 days. 4. About 10 out of every 10,000 drums shipped were received in a damaged or defective state. This represented a damage rate of 0.1 percent. About 50 percent of these damaged drums leaked as a result of punctures or split seams. These were caused by improper loading and defective drums. Forklifts operated by stevedores also caused punctures. Redrumming was accomplished at the ports. 5. About 65 percent of the herbicide was shipped to the 20th Ordnance Storage Depot, Saigon, and 35 percent was shipped to the 511th Ordnance Storage Depot, Da Nang. Under the normal handling procedures, drums were unloaded at Da Nang and Saigon from the cargo vessel directly into semi-trailers and were placed in an upright position. The trailers were driven to the various units of the 12th Air Commando Squadron (primarily at the bases of Da Nang, Phu Cat, or Bien Hoa) for disposition. 6. Normally the contents of the drums were transferred into blocked F-6 trailer tanks through a suction tube without removing the full drums from the semi-trailers. Each F-6 trailer held 4,298 gal or about 78 drums of herbicide. If blocked F-6 trailer tanks could not accommodate the total inventory, the drums were stacked in pyramidal style until needed. 7. The transfer of the herbicides from the 55-gal steel drums to storage tanks or aircraft tanks required some precautionary measures. Personnel charged with the supervisory responsibilities of handling the herbicides were indoctrinated in appropriate safety precautions including the use of gloves and face shields as needed. Personnel handling the chemicals were encouraged to "take normal sanitary precautions and to maintain personal cleanliness and to avoid skin and eye contact with the material. Contaminated clothing were to be washed before re-use. Spillage on the skin or in the eyes was to be rinsed copiously with clea1" water" (14). 8. When the herbicide was pumped from the drums into the F-6 trailers about 0.5 to 1.5 gal remained in the drum. Hence the drum was placed on a drain rack and the "drippings" were collected from many drums in a pan-type receptacle and used for spraying base perimeter areas. 1-16 �9. Empty drums were given to the military forces (Vietnam, U.S. and Free World Military Assistance Forces) for use as barriers in defensive positions. The drums were filled with sand or concrete and used in the construction of bunkers or in foundations for runways and barbed wire perimeters (12). 10. Surface areas contaminated by spillage of the herbicides were flushed with diesel fuel or water with diversion of the drainage into settling basins or pits for incorporation into the soil. 11. The F-6 trailers were tied to plumbing and pumps so that the herbicide could be delivered to the aircraft without moving the trailers. 12. As previously noted, Orange was insoluble in water, while Blue and White were not. When Orange was mixed with either Blue or White, a gummy substance formed. The F-6 trailers were therefore color-coded to correspond to the drum color-codes and used exclusively for the herbicide to which the code applied. 13. The aircraft spray tanks, positioned in the center of the airplane, and the spray system were purged before the type of herbicide carried was changed. Particular attention had to be given to sequences involving Blue and White. .A mixture of these two herbicides resulted in the formation of a precipitate consisting of the sodium salt of 2,4-D. 14. Most of the personnel involved in the actual handling of the herbicide drums were Vietnamese. However, a USAF flight mechanic or crew chief was responsible for insuring that the aircraft wa^ properly loaded and the spray system functional. A flight mechanic was also the console operator for the spray unit. The pilot and co-pilot were officers while the flight mechanics and crew chiefs were usually enlisted personnel. 15. For record keeping purposes a herbicide "mission" consisted of several aircraft; if only one aircraft was used the operation was termed a sortie.' All missions within a target formed a project. 16. Aircraft takeoffs were normally before sunrise. From a tactical point of view, the arrival of the aircraft at the target area just prior to sunrise permitted the aircraft to approach the target from the direction of the rising sun. This afforded some degree of protection from enemy ground fire. From the standpoint of herbicidal action, application by aerial spray was most effective if accomplished prior to 0800 hours while inversion conditions existed, in the absence of precipitation, and while the wind was calm or not exceeding a velocity of 8 knots. This insured the proper settling of the spray on the target area. 1-17 �17. Within the aircraft, it was not uncommon to have herbicide leakage from around the numerous hose connections joining the spray tank and pumps with the wing and aft spray booms. In hot weather, the odor of herbicide within the aircraft was decidedly noticeable. Periodically, the spray tank and console were removed (especially with the portable A/A 45Y-1 system) and the interior flushed with surfactant or soap and with water. Because of the corrosive nature of some herbicides, it was necessary for the aircraft to also be repainted periodically. 18. In the 1966 through 1968 period, more than one sortie per day was often common. For example, during the first six months of 1968, the 24 UC-123B aircraft assigned to RANCH HAND averaged approximately 39 sorties per day. IV. PERTINENT DEPLOYMENT AND BIOLOGICAL FACTORS OF THE HERBICIDES The previous section dealt with those factors that would Influence the frequency of "physical contact" with liquid forms of the herbicide. As noted, the individuals most likely to be exposed to liquid herbicide were those charged with transport, handling, and disseminating responsibilities. This section will deal with some factors that would have influenced the likelihood of contact with the herbicides once they had been sprayed. A. Use Patterns of Individual Military Herbicides 1. Herbicides Orange, Orange II, Purple, Pink and Green Herbicides Orange, Orange II, Purple, Pink and Green were effective defoliants and herbicides on a wide array of woody and broadleaf herbaceous species. Grasses, bamboos, and other monocoiyledonous plants were less affected. The effects of these military herbicides on the forests of South Vietnam has been well documented (5, 9, 11, 13, 14, 21, 29, 32, 34). Darrow (13), and Harrow et al (14, 15) showed that at the normal use rates (3 gal/A) these herbicides, when applied to mixed woody vegetation, caused a browning and discoloration of the foliage within a period of one or two weeks. Foliage of the more susceptible species turned brown rapidly, and subsequent leaf drop occurred over a period of one to two months. Under tropical conditions, maximum defoliation occurred two to three months after the spray application. At 3 gal/A the maximum average defoliation in a single or multiple canopy was 88 and 75 percent respectively for rainy season application, or 82 and 67 percent respectively for dry season application. Under tropical forest conditions, satisfactory levels of defoliation persisted for four to twelve months or more. The National Academy of Science (11) reported that from August 1965 through February 1971, 2,962 herbicide mission^ (out Q? a total of 6,237 missions for all herbicides and all use:.) were for forest defoliation uring Orange. These 2,962 missions �accounted for 90 percent of all Herbicide Orange (including Orange II) used in South Vietnam. Likewise 90 percent of all Purple, Pink and Green sprayed in South Vietnam was for forest defoliation (26). Orange and Orange II (and Purple) were also used in control of broadleaf crops (8, 11, 34). For convenience and simplification in programming crop destruction and defoliation targets, application rates were routinely 3 gal/A (14). Annual crops; e.g., beans, gourd, jute, peanuts, and ramie, were rapidly killed by an application of Orange. Root or tuber crops; e.g., manioc, potatoes, taro, and yams, showed great reduction in yield when treated with Orange during early growth stages. Perennial and woody tropical crops; e.g., jackfruit, papaya, castor bean, and mango were susceptible to Herbicide Orange (14). From August 1965 through February 1971, crop destruction missions with Orange accounted for 8 percent of the Herbicide Orange applied (11). The remaining 2 percent of Herbicide Orange used in South Vietnam was used around base perimeters, cache sites, waterways, and communication lines (11). 2. Herbicide White Herbicide White was effective principally on broadleaf herbaceous and woody plants. Conifers (pine trees) were especially susceptible to White. However, the herbicidal action on woody plants was slow and full defoliation did not occur for several months after spray application (14). Since White was water soluble, it was frequently used in field situations where drift was to be held at a minimum (e.g., near rubber plantations). White was sprayed during 1,324 defoliation missions (21 percent of all missions) and of the total volume of White used in South Vietnam, 99 percent was for defoliation (11). The remaining one percent of White was used primarily in base perimeter applications. White was not recommended for use on crops because1' of the persistence of picloram in soils (14). 3. Herbicide Blue Herbicide Blue was the herbicide of choice for other crop destruction missions; e.g., on cereal or grain crops. For crop destruction missions, the basic rate of 3 gal/A was used. Helicopter applications were usually at the rate of one gal/A for control of grain crops. (15). Forty-nine percent of all Blue (580,000 gal) was used in crop destruction missions conducted from August 1965 through February 1971 (11). The remaining Blue was used in defoliation or in control of grass around base perimeters pi)- As a defoliant, Blue caused a rapid browning or desiccation with accompanying shriveling and leaf fall. Noticeable browning or discoloration was evident in one day, with maximum defoliation occurring within two to four weeks (14). 1-19 �B. Canopy Penetration of Defoliants As previously noted, 90 percent of all Herbicide Orange (and probably Purple, Pink and Green) was for defoliation in the forests and mangroves of South Vietnam. The quantity of herbicide that reached the forest floor is not known. However, such factors as canopy composition and time of season of application would have influenced this value. Huddle (23) recorded the following 1968 statement by Dr C.E. Minarik (Dr Minarik was at that time Director, Plant Sciences Laboratories, Fort Detrick, Maryland): "Three gallons per acre is employed. We would prefer to use less if we could get uniform deposition, but in these dense jungle areas where there may be 300 tons of vegetation per acre, this is the minimal effective volume. The three gallons contain 24 pounds of herbicide on an acid basis. Thus high dosage rate is also a requirement since much of the vegetation consists of trees 100 to 150 feet tall." In the evaluation tests of the C-123/A/A 45Y-1 Spray System, Harrigan (22) and Klein and Harrigan (27) found that in mass distribution studies (following aerial dissemination) 87 percent of the Orange Herbicide intercepted by collecting devices had a mass median diameter between 100 and 500u. The mean diameter was 367u. Harrigan (22) concluded that with altitude delivery conditions at 130 knots and 150 feet altitude, most of the Orange released would have settled onto the forest canopy in a swath approximately 260^20 feet wide within which effective defoliation was produced. Hurtt and Darrow (25) showed that the minimum biological effective deposition rate under the climatic conditions of South Vietnam was 1.0 gal/A at a mass median diameter of 350y. The minimum biological effective rate was defined as "that rate which promoted leaf fall and inhibited growth" (25). In canopy penetration studies, Tschirley (33) found (with phenoxy herbicide formulations similar to Orange) that the volume of spray reaching lower sampling levels varied proportionately with the amount deposited on the top line above the canopy. On the average, about 21 percent of the spray penetrated the upper canopy and about 6 percent penetrated to ground level. He also found that the percentage penetration remained relatively constant for drop densities greater than about 100 per square inch. Spray drops having mass median diameters of 400 to 500y would approximately equal 100 drops per square inch. Moreover, the percent spray penetration through forest canopies was inversely related to canopy density (33). 1-20 �No data were available on the number of defoliation missions conducted during the wet or dry seasons. However, as noted earlier, defoliation of forest canopy was greatest during the rainy season, when the vegetation was in full-leaf and actively growing. V. ESTIMATED QUANTITIES OF INDIVIDUAL CHEMICALS SPRAYED IN SOUTH VIETNAM For this report, the total quantities of individual chemicals become important only in reference to their potential association with dose and duration'of exposure to the population at risk. Although the National Academy of Science (11) primarily defined the population at risk as Vietnamese (especially Montagnards), our concern at this time is with U.S. military forces. The chemicals of concern are 2,4-D, 2,4,5-T and TCDD. The extreme toxicity of TCDD, however, makes it the prime chemical of concern. The toxicology of these chemicals is discussed in detail in Chapters IV and VI. The previous scientific assessments of these chemicals as applied in South Vietnam are addressed in Chapter V. A. Herbicide Orange and its Components 2,4-D, 2,4,5-T and TCDD 1• Concentrations of TCDD in Orange, Purple, Pink and Green Figure 2 shows the structure of TCDD and gives a brief description of some of its physical and chemical characteristics. Table 6 shows the available data on the concentration of TCDD (parts per million, ppm) in samples of Herbicides Orange and Purple. As noted, the mean concentration of the surplus Herbicide Orange remaining after termination of its use in South Vietnam was a value derived from data on the analyses of 492 samples. Some of these data have been previously published (4, 11). Craig (12) has reported that the Orange Herbicide maintained at the Naval Construction Battalion Center (NCBC), Gulfport, Mississippi, was probably authorized and procured during the 1968-69 fiscal year. The Orange returned from Vietnam in 1972 (to Johnston Island) was procured no earlier than late FY 64, since the first shipment of Orange did not arrive in Vietnam until early 1965, and a six months lead time was typical. Note that the mean TCDD concentration was 1.91 ppm for the Johnston Island inventory and weighted means of 1.77 and 2.11 for samples analyzed from the NCBC inventory. It is important to note the range of TCDD concentration of TCDD in both surplus Orange inventories. The maximum concentration of TCDD in Orange samples collected at NCBC was 15 ppm, while the maximum concentration of TCDD reported in samples from Johnston Island was 47 ppm. Only 4 of 200 samples from Johnston Island exceeded TCDD levels found in the NCBC inventory (4). The values of these 4 samples were 17, 22, 33, and 47 ppm (4). 1-21 �A. Structure 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) B. Physical Characteristics molecular weight melting point, °C decomposition point, °C C. 322 303 - 305 980 - 1,000 Chemical Characteristics Solubility, grams/liter ortho-dichlorobenzene chlorobenzene Orange Herbicide benzene chloroform acetone 1.40 0.72 0.58 0.57 0.37 0.11 . normal-octanol 0.05 lard oil methanol water 0.04 0.01 2 x 10 FIGURE 2. Structure and physical/chemical characteristics of 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD or dioxin. 1-22 �TABLE 6. Concentration, ppm, of TCDD in samples of Herbicides Orange and Purple.3 Number of Samples Orange Purple Range of TCDD (ppm) Johnston Island b Inventory, 1972 200 (4)c 0.05-47 1.91 Johnston Island Inventory, 1974 10 0.07-5.3 1.68 NCBC, Gulfport d Inventory, 1972 42 0.05-13.3 1.77 NCBC, Gulfport Inventory, 1975 238 0.02-15 2.11 Source of Samples Mean TCDD, Concentration (ppm) Eglin AFB Archived Sample 45 Eglin AFB Inventory, 1972 0.04 The Weighted Mean Concentration of TCDD in Orange = 1.98 ppm Analyses for TCDD performed by Interpretive Analytical Services, Dow Chemical U.S.A., Midland Michigan; Aerospace Research Laboratories, Wright-Patterson AFB, Ohio; and The Brehm Laboratory, Wright State University, Dayton Ohio. Surplus Herbicide Orange was shipped from South Vietnam to Johnston Island for storage in April 1972. c Four of 200 samples may have been Herbicide Purple, see text. d The Naval Construction Battalion Center (NCBC) Gulfport, Missippi served as a storage site for Surplus Herbicide Orange from 1969 to 1977. e Herbicide Purple was extensively used in the evaluation of aerial spray equipment on Test Area C-52, Eglin Air Force Base Reservation, Florida, 1962-1964. 1-23 �Only one sample of Herbicide Purple has been analyzed (see Table 6). The age of the sample was not known except that it was representative of the Purple applied to Grid 1 (ARPA Grid), Test Area C-52A, Eglin AFB, Florida (Personal information, A.L. Young, and references 34 and 36), and thus, may have been from the 1962-1964 time period. The 1971 Report on 2,4,5-T by the Executive Office of the President (18) presented data on TCDD concentrations found in the analysis of Technical 2,4,5-T from one manufacturer. The data were for samples manufactured yearly from 1958 through 1969, and ranged from 1 to 32 ppm. The highest levels of TCDD were found in samples manufactured in 1965 (32 ppm) and 1968 (25 ppm). If these two samples had been used in formulating Herbicide Orange, the concentrations in the Orange would have been 16 and 12.5 ppm, respectively. If the lowest TCDD containing samples for the same two years would~Fave been used (i.e., samples containing 5 and 1 ppm TCDD) the concentrations in the Orange would have been 2.5 and 0.5 ppm, respectively. The one sample of Purple reported in Table 6 contained 45 ppm. Thus, the Technical 2,4,5-T used in that sample may have contained 90 ppm TCDD. When the Orange Herbicide was shipped to Johnston Island from South Vietnam, redrumming of the herbicide in South Vietnam was accomplished as necessary (12). The project (PACER IVY) involved U.S. military personnel. One of the individuals participating in the redrumming operation at Da Nang (redrumming also occurred at Phu Cat and Bien Hoa) has stated that drums of Purple were found (although fewer than 20) and redrummed into Orange-banded drums (personal communication, Or Michael D. Neptune, now with the U.S. Environmental Protection Agency, Washington, D.C.). In addition, an analytical chemist involved in the analyses of Orange samples for 2,4-D and 2,4,5-T, reported finding significant quantities (15 percent) of the iso-butyl ester 2,4,5-T in a few of the samples collected from Johnston Island (unpublished data, personal communication, Dr Eugene L. Arnold, now with the Clinical Sciences Division, USAF School of Aerospace Medicine, Brooks AFB, Texas). Thus, the 4 samples of Orange Herbicide containing TCDD concentrations greater than 15 ppm, may have been Purple. If these were, in fact, from drums of Purple, then the mean concentration of TCDD in 5 samples of Purple would have been 32.8 ppm. The mean value of 32.8 ppm may or may not represent the TCDD concentration of the Herbicide Purple used in South Vietnam from 1962 through 1964. Data from the 1971 Report on 2,4,5-T (18) suggests that Purple manufactured in 1958 through 1963 would have had a mean concentration of approximately 5 ppm (4.711.2 as the mean and standard deviation for the 6 samples reported for the years 1958 through 1963). However, the persistence of TCDD in soils of the two grids used for the testing and evaluation of the early RANCH HAND spray systems may indicate that Purple indeed had concentrations of TCDD from 17 to 47 ppm. Young (35) and Young et al (37) reported finding concentrations of 710 parts per trillion TCDD in the top 6 inches (15 cm) of soil collected in 1974 from the equipment-testing 1-24 �grid known to have received at least 1,894 Ib of Purple per acre during the 1962 through 1964 period. The test grid had received 16,164 gal of Purple. On an adjacent test grid, 1,168 pounds of Orange per acre had been disseminated during the 1964-1966 programs evaluating the A/A 45Y-1 Spray System. The'levels of TCDD in the soil treated with Orange at comparable depth was 30 parts per trillion. All soil samples were analyzed in 1973. Young et al (36) have reported the half-life of TCDD to be less than one year when in the presence of the phenoxy herbicides. Persistence data suggested that the levels of TCDD in Purple and Orange were significantly different. Further evidence of this is recorded by the National Academy of Science (11) for TCDD residue found in the soils of the Pran Buri Calibration Grid. They reported finding levels from <0.0012 to 0.233 ppm TCDD in the top 6 inches of soil from this testing ground. They concluded that since the grid had received approximatly 1,000 Ib/A 2,4,5-T in 1964-65, the original concentration of the TCDD in Orange would have ranged from <3 to 50 ppm. The NAS Committee (11) assumed that the material applied to the Pran Buri Calibration Grid was Orange. Darrow et al (15), responsible for the original calibration studies, reported that the Pran Buri Calibration Grid received 6,000 gal Purple, 3,800 gal Pink (all 2,4,5-T) and only 825 gal Orange. Since the majority of herbicide applied on this grid was either Purple or Pink, it further supports the contention that the four high-TCDD-containing samples from Johnston Island were Purple. Accepting the mean concentration of TCDD in Purple as 32.8 ppm and recognizing that Pink and Green contained essentially twice the active ingredient (8.16 Ib acid equivalent 2,4,5-T per gal) as Purple (4.0 Ib acid equivalent 2,4,5-T per gal), the mean concentration of TCDD in Pink and Green would have been twice that of Purple, or 65.6 ppm. Also, from the above discussion, it can be concluded with reasonable certainty that the weighted mean concentration for aVl_ Herbicide Orange sprayed in South Vietnam was 1.98 ppm: individual lots may have contained higher (<15 ppm) or lower (>_ 0.02 ppm) concentrations of TCDD, but the weighted mean was 1.98 ppm. 2- Concentrations of 2,4-D and 2,4,5-T in Orange The original military specifications for Herbicide Orange were published on 19 July 1963 as specifications MIL-H-51158 (MU) and MIL-H-51147 (MU). As written in the specifications, for the n-butyl ester of 2,4-D: "The total acid equivalent of the herbicide shall be not less than 78 nor more than 80 percent when tested as specified. The free acid content of the herbicide shall not be greater than 1.0 percent." For the n-butyl ester of 2,4,5-T the specifications noted: "The total acid equivalent of the herbicide shall be not less than 80 nor more than 82 percent when tested as specified. The free acid content of the herbicide shall not be 1-25 �greater than 1.0 percent." Orange was to be a 50:50 mixture of the products from the two specifications. These specifications were updated on 7 November 1966. Fee et al (20) and Hughes et al (24) have extensively analyzed*Herbicide Orange samples (from Johnston Island and NCPC, Gulfport, Mississippi) for composition. Table 7 .is a comparison of different manufacturers' lots for percent composition. Although the actual mean composition varied from the "theoretical" specification, the analytical method employed to test the total acid equivalent of the herbicide permitted some fluctuation in content. The parent acid portion of the herbicide molecule will remain as the herbicidally active portion of its respective ester form, while the ester appendage to the parent acid form will serve to satisfy some additional properties, such as decreased water solubility and increased surface penetration and/or translocation. The butyl ester of 2,4-P contained 79.4 percent acid 2,4-D and the butyl ester of 2,4,5-T contained 80.2 percent acid 2,4,5-T. From the data in Table 7, the mean actual weight of active ingredient per gal of Orange was 4.14 and 4.00 pounds for 2,4-D and 2,4,5-T, respectively. These values have been accepted also for the active ingredients in Herbicide Purple. 3. Quantities of Herbicides and TCDD Disseminated in South Vietnam •~-~~ Using data in Tables 2 and 3 (herbicide procurement records), Table 6 (mean TCDD concentration in Orange) the value of 32.8 ppm TCDD for Purple,the value of 65.6 ppm TCDD for Pink and Green, and Table 7 (mean, actual composition of Herbicide Orange), an estimate of the quantities of herbicides and TCDD disseminated in South Vietnam from January 1962 through February 1971, can be determined. These "estimated" quantities are in Table 8. The National Academy of Science Committee on the Effects of Herbicides in South Vietnam (11) estimated that between 220 and 360 pounds of TCDD were released over South Vietnam during the period August 1965 to February 1971. The estimate of 368 pounds in Table 8 for TCDD falls very close to their estimate. The important difference is that 143 pounds of the TCDD reported in Table 8 (or approximately thirty-nine percent of all the TCDD) was contained in Purple, Pink, and Green and was sprayed on 90,000 acres in Vietnam from 1962 through 1964» a time period when only a small force of military personnel were in South Vietnam. Herbicide Orange was sprayed on 3.5 million acres from 1965 through 1970. However, 90 percent of the Orange was sprayed on 2.9 million acres of inland forests and mangrove forests. 1-26 �TABLE 7. Composition, Percent, of Selected Samples of Herbicide Orange in Relation to Military Specifications. NCBC Inventory Number9 ASN 10 ASN 14 Mean Composition Approximate Military Specification^ Component ASN 8 Number of Gallons 123,695 383,955 145,860 Level of TCDD <0.02 ppm 0.30 +0.06 ppm <0.02 ppm n-Butyl ester 2,4-D 42.6% 46.2% 43.7% 44.2% 49.5% n-Butyl ester 2,4,5-T 39.3 44.9 42.2 42.1 48.8 Other Butyl esters of chl orophenoxyaceti c acids 7.96 4.01 ^ Octyl esters of "-1 chlorophenoxyacetic acids 5.76 0.25 Acid, 2,4-D 0.78 0.19 Acid, 2,4,5-T 0.84 Inert Ingredients0 2.76 9.05 7.0 *• 2.0 — 0.65 0.5 0.1 0.13 0.78 0.6 1.0 4.32 3.62 3.6 0.6 t—t Selected samples of Herbicide Orange were collected from the surplus inventory maintained at the Naval Construction Battalion Center (NCBC), Gulfport,, Mississippi. Samples represented lots produced by different manufacturers. Analyses for TCDD and samp]_e composition were performed by the Aerospace Research Laboratories, Wright-Patterson AFB, Ohio. [_ See Reference by Hughes et al. ( 4 . ] 2)] ^Military specifications for manufacture of Herbicide Orange were based on Specifications MIL-H-51147A (MU) and MIL-H-51148A (MU) dated 7 Nov 1966. c lnert ingredients included butanol, toluene, butylchloride, dichlorophenol, trichlorophenol, butoxydichlorobenzene, and butoxytrichlorobenzene. �TABLE 8. Estimated quantities of herbicides and TCDD disseminated in South Vietnam from January 1962 - February 1971. Chemical Pounds 2,45-Da 55,940,150 2,4,5-Tb 44,232,600 TCDDC 368 Picloram 3,041,800 Cacodylic Acid6 3,548,710 Total of Herbicides 106,763,260 2,4-D was an active ingredient in Herbicides Orange, Purple and White. From data in Table 7, the acid equivalents for 2,4-D in Herbicide Orange and White were calculated to be 4.14 Ib/gal and 2.00 Ib/gal, respectively. The acid equivalent for 2,4-D in Herbicide Purple was assumed to be 4.14 Ib/gal. 2,4,5-T was an active ingredient in Green, Pink, Purple and Orange. Approximately 276,000 gal of Green, Pink and Purple were sprayed in South Vietnam prior to 1965, when it was replaced by Herbicide Orange. Herbicides Green and Pink contained 8.16 Ib/gal 2,4,5-T. Herbicides Purple and Orange contained 4.00 Ib/gal 2,4,5-T (Table 7). G The mean TCDD concentration in Herbicide Purple was estimated at 32.8 ppm. The mean TCDD concentration in Herbicides Pink and Green was estimated at 65.6 ppm. The mean TCDD concentration in Herbicide Orange was estimated at 1.98 ppm. Picloram was an active ingredient of Herbicide White. e Cacodylic acid was the acitve ingredient of Herbicide Blue. The Herbicide Blue formulation contained 15.4 percent arsenic in the pentavalent organic form. The value includes 10,000 Ib cacodylic acid disseminated in South Vietnam from 1962-1964. 1-28 �B. Military Projects that Involved Handling Herbicides Orange, Purple, Pink or Green. Herbicide Orange was first manufactured in late 1964. It arrived in Vietnam for use in Operation RANCH HAND in early 1965. Prior to Orange, Herbicides Purple, Pink and Green were used but in far less quantities and on a limited area. All of the quantities of Orange returned from Johnston Island in 1972 and those stored at the Naval Construction Battalion Center since late 1968 were destroyed by at-sea incineration in 1977. From the first aerial spray test in 1961 through the incineration project in 1977, numerous U.S personnel directly handled the herbicide in support of specific project goals. Table 9 was assembled after an extensive search of available documents and from personal contact with eleven different individuals that had participated in one or more of the listed projects. Other than Operation RANCH HAND the most extensive handling of Herbicide Orange occurred during Project PACER HO. At the time of this latter project, analytical techniques were sufficiently developed to permit the environmental monitoring of TCDD at the parts per trillion level during all stages of the project. These data permitted an assessment of the actual exposure of personnel involved in the handling of the herbicide. Chapter II is devoted to Project PACER HO, the disposal of the surplus Herbicide Orange. VI. 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 specturm of vegetation, and proven safe to humans and animals. * The major herbicides used in South Vietnam were the 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). An estimated 107 million pounds of herbicides were aeriallydisseminated on 6 million acres in South Vietnam from January 1962 through February 1971. Approximately 94 percent of all herbicides sprayed in Vietnam were 2,4-D (56 million pounds or 53 percent of total) or 2,4,5-T (44 million pounds or 41 percent of total). The 44 million pounds of 2,4,5-T contained an estimated 368 Ib of the toxic contaminant, 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD or dioxin). Ninety-six percent of all 2,4,5-T was contained in Herbicide Orange; the remaining 4 percent in Herbicides Green, Pink and Purple. . However, Herbicides Green, Pink and Purple contained approximately 40 percent of the estimated amount of TCDD disseminated in South Vietnam. 1-29 �TABLE 9. Project Data on the major military projects involved in the handling and/or spraying of Herbicides Orange, Purple, Pink or Green in support of military programs in South Vietnam. Dates Brief Description Selected References Project AGILE 1960-1968 Selection of herbicides, and development and evaluation of defoliation techniques. Brown, 1962 (7) Coates et a!, 1962 (10) Darrow et al, 1966 (15) Demaree and Creager, 1968(16) Operation RANCH HAND 1962-1971 Aerial spraying of herbicides in South Vietnam. Anonymous, 1961 (3) Fair, 1963 (19) Ellison, 1967 (U) Darrow et al, 1969 (14) Huddle, 1969 (23) McConnell, 1970 (29) USAF Projects 2525, 5172 5186, 5957 1962-1970 Development and testing of aerial spray equipment Biever, 1969 (6) CO o Redrumming and movement of surplus herbicide from South Vietnam to Johnston Island Craig, 1975 (12) 1972-1977 Maintenance of herbicide inventory and research on options for disposal Young, 1974 (35) Anonymous, 1974 (4) Lavergne, 1974 (28) Newton, 1975 (30) Young, et al, 1976 (37) 1977 Dedrumming of herbicide inventory and at-sea incineration of Herbicide Orange Ackerman et a l , 1978 (1) Project PACER IVY 1971 AFLC Project on Disposition of Herbicide Orange Project PACER HO Klein and Harrigan, 1969(27) Harrigan, 1970 (22) �Green, Pink and Purple were sprayed as defoliants on less than 90,000 acres from 1962 through 1964, a period when only a small force of U.S. military personnel were in South Vietnam. Ninety percent of all the Herbicide Orange (containing 38.3 million pounds of 2,4,5-T and 203 Ib of TCDD) were used in defoliation operations on 2.9 million acres of inland forests and mangrove forests of South Vietnam. The handling, transport and storage procedures employed for the herbicide generally precluded physical contact with the herbicides by most military personnel assigned to Operation RANCH HAND. However, flight mechanics (console operators for the internal spray systems) and crew chiefs (responsible for loading the aircraft) were the most likely military personnel exposed to the herbicides. The methods employed in spraying the herbicides and the geographical areas designated for dissemination of the herbicides generally precluded direct physical contact with the herbicide by military personnel assigned to other military programs. 1-31 �CHAPTER I LITERATURE CITED 1. Aekerman, D.G., H.J. Fisher, F.J. Johnson, R.F. Maddalone, B.J, Mathews, E.L. Moon, K.H. Scheyer, C.C, Shin, and R.F. Tobias, 1978. A£-4ea ^nc^tneAa-tuw oft HcAb-tcu.de Orange onboard the. M/T l/a£canai. Environmental Protection Technology Series EPA-600/2.J8-Q86. Office of Research and Development. U.S.- Environmental Protection Agency, Research Triangle Park, North Carolina. 263 p. 2. Advisory Committee on 2,4,5-T. 1971. Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency. 76 p. 3. Anonymous. 1961. Memorandum for Assistant Secretary of Defense. Subject: Summary of current status project "RANCH HAND" chemicals. Department of the Air Force, Office of the Under Secretary, Washington, D.C. Win. 4 p, 4. Anonymous. 1974, Disposition of Orange Herbicide by incineration, Final Environmental Statement. Department of the Air Force, Washington, D.C. 737 p, 5. Bethel, J.S., K.J. Turnbull, D. Briggs, and J. Flores. 1975. Military defoliation of Vietnam forests. Am&t-tcan F0<te-i£6 81(1):26-30, 56-61. 6. Biever, H. 1969. Defoliant history of Test Area C-52A. Working Papers. Armament Development and Test Center, Eglin AFB, Florida. December 1969. 7. Brown, J.W. 1962, Uefle&t£t0na£ Apbcuj te4tt> -in South U.S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland. 119 p. Available from' the Defense Documentation Center, Defense Logistics Agency, Cameron Station, Alexandria, Virginia, DDC Number AD 476961. 8. Carrier, Hickey's in South Science, J.M. 1974. The location of herbicide missions and Informants in South Vietnam. The Effects of Herbicides Vietnam, Part B. Working Papers. National Academy of Washington, D.C. 15 p. 9. CAST. 1975. Effects of herbicides in Vietnam and their relation to herbicide use in the United States. Council for Agricultural Science and Technology. Report No. 46. Department of Agronomy, Iowa State University, Ames, Iowa. 14 p. 1-32 �10. Coates, J.H., L.M. Sharpe, and H. Pollack. 1962. The 4,to£iL6 oft ehenu.ca£ e.on&io£ ofi vegetation -in le&ttuw to need-i. Technical Notes 62-68. Institute for Defense Analyses, Department of Defense, Washington, D.C. 30 p. 11. Committee on the Effects of Herbicides in South Vietnam. 1974. Part A. Summary and conclusions. National Academy of Science, Washington, D.C. 398 p. 12. Craig, D.A. 1975. Use of Herbicides in Southeast Asia. Historical Report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 p. 13. Darrow, R.A. 1973. Foliage characteristics and defoliation/ herbicidial responses in a Thailand Forest. Weed Sex.. Soc. Am. Ab*#l. 66, pp 29-30. 14. Darrow, R.A., K.R. Irish, and C.E. Minarik. 1969. HeA.b.icxxie-6 U&ed -in Soutkmut Aaxa. Technical Report SAOQ-TR-69-11078. Directorate of Air Force Aerospace Fuels, Kelly AFB, Texas. 60 p. 15. Darrow, R.A., G.B. Truchelut, and C.M. Bartlett. 1966. OCONUS de^o-tcatuM tut p/tog/iam. Technical Report 79. Crops Department, Biological Sciences Laboratory, U.S. Army Biological Center, Fort Detrick, Frederick, Maryland. 126 p. 16. Demaree, K.D. and R.A. Creager. 1968. Defoliation tests in 1966 at Base Gagetown, New Brunswick, Canada. Technical Memorandum 141. Department of the Army, Fort Detrick, Frederick, Maryland. 17. Ellison, R. 1967. C-123s defoliate jungle stronghold of Viet Cong. Aviation Week and Space Technology 86(19) :82-86. 18. Executive Office of the President. 1971. Report on 2,4,5-T. A report of the Panel on Herbicides of the President's Science Advisory Committee. C.M. MacLeod, Chairman. Office of Science and Technology, Executive Office Building, Washington, D.C. 69 p. 19. Fair, S.D. 1963. No place to hide. How defoliants expose the Viet Cong, Asuny 14:54-55. 20. Fee, D.C., B.M. Hughes, M.L. Taylor, T.O. Tiernan, and C.E. Hill. 1975. Ano£t/-ttca£ Methodology faofi HeA.bx.cx.de 0/wuage. l/o£. II. V&teAmination o$ Onig-in o& USAF S-tocfc6. Technical Report ARL-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB, Ohio. 30 p. 21. Flamm, B.R., and J.H. Cravens. 1971. Effects of war damage on the forest resources of South Vietnam. J. Poie^u/ 69(11):784-789. 1-33 �22. Harrigan, E.T. 1970. Catibtuvtian Jut oh the. UC-123K/A/A45y-l Sptai/ Sy*te.m. Technical Report ADTC-TR-70-36. Armament Development and Test Center, Eglin AFB, Florida. 160 p. 23. Huddle, P.P. 1969. A Technology Assessment of the Vietnam Defoliant Matter - A Case History. Report to the Subcommittee on Science Research and Development of the Committee on Science and Astronautics. U.S. House of Representatives, Ninety-first Congress. Prepared by the Science Policy Research Division, Legislative Reference Service, Library of Congress, Washington, D.C. 73 p. 24. Hughes, B.M., D.C. Fee, M.L. Taylor, T.O. Tiernan, C.E. H i l l , and R.L.C. Vlu. 1975. Analytical Methodology iofi HeA.bi.<Ude. Oiange,. Vol. I. V<LteAmina£ian o^ Chemical Compo&ition. Technical Report ARL-75-0110. Aerospace Research Laboratories, Wright-Patterson AFB, Ohio. 357 p. 25. Hurtt, W., and R.A. Darrow. 1968. &ioloai.o.al eXXecttueneiA oX Stult SifilLud and Osianae.. Technical Report AFATL-TR-68-122. Air Force Armament Laboratory, Eglin AFB, Florida. 31 p. 26. Irish, K.R., R.A. Darrow and C.E. Minarik. 1969. manual fax. vegetation control in Bouuth<La&t k&ia. Miscl . Public. 33. Department of the Army, Fort Detrick, Frederick, Maryland. 71 p. 27. Klein, R.E., and E.T. Harrigan. 1969. CompasuAon Tut 06 Ve.Kolia.nt!>, Technical Report ADTC-TR-69-30, Vol. I. Armament Development and Test Center, Eglin AFB, Florida. 356 p. 28. Lavergne, E.A. 1974. Study oh teaAlbilitu oh HeAbicMie. O^ianpe. c.hlo>u.noluAJA . Technology Series Report EPA-600/2-74-006. Office of Research and Development. Environmental Protection Agency, Washington, D.C. 67 p. 29. McConnell, A.F. 1970. Mission: RANCH HAND. - MJI UYiivvuitg Re.vi.ew 21(2):89-94. 30. Newton, M. 1975. Environmental impact of "Agent Orange" used in reforestation tests in Western Oregon. Weed Sex.. S<?c. Am., Abstr. 144, 52 p. 31. Peterson, 6.E. 1967. The discovery and development of 2,4-D. Ag*. Hlt>t. 41:243-253. 32. Tschirley, F.H. 1969. Defoliation in Vietnam - The ecological consequences of the defoliation program in Vietnam are assessed. Science. 163:779-786. 1-34 �33. Tschirley, F . H . 1968. Reiponie ofa &iopic.at and bmb&Lopic.aJL woody p£aitt6 to c.kmic.aJL -fiea£rnen£6 . Research Report CR-13-67. Agricultural Research Services, U . S . Department of Agriculture, Washington, D . C . 197 p. 34. Westing, A.H. 1976. Ec.otoQ-ic.aJL consequence* o& the. second Indochina. Wo/i. Stockholm International Peace Research Institute. Almgrist and Wiksel Internation, Stockholm, Sweden. 119 p. 35. Young, A.L. 1974. Ec.otoQic.aJL AtudieA on a keAbi.oJ.de. - equipment teAt oA.ua, (TA C-52A). Air Force Armament Laboratory, Eglin AFB, Florida. 141 p. 36. Young, A . L . , C . E . Thalken, E . L . Arnold, J . M . Cupello, L . G . Cockerham. 1976. fate. o& 2,3,7,S-te£uiQ.hlo'LOdA.be.nzo-p-dioiu.n (TCW) -en tke. nwiA.onm2.nt', Aummany and dzzontamination H.e.commz.ndatiom, . Technical Report USAFA-TR-76-18. Department of Chemistry and Biological Sciences, USAF Academy, Colorado. 41 p. 37. Young, A . L . , C.E. Thalken, and W . E . Ward. 1975. S^udcei o& the. <LC.oloQic.aJL impact o& ^epetctcue aerial apptication* o& heAbiciideA on the. <Lc.o*yAtw ofi TeAt AA.ea C-52A, Egtin AFB, fi.oni.da. Technical Report AFATL-TR-75-142. Air Force Armament Laboratory, E g l i n AFB, Florida. 127 p. 1-35 �CHAPTER II DISPOSAL OF HERBICIDE ORANGE I. INTRODUCTION During the summer of 1977 the United States Air Force (USAF) disposed of 2.22 million gallons (gal) of Herbicide Orange by high temperature incineration at sea. This operation, Project PACER HO, was accomplished under very stringent criteria of U.S. Environmental Protection Agency (EPA) ocean dumping permits. Numerous conditions of these EPA permits required the USAF to conduct extensive environmental and occupational monitoring of the land-transfer/loading operations and shipboard incineration operations. The results of EPA permit compliance monitoring for ship board operations are reported elsewhere (1). The purpose of this chapter is to summarize the historical background leading to Project PACER HO, to briefly describe the land-transfer operations and to present a summary of industrial hygiene and ambient air monitoring accomplished during the land-based operations. At the time of this writing not all occupational and environmental monitoring data are -available; thus, the final reports of land-based monitoring for project PACER HO have not yet been published. II. 'HISTORICAL BACKGROUND In April 1970, the Secretaries of Agriculture; Health, Education and Welfare, and the Interior jointly announced the suspension of certain uses of 2,4,5-T. These suspensions resulted from published studies indicating that 2,4,5-T was a teratogen. Subsequent studies revealed that the teratogenic effects had resulted from a toxic contaminant in the 2,4,5-T, identified as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Subsequently, the Department of Defense suspended the use of Herbicide Orange (3). At the time of the suspension, the Air Force had an inventory of 1.37 million gal of Herbicide Orange in South Vietnam and 0.85 million gal at the Naval Construction Battalion Center (NCBC) Gulfport Mississippi. In September 1971, the Department of Defense directed that the Herbici.de Orange in South Vietnam be returned to the United States and that the entire 2.22 million gal be disposed of in an environmentally safe and efficient manner. The 1.37 million gal were moved from South Vietnam to Johnston Island, Pacific Ocean, for storage (Project PACER IVY) in April 1972. The average concentration of TCDD in the Herbicide Orange was about 2 parts per million and the total amount of TCDD in the entire Herbicide Orange stock was approximately 44.1 pounds. Various techniques of destruction and recovery of the herbicide were investigated from 1971 to 1974 (AFLC Project on Disposition of Herbicide Orange). Destructive techniques included soil biodegradation, high temperature incineration, deep well injection, burial in underground nuclear test cavities, sludge burial and microbial reduction. Techniques to recover a useful product included use, return to manufacturers, fractionation and chlorinolysis. II-l �Of these techniques, only high temperature incineration was sufficiently developed to warrant further investigation. The other methods were rejected because of several considerations, including long lead times for development, inadequate assurance of success, and the lack of industrial interest. In December 1974, the USAF filed a final environmental impact statement (3) with the President's Council on Environmental Quality on the disposition of Herbicide Orange by destruction aboard a specially designed incineration vessel in a remote area of the Pacific Ocean, west of Johnston Island, The EPA held a public meeting in February 1975 to consider an ocean incineration permit application submitted by the USAF in accordance with the Marine Protection, Research and Sanctuaries Act of 1972 as amended, 33 U.S.C. 1401 et seq. During this meeting, testimony was presented which indicated that techniques for chemically reprocessing the herbicide to remove unacceptable quantities of TCDD might have been developed. The EPA indicated that the option for reprocessing should be further explored as a means of disposition prior to making a decision to destroy the herbicide via incineration (7). Subsequently, the USAF undertook an investigation into the feasibility Of reprocessing Herbicide Orange. Pilot plant studies were conducted from the fall of 1975 to July 1976 on selective activated carbon adsorption of TCDD from herbicide. This reprocessing method was shown to be technically and environmentally feasible; however, a feasible and environmentally acceptable method of safely disposing of the TCDD-laden activated carbon was not demonstrated. The USAF concluded in February 1977 that the option of reprocessing was not feasible, timely or cost effective since a technique for the ultimate disposal of the activated carbon was not currently available Or anticipated in the foreseeable future. Consequently, on 9 March 1977, the USAF requested reconvening the EPA public hearings. As a result of the public hearing held on 7 April 1977, the EPA issued a research permit to the USAF and Ocean Combustion Services, B.V. (OGS) (6). This permit authorized the transport of the Herbicide Orange from the Naval Construction Battalion Center, Gulfport MS to a designated site in the North Pacific Ocean for the purpose of atsea incineration in accordance with the provisions of the Marine Protection, Research and Sanctuaries Act of 1972, as amended. The vessel contracted for the at-sea incineration was the Dutch-owned ship, M/T Vulcanus, a ship registered in Singapore and previously used in the North Atlantic Ocean and the Gulf of Mexico to destroy chlorinated hydrocarbon wastes (12). A total of three herbicide loadings were required to incinerate the total stocks of Herbicide Orange: one loading from Gulfport MS and two loadings from Johnston Island. III. DESCRIPTION OF LAND-BASED OPERATIONS The operations at both storage sites were similar in many ways. At both sites, the 55-gal drums of Herbicide Orange were transported II-2 �short distances from their storage location to a centralized facility. The herbicide was drained from the drums and transferred to the M/T Vulcanus. Following emptying, the drums were rinsed with diesel fuel, and subsequently crushed. The rinsing from empty drum cleaning was combined with the herbicide and transferred to the ship for later incineration at sea. A. NCBC, Gulfport MS The centralized dedrumming facility at the NCBC was a temporary, enclosed facility measuring approximately 35 feet by 35 feet with an interior ceiling height of approximately 10 feet. A ventilation system capable of providing approximately 57 air changes per hour was equipped with in-line activated charcoal filters to reduce vapor emissions to the outside air. Within this enclosed facility were four identical processing lines. Each line consisted of a self-closing entry door to admit full drums, a roller conveyor along which drums were moved in an upright position, a position equipped with a heavy duty electrically operated deheading cutter, a suction wand to remove the greatest portion of the herbicide from a deheaded drum, a spray device beneath the conveyor over which the deheaded and emptied drum was inverted and rinsed with two gal of diesel fuel, a commercial, heavy duty drum crusher and a selfclosing exit door through which the crushed drums were passed. Once each drum was deheaded the con.tents were removed by the suction wand, leaving approximately three gal of liquid in the drum. The drum was then manually inverted and the remaining herbicide was collected in an open trough beneath the conveyor. Each drum was permitted to drain into the same trough for a minimum period of five minutes after which it was sprayed with two gal of diesel fuel, allowed to drain while still inverted for a minimum of two minutes, and then crushed end-to-end to approximately one-third its original volume. The rinsed and crushed drum was passed through the exit door and stacked with all other crushed drums. The liquid herbicide from the suction wands, and the herbicide and diesel fuel rinsing from the below-grade, open trough were pumped to 10,000 gal capacity rail tank cars. Air displaced from the tank cars during filling was filtered through an activated charcoal filter. The rail cars were moved along a rail spur approximately two mi.les to a dockside location where the herbicide was transferred to the incinerator ship, M/T Vulcanus. Displaced air from the ship's cargo' tanks was also filtered through activated charcoal. A total of 15,480 drums of Herbicide Orange was processed in this fashion at the NCBC between 24 May 1977 and 10 June 1977. Two 8hour shifts of approximately 55 men each accomplished the dedrumming/transfer operations. These men were all USAF officers/technicians from the five Air Logistics Center of the Air Force Logistics Command located at Kelly AFB, Texas; Hill AFB, Utah; Robins AFB, Georgia; Tinker AFB, Oklahoma and McClellan AFB, California. All workers were provided daily changes of II-3 �freshly laundered work clothes and men working within the dedrum facility were provided protective clothing including cartridge respirators, face shields, rubber aprons and rubber gloves. With only few exceptions the men rotated through all jobs involved in the dedrumming/transfer operations. All personnel were given pre-operational and post-operational physical examinations consisting of a complete medical history, complete neurological examination and the following laboratory procedures: 1. Complete hemoglobin, including hematocrit and platelet count 2. Prothombin time 3. Serum lipids 4. Serum glutamic oxaloacetic transaminase (SGOT) or 5. Serum glutamic pyruvate transaminase (SGPT) 6. Serum bilirubin 7. Blood glucose 8. Complete urinalysis 9. Chest x-ray B. Johnston Island The centralized dedrum facility at Johnston Island was a temporary, open facility measuring approximately 30 feet by 90 feet consisting of a concrete pad, roof and moveable canvas walls to exlude rain. This open facility was located adjacent to the Herbicide Orange storage site on the northwest end of Johnston Island. Nearly constant east winds ranging from 10 to 20 miles per hour provided natural ventilation and carried released vapors away from occupied areas. Two processing lines consisting of fabricated metal racks and open troughs were located in the west two-thirds of the facility. The east one-third contained pumps and drive-through for fuel trucks that were used to transport the dedrummed herbicide to the M/T Vulcanus. Full drums of herbicide were transported to the dedrum facility in sets of four using forklifts equipped with specially designed clamps. The drums were placed on the inclined metal racks in four groups of 12 drums each. Each set of 12 drums was handled independently by the dedrumming crew. Once a set of 12 drums was on the rack and the forklifts had withdrawn, a crew member would punch one hole near the top of each inclined drum as a vent hole to allow the crew's supervisory personnel to check the contents. Any drums containing other than Herbicide Orange were removed from the line and held for further testing. Three or more closely spaced holes were then punched in the bottom of each drum and the contents allowed to drain into II-4 �the open troughs. Once the herbicide had stopped flowing from the drums, they were allowed to drain for a five minute period after which the interior of each drum was rinsed twice with a total of two gal of diesel fuel. The diesel fuel rinsing drained into the open troughs, combining with the herbicide. After the 12 drums in each set had drained for a minimum of two minutes they were transported to a nearby drum crusher which consisted of a large weight suspended between two vertical I-beams. One drum at a time was crushed along its longitudinal axis and when approximately 30 drums had been crushed they were removed, banded, and stacked together near the crusher. The liquid herbicide and diesel fuel rinsing from the drums flowed into the two open troughs to a below-grade sump. The material was pumped from this sump into modified fuel tankers that transported 3,000 gal lots to dockside where the material was pumped aboard the M/T Vulcanus. A total of 24,795 drums of herbicide was processed in this fashion between 27 July 1977 and 23 August 1977. Two 10-hour shifts of approximately 50 men each were used. The workers were civilian employees of a contractor engaged to perform the dedrumming operations. USAF officers monitored all operations. As at NCBC, all workers were provided daily changes of freshly laundered work clothes, and men working within the dedrum facility were provided protective clothing consisting of cartridge respirators, face shields, rubber aprons, rubber gloves and boots. Unlike at NCBC, men on each crew remained in the same job through the dedrumming/transfer operations. A requirement of employment was preand post-operational physical examinations similar to those given the workers at the NCBC. IV. LAND-BASED OPERATIONS MONITORING PROGRAMS Detailed plans for environmental and occupational monitoring at both sites are contained in Annexes 4 and 5, kJtii Fo/ice. LciQiAticA Command VnoQfummhiQ Plan 75-19 faon the. V<it>pa&cut o& Oi&nge HeA.bi.cu.de (2). This section outlines only the industrial hygiene and ambient air monitoring programs conducted at each site. These aspects of the environmental and occupational monitoring at each site were very similar. Essentially, the same equipment, methods and procedures were used at both sites. The only significant difference between the two operations was that all sampling at the NCBC site was accomplished by members of the US Air Force Occupational and Environmental Health Laboratory (USAF OEHL), Brooks AFB, Texas, while all sampling at the Johnston Island site was conducted by Battelle Columbus Laboratories (BCL), Columbus, Ohio,under contract to the USAF. An environmental engineer from the USAF OEHL served as Project Officer and monitor of the BCL contract. In general, the industrial hygiene sampling program consisted of daily air samples within the dedrum facilities with rapid analysis (approximately 24-hour turn around time) for 2,4-D and 2,4,5-T. Samples collected for analysis of TCDD were analyzed after-the-fact. The ambient air sampling at various locations and distances from the dedrum II-5 �facilities included samples for 2,4-D, 2,4,5-T and TCDD analyses, as well as biomonitoring using rapidly growing tomato plants as indicator organisms. Pre-operational and post-operational background sampling was also accomplished. A. Monitoring Equipment and Procedures Two different methods were employed for industrial hygiene and ambient air sampling for 2,4-D, 2,4,5-T and TCDD. These procedures have been developed and field tested by the USAF OEBL. 1. 2,4-D and 2,4,5-T . , Sampling for 2,4-D and 2,4,5-T was accomplished utilizing Chromosorb*R' 102 as an adsorption medium, a granular polymer well suited for collection of chlorinated hydrocarbon vapors in air (10,11). The polymer was packed in micro-pipet tubes which were then wrapped in new aluminum foil and stored in rubber stoppered test tubes. The sampling apparatus consisted ofRa Mine Safety Appliance Model G Personnel Sampling Pump., The Chromosorb^ ' 102 tubes were connected to the pumps with Tygon^' or latex rubber tubing. A flow rate of 0.50 liters/minute (1/min) for periods ranging from five to ten hours was used, yielding an air sample volume of approximately 150 to 300 liters. This sampling time corresponded to the length of approximately one-half shift and was expected to yield sufficient adsorption efficiency to permit easy analysis. Flow rates were checked hourly with a calibrated rotameter to insure that 0,50 1/min flow rate was maintained. Where possible the pumps were maintained on constant "high" recharge by providing connections to available 110-volt power supply. When the Chromosorb(R' 102 tubes were removed from the field for lab analysis the individual tubes were wrapped in aluminum foil and returned to their respective rubber stoppered test tubes. 2. TCDD Air sampling for TCDD was accomplished using benzene as a collection medium. The sampling apparatus consisted of a train of four Greenberg-Smith impingers. The first two impingers were fritted and each contained approximately 350 ml of benzene. The third and fourth impingers were modified by removal of the fritts and contained activated carbon to adsorb vaporized benzene. The two benzene impingers were wrapped with aluminum foil providing a light barrier that would prevent any photodecomposition of the TCDD collected in the sample. Following the four impingers, an in-line paper filter was attached with TygonW tubing to prevent carbon particles from entering the Mi Hi pore pump. The pumps were operated directly from 110-volt AC power and the flow rate was one 1/min. The duration of sampling ranged from three to five hours, yielding an air sample volume from 180 to 300 liters. Flow rates were checked hourly using a calibrated rotameter and total volume of air sampled was calculated from these hourly flow rates. The maximum running time of five hours was dictated by ambient temperatures ranging from 71 to 92 degrees F and the saturation limitations of the carbon to adsorb the benzene vapors. Samples were removed from the sampling sites with II-6 �impinger trains intact in special wooden holders. The benzene was drained into new brown glass jars in a "clean" laboratory area. The impinger glassware was rinsed with benzene into the sample container to collect any materials adhering to the impinger walls. All impinger glassware was rinsed three times with acetone and once with benzene prior to reuse in the field. 