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DOW372213 Table 12 . Results of Lifetime Feeding Studies with 2 ,7-DCDD and 2,3,7,8-TCDD. ug/kg/Day 2 Years ppt in Diet Total 1 3 / 1 2 2 ,7-DCDD in Rats and Mice a 5 x 10 9 > 10 x 10 9 ? Lifetime Observation Neg. Rats (M + F) Neg. Mice (F) Mice (M) Extremely weak poten tial for causing cancer 2,3,7,8-TCDD in Rats b '0.001 22 0.01 210 0.1 2200 0.73 No significant effect 7.3 Signs of toxicity 73 Increased mortality Multiple toxicologic effects Carcinogenic a U.S. National Cancer Institute, 1979. 13 Kociba et al., 1978. 5 0002259 Table 13. Comparison of Levels of 2,3,7,8-TCDD in Diet, Liver, and Fat of Rats Fed the Compound for 2 Years. a ppt TCDD in Diet 2 Years 22 ppt in Tissues at 2 Years Liver Fat 540 540 TCDD in Liver vs Fat 1:1 TCDD in Liver vs Total Fed b 2.4% 210 5,100 1,700 3:1 2.8% 2200 24,000 8,100 3:1 2.3% ( C fv I?.? a Kociba et al., 1978. "k Calculated from individual weight of liver and parts per trillion TCDD in liver of three female rats at each dosage level at the end of the 2-year feeding period. 0002260 OOW37221S Figure 1. Traces of 2,3,7,8-tetrachlorodibenzo-p-dioxin can form during manufacture of 2,U,5-trichlorqphenol from 1,2,U,5tetrachlorobenzene under alkaline conditions at high temperature and pressure. (Note: According to a number ing system sometimes used in.Europe, this isomer may be designated as 2,3,6,7-TCDD.) 0002261 Figure 2 Condensation of 2,b,6-trichlorqphenate leads to mixture of 1,3,6,8- and 1,3,7,9-TCDDs fcy Smiles rearrangement of intermediate. (Gray, et al., 1975; Kende and DeCamp, 1975.) d 0002262. DOWG7221 Figure 3* Condensation of 2,^-dichlorophenol to fora 2 ,7-dichlorodibenzo-p-dioxin is not likely to occur under the acid condi tions, low temperature and pressure used for manufacture of DCP by chlorination of phenol. 97 7 7 0002263  TZZA FM O n MONO l DI ^ TRI i TETRA T PENTA T HEXA T HEPTA t OCTA Chlorination not likely 2.3.7.8-TCDD Dechlorination Figure U. Formation of tetrachlorodibenzo-p-dioxins try progressive chlorination of lover analogs or progressive dechlorina tion of higher analogs* 0002264 I>o IloO << 4-~ J2> CARCINQGENICITY OF 2,4-DICHLOROPHENOXYACETIC ACID 0 d o w 352806 m m 0 . 2 2 7 77 Melvin Dwaine Reuber, M.D. NCI Frederick Cancer Research Center Frederick, Maryland 21701 June 12, 1979 0002136 4f& ABSTRACT 2,4-Dichlorophenoxyacetic acid (2,4-D) is carcinogenic in male and female rats and probably also in mice. Wale and female rats ingesting 2,4-D developed increased incidences of malignant neoplasms. Lymphosarcomas A A fV 1 were increased in rats of both sexes, and neoplasms of the mammary gland in female rats. Male rats also had carcinomas of the endocrine organ. 2,4-0 isooctyl ester was carcinogenic for the lymphoreticular system in female mice i c c 7783 0 .0 0 2 X 3 7 Detailed histologic examination was made of tissues from six male, rats and six female rats from the 1250 ppm 2,4-D groups and the control groups. Liver, kidney, spleen, and ovary or testes, as well as tumors and other gross lesions, were sectioned histologically from the rats on all other doses of 2,4-D. o n n <*n r M O O At the end of 104 weeks the following rats were alive: 17 (0 ppm), 12 (5 ppm), 18 (25 ppm), 17 (125 ppm), 19 (625 ppm), and 16 (1250 ppm).. Body weights and the organ-body weight ratios for liver, kidney, heart, and testes were reported as showing no differences among the various groups. These results have little meaning, however, since the rats that died with the most chance of being abnormal were not included. The spleen-body weight ratio was slightly elevated in rats given 625 ppm or 125 ppm 2,4-D (p * <0.05): Terminally, the red blood cells of rats given 5 ppm, 625 ppm, or 1250 ppm 2,4-D showed macrocytosis, slight polychromasia, and slight to moderate hypochromasia. These changes were not present or were of a minor degree in control rats. Malignant Neoplasms at All Sites in Hale Rats I There were increased incidences of malignant neoplasms in male rats given all doses of 2,4-D (Table 1). Nine of 25 rats (365) given 25 or 125 ppm (P 0.0053) and 9 of 23 (395) given 1250 ppm of 2,4-D (p 0.0033) developed carcinomas and sarcomas. The incidences of these neoplasms were dose related (p * 0.039). For some unexplained reason, the rats receiving . 625 ppm, 6 of 24 (255), had fewer malignant neoplasms than male rats in other groups (p 0.043). Thirty-six of 122 (305) 2,4-D-treated male rats had carcinomas and sarcomas (p = 0.0038). 7784 000213S ut Carcinomas at ATI Sites in Female Rats Carcinomas at all sites were seen in 43 (10 of 23) (p * 0.065) of female rats on 125 ppm, 585 (14 of 24) (p = 0.0059) given 625 ppm and 525 (13 of 25) (p 3 0.017) receiving 1250 ppm 2,4-D (Table 3). Carcinomas were found in the reproductive system, particularly the mammary gland, and occasionally in endocrine organs. Neoplasms of the Mammary Gland in Female Rats The incidence of mammary gland tumors were higher in treated female rats, particularly on gross examination, which.is generally quite reliable. The number of rats with such tumors was less in all but one group after histologic examination (Table 5). On gross examination, 18 of 21 female mice (855) ingesting 25 ppm (P 3 0.0064) 20 of 24 rats (835) ingesting 625 ppm (p 0.0080) and 16 of 23 rats (705) given 1250 ppm of 2,4-D had neoplasms of the mammary gland, compared to 10 of 22 (455) control female rats. The findings are quite different for the numbers of histological sections examined by pathologists at FDA and sections available for my examination. Comments It is worth noting that histological examination of tissues from this study was inadequate. Microscopic neoplasms would have been overlooked at the 5 to 625 ppm doses of 2,4-D.(only gross neoplasms were sectioned histologically) and also even at the highest dose because only six rats of each sex were examined in detail. Also histological sections were not available for 19 mammary gland neoplasms described at the time of necropsy, i and no exaplanation was given for this discrepancy. * Despite the shortcomings of this study, it must be considered as an acceptable study. . 0002140 4 Conclusions 2,4-D is carcinogenic for male and female rats. FDA 2,4-D DOG STUDY r o 7 P n A A r-i Beagle dogs,' 6 to 8 months old (3 males and 3 females per group) ingested 0, 10, 50,i 100, or 500 ppm 2,4-D in the diet for 104 weeks (1-3). ' Organ weights were taken for brain, heart, liver, kidneys, spleen, thyroid, adrenals, and testes. Tissues from all dogs were studied grossly and microscopically. ' f' Many of the dogs ingesting 2,4-D lost weight. There were scattered lesions such as atrophy of the testes and prostate, interstitial nephritis, hemangioma of the adrenal, atrophic or cystic pituitary, atrophy of the thyroid, and hypoplasia of the bone marrow. Most of the lesions were seen in endocrine organs. Control dogs generally did not have lesions. Comments Previous experience at FDA has shown that long-term chronic dog studies should be carried out for six years or longer in order for neoplasms to develop. Since they occurred predominantly in 2,4-D treated dogs, lesions may have progressed to neoplasms had the dogs been treated -for a longer period of time. Summary A two-year feeding study in dogs cannot be considered a carcinogenicity study. INNES, ET AL. 2,4-D, OR ITS ESTERS, ORAL MOUSE STUDIES 1. 2.4-D ^ The maximum tolerated dose of 2,4-D was given to two hybrid strains of mice, (C57BL/6 x C3H/Anf)F>j designated as "strain A" and (C57BL/5 x AKR)F} 000214- Three of IS strain A female mice (175) and 4 of 87 pooled controls (55) had reticulum cell sarcomas (p * 0.0955) (Table 7). In this study, there was an increased incidence of reticulum cell sarcoma in 2,4-D butyl ester-treated strain A female mice. 4. 2.4-0 Isooctyl Ester "Strain A" and "strain B" male and female mice received 46.4 mg/kg of 2,4-dichlorophenoxyacetic acid, isooctyl ester, in 0.55 gelatin by stomach tube. After 28 days they ingested 130 ppm 1n the diet for approximately 18 months. Neoplasms in treated mice were found mainly in the liver and lung. One strain A treated male mouse had an "angioma" of the liver and another an "angioma" of the spleen. In this study, neoplasms of the liver were slightly increased in 2,4-D isooctyl ester-treated strain'A male mice and tumors of.the lung in strain A female mice. Two male mice had rare tumors, "angiomas" of the liver and spleen. INNES, ET AL. 2,4-D, OR ITS ESTERS, SUBCUTANEOUS MOUSE STUDY - A single subcutaneous injection of 2,4-D, or its esters, was given in the nape of the neck to two hybrid strains of mice, (C57BL/6 x C3H/Anf)F| designated as "strain A" and (C57BL/6 x AKR)F-j designated as "strain B" at approximately the 28th day of age (4-5). There were 18. treated mice and 18 untreated controls of each strain and each sex. They were killed after * f% approximately 18 months. Mice received the following doses: 215 mg/kg or 464 mg/kg of 2,4-D in DMS0; 100 mg/kg of 2,4-D isopropyl ester, 25.5 mg/kg of 2,4-Q butyl ester or 21.5 mg/kg of 2,4-D isooctyl ester in corn oil. 000214A A A on > REFERENCES 1. Hansen, W.H., Quaife, M.L., Habertnan, R.T., and Fitzhugh, O.G.: Chronic toxicity of 2,4-dichlorophenoxyacetic acid in rats and dogs. Toxicol. Appl. Pham. 20:122-192, 1971. 2. FDA Memorandum;-"Pathological Changes in Rats Fed.2,4-Dichlorophenoxy Acid for Two Years" from Robert T. Haberman to A. J. Lehman dated March 20, 1964. 3. FDA Memorandum: "Pathological Changes in Dogs Fed 2,4-Dichlorophenoxy Acid for Two Years" from Robert T. Haberman to A. 0. Lehman dated November 5, 1963. 4. Innes, J.R.M., Ulland, B.M., Valerio, M.G., Petrucellt, L., Fishbein, L . , Hart, E.R., Pallota, A.J., Bates, R.R., Falk, H.L., Gart, J.J., Klein, G.M., Mitchell, I., and Peters, J.: Bioassay of pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. Nat'l. Cancer Inst. 42:1101-1114. 1969. ' Bionetics Research Labs, Inc.: Evaluation of carcinogenic,, teratogenic, and mutagenic activities of selected pesticides and industrial chemicals. Vol. 1 Carcinogenicity. National Technical Information Service, U.S. Department of Commerce, 1968. 0002U 6 TABLE 2. NUMBER OF MALE RATS INVESTING 2,4-D WITH LYMPHOSARCOMAS AND NEUROSARCOMAS (FOA Rat Study) -- C--------Dos (ppm) Lymphosarcoma Subcutaneous Neurosaromas Total 0 5 25 125 625 . 1250 5-1250 0/25 (OX) 2/25 (ex) 4/25 (16X) p-0.055 5/25 (20X) p-0.025 3/24 (13X) 6/23 (26X) p-0.0082 20/122 (16X) p-0.018 0.034*/ 0/25 (OX) 0/25 (OX) 0/25 (OX) 1/25 (4X) 1/24 (4X) , 1/23 (4X) 3/122 (2X) 0/25 (OX) 2/25 (8X) 4/25 (16X) p-0.055 6/25 (24X) p-0.011 4/24 (17X) p-0.050 7/23 (30X) p-0.0033 23/122 (19X) p-0.0092 0.013*/ a/ Test for positive trend 0002-14 8F82CeMOa CO ': ? .Q 0002150 TABLE 4 . NUMBER OF FEMALE RATS INGESTING 2,4-D WITH LESIONS OF THE LYMPHORETICULAR SYSTEM (FDA Rat Study) _L__________ Dose (ppm) Hyperplasia Lymphosarcoma Hyperplasia and Lymphosarcoma 0 5 25 125 625 1250 5-1250 , 0/22 (OX) 0/20 (OX) 2/22 (9X) 2/23 (9X) 4/24 (17X) p0.065 2/25 (81) p-0.28 10/114 (9X) p-0.16 0/22 (OX) 5/20 (25X) p=0.018 6/22 (27X) p*0.011 6/23 (26X) pe0.012 12/24 (50X) p0.00007 6/25 (24X) p-0.016 35/114 (31X) p"0.00073 0/22 (OX) 5/20 (25X) p-0.010 8/22 (36X) p0.0018 8/23 (35X) p-0.0023 16/24 (67X) p<0.00001 8/25 (32X) p-0.0034 45/114 (39X) p=0.00005 0.039^ 0.0004-/ a/ Test for positive trend b/ Departure from linear trend . .. . .. . 0282S eM O c _ Jr l TABLE 6. NUMBERS OF MALE AND FEMALE MICE INGESTING 2,4-D ISOPROPYL ESTER WITH TUMORS OF THE LUNG Strain "X" Hatched "X- Pooled "X" "Y" Matched "Y* Pooled My Dose (ppm) 0 0 111 0 0 111 Male 2/17 (12X) 5/79 ( 6%) 4/18 (222) p - 0.0584 2/10 (112) 9/90 (102) 2/18 (1IX) Female 1/18 (62) 3/87 (32) 1/18 (62) 0/17 (02) 3/82 (42) 0/17 (02) i J ! ; < Nr-- S' i 228S2eMoa r TABLE 8. NUMBER OF "STRAIN B" MALE AND FEMALE MICE GIVEN A SINGLE SUBCUTANEOUS INJECTION OF 2,4-D ISOOCTYL ESTER WITH RETICULUM CELL SARCOMAS Dose (mg/kg) 0 1 Males 0/161 ( OX) 2/18 (1IX) p 0.0096 Females 5/157 ( 3X) 5/17 (29X) p - 0.0009 r-> rs N. .. *A -a -I ^ssseynoa CD r TABLE 10. NUMBERS OF MALE AND FEMALE MICE INGESTING 2.4-0 ISOOCTYL ESTER WITH NEOPLASMS AT ALL SITES Strain MX" Matched "X" Pooled "X" Y* Hatched "Y" Pooled My N Dose (ppm) 0 0 0 0 Male 6/17 22/79 5/17 5/18 16/90 3/18 i Female 3/18 8/87 4/17 2/17 7/80 1/17 gsU O O O TADLE 12. NUMBER OF "STRAIN A" MALE AND FEMALE MICE GIVEN A SINGLE SUBCUTANEOUS INJECTION OF 2,4-0 ISOOCTYL ESTER WITH NEOPLASMS AT ALL SITES Dose (mg/kg) 0 1 Males 27/141 ( 6/18 ( %) t) Females 9/154 { s 2/18 ( t) t) z o oo ~i C*D ' 8282fiMOa " \ loi Analytical m ethods have been im plem ented to m easure levels of polychlorodibenzo-pdioxins, and polychlorodibenzofurans in pentachlorophenol solutions used in the Boulton w ood treatm en t process. W hen norm alized against th e pentachlorophenol (PCP) c o n te n t, th e octachlorodibenzo-p-dioxin level w as 34% higher in the recirculating PCP solution th an in th e fresh PC P solution, and in th e sludge it w as 90% higher. A smaller c o n cen tratio n increase w a s o b served fo r th e heptachlorodibenzo-p-dioxin in the recirculating solution, b u t a sim ilar in cre ase w as observed in th e sludge. The determination of polychlorodibenzo-p- ^ z dioxins in pentachlorophenol and wood C] treatment solutions * - ^ J . LAMBERTON. D. GRIFFIN. B. ARBOGAST. R. INMAN and M. DEINZER Environmental Health Sciences Center, Department of Agricultural Chemistry, Oregon State University, Corvallis, Oregon 97331 --* Cj introduction The United States production of technical pentachlorophenol (PCP) is around 50 million pounds per year.01 Most of the PCP is used in the wood products industry for protection against insects and fungus.12' Technical PCP may also be used as a slimicide, bacteriocide, and molluscacide, and it has frequently been added to paints for its biocidal properties.12' Analysis of technical PC P shows that numerous contaminants are present in fairly high concentrations. These contaminants arise during the manufacturing process/3' The actual PCP content in the technical product ranges between 85%-90%. T etrachlorophenol and trichlorophenol are present at 5%-12% and < 0.1% respectively. In addition there are chlorinated dibenzo-p-dioxins, dibenzofurans, diphenyl ethers, and a variety of hydroxypolychlorodiphenyl ethers/3' The extremely toxic 2,3,7,8-tetrachloro-p-dioxin has not been detected in P C P /4' The presence of these impurities in technical PCP raises concerns about their possible health hazards. The acute toxicity of octachlorodibenzo-p-dioxin (OCDD) (LDjo < 1000 mg/ kg)<3' is much less than that of PCP (LD 27-100 mg/kg)<6' itself. The acute toxicity of hexachlo- Copyright 1979vAgMijc*p, SIC CJ\ CJT cc rodibenzo-p-dioxin isomers (HCDD), however, has been reported to be about 100 p g /k g'51 or about 10,000 times as toxic as OCDD. Terato genic response in the pregnant rat is in the range of 100 jig/kg/day for HCDD, while OCDD showed embryo toxicity at 500 mg/ kg/day and no teratogenic effects at this level.13' Dioxins are readily formed from chlorophenols under basic conditions at elevated temperatures/7'*' Dioxins can also form from phenols: a) at elevated temperatures in the absence of base/*' b) from the corresponding copper salts;"0'1" and c) under conditions which give rise to free radical species."*' The wood products industries frequently treat wood with 5% PCP solutions at elevated temperatures in the Boulton drying p ro c e s s /13' We were concerned whether these conditions, possibly under the catalytic influence of metallic surfaces, had caused the form ation of additional quantities of chlorinated dioxins. In the course of this study we implemented methods from the literature for determining polychlorodibenzo-p-dioxins (Clx-DBDs) and polychlorodibenzofurans (Clx-D BFs), polychlorodiphenyl ethers (Clx-DPEs) in technical PCP as well as in solutions of PCP used in the treatm ent processes of wood |us)rial Hygisn# Association Am. M . Hyi Astoc. J (40) September, 1979 7796 dii zo-ptulton noi (PCP) ating PCP A sm aller in in th e . however, -12/ kg'" or D. 'ato- -ange OCDD g'day and m chlorot elevated orm from res in the esponding ons which The wood wood with ratures in We were , possibly r surfaces, dditional plcmented termining BDs) and -D B F s), ^PEs) in s of PCP of ->od timber. 1979 -';--vUi' - neutrals (Clx-DBDs, Clx-DBFs, and Clx-DPEs) prevented confusion between the HO-Clx-DPEs and Clx-DBDs during gas chromatographic analyses. ! -I.1 m eth o d s and m aterials instrum entation Because of a high level of interference from the aromatic solvents and wood residues in the . solutions and sludge, a standard GLC system was not adequate. Therefore, a specially modified gas chromatographic system that could handle the solvents and various extractants was used for the analyses of the v a rio u s fra c tio n s (F ig u re 1). A V arian ^ Aerograph model 204 equipped with a Dohrman ^ Division of Envirotech microcoulometric Figure 1 --Top view of modified Varian Aerograph 204 with column oven cover removed. The microcoulometric cell and furnace are located in the upper right hand corner. detector system consisting of a C-200-B X . microcoulometer, T-300-S titration cell, and S - ,--' 400 furnace (Figure 2) was used. A precolumn vent assembly*14' was installed in the gas chromatograph oven for venting the solvent (Figure 3). The inlet Nj and exit valves were products. The actual samples analyzed came C lippard M inim atic No. M JV-2 electric from wood treatment plants and consisted of the solenoid operated valves. The solenoids were starting and recirculated treating solutions controlled by a Magnecraft No. W 399ACQ which were 5% technical PCP in aromatic SOX-2 delay timer adjustable from 0.1 to 2 solvents. Sludge from the bottom of the minutes with a 10-turn 650 Kfl potentiometer. A recirculation tank was also analyzed. Ion delay of 1.75 min was normally used. The exchange separation of PCP and hydroxypoly- specially designed precolum n vent system chlorodiphenyl ethers (HO-Clx-DPEs) from the allowed for venting of chlorinated solvents and To Vent 74; To r \ C oulom etne Ceil VJ !----------------------1 I Fvrnoce j ,C; ! 8 5 0 *-9 0 0 * C I a I l ------------------------ -1 (K) (6 > ( H ) \_____ _ nanl L To G.C. A n o ly r col C olu m n I____________ I To 0 g R o to m e te r To Ng R otam eter Figure 2 - Combustion unit assembly. A. 1 2 /5 quartz ball joint; B. 1 8 /7 quartz ball end socket joints; C. outer tube-6 mm X 5 mm I.D. quartz tube, inner tube-4 mm X 2 mm I.D. quartz tube; D. silicone plug; E. 2-way ball valve; F. quartz wool; G. 1/ 4 " S.S. Swagelok plug drilled to fit 1/1 6 " tube; H. 1 X l / 1 6 x .0 0 9 I.D.S.S.tube welded into plugs as shown; 1.1/ 8 " S.S. Swagelok plug drilled to fit 1 /1 6 " tube; J. 1 /1 6 " S.S. tube welded into side of plug I; K. 1 /8 " Whitey toggle valve. Graphite ferrules were used throughout assembly. Antenori industrial Hygiene Association JOURNAL (40) 9/79 0007602 . ; .rt i- V* M.it* ':*- `"'.S ? :' 5 ' :E'1 isk; ;s#?pr: G. C. In jection Port Ci Figure 3 - Precolumn vent assembly. (A) glass injection port liner; (B) precolumn packed with 7% Dexil 300 GC on 1 0 0 /1 2 0 Chromosorb W AW. 6" X 1/ 8 " O .D. stainless steel tube; (C) vent assembly, 3 " X 1/ 8 " O.D. X 0.03" I.D. S.S. tube with 1 /1 6 " X 0 .0 4 " I.D. S.S. tubes brazed into it; (D) 1 /8 " S.S. Swagelok plug drilled to fit 1 /8 " tubes and brazed. petroleum distillates from the gas chromato graphic system during analysis for the chlorinated contaminants. A 4' X 1/8" glass column or a 5' X 1/8" stainless steel column with 1% Dexsil 300 GC (Dexsil Chem. Corp.) on 100/120 mesh acidwashed Chromosorb W AW (Johns-Manville Corp.) was used for GLC analyses. The column oven was programmed from 200-350 C at 8C/m in with an initial 5 min program delay. Mass spectral analyses were performed on a Varian CH-7 GC-MS with a System Industries 150 data system. The samples were introduced into the MS via a single stage glass jet separator from a Varian 1200 gas chromatograph using the columns previously described. The identity of the Clx-DBDs, Clx-DBFs, and Clx-DPEs were confirmed by comparison to published spectra and spectra of authentic samples. The presence of HO-Clx-DPEs was confirmed by GC-MS using the methyl ethers formed from the reaction with diazomethane. Concentrations of CL-DBDs, Cb-DBDs and Cli-DBD were also determined by mass spectrometry. This was achieved by integrating the molecular ions of the Clx-DBDs and comparing them to the integrated area of the ClDBD m olecular ion reco n stru cted gas chromatogram. This method is referred to as Selected Ion Sum mation Profile Analysis (SISPA). sam ple preparation Several methods commonly used to extract ClxDBDs in PC P were evaluated. An ion exchange m e th o d " 5'161 was a d a p te d w ith som e m odifications. In order to reuse the ion exchange columns, the phenolic material was stripped from it, and the colum n was regenerated. Samples were obtained from two local wood treatment plants. Several small pieces of the solid technical PCP weighing about 1 kg were ground in a ball mill, and 5 gm of the resultant powder was dissolved in 100 mL of benzene. Five milliliters of this solution or of the 5% PCP wood treatm ent solutions were used for analyses. Sludge samples (5-10 gm) were 0007603s i t An. Ind Hyg. Assoc. J (AO) September. 1979 dissolved in K suction throu. filter was rinse mL of 1:1 ben evaporated to anhydrous N evaporated tc benzcnetmeth Dowex 2 Ik to the hydro:NaOH. Before was washed methanol, anc The column v using the same 0.24 M HC1 ii NaOH washe milliliters or le was added to the resin. The benzene:m e benzenermet: neutral compr steam bath. The residue in 5 mL benze. samples from required add: chromatograp in 25 mL be concentrated removed by p benzene was benzene extrac distilled water. mL isopropar. break the cm Americin ktduslnal h- gating >s and he Cis ti g a s i to as .alysis =t Clx:hange some e ion al was was wood of the z were sultant nzene. 3PCP 3 for we - 379 TABLE I Polychlorodibenzo-p-dioxin and Polychlorodibenzofuran Concentrations* (ppm) in Boulton Wood Treatment Solutions G C -M S Method Microcoulometric Method Sample C I.-D B D c l -d b f + C I.-D B D CL-DBF1 + CI.-DBD C I.-D B F " + C I.-D B D Fresh 5% Treatment Solution 26 S5 519 76 1036 136 Recirculated 5% Treatment Solution 29 57 610 50 1385 62 Sludge 21 47- 963 1965 AConcentrations are normalized against PCP content of the solutions. "Average of four determinations. dissolved in 100 mL of methanol and filtered by suction through a Whatman no. 42 filter. The filter was rinsed with 100 mL of benzene and 200 mL of 1:1 benzene : methanol. This extract was evaporated to less than 100 mL and dried over anhydrous NajSOa. The solution was then evaporated to dryness and dissolved in 5 mL benzene:methanol ( 1:1). Dowex 21k ion exchange resin was converted to the hydroxide ion form using 100 mL IN NaOH. Before loading each sample, the column was washed with 150 mL water, 100 mL methanol, and 50 mL benzene:methanol (1:1). The column was regenerated between samples using the same procedure but included a 400 mL 0.24 M HC1 in benzene.