3. Biomonitoring Immature, rapidly-growing, potted tomato plants, Lycop&ti>J.con ej>c.vJte.Yvt(m, ranging in size from 6 inches to 18 inches were used as indicator organisms for detecting the presence of Herbicide Orange vapors in air at various locations around the land-based dedrumming, transfer and loading operations. Young tomato plants are known to be very sensitive to phenoxy herbicide vapors (9). The symptoms typical of exposure to Herbicide Orange vapors, known as epinastic growth, is described as a curling and/or twisting of the apical portions of the plants. Depending on level of exposure these symptoms would appear within 24-hours after exposure. Normal procedures included observations, at least once daily, of the tomato plants to record the presence of the epinastic growth symptoms and to water the plants. Relative rating scales were used to describe the levels of damage noted. It was not possible to quantitate the levels of vapor exposure, but the extent to which low parts-pertrillion (ppt) herbicide vapor levels were carried by prevailing winds could be determined. B. Analytical Procedures and Methodologies The analytical procedures and methodologies used throughout Project PACER HO were developed, refined, tested and repeatedly used throughout the variety #f field exercises conducted by the USAF OEHL over the five year period from 1972 to 1977. 1. 2,4-D and 2.4,5-T Analysis of Chromosorbv(R^ 102 air samples was provided by ' two different laboratories. In the case of the NCBC, samples were analyzed by the U.S. Department of Agriculture Laboratory, Gulfport MS under an interservice agreement. All Johnston Island air samples for 2,4-D and 2,4,5-T were analyzed by the staff of the Battelle Columbus Laboratory team. The methods for analyses of herbicide will be reported elsewhere (4,5). 2. TCDD The Brehm Laboratory, Department of Chemistry, Wright-State University, Dayton Ohio, analyzed all benzene impinger samples for TCDD as well as many other types of samples and substrates in support of Project PACER HO. The Brehm Laboratory has been under contract with the USAF for II-7 �several years and has developed unique analytical capabilities in trace analysis for TCDD in a variety of substrates. The analytical methods employed for this project by the Brehm Laboratory have recently been published (8). V. LAND-BASED MONITORING RESULTS Detailed results of environmental and occupational monitoring at both sites will be reported elsewhere (4,5). This section outlines only the industrial hygiene and ambient air monitoring results for each site. Suffice to say that all other available data have indicated that there were no adverse environmental impacts on air, water or land resources at either site as a result of land-based dedrumming, transfer operations. A. NCBC, Gulfport MS The results of the industrial hygiene and ambient air monitoring programs at the NCBC are summarized below: 1. Industrial Hygiene The industrial hygiene air sampling results for 2,4-D, 2,4,5-T and TCDD are presented in Table 1. Five operational industrial hygiene samples were collected during each shift from the four corners within the enclosed dedrum facility. Four of these samples were for 2,4-D, 2,4,5-T using Chromosorb'R) 102, while the fifth sample was a benzene impinger in one corner of the facility collected for TCDD analysis. The ChromosprbW 102 and benzene impinger samplers were placed in low traffic areas near the four corners of the enclosed facility to prevent interference with work activity within the facility. As shown in Table 1, vapor concentrations of the n-butyl esters of 2,4-D and 2,4,5-T ranged from 7.76 - 141.15 ug/m3, respectively. The uniformity of concentrations of herbicide vapors within the dedrum facility is demonstrated by the lack of significant variability of 2,4-0/2,4,5-T data among the four sampling locations. All noted levels were well below the time weighted average Threshold Limit Value (TLV) of 10,000 yg/m3 for either 2,4-D or 2,4,5-T as adopted by the American Conference of Governmental Industrial Hygienists (ACGIH). No TCDD was detected in any of the 27 benzene impinger samples. The minimum detectable concentrations for TCDD ranged from 22.4 to 35.9 ng/m3. 2. Ambient Air Ambient air samples for 2,4-0/2,4,5-T and TCDD analysis were collected from three different locations. In addition, 29 groups of four tomato plants each were positioned around the dedrumming/transfer operations. The results of these monitoring efforts are presented below: a. 2,4-D, 2,4,5-T and TCDD. Sampling stations at two locations on the NCBC were established, one at the base fire station approximately 900 feet SW of the dedrum facility and one at the PACER HO Operation Center approximately 1,500 feet E of the dedrum facility. A II-8 �TABLE 1. Results of industrial hygiene air samples collected inside the dedrumming facility Project PACER HO NCBC, Gulfport, MS, 24 May - 10 June 1977. Sample Location Dedrum Facility Naval Construction Battalion Center (NCBC) • SE Corner NE Corner SW Corner NW Corner 28 28 14 14 NBEa2,4-D (yg/m3) Range Std Dev Mean 8.7-141.15 31.45 52.99 7.86-136.35 34.55 53.72 7.76-134.9 36.25 54.58 15.18-105.11 27.01 51.5 NBEa2,4,5-T (yg/m3) Range Std Dev Mean 5.52-65.11 14.98 26.40 5.70-76.36 18.57 29.93 3.01-79.62 21.02 32.39 7.59-51.31 12.79 25.93 0 0 0 Parameter No. of Samples TCDD No. of Samples Mean a 27 K NDb NBE is normal-butyl ester. ^ND is non-detectable at minimum detectable concentrations that ranged from <22.4 to <35.9 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 yg/m3. (See text) II-9 �third location on the wharf approximately 300 feet north of the ship loading point was also sampled. The results of analyses of these samples are presented in Table 2. As expected, the levels of 2,4-D, 2,4,5-T were significantly (45 to 150 times) lower than were found within the dedrum facility. Filtering of exhaust air from the facility, downwind diffusion/ dispersion of released vapors, and the lack of any significant spillage of herbicide outside the facility no doubt accounted for these significantly lower levels. No TCDD was detected at any of the three ambient air sampling stations with the minimum detectable concentrations ranging from approximately 22 to 55 ng/m3. b. Biomonitoring. Tomato plants were placed in groups of four in two concentric rings around the dedrum facility at 500 feet and 1000 feet distances. Moderate to severe plant damage was noted along the axis of prevailing winds in the inner ring (500 feet). Slight to moderate plant damage was noted in the corresponding outer (1000 feet) ring. In addition, several sets of four plants were set up along the NCBC perimeterfence. In two cases test plants along the base perimeter showed only minimal damage. One set of plants was also placed on the dock 300 feet inland from the loading operations. No damage was noted at this location. B. Johnston Island The results of the industrial hygiene and ambient air monitoring programs at Johnston Island are summarized below. There were two distinct loading operations during the Johnston Island phase of the project. The first dedrum/transfer (first loading) operation was conducted from 27 July 1977 to 5 August 1977, and the second loading from 17 August 1977 to 23 August 1977. 1. Industrial Hygiene The industrial hygiene sampling of the Johnston Island operations differed from the sampling at the NCBC. The facility was larger and open to natural ventilation and the dedrum operations were far different as described earlier. Because of these and other factors the industrial hygiene sampling program was modified to include true "breathing zone" samples for 2,4-D, 2,4,5-T from selected worker positions. In general, there were three worker positions evaluated using the Chromosorb\R' 102 tubes. These positions were selected after an analysis of all positions revealed that these worker locations represented the greatest possibility of receiving a significant exposure. The first was the position occupied by those workers who punched the vent holes in each drum. When the vent holes were punched internal pressure in many drums was released, and there was a possibility of elevated exposures to workers in these positions. The second worker position evaluated was that occupied by the workers who punched the several drain holes in each drum, and the third position was the operator of the sump pump. In the latter two cases, these workers were close to open troughs of flowing Herbicide Orange. In addition to these "breathing zone" samples, air samples within the dedrum facility were also collected for ^,4-D, 2,4,5-T and TCDD. Tables 3, 4 and 5 present the results of these sampling programs. 11-10 �TABLE 2. Results of ambient air samples collected at Gulfport MS, Project PACER HO, 24 May - 10 June 1977. Sample Location NCBC, Gulf port, MS Parameter No. of Samples Fire Station Ops Center Wharf 28 29 30 NBEa2,4-Diugym3l Range Std Dev Mean 0.09-5.76 1.20 1.17 0.13-3.88 1.00 1.09 0.07-2.41 0.53 0.52 NBE a 2,4,5-T (yg/m 3 ) Range Std Dev Mean 0.04-3.36 0.85 0.52 0.34-1.97 0.49 0.34 0.01-1.45 0.32 0.21 TCDD No. of Samples Mean 27h NDb 27. NDb 23 h NDb NBE is normal butyl ester. } ND is non-detectable at minimum detectable concentrations that ranged from <21.9 to 55.2 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 yg/m3. (See text) 11-11 �TABLE 3. Results of industrial hygiene air samples collected inside the dedrumming facility* Project PAGER HO Johnston Island* first loading 27 July - 5 August 1977. Sample UeatiSn Dedfum Facility deHhStbfi Islands First Loading Parameter SW Corner NW Corner E Wall 3 3 3 NBE a 2,4-D (ug/m3) Range Std Dev Mean 12.8-16,0 1,77 14.84 4.79-13*33 7,30 9.99 0.50-2.58 1.37 1.03 NBla2,.4,5-t (ug/m3) Ramge Std Dev Mean 192-8.84 1.05 8J2 2.26-8.28 3.24 4.58 - 0 0 No, of Samples TGDD No. of Samples Mean NDb a NBE is normal butyl ester, bND is non-detectable ait ifiihlfiiufri deteetSble concentrations that ranged from <8.06 to <13.89 rig/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 vig/m3. (Sfee text) 11-12 �TABLE 4. Results of industrial hygiene air samples collected inside the dedrumming facility Project PACER HO, Johnston Island, second loading, 17 - 23 August 1977. Sample Location Dedrum Facility Johnston Island, Second Loading Parameter SW Corner No. of Samples NBEa2,4-D (ug/m3) NW Corner 1 1 18.78 6.60 7.35 2.27 5 NDb 0 NBEa2,4,5-T (uq/m3) TCDD No. of Samples Mean NBE is normal butyl ester. 'ND is non-detectable at minimum detectable concentrations that ranged from <6.64 to <23.41 ng/m3. 11-13 �TABLE 5. Results of .industrial hygiene "breathing zone" samples collected inside the dedrumming facility Project PACER HO, Johnston Island. p'arsfrtete'r ' Sample' locations Dedrum Facility Johnston Island, (See Text) Veftt Drain Pump Punchers Ptine fret's O^efatof First Leading (27 July - 5 August 1977} Nd. of Samples NBEa2,4-D (ug/m3) Range Std Dev Mean NBEa2.,4,5-T (ug/m3) Range Std Dev Mean 8 10 5 2.14-30.8 8.35 17.92 7.64-19.18 5.73 19.18 . 6.11-26.78 8.18 14.36 0. 57-16. t 4.52 8.70 3.79-13.6 2.95 9.54 2.43-11.48 3.61 ' 6.32 Second Loading (17 - 23 Atigust 1977) No., of Samples 12 7 NBEa2,4-D (yg/m3) Range Std Dev Mean 8.38-40.28 10.47 23.20 .NBEa2,,4,5-T 4yg/m3J Range Std D£v Mean 6.49-22.22 6.06 13.-21 0 15.96-38.0 8.53 23.04 - 8.82-22.53 5.20 13.68 - NBE is ndrltial butyl ester. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10*000 yg/m3. (See text) 11-14 �The levels noted within the dedrum facility at Johnston Island were on the order of two to five times lower than those noted at the NCBC, Gulfport MS. These lower concentrations probably resulted from much greater dilution by natural ventilation of the open facility at iiohnston Island. Needless to say, the noted levels of 2,4-D and 2,4,5-T were well below the ACGIH TLV of 10,000 ug/m3. No TCDD was detected in any of the samples analyzed. 2. Ambient Air Ambient air samples for 2,4-0/2,4,5-T and TCDD analyses were collected from three different locations. In additon, 14 groups of four tomato plants were positioned at selected locations around the dedrum/transfer operations. The results of these monitoring efforts follow. a. 2,4-0/2,4,5-T and TCDD. One downwind and two upwind sampling stations were established. The downwind site was located approximately 300 feet west of the dedrum facility. The two upwind sites were the fire station approximately 4,000 feet SE and the weather station approximately 6,000 feet ESE of the dedrum facility. The results of downwind and upwind ambient air sampling sites are presented in Tables 6 and 7, respectively. As was expected, the levels of 2,4-0/2,4,5-T noted at the downwind site were somewhat lower than those levels noted within the dedrum facility. The relatively higher levels noted for the second loading as compared to the first loading are not explainable. These levels, however, are well below the TLV. No TCDD was detected in any of these samples. b. Biomonitoring. Tomato plants were placed at 14 biomonitoring stations on Johnston Island. Four of these sites were downwind of the dedrumming facility and the remaining ten locations were all upwind. Throughout the two periods of dedrumming operations all the downwind sites displayed slight to severe herbicide induced damage. There was only slight damage noted on two days at one of the upwind sites. The results of the tomato plant bioassay indicate that during the dedrumming operations concentrations of Herbicide Orange did not occur upwind of the dedrumming facility at sufficient concentrations to affect the tomato plants. VI. SUMMARY AND CONCLUSIONS As part of the environmental and occupational monitoring programs, the USAF accomplished industrial hygiene and ambient air sampling of all land-based dedrumming/transfer operations of Project PACER HO, the USAF project to dispose of 2.22 million gal of Herbicide Orange. The results of these sampling programs revealed that under the worst case noted, the levels of 2,4-D and 2,4,5-T vapors were well below the TLV for each of these materials. The noted levels were at least two and in most cases three orders of magnitude below the TLVs. TCDD was not detected in any air samples. 11-15 �TABLE 6. Results of downwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. Downwind Ambient Air Sampling Parameter No. of Samples NBEa2,4-D (yg/m3) Range Std Dev Mean NBEa2.3»5-T Cug/m3) Range Std Dev Mean First Loading (27 Jul-5 Aug 77) Second Loading (17-23 Aug 77) 14 1.92-25.5 5.99 6.21 5.79-32.67 7.73 12.51 0.82-17.1 4.33 3.27 1.89-14.0 3.46 5.12 TCDD No, of Samples Mean NDC NBE is normal butyl ester. ND is non-detectable at minimum detectable concentrations that ranged from <11.68 to <21.0 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 pg/m3. (See text) 11-16 �TABLE 7. Results of upwind ambient air samples collected at Johnston Island, Project PACER HO, 27 July - 23 August 1977. Wharf Station Weather Station First Loading'3 No. of Samples NBEa2,4-D (jjg/m3) Range Std Dev Mean NBEa2,4,5-T (jjg/m3) Range Std Dev Mean TCDD No. of Samples Mean a 11 Second 0 Loading 11 First 13 Loading 11 7 Trace-0.67 0.39 0.25 ND-2.54 0.77 0.23 0 0 Trace 0.34 0.10 0 0 0 0 1 NDd 1 NDe 0 0 Trace-1.09 0.42 0.29 Second Loadi ngc 0 0 NBE is normal butyl ester. b First Loading 27 July - 5 August 1977. C 5econd Loading 17-23 August 1977. d ND is non-detectable at the minimum detectable concentration of <8.52 ng/m3. e ND is non-detectable at the minimum detectable concentration of <20.34 ng/m3. NOTE: The time-weighted Threshold Limit Value for either 2,4-D or 2,4,5-T is 10,000 yg/m3. (See text) 11-17 �Biomonitoring using tomato plants revealed that low-level vapors of Herbicide Orange were dispersed and diffused downwind of the land-based dedrumming/transfer operations at both sites. No adverse environmental impact resulted from these operations. Approximately 200 personnel carried out the dedrumming activities at the NCBC, Gulfport MS and at Johnston Island. Comparisons of available pre- and post-operational medical examinations of military personnel involved have revealed no apparent physical effects as a result of these activities. II-18 �CHAPTER II LITERATURE CITED 1. Ackerman, D.G., H.J. Fisher, R.J. Johnson, R.F. Maddalone, B.J. Mathews, E.L. Moon, K.H. Scheyer, C.C. Shin, and R.F. Tobias. 1978. At-4ea -tnc-tneAotton ofa HeAb4.cJ.de. Orange onboard the. M/T l/u£canoA. Environmental Protection Technology Series EPA-600/2-78-086. Office of Research and Development. U.S. Environmental Protection Agency, Research Triangle Park, North Carolina. 263 p. 2. Anonymous. 1977. A/iA Force Log<it>ticA Command programnujtg p£an 75-19 fan the. di&po&aJL o<j Orange Herfa.tc-t.de. San Antonio Air Logistics Center, San Antonio, Texas. Annex 4, pp 1-17, Annex 5, pp 1-23. 3. Anonymous. 1974. fl-iipo-A-ctcon o& Orange HeAb^cu.de by -imu.neAott.on. Final Environmental Statement. Department of the Air Force, Washington, D. C. 737 p. 4. Anonymous. 1977. Land-bo6ed env-tronmentat monitoring at Johnston Uland. Parts I and I I . Project PACER HO. USAF Contract No. F08635-76-D-0168 Battelle Columbus Laboratories, Columbus, Ohio. IH press. 5. Anonymous. 1978. Lewd-boused env-tAonmentod monitoring at tke. Navat Co»t6.t>t.uCxfcton Bouttation CwteJi, GutfipoKt, Mx6i4-c4AxCpp/c.. Technical Report of the U.S. Air Force Occupational and Environmental Health Laboratory, Brooks AFB, Texas. Jji press. 6. Anonymous. 1977. Mo/toie Pio.£ecxtt.on, Reieotch, and Act (Ocean Pampxjag) Re^ eaA.cn peAm-ct No. 770VH001R, United State* Protection Agency, Washington, D. C. , 15 p. 7. Anonymous. 1975. Ocean dumping, receipt of application and tentative determination. U.S. Environmental Protection Agency. Reg-cAteA 40(57): 13026- 13028. 8. Erk, S.D., M.L. Taylor and T.O. Tiernan. 1978. Env/^ionmenta£ moyUtotsing -en con/unctcon w^t^i -tnctneAa^tcon o& HeAb-tttde Orange at *ea. Activities of the Brehm Laboratory, Wright State University Dayton, Ohio. Presentation to the 1978 National Conference and Exhibition on Control of Hazardous Material Spills, Miami, Florida. 31 p. 9. M u l l i s o n , W . R . 1951. The tomato as a test plant for growth regulators. Bot. Gaz. 112:521-524. 10. Thomas, T.C. and J . N . Seiber, 1974. Chromosorb(R) 102, an efficient medium for trapping pesticides from air. Bu££. Env-cAon. Contain. and ToKicol. 12(1): 17-25. 11-19 �11. Thomas, T,C. and J.W. Jackson. 1978. A technique for sampling 2,4-D; 2,4, 5-T herbicides from air. J. A-UL VoUi-. Control tin press. 12. Wastler, T.A, , C,A. Offutt, C.K. Fltzsilflmons and P.E, Des 1975. V4J>p0Aa£ Ojf 0tycM.o£ki0JUn& um/tfci by 4.nc*Ln&t£Utin Environmental Protection Tedhnglociy Series EPA«430/9-7 Office ef Water and Hazardous Materials. Environmental Proteetion Agehcy* Washifigtdn, D,C, 223 p; II-20 �CHAPTER III ENVIRONMENTAL FATE OF 2,4-D, 2,4,5-T AND TCDD I. INTRODUCTION Chapter I was devoted to the topics of types and quantities of herbicides sprayed in South Vietnam and their handling and application. Emphasis was placed on those factors that may have influenced human exposure to the herbicides prior to actual spray applications. This chapter will focus primarily on the fate of the phenoxy herbicides sprayed in South Vietnam and on the contaminant TCDD. This is appropriate since 94 percent of all herbicides disseminated in South Vietnam were phenoxy herbicides (53 percent 2,4-D and 41 percent 2,4,5-T). The extreme toxicity of the contaminant, and its associated biological effects, require that all available data be reviewed in an attempt to determine the potential adverse human effects this compound may have had on the population at risk in South Vietnam. What happens to the individual compounds physically, chemically and biologically in the environment will significantly influence the route of exposure, the duration of exposure and the total dose (or level) of that exposure to the population at risk. Again, as noted in Chapter I, the population at risk will be confined to personnel of the U.S. military forces. The expression of units of weight, area, or volume has not been standardized between various publications cited in this Chapter. II. THE ENVIRONMENTAL FATE OF THE PHENOXY HERBICIDES A. Physical/Chemical Factors Influencing Disappearance of Herbicides 1. Fate in Air Harrigan (27) reported that in a test program evaluating the dissemination characteristics of the A/A 45 Y-l Spray System, the mean recovery of Herbicide Orange by ground sampling methods from six missions flown under operational parameters typically used in South Vietnam was 87 percent. The remaining 13 percent may have been undetected due to sampling technique or may havev failed to impact the sampling array due to drift or volatility. The mean particle size for the six missions flown was 367 micron (y). Harrigan (27) 1n the above test program with Herbicide Orange, found the following droplet size distribution in the mean percent mass recovered: Particles less than lOOy Particles 100 to 50Qy Particles greater than 500y III-I 1.9 percent 76,2 percent 21.9 percent �The recovery of 87 percent of the Herbicide Orange disseminated is in agreement with Plimmer (50) who reported that deposition of 80 percent of particles greater than 200u in size takes place in short downwind distances, whereas those of diameter less than 5y may drift for miles. The aerial application of Herbicide Orange also presented an opportunity for volatilization since spray drops evaporate during their fall. This was recognized by Grover et al (25), who examined the relative potential for drift of volatile and nonvolatile formulations of 2,4-D under conditions of typical agricultural application. The ground application system employed by Grover et al resulted in only 2.8 percent of the total spray having a particle size less than 200y. The mass of the formulation drifing- as droplets was similar (3 to 4 percent) for either the volatile (n-butyl ester) or nonvolatile (dimethylamine) formulation of 2,4-D. However, for the butyl ester, in addition to droplet drift, within the first 30 minutes after spraying 25 to 30 percent of the material was collected as vapor drift in air samplers up to 246 feet (ft) downwind from the point of application. The data by Grover et al (25) may suggest that although high percentages of Orange particles were intercepted by the vegetation, a significant amount of the material may have rapidly volatilized and moved in the air within the jungle canopy. This is in accord with what Brown (12) had first proposed in 1962 when he recommended the use of the esters of 2,4-D and 2,4,5-T for defoliation in South Vietnam. Better effect can be achieved on a susceptible tree if all its leaves receive a few drops of chemical as opposed to only one side or only the very top of the tree receiving all the chemical. In this connection, forms of the chemical known as volatile esters were requested subsequently in order to achieve more uniform coverage within a forest canopy. 2. Fate on Vegetation Approximately 85 percent of all the 2,4-D and 2,4,5-T sprayed in South Vietnam was'with the C-123/A/A 45 Y-l Spray System [estimate based on data by Irish et al (31), National Academy of Science (15), Craig (16), and Chapter I.] Klein and Harrigan (36) found that in five standard Orange missions the statistical mean value for maximum swath width having a deposition rate that would result in acceptable defoliation was 260t 20 ft. Thus, a typical 1,000 gallon (gal) sortie in South Vietnam would have effectively defoliated an area of approximately 346 acres (A). Data by Tschirley III-2 �(58) suggested that a multicanopy forest would intercept at least 94 percent of all the spray droplets. It is therefore reasonable to assume that if the entire 1,000 gal of Orange fell within the 346 A area, 940 gal of Orange would have been deposited on the canopy vegetation, and 60 gal deposited at ground-level on the soil or small herbaceous understory. The actual ground-level deposition may then • have been 0.17 gal/A or 1.4 pounds (Ib) of 2,4-0/2,4,5-T per acre (60 gal/346 A = 0.17 gal/A x 8.14 Ib active ingredient/gal = 1.4 Ib 2,4-0/2,4,5-T per A). In the United States, mixtures of these phenoxy herbicides are routinely applied at 2 Ib/A. If time after application was the same, then military personnel moving through defoliated forests in South Vietnam probably would have encountered the same amount of herbicide as would a rancher in the United States walking through defoliated brush-infested ranch land. Once the herbicide is intercepted by the vegetation, numerous physical and chemical barriers influence the amount of herbicide that is absorbed, transported and accumulated. In Volume 2 (Weed Control) of a special series on the principles of plant and animal pest control, the National Academy of Science (49) reviewed the physical and chemical barriers which intervene between application of a herbicide and its ultimate effect on the plant. They found that, in general, both the upper and lower leaf surfaces absorb herbicides. Usually, the lower epidermis is penetrated more readily, but not all areas of either surface are equally permeable. The penetration of the phenoxy herbicides into most foilage is by diffusion through the cuticle (cuticular entry). Warm temperatures that are not excessive and high humidity may actually promote the entry. Because the cuticle and the cell walls upon which the cuticle is deposited contain chemically nonpolar materials that are slightly electronegative, nonpolar herbicides (e.g., Orange and Purple) tend to be absorbed into leaves faster than polar herbicides. Cuticular penetration by the esters of 2,4-D or 2,4,5-T may occur within 30 minutes of their application. 3. Fate in Soils Hamaker (26) has reviewed the physical and chemical factors that influence fate of herbicides in soil. These include soil adsorption, hydrodynamic dispersion and diffusion, adsorption dynamics and evapotranspiration. The phenoxy herbicides, for example, have low adsorption coefficients and thus tend to leach in a soil profile. The actual amount of leaching, however, will vary from soil to soil, mainly in response to the organic carbon content. Moreover, only the herbicide free in the soil water will be carried down by descending water. Crosby (18), in reviewing nonbiological degradation of the phenoxy herbicides in soil, reported that the isopropyl, butyl and isooctyl esters of 2,4-D had a half-life of about 100 hours (h) III-3 �in neutral soil water (although hydrolysis was almost instantaneous in the presence of a base or a suspension of any of several soils at pH 7.0-7.5). Moreover, many of the phenoxy herbicides, e.g., 2,4-D, will readily undergo oxidation, reduction and substitution (notably hydrolysis) in aqueous solutions when activated by sunlight in air. The end product of the photodegradation of 2,4-D is humic acid (17). Although 2,4,5-T absorbs some ultraviolet light in sunlight, the amount is small and this herbicide is relatively unreactive (only 7 percent was hydrolyzed in 48 h). However, the presence of ferric salts or zinc oxides in the soil water will result in an increase in photolysis rate (17). Another nonbiplogical factor that determines the soil persistence of the phenoxy herbicides is their tendency to volatilize from the soil complex. Plimmer (50) noted that some volatilization will occur whether or not water is evaporating from the soil. However, a reduction in soil moisture content will decrease the pH of soil. This will favor the undissociated form of 2,4-D and 2,4,5-T and their potential for vapor loss may be increased. B. Biological Degradation of the Phenoxy Herbicides 1. Fate in Plants Loos (40) has recently reviewed the degradation of phenoxy herbicides in plants. In general, because of the widely different degradative pathways, the phenoxy herbicides do not persist in plants. However, Muzik (44) has reported that in some plants, for example, tomato, unmetabolized 2,4-D may be bound to cellular membranes and persist for two or three months. 2. Fate in Soils There is considerable evidence available to show that the phenoxy herbicides are rapidly decomposed in soils (5). Goring et al (23) in reviewing principles of pesticide degradation in soil noted that 2,4-D may undergo at least 6 different types of oxidation reactions, 1 reductive reaction, 1 hydrolytic reaction and 4 conjugative reactions. Because of this ability to readily undergo transformation, 2,4-D has been classed as a non-persistent pesticide since the estimated time required for 50 percent to disappear from soil was <0.5 months. However, 2,4,5-T has been classed as a slightly persistent pesticide since the time required for 50 percent disappearance was 0.5 to 1.5 months. III-4 �If 2,4-D were applied to a moist loam soil under summertime temperature at a rate of 0.5 to 3 pounds/acre (Ib/A), it would disappear in 7 to 30 days (37). If applied at rates of 4 to 55 Ib/A, it would probably disappear in one to three months (22). If 2,4-D were applied to the soil at a concentration of 500 ppm and disappeared at a rate proportional to the breakdown of 55 Ib/A, the calculated time would be 5.6 years. However, there is evidence that a more realistic time for inactivation of 500 ppm would be less (4). Persistence of 2,4,5-T in soils is usually two to three times longer than 2,4-D (22), and very few organisms have been identified as having the ability to breakdown the 2,4,5-T molecule (2). Newton (46) has calculated from studies on the kinetics of degradation by microorganisms that 2,4,5-T has a half-life of seven weeks in the forest floor. Investigations by Winston and Ritty (59) and Reigner et al (51) indicated that both 2,4-D and 2,4,5-T are decomposed to form carbon dioxide, inorganic chlorides and water; objectionable chlorophenols are not end-products of this decomposition. Further supporting evidence has been provided by Reinhart (52). The upper half of a 60 acre timber watershed in northern West Virginia was logged and treated with 2,4,5-T ester to kill all vegetation. The volume of herbicide that was applied was 1,325 gal on 30 acres (418 liters/ha). Almost 790 gal of this were potential contaminating materials: about 740 gal of diesel oil and 50 gal of a commercial formulation of 2,4,5-T (313 pounds acid equivalent). Reinhart found n£ odor contaminants (phenols or catechols) in the numerous water samples taken from the stream draining the treated watershed. In relation to the effects of herbicides on the soils of South Vietnam, the National Academy of Science published a report by Blackman et al (11) on persistence and disappearance of herbicides in tropical soils. The 1974 report stated a number of general conclusions, namely: 1. The behavior of herbicides in the soils of South Vietnam was similar to that reported for soils elsewhere. 2. Only where 2,4-D and 2,4,5-T were applied in very massive doses; e.g., at the Pran Buri Calibration Grid in Thailand at rates in the magnitude of 1,000 Ib/A, were there still residues (10 years following application) in concentrations above the threshold likely to induce phytotoxic symptoms in some plant species. 3. When applied to mangrove soils at total doses approaching 10 Ib/A of 2,4-D and of 2,4,5-T, the level of herbicide residue at the end of 30 weeks had no effect on the establishment of two major mangrove species. 4. In geographical areas subjected to one or two military herbicide missions 1.5 years before sampling, no soil phytotoxic residues could be detected. III-5 �5. Soils that received a directed application of Herbicide Orange at the rate of 27 Ib/A safely supported the growth of crops sensitive to 2,4-D or 2,4,5-T four to six months following application. 6. Claims that the herbicides rendered the soil sterile were without any foundation. Byast and Hance (14) have studied the degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Although care must be exercised in extrapolating laboratory results to field situations, their results suggested that the four Vietnamese soils studied were inherently capable of degrading 2,4,5-T at levels roughly twice the rate of military application in Vietnam. In support of feasibility tests for the soil disposal of surplus Herbicide Orange, the Air Force established a field study in 1972 on the Air Force Logistics Command Test Range, Hill Air Force Base, Utah. The study consisted of replicated plots subsurface injected with concentrations of either 1,000, 2,000, or 4,000 Ib herbicide/A. Soil samples were taken by Stark et al (56) three times throughout 1973, and microbial species present (bacteria, actinomycetes and fungi) were determined. Bacterial counts were higher for soils with greater concentrations, of the herbicide and with greater moisture content; i.e., those samples collected in midwinter from the 4,000 Ib/A plots. Herbicide Orange, in any concentration, had no significant effect on mycoflora. Arnold et al (4) monitored the herbicide levels in these plots. They sampled the plots on eight occasions from 1973 through 1975 and determined the concentrations of the n-butyl esters and free acids of both 2,4-D and 2,4,5-T. They suggested that at such massive application rates (soil concentrations greater than 10,000 ppm) and in an alkaline desert environment, the half-life of 2,4-0 and 2,4,5-T appeared to be in the range of 150 to 210 days. The cooperative studies by Stark et al (56) and Arnold et al (4) have shown that the application of 2,4-D and 2,4,5-T at massive rates not only did not sterilize the soil, but indeed stimulated the growth of certain microflora, and this stimulation may have contributed to the degradation of the herbicide. C. Accumulation and Metabolism of Phenoxy Herbicides in Animals A detailed review of the toxicity, distribution and fate of 2,4-D and 2,4,5-T in animals is provided in Chapter IV. Some general observations on the metabolism of the phenoxy herbicides have recently been published by Leng (39). She reported that residues of the phenoxy herbicides in treated food or feed crops were readily absorbed in the gut of animals and were excreted rapidly in the urine, largely as unchanged phenoxy acid. Some conjugation occurred, particularly at higher dosage levels, but the basic structure of the herbicide was III-6 �not readily altered in animals. The ether linkage can be cleaved by bacterial action in the rumen but the rate of cleavage depended on the chemical structure of the phenoxy compound. The rate of clearance of residues from the body was dependent upon dosage level, particularly if the renal threshold was exceeded. Leng (39) concluded that residue levels were considerably lower in muscle, milk, and cream than in liver and kidney, but that all residue levels rapidly declined after withdrawal of animals from treated feed. Residues of phenol metabolites were present in milk, liver and kidney of animals fed high doses of 2,4-0 and 2,4,5-T. III. THE ENVIRONMENTAL FATE OF TCDD A. Analytical Limitations Statements on the fate of TCDD in the environment are predicated upon the detection in environmental substrates. Prior to 1973, the detection limit for TCDD, was 0.1 ppb for soils and 0.05 ppm for biological tissue (60). As noted by Kearney et al (35) and Dost et al (23), a 1 Ib/A application of 2,4,5-T containing 0.1 ppm TCDD applied directly to the soil could result in a maximum of 0.1 parts per trillion (ppt) in the top 15 cm of soil. Likewise, Baughman and Meselson (9) have calculated that environmental monitoring of food chains for buildup of TCDD would require a level of detection of 1 ppt. For a 1 gram sample of biological tissue, this would require a limit of detection of 1 picogram (pg) (10-^2 gram). Highly sophisticated instrumentation is required to obtain these low detection limits. However, another one of the limiting factors, even with appropriate instrumentation, has bee,n the need for cleanup techniques applicable to a wide variety of environmental samples. Recently (1977), Hummel (30) has reported on a technique suitable for permitting the detection of ppt residue levels of TCDD. Using this technique, Hummel has analyzed a wide array of environmental substrates. These have included analyses of whole fish, fish muscle, rat and mouse liver, mouse pelts, bird liver and stomach, insects, diving beetles, seeds, soil, water, and bovine and human milk. Largely due to the analytical limitations" noted above, the quest for environmental data on TCDD began with laboratory experiments. The use of radiolabeled preparations were invaluable in these studies. There has been considerable interest placed on the analysis of TCDD in field samples; e.g., fish and human milk from South Vietnam (7), bovine fat, liver and milk from the Western United States (3,41), and rice from Arkansas (30, 55). B. Laboratory Studies of TCDD Two model ecosystem studies (33, 42) have been conducted in an attempt to simulate the mode of entry of TCDD into water with the III-7 �subsequent exposure of several organisms representing parts of natural food chains. These systems were not designed to determine the effects of TCDD on the organisms but rather, how does TCDD behave when subjected to likely environmental conditions. Matsumura and Benezet (42) introduced 14C-TCDD in the form of residues on sand into an aquatic ecosystem containing brine shrimp, mosquito larvae and fish. The results indicated that the rate of pick-up was extremely low in brine shrimp and fish under the experimental conditions, Mosquito larvae, which were bottom feeders, showed a faster rate of TCDD pick-up. They concluded that because of TCDD's low solubility in water and its low partition coefficient in liplds, it was not likely to accumulate in as many biological systems as DDT. Isensee and Jones (33) exposed several organisms to ' C-TCDD for up to 31 days to determine the distribution and bioaccumulation potential in the aquatic environment. TCDD accumulation by all organisms was directly related to water concentration (0.05-1330 ppt) and ranged from 2.0 x 104 to 2.6 x 104 times the water concentration for snail, mosquito fish and daphnids and averaged 4.9 x 103 for duckweed, algae and catfish. No metabolities of TCDD were found in submerged soil, water, snails, mosquito fish or catfish. Isensee and Jones further noted that most (85-99 percent) of the 14C-TCDD originally added to the ecosystem remained in the soil at the end of the experiment. Total recovery for the ecosystem averaged 92.2 percent, indicating that TCDD was very stable during this study. From the model ecosystem data, it has been concluded that TCDD is taken up by an organism and retained (bioaccumulation). The accumulation results in TCDD concentrations in the environment (bioconcentration). The food chain studies do not suggest, however, that TCDD is biomagnified; i.e., organisms at successive trophic levels do not exhibit an ascending order of TCDD concentrations in their tissues. Neither of the model ecosystem studies reported toxic effects from the bioconcentration of the TCDD. Both studies, however, were of short duration and the water concentration was generally low, although in one experiment by Isensee and Jones (33) the water concentration exceeded 1 ppb and'mosquito fish and catfish accumulated concentrations greater than 1.4 ppm TCDD for 3 and 6 days, respectively. Miller et al (43) conducted chronic toxicity tests to assess the hazard to aquatic organisms exposed to TCDD in water or food. They evaluated three species of fish: guppies, coho or silver salmon, and the rainbow trout; and three aquatic invertebrates: a snail, a worm and mosquito larvae. Their conclusions were that TCDD in water or food was toxic to fish. The effects of exposure for 2496 h of young salmon to TCDD in water at levels greater than 23 ng/g (23 ppb) were irreversible, and death resulted in 10-18 days. Duration of exposure was less important than level of exposure except as threshold response level was approached. The critical exposure III-8 �period was somewhat less than 24 h in static water toxicity tests in which TCDD concentrations changed markedly with time. Small fish were more sensitive than large fish on an equivalent exposure level basis. TCDD in food at 2.3 ppm markedly reduced growth of young rainbow trout (10/aquaria) exposed to 6.3 yg TCDD per tank per week for 4 weeks. TCDD at 0.2 ppb had no effect on pupation of the mosquito larvae, but reduced the reproductive successes of the pulmonate snail and the oligochaete worm. Morris and Miller (48) have conducted additional bioassay tests with guppies. Exposure of guppies to concentrations of TCDD equal to or greater than 0.1 ppb for 120 h caused complete mortality in approximately 30 days. Duration of survival was significantly and positively correlated with body lengths. Beatty et al (10) administered larval and adult forms of the American bullfrog doses of TCDD varying from 25 to 1,000 yg/kg. Doses of TCDD as high as 1 mg/kg failed to have any significant effect upon survival or completion of metamorphosis in tadpole. Doses of TCDD up to 500 yg/kg had no effect on survival of adult frogs. Histopathological examination of various tissues from the metamorphosed tadpoles and adult frogs revealed no abnormalities. In one of the first laboratory studies of TCDD in soil, Helling (28) found that TCDD was immobile when evaluated by soil thin-layer chromatography. In laboratory leaching studies, Matsumura and Benezet (42) found that virtually no TCDD leached from soil columns of sand or sandy loam. Kearney et al (34) have determined the persistence of TCDD after 20, 40, 80, 160 and 350 days in Hagerstown and Lakeland soils receiving 1, 10 and 100 ppm TCDD. After 1 year, 56 and 63 percent of the originally applied TCDD was recovered in the Hagerstown and Lakeland soils, respectively. Thus, the half-life was estimated to be about 1 year. Furthermore, TCDD could not be detected after 70 days in soils receiving 10, 100 or-1,000 ppm 2,4,5-trichlorophenol, suggesting that TCDD was not biosynthesized by microbial condensation reactions. The long half-life of TCDD suggested to Kearney et al (34) that it was not readily metabolized by soil microorganisms. This observation was in keeping with what Matsumura and Benezet (42) found. They evaluated 100 microbial strains, which had previously shown the ability to degrade persistent pesticides, for their ability to degrade TCDD. Only 5 of 100 organisms showed some ability to degrade this compound, suggesting that microbes capable of degrading TCDD were rather rare in nature. Helling et al (29), in reviewing the previous studies, concluded that persistence of TCDD was not surprising since it is an insoluble, nonpolar, chlorinated molecule, devoid of biologically labile functional groups. III-9 �Isensee and Jones (32), in laboratory studies determined the uptake of TCDD from soil by two crop species. Lakeland Sandy loam, a soil with low adsorptive capacity, was treated with ^C-T at the rate of 0.10 and 0.06 ppm, respectively. Oats or soybeans were grown in this soil and their tops were harvested at intervals to maturity. All tissue '^C-activity was expressed on the basis of the original compound. Oats and soybeans accumulated in their tissue less than 0.15 percent of the TCDD present in the soil. Isensee and Jones (32) also evaluated the fate ot TCDD when applied to foliage. Uniform quantities of '^C-TCDD were applied to the center leaflet of the first trifoliate leaf of 3-week-old soybean plants. The first leaf blade of 12-day-old oat plants was treated with '4C-TCDD only. Results indicated that TCDD was not translocated beyond the treated leaflet. An average of 94 percent of the TCDD remained on soybean leaves for 21 days, but the amount continuously decreased on oat leaves. Although Isensee and Jones suggested that volatilization was a key factor in the disappearance of TCDD from foliage, Crosby and Wong (19) have suggested that photodegradation of the dioxins was a plausible explanation. In an uptake study similar to that of Isensee and Jones (32), but using sorghum, Cupello and Young (20) found that the rate of uptake of TCDD from a Ulysses sandly loam soil was approximately one millionth of one percent of the amount of TCDD in the soil. Nash and Beall (45) have recently (1978) completed a study on the fate of TCDD in the plants, soil, water and air of a microagroecosystem. Tritium-labeled TCDD at concentrations of 44 or 7,500 ppb was applied to a bluegrass turf microagroecosystem using an emulsifiable concentrate form of the isooctyl ester of 2-(2,4,5trichlorophenoxy) propionic acid (Silvex) as a carrier. They found that: 1. TCDD concentrations in water leached through soil were below the analytical detection limit (10~'6 g/g water). 2. TCDD concentrations on grass were initially 20 ppt (10-12 g/g grass), but after four weeks were at or below 1 ppt. The half-life was approximately six days. 3. TCDD concentrations in or on soil were less than 0.2 ppt and most (80 percent) was near the soil surface (0-2 cm). 4. TCDD concentrations in air were (immediately after application) less than 100 fg/m3 (femtogram - 10~'5g/m3) and after four weeks decreased to <3 fg/m^. 5. TCDD, or its degradation products, concentrations in earthworms were less than 0.3 ppt. 111-10 �6. The major repositories for TCDD were the soil and thatch. Nash and Beall (45) concluded that volatilization (approximately 10 percent) of TCDD was a major pathway of dissipation from the microagroecosystem chamber. However, once TCDD was volatilized it dechlorinated in the direct sun and apparently even in shade outdoors or when the sun was filtered with glass in the chambers. Thus, TCDD is sensitive to photodechlorination in the vapor phase even without the presence of ultraviolet light. C. Field Studies of TCDD 1. Residue in Aquatic Ecosystems Several monitoring studies for TCDD in aquatic organisms have been conducted. Baughman and Meselson (8) reported finding TCDD concentrations of 70 to 810 parts per trillion (ppt) in fish from rivers of interior Vietnam and concentrations of 18-79 ppt in fish, and shellfish along the seacoast of South Vietnam. Their samples were collected in 1970 and analyzed 2-1/2 years later by their method and instrumentation. Zitko (65) and Zitko et al (66) did not detect dioxins in a wide assortment of aquatic organisms collected from the St. John River, New Brunswick or the Bay of Fundy, Canada. Their detection limits, however, were between 0.1 and 1.0 ug/g of tissue. Shadoff et al (55) have examined fish (bass and catfish) from a reservoir in a rice-growing region of Arkansas, where 2,4,5-T had been used annually for more than 20 years. Likewise, fish (walleyes and catfish) were obtained from a reservoir in West Texas where 2,4,5-T had been used for brush control over the past 20 years. No TCDD was detected in any of the samples with a minimum range of 10 ppt. Young et al (62) reported on species diversities and food chain studies conducted in two aquatic ecosystems draining a unique one-square mile military test area (Test Area C-52A, Eglin AFB, Florida) that received 161,000 pounds 2,4,5-T and 170,000 pounds 2,4-D herbicide during the period 1962-1970. Significant levels (10710 parts, per trillion) of TCDD were found in 1973 within the top six inches of the test area soil. Erosion of soil occurred into a pond on the test area and into a stream immediately adjacent to the area. TCDD levels of 10-35 ppt were found in 1974 in silt of the aquatic systems, but only at the point where eroded soil entered the water. Species diversity studies of the stream were conducted in 1969, 1970, 1973 and 1974. Insect larvae, snails, diving beetles, crayfish, tadpoles and major fish species (by body parts) from both aquatic systems were analyzed for TCDD. Species diversity studies indicated no significant change in the composition of ichthyofauna between these dates or a control stream. Concentrations of TCDD (12 ppt) were found in only two species of fish from the stream, sail fin III-ll �shiner and mosquito fish. The sample of mosquito fish consisted of bodies with heads and tails removed. Two samples of sailfin shiner were analyzed: one containing viscera only and the other bodies less heads, viscera and caudal fins. Only the viscera contained TCDD. Samples of skin, muscle, gonads, and gut were obtained from spotted sunfish!, from the test grid pond. Levels TCDD in those body parts were 4, 4, 18 and 85 ppt, respectively. Grass pathological observa tions Qf the sunfish revealed no significant lesions or abnormal itit r.. 2. Residues 1n Sails * The National Academy of Science (15) reported finding TCDD concentrations of <1.2 to 23.3 parts per billion (ppb) in soil of the Pran Buri Calibration Grid (Thailand), an area used in calibrating RANCH HAND aerial equipment. Wool son et al (60) found no residues in 1971 in Lakeland sand which had received 947 Ib/A of 2,4,5-T during 1962-1964. These unusually high doses resulted from testing of aerial application equipment at Eglin AFB, Florida. Although analysis of the applied material was not conducted, 2,4,5-T made prior to 1968 probably contained enough TCDD to be detected throughout the 1-yard of soil profile sampled. Wool son et al suggested that the lack of detectable residue was due probably to its decomposition on or in the soil and/or to its transportation by wind erosion. Young et al (64) conducted four years of field studies on the persistence of Herbicide Orange and TCDD when applied at massive rates to soils. Herbicide Orange "biodegradation" plots were established in Utah (Air Force Logistics Command Test Range) and in Florida (Eglin AFB Reservation) using simulated subsurface injection techniques to place the herbicide 4 to 5 inches beneath the soil surface in bands 2.5 or 6 inches wide for Utah or Florida, respectively. An application rate of 4,000 Ib herbicide/A resulted in initial TCDD residues of approximately 148 ppb and 0.375 ppb in the Utah and Florida plots, respectively. Figure 1 is a semi-logarithmic plot of the soil concentration of Herbicide Orange while Figure 2 is a semilogarithmic plot of the soil concentration of TCDD in the same field tests. Using Figures 1 and 2, the half-life data were calculated as 300 and 220 days for Orange, and 320 and 230 days for TCDD for Utah and Florida, respectively. It should be emphasized again that these data were from field plots where the herbicide and TCDD were injected as highly concentrated herbicide in narrow bands beneath the soil surface. Data on soil penetration of TCDD within the soil profile of Utah biodegradation plots receiving either 1,000, 2,000 or 4,000 Ib/A are shown in Table 1 (Unpublished data: Young, A.L., and E.L. Arnold. 1978. Report on TCDD soil penetration studies, USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas). Note that in Table 1, 98 percent of all TCDD was detected in the 0-6 inch increment of soil, the increment into which the herbicide was applied. Even in the plots receiving 4,000 Ib/A, the TCDD detected in the 6-12 111-12 �Hill AFB, Utah .Eg!in AFB, Florida 10,000 c o t. O) Q . 03 Q . 1 ,000 u -S O) a: -M (O 4-> C cu u c o o (X) 100 r ?1 200 400 600 800 1,000 Time (Days After Incorporation) FIGURE 1. Semi-logarithmic plot of soil concentrations (parts per million) of herbicide in Herbicide Orange biodegradation studies at Eg!in AFB, Florida, and Hill AFB, Utah. Source: Reference (64 ) 111-13 1,200 �20,000 Hill AFB, Utah Eglin AFB, Florida 10,000 *<• 1,000.. Q . in •»-> i. (T3 Q. Q O O c O £ •4-> C O) O c O O 100-. O 00 10 200 400 600 800 1,000 Time (Days After Incorporation) FIGURE 2. Semi-logarithmic plot of soil concentrations (parts per trillion) of TCDD in Herbicide Orange biodegradation studies at Eglin AFB, Florida, and Hill AFB, Utah. Source: Reference (.64). 111-14 1,200 �TABLE 1, Concentrations of TCDD, parts per trillion, in the Herbicide Orange biodegradation plots, AFiC Test Range, Utah, four years after applications.3 Original Rate of Herbicide Orange Applied Depth (inch) 1,000 Ib/A 2,000 Ib/A 4,000 Ib/A 0 -6 650 1600 6600 6 - 12 11 90 200 12 - 18 NAb NAb 14 a Samples collected 6 November 1976. Plots established 5 October 1972. ^Samples not analyzed. Source: Unpublished data (Young, A. L., and E. L. Arnold. 1978. Report on TCDD soil penetration studies. USAF Occupational and Environmental Health Laboratory, Brooks AFB, Texas). 111-15 �inch increment may have been there because of the mass movement of the herbicide at the time of application rather than through the movement of percolating water. These penetration data are similar to those reported by Young et al (64) for the Florida biodegradation plots (noted earlier) although the Florida site received an annual rainfall of 60 inches (vs 10 Inches annual rainfall in Utah). Young et al (63) reported TCDD data from soil analyse-* of the Eglin AFB, Florida, Spray Equipment Calibration Grid (Grid 1 5 Test Ana C-52A). As noted in Chapter I, this grid received 1,894 pounds of Purple per acre during the 1962 through 1964 period. TCDr concentrations in a soil profile from samples collected ten years after the last application of Purple are shown in Table 2. 3. .Residues in Animals The current search for TCDD in beef fat and liver in the United States may provide an indication of the possible fate of TCDD in South Vietnam. In September 1974, the Environmental Protection Agency established a Dioxrn Implementation Plan which consisted of a short term monitoring program (Part I) and a broad research plan which would take 4 to 5 years to complete (Part II) (3). Part I of the program was initiated in February 1975. The guiding principles for the sample program were: (a) the samples should be representative of beef actually prepared for human consumption and (b) the samples should be from cattle grazed on lands treated with 2,4,5-T. Control samples were to be taken from cattle grazed on non-treated areas within the same state. Between February and March 1975, 85 beef fat (peritoneal and kidney) and 43 liver samples were collected (3). Approximately 25 percent of these samples were collected from non-treated areas. One laboratory prepared all sample extracts, and identical aliquots were sent to all participating analytical laboratories. In June 1976, analytical results for these samples were announced by the EPA Dioxin Project Manager (53). TCDD was present (range of 20-60 ppt) in a small percentage (3.5 percent) of the beef fat samples taken from cattle with a known exposure of 2,4,5-T. All of the beef liver samples analyzed were negative, at a detection limit of 10 ppt TCDD. Phase II of the Dioxin Implementation Plan began in 1978, with the intended goal of providing EPA with information on the range and possible bioaccumulation of TCDD in the environment (3). Analyses of human fat and liver tissue and human milk, and additional samples of beef fat and liver were to recieve the highest priority. Mahle et al (41) have recently completed a surveillance of bovine milk samples from the states of Oklahoma, Arkansas and Missouri. Twenty-five samples were collected from cows grazing on pastures on rangeland treated with normal applications of 2,4,5-T. These samples and control samples were analyzed for TCDD by gas chromatography-mass spectroscopy (GC/MS). They found no TCDD in bovine milk 111-16 �TABLE 2. Concentration of TCDD in soil profile of Grid 1, Test Area C-52A, Eg!in AFB, Florida.3 Depth of (inch) Parts per Trillion (ppt) TCDD 1 1 -2 160 2 -4 700 4 -6 44 6-36 a 150 NDb Grid 1 received 1,894 pounds of Herbicide Purple per acre during 19621964. The soil samples were collected and analyzed in 1974. detected, minimum detection limit - 10 ppt. Source: Young et al . (63). 111-17 �from control or treated areas with a detection limit of 1 ppt. In reforestation tests in Western Oregon, Newton and Snyder (47) applied Herbicide Orange at the rate of 2-4 Ib/A. Analysis of resident mountain beaver captured inside the treated area two months after treatment showed no TCDD in livers, with a minimum detection limit of 3 ppt, and the animals appeared to be in good health in all respects. Wool son et al (60) examined extracts of 19 bald eagles from locations throughout the United,States for TCDD and higher dioxin residues. No dioxins were detected at a minimum detection limit of 50 ppb. Baughman (7) analyzed samples of human milk for TCDD from areas of South Vietnam heavily treated with 2,4,5-T during the military herbicide program. Levels of 40-50 ppt in human milk were found in samples collected in 1970 and analyzed four years later. Shadoff et al (55) analyzed samples of human milk obtained from mothers residing near the North Concho River Basin of West Texas, an area where large acreages of the watershed had been sprayed repetitively with 2,4,5-T herbicides for brush control over the past 20 years. No TCDD was found in any of the milk samples at a minimum detection limit below 10 ppt. 4. Air Force Studies Chapter I and earlier sections of this chapter have referenced studies conducted on the Spray Equipment Calibration Grids, Test Area C-52A, Eglin AFB, Florida. The soil residue studies and the aquatic studies have previously been described. Test Area C-52A offered a unique opportunity to follow the fate of TCDD in the many components of the ecosystem. Young (61), Young et al (62, 63, 64), and Bartleson et al (6) have reported on various investigations conducted on this test area. The following is a brief synopsis of the magnitude of the contamination and the subsequent effects upon the wildlife of the test area. In addition to these references, data by Young, Thalken and Harrison (unpublished - USAF Occupational and Environmental Laboratory, Brooks AFB, Texas) of recent investigations at the test site have been incorporated into the synopsis. Field investigations were conducted during 1973-1978 on the 3.0 km2 test area containing 4 different calibration grids that received a total of approximately 73,000 kg 2,4,5-T and 77,000 kg 2,4-D during the period 1962-1970. No residues of 2,4,5-T or 2,4-D were detected (detection limit of 10 ppb) in any soil samples collected during 1971-1972. However, residues of the contaminant, TCDD, were still present in 1978. Fifty-four soil samples were collected to a depth of 015 cm from throughout the test area. TCDD levels ranged from <10 to 111-18 �1,500 parts per trillion (ppt). The median concentration was 30 ppt while the mean was 165 ppt. The ecological survey extending over a five-year period documented the presence of more than a 123 different plant species, 77 bird species, 71 insect families, 20 species of fish, 18 species of reptiles, 18 species of mammals, 12 species of amphibians and 2 species of molluscs. At least 170 biological samples were analyzed for TCDD, including 30 species of animals. No TCDD was found in any of the plant species examined. However, TCDD was found in nine species of animals including two rodent species: beachmice (300-1,500 ppt, liver) and hispid cotton rat (<10-210 ppt, liver); three species of birds: meadowlark (100-1,020 ppt, liver), mourning dove (50 ppt, liver), and Savannah sparrows (69 ppt, liver); three 'species of fish: spotted sunfish (85 ppt, liver), mosquito fish (12 ppt, whole body), and sail fin shiner (12 ppt, whole body), and one reptile, the six-lined racerunner (360-430 ppt, muscle). Gross pathology was done on all species collected for TCDD residue analyses. Histopathological examinations were performed on over 300 adult or fetal beachmice or hispid cotton rats from the test area and a control field site. 