-methanol (1:1) and IN NaOH washes before the water rinse. Five milliliters or less of sample solution noted above was added to a 100 mm X 19 mm column bed of the resin. The column was eluted with 75 mL of benzene:m ethanol (1:1). The collected benzenetmethanol fraction containing the neutral compounds was then evaporated on a steam bath. The residue from technical PCP was dissolved in 5 mL benzene without further treatment, but samples from the sludge or aromatic solvent required additional cleanup before column chromatography. These samples were dissolved in 25 mL benzene and shaken with 25 mL concentrated HjSO. The benzene layer was removed by pipette, and an additional 25 mL benzene was used to reextract the acid. The benzene extracts were washed twice with 50 mL distilled water. Ten milliliters of 2% NaCl and 2 mL isopropanol were added after shaking to break the emulsion. The extracts containing Amencan Industrial Hyjient Association JOURNAL m 9/79 aromatic solvent were then concentrated to approximately 5 mL while the sludge extract was concentrated to 2 mL. One-half of the extracts was placed on top of a 30 gm (19 mm I.D.) column, slurry-packed with activated alumina (Fisher A-540; 4 hr. at 450C). Approximately 0.3 mL benzene and 10 mL pentane were used to rinse the sample onto the column. The column was eluted with 400 mL of pentane, 100 mL of 10% benzene in pentane, and 400 mL of 25% benzene in pentane. The ethers and aromatic petroleum solvent were found in the first two fractions, and the dioxins and furans were in the 25% benzene fraction. standards Standards for CL-DBDs and CL-DBDs were not available, so Clg-DBD standards were used. Temperature programs were set such that the retention times of the analytes of interest were as close as possible to the Clg-DBD standard._A correction factor was applied to account for the differences in percentage chlorine. The ratios of the molecular weight of Clg-DBD divided by eight to the molecular weights of Clx-DBD divided by "x" were used. Using the ratio method described above, comparison of a 1,2,3,4-CLDBD standard to Clg-DBD in the 100-200 ng range was within 5% of the expected value. results and discussion Complete separation ol the neutral Clx-DBDs from the phenolic HO-Clx-DPEs which was required for quantitative gas chromatography was achieved by ion exchange chromatography. The Clx-DBDs were eluted from the Dowex 21K 0007604 o "fas-ife- cn :ti ro : rj- M ; .^ : :T'rt - v: & & -t.'. * -#... ` .A.* r._0.- iafrpr * .. . -* t - -. I -t u rU^ -v kr:*- -Vi..---. - i . #. I.**1 column with 25 to 50 mL 1:1 benzcnc:methanol. When aromatic solvents were present some tailing occurred, and a total of 75 mL of solvent was used to assure complete elution of CLDBDs. The separation of the Clx-DPEs from the ClxDBDs and Clx-DBFs in the technical PCP was achieved by alumina column chromatography. Pentane containing 10% benzene was used as an eluting solvent to remove the Clx-DPEs. The gas chromatogram showed that very little Cl?-DBDs or CL-DBDs were present in this fraction. The absence of a molecular ion cluster based at (m/e 388) resulting from CL-DBDs in this fraction confirms that these compounds were also retained by the alumina column. Elution of the dioxins and furans was achieved subsequently by increasing the polarity of the solvent system to 25% benzene in pentane. The recovery for the analytical procedure was measured using CLDBD and found to be 85%. The limit of sensitivity for the method is 10 ppb. Since the microcoulometric detector response is based on the amount of chlorine present, the levels of chlorinated com ponents were determined by comparing their response with that observed for CL-DBD. When PCP was measured against this standard the results obtained were in good agreement with those in which PCP was used as the standard. Analysis of Clx-DBDs in PCP samples which were obtained from two Boulton wood treatment plants are shown in Table I. All the contaminants were measured relative to PCP. The chromatograms showed the presence of three CL-DBDs, two CL-DBDs, and one CLDBD. The interference from a CL-DBF in the determination of CL-DBDs gives a value that is a b o u t a tw o -fo ld fac to r high in the microcoulometric method when compared to the GC-MS method (Table II). The GC-MS is considered to be more reliable in the case of such interferences because it uses the molecular ion or other specific fragment ions of the compounds in the q u an titativ e determ ination. These measurements are used for the CL-DBDs to make the com parison shown in Table I. Analyses of the other Clx-DBDs by GC-MS shows that Clx-DBFs are generally not resolved from the Clx-DBDs under our gas chromato- t a b l e II Determination of Clx-DBFs and Clx-DBDs Residues* (ppm)11by G C /M S Single Ion 1.5% of thc thc ion clus- Monitoring Profile Analysis (SISPA) parent ion oi C h lo rin e s 6 7 8 D io xin 29 524 1306 Furan 20 91 18 Total 49 615 1324 M C D c Value 55 610 1385 ` From recirculated 5% treatment solution. ` Concentrations normalized against Pentachlorophenol (PCP) content in the solution. cGas Chromatography - microcoulometric detector. was compart .<56 using isolution. The appa Cl-DBD c solution rei: due to conve ;X0-HO-CW-D graphic conditions (Table II). However, the relative ratio of CIt-DBFs to CL-DBDs is lower than the CL-DBFs/CL-DBDs ratio and lower still for CL-DBF/CL-DBD. All analyses for the ^ up od treati; j other factor i PGP in the ! so'upion. T t distinction Clx-DBDs, however, are somewhat higher when measured by the microcoulometric detector, and the sum of the Clx-DBD and Clx-DBF concentrations measured by GC-MS cor respond more closely to the values measured by relatively h: DBD in the may tend t solubility in the microcoulometric detector. In contrr. When normalized against the PCP content, ; process, it the CL-DBD level was 34% higher in the I DBD/PCP recirculating PCP solution than in the fresh PCP the Cellon v solution. The CL-DBD content of the sludge was where this- 90% higher relative to the fresh solution and 42% dramatic di higher when compared to the recirculating 5% ; DBD from PCP solution. Similarly, the CL-DBD levels ` a low vola were 18% higher in the recirculating PCP j ether, mus solution and 86% higher in the sludge when possible rc compared to the fresh PC P solution. Because \ dioxin fon replicate analyses were not performed for CL- j relative sol DBD content in the three samples, not much can j these two p be said about their relative concentrations dioxin in er except that major differences were not observed between the samples. In a similar study,"1' it has been shown that an I1 the process incorporai accumulati increase in the level of CL-DBD in the processing solution occurs. The present method, appears to accumulate however, should be more accurate since a clean and more t separation between the predioxin, 2-HO-CL- hand, appr DPE, and CL-DBD was achieved by ion exchange chromatography; thus, the use of a rather variable correction factor was not and this co theCL-DB impregnati required to account for thermal conversion of the 2-HO-CL-DPE to CL-DBD. Interferences from CL-DBF can occur during gas chromatographic analyses of OCDD. This component, however, was determined to be < ( ( acknowle We are ino L.D. McF 000760520 Am. bid. Hft. Assoc. J (40) September. 1979 Amencifl lpdus~ ll) s on O 1 Valu* 55 6)0 ' 365 lend (PCP) ver. the is lower d lower s for the er when tor.and x -D B F S corured by ornent, il ,e 'CP :ge was id 42% ing 5% levels PCP when ecause Jr CUchcan ations served hatan n the -thod, clean D-Cl*y ion : of a is not on of unng 1979 l.57r of the OCDD by GC/M S/SISPA using the ion cluster based at m/e 440 which is the parent ion of Clj-DBF. The CU-DBF ion cluster was compared to the CU-DBD ion cluster at m/e 456 using the recirculated 5% treatm ent solution. samples of the PCP residues. This work was supported by the U.S. Public Health Service Grant ES-00210 from the National Institute of E nvironm ental H ealth Sciences. This is Technical Paper No. 4454, Oregon Agricultural Experiment Station. The apparent increase in the CU-DBD and CI--DBD content in the recirculated PCP solution relative to the fresh solution could be due to conversion of the appropriate predioxins, 2-HO-CU-DPE and 2-HO-CU-DPE, during the wood treating process. It could also be due to other factors such as selective deposition of the PCP in the wood leaving a dioxin enriched solupion. The present data does not allow a distinction between these alternatives. The relatively higher levels of CU-DBD and CUDBD in the sludge suggests that these dioxins may tend to concentrate because of their low solubility in the petroleum distillates. In contrast to these results for the Boulton process, it has been reported that the CltDBD/PCP ratio is 36/1000 in the sludge from the Cellon process compared with technical PCP where this ratio is about 1/1000.031 This dramatic difference in the relative level of CltDBD from the sludge of this process which uses a low volatile carrier vehicle, e.g. di-isopropyl ether, must again be viewed in terms of the possible roles of the chemical mechanisms of dioxin formation in these two processes, the relative solubility of the dioxin formation in these two processes, the relative solubility of the dioxin in each of the solvents, and the effects of the process with respect to the amount of dioxin incorporated into the wood. However, CU-DBD accumulation in the sludge from either process appears to be significant, and the CU-DBDs also accumulate in the sludge. The lower chlorinated and more toxic CU-DBD isomers, on the other hand, appear not to concentrate in the sludge, and this could be due to the higher solubility of the CU-DBD isomers which may also allow their impregnation into the wood. acknowledgem ents We are indebted to J.H. Baxter and Co. and the L.D. McFarland Co. in Eugene, Oregon, for references 1. U.S. International Trade Commission: Synthetic Organic Chemicals United States Production and Sales. USrtC Publication 775:185 (1974.). 2. Bevenue. A. and H. Beckman: Pentachlorophenol: A Discussion of its Properties and its Occurrence as a Residue in Human and Animal Tissues, Residue Rev. 79:83-134(1967). 3. Schwetz. B.A., P.A. Keeler and P.J. Gehring: The Effect of P urified and Com m ercial Grade Pentachlorophenol on Rat Embryonal and Fetal Development. Toxicol. Appl Pharmacol 23:151161 (1974). 4. Buser. H.R.: Polychlorinated Dibenzo-p-dioxins. Separation and Identification of Isomers by Gas Chromatography-Mass Spectrometry. J. Chromatogr. 7 74:95-108 0 9 7 5 ). 5. 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-pdioxins. po. 55-69: In. Chlorodioxins -- Origin and Fate. E.H. Blair. Ed.: American Chemical Society. Advances in Chemistry Series. 120 (1971). 6. Registry of Toxic Effects of Chemical Substances. Vol. II. National Institute for Occupational Safety and Health, Cincinnati. OH. 648 (1977). 7. Langer, H .G .. T .P . Brady and P .R . Briggs: Formation of Dibenzo-p-dioxins and Other Condensation Products from Chlorinated Phenols and Derivatives. Environ. Health Perspect. 5:3-7 (1973). 8. Gray. A .P.. S.P. Cepa. J J . Solomon and O. Aniline: Synthesis of Specific Polychlorinated Dibenzo-p-dioxins. J. Org. Chem. 47:2435-2437 (1976). 9. Fanta. P.E.: Ullman Synthesis of Biaryls, 19451963. Chem. Rev. 64:613-632 (1964). 10. Gilman, H. and J .J. Dietrich: Halogen Derivatives of Dibenzo^p-dioxin. J. Amer. Chem. Soc. 79:14391441 (1957). 11. Pohland, A.E. and G .C . Yang: Preparation and Characterization of Chlorinated Dibenzo-p-dioxins. J. Agr. Food Chem. 20:1093-1099 (19721. 12. Sandermann. W .. H. Stockmann and R. Casten: Pyrolysis of Pentachlorophenol. Chem. Ber. 90:690692(1957). 13 Arsenault. R.: Pentachlorophenol and Contained Chlorinated Dibenzodioxins in the Environment. Proceedings of the American Wood-Preservers Association, pp. 1-26. Atlanta. Georgia. April 25-28. 1976; 1625 I Street NW, Washington. DC. OT C3 Amencin Industrial Hygiene Association JOURNAL m 9/79 000760G .-- 14. Warner. C .R ., M .C . Johnson. D.G . Prue and B.T. Kho: A Device which Permits Selective Purging of th Solvent and Other Components from a GasLiquid Chromatographic Column. J. Chromatogr. 82:263-268 (1973). 15. Crum m ett. W .B. and R .H . Stehl: Determination of Chlorinated Diben20-p-dioxins and Dibenzofurans in Various Materials. Environ. Health Perspeti 5:15-25 (1973). 16. Jensen. S. and L. Renberg: Contamination in Pentachlorophenol: Chlorinated Dioxins ana Predioxins. Ambio 2:62-65 (1972). Accept) January 31. 1979 -J CJ1 05 Cl Short-t! monitor fiazardc which n industri materia reviews applicai industri from th ` : are dis< researc ! discuss Sho Injuries from liquids under pressure injected into tissue may lead to amputation . . . "TechData Sheet 79-01" (February, 1979), from the Civil E ngineering L aboratory, Naval Construction Battalion Center, PortHueneme. CA 93043, gives "Safety Precaustions for Use of Airless Spray Equipment". The high pressures (up to 4,000 psi) pose a potential hazard to, users. As stated in the bulletin, "Paint applied by airless spray emerges from the gun at sufficiently high pressures to penetrate the flesh. This has frequently resulted in amputation of hands or fingers, either from direct-impact injury or the toxic effects of the paint, despite medical treat ment. Invariably, the cause of such serious injury is lack of knowledge about the uniquely devastat ing effect the airless injection injury can have. Accidents most frequently occur during the wiping of gun tips or their removal for cleaning. The injured person may feel only a pinprick (possibly with no loss of blood) despite serious tissue damage, which may subsequently lead to amputation. "In an effort to prevent such accidents, the Consumer Product Safety Commission has ruled that all airless spray guns sold must be equipped with tip guards to keep hands away from the tip. A recent Civil Engineering Laboratory survey revealed that continuous dripping and spitting of the paint that result in nonuniform application. Also, beca use of the dripping painters tend to wipe off the guard close to the spray tip, which is not safe. The survey also revealed some painters thought the plastictip guards were there to protect the tip of the spray gun rather than th user". (T hese co m m en ts apply to a l l ty p es of paint/grease guns operating at high pressure. The seriousness of this minor-appearing injury was previously reported in "Notes" June, 1975, based on an article in Jama. March 3, 1975). Reprinted from Navy Environmental Health Center Occupational Health holes j air r ! sha. i JOAN M Institute 55 0 Firs | introd In ere?.: I domes to a rr shale c To mi [ accom ( made arise. pilot y raateri similat coal t: 0007607 7802 The which arom poun napht anah biolo; Thus occur based <22 An. bid. H/t- Assoc.J (40) September, 1979 Amenta *ro- PUBLISHER-SCOPE: JOURNAL CODE: MJ LO C A TIO N PHOTOCDPV DCAIic p t r n n ----- j DATE -------P!" " JO U R N A l VOLUME AUTHOR TITUE 6906B S 33 pQLY CHL0R0 DI BENZO- P-DIOXINS IN - - r_- THE DETERMINATION OF POLY SOLUTIONS IN: * 25 pages PENTA CHLORO PHENOL AND WOOD TREA1 ZER M; GRIFFIN D LAMBERTOM_d: / R B O G A S l ^ B : ^ N M A N ^ . ^ ^ , CCENNTT . . (OJRREEGG.. STAFF. and Reading Roam table. o ep ` a g r i c . c h e m . . e n v i r o n h e a l i h SCI UNIV.. CORVAI.LIS. OREG. ' U1S9A7 .9 . 8 1 6 - 8 2 2 . C o d e n : AIMA* AM IMP HY ASSOC J l a n Q u a g e : ENGLISH 'U imoiu,entec! to m easure l e v e l s of pp ooAI. vnccnmnl ylootriroc.r,AtritlU. *eenn`"r*oo"--p"p-'-d``dirtOonKl i!Mn S .. aa n" d pP o I yc:li I o r o O I Mwmo moJf ut rr ea an tsm e n- tn 3 N T M teak ^ n t a c n l o r o p n e n o . s o l u t ons u se d ** When n o r m a l i z e d a g a i opHeno. (PCP) 0 . p . fl, o x l n le v e l was content. o n ' t l * . ... t n e 1r e s , , 3 -17. h i g n e r i n t h e '| C | ^ U PCP s o l u t i o n , a n d i n * " - c o n c e n tra tio n increase a s im ila r n.ci --- these im p u ritie s in tec .p o ss ib le H ealtn Hazards. it was 90% H ig n e r. A sm aller no hb cs ee r v e d for the p resen c e of clftOU F 1r CHARGE ----> ___ / ' // / -_--_- * " THE LIBRARY DEPTS. 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This institution reserves the right to refuse to accept a copying order if, in its judgement, fulfillm ent of the order would involve violation of copyright law.______________________________________________________________________ TO X o o o />2lU Z . \ DEBATING SCIENTISTS OF 8 NATIONS AGREE PESTICIDE 2,4,5-T IS SAFE! !opy for Personal Use Only .ThLiorary, University of California atRerkelrv poses no cancer hazard to humans or animals rrt does not cause abortions in women (as claimed) poses no practical environmental harm i Safe...that's the consensus of 59 scientists. Top world experts on pesti cides who journeyed to Washington. D.C. recently from seven countries and 20 U.S. States to 'dispute and resolve' the controversy surrounding the currently banned 2,4.5-T, the pesticide widely used to protect forests, cattle and weeds. Scientific findings of the unique, first-ofits kind Dispute Resolution Conference sponsored by the American Farm Bureau Federation run counter to the position of the Federal EPA which placed a tempo rary ban on most uses of the pesticide in March. To those who still believe 2,4,5-T should be banned we commend the just re leased Conference report. Especially the scientific conclusion on the Alsea, Oregon epidemiology study on which the EPA based its action to ban. The scientists agreed that the report has such deficiencies that no conclusions are possible with regard to alleged abortioncausing effects of 2,4,5-T. We compliment the scientists who took the necessary time to resolve issues now restricting the use of this important agricultural tool. The participants came from the fields of medicine, toxicology, chemistry, ecology, agriculture. They represented the scientific concern of Canada, Italy. Sweden, New Zealand, Switzerland, France, Germany and the U.S.A. But we wonder why many of the evervocal minority who seek a final end to the use of 2,4,5-T and other pesticides declined to atte nd or to join in the scientific dispute ... certainly an ideal forum for arguing their case with the world's scientific experts. The full Dispute Resolution Conference report has been mailed to Members of Congress and other government officials ...we urgently direct their attention to this decisive, truly scientific consensus ...that 2,4,5-T is safe when properly applied. It's important to understand that there is still no scientific reason to prevent the pesticide's use by literally hundreds of thousands of Americans who have de pended on it for 30 years...in growing their crops, cattle, timber. For more information on the Conference findings, write American Farm Bureau Federation, 225 Touhy Ave., Park Ridge, IL 60068. THE DOW CHEMICAL COMPANY MIDLAND, MICHIGAN 486X0 7805 'Trademark ol The Dow Chemical Company 1330 S C IE N C E . V O L . 205 5e^. I 3 It 3 Uj 794930 if!& D REPORT D O W C H E M I C A L U.S.A. R E S TH `i : . ' .w thin 1h Dow Chemical Com pany only andE n v i r o n m e n t a l R c s e a r . i l / T u x l i O l u g y R e s e a r c h hl-tMf-lEwTnI-It8K--W-I'I676M69r8--11-T90a--63)0Paorft11! August 20. 1979 LIB SB IUk i II>4 1j 7|-7l0|1|7 . i i Reversal of Immunologic and Toxicologic Effec ta of a Single Exposure of \ i 2.3,7.8-Tetrachlorodibenxo-p-dloxln In Mice I i; 2 I R. P . S h a r m a . R . J. Kociba and P. J. Cehriag ^ m airuuli) IV RAGES IN FULL REPORT 5! r ^ KVV-v' ^ ] ^ jk A Z J L 'jJ /, i & Ai O H b c A u i > 4 . % M 4'~-- 7 < k)ATA REFERENCESS IbbaMafkc a n ^ p ^ i \J Th P* : U m t c im s *> S p in a l V EW w r ev iew I NBK-21-1, NBK-21-3, NBK-21-4 :(A4*r a-- (a -- l i -- n p e > a-- --M-- tmma.) 4* tin t statu* ( I 1 I a-- Ma* CU) - I DESCRIPTIVE SUMMARY WITH CONCLUSIONS: A single oral dose of 10 ug/kg 2,3,7,8-tetrachlorodibenxo-p-dioxin (TCOD) to CD1 mice produced toxic signs involving weight changes and microscopic lesions in liver and thymus, and in hematologic functions. Such exposure also increased spontaneous lymphocyte transformation in cultured splenic cella prepared from treated animals, and reduced transformations induced by mitogenic agents. The effects were apparent after 2 weeks of exposure and persisted until 4 weeks after the treatment. The animals | sacrificed 8 weeks after the exposure showed a definite diminution of severity of , the toxic signs. While the liver lesions persisted at this time, these were less severe <n n*r-r<' And ch* organ weights were approaching normal. Lesions in thymus | were not discernible, even though atrophy of this organ perslstad. lad blood cell ; counts reverted to control values after 8 weeks of TCDD exposure while lymphopenia j and neutrophilia continued. At 8 weeks, lymphocyte transformations, either spoetane j ous or mitogen induced, were similar to those in the control group. The results I suggest that the immunologic and toxic effects of TCDD are reversible in mice in a relatively short period. I RECEIVED < * ICV"^C jU M A X A N D D lfT R ie U T lO W ft* C-MM WiSGQ - ~ -,-ra 8 9 9 R q! * REVERSAL OF IMMUNOLOGIC AND TOXICOLOCIC EFFECTS OF A SINGLE EXPOSURE OF 2 3,7,8-TETRACHLORODlBENZO-p-DIOXIN IN MICE R. P. Sharna, R. J. Koclba and P. J. Gehrlng Toxicology Raaaarch Laboratory Health and Environmental Science*, USA Dow Chaaical U.S.A. Midland, Michigan 48640 Problaa No. 177-0007600 J Pile No. HET-K-66681-(36) Part II of IX f V C003S70 REVERSAL OF LMKl'NOLOGIC AND TOXICOLOGIC EFFECTS OF A SINGLE EXPOSURE OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN IN MICE ABSTRACT A single oral dose of 10 ug/kg 2,3,7,8-tetrachlorodibenro-p-dloxln ^ (TCDD) to CD1 alee produced toxic signs involving sight changos sad microscopic lesions in liver and thymus, and in hematologic functions. Such exposure also increased spontaneous lyephocyte transformation in cultured splenic cells prepared from treated anleals, and reduced trans formations induced by mitogenic agents. The effects vara apparent after 2 weeks of exposure and persisted until 4 weeks after the treatment. The aninals sacrificed B weeks after the exposure sh owed a definite diminution of severity of the toxic signs. While the liver lesions i persisted at this tins, these were less severe la nature and the organ weights were approaching normal. Lesions in thymus were not discernible, even though atrophy of tnls organ persisted. Red blood cell counts reverted to control values after 8 weeks of TCDD exposure while lympho penia and neutrophilia continued. At weeks, lymphocyte transformations, either spontaneous or altogen induced, ware similar to those in the con trol group. The results auggsst that tha immunologic end toxic effects of TCDD are reversible in nice in a relatively abort period. a oc a 05 CO 7809 0003871 * -- i L.--** 1686770 REVERSAL OF IMMUNOLOGIC AND TOXICOLOGIC EFFECTS OF A SINGLE EXPOSURE OF 2,3,7,8-TETRACNLORODIBENZO-p-DIOXIN IN MICE INTRODUCTION 2,3,7,8-tetrachlorodlbenso-pdloxln (TCDD) 1 a highly toxic contaminant < formed in the manufacture of trichlorophenol and in a number of other related compounds (Sehuets et a l . , 1973; Elvidge, 1971; Uoolaon at a l . , 1972). It is one of the most toxic chemicals known (other than biotoxins). The studies on various toxic effects of TCDD heve inclmdod its teratologic effects (Courtney and Moore, 1971), general toxicologic effects (Harris et al., 1973), pathologic effects (Gupta et al., 1973), and other clinical effects (tinkle et al., 1973). It is a potent in* duccr of hepatic microsomal enxymss (Poland and Kendo, 1978) and causes induction of -aminolevulinic acid synthetase (Poland end Glover, 1973). A 13-veek oral feeding study in rats with different levels of TCDD and related toxicologic effects has keen reported (Koclbe et al., 1978). In addition to other toxicologic effects, there have been implications that TCDD may adversely affect the immunological eystan. This rhsmlrsl was shown to be particularly toxic to lymphoid organs, l.a. thymus (BumHoi et a l . , 1972). Gupta et al. (1973) reported that TCDD was toxic to the thymus of rats, mice and guinea pigs, desplts sons selectivity of other organs in these species. Vos et al. (1973) reported the issmino- suppressive affects of TCDD in rats and mice and subsequently Vos mad Moore (1974) suggested that the affects wore primarily oo cellular 0093872 -3 - i u n e function. Thigpen et el. (1975) showed e decreased host sen sitivity to bacterial but not to viral infections. In our recent etudles (reported separetely) we have found a decreased responsiveness in nice and rabbits exposed to TCDD when their lanuae function Is ehsl- lenged with1an antigen. Using a splenic cell culture system, however. an Increased OKA synthesis In lymphocytes from animals exposed to TCDD * was observed. The objective of this study was to determine if the toxicologic effects, particularly the Immunologic ones, are reversible with time. This report describes the time-related effects of e single exposure to TCBD for periods up to g weeks in nice. S ./3 J L a *. - 0003873 -4 - * METHODS Male, CD-I Mice were obtained fron Charles River (Vlllmlugton, Maas.) and vere acclimated for one week. The animals were housed 2 per cs|C, provided free access to feed and water and kept in an environmental 1ycontrolled room with a 12 hour light/dark cycle. At the start of the t experiment, the animals weighed approximately 28 g each. The nice were divided into two groupa; half were dosed with a solution of TCDD in corn oil (containing 5Z acetona) at the rate of 10 tig TCBO/kg and the other half was dosed with an equal volume of vehicle alone . Croups of > 4 animals were sacrificed at 2, 4 and 8 weeks after the treatment. The animals were sacrificed by decapitation and their b lo od was collected for total and differential blood cell counts and also to obtain serum. The liver, kidney, spleen and thymus were obtained and weighed. Spleen was used for lymphocyte cultures whereas other organs were placed in formalin fixative, processed routinely and examined for nicroscoplc alterations. Splenic cell cultures were conducted as described gloowhore (Sharaa at al., 1977). Suspensions of cells obtalnad from these orgeaa were cul tured in triplicate with or without the presence of either phytehamegglutInin (PHA) or pokeveed nitogen (PHM) in micro-culture platee. After 48 hours of incubation in humidified air containing 51 CO^, the cultures ve r s pulsa labeled with 0.5 iiCl of ^H-thymidlne and the incubation c o n tinuad for an additional 16 hours. The cells were harvested, vnebed and 1 2 0003874 *- Ti t=T oai ooi O il to l -5 - activity counted in a liquid scintillation spectrometer The counts - were converted to disintegrations per slnute using a standard quench curve. Alter averaging the replicates for each culture, the stimulation indices with both mitogens (dps with a aitogen/dpe with no altogen) were calculated and the values for animals in aach treatment group were averaged. * Serua 1amunoglobulins were estimated by e l e c t r o - l i u n n s a s a y using carbamylated rabbit anti-souse Immunoglobulin in an agarose gel system. The system was modified from BJerrua et al. (1973) and haa been deacrlbed in detail previously (Sharna et al., 1977). The results are expressed as mean of observations with either standard deviation or standard error of the mean. Values in control and treat ment groups were compared by a t-test (Steel and Torrle, 1960), using a prespecified p*.05 value. 0003875 '-J- jL. -6 - OflW 1686774 RESULTS _,, j.- None of che mice died due co the treatment or showed any overt clinical signs of TCDD toxicity. The growth of treated animals was comparable to that of controls. The body weights, organ weights and orgaa/body weight ratios for selected organs are listed in Table 1. A reduction in f thymus/body ratio was observed in the group sacrificed 8 weeks after TCDD exposure. The only other organ showing a change was liver, its absolute weight and the liver/body weight ratio being increased con sistently at all intervals albeit a significant difference occurred only 4 weeks after treatment. The increase in liver weight wee nearly 18X at the 2-week Interval, rising to 26Z 4 weeks after the exposure, bet decreasing to only 111 by 8 weeks after TCDD treatment. Microscopic examination revealed treatment-related changes in liver and t'. vcui. The lesions wars similar to those described elsewhere (therms et al., 1977) and the incidence at different time intervale is onmenrieed in Table 2. Liver lesions included degenerative or necrotic changes at 2 and 4 weeks after the treatment whereas fibrosis with increase cytic infiltration was apparent at 8 weeks. As seen in Table 2 a progressive Increase in the Incidence of liver effects was observed the tissues were evaluated in a blind randnu faahion. The thymus effects Included regression of c o r n e r ! area, depletion of thynocytee and oc casional pyknotlc foci, but these were seen only in saaples obtained at 4 weeks after the TCDD treatment. Thymic changes were evident 4 w e b s after dosage but not at 2 weeks or 8 weeks (Table 2). Mo changes were observed in the kidneys. 76 PV*' 4jfc 0003876 -7- Hematological examination (Table 3) revealed a significant reduction in the total number of erythrocytes at the 4-week Interval. This change was not noted at 8-week poet-exposure period. Total leukocyte counts showed no change at any of the saapling periods. A consistent increase in the nuaber of segmented neutrophils and a corresponding decrease in the nuaber of lymphocytes was observed at all intervals. Splenic lymphocyte cultures at different time intervals showed that at 2 and 4 weeks after TCDD treatment there was a marked increase in the ORA synthesis in uastiaulated cultures. Although the extant of the increase was similar at these two periods, there was a greater variation at the 4-week period making the difference statistically non-significant (Table 4). At 8 weeks after the treatment, the uptake of t r l t i w was similar for both control and treated groups. When these cultures were supplemented with either TEA or PWM, the resultant mitogen-induced stimulation was decreased at 2 and 4 weeks after treatment with TCDD, but the control and treated groups were similar in this respect at the 8-week post-treatment period. Estimation of serum 1wain oglobulins indicated that a single dees of 10 wg/kg TCDD did not produce any narked change in this parameter at either of the periods of sample collection (Table 5). a :i CteJ 05 j N I] N J, Cfti ^ 7815 0003877 -s- DISCUSSION The results presented Indicate that TCOD in a single dose of 10 ug/kg is toxic to nice and the toxicity as evidenced by decreased thyaus weight increased liver weight, alterations in hematological paraaeters, and histopsthological lesions, prlaarily in liver. In general, these toxic effects seen to be reversible and the reversal nay be evident as early as 8 weeks after the exposure to TCDD. The severity of pathologic changes in the liver was aost pronounced at 1 weeks after the treataent. At 8 weeks after TCDD exposure, the liver lesions were aore of a chronic type, l.e., fibrosis. The incidence of thymic changes was the highest at 4 weeks post-treataent, and even though a decreased weight of the thyaus was noted after 8 weeks, the histologic changes were not observed at this interval. In another study, where the alee were given repeated weekly doses of different amounts of TCDD, a lack of effects on the thyaus after 8 weeks of ex posure was noted (Sharma at al., 1977). These findings indicate that the toxic effects of TCDD to this organ are not only reversible but even with continued exposure there is an adaptation. The single dose oral LD50 of TCDD in male mice is reported to be 114 yg/kg (Vcs at al., 1974). Following a single administration aore than half of the total body bur**n for TCDD Is accsunted for in the liver in a sensitive strain of aouse (Poland and Clover, 1976). According to the data presented by these authors, the concentration of TCDD In the liver 0005878 7816 DOW 1 CS6777 o -9 - of CS7BL/6J alee disappeared at a half-life rate of approximately one week. This value is considerably smaller than the whole body half-life value obtained in rata (Piper et al., 1973; Rose et al., 1976). In view of these observations a major fraction of TCDD in the liver of mice may have been eliminated in 8 weeks (while it may be more persistent in t other species, e.g. rats). If this interpretation is correct, the toxic effects of TCDD in the liver and other organs will be expected to diminish in this time interval. The most convincing effect of TCDD exposure was seen on the splenie lymphocyte transformation in cultures. A single dose of TCDD caused a considerable Increase in spontaneous blast formation in splenic cultures obtained from treated animals. This finding was aiso observed when the exposure of T C D D was repeated once a we e k for 8 weeks (Shares et al., 1977). Although the implication of this finding is not clear, nor is i f mec^r.irs understood currently, this affect is most striking 2 weeks after administration of 10 tig TCDD/kg. After 4 weeks, the effect p e r sisted, although there was a great individual variation in these pa rameters. In another study (data not reported) we exposed adult (4-5 months old) mice to the same dose of TCDD (vs. growing mice in present report) and a similar, although less extensive, effect was seen when the spleens were cultured 2 weeks after the treatment. Another important consideration of this increase in spontaneous lymphocyte treesformation is a decreased responsiveness to mitogenic agents giving rise to m a i l e r scinulacion indices in treated animals. This is indicative of a decreased V n Di V -10- \ 686778 responsivenees of the immune system to antigenic stimulation, alao c o n f i n e d In another atudy (Sharma et al., 1977). The eajor finding of thia atudy is that both the spontaneoua blaat formation and tha mitogen induced stimulation return to normal in 8 weeks after TCDD exposure. This further supports the argiaaent of the reversal of toxic effects of TCDD in mice within a relatively short duration. 1 It la difficult to extend these findings at present to a n accidental huaan exposure to TCDD since it Is well known that considerable species variation occurs in response to this toxic chemical. But tha differ ences in physiological disposition would alter the rata at which the chemical is cleared froe the body and if a aublethal exposure occurs the effects probably would reverse with time. The immunologic effects in particular, were not lasting whereas the hepatic effects persisted longer than the imeunologic effects. Perhaps accumulation of TCDD in the liver mav >>e considered a defensive etUiu.*, at least insofar as the immune* logic alterations are concerned. ACKNOWLEDGMENTS The valuable assistance of Roselle Lisowe and Ruts Kalnins during these studies is gratefully appreciated. 1686779 -11- Thl* report was prepared and submitted by the following Staff Member: Cip-Sz^WwA T p T n h a rn a \,v , R. J.'>ociba, D.V.M., PIup . Aaaociate Scientist Croup Leader in Pathology r / D ate:^ |f> y ^ ft+ P i f Oats: P. il Gehring,, i u ;h ., ph.D. Reviewed By: B. A* Schwets, ])VNf Pb*D< Director, Toxicology Research Laboratory Hea lth and Environmental Sc lanca a, USA Date? 0003S81 7819 REFERENCES B J e m a , 0.J . , Inglld, A., Lovenstein, H. and Weeke, . (1973). Carbaaylated antibodies used for quantitation of huaan IgC. A routine nethod. In, Quantitative T m u n o e l e c t r o p h o r e s i a . (Axelsen, M.H., troll, J. and Weeke, B., Eds.) Universltetsforlaget, Oslo, pp 145-148. I Buu-Hol, N.P., Chann, P.H., and Saaque, C. (1972). Orgeat as targets pf dioxin (2,3,7,8-tatrachlorodlbenao-p-dioxln) Intoxication. Naturvlssaaachaftan 59, 174-175. Courtney, K.D. and Moore, J.A. (1971). Teratology studies with 2,4,5trlehloropheayoxyscetlc acid and 2,3,7,8-tetrachlorodlbenso-p-dloxln. Toxicol. Appl. Fharaaeol. 20:396-403. Elvldge, D.A. (1971). Tbs gas chroaatographic detaralnatlon of 2,3,7,8tetrachlorodlbeaso-p-dioxin In 2,4,5-trichloropbenoxy acetic acid c r u.rs end 2,4,5-tTichlorophenol. Analyst 96:721-727. Gupta, B.M., Vos, J.C., Moore, J.A., Zlnkle, J.C., and Bullock, B.C. (1973). Pathologic effects of 2,3,7,8-tetrachlorodibaaso-p-dioxla In laboratory aalaals. Environ. Health Perspect. 5:125-140. Harris, M.V . , Moors, J.A., Vos, J.G., and Gupta, B.N. (1972). General biological affects of TCDD in laboratory anteals. Environ. Health Perspect. 5:101-110. CO 000 7820 DOW 1686781 -13- Hocib, R.J., K f d i r , P.4., Park, C.N., and Cehring, P.J. (1976). 2,3,7,8-tetrachlorodibenxo-p-dioxln (TCDD): Results of a 13-week oral toxicity study In rats. Toxicol. Appl. Pharmacol., 35:353-374. Piper, W.N., Rose, J.Q., and Cehring, P.J. (1973). Excretion and tissue distribution of 2,3,7,8-tetrachlorodlbenso-p-dioxia In the rat. .; Environ. Health Perspect. 5:241-244. Poland, A. and Glover, E. (1976). S t e r e o a p e d f l c , high affinity binding of 2,3,7,8-tetrachlorodibemo-p-dioxin by hepatic cytoaol. J. Biol. Chen., 251:4936-4946. Poland, A. and Kende, A. (1976). 2,3,7,6-tetrachlorodlbenso-p-dloxln: envlronaental contaalnant and nolecular probe. Federation Free. 35:2404-2411. Rose, J.Q., Rassey, J.C., Ventxler, T.H., Hunaal, R.A., and Cehring, P.J. (1976). The fate of 2,3,7,8-tetraehlorodlbcnso-p-dloxia following single and repeated oral doses to the rat. Toxicol. Appl. Pharmacol. 36:209-226. Schwets, B.A., Norris, J.M., Sparschu, C.L., Rowe, V.K., Cehring, P.J., Eaerson, J.L. and Cerblg, C.C. (1973). Toxicology of chlorinated dlbenco-p-dloxlns. Environ. Health Perspect. 5:87-99. Sharma, P-. P . , Koclba, R. J. and Cehring, P. J. (1977). laaunologlc studies Involving 2 , 3 , 7 :6-tetrachlorodlbento-p-dloxln (TCDD) in nice and rabbits. The Dow Chealcal Conpany., research report. Kor. S 3 321 14 Steel, B.G.D., and Torrle, H.H. (1960). Principles and Procedures of ttitltlci. McCrev-Hill Book Co., Inc., Mtw York. - Thigpn, J.E., Faith, B.E., McConnell, E.E., and Moor, J.A. (1975). Increased susceptibility to bacterial infection as a saquela of exposure to 2,3,7,8-tetrachlorodlbenao-p-dloxln. Infect. laaua. 12:1319-1324. Vos, J.C. and Moore, J.A. (1974). Suppression of cellular laaunity in rats and nice by asternal treataeat with 2,3,7,8-tetraehlorodlbensop-dloxln. Int. Arch. Allergy 47:777-794. Vos, J.C., Moore, J.A., and Zinkl, J.C. (1973). tffect of 2,3,7,8tetrachlorodibenso-p-dioxin on the isaune aystea of laboratory anlaals. Environ. Health Psrspect. 5:149-162. Vos., J . C . , Moore, J. A., and Zinkle, J.C. (1974). Toxicity of 2,3,7,8tetraehlorodlbanso-p-dioxln (TCDD) in C57B1/6 nice. Toxicol. Appl. Phazaacol 29:229-241. Woolaon, E . A . , Thoaas, I. P., and Easor, P.D.J. (1972). Survey of polychlorodlbenxo-p-dioxin content in aelectad pesticides. J. Agrie. Pood Chea. 20:351-354. Zinkl, J.C . , Vos, J.C., Moore, J.A., and Gupta, B.M. (1973). Baaetolnglc and clinical chemistry effects of 7|1|T|HrsrrsrblnTnd1bsnto p dioxin in laboratory anlaals. Environ. Health Perspect. 5:111-118. 0GO \fM o 7823 Tabla 1 ih u i witcm or m a n un Mw M a i n a a t wiimn *a motwiwcut oui. uval i ose * or tcoo (io s/k) Tta A ita r T ra a ta ai: -1 --**cL > A Crono C o n tro l* TroataO C o n tro l* T ro n to * C ontrol T taatat lily W a lg fc t -lai . U nan lai -ia/i Organ V olg h ta a a l T lta lr l a la t io s ta Ootp Walght lo a n _ i t i -- fa /lo A a l L lra r lai ju m a K lO n o ra U ). ))t< HO 0.00*0.01 0 .0 3 *0 .0 1 0.17*0.03 0.11*0.00 0.33*0.10 1.90*0.29 3.00*0.30 0.31*0.00 0.13*0.03 0.09*0.01 0.23*0.03 2.24*0.30 0.44*0.02 0.40*0.07 1 .4 0 *0 .1 0 1.74*0.14 i s t i 0.07*0.02 0.20*0.14 0.09*0.02 0.23*0.00 2.03*0.03 3.00*0.21 0.30*0.03 1.40*0.01 19*4 0 .0 7*0 .0 2 0 .1 0 * 0 .(* 0.12*0.03 0.31*0.00 2 .3 3*0 .2 9* 0 .30*0.44* 0 .44*0.09 1.41*0.07 0*3 42*3 0.00*0.01 0 .0 3 *0 .0 2 0.19*0.02 0.10*0.03 O .lltO .C l* 0.10*0.02 0.24*0.10 0.23*0.03 2.03*0.24 2.20*0.10 3.07*0.31 0.40*0.07 3.47*0.41 0.43*0.20 1.49*0.11 1.32*0.20 N m t S o f 4 lo p o r g ra n p , arara p t a s ta * a t t o r n io * . Oolp 1 la por t r l a t i t a t a a l y H f a aal O a v la tlo a f v etto c s o tto l a t p<.05 ( I a tt' a to a t). 4 TabIt 2 INCIDENCE OF LESIONS IN LIVER AND THYMUS O F MALE MICE TREATED WITH A SINGLE DOSE (10 |ig/kg) O F TCDD AND SACRIFICED AFTER DIFFERENT INTERVALS* Duration After Exposure (Weeks) Control Liverb Thysus 2 0/3 1/3 A 2/3 1/3 8 2/3 0/3 Treated Liver6 Thysus1 3/A 0/A A/A 2/A 3/3 0/3 *Tht t K C l o n i vara exsslned sleroscoplcally in a blind randas fashion. Kidney abonad no rasarkable changa la any of eha groups. Tha nusbsrs lndlcata eha incidanca of laalooa/total ouabar of ansala arsainad in that group. &Ths lesiona in control liver included vacuollaatlon or alight degeneration of hepatic cells. cThe liver in these groups generally ahoved alight to extensive diffuse degeneration or necrosis. ^Cortical atrophy vith depletion of thyaocyt e s -aeon in thysua of ansala sacrificed A weeks after tha traatsant. Tabi* 3 I SELECTED HEMATOLOGICAL Pa U M E T E X S IN TON) EXPOSED MICE Hooka after TCDO 2 4 8 Croup t.B.C. El0^/can Control Treated S.0211.11 8.2110.55 Control Treated 8.7510.40 7.9410.21* Control Treated 8.7510.56 8.0810.38 V.B.C. 2.711.1 2.910.7 1.710.4 2.211.3 2.010.2 2.110.9 Neutrophils Perant Incidence of Lrnphocytea Monocvtes 1517 (405)* 2514* (725) 8317 (2241) 7213* (2088) 2l2 213 1713 (289) 41118* (902) 8011 (1360) 56118* (1232) 111 3ll 2117 (420) 7513 (1500) 55122* (1155) 41122* (861) 312 3ll Eosinophils OlO 112 212 111 112 111 * laiteat* significantly diffrant froa respective control group. * saluas In parentheses Indicata the nun ber par can. I <* CO to CJ1 o o o C) Od CO I t/ Tabi* 4 THTHID1MB UFTAEE BT SPLBKIC LIWfH OCTT gS AMD TRE MITOGEN STIMUIAT10N IMDICES IN M1CB AFTER A SINGLE ORAL DOSE OF IO D g /k g TCDD ( Tlaa Aitar Expoaura 2 aaka 4 aaaka aaka Group Coatrol TCDD Traatad Coatrol TCDD Traatad Controla TCDD Traatad DFM/106 Calla Hithoot any Kltoaao Stlaulation Index** vlth FRA PWM 37,522*12,965 1.99*0.81 12.59*10.43 339,234*119,811* 0.51*0.32 1.08*0.45 30,794*7,638 11.06*2.58 14.11*1.78 268,569*242,920 5.29*2.21 7.06*2.58* 22,860*12,603 1.51*0.51 4.63*0.79 20,386*15,243 1.83*0.47 3.85*1.51 * Maaa * SD of 3-4 m l a a l a par groap. k Stlaadatlon l a i d p a fa tfca praaaaca of n i togati d l v U a d by d p n withont aajr afttogan. faticata algnltlcaat dlffaraaca (p<.03) fra raepectftae control group. 0003888 9 9 t -1 9 - Table 5 SERUM IMMUNOGLOBULINS IN MICE AFTER A SINGLE DOSE OF 10 pg/kg TCDD Group Control Treated Senta Ig Levels* After 2 weeks A weeks B weeks 1.370.23 1.13tO.19 1.13t0.10 1 . 2 0 t O . 13 1.240.07 1.090.16 *The velues ere retlos of totel serua laaunoglobulin to thet of e pooled aouse serua seaple. Mean SD of 3*4 observations per group. 103 CO 03 CO Nl \ 0003889 ^v 5>>0*7S . 8*t7' OO O Therefore, to all mtrnts and purposes, we are unable to ro itinely monitor the environment for the | ossible presence of dangerous levels of i ioxin. We are thus in sotpe senses at th : mercy of the pro Letters ficiency, prudence, care, and honesty of the manufacturers ar d applicators. Our federal and i late bureaucracies have demonstrated I ttle ability to cope comprehensively am! consistently with the regulation pud r lonitoring o f pesti cides. Moreover, ex sting regulations-- crograms per hectare or so (approxi- ' even if these are cor sidered adequate- mately 75 picograms per kilogram in the are only indifferent)) enforced in many This is in response to the full-page ad topsoil) would become incorporated into parts of the countr). And there are a vertisement by the Dow Chemical Com the ecosystem and remain there for a number of indication ;to suggesfthat ef pany (28 Sept., p. 1330) extolling the un considerable period o f time. In fact, the forts to protect our <nvironment are, in tarnished virtues of 2,4,5-T. !I write as ' environmental half-life of this remaining the face of growing 'fuel shortages and one of the 39 " top world experts" that dioxin is-in the neighborhood of 3 years rising inflation, regressing rather than ad were brought together last June`at the or even longer, in other words, perhaps vancing. ! Dow-financed conference upon ' whose 40 micrograms per hectare will still .be In conclusion, perjmit me to suggest " consensus" the substance of this proc present after 4 years, when the next that we leave ourselves too small a mar lamation was based. I participated in the batch is introduced. [ gin o f safety *hen we use 2,4,5-T. We ecology workshop, and therefore ai)v As the applications continue, a steady- are, through the use qf 2,4,5-T. forced to dress myself to the unqualified asser-i state contamination level will in tim deal with a substance--dfcxin--that is lions that 2,4,5-T is " safe!" and that it be attained that fluctuates between 70 dangerous at the ven| limits of detection " poses no practical environmental and 170 micrograms per hectare (or and beyond. Even wi h 2.4.5-T prepared hartn-i 50 to 130 picograms per kilogram of and applied as re ommended, such Our group was primarily concerned topsoil). Various local -animals would doses are,w%ithin about one or two ord"ers with the unavoidable dioxin (2,3,7,8-tet- . as a result be expected to attain body q f magnitude of lethality for some ani- rachlorodibenzo-p-dioxin)- contaminant ' burdens of perhaps 10 to 100 times this 'mals, and even eloper for nonlethal. o f 2.4.5-T and with the potentialenvironmental threat posed by this pernicious poison. As I recall, the group concluded, inter alia, that it has not -yet been estab lished whether dioxin is presently accu mulating in the environment or whether routine use of 2,4,3-T might lead to ad verse biological effects. Moreover, we recognized the need for a routine pro gram of environmental monitoring for concentrption in the soil, that is,- of though still toxic, concentrations. And, perhaps I .3 to 13 nanograms per kilo o f course, that does! not take into ac gram! Indeed, three of several dozen count man's occasional lapses and other cows recently tested in Texas that had foibles. I been grazing on rangelands previously A rT h u r H . W e s t in g treated with 2,4,5-T were found to have School ofNatural Sci nee, up to 60 nanograms per kilogram of d io x-' Hampshire College. in in their fat. It is disquieting, moreover. Amherst. Massachusetts 01002 that macaques have been reported to succumb in a year or less when subjected^/ ''T h e question raised by Westing of potentially dangerous levels of dioxin, to a dioxin body burden in the neighbor- 2.3.7,8 tetrachlorod benzo - p - dioxin but at the same time concluded that pres hood of 340 nanograms per kilogram, a (TCDD) accumulating in the environ ently available analytical methods of de? value thus only 26 to 260 times what can ment would hardly fcppear justified in lection were inadequate to carry out be expected as a result of routine right- -t light of recent studieson this subject. In such a program. So much for " safe!'