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 animal was from a control or test area. All microscopic changes were recorded including those interpreted as minor or insignificant. The tissues v/ere then reexamined on a control and test basis, which demonstrated that the test and control mice could not be distinguished histopathologically. Similar histopathological studies were conducted on the fish and racerunner, and again no significant abnormalities were found. As a concluding remark, Young et al (64) noted that Test Area C-52A offered a unique opportunity to examine the effects of long-term, low-level exposure of biological systems to TCDD. As previously noted, histopathological examination in body organs from adult and fetal beachmice revealed only lesions which are normally observed in microscopic surveys or large numbers of field animals. The absence of liver lesions in animals that had liver levels of TCDD from 200 to 1,500 ppt was most significant in view of the quantities of TCDD that must have been applied to the test site. Although these pathologic studies were initiated in 1973, beachmice had been collected from the test area as early as 1970 for gross pathological observations. They believed the animals examined in 1973-1974 from Grid 1, the area of greatest contamination (having received 1,894 pounds of Purple per acre in 1962-1924) may have been between 24 and 40 generations removed from the mouse population first noted in 1970. Thus, these studies conducted on the mice of Test Area C-52A suggested that long-term, low-level exposure to TCDD under field conditions may in fact not be teratogenic, mutagenic nor carcinogenic. 111-19 �D. Environmental Production of TCDD In 1971, Buu-Hoi et al (14) reported that small quantities of TCDD were formed upon the pyrolysis of 2,4,5-T acid, its butyl ester, or from vegetation defoliated by these products. In their article, Buu-Hoi provided mass spectral data for TCDD (compound I in his text). In reference to these spectral data, they stated (as translated from French): There is no need to use the precise analytical techniques described in the foregoing in the case of pyrolysis of 2,4,5-trichlorophenoxyacetic acid (500-600°), because simple fractional sublimation of the pyrolysate, prewashed in diluted aqueous soda, will yield about 5 percent of compound (1). This yield is increased to 15 percent as a result of the pyrolysis of trichloro-2,4,5 sodium phenoxyacetate. The conclusion (and this has been verified) is that quantities of "dioxin" (I) are formed during the combustion, more or less forced, of materials coming from plants pretreated by 2,4,5trichlorophenoxyacetic acid, and its derivatives (2,4,5-trichlorobutyl phenoxyacetate, the base of the "Orange" defoliant, leads naturally to free acid as a result of hydrolysis attributable to humidity, or to bacterial or fungal degradation), and this is all the more so because alkaline ash appears as a result of such combustion. One then can conceive the possibility of danger, in the long or short term, to public health in areas such as South Vietnam where the people use materials that are principally of plant origin, and are local, as fuels in their homes (wood, charcoal, dry leaves and branches), and which, as a result of the intensive defoliation that took place since 1964* could contain 2,4,5-trichlorophenoxyacetic acid. In 1972, Saint-Ruf (54), a colleague of Buu-Hoi, reported on the formation of "dioxin" from the pyrolysis of Si 1 vex. He reported that although the quantity of TCDD was less than that observed from the pyrolysis of 2,4,5-T, it was nevertheless sufficiently important to render the use of Silvex extremely dangerous for man and animals, especially in areas where treated vegetable matter was likely to be used as domestic foodstuff. The data of Buu-Hoi et al (13) and Saint-Ruf (54), and their conclusions* were challenged by Langer et al (38) in 1973. Langer et al investigated conditions which might produce dioxins from salts and esters of 2,4-D, 2,4,5-T and Silvex. No dioxins were detected even when the sodium salts of 2,4,5-T and Silvex were heated to 300°C and 111-20 �3500C, respectively. However, if a mixture of 0.25 gram (g) 2,4,5-T acid, 2 g HoO and 10 g koC03 was refluxed at 100°C for 3 hr, then heated at 200°C for 15 hr, then at 400°C for 43 hr, a total yield of 0.13 percent TCDD could be detected. Furthermore, Lanaer et al found that the mass spectrum reported by Buu-Hoi et al (and used by Saint-Ruf) for TCDD was in fact not the mass spectrum of TCDD. They suggested that the mass spectrum obtained by Buu-Hoi et al was that of a polymeric matter similar to that found in their own studies. Langer et al concluded that it was extremely unlikely that dioxin {TCDD) could be produced in the field by burning plant material treated with 2,4-D, 2,4,5-T, Silvex or their derivatives. Recently (1977), Stehl and Lamparski (57) reported finding small amounts of TCDD in trapped residue from self-supported fires of grass and paper treated with different compounds containing 2,4,5-T. Under controlled, but as "natural" as feasible conditions, they analyzed the combustion products of the grass or paper after treatment with 13.3 kg 2,4,5-T per ha (12 Ib/A). Stehl and Lamparski felt that the most meaningful way to express their data was in parts per trillion (ppt) of TCDD formed per parts per million (ppm) of 2,4,5-T burned. The average of all their experiments was 0.6 ppt of TCDD formed per 1 ppm of 2,4,5-T burned. The TCDD burden added to the environment by the combustion of natural materials treated with 2,4,5-T would be no . larger than 1 ppt of TCDD per 1 ppm of 2,4,5-T residue burned. Ahling et al (1) has reported similar results when 2,4,5-T residue on wood chips is burned at 500°C; 6 ppt of TCDD formed per 1 ppm 2,4,5-T residue burned. They suggested that this would correspond to a formation of about 1 microgram (yg) TCDD per m2 in forest fire directly after application of the herbicide formulation. However, Cutler (21) recently (1978) has suggested that the burning of forested areas treated with 2,4,5-T may be of little concern, since TCDD decomposes at temperatures above 800°C (and 2,4,5-T decomposes at temperatures above 500°C), considerably below the temperatures of 1200°C or more achieved in the field with a free exchange of air. E. Photodegradation of TCDD In perhaps what can be termed as one of the most significant studies on the environmental degradation of TCDD, Crosby and Wong (19), in 1977, found that herbicide formulations (including Orange) containing known amounts of TCDD and exposed to natural sunlight on leaves, soil or grass, lost most or all of the TCDD in a single day, due principally to photochemical dechlorination. Despite the known persistence of pure TCDD, it was not stable as a contaminant in thin herbicide films exposed to outdoor light. Crosby and Wong (19) have established three requirements for significant dioxin breakdown in the environment; namely, dissolution in a light-transmitting film, the presence of an organic hydrogendonor such as a solvent or pesticide and ultraviolet light. They 111-21 �noted that all three conditions are normally met during the practical application of 2,4,5-T or other TCDD-containing chemicals. Thus, their data suggested that environmental residues of TCDD often will be considerably less than previously expected. Nash and Beall (45) concluded from their studies of the fate of TCDD in a microagroecosystem chamber, that once TCDD was volatilized, it dechlorinated in the direct sun and apparently even in shade outdoors or when the sun was filtered with glass in the chambers. They concluded further that TCDD was sensitive to photodechlorination in the vapor phase even in the absence of ultraviolet light. IV. SUMMARY Available data indicate that the vast majority of the phenoxy herbicides would impact 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 occur. Most of herbicide probably would undergo rapid degradation (weeks) within the cellular matrix of the vegetation. However, some of herbicide may remain unmetabolized and would be deposited on the forest floor at the time of leaf fall. Soil microbial and/or chemical action would likely complete the degradation process. Herbicide droplets that impacted directly on soil or water would probably hydrolyze rapidly (within hours). Biological and nonbiological degradatiye processes would further occur to significantly reduce these residues. Some volatilization of the esters of 2,4-D and 2,4,5-T would occur during and immediately after application. The volatile material most likely would dissipate within the foliage of the target area. Photodecomposition of TCDD would minimize the amount of biologically active volatile residues moving downwind of the target area. Accumulation of phenoxy herbicides in animals may occur following ingestion of treated vegetation. The magnitude of this accumulation would likely be at nontoxic levels. Herbicide residues in animals would rapidly decline after withdrawal from treated feed. Most TCDD sprayed into the environment during defoliation operations would probably photodegrade within 24 hours of application. Moreover, recent studies suggest that even within the shaded forest canopy, volatilization and subsequent photodecomposition of TCDD would occur. Since translocation into vegetation would be minimal, most TCDD that escaped photodegradation would enter the soil-organic complex on the forest floor following leaf fall. Soil chemical and microbial processes would further reduce TCDD residues. Bioconcentration of the remaining minute levels of TCDD may occur 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 accumulate in these animals would have a biological effect. 111-22 �The environmental generation of TCDD from 2,4,5-T residues, through thermal or photolytic processes, would be highly unlikely and of no consequence. 111-23 �LITERATURE CITED CHAPTER III 1. Ahling, B., A. Lindskog, B. Jansson and G. SundStrom. 1977. Formation of polychlorinated dibenzo-p-dioxins and dibenzofurans during composition of a 2,4,5-T formulation. Ckuma&ph&m 33:461-468. 2. Aly, O.M. and S.p. Faust. 1964. Studies on the fate of 2»4-D and ester derivatives in natural surface waters. J. Ag/w.c.. Food Chem. 126.541-546. 3. Anonymous. 1977. flioxot: Position document. (Draft) Dioxin Working Group. April 1977. U.S. Environmental Protection Agency; Washington, D.C. Mim. 17 p. 4. Arnold, E.L., A.L. Young and A.M. Wachinski. 1976. Three years of field studies on the soil persistence and movement of 2,4-D, 2,445-T and TCDD. Weed SCA.. Soc. Am. Meet. Atotfc. 206. p 86. 5. Audus, L.J. 1960. faioAabiotogJjCLOut breakdown. o&ft.eA.b-ccx.dei-in 4o^tA. P 1-19. In_ Herbicides and the soil. E.K. Woodford and G.R. Sugar (Eds.). Blackwell Sci. Pub, , Oxford, England. 6. Bartleson, D.D., O.D» Harrison, and J.D. Morgan. 1975. Stadia ol WiJtttJUAe. exposed to TCW zon&min&tiid 4o^ta. Technical Report AFTL-TR-75-49. Air Force Armament Laboratory, Eglin Air Force Base, Florida. 53 p. 7. Baughman, R.W. 1976. Tetrachlorodibenzo-p-dioxins in the environment. High resolution mass spectrometry at the picrogram level. P-U-6. Ab^^t. Int. 36(7):3380B. 8. Baughman, R.W. and M.S., Meselson. 1973. An analytical method for detecting TCDD (dioxin): Levels of TCDD in samples from Vietnam. EnviAdn. Health P&upa&t, 5:27-35. 9. Baughman, R.W. and M.S. Meselson. 1973. An improved analysis for tetrachlorodibenxo-p-dioxins. Advdn. Chem. SeA. 120:92-104. 10. Beatty, P.W., M.A. Helscher, and R.A. Neal. 1976. Toxicity of 2,3,7,8-tetrachlorodibenxo-p*dioxin in larval and adult forms of Rana catesbelana. 6«££. Envvwn* Cdnfaw. Taxi-tol, 16(5) :578-581 . 11. Blackman, G.E.,, J,D. Fryer, A. Lang and M. Newton. 1974. The effects of herbicides in South Vietnam. Part B» Working PapersPersistence and disappearance of herbicides in tropical soils. Nat. Acad. Sci., Washington, D.C. 56 p. 111-24 �12. Brown, J.W. 1962. l/egexUttoiaa£ bpnay ttete in South Vietnam. U.S. Army Chemical Corps Biological Laboratories, Fort Detrick, Frederick, Maryland, 119 p. Available from the Defense Documentation Center, Defense Logistics Agency, Cameron Station, Alexandria, Virginia, DDC Number AD476961 . 13. Buu-Hoi, N.P., G. Saint-Ruf, P. Bigot and M. Mangane. 1.971. Preparation, properties and identification of dioxin (2,3,7,8tetrachlorodibenzo-p-dioxin) in the pyrolysate of defoliants containing 2,4,5-T and its esters and in contaminated vegetation. C.R. Hebd. Seances Acad. Sex.., Se/t. 0. 273:708-7111. (French) 14. Byast, T.H. and R.J. Hance. 1975. Degradation of 2,4,5-T by South Vietnamese soils incubated in the laboratory. Bo££. Envision. Contam. Tovuiat. 14(1): 71 -76. 15. Committee on the Effects of Herbicides in South Vietnam. 1974. Part A. Summary and conclusions. National Academy of Science, Washington, D.C. 398 p. 16. Craig, D.A. 1975. U6e orf HeAbx.cx.de* in Southeast Mia. Historical Report. San Antonio Air Logistics Center, Directorate of Energy Management, Kelly AFB, Texas. 58 p. 17. Crosby, D.G. 1976. Herbicide. pkotade.c.ompo*ition. P 835-890. In. Herbicides - Chemistry, Degradation and Mode of Action. Vol. 2. P.C. Kearney and D.D. Kaufman (Eds.). Marcel Dekker, Inc., New York. 18. Crosby, D.G. 1976. Nonbio£ogic.cLt de.Qtiadcvtion o& heAbicideA in tk<L boiJL. P 65-97. _In_ Herbicides - Physiology, Biochemistry and Ecology. Vol. 2. L.J. Audus (Ed.). Academic Press, New York. 19. Crosby, D.G. and A.S. Wong. 1977. Environmental degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Science 195:1337-1338. 20. Cupello, J.M. and A.L. Young. 1976. Radiochemical bioassay of TCDD uptake in plant material. Extract. Annual Research Progress Report No. 11. Dean of Faculty, United States Air Force Academy. 22 p. 21. Cutler, M.R. 1978. Remarks to the National USDA/EPA Symposium on the use of herbicides in forestry. USDA-SEA, Washington, D.C. 7p. 22. DeRose, H.R. and A.S. Newman. 1947. The comparison of the persistence of certain plan growth-regulators when applied to soil. PA.OC. Sci. Soa. Am. 12:222-226. 23. Dost, F. , J. Witt, M. Newton and L.A. Norris. 1975. Statement on 2,4,5-T and TCDD. J. fofi. 73(7) :410-412. 111-25 �24. Goring, C.A.I., D.A. Laskowski , J.W. Hamaker and R.W. Meikle. 1975. Psu-ndipleA o& pe6£tcxde d&gsuidation -in Ao-it. P 135-172. In_ Environmental Dynamics of Pesticides. R. Haque and V.H. Freed (Eds.) Plenum Press, New York. 25. Grover, R. , J. Maybank and X. Yoshida. 1972. Droplet and vapor drift from butyl ester and dimethyl ami ne salt of 2,4-D. Weed Sex. 20(4): 320-324. 26. Hamaker, J.W. 1975. The, witeA.pi&tation ofi *oi£ le.ac.kt.ng expetxmentb. P 115-133. ln_ Environmental Dynamics of Pesticides. R. Haque and V.H. Freed (Eds.). Plenum Press, New York. 27. Harrigan, E.T. 1970. CaUbxation TdAt o& tkz UC-123K/A/M5V-1 Sp>uu/ SyAt&m. Technical Report ADTC-TR-70-36. Armament Development and Test Center, Eg! in AFB, Florida. 160 p. 28. Helling, C . S . 1971. Pesticide mobility in soils. II. Applications of soil thin-layer chromatography. P/toc. SoJJt Sex. See. Am. 35(5):737-743. 29. Helling, C.S., A.R. Isensee, E.A. Woolson, P.D.J. Ensor, G.E. Jones, J.R. Plimmer and P.C. Kearney. 1973. Chlorodioxins in pesticides, soils and plants. 3. tnvJUion. Qual. 2(2) :171-178. 30. Hummel, R.A. 1977. Clean-up techniques for the determination of parts per trillion residue levels of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). J. Ag^c. Food Chm. 25(5) : 1049-1053. 31. Irish, K.R., R.A. Darrow and C.E. Minarik. 1969. InfiotLmcution manual fan. v&geJution c.on&ioi Ln SouuthnaAt faia. Miscl. Public. 33. Department of the Army, Fort Detrick, Frederick, Maryland. 71 p. 32. Isensee, A.R. and G.E. Jones. 1971. Absorption and translocation of root and foliage applied 2,4-dichlorophenol , 2,7-dichlorodibenzop^dioxin, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Ag/txc. Food Ckm. 19(6):1210-1214. 33. Isensee, A.R. and G.E. Jones. 1972. Distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic model ecosystem. BnuAon. Sex. Techno£. 9(7) :668-672. 34. Kearney, P.C., E.A. Woolson and C.P. Ellington, Jr. 1972. Persistence and metabolism of chlorodioxins in soils. Envision, Sex.. Uchnol. 6(12):1017-1019. 35. Kearney, P.C., E.A. Woolson, A.R. Isensee and C.S. Helling. 1973. Tetrachiorodibenzodioxin in the environment: Sources, fate, and decontamination, tnv-uion. H&alth PvAApe&t. 5:273-277. 111-26 �36. K l e i n , R . E . and E . T . Harrigan. 1969. Comp<vuAon Tut o Technical Report ADTC-TR-69-30, Vol. I. Armament Development and Test Center, Eglin AFB, Florida. 356 p. 37. Klingman, G.C. 1961. Sons, Inc., New York. 38. Langer, H . G . , T.P. Brady and P.R. Briggs. 1973. Formation of dibenzodioxins and other condensation products from chlorinated phenols and derivatives. EmuAon. Health P&t6pee£. 5:3-7. 39. Leng, M.L. 1977. Companative. m&taboti&m o& phznozy k<>Abi.cA.d<Lt> <in cwxjTia&6. P 53-76. Jj^ Fate of Pesticides in the Large Animal. Academic Press, Inc., New York. 40. Loos, M.A. 1975. Phe.noxyalkano-ic. acMt>. P 1-128. In_ Herbicides Chemistry, Degradation and Mode of Action. Vol. 1. P . C . Kearney and D.D. Kaufman ( E d s . ) . Marcel Dekker, Inc., New York. 41. Mahle, N . H . , H.S. Higgins and M.E. Getzendaner. 1977. Search for the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin in bovine m i l k . Ball. EmuAon. Contam. Tazicol. 18(2) :123-130. 42. Matsumura, F. and H . J . Benezet. 1973. Studies on the bioaccumulation and microbial degradation of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Envision. Health PeA6pee£. 5:253-258. 43. M i l l e r , R.A., L.A. Norris and C.L. Hawkes. 1973. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in aquatic organisms. Env^ion. Health Pnupuct. 5:177-186. 44. Muzik, T.J. 1976. Influence o& e.nv4Aonme.ntal factor on toKiCsity to plant*. P 203-247. j£ Herbicides - Physiology, Biochemistry and Ecology. Vol. 2. L.J. Ausus (Ed). Academic Press, New York. 564 p. 45. Nash, R.G. and M.L. Beall, Or. 1978. EnviJiom\e.ntal dlbtfu-bution aft 2,3,7,$-te&ULC.hlotLO(LLb<>.nzo-p-dioXsin (TCVV) apptiid with 4-cCvex to tunfa <in m*ioAoa.QSLoe,c.oAyAtm. Final Report EPA-1AG-D6-0054, Agricultural Environmental Quality Institue, U . S . Department of Agriculture, Beltsville, Maryland. 46. Newton, M. 1971. Disappearance of 2,4,5-T from forest ecosystems. Weed Sex.. Soc. Am. Meet. Abttn. 57. pp-30. 47. Newton, M. and S.P. Synder. 1978. Exposure of forest herbivores to 2,3,7,8-tetrachloro-p-dioxin (TCDD) in areas sprayed with 2,4,5-T. Bull. EnviAon. Con-tarn. Tozicoi. (In Press) Weed control: <u a .icx.ence. 421 p. 111-27 John Wiley and �48. Norris, L . A , and R.A. Miller. 1974. The toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in guppies (Poecilia reticulatus Peters). Eatt Env.iAon. Contam. TOXA,O.O£. 12(1):76-SO. 49. Palm, C . E , (Chairman). 1968. Weed Control, Vol. 2. Principles of Plant and Animal Pest Control. Nat. Acad. Sei,, Washington, D . C . 471 p, 50. Plimmer, J,R. 1976. Vet&tA&Uy, P 891-934. In Herbicides Chemistry, Degradation and Mode of Action. P,c71<earney and D.D. Kaufman (Eds,), Vol. 2. Marcel Dekker, Inc., New York, 51. Reigner, I . e . , w . E . Sopper and R . R , Johnson. 1968. W i l l the use of 2,4,5-T to control streamside vegetation contaminate public water supplies? J, FOA, 66(12) :914-918. 52. Reinhart, K . G . 1965. Herbicidal treatment of watersheds to increase water yield. N. East Weed Contr. Conf. Proc. 19:546-551. 53. Ross, R . T . 1976. 2,4,5/Dioxins: Analytical Collaborators Meeting, June 15, 1976. June 25, 1976, Memorandum of the United States Environmental Protection Agency; Washington, D . C . p 3, 54. Saint-Ruf, 6. 1972, Formation of dioxin in the pyrolysis of sodium a-(2,3,7,8-trichlorophenoxy)'propionate. No^u/tM^am chosen 59(12) :648. (French) 55. Shadoff, L.A,, R,A, Hummel and L. Umparski, 1977. A search for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in an environment exposed. annually to 2,4,5-triehlorophenoxyacetic acid ester (2,4,5-T) herbicides. 8u££, Env-cton. Contam. Tou.o.oi. 18(4) :478-485. 56. Stark, H . E . , J.K. McBride and G.F. Orr, 1975. Soil biodtQfm.dcuU.on o£ H&ib-ctiide. 0/tang&. 1. bti.cAob<iai and <Lc.atoQ-ic.ak 4tudy a£ the. U.S, MA. FoA.ce Log<u>£icA Command TeAt HIU MA Poize. 6o4e, U-tofe. Final Report, TECOM Project No. 5-CO213'00'015, U.S, Army Dugway Proving Ground, Dugway, Utah. 73 p. 57. Stehl, R.K, and LL Umparski, 1977. Combustion of several 2,4,5trichlorophenoxy compounds: Formation of 2,3,7,8-tetrachlorodibenzop-dioxin. Science 1 97 ( 4307) :1Q08- 1009, 58. Tschirley, F.H. 1968. Reiponie ofi tfiopicjaJi and Au.b&iap<ic.at woody plante to chmic.aUi- Vieatinnnte , Research Report CR-13-67. Agricultural Research Services, U.S. Department of Agriculture, Washington, D.C. 197 p, 59. Winston, A,W., Jr. and P.M, Ritty. 1972. What happens to phenoxy herbicides when applied to a watershed area. Ind, l>eg. Manage. 111-28 �60. Woolson, E.A., P.D.J. Ensor, W.L. Reichel and A.L. Young. 1973. Dioxin residues in lakeland sand and bald eagle samples. Advan. Ckem. Svi. 120:112-118. 61. Young, A.L. 1974. Ecological &tudi&> on a heAb-icide. equipment tut ouita (TA C-52A) Egtin AFB ReAeA.vatt.an, Florida. Tech Rep. AFATL-TR-74-12. Air Force Armament Laboratory, Eglin Air Force Base, Florida. 141 p. 62. Young, A.L., P.J. Lehn and'M.F. Mettee. 1976. Absence of TCDD toxicity in an aquatic ecosystem. Weed Sex. Sec. Am. Meet. Afa-6-tt. 107. p 46. 63. Young, A.L., C.E. Thalken and W.E. Ward. 1975. Studies o& tke. ecological impact o& sie.pe£itivn a&ual appLic&tionA of, h<Lnbtcid(L& on tho, <tcoAyt>tw o& tut ouzo. C-52A, Egtin AFB, FloiLda. Technical Report AFATL-TR-74-12. Air Force Armament Laboratory, Eglin AFB, Florida, and Department of Chemistry and Biological Sciences. U.S. Air Force Academy, Colorado 80840.- 127 p. 64. Young, A.L., C.E. Thalken, E.L. Arnold, J.M. Cupel lo and L.G. Cockerham. 1976. Fate otf 2,3,7,B-t<i&iachlosiodibe.nzo-p-dAQxJ.n (TCW) -en tke. znvJJionmtLnt: Sumnasiy and de.contam<ination ie.comme.ndationA. USAFATR-76-18. Department of Chemistry and Biological Sciences, USAF Academy, Colorado 80840. 41 p. 65. Zitko, V. 1972. Absence of chlorinated dibenzodioxins and dibenzofurans from aquatic animals. 8o££. EmuAoia. Contain. Topical. 7(2/3):105-110. 66. Zitko, V., 0. Hutzinger and P. U.K. Choi. 1972. Contamination of the Bay of Fundy-Gulf of Maine area with polychlorinated biphenyls, polychlorinated terphenyls, chlorinated dibenzodioxins, and dibenzofurans. EnvxAon. Health PeAAp&ct. 1:47-50. 111-29 �CHAPTER IV ' THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS I. INTRODUCTION This review cites a major portion of the world scientific literature dealing with the toxicological aspects of 2,4-D, 2,4,5-T and TCDD in various laboratory and domestic animal species. The primary purpose was to provide a broad overview of research investigations performed to date. With this information, a more critical evaluation could be made of reported human exposures to actual and theoretical levels of 2,4-D, 2,4,5-T and TCDD as presented in other chapters of this report. In an attempt to organize this chapter the following format and sequence was followed. Each of the compounds in question was reviewed for a) acute and short-term toxicity; b) subacute and chronic toxicity; c) absorption, distribution and excretion data; d) embryotoxic, fetotoxic and teratogenic potentials; e) carcinogenic and tumorigenic potentials; and f) mutagenic and cytogenetic potentials. Where possible, the cited data were tabu! ari zed for ease of comparison and interpretation and a summary of the tabular data presented in the text. In the review of acute and short-term toxicity the primary effort was directed toward finding references establishing for each compound a no effect dose, a dose lethal to 50 percent (LD50), and a dose lethal to 100 percent (LDiOO) of the laboratory or domestic animal species studied. After the acute toxic doses were known the subacute and chronic levels were then addressed. The highest "no effect" level of repeated dosing as well as the lowest repeated dosing level causing symptoms of toxicity were collected. These two sections were followed by discussion of cited literature dealing with the absorption, distribution in various body compartments and tissues, and excretion of the 2,4-D, 2,4,5-T and TCDD. In a 1977 review of the TeAatogenxc E^e.c.t& oft Wilson (145) stated: Many chemicals with which man comes into contact are known to be overtly or potentially harmful, causing structural or functional change immediately and these effects are recognized as acute toxic responses to these chemicals. On the other hand, chemicals known to be overtly or potentially harmful, causing structural or functional change and effects, only, after some intermediate time or considerable lapse of time following exposure, are recognized IV-1 �as chemicals producing a chronic toxic response. This latter group with its subacute effects is where most of the chemicals fall the at interfere with reproduction. Reproduction effects are rarely the first or only toxic manifestations, but occasionally an embryo or fetus in ut&io is the primary or the only individual expressing the effects of the toxic material, indicating that the conceptus may have extraordinary sensitivity to certain chemicals or compounds. These so called teratogens may be naturally occurring or manufactured materials and despite its sequestered location deep within the maternal body, the embryo or fetus sometimes receives a toxic, i.e., teratogenic dose, albeit only a small fraction of the maternal dose. Dencker (31) in the introduction to his 1976 study TVcAAue Localization o& Some. T0Aatog<Lm> at EanJiy and Late. Gestation ReJLat&d to f<Ltai Ejects stated: Our knowledge concerning the mechanisms by which chemicals cause fetal damage is very sparse, and only in a few instances have generally accepted theories been presented. Most often there is no specific effect for a given chemical; rather it seems. that one agent produces a wide spectrum of mal forma tions - which indicates a nonspecific mechanism of action. Moreover, different chemicals may often produce the same type of malformation. This confusing aspect may partly be explained by the fact that the different organs have certain sensitive periods; in their development, when they are especially susceptible to external influences. With these comments in mind the literature dealing with embryotoxic, fetotoxic and teratogenic potentials of 2,4-D, 2,4,5-T and TCDD was reviewed to provide as much detail as possible as to the number of animals used, reproductive state, route of administration and toxic response as well as dose and formulation of each compound being tested. A Similar approach was used to review the carcinogenic, tumorigenic, mutagenic and cytogenetic potentials for each of the compounds. Care was taken to try and establish numbers of test animals, method and route of administration and the specific effect of a particular formulation or purity of 2,4-D, 2,4,5-T or TCDD on those animals. Where specific animal data were not available, studies dealing with animal tissue cultures or bacterial mutant strains were referenced. For simplicity in format, dosage levels of the various chemicals were expressed as mg/kg, but it should be understood that this refers to milligrams of a specific chemical or formulation per kilogram of body weight of the test animal unless otherwise specified. IV-2 �II. REVIEW OF 2,4-D TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of 2,4-D The following review of 2,4-D toxicity was based primarily on three review articles: Rowe and Hyman (115); Dalgaard-Mikkelsen and Poulsen (29); and the International Agency For Research on Cancer (IARC) Monograph, Vol 15 (66) although other recent publications on the subject have been cited. Bucher (18) in 1946 was among the first to report the results of experiments with small animals using 2,4-D. Temporary myotonia lasting from eight to twenty-four hours or more following a single injection of 150 to 250 mg/kg was observed in mice, rats, rabbits and dogs. In 1947, Hill and Carlisle (63) published the results of acute oral studies, following single doses of 2,4-D and found the U^Q for mice to be 375 mg/kg; for rats, 666 mg/kg; for rabbits, 800 mg/kg; and for guinea pigs, 1,000 mg/kg. The largest single oral dose administered to monkeys without serious after-effect was 214 mg/kg or 428 mg/kg given intraperitoneally. An oral, plus an intraperitoneal injection in monkeys for a total dose of 500 mg/kg 2,4-D caused nausea, vomiting, lethargy, muscle incoordination and head drop. These workers observed that all species reacted similarly and that there were no significant differences in potency between crude and purified preparations, or between the sodium or ammonium salts. Deaths from large doses were apparently due to ventricular fibrillation. When death was delayed, myotonia, stiffness of extremities, ataxia, paralysis and coma were observed. Parenteral administration of 150-200 mg/kg 2,4-D caused symptoms of myotonia in mice. In those acutely intoxicated, dilatation of the blood vessels of lungs, liver and kidneys was observed by Bucher (18). Rats and guinea pigs administered lethal doses of 2,4-D exhibited congestion of the viscera. Enlarged, swollen, kidneys and microscopically .massive cloudy swelling of the proximal convoluted tubules with cast formation was noted by Hill and Carlisle (63). Florsheim and Velcoff (43) reported a decrease in both thyroid and body weights in male rats given single subcutaneous injections of 2,4-D at 100 mg/kg. Guseva (57) found the LDso for the subcutaneous injection of 2,4-D in mice to be 220 mg/kg. At 10-100 mg/kg 2,4-D in rats and mice no impairment of motor activity was seen nor did those doses alleviate strychnine spasms. In cats, 20-30 mg/kg 2,4-D given intravenously was hypotensive and that effect was not impaired by atropinization. 'Baker et al (6) administered 112 grams (g) of grass mixed with horsemeat and dog meal divided over three consecutive meals to two IV-3 �healthy one-year-old mongrel dogs. The grass had been treated two days earlier with the equivalent of 4 pounds per acre (Ib/A) of 2,4-D butyl ester, which was twice the recommended rate. The dogs readily ate the food and no ill effects were observed during the following 96 hours. Each animal was then treated with 500 mg/kg 2,4-D in a single oral dose. No deleterious effects were seen in the next 96 hours of observation. One animal, killed and necropsied at 96 hours post administration, failed to reveal any macroscopic lesions and the other animal remained healthy for 82 days following the second treatment, at which time the experiment was terminated. In dogs, Drill and Hiratzka (33) found that toxic symptoms were often delayed up to six hours following a single oral administration of lethal doses of 100, 250 and 400 mg/kg 2,4-D. The deaths were delayed and occurred two to nine days after the compounds were administered; The acute oral LDgg for (98.5 percent purity) 2,4-D was in the range of 100 mg/kg or higher. Death appeared to be due in most cases to hepatic congestion or pneumonia. Pathological changes were limited to the gastrointestinal tract, lungs and liver, and followed the development of anorexia, weight loss and myotonia (33). Dogs exhibited more evidence of hepatic congestion and moderate hepatic necrosis than was seen in other animals studied by Bucher (18). Drill and Hiratzka (33) concluded that the no effect level for a single oral dose of 2,4-D in dogs was 25 mg/kg. In a study by Shavgulidze et al (125), the single oral LDioO of 2,4-D sodium salt in sheep was 900 mg/kg. Death occurred in 2-4 days following the clinical signs of asthenia, depression, ataxia, hypothermai, dyspnea, muscle paralysis, anorexia and intense photophobia in those animals dosed at 500-1,000 mg/kg. The no effect single, oral dose of 300-400 mg 2,4-D was detoxified in 9-12 days with no traces remaining in the tissues. McLennan (88), reported on the accidental oral administration of 2,4-D in two cows. He noted that the death of one animal occurred within 12 hours following a calculated dose of 150-188 mg/kg. The toxic dose for the animal that survived, was calculated at between 105 and 132 mg/kg. In contrast Rowe and Hymas (115) noted that the 1050 of 2,4-D for cattle ranged between 500 and 2000 mg/kg body weight while a single dose of 1000 mg/kg may or may not cause illness. Rade-leff (110) cited a report in which cattle given one dose of 250 mg/kg 2,4-D showed signs of toxicity. In calves six to eight weeks old, Bjorklund and Erne (15) found that single doses of 100 to 200 mg/kg 2,4-D produced reversible signs of toxicity. Toxic symptoms summarized by Rowe and Hymas (115) included the following general observations in animals treated with acute toxic doses of 2,4-D: loss of appetite, loss of weight, depression, roughness of coat, general tenseness and muscular weakness particularly of the posterior quarters. Post mortem findings usually included irritation of the stomach of small animals and of the abomasum of ruminants, minor evidence of liver and kidney injury and in some instances congestion of the lungs. IV-4 �From data presented in Table 1, the acute 1059 as a rule was in the order of 300-800 mg/kg 2,4-D for rats, mice, guinea pigs, rabbits and cats. Analyses of data from the limited available studies supported the conclusion that the dog may be slightly more susceptible to oral doses of 2,4-D than other animals. Monkeys, sheep and cattle appeared to be somewhat more tolerant. The experiments referenced in Table 1 have also provided information on the acute oral administration of various salts and esters of 2,4-D as pure chemicals and as commercial preparations. No significant differences in the toxicity of the salt and ester forms of 2,4-D were seen when compared to the free acid (63,115). Hill and Carlisle (63) stated: In any assessment of the acute toxicity of a chemical based on data obtained from laboratory animals it should be borne in mind that considerable variation in species susceptibility may occur and that the data obtained cannot always be translated into toxic doses for humans. In the studies referenced in this section it can be concluded as Hill and Carlisle did in their studies that: all of the laboratory animals tested reacted in a similar fashion from signs and symptoms which developed and from the pathological lesions which were present at autopsy. Assuming that man is no more resistant or susceptible than the rabbit or monkey, then the largest tolerated dose for a 75 kg man would be 15 grams of 2,4-D. With the exception of dog and monkey, all of the laboratory animals used in the cited references lacked the vomiting reflex so that they were unable to relieve themselves of irritating material by vomiting. The experiments conducted in monkeys indicate that the material is a gastric irritant in large doses, so that the possibility of the occurrence of acute poisoning in humans would seem relatively remote because of the large dose which man could presumably tolerate. Assuming that man is no more susceptible than the most susceptible animal test, the mouse, then the calculated oral LDso for man would amount to approximately 28 grams (63). It is generally accepted that the oral LD5Q of 2,4-D for man is around 500 mg/kg while the accepted LD^Q of aspirin for man is around 1,500 mg/kg. B. The Subacute and Chronic Toxicity Potentials of 2,4-D Repeated once or twice daily, subcutaneous injections of 50 to 90 mg/kg 2,4-D in mice for three weeks to ninety days did not elicit a characteristic chronic syndrome of toxicity or any notable histological IV-5 �TABLE 1 . Animal Number Used Mouse 450 Summary of literature data on the no-effect, and LD levels of the acute toxicity of 2,4-D in animals Route of Administration DoseToxicity Single Dose mg/kg Reference § Gavage LD 375a'b 63 NSC Intraperitoneal LD 375a 63 NS Subcutaneous LD 220a 57 NS Subcutaneous LD 280b 18 NS Oral in olive oil LD 368a 115 NS Oral in olive oil LD 541d 115 NS Oral in corn oil LD 713e 115 NS Oral LD 380f 81 50 50 50 50 50 50 50 50 Rat L 150 Gavage LD 666b 63 NS Intraperitoneal LD 666b 63 NS Oral in water LD 805b 115 NS Oral in olive oil LD 375a 115 NS Oral in olive oil LD 700d 115 NS Oral in corn oil LD 620e 115 NS Oral LD 1 ,500f 119 NS Oral LD 2,000b 119 NS Oral LD 900f 81 125 Gavage LD 1 ,000b 63 NS Intraperitoneal LD 666b 63 NS Oral in water LD NS Oral in olive oil 50 50 50 50 50 50 50 50 50 Guinea Pig u 50 50 IV-6 50 LDcn 551-2000b 469a 115 115 �Table 1 continued NS Oral in olive oil LD50 550° H5 NS Oral in corn oil LD 50 848e 115 70 Gavage LD 50 800 63 NS Intraperitoneal LD 50 400b 63 NS Intravenous LD50 400b 63 NS Oral in corn oil LD 50 424e 115 NS Oral LD 50 820 81 • Oral LD 100d 33 1 Mh/2 F Oral No effect 25a 33 2 Oral No effect 500' 6 Oral No effect 214a 63 Intraperitoneal No effect 428b 63 Oral LD 900 125 No effect 300-400b 125 Rabbit Cat Dog 4 Fg 50 Monkey Sheep NS 100 Oral LD 100 Oral Cattle LD 50 150-188' (a calculated dose) 500-2000^ 115,132 a 2,4-D acid Sodium salt of 2,4-D C NS - number of animals in study not stated or unavailable from literature source. Isopropyl ester of 2,4-D e Mixed butyl esters of 2,4-D IV-7 �Table 1 continued Butyl ester calculated as 2,4-D 9 F - Female h M - Male n 20% w/v amine salt of 2,4-D in aqueous solution J Form not stated in available literature source IV-8 �changes. Levels of 70 mg/kg or more retarded growth, probably by reducing food intake. Mice undergoing this treatment became pregnant and bore apparently normal litters (18). Guseva (57) found that 22 daily subcutaneous injections of 0.1 mg 2,4.-D in mice caused no toxic effects. In subacute studies with rats, Hill and Carlisle (63) fed a diet containing 1,000 mg 2,4-D/kg for 30 days without severe harmful effects. Some visceral congestion and kidney edema with degenerative changes in the tubules were noted. No adverse effects were seen in groups of 5 or 6 young female rats given 2,4-D by intubation five times a week for four weeks at doses of 3, 10, and 30 mg/kg in olive oil. At doses of 100 mg/kg 2,4-D, varying degrees of gastrointestinal irritation, slight cloudy swelling in the liver and depressed growth rates were noted. At 300 mg/kg 2,4-D, the animals failed rapidly and died. The principle lesion observed at post mortem was a severe gastrointestinal irritation. In another study, matched groups of five, young, adult, female rats were placed on diets containing 100, 300, 1,000, 3,000 and 10,000 mg 2,4-D/kg of diet for 113 days. The no effect levels were 100 and 300 mg/kg of diet. At 1000 mg/kg, adverse effects were characterized by a depressed growth rate, excessive mortality, slightly increased liver weights and slight cloudy swelling of the liver. The animals on the 3,000 and 10,000 mg/kg levels in their diets were destroyed after twelve days as they were not eating and were rapidly losing weight. Increased liver and kidney weights were noted with unstated minimal pathological changes (115). No drastic damaging effects were noted when male Long-Evans rats were given 2,4-D equivalent to 2-5 g/kg over a 4 to 7-week feeding period. Response to herbicide treatment was dependent on animal age and on the duration of time that the chemical was fed. Little or no effect was noted on liver weight. Herbicide-induced enlargement of the liver was associated with increases in most of the major cellular components on a per liver basis. Isolated liver nuclei were 20-30 percent more active in the -cn-v-c^to RNA synthesis than in the control nuclei (22). Schwetz et al (122) found that oral doses of 12.5 to 87.5 mg/kg 2,4-D did not adversely affect the weight gain of rats during pregnancy. In a preliminary study, non-pregnant rats tolerated 75 mg/kg 2,4-D for 10 days while 100 mg/kg killed two rats and produced overt signs of toxicity in three survivors, Hansen et al (58) conducted a study with rats starting at 3 weeks of age, using groups of 25 female and 25 male animals, fed 0, 5, 25, 125, 625 or 1,250 mg 2,4-D/kg of diet for 2 years. During the study no significant differences in survival rates between controls and test animals were noted. The mean body weights of the different groups of IV-9 �males and females and the organ-to-body weight ratios for liver, kidney, heart, spleen and testes were not significantly different (P>0.025). The only exceptions were in two male rats, one at 625 mg/kg, and a second at 125 mg/kg dosage level in the diet. These two animals had slightly enlarged spleens. Mean values for hemoglobin, hematocrit, and total white blood cell count of controls and of rats at each dose level, at the same time interval, were similar and within normal range. The maximum no effect level for the rats in this study was greater than l»250 mg 2,4D/kg of diet, Hansen et al (58) in another study fed 0, 100, 500 or 1,5QQ mg 2,4-D/kg of diet to groups of 20 male and 20 female rats, t^o effect was observed at the 100 and 500 mg/kg of diet levels. At the 1,500 mg/kg level, there was no effect on fertility nor on the average number of pups per litter; however, significant effects on the average number of pups weaned and also on their weaning weights were noted. The no effect level is at least 500 mg/kg but less than 1,500 mg/kg of 2,4-D in the diet. Bjorklund rats at 1,000 mg/1. same dose level for health and diarrhea and Erne (15) administered 2,4-D in drinking water to Progeny from treated females were maintained on the 2 years with signs of growth inhibition, poor general as the main effects. Kay et al (72) found no significant adverse effects in a study using 112 New Zealand strain albino rabbits where 15 ml of each of three commercially available formulations of 2,4-D (dimethylarrrfne salt and the isooctyl and butyl esters) were administered 5 times a week for 3 weeks to the intact and abraded skin at 0.626 percent and 3.13 percent concentrations. Body weights, survival, hematological values, clinical chemistry values and organ/body weight ratios were all within normal ranges. Local skin inflammatory reactions occurred in all groups of animals including controls. This was especially severe in those applications where the 2,4-D esters were diluted with an unspecified oil. The water dilutions of all three forms produced less local skin inflammation. Historically, the treated animals had an increased incidence and severity of subepithelial fibre-sis and accompanying mononuclear infiltration in the skin. No peripheral or central nervous system tissues or microsections of other tissues disclosed any adverse findings. Hansen et al (58) conducted a study using groups of 3 male and 3 female beagle dogs being fed 0, 10, 50, 100 or 500 mg/kg 2,4-D in the diet (96.7 percent pure, with no detectable TCDD by GLC with a sensitivity of 1 mg/kg) for 2 years, starting at 6-8 months of age. Twenty-eight dogs surviving the 2-year period were clinically normal, in fair to good condition, with a no effect level greater than 500 mg/kg in the diet. One female at the 100 mg/kg level was emaciated at the end of the experiment; however* no significant lesions were noted. A male animal that died after 10 months on the study at the 10 mg/kg 2,4-D lev/el, shbwed a slight atrophy o,f the testes and moderate depletion of cellular elements in other tissues. IV-10 �Drill and Hiratzka (33) orally dosed (via capsule in a piece of canned dog food) adult mongrel dogs of both sexes with either 2, 5 or 10 mg/kg 2,4-D 5 days a week for 13 weeks. The 2,4-D was a commercial product of 98.5 percent purity (label stated). All dogs survived this study and no significant symptoms of toxicity were seen and no changes in body weight, organ weights, or blood count were noted. In a separate study, three of four dogs given daily doses of 20 mg/kg 2,4-D died between days 18 and 49. The signs observed in these animals differed somewhat from those seen in the acute studies. The chronically treated animals displayed stiffness of hindlegs and ataxia, weakness, difficulty in chewing and swallowing and occasionally bleeding from the gums. Weight loss occurred after 7-12% days and a terminal fall in lymphocyte count occurred prior to death. * The authors stated, "Death during the repeated administration of 2,4-D was not related to pathological changes in the liver, kidneys, or other organs examined." Seabury (123) treated three dogs experimentally infected with histoplasmosis, by intravenous injections of sodium 2,4-D at the rate of 1.17, 2.6, and 3.2 mg/kg per injection for 32-37 days without evidence of chronic toxicity. Bjorklund and Erne (15) treated young pigs at varying intervals up to 103 days with 50, 100 or 300 mg/kg of the commercial triethanolamine salt or butyl ester of 2,4-D. Exhibited symptoms of intoxication and pathology were analagous to those seen in laboratory animals. Clinical signs of anorexia and retarded growth were found in one animal given 51 doses of 50 mg/kg triethanolamine salt over 103 days. Pigs fed 500 mg/kg of diet triethanolamine salt of 2,4-D for up to 12 months developed locomotor disturbances of increasing severity after about one month. Animals sacrificed after 2-12 months had normal organ weights and no gross pathological changes. Clinical chemistry observations included lowered hemoglobin and hematocrit values, elevation of glutamic-oxaloacetic transaminase and reduced albumin and albumin: globulin ratios in the treated animals [see IARC Monograph, Vol 15 ( 6 ] 6). Shavgulidze (125) observed transient hematological changes in sheep receiving daily doses of 18 mg/kg 2,4-D sodium salt for 120 days. Mitchell et al (90) fed a cow 5.5 g of 2,4-D acid daily for 106 days with no apparent harmful effects on the health or milking performance. Post mortem examinations revealed no pathological changes in the liver, kidneys or body fat. By biological assay the presence of 2,4-D was demonstrated in the blood serum; however, 2,4-D was not found to be secreted in the milk nor was it found in the blood serum of a calf fed milk from this cow. Palmer (99) found that yearling steers needed to be given 15 daily doses of 250 mg/kg of the alkanolamine salt of 2,4-D before signs of toxicity occurred. He found that 112 daily doses of 50 mg/kg of this 2,4-D salt had no deleterious effect on the steers. IV-11 �D stated: Rowe and Hymas (115) in reviewing the chronic toxicity of 2,4The results of repeated oral administrations indicate that 2,4-D can be tolerated without adverse effects in doses only slightly smaller than those which cause toxic effects when given only once. This fact demonstrates that 2,4-D has a low degree of chronic toxicity. The same general observations of toxicity were noted in animals receiving chronic toxic doses of 2,4-D, as were seen in animals given single toxic doses. These were loss of appetite, loss of weight, depression, roughness of coat, general tenseness, and muscular weakness particularly of the posterior quarters. Post mortem findings usually included irritation of the stomach and gastrointestinal tract of small animals and abomasum of ruminants with only minor evidence of gross and histopathological injury in the liver and kidneys. Study of the data presented in Table 2 indicated that mice tolerate subcutaneous injections of 2,4-D at 50-70 mg/ka with no effect, while 70-90 rug/kg retards growth. Rats tolerated 1,000-1,250 mg/kg 2,4-D in their diet and 75 mg/kg orally without toxic effects. At levels of 1,000 mg/kg 2,4-D in the water and 1,500 mg/kg in the diet and 100 mg/kg orally, toxic signs were noted. Rabbits showed no gross differences between test and control animals, as far as skin irritation, when 3.13 percent solutions of various formulations of 2,4-D were placed on their intact or abraded skin. Dogs tolerated 500 mg/kg diet or 10 mg/kg orally with no toxic signs, while 20 mg/kg caused death in three of four animals. Oral doses of 300 to 500 mg 2,4-D/kg of diet were toxic to pigs. Rowe and Hymas (115) stated: Cattle demonstrate a similar susceptibility to 2,4-D as do the small laboratory animals. Cattle are distinctly more tolerant of 2,4-D than are dogs. Cattle can probably tolerate 30-50 mg/kg/day [(99)] for long periods without adverse effects. Daily doses of 100-250 mg/kg (99) would have to be continued for a week or longer to cause ill effects in cattle. A single dose of 500-1,000 mg/kg is not likely to cause problems; however, if repeated, serious effects and deaths are likely to occur. The chronic toxicity of 2,4-D did not differ greatly from the acute toxicity. At only slightly lower doses the same general signs, symptoms, and pathology were seen. Hansen et al (58) made the following statement on chronic exposure of humans to 2,4-D residues. (The cited values were for 1971. They have now been lowered slightly; however, in this case they were used to establish a worst-case situation.) IV-12 �TABLE 2 . Summary of literature data on the subacute and .chronic toxicity of 2,4-D in animals Route of Administration Effect Dose NSa 1-2 daily s.c. injections for 3 weeks to 90 days No effect 50-70 mg/kgb 18 1-2 daily s.c. injections Retarded growth 70-90 mg/kgb 18 NS Mouse No. Used NS Animal 22 daily s.c. injections No effect 0.1 mg/injc 57 NS 30 days in diet No severe effect 1000 mg/kg dietb 63 6Fd 5 doses/wk for 4 wks by intubation No effect 30 mg/kge 115 5 doses/wk for 4 wks by intubation Liver, G.I. growth effect 100 mg/kge 115 5 doses/wk for 4 wks by intubation Fatal in days 300 mg/kge 115 5 F 113 days in diet No effect 300 mg/kg diet6 115 5 F 113 days in diet Liver and growth effects, deaths 1000 mg/kg diet 115 Slight effect Total 2-5 g/kgc No effect 75 mg/kg' Rat 6 F 6 F 44 Mf 5 F 4 or 7 wks in diet 10 daily doses via stomach tube Referent t- 22 122 �Table 2 contumed 10 daily doses via stomach tube 2 died, overt toxicity m 3 100 mg/kgc 25 F/25 .M In diet for 2 yrs No effect 1250 ing/kg d i e t 58 W F/20 M In diet for 3 generation reproduction study in adults No effect 500 rag/kg diet 0 58 No effect on fertility or litter size. Lower no. pups weaned, lowered weight 1500 mg/kg diet 0 58 growth inhibition, poor health, diarrhea 1000 rag/ 1 water 15 No effect at gross exam. Some histopath effects in 2,4-D/oil treated animals. 3.13% solution9 72 5 f 20 F/2D M NS Rabbit 22 F/22 M In diet for 3 generation reproduction study in adults In drinking water for 2 yrs. 5 times/wk for 3 wks to intact and abraded skin Dog 122 3 F/3 M In diet for 2 yrs No effect 500 nig/kg diet c 58 3 M Oral dose via capsule 5 days/wk for 13 wks No effect on gross 10 mg/kg 33 Death in 3 at 18-49 days. Severe signs in 1 animal surviving 20 rag/kgc 33 1 F/3 M Oral dose via capsule 5 days/wk for 13 wks �Table 2 continued 1.1 mq/kg° 2.6 mg/kgj 3.2 mg/kg° 123 123 123 Toxicity, anorexia, retarded growth 300 mg/kgh 15 Locomotor problems, normal organ weights, no gross pathology 500 mg/kg1 15 Transient hematological and biochemical changes 18 mg/kg 3F Daily I.V. doses for 32 days at two higher levels, 37 days at lower level No effect NS Oral doses up to 103 days Pig NS In diet up to 12 months Sheep NS Daily oral doses for 120 days Cattle 125 1 Daily oral dose for 106 days No effect 5.5 gc 90 NS, S 15 daily oral doses Toxicity 250 mg/kg1 99 NS, S 112 daily oral doses No effect 50 mg/kg1 99 I en NS - number of animals in study not stated or unavailable from literature source Sodium salt of 2,4-D C 2,4-D acid F - Female e Butyl ester calculated as 2,4-D f M - Male ^Sodium salt, isooctyl ester and butyl ester of 2,4-D each applied separately on individual animals under the conditions described and concentration listed. Amine salt and butyl ester of 2,4-D used separately in animals at dose indicated. Vmine salt , Female lactating b S - Steer �Adequate data are not available to enable one to state conclusively what the total level of 2,4-D residues may be in foods ingested by the human population. An estimate of the greatest amount that might possibly be invested cart be made by use of the legal tolerances established by the FDA for 2*4-0 in various crops. They are 5 mgVkg pri 4 fruit Crops (apples^ citrus fruits* pe^ars and qUirices} aHd 0;5 mg/ky 6h 4 fcjraih crops (barley'} bats, fy'e ana wheat); if it is assumed that all the crop fbr Which a tdie^aHce exists always carried the" maximum amount of 2j4-D permitted; it can bis calculated that approximately Ch3 mg/kg of 2*4-0 Would be cbntribut^d to the total diet (fruit crops = 6 pertertt of the dietary intake of man ahd grain drops = 9 percent); tohe'h the maximum estimated human exposure to 2,4-D via the diet is cbtiipared to the dbsages given rats in the pr~e'sent study, it is apparent that there is an extremely wide margin of safety between 0.3 mg/kg of diet in man and the Ij250 mg/kg of diet fed to rats; C; Absorption, Distribution and Excretion of 2*4-0 Different degrees of sdditim 2,4-D poisoning were produced by Elb artd Yiitalo (35) in adult male Sprague-Dawley rats when 250 mg/kg • 2,4-0 was administered &y subcutaneous injection-. After varioU's intervals the cOncen'tratibn of intravenous 14fe-2,4-D Was cbm'pafed to the level fburtd iti the e^referbspihai fluid (CSF) and brairti At 4.5 hours 'when the sodium 2 i4-D, radioactivity in plasilia had diminished to 67 percent of control levels* ah 11-fold increase in the brain and a 39-fold increase i'n the CSF were s^fert compared to a 4.5 fold increase in the liver. All physiological and t'oxied logical parameters of this 1977 study had not been fully analyzed; however, during acute 2,4-D poisoning, the levels found in the brain were greatly increased. This increase appeared to be closely associated with the toxic symptoms. In ratsi pigs and cal'vies, 2^4-D administered in doses of 50-100 mg/kg brail y as salts ty&?$ readiiy absorbed arid eHifriMt&di mainly in the ail uHriev Wth.JpjAsflft hajf-li'ves varying from 3-12 hMrs .(^-,3^). The rate of 2-i4-D elimfnatib'n ih rats was dosage dependent-. Fbl Vowing administration of 14C-2-,4-D-, Khartna ahd Faftg (73) fouWd radioactivity in all organs and tissues examined [see IARC monograph i Vol 15 (66)]. Berridt arid Koschier (9) using J G - labeled 2-,4-D in rat and rabbit renal ddrtical tissue sliees-, JLH vijtio, noted that 2,4-D was transporlfecl. By the ciassitil retii.! organic aMon transport process j howeVer-, 'otfter 'mechanisms of tra;ns'p'ort may also have b'een involved. This study tnay h^Vp 'ek'pVafn brYe of tfe frtech'an'isms contributing to the relatively ! ;f%^j el dls'app'e'aran'c^ '6f 2\4-D 'frdm iftbst species and th'e loW levels of bi'oVO'gical deposition of this compbund. IV-16 �The esters of 2,4-D were hydrolyzed in animals and the phenoxy acids were excreted predominantly as such in the urine of rats after oral administration, although a minor portion of them may have been conjugated with the amino acids glycine and taurine and with glucuronic acid (54). No 2,4-dichlorophenol was detected, however, in the urine of C57BL/6 mice treated subcutaneously with 2,4-D or its butyl or isooctyl esters. The rates of disappearance from plasma of 2,4-D and its butyl and isooctyl esters following single subcutaneous injections of 100 mg/kg of the compounds to female C57BL/6 mice were: butyl ester > isooctyl ester > 2,4-D (147), [see IARC monograph, Vol 15 (66)]. After oral administration of 0.05 mg/kg 2,4-D to rats, Fedorova and Bel ova (42) found that traces were detected in the milk of lactating animals for six days. Within 24 hours after administration of 2,4-D to pregnant rats, 16.8 percent of the dose was detected in the uterus, placenta, fetus and amniotic fluids, [see IARC monograph, Vol 15 (66)]. Bjorklund and Erne (15) found that 2,4-D passed the placental barrier in pigs. Clark et al (23) fed 2,4-D acid (99 percent purity) to groups of 3, adult beef cattle and adult sheep at levels of 0, 300, 1,000 and 2,000 mg/kg of feed for 28 days. Animals were killed and tissues sampled one day after the last dose, others one week later. Residues of the 2,4D and its phenol metabolites were determined in muscle, fat, liver and kidney. Muscle and fat contained the lowest levels while kidneys and liver contained the highest residue level. Withdrawal from treatment for one week before killing resulted in a significant reduction in tissue residue levels. With the exception of the kidneys, 2,4-D residues averages less than 1 mg/kg in the tissue analyzed. The kidney tissue level averaged 7.82 mg/kg with 0.37 mg/kg present after a 7-day withdrawal period. No 2,4-D was detected in fat or muscle of any animals at a detection limit of 0.05 mg/kg. All treated animals showed some anorexia, weight loss or poor weight gain depending on the level 2,4-D present in the feed, due to lowered palatability. During the 7-day withdrawal period, feed consumption in all groups returned to normal. 