! of-way treatment vestigations o f the areas of heavy 2,4,5-T Some specific data will prove instruct Injurious of even fatal amounts of di agricultural use in Ti xas and Arkansas live. i j oxin cannot be seen, smelled, or other reveal that the sear:h for TCDD has The 2,4.5-T on the market today con wise sensed by humans or, presumably, been negative. tains between 50 and 100 micrograms per by the other animals at risk. And -sucji In one of the eariic st studies, ShadofT kilogram o f dioxin, if one assumes all concentrations cannot even be recog et at. (/) reported the results of a search goes well during the manufacturing prof nized -in environmental samples (soil, for TCDD in an area af Arkansas where cess. Additionally, the quantity ofdioxin plant, animal) by any but the most ex 2,4,5-T had been used for weed control can be slightly augmented in i aturd quisitely sophisticated equipment oper- on rice for the past 2Q years. Water from through the combustion of 2,4.5-T with * ated by the most highly trained techni the rice fields was held and reused year a yield of perhaps 1 milligram per kil<> cians. Indeed, only a handful of laborato after year. The reservpir used for holding gram and possibly more. The amount of ries in the entire world--presently five in this recycled water ufas studied for any dioxin applied to a right of way as a re-1 the United .States and one each in Switz- accumulation o f TCEjD. Samples of two 'suit of a typical application of 5 kilo erland and Holland--have the capability .species o f fish, boltpm sediment, and grams of 2,4,5-T per hectare would be in to test environmental samples lor con water were examined for TCDD. but the neighborhood of 380 micrograms per centrations of dioxin as small as I to 3 none was detected sing an analytical hectare. Between half and three-quarter^ .nanograms per kilogram. Those several method sensitive to 0 parts per trillion o f this dioxin would rather quickly de-> that make a business of analyzing -such (ppt). A similar stud] was conducted in compose or be widely dispersed (that is, samples charge 5750 to SIOOO or even an area of Texas whie e large portions of within days) and the remaining 100 mi' more for each single analysis performed. a watershed bad l+en sprayed with it)} L- \%r: - V* B, : t ! Hit.- vri*q40t Can****' ** k(n| fk+ltcnnrntvpr#Cj[ Cud* No 291 onAuden' Sonco Cold 2.4.5- T for rangeland brush control over live oak trees 6 months after spraying 20-year period. Water from this water were less than I part per million (ppm). shed was collected in a reservoir. Sam Burning sprayed trees is not a regular ples of two species o f fish, bottom sedi practice, but it may be done whereat is ment, and water from this reservoir were desirable to remove unsightly dead trees. .. examined for TC D D . Again, none was Burning, if desired, is generally done detected at the 10 ppt level. when the trees and brush have become In 1979, Garcia et al. (2) in Texas re dry enough to burn easily. This period of ported on Studies o f the search for time varies from 2 to .5 years after TCDD in the American coot. These stud spraying. Under laboratory conditions, it. 6. T. E. Dixon. **2.4.5TVtitvrt crayfish study in Louisiana'' (Envaonmcnial Protection Agency. Washington. D.C.. 1979). 7. Pat. Taj. them. Newt, 24 October 1979. pp. 16-17. ? 6. j. R. Baur. R. W. Bovey.1.1>.Smith. WeedSet. 17. 367(1969). 9. R. H. Stehl and L. L. Lampartki. Science 197. 1006(19771. 10. FIFRA Scientific Advisory Panel, "Review of noticesof intent to hold FIFRA section6 tbX2) hearingon 2.4.3-T and silvet'* (Environmental Protection Agency. Washington. DC. 1979). tl. R. L. Rawls.ChemEny. AVmj 57.21(12 Febru ary 1979). i ies revealed the absence o f TCDD and has been determined by. Stehl et al. (9) the presence of 2.4.5-T only at the part that 0.00016 percent of the 2,4.5-T in per billion (ppb) level in coot body lis- plant parts when burned might 4k con * 1 , vened -to TCDD. Thus, if there were I Atlantic ilot Springs? Additional studies by Garcia searching ppm 2,4.5-T in the wood being buriled, for TCDD in soiis..lafce sediments, tur , the amount of TCDD produced would be Richard A. Kerr, in his article "How tles, and fish from watershed areas ;in insignificant. is new odean crust formed?" (Research Tpxas where 2.4.5-T hast been applied The speculative assumption made by News, 14 Sept., p. 1115). repeatedly Jsve been negative (J1. None of the san- Westing that TCDD will occur in the en states incorrectly that no submarine hot pies collected over a 13-month period vironment is not supported by the actual springs hive been found in the Atlantic. contained TCDD. studies noted above and conducted in The TACj (Trans-Atlantic Geotraverse) Newton and Snyder (4) conducted the field in the search for TCDD in areas Hydrothehnal Field, a site of hot springs studies searching t ? TCDD in mountain under heavy agricultural use. The re and hydrothermal metal deposits, was heaven feeding i i i areas sprayed with sponses reported jjpiVthc Federal In discovered on the mid-Atlantic ridge at 2.4.5- T . The livers o f the animals weresecticide. Fungicide, and Rodenticide latitude 26"N in 1972 and is well docu analyzed for TCDD. lite ^ninimum de Act (FIFR A ) Scientific Advisory Panel mented irt the scientific literature (/). It is tectable levels ranged frotji 3 to 17 ppt. . Report of 27 September 1979 (/fl) also the first Relive submarine hydrothermal At this level only one sample showed ' support this position. Specific questions field found on any oceanic ridge, a possible positive reading'at--3 ppt by EPA to the panel were related to j Peter A. Rona (readings at that level can 'be caused issues about exposure in rice-growing Atlantic Oceanographic and or influenced by several faetn). All areas, and the panel's response was that Meteorological Laboratories. .other samples were negative. " . . . insufficient data was presented id National Oceanic and Atmospheric ' The negative findings in the search for made available to the Panel in support Administration. .-TCDD in the'environment from agricul .o f the argument that human exposure. Miami. Flithda 33149 * tural uses o f 2,4.5-T support the studies ^ fromi spray drip1and the water environ-: '.j . BeTerem o f Crosby el al. (5), in which they found rqent is likelvto be broad or substantial." 1 . 1. M. R. io tt. R. B. Scon. P. A. Ron*. L. W. that the herbicide formulations exposed Considering the field studies men Boiler. A. i. Najwllk. (intphxi. Rex. Lrlt. I. td natural sunlight on leaves, soil, and tioned here and the evaluation made by . 153 1074); R. B. Scon, P. A. Rom. B. A. McGrefr. M. R. Scoll, Naimrr tLuaJimi lit. glass plates lost most or all of the TCDD the FIFRA Scientific Advisory Panel. H- -101 (1974); P. A. Ron*. B. A. McGregor. P. A. during a single day. would appear that well-qualified scien-' Betzer. G. W. Bolger, D. C. Krause.Orrp-Sra Rrx. 22.1611 (19751: B. A. McGrefor and P. A. One might consider the detection of tists have concluded that the likelihood 2.4,5-T in rural areas as an indicator of o f TCDD residues occurring in the envi-. Roa,J. Grnphrt. Rn. SO. 3107 (17751: P. A. Roa, X. N. Harbison. B. G. Baasinscr. R. B. t-ISron. A. i.'Nalwalk. Grot. Sac. Am. Bull. S7. the possible presence o f TCDD. Two re ronment from applications o f the herbi-.. , 61119761: R. P. Lowell andP. A. Rona.Earth ,, Planer. Sri. Lett. 12. IS (19761: P. A. Rona. cent reports on this subject lend further .credence to the unlikely occurrence of cide 2.4,5-T`is extremely remote. In light o f the recent studies reported ,GopfIi Hex. Lett. S. 99} (I97B). *. ie TC D D in the environment where 2,4.5-T from the United Statei and Europe on In my article. I stated that " . . . no hot has been used. One study wais reported chlorinated dioxins resulting from com-' springs h^ve been found in the Atlantic. by the Environmental Protection Agency bustion, one would conclude that in Jeer-! [but| several different kinds of evidence (6) in which 48 samples o f catfisWcray- tain geographic areas TCDD might be! indicate that they are there." The evi fish, reservoir water, and sediment were found in environmental samples from! dence in the TAG area does not as yet examined. Only one sample of surface sources other than the herbicide ( //) . j include any visual or photographic ob- water contained 0.03 ppb o f 2.4.5-T. In * Etcvl H . Blair: ' seryafions. As long as unequivocal direct another study (7) regarding their food Health and Environmental Sciences. observations are unavailable, the con monitoring program recently released by Corporate Research and Development; sensus seems to be that active hot the Food and Drug Administration, no Dote Chemical Company, springs in the TAG area are a strong pos 2.4j5-T was reported in either 1978 or Midland. Michigan 4R640 sibility, but they have not been found. 1979. --Ritha r d A. K frr The question o f burning spraye I vege tation frequently is raised in dis ussing forestry uses r f 2,4,5-T. It is vei f diffi cult to design any kind of resear :h that adequately covers all the facto s that need to be considered. Studiel con ducted in Texas by Baur et al. (8) have shown that residues o f2,4,5-T in sprayed Krfrrmc Md Ni t 1. L. A. Shaduff, R. A. Hummel. L, Lampanki.J. H. Davidun, Butt. Environ. Comtam. Tnxtmii It. 476 (1977). ; 2. 1. D. Gnrcia and M. J. Rhode*. ibid. 23. 231 (1979). 3. j. D. Gircit, personal communicatKyi. . 4. M. Newton and S. P. Snyder. Bun. iniiwfl, Contam. Toxicol. 20. 74) (IV74). 3. O. G. Crmby and A. S. Wong. Science |9S, 13)7 (1977). Erratami tii thr report by Ballou et ml. `Tumor tocaiioo detened with tadioactivcly labeled mouv ctaal antibody and external acmivraphy*' C16 Nov., p.644).theabstractaboutdhavetrad: Mirria teemtncmrrinnmat tre incited in mire b external y+mr scintiprmjihy with m MV-labeled numochmat 'antibodyspecificInthetumori. Thespecificityofike method wot bwremied by xtchtroetinf the radiation produced be' lti14abeled indifferent reonacUmml antipode of the tome immmm*phibabnclass msthe irnmce-specifix antibody. SCIENCE. VOL. 3ft wt ...I* . .. i l-l-. a, \ . I cost SYSTEM puts affordz Up to 50 tir performs hi with either advantages A compie maintennnr spending in expansion u processors complex prr SYSTEMS i you perform scalar procc 7830 / 5 / 7831 / - Q o ) J\ HN07 OOW 088793 H ealth Hazards STUDY OF 121 D IO X IN -EXPOSED W O RKERS \ SHOW S NO EXCESS DEATHS OVER 30 YEARS A mortality analysis of 121 employees of Monsanto Com pany who were exposed to dioxin 30 years ago revealed "no apparent excess in deaths from cancer or cardiovascular disease." Monsanto announced October 22. According to Barney Wander, director of environmental planning at Monsanto, workers at the Nitro. W.Va.. plant were exposed to the dioxin TCDD March B. 1949. when a relief valve on an autoclave opened, releasing substances used in a trichlomphenvl process. Tt'DD is a trace contaminant which can occur during the production of industrial chemicals such as the herbicide 2.4.9-T. which the Nitro plant was manufacturing. Wander said the follow-up study involved tracing the 121 employees who developed chloracne after the 1949exposure to dioxin. The workers, he said, either worked with dioxincuniaming substances regularly or were involved in the clean-up process. Plant medical records, workmen's com pensation files, and death certificates, for all 121 were traced. According to Monsanto's announcement, the total number of deaths for the group was 32, less than the 46.41 expected when compared to the national average. According to the authors of the study, Judith A. Zack and Raymond R. Suskind, little information on the long-term effects of exposure to dioxin -was available until now, although the short-term effects have been described. Zack is an epidemiologist with Monsanto, and Suskind is director of the Institute of Environmental Health at the University of Gncinnati Medical Center. According to Monsanto, the 1949 accident is the earliest recorded incident of dioxin exposure involving a population of this size. Monsanto said in its announcement that this study will become part of larger analysis of information being con ducted by Suskind and a medical team from the Institute of Environmental Health. The larger study will include not only those workers exposed to dioxin in the accident, but those who worked in the 2.4.5-T operation between 1948, when the unit was started, and 1969, when Monsanto ceased produc tion of the product. Acontrol group of employees who worked in other areas of the plant also will be studied for comparative purposes. According to Wander, a report of the findings of the first study will appear in a forthcoming issue of the Journal ofOc cupational Medicine. Monsanto's announcement indicated that the more comprehensive study will be published late this year or early in 1980. 0000SG7 I /oio i lo ia . 7833 r Mechanism of Toxic Action on Some Target Organs Arch. Toxicol.. Suppl. 2. 291-302 (1979) by Springer-Verlag 1979 Estimation of the TCDD Toxic Potential in the Light of the~e'veso Accident G. Reggiani ~:Rqearch Board. F. Hoffmatm-La Rocfaa 4 Co.i B u d . Ssritagf&ad) m I o err o CO fr* to O CO Ob II, Abstract.[Trwwo y\ ears after the TCDD release in Seveso from the assessment of the acute and midterm effects on the health of the population exposed to known level of contamination the following facts emerged. Chloracne appeared mainly in a small number of children mostly with mild severity and quick recovery. Immunoresponse remained normal and susceptibility to infectious diseases was not increased. Peripheral nervous system was not affected in these children nor were the hepatic functions and the urinary porphyrin excretion. Fetal losses and birth defects remained within the expected rates. Newborn growth and development proceeded normally. Chromosome examinations did not reveal abnormalities in number and patterns. A case of intercurrent death provided for the first time TCDD tissue levels in man 6 months after the exposure. | Key words: TCDD Toxic effects in man -- Tissue levels. Introduction The most publicized accidental release of TCDD, which occurred two years ago in northern Italy neaf the town of Seveso. has prompted a careful health survey of the exposed populatfch. The acute and midterm effects registered have provided a sub stantial amount of information, which may supply some new facts to the still limited knowledge of the TCDD toxic effects on the human beings. The significant aspects which have so far emerged from the observations col lected relate to the skin and neurological lesions, to the functions of the liver, to the growth of newborn and child, to the rate of birth defects and abortions, to the cytogenetical abnormalities, to the immunological deficiency and frequency of infec tious diseases. 7834 00032RR Dermatological findings Particular attention has been paid from the beginning to the skin lesions in general and to chloracnc in particular. Careful and thorough survey especially o f the youn ger groups of the population have been carried out (Tabic I). The repealed screenings have revealed that only a very small section o f the most sensitive group o f the population has been affected by a sufficiently high quantity of ct9 o e o oG ca '3 J eno 1 ott s J .c i Xa U V v/l cc 25*J u27. U 'J O E1J 3 7 </* 3** oe cc vc*> c 'y7 'V7. uu i y Eo *? 3^ * c0 c IA e '7u u ED t? 3t i auitr/i.t 5n r. 2it c `j j >y 3oa 3 . c r>~ s i/t Wt e eI o u / 7* E y Eu 7 i* 9 e Oe c 3 .3 ' a euo U t= / a z <t i >, c. a fX uI. 3 t-'Himmkm of llit TCDD Toxic Polenli.il the chemical so as to produce chloracne. The lesions have been o f mild severity and recovery occurs rather quickly in the great majority o f cases. Only a few o f the cases, which were directly exposed to the chemical at the moment or the explosion, present, still today, the typical skin lesions (Fig. I) and only in those parts o f the body (earlobe, armpit) which can be considered a defining characteristic o( the exposure to some halogennted aromatic compounds. The most recent screening of the schoolchildren has disclosed only 6 new cases o f chloracne and a reexamination of 75 chloracne cases discovered during former screenings and followed up for 24 months (9) has shown that there is a shift in the severity o f the skin lesions towards improvement and healing. The frequency o f new cases o f chloracne is therefore decreasing and the affected cases are improving. The incidence o f the puberty acne and its course were not affected by the pres ence of T C D D in the environment. The frequency o f chloracne cases disclosed by the survey amounted to 0.6--1.2% through the whole period and remained therefore close to the 0.1--0.4% o f chloracne cases identified during the same period o f time in other cities o f northern Italy. All cases with skin lesions pointing to T C D D exposure and absorption were given thorough clinical, paediatric, neurological and haematological examinations. Data so far available do not reveal any systemic pathological conditions. In some o f the cases with declared chloracne. the immune capability was also tested as will be related later. o in in Co K> 00 Ul in co 03 *> 0003259 ><4 Clinical Chemistry Findings li. Ktfiiani A large buttery of laboratory tests have been included in the design o f the survey in order to c o v e r those functions which were possibly impaired by the T C D D effect as an integral part of the inspections by other means and devices of the anatomical and functional conditions of organs and systems. The available data (8) refer to a group o f adults members o f the concerned population who have been submitted to repeated laboratory analysis, i.e. 447 inhabi lants of the Zone A with an average T C D D contamination o f SO ug/m 2, 362 o f the Zone B with an average of 3 ug/m2 and IS6 plant workers o f the factory where the accident occurred. No damage o f the blood cells and their forming organs as well as o f the kidney functions could be detected. As for the hepatic functions the serological analysis revealed in about 10% o f the Zone A and Zone B groups a slight raise in the values of two enrymes which express hepatocellular damage (SG O T and SG PT) and o f one enzyme (yG T ) which may express cholestases, liver regeneration and drug metab olism induction. Alkaline phosphatase and serum bilirubin were not increased. In the group of the lemesa plant workers and employees there were a few cases with a modest rise of the transaminases and yG T values but there was no prevalence for the highest risk workers who actively participated in the production. A transient hepatomegaly has been found in about 8% o f these people, which was never accom panied by jaundice or any other overt and extensive signs o f liver insufficiency. Chloracne was not observed in these cases. Urinary porphyrin excretion from T C D D exposed people in Seveso did not show any increase. Recently an abnormal porphyrin pattern without increase of the excretion ( I I) has been observed in a small group o f people by the thin layer chromatography method. It is not yet clear wheth er these changes are related to an enzyme defect or to a deficiency in the enzymatic activity. From December 1976 until M ay 1977. 47 employees o f the Roche Companies in Basel und Zurich and 12 employees o f a commercial cleaning firm in Zurich, all males in normal health conditions in the age o f 26--52 year, were sent to Seveso to carry out the decontamination o f the soil and the houses o f one part o f the Zon^A (~ 20 ug/m2 T C D D ). They were provided with protective overalls, gloves, boots and gasmasks and instructed to keep strictly to the hygiene rules and measures as laid down for them. The medical examination o f these people carried out before, during and after the assignment in Seveso did not reveal any dysfunctions o f systems or organs nor skin lesions of any kind. Blood count and liver function tests (alkaline phosphatase, G O T and G PT) always remained normal. Neurological Findings Samples o f the population involved, comprising a total o f 625 adults and 224 chil dren below the age of 15, 52 o f which had skin lesions, were examined by neurolo gists. Clinical symptoms of acute pathology o f the central and peripheral nervous system were not observed except those that could be explained by factors other than TCDD. 0920000 9es DOW 553286 Ilslimalm o f ihc TCDD Toxic Potential I*1! Suhclinical sinus of impairment (reduction of the sensory and motor conduction velocity o f one or two nerves) were delected in 10% of the people belonging to the area of highest T C D D contamination, which could not be related to any other factor. There was no correlation between the occurrence o f the skin lesions and the neurological findings. Comparison with a suitable control group after random or active selection has not been performed. Abortions, Congenital Malformations, Child Growth The incidence o f abortions, o f the congenital malformations and the somatic and mental development o f the children born after the accident have also been included in the health survey ( I , 12). The women pregnant at the fnomenl o f the accident and those in child-bearing age are considered a high'risk group o f the population. Until now, the incidence o f abortions o f the second six months or 1976 and o f the whole 1977 has remained unchanged in respect o f the incidence o f the previous years (Table 2) and well below the incidence o f 15--20% o f ail pregnancies generally, ac cepted for the western countries. A closer analysis o f the chronology o f the abortions for the months which immediately followed the accident and in correlation with their geographical distri bution in the three Zones at different level o f contamination shows that (Table 3) during the third quarter o f 1976 the incidence of abortions for Zone A (~ 50 ug/m2) was 16.46%; for Zone B (~ 3 pg/m 2) it was 10.63 per cent and for the more distant Zone R (sporadic contamination) it was 9.67. The incidence increased in the fourth quarter o f 1976: 21.27 per cent Zone A ; 16.72 per cent Zone B and 11.60 per cent Table Z. Incidence of stillbirths, spontaneous and induced abortions talc per number of calculated pregnancies Tnwnship 1973 % 1974 % 1975 % 1976 % J977 / Cesano Desio Meda Sevcso Avcragc Harlassina fovisio Leniate Muggin Nova M. Scrcgnn Vnrcdo Avcragc Scveso region 9.01 13.34 6.23 10.79 10.39 6.18 9.46 1-82 11.43 1344 13.28 10.30 9.41 9.9 10.89 13.43 3.88 7.84 9.51 5.43 11.05 t.85 1372 15.03 11.09 9.09 9.60 955 8.81 14.38 5.70 12.83 10.23 4.12 14.61 1.87 4.H6 11.72 9.85 11.36 9.05 9.64 9.15 8.91 11.14 9.55 8.97 12 82 1.59 12.67 15 36 8.75 7 02 9.59 9.57 4.06 14.54 8.70 12.09 11.09 3.03 16.56 5.85 I l IX 12.64 12.07 0.34 10.69 10.89 (Worldwide: 15-20% - Lombardy region: 12-15% ) 2% G. Rtffiani Si 'CC55s? c1ia3<_3"rIs-* <n-- S- --; ? n r c ac 4* ,> 3 f*' Q ori --9- i3* ^sCr--* *--* a--*t j&r>, *^*1 >pcj ^* S V 9, C~ 52 3T ^h <_ -- nm M 2 s* 52 i 3>% *- a & -- <C:. r*~. *1 ----wr**.. *-- ra*c --*. <( rt wa -c i< .