2,4-D was rapidly eliminated from animals, mainly in the urine with plasma half-lives of 3-12 hours following a single dose. Generally, it accumulated in animal tissues when given at high doses or repeated lower doses. However, these residues declined rapidly with a half-life of 1 to 2 weeks. Because of its excretion by the kidney, kidney tissue levels were as much as twenty times greater than the level seen in other organs and tissues. D. Embryotoxic, Fetotoxic and Teratogenic Potentials of 2,4-D The embryotoxic, fetotoxic and teratogenic potentials of 2,4-D appeared to be extremely variable with observable effects dependent upon concentration, degree of purity and method of administration with some effects only occurring with doses that approached maternal toxicity. IV-17 �Bionetics Research Laboratories (12, 13, 14) reported that either the acid* or the isopropyl, butyl and isooctyl esters of 2,4-D, administered orally or subcutaneously at days 6-14 of gestation, increased the incidence of anomalous fetuses among BL6, AKR and C3H strains of mice but not among B6AK arid AIHa strains. No single strain showed a positive response to all formulations. No single formulation caused a positive response among all strains of mice. Thus, the reported effects were highly strain-specific, in addition, the Bionetics study involved parenterai administration using dimethyl sulphoxlde (DMSO) as a vehicle, which complicated the interpretation of the data* since DMSO has been shown to be a teratogen in several species of laboratory animals when administered by the route used in the Bionetics study [see Schwetz et al (122)]. Schiller (118) found no difference in fertility (defined as the number of rats weaned per female mated) of test and control animals in one experiment where rats were fed potatoes which had been treated with 2,4-D. In a combined second and third experiment, fertility of the P, Fp Fo and Fo generations was 7.2, 5.8, 6.8 and 6.1 for controls versus 7.2* 7.1, 5.4 and 5.1, respectively, for test rats. The differences between control and test groups were not significant (P>0.05). The content of 2,4-0, its form, or purity in the potatoes was not given. When 1,000 mg/1 2,4-D was given throughout pregnancy to S'pragueDawley rats via the drinking water, the gestation and parturition were normal. The litter size was not significantly reduced and no anomalies were seen in the pups (15). Hansen et al (58) stated that in unpublished work performed by T.B. Gaines and R.D, Kimbrough, female rats were fed 2,4-D acid at 0, 1,000, and 2,000 mg/kg of diet for 95 days, mated with untreated males and continued on their respective diets throughout gestation and lactation, At the highest dosage level, females gave birth to pups that were small at birth and 94 percent died before weaning. Some deaths also occurred in pups of females fed the lower level. Starting with rats three weeks of age, groups of 25 female and 25 male animals were fed for two years either 0^ 5* 25, 125, 625 or 1,250 mg 2,4-D/kg of diet. No significant effects on growth rate, survival rate, organ weights or hematologic values were noted (58). Hansen et al (58) also noted in a three generation, six litter rat reproduction study, no deleterious effect of dietary 2,4-D acid at 100 or 500 mg/kg was evident. At 1,500 mg/kgi, however, 2,4-D, while apparently affecting neither fertility of either six nor litter size, sharply reduced the percent of pups born surviving to weaning and the weights of weanlings. I* studies by Schwetz et al (122), the acid of 2,4^0, the propylene glycol butyl ether ester of 2,4-D and the isooctyl ester of 2,4-D were evaluated for effects on fetal development, neonatal growth IV-18 �and survival when administered at 12.5, 25, 50, 75 and 87.5 rag/kg orally to pregnant Sprague-Dawley (Spartan strain) rats during organogenesis (days 6-15 of gestation). Fetuses were delivered by Caesarean section on day 20 of gestation and were examined grossly, measured and weighed. Fetotoxic responses seen at high dose levels 50, 75 and 87.5 mg/kg included subcutaneous edema, delayed ossification and wavy ribs. Teratogenic responses were not seen at any dose level. 2,4-D did not affect fertility, gestation, lactation or viability of the newborn. The esters of 2,4-D decreased viability of the newborn and lowered lactation indices (Lactation Index: pups weaned/pups alive on day 4 X 100). In a second part of the experiment in which litters delivered naturally, 2,4-D and its esters had little or no effect on fertility, gestation, viability or lactation indices. There were no observable effects on neonatal growth and development. Khera and McKinley (74) observed minimal 2,4-D induced fetopathy and an increased incidence of skeletal anomalies in rat pups following single daily oral doses of 100-150 mg/kg 2,4-D from days 6 to 15 of gestation. The observed skeletal defects did not appear to be incompatible with postnatal survival. Following treatment of dams with the acid of 2,4-D and the butyl and isooctyl esters of 2,4-D, weight gain and viability of the offspring were within control limits. The findings of their study suggested that postnatal parameters were unrelated to the teratologic potential of the chemicals. No consistent embryotoxic effects were noted when 2,4-D acid was administered orally to hamsters at doses of up to 100 mg/kg on days 6-10 of gestation (24). Binns and Johnson (10) showed that 2,4-D did not have a teratogenic potential in sheep. Starting one day after breeding ewes were given 2 g/day of 2,4-D acid in an alfalfa meal/water mixture via stomach tube for 30, 60, or 90 days. No clinical signs of toxicity nor histopathologic lesions were seen in the ewes and no congenital anomalies nor histopathologic lesions were seen in the lambs. Ewes were reported to have had increased rates of stillbirths and bucks displayed reduced sexual activity and decreased sperm quality when pastures were grazed soon after treatment with 3 Ib/A of the 2,4- • dichlorophenoxybutric acid (2,4-DB) (116). When a diet containing 500 mg/kg 2,4-D was fed to a sow during the entire pregnancy, the sow was anorexic and the newborn piglets were underdeveloped and apathetic with 10/15 dying within 24 hours. When the survivors were continually fed 2,4-D at 500 mg/kg of diet until they were 8 months of age marked growth depression, persistent anemia and moderate degenerative changes of the liver and kidneys were noted (15). Erne (40) fed pregnant reindeer birch leaves that had been sprayed with a mixture of 2,4-D and 2,4,5-T at a daily dose of 1 mg IV-19 �phenoxy herbicide per kg body weight. There were no clinical or histopathological changes noted in any of the female reindeer and no fetal anomalies were seen. From the data presented in Table 3 the no effect level for embryotoxic, fetotoxic and teratogenic signs in the rat was approximately 1,000 mg/1 of the sodium salt of 2,4-D, while the no effect level from 2,4-D acid in the rat diet ranges from 1,250 to 1,500 mg/kg of food. Oral doses of 2,4-D acid and the butyl and isooctyl esters cause no effect at daily doses of 87.5 mg/kg. At 100 to 150 mg/kg 2,4-D acid and esters produced embryo and fetotoxic responses in rats and hamsters. In pigs 500 mg 2,4-D acid/kg diet caused the sow to be anorexic and produced weakened piglets with 10 to 15 dying within one day after birth. When the five surviving piglets were fed the same diet for eight months they showed a marked depression in growht. Sheep have tolerated oral doses of 2,4-D acid for 30-90 days at 2 grams per day levels, while reindeer experienced no adverse effects from daily oral doses of 1 mg/kg for 30-54 days. E. Carcinogenic and Tumorigenic Potentials of 2,4-D Studies of the carcinogenic properties of 2,4-D in mammalian biological systems are limited at best. However, in an extensive study by Innes et al (67), the tumorigencity of some 130 test compounds were tested in mice. Included in the test compounds were the 2,4-D acid and the isopropyl, butyl and isooctyl esters of 2,4-D. They were given orally, at a daily dosage rate of 46.4 mg/kg. An additional test using the dosage rate of 100 mg/kg for 2,4-D acid was included. These doses were given by stomach tube starting at 7 days of age and continued until the mice were 4 weeks of age. After weaning, the test compounds were mixed directly into the diet and the same dosage rate maintained for approximately^ 18 months of observation. The tumor incidence in any group or combination of groups in which 2,4-D was tested was not significantly different from that in control animals. Groups of male and female mice were given single subcutaneous injections of 215 mg/kg 2,4-D in dimethyl sulphoxide (DMSO) on the 28th day of life and observed up to 78 weeks of age. Tumor incidences in any group or combination of groups were not significantly different from that in controls. No increase in the incidence of tumors was observed in similar groups of mice treated with single subcutaneous injections of 21.5 mg/kg butyl or 100 mg/kg isopropyl esters of 2,4-D, both 99 percent pure. Mice treated with 21.5 mg/kg isooctyl ester of 2,4-D, 97 percent pure, had 5/17 females of one strain developing reticulum-cell sarcomas (12). Walker et al (141) demonstrated that six, daily, intraperitoneal, injections of highly purified 2,4-D (99.0 percent) at the rate of 62 mg/kg effectively inhibited development of the Ehrlich ascites tumor maintained in BALB/c mice. IV-20 �TABLE 3. Animal Rat Number Used . PFa NSb Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4-D in amimals Route of Administration Response Dose fteferen< 1000 mg/1 water0 15 Diet for 95 days then mated and Small birth wt. 94% died continued through gestation before weaning. and lactation. 2000 mg/kg dietd 58 Some reduction in birth wt. Some deaths in pups. PF NS Drinking water during pregnancy. No effect 1000 mg/kg dietd 58 25 Fe ro In diet for 2 yrs. No effect 1250 mg/kg dietd 58 25 Mf In diet for 2 yrs. No effect 1250 mg/kg dietd 58 PF NS In diet 3 generations. Six litter reproduction study. No effect 500 mg/kg diet No effect on fertility of either sex nor litter size. Reduced % of pups born and surviving to weaning - lowered weaning weights. 1500 mg/kg diet No effect on fertility, gestation, lactation or viability of newborn. 87.5 mg/kg9 122 Fetotoxic as edema, delayed ossification, wavy ribs. No teratogenicity. 87.5 mg/kg- 122 19 PF 119 Fetuses Daily oral dose - days 6-15 of gestation. Daily oral dose to females days 6-15 of gestation. H 58 58 �Table 3 continued PF NS Daily oral dose - days 6-15 of gestation Minimal fetopathy, increased , skeletal anoma'Mes 150 mg/kg PF NS Daily oral dose - days 6-15 of gestation No consistent embryotoxic effects 100 mg/kg1 24 Via stomach tube 30, 60 or 90 days No effect 2 g/dayl 10 In diet throughout pregnancy, Female anorexic. 10 of 15 piglets died in 24 hrs. 500 mg/kg diet 15 Growth depression, anemia, moderate liver and kidney lesions. 500 mg/kg diet0 15 No effect 1 mg/kg 40 74 Hamsters Sheep PF Pig 1 PF 5 Newborn ro ro In diet for 8 months Reindeer 15 PF In diet for 1 - 1 . 5 months PF - pregnant female NS - number of animals in study not stated or unavailable from literature source c Sodium salt of 2,4-D d 2,4-D acid p F - Female f M - Male 9 2,4-D acid or molar equivalents of propylene glycol butyl ether ester of 2,4-D or the isooctyl ester of 2,4-D 2,3-D acid or butyl or isooctyl esters of 2,4-D �Hansen et al (58) studied groups of 25 male and 25 female Osborne-Mendel rats that were fed for two years on diets containing 2,4D at 0, 5, 25, 125, 625 or 1,250 mg/kg levels. The 2,4-D was 96.7 percent pure and contained no detectable levels of 2,7-dichloro or 2,3,7,8tetrachlorodibenzo-p-dioxin (limit of sensitivity of method of analysis was 1 mg/kg). No target organ tumors were observed and the individual tumor types were randomly and widely distributed and of the type normally found in aging rats of that strain. Statistical analysis of the randomly distributed tumor types indicated a tendency for the proportion of females with tumors to increase with 2,4-D dosage and a trend toward dose related increases in the proportion of males with malignant tumors. The number of treated rats with malignant tumors over controls was found only in males receiving the highest dosage level. A review of the summary of the literature on the carcinogenic and tumorigenic potentials of 2,4-D in animals presented in Table 4, revealed that 2,4-D acid, and the isopropyl, butyl and isooctyl esters of 2,4-D did not adversely affect nor increase the incidence of tumors in test animals when fed at levels of 46.4 to 100 mg/kg of diet to mice or 1,250 mg/kg of diet to rats for 18 to 24 months. Those tumors that did occur were not necessarily in target organs and were the type tumors normally seen in aging laboratory animals of the species and strain being studied. Single subcutaneous injections of 21.5 to 215 mg/kg of 2,4-D acid, isopropyl and butyl esters of 2,4-D in DMSO did not produce carcinogenic or tumorigenic responses in male or female mice, A single subcutaneous injection of 21.5 mg/kg of the isooctyl ester of 2,4-D in DMSO did produce an increased incidence of reticulurn-cell sarcomas in treated female mice. It should be noted that DMSO itself is now considered to be a potential carcinogen. At 62 mg/kg, 2,4-D acid injected intraperitoneally in mice inhibited the development of Ehrlich ascites tumor being maintained in mice. F. Mutagenic and Cytogenetic Potentials of 2,4-D in Animals Most of the mutagenic studies of 2,4-D have been conducted in bacterial cultures or in plant and animal tissue cultures; however, Styles (133) investigated the cytotoxic effects of 2,4-D on .01 u-cvo and Jin v-cfio test systems and found no increase in mutation rate and no evidence of mutagenicity in the test rats. He found serum from orally dosed rats was not mutagenic to Sa&none&ta. typkunusuium. Complete details of this study were not readily available. Pilinskaya (101) observed that treatment of cultured human lymphocytes with 2.5 X 10~7 M (0.02 yg/ml) 2,4-D increased the number of chromatid aberrations (single acentric fragments) and, to a lesser extent, the chromosomal aberrations (paired acentric fragments). In mice, Pilinskaya (101) found toxic concentrations (100-300 mg/kg) of 2,4-D administered as a single oral dose significantly increased the frequency of aberrant metaphases (2-4 fold) with single fragments being the aberration seen. IV-23 �TABLE 4 Animal Number Used Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4-D in animals Route of Administration M - F NSe Single subcutaneous injections in DMSO No effect 46.4 mg/kg dietc 67 100 mg/kg diet 67 No effect 215 mg/kgd 12 No effect h 18 Ma/18 FD of Stomach tube, beginning at 7 two hybrid days of age for 21 days, then strains in diet for 18 months Dose No effect Mouse Response 21.5 mg/kgf 12 Reference 9 No effect f\ f J_ . T . T 6 daily . intraperitoneal injections Rats 25 M/25 F Diet for 2 years 12 5/17 females developed reticul urn-cell sarcomas ro 100 mg/kg 21.5 mg/kgh 12 C O m» / I «^ Erlich ascites tumor No target organ tumors, random type tumors normally seen in aging rats M - Male F - Female C 2,4-D acid and isopropyl, butyl and isooctyl esters of 2,4-D 2,4-D acid e NS - number of animals in study not stated or unavailable from literature source 1250 mg/kg diet Butyl ester of 2,4-D ^Isopropyl ester of 2,4-D h lsooctyl ester of 2,4-D 58 �Jenssen and Renberg (68) found there was no detectable increase of micronuclei in the erythrocytes of mouse bone marrow after intraperitoneal administration of 100 mg/kg 2,4-D. Because of the high experimental resolution power of the test system used in this study it was particularly suitable for the detection of weak chromosome breaking activity of 2,4-D in mammals. The lack of penetration of 2,4-D into the cells was in accordance with the rapid excretion that is known to occur in the mammalian body. This experiment did 'not in the authors opinion, consititute a reliable measure of the mutagenic potential of 2,4-D; however, in practice the lack of penetration of this substance into the cells indicated it does not constitute a cytogenetic hazard to man. Epstein et al (37) found that 2,4-D did not increase dominant lethal mutations in mice when given as a single intraperitoneal injection of 125 mg/kg or when given orally on five successive days for a total dose of 75 mg. In host-mediated assays Zetterberg et al (146) using strains TA1530 and TA1531, or Sac.c.kaAomyc&> D4, found no mutagenic effects in the organisms when host adult male mice were given 6 mg 2,4-D (200 mg/kg) by gavage. Bongso and Basrur (17) exposed embryonic bovine kidney cells and bovine peripheral blood cells, /en v-ttao, to concentrations of 1-1,000 iag/ml 2,4-D for 6-96 hours resulting in stimulation of mitosis. ChromosoMid' aberrations were not detected in the peripheral blood cells, but nucleolar irregularities and polyploid mitotic stages were observed in the kidney cells. Andersen et al (4) evaluated 110 herbicides for their ability to induce point mutation in one or more of 4 different microbial systems. The herbicide 2,4-D was included in this study. The authors did not state the purity of the compounds being tested. The 2,4-D did not cause point mutations in these microbial systems in comparison with known mutagens such as 5-bromouracil or 2-aminopurine. These observations of no mutagenicity of 2,4-D in Eiah<yu.dUa c.oti WP2 her+ or her" or in S<t£mone££a typhimuruwn strains TA1535, TA1536, TA1537 or TA1538 were also confirmed in works by Nagy et al (94), Shirasu (126) and Shirasu et al (127). A review of the literature on the mutagenic and cytoger.ic potentials of 2,4-D in animals generally supported the premise that 2,4-D was not highly cytotoxic in laboratory animals. It did not increase mutation rates nor stimulate a mutagenic response in rats and mice. In various Ln vJutiio and Jin vuvo test systems 2,4-D did cause chromatid aberrations and nucleolar irregularities in cultured human lymphocytes, bovine kidney cells and tissues of mice given single toxic doses. No mutagenic responses were seen in several studies using microbial systems for the detection of mutagenic and cytogenic responses to 2,4-D. IV-25 �III. REVIEW OF 2,4,5-T TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of 2,4,5-T Detailed accounts of the experimental procedures used to study the acute and short-term toxicity potentials of 2,4,5-T were not available, References to acute toxicity of 2,4,5-T in small laboratory animals referred to a summary article on toxicological information on 2,4-D and 2,4,5-T by Rowe and Hymas in 1954 (115). Their literature review made reference to the 1953 work of Drill and Hiratzka (33) where acute and chronic oral toxicity studies on 2,4-D and 2,4,5-T were conducted with dogs. Apparently, the earlier research with small laboratory animals dealt primarily with 2,4-D, although some 2,4,5-T studies conducted in 1950 discussed effects on horses, dairy and beef cattle, sheep, swine and chickens immediately pastured on freshly treated alfalfa. Unfortunately, Rowe and Hymas did not detail the methodology for obtaining all the data that appeared in their article. Table 5 presents the available data from the literature dealing with the acute toxicity of 2,4,5-T in laboratory animals. Drill and Hiratzka (33) administered commercial 2,4,5-T of 98.9 percent purity (TCDD level not stated) in a single oral dose of 50, 100, 250 and 400 mg/kg to a total of 10 adult mongrel dogs of both sexes, The 400 and 250 mg/kg dosages were given to individual male dogs and both died in 2 and 3 days, respectively. One of four female animals died at the 100 mg/kg dose level; however, no other signs of toxicity were noted. All three males and one female in the 50 mg/kg dosage level survived with no signs of toxicity. The acute oral LDso for 2,4,5-T acid was in the range of 100 mg/kg or higher for dogs. Toxic doses of this level produced only mild signs of muscle spasticity. Bjbrklund and Erne (15) fed single oral doses of 100 mg/kg 2,4,5-T to pigs causing anorexia, vomiting, diarrhea and ataxia. At autopsy, hemorrhagic enteritis and congestion of the liver and kidney were found [see IARC Monograph, Vol 15 (66)]. Study of the data in Table 5 indicated that the various forms of 2,4,5-T all fall in the same range of acute toxicity for mice, rats, guinea pigs and rabbits. The dog appeared to be somewhat more susceptible, The LDso values for 2,4,5-T and its common derivatives were in the range of 380 to 940 mg/kg for the small laboratory animals. When given orally to dogs, even in fatal cases, 2,4,5-T produced only weak signs in the form of ataxia and stiff movements of the hindlegs. B. The Subacute and Chronic Toxicity Potentials of 2,4,5-T Highman et al (62) conducted a study using 978 mice including control animals. On days corresponding to days 6 through 14 of pregnancy, groups of pregnant and nonpregnant CD-I mice and male and nonpregnant IV-26 �TABLE 5 . Animal Mouse Number Used Summary of literature data on the no-effect LD5Q and LD1QO levels of the acute toxicity of 2,4,5-T in animals Route of Administration Dos e-Toxi city Single Dose nig/ kg Reference NSa Mb Oral in olive oil LD50 389C 1 15 NS Fd Oral in olive oil LD50 e 551 1 15 NS F Oral in corn oil LD50 940f 1 15 NS M Oral in olive oil LD50 500C 1 15 NS M & F Oral in ol i ve oil LD 50 495e 115 NS F Oral in corn oil LD50 481f 1 15 NS F Oral in ol i ve oil LD50 7509 1 15 NS M & F Oral in olive oil LD50 381c 1 15 NS F Oral in olive oil LD50 449e 1 15 NS F Oral in corn oil LD en 750f 1 15 NS M Oral in corn oil LD 712' 115 Rat ro Guinea Pig Rabbit 50 �Table 5 continued 1 M Oral in capsule LD 2501 33 4 F Oral in capsule LD 100C 33 1 Dog Oral in capsule F 3 M 100 50h No effect 33 Pig NS Oral Anorexia, vomiting, diarrhea, ataxia, hemorrhagic enteritis, liver and kidney congestion. 100 NS - number of animals in study not stated or unavailable from literature source. b M - Male C 2,4,5-T acid ro oo F - Female e lsopropyl ester of 2,4,5-T f Mixed butyl esters of 2,4,5-T 9 Mixed amyl esters of 2,4,5-T One of four animals died on 7th day 15 �female dihybrid cross Fg mice received, by gavage, 2,4,5-T acid doses ranging from 30 to 140 mg/kg. Some groups received a technical preparation of 2,4,5-T (97.9 percent pure, containing < 0.05 mg/kg TCDD or a purified preparation of 2,4,5-T (99 percent pure, containing < 0.05 mg/kg TCDD). Mice killed when they became moribund and at 1, 2, 4, 6, 8 and 11 days after beginning treatment. Sick or moribund mice sacrificed after 2-9 doses of 2,4,5-T often showed severe myocardial lesions, hypocellularity of the bone marrow and depletion of lymphocytes in the thymus, spleen, or lymph nodes. They also showed marked hematologic and blood chemistry changes. Treated mice remaining healthy showed few or no lesions and no blood chemistry changes, but often developed a mild anemia attributable to a hemolytic effect of 2,4,5-T. The incidence of animals becoming moribund was less than 1 percent in the CD-I mice, including those given 140 mg/kg, and ranged from 53 to 82 percent in groups of male and female F2 mice receiving 120 mg/kg 2,4,5-T. The incidence of moribund mice tended to be higher in male than in female F2 mice and in those given the purified compound. The findings of this study indicated that impairment of maternal health by severe lesions early in gestation were not the primary cause of an increased incidence of fetal abnormalities observed in mice given 2,4,5-T. The lesions appeared to be due primarily to 2,4,5-T, rather than to contaminants in the technical preparation. Finally, the importance of using more than one strain of mouse in toxicological studies was vividly illustrated. Highman et al (60) using 378 pregnant dihybrid cross F£ female mice gave either 60 or 120 mg/kg 2,4,5-T via gavage on days 6 through 14 of gestation. Both technical (97.9 percent pure with less than 0.005 mg/kg TCDD) and a more purified preparation (99 percent pure with less than 0.005 mg/kg TCDD) of 2,4,5-T were used in this study. Mice were killed when they became moribund and at 6, 24, and 30 hours, as well as at 4, 6, 8 and 11 days after beginning treatment. Mice given 60 mg/kg and many given 120 mg/kg 2,4,5-T appeared normal at the time they were terminated either in early or late pregnancy and showed few or no pathologic changes. Mice that became ill or moribund often showed severe lesions and few survived past 11 days. The histopathological lesions included myocardial rarefaction and necrosis, thymus cortical atrophy, splenic atrophy and hypocellularity in bone marrow and lymph nodes. Groups of 10 male and 10 female rats per test dose were fed for 90 days on diets containing 2,4,5-T at the daily dosage levels of 0, 3, 10, 30 or 100 mg/kg body weight. The 2,4,5-T acid was from commercial production and contained less than 1 mg/kg TCDD. No effects were noted in the animals fed 3, 10 or 30 mg/kg doses. Changes found in both sexes fed 100 mg/kg included depression in body weight gain, slight decrease in food intake and elevated serum alkaline phosphatase levels. Male rats at this dose had slightly increased serum glutamic-pyruvic transaminase levels and slight decreases in red cell counts and hemoglobin. Inconsistent hepatocellular swelling was observed upon histopathological examination in some livers. [See World Health Organization (WHO) Monograph 71.42 (143), and IARC Monograph, Vol 15 (66)]. IV-29 �Groups of 10 male and 10 female rats per test dose were fed for 90 days on diets containing 0, 100, 300, 1,000 an0 3,000 mg/kg of food, of the mono-, di-, and tripropylene glycol butyl either esters of 2,4,5-T. The 2,4,5-T acid equivalent was 62 percent. No effect levels were 100 and 300 mg/kg of diet. At 1,000 mg/kg level slight cloudy swelling of the parenchyma! cells with central lobular necrosis was noted in two of ten animals examined. Kidney weights were increased wtfth mild cellular changes noted such as cloudy swelling of renal tubular epithelium. At 3000 mg/kg a significant retardation in growth was noted in males but not females, liver and kidney weight increased in males, livers were large and light in color in both sexes, generalized cloudy swelling of liver cells and slight central lobular necrosis and cloudy swelling of renal tubular epithelium [see WHO Monograph 71.42 (143) and IARC Monograph, Vol 15 (66)]. Konstantinova (81) conducted experiments with pregnant rats using 12-13 animals per treatment group and dosing them via stomach tube for the entire gestation period with 0.01, 0.1, 0.42 and 4.2 mg 2,4,5T/kg body weight. The butyl ester of 2,4,5-T was used in this study; however, source and purity were not stated in the translation. No effects were noted at the 0.01 mg/kg dose. The threshold level in this study was at the 0.1 mg/kg dose. At 0.42 mg/kg a general toxic effect on pregnant females was noted and embryotoxic effects were of an irregular character and difficult to evaluate. The 4.2 mg/kg produced a significant increase in total embryo fatalities, a decrease in the number of live offspring (average one less per female) and a decrease in the weight of the offspring. Rip and Cherry (111) fed a group of 12, four-week-old, male, Long-Evans rats, analytical standard grade 2,4,5-T (containing no detectable TCDD at a sensitivity of 0.05 mg/kg) mixed with the diet at a rate of 10 mg/animal/day for 1-11 days. Feeding of the 2,4,5-T caused liver enlargement with no effect on the weight of the kidneys, spleen, or body weight of the animal. Increases in relative liver weight were dose dependent and were observed after the first or second feeding. Enlargement was associated with substantial increases in total RNA and total protein per liver. The increases were not restricted to any particular subcellular fraction, but appeared to represent a general induction of RNA and protein synthesis. Total DNA content per liver was not affected. The enlargement response was reversible on the removal of the 2,4,5-T from the diet. This increase did not appear to be directed toward the synthesis of 2,4,5-T metabolizing enzymes. 2,4,5-T did not stimulate production of enzymes known to be produced by hepatotoxic compounds. This suggested that 2,4,5-T did not have a strong hepatotoxic activity and in fact it demonstrated activity similar to a structurally related compound chlorophenoxyisobutyrate (CPIB) which, like 2,4,5-T, induced liver enlargement and stimulated RNA and protein synthesis while inducing a strong self-metabolizing influence in the liver. IV-30 �Drill and Hiratzka (33) administered 2,4,5-T acid via capsule to adult mongrel dogs, five days a week over a 13 week period. There was 1 male and 1 female in the 0, 2 and 5 mg/kg groups, a male and 2 females in the 10 mg/kg group and 2 males and 2 females in the 20 mg/kg group. All dogs in the 0, 2, 5 and 10 mg/kg dosage levels survived'the 90-day test period. At 20 mg/kg the dogs died between days 11 and 75. The no effect level was 10 mg/kg per day. The histopathological examination of the 20 mg/kg dosed dogs was not remarkable and did not reveal a morphological cause of death. The prominent effects were weakness, slight stiffness in the hind legs, difficulty in swallowing food and in one dog, bleeding from the gums. Study of the data in Table 6 indicated that the various forms of 2,4,5-T fell in the same general range of chronic toxicity for mice, rats and sheep. The exception to this statement were data presented by Konstantinova (81) using the butyl ester of 2,4,5-T of unstated purity. In mice there appeared to be a definite strain difference in susceptibility to 2,4,5-T toxicity. The no effect level in mice ranged from 30-120 mg/kg with an overlapping of adverse effects from 60-140 mg/kg in various strains of mice. Rats appeared to be tolerant to about 10 times the 2,4,5-T calculated dose when administered as mg/kg of diet as opposed to mg/kg body weight of the animal. The no effect level for rats fed 2,4,5-T chronically were approximately 30 mg/kg body weight and 300 mg/kg diet. Threshold toxicity levels for rats were approximately 100 mg/kg body weight and 1,000 mg/kg diet. C. Absorption, Distribution and Exretion of 2,4,5-T Single subcutaneous administration of 100 mg/kg 2,4,5-T to mice resulted in 23 percent of the dose being recovered in the body over a 24hour period (147). In rats, 85.8 percent of a single intravenous dose of 100 mg/kg was found in the urine within 6 days (117). Single oral doses to rats of 100 mg/kg of the triethanolamine salt of 2,4,5-T were readily absorbed, distributed and eliminated; excretion was primarily via the kidneys (38). Seven days after oral administration of 50 mg/kg 2,4,5-T (99.6 percent pure) to rats, 56-69 percent of the dose was recovered in the urine; 70-85 percent of the recovered dose was unchanged 2,4,5-T, and approximately 15-30 percent was found as the glycine and taurine conjugates and as 2,4,5-trichlorophenol; the two conjugates were excreted in nearly equal amounts (16, 55). Similar results were obtained in mice, except that the quantity of the taurine conjugate was greater (54). The biological half-life of 5 mg/kg 2,4,5-T administered orally to dogs (77 h) was longer than that in rats (4.7 h). When the dose of 2,4,5-T to rats was increased to 200 mg/kg the biological half-life was prolonged to 25 h, indicating that the excretory capacity of the animals could be exceeded (104). IV-31 �TABLE 6. Animal Number Used Summary of literature data on the subacute and chronic toxicity of 2,4,5-T in animals Route of Administration Reference Dose Varied by strain 30-140 mg/kgd 62 <1% moribund 53-82% moribund 111 or moribund No effect 140 mg/kgd 120 mg/kgd 60 mg/kgd 90-120 mg/kgd 62 62 62 62 Varied 60-120 mg/kgd 60 Most all animals outwardly6 normal 60 mg/kgd 60 Many animals outwardly normal Mouse Effect 120 mg/kgd 60 Daily dose per animal in feed for 90 days No effect 30 mg/kg 66 Daily dose per animal in feed for 90 days Anorexia, depressed weight gain 100 mg/kgf 66 90 days in diet No effect 300 mg/kg9 66 . 978; F PFD Mc Gavage, dosed daily for 6-14 days, corn oil vehicle CD-I strain F£ dihydrid NCTR strain CRBL strain 378; PF Gavage, dosed daily for 6-14 days, corn oil vehicle CO no Q Rat 10 M/10 F 10 M/10 F 10 M/10 F �Table 6 continued 10 M/10 F 90 days in diet 10 M/10 F 90 days in diet Toxicity; no deaths due to treatment; histopath changes were noted 1000 nig/kg diet9 66 Growth retardation in males not females', no deaths due to treatment; histopath changes were noted 3000 mg/kg diet9 66 12 or 13/PF Stomach tube, daily dosing, entire gestation. No effect 0.01 mg/kg 8T 12 or 13/PF Stomach tube, daily dosing, entire gestation. Threshold level 0.1 mg/kg 81 12 or 13/PF Stomach tube, daily dosing, entire gestation. Irregular effect on dam and fetuses . 0.42 mg/kg 81 12 or 13/PF Stomach tube, daily dosing, entire gestation. One less live pup per female, ^ toxic signs noted. 4.2 mg/kg 81 I co co 12 M In diet to provide daily dose indicated Liver enlargement only 10 mg/kg Via capsule for 90 days Via capsule for 90 days Oral for 35 days No effect All died No effect 10 mg/kgv 20 mg/kgJ 100 mg/kg 1 111 Dog Sheep 1 M/l F 2 M/2 F NSk F - Female PF - Pregnant female C M - Male 33 33 29m �Table 6 continued d Both technical and purified 2,4,5-T acid containing <0.05 and 0.005 mg TCDD/kg. e Histopathological information presented in text. 2,4,5-T acid from commercial production containing <1 mg TCDD/kg. 9 Mono-, di-, and tripropylene glycol butyl ether esters of 2,4,5-T, 62% 2,4,5-T acid equivalent, h Butyl ester of 2,4,5-T, purity not stated. Analytical standard grade 2,4j5-T acid containing <0.05 mg TCDD/kg. •Commercial 2,4,5-T acid, 98.9% purity, TCDD level not stated. NS - number of animals in study not stated or unavailable from literature source. Form not known. < CO m From table in reference (29): �Similar results were obtained with single intravenous injections of 5 or 100 mg/kg 14C-2,4,5-T in rats, where 2.3 and 7.6 percent of the radioactivity were excreted in the feces, respectively, suggesting that at the higher dose biliary excretion of 2,4,5-T and/or Hs degradation products was involved in the overall elimination of 2,4,5-T from the body (117). Marked differences in the pharmacokinetics of 2,4,5-T were seen with different species, ages and doses: clearance of 2,4,5-T from the plasma and body of dogs, mice and man was slower than that in rats. The volume of distribution after a single oral dose of 5 mg/kg also differed: in man, 0.079; in rats, 0.14; and in dogs, 0.22 I/kg (49, 104). A single dose of 100 mg/kg 2,4,5-T to pregnant mice was almost entirely eliminated in 72 h; however, after 4 daily administrations of the same dose, 2,4,5-T accumulated in maternal tissues and fetuses, and by 48 h 2,4,5-T was still detectable throughout the fetuses (34). No radioactivity was found in NMRI strain mouse embryos in an early stage of gestation after administration of ^C-2,4,5-T to the dams. When given in late gestation, the fetal tissue had a level similar to that in maternal tissue (83). Selective uptake of 2,4,5-T into the yolk sac epithelium and absence of placental transfer in early pregnancy were effects similar to those seen with trypan blue in mouse embryos (84). No radioactivity was detected in hamster embryos in a late stage of gestation after similar administration of 2,4,5-T to the dams (31).* The biological half-life of l4C-2,4,5-T was significantly longer in newborn than in adult rats (41, 64). Radioactivity was found in all tissues examined as well as in milk and fetuses after a single oral administration of 0.17-41 mg/kg l4C-2,4,5-T to pregnant rats (41). Care must be taken when making all inclusive or generalized statements on the absorption, distribution and excretion of 2,4,5-T due to the demonstrated and marked differences in the pharmacokinetics of 2,4,5-T seen in laboratory animals. Such variables as age, dosage levels, routes of administration and chemical formulations all contributed to variations in response. Single doses of 2,4,5-T appeared to be rather quickly eliminated, primarily unchanged, via the urine and feces in a few hours up to about 7 days. High doses and repeated lower doses of 2,4-D or 2,4,5-T accumulated in animal tissues. The liver appeared to take a more active role in the metabolism and excretion of higher or chronic doses of 2,4,5-T than when single lower level doses were administered. IV-35 �D. Ernbryotoxic, Fetotoxic and Teratogenic Potentials of 2,4,5-T When reviewing the literature dealing with the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T, care must be taken to note the levels of TCDD contamination that may have been present in the 2,4,5-T tested. The TCDD contamination may very well have ranged from undetectable levels, using analytical technology available at the time, to 30 ppm or more. In an extensive 1977 review article of the teratogenic effects of environmental chemicals, Wilson (145) stated that 2,4,5-T had been intensively examined in pregnant animals of six different species. A low level of teratogenicity had been demonstrated in three rodent species: rats, mice and hamsters. Tests in pregnant rabbits, sheep and rhesus monkeys have been negative. Wilson discussed these studies in detail in the test, Handbook o& JnnatotoQg (144). Doses of more than 30 mg/kg 2,4,5-T (containing <0.1 mg/kg TCDD) increased the frequency of cleft palates in some strains of mice. When similar doses were administered to pregnant mice on days 6-15 of gestation some fetal growth retardation was observed (13). Courtney et al (26) first reported that under laboratory conditions 2,4,5-T was implicated as being teratogenic and fetotoxic. The 2,4,5-T used in the study was later found to contain 30 mg/kg TCDD. The 2,4,5-T was administered either orally or subcutaneously at a dose rate of 113 mg/kg per day on days 6-14 of gestation in C57B1/6 mice and days 6-15 in AKR mice. Oral administration caused an increased incidence of cleft palate and fetal mortality in both strains and cystic kidneys in the C57B1/6 mice. Subcutaneous injection resulted in significant increases in the incidence of cleft palate and cystic kidneys in the embryos of both strains of mice and evidence of fetal mortality in the C57B1/6 mice. Roll (112) found embryotoxic and teratogenic effects in NMRI mice exposed to 2,4,5-T, containing 0.05 ppm TCDD, administered orally at 20 to 130 mg/kg daily from 6 to 15 days of gestation. At 90 or 130 mg/kg/day the percentages of resorptions and/or dead fetuses were markedly increased relative to the controls. These levels also produced maternal toxic effects. Dose related reductions in fetal weight were observed at levels of 20 mg/kg/day and above. Cleft palate increased among fetuses exposed to 35 mg/kg/day or more. The teratogenic no effect level in mice for this particular sample of 2,4,5-T was considered to be 20 mg/kg/day. This was later confirmed with specially prepared samples of 2,4,5-T with no detectable (<0.02 mg/kg) amount of TCDD (112, 113). Neubert and Dillmann (96) found that samples of 2,4,5-T acid containing less than 0.02 mg/kg TCDD produced embryotoxic effects in NMRI mice in the form of fetal weight reductions at levels as low as 10 and 15 mg/kg per day, given orally from day 6 to 15 of gestation. The butyl ester of 2,4,5-T showed similar embryotoxic effects in mice when administered IV-36 �in the same manner. Cleft palates were produced using single doses of 2,4,5-T acid at 150-300 mg/kg. The maximum teratogenic effect was seen when mice were dosed on day 12 or 13 of gestation. A potentiation of the teratogenic effects (cleft palate) of 2,4,5-T and TCDD was obtained when teratogenic doses of one of the substances was combined with threshold doses of the other. However, for clear-cut potentiation of the effect of 30-60 mg/kg 2,4,5-T acid more than 1.5 mg/kg TCDD was required. When the level of TCDD drops below 1 mg/kg it was predicted that there would be no additional contribution to the embryotoxic effects of 2,4,5-T (i.e., cleft palate) in NMRI mice. It was concluded that in some of the 2,4,5-T preparations there must have been other contaminants present which exaggerated the teratogenic effect to some extent. Such contaminants may have been present in more than trace amounts. For example, 2,4,5trichlorophenol, did not contribute significantly to the teratogenic effect. The effect of 2,4,5-T and TCDD were studied in random bred CD-I and inbred DBA/2J and C57B1/6 strains of mice by Courtney and Moore (27). Two different samples of 2,4,5-T and one sample of TCDD were used. The 2,4,5-T technical grade contained 0.5 mg/kg TCDD and the analytical grade contained less than 0.05 mg/kg TCDD. Compounds were administered subcutaneously from day 6 to day 15 of pregnancy as solutions in 100 percent dimethyl sulfoxide (DMSO) in a volume of 100 yl/animal/injection. Both samples of 2,4,5-T and TCDD produced cleft palate in all three strains of mice when 2,4,5-T was administered at levels of 100 mg/kg and TCDD at 3 ug/kg. Kidney malformations were produced by both 2,4,5-T samples in CD-I mice and TCDD produced marked kidney anomalies in all mice strains. When 100 mg/kg 2,4,5-T and 1 yg/kg TCDD were administered in combination to CD-I mice, the activity was not potentiated at the dose levels employed. Bage et al (5) injected NMRI mice subcutaneously with 50 and 110 mg/kg 2,4,5-T containing less than 1.0 ppm dioxin on each of days 6 through 14 of gestation. At 110 mg/kg 2,4,5-T was teratogenic, causing cleft palates, rib and vertebrae anomalies as well as being fetotoxic causing 25 to 35 percent resorptions. Highman et al (61) recently reported that it was possible to detect a retardation in renal alkaline phosphatase in fetal kidneys from fetuses of mice given doses of 2,4,5-T by gavage at the rate of 60-120 mg/kg on days 6-14 of pregnancy. This retardation in renal alkaline phosphatase levels was suggested as the cause for the delay in renal functional development and indirectly supported the view that 2,4,5-T caused retarded development, rather than true teratogenesis. In this study a reduction of fetal weight and an increase in the incidence of cleft palate were seen in fetuses from treated females. Frohberg et al (45) administered 0, 20, 40, 80 and 120 mg/kg 2,4,5-T acid or butoxyethyl ester containing <0.1 mg/kg dioxin, by the oral route to NMRI mice. Oral doses of 80-120 mg/kg 2,4,5-T acid or 120 IV-37 �mg/kg butoxyethyl ester were required to produce 3 malformations and fetal deaths. In the inhalation experiments, 216 mg/m of the butoxyethyl ester showed a slight maternal toxic and fetotoxic and teratogenic effect. Ten exposures to 374 mg/m killed 5 of 15 dams, while 392 mg/m3 from day 11-15 of gestation was toxic for the dam and caused fetal deaths. Sparschu et al (130) orally administered commercial grade 2,4,5-T containing 0.5 mg/kg TCDD, to rats in daily doses of 50 and 100 mg/kg on days 6 to 15 and 6 to 10 of pregnancy, respectively. At the lower dosage level minimal fetal effects were seen with a slightly higher incidence of delayed ossification of the skull bones being observed. The higher level was toxic to the dams and caused a high incidence of maternal deaths. Only 4 of 25 rats survived with three showing complete, early, fetal resorptions and one had a litter of 13 viable fetuses which showed toxic effects but no evidence of teratogenic anomalies. Khera and MeKinley (74) found that 2,4,5-T, containing less than 0.5 mg/kg TCDD, induced some fetopathy and increased the incidence of skeletal anomalies in Wistar rats following single daily oral doses of 100-150 mg/kg on days 6-15 of gestation. The butyl ester produced no grossly observable teratologic effects when given at doses of 50 or 150 mg/kg. Various formulations of 2,4,5-T given to pregnant female rats demonstrated that a teratologic potential existed, in the form of skeletal anomalies, when repeated doses of 100 mg/kg or greater were given. At 25 mg/kg 2,4,5-T negative results were noted while at 50 mg/kg effects were noted but were not significant (P=0.05) when compared to control animals. The butyl ester produced no grossly observable anomalies and no adverse effects on the postnatal survival when pregnant females were treated at 50 and 150 mg/kg. Three of 8 females died at the 150 mg/kg dose. Skeletal deformities noted were not incompatible with life and no adverse change in reproductive performance or behavioral characteristics were detected. In the authors opinion, th£ predictive value of postnatal studies in relation to the detection of the teratogenic potential of test compounds may not be raliable on its own. Courtney et al (26) found that when 4.6, 10 or 46.4 mg/kg/day of 2,4,5-T was given orally on days 10-15 of gestation to Sprague-Dawley rats, kidney anomalies and other embryotoxic signs were seen at all levels. Some rat fetuses were reported to have had hemorrhagic gastrointestinal tracts. At the highest level there was a 60 percent fetal mortality and a higher incidence of abnormalities in the survivors. Courtney and Moore (27) reported that in CD rats, 2,4,5-T orally administered at 10, 21.5, 46.4 and 80.0 mg/kg was neither teratogenic nor fetotoxic. Prenatal administration of 2,4,5-T did not effect the postnatal growth and development of the CD rat. Sokolik (129) orally administered 2,4,5-T acid at dosage levels of 100 and 400 mg/kg per day and the butyl ester of 2,4,5-T at dosage levels of 50 and 200 mg/kg per day to rats on days 1 to 14 or 1 to 16. The purity of the 2,4,5-T in either form was not given. At 100 mg/kg IV-38 �2,4,5-T produced embryos with a combination of deformities including absence of the lower jaw, changes in the hind limbs and exophthalmos. At the level of 400 mg/kg one embryo was found with tridactyly of the upper limb combined with syndactyl, while another embryo had brachydactylia of the upper limb. Both levels of the butyl ester of 2,4,5-T were more toxic than 2,4,5-T acid, causing 30 percent embryonic mortality at 200 mg/kg. The lower dose of 50 mg/kg caused high mortality among the embryos as well. At the higher level the butyl ester induced cleft palate, hydronephrosis, hydrocephalus and extensive gastrointestinal hemorrhages along with hind limb brachydactylia. Cleft palate was the primary anomaly seen at the 50 mg/kg dosage level. Sokolik (129) concluded that the identical teratogenic action of the two preparations was probably attributable to the presence of dioxin, while the quantitative differences between the effects were attributable to differences in the concentration of the dioxin. Konstantinova (81) conducted experiments in white rats where 2,4,5-T butyl ester, purity not stated, was given orally to pregnant females for the entire period of the pregnancy at 0.01, 0.1, 0.42 and 4.2 mg/kg. The lowest level found to cause no effect was 0.01 mg/kg in the water. The threshold level was considered to be 0.1 mg/kg, with 0.42 mg/kg showing a general toxic effect on the pregnant female rat; however, the changes noted in the embryos had an irregular character. The highest dose level, 4.2 mg/kg, had a general toxic effect causing nervous system dysfunction in the female rats, changes in peripheral blood and a relative ( increase in the weight of internal organs. The embryotoxic effects were increased embryo deaths, lowered offspring weight, hydrocephaly and peritoneal cavity hemorrhages. In FW49 rats given daily oral doses of 25 to 150 mg/kg of either the TCDD-free or commercial grade 2,4,5-T (<0.1 ppm TCDD) showed no evidence of teratogenic effects (113). Emerson et al (36) confirmed the lack of teratogenic and fetotoxic effects of 2,4,5-T when containing only 0.5 ppm TCDD and when given in daily doses, by gavage, at the levels of 1, 3, 6, 12 or 24 mg/kg in Sprague-Dawley rats. Moreover, doses up to 24 mg/kg 2,4,5-T containing 1 mg/kg TCDD had no teratogenic effect in rats when given on days 6-15 of gestation. King et al (76) found no cleft palates when 93 embryos of Sprague-Dawley rats were injected in uteAo with purified 2,4,5-T on any one day ranging from 12 to 16 days of gestation at dosages of 50 to 125 yg/embryo. Two cleft palates were produced when technical grade 2,4,5-T was injected on day 15 of gestation into 118 embryos using the same techniques. In the control rats 45 females delivered 442 normal fetuses, with a 3.5 percent resorption rate and average liter size of 9.8 Commercial samples of 2,4,5-T containing TCDD in concentrations of 0.1, 0.5, 2.9 or 45 mg/kg caused fetal death and teratogenic effects IV-39 �in Syrian golden hamsters when given orally on days 6 through 10 of pregnancy at dosage levels of 20, 40, 80 or 100 mg/kg. As the dosage of 2,4,5-T increased and the TCDD content elevated, the effects were also increased. Pure 2,4,5-T containing no detectable TCDD produced no malformations when the dosage level was less than 100 mg/kg. Absence of eyelids (bulging eyes) and delayed ossification of the skull and exencephaly accounted for the main teratological abnormalities caused by 2,4,5-T containing TCDD. Hemorrhagic gastrointestinal tracts in the hamster fetuses appeared to be directly related to 2,4,5-T administration and could not be clearly linked to dose level of the compound or the dioxin content* These hemorrhages along with a marked edema noted in some of the fetuses reflected a toxic effect on fetal organs as opposed to a teratological effect (24). Gale and Perm (47) gave pregnant golden hamsters intravenous doses of 2,4,5-T on day 8 of gestation at the level of 2 mg/kg and found a 9 percent resorption rate in test animals compared to a 6 percent resorption rate in control animals. No malformed embryos were detected in this study; however, the purity of the 2,4,5-T was not given. New Zealand rabbits given oral doses of 0, 10, 20 or 40 mg/kg 2,4,5-T (containing <0,5 mg/kg TCDD) on days 6-18 of pregnancy showed no evidence of embryotoxic or teratogenic effects in their offspring (36). Dougherty et al (32) found that technical grade 2,4,5-T, containing 0.05 mg/kg TCDD was not teratogenic in rhesus monkeys, Macaco. mu£at£a, when given at 0, 0.05, 1.0 or 10 mg/kg, nor did 1t interfere with normal development of the young. Groups of 10 pregnant females were treated dally with stomach tube from days 22-38 of pregnancy. There was no evidence of toxicity to the females at these levels. In the 1971 Report of the Advisory Committee on 2,4,5-T (2), a preliminary study was cited where pregnant rhesus monkeys were orally dosed with 2,4,5-T, containing 0.05 mg/kg TCDD, at levels of 5, 10, 20 and 40 mg/kg three times weekly for 4 weeks between days 20-48 of pregnancy. After 100 days of gestation 12 fetuses were removed by hysterectomy and examined. All were found to be developmental^ normal and their weight range was not significantly different than the control animals of the same age. B1nns and Balls (11) found no congenital deformities 1n lambs from ewes daily fed 100 mg/kg 2,4,5-T add or the propylene glycol butyl ester of 2,4,5-T from the 14th to the 36th day of gestation. A third group of ewes fed 113 mg/kg of 2,4,5-T also showed no congenital deformities when fed at different periods during gestation. A summary of the literature on the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T in animals is presented in Table 7. In reviewing the literature it was evident that embryotoxic and teratogenic responses occurred in some strains of mice* rats and hamsters when repeated oral doses of 20 to 400 mg/kg 2,4,5-T was administered. Embryotoxicity and teratogenic studies in pregnant rabbits, sheep and rhesus monkeys have been negative. The embryotoxic and teratogenic potentials IV-40 �TABLE 7 Animal Number Used Mouse NSa PFb NS PF C57B1/6 strain AKR strain Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of 2,4,5-T in animals Route of Administration Response Dose Daily oral dose, days 6-15 of gestation Increased frequency of cleft palate in some strains. Fetal growth retardation. >20 trig/kg0 13 113 mg/kge 26 Daily oral or s.c. dose, days 6-14 of gestation Oral increased cleft palate and fetal mortality, both strains. Reference Cystic kidneys in C57B1/6 s.c. increased incidence of cleft palate and cystic kidneys in both strains. Increase in fetal mortality in C57B1/6 strain. NS PF Daily oral dose, days 6-15 of gestation No effect 20 mg/kgr 112 Cleft palate 35 mg/kg9 112 Marked increase in resorptions and "4" dead fetuses. 90-130 mg/kg9 112 �Table 7 continued Daily oral dose, day 6-15 of gestation Fetal weight reduction 10-15 mg/kg f ' h 96 Single dose during midgestation Cleft palate, maximum teratogenic effect day 12 or 13 of gestation 150-300 mg/kg 96 Daily oral dose, days 6-15 of gestation Cleft palate 30-60 mg/kg1 96 NS PF CO-1 strain OBA/2J strain C57B1/6 strain s,c. days 6-15 of gestation in a solution of DMSO at 100 yl/animal/injection Cleft palate, all three strains 100 rag/ kgJ 96 NS PF s.c. days 6-14 of gestation 50 mg/kgK 5 NS PF Kidney malformations in CO-1 strain No effect 110 mg/kg1 Teratogenic,cleft palate, rib and vertebrae anomalies, fetotoxi c ro 5 1 NS PF Savage, daily,days 6-14 of gestation Retardation in renal alkaline phosphatase in fetal kidneys, no true teratogenes i s, reduced fetal weight, cleft palate 60-120 mg/kg MS Daily oral dose,days 6-15 of gestation Toxic to females, malformations and fetal death 80-120 mg/kg 120 rag/kg1 45 Inhalation of aerosol for 10 exposures Slight maternal toxicity, fetotoxic, teratogenic 216 mg/m 3,n 45 PF m 61 �Table 7 continued Inhalation of aerosol for 10 exposures 5-15 females died 374 mg/m 3,n 45 Inhalation of aerosol for 5 exposures Toxic to females, fetal deaths 392 mg/m 3,n 45 Daily oral dose,days 6-15 of gestation Minimal fetal effects 50 mg/kg 130 Daily oral dose, days 6-10 of gestation Toxic to females, high maternal death, 4 of 25 survived, 3 had complete fetal resorptions, 1 had a litter of 13 live fetuses, toxic but no anomalies 100 mg/kg 130 Daily Oral dose,days 6-15 of gestation Fetopathy and skeletal 100-150 mg/kgp anomalies No effect at 50 and 100 mg/ 5fl , 0 ma/kaq , 9/ 9 kg. 150 mg/kg killed 3 of 8 females. 4.6, 10 or 46,4 Kidney anomalies, embryotoxi c mg/kge Rat 25, PF per test group NS PF Wistar strain Daily oral dose,days 6-15 of gestation NS PF Sprague-Dawley strain Daily oral dose,days 10-15 of gestation 74 74 26 At 46.4 mg/kg, 60% fetal mortality, many abnormalities in survivors NS PF Daily oral dose,days 1-14 of gestation Many deformities 100 mg/kg 129 Many limb abnormalities 400 mg/kg 129 �Table 7 continued Daily oral dose, days 1-16 of gestation Embryo mortality, cleft palate 50 mg/kg 129 30% embryo mortality and many anomalies 200 mg/kgs 129 No effect 0.01 mg/kgs 81 Threshold level 0.1 mg/kg 81 Toxic to female, irregular embryotoxic effect 0.42 mg/kgs 81 Toxic to female, nervous signs, embryo deaths 4.2 mg/kgs 81 Daily oral dose during pregnancy No effect No effect 25-150 mg/kgC5t 1-24 mg/kg0 Sprague-Dawley strain Gavage, daily during pregnancy No effect 24 mg/kgu 36 93 embryos Sprague-Dawley strain One -ui uteA.o injection on any one day from 12-16 days of gestation No effect 50-125 yg/kg' 76 Daily oral dose, days 6-10 of gestation No effect NS PF NS PF Daily oral dose throughout pregnancy NS PF 113 36 Golden Hamsters NS PF <100 mg/kg Fetal death, teratogenic NS PF Single intravenous dose on day 8 of gestation 20-100 mg/kg w No malformed embryos, 9% resorption - Test 6% resorption - Control 2 mg/kg r 2424 47 �Table 7 continued Rabbit NS PF Daily oral dose on days 6-18 of gestation No effect 40 mg/kgu 36 Daily stomach tube dose from 22-38 days of gestation No effect 0.05, l.O9 or 10 mg/kg 32 NS PF 3 oral doses weekly for 4 weeks between days 20-48 of gestation No effect on 12 fetuses removed by hysterectomy at 100 days gestation 5,10,20, 40° mg/kg 1 NS PF Daily dose from 14-36 day of gestation No effect 100 mg/kgr'x 11 NS PF Dosed at various periods of gestation No effect 113 mg/kg' 11 Monkey 10, PF per group Sheep en NS - number of animals in study not stated or unavailable from literature source. PF - pregnant female C 2,4,5-T acid, containing <0.1 mg/kg TCDD. s.c. - subcutaneous injection. e 2,4,5-T acid containing 30 mg/kg TCDD. f 2,4,5-T acid containing <0.02 mg/kg TCDD. 9 2,4,5-T acid containing 0.05 mg/kg TCDD h Butyl ester of 2,4,5-T, containing <0.02 mg/kg TCDD. ''z^.S-T acid, containing 1.5 mg/kg TCDD. J 2,4,5-T acid, technical grade, containing 0.5 mg/kg TCDD or analytical grade, containing <0.05 mg/kg TCDD. k 2,4,5-T acid, containing <1.0 mg/kg TCDD. 1 2,4,5-T acid containing <0.05 mg/kg TCDD. m'2,4,5-T acid, containing <0.1 mg/kg TCDD. n ButoxyethyTester of 2,4,5-T, containing <0.1 mg/kg TCDD. °2,4,5-T acid, containing 0.5 mg/kg TCDD. P 2,4,5-T acid, containing <0.5 mg/kg TCDD. q Butyl ester of 2,4,5-T, containing <0.5 mg/kg TCDD. r 2,4,5-T acid, purity not stated. s Butyl ester of 2,4,5-T, purity not stated. ^ 4 5 acid, free of TCDD. Detection level not stated. ,,^ ^2,4,5-T acid, containing 1.0 mg/kg TCDD. v Purified 2,4,5-T acid, purity not stated. w'?,4,5-T acid with oil, 0.5, 2.9 or 45 mg/kg TCDD. K Propylene glycol butyl ester of 2,4,5-T. �of 2,4,5-T in susceptible animals varied with the content of the contaminant TCDD. Levels of TCDD greater than 1 mg/kg were required to enhance the embryotoxic and teratogenic potential of 2,4,5-T. E. Carcinogenic and Tumorigenic Potentials of 2,4,5-T The industrial production of 2,4,5-T always results in some TCDD contamination, although admittedly at very low levels (<0.01 ppm) with current technology. Nevertheless, in the following review, the effects of various levels of TCDD associated with the 2,4,5-T being tested must always be considered. Innes et al (67) and the Bionetics Research Laboratories (12) reported that in groups of male and female mice receiving commercial 2,4,5-T (98 percent pure) there were no increases in any type of tumor in any group or combination of groups when compared to control animals. The treated mice were given 2,4,5-T at the dosage level of 21.5 mg/kg in 0.5 percent gelatin by stomach tube at seven days of age daily up to 28 days of age, followed by 60 mg/kg of diet until the mice were 78 weeks of age. Muranyi-Kovacs et al (92) conducted a two month study in XVII/G and C3HF mice. Beginning at six weeks of age the mice were given 2,4,5-T (containing <0.05 ppm dioxins) in the drinking water at a dosage of 100 mg/1. Following the initial two month treatment the exposure was continued throughout the animals life span by mixing 2,4,5-T directly with the diet at a concentration of 80 mg/kg. The average survival times for the XVII/6 mice was 555 days in 20 treated males and 632 days in 19 treated females, compared to 516 days in 32 control males and 40 control females. No significant differences were found in the incidences of tumors in the XVII/G strain of mice between the treated and control mice. The XVII/G strain of mice have a known high spontaneous incidence of lung tumors. In test groups of 22 male and 25 female C3HF mice studied, the average survival times were 523 days in treated males and 621 days in treated females, compared to 641 days in 43 control males and 661 days in 44 control females. The total number of tumors was 13/22 in treated males and 13/15 in treated females, which was significantly different from that in the female controls of 9/44 (P<0.01). No significance was seen when test males with tumors were compared to control males with a tumor Incidence of 22/43. The C3HF strain of mice has a known high spontaneous incidence of hepatomas. In a 1968 study (12) groups of 18 male and 18 female mice from two different crossbred strains were given single subcutaneous injections of 98 percent pure, 2,4,5-T at a dosage level of 215 mg/kg in DMSO at 28 days of age and observed up to 78 weeks of age. Tumor incidences in treated mice of any groups or combination of groups were not significantly different from any groups or combination of groups of control animals that numbered 141, 154, 157 and 161. The control animals were either untreated or were injected with DMSO, 0.5 percent aqueous gelatin or corn oil. IV-46 �Walker et al (141) demonstrated that six daily intraperitoneal injections of highly purified 2,4,5-T (99.0 percent) at the rate of 62 mg/kg effectively inhibited development of the Ehrlich ascites tumor being maintained in BALB/c mice. When the dosage of 2,4,5-T was increased to 80-85 mg/kg per day for six injections, the extent of inhibition of tumor development was doubled. From data presented in Table 8 it appeared that 2,4,5-T was not carcinogenic in most strains of mice tested at the oral dosage ranges of 21.5 mg/kg or 60 to 100 mg/kg in the diet or drinking water. Single subcutaneous doses of 2.5 mg/kg 2,4,5-T did not induce tumor formation in mice and 62 to 85 mg/kg 2,4,5-T in six daily intraperitoneal injections actually inhibited Ehrlich ascites tumor development being maintained in BALB/c mice. The only exception noted was the results reported by Muranyi-Kovacs et al (92), where treated C3HF female mice had a significantly higher incidence of tumors than did the control females. These authors stated: The carcinogenesis observed in our experiments should be attributed to 2,4,5-T per se. Nevertheless, a problem in assessing the significance of this effect was the choice of statistical analysis. Since the average survival time was different in some experimental groups, the choice of the experimental animal in assessment of carcinogenic potential is very important. For practical reasons rodents, particularly mice, are often used without scientific justification for such a choice. The problem of species specificity in the metabolism of chemical carcinogens is a known variable. The work by Gehring et al [ 4 ) on 2,4,5-T showed that the (9] kinetics of excretion of 2,4,5-T was extremely variable from one species to another. The half-life of 2,4,5*T in the plasma after a dose of 5 mg/kg was found to be 4.7 h in the rat, 77 h in the dog and 23 h in man. So the mouse being a rodent may not be the ideal experimental model for testing the carinogenicity of 2,4,5-T. Muranyi-Kovacs et al (92) further noted that in their opinion 2,4,5-T should be placed in the C group of chemical substances whose activity has been insufficiently assessed and in C2 and C3 priority groups requiring additional data, Implying that further testing in greater numbers of animals and in other species such as the rat and the dog was necessary. F. Mutagenic and Cytogenetic Potentials of 2,4,5-T As with 2,4-D, most of the mutagenic studies involving 2,4,5-T have been conducted in bacterial cultures or in plant and animal tissue cultures; however, Styles (133) investigated the cytotoxic effects of 2,4,5-T on -in vivo and Jin vi&io test systems and found no increase in IV-47 �TABLE 8. Animal Number Used Mouse . 18 Ma/18 FD of two hybrid strains 20 M/19 F XVH/G strain Summary of literature data on the carcinogenic and tumorigenic potentials of 2,4,5-T in animals Route of Administration Stomach tube, beginning at 7 days of age for 21 days, then in diet for 18 months Starting at 6 weeks of age for 60 days in drinking water, then in diet for life span Response No effect Dose . Reference 21.5 mg/kgc by stomach tube 60 mg/kg diet0 No effect 100 mg/ld for 60 days 80 mg/kg diet 5 i 22 M/25 F C3HF oo Starting at 6 weeks of age for 60 days in drinking water, then in diet for life span No effect in males, more tumors treated in females than in controls 12, 67 12, 67 92 H 92 100 mg/ld for 92 60 days 80 mg/kg diet H 18 M/18 F Single subcutaneous injections No effect 215 mg/kge in DMSO 8 sex not stated BALC/c Six daily intraperitoneal injections Inhibited Ehrlich ascites 62 mg/kg 92 tumor Doubled inhibition *M - Male 3 F - Female "2,4,5-T acid from a commercial source, TCDD level and purity not stated 80-85 mg/kg 12 141 f 141 2,4,5-T acid, containing <0.05 mg/kg TCDD "2,4,5-T acid, 93 percent pure, in dimethyl sulphoxide. 2,4,5-T acid, 99 percent pure, TCDD level not stated. �mutation rate and no evidence of mutagenicity in the test rats. He found serum from orally dosed rats was not mutagenic to SatmonMa. typhunufu.im. However, complete details of this study were not available. Jenssen and Renberg (68) found there was not a detectable increase of micronuclei in the erythrocytes of mouse bone marrow after intraperitoneal administration of 100 mg/kg 2,4,5-T containing less than 1 mg/kg TCDD. Because of the high experimental resolution power of the test system used, it was particularly suitable for the detection of weak chromosome breaking activity of 2,4,5-T in mammal cells. The lack of penetration of 2,4,5-T into the cells was in accordance with the rapid excretion that is known to occur in the mammalian body. This experiment did not, in the authors opinion, constitute a reliable measure of the mutagenic potential of 2,4,5-T; however, in practice, the lack of penetration of this substance into the cells indicated it did not constitute a cytogenetic hazard to man. In an abstract Buselmaier et al (19) reported on a large number of pesticides evaluated for mutagenic activity in mice with the hostmediated assay and to a smaller extent the dominant lethal method. These test systems took into account the mammalian metabolism and covered two different spectra of mutations: point mutations and the dominant lethal mutations which were thought to be the result of chromosomal aberrations. Back mutation systems of SaJLmonntta typhMnusuum G46 His" and SeA/uttut mot.ce4ce.n4 a21 leu" and Se/tAatLa. matce6cen4 a31 His" were used. In the host-mediated assay there was no significant increase in mutation rates after unspecified levels of subcutaneous injections of the acid or nbutyl ester of 2,4,5-T. All spot tests for this herbicide Jbi vWio was also negative. When the n-butyl ester, unspecified purity, was given to test mice by a single intraperitoneal injection, at a dose of 100 mg/kg, no increases in dominant lethal mutations were seen. Da'rving and Hultgren (30) reported that commercially available 2,4,5-T, with a TCDD concentration guaranteed on the label to contain less than 0.1 mg/kg, affected chromosomal and reproductive mechanisms in bone marrow cells from two different strains of mice. The authors concluded, however, that chromatid inter- or intra-exchanges were never observed. The study was not carried-out for sufficient time to demonstrate the effects on future generations of somatic cells. Majumdar and Hall (85) investigated the effect of 2,4,5-T -. containing no detectable TCDD, on male and female Mongolian gerbils. Test animals ranging from 50-80 days of age were given 5 consecutive daily intraperitoneal injections of 50, 150, 250, 350 or 500 mg/kg. No effects were seen on the chromosomes of bone marrow cells at doses of 150 mg/kg or less. At levels of 250 mg/kg and above, significant increases in chromatid gaps, chromatid breaks and chromatid fragmentation were observed. No exchange figures or isochromosome gaps or breaks were reported. IV-49 �Fujita et al (46) conducted studies to examine the cytogenetic effects of high purity 2,4,5-T (0.09 mg/kg TCDD) at levels of 10-' to 10-14 M on human lymphocytes in vWio. Breaks, deletions and rings were observed. Chromatid breaks increased with increasing concentrations of 2,4,5-T; however, it was not possible to distinguish if this effect was due to cellular toxicity or to a potential genetic alteration. Andersen et al (4) evaluated 110 herbicides for their ability to induce point mutation in one or more of 4 different microbial systems. The herbicide 2,4,5-T was included in this study. The authors did not state the purity of the compounds being tested. The 2,4,5-T did not cause point mutations in these microbial systems in comparison with known mutagens such as 5-bromouracil or 2-aminopur1ne. These observations of no mutagenicity of 2,4,5-T in E&che/tichia. aotL WP2 her* or her" or in Salmonella. typhirrwuum strains TA1535, TA1536, TA1537 or TA1538 were also confirmed in works by Nagy et al (94), Shirasu (136) and Shirasu et al (127). A review of the literature on the mutagenic and cytogenic potentials of 2,4,5-T in animals generally supported the premise that 2,4,5-T, like 2,4-D, was not highly cytotoxic in laboratory animals. The herbicide did not increase mutation rates nor stimulate a mutagenic response in rats and mice. In various in \)Wio and in vivo test systems 2,4,5-T did cause chromatid aberrations in cultured human lymphocytes and affected the chromosomes and reproductive mechanisms in mouse and hamster bone marrow cells. It was not determined whether these affects were due to cellular toxicity or to a potential for genetic alteration of future generations of somatic cells. No mutagenic responses were seen in several studies using microbial systems for the detection of mutagenic and cytogenic responses to 2,4,5-T. IV. REVIEW OF TCDD TOXICITY IN ANIMALS A. The Acute and Short-Term Toxicity Potentials of TCDD Studies on the extremely high acute toxicity of TCDD, the most toxic of the chlorinated dibenzo-p-dioxins, have been conducted by Carter et al (21), Greig et al (53), Gupta et al (56), Harris et al (59), King et al (76), Kociba et al (77), McConnell et al (87), Schwetz et al (121), Vos et al (140) and Zinkl et al (148). Schwetz et al (121) noted that perhaps the most striking fact about TCDD was its ability to cause death after a single'oral dose at levels as low as 0.6 yg/kg in male guinea pigs or 1000 yg/kg in the dog. Lethal doses to rabbits were in the same dose range with either oral (115 yg/kg), intraperitoneal (>252 yg/kg), or skin (275 yg/kg) administration. In mice, single oral doses of 1 to 130 yg/kg produced some deaths, however, no dose-response relationship was established. Schwetz et al (121) noted that approximately half the deaths in mice occurred between 13 and 18 days after treatment. IV-50 �Poland and Kende (108) considered TCDD to be one of the most potent low molecular weight toxins and teratogens known. They noted that most poisons act rapidly and kill by impairing the physiologic function of the nervous system. TCDD in contrast, is a "cellular poison." In the rat, deaths appeared to have resulted from hepatic necrosis and ensued weeks after a single oral dose. Harris et al (59), Schwetz et al (121), and Vos et al (140) also noted TCDD produced hepatic cell necrosis that was the probable cause of death in the rats in their studies. They also noted that in mice and guinea pigs, hepatic cell necrosis and liver insufficiency occurred only minimally. Putnam and Courtney (109) treated female Wistar rats with single oral doses of 100 yg/kg TCDD and found it caused a biphasic decline in body weight with a cessation of food and water consumption and urine production. The first phase started immediately after dosing and lasted 7-10 days followed by a recovery from 4-6 days during which time the rats ate and drank and regained about 10-15 percent of their body weight. This was followed by a second phase which occurred at 16-24 days after treatment with a weight loss of about 15-30 percent. If the loss of body weight exceeded 30 percent the rats usually died. Daily administration of water, electrolyte solution, or a balanced liquid diet did not alter or reverse the biphasic response. Cunningham and Williams (28) treated groups of 12 to 16 weanling male Wistar rats with single oral doses of 0 or 10 yg/kg TCDD. This was close to the lethal dose for when this amount was given orally to rats each day in a preliminary experiment, all died within 2 to 4 days. The lowest level in a single dosage that caused an increase in liver weight of rats in the preliminary study was 0.1 yg/kg. The TCDD had no effect on the rate of incorporation of ^H-acetate into liver lipids; however, it may have restricted the transport of lipids out of the liver. The storage of lipids reached a maximum at about 3 days after the TCDD was given and was accompanied by a significant increase in the incorporation of 14C-leucine into liver proteins. The increased synthesis of all proteins may have resulted from an induction of liver enzymes by TCDD. Harris et al (59) found the mortality pattern was very near the same in rats and guinea pigs when TCDD was given as a single oral dose or divided into daily or weekly doses over a 4 to 5 week period. He noted that this mortality pattern could be interpreted as demonstrating a cumulative toxicity from the TCDD. In rats, guinea pigs and mice, changes in the weight of the thymus appeared to be the most sensitive indicator of TCDD exposure according to work by Harris et al (59). These decreases in thymus weight occurred with doses of TCDD that had no effect on body weight. Van Miller et al (137) produced high levels of TCDD in the skin of rhesus monkeys by giving a single intraperitoneal injection of 400 yg/kg TCDD and produced clinical signs of alopecia and acne. IV-51 �A summary of the literature on the LDcn levels of the acute toxicity of TCDD for animals is presented in Table 9. TCDD was found to be an extremely toxic compound with an oral LDso range of 0.6 yg/kg in male guinea pigs to 115 yg/kg male and female rabbits. Male rats appeared to be more sensitive than females when TCDD toxicity was studied in the Sherman (Spartan) strain rat. In rabbits, essentially similar dosage levels of TCDD caused death following either intraperitoneal, oral or skin administration. Limited studies on dogs suggested that tltsy were less sensitive to TCDD than were the other laboratory animal species studied. In all species studied however, reduction in body weight was a common finding following TCDD treatment while other signs of toxicity were species dependent. B. The Subacute and Chronic Toxicity Potentials of TCDD Subacute and chronic doses of TCDD produced a variety of toxic effects, including hepatic necrosis, thymic atrophy and lesions of the myocardium in rats (20, 56), thymic atrophy, depletion of lymphoid organs and hemorrhage and atrophy of adrenal zona glomerulosa in guinea pigs (56) and hepatic necrosis in rabbits (120). The main target organs of TCDD in rats, guinea pigs and mice appeared to be the liver and thymus (56, 69, 70, 139, 140). The degree of hepatic involvement appeared to be dose dependent and the severity of the changes produced varied between species (56). A single oral dose of 126 yg/kg TCDD resulted in loss of body weight and death with an enlarged fatty liver after 21 days in C57B1/6 mice. A progressive necrotic centilobular liver lesion was seen (71). Vos and Moore (139) studied pre- and postnatal effects of TCDD in*groups of 5, 6 and 5 pregnant C57B1/6 mice dosed at 0, 2 or 5 yg/kg TCDD on days 14 and 17 of gestation and postnatally on day 1, 8 and 15. All neonates were weaned on day 23 and used for a skin graft experiment. This treatment resulted in a severe depletion of lymphocytes in the thymic cortex of the offspring. Cellular immunity was impaired and allograft rejection times were prolonged. Murray et al (93) conducted a three generation reproduction study to evaluate the effects of chronic, low-level ingestion of TCDD in Sprague-Dawley rats administered daily doses of 0, 0.001, 0.01 or 0.1 yg/kg provided via the diet for 90 days. No signs of toxicity were noted in either male or female rats during the TCDD feeding study. Vos et al (140) found the most significant findings in both mice and guinea pigs treated with sublethal doses of TCDD were in the lymphoid system where there was a supression of cell mediated immunity at doses of 2 and 5 yg/kg TCDD. Thigpen et al (134) found that low levels of TCDD did not produce overt clinical or pathological changes, however, these low levels IV-52 �Summary of literature data on the no-effect, LD5Q and levels of the acute toxicity of TCDD for animals TABLE 9 . Animal Number Used Route of Admin. Dose-Toxicity Single Dose yg/kg Reference >50 59 1-130 T21 Mouse 10 CD-I strain C57Bl/6Sch strain LD NSa Oral A few sporadic deaths 29 Mb C57B1/6 strain Oral LD 150 50 M NS T ) Oral Intraperitoneal LD 120C 138 5 M Oral No effect 8 121 5 M Oral No effect 16 121 10 M Oral LD 32 121 Oral 25 M Sherman (spartan) strain LD 22 121 Oral LD 45 121 100 100 50 Rat NS F strain 100 50d 50d �Table 9 continued Guinea Pig NS M Oral NS M Hartley strain 50 .6 2.1 121 121 Rabbit NS M/F 5 M/F 5 M/F 5 M/F 5 M/F New Zealand albino Oral Topically to skin Intraperitoneal Intraperitoneal Intraperitoneal LD 50e LD 50e No effect 2 of 5 died 3 of 5 died 115 121 275 121 32 121 >252 121 500 121 300 121 3000 121 30 121 100 121 <70 87 Dog 2 M 2 M 2 F 2 F Beagles 100 No effect No effect 1 F Rhesus I en Oral Oral Oral Oral Oral LD No effect LD Monkey 50f NS - Number of animals in study not stated or unavailable from literature source M - Male 3 "3H-TCDD A calculated LD50 cn "Responses to individual doses when ID™ could not be calculated Correlated the acute LD™ of TCDD with the clinical and pathological manifestations - not true calculated '50 LD50 �reduced host defenses. When 1 ug/kg was given orally once a week for 4 weeks to mice before infection with SaJtmon&JUa be/m, an increased mortality and decreased time from infection to death was noted. Weissberg and Zinkl (142) and Zinkl et al (148) noted hematological changes in mice, rats and guinea pigs treated with TCDD including lymphopenia and thrombocytopenia at dosage rates of 0.004 to 10 ug/kg for various repeated doses. Goldstein (50) gave TCDD orally to mice once a week over a 4 week period at a dosage of 25 ug/kg and found a 2,000-fold increase in the levels of 8- and 7-carboxyporphyrins in the liver. In a 13-week feeding study by Kociba et at (77), Sprague-Dawley rats of both sexes were given 0.001 or 0.01 ug/kg TCDD five days per week. A slight increase in relative liver weight occurred in those animals receiving 0.01 ug/kg TCDD. A steady state concentration of TCDD was attained in body tissues by the end of the study. Vos and Moore (139) in a pre- and postnatal study, treated groups of 5, 4 and 6 pregnant Fisher-334 rats with 0, 1 or 5 ug/kg TCDD prenatally on days 11 and 18 of gestation and postnatally on days 4, 11 and 18 via gastric intubation. Most of the neonates in the 5 ug/kg group 'died. Only the spleens of 25-day-old male animals from the 0 and 1 ug/kg groups were used for immunologic studies. At 1 ug/kg the pups had a depressed body and spleen weight. At 5 ug/kg, in those pups that survived, the body and spleen weights were depressed and the thymus was severely affected with marked depletion of lymphocytes in the thymic cortex. Cellular immunity was impaired with allograft rejection times being prolonged. Schwetz et al (121) found that solutions of 0.04 ug TCDD/ml of benzene was acnegenic in a rabbit ear bioassay study where the solution was applied to the inside of the ear 5 days per week for four weeks. Norback and Allen (98) fed fat, containing unspecified concentrations of chlorinated dibenzo-p-dioxins in the diet, to Macaco, mulatta monkeys for 100 days and found the monkeys developed alopecia, subcutaneous edema, anemia, progressive leukopenia and hypoproteinemia. Enlargement of the liver, hydropericardium, gastric hyperplasia and ulceration as well as hyperplasia of the lymph tissue and bone marrow was noted in the treated monkeys. Allen et al (2) found that female rhesus monkeys given a diet containing 500 ng/kg TCDD for 9 months became anemic within 6 months and pancytopenic after 9 months of exposure. Marked thrombocytopenia was associated with widespread hemorrhage. Death occurred in five of the eight animals between months 7 and 12 of the experiment at total IV-55 �exposure levels of 2-3 yg/kg TCDD body weight. At autopsy, in addition to the hemorrhage, there was a distinct hypocellularity of the bone marrow and lymph nodes. Death of these monkeys was attributed to complications from the severe pancytopenia. McNulty (89) fed one rhesus monkey a diet containing 2 yg/kg TCDD and another monkey a diet containing 20 yg/kg TCDD. The first animal died within 76 days, while the second animal died in 12 days. McNulty noted that although responses in two animals scarcely provided data for a dose-response curve, two conclusions could be drawn: (a) a total TCDD dose of less than 10 yg/kg of body weight accumulated over a few weeks period, and (b) young rhesus monkeys were among the most TCDDsusceptible animals of those that have been tested. A summary of literature data on the subacute and chronic toxicity of TCDD in animals is presented in Table 10. Subacute and chronic doses of TCDD produced a variety of toxic effects, including hepatic necrosis in mice, rats and rabbits; thymic atrophy in mice, rats and guinea pigs with adrenal gland hemorrhages and depletion of lymphoid organs also being seen in guinea pigs. Repeated oral doses of 0.001 to 10 yg/kg TCDD for four to 13 weeks did not significantly affect weight gain nor were signs of toxicity noted in mice and rats. Suppressed immune responses and changes in liver enzymes were noted, however, in mice. Repeated doses of TCDD as low as 1 yg/kg caused guinea pigs to become moribund and repeated doses of 0.04 yg/kg decreased lymphocyte counts. Rabbits developed acne of increasing severity when doses of 0.04 to 400 yg/kg were applied repeatedly to the internal surface of the ear. A total oral dose of 2-3 yg/kg over a nine month period produced severe hematological changes and death in rhesus monkeys. C. Absorption, Distribution and Excretion of TCDD Following a single oral administration of 50 yg/kg ^C-TCDD to rats, Piper et al (102, 103) found that almost 30 percent was eliminated in the feces during the first 48 h. The half-life for the disappearance of 14c activity from the body was 17.4 ± 5.6 days. After this time the excretion of ^C activity via the feces was from 1-2 percent per day. As the l^C-TCDD was absorbed into the body tissues most of the activity was localized in the liver and fat at levels about 10 times higher than that in other tissues. A total of 53.2 percent of the dose was eliminated via the feces and 13.2 percent via the urine, while 3.2 percent was expired into the air when measured over a 21 day period. Rose et al (114) found that, following daily oral administration of 0.01, 0.1 or 1.0 yg/kg l^c-TCDD five times per week for seven weeks to Sprague-Dawley rats» the major route of excretion was via the feces. Urine contained 3-18 percent of the cumulative dose of 14C activity after the seven week treatment. The half-life of 14C activity in the rats studied was 23.7 days. IV-56 �TABLE 10. Animal Mouse Number Used Summary of literature data on the subacute and chronic toxicity of TCDD in animals Route of Administration 377 Ma Once per week by gastric tube C57Bl/6JFh for 4 weeks (J67) strain Specific Pathogen free Effect Dose No effect on weight gain 0.5, 1, 5 and 10 yg/kg 134 Significant decrease in weight gain 20 ug/kg 134 Reference NSC F CD-I 134 1 yg/kg and Significant increase in mortality of mice challenged greater wi th Salmonella bern 5-6 per group C57Bl/6Sch FD strain d C57B1/6 M strain 134 No effect on mice challenged 0.5 ug/kg wi th Salmonella bern en No effect, on mice challenged 0.5, 1, 5, 10 and 20 ug/kg with Herpesvirus suis 134 2 yg/kg Oral dose given days 14 and 17 of gestation and postnatal ly on day 1, 8 and 15 No effect on weight gain Single oral dose after 8 weeks of age Hematological changes at 1 week after dose; normal at 3 weeks 1, 10 or 50 yg/kg 2000 fold increase in carboxyporphyrins in the liver 25 yg/kg 12 M Oral dose once per week for C57B1/6 strain Four weeks Suppressed cellular immunity 2 or 5 yg/kg 140 140 143 50 �Table 10 continued Rat NS M/F Sprague-Dawley strain Daily oral dose for 90 days No signs of toxicity NS M/F Sprague-Dawley Daily oral dose, 5 days per week for 13 weeks No toxicity, slight increase 0.001 or 0.01 in relative liver weight at yg/kg 0.01 yg/kg NS F CO strain Daily oral dose for 30 days Liver enzyme changes and hematological changes 10 yg/kg 148 Weekly oral doses for 8 weeks Moribund at 3 to 5 weeks 1.0 yg/kg 148 Significant decrease in lymphocyte counts 0.04 yg/kg 148 Applied to inside of ear, 5 days per week for 4 weeks in a .1 ml volume Acne with increasing severity as dose was increased 0.04 to 400 yg/kg 121 NS Macaca mulatta Fed fat containing 64% mass tetrachlorinated compounds in diet for 100 days Multiple toxic signs Unknown 8 F Macaca mulatta Fed in diet for 9 months Hematologic changes, 5 animals died 500 ng/kg of diet 2-3 yg/kg total exposure 0.001, 0.01 or 0.1 yg/kg 93 77 Guinea Pig NS F Hartley strain en 00 Rabbi t Monkey 98 �Table 10 continued 2 Macaca mulatta d Fed in diet Death in 12 days Death in 76 days M - Male b F - Female C NS- Number of animals in study not stated or unavailable from literature source Ul 20 yg/kg diet 2 jig/kg diet 89 89 �Allen et al (2) treated 40 male Sprague-Dawley rats with a single intragastric dose of 50 yg/kg of 14C-TCDD. One-half of the animals died within 25 days, 25 percent being accounted for during the first 3 days. The total amount of radioactivity in the urine was 4.5 percent of the total dose, with the highest daily levels being excreted toward the end of the experiment. A large percentage of the remaining radioactivity was localized in the liver and of this over 90 percent was located within the microsomal fraction. Fries and Marrow (44) found the half-life to be 12-15 days in rats given 7 or 20 yg/kg Mc-TCDD of diet (equivalent to 0.5 or 1.5 ya/kg per day) for 42 days. *, Vinopal and Casida (138) administered 3H-TCDO by a single intraperitoneal injection to male mice at the LDso dose of 120 yg/kg and found that it was not measurably converted to water soluble products and was eliminated primarily in the feces. Traces of tritium activity were detected in the urine. A large proportion of the administered dose persisted in the unmetabolized form in the liver, partially concentrated in the microsomal fraction for 11 to 20 days after treatment. The 3H-TCDD was not metabolized by liver microsomal fractions from mice, rats or rabbits. Gasjewicz and Neal (48) studied the tissue distribution and excretion of 14C-TCDD in adult male guinea pigs for up to 15 days following its intraperitoneal injection of 2.0 yg/kg. On day 1 the highest levels of radioactivity were located in the adipose tissue 2.36 percent, adrenals 1.36 percent, liver 1.13 percent, spleen 0.70 percent, intestine 0.42 percent and skin 0.48 percent. 14 other tissues examined contained less All than 0.3 percent. The level of C-TCDD 1n the liver Increased to 3.23 percent on day 15. An increase in 14C-TCDD was also noted in the adrenals, kidneys and lungs while adipose tissue and skin decreased in radioactivity. For the 15 days of the experiment the total urinary and fecal excretion of radioactivity was less than 1 and 5 percent respectively. The effects of 1.0 yg/kg TCDD upon plasma levels of Na, K, Cl, 003, Fe, Ca, inorganic P, alkaline phosphatase, SGOT, SGPT, LDH, glucose, urea nitrogen, creatinine, uric acid, total protein, albumin, cholesterol, triglycerides and bilirubin were determined periodically up to 14 days and compared to pair-fed control animals. Statistically significant increases in plasma albumin, total protein, Fe, urea nitrogen, cholesterol and triglycerides were observed in the TCDD-treated guinea pigs. The primary route of excretion for TCDD in animals appeared to be the feces, with urinary excretion occurring at a much reduced rate. Liver and fat accumulated about 10 times higher levels of TCDD than did other body tissues. The half-life for TCDD in rats following a single or repeated exposure was 12-24 days after termination of treatment. Large proportions of an administered dose of TCDD remained unmetabolized in the liver microsomes and were slowly excreted over an extended period. IV-60 �D. Embryotoxic, Fetotoxic and Teratogenic Potentials of TCDD The embryotoxic and teratogenic effects of TCDD in mice have been described by Courtney and Moore (27), Neubert and Dillmann (96), Neubert et al (97), and Smith et al (128) where doses as low as 1-10 ug/kg» given in a single or repeated dose, caused significant increases in the frequency of cleft palate and kidney anomalies. Neubert (95), and Neubert et al (97) noted a dose-response relationship for producing cleft palates in mice with TCDD. They also observed increased incidences in the frequency of cleft palate in mice,1 apparently caused by the synergistic effect of combining 'sub-threshold and 'threshold1 levels of TCDD with similar low levels of other known teratogens such as the weak teratogen 2,4,5-T when administered during days 6-15 of gestation. Moore et al (91) confirmed that exposure to TCDD via the milk was a major factor in the development of renal hydronephrosis in mouse pups when the nursing dam received a single oral dose of 1, 3 or 10 ug/kg TCDD at parturition. This effect was also seen in mouse pups nursed by a foster mother treated with TCDD during pregnancy or at the time of parturition. The common etiology of these kidney anomalies, whether prenatal or postnatal, was TCDD interference with development of the metanephric kidney and/or subsequent maturation. The incidence and degree of hydronephrosis was a function of dosage and length of target organ exposure. The dose effecting 50 percent of the test organisms (£050) for cleft palate production in NMRI mouse pups was estimated by Neubert et al (97) to be 40 ug/kg TCDD per day. The no effect level during days 6 to 15 of gestation was estimated at 2 ug/kg TCDD per day with no pronounced fetal mortality occurring when 3 ug/kg TCDD was given from day 6 to day 15 of gestation. Becker (8) has concluded that the influence of a teratogenic substance closely related to the critical developmental period of a particular tissue or organ. After this critical period passed, damage to other tissues may have occurred even if no significant malformations were observed. Unspecified doses of TCDD produced an extremely fatty degeneration of the liver in adult female rats when they were treated on days 13 to 15 of gestation. Fatty inclusions were seen in the liver of embryos from these treated females; however, no structural anomalies were noted in any of the embryo livers. Courtney and Moore (27) produced cleft palates in three strains of mice by giving 1 or 3 ug/kg TCDD subcutaneously on days 6 to 15 of pregnancy, while Courtney (25) found TCDD to be the most fetotoxic and teratogenic of several dioxin compounds when given at 25, 50, 100, 200, and 400 ug/kg per day orally and 25, 50, 100, 200 ug/kg per day subcutaneously IV-61 �in CD-I mice on days 7 to 16 of pregnancy. Fetal mortality increased with the dose: up to 97 percent in orally treated dams and up to 76 percent in dams receiving subcutaneous administration of the highest levels of TCDD. Other anomalies observed were hydrocephalus, lack of eyelid formation (open eye) and clubfoot with edema and internal hemorrhages being noted in fetuses of dams receiving the highest doses. Smith et al (128) administered 0.001, 0.01, 0.1, 1.0 and 3.0 wg/kg TCDD per day to CF-1 mice by gavage from days 6 to 15 of pregnancy. Only at the 1.0 yg/kg dose was the percentage of resorption sites per implantation sites significantly higher than in the control animals. At 3.0 ug/kg, cleft palate occurred in 71 percent of the treated litters and at 1.0 yg/kg, 21 percent had cleft palate. Renal anomalies*occurred in 28 percent of the litters treated at 3.0 yg/kg and in 5 percent of the litters treated at 1.0 yg/kg. No significant anomalies were seen at the other dosage levels. Embryo lethal effects have occurred in rats under experimental conditions imposed by Sparschu et al (131). Courtney and Moore (27) have observed kidney anomalies in rats, while Khera and Ruddick (75) observed intestinal hemorrhages and general edema in rat fetuses when oral or subcutaneous doses ranging from 0.03 to 16.0 yg/kg TCDD were administered daily to dams on days 6 to 15 of gestation. Sparschu et al (131) administered 0.03, 0.125, 0.5, 2.0 and 8.0 yg/kg TCDD per day to Sprague-Dawley rats on days 6 to 15 of gestation. At 8.0 yg/kg per day all fetuses were resorbed. Fetal weights were significantly (p<0.05) depressed at the 0.125 and 2 yg/kg per day level. Internal hemorrhages were observed at the 0.125, 0.5 and 2.0 yg/kg per day level. No adverse effects were noted in the fetuses of dams treated at the 0.03 yg/kg per day level. The authors suggested that 0.03 yg/kg per day was the no effect level for fetal and embryotoxic effects in rats. Khera and Ruddick (75) studied the perinatal effects of TCDD in Wistar rats in a two part experiment by giving daily oral doses of 0.125, 0.25, 0.5 and 1.0 yg/kg TCDD on days 6 to 15 of pregnancy. Visceral lesions were observed at 0.25 yg/kg per day and above with slight decreases 1n fetal weight also being observed. Postnatal effects of prenatal exposure to TCDD were studied by allowing offspring of treated dams to be reared by untreated dams until weaning. At maternal levels of 0.5 and 1.0 yg/kg per day, reduced survival, lowered body weight and reduced reproductive ability in the offspring were observed. At levels of 0.125 yg/kg per day no fetotoxic effects were observed. In the second part of the Khera and Ruddick study (75), rats were treated with daily oral doses of 1, 2, 4, 8 and 16 yg/kg TCDD on days 6 to 15 of pregnancy. At doses of 1.0 and 2.0 yg/kg per day, visceral lesions, reduction in fetal weight, and lowering of the number of live fetuses per litter were observed. Doses of 1 yg/kg per day or more IV-62 �produced maternal toxicity with all doses of 4 ug/kg or more producing 100 percent embryo lethality. The fetotoxic no effect level in Wistar rats appeared to be 0.125 ug/kg per day with any level of 0.25 ug/kg per day or more on days 6 to 15 of pregnancy adversely affecting fetal rat development. Courtney and Moore (27) administered TCDD to CD rats at the rate of 0.5 ug/kg per day subcutaneously in solutions of 100 percent DMSO on days 6 to 15 of gestation. Kidney anomalies were seen in 67 percent of the litters of treated females. At this level, TCDD did not affect fetal mortality or fetal weight, nor were cleft palates observed in any of the fetuses. • A summary of literature on the etnbryotoxic, fetotoxic and teratogenic potentials of TCDD in animals is presented in Table 11. It was apparent that TCDD caused birth defects and embryo mortality. Repeated daily oral doses of 0.1 to 2 yg/kg in pregnant mice produced no effect on the embryos; however, 3 ug/kg was the threshold level for production of cleft palate and kidney abnormalities. Single or repeated oral doses of 6.5 to 40 ug/kg TCDD were required to produce cleft palate in 50 percent or more of some strains of mouse embryos being studied. Daily subcutaneous injections of 1 to 3 ug/kg TCDD produced cleft palate and kidney abnormalities in 50 percent or more of three different strains of mouse embryos studied. Repeated oral doses of 25 to 400 ug/kg TCDD produced increasing fetotoxic and teratogenic responses in mice. Repeated daily oral doses of 0.03 to 0.125 ug/kg TCDD produced no effect in some strains of rat embryos while repeated oral doses of 0.125 to 2 ug/kg TCDD depressed fetal weight, lowered fetal survival and caused internal hemorrhages in fetuses. Repeated daily oral doses of 4 to 8 ug/kg TCDD produced 100 percent fetal mortality in rats. Signs of embryo toxicity and fetal death occurred in rats more frequently than did any signs of teratogenicity. When teratogenic lesions did appear in rats, kidney abnormalities were more common than cleft palate. E. Carcinogenic and Tumorigenic Potentials of TCDD In a preliminary report by Toth et al (135), 50 ten-week old male random bred Swiss H/Riop mice received gastric intubations of 7 ug/kg TCDD in sunflower oil for 12 months. No tumors were observed in 19 mice receiving post mortem examinations at the end of the treatment period. The livers from three animals showed histological evidence of cirrhosis and eight animals had developed dermatitis and showed histological evidence of increased amyloid in the tissues. Weekly doses of 0.007 and 0.7 ug/kg TCDD were given for 12 months to similar groups of mice. No pathological lesions were observed in five animals killed two months after the end of treatment. All surviving mice were kept for life-span studies and observation for the development of tumors. IV-63 �TABLE 11. Summary of literature data on the embryotoxic, fetotoxic and teratogenic potentials of TCDD in animals Mouse 700 total /PFa NHRI strain, 7000 fetuses examined 100 total/PF Route of Administration Response Dose yg/kg References Daily oral dose, days 6-15 of gestation No effect 96 CP - ED5Qd 2 (estimated) 3C 6.5 96 96 Daily oral dose, days 9-13 of gestation , Animal Number Used CP CP - ED5Qd 9C <9 96 96 Single oral dose, day 13 of gestation CP 15C 97 97 K D CP - Threshold CP 40 ED - 50 5C Single oral dose, day 11 of gestation 01 35 litters total from CD-I, DBA/2 J, and C57B1/60 strains CP Daily doses given subcutaneously on days 6-15 of gestation CD-I - CP effect, 1 litter only - CP, Threshold - CP, ED50 - KAe, Threshold - KA, ED5Q CP 15 ED * 50 DBA/ 20 - CP, Threshold 1 3C >3 c lc 1 -3 3C 97 97 27 27 27 27 27 - CP, ED5Q >3 - KA, ED5Q >3 27 27 27 27 C57B1/6J - CP, Threshold 3C >3 c 3C <3 27 27 27 27 - KA, Threshold - CP, ED50 - KA, Threshold - KA, ED50 c 3C �Table 11 continued 17 PF 19 PF Daily oral dose, days 7-16 of gestation Fetotoxic, teratogenic, increasing w/dosage up to 97% at highest dose 25, 50, 100, 200, and 400 25 Daily dose given subcutaneously on days 7-16 of gestation 31 PF CD-I strain Fetotoxic, teratogenic, increasing w/dosage up to 76% at highest dose 25, 50, 100, and 200 25 Daily oral dose, by gavage, days 6-15 of gestation No effect 0.1 128 Increased fetal resorption sites, 21% CP, 5% KA 1 128 71% CP, 28% KA 3 128 14 PF 18 littersf Single oral dose, day 10 of gestation No effect, CP 34% KA 1 91 16 littersf Daily oral dose, days 10-13 of gestation 1.9% CP 58.9% KA 1 91 14 littersf Daily oral dose, days 10-13 of gestation 55.4% CP 95.1% KA 3 91 NS9/fetuses Females given oral dose at parturition Renal hydronephrosis, 12, 71 or 75% depending on dose Daily oral dose, days 6-15 of gestation No effect 0.03 131 Depressed fetal weight 0.125 and 2 131 Internal hemorrhages in fetuses 0.125, 0,5 or 2 131 All fetuses died 8 131 cr> en ,il, 3 or 10 91 Pat 51 total/PF Sprague-Dawley (Spartan) strain �Table 11 continued 103 total/PF Daily oral dose, days 6-15 of gestation 0.25 75 0.5 and 1 75 Visceral lesions, reduced fetal weight, increased fetal death with maternal toxicity 1 and 2 75 100% embryo death en 75 Reduced fetal survival, lower body weight and lowered reproductive ability in progeny Daily subcutaneous dose, days 6-15 of gestation 0.125 Slight decrease in fetal wei ght 6 PF 48 fetuses CD strain No effect 4 75 No effect on fetal mortality 0.5 or CP 67% KA PF - Pregnant Female CP - Cleft palate b °Lowest dose with which a significant teratogenic effect has been produced. In some cases this is the only dose level tested and does not necessarily represent the lowest dose which could result in teratogenic effects.. EDg0 - Dose required to produce an effect in 50% of animals g KA - Kidney abnormalities f C57Bl/6 strain 9 NS - Number of animals in study not stated or unavailable from literature source Dose given in 100 percent dimethylsulfoxide solution (DMSO) 27 �Van Miller et al (136) recently reported the results of a two year study where ten groups of 10 male Sprague-Dawley rats were fed a laboratory diet-containing 0, 1, 5, 50, 500 or 1,000 yg/kg TCDD of food or 1, 5, 50 or 500 ng/kg TCDD of food for 78 weeks. All rats receiving the 50, 500 or 1,000 yg/kg TCDD of food died between the second and fourth week of treatment. In seven remaining groups, only one animals died before the 30th week and that death occurred in the 500 ng/kg TCDD of food at the 17th week. In the 1 and 5 yg/kg TCDD of food groups, all animals died between the 30th and 90th weeks of the experiment. The number of animals dead at the 95th week of the experiment were: 0 dose 6/10, 1 ng/kg 2/10, 5 ng/kg 4/10, 50 ng/kg 4/10, 50 ng/kg 4/10 and 500 ng/kg 5/10. Those animals surviving after the 95th week were killed and subjected to complete necropsy examinations. In all rats surviving past the 65th week laparotomies were performed and biopsies of any tumors were taken. After the 78th week on treated diets, the rats were placed on the same diet used to feed the control animals. Tumorigenie and toxic effects were observed in rats from the lowest six dosage groups. The overall incidence of neoplasms in these six experimental groups was 23/60 (38 percent) compared with 0/20 (0 percent) in both the 1 ng/kg and the control groups. Neoplastic nodules and cholangiocarcinomas of the liver were observed in 40 percent of the rats ingesting 5 yg/kg TCDD of food; two animals had both neoplastic nodules of the liver and cholangiocarcinomas, Van Miller et al (136) also found that tumors developed in 24/50 (46 percent) of the rats ingesting 5, 50 or 500 ng/kg TCDD-of food and 1 or 5 yg/kg TCDD of food, compared to none (0/10) in the control animals. The tumors seen were carcinomas of the ear duct, kidney and liver. Three retroperitoneal histiocytomas were described as metastasizing to the "lungs, kidney, liver and skeletal musculature." Three of the ten deaths which occurred in the 5 yg/kg TCDD of food dose group were attributed to aplastic anemia. One animal in the 500 ng/kg TCDD of food group had a severe liver infarction. Kociba et al (78) conducted a chronic study of TCDD toxic effects to Sprague-Dawley rats fed 0.1, 0.01 or 0.001 yg/kg TCDD daily for two years to groups of 50 rats of both sexes. Eighty-six animals of each sex served as controls. Discernible increases were noted in the incidence of hepatocellular carcinomas of the liver and of squamous cell carcinomas of the lung, hard palate/nasal turbinates and tongue in rats fed at the rate of 0.01 yg/kg. They also reported decreased incidences of pituitary, uterine, mammary gland, pancreatic and adrenal gland tumors at the 0.01 yg/kg level. The squamous cell carcinoma of the hard palate observed in one female rat receiving this dose was considered unrelated to TCDD treatment since a similar tumor had occurred in other unrelated studies. At 0.001 yg/kg TCDD, no significant lesions were seen in male rats and the only lesion of significance in female rats at the 0.001 yg/kg TCDD was swollen hepatocytes, considered to be a reversible lesion. IV-67 �Many chemically nonreactive carcinogens ai-e eiizymaLiu-a I ly converted to biologically active carcinogens. The enzyme aryl hydrocarbon hydroxylase (AHH) has been strongly implicated in this process (86). Kauri et al (32) studied AHH induction in human lymphocyte cultures by TCDD. The authors stated; TCDD itself is not a potent carcinogen in mice; however, the synergistic action of TCDD with 3-methylcholanthrer.e (MC) produces cancer in different strains of mice in direct proportion to the degree of elevation of the induced hy/droxylase activity and associated cytochrome content. Their study showed a positive correlation between basal enzyme activity and enzyme levels maximally inducible by either TCDD or MC. They also found that TCDO WAS about 40 to 60 times more potent than MC as an inducer of hydroxy/lase activity in cultured human lymphocytes. The implication, of TCDD in AHH inducibility had also been reported t>y Poland and Glover (105, 106) and Poland et al (107) in their studies on chick embryo livers. They found that all dioxins which were potent inducers have halogens at three of the four lateral ring positions and at least one noft-halagenateti carbon atom. When TCDD potency, as an inducer of hepatic AHW activity » was compared with that of MC by a computer bioassay technique, data reflected that TCDD may be 28,640 times as potent as MC on a molar basis. Allen et al (2) conducted a study in which female rhesus monkeys were fed diets containing 500 ppt TCDO for nine months. Anemia, thrombocytopenia and leuikapenia were the most debilitating changes noted. The altered, lymphopoiesis cotild be associated with immune suppression. Epithelial changes, including hypertrophy, hyperplasia, and metaplasia were reported in these TCDD exposed monkeys. A summary of the literature on the carcinogenic and tumori genie potentials of TCDD in animals is presented in Table 12. It was noted that in a preliminary study where O.Q07, O..Q7 and 7 yg/kg TCDD was given in weekly oral doses for 12 months to. mice* no tumors were produced. In "'ats, levels o>f 1 and! 5 wo/kg TCDO of diet and; 1, 5, 50 and 500 ng/kg 1CD0 of diet fed for 78 weeks produced an overall tumor incidence of 38 percent in the test amiraals. At 0.001 ug/kg TCDD, given via the diet to rats far 2 years, nS effect was produced. A level of 0.01 yg/kg TCDD given via the diet to rats* for 2 years, produced liver nodules and hyperplasia of the epithelium of the lungs. An increase in liver and lung carcinomas was seen when O.I ug/kg TCDD was fed to rats for 2 years via the diet. An interesting unexplained observation, however, was the reduction of pituitary, uterine, niaranary/,, pancreas and adrenal tumors. Monkey/s fed 500 n§/k§ TCDO of diet for 9 months did not develop tumors but died of marked hetnatological alterations. IV-68 �TABLE 12. Animal Number Used Summary of literature data on the carcinogenic and tumorigenie potentials of TCDD in animals Route of Administration Response Dose Gastric intubation weekly for 12 months starting with 10 week old animals No effect in 5 animals examined 2 months after treatment ended 0.007 ug/kgb 135 No effect in 5 animals examined 2 months after treatment ended 0.07 ug/kgb 135 No tumors in 19 animals examined at end of treatment 7 yg/kgb 135 All died in 2 to 4 weeks 50, 500 or 1000 ug/kg diet 136 All died in 30 to 90 weeks 1 and 5 yg/kg diet 136 50% dead at 95th week 500 ng/kg diet 136 50 ng/kg diet 136 Mouse 50 M per group Swiss H/Riop strain I CTi Reference Rat 10 groups of 10 M In diet for 78 weeks 38% tumors < 40% dead at 95th week 40% dead at 95th week '20% dead at 95th week No tumors 5 ng/kg diet 1 ng/kg 136 < 60% dead at 95th week Controls 136 �Tafrle 112 continued 10; Met fswr 2 years No, effect Live* - 540; ng/TOW' k§e Fat - 540 ng; JIQT lH€.reasedi urinary ex.Ofeti?<m erf? ptiqDfry/irins in; females Liver - noduTes 5® ararfcmaTs LQn! tag/kg Liver - 5,100 ng Fat - IJQQ n§ d Increaseefc incidence off eell carcinQmas evidence' ©f Ltterlnfi * mcanmary pancreas aiatdi adrenaJ Fat ~-J - 24,800 nig: TCBi^ltg; - ajtfO ng: o Monkey 8 Maeaca ff diet far § msrrtfos M - Male ""Preliminary report remaining animals to be kept for life span study and observation for tumor development wfthiro 6roanthis Pancytopenia after 5 months Rarked thrombocytapenia Tissue hemorrhages 5 of 8 died between 7 and 12 months Epithelial tissue changes ag/kg diet This is the dose supplied to each animal via the diet: 0.001 yg TCDD/kg body weight = 22 ng TCDD/kg diet 0.01 yg TCDD/kg body weight = 210 ng TCDD/kg diet 0.1 ug TCDD/kg body weight = 2200 ng TCDD/kg diet : F - Female "Terminal samples of liver and fat indicating accumulated levels of TCDD/kg of tissue after two years of treeiment at the respective dosage levels Total exposure, 2-3 yg/kg body weight �F. Mutagenic and Cytogenetic Potentials of TCDD Again, as with 2,4-D and 2,4,5-T, most of the mutagenic studies involving TCDD have been conducted in bacterial cultures or in plant and animal tissue cultures. Khera and Ruddick (75), however, have conducted dominant lethal tests in which male Wistar rats received TCDD, orally, at dosages of 4, 8 or 12 yg/kg per day for seven days. TCDD did not induce dominant lethal mutations during or in the 35 days following treatment. This 35 day period corresponded to the postmeiotic stages of spermatogenesis Green and Moreland (52) conducted a short-term investigation of several dioxins, using male Osborne-Mendel rats, to determine what potential these substances had to cytogenetic damage in rat bone marrow. In one study, all of the dioxins were tested via gavage in the rats for five consecutive days at 10 yg/kg per day. A second study involved TCDD being given separately by two routes. A single oral dose of 20 yg/kg TCDD or oral doses of 10 yg/kg TCDD for five consecutive days, and in other rats, single intraperitoneal doses of 5, 10 or 15 yg/kg TCDD were given. No evidence was found that any of the substances tested produced cytogenetic damage in the bone marrow of male rats under the conditions of the experiment. However, when rats of both sexes were treated twice weekly with TCDD at a dosage level of 4 yg/kg for 13 weeks, a significant increase in the number of chromosome aberrations was found by 6r,een (51). Hussain et al (65) evaluated the mutagenic activity of TCDD (99 percent pure) of three different microbial test systems. In the first study, TCDD significantly increased the incidence of reverse mutations in EAdieAdua. coti Sd-4 when 2 yg/ml TCDD caused the bacteria to change from streptomycin dependence to streptomycin independence. This dosage was the only dose at which mutations were clearly observed. In a second study, Hussain et al (65) examined reverse mutation from histidine dependence to histidine independence in So£mone££a typkmuA^u strains TA1530 and TA1532. TCDD caused positive changes in TA1532 strain but negative results were seen in TA1530 strain which indicated that TCDD may act as a frameshift mutagen in this bacterial strain. In a third study conducted by Hussain et al (65) slight prophage induction in E4cAa>u.cA-ta c.otl K-39 was observed. However, in this study the solvent DMSO was used which.itself causes cellular effects. Seiler (124) using plate assays to study the mutagenicity of TCDD found a positive response in Satmonatta. typkunufuum strain TA1532, doubtful responses in strains TA1531 and TA1534, and negative responses in strains G46 and TA1530. Metabolic activation systems were not included in any of these microbiological assays. Beatty et al (7) conducted a study with -en \>Ww cultures of the mammalian cell types Hela, Balb-3T3, virus (SV-40) transformed 3T3 IV-71 �mouse fibroblasts, human foreskin fibroblasts and human lymphocytes. In all cases TCDD added in a final theoretical concentration of 10'° to the culture medium prior to the addition of cells resulted in no significant inhibition of growth measured after a period of four days. Electron microscopic examination of the TCDD-treated cells did not reveal any changes in morphology as compared to untreated cells. Incubation of human fibroblasts and SV-101 cells with 14C-labelled TCDD showed that incorporation of the TCDD into the cells did occur. Kondorosi et al (79) found that TCDD did not impair the transfectivity of QB-RNA, thus confirming the assumption that TCDD did not react chemically with nucleic acid. Whatever mutagenic property it had must have occurred by the forming of a physical complex by "intercalation" in DNA, leading, to frameshift mutation. In summarizing the limited literature dealing with the mutagenic and cytogenic potentials of TCDD in animals, it was noted that daily oral doses of 4, 8, or 12 yg/kg TCDD given to rats for seven days did not induce dominant lethal mutations. Five daily oral doses of 10 yg/kg TCDD and a single oral dose of 20 yg/kg TCDD did not produce cytogenetic damage in bone marrow cells of male rats. Chromosome aberrations were detected when male and female rats were dosed twice weekly at 4 yg/kg TCDD for 13 weeks. Using microbiological systems, TCDD has been shown to induce mutagenic changes in some strains of bacteria. V. SUMMARY OF THE LITERATURE REVIEW OF THE TOXICITY OF 2,4-D, 2,4,5-T AND TCDD IN ANIMALS In summarizing the literature on the toxicity of 2,4-D, 2,4,5-T and TCDD in animals, the following general statements provided a concept of the overall toxicity of each compound as they related to each other and the effects they produced in experimental animal studies. Where possible, inclusive statements were given rather than individual species responses. A. 2,4-D 1. The LDcn for single oral doses of 2,4-D in animals ranged from 100-2,000 mg/kg with the majority of LDso values in the 300-800 mg/kg range. 2. Signs of chronic 2,4-D toxicity did not differ greatly from those seen in acute toxicity. No effect levels, seen when 2,4-D was given in repeated oral doses, ranged from 30 to 75 mg/kg. 3. Being a strong acid, 2,4-D was rapidly eliminated from the body mainly via the urine. The plasma half-life of a single oral dose was in the 3-12 h range. After high doses or repeated lower doses, 2,4-D accumulated in the tissues; with residue levels rapidly declining as evidenced by a half-life of 1 to 2 weeks. IV-72 �4. No teratogenic signs were seen in rats fed repeated doses of 1,250 to 1,500 mg/kg 2,4-D of diet, nor when repeated daily doses of 8.75 mg/kg were given. Embryo toxic and fetotoxic responses appeared in rats and hamsters at repeated daily oral doses of 100 to 150 mg/kg. 5. Tumors were not produced in mice fed 46.4 to 100 mg/kg 2,4D of diet nor in rats fed 1,250 mg/kg 2,4-D of diet for 18 to 24 months. Single subcutaneous injections of 21.5 to 215 mg/kg 2,4-D did not produce carcinogenic or tumorigenic responses in mice. 6. The 2,4-D was not highly cytotoxic in laboratory animals and did not cause increased mutation rates nor did it stimulate a mutagenic response in rats and mice. No mutagenic or cytogenic responses were seen in several studies using microbial systems for the detection of such toxicity. B. 2,4,5-T 1. The acute toxicity for 2,4,5-T was in the same general range as for 2,4-D in most animal species. The 1050 values for single oral doses of 2,4,5-T ranged from 380 to 940 mg/kg in small laboratory animals. 2. Chronic toxicity studies in mice using repeated oral doses of 30-120 mg/kg 2,4,5-T produced no effect. An overlapping of adverse effects were seen, however, in doses of 60-140 mg/kg 2,4,5-T, depending on the strain of mouse studied. The no effect level for rats orally administered repeated doses of 2,4,5-T was approximately 30 mg/kg while as much as 300 mg/kg of diet could be fed with no adverse effects being noted. Threshold toxicity levels for adverse effects of repeated oral doses of 2,4,5-T in rats was approximately 100 mg/kg or 1,000 mg/kg of diet. 3. Single doses of 2,4,5-T were eliminated in animals, primarily unchanged, via the urine and feces over a period of a few hours up to about 7 days. 4. It was evident that 2,4,5-T induced embryotoxic and teratogenic responses in some strains of mice, rats and in hamsters when repeated oral doses of 20 to 400 mg/kg were administered. However, doses of 20 to 150 mg/kg in the same laboratory animal species produced a negative or no effect response. This indicated a great species and strain variation in response to 2,4,5-T as well as the fact that the embryotoxic and teratogenic potential of 2,4,5-T varied with the concentration of TCDD present. Levels of TCDD greater than 1 mg/kg were required to enhance the embryotoxic and teratogenic potential of 2,4,5-T. Embryotoxicity and teratogenic studies in pregnant rabbits, sheep and rhesus monkeys have been negative. 5. In most strains of mice, oral doses of 21.5 mg/kg or repeated doses of 60 to 100 mg/kg in the diet or drinking water and single subcutaneous doses of 2.5 mg/kg of 2,4,5-T did not induce tumor formation. IV-73 �6. In animals, 2,4,5-T, like 2,4-D was not highly c>totoxic and did not increase mutation rates nor stimulate a mutagenic response in rats and mice. It produced, however, chromatid abnormalities in cultured human lymphocytes and affected the chromosomes and reproductive mechanisms in mouse and hamster bone marrow cells. These effects may have been due to cellular toxicity rather than genetic alterations. No mutagenic responses were seen in several studies using microbial systems for the detection of such toxicity. C. TCDD 1. TCDD was an extremely toxic material with a single oral dose LDgg range of 0.6 yg/kg in male guinea pigs to 115 yg/kg in rabbits. 2. Chronic toxicity was manifested by hepatic necrosis, thymic atrophy and depletion of lymphoid organs. In mice and rats, repeated oral doses of 0.001 to 10 yg/kg for four to 13 weeks produced a no effect response for weight gain and no signs of toxicity were noted. Repeated oral doses as low as 1 yg/kg caused guinea pigs to become moribund and a repeated dose of 0.04 yg/kg decreased lymphocyte counts. Acne of increasing severity was produced in rabbits when doses of 0.04 to 400 yg/kg were applied repeatedly to the internal surface of the ear. A total oral dose of 2-3 yg/kg over a nine month period produced severe hematological changes and death in rhesus monkeys. 3. The primary route of excretion for TCDD in animals appeared to be the feces, with urinary excretion occurring at a much reduced rate. Liver and fat accumulated about 10 times higher levels of TCDD than did other body tissues. The half-life for TCDD in rats, following repeated exposure, was 12-15 days after termination of treatment. 4. It was apparent that TCDD caused birth defects and embryo mortality. Repeated daily oral doses of 0.1 to 2 yg/kg TCDD in pregnant mice produced no effects on the embryos; however, 3 yg/kg was the threshold level for production of cleft palate and kidney abnormalities. Single or repeated oral doses of 6.5 to 40 yg/kg TCDD were required to produce cleft palate in 50 percent or more of some strains of mouse embryos. Daily subcutaneous injections of 1 to 3 yg/kg TCDD produced cleft palate and kidney abnormalities in 50 percent or more of three different strains of mouse embryos. Repeated daily oral doses of 0.03 to 0.125 yg/kg TCDD produced no effect in some rat strains while doses of 0.125 to 2 yg/kg TCDD depressed fetal weight, lowered fetal survival and caused internal hemorrhages in fetuses. When teratogenic lesions appeared in rats, kidney abnormalities were more common than cleft palate. 5. No tumors were produced in a preliminary study where 0.007, 0.07, or 7 yg/kg TCDD was administered to mice in weekly oral doses for 12 months. When levels of 1 and 5 yg/kg TCDD of diet and 1, 5, 50 and IV-74 �500 ng/kg TCDD of diet were fed to rats for 78 weeks, an overall tumor incidence of 38 percent was present in the test animals. No effects were produced when 0.001 yg/kg TCDD was given to rats via the diet for 2 years. A level of 0.01 yg/kg TCDD given via the diet for 2 years produced liver nodules and hyperplasia of the lung epithelium. A level of 0.1 yg/kg TCDD in the rats' diet for 2 years produced an increase in liver and lung carcinomas. Monkeys fed 500 ng/kg TCDD of diet for 9 months did not develop tumor but died of marked hematological alterations. 6. Daily oral doses of 4, 8 or 12 yg/kg TCDD given to rats for seven days did not induce dominant lethal mutations. Five daily oral doses of 10 yg/kg TCDD and a single oral dose of 20 yg/kg TCDD did not produce cytogenic damage to bone marrow cells of male rats. 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McConnell, E.E., J.A. Moore and D.W. Dalgard. 