*'*S-- '-- r, oeoo-- r~ x 3Q i M f l - t f l f- -- 5>c? |f o .jV aa r ,iTn, r i*i t-fr ' te a <. . T --s: rM. 5-- <CnI *?t npt v2 | e- I*h- so ** ac C, ll -cS e3 si S C 3 sI A S 1 ! I If I I 1 1' (= g 6S. 1 1 d j I 1 1 l in the third Zone. A t First sight the figures could be interpreted as showing a relation between incidence of abortions and degree of contamination. Yet if the analysis is extended to the following months, i.e. the first and second quarter o f 1977, there is a marked drop to 12 per cent for the Zone A and an increase of almost 100 per cent (from 11.56% to 19.54%) for the Zone with practically no contamination. The fluctuations observed are therefore related to factors other than the T C D D contami- 0003261 Eitimttion of live TCDD Tonic Potential Tabic4. Frequency of innlforniiilions illIhcScvcvo rone Hole of nialfrniiiiiiK per 100 livo-birilw Seeeto /one 11 tlislricln I .oinbarily feyil' Collaborative perinatal proie USA I9S K ~1965 1976 ( 12 months) l ive-births: 209 Malfotmationt registered: 4 0.12% 1977 (12 months) Live-births; 2774 Malformations registered: 58 1.36% Malformations expected: 6 9 -8 3 2.9% (Fara. 1974) live-births: 502H2 Malformations: 5861 7.67% (Hetnonen, Sloanc and Shapiro. 1977) 291 Wot Id-wide 2.5-5% (Fraser. 1977) nation of the territory. Besides it should be noted again that the frequency of abor tions in the contaminated areas remained well within the normal incidence in Europe (14). The same can be said for the congenital malformations. Prom the data available (Table 4) there were only 4 congenital malformations (two cases of mongolism and two cases of hypospadia) out of 3902 births in 1976, while in 1977 38 malforma tions were notified out of 2774 births, i.e. 1.36 per cent The number on record for the first quarter of 1978 is 3 and the tivebirth* for the tame period 674 which gives a frequency o f 0.74%. These figures are well below those provided in the literature including Italian statistics. In the western countries, it is generally accepted that the average incidence of congenital malformations varies between 2.3 and 3 per cent (14). In Italy, figures of 2.97 per cent for the Lombardy region and 2.32 per cent for the country as a whole have been reported (2). If the observed birth defects are taken to include deformities and anomalies of the visceral systems. Le. malformation of the cardio vascular and genitourinary system, of the respiratory and gastrointestinal tracts, then the rate increases to 7.67% of malformations per 100 liyebirths (4). It is therefore probable that the incidence of malformations registered in Scveso do not reflect the real frequency and that the higher figures collected for 1977 in respect to 1976 are the result of deeper inquiries and do not reflect a real increase. The polymorphism of malformations observed (Table 3) in the newborns of the Seveso region is another element in favour of the assumption that there is no causal relationship between the malformations registered and the chemical. Morphological hcterogenicity of anomalies scarcely supports the presence of a single causal agent It corresponds to the normal distribution of congenita) spontaneous anomalies. The morphological differences of these anomalies is from this point of view convinc ing. Tabic 5* Noniiilal malformation* in Sevcso Typo and piiilcrn l*76 IV77 Pufmonar aplasia AncniTphiilhi Atresia of auditory meatus Concernlal cardiopathy Vesical ectopia UaMrical exstrophy Hydrocephaly Htpospadia - Hrpispadia Alnlonunal malformation Anal malformation Pcdatir malformation Meningocele Neoplasia Detect osteogenesis Down syndrom Syndactyly Cleft palate Diaphragmatic hernia Total * - 4>. - - - ~ 2 - 4 1 1 1 1 1 2 2 1 10 1 2 i 2 j - - 38 IV78 first quarter 2 - 1 -- - - - - 1 1 3 O. Regiiini Table 6. Neonatal malformations in Scvcso. Geographical distribution - association with environmental facto* Zone Zone A ( > 50 pg/m:) Zone U ^ itg/m'l Zone R ( < O ifig /m 1) Districts surrounding the contaminated zone 1977 0 0 9 29 A further aspect examined, the geographical distribution o f the mothers o f the malformed children in the Zones contaminated by T C D D provided another point against an association with this environmental factor (Table 6). A representative group o f children bom after the accident and which today are 1 2 -1 8 months o f age, have been kept under observation. N o deviation from the normal standard o f somatic and mental growth could be detected so far. Infectious or nutritional diseases have not occurred in these children more than it would have been expected in a comparable control group. Embryomorphological Studies Embryomorphological studies were performed in' Lbeck on 34 cases of abortions. In 30 cases the pregnancy had been interrupted at (he gestational age o f 5/6 to 0003262 Estimation of the T C PD Toxic Potential 299 15/16 weeks. In 3 cases a spontaneous abortion followed an intrauterine death at the developmental stage o f 8, 13 and 18 weeks respectively. In a 4th case o f sponta neous abortions no embryo was found ( I) . Direct examination and radiophotography did not detect any gross or clear signs of abnormal development. Histological examination and morphological evaluation of the lymphatic and other visceral systems did not provide any sign o f damage brought about by the action o f an exogenous agent 00 is O Cytogenetic Examination Cytogenetic examination o f the same fetal tissues as well as o f the maternal blood revealed no abnormality in the number o f chromosomes. Abnormal pattern in the chromosomes o f the foetal tissues (embryonic, placental and umbilical cord cells) were equal to those o f the cells o f the amniotic fluid corresponding to the same gestational period. Chromosome examinations have been performed also (3 ) on the blood o f people belonging to the group o f acute and chronic exposure; o f the Icmesa plant workers; on children with and without chloracne. The average frequency o f gap* (3.68% ) and breaks (2.04% ) found are well within the accepted standard frequency ( ~ 5%). cn cn co A3 GO 00 Immunological Studies Immunosuppression and decreased resistance to infections have been observed in several animal species exposed to T C D D (13). Immune capability was therefore examined ( I ) in two groups o f 20 children each, with and without akin specific lesions, in comparison to a third suitable group o f 43 children- The serum immuno globulins, the circulating level o f complement, the ability o f the lymphocytes subpop ulation T and B cells to react to non-specific mitogens were examined. There was no difference between the contaminated and the control group. H ie number o f notifications for infectious diseases submitted in 1977 to the health offices shows no increase for those where vaccination is currently performed (poliomyelitis, diphteria), thereby confirming a normal immunological reaction o f the population to antigen stimulation. As for the other infectious diseases the inci dence o f the Seveso Zones does not exceed the average of-the whole Lombardy region. T C D D Tissue Levels In M an The metabolism o f T C D D has been rather extensively studied in laboratory animals. Tissue distribution and excretion have been obtained by studying the radioactivity derived from T C D D -14C (7 , JO). Over 50% o f the absorbed dose is excreted in the faeces and the half-life o f radioactivity in the body is approximately 4 weeks for the .100 O. Reggiani rai. Analysis of tissues indicates that T C D D is located mainly in the liver and in the fat and that concentrations in the order of 20 to 30 limes lower than that in these tissues have been found in other tissues: spleen, bone, heart, lungs, testes, kidneys, muscle, skin, pancreas, brain, stomach and adrenals in decreasing order. Continual administration of the same daily dose shows that T C D D accumulates to some de gree. but that a steady state is attained within a couple o f months and that beyond this time further accumulation and consequential toxicity do not occur (S, 6) and that that period is independent o f the administered dose for the range o f 0.001--0.1 pg/kg/day. T C D D undergoes little if any metabolism. There is a complete lack o f information on the metabolism o f T C D D in man. The autopsy of a 33 years old woman, who died o f pancreas carcinoma 7 months after a T C D D exposure, has offered the opportunity o f a tissue levels analy sis in man. She lived with the other members o f the fam ily, two adults and two children, close to the factory, in a Zone where the concentration o f T C D D in the soil was 162--1847 ug/m2 and stayed there from the day o f the explosion, July lOih until July 26th. when she was evacuated together with other 700 people. The two children living with her suffered severe skin lesions caused by the caustic compo nents o f the material released from the reactor and after the bums and blisters o f the first few days had faded away, developed a rather severe chloracnc, signs o f which are still present today in their armpits and earlobes. Neither she, nor the parents of the children developed chloracne. The whole family absorbed, almost certainly, T C D D through the food, probably vegetables, and farm animals during the first days after the accident. In September 1976. two months later, she was reported ill by the house physi cian and sent to hospital with thrombophlebitis. A t the hospital she was found to have jaundice and a laparatomy performed December 7th revealed a liver greatly enlarged with nodules of varying size invading the whole organ. Death occurred twm months later and the autopsy revealed a carcinoma o f the head o f the pancreas as large as a hen's egg. The T C D D analysis (Table 7) in the tissues has been performed at the Micromass Laboratory at Altrincham, U .K ., where concurrently samples of Tabic 7. T r O I l levels in the human both nw Sample Origin Ouanlilv l.imil of detection Kt-covcry : k. 7. n * Lh er l-'al Pancreas lung Kitlncy Drain Aulupsi n ig nig n 'g IO g ng 10 ppt n ip p i )Oppt in ppt n ip p i n ip p i 64% 5V% 59% 60% 60% 60% * Vacuum Generator Micrmnass (.tborainry, Aluincham (Manchester IJ K.) ** Total hHly weight: 70 kg - culcuhttcd total amount at time of death: 40 pg TCDD" 0.15 pph I.H4pph MM pph 0.06 pph II.114 pph 0.06 pph EdinMiion of the TCDD Tonic Potential 101 the same tissues obtained from the autoptic material of three cases certainly not related to a T C D D exposure was performed. No T C D D was found in these cases. The total amount of T C D D found in the body of the woman who died o f the pancreas carcinoma has been calculated at 40 ug. An interpretation o f these values and speculations about the T C D D doses which leads to skin, liver and neurological lesions in the adult, are probably meaningless at this stage o f our knowledge. A causal relationship with the malignancy can be excluded owing to the lapse o f time required for tumor growth to reach the size, weight and diffusion o f this case. The exposure to T C D D has occurred at a time when the growth o f the tumor had already reached the stage o f occult spreading throughout lymphatic and blood vessels to the adjacent tissues and organs. Conclusions The Scvcso accident has provided for the first time in the history o f the known episodes o f human exposure to T C D D a careful and thorough evaluation o f the levels o f concentration o f the toxin in the environment In this respect it can be compared with the field investigations, which have been conducted by the scientists of the U.S. A ir Force in Florida on rodents, insects, aquatic organism* and plant species (15). The level, intensity, duration o f exposure to T C D D cannot be defined for each individual, but a population o f several thousands people has been exposed for two years by now to a known level o f T C D D . Attempts have even been made by the Italian medical commission in charge o f the health monitoring to find out wheth er the incidence o f toxic effects (chloracne) is higher for the group o f population living in the Zone at highest contamination and to estimate therefore a dose response relationship. The effects observed in this population and the present health conditions can therefore be related to a defined level o f exposure. Secondly the Seveso accident has not revealed up to now toxic effects in hu mans, 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 o f impairment. Transient signs o f involvement o f the liver without apparent functional disorders have occurred. N o other organs or functions have been im paired. There has Bfcfcn no derangement o f gestation, no foetal lethality and loss, no gross malformations, no growth retardation at term and no cytogenetic abnormali ties. The immunocapability o f the population, not even o f the children with chlo racne. has not been affected. The T C D D levels o f exposure in Seveso have therefore brought about a biological response in the human beings, which is significant from a scientific point of view even if it is still o f low importance from a clinical point o f view. It has in fact little impact on the normal life o f the population. The T C D D levels have been sufficiently high lo produce the typical skin lesion, but not high enough to produce an easily perceivable toxicological effect on other organs and functions. One could conclude that man has a higher degree o f tolerance to T C D D than a direct extrapolation from animal data would suggest. DOW 553289 05 coOD cn IN CO CM CO o o References G. Reggiani 1. Fara. G. M.: Rapporto prcliminarc iuIIo slstodi salute nella ton* inquinala da T C D D . Quaderno di Documentation! 28, 5 (1977) 2. Fara. (i. M . Marubini. E.: Monitoring birth defects: an Italian project. 3rd Conference of the European Teratology Soc.. Helsinki, 1974 3. Greim. II.: Keine bleibcndcn Schiden in Seveso. limschau 1, 53 (1978) 4. Ilcinonen. O. P . Slone. D.. Shapiro. S.: Birth defects and drugs in pregnancy. Uttleton. Massachu setts: Publishing Sciences Group, Inc. 1977 5. Kociba. R. ).: TCDD: Results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 3J, 553-574 (1976) 6. Kociba. R. 1.: Results of a two year chronic toxicity and oncigcnvcity study of T C D D in tats. Accepted for publication by Toxicol. Appl. Pharmacol. 7. Piper. W. N.: Excretion and distribution of TC D D in the rat. Symposium on Chlorodioxins. Origin and Fate. Washington. September 1971. Advances in Chemistry Scries 120 g. Pocchiari. F.: Workshop on the long-term hazards of chlorinated dioxins and dibenzofurans. Int. Agency for Research on Cancer, Lyon, January 1978 9. Pocchiari, F.: Human health effects from accidental release of T C D D at Seveso (Italy). Interna tional Conference on Health Effects of Halogenatcd Aromatic Hydrocarbons- New York: Acad emy of Sciences 1978 10. Rose. J. Q.. Ramsey. J. C,, Wentzler. T. H.. Hummel. R. A.. Gehring. P. J.: The fate of T C D D following single and repeated oral doses to the rat. Toxicol. AppL Pharmacol 34. 209-226 (1976) 11. Sink. J. J. T. W. A.: Porphyrins in urine as indication for exposure to chlorinated hydrocarbons. International Conference on Health Effects of Halogenatcd Aromatic Hydrocarbons. New York: Academy of Sciences 1978 12. Tuchmann Duplcssis. H.: Pollution de 1`environnement et descendance. A propos de I'acddent dc Seveso. Med. ct Hyg. 34, 1758-1766 (1978) 13. Vos. J. G.: Studies on T C D D induced immune suppression and decreased resistance to infections. Toxicology 9, 75 -86 (1978) 14. Wilson. J. G.. Frazer, F. C.: Handbook of teratology. Vol. I. p. 324. New York, London: Plenum Press 1977 15. Young, A. L.: Studies o f the ecological impact of repetitive aerial applications of herbicides on the ecosystem. Air Force Laboratory, Eglin Air Force Base. Florida, 1975 0 8 i *nzrooo DOW 553290 Free Communications and Poster Sessions \ ich I (o*] DOW 423557 G Elsevier Scientific Publishing Com pany, A m sterdam -- Printed in The Netherlands - T . I el - ! EFFECTS OF 2 ,3 ,7 ,c TETRACHLORODIBENZO-p-DIOXIN (TCDD) ON EARLY LIFE STAGES OF THE PIKE (Esox lucius L.) -- THEO HELDER Deportm ent o f Special A nim al Pathology and Institute o f Pharmacology and Toxicology, University o f Utrecht, Biltstraat 172, Utrecht (The Netherlands) (Received Septem ber 21st, 1 9 7 9 ;accepted in fin a l form O ctober 23rd , 19 7 9 ) O O O' o U ABSTRACT Freshly fe rtilize d pike eggs were exposed to 2,3 ,7 ,8 -tetra ch lo ro d ib en zo -p -d io x in (T C D D ) a t concentrations o f 0.1 , 1.0 and lO p p t (n g /Iite r) fo r 96 hours. A t all concentrations exam ined egg developm ent was retarded by 23% , and the growth o f fry was also significantly retarded fo r a long period a fte r exposure. A dose-related m o rta lity was ob served. Highest m o rta lity rates occurred d w in g resorption o f the yo lk and reached alm ost 10 0 percent a t a concentration o f lO p p t. D eath was preceded by developm ent o f severe generalized edemas. H istopathologically edemas and hemorrhages were observed, together w ith alterations o f bloodvessel w ails. In the liv e r, tw o stages o f pathological changes were distinguished. T he first was characterized b y a d ilatio n o f sinusoids and a slight swelling o f hepatocyte nuclei; in the second stage the nuclei w ere enlarged up to tw ice the norm al diam eter. Hepatocytes were degenerated and varied in size and shape and liver architecture was a alm ost com pletely lo st. i l f 1 I ! j 1 | IN T R O D U C T IO N From industrial sources 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) may occur as an environmental contam inant, particularly when 2,4,5-trichlorophenol is used as raw m aterial. When the production is not carefully super vised, conditions may arise which are favourable for the form ation of TCDD (Gribble, 1974), and then through effluents TCDD may enter the aquatic environment. Municipal incinerators can provide other sources of TCDD (Olie et al., 1977). TCDD has a very low solubility in w ater (0.2 ppb) and is m ainly adsorbed onto sedim ents where it is not readily available for m icrobial degradation; being very persistent it m ay accum ulate in m ud (Kearney e t al., 1973; Ward and Matsumura, 1977). In laboratory animals (Schwetz et al., 1973) TCDD is extrem ely toxic, having a LD S0 in the pg/kg range. It produces chloracne in m an and rabbits (Kimmig and Schulz, 1957; Jones and Krizek, 1962) and causes several pathologic changes in th e liver of different species (Gupta et al., 1973; Jones 7842 0002621 and Butler, 1974; Vos e t al., 1974). Edemas were reported in chickens (Schwetz et al., 1973), in mice (Vos et al., 1974) and in rat fetuses after maternal treatm ent (Khera and Ruddick, 1973). Toxicity testing with salmonid fish and guppies has been carried out (Miller et al., 1973; Norris and Miller, 1974; Hawkes and Norris, 1977) but there are no reports concerning effects on early life stages. De Hullu and Poels (1976) observed a high toxicity for trout fry exposed to water of the river Rhine. The occurrence o f severe edemas in exposed fry led to the speculation th at TCDD, or similar contam inants, could have been the agent; although so far TCDD has n o t been detected in Dutch waters. This prom pted us to study the effects of TCDD on early life stages of fish. The present study deals with the pike (Esox lucius L.) MATERIAL AND METHODS A sample of TCDD (Lot number 851:144-11, 98.6% purity) was kindly supplied by Dow Chemical (Nederland) B.V. A stock- solution of TCDD in dimethylsulfoxide (DMSO, BDH, AnalaR, purity 99%) a t a concentration of 25pg/m l was prepared, subsequent dilutions were m ade in acetone (BDH, Aristar, purity 99.8%). Pike eggs and m ilt were obtained from clinically healthy pike caught by netting in the Pikmeer near Grouw, The Netherlands. The eggs were fertilized by the dry m ethod (Huisman, 1975). A fter 5 min the eggs were divided into 5 groups of approxim ately 5000 eggs each and transferred to glass jars con taining 11 of tap w ater; 0.05 ml of TCDD in acetone was added to four jars to provide concentrations o f 10.0, 1.0, 0.1 (ng/I) and 0.0 ppt TCDD; one group remained untreated. A fter transportation to the laboratory, which to o k 2 h, the eggs were transferred to glass tanks, containing 201 of tap w ater (pH 6.9, hardness 94--102 ppm C aC 03). TCDD--acetone solutions and ace tone were again added, to provide the same concentrations as used in the glass jars. The m edium was changed a fter 48 h, and a fter 96 h was replaced with tapw ater which was then changed every 24 h. No control with DMSO was used. In other work (Helder, m anuscript in preparation) it was dem on strated th at concentrations of DMSO at the same level as used in this study (maximum 0.4 ppm ) are n o t toxic. T he eggs were incubated on perforated alum inum trays suspended in tanks which were continuously aerated; water tem perature was kept at 14 0.5C. A fter hatching, wooden boards were placed in the aquaria to which the fry became attached (Huet, 1975). The fry were fed with live zooplankton and after developm ent to the swimming stage all groups were reduced at random to 500 fish. Reduction of the 10-ppt group was not necessary because of the high m ortality rate. For measuring length and light microscopy studies, 10 fishes were taken from each group, 2--3 tim es a week, anaesthetized in buffered tricaine m ethane sulphonate (MS 222 , Sandoz, Basel), fixed in Bouin Hollande fo r 2 4 h , stc in of ma ist; o5? re cit fib grc t o ati c o Ta c n tre ui 00 in Nc we we ha th i mi me str stz ed TA BO Da; fe r reported in chickens -id in ra t fetuses after ' -:arried out (Miller s , j.9 7 7 ) b u t there are ullu and Poels (1976) bt of the river Rhine. , the speculation that .gent: although so far npted us to study the - study deals with the % p urity) was kindly solution of TCDD in at a concentration of ie in acetone (BDH, althy pike caught by e eggs were fertilized ggs w ere divided into red to glass jars con gas ad d ed to four jars 0.0 ppt TCDD; one e laboratory, which :ing 201 o f tap w ater lutions and aceiljuns as used in the =r 9 6 h was replaced control with DMSO tion) it was demonas used in this study s suspended in tanks k ep t a t 14 0.5C. ria to which the fry stored in 70% ethanol and after m easurem ent of length they were em bedded in Paraplast. Transverse sections 7 pm thick were taken serially a t distances of 100 pm and stained with Mayers haemalum and eosin. Clinical abnor malities and m ortalities in all stages of developm ent were recorded. For sta t istical analysis the f-test was used (Brown and Hollander, 1977). RESULTS Clinical observations Embryonic development was considerably retarded in all TCDD-treated groups. Control groups hatched during the 8th and 9th day after fertiliz ation, whereas the exposed groups hatched during the 10th and 11th day. Tail-hatching, i.e. hatching with the tail first, was observed for all TCDDtreated groups. In the 0.1 p p t group the incidence was less than 10 percent, in the 1 ppt group about 45 percent and in the 10 ppt group over 90 percent. N ot a single case was observed in the controls. A num ber of tail-hatched fry were too weak to free themselves from the egg shells and died. Exposed fry were also less pigmented and smaller in size (Fig. 1). For a long period after hatching (Table 1) body lengths were significantly (p < 0.05) shorter than those of controls. In all groups, however, the transition to the stage of swim ming fry occurred 15 days after fertilization. Mortalities were recorded over three periods. Before hatching, the egg m ortality was of the same order in all groups. In the yolk-sac fry stage, striking dose-related mortalities were observed which continued during the stage of swimming fry, although to a much smaller extent (Table 2). Closely related to the m ortality rates was th e incidence of generalized edemas in the TCDD-treated fry. In the 0.1 p p t group it was less than 5 TABLE 1 BODY-LENGTH (mm)* OF PIKE FRY AFTER 96-hours EXPOSURE TO TCDD Day after Control fertilization Acetone control Dose TCDD -- --1 1 0.1 ppt l.Oppt ' - lO.Oppt cc o OCHO 00 00 11 9.0 0.15 0.13 8.5 0.16b 7.8 7.3 0.151 15 11.6 0.16 11.4 0.16 10.7 0.21* 10.1 0.10f 9.3 0.151 18 13.3 0.15 13.1 0.10 12.8 12.3 0.15f 11.3 0.151 21 14.6 0.17 14.4 0.22 13.9 0.10d 13.6 0.16* 12.8 0.201 23 14.6 0.26 15.0 0.15 14.1 0.44 14.4 0.29 13.6 0.30' oO 423559 DOW 423560 .1 mm. Vj ppt ____ 1 PP T 0.1 ppt - -^ -r ~ -' control Fig. 1. Appearance of pike yolk sac fry, exposed to different concentrations of TCDD, 11 days after fertilization. Arrows indicate the onset of edema. TABLE 2 MORTALITIES DURING EARLY LIFE STAGES OF PIKE AFTER 96 HOURS EXPOSURE TO TCDD Egg Yolk sac fry Swimming fry Control a 1671/5000 303/3279 10/420 b 33.4 9.2 2.9 c 33.4 39.5 41.1 Acetone-control ab 1713/5000 457/3237 11/420 34.3 14.0 2.6 c 34.3 43.5 45.0 Dose TCDD 0.1 ppt .A 1.0 ppt b ea bc 10.0 ppt a bc 1760/5000 662/3200 23/420 35.0 35.0 14.2 48.8 5.8 51.2 1671/5000 1609/3279 27/420 33.4 33.4 1714/5000 48.8 65.9 3061/3236 6.4 68.0 108/135 34.3 34.3 94.1 61.8 80.0 98.9 a *= Number death/total. b = percentage death in given stage; e = cumulative mortality in percent. 00i 7845 .entrations o f TCDD, 11 percent, in the 1.0 p p t group 40 percent, and in the l O p p t group nearly all fry developed severe edemas. No edem atous fry were observed in the control groups. Histopathology In the clinically edamatous fry the histopathologic changes included edemas and haemorrhages, alterations of blood vessel walls and liver damage. These changes always occurred together. Extensive accumulations of protein-containing fluid could be observed throughout the body, particularly in the soft connective tissue of the hypodermis, behind the eyes and between fibres of the hypaxial musculature. Consequently, the abdominal wall was com pletely disorganized and com pared with the normal state its thickness was increased up to 3 times its norm al thickness (Fig. 2). The pericardial and abdominal cavities were also extremely dilated due to the excess of fluid (Fig. 3) and in severe cases the fluid contained blood cells. Many capillaries and smaller bloodvessels in the edem atous regions were disrupted and the nuclear size of endothelial cells was slightly increased. In the heart, the endothelial linings of both atrium and ventricle were discontinuous and occasionally red blood cells could be observed beneath the endothelium. In pathological changes found in the liver, tw o stages could be dis tinguished. In the first, there was a severe dilation of the liver sinusoids and nuclear size at the hepatocytes was slightly increased (Fig. 4). In the OOVV 423561 s11 f v ) fi/ !v. i. Fig Fig. 3. Transvene sections of nomal and exposed (lOppt) fry 18 days after fertilization. Note the extreme edemas and the dilated liver sinusoids in the fry. 1 --spinal cord; 2 = chorda dorsalis; 3 --swimbladder; 4 = digestive tract; 5 --yolk; 6 = liver. second stage, liver architecture was com pletely lost and the normal double row lamina and rosettes (Elias and Bengelsdorf, 1952) (Fig. 5) were hardly recognised. The parenchymal cells showed a severe degeneration with pale staining cytoplasm and a wide variation in size and stage. Nuclei were en larged up to 1.5--2.0 tim es the norm al diam eter, cell borders were badly outlined or lost and a few necrotic foci could be observed (Fig. 6). DISCUSSION During this study it has been dem onstrated that following 96 hour ex posure o f fresh pike eggs to TCDD a t a concentration as low as 0.1 p p t retards the development of the embryos; this concentration is beyond the detection limits of GC/MS analysis (Dow Chemical, 1978). Under normal conditions, the average incubation period of pike eggs is 120 degree days (H uet, 1975), which was that required for the controls. The TCDD-exposed eggs had an incubation period averaging 147 degree days, th a t is a delay of a b o u t 23%. The appearance of tail-hatching is n o t directly associated w ith ol Z S 2 G DOW 423563 \ V' i'yT'i 'V*. i days after fertilization, e fry. 1 --spinal cord; ; 6 = liver. ' normal doublei ig. 5) were hardly eneration with pale ge. Nuclei were enoorders were badly (Fig. 6). owing 96 hour exas low as 0.1 p p t tion is beyond the 78). Under normal is 120 degree days The TCDD-exposed . th a t is a delay of ly associated w ith Fig. 4. Liver o f pike fry exposed to 10 ppt TCDD, 16 day* after fertilization. Parenchymal cells are slightly degenerated with minor nuclear enlargement, and dilated liver sinusoids. X 360. '>U" " 4 ** " ">K* 18 d iyi 000262 ... 7 8 4 8 DOW 423564 ft m M M'ft*6 a Y i - IS Fig. 6. Liver of pike fry exposed to 10 ppt TCDD, 18 days after fertilization. Severe degeneration of hepatocytes, with marked nuclear enlargement and complete loss of liver architecture, x480. the longer incubation periods, because this phenomenon was of the same order in all exposed groups. It is, however, related to the degree of under development of the pike fry. In fish culture, tail-hatching is generally con sidered to be a sign of weakness. Developmental retardation is also shown by the growth rates. These were significantly (p < 0.05) lower than the controls during the whole experiment, except for the 23rd day after incu bation for the 0.1 and 1.0 ppt groups. Mortality in the egg stage is not influenced by TCDD. A loss of 30--50 percent of pike egp, because they are not fertilized, is considered normal under hatchery conditions (Huet, 1975). The high mortality among yolk sac fry is partly associated with the inability of tail-bom fry to free them selves from the egg shells, but to a much higher degree with the occurrence of generalized edemas. Edema formation is a consistent feature of TCDDpoisoning (Khera and Ruddick, 1973; Schwetz et al., 1973; Vos et al., 1973). Also the presence of hydropericardium and degenerative changes of bloodvessels are in concordance with previous reports, concerning ani mals fed with fat-containing TCDD (Allen, 1964; Allen and Carstens, 1967). Pathological changes that were found in the liver, i.e. focal necrosis and degeneration with pleiomorphism of parenchymal cells and nuclear enlarge ment, have also been observed in mammalian laboratory animals (Gupta et al., 1973; Jones and Butler, 1974). However, multinucleate hepatocytes, as observed in mice (Vos et al., 1973; Jones and Greig, 1975) were not 0 102528 7849 ter fe rtiliz a tio n . Severe : and com piete lots o f on was of the same he degree of underng is generally con.ation is also shown ?' lower than the ^ day after incu- D. A loss of 30--50 : considered normal vitality among yolk -n fry to free themwith-the occurrence t feature of TCDD- 1973; Vos et al., ^generative changes rts, concerning anind Carstens, 1967). . focal necrosis and rnd nuclear enlarge?ry animals (Gupta 'cleate hepatocytes, i, 1975) were not 423565 found during the course of this study. Disturbances of liver architecture have been described for monkeys (Allen and Carstens, 1967). The methods used in the present study were inadequate for detecting thymus atrophy, as is observed in mammalian species (Gupta et al., 1973; Vos et al., 1973) and in juvenile rainbow trout (Helder, in preparation). There was no sign of fin erosion (Norris and Miller, 1974). The extreme toxicity of TCDD to early life stages of pike may be due to the high lipophilia of the compound. The lipophilia of the TCDD, calculated according to the method of Rekker (1977) has a value of 7.1 expressed as log P octanol. This property could probably account for the high mortality in the yolk sac fry stage, during which the resorption of TCDD-containing yolk is increasing rapidly. This study reported here may encompass the most vulnerable part of the life cycle of fishes and should have a wider application in the assessment of water quality. .^ iQ ACKNOWLEDGEMENTS This study was made possible by the financial support of T.N.O., Nether lands. The author is indebted to Mr. F. W. J. J. Snel for his excellent histotechnical work. I wish to thank Prof. H. van Genderen and Dr. P. Leeuwangh for their helpful advice and critical review of the manuscript. This work would not have been possible without the help of Mr. F. de Haan, professional fisherman, Grouw and the Organization for Improvement of Inland Fish eries, Nieuwegein. Editor's comment The Dow Chemical Company has suggested that dioxins are ubiquitous in the environment, arise as a result of various combustion processes and are widespread. The processes invoked as providing sources of dioxins range from fossil-fuelled plants, cigarettes and charcoal-broiled steaks; in order to identify a common ground Dow put forward a theory of the trace chemistries of fire which requires the production of very small quantities of dioxins from a large number of processes at very low yields which is disputed by Professor Rappe, see Nature, London, Vol. 281, No. 5733, 25th October 1979, p. 619. . I. Hamilton REFERENCES Allen, J. R., Am. J. Vet. Res., 25 (1964) 1210-1219. Allen, J. R. end L. A. Carstens, Am. J. Vet. Res., 28 (1967) 1513-1526. 0002s:g 7850 423566 Brown B. W. and M. Hollander, Statistic*, a Biomedical Introduction, John Wiley and Sons, New York, 1977, pp. 109--129. Dow Chemical Company, Michigan --The Chlorinated Dioxin Task Force, The trace chemistries of fire --A source of and routes for the entry of chlorinated dioxins into the environment (1978). Elias, H. and H. Bengelsdorf, Acta Anatomies, 14 (1952) 297--337. Faith, R. F. and J. A. Moore, J. Toxicol. Environ. Health, 3 (1977) 461--464. Gribble, G. W., Chemistry, 47 (1974) 1 6 -1 8 . Gupta, B. N., J. G. Vos, J. A. Moore, J. G. Zinkle and B. C. Bullock, Environ. Health Persp., 6 (1973) 1 25-140. Hawkes, C. L. and L. A. Norris, Trans. Am. Fish. Soc., 106 (1977) 641--645. Huet, M. Textbook of Fish Culture, Fishing News (Books) Ltd., West Byfleet, 1975, pp. 1 5 1 -1 6 3 . Huet, M., In EIFAC Technical Paper No. 25, Workshop on controlled reproduction of cultivated fishes, FAO, Rome, 1975, pp. 147--163. Huisman, E. A., in EIFAC Technical paper No. 25, Workshop on controlled reproduction of cultivated fishes, FAO, Rome, 1975, pp. 101--110. Hullu, E. de and C. L. M. Poels, Internal Report, KIWA, 1976. Jones, E. L. and H. Krizek, J. Invest. Dermatol. 39 (1962) 611--618. Jones, G. and W. H. Butler, J. Pathol., 112 (1974) 93--97. Jones, G. and J. B. Greig, Experientia, 31 (1975) 1315--1317. Kearney, Ph. C., A. R. Isensee, C. S. Helling, E. A. WooIson and J. R. Plimmer, Adv. Chem. Ser., 120(1973) 105-111. Khera, K. S. and J. A. Ruddick, Adv. Chem. Ser., 120 (1973) 7 0 -8 4 . Kimmig, J. and K. H. Schulz, Naturwissenschaften, 44 (1957) 337--338. Miller, R A ., L. A. Norris and C. L. Hawkes, Environ. Health Persp. 5 (1973) 177--186. Norris, L. A. and R. A. Miller, Bull. Env. Contam. Toxicol, 12 (1974) 76--80. Olie, K., P. L. Vermeulen and O. Hutzinger, Chemosphere, 8 (1977) 455--459. Rekker, R. F., The Hydrophobic Fragmental Constant, Elsevier, Amsterdam, 1977. Schwetz, B. A., J. M. Norris, G. L. Sparschu, V. K. Rowe, P. J. Gehring, J. L. Emerson and C. G. Gerbig, Adv. Chem. Ser., 120(1973) 55--69. Vos, J. G., J. A. Moore and J. G. Zinkl, Environ. Health Persp., 5 (1973) 149--162. Vos, J. G., J. A. Moore and J. G. Zinkl, Toxicol. Appl. Pharmacol., 29 (1974) 229--241. Vos, J. G., J. G. Kreeftenberg, H. W. B. Engel, A. Minderhoud and L. M. van Noorle Jansen, Toxicology, 9 (1978) 75--86. Ward,' C. and F. Matsumura, Techn. Report WIS WRC 77-01, University of Wisconsin, Water Resources Center, Madison, 1977. 00025D0 7851 7852 I ' ./s 4 , (J . is * ,iz > . A m i m .a U OdAa-. 6xt*(Str X p Pf pf4~ DOW37394S MN 0 5 3 3 05 Hormonal Alterations in Female Rhesus Monkeys Fed a Diet Conta ining 2,3,7,8-tetrachlorod ibenzo-p-di ox in ` 7853 by D. A. BARSOTTI, L. J. A3RAHAMS0N, and 0. R. ALLEN QQ024i8 j -. , .* 1 - r T u *! r* .................. | r -,- .. -- 0 ,, * r*f Oa * - ! n r - / 1 !i<n r/er5i t v o f h 'i s o t n .s ia 'M e d ic a T 'S c h o o l' v - .'- 'd r \ Considerable attention has been drawn to potential hazards of 2,3,7,8-tetrachlorodibenzo-o-dioxin (TCDD). Varying amounts of TCDD. are produced in side reactions during the synthesis of poly chlorinated phenols, 2,4,5-trichlorophenoxyacetic acid herbicide (2,4,5-T) and related compounds. Industrial accidents resulting from overheating reaction mixtures of trichlorophenol have led to. explosions and release of large quantities of TCDD. The most recent accident occurred in northern Italy where an entire town was exposed to TCDD and other phenolic compounds. Soil samples from the contaminated area contained levels of TCDD in excess of 50 ppm (RAWLS and O'SULLIVAN 1975). The widespread use of 2,4,5-T has been another source of human exposure to TCDD. In Vietnam, a mixture of 2,4,5-T and 2,4-D (Agent Orange) which con tained TCDD at levels as high as 49 ppm was used extensively as a defoliant. Widespread injurious effects on man and lower ani mals have been reported, as a result of exposure to this mixture. Clinical reports from Vietnam suggest reproductive abnormalities in the human and animal population "of the sprayed areas. More recently, CARTER et al. (1975) reported, a high incidence of abor tions in horses that lived in an arena that had been sprayed with waste products from trichlorophenol production containing high .1 evels of TCDD. Reduced spermatogenesis has also been reported, in monkeys exposed to commercial fat products adulterated with TCDD (ALLEN and CARSTENS 1967). OOW373946 5 I I i i I Since there seems to be little doubt that TCDD exposure influ ences reproduction, further clarification of this question was deemed important. The presently reported experiment addresses this problem through the use of nonhuman primates that have been exposed to low levels of TCDD. MATERIALS AND METHODS Sixteen (16) adult female rhesus monkeys (Macaca mulatta), weighing an average of 5.6' kg, were individually housed in a controlled environment simulating the light, temperature and humidity of a 20 day period of the breeding season of their native India. The animals were fed a diet containing Purina Monkey Chew (Ralston-Purina Co., St. Louis, MO) supplemented with fruit twice weekly and water ad 1ibiturn. . ( 725$0024,9 I For six months.the menstrual cycles',of these females'.were evaluated as to length, intensity and duration. -In addition, 5 cc of blood were obtained daily via femoral venipuncture . 'throughout an entire menstrual cycle for the determination of serum 173-estradiol and progesterone levels (BIELERT et al. 1976). Following the period of control evaluations, the ani- mals were divided into two groups. One group of eight animals "was fed a diet to which 500 parts per trillion (ppt) TCDD had . been adtfed. for nine months. The food-was prepared by the addi W'i l j / ' rf'* * .JL ' l 3tl ) .O'** \^ W o ' tion of the proper amounts of c o m oil-suspended TCDD to ground Purina chow. The TCDD-containing chW^ias^uniformly"mixed and subsequently repslletted. The other group received a similar diet devoid of TCDD. A 200 q portion of the food was given daily. Food intake was monitored in all animals throughout the nine months. Hemograms and clinical chemistries, includ- ' m 1 -, ing total serum lipid, serum cholesterol, serum glutamic pyru vic transaminase (SGPT), total serum protein and serum albumin globulin ratios were performed on a monthly basis. After 1, 3 and 6 months on the diet, blood.samples were obtained through out. the menstrual cycle in order -to establish serum progester one and estradiol levels. After being on the experimental diet TD , O -. C O ' <1 -to; for 6 months, the animals were Housed with control males at the appropriate time of the menstrual cycle (VALERIO 1969). Preg CD ** nancy was confirmed by obtaining a 5 ml blood sample 20 days following breeding, and serum from this, blood was then used in the immature mouse 'bioassay for monkey chorionic gonadotropin as described by WILSON et al. (1972). Further clarification of pregnancy was obtained at 30 days by rectal palpation of the uterus. r E f3%i 1c5ei RESULTS The morphological changes that v/ere produced in the TCDD exposed females have previously been reported (ALLEN et al* 1977) and will be sunmarized here. Within 6 months following, exposure to 500 ppt of TCDD in the diet, which amount to approxi mately 2 yg per^kg_body weight, the animals became anemic. A severe pancyt'openia was apparent wi thin 9 months and after the animals had consumed approximately 3 yg of TCDD per kg body weight. Widespread hemorrhage was related to the marked throm bocytopenia. Death occurred in five of the eight animals between the seventh and twelfth month of the experiment. At necropsy, in addition to the extensive hemorrhage, there was a distinct hypocellularity of.the bone marrow and lymph nodes. Hypertrophy-byperplasia and metaplasia of the epithelium and bronchial tree, bile ducts, pancreatic ducts, salivary gland ducts and palpebral conjunctivae were observed. Squamous metaplasia and keratinization of the sebaceous glands and hair follicles were present in the skin. A severe hyperplastic gas tritis with ulceration of the gastric mucosa was present in the animals that died. Death was attributed to complications arising frem the severe pancytopenia. 0 0 0 2420 The length of the menstrual cycles of the monkeys as well as the intensity and duration of menstruation were hot altered appre ciably during the initial 6 months of the experiment. The length of cycle for the experimental animals was 27.8 + 3 . 9 days and.the duration was 3.9 + 1.4 days. Control animals had a cycle of 27.3 +_ 4.3 days and a duration of 3.4 +_ 1.3 days. Individual pretreatment steroid patterns were used as controls for comparing estradiol and progesterone levels at 1, j and 6 months of the study. One animal had an unsuccessfuPbreeding his tory and thus was eliminated from the steroid evaluation.- The steroid analysis at 6 months showed alterations in 5 of the 7 ani mals. The values of the remaining 2 animals were unaltered at 6 months. Progesterone levels in 3 animals (Nos. 38, 49, 7) decreasr ed to 72.4, 51.9% and 47.3% of their pretreatment values, respec tively.. In the same time period, estradiol levels in- two of these . animals (7 and 49) also decreased to 50.4% and 43.2% of the con trol, respectively, while estradiol remained unchanged in the re maining animal (38). The additional 2 animals studied (23 and 32) revealed anovulatory patterns of both steroids. Progesterone remained below 400 pg/ml serum and estradiol never rose above 80 pg/ml serum throughout the cycles. `.Examples of-these steroid pat- DOW373948 lcgic data on serum progesterone at 0, 3 and 6 months cn the diet. The data was normalized to the progesterone peak (day 0) + day 1 and 2. * = Anovulatory cycle. Progesterone values were below 400 pg/ml serum indicating an anovulatory cycle. 000242 L DOW373949 Fig. 2. Animals 9, 7 and 23 serve as examples of the radioimmunologic data on serum 17B-estradiol at 0, 3 and 6 months on the diet. The data were normalized to the preovulatory estradiol peak (day'0) + day 1 and 2. * = 170-estradiol values v/ere below 80 pg/ ml serum indicating an anovulatory cycle. After the radioimmunologic evaluations were completed at the end of the sixth month, the animals were bred. All of the control animals conceived and delivered healthy infants. The two experi mental animals (9 and 41) which maintained normal estradiol and progesterone levels throughout the six month .period conceived. However, only animal 41 was able to carry her infant to term. Animal 9 expelled a dead, partially resorbed fetus on the 62nd day of gestation. Animal 38 which showed a decrease in proges terone levels in the serum but not estradiol also conceived, but subsequently aborted' on the 46th day of gestation. Animals 7 and 49 that had decreases in both of the steroid hormones failed to conceive following repeated breedings. Such was also the case with Animal 23 which showed an anovulatory pattern as indicated by serum steroid levels. Animal 32 which also showed an anovula tory steroid'llevel was not bred due to her debilitated state. Following conception, Animals 9 and 38 showed intense and pro- longed implantation bleeding. Menstruation became intense and 7857 sporadic in Animals 7 and 49. The loss of blood resulting frcm the excessive implantation bleeding and menstruation markedly decreased the hemoglobin levels of these already anemic animals. 0002422 * 1 I 9 1 9 1 1. 