1978. Toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in Rhesus monkeys (mo.co.ca mulatto.) following a single oral dose. Tox^iaol. App£. PftoAmaco£. 43:175-187. 88. McLennan, M.W. 1974. 2,4-D toxicity in dairy cattle. AuA&ialia.n Vvt. 3. 50(12) :578. 89. McNulty, W. 90. Mitchell* J.W., R.E. Hodgson and C,F. Gaetjens. 1944. Tolerance of farm animals to feed containing 2,4-D. 3. Aiu/n. Sex. 5:226-232. 91. Moore, J.A., B . N . Gupta, J.G. Zinkl and O.G. Vos. 1973. Postnatal effects of maternal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Emu/urn. Heo£#i Pe/wpec*. 5:81-85. 92. Muranyi-Kovacs, I . , G. Rudali and J. Imbert. 1976. Bioassay of 2,4,5-trichlorophenoxyacetic acid for carcinogenicity in mice. B/tc£. J. CanceA. 33:626-633. 93. Murray, F . J . , F.A. Smith, K.D. Nitschke, C . G . Humiston, R.J. Kociba and B.A. Schwetz. 1977. Three-generation reproduction study of rats ingesting 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxxco£. App£. 1977. PCB poisoning in Rhesus monkeys. Ptujmate. New*. 35(1) -.139-140. 94. Nagy, Z., I. Mile and F. Antoni. 1975. The mutagenic effect of pesticides on Eacke/u.akca c,oti WP2 try. Acta Mxc/Lofaxo£. Acad. Sox. Hang. 22:309-314. 95. Neubert, D. 1976. Some remarks to the toxicity of polychlorinated dibenzo-dioxins. In. Proceedings of the Congress of the European Teratology Society, Gaspiano, 1976 (in press). 96. Neubert, D. and I. Dillmann. 1972. Embryotoxic effects in mice treated with 2,4,5-trichlorophenoxyacetic acid and 2,4,7,8-tetrachlorodibenzo-p-dioxin. Maun(/n-Sdtnu.edebe/u} '& M.c.k. Phmasikot. Exp. ?outkot. 272 (3): 243-264. 97. Neubert, D . , P. Zens, A. Rothenwallner and H.-J.Merker. 1973. A survey of the embryotoxic effects of TCDD in mammalian species. . Heo&th PeMpect. 5:67-79. 98. Norback D.H. and J . R . Allen, 1973. Biological responses of the nonhuman primate, chicken and rat to chlorinated dibenzo-p-dioxin ingestion. EnvxAon. Heotth P&tApect. 5:233-240. 99. Palmer, O.S. 1963. Chronic toxicity of 2,4-D alkanolamine sales to cattle. J. Am. l/et. Med. A^oc. 143(4): 398-399. IV-83 �TOO. Palmer, J.S. and R.D. Radeleff. 1964. The toxicologic effects of certain fungicides and herbicides on sheep and cattle. Ann. M. V. Acad. Su. 111:729-736. 101. Pilinskaya, M.A. 1974. Cytogenetic effect of the herbicide 2,4-D on human and animal chromosomes. J&Ltot. Genei. 8(3) :202-206. (Russian) . 102. Piper, W.N. and J.Q. Rose. 1971. The excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Amer. Chem. Soc., Abstr. Pap., 162nd Nat. Meet., Pe-6-ttc. Cheat. Sue.., No. 88. 103. Piper, W . N . , J.Q. Rose and P.O. Gehring. 1973. Excretion and tissue distribution of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Aduan. Chum. SeA. • 120:85-91. 104. Piper, W . N . , J.Q.. Rose, M.L. Leng and P.J. Gehring. 1973. The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral administration to rats and dogs. Tox^co£. App£. PkoAmacoi. 26:339-351. 105. Poland, A. and E. Glover. 1973. Studies on the mechanism of toxicity of the chlorinated dibenzo-p-dioxins. EnviAon. H&ctiLth 5:245-251. 106. Poland, A. and E. Glover. 1974. Comparison of 2,3,7,8-tetrachlorodibenzo-p-dioxin, a potent inducer of aryl hydrocarbon hydroxylase, with 3-methylcholanthrene. Mo£ec, Pkasunacol. 10:349-359. 107. Poland, A., E. Glover and A.S. Kende. 1976. Stereo-specific, high affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin by hepatic cytosol. J. Etc. Chem. 251 (16):4936-4946. 108. Poland, A. and A. Kende. 1976. 2,3,7,8-tetrachlorodibenzo-pdioxin: environmental contaminant and molecular probe. fe,d. Vtioc.. 35(12): 2404-2411. 109. Putnam, J. and K.D. Courtney. 1976. Metabolic studies with TCDD (Dioxin) treated rats. Tox^cot. App£. ?haJuna.c.ol. 31(1):170. 110. Radeleff, R.D. 1970. Herbicides, desiccants, plant growth regulators and fungicides. P 264-297. Jji^ Veterinary Toxicology. Lea and Febiger, Philadelphia PA. 111. Rip, J.W, and J.H. Cherry. 1976. Liver enlargement induced by the herbicide 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). J. \gtUc.. food Ckm. 24(2):245-250. 112. Roll, R. 1971. Studies of the teratogenic effect of 2,4,5-T in mice. Food CaAm<Lt. Tox^cot. 9(5) :67l-676. 113. Roll, R. 1973. Toxicological evaluation of special organochlorinated compounds. Enu^Aon. Qvuti. Sa^. 2:117-124. IV-84 �114. Rose, J.Q., J.C. Ramsey, T.H. Wentzler, R.A. Hummel and P.O. Gehring. 1976. The fate of 2,4,7,8-tetrachlorodibenzo-p-dioxin following single and repeated oral doses to the rat. TOJO.CO£. App£. Phasunacot. 36:209-226. 115. Rowe, V.K. and T.A. Hymas. 1954. Summary of toxicological information of 2,4-D and 2,4,5-T type herbicides and an evaluation of the hazards to livestock associated with their use. AmeA. J. Rei. 15: (57)622-629. 116. Sadykov, R.E., V.K. Rabochev and Yu. N. Strokov. 1972. The effect of 2,4-DB used for the treatment of pastures on the reproductive function of sheep. IkivotnovodAtvo 34(2):73-74. (Russian) 117. Sauerhoff,. M.W., W.H. Braun, G.E. Blau and P.O. Gehring. 1976. The dose-dependent pharmacokinetic profile of 2,4,5-trichlorophenoxyacetic acid following intravenous administration to rats. Toxxco£. App£. PkaAmac.o£.. 36:491-501. 118. Schiller, K. 1964. Beeinflussung der Fertilit'at von Ratten durch Stoffe mit Sonderwirkung im Kartofelbau. 14(2):111-114. (German). 119. Schillinger, J.I. 1960. Hygienische Wertung von landwirtschaftlichen Erzeugnissen, angebaut unter Anwendung von Herbiziden. J. Hyg. EpUe.miol. 4:243-252. (Czech). 120. Schulz, K.H. 1968. Zur Klinik und Atiologie der Chlorakne. Arbeitsmed. Soz-uLtmed. M.b<i£ti>hyg. 3:25-29. (German). 121. Schwetz, B.A., J.M. Morris, G.L. Sparschu, V.K. Rowe, P.J. Gehring, J.L. Emerson and C.G. Gerbig. 1973. Toxicity of chlorinated dibenzo-p-dioxins. EMUAOPI. Heo£#i Pmpeet. 5:87-99. 122. Schwetz, B.A., G.L. Sparschu, P.J. Gehring. 1971. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and esters of 2,4-D on rat embryonal, fetal and neonatal growth and development, food CoAmnt. To?o.c.o£.. 9:801-807. 123. Seabury, J.H. 1963. Toxicity of 2,4-dichlorophenoxyacetic man and dog. M.ch. EmuAon. HeotCfc. 7:202-209. acid for 124. Seiler, J.P. 1973. A survey on the mutagenicity of various pesticides. Expe*x.mtui 29:622-623. 125. Shavgulidze, M.M., V.I. Nanobashvili and M.N. Mirianashvili . 1976. Toxicity of the herbicide 2,4-D. -Vzt<iru.naMycL 4:103-104. (Russian). 126. Shirasu, Y. 1975. Significance of mutagenicity testing on pesticides. Envlton. Qual. Satf. 4:226-231. 127. Shirasu, Y., M. Moriya, K. Kato, A. Furuhashi and T. Kada. 1976. Mutagenicity screening of pesticides in the microbial system. Mutation R&6. 40:19-30. IV-85 �128. Smith, F.A., B.A. Schwetz and K.D. Nitschke. 1976. Teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in CF-1 mice. Tox^col. App£. 38:517-523. 129. Sokolik, I. Yu. 1973. Effect of 2,4,5-trichlorophenoxyacetic acid and its butyl ester on embryogenesis of rats. Byu£t. Efe^p. &to£. Med. 76(7):90-92. (Russian). 130. Sparschu, G.L., F.L. Dunn, R.W. Lisowe and V.K. Rowe. 1971. Study of the effects of high levels of 2,4,5-trichlorophenoxyacetic acid on fetal development in the rat. Food Co4me£. To3u.co£. 9:527-530. 131. Sparschu, G.L., F.L. Dunn and V.K. Rowe. 1971. Study of the teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat. Food Commit. To3u.eo£. 9:405-412. 132. Stupnikov, A. A. 1966. Comparative toxicity of chemicals and mineral fertilizers. Translations on USSR Agr. No. 163, U. S. Dept. of Commerce, Joint Publications Research Service, Lesnoye Khozyaystvo 7:29-31. 133. Styles, J.A. 1973. Cytotoxic effects of various pesticides -in vivo and In vi&io. Matat. Re4. 21(1):50-51. 134. Thigpen, J.E., R.E. Faith, E.E. McConnell and J.A. Moore. 1975. Increased susceptibility to bacterial infection as a sequela of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Infect. Immun. 12:1319-1324. 135. To'th, K. , J. Suga'r, S. Somfai-Relle and J. Bence. 1977. Carcino bioaAAay o^ the, keA.bi.cide., 2,4,5-tnic.h£oiaphe.nox.yy<ithanol (TCPE) with cU.MeAe.nt 2,3,7,8~teJ^chZaKa<iib<inzo-p-<LiQ.iu.n (cU.o\in) c.onte.nt. in SMU>& mice. \r\_ International Conference on Ecological Perspectives on Carcinogens and Cancer Control. Cremona, 1976, Basel, Karger AG (In press). 136. Van Miller, J.P., J.J. Lalich and J.R. Allen. 1977. Increased incidence of neoplasms in rats exposed to low levels of 2,3,7,8tetrachlorodibenzo-p-dioxin. ChwoAph&ie. 9:537-544. 137. Van Miller, J.P., R.J. Marlar and J.R. Allen. 1976. Tissue distribution and excretion of tritiated tetrachlorodibenzo-pdioxin in non-human primates and rats. Food Cotmet. Toxicol. 14:31-34. 138. Vinopal, J.H. and J.E. Casida. 1973. Metabolic stability of 2,3,7,8-tetrachlorodibenzo-p-dioxin in mammalian liver microsomal systems and in living mice. M.oh. Emu/ton. Contain. Tox*cco£. 1:122-132. IV-86 �139. Vos, J.G. and J.A. Moore. 1974. Suppression of cellular immunity in rats and mice by maternal treatment with 2,4,7,8-tetrachlorodibenzo-p-dioxin. Int. M.ch. MZeAgy App£. Immanol. 47:777-794. 140. Vos, J . G . , J.A. Moore and J . G . Z i n k l . 1974. Toxicity of 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) in C57B1/6 mice. Toxico£. App£. PhaAmacol. 29:229-241. 141. Walker, E . M . , Jr., R . H . Gadsden, L . M . Atkins and G . R . Gale. 1972. Some effects of 2,4-D and 2,4,5-T on Ehrlich ascites tumor cells Jun VM.VO and <in \jWio. Ind. Mecf. 41(l):22-27. 142. Weissberg, J.G. and J.G. Zinkl. 1973. Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin upon hemostasis and hematologic function in the rat. Environ. Health Pmpea*. 5:119-123. 143. World Health Organization. 1971. 1970 Evaluations on some pesticide residues in food. The Monographs. WHO/Food Add./71.42, pp 459-477. 144. W i l s o n , J . G . 1977. Envvtomzntal c.kzm.c.atb . P 357-386. In_ Handbook of Teratology. J . G . Wilson and F . C . Fraser ( E d s . ) . Vo-1 . 2. Plenum Press, New York. 145. W i l s o n , J . G . 1977. Teratogenic effects of environmental chemicals. Fed. Ptoc. 36 (5): 1698-1 703. 146. Zetterberg, G. 1977. Experimental results of phenoxy acids on microorganisms. ln_ Chlorinated Phenoxy Acids and their Dioxins: Mode of Action, Health Risks and Environmental Effects. C. Ramel , ( E d ) , Ecol. Bull. (Stockholm), 27: ( i n press). 147. Z i e l i n s k i , W . L . , Jr. and L. Fishbein. 1967. Gass chromatographic measurement of disappearance rates of 2,4-D and 2,4,5-T acids and 2,4-D esters in mice. J. Ag/u.c. food Cfiem. 15:841-844. 148. Z i n k l , J . G . , J . G . Vos, J . A . Moore and B . N . Gupta. 1973. Hematologic and clinical chemistry effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in laboratory animals. Env&ion. H&altn PeAApe,c.t. 5:111-118. IV-87 �CHAPTER V 2,4,5-T/TCDD EPISODES I. INTRODUCTION The current controversy over the potential adverse human effects of 2,4,5-T and TCDD stem from a chain of events that occurred in the 1960s. The presence of TCDD as a contaminant, potent acnegen and acute toxin in the production of 2,4,5-trichlorophenol was documented in 1957 by Kimmig and Schulz (58). However, it was not until 1964 that concern over the levels of TCDD in 2,4,5-T herbicide was reported. In that year, the Dow Chemical Company experienced contamination problems during its expansion in production of 2,4,5-T to meet the requirements for Herbicide Orange by the U.S. military. They closed their production facilities and made extensive modification in the reaction conditions for the synthesis of trichlorophenol. By late 1965, the new technology developed by Dow Chemical Company permitted production of 2,4,5-T containing no more than 1 ppm TCDD (36). Simultaneously, in 1964, the National Cancer Institute contracted for a screening study of a number of pesticides to determine if they were tumorigenic, teratogenic or mutagenic. Among the pesticides evaluated was 2,4,5-T herbicide. By 1967-68, preliminary data on 2,4,5-T from this screening study indicated that 2,4,5-T was teratogenic (15). The data were apparently provided to the press prior to actual publication. [When the manuscript eventually appeared in the scientific literature, in 1970, it contained a footnote indicating that the original sample of 2,4,5-T used in the screening tests contained approximately 30 ppm TCDD (23).] The press releases in the U.S. on the teratogenicity of 2,4,5-T were occurring in the same time period that South Vietnamese newspapers were publishing reports of an alleged increased occurrence of birth defects in areas sprayed with Herbicide Orange. These releases elicited far-reaching reactions from governmental agencies, segments of the scientific community and various lay groups concerned with environmental problems (2). On October 29, 1969, the President's Scientific Advisor announced that a series of coordinated actions was being taken by several governmental agencies to restrict the use of 2,4,5-T herbicide. Additional animal experiments performed early in 1970 confirmed that pregnant mice did deliver some malformed offspring. The question then was one of whether, or to what extent, such animal data could be extrapolated to man. On April 14, 1970, the Secretary of Health, Education and Welfare (HEW) advised the Secretary of Agriculture that: "In spite of these uncertainties, the Surgeon General feels that a prudent course of action must be based on the decision that exposure to this herbicide may present an imminent hazard to women of child-bearing age." Accordingly, on the following day, the Secretaries of Agriculture; HEW, and Interior jointly announced the suspension of 2,4,5-T for "all uses around the home, recreation areas, and similar sites" and "all uses on crops intended for human consumption." Immediately thereafter, the Department of Defense suspended the use of Herbicide Orange in South Vietnam (7). V-l �Numerous incidents involving suspected 2,4,5-T/TCDD poisoning of humans or livestock have been reported since this initial controversy. The most recent alleged episode involved veterans of the Vietnam Conflict. In March 1978, WBBM, a CBS-owned television affiliate in Chicago, Illinois, aired a special report on "Agent Orange: Vietnam's Deadly Fog". In the film, a number of past episodes allegedly involving 2,4,5-T and TCDD were examined. This chapter will review the available scientific data on these and other episodes, including industrial episodes, assessments in South Vietnam and the incident that occurred in Seveso, Italy, in July 1976. The medical data on many of these episodes are reviewed in Chapter VI. The expression of units of weight, area, or volume has not been standardized between the various publications cited in this Chapter. II. INDUSTRIAL EXPERIENCES A. Industrial Processes The herbicide, 2,4,5-T, was first commercially produced in the United States in 1944 (79). The quantity o'f 2,4,5-T produced and used in the United States and in world agriculture increased steadily until 1968-69, after which a sharp decline in its use occurred. Table 1 shows total U.S. Production data for 2,4,5-T, and how it was subsequently used, during the period 1961 through 1969. Approximately 34 percent (53 million pounds) of the total U.S. production was procured by the Department of Defense for use in South Vietnam. However, 8.9 million pounds of the 53 million pounds were not sprayed in South Vietnam, but rather destroyed by at-sea incineration in 1977 (see Chapter II). During the same period, 1961 through 1969, 50.6 percent (78.1 million Ib) of the total U.S. 2,4,5-T production was used in domestic herbaceous and woody plant control programs. The synthesis scheme for the industrial production of 2,4,5-T herbicide is shown in Figure 1. Forth (40) has described two different processes for the manufacture of the herbicide. The "Dow" process is a pressureless, high temperature process (>160°C but <200°C) requiring the alkaline hydrolysis of 1,2,4,5-tetrachlorobenzene to sodium trichlorophenate in the presence of ethylene glycol (an alcohol) and caustic soda (e.g., sodium hydroxide). The second process, the "Boehringer" process uses high pressure (19.5 atmospheres) but low temperature (157°C) conditions in the presence of methanol, caustic soda, and 1,2,4,5-tetrachlorobenzene. Both processes will result in the formation of sodium trichlorophenate. The sodium trichlorophenate can be acidified to form trichlorophenol or may be used directly in the production of 2,4,5-T by adding chloroacetic acid. The production of the n-butyl ester (NBE) of 2,4,5-T is accomplished by V-2 �TABLE 1. Total United States production and use of 2,4,5-T herbicide for the period 1961 through 1969.a Use Million Pounds Herbicides Green, Pink and Purple^ Percent of Total 1.6 1.04 Herbicide Orange0 51.4 33.27 Exports 23.4 15.15 Domestic Use 78.1 50.55 154.5 100.01 Total a Total production and export data were from The Pesticide Review, 1970 and earlier issues, U.S. Department of Agriculture, Agricultural Stabilization and Conservation Service, U.S. Government Printing Office, Washington D.C. Data expressed in acid equivalents. Data based on estimated number of gallons of Herbicides Green, Pink and Purple used in South Vietnam, 1962-1964. c Data based on estimated number of gallons of Herbicide Orange used in South Vietnam (10,645,904 gallons) plus the surplus 2,215,125 gallons remaining after termination of Operation RANCH HAND. V-3 �methanol or ethylene glycol caustic in«0 / socja Cl' " X "Cl 1 c n O - 180° C \" 160° 1,2,4,5-tetrachlorobenzene - where R = CH or OUCH Chloroacetic acid,^ sodium hydroxide sodium trichlorophenate > 230°C 0 0-CH^C-OH 2,4,5-T butanol, anhydrous hydrochloric acid 0-CH2C-0-CH2CH2CH2CH3 Cl n-butyl ester 2,4,5-T FIGURE 1. Synthesis scheme for production of the n-butyl ester 2,4,5-T (NBE 2,4,5-T) and site where formation of TCDD may occur. V-4 �esterification using butanol and anhydrous hydrochloric acid. TCDD is formed only, during the formation of the phenol. Dimerization of the sodium trichlorophenate to form TCDD will occur in the reaction vessel during the alkaline hydrolysis of 1,2,4,5-tetrochlorobenzene. Maintaining low temperatures, J160°-'I800C, will minimize the formation of TCDD. The reaction temperatures during the "Dow" process may become difficult to maintain. If the temperature of the hydrolysate rises above the normal ,180°C, an exothermic reaction occurs after any residual solvent, e.g., glycol, is removed by distillation. This reaction, attributed to the decomposition of sodium-2-hydroxethoxide, starts at a temperature of 230°C and continues to 410°C. The heat generated by this reaction assists in the formation of TCDD through the dimerization of two molecules of sodium trichlorophenate. The rapid temperature increase in the reaction vessel, results in a pressure increase; failure to release the pressure has resulted in some of the industrial accidents that have been reported (46). B. Industrial Episodes In the years since the first commercial production of 2,4,5-T herbicide (1946-47), there have been numerous industrial episodes involving exposure to TCDD (and/or other chlorinated dibenzo-p-dioxins). The exposure to TCDD normally occurred during the handling of contaminated intermediate products (e.g., trichlorophenol, TCP). Fifteen of 23 episodes recorded in the literature were apparently associated with this "occupational" exposure. However, on eight occasions, explosions occurred, generally during the production of sodium trichlorophenate, and personnel were exposed to TCDD at the time of the accident, during the clean-up of the accident or from subsequent contamination of the workshop environment. The first reported industrial accident occurred in Nitro, West Virginia, in 1949 (51). A total of 228 people were poisoned by the reactor residue during and/or immediately after the accident. No measures were taken to decontaminate the factories or to control the residue from the reaction vessel. In 1953, an accident occurred at a TCP factory in Ludwigshafen, Federal Republic of Germany (43). The 55 workers that showed chloracne and other acute toxic effects were exposed to the residue of the reaction vessel either during the accident or in the subsequent clean-up work. By 1957, Kimmig and Schulz (55) implicated TCDD as the causative agent of at least the chloracne seen in these workers. The most thoroughly documented episode of occupational exposure has been by Oirasek et al (54,55) and occurred in Czechoslovakia between 1965 and 1969. In 1965, two technicians developed chloracne during the evaluation of a new production process for the manufacture of 2,4,5-T. At the time, it was assumed that they were exposed because of careless work and defective equipment under the pilot plant conditions. During the following two years, after the plant became operational, an additional V-5 �76 people developed chloracne. An investigation and study of the entire problem finally resulted in a shutdown of the operation and abandonment of the production in 1968. Jirasek et al have carefully described the afflicted individuals and have continued to monitor their health since the onset of the disease. The above two episodes and other episodes involving occupational chloracne associated with the manufacture of chlorinated phenols are presented in Table 2. The clinical features of the affected cases described in the 23 episodes listed in Table 2 are described in Table 3. Note that most features observed were inconsistent with the exception of chloracne. Certainly, extended exposure to the major chemicals [TCP, pentachlorophenol (PCP), 2,4-D or 2,4,5-T] must have complicated the observed clinical features. In addition to TCDD, it should also be noted that TCP may also contain significant concentrations of hexachlorodibenzop-dioxin, while PCP may contain hexa-, hepta- and octachlorodibenzo-p-dioxin. An extensive review of occupational chloracne has been prepared by Crow (24) and Kimbrough (56). With one exception, men have been almost exclusively affected by the disease due to the occupational position they have historically maintained in the factories producing the chlorinated phenols. The few women and children that have been afflicted were exposed because of contact with clothing worn by the men. Goldmann (43) reported that a female animal nurse developed chloracne by contact with contaminated test animals. The one industrial incident where a large number of women were afflicted with chloracne was reported by Braun (17) in 1959. Braun examined 114 women and 9 men, who in the course of a year became afflicted with chloracne in a condenser factory where chloronaphthalenes of different degrees of chlorination were used as dip-waxes. The reporting of this incident is important because of contradictory statements in the literature on the differential sensitivity of different people (including^-sexes) to chloracne. From the examination of the women Braun concluded the following: 1. Age was of no importance within the range of 20 to 45 years. 2. Strongly adiposed persons were more likely to be afflicted. 3. Seborrhoic types with greasy skin and open pores and scars of previous Ac.ne vutg<wit> were sooner and more severely afflicted. 4. Non-affliction was definitely extremely rare under the circumstances. Only four women (2 of them sisters) with a very smooth and fine skin through which the veins showed bluish when they were at rest remained free from the alterations due to the disease. 5. The occurrence of chloracne had no real relationship to hair color and skin pigmentation. V-6 �TABLE 2. Industrial incidents Year Country associated with the manufacture of chlorinated phenols. Primary Production Source of Manufacturer/Location3 Productr Exposure Monsanto/ Nitro, West Virginia . / / Nordrhein, Westfalen / / TCP Explosion PCP, TCP 1952- West Germany 53 1953 West Germany Years from Number Incident to of Last Cases Observation0 Reference 228 4 Occupational 17 1 TCP Occupational 60 12 DUcrir 1 liyt:i / TCP Occupational 37 46 BSAF/ Ludwigshafen TCP Explosion 55 24 51, 43 Boehringer, Ingleheim/ Hamburg TCP, 2,4,5-T Occupational 31 9 58, 12 1956 France Rhone Poulenc/Grenoble TCP Explosion 17 2 30 1956 United States Diamond Alkalai/ Newark, New Jersey 2,4-D, 2,4,5-T Occupational 29 13 1956 United States tlUUKci/ TCP Occupational (?) 46 1960 United States Diamond TCP Occupational (?) 46 1949 United States 1949 West Germany 1952 West Germany 1954 West Germany Ch amv»ni~ \r t 51, 73 11 16, 68 �Table 2 (continued) 1962 Italy 1963 Netherlands 1964 USSR 1964 United States / 5 TCP Explosion Philips-Duphar/ Amsterdam / / TCP Explosion ' 50 2,4,5-T Occupational 128 Dow Chemical/ Midland, Michigan 2,4,5-T Occupational 60 6 38 TCP Occupational 78 6 54, 55 67 1965- Czechoslovakia Soolana/ 69 Rhone Poulenc/ Grenoble 47, 51 14 14, 26 51 50, 74 1966 France TCP Explosion 21 1968 United Kingdom Coalite and Chemicals TCP Products/ Bolsover, Derbyshire Explosion 79 9 51, 60 1970 Japan PCP, 2,4,5-T Occupational 25 3 64 1972 USSR TCP Occupational 1 1 81 2,4,5-T Occupational 50 40, 46 00 1973 Austria / / Linz Nitrogen 46 Un i-l-i: / worKs/ 1974 West Germany Bayer/Uerdingen 2,4,5-T Occupational 5 40, 46 1975 United States Thompson-Hayward/ TCP Occupational - 46 Kansas City, Kansas �Table 2 (continued) 1976 Italy ICMESA/Meda TCP Explosion 134 2 69, 80 a The name of the factory or company and its location was cited whenever it was available because considerable confusion exists in the published literature as to what incident is addressed. The absence of data indicates the information was not available. b TCP = trichlorophenol, PCP = pentachlorophenol Frequently individuals involved in an incident, and who were examined initially, may have also been examined at a later date. The years that lapsed from the exposure until the most recent examination are cited in this column. The absence of a number indicates that only an initial examination was reported in the referenced literature. �TABLE 3. Some clinical features observed in cases of chloracne associated with production of 2,4,5-T and other chlorinated phenols. Frequency observed Clinical Features Consistent Chloracne Additional Notes In worst cases, chest and inguinal area affected and scarring generally increased. Occasional Prophyria cutanea tarda Increased excretion of urinary uroporphyrin or coporporphyrin or both. -^ o Inconsistent Hyperpigmentation of the skin Usually prominent on face and consisted of grayish or brownish tone to the complexion. Hirsutism Noticeable between the outer edge of the eyebrow and the temple hair margin. Enlarged, tender liver Excessive mechanical fragility of the skin Neuromuscular symptoms Severe pains in the chest and pain and weakness in extremities �Table 3 (Continued) Mucous membrane irritation Itching of the eyes and frequent tearing, hyperemia of the nasal mucosa, and inflammation of the buccal mucosa. Irritability Nervousness and insomnia. �6. A simultaneous psoriasis or a systemic eczema in the previous case history, or a pregnancy, did not make any difference and had no perceptible effect on the course of the chloracne. One woman reported that she had noticed a worsening of her chloracne after her delivery. 7. During the menstrual period, the pimples on the cheeks of some of the women were temporarily more prominent. 8. Dirty and untidy women took ill sooner and more severely than those who placed great importance on cleanliness and hygiene. The persistence of dioxins in the environment of an industrial plant has been documented by Jensen (53). He reported on two cases of chloracne in employees of an outside contractor that had been working on a piece of equipment exposed (but thought to have been decontaminated) to TCDD three years earlier in an industrial explosion in Derbyshire, England in 1968. A young son of one of these employees also develeoped chloracne. The presumed source of the child's contamination was the father's working clothes. An episode involving the deliberate synthesis of TCDD has been reported by Oliver (65). This episode involved three young (male) scientists working with pure TCDD in the laboratory. Two of the men were exposed to the dioxin while attempting to synthesize it by heating trichlorophenol in an alkaline solution in the presence of a catalyst or by heating prepared potassium trichlorophenate in a closed system. Both men wore overalls and plastic gloves and allegedly took the utmost care to avoid inhalation or skin contact. The third man was a colleague of the other men and had been working with the diluted dioxin standards they had prepared. His work also had been done with the utmost caution and with special care to avoid personal contamination. Three clinical features were common to the three men, namely, chloracne, hyperpigmentation and hypercholesterolemia (increased levels of cholesterol). None of the three patients had evidence of acquired porphyria. However, two patients developed hirsutism (excessive facial hair) two years after the exposure. These same two also reported that when the hirsutism developed, other symptoms occurred, e.g., loss of appetite, oppressive headaches, and an unusual loss of vigor and drive with excessive fatigue. Oliver concluded from the evidence that those accidentally exposed to dioxin (TCDD) may be subject to delayed toxic effects for at least two years. III. VIETNAM EPISODE As noted in Chapter I, approximately 53 million Ib of 2,4,5-T were in the 13.2 million (gal) of Orange, Purple, Pink and Green procured by the Department of Defense. However, 8.9 million lb of 2,4,5-T were in the surplus Herbicide Orange. Thus, approximately 44 million pounds of 2,4,5-T were sprayed in South Vietnam from 1962 through 1970. As noted in Chapter I, an estimated 368 lb of TCDD were probably present in Herbicides Orange, Purple, Pink and Qreen. V-12 �Irish et al (52) have stated that among all the controversial subjects that were part of the conflict in Vietnam, the use of vegetationcontrol chemicals received an undue amount of publicity that was generally critical. They noted that the Department of Defense was not insensitive to the critics' pronouncements; justification for continuation of the RANCH HAND program had been periodically reviewed. The conclusions of all the evaluations prior to 1969 recognized that defoliation had reduced the incidence of ambushes, saved lives and disrupted enemy tactics. The issues of long-term ecological damage or potential adverse human health effects due to the herbicides were little discussed until the late 1960s. These issues when viewed in context with the realities of the military conflict were of minor concern, especially since the available scientific data did not support the justification for greater concern. It should be noted that in 1967 the Department of Defense had contracted with the Midwest Research Institute (MRI), Kansas City, Missouri, for an in-depth report on the assessment of ecological effects of extensive or repeated use of herbicides (49). Following its publication in December 1967, both the National Academy of Science (NAS) and the American Association for the Advancement of Science (AAAS) reviewed the document and concluded that MRI had "done a creditable job of assessing the scientific literature related to herbicides and their ecological effects." However, both organizations felt that the report represented "only a first step in investigating further the ecological effects of intensive use of herbicides" (3). Some of the conclusions that the MRI reported (49) included: 1. The greatest short-term or long-term direct ecological consequence of using herbicides in Vietnam or anywhere else is the destruction of vegetation. As long as soil sterilization is not an objective, destruction of vegetation by herbicides is a selective process, denuded earth does not occur especially in forest spraying. Furthermore, the end result of the use of herbicides from an ecological standpoint is that the ecosystem is set back to an earlier sere, i.e., an earlier stage of plant succession. 2. The long-term effects on wildlife may be beneficial or detrimental. Studies in other countries have shown that herbicidal treatment of forested areas improves wildlife habitat and is favorable to animal populations. The extent and pattern of herbicide treatment in Vietnam have no precedent; therefore, it is difficult to predict effects on wildlife with any accuracy. 3. The herbicides used in Vietnam will not persist at a phytotoxic (plant toxic) level in the soil for a long period of time. On the basis of the average temperatures and rainfalls in Vietnam, it would be reasonable to expect that the chlorophenoxy acid esters will be dissipated quickly. 4. The possibility of lethal toxicity to humans, domestic animals or wildlife by use of the herbicides used in Vietnam is highly unlikely and should not be a matter of deep concern. V-13 �5. Herbicides seldom persist in animal or insect tissues. Toxic transfer to the next higher animal in the food chain is minimal. In fact, biological concentration does not occur with most herbicides, since they are readily excreted from animals. In September 1968, the U.S. Department of State released an assessment of the ecological consequences of the defoliation program in Vietnam. Tschirley (75), a plant ecologist and the author of the Department of State report, published his assessment in Sconce (the Journal of the AAAS) in February 1969. The major conclusions reached by Tschirley after his fourweek visit to South Vietnam included: 1. The defoliation program has caused ecologic changes. These changes are not irreversible, but complete recovery may take a long time. Regeneration of the mangrove forest to its original condition is estimated to require about 20 years. 2. The effects of defoliation on animals is not known, but it does not appear to have been extreme. There is no evidence to suggest that the herbicide used in Vietnam will cause toxicity problems for man or animals. In March 1969, the Society for Social Responsibility in Science, sponsored a five-week trip, for two zoologists to Vietnam with the objective of supplementing Tschirley's observations. The subsequent report, written by Orians and Pfeiffer (66), was published in May 1970. Their conclusions included: 1. The ecological consequences of defoliation were severe, especially in areas receiving repetitive applications of defoliants. 2. Evidence was found of moderate to severe defoliation of trees and herbs in areas many miles removed from sites of application. 3. Little evidence of toxic effects of the herbicides to animals was found, although one report was received (through an interview) of many sick and dying birds and mammals in forests following defoliation. The report was not investigated. 4. No evidence was found that the herbicides had direct adverse effects on human health. The defoliation program however, has had tremendous psychological impact upon the Vietnamese people, and the crop destruction program may have impacted on the availability of food for women, children and elderly people in the highland regions of South Vietnam. The first Herbicide Orange and July 5, 1969 reports resulted reports of human birth defects allegedly attributed to appeared in Vietnamese newspapers between June 26, 1969 (2). The public and scientific furor caused by these in two surveys of South Vietnamese hospital records V-14 �conducted independently by Cutting et al (25) and Meselson et al (63). An evaluation of both documents in 1971 by an advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency (2) concluded with the following summary: Summarizing the Vietnam data on human embryotoxicity, it can be said that (1) the sample of births surveyed was from year to year a variable but usually very small fraction of the total number, (2) it was quite unrepresentative of the geographic and ethnic distributions, (3) the heavily sprayed and otherwise exposed areas were greatly underrepresented, and (4) the birth records were not trustworthy and, therefore, the rates of stillbirth, and especially of congenital malfoVmation, derived from them were equally unrealiable. For example, the overall congenital malformation rate found in South Vietnam, 4.91 per 1000 livebirths, is about half of what was reported in other studies in various parts of Asia, and possibly a quarter of what might actually exist at term. A further indication that the newborn children were not carefully examined is the absence of Down's syndrome in the list of specific malformations compiled by the Army survey [Cutting et al (25)] despite the fact that some Oriental populations have been reported to have an incidence of this condition not unlike that in Western populations. Finally there is, and can be, no precise knowledge or reasonable approximation of the exposure to 2,4,5-T (and hence, TCDD) experienced by pregnant Vietnamese women, including what amounts they ingested or absorbed and when this may have occurred during pregnancy. Thus, any attempt to relate birth defects or stillbirths to herbicide exposure is predestined to failure. It can only be concluded that the birth records that have been surveyed, and probably any that will be surveyed in the future, for South Vietnam for the period 1960-1970 cannot answer positively the questions about possible adverse prenatal effects following human exposure to 2,4,5-T. It must be emphasized, however, that the searches that have been made almost certainly would have revealed any marked increase in the incidence of birth defects or the introduction of a striking defect such as that produced by thalidomide. In spite of considerable effort, no such occurrences were found. Following the publication of the above two surveys, some additional reports of birth defects in South Vietnam were released. One of these was by Tung et al (77) of North Vietnam (Democratic Republic of Vietnam). They reported that out of a total of 903 South Vietnamese taking shelter in the North and grouped in hospitals and lodgings in Hanoi, 19 adult women, including 4 mothers, and 70 children between the ages of 6 and 14 had been directly hit by herbicidal sprays while living in South Vietnam. The report went on to state that of the above four mothers, two had given birth to children with Down's Syndrome (Trisomy 21). In addition, among V-15 �the 70 children between the ages of 6 and 14, numerous cases of deformations were evident, e.g., ocular lesions, exaggerated lumps on the forehead, valgus feet (i.e., feet that are bent outward) and a high frequency of chromosomal aberrations in lymphocytes and leucocytes. Following a summary of their data, Tung'et al (77) concluded by stating: Though still limited in number, our clinical observations confirm the results obtained on animals by American researchers. The massive and prolonged utilization of defoliants besides permanent ocular lesions, can cause chromosomic alterations among a population obliged to cling to ancestral soil and these alterations can provoke among* their progeny congenital malformations the importance of which remains to be determined. In the abominable history of wars, have we ever seen such an inhuman fate reserved for the survivors except in the case of atomic war? In reviewing the report by Tung et al (77), the Dow Chemical Company (8) noted that basically, the whole study was a result of a seemingly hit and miss clinical examination of some refugees from South Vietnam who had lived in regions where defoliants had been applied. There was no record of exposure except that most had been sprayed at one time or another with something. Dow further stated: "Trying to correlate cause and effect from the published data is completely frustrating and futile. There is no doubt that these authors saw some ill people, but to reach the conclusion that their problems were caused by 2,4,5-T rather than the ravages of war is speculation." A study similar to Tung et al (77) was reported by Rose and Rose (71) in 1972. They interviewed 98 refugees in Hanoi who claimed to have been repeatedly sprayed with defoliants while in South Vietnam. Abortions were reported for humans and domestic animals and monstrous births were said to have occurred. Deaths evidently occurred among human, domestic animals, fish and fowl. The charge by Tung et al that TCDD in 2,4,5-T was reponsible for much of the Down's Syndrome seen in South Vietnam was also made by Grumner as reported by Honoroff (48). Grumner, apparently of Rockstock University, German Democratic Republic, claimed to have observed high incidences of children with Down's Syndrome while on a trip through North and South Vietnam. Honoroff quoted Grumner: Provided that it be understood that this estimate cannot be anything but a cautious one, it may be assumed that there are at least 25,000 children with hereditary defects in South Vietnam. This does not include all the unborn babies whose mothers were sprayed during the missions that were flown in recent months. It does not include those who were stillborn, or died soon after birth, on account of their serious chromosomatic defects. Even V-16 �after the war, it will probably be possible to arrive at only an approximation of the entire scale of this crime since one will only be able to examine the survivors when the time comes. In 1973, Tung et al (78) reported an increase in the number of persons with primary liver cancer in proportion to all cancer patients admitted to Hanoi hospitals during the period 1962-1968 (790 liver cancer cases out of 7,911 cancer cases, 10 percent) as compared to the period 1955-1961 (159 liver cancer cases out of 5,492 total cancer cases, 2.9 percent), which was prior to the start of herbicide spraying. The authors attributed this increase to exposure as a result of the spraying of herbicides containing TCDD in South Vietnam during the 1960s [however, a recent IRAC monograph (50) noted that limitations in the reporting of the study make impossible an adequate assessment between the incidence of liver cancer and herbicide spraying in South Vietnam]. A further factor of importance has -been suggested by Ford et al (39). They noted that at least in Thailand, consumption of aflatoxin-contaminated food was highly correlated with liver cancers. Aflatoxin is a naturally occurring contaminant of cereal crops. In 1974, the National Academy of Science (NAS) (21) announced the results of studies conducted in South Vietnam in 1972 and 1973. The NAS Committee could find no conclusive evidence of association between exposure to herbicides and birth defects in humans. Available records of two major Saigon hospitals and evaluation of records in a third, as far as they went, showed no consistent pattern of association between rates of congenital malformations and annual amounts of herbicides sprayed. The Committee recognized, however, that the material was not adequate for definite conclusions. The Committee was also unable to confirm or deny reports that some humans (especially the Montapnards) and domestic animals became ill or died after exposure to herbicide sprays or after eating treated plants or drinking contaminated water. The Committee also attempted to assess the social, economic and psychological effects of the herbicide program. The impact of the program on the population "appeared relatively trivial as compared with other aspects of the upheaval in that country." Evidence was obtained that numbers of families moved away from their traditional homes because of the herbicide spray program but few were actually identified. In a letter of transmittal for the NAS report (21), the President of NAS stated: "On balance, the untoward effects of the herbicide program on the health of the South Vietnamese people appear to have been smaller than one might have feared." IV. EASTERN MISSOURI HORSE ARENA EPISODE In August 1972, the Missouri Division of Health, St. Louis, Missouri, and The Center for Disease Control, Atlanta, Georgia (59) reported an investigation of a horse arena in eastern Missouri where 54 of 57 horses exposed to the arena had died of an illness characterized V-17 �by skin lesions, severe weight loss and heptotoxicity. Birds, dogs, cats, insects and rodents were also found dead in and around the arena, and one 6-year-old girl exposed developed epistaxis, gastrointestinal complaints, and severe hemorrhagic cystitis (characterized by blood in the urine). Analysis of urine cultures for bacterial and viral pathogens was negative. Three other persons developed milder illnesses consisting primarily of transient headaches and nausea after exposure to the arena. The toxic substance(s) responsible for the illness was not at that time identified. In the investigation of the illness, Lobes et al (59) found that the outbreak coincided with treatment of the arena floor for dust control with approximately 2,000 gal of salvaged motor oil. The treatment occurred on May 26, 1971. On May 30, the stable owners reported that "hundreds" of birds were found dead on the floor of the arena barn. Within the next few weeks, cats, dogs, rodents and horses began to die. The four people cited above [2 adults and 2 children (both girls)] had more than occasional exposure to the arena barn during the six months following the oil spraying. These individuals were first examined in mid-August 1971, The report (59) also noted that similar horse illnesses and deaths occurred in two other horse arenas in the eastern Missouri area sprayed by the same salvage oil company. The three arenas had been sprayed within one month of each other. Subsequent to investigation, soil from all three arenas was excavated and disposed. No further problems occurred following the excavations. In 1974, laboratory analysis of soil samples taken from the initial arena implicated 2,4,5-trichlorophenol (TCP) and TCDD as the probable toxic substances (29). The actual levels of TCDD in these soils however were not published until 1975, when Carter et al (19) provided more details on the exposure and the probable source of the TCDD in the salvage oil. The horse arena soil was found to contain 31.8 to 33 yg of TCDD per gram (ppm) of soil. In addition, further investigations revealed that the sludge used to spray all three arenas came from a common storage tank at the salvage oil company. It was suspected that TCDD and TCP were in distillate residues collected by the salvage company from a hexachlorophene producer in southwestern Missouri. Between February 1971 and October 1971 the salvage oil company obtained and stored 18,000 gal of the distillate in a storage tank from which the sludge for spraying the three arenas was obtained. In late 1971 the hexachlorophene plant and subsequently the salvage oil company both discontinued operations. The residue remaining in the tank originally used to store the distillate residue at the plant site was sampled in 1974. It contained TCDD in concentrations of 306 to 356 yg/g (19). Case (20) has described some of the clinical studies performed on the horses involved in this episode. Kimbrough et al (57) has recently (1977) detailed the epidemiology and pathology associated with the poisoning episode. V-18 �Commoner and Scott (22) have reviewed the Missouri Horse Arena Episode in an attempt to provide consultative data to the Italian Government in the wake of the Seveso, Italy episode. Their,review focused on the human reactions (symptoms) to accidental TCDD exposure and the problem of soil degradation of TCDD. They also provided an excellent chronological account of the episode. Beale et al (13) have recently re-examined the young girl who had developed hemorrhagic cystitis following repeated exposure to TCDD in one of the horse arenas sprayed with the waste oil. In the 5-year interval since exposure, the patient had grown normally, and both her height and weight were above the 75th percentiles. Detailed physical, chemical and neurological examinations were also conducted and found to be normal. The same studies were done on the patient's sister and mother, exposed simultaneously, but less extensively to dioxin, and the results were also normal. Beale et al (13) concluded: "Our experience demonstrates that people exposed to dioxin can recover completely with no apparent sequela from the toxin. It remains to be determined whether -the exposure to dioxin in these children will result in abnormal pregnancies or affect their offspring." V. THE SEVESO, ITALY EPISODE Perhaps the most publicized chemical accident in modern times is the TCDD episode in Seveso, Italy. This episode has attracted worldwide interest and concern. Hundreds of scientists, physicians and veterinarians have participated in either on-site inspections, conferences, or consultations into the various facets of this episode. Although the Seveso, Italy episode did not involve 2,4,5-T herbicide, it did involve the production of trichlorophenol. The trichlorophenol was in this case used in the production of hexachlorophene. Nevertheless, this episode represents to many people the inherent danger associated with the industrial production of 2,4,5-T. Data on levels of TCDD found, the magnitude of the contamination and the extent of human and animal illness have just recently begun to appear in the scientific literature. The following scenario of the episode has been assembled from this literature. The episode of TCDD poisoning occurred on 10 July 1976 in Seveso, Italy, a small town 40 kilometers (km) north of Milan (40,46). The source of the TCDD was a chemical factory that produced trichlorophenol through the alkaline hydrolysis of tetrachlorobenzene (see Figure 1). When the temperature in a steam-heated reaction vessel rapidly increased, a safety disk ruptured sending a plume of trichlorophenol, TCDD and other products 30 to 50 meters (m) high above the factory. The cloud apparently rose into the air, cooled and came down over a cone-shaped area about 2 km long and,700 m wide. V-19 �The chemical plant involved was the Givaudan ICMESA (Swiss-owned) chemical plant. At the time of the incident, there were some 2,000 kg of reagents and reaction products in the reactor (sodium trichlorophenate, soda, sodium chloride, ethylene glycol, tetrachlorobenzene and secondary reaction products) (9). Based on determinations made by production officials it was estimated that 4,000-500 kg of reaction product was discharged into the atmosphere (9,27). The amount of TCDD dispersed with the other reaction products has been estimated to have ranged from 650 grams to 1,700 grams (27,69). A sample of the escaped product taken from the reactor head for analysis revealed the presence of 3.5 percent TCDD (35,000 parts per million TCDD) (9). The accident occurred on a Saturday. By the following Monday, a site inspection of the area revealed phytotoxic effects (brown discoloration and drop-like perforation of the leaves) for a distance of some 1,000-1,300 m in a triangle with a base of approximately 400 m and a vertex of 100 m centered on the factory (9). Several measurements of TCDD on vegetation in this area and areas adjacent to the factory were in the 1 to 15 ppm range, with one reading as high as 50 ppm (69). • Reggiani (69) reported that animals (birds, rabbits and chickens) were beginning to die 2-3 days after the accident. A few children and some adults who had been directly seized by airborne dust consisting of the reactor content were complaining of nausea and presenting skin lesions of various aspects and extension but mainly redness and swelling. Some of the children were hospitalized and the physicians in charge warned that beyond overt signs of injury pointing to the action of caustic material causing burns and blister formation, they also had to consider the possibility of a contact or ingestion of a still unknown quantity of TCDD. In the meantime numerous Italian laboVatories and the Givaudan Laboratories cooperated in mapping out the polluted zones, determining TCDD on soil, vegetation and buildings by gas chromatographic-mass spectrometric techniques (9,41,69). In addition, the Regional Veterinary Service assisted in drawing up the map, working from animal death patterns and TCDD levels in the liver of surviving animals. Highest TCDD levels were found in herbivorous animals (41). About 1,000 assays led to the area being divided into two zones. Giovanardi (42) reported that the first zone, Zone A, was a triangularshaped area covering approximately 1000 hectares (ha). This area, located south south-east of the ICMESA factory and downwind at the time of the accident, had estimated soil levels of TCDD greater than 0.001 ppm [Reggiani (69) later described this area as having TCDD levels greater than 10 ppb]. The 700 inhabitants of this area were evacuated in three stages, on 26 July, 28 July and 2 August 1976. In the second zone, Zone B, soil levels of TCDD were detectable but less than 0.001 ppm [Reggiani (69) defined the soil levels of TCDD as between 0.1 and 10 ppb]. This area covered approximately 250 ha and was divided between a large urban center and an extensive rural area with some small residential V-20 �aggregates (42). This area had a population of 4,900 and was not evacuated. For the people in Zone B, recommendations were issued to reduce the possibilities of exposure in particular for the children and the women (69). A third zone, Zone C or "Respect Zone," covering a total of about 1,430 ha was also delineated. Occasional concentrations of less than 0.1 ppb TCDD in soil were found in this zone. The population of this area was approximately 40,000 people (69). By late August 1976, an extensive surveillance system of the health of the population was established covering the acute and mid-term effects of the exposure as well as the long-term effects. General and special medical examinations, laboratory tests at given intervals, course and outcome of pregnancies, examinations of abortions, rate of stillbirths, followup of newborns, morbidity and mortality of the population, and a cancer registry were all set up to detect any abnormality of the health of the community for which an exposure to TCDD could be postulated. The medical health surveillance program was extended to 11 districts with a total population of 216,000 (9,14,37,69). Periodically, reports of clinical damage to the population of Seveso have appeared in the press and scientific literature (38,40,41, 46,80). However, the most complete analyses of health data have been recently published by Reggiani (69). He concluded: The Seveso accident has not revealed up to now toxic effects in humans, which have not been observed in other episodes. Chloracne, the typical skin lesion, has occurred in children with tendency to spontaneous and rapid healing. The peripheral nervous system has perhaps been attacked and reacted with subclinical signs of impairment. Signs of involvement of the liver without apparent functional disorders have occurred. No other organs or functions have been impaired. There has been no derangement of the gestation, no foetal lethality and loss, no gross malformations, no growth retardation at term and no cytogenetic abnormalities. The immunocapability of the population, not even of the,children with chloracne, has not been attained. VI. GLOBE, ARIZqNA_EPISODE_ Globe, Arizona was another site of possible human exposure to TCDD. In 1969, the U.S. Forest Service applied 3,680 Ib of 2 (2,4,5-trichlorophenoxy) propionic acid (Silvex) and 120 Ib 2,4,5-T in the Kellner Canyon-Russell Gulch spray project near Globe (76). The reports of harmful effects to animals and people from the spraying began during and immediately after the spray treatment. The complaints included damage to vegetation off the spray project area, deformed animals and human illnesses. Although the Forest Service investigated the allegations, many of the local citizens were dissatisfied with the V-21 �reports and the case continued to fester until, in February 1970, it attained national attention. Television newscasts showed deformed animals alleged to have been caused by the herbicides. On February 13, 1970, a public hearing was held in Globe. As Tune Ma.ga.zwie. (4) reported, the local veterinarian insisted that he had noticed nothing out of the ordinary in local animals. Doctors too were puzzled. Said one: "I keep trying to see the relationship between the spraying and the illnesses, but I have simply not found anything." T-unn (4) also reported that: "The investigators holding the public hearing ended up perplexed and incredulous. In a paranoid outburst, the investigators were accused of being impostors, really representatives of chemical manufacturers in clever disguise." To look further into this episode, The Office of Science and Education, USDA, established an investigating team to assess the allegations against the Kellner Canyon-Russell Gulch Spray Project. Tschirley et al (76) published the results of the investigating team following on-site inspections of the spray project area, February 16-20, 1970. Tschirley et al, attempted to assess numerous parameters that would contribute to a comprehensive assessment of the episode. Some of these parameters included: (1) assessment of herbicide damage to plants off the project area, (2) effects of plant diseases, (3) effects of air pollution, (4) residue analyses of soils, plants and animal tissue, (5) observations of fish and wildlife, (6) evaluations of the health of domestic animals and (7) interviews with many of Globe's citizen and physicians. Some of the conclusions reached by Tschirley et al (76) were: 1. There was clear evidence of drift of herbicide outside the project area. 2. There was evidence of woody plant mortality from root rot, and also visible damage to certain yard trees from several kinds of birds and insects. 3. Reports from wildlife specialists indicated no significant effects on birds, deer and other wildlife. 4. With the exception of soil from the site where the herbicide was loaded aboard the helicopter, no residues of 2,4-D, 2,4,5-T, Si 1 vex or TCDD were found in any of the substrates analyzed. 5. Information obtained from owners of livestock and observations of animals did not indicate any illnesses that do not commonly occur in other regions. No association was found between the herbicides and the deformed animals shown on the television newscasts. 