1 I I i 1 1 1 1 1 ! Of the seven animals that v/ere evaluated for their reproduc tive capabilities following TCDD exposure,'only Animal 41 was able to carry her infant to term. Throughout the course of the experiment, this animal showed only minimal effects of TCDD expo sure even though her level of consumption was equally as great or greater than-that of those animals that experienced morbidity and mortality. Five of the seven animals died between the seventh and twelfth month of the experiment. The two surviving animals, 41 and 23, continued' to be evaluated following the discontinuation of 'its TCDD diet. Animal 41 gave birth to a well developed infant. The infant remained healthy during the 4 months of nursing as det ermined by gross appearance and hematologic and inrcunologic evalu ations. After the infant was weaned-and a normal menstrual cycle was reestablished, the serum estradiol and progesterone were once again evaluated and found to be normal. a 9 f.c itM d o Animal 23 showed a-decided leukopenia and thrombocytopenia after 9 months of TCDD exposure (3.3 x 103 white blood cells per mm3 and 28 x 103 platelets per mm3 ). After 8 months on a.control diet, the white blood cell counts returned to normal, and the platelets increased to 364 x 103 per mm3 blood. Within 6 months the level of serum estradiol and progesterone had also reesta blished, a pretreatment level. The animal was subsequently bred and gave birth to a well developed infant 5 1/2 months later. ' -DISCUSSION. There appears to be little doubt that the consumption of a diet containing 500 ppt of TCDD over 9 months by nonhuman primates is capable of causing reproductive abnormalities. The seven female monkeys that were included in this experiment had a history of nor mal menstrual cycles and had given birth to normal infants during the year prior to this study. In addition, the levels of serum estradiol and progesterone throughout the menstrual cycle of these 'animals were similar to those of animals of the untreated breeding 'colony. Thus, it can be said that prior to-TCDD exposure the ani- ^ " mals utilized in this experiment had a normal reproductive history. ^ Following 6 months of TCDD ingestion, during v/hich time the food intake v/as similar to that of the controls, five of the seven animals showed a varying degree of modification in their serum estradiol and progesterone. Even though the total intake of TCDD was essentially the same for each animal, it was not surprising '* .that all monkeys did not respond in a similar manner. Much like the human population, nonhuman primates are a rather heterogeneous group and considerable amounts of biological variation are to be expected. . , . '7858 The question arises as to how much influence the general toxic effects which include a moderate to severe pancytopenia had on the reproductive capabilities of these animals. During the period when the majority of the animals were not obviously ill, some of the animals began to show modifications in their serum estradiol and progesterone levels. These animals also had difficulty. breeding and maintaining pregnancy. Thus, it appears that the reproductive problems arose prior to the more debilitating toxic manifestations. A possible link exists, between the- reproductive dysfunction associated with TCDD exposure and the increased metabolism of steroids. The fact that many chemicals increase hepatic micro somal -enzymes as well as accelerate metabolism of steroids has- been documented (HART and FOUTS 1953; COliilEY 1967; PEAKALL 1967; WELCH et al. 1967). TCDD administration to 'pregnant rats caused a marked elevation of some maternal hepatic microsomal enzymes and a unique transplacental induction of microsomal enzymes in fetal and newborn systems (LUCIER et al. 1975; BERRY et al. 1976,1977). This TCDD induction mechanism could cause an alteration in the intricate balance of steroids leading to the high incidence of fetal mortality and early and late resorptions (SPARSCHU et al. 1971) and morphologic suppression of reproductive organs in rats (KOCIBA et al. 1976) as well as the altered steroid patterns and fetal wastage reported here. t DQW37395"1 Indications are that if the TCDD exposed animals survive the toxic effects and are allowed to recover, they will once again establish a normal menstrua*! cycle and be capable of breeding and reproducing. In the one severely affected animal that did survive, a normal menstrual cycle as well as pretreatment levels of estra diol and progesterone were established within 6 months following the discontinuation of TCDD exposure. This animal became pregnant, experienced an uneventful gestation and gave birth to a wall dev eloped infant. Similar reproductive abnormalities have also been reported in nonhuman primates exposed to other chlorinated aromatic hydrocar bons (ALLEN and BARSOTTI, 1976; ALLEN and NORBACK, 1976; BARSOTTI et al. 1976). In these experiments, the rhesus monkeys were expo sed to low levels of polychlorinated biphenyls for 18 months. Dur ing this time the animals experienced menstrual irregularities and "increased excretion of urinary 17-ketosteroids (BARSOTTI and ALLEN 1975). Subsequently, they had difficulties in conceiving and experienced a high incidence of early abortions. However, when the animals were removed from the experimental diet for one year, menstrual cycles and their breeding and gestational performance were similar to those of the controls. 'V./ V. ACKNOWLEDGEMENTS This investigation was supported in part by U.S. Public Health Service grants ES00958, ES01339 and RR0C167 from the National Institutes of Health and the University of Wisconsin Sea Grant Pro gram. A portion of this research was conducted at the University of Wisconsin-Hndison Biotron, a controlled environmental research facility supported by the National Science Foundation and the Uni versity of Wisconsin. Primate Center Publication No. 17-035. 0002424 REFERENCES' ALLEN, O.R., and L.A. CARSTENS: Amer. Vet. Res. 28, 1513 (1967). ALLEN, J.R., and D.A. BARSOTTI: Toxicology 6., 331 (1976). ALLEN, J.R., and D.H. NORBACK: Proc. Nat`l. Conf. on Polychlori nated Biphenyls, EPA-560/6-75-004, p. 43 (1576). ALLEN', J.R., D.A. BARSOTTI, J.P.. VAN MILLER, L.J. ABRAHAMSON, and J.J. LALICH: Food Cosmet. Toxicol. I5_, 401 (1977). BARSOTTI, DjA., and J;R. ALLEN: Fed. Proc. 34, 338 (1975). BARSOTTI, D.A., R^J. MARLAR,. and J.R. ALLEN: Food Cosmet. Toxicol. 14, 99 (1976). DOVV973952. BIELERT, C., J.A. C2AJA, S. EISELE, 6. SCHEFFLER, J.A. ROBINSON, and R.W. GOY: J. Reprod. Fertil. 46, 179 (1976).. BERRY, D.L.,. P.K. 2ACHARIAH, M.J. NAMKUNG, and M.R. JUCHAU: Toxicol. Appl. Pharmacol. 36, 569 (1976). BERRY, -D.L., T.J. SLAGA, N.M. WILSON, P.K. 2ACHARIAH, M.J. NAMKUNG, W.M. BRACKEN, and M.R. JUCHAU: Biochem. Pharmacol. 26, 1383 (1977). CARTER, C.D., R.D. KIMBROUGH, J,A.. LIDDLE, R.E. CLINE, M.M. 2ACK, and W-F- BARTHEL: Science 188, 738 (1975). . . ... CONNEY, A.H.: Pharmacol. Rev. 19., 317 (1967). HART, L.G., and J.R. FOUTS:.Proc. Soc. Exp. Biol. Med. 114, 388 (1963). ....... ... ,, . . . .. i* * I ' .... .KOCIBA, R.J., P.A. KEELER, C.N. PARK, and P.J. GEHRING: Toxicol. Appl. Pharmacol. 35, 553 (1976). LUCIER, G.W., B.R. SONAWANE, O.S. MCDANIEL, and G.E.R. HOOK: Chem.Biol. Interactions 21 15 (1975). PEAKALL, D.B.: Nature 21, 505 (1967). RAULS, R.L., and D.A. O 'SULLIVAN: Chem. Eng. News, August 23, 1976, p. 27 (1976). SPARSCHU, G.l., F.L. DUNN, and V.K. ROUE: Food Cosmet. Toxicol. 9, 405 (1971).. VALERIO, D.A.: Lab. Animal Handbook , 223 (1969). . . . /. 7 0 6 0 WELCH, R.M., W. LEVIN, and A.H. CONNEY: J. Pharmacol. Exp. Ther. 155, 167 (1967). , WILSON, J.G., R. FRADKIN, and A. HARDMAN: Teratology 000242 5 59 (1972). /<>1 Part V. Carqnocenictty LONG-TERM TOXICOLOGIC STUDIES OF 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) IN LABORATORY ANIMALS R. J. Kociba, D. G. Keyes, J. E. Beyer, R. M . Carreon, and P. J. Gehring Toxicology Research Laboratory Health and Environmental Research Dow Chemical, US.A. Midland, Michigan 48640 * Introduction The compound 2,3,7,8-tetrachlorodibenzo-p-dioxin (T C D D ) is a highly toxic impurity that may be formed as an unwanted contaminant under certain conditions during the production of 2,4,5-trichlorophenol. T C D D has been considered one of the causes of chloracne, which has been associated historically with the industrial production of 2,4,5-trichlorophenol and other products made from 2,4,5-trichlorophenol. Over the past decade, a number of toxicologic studies with T C D D were conducted to assess the potential for acute toxicity and teratogenesis. Results of these earlier studies have been summarized in a previous publication by Kociba et al.' In that publication, we also reported the results of a subchronic study in which rats were given 1.0, 0.1, 0.01, 0.001 or 0 Mg T C D D /kg 5 days/week for 13 weeks. Doses of 1.0 Mg T C D D /kg/d ay caused multiple toxicologic effects, with significant morphologic changes in the liver, thymus and reproductive organs. A dose level of 0.1 Mg T C D D /kg/d ay caused lesser degrees of toxicity, and rats given 0.01 or 0.001 Mg T C D D /k g /5 days/week for 13 weeks had no alterations considered of any toxicologic significance. Since that time, studies have been in progress to assess the potential for chronic toxicity associated with long-term exposure to TC D D . Allen et al.1 reported on a subchronic study in which monkeys consumed a diet containing 500 ppt of T C D D for up to 9 months. It was calculated that these monkeys ingested 2-3 MgT C D D /k g over the course of the 9-month study. Clinically, these monkeys had changes sim ilar to those described by McConnell et al? as well as some hematologic depression and hemorrhages in various tissues. Hypertrophy, hyperplasia and/or metaplasia were noted in the epithelium of the bile ducts, salivary glands, bronchi, pancreatic ducts, sebaceous glands, skin, gastric lining and urinary tract of these monkeys given diets , containing 500 ppt o f T C D D . In 1969, Innes et al? reported on a long-term carcinogenic study conducted in mice given 2,4.5-trichlorophenoxyacetic acid contaminated with a level of T C D D , sufficient to supply approximately 0.27 Mg T C D D /kg /d ay. Neither of the two strains of mice had an increase in tumors as a result of the treatment. DiGiovanni et al? reported on a study in which T C D D was reported to be only a weak tumor initiator in the two-stage system of mouse skin carcinogenesis with 7,12-dim ethylbenz(a)anthracene (D M BA). Van M iller and Allen* issued a preliminary report on a study of small groups of male rats fed diets containing T C D D for 65 weeks. A ll 10 rats of each group receiving 1.0, 0.5 or 0.05 ppm T C D D in the diet died within 4 weeks, with acute toxic effects. 397 0077-S923/79/0320-0397 IOI.75/OC 1979, NY AS m M07 3280 OOW 0 9 0 3 0 6 CTi O no i COC 1 wnariyii -I* <3: o v\ C) crO oO i .-,,*.%-u.y:-.?; ......-- - --: w r Groups of male rats on diets containing 3000 or 1000 ppt of T C D D had increased m ortality, decreased weight gain and liver toxicity, (n a more recent publication by this same group of researchers. Van M iller et a l.' reported lung and/or liver tumors in those rats given 1000 or 5000 ppt o f TC D D . Neoplasms of various organs, but not of lung or liver, were reported in rats given diets containing 300, 30 or 5 ppt of TCDD . Van M iller et al. reported a zero incidence of tumors in the group of rats given 1 ppt of T C D D and in a group of 50 control rats. In view of the need for an evaluation of the chronic toxicity and potential for carcinogenicity of T C D D , the study summarized herein was conducted. For the sake o f brevity, only the more salient data are included in this presentation. The full report can be found in a recent paper by Kociba et al.* Materials and Methods The TC D D sample was prepared by the Dow Chemical Company, Midland, Michigan, USA, and had a purity exceeding 99%. On the basis of results from a previous 13-week long toxicity study1in rats dosage levels of 0.1,0.01 and 0.001 ug of T C D D /kg /d ay were given in the diets. The test diets were adjusted at intervals to provide these dose levels of T C D D as required by body weight and food comsumption determinations. Groups of 100 rats (50 male, 50 female) of theSprague-Dawley strain were maintained on diets supplying these dose levels for two years. The control group consisted of 85 males and 86 females. Samples of the prepared diets were analyzed periodically to ascertain if the dietary levels o f T C D D were being maintained as scheduled. Parameters monitored during the course of the study included body weights, food consumption, hematologic parameters including to ut erythrocyte count (R B C ), total and differential leukocyte counts (W B C ), thrombocyte and reticulocyte counts, packed cell volumes (P C V ) and hemoglobin concentration, routine urinalyses, and urinary excretion of creatinine, coproporphyrin, uroporphyrin and delta-amino levulinic acid (d elta-A L A ). Serum samples were collected preterminally or terminally for determination of urea nitrogen (B U N ), glutamic pyruvic transaminase (SG PT), bilirubin (total, direct and indirect), cholesterol, triglycerides, alkaline phosphatase (A P ), gamma-glutamyl transferase (G G T ), total protein, albumin and globulin. A ll rats killed after two years, as well as those dying or culled during the study, were subjected to a complete necropsy examination by a veterinary pathologist. Representative tissues from all organ systems of the body of each rat were preserved in formalin fixative, with subsequent preparation of paraffin-embedded sections for light microscopy examination. Additional sections o f livers collected at terminal necropsy from three female rats per group were processed for transmission electron microscopy. The weights of the liver, kidney, brain, heart, thymus, spleen and testes or ovaries/uterus were recorded at the time of terminal necropsy. Liver and fat samples collected from three females per group at terminal necropsy 4 were subsequently analyzed for T C D D content, using gas chromatography low- resolution spectrometry. The various data were statistically analyzed according to the procedures listed in ~ the previously cited publication by Kociba et al.,' which includes the full details of this . * research. * o ^ R esults Analyses of the test diets indicated that rats given 0.1, 0.01 and 0.001 Mg. T C D D /kg /d ay were ingesting an average of 2.193, 208 and 22 ppt of TC D D , respectively, in the diet for two years. 000 mmm. m rnrnm " '" J rrf. i - r ~ >t w w r ; tu-we.;.'.. w u i jfnpW m i in i iii": The rate of mortality was increased in female rats given 0.1 fig T C D D /kg /d ay. The body weights o f male and female rats given 0.1 jig T C D D /kg/d ay and also the females given 0.01 ug T C D D /k g /d a y were decreased. However, these parameters were not affected at the lower dose levels. Hematologic data indicated some effects at the high dose level o f 0.1 ug ' T C D D /kg/d ay: this included slight decreases in PCV and Hgb of males and decreases in PCV, total RBC and W BC counts and Hgb values of females; reticulocyte counts s appeared to be slightly increased. No effect was noted on thrombocyte counts and <' WBC differentials at all the dose levels studied. No hematologic changes were noted in rats given 0.01 or 0.001 ug T C D D /k g /d ay. Changes in urinary parameters were limited only to females given 0.1 or 0.01 ug T C D D /kg/d ay; these groups had increased urinary excretion o f coproporphyrin and uroporphyrin. Urinary excretion of delta-ALA was increased only in females given 0.1 tig T C D D /kg/day. Table 1 M ajor G ross N ecropsy F indings in Rats Attributed to Ingestion of Diets Containing T C D D for T wo Years Dose U vei of T C D D (jjg/kg/diy) 0.1 0.01 Emaciation (M , Fe) Icterus (Fe) Liver toxicity (M . Fe) Liver nodules (Fe) Thymic atrophy (Fe) Splenic atrophy (M . Fe) Anemic paleness of tissue and focal hemorrhages (Fe) Increased incidence of mesenteric periarteritis (M . Fe) Pulmonary congestion, edema, effusion and mass formation (Fe) Decrease in incidence of pituitary enlargement, endometrial hyper plasia, uterine polyps, and sub cutaneous masses (Fe) Decrease in severity of chronic renal disease (M ) Increased incidence of keratinized proliferative lesions near pharynx (M , Fe) Liver toxicity (M . Fe) Liver nodules (Fe) Mesenteric periarteritis (Fe) 0.001 ( --) Indicates observation was not noted at that dose level, or incidence was com parable to control data. Tabulation excludes voluminous data considered unrelated to treatment with T C D D . O f all the clinical chemical parameters evaluated, the only treatment-related changes were noted in females given 0.1 ng T C D D /kg /d ay, which had increases in SGPT, AP, and G G T activities. For the sake of brevity, treatment-related gross and microscopic observations on tissues have been summarized in Tables 1 and 2. Gross necropsy examination indicated that the target organs of chronic T C D D toxicity in rats were prim arily the liver, lymphoid organs, vascular and respiratory systems (Table 1). Light microscopy examination revealed the liver to be the organ most consistently affected, and rats DOW 090307 C c c.~ r-- C O 17c n Q s Ou m am m y ' , . .*--._ y W ' - J v * r^ - Annals New York Academy of Sciences Table 2 Major Histopathologic Findings in Rats Attributed to Ingestion A t H i r r i r ' A v i ' p * i L i i k i / r T P P i r\ c a b T u / a -- Dose Level of T C D D Oig/kg/day) 0.1 0.01 0.001 Liver Hepatic degenerative infiamatory, ne crotic and proliferative changes. Cytomegaly, distortion of lobular pattern and atropy of cords, cyto plasmic vacuolation, fatty change, altered tinctorial properties, he patic necrosis and inflammation, multinucleated hepalocytes, foci or areas of hepatocellular alteration (swollen hepalocytes), pigment ag gregates, bile duct hyperplasia, periportal inflammation, fibrosis Hepatocellular carcinoma (Fe) Hepatocellular hyperplastic (neoplas tic) nodules (Fe) Livers had much lesser degree of change. Hepatocellular nodules (Fe) No changes. Livers of females had statistical increase in foci or area of hepatocellular al teration (swollen hp atocytes), and livers of males had statistical decrease in area of hepatocellular altera tion (swollen hpato cytes). Reproductive Decreased incidence of uterine hyper plasia, cyst formation and polyp formation (Fe) Questionable decreased incidence of uterine polyps (Fe) Mammary Tissue Decreased incidence of mammary tu mors Endocrine Pituitary--decreased incidence o f pi tuitary hcmangiectasis and ade noma formation (Fe) Adrenal--decreased incidence of medullary hyperplasia (M , Fe) and pheochromocytoma (M ) O Increased incidence of cortical ne crosis and hemorrhage (Fe). corti cal adenoma and hemalocysl (M ) Pancreas--decreased incidence of acinar adenoma ( M ) Thyroid--occurrence of various iso lated follicular changes of ques tionable significance ( M ) Parathyroid--decreased incidence of parathyroid hyperplasia, secondary to chronic renal disease (M ) Lymphoid Isolated cases of thymic/splenic atro phy (Fe) o CTi ro ! Respiratory Focal alveolar hyperplasia, pigment aggregates, accumulation of alveolar macrophages and cholesterol clefts, pulmonary edema, focal in terstitial inflammation and fibrosis Focal alveolar plasia (Fe) hyper- _. r> v-< w o i I DOW 090308 T able 2 (Continued) Dose Level of T C D D (Mg/kg/day) _______________ 03_________________________0 l__________________ 0001_________ Keratinizing .squamous metaplasia ' (Fe) Keratinizing squamous cell cardnoma (Fe) -- -- -- -- r` Cardiovascular Isolated cases of hemorrhage in CNS -- -- (Fe) increased incidence of mesenteric and Increased inddence of thoradc periarteritis, with cases of periarteritis (M ) -- thrombosis or hematoma forma tion (M . Fe) Increased incidence of myocardial -- -- degenerative changes (Fe)_______'_________________________________________ _ ( --) Indicates observation was not noted at that dose level, or inddence was comparable to control data. Tabulation exdudes voluminous data considered unrelated to treatment with TCDD. 000 i 0G3 given 0.1 or 0.01 Mg TC D D /kg/d ay had a dose-dependent inddence of hepatic degenerative, inflammatory and necrotic changes listed in Table 2. Female rats given 0.1 fig T C D D /kg/day also had a statistical increase in lesions described morpholog ically as hepatocellular cardnomas (no metastasis) and hepatocellular nodules; females given 0.01 Mg T C D D /kg/d ay had an increase in hepatocellular nodules. Histopathologic examination of livers from rats given 0.001 fig TC D D /kg/day revealed none of the treatment-related effects that were noted at the higher dose levels. On a statistical basis, the incidence rate of a slight hepatocellular alteration (focal swollen hepaiocytes) was increased in females and decreased in males of the group given 0.001 fig T C D D /kg/day; these statistical variations were not considered indicative of any toxicologic significance. Ultrastructural examination of hpatocytes from females of the 0.1 Mg TC D D /kg/d ay revealed the most consistent change to be in the rough endoplasmic reticulum (R E R ) which showed proliferation, distortion and fragmentation. Smooth endoplasmic reticulum (SER) and mitochondrial structures were within control limits. Other changes in hpatocytes from this group given 0.1 Mg T C D D /kg/day included focal cytoplasmic vacuolization, increased lysosomal activity with residual body formation and an occasional multinudeated hpatocyte. In hepaiocytes from females given 0.01 fig T C D D /kg/day, the most notable changes included a lesser i degree of proliferation of the RER and some proliferation of the SER. This was . accompanied by a slight increase in the number of hepatocytes with lipid droplet ' accumulation. The hepatocytes from females given 0.001 ig TC D D /kg/d ay were ` within the limits of variation seen in the hepatocytes of the control group. There was no general increase in lipid droplet content of the hepatocytes, although an occasional hpatocyte had some lipid droplet content. As predicted from previous studies, gross and histopathologic examination of some rats given 0.1 tig TC D D /kg/day had isolated cases of thymic and/or splenic atrophy. - Lower dose levels did not produce this effect. * The respiratory system of both male and female rats was afTected by ingestion of 0.1 Mg T C D D /kg/day. with focal alveolar hyperplasia, pigment aggregates, alveolar macrophages, cholesterol clefts, edema, focal interstitial inflammation, fibrosis. keratinizing squamous metaplasia, and keratinizing squamous cell carcinomas. A t 0.01 Mg TC D D /kg/day. these effects were not noted, except for focal alveolar hyperplasia in female rats. At the dose level of 0.001 Mg T C D D /kg/d ay, there were no treatment-related changes in the respiratory tract. Examination of the reproductive organs of females given 0.1 (and possibly those given 0.01 Mg T C D D /kg/d ay) revealed a decreased incidence of the uterine changes typically encountered as spontaneous lesions in this strain of rat. The high dose group of females also had decreased incidences of both mammary and pituitary tumors. Adrenal changes noted only at the high dose level of 0.1 Mg T C D D /kg/d ay included a .' V Table 3 Increased Incidence of Tumor and Tumor-like Lesions Attributed to Ingestion by Rats of Diets Containing TCDD for Two Years Hard Palate/Nasal Turbinates Stratified squamous cell carcinoma Tongue Stratified squamous cell carcinoma Lung Squamous cell carcinoma Dose Level of T C D D 0<g/kg/day) 0 0.1 0.01 0.001 Number o f Males with Lesion/Number in Group 0/85 4/50* 0/50 0/50 0/85 3/50* 1/50 1/50 0/85 1/50 0/50 0/50 Hard Palate/Nasal Turbinates Stratified squamous cell carcinoma Tongue Stratified squamous cell carcinoma Lung Squamous cell carcinoma Liver Hepatocellular carcinoma Hepatocellular nodule(S) Number o fFemales with Lesion/Number in Group 0/86 4/49* 1/50 0/50 1/86 2/49 0/50 0/50 0/86 7/49* 0/50 0/50 1/86 8/86 11/49* 23/49* 2/50 18/50* 0/50 3/50 'Statistically increased above control data, p < 0.03. Table excludes additional data on incidence of tumors considered unrelated to treatment with TC D D . 