6. Human illnesses had been reported by several residents in the Globe region. Many of the residents with complaints were interviewed V-22 �by a medical member of the panel. The complaints were those that commonly occurred in the normal population; no cases of chloracne were reported. One individual had an eye irritation from steam cleaning an empty herbicide drum. Nine doctors serving the area of Globe were interviewed and there was general agreement that there had been no significant increase in human illness related to the spraying. Tschirley et al (76) summarized their panel report by stating: "Significant in evaluating the Globe situation was the emotional peak of its inhabitants. The complaints offered were those occurring in normal populations, with many of them (especially in the adults) being quite subjective. With the exception of the skin rash and eye irritation experience by one subject, it is highly unlikely that the ailments described were related directly to the spraying. However, the psychosomatic effect of an aroused public very likely has played a role. It is also important to note that except for three subjects all of the complaints dated only from the June 1969 spraying, despite the Forest Service having sprayed the same area three other years." A subsequent report was published by Roan and Morgan (70) of analytical results of selected human tissue collected by Tschirley et al (76) and of an epidemiologic study of the hospital records. Roan and Morgan concluded: We cannot find any evidence that there was long-term exposure of residents of the Globe, Arizona area to chlorophenoxy herbicides, or significant contamination of water supplies in this area with 2,4-D, 2,4,5-T, Si 1 vex or metabolites of these herbicides. Nor have we found contaminants such as TCDD that may be associated with one or more of the above technical grade products. Statistics on reproductive mortality and morbidity for the period 1960 through the first six months of 1970, from one hospital serving this area, do not indicate any trends that are suggestive of adverse influences on human reproductive function that might be associated with herbicide use during the years 1965, 1966, 1968 and 1969. Even though the analytical data available to us apply only to the years 1969 and 1970, the rate of disappearance of these compounds in the environment leads us to believe that gross, protracted contamination was probably absent in prior years as well. Although samples of. human tissues and body fluids, obtained through the cooperation of the medical profession in the area, are few in number, we believe the analytical results (which were all negative) are very probably representative. V-23 �VII. THE SWEDISH LAPLAND EPISODE In the spring of 1970, Swedish newspapers reported an accumulation of sudden deaths of reindeer grazing in the Visttrask area of Lapland. Approximately 30 reindeer, mainly young animals, died within a week after a heavy, wet snowfall, without any previous signs of illness. It was also reported that about 10 reindeer cows aborted their fetuses. Examination of several reindeer by veterinarians showed inanition (empty stomachs). When given additional feed, the deaths stopped. The case was of particular interest since it was learned that the area where the reindeer grazed had been treated with a mixture of 2,4-D plus 2,4,5-T (2). Analyses performed on liver and kidney samples (33,35) from one of the cows and three aborted fetuses noted above indicated traces of 2,4-D (0.2 to 0.5 ppm) and 2,4,5-T (0.3 to 1.0 ppm). Tree leaves contained 25 and 100 ppm of 2,4-D and 2,4,5-T respectively. However, no herbicides could be found in the ground vegetation. Although it was generally accepted that the deaths of the reindeer were attributed to starvation rather than exposure to 2,4,5-T and/or TCDD, Erne (34) initiated a controlled experiment on female reindeer and phenoxy herbicides. Erne's experiment involved thirty pregnant reindeer, where half of the animals were given birch leaves from an area that had been aerially sprayed with one of the products that was used in the Visttrask area, the rest received untreated birch leaves. The average daily intake of leaves for both test and control groups was about 1 kg per animal, which for the test group corresponded to a daily dose of phenoxy acid of 1 mg/kg body weight. After the feeding experiment, the reindeer were sacrificed and necropsied just before the expected parturition. During the course of the investigation, no clinical hematological or chemical signs were observed of injurious effects attributable to the sprayed leaves. The necropsy of the sacrificed animals showed nothing at all remarkable. All were pregnant (except one in the control group) and all the embryos were alive and normally developed. In histological investigations of the female reindeer and the fetuses, no pathological changes were observed that could be attributed to the prolonged consumption of sprayed leaves as fodder. Thus, Erne (34) concluded that the toxic manifestations noted in the Lapland incident were probably not caused by ingestion of herbicides. Immediately following the report of reindeer deaths (and concurrent with press reports on alleged health effects from 2,4,5-T and TCDD in Vietnam), two cases of congenital malformations in human infants were also attributed to alleged exposure of pregnant women during application of phenoxy herbicides in Lapland forests (2). However, competent medical scientists at the Institute of Hygiene and the Teratological Laboratories of the Karolinska Institute of Stockholm and at the Institute of Human Genetics at Munster, Germany, were unable to find temporal or clinical evidence to suggest that the occurrence of these human birth defects was more than coincidentally related to the herbicide operations. V-24 �The publicity given to the Lapland incident resulted in additional reports of alleged adverse human health effects due to the phenoxy herbicides. For example, in early 1972, Swedish newspapers reported excess lung cancer mortality among railroad workers exposed to 2,4-D and 2,4,5-T. These reports prompted the Swedish National Board of Occupational Safety and Health to request an epidemiological evaluation of the stated excess mortality and its relation to herbicide exposure (10). The subsequent investigation as reported by Axel son and Sundell (10) in 1974 found that a slightly dose-dependent and significantly increased tumor incidence and mortality among workers exposed to the herbicide amitrol (3-amino-l,2,4-triazol) whereas those exposed to 2,4-D or 2,4,5-T had about normal tumor incidence and mortality. The study comprised 2,978 person-years at observation in the total cohort. The study has been recently reanalyzed with a case-control approach and through stratification on amitrol when considering the effect from phenoxy acids and vice versa (51). The results showed a possible and previously masked tumor inducing effect also from phenoxy acid. By 1976 an Intense debate was in progress in Sweden over the use of phenoxy herbicides. This debate prompted Harden (45) to examine the occupational history of 87 patients who had malignant mesenchymal tumors and who had visited the oncological clinic in Umea during the years 1970-76. Nine of the 87 patients were forestry workers, four worked in farming and forestry and six in sawmills or the pulp industry. The implication by Harden was that these 19 individuals were in occupations where exposure to phenoxy herbicides was relatively common. Based on the official statistics of Sweden, the expected fraction of tumors has been calculated for these occupations: the expentancy was 11 cases versus the 19 observed. Harden (45) however, cautioned making any conclusion about the possible casual connection between exposure to phenoxy acids and contaminants and the occurrence of malignant tumors, solely on the basis of the reported cases. In February 1977, the debate climaxed when the Royal Swedish Academy of Sciences organized a conference on "Chlorinated Phenoxy Acids and their Dioxins, Mode of Action, Health Risks and Environmental Effects" (31). The conference participants concluded that (1) there was no evidence that dioxins could be formed in nature, (2) there was no evidence of bioaccumulation of TCDD at levels of application used in Sweden and (3) that if the concentration of TCDD can be kept below 0.1 ppm in all phenoxy formulations the risks involved can be disregarded and the safety factors based on the phenoxy acids themselves. In the March 1978 WBBM television report on "Agent Orange: Vietnam's Deadly Fog", reference was made to a report from Sweden on birth defects (e.g., spina bifida) in children born to 65 women allegedly exposed to 2,4,5-T herbicide. The only reference to such an incident was that reported by Hailing (44) in 1977. Hailing studied the malformations V-25 �in children born to mothers exposed to hexachlorophene soap during early pregnancy. All of the mothers were employed as nurses in a hospital and thus came in contact with the hexachlorophene in performance of this job. A group of 65 children born to this group showed six slight malformations and five severe malformations, whereas only one slight case in 68 children was observed in the unexposed group. VIII. TE AWAMUTU, NEW ZEALAND EPISODE The New Zealand episode had many similarities to the episode in Sweden; once the initial report was publicized, additional cases were forthcoming. In January 1972, Sare and Forbes (72) reported the following in the New Zealand Medical Journal: "Sir, - Two babies, born within a month of each other at our local maternity hospital, had congenital defects incompatible with life. Both had a gross myelo-meningocele. Post-mortem was performed on only one and other congenital abnormalities were brought to light. What intrigued us was that the families concerned live on adjoining hilly country farms, where for several years aerial spraying has been carried out with a chemical called 2,4,5-T, designed to kill useless vegetation. Inquiries into the nature of this chemical revealed that it contains an impurity called dioxin, which is apparently one of the most powerful poisons ever discovered. It has been investigated in the United States, partially banned in all states, and totally in others. It was likewise banned in Vietnam when its potential danger was discovered " The suggested relationship between 2,4,5-T/TCDD and the two deformed babies quickly received national and international attention. Accusations that 2,4,5-T/TCDD were indeed responsible for the congenital defects soon appeared in articles in the United States (1, 32). The circumstances surrounding these cases at Te Awamutu were thoroughly investigated by a subcommittee of the Agricultural Chemicals Board of New Zealand (6). In the subcommittee report it was noted: The women who gave birth to deformed babies had both been exposed to 2,4,5-T during pregnancy, one person by assisting at the airstrip during spraying and the second person by helping to free the spray truck which was stuck on the property and was exposed to 2,4,5-T when spraying was done to lighten the load. It was not possible to ascertain the degree of exposure in either case. V-26 �The deformity common to both babies is spina bifida, caused by a failure of the end of the neural tube to close completely during early development. This deformity is one of the commonly occurring deformities, with overseas averages of about 1 per 1,000 total births. In New Zealand during the period 1964-70, 515 live births and 151 stillbirths affected with spina bifida were recorded. In the light of present embryological knowledge it may be stated that the neural tube is usually closed by the fourth week after conception and definitely by the sixth week. Medical records show that in one case, exposure to 2,4,5-T during the spraying operation occurred after the neural tube would have normally closed. It is concluded that in one of these cases the reported exposure to 2,4,5-T could not have caused the birth deformity. It is not possible to state definitely in the second case whether exposure to 2,4,5-T was in any way a factor causing the deformity, and thus the subcommittee was unable to arrive at any information of value to the general topic of 2,4,5-T toxicity to human foetuses. In April 1977, the New Zealand televison program "Dateline Monday" suggested that the occurrence of "clusters" of neural tube defects in the South Taranaki, Northland and Waikato areas of New Zealand were related to the use of 2,4,5-T (61). The New Zealand Department of Health, Division of Public Health, appointed a committee of experts to investigate the allegations. In the Committee report, McQueen et al (61) noted that the three "clusters" represented 20 cases of birth defects. Seven of the cases were anencephaly (congenital defect of the cranial vault) and 13 were spina bifida (congenital defect of the bony encasement of the spinal cord). McQueen et al noted that although this group of defects may well have occurred entirely by chance, the possibility of a common causal factor must be considered. After a thorough investigation of each of the 20 cases reported, McQueen et al (61) concluded: It is obvious from an inspection of the data for the three "clusters" that 2,4,5-T cannot reasonably be implicated in the causation of neural tube defects. It is true that in one or two cases there may have been some "exposure" to 2,4,5-T around the critical period. However, considering 2,4,5-T is the most used pesticide in New Zealand, this is'not unexpected. In short, the data permit the conclusion that there is no evidence to implicate 2,4,5-T as a causal factor in human birth defects. As a final note in relation to this episode, the following brief article appeared in the New Zealand Medical Journal (5): V-27 �Publicity on certain chemicals as causation of malformations of the human fetus has been widespread. Some of the publicity has been sensation mongering and not all the remarks from the profession have been in keeping with a balanced assessment of scientific evidence. It is proper that there should be intelligent public awareness of the various environmental hazards that may come from the use of chemicals in farming ....however, those who would write of their experiences in medical journals must remember that disasters are the staple of the sensation mongers in the news media industry. Until recent publicity there had been no suggestion that 2,4,5-T, which has been used for over 20 years in New Zealand, was responsible for congenital malfunctions either in man or in farm animals. It is the duty of the physicians (and scientists) who have any concern for science to attempt to make valid observations which can be repeated. In the problem at issue, fetal malformations are natures common mistakes which we have no desire to perpetrate or to increase, although they are the inevitable price that is paid for our place on the evolutionary scale. There are extensive gaps in our knowledge but they will be filled only by patient work. Unresolved problems of fetotoxicity can only be solved by accurate record keeping at all stages of pregnancy. IX. DISCUSSION OF LITERATURE AND CONCLUSIONS The episodes described in this chapter have provided much of our knowledge of the adverse effects to human health of the phenoxy herbicides, other chlorinated phenols and the associated dioxins. The only episodes however where TCDD was actually confirmed as a caustive agent were those involving some of the industrial accidents, the Eastern Missouri horse arena episode and the Seveso, Italy episode. Mercier (62) estimated that in the industrial accident in 1963 at the Philips-Duphar Company, Amsterdam, The Netherlands, up to 200g of TCDD were released into a factory hall. The incident in the horse arenas in Missouri may have involved 5,000g of TCDD (69). The quantity of TCDD involved in the Seveso, Italy episode has been estimated at 650-1,700g (69). In these three episodes, the TCDD was confined to a relatively limited area. The exposure of the people involved was from days (Philips-Duphar) to weeks (Seveso) to months (Missouri). Nevertheless, no human deaths were reported, although in both Missouri and Seveso, numerous animal deaths did occur. The clinical experience from these three episodes (and the other industrial episodes involving at least 1,000 individuals) support the opinion that patients without chloracne are extremely unlikely to have suffered the toxic effects of TCDD. In general, only in the most severe cases of chloracne has symptomatology persisted, admittedly for many years in a few instances. V-28 �The available scientific literature suggests that the episodes in Arizona, New Zealand and Sweden were primarily the result of emotionalism associated with zealous press coverage. Although each incident began subsequent to field applications of phenoxy herbicides, it was highly unlikely that the symptoms reported were attributable to actual pesticide or TCDD exposure. The behavior in the environment of 2,4,5-T and TCDD following normal field applications (see Chapter III) lends little credence to accusations that significant bioaccumulations occurred in humans to initiate the t-oxic symptoms reported. Furthermore, the absence of confirmed illness in domestic livestock or wildlife in these three episodes also addresses the issue of whether an actual toxic exposure occurred. Chapter IV defined the concentrations of herbicide and TCDD that were toxic to animals. The magnitude of the dosage required to elicite toxic symptoms in animals might be obtained only under the most extreme cases (e.g., spills or sequential repetitive applications). These extreme situations were not noted in the episodes in Arizona or New Zealand. The human responses associated with these episodes show a similarity to what occurred in Michigan involving exposure to polybrominated biphenyls (PBB). In 1973 and 1974, more than 10,000 Michigan farm residents were exposed to PBB when several hundred pounds were accidentally introduced into a nutritional supplement that was subsequently fed to numerous herds of dairy cattle. Budd et al (18) conducted an epidemiological study in an effort to determine whether or not exposure to PBB had caused illness in Michigan residents. Three groups were invited to participate in a prospective cohort study: (1) all persons who had been identified as living on PBB-contaminated farms at the time of quarantine; (2) all persons who had received food products directly from such farms; and (3) workers and their families who had been exposed occupationally to PBB in a chemical manufacturing plant. All subjects were administered a questionnaire requesting information on the occurrence in the years before and since 1973 of 17 symptoms and conditions potentially related to PBB. Venous blood samples were also obtained on the subjects. An evaluation of dose-response relationships revealed that symptom-prevalence rates were higher in persons with no detectable PBB in serum than in those with measurable quantities. These observations suggested that factors other than PBB absorption were responsible for the production of symptoms and that selection factors (e.g., selecting from a list of given symptoms by the subject) may have played an important role in the observed distribution of complaints. The episodes in Arizona, New Zealand and Sweden all occurred in the same time period; a period when numerous articles appeared in the world press on the alleged human health effects of Herbicide Orange and TCDD in South Vietnam. The effects these articles had on the actual episode can only be speculated. V-29 �The wide publicity that was given to the use of defoliants, especially Herbicide Orange in South Vietnam, appeared to have exceeded concerns of human health or the environment. Political issues may certainly have been a major reason for much of this publicity. Consider, for example, the data in Table 1 of this chapter; more 2,4,5-T and hence TCDD, was disseminated in the United States during the same period than in South Vietnam. If the assessment of canopy penetration is reasonably accurate in Chapters I and III,then the actual groundlevel deposition of Herbicide Orange in South Vietnam (1.4 pounds 2,4-0/2,4,5-T per acre) would have been approximately equal to the concentrations of herbicides encountered at ground-level following brush applications in the United States. The Committee on the Effects of Herbicides in South Vietnam of the the National Academy of Sciences (21) attempted to assess the effects of propagandists activities on the attitudes of the South Vietnamese towards the use of herbicides. The following statements are quotations from the 1974 report: Our findings indicate that there is a major dichotomy between,the views of the rural population and those of the urban middle-sector regarding the use of herbicides in SVN. Contrary to what might be expected, the herbicide missions are much less emotional issue among the peasants, who bore the brunt of the effects, than it is among urban intellecturals for whom it has become a symbol. Despite extensive propaganda and counter-propaganda campaigns waged by the RVN and the NLF, peasant views regarding herbicide effects seem to be based upon their own experience. The RVN stressed that herbicides were used as a military measure to deprive the guerrillas of their hiding places, that the herbicides might damage crops but could also have beneficial effects, and that people and livestock would not be adversely affected by spraying. NLF statements emphasized the dangerous nature of herbicides. They claimed that the chemicals caused the death of people as well as livestock and crops, resulted in increased numbers of miscarriages and stillbirths, and caused numerous diseases, especially leprosy and conjunctivitis. Further, it was said that the U.S. had deliberately introducted "chemical bacteria" into the spray which could penetrate peoples bodies and cause disease. The fact that the villagers did not appear to subscribe blindly to the propaganda claims of either side does not mean that they lacked political opinions nor that they were uninfluenced by information derived through the mass media. Rather it seems to mean that their opinions on this issue came mainly from their own observations. V-30 �The degree to which the above referenced propaganda influenced world opinion is illustrated by Dmitriyev (28) in articles published in 1974 in a Russian medical journal. The following quotation is a translation from that journal: Often in South Vietnam, chemical substances were used not only in the forest regions but also close to populated areas; this resulted in injury to a considerable part of the peaceful population. According to the data of the Provisional Revolutionary Government of the Republic of South Vietnam in 1961-1969, 1,293,000 persons were subjected to the effect of poisonous chemicals. In the first ten months of 1970, 185,000 cases of persons being poisoned were recorded. Three hundred persons died and a significant number of those injured became chronic patients. Persons injured by herbicides and defoliants noted perceiving a sharp odor of chlorine or DDT, sharp pain, burning in the nasopharynx and sneezing (91%), crying and vomiting (73%), headache and vertigo (38%), a burning sensation in the area of the eyelids and the skin (41%). These clinical symptoms were apparent after a 2.4-hour incubation period. Improvement in the patients, if they did not die, began after 3-4 days. However, they continued to suffer from asthenic symptoms in the form of sleeplessness, sexual weakness, and weakening of the vision. Similar quotations are available in American or European literature. Mercier (62), in reviewing the literature on TCDD for a conference in Milan, Italy in 1976, stated of the National Academy of Science Report (21): Considerable information is contained in a NAS report (1974) about the effects of herbicides, and especially 2,4,5-T, so-called "Agent Orange" and the contaminant TCDD on humans, animals and vegetation in Vietnam where they have been used during military herbicide operations. It contained reports of death to children, diarrhea, skin rashes looking like insect bites, and abdominal pain following spray missions. The use of the materials also significantly increased the incidence of congenital malformations among children. The point to be made is that the scientific studies that have been conducted in Vietnam; Globe, Arizona; Eastern Missouri, Sweden; New Zealand; Seveso, Italy; and the numerous industrial accidents do not document deaths of children or adults due to the herbicides or TCDD, nor do they substantiate increased incidence of congenital malformations among children. The reports published by North Vietnamese scientists provide insufficient data on which to draw contrary conclusions. V-31 �X. SUMMARY Increased industrial production of the phenoxy herbicides parallelled the rapid acceptance of these materials in world agriculture. The demands upon the industrial production however, resulted in at least 23 industrial incidents involving'over 1,100 people (almost all adult males). Although medical examinations were initially conducted on these individuals, few long-term studies are available. The use of herbicides by the United States military in South Vietnam precipitated numerous allegations of adverse health effects upon the human population. Review of the scientific literature of the few available studies conducted in Vietnam do not confirm the allegations. Episodes of TCDD poisoning in Eastern Missouri in 1974 and in Seveso, Italy in 1976 resulted in adverse effects to primarily women and children. Although the acute symptoms of poisoning have dissipated, long-term effects remain to be determined. Episodes of alleged poisoning from 2,4,5-T and TCDD in Globe, Arizona (1969-70), Sweden (1970) and New Zealand (1972) occurred in a period of time when intense publicity was given to the use of herbicides in South Vietnam. The available scientific studies of these incidents suggest that factors other than herbicide exposure may have been responsible for the symptoms reported. V-32 �CHAPTER V LITERATURE CITED 1. Adamson, L. 1974. Spray Now - Pay Later? EmuAon. Action, p 9-13; July 6, 1974. 2. Advisory Committee on 2,4,5-T. 1971. Report of the Advisory Committee on 2,4,5-T to the Administrator of the Environmental Protection Agency. U.S. Environmental Protection Agency, Washington, D.C. Mim. 76 p. 3. Anonymous. 1968. A preliminary assessment of herbicides and defoliation. EnuxXon. Sex.. Te.chno£. 2(3): 176-181. 4. Anonymous. 1970. Globe's Mystery. T^nie 95(8):42. 5. Anonymous. 1972. Fetotoxicity. N.Z. Med. 3. 75(480):304-305. 6. Anonymous. 1972 Report of the Subcommittee on 2,4,5-T. Agricultural Chemicals Board; Wellington, New Zealand. Pp 1-10. 7. Anonymous. 1974. Disposition of Orange Herbicide by incineration. Final Environmental Statement. November 1974. Department of the Air Force, Washington, D.C. 737 p. 8. Anonymous. 1974. Comments of the Dow Chemical Company on the paper by Lucile Adamson, Spray Now - Pay Later? Published in Environmental Action July 1974, p 9-13. The Dow Chemical Company, Midland, Michigan. 79 p. 9. Anonymous. 1977. 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Congenital malformations, hydatidiform males and stillbirths in the Republic of Vietnam, 1960-1969. Document No. 903.233. Government Printing Office, Washington, D.C. 26. Dalderup, L.M. 1974. Safety measures for taking down buildings contaminated with toxic materials. II. T. Sec. Ganaeife. 52:616-623. (Dutch) V-34 �27. di Domenico, A. 1977. Valiuta.z4.one. dsJULa. TCVD net teM.e.no. Rapporti ISTISAN 1977/4. Istituto Superiore di Sanita, Roma. 101 p. (Italian) 28. Dmitriyev, V.I. 1974. Harmful effects of chemical substances used by the U.S. Army in Indochina l/oen. Med. In. 1:88-90. (Russian) 29. Donnell, H.D., and P. Phillips. 1974. Illness associated with TCDD contaminated soil - Missouri. Moib. Mont. 23(34) :299. 30. Dugois, P., J. Marshal, and L. Colomb. 1958. Chloracne caused by 2,4,5-trichlorophenol. M.ch. Mo£. Piotf. 19:626-627. (French) 31. Emmelin, L. 1977. Conference on phenoxy acids. Current Sweden Environment, Planning and Conservation. Swectcih Institute., Bait No. 72. Him. 5 p. 32. Environmental Defense Society. 1972. The case against 2,4,5-T. W. Z. EHVMACW. 2:16-21. 33. Erne, K. 1972. lexicological aspects of phenoxy herbicide usesome recent results. Jn_ Weeds and Weed Control. Swed. Weed Conf. 13.-C1-C2. Weed Afa^. 22(2):38, 1973. 34. Erne, K. 1973. Toxicity studies with phenoxy herbicides on reindeer. Swen. t/e^. 24:273-275. (Swedish) 35. Erne, K. 1974. Phenoxy fietiu.cute. te^-tdueA <tn Swe.d>ti>k f^h and wu£cttc($e. P 192-195. .In. Environmental Quality and Safety Supplement. Vol. III. Pesticides, International Union of Pure and Applied Chemistry. 3rd International Congress. Helsinki, Finland; 3-9 July 1974. F. Coulston and F. Korte (Eds.). 36. Executive Office of the President. 1971. Report on 2,4,5-T. A report of the Panel on Herbicides of the President's Science Advisory Committee. Executive Office of the President, Office of Science and Technology. Washington, D.C. 69 p. 37. Fara, G.M. 1976. Health Surveillance program. Medical - epidemiclogical commission. Milan, Italy, August 27, 1976. Mim. 10 p. 38. Firestone, D. 1977. The 2,3,7,8-tetrachlorodibenzo-para-dioxin problem: A review, jn. Chlorinated Phenoxy Acids and Their Dioxins: Mode of Action, Health Risks and Environmental Effects. Ec.o£. 8od£. (Stockholm), 27 (In press). 39. Ford, R.E.* B.J. Jacobsen, and D.G. White. Myc.atoxA.nt> - e.nv<inom<>.ntaJt contaminants Jbi natusui. Illinois Research, Winter 1978, pp 10-11. 40. Forth, W. 1977. 2,3,7,8-tetrachlorodibenzo-l ,4-dioxin (TCDD): The Seveso incident. Oeoticfie^ biztitblatt 44(3) -.2617-2628. (German) V-35 �41. Garattini, S. 1977. TCDD poisoning at Seveso. Blome.dicA.ne. 26:28-29. 42. Giovanardi, A. 1976. Decontamination program for the dioxin - contaminated areas of Seveso and Media. Giuhta Regionale Delia Lombardia. Ministero Delia Sanita. Milan, Italy. August 18, 1976. Mim. 17 p. 43. Goldmann, P.J.. 1973. Severe acute chloracne, a mass intoxication due to 2,3,6,7-tetrachlorodibenzodioxin. HawtaAzt 24:149-152. (German) 44. Hailing, H. 1977.' Suspected link between exposure to hexachlorophene and birth malformed infants. LakaKtidnAnge,n 74:542-546. (Swedish) 45. Hardell, L. 1977. Malignant Mesenchymal tumors and exposure to phenoxy acids - a clinical observation. LafeoAXufrutngen 74(33) :27532754. 46. Hay, A.W.M. 1977, Tetrachlorodibenzo-p-dioxin release at Seveso. 1(4): 289- 308. 47. Hofman, M.F., and C.L. Meneghini. 1962. A proposito delle follicolosi da idrocarburi clorosostituito (acne clorica). G. Ital. VeAm. 103:427-450. (Italian) 48. Honoroff, I. 1973. Down's Syndrome - it can happen here. A Report to the Consumer 111(50) :l-4. Sherman Oaks, California. Mim. 4 p. '49. House, W.B., L.H. Goodson, H.M. Gadberry, and K.W. Dockter. 1967. Assessment of ecological effects of extensive or repeated use of herbicides. Midwest Research Institute (Kansas City, Missouri). Sponsored by Department of Defense. ARPA Order No. 1086. 369 p. 50. International Agency for Research on Cancer. 1977. IARC Monographs on the Evaluation of Carcinogenic Risk of Chemicals to Man. Vol. 15. Some Fumigants, the Herbicid.es 2,4-D and 2,4,5-T, Chlorinated Dibenzodioxins and Miscellaneous Industrial Chemicals. World Health Organization; Lyon, France. 354 p. 51. International Agency for Research on Cancer. 1978. IRAC Internal Technical Report No. 78/001. (Draft). Coordination of Epidemiological Studies on the Long-Term Hazards of Chlorinated Dibenzodioxins/Chlorinated Diobenzofurans. World Health Organization; Lyon, France. 48 p. 52. Irish, K.R., R.A. Darrow and C.E. Minarik. 1969. Incarnation manual faott, vegetation c.onfao£ in Southeast ktxia.. Misc. Public. 33. Department of the Army, Fort Detrick, Frederick, Maryland. 71 p. 53. Jensen, N.E. 1972. Chloracne: Three cases. PJLOC.. R. See. Med. 65(8):687-688. V-36 �54. Jirasek, L., J. Kalensky, and K. Kubec. 1973. Acne chlorina and porphyia cutanea tarda during the manufacture of herbicides. Ce-afe. 48(5): 306-31 7. (Czech) 55. Jirasek, L., J. Kalensky, K. Kubec, J. Pazderova, and E. Lukas. 1974. Acne chlorina, porphyria cutanea tard and other manifestations of general intoxication during the manufacture of herbicides. Part II. Cwfe. VvumaXat. 49(3):145-157. (Czech) 56. Kimbrough, R.D. 1974. The toxicity of polychlorinated polycyclic compounds and related chemicals. Ctc£. Rev. Topical. 2:445-498. 57. Kimbrough, R.D., c.D. Carter, J.A. Liddle, R.E. Cline, and P.E. Phillips. 1977. Epidemiology and pathology of a tetrachlorodibenzodioxin poisoning episode, kick. EnviJion. Heo&tfi 28:77-85. 58. Kimmig, J. and K»H. Schulz. 1957. Occupational acne (so-called chloracne) due to chlorinated aromatic cyclic ethers. VeAmcutolog^cja. 115:540-546. (German) 59. Lobes, L.A., R.E. Koehler, W.F. Barthel , R.A. Feldman and J.V. Bennett. 1972. Toxic illness, Lincoln Gouty, Missouri. CDC No. EPI-72-13-2. U.S. Public Health Service. Center for Disease Control. Atlanta, Georgia. Mim. , 16 p. 60. May, G. 1973. Chloracne from the accidental production of tetraclorodibenzodioxin. &*.. J. Ind. Med. 30:276-283. 61. McQueen, E.G., A.M.O. Veale, W.S. Alexander, and M.N. Bates. 1977, 2,4,5-T and human birth defects. New Zealand. Dep. Health, Div. Publ. Health. Mim. 41 p. 62. Mercier, M.J. 1976. 2,3,7 ,S-t&tMLC.ktotiodib<inzo-p-d<LoxAn, an \jJim. P 141-157 In Proceedings of the expert meeting on the problems raised by TCDD pollution. A. Berlin, A. Buratta and M.Th. Vander Venne (Eds.). Milan, Iialy, 30 September and 1 October. 63. Meselson, M.S., A.H. Westing, and J.D. Constable. 1971. Background material relevant to presentations at the 1970 annual meeting of the AAAS concerning the Herbicide Assessment Commission for the American Association for the Advancement of Science. Washington, D.C. Min. 47 p. 64. Mivra, H., A. Omori , and M. Shibue. 1974. The effect of chlorophenols on the excretion of porphyrins in urine. Jpn. J. Ind. Health 16(6): 575-577. (Japanese) 65. Oliver, R.M. 1975. Toxic effects of 2,3,7,8-tetrachlorodibenzo-l ,4dioxin in laboratory workens. &Ut. J. Ind. Med. 32(1): 49-53. V-37 �66. Orians, G.H. and E.W. Pfeiffer. 1970. Ecological effects of the War in Vietnam. Science 168:544-554. 67. Pazderova, J., E. Lukas, M. Nemcova, M. Spacilova, L. Jirasek, J. Kalensky, J. John, A. Jirasek, and J. Pickova. 1974. Chronic poisoning by chlorinated hydrocarbons formed in the production ot 2,4,5-trichlorophenoxyacetate. P*AC. Lefe. 26(9):332-339. (Czech) 68. Poland, A. P., D. Smith, G. Metter, and P. Possick. 1971. A Health survey of workers in a 2,4-D and 2,4,5-T plant. M.c.k. 22:316-327. 59. Reqgiani, G. 1978. The estimation of the TCDD toxic potential in the light of the Seveso accidpnt. Pane*' presented at the 20th Congress of the European Society ot 1:0x1 coiogy. Berlin ^West), June 25-28, 1978. 70. Roan, C.C., and D.P. Morgan. 1972. Alleged effects on human health of the use of herbicides in the area around Globe, Arizona. Arizona Community Pesticides Studies Project, March 6, 1972. University of Arizona, Tucson, Arizona. Mim. 7 p. 71. Rose, H.A. , S.P.R Rose. 1972. Chemical spraying as reported by refugees from South Vietnam. Science 177:710-712. 72. Sare, W.M. and P.I. Forbes. 1972. Possible dysmorphogenic effects of an agricultural chemical: 2,4,5-T. Mew Zeo&md. Mc.d. J. 75(476): 37-38. 73. Suskind, R.R. 1976. A review of occupational exposures to dibenzop-dioxins. Presentation to a Conference on Dibenzodioxins/Dibenzofuran. November 18, 1976. Rougemont, N.C. 74. Telegina, K.A. and L.I. Bikbulatova. 1970. Affliction of the follicular apparatus of the skin in workers occupied in the production of butyl ether of 2,4,5-trichlorophenoxyacetic acid. l/e6^Cn. Vwmeutol. 44:35-39. 75. Tschirley, F.H. 1969. Defoliation in Vietnam. Science 163:779-786. 76. Tschirley, F.H., W. Binns, C. Cueto, B.C. Eliason, H.E. Heggestad, G.H. Hepting, P.F. Sand, and R.F. Stephens. 1970. Investigations of spray project near Globe, Arizona. Investigation conducted February 1970. U.S. Department of Agriculture, Office of Science and Education. Mim. 29 p. 77. Tung, T.T., T.K.anh, B.Q. Tuyen, D.X. Tra, and N.X. Hugen. 1971. Clinical effects of massive and continuous utilization of defoliants on civilians. lAtetnomeAe Studies 29:53-81. V-38 �78. Tung, T.T., T.T., An, N.D. Tarn, P.H. Phiet, N.N. Bang, T.T. Bach, H. vanSon and O.K. Son. 1973. Le cancer primaire due foie au Viet-nam. CklnuAQ^n 99:427-436. (French) 79. United States Tariff Commission. 1946. Synthetic organic chemicals: U.S. production and sales, 1944. Report No. 155, Second Series. U.S. Government Printing Office, Washington, D.C. 80. Walsh, J. 1977. Seveso: The questions persist where dioxin created a wasteland. Science 197(4308) :1064-1067. 81. Zelikov, A. Kh. and L.N. Danilov. 1974. Occupational dermatoses (acnes) in workers engaged in production of 2,4,5-trichlorophenol. Sov. Med. 7:145-146. (Russian) V-39 �CHAPTER VI HUMAN EFFECTS OF HERBICIDE ORANGE I. INTRODUCTION This chapter will discuss the human effects of Herbicide Orange. There has been considerable medical literature published on the constituents of Herbicide Orange, i.e., 2,4-D, 2,4,5-T, and the contaminant TCDD. The pharmacokinetics of these chemicals will be discussed along with their adverse effects. Most of the reports in the literature have involved occupational experiences. However, several episodes involving general populations from selected localities throughout the world will also be discussed. II. PHARMACODYNAMICS Little work has been done regarding the pharmacodynamics of 2,4-D, 2,4,5-T or TCDD in humans. The available studies are summarized below. A. Percutaneous Entry of Phenoxy Herbicides Feldmann and Maibach (27) in 1974 in studies using radioactive tracers showed that 2,4-D was able to penetrate the skin. Indirect evidence resulting from the numerous occupational exposures to 2,4-D and 2,4,5-T (and TCDD) in industry and herbicide spraying described later further supports percutaneous entry. B. Ingestion of Phenoxy Herbicides Kohli et al (46, 47) in two separate studies gave purified 2,4-D and 2,4,5-T in capsules to human volunteers. Each herbicide was orally administered to six men as the acid at a dose level of 5 mg herbicide per kg body weight (mg/kg). The 2,4-D was quickly absorbed and appeared in the plasma within one hour after ingestion. Seventy-five percent of the administered dose was excreted unchanged in the urine within 96 hours (h). The 2,4,5-T was also readily absorbed, being present in the plasma one hour after ingestion. After 96 h, 63 percent to 72 percent of the herbicides had been excreted unchanged by the kidney. Plasma levels peaked between seven and twenty-four hours for both 2,4-D and 2,4,5-T and the half-lives for plasma clearance were 33 and 18 h respectively. In a study by Saueroff et al (70) in 1977, five male humans ingested 5 mg/kg of 2,4-D. Essentially all was absorbed from the gastrointestinal tract. It was eliminated from the plasma with an average half-life of 11.6 hours and from the urine with an average half-life of 17.7 hours. Eighty-two and three tenth percent was excreted unchanged and 12.8 percent in a conjugated form for a 95.1 percent total recovery. Utilizing this rate of clearance, 99 percent of the steady state would be reached in about three days making body accumulation of repeated exposure unlikely. Gehring et al (28) in 1973 and Matsamara (52) in 1970 found similar results in comparable studies of 2,4,5-T. It should be noted VI-1 �that no short-term adverse effects were found in any of the above studies with 5 mg/kg being the highest dose. The fate of Silvex [2-(2,4,5-trichlorophenoxy) propionic acid] was studied by Saueroff et al (71). Seven men and one woman ingested a dose of 1 mg/kg. Peak plasma levels were reached two to four hours after ingestion. The Silvex was excreted in the urine both in the unchanged and conjugated forms. The mean recovery in the urine was 64 percent of the orally administered dose after 24 hours and 79.8 percent after 144 hours. Recovery of Si 1 vex in the feces accounted for not more than 3.2 percent of the administered dose. The half-life for plasma clearance was biphasic, 4.0 +. 1.9 h and 16.5 ±. 7.3 h for initial and terminal periods respectively. No adverse effects were noted. Park et al (60) described clinical and pharmacokinetic observations in a 39-year-old male following ingestion of the amine salts of 2,4-D and [2-(2-methyl-4-chlorophenoxy) propionic acid] (MCPP). Following forced alkaline diuresis, the plasma half-half of 2,4-D was greatly reduced from 220 to 4.7 h. The renal clearance of 2,4-D increased up to greater than 100-fold during the period of alkaline diuresis. I'lrinary recovery studies confirmed absorption of about 70 ml of the herbicide. ,fthough the patient initially demonstrated a mild proximal neuropathy and myopathy, full recovery occurred in two months. C. Tissue Analyses for the Phenoxy Herbicides Levels of phenoxy herbicides found in human tissue or body fluids following ingestion of a fatal dose are shown in Table 1. The data are reported in parts per million: it was assumed that all tissues (removed during the autopsy) were analyzed on a fresh weight basis and that 1 ml of blood or urine was equal to 1 g. Frequently, no description of the handling procedures was reported; thus, fluid may have been present within the organ (e.g., liver) at the time of the analyses. This fluid contamination may have resulted in high values for a given tissue. Coutselinis et al (20) in 1977, reported on the analyses of herbicide in the organs of a woman who died 16 hours after ingesting a large dose of a mixture of 2,4-0 and 2,4,5-T. Levels of the two herbicides found in selected tissues at the time of death are shown in Table 1. The formulation ingested was a mixture in a 3:2 ratio, 2,4-D to 2,4,5-T. Nielson et al (56) reported a more complete investigation of herbicide residue in body tissue resulting from an autopsy of a 23-year-old male who ingested 2,4-D. The levels of 2,4-D in parts per million for selected tissue are also shown in Table 1. The herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA) has been associated with two deaths. Johnson and Koumides (39) reported the death of a 65-year-old man following the ingestion of 250 mg MCPA/kg body weight. VI-2 �TABLE 1 Levels (part per million) of Phenoxy Herbicides in Human Tissue or Body Fluid Following Ingesfion of Fatal Dosea Patiert Herbicide Coutselinis et al. (20) Young Wcmsn 2,4-D/ 2,4, 5-T "Large" Nielson et al. (56) 23 Yr Old Han 2,4-D Time Level of Herbicide (Parts Per Million) in Tissueb Ingestion Gastric Fatty - Death Blood Urine Liver Kidney ! Brain Spl een Muscle Heart Washings Tissue ;• 18 hrs 826 210 82 12 182 48 5 22 Dose (mg/kg) >80 Source i Dudley and Thapar (23) 669 264 183 63 13 2,4-D 76 Yr Old Man >2,000C 5 days 58 408 194 i 20 hrs 250 180 1 1 2,500 j 800 t I 440 20 hrs 230 970 i . i ! 32 Yr Old Han '• MCPA : I 118 93 ! MCPA 83 : 70 134 ( i 65 Yr Old Han Johnson and Koumides (39). Popham and Davies (63) 24 hrs 146 154 33 3,000 i I ^Tissue/fluid removed during autopsy. °Ip convert parts per million to mg/dVor to mg/100 ma, multiply tabulated values by 0.1 . e Tne 55 kg patient consumed one pint of a presumed ester formulation (kerosene-like, water insoluble formulation) of 2,4-D. tster formulations containing the least amount of active ingredient 2,4-D are two pound/gallon formulations. If a pint contained 113 g 2,4-D acid, then the dose would have been >2,000 mg 2,4-D/kg body weight. * �Popham and Davis (63) report the death of a 32-year-old man following a MCPA dose of 440 mg/kg. The levels of MCPA found in selected organs or body fluids following death are reported in Table 1. The high level of MCPA in the urine suggests that this herbicide, like 2,4-D, was rapidly excreted unchanged by the kidney. D. Pharmacodynamics of TCDD There is almost a complete lack of information concerning the Pharmacodynamics of the dioxins in man. In November 1977, Fanelli (2)-in a letter to the Mario Negri Institute of Pharmacologic Research in Italy found no TCDD in samples of the liver, mesenteric fat, or cerebral fluid in the necropsy of a woman who had been included in a follow-on study of the Seveso, Italy, TCDD episode. The lower limits of detection were 0*4 ng TCDD/ml of fluid and 0.25 ng/gm of tissue. The cause of death was not given. Reggiani (65) described the case of a 55-year-old woman who died of pancreatic carcinoma with liver involvement seven months after the Seveso episode. Children living with her suffered severe caustic burns of the skin and, subsequently, chloracne. Neither the patient nor the mother of the children developed chloracne. It is almost certain that the entire family ate food contaminated with TCDD. TCDD detected in the analysis of tissue obtained during the autopsy is shown in Table 2. No TCDD was found in the same tissues taken from autopsies of three persons who were certainly not related to a TCDD exposure. The samples were run concurrently with those of the case described above. III. ADVERSE EFFECTS A. Limitations of Referenced Studies There is considerable information in the world literature regarding the adverse effects of 2,4-D, 2,4,5-T, 2,4,5-trichlorophenol (TCP) and TCDD in humans. Most of it is the result of studies on worker experience, industrial accidents or individuals poisoning. Unfortunately, there are very few controlled studies and only generalizations can be made regarding a causeeffect relationship. In most cases all that can be said is that an association exists. There are several other important limitations of the studies that must be kept in mind when reviewing them. These include: 1. The populations were biased toward the adult male of working age. 2. Examinations were post-exposure, and therefore, pre-existing disease often was not known or reported. 3. Exposures frequently were to mixtures and, therefore, one cannot be certain which chemical produced which effect. 4. An accidental or intentional ingestion or an exposure from an industrial accident would result in a dose much higher than would be expected in the general population in the region of a herbicide spraying program. VI-4 �TABLE 2 TCDD Levels in a Human Body Date Sampl e b 28, 7, 7 Liver Limit of Origin Quantity Detection Recovery TCDDa 10 g 10 PPT 64% 0.15 PPB Autopsy 10 g 10 PPT 59% 1.84 PPB Pancreas Autopsy , Autopsy 5g 10 PPT 59% 1.04 PPB Fat Lung Autopsy 10 g 10 PPT 60% 0.06 PPB Kidney Autopsy 10 g 10 PPT 60% 0.04 PPB Brain Autopsy 10 g 10 PPT 60 0.06 PPB i a - Total body weight:, kg 70 - Calculated total amount at time of death: 40 yg b - Vacuum Generator Micromass Laboratory, Altrincham (Manchester, U.K.) Source: Reggiani (65). VI-5 �5. Although the routine occupational exposure would in most cases be at a dose rate lower than that of accidents, the exposure would be prolonged effectively raising the total dose. 6. The actual dose received in most instances was not known. B. Phenoxy Herbicides That Do Not Contain TCDD As was explained in a previous chapter, TCDD is a contaminant of phenoxy herbicides made from TCP. TCP is not a precursor of 2,4-D. This permits the evaluation of health effects of 2,4-D (or 2,4-D-like herbicides) as an entity separate from TCDD. 1. Experimental Exposure to 2,4-D There have been at least three reports of no-effect exposure where the precise dose was known. Assouly (4) in 1951 reported on a man who ingested 0.5 g of 2,4-D daily for three weeks without adverse effects. Kohli (46) in 1974 in his pharmacodynamic study of 2,4-D reported no effect after a single oral dose of 5 mg/kg. In 1962, Seabury (74) treated two cases of disseminated coccidiomycosis with 2,4-D. The first patient received a total of 40 mg of the sodium salt by intramuscular injection over a period of four days. The patient died on the fifth day without evidence of 2,4-D toxicity. In the second patient, approximately 13 g were given intravenously over a period of one month, the last 2 g in one dose. No adverse effects were noted. When the dose was increased to 3.6 g over a period of two hours the patient became semi-stuperous and exhibited fibrillary movements about the mouth and in both hands and forearms. The stupor deepened to a point where the patient responded only to painful stimuli. Forty-eight hours after the dose was given he returned to his pre-reaction state. There was no evidence of neurologic or muscular change in the next seventeen days after which he died from the primary disease. 2. Exposure in the Production of 2,4-D or MCPA Bashirov (8) in 1969, examined 292 workers including 44 women employed in the production of the amine salt and the butyl ester of 2,4-D. This report is of particular significance in that the butyl ester is the form found in Herbicide Orange. Table 3 shows the various responses along with the percentage of occurrence. Several organ systems were involved with emphasis on headaches, the asthenic syndrome, and gastrointestinal complaints. Fifty persons from the above group were selected for controlled studies involving the liver and stomach. Bashirov indicated that there were significant differences between the control and test groups in amount of gastric secretion and the antitoxin and carbohydrate functions of the liver. In addition, they noted a correlation between the length of service and the changes in the functional state of the stomach. The authors did not state their level of confidence. Telegina and Bikbulatova (78) in 1970 reported on 158 workers employed in the production of MCPA. Telegina and 'Bikbulatova found contact VI-6 �TABLE 3. Distribution of symptoms in 292 workers employed in the production of the amine salt and the butyl ester of 2,4-D. Percent of workers describing symptoms Symptoms 1. Weakness, fatigability, headaches 63 2. Asthenic Syndrome with vegetative dysfunction 61 3. Anorexia, bitter taste in mouth, dyspepsia abdominal pains, constipation 51.7 4. Vertigo 33 5. Dyspnea on exertion 26.7 6. Tachycardia, precordial pain 17.8 Source: Bashirov (.8) VIr7 �dermatitis or history of same in 55 individuals in the first examination and in 65 in a second examination a year later. Irritation of mucous membranes was also found in a majority of these individuals. 3. Accidental or Intentional Exposure to 2,4-D, MCPA, 2.4-DP or MCPP Another major group of persons exposed to 2,4-D or the analogs MCPA, 2,4-DP [2,4-dichlorophenoxy) propionic acid] and MCPP, are those involved with accidental or intentional ingestion of the substance. Table 4 is a summary of many such cases along with the estimated dose of herbicide (where available), major effects and outcome of the intoxication. Popham and Davies (63) reported the case of a 32-year-old man who ingested an estimated dose of 440 mg MCPA/kg. There were signs of severe meningoencephalitis including grand mal and focal seizures with death within hours. Necropsy showed no evidence of damage to the gastrointestinal tract, but the liver showed signs of early necrosis. The brain and meninges showed marked congestion but otherwise were normal. Johnson and Koumides (39) described a similar MCPA episode without the severe central nervous system signs and with death in hours. The dose was estimated at 250 mg/kg. Nielson et al (56) published a paper describing a 23-year-old man who committed suicide by ingesting an unknown amount of 2,4-D. Tissue analysis indicated, however, that at least 80 mg/kg must have been absorbed. Unlike the cases of Johnson and Koumides (39) and Popham and Davies (63), this subject was in good physical health prior to the ingestion. The others were suffering from chronic illnesses. There was evidence that this subject had at least one convulsive episode before dying, implicating the central nervous system. In the necropsy, small amounts of 2,4-D were found in the brain tissue (see Table 1). There was also evidence of degeneration of ganglionic cells in the brain. If the degeneration was due to 2,4-D and not hypoxia, it would have indicated that the cellular elements of the central nervous system were quite sensitive to 2,4-D as the tissue analysis showed the brain to have a much lower level of herbicide when compared with other organs of the body. Other episodes of poisoning by 2,4-D or MCPA have been reported by Jones et al (40), Berwick (12), Brandt (15), Dudley and Thapar (23), and Park et al (60). Findings, other than those involving the central nervous system, included abnormal enzyme levels, anemia, thrombocytopenia, skeletal myositis with myoglobinuria, myocardial irritability, loss of color vision, peripheral nervous system disorders, pulmonary edema, and renal disorders. The subject reported by Dudley and Thapar (23) died; the remainder survived with varying degrees of recovery. The case reported by Brandt (15) had a complete recovery after an estimated dose of 300 to 600 mg/kg; however, this individual had ingested a mixture of 2,4-D and 2,4-DP. The case reported by Berwick (12) was noteworthy because the individual involved accidentally ingested a dose of 110 mg 2,4-D/kg. The herbicide was formulated as the isooctyl ester of 2,4-D. Although the individual demonstrated numerous symptoms (Table 4), he fully recovered. VI-8 �TABLE Distribution of Adverse Effects in Case Reports Following the Ingestion of Non-TCDD Containing Phenoxy Herbicides c to c O O — 4_> .— 4J ._ *-* in >- — U 4) O *J OJ fD -C <U 4-1 O 4-1 X aj *- — i_ Q. (LI (0 Q- i/l — 4-t Q_ ••-» r— 4_l O I- ( U - C Q - O u i O <U O O (0 <0 O _c ra ^D ~ " I 0) *J 5 41tOmg/kg MCPA 1965 250mg/kg . ^ " 2 Q 4 toSI -C - 1 Nielson et al . (56) 1965 >80mg/kg 2.4-D Jones et al . (40) Berwick (12) g ( ? Z l G, O Z + 1967 <1 900mg/kg MCPA + 1970 HOmg/kg 2,4-D + + Brandt (15) 1971 300-600 mg/kg 2,4-D/ 2,4-DP Dudley and Thapar (2k) 11--A -2000mg/kg 2.4-D Total Number o f Reports Listing Effect Unkn 2.A-D/ MCPP 2 ^ (J (U d> 4-* a.— ^n < CL O -o 1_ 01 Death- + Death Death + •*- + + + + + * + + - + + 1 ) 8 2 + 2 Ful 1 Recovery + -t + Ful 1 Recovery + + + 6 Residual * * Peripheral Sensory Defect Death Full Recovery + 3 Outcome (_> | + 1377 - + + (fcn.) O 03 + •I- Park et ai. *j .— >-C 1 MCPA • Johnson and Koumides (39) tfl >- Q. 1964 Popham and Davies (63) Q o — Senses ion c •Q 0) -i-" 3 ^ 1 1 �The patient was routinely observed over a three year period and no signs of peripheral neuropathy occurred. 4. Exposure to 2,4-D in Spray Operations A fourth group of exposed individuals is those who were involved in spraying operations contacting either the spray or the liquid. Table 5 summarizes these cases where individuals were exposed to 2,4-D. In 1959, Goldstein et al (31) first reported on three patients who developed peripheral neuropathies manifested by pain, paresthesias and paresis. There had been previous skin contact with liquid 2,4-D indicating a probable percutaneous route of entry. Recovery from the neuropathy was incomplete for the three patients during the periods of observation which were 1, 2 and 3 years, respectively for a 65-year-old male, 50-year-old female and a 52-year-old male. The 65-year-old male was exposed during the course of spraying a field with an ester of 2,4-D wetting his arms and legs. He was reported to have been in ill health prior to exposure. The 50-year-old female was exposed twice, one year apart, to an ester of 2,4-D wetting hands and legs. The 52year-old male was exposed first when he spilled 60 ml 2,4-D ester on his arms and failed to wash it off. His second exposure was two months later, wetting his legs with the same formulation. In 1961, Monarca and di Vito (55) reported a case where the entry route may have been at least partially respiratory, the subject having stayed downwind during much of the spraying operation. The immediate toxic symptoms consisted of asthenia, autonomic hyperactivity, gastrointestinal irritation and alterations of the central nervous system. Some days later he developed a hemorrhagic enterocolitis. After a period of five months recovery was complete except for hyporeflexia of the lower limbs. Berkley and Magee (11) described the development of peripheral neuropathy in a 39-year-old farmer who had significant hand contact with 2,4-D. At the end of one year the only residual effect was mild hypoalgesia on the fourth and fifth fingers of the right hand. Todd (80) reported a case in which the subject presented with anemia and leukopenia as well as peripheral neuropathy. The subject had two separate contacts with liquid 2,4-D experiencing gastrointestinal symptoms each time. The neuropathy lasted almost two years. In 1966, Tsapko (81) reported headache, retrosternal pain, general weakness, vertigo, nausea, vomiting, and mild leukopenia in a group of field workers who entered an area immediately after it was sprayed with 2,4-D. Kotlarek-Haus et al (48) described an autoimmune hemolytic anemia in a pesticide applicator. However, DDT, Lindane and Fenthion were routinely sprayed by this individual, as well as was 2,4-D VI-10 �TABLE 5 Distribution of Reported Adverse Effects Following Exposure of Field Workers and Applicators to 2,4-D 4-1 VI to 4_t in — u O a Yc-> of if.^.. of Source Number numoer Episode Of Cases ? b~D i,t u Formulation Primarv — rrimary Rnnfp o r ( ) / nouie nf z Exposure O 3 nc/)i/l 2>. " OQ + 1<<55 3 Ester Percut Monarca anddi Vito (55) HoO 1 Sodium Sal t inhal o 0) (0 -C CL Q. _ O J: U3 ua) °-z 4-. O c— *> <U <0 a . * ->- — - 4J (/itO JEQ. >-O n j u QJ ra i- IQ z UuO 1 N/Ab Percut + Berkley and Magee (11) IS61 1 Amine Salt Percut Tsapko (81) 1S66 Group Sodium Salt Percut Wai Us et al. ( ? 8) 1?S6 1 N/A Inhal Paggiaro et al . (58) 1972 1 Ester Inhal Total Number of Reports Listing Effect Percut = Percutaneous; Inhal = Inhalation Formulation description not available. ">- 10 Q. O 41 _1-c -—o . c «Q. <g 1- z + + °1 >—O <u o i-v) -O Remarks + + + + + One case of neuropathy for 3 Yr 5 Mos Residual hyporeflexa 2 Yrs + 3 Vrs Full recovery but neuropathy lasted two Yrs 1 Yr + + + oju + + + ID e ° °- ° + + + «-^ ° •* + + Todd ( 0 8) b <u c C + Goldstein et a!. ( ! 3) a C Mild hyperalgesia in two finger N/A No comment 2 Yrs + + + Full recovery 1 Mo Ful 1 recovery �Sare (69) reported on a subject who complained of diplopia toward the end of days in which he sprayed 2,4-D. In 1974, Barthel (7) related three cases of pulmonary fibrosis in workers engaged in weed control programs using MCPA. It is more probable, however, that the fibrosis was related to the carrier substances which were slatemetal, kaolin, and talcum. In a letter to the editor, Taylor (76), reported a suicide in a young farmer who became depressed over an illness possibly resulting from exposure to 2,4-D and 2,4,5-T. The illness was not specified nor was it clear whether the depression was a primary response to the herbicides or entirely secondary to the illness. However, this was the only reference found in which a psychiatric disorder was attributed to 2,4-D. Paggiaro et al (58) described an individual intoxicated by inhalation of 2,4-D. The individual manifested headaches, constipation, urinary incontinence, myalgia, muscular hypotonia, proteinuria and tachyarrhythmia. Despite these numerous symptoms, the patient fully recovered in one month. Palva et al (59), in 1974, reported a case of aplastic anemia in a 64-year-old farmer after exposure to MCPA. Recovery was complete after five months. C. Trichlorophenol (TCP), 2,4,5-T and TCDD Since TCDD is formed in the production of TCP (see Chapter V), both TCP and 2,4,5-T are contaminated with TCDD. As a result, TCDD must be considered when discussing either TCP or 2,4,5-T. Although other dioxins are usually formed in the production of pentachlorophenol (PCP), small amounts of TCDD may also be produced and, therefore, exposure to PCP will be included in this section. 1. Industrial Exposure and Symptomatology Since the first commercial production of 2,4,5-T there have been numerous industrial episodes involving exposure to TCP, 2,4,5-T and TCDD. Chapter V discussed these industrial episodes in depth. Fifteen of the 23 episodes recorded in the literature were apparently occupational exposures that occurred during industrial production of chlorinated phenols. However, on eight occasions, explosions occurred and personnel were exposed during the clean-up of the accident or from subsequent exposure to an improperly decontaminated workshop. The symptomatology reported for various occupational episodes are presented in Tables 6, 7 and 8. Table 6 is a summary of the organ systems affected during episodes of occupational exposure to chlorinated phenols and/or TCDD. Table 7 is a summary of the signs, symptoms and disorders reported for these episodes. Table 8 is a summary of special clinical studies conducted in support of physical examinations given to selected VI-12 �TABLE 6 Organ Systems Reported Affected After Occupational Exposure to PCP, TCP, 2,4,5-T or TCDD <u z (1) u> .c 3 Q. O Source Chemical Baader and Bauer (6) Bauer et al. (9) PCP TCP/2,4,5-T Bleiberg et al. (14) TCP/2,4.5-T/2.4-D Po.land et al . (62) TCP/2,4,5-T/2.4-D Dugois et al. ( 4 2) TCP Phenoxy Acid TCP/2,4,5-T Hardell (33) Kimmig and Schulz (44) • c a 17 8 21 48 c <a o CP o. 3 20 PCP/2.4.5-T 10 23 76 PCP/2.4.5-T PCP/2,4,5-T PCP/2.4.5-T 53 + TCDD 2,4,5-T TCP Same p]ant as Bleiberg 1964 after improved conditions. Chloroform odor from skin. Same plant as Bauer 1961. 10 No significant difference in findings from control. 2 persons had porphyria without acne. Subjects taken from group examined in Jiracek et al. (37) 13 3 489 278 20 Comment Examined June 1950, 16 months after last exposure 31 workers exposed. Examined 5 years after exposure ceased. 7 17 31 3 Number of cases in which organ system affected1" a E O E O w ±J c/1 2,4,5-T Kramer ( 9 4) Jirasek et al, (37) Jirasek et a!. (38) Pazderova et al (61) Miura et al. (54) Oliver (57) Ter Beek et al. (79) Zelikov and Danilor (88) -Q E 3 Lab workers synthesizing TCDD. 4 40 36 11 24 10 0 10 Number entries in table reflect the number of cases in which a disorder of the organ system was reported. * -»• = Organ system involvement reported; however, number of cases not given. Numbers do not include cases represented by "+" and totals may represent some double counting due to overlap of studies by Jirasek et al . and Pazerova et al. �TABLE 7 Signs. Symptoms, and Disorders Reported After Occupational Exposure to TCP. 2,4,5-T or TCDD c O m to > > 10) -C U ~O 0) 4^ U . U 0) o> . — O> <B — .- (D I/1JI . - ! _ 1" u ~ 0)C Q ) > ~ ( 0 0) 4J 0) (0 T3 4->£ (0 >-<0 L - O (Din Z in- L. 4-1 V (0 CU1 <u • — £ 4J — l_ > . Ol 3 .— in Q.L. . _ 1O V) •— Q <u (0 -C c in »- — ( 3 (0 0 l _ ID .— C lf> Q. Q . d > X tt) (0 O ^ : (U 0) O O 0) (U J-> I/I (D Ifl C Ul — 0) OQ_Q S_ Source 8 4a Bauer et al . (9) 2 6 9 17 Poland et al. (62) 8 Dugois et al . (24) 4-J 4) 18 20 1 7 30 48 i + 22 i + + t (33) 9 5 87 Kimmig and Schulz (44) 31 + Kramer (49) Jirasek et al. (37) i | +• Pazderova et al. + + 12 + Jirasek et al . (38) 2 19 2 i + 78 + + 23 + 3 3 i + + (£]) + + Miura et al . (54) 2 \ 27 53 8 + + 1 1 1 + O l i v e r (57) 2 Ter Beek et al . (79) 1 + 1 + 17 6 15 18 0 47 75 2 3 1 + 1 3 Zelikov and Danilov (88) cases + : 4 3 Total number of reportedc 6 17 2 Q i 8 +b a »-.— CO 5 1 Hardell (0 O) •— C X— 11 3 Bleiberg et al . (14) -fc- 0 — ^ Baader and Bauer (6) < ( - C O - ( U Z Q U « _ >. — 3 •> • O >~l- fD u — + + 47 17 275 0 91 + 6 23 6 Number entries in table reflect the number of cases in which sign, symptom or disorder was reported. + = Sign, symptom or disorder reported but number of cases not given. c Numbers do not include cases represented by "+" and totals may represent some double counting due to the overlap to studies by Jirasek et al. and Pazderova et al. �TABLE 8 Special Clinical Studies Following Occupational Exposure to TCP, 2,4,5-T or TCDD Liver Funct Source Renal Funct Baader and Bauer (6) 6a 2 2 1 6 1 1 1 B 1 ood Elements 1 Poland et al. Proteins 2 Bauer and Schulz (9) Lipids (62) Kramer ( 9 . ^) a 0 2 7 1 k 5 ]k +b 11 11 Oliver (57) Total number of cases with abnormal study B 1 ood Pressure 6 Jaracek et al . (37) Pazderova et al . (61 )c EEG 6 Carbohydrates 37 8 9 3 28 5 18 **7 9 ' 13 15 Number entries in table reflect the number of cases in which the special study was reported as abnormal. °+ * Special study reported as abnormal but number of cases not given. C The results include studies reported in Jiracek et al. (AO). The two studies complement each other. - Numbers do not include cases represented by "+". 18 �individuals following or during occupational episodes. The data in these tables are probably representative of the over 520 individuals that were reported in Chapter V to have been medically examined following the various occupational episodes. Approximately 600 individuals were adversely affected by exposure to TCDD following eight reported industrial accidents (see Chapter V). These individuals were either involved in the accident, responsible for clean-up after the accident, or returned to work in the plant following the accident. Table 9 is a summary of organ systems affected after .an exposure to TCP and TCDD following these industrial accidents. Table 10 is a summary of the signs, symptoms and disorders noted in the individuals following exposure to TCP and TCDD. Table 11 is a summary*of the few available data on special clinical studies on those individuals involved in the industrial accidents. Armstrong et al (3) and Robson et al (67) have reported on extensive medical data (organ systems affected, symptoms, disorders and clinical examinations) from newborn infants exposed to sodium pentachlorophenate in a hospital episode of PCP poisoning. Since these data involved newborn infants and PCP in a hospital environment, they were not included in the Tables. The data in Tables 6 thru 11 list the effects reported in the industrial environment where TCDD may be produced in the course of trichlorophenol production. The absolute numbers must be looked upon with caution for the reasons expressed earlier. There were no controls and preexisting conditions in most cases were not described in the articles. This limitation is demonstrated nicely by the study of Reggiani in 1977 (64) on the workers of the ICMESA plant in Seveso, Italy. As will be explained in more detail later there appears to be minimal if any development of systemic disorders if chloracne or a history of the same is not also present (64, 65). (Personal communication: Crow, K. D., Princess Margaret Hospital, Swindon, England. Holder, B. B., Dow Chemical Company, Midland, Michigan.) Out of 176 ICMESA workers examined immediately after the accident and more thoroughly four weeks after, only one displayed a doubtful case of chloracne. Yet, there were 29 subjects with liver disorders, 28 with lower respiratory problems, and nine with disorders involving the heart. In this case, the caustic products and TCDD exited the plant through a stack resulting in minimal, if any, exposure to the workers in the plant. This is contrasted with other accidents where the formed material remained in the plant providing major exposure to TCDD. If the premise that chloracne will be present before or during the time systemic symptoms are present is accepted, the abnormalities seen in this case must be due to some etiology other than TCDD. It is a matter of speculation as to why the one worker developed chloracne. There are at least two possibilities. He may have had a low threshold or the chloracne may have preceded the incident, being present as a result of his routine work. This raises the question of how many of the systemic problems listed were also completely or partially unrelated to TCDD. From the data available, the question cannot be answered. VI-16 �TABLE 9 Organ Systems Reported Affected After Exposure to TCP and TCDD Following an Industrial Accident tn >- a> tfi <U i" O > 3 O > ui CO »- <0 U O > <1) Z I4) <1) </> <U — u — co +•> C •— +J U «o E (0 I - X ui i— c — ja Q. E >* i- i- Source in tO "O _a E 3 z 3 c .^ ~ ^ a) > • •— 1 - (/) a) c£ a) 2: c i- a> 3 o 1 O — ^ Dugois et al. (25) 21 83 83 13 1 Goldman U9,30) 42 42 6 1 Anonymous (2) (Seveso, Italy) 176 1 29 Suskind (75) 228 + + Number of cases in which organ system affected0 550 147 48 i- L. z z <u >- c <1> i/> — <u E i / i f D E - C E O E 10 — < l ) ( D o 3 u C ro ^ *-• -tJ .— j-i o 4-1 iCirt Uui 4JO1 to a/ > > ^ ( u > - 3 > » < u ) 0t Q.CO <co or o. u Q. 0) to Comments 21a Jensen and Walker (36) - 3 +b Includes family members of exposed worker 1 -xj a 7 4 1 6 6 28 35 1 3 + + 4 1 6 Includes 14-yr old son of employee Includes only workers at ICMESA Plant 9 + 2 1 6 10 1 3 Number entries in table reflect the number of cases in which a disorder of the organ system was reported. b + = Organ system involvement reported. c Number of cases not given. Numbers do not include cases represented by "+". �TABLE 10 Signs, Symptons and Disorders Reported After Exposure to TCP and TCDD Following an Industrial Accident C C <D E O in t/1 (U V) JT 0 ( a) Source U O <U •— — +j 0 0)O> »— 3 ( 0 3 1 - a) z ^ >- a) zz tn — (D °- Q) U) <U <U •— -C 1 . E <u = •*-* fl3 L . O °- O1 —i_ °-° 1 CD (75) Number of cases reportedc a ( •— C C u ** 4 J to "^ 6 O f C W — 0) § - ja L. <o 7 k2 3 1 5 + 5 ; + + 11 + + + + 0 Bk ]k 0 + + 1 6 7 + 00 ^ a. O +b 21 a 1 »3 D O •""" 13 Reggiani (64) Suskincf -*- i4-»tfl > - C l - Dugois, P. et al . (25) Goldmann (29,30) (X d) 1 Number entries in table reflect number of cases in which sign, symptom or disorder was reported. °+ = Sign, symptom or disorder reported. c Numbers do not include cases represented by "+". �TABLE 11 Special Clinical Studies After Exposure to TCP and TCDD Following an Industrial Accident Llver Funct Renal Funct Carbohydrates 13a 1 Goldmann ( 9 3 ) 2,0 + + Reggian! (64) +b Suskind (75) Total number of cases with abnormal studyc + Lipids 3 Source May, G. (53) 13 Blood Pressure + 17 + 1 3 a 0 ' 17 Number of entries in table reflect the number of cases in which the special study was reported as abnormal. ^Special study reported as abnormal but number of cases not given. c Numbers do not include cases represented by "+H. �Still, even with the limitations the data do allow for an evaluation of trends. Chloracne is by far the most common finding. Also appearing frequently are disorders involving the liver, nervous systems, and mental state, the latter primarily in the form of asthenia. Early symptoms such as respiratory tract and mucous membrane irritation as well as headaches and nausea probably result from the primary substance and not TCDD (75). Lipids are frequently evaluated, but due to the normal large day-to-day variation within an individual, the findings are difficult to evaluate. Chloracne, asthenia, and liver disease in the form of porphyria cutanea tarda will be discussed in greater detail. a. Chloracne. Chloracne is the hallmark of exposure to the highly chlorinated dibenzodioxins and dibenzofurans. Kimmig and Schulz (44, 45) in 1957 and Schulz in 1968 (73) showed that it was TCDD and not TCP that produced Chloracne. The history of chloracne since its first description in the late 1800's has been well documented, in numerous review articles (16, 21, 43, 44, 74, 77). The problem peaked about the time of World War II as the result of a large production of chlorinated napthalenes. It also has been a major problem with the polychlorinated biphenyls and the associated dibenzofurans, particularly in Japan. The incidence of chloracne has been decreasing as production techniques and housekeeping methods have improved resulting in a reduced level of TCDD. Chloracne is a disorder of the pilosebaceous mechanism with the overproduction of keratin in the sebaceous ducts. This results in the development of the comedone or blackhead seen in all types of acne. In mild cases this may represent the full extent of the disorder. However, the natural progression is the formation of cysts and in severe cases to the development of inflammatory lesions and scar formation. Inflammation, however, tends to be less prominent than that found in acne vulgaris (common or juvenile acne). Frequently associated with the chloracne are hyperpigmentation and hirsutism manifested by excessive facial and body hair. In the mildest cases acne may only appear in the area of the outer canthus of the eye and pre- and post-auricular regions. In somewhat more severe cases, the rest of the face and neck may be involved with a sparing of the nose. In even more pronounced cases, the trunk and extremities, except for the hands and feet, may be affected. A preferential site of involvement not usually seen in other forms of acne is the genital region. In the worst cases the skin of the entire body gives the appearance of a homogeneous covering of comedones and small cysts. Severity of the acne does not necessarily reflect the degree of exposure to TCDD (14). Acne may appear as early as two to three weeks after the first exposure; however, there may be a delay of several months. The delay could represent a time for the development of a skin burden of TCDD. (Personal communication: Holder B. B., Dow Chemical Company, Midland, Michigan.) This burden would represent a threshold below which acne does not appear. VI-20 �Oliver in 1975 (57) reported two laboratory workers who developed very greasy skin, one of whom developed acne. This picture is contrary to the usual finding in chloracne where the skin is typically very dry. (Personal communication: Crow, K. D., Princess Margaret Hospital, Swindon, England.) Why these workers developed the greasy skin cannot be explained and must at this time be considered an anomaly. Experience from the industrial episodes (and from the Seveso, Italy episode) confirm that mild chloracne may clear quickly (e.g., in months). Severe chloracne is known, however, for its recidivism. Cases with active lesions have persisted for up to fifteen years after exposure ceased (53). Many of the studies of systemic effects used populations presenting with chloracne as a point of entry. This probably is not a major weakness, however, because chloracne is one of the earliest indicators of disease (13). Nevertheless, it could have resulted in an artifically lowered incidence of systemic effects present without acne. b. Porphyria Cutanea Tardia. Porphyria cutanea tarda (PCT) is a disorder of heme pigment metabolism characterized by skin sensitivity, accumulation of excess pigment in the liver, and the build-up of the various porphyrin pigments. Skin findings include skin fragility, bullous lesions, pigmentation, and photosensitivity. It may be either hereditary or acquired. The latter is usually associated with hepatic disorders. Bleiberg et al (14), in 1964, discovered eleven cases of PCT in workers involved in the production of 2,4,5-trichlorophenol. In 1973, Pazderova et al (61) reported on twenty-three additional cases. Liver biopsies were obtained in five subjects and liver tissue from necropsies in two. All showed fluorescence under ultraviolet light indicating high levels of porphyrins. In 1971, Poland et al (62) studied the workers described by Bleiberg and noted only one asymptomatic urine uroporphyrin. Changes in the plant had greatly decreased the exposure to TCP and TCDD. The hyperpigmentation and skin lesions found in PCT are independent of those found in chloracne. Pre-existing liver disease appears to predispose a subject to PCT when challenged by another agent such as TCDD. Based on the majority of studies, systemic disease does not result unless chloracne is present either before or during the course of the disease. However, Bleiberg (14) found porphyria was present in two cases without acne, and Oliver (57) noted that one laboratory worker synthesizing TCDD developed rather severe systemic symptoms but never any acne. It is accepted that chloracne can result from external exposure to TCDD. The role of systemic absorption of TCDD in the development of chloracne has been a matter of debate. Similarly, it is even less clear what role percutaneous absorption of TCDD plays in the development of systemic disease. Regardless, the basic observation is true enough so that an etiology other than TCDD should be diligently searched for in any case where symptoms developed without acne. VI -21 �c. Asthenia. Many asthenic and other vegetative symptoms have been described in 2,4,5-T, TCP and TCDD intoxication. For purposes of this report, asthenia includes the following: headache, apathy, fatigue, anorexia, weight loss, sleep disturbances, decreased learning ability, decreased memory, dyspepsia, sweating, muscle pain, joint pain and sexual dysfunction. True pathology is closely interwoven with the depression which undoubtedly exists as a result of other disorders, particularly the disfigurement of chloracne, therefore causing difficulty in interpretation of these symptoms. This problem is well demonstrated in a report on polybrominated biphenyl (PBB) exposure in the April 7, 1978, issue of the Center for Disease Control MoibM<ti;y and mofttattty Weefe£</ Repoit (17). Several hundred pounds of PBB were accidently introduced into animal feed. Three cohorts were studied, the first involving all persons who had been identified by the Michigan Department of Public Health as living on PBB contaminated farms at the time of quarantine, the second including persons who had received food products directly from such farms, and the third included workers (and their families) who had been exposed occupationally to PBB in a chemical manufacturing plant. Two additional groups with low level PBB exposure were also evaluated. Highest PBB levels were found in those groups in whom one would expect the exposure to be the greatest. However, symptoms occurred most frequently in volunteers and in persons from nonquarantined farms with low level PBB contamination. Symptoms were least prevalent in quarantined farm families and in chemical workers, just the opposite of what one would expect. Symptoms and conditions included fatigue, rashes, joint pains, hepatitis, diabetes, benign tumors, and cancer. The point to be made is that signs and symptoms of asthenia are common and need not be related to chemical exposure. There is little question that asthenic symptoms can develop following TCDD exposure. In an early plant accident in which exposure is felt to have been massive, workers developed fatigue and severe muscle pain (75). Impotency was present. As it was one of the first such episodes, the symptoms of TCDD or TCP exposure had not been delineated, and therefore the effect of suggestion would have been minimal. It needs to be emphasized, however, that the exposure was massive and the symptoms did clear. One must be very careful in transposing the results of this accident to another where exposure was much less. One is on particularly tenuous ground if he attempts to attribute the symptoms to the exposure levels found in herbicide spraying. 2. Special Case Studies a. Exposure resulting from spraying operations. The study by Londono in 1966 (51) is of special interest in that he reported on five subjects who were involved in herbicide spraying as opposed to industrial exposure. The herbicides included the butyl ester of 2,4-D and the methyl ester of 2,4,5-T. All five developed chloracne. One of the workers manifested the acne seventeen days after the onset of spraying, three after two months, and one after eighteen months. No clinical systemic disease was reported, although liver function tests were mildly abnormal. b. Controlled study on 2,4,5-T plant workers. In contrast with the episodes just described, which were in effect case studies, Kramer in VI-22 �1970 and revised in 1974 (49) in an unpublished report described a control study on the health of employees exposed to 2,4,5-T at Dow Chemical Company. The control population of 4,600 non-exposed Dow employees did not vary significantly from the general population. Fifty clinical parameters were investigated including both history and laboratory studies. Parameters included were those which would be indicative of disorders of the central nervous system, mucous membrane irritation, pulmonary disease, cardiovascular disease, gastrointestinal and hepatic disorders, renal disease, asthenia, and psychiatric disorders. No significant differences were found between the study and control groups. 3. General Population Exposures The discussion up to this point has generally related to the occupational hazards of TCP, 2,4,5-T and TCDD. In recent years there has been considerable interest expressed by a number of groups concerning the public health aspects of the phenoxy herbicides and TCDD. The remainder of this section will concentrate on several incidents in which the general population was involved. Chapter V has provided more extensive details on each of these episodes. a. South Vietnam Episode. In the latter part of 1969 newspapers in South Vietnam reported that there were an unusually large number of malformed babies being born among the Montagnards, This was followed by a publication by Tung et al (85) of the Democratic Republic of Vietnam in T971 in which they reported 179 people who had lived in sprayed areas from two months to five years or had been in direct contact with the spray. He did not specify the type or composition of the spray material. Disorders were divided into three major groups: asthenia, ocular syndrome and genetic effects. The general asthenia was accompanied by insomnia, headache, sexual impotence and menstrual problems in females. A specific form of the asthenia, visual asthenia,was characterized by early onset of eye fatigue (5-15 minutes) when reading. The ocular syndrome consisted of the visual asthenia (mentioned above) as well as a decrease in visual acuity and corneal scarring. The genetic syndrome consisted of chromosomal alterations in seriously affected adults, congenital malformations (particularly Trisomy 21) in the new born, and unclassifiable multiple congenital malformations with multiple chromosomic alterations. In 1973, Tung et al (86) reported an increase in the number of persons with primary liver cancer in proportion to all cancer patients admitted to Hanoi hospitals during the period 1972-1968 (790 liver cancer cases out of 7911 cancer cases, 10 percent) as compared to the period 19551961 (159 liver cancer cases out of 5492 total cancer cases, 2.9 percent), which was prior to the start of herbicide spraying. The authors attributed this increase to exposure as a result of the spraying of herbicides containing TCDD in South Vietnam during the 1960's; however, a recent IARC monograph (34) noted that limitations in the reporting of the study make impossible an adequate assessment between the incidence of liver cancer and herbicide spraying in South Vietnam. VI-23 �A National Academy of Science (NAS) committee (18) was established in 1972 to investigate the effects of herbicides in Vietnam. in their report, published in 1974, they described an earlier study by Cutting et al (22) in 1970 reviewing congenital malformations, hydatidiform moles, and stillbirths in 22 hospitals in the Republic of Vietnam for the periods between 1960-1965 and 1966-1969. The first time period involved light spraying of Herbicide Orange, the second, heavy. Neither the NAS nor Cutting et al were able to demonstrate any influence of the herbicides on the development of these disorders. It must be pointed out that all studies in Vietnam were limited by poor and incomplete reporting, and most important by the politics of the area. Large segments of the population in question were not available to the investigating groups. b. Eastern Missouri Horse Arena Episode. Following the spraying in 1971 of three horse arenas in Lincoln County, Missouri, with salvage oil contaminated with TCDD, a number of people who worked or played in the arenas developed medical disorders (10, 19, 43, 50). The most serious war> a six-yearold girl who developed hemorrhagic cystitis and focal pyelonephritis. The urinary tract symptoms were preceded by headache, epistaxis, diarrhea and a general malaise. Her urinary tract symptoms cleared after a few days. Three months later examination was normal except for punctate hemorrhages of the bladder seen on cystoscopy. The father of the child had developed headaches and nausea while working in the arena. Her mother reported severe headache, nausea, diarrhea and abdominal pains, and arthralgia. A ten-year-old sister developed easy fatigability, epistaxis, headaches, abdominal pain, and diarrhea. All developed at least mild acne lesions (64). Follow-up studies on the mother and two children performed five years later were normal (10). Chloracne developed in two three-year-old boys who played in another arena (19, 43). One case lasted more than a year. Commoner and Scott (19) in 1976 mentioned one additional case of chloracne in a veterinarian who obtained samples in a third arena. The symptoms in all seven of the humans were relatively mild with the most severe being the hemorrhagic cystitis and focal pyelonephritis seen in the six-year-old girl. The symptoms had cleared on re-examination several years later. Exposure, at least to the four children must have been significant in that they regularly played in the soil of the arenas. Contrast this with the disastrous effects the dioxin had on the horses and other animals that were in the arena often for only a short period (43). This gives strong support to the contention that man is relatively resistant to TCDD or absorbs it to a much lesser extent. c. The Seveso, Italy Episode. The details of the Seveso, Italy, incident where an industrial accident resulted in the exposure of the general population to a cloud of TCP and other toxicants are described in the previous chapter. This incident is most significant in that it represents the first episode where a cross section of a community received a definite exposure to TCDD, although the degree of exposure can only be estimated. As in any such situation confusion reigns, and a great deal of information is passed VI-24 �consisting of a mixture of truths, half-truths, and untruths. This confusion was amplified by the fact that the caustic nature of the cloud produced serious irritative effects including skin burns in many of the people who came in direct contact with the cloud. Very early after the incident an organized program was established to follow the general populace to determine what if any effects, both long and short-term, resulted (26). The findings for the first two years have been reported and are summarized as follows (2, 64, 65, 83, 84): (1) Chloracne--A massive screening program was initiated in 32,000 school children below the age of ten. Seventy-nine cases of chloracne were confirmed, only eight of which were severe. Many of the victims were not present in the area until weeks or months after the accident, indicating that the TCDD remained in the environment outside the zone of evacuation at a level high enough to produce chloracne. Except for the eight severe cases, the acne cleared in a few months. Two years after exposure some of the severe cases still showed active lesions and had severe scarring (65). In October 1977, six new cases were found in children who returned to their homes after decontamination, bringing the total to 85. No systemic abnormalities have been found in the children with acne. It is of interest that nearly all cases of acne were found in children. There are several possible explanations" for this. A massive systematic, search for chloracne was undertaken for children under the age of ten. No such search was undertaken for adults. It may be that children are more sensitive than adults. This is a phenomenon frequently seen with chemicals and drugs. It may also simply be that the children's daily routine results in greater exposure. (2) Spontaneous Abortion and Fetal Malformation—Information concerning the birthrate, abortions and fetal malformations for the two ylars following the accident revealed no significant changes (2, 64, 65, 83, 84). There were several problems regarding the evaluation of the results. First and most important was the lack of reliable background data for the area. Worldwide figures and figures from the Lombardy region of which Seveso is a part were used. Data were also biased by the fact that therapeutic abortions were offered to women who were pregnant at the time of or immediately after the accident. Nevertheless, it was the opinion of the evaluators that significant increases in spontaneous abortions and fetal malformations could not be demonstrated. Chromosomal studies were performed on the fetuses of thirty pregnancies interrupted between August 13 and December 10, 1976. No abnormalities in number of pattern beyond the expected rate were seen (2, 64, 65, 83, 84). (3) Immunology—No differences in imrnunoglobulins and B lymphocytes were found between a study and a control group of children, even though twenty of the children in the study group had chloracne (65). Hospital admissions and disease classification data evaluation revealed no significant changes from the previous year. There was an increase in VI-25 �infectious diseases compared to the previous year, but this increase was also seen in the nonex.posed districts (2, 64, 65). (4) Summary—Except for the initial irritative effects of the caustic substances and the presence of eighty-five cases of chloracne, no adverse effects to the chemicals in the toxic cloud have been confirmed. It must be remembered, however, that in many instances the findings were not conclusive and that long-term effects such as cancer and hidden congenital malformations have not yet had time to manifest themselves. It will be several years before all the data are published on the Seveso incident. d. Globe, Arizona Episode. In 1969, a number of residents in the Globe, Arizona area alleged that numerous physical ailments resulted from the spraying of the surrounding area with 2,4-D, 2,4,5-T and Silvex. Symptoms and disorders mentioned included headaches, fatigue, chest and arm pain, worsening of pre-existing nasal allergies and asthma, loss of the sense of smell and taste, severe diarrhea, spasms of the arms and legs, anemia, irregular and painful menses, spontaneous abortions, fetal malformations and cancer (82). An investigation in 1970 by Tschirley et al (82) was unable to connect the disorders with definite exposure; however, he recommended further studies. As a result of this recommendation Roan and Morgan (66) in 1972 reported on the results of an epidemiological study of the hospital records in the area and a pesticide analysis of several body tissues and fluids including adipose tissue. The technique of analysis allowed minimum detection of TCDD at 2 ng/gm tissue and of 2,4,5-T, 2,4-D or Silvex at 0.01 ng/gm tissue. No 2,4,5-T, 2,4-D, Silvex or TCDD was found. They concluded that the probability of chronic human exposure in the area in question was very minimal. e. The Swedish Lapland Episode. As noted in Chapter V, this episode involved a series of public debates on the risks to humans (and animals) of the phenoxy herbicides, especially 2,4,5-T. In the March 1978 WBBM television report on "Agent Orange: Vietnam's Deadly Fog," reference was made to a report from Sweden on birth defects (e.g., spina bifida) in children born to 65 women allegedly exposed to 2,4,5-T herbicide. The only reference to such an incident was that reported by Hailing (32) in 1977. Hailing reported on the presence of malformations in children born to mothers exposed to hexachlorophene soap during early pregnancy. A group of 65 children born to this group showed six slight and five severe malformations. This contrasted with one slight malformation in 68 children born to a group of nonexposed mothers. It needs to be emphasized that the chemical in question was hexachlorophene and not phenoxy herbicide. f. Te Awamutu, New Zealand Episode. In 1972 Sare and Forbes (68) reported on two babies born within a month of each other in the same hospital, each presenting with meningomyelocoeles. They lived in farm country where spraying with 2,4,5-T had been routinely carried out for several years. The possibility that the malformations may have been related to the herbicide was suggested. Because of this report and other allegations that neural tube deformities were the result of 2,4,5-T exposure, a thorough, although retrospective, investigation of the problem was undertaken by the VI-26 �Department of Health in 1977. The investigating committee (1) concluded that there was no evidence to implicate 2,4,5-T as an etiologic factor. D. Cancer There are a number of individual case reports and geographically limited studies of herbicide workers, both in manufacturing as well as application, that suggest an associative relationship between exposure to either 2,4,5-T, TCP or TCDD and subsequent development of a variety of neoplasms. Of 75 workers exposed in a TCP factory accident in 1953, most were affected by chloracne, 42 were listed as severe. All of the 75 workers could be traced 25 years later and whfle the mortality rate was no higher than expected, there had been 6 deaths due to cancer versus the 4 that could have been expected from national averages. Three of the deaths were due to stomach cancer in the 60-69 year age group, which was significantly more than expected (35). One worker involved in an accident in a 2,4,5-T producing factory in the Netherlands in 1963 died of carcinoma of the pancreas in 1964 (35). Because of the extremely short time span between the exposure and the death, it is not likely that the two are related. In 1973 Tung (86) reported an increased incidence of hepatic cancer in Vietnamese allegedly exposed to the spray of Herbicide Orange. A lack of details in the reporting make evaluation of the claim difficult. Jirasek et al (37, 38) and Pazderova et al (61) reported the presence of two bronchiogenic carcinomas at ages 47 and 59 during the first five years of the follow-up of 75 workers occupationally exposed to 2,4,5-T and pentachlorophenol. They noted that only 0.12 lung cancer deaths were expected from national mortality statistics. Again, the latent period was short and no smoking statistics were given. In 1972, newspapers in Sweden reported an excess mortality due to lung cancer in railroad workers exposed to herbicides. As a result, Axelson and Sundell (5) initiated a controlled study and in 1974 reported that although there appeared to be an increased incidence with Amitrol there was no significant increase with 2,4-D or 2,4,5-T. However, a re-evaluation of the data indicated a possible and previously masked tumor inducing effect from the phenoxy acids (35). A similar study on workers involved with spraying 2,4-D and 2,4,5-T on brushwood in Finland showed no increase in overall mortality. There were, however, four cases of cancer in the age group younger than 45 years as opposed to the expected two(35). Hardell (33) reported that of 87 mesenchymal tumors seen from 1970-1976 in the Department of Oncology, Regional Hospital, Umea, Sweden, 19 had been in men whose employment (farmers and forestry workers) may have resulted in exposure to the phenoxy herbicides. The expected mesenchymal VI-27 �cancers for this group was eleven. Seven cases with known sporadic herbicide exposure 10-20 years before diagnosis were presented. Hardell noted the difficulty in establishing a causal relationship but suggested that the deviation from national averages for these relatively uncommon tumors could perhaps be linked to extensive use of the phenoxy herbicides in the Umea region. Five leukemia deaths have been reported in the area of Meda, Italy, since the Seveso episode in July 1976. No more than 1.4 were expected. One of the cases was found to predate the accident (35). Additionally, the interval from exposure to diagnosis appears to be too short to ascribe causation. The case of pancreatic carcin'oma in the 55-year-old woman described by Reggiani (65) and mentioned previously in the section on the pharmacodynamics of TCDD was also felt not to be related to the exposure to TCDD. To quote Reggiani "A causal relationship with the malignancy can be excluded owing to the lapse of time required by tumor growth to reach the size, weight and diffusion of this case. The exposure to TCDD has occurred at a time when the growth of the tumor had already reached the stage of occult spreading throughout lymphatic and blood vessels to the adjacent tissues and organs." (65) As noted above, these studies should be viewed only as preliminary evidence of a statistical relationship between exposure to the phenoxy herbicides, TCDD and TCP and subsequent cancer development. Except in cases such as the angiosarcoma caused by vinyl chloride where the type of cancer is rare and the association with exposure irrefutable, it is virtually impossible to differentiate a cancer caused by a specific chemical agent from a similar cancer caused by some other etiology. This is certainly true with the retrospective studies currently available and may be true even with meticulously controlled prospective studies. There are, however, a number of cohort studies either ongoing or planned which may help clarify the present uncertainty concerning the role of the phenoxy herbicides in cancer causation in humans (35). IV. CONCLUSIONS A. Pharmacodynamics 1. 2,4-D and 2,4,5.-T are readily absorbed via the cutaneous, and gastrointestinal routes, distributing throughout the body. The respiratory tract may also be a point of entry although of lesser importance, 2. Liquid phenoxy-herbicide contact to the skin can produce systemic reactions. 3. 2,4-D and 2,4,5-T have relatively short half-lives in the human body and persistent body burden is unlikely to develop, at least in short-term or intermittent exposures. VI-28 �Other than the knowledge that TCDD may enter the body ly, the pharmacokinetics of TCDD in man are essentially unknown. Sasud on the way ot;her_p«-c.t,icides are handled, it is reasonable to assume that t^e use of 2,^,5-T has resulted in considerable skin-liquid contact. In spite of this, reports of 2,4,5-T toxicity and therefore TCDD toxicity are minimal considering the degree of use. This may indicate that man is more resistant to the effects of 2,4,5-T and TCDD than other animals, but it could also indicate that percutaneous absorption is less. The apparent relative lack of toxicity or percutaneous absorption is further supported by the Missouri incident where there was a marked difference between the degree of toxicity in man and animals. B. Effects of the Herbicides 1. The use of 2,4-D and 2,4,5-T worldwide since the middle 1940s, with minimal reports of adverse effects indicate that they jre generally saf" chemicals if properly used. Large total doses or 2,4-D have been given tc humans in controlled circumstances without adverse effects. 2. The ne-vous system is p-: v - r icui at i,> sensitive to 2,4-D. If peripheral neuropathy developed following exposure to 2,4-D, it normally disappears in a matter of months. However, in some reported incidents, it "nd persist for on? to three years. 3. Symptoms present within the first few days after exposure are probably due to the herbicide and not TCDD. 4. Adverse effects of 2,4-D and 2,4,5-T should manifest themselves shortly after exposure. Symptoms arising for the first time, months to years after the last exposure are probably due to an etiology other than 2,4-D and 2,4,5-T, 5. The hematopoetic system may be an important target organ for 2,'-i-D in some people. C. Effects of TCDD 1. If there is not a history of chloracne, it is highly unlikely that systemic changes will be due to TCDD. However, the acne may be minimal and, therefore, the historical search must be meticulous. 2. The presence of active chloracne months to years after exposure does not necessarily mean continuing exposure. 3. Skin lesions of porphyria cutanea tarda are independent of those associated with chloracne. 4. The development of porphyria cutanea tarda following exposure to TCDD suggests an adverse liver response to the TCDD. VI-29 �5. Although asthenia is difficult to interpret, it probably represents a symptom of TCDD intoxication. 6. A rise in serum lipids may occur after exposure to TCDD. However, because of large individual variations, the finding is difficult to interpret. 7. Claims of carcinogens!s, teratogenesis, and mutagenesis in man have not been confirmed at this time for the phenoxy herbicides or TCDD. However, the topic remains open. • 8. The preliminary information from the Seveso episode and the study by Kramer on the health of 2,4,5-T workers indicate that incidental nonoccupational exposure to small amounts of TCDD is unlikely to produce symptoms. 9. The long-term effects of large acute doses of TCDD or small intermittent or chronic exposures are not known. V. SUMMARY The pharmacodynamics and adverse effects of the phenoxy herbicides, trichlorophenol and TCDD were reviewed, primarily through reports of occupational exposure and accidents as well as reported exposures to the general public. A number of organ systems may be involved if the dose is significantly high with emphasis on the skin, liver, CNS and peripheral nervous system. Adverse effects of 2,4-D and 2,4,5-T should manifest themselves shortly after exposure. Symptoms arising for the first time months to years after the last exposure are probably due to an etiology other than 2,4-D and 2»4,5-T. The hallmark of TCDD is chloracne and its absence makes it unlikely that systemic disorders present are related to TCDD. Asthenic and vegetative symptoms are often present in overexposure but are difficult to interpret. They would normally be expected to clear with time. There is no conclusive evidence at this time that the phenoxy herbicides or TCDD are mutagenic, teratogenic or carcinogenic in man. VI-30 �LITERATURE CITED CHAPTER VI 1. Anonymous. 1977. 2,4,5-T and Human Birth Defects. Report prepared in the Division of Public Health, Department of Health, New Zealand. Mim. 42p. 2. Anonymous. 1977. 28th Technical Report to the Seveso Authority. Mario Negri Institute of Pharmacological Research. Milan, Italy. November 1977. 3. Armstrong, R.W., E.R. Eichner, E.D. Klein, W.F. Barthel , J.V. Bennett, V. Jonsspn, H. Bruce, and I.E. Loveless. 1969. Pentachlorophenol poisoning in a nursery for newborn infants. II. Epidemiologic and toxicologic studies. J. PzdLLa&L. 75(2) -.317-325. 4. Assouly, M. 1951. Desterbants selectifes et substances de croissance. Apercu technique. Effet pathologique sur Thomme au cours de fabrication de Tester du 2,4-D. AtcA. Mo£. P/torf. 12:26-30. 5. Axelson, 0. and L. Sundell. 1974. Herbicide exposure, mortality and tumor incidence. An epidemiological investigation on Swedish railroad workers. Wo/ife, Enutton. , Heotth ll(l):21-28. 6. Baader, E.W. and H.J. Bauer. 1951. Industrial intoxication due to pentachlorophenol. Ind. Med. SotgeAt/ 20(6) : 286 -290. 7. Barthel, E. 1974. Pulmonary fibroses in persons occupationally exposed to pesticides. Z. &ikA.. ktmungAoig 141:7-17. (German) 8. Bashirov, A. A. 1969. The state of health in workers manufacturing the herbicides, the amine salt and the butyl ester of 2,4-D acid. (//uzcAebnoe t?e£o Wo. 10:92-95. (Russian) 9. Bauer, H., K. H. Schulz and U. Spieqeioerg. 1961. Occupational intoxication in tne manufacture of chiorophenol compounds. A/tcA. GzweA.be.path.ot. 18:538-555. (German). 10. Beale, M.G., W.i. Shearer, M.M. Karl and A.M. Roboon. 1977. Longterm effects of dioxin exposure. Ltr to Editor. Lancat(l) (8014) :748. 11. Berkley, M.C. and K.R. Magee. 1963. Neuropathy following exposure to a diethylamine salt of 2,4-D. A/ich. Intern. Med. 111:351-352. 12. Berwick, P. 1970. 2,4-Dichlorophenoxyacetic acid poisoning in man. Some interesting clinical and laboratory findings. 3. Am. Med. VI-31 �13. Birmingham, D.J. 1964. Occupational dermatology: current problems. 3:38-42. 14. Bleiberg, J., M. Wallen, R. Brodkin and I.L. Applebaum. 1964. Industrially acquired porphyria. M.c.h. Vejuncutat. 89:793-797. 15. Brandt, M.R. 1971. Herbatox poisoning, a brief review and a report of a new case. Ugtekn. La&g. 133(11) :500-503. (Danish) 16. Braun, W. 1970. Chloracne. TkeA.. Umck. 27(8) :541-546. (German) 17. Budd, M.L., N.S. Hayner, H.E.B. Humphrey, J.R. Isbister, H. Price, M.S. Reizen, G. van Amburg and K.R. Wilcox. 1978. Polybrominated biphenyl exposure - Michigan. Mo-tb. Matt. 27(14) :115-116, 121. 18. Committee on the effects of herbicides in South Vietnam. 1974. Part A. Summary and Conclusions. National Academy of Science, Washington, D.C. 398p. 19. Commoner, B. and R.E. Scott. 1976. Accidental contamination of soil with dioxin in Missouri: Effects and countermeasures. Center for the Biology of Natural Systems. Washington University; St. Louis, Missouri. Mim. 27p. 20. Coutselinis, A., R. Kentarchou and D. Boukis. 1977. Concentration levels of 2,4-D and 2,4,5-T in forensic material. Foimdxlc Science 10:203-204. 21. Crow, K.D. 1970. 56:79-99. Chloracne. Trans. St. John's Hosp. Vexmcutol. Sco. 22. Cutting, R.T., T.H. Phuoc, J.M. Ballo, M.W. Benenson and C.H. Evans. '1970. Congenital malformations, hydatidiform moles and stillbirths in the Republic of Vietnam, 1960-1969. Document No. 903.233. Government Printing Office, Washington, D.C. 23. Dudley, A.W. and N.T. Thapar. 1972. Fatal human ingestion of 2,4-D, a common herbicide. M.c.k. ?cuthol. 94(3):270-275. 24. Dugois, P., J. Mare'chal and L. Colomb. 1958. Chloracne caused by 2,4,5-trichlorophenol. hick. Mat Ptorf. 19:626-627. (French) 25. Dugois, P., D. Amblard, M. Aimard and G. Deshors. 1968. A collective and accidental Chloracne of a new type. Bo££e£tn de. to. Soc^e^e'Ctcn-tque do. VexmcutoioQle. <tt SyphJJUgHaphie. 75:260-261. (French) 26. Fara, G.M. 1976. Health surveillance program. Medical -Epidemiological . Commission. Milan, August 27, 1976. Mim. lOp. 27. Feldmann, R.J. and H.I. Maibach. 1974. Percutaneous penetration of some pesticides and herbicides in man. Tozicoi. App£. Phasunacoi. 28(1):126-132. VI-32 �28. Gehring, P.J., C.G. Kramer, B.A. Schwetz, J.Q. Rose and V.K. Rowe. 1973. The fate of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) following oral administration to man. TOJO.CO£. App£. VhaJwac.ot. 26:352-361. 29. Goldmann, P.J. 1972. Extremely severe acute chloracne due to trichlorophenol decomposition products. A contribution to the perna problem. Atfae^&med. Soz-ta6ned. Afi.b<ut&hyg. 7(1):12-18. (German) 30. Goldmann, P.J. 1973. Severe acute chloracne, a mass intoxication due to 2,3,6,7-tetrachlorodibenzodioxin. Huutaft.zt laJit. 24:149-152. (German) 31. Goldstein, N.P., P.H. Jones and J.R. Brown. 1959. Peripheral neuropathy after exposure to an ester of dichlorophenoxyacetic acid. J. Am. Med. A44oc. 171:1306-1309. 32. H a i l i n g , H. 1977. Suspected l i n k between exposure to hexachlorophene and birth malformed infants. LakafcUdninge.n 74:542-546. (Swedish) 33. Hardell, L. 1977. Malignant mesenchymal tumors and exposure to phenoxy acids - A clinical observation. Lafea/t£cdcu.ngen 74(33): 2753-2754. (Swedish) 34. International Agency for Research on Cancer. 1977. I ARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Man. Vol. 15. Some ^umiQantA, the. koAb<icMizA 2,4-1? and 2,4,5-T, c.kio^inate.d and mit>c.<Mane.ouA induA&tijot chenu.co£a. Lyons, France. 35. IARC. 1978'. IARC Internal Technical Report No. 78/001. Coordination of epidemiological studies on the long-term hazards of the chlorinated dibenzo-dioxins/chlorinated dibenzofurans. Joint NIEHS/IARC Working Group Report. Lyon, France. 36. Jensen, N.E. and A.E. Walker. 1972. Chloracne: Three cases. Royal Soc. Meet. 65:687-688. P/coc. 37. Jirasek, L., J. Kalensky and K. Kubec. 1973. Acne chlorina and porphyria cutanea tarda during the manufacture of herbicides. Vesunatal. 48(5): 306-31 5. (Czech) 38. Jirasek, L. , J. Kalensky, K. Kubec, J. Pazderova and E. Lukas. 1974. Acne chlorina, porphyria cutanei. tarda, and other manifestations of general intoxication during the manufacture of herbicides. II. C&sfe. Vfwnalol. 49(3):145-157. (Czech) 39. Johnson, H.R.M. and 0. Koumides. 1965. A further case of M.C.P.A. poisoning. B/i. Meet. J. 2:629-930. 40. Jones, D.I.R., A.G. Knight and A.J. Smith. 1967. Attempted suicide with herbicide containing MCPA. M.c.k. Env<tiion. H&atth. 14:363-366. VI-33 �41. Kimbrough, R.D. 1972.. Toxicity of chlorinated hydrocarbons and related compounds. hick. Evwifian. Health 25(2) :125-131 . 42. Kimbrough, R.D. 1974. The toxicity of polychlorinated polycyclic compounds and related chemicals. CJut. Rev. Toiu,c.ol. 2:445-498. 43. Kimbrough, R.D. , C.D. Carter, J.A. Liddle, R.E. Cline and P.E. Phillips. 1977. Epidemiology and pathology of a tetrachlorodibenzodioxin poisoning episode. hick Env-inon H&cuLth 28:77-85. 44. Kimmig, J. and K.H. Schulz. 1957. Chlorinated aromatic cyclic ether as cause of so-called chloracne. Na£uAw^en4cha££en ,44:337-338. (German) 45. Kimmig, J and K.H. Schulz. 1957. Occupational acne caused by chlorinated aromatic cyclic ethers. V&ima£ol.OQ4.ca 115:540-546. (German) 46. Kohli, J.D. , R.N. Khanna, B.N. Gupta, M.M. Dhar, J.S. Tandon and K.P. Sircar. 1974. Absorption and excretion of 2,4-dichlorophenoxyacetic acid in man. Xeno b-iotic.a 4(2):97-100. 47. Kohli, J.D. , R.M. Khanna, B.N. Gupta, M.M. Dhar, J.S. Tandon and K.P. Sircar. 1974. Absorption and excretion of 2,4,5-trichlorophenoxyacetic acid in man. 'hich. Int. Phasunacodyn. TheA. 210:250-255. 48. Kotlarek-Haus, S., W. Dzierkowa-Borodej and B. Lawinska. 1971. Autoimmune hemolytic anemia after handling insecticides and herbicides with simultaneous detection of the Australian antigen (AT) in the serum, fotia. Hamcutol. 95(3)240-253. (German) 49. Kramer, C.G. 1970, revised 1974. Health of employees exposed to 2,4,5-T. Dow Chemical Company, Midland, Michigan. Mim. Unpublished data. 19p. 50. Lobes, L.A. , R.E. Koehler, W.F. 1972. Administrative report to Control (CDC) on toxic illness, EPI-72-13-2, U.S. Public Health Barthel , R.A. Feldman and J.V. Bennett. the director of the Center for Disease Lincoln County, Missouri, CDC No. Service, CDC, Atlanta, Georgia. 51. Londono, F. 1966. Occupational acne: Five cases produced by weed killers. Medicxjoo. Cwtanae. 3:225-232 (Spanish) 52. Matsumura, A. 1970. The fate of 2,4,5-trichlorophenoxyacetic acid in man. Jap. J. Ud. Hea&th 12(9):20-25. (Japanese) 53. May, G. 1973. Chloracne from the accidental production of tetrachlorobenzodioxin. B-t. J. Ind. Mad. 30:276-283. 54. Miura, H., A. Omori and M. Shibue. 1974. The effect of chlorophenols on the excretion of porphyrins in the urine. Sangyo Igafeu 16(6) :575,, (Japanese) VI-34 �area, G. and G, di Vito. 1961. Acute poisoning from weed killer ,4-dichlorophenoxyacetic acid), fotia Med. 44:480-485. 56. Hielson, K. , B. Kaempe and J. Jensen-Holm. 1965. Fatal poisoning in in man by 2,4-dichlorophenoxyacetic acid (2,4-D): Determination of the agent in forensic materials, kcta ?kamac.ol. TOJO.CO£. 22:224-234. 57. Oliver, R.M. 1975. Toxic effects of 2,3,7, 8-tetrachlorodibenzo-l ,4dioxin in laboratory workers. St. J. Ind. Meet. 32(l):49-53. 58. Paggiaro, P.L., E. Martino and S. Mariotti." 1974. A case of 2,4-dichlorophenoxyacetic acid (2,4-D) intoxication. Meci. Lav. 65 (3-4.) :128-135. (Italian) 59. Palva, H.L.A., 0. Koivisto and I. P. Palva. 1975. Aplastic anaemia after exposure to a weed killer, 2-methyl-4-chlorophenoxyacetic acid. Haemato£. 53(2) :105-108. 60. Park, J., I. Darrien and L.F. Prescott. 1977. Pharmacokinetic studies in severe intoxication with 2,4-D and mecoprop. Ctot. To>u.c.o£. 18:154-155. 61. Pazderoz, J., E. Lukas, M. Nemcova, M. Spacilova, L. Jirasek, J. Kalensky, J. John, A. Jirasek and J. Pickova. 1974. Chronic poisoning by chlorinated hydrocarbons formed in the production of 2,4,5-trichlorophenoxyacetate. Piac.. Ltk. 26(9) :332-339. (Czech) 62. Poland, A.P., D. Smith, G. Wetter and P. Possick. 1971. A health survey of workers in a 2,4-D and 2,4,5-T plant. A/icfo. Envision. \\<LaJWi 22:316-327. 63. Popham, R.D. and D.M. Davies. B*. Med. J. 1:677-678. 1964. A case of MCPA poisoning. 64. Reggiani, G. 1977. Medical problems raised by the TCDD contamination in Seveso, Italy. Presentation to the 5th International Conference on Occupational Health in the Chemical Industry (Medichem), San Francisco, California, September 5-10, 1977. 65. Reggiani, G. 1978. The estimation of the TCDD toxic potential in the light of the Seveso accident. Paper presented at the 20th Congress of the European Society of toxicology. Berlin (West), June 25-28, 1978. 66. Roan, C.C. and D.P. Morgan. 1972. Alleged effects on human health of the use of herbicides in the area around Globe, Arizona. Arizona Community Pesticide Study Project, March 6, 1972. University of Arizona, Tucson, Arizona. Mim. 7p. 67. Robson, A.M., J.M. Kissane, N.H. Elvick and L. Pundavela. 1969. Pentachlorophenol poisoning in a nursery for newborn infants. I. Clinical features and treatment. J. PzdMi. 75(2) :309-316. VI-35 �68. Sare, W.M. and P.I. Forbes. 1972. Possible dysmorphogenic effects of an agricultural chemical: 2,4,5-T. W.Z. Med. J. 75(476):37-38. 69. Sare, W.M. 1972. The weedicide 2,4-D as a cause of headaches and diplopia. N.A. Med. J. 75(478) :173-174. 70. Sauerhoff, M.W. , W.H. Braun, G.E. Blau and P.J. Gehring. 1977. The fate of 2,4-dichlorophenoxyacetic acid (2,4-D) following oral administration to man. Toiu.c.ot.ogy 8:3-11. 71. Sauerhoff, M.W., M.B. Chenoweth, R.J. Karbowski, W.H. Braun, J.C. Ramsey, P.J. Gehring and G.E. Blau. 1977. Fate of Silvex following oral administration to humans. J. ToiU.c.ol. Envision Hea&tk 3:941-952. 72. Schulz, K.H. 1957. Clinical and experimental studies on the etiology of chloracne. M.C/I. K&tn. Ex.p. V<ywatot. 206:589-596. (German) 73. Schulz, K.H. 1968. Clinical picture and etiology of chloracne. . Sozxu&ned. Atbexx&hyg. 3(2):25-29. (German) 74. Seabury, J.H. 1963. Toxicity of 2,4-dichlorophenoxyacetic acid for man and dog. A/tch. EmuAon. Health 7:202-209. 75. Suskind, R.R. 1978. Chlorinated hydrocarbon acne. Presentation at the American Occupational Health Conference, New Orleans, Louisiana, April 10-14, 1978. 76. Taylor, C.C. 1974. Chemical toxicity and mental disorder. Am. J. 131(5):609., 77. Taylor, J.S. 1974. Chloracne - A continuing problem. Cu£u 13:585-591 78. Telegina, K.A. and L.I. Bikbulatova. 1970. State of the skin in persons in contact with methoxone during its industrial production. l/ei£n. Vvwatol. VzneAol. 44(8):76-79 (Russian) 79. Ter Beek, R. , R. Bokhorst, M.V.D. Plas, K. Olsthoorn, P. Vergragt and G. Van Der Zwan. 1973. Dioxin, a dangerous contaminant in a much used herbicide. Cfiem. We.efefa£ 69(23) :57 (Dutch) 80. Todd, R.L. 1962. A case of 2,4-D intoxication. J. Iowa Med. Soc.. 52:663-664. 81. T.sapko, V.P. 1966. The herbicide 2,4-D as a health hazard in agriculture. GA&. Saute. 31:449-450. 82. Tschirley, F.H., (Chairman) W. Binns, C. Cueto, B.C. Eliason, H.W. Heggestad, G.H. Hepting, P.F. Sand and R.F. Stephens. 1970. Investigation of spray project near Globe, Arizona. Investigation conducted February 1970. Mim. , U.S. Dep Agric Office of Science and Education. Mim. 29p. VI-36 �83. Tuchmann-Duplessis, H. 1977. Embryo problems posed by the Seveso accident. Le. Conc.onfu> Medico£ No. 44, November 26, 1977. Translation. Mim. 17p. (French) 84. Tuchmann-Duplessis, H. 1978. Environmental pollution and offspring: With relation to the accident at Seveso. MecL &t Hyg. 36:1758-1766. (French) 85. Tung, T.T., T.K.A.B.Q. Tugen, D.X. Tra and N.X. Huyen. 1971. Clinical effects of massive and continuous utilization of defoliants on civilians. W.etnome4e Stu.di.tA. 29:53-81. 86. Tung, T.T. , T.T. An, N.D. Tarn, P.M. Phiet, N.N. Bang, T.T. Bach, H. vanSon and O.K. Son. 1973. Le cancer primaire du foie au Vietnam. e. 99:427-436. (French) 87. Wallis, W.E., A. Van Posnak and F. Plum. 1970. Generalized muscular stiffness, fasciculations and myokymia of peripheral nerve origin. A/icfc. NeuAo£. 22:430-439. 88. Zelikov, A. Kh and L.N. Danilov. 1974. Occupational derma toses (acnes) in workers engaged in production of 2,4,5-trichlorophenol . Sov. Meet. 7:145-146. (Russian) *U.S. GOVERNMENT PRINTING OFFICE: 1980-671-1H3/2S VT- 1 }? � 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. 046 Folder The folder containing the original item. 1165 Series The series number of the original item. Series III 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. John A. Calcagni Charles E. Thalken James W. Tramblay Description An account of the resource <strong>Corporate Author: </strong>United States Air Force Occupational and Environmental Health Laboratory, Aerospace Medical Division (AFSC), Brooks Air Force Base, Texas Date A point or period of time associated with an event in the lifecycle of the resource 1978-10-01 Title A name given to the resource The Toxicology, Environmental Fate, and Human Risk of Herbicide Orange and its Associated Dioxin Subject The topic of the resource Ranch Hand biodegradation herbicide disposal herbicide toxicology ao_seriesIII 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. 051 Folder The folder containing the original item. 1346 Series The series number of the original item. Series III 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. Description An account of the resource <strong>Corporate Author: </strong>Office of Environmental Medicine, Veterans Administration, Washington, D. C. Source A related resource from which the described resource is derived Human and Environmental Risk of Chlorinated Dioxins and Related Compounds Date A point or period of time associated with an event in the lifecycle of the resource 1982 Title A name given to the resource Long-Term Studies on the Persistence and Movement of TCDD in a Natural Ecosystem Subject The topic of the resource dioxin Eglin AFB ecological fate biodegradation ao_seriesIII