0001004 decreased incidence of medullary hyperplasia and pheochromocytoma formation, and an increased incidence of cortical necrosis, hemorrhage, hemalocyst and adenoma formation. The incidence of pancreatic acinar adenoma was decreased in males given 0.1 Mg . T C D D /kg/d ay. This group of males also had a decrease in the incidence rate o f ' secondary parathyroid hyperplasia: this was due to a decrease in the severity of chronic renal disease noted in males given this high dose level of 0.1 Mg T C D D /k g /d a y . Examination of the cardiovascular system revealed several effects assumed to be due to ingestion of 0.1 Mg T C D D /kg /d ay. These effects included isolated cases of hemorrhage in different organs of the body, and an increase in the incidence of periarteritis and myocardial degenerative changes, both of which occur spontaneously 7866' Kociba et al.: Long-Term Toxicologie Studies of TCDD 403 Table 4 Decreased Incidence of Tumors attributed to Ingestion by Rats of Diets Containing TC D D for Two Years Pancreas Acinar adenoma Pheochromocytoma Dose Level of T C D D (^g/kg/day) 0 0.1 Number o f Mates with Lesions/ Total Number in Croup 14/85 28/85 2/50* 4/50* Uterus Benign tumor Mammary Gland Benign tumor Pituitary Adenoma Number of Females with Lesionf Total Number in 30/86 8/49* 73/86 - 43/86 24/49* 12/49* Statistically decreased from control data, p < 0.0S. Data at lower dose levels comparable to controls, except for possible decrease in incidence of benign uterine tumors in females given 0.01 Mg TCDD/kg/day. in this strain of rat. None of these observations were noted at the two lower dose levels, except for an increase in incidence of periarteritis in male rats given 0.01 Mg T C D D /k g /d a y . Other treatment-related effects included an increase above the background incidence of squamous cell carcinomas of the tongue and hard palate/nasal turbinate region of rats given 0.1 Mg T C D D /kg/d ay. Historically, squamous cell carcinomas have occurred in these organs of this strain of rat at a spontaneous incidence rate of 1-3%. Only the high dose level of 0.1 Mg T C D D /kg/day increased the incidence of this type of neoplasm. All the data on incidence rates of neoplasms that were increased or decreased by treatment with the high dose level of 0.1 Mg TC D D /kg/d ay are summarized in T ables 3 and 4. Review of organ weights recorded at the terminal necropsy indicated the following effects considered related to treatment (a) an increase in absolute and/or relative liver weight of rats given 0.1 or 0.01 Mg T C D D /kg/d ay and (b) a decrease in weight of the thymus of females given 0.1 Mg T C D D /kg/day. There were no organ weight changes at the dose level of 0.001 Mg TC D D /kg/d ay. T able 5 lists the T C D D content of liver and fat samples collected from female rats at terminal necropsy after two years of ingesting diets containing T C D D . Table 5 T C D D Content in Rat Livers and Fat Samples Collected after Two Years Ingestion of Diets Containing T C D D Dose Level o fT C D D (Mg/kg/day) 0.1 0.01 T C D D Content (ppt) Fat Liver 8100 1700 24000 5100 SOCORO MOO O CJ o r-O C li -S<` VC k ;m m m - ' v -`: ^ .M T - ^ r r y rr ^nT-svr^r^ w .-~.-- Mhos* D is c u s s io n The results of this study of rats ingesting T C D D for a lifetime serve as a basis for assessing the long-term chronic toxicity of T C D D . Continuous ingestion of a high dose level of 0.1 Mg T C D D /k g /d ay (approximately 2200 ppt in diet) predictably caused multiple toxicologic effects. Liver toxicity was the most consistent observation, and this was accompanied by morphologic changes of the lymphoid, respiratory, and vascular tissues of the body. The incidence of hepatocellular carcinomas of the liver and squamous cell carcinomas of the lung, hard palate/nasal turbinates or tongue was increased at this dose level. Conversely, the incidence of tumors of the pituitary, uterus, mammary gland, pancreas and adrenal medulla was decreased at this high dose level of treatment. Similarly, the incidence of other spontaneous lesions such as chronic renal disease was also decreased at this high dose level. Lifetime ingestion by rats of 0.01 Mg T C D D /kg/d ay (approximately 210 ppt in diet) caused a lesser degree of toxicity, primarily of the liver. However, there was no increase in the incidence of neoplasia at this dose level. Rats ingesting 0.001 Mg TC D D /kg/d ay for two years had no adverse effects in spite of the fact the liver and fat each contained 540 ppt of TC D D at termination of the study. Thus, these data indicate a good dose response for the long-term toxicity of T C D D . Whereas higher doses were predictably toxic and did alter the incidence of tumors, an intermediate dose level caused lesser toxicity but no neoplasia. A lower dose was tolerated for a lifetime with no adverse effects noted in any of the parameters evaluated. i o O o oc c CO o References 1. Kociba. R. J,, P. A. Keeler. C. N. Park A P. J. Gehring. 1976. 2.3.7,8-Tetrachlorodi- benzo-p-dioxin (T C D D ): Results of a 13-week oral toxicity study in rats. Toxicol. Appl. Pharmacol. 35: 553-574. 2. Allen, J. R.. D. A. Barsotti, J. P. Van Miller. L. J. Abrahamson, a J. J. Lalich. 1977. Morphological change in monkeys consuming a diet containing five hundred parts per trillion of 2J,7,8-lelrachloro-dibenzo-p-dioxin. Food Cosmet Toxicol. 15:401-410. 3. McConnell. E. E,, J. A. Moore A D. W. Dalgard. 1978. Toxicity of 2.3,7,8- teirachlorodibenzo-p-dioxin (T C D D ) in rhesus monkeys (Macaca mulatta) following a single oral dose. Toxicol. Appl. Pharmacol. 43: 175-187. 4. Innes. J. R. M .. B. M . Ullano. M . G. Valerio. L. Petrucelli. L. Fishbein. E. R. Hart. A . J. Pallotta, R. R. Bates. H. L. Falk. J. J. Gart. M . Klein. 1. Mitchell A J. Peters. 1969. Bioassay o f pesticides and industrial chemicals for tumorigenicity in mice: A preliminary note. J. N at. Cancer Inst. 42: 1101 --1114. 5. DiGiovanni. A. Viaje. D. L. Berry. T . J. Slaga A M. R. Juchau. 1977. Tumor initiating ability of 2.3,7,8-tctrachlorodibenzo-p-dioxin (T C D D ) and Arochlor 1254 in the twostage system of mouse skin carcinogenesis. Bull. Environ. Contam. Toxicol. 18: 55 2557. 6. Van Miller. J. P. A J. R. Allen. 1977. Chronic toxicity of 2.3.7.8-tetrachlorodibenzop-dioxin in rats. Fed. Proc. 36: 396 (Abstract 673). 7. Van Miller. J. P.. J. J. Lalich A J. R. Allen. 1977. Increased incidence of neoplasms in rats exposed to low levels o f 2.3,7.8-letrachlorodibenzo-p-dioxin. Chemosphere 9: 537- 544. 8. Kociba. R. J,, D. G . Keyes. J. E. Beyer. R. M . Carreon, C. E. Wade. D . A . Dittenber. R. P. Kalnins. L. E. Frauson. C. N . Park, S. D. Barnard, R. A . Hummel A C. G. Humiston. Results of a two-year chronic toxicity and oncogenicity study o f 2.3.7.8- tetrachlorodibenzo-p-dioxin (T C D D ) in rats. Toxicol. Appl. Pharmacol. In press. 1978. i 12345678 * cc CD o o C: OL i oui 1 Speru! Report T W E L V E WISH M EN G IV E 2 ,4 -1 ) A N D 2 ,4 ,5 --T A CLEAN 01LL OF HEALTH In spile of > deluge of emotional, sensational and often wildly inaccurate claims in the mass media earlier this year, a 12-man government-appointed Victorian group of experts has given the phenoxy herbicides 2,4-D and 2,4,5-T a clean bill of health. Victoria's Consultative Council on Congenital Abnorm alities in the Yarram District has produced a sober, factual and detailed 55-page report which quietly, yet very effectively, demolishes the extravagant and fright ening misconceptions which were purveyed in the media following a claim by two Yarram doctors that a greaterthan-normal number of babies with birth defects had been bom in the Yamm district of Victoria in 1975-76. It was widely suggested that the six cases concerned had resulted from exposure of the mothers to heavier-thanusual spraying of the herbicides 2,4-D and 2.4,5-T in the dairying district of Y am n during 1975. The other phenoxy herbicide, 2,4,5-T is used mainly to control woody plants, such as blackberries, gorse and ecalypts. Of the 96 plants which are proclaimed noxious weeds ii( Victoria 2,4-D is recommended for the control of 49 and 2,4,5-T for the control of 13. However, 2,4,5-T is not used to control weeds in food crops in Victoria. The Consultative Council investigated a total of eight birth defects which occurred in the Yamm area betwaen 1975 and 1978. Two of these were unearthed by the inquiry. Two cases were finally eliminated from consid eration because they involved the deaths of two 22-week and 27-week premature babies, neither of whom had any congenital deformity. The Council stated that "births of this nature are by no means uncommon and survival is rare when birth occurs before 28 weeks of gestation." 'In brief, the Council concluded thafthe analysis of all oKilobit information shows no evidence that these birth defects were caused by exposure to 2,4-D or 2,4,5-T and that normal agricultural use of 2,4-D end 2,4,5-T has not been shown to cause birth defects in domestic animais, nor is there evidence to connect such use with human birth abnormalities. " Immediately following the release of the report (which has attracted microscopic media attention, compared with the original outcry) Victoria's Health Minister, Vasey 1laughton, said: "In the light of this evidence, I have no hesitation in lifting the restriction I placed on the use of 2,4,5-T by Government Deportments earlier this year and the recommendation that it should not be used. " A Mountain of Data I The delightfully non-technical report represents a sifting of a mountain of world-wide data on the phenoxy herbicides and their dangers, as well as the results of detailed investigations in the Yarram area by the Consultative Council and the Victorian Health Depart ment. No Link With Herbicides Two other births were also eliminated from consider ation. One because the entire pregnancy and birth occurred outside the Alberton Shire, which includes Yamm, and the other because the birth occurred in 1978, well after the period in question. Neither ease appeared to have any connection whatsoever with herbicide spraying. Of the remainder, the Council made a number of points. Over the period 1960 to 1977 there were six deaths of newborn children, due to birth defects, among 2,247 births ' a rate of 2.7 per 1,000, which was comparable for that of the whole of Victoria, for the Central Cippsland area and for the world. The existence of four deaths of newborn babies with birth defects out of 278 births in the Yamm district from 1975 to 1977, or of three such births among the 93 deliveries by the two Yamm doctors, was not impossible on the grounds of chance alone, the Council found. The probability was, the experts said, that one medical.practice in Victoria could have a "duster" of birth defect cases such as occurred in Yarram in 1975-76 by chance alone. The report is bound to be widely sought by Australian i and international authorities because the phenoxy herbicides, although the most widely used herbicide in Australia since the early 1950s, have become a major focus for the world-wide storm of protest against the careless or excessive use of dangerous or potentially i dangerous agricultural chemicals. i Detailed ease records showed that none of the eight women wts "specifically exposed to herbic ides during pregnancy" -- meaning that none handled the materials directly, nor was near enough to spraying to be contaminated with spray moisture. Only three women even reported smell ing spray used In their vidnlty during pregnancy. I Current Australian usage of 2,4-D is about 2,500 tonnes ! a year, while 2,4,5-T is used at a rate of about 250 i ii tonnes a year. Control of annual and perennial broadleaf weeds in cpreal crops and pastures is the main use of 2,4-D, although it is also used on roadsides and in Nor was usage of the herbiddes greater in 1975. In fact usage was down on 1974 and there was no difference in the pattern of hrrbidde use around Yarram from that in other parts of Cippsiand, where the sprays are widely used on ragwort and blackberry. other non-crop situations. nD (continued centre column next page) * e - t U -------------------------------------------------------------- . DOW 104082 WOOL CLIPS ( The Australian Wool Corp oration reports that while the EEC and Japan continue to take the major proportion of the Australian dip, their significance as Australian wool customers has been declinin'. The big tains are beint made by the Comccon and developing countries at the expense of the tradit ional wool consuming nations. SIZZLINGLY LATE How does one put "sizzle" into cheese sales? PIN doeai't know, but the Aust ralian Dairy Corporation is promising to do it this sprint "with a strong sevenweek campaign using the unique presentation style of this popular culinary entre preneur" (none other than Peter Russell-Clarke). Only problem: The ADC's announcement of the campaign, "to be bunched mid-September," lobbed into PIN's office on Oct. 2. HELP YOURSELF Noted in a Western Europ ean survey of the purchasing habits of Continental house wives towards fruit and vegetable buying -61.5% prefer personal pick and self-weighing methods, 34% prefer the traditional counter service and only 1.9% prefer self-service prepack buying. We wonder if the results would be any different in Australia? PIG SHORTAGE Victorian Department of Agriculture economist John O'Connor, has warned that the exodus of pig producers from the industry and the reduction in the herd could lead to higher prices later in the year. Mr O'Connor's advice to pig producers is to stay with the industry, avoid a major drop in production and take advantage of increased prof itability which may accrue from the "superporker" marketing programme. TWELVE WISE MEN AND 2,4,5-T (contd) Thus the Council ruled that the cluster of major birth defects around Yarram was neither sufficiently abnormal to be of significance, nor related to the pattern of 2,4-D or 2,4,5-T usage in the arte. In addition, a check of veterinarians in Gippsland found that none of the 33 vets questioned attributed any deaths, illnesses, birth defects or infertility in farm animals to the use of herbicide sprays in the five-year period 1973-77, even though animals are normally far more commonly exposed to such sprays than pregnant women. The Council makes no attempt to' obscure the fact that 2,4-D and 2,4.5-T do cause birth defects in some laboratory animals when fed at certain levels. The Council ealcubted a conservative level of herbicide intake for humans which, on the basis of the animal experimente, would have no effect. It found that in the case of 2,4,5-T, which contains traces of the deadly impurity TCDD, a 60kg pregnant woman could safely consume 114 kilograms of sprayed blackberries, 60.000 litres of water polluted with 2.4.5-T residue, 12.000 litres of milk containing herb icide residue at the maximum permitted level, or 6,000 kilograms of meat containing 2,4,5-T residue - -and she could consume each of these quantities PER DAY! And for 2,4-D In the case of 2,4-D, a corresponding 60kg pregnant woman could safely consume 300kg of sugar cane sprayed with the herbicide, 300kg of citrus fruit, 750kg of raw offal, 7,500kg of meat, 7,500kg of raw cereals, 15,000kg of potatoes, 15,000 litres of water, 30,000 litres of milk, 30,000kg of milk producb or threequarters of a kilogram of conventional 2,4-D spray mb as used on ragwort --and all, again, on a PER DAY basis with concentrations rarely, if ever, reached on food crops in Australia. And the Council adds the comment: "It is of note that the women surveyed in connection with the Yarram investigation were not exposed to levels of the herbicide which remotely approach the exposures used in the above calculation. " The Council therefore reached the final conclusion that the cluster of birth defeeb at Yarram was not such as to suggest a specific local cause; there was no evidence that the drfeeb were caused by exposure to 2,4-D or 2.4.5- T, and that the normal agricultural use of the two herbicides "has not been shown to cause birth abnorm alities in domestic animals, nor is there any evidence to connect such use with human birth abnormalities." The Council therefore recommended no change in existing legislative controls on the use of the two herbicides, but urged the establishment by the Victorian Government of a unit to conduct research into the cause of birth defeeb "with special emphasis on extra-genetic factors." The unit would have the task of designing an affective system for the notification and surveillance of birth defeeb. CROOKED: Streams become crooked by following the path of least resistance. So do people. GOING WELL \ PIN notes that the still-new McMillan Rural Studies Centre In Victoria has attracted 6% of Gippsland farmers to its courses in the past 14 months. The courses, which cover a wide field, average two to three days and work out roughly 3G% managementoriented, and the rest either technical or adult education in their emphasis. ODD Noticed in the circular from'the Australian Meat and. Livestock Corporation, an inquiry from New Zealand for 30 tonnes of cows' horns, and an inquiry from Angola for industrial fat for the production of soap. CROCODILES Under the leadership of Professor Harry Messel, Sydney University has commenced a feasibility study into commercial croc odile fanning in the North ern Territory. The project is expected to provide inform ation on the biological char acteristics and life cycle of salt water crocs, help prev ent extinction of the species and provide the basis for a new industry. The work base is at Beatrice Hill Research Station, near the Adelaide River, not far from Darwin. FLEXITIME The Queensland Depart ment of Primary Industry has introduced flexitime working hours for salaried field and technical staff on a sb months' trial basis. Officers will now take time off as compensation for working excess hours on week days. BLANKETED We quote verbatim from an announcement in a grower organisation paper: "Wool Blankeb.-- file Association has a supply of wool and acrylic single bed blankets... Price is $9.50 plus freight." Paying to promote wool, criticising wool promotion efforb and then advertising the competitive product! 77I s I "II m ( IARC Monographs on the 'Evaluation o f the Carcinogenic Risk 303 o f Chemicals to Humans (1979), Volume 20 PENTACHLOROPHENOL A review on pentachlorophenol i s a v a ila b le ( H e rd e r, 1977) 1. Chemical and P hysical Data 1 .1 Synonyms and trad e names Chem. A b str. Services Reg. No.: 87-86-5 Chem. A bstr. Name: Pentachlorophenol Synonyms: Chlorophen; PCP; pen ch lo ro l; penta; pentachlorofenol; pentacblorofenolo; pentachlorophenate; pentaehlorphenol; 2 ,3 ,A,5 ,6-pentachlorophenol; pentanol Trade names: Chem-Tol; C ry p to g ll o l; Dowcide 7; Dowicide 7; Dowicide G; Durotox; EP 30; Funglfen; Grundier Arbezol; L auxtol; Lauxtol A; Liroprem; Pentaeon; P en ta-K il; P entasol; Penwar; Peratox; Permacide; Permagard; Permasan; Permatox; Permit e ; S an to b rlte; Santophen; Santophen 20; S inituho; Termi- T r o l; Thompson's Wood F ix; Weedone 1.2 S tru c tu ra l and molecular formulae and m olecular weight C6HC150 Mol. wt: 266.3 1 .3 Chemical and ph y sical p ro p e rtie s of th e pure substance From G rasseH i & Ritchey (1975), u n less otherw ise sp e c ifie d 0 0 D escrip tio n : White c r y s ta ls () B o ilin g -p o in t: 309-310C (decom position) a t 754 mm IARC Monographs on the Evaluation o f the Carcinogenic Risk 349 o f Chemicals to Humans (1979)t Volume 20 2 ,4 ,5 - AND 2 ,4 ,6-TRICHLOROPHENOLS 1. Chemical and Physical Data 1.1 Synonyms and trad e names 2 .4 .5 - T rlchlorophenol Chem. A bstr. S ervices Reg. No.: 95-95-4 Chem. A bstr. Name: 2 ,4 ,5-T richlorophenol Trade names: C ollunosol; Dovicide 2; Dovlcide B; N urelle; v- P reventol I 2 .4 .6- Trichlorophenol ^C hem . A bstr. Services Reg. No.: 88-06-2 Chem. A bstr. Name: 2 ,4 ,6-Trichlorophenol Synonym: T ric h lo rfe n o l Trade names: Dovlcide 2S; Omal; Phenachlor 1.2 S tru ctu ral and molecular formulae and molecular veights 2 ,4 ,5-Trichlorophenol ja _ C5H3CI3O 2 ,4 ,6-Trichlorophenol Cl Mol. v t : 197.5 OH Cl C6H3C130 Mol. v t: 197.5 t $ 'J{ 1.3 Chemical and physical p ro p erties of the pure substances 2 ,4 ,5-Trlchlorophenol From Wlndholz (1976), u n less otherw ise s p e c ifie d (a) D escrip tio n : Grey fla k e s (Hawley, 1977) B o ilin g -p o in t: 248C (746 mm); 253C (760 mm) M eltin g -p o in t: 67C Spectroscopy d ata: 299 nm (Ej = 140); 292 nm (Ej = 145) In methanol; in f r a - r e d , n u clear magnetic resonance and mass sp ectral data have been tabulated (G rasselll & Ritchey, 1975). S o lu b ility : g/100 g solvent a t 25C: acetone, 615; benzene, 163; carbon te trach lo rid e, 51; d ieth y l eth er, 525; denatured ethanol, 525; methanol, 615; liq u id petrolatum (a t 50C), 56; soya bean o il, 79; toluene, 122; w ater, < 0.2 V o l a t i l i t y : Vapour p ressu re i s 1 mm a t 72C (P erry & C h ilton, 1973). (a) S t a b i l i t y : S tab le up to I t s m e ltin g -p o in t (h) R e a c tiv ity : Can be converted to sodium s a l t by re a c tio n w ith sodium carbonate; th e hydroxyl group forms e th e rs , e s te rs and s a lt s w ith m etals and amines; arom atic p o rtion undergoes sub s titu tio n reactions such as n itra tio n , alkylation, acetylation and halogenation; chlorine atoms can be hydrolysed to produce polyhydroxyl benzenes, by reactio n w ith bases a t elevated tem peratures and p ressu res; o xidative decomposition occurs w ith stro n g o x id izin g agents (Howard & Durkin, 1973). 2 ,3 ,7 ,8 -T etrach lo ro d lb en zo -p an z-d io x ln may be formed as a by product during the sy n th esis of 2 ,4 ,5 -trich lo ro p h en o l by the h y d ro ly sis of 1 ,2 ,4 ,5-tetrachlorobenzene using methanol and sodium hydroxide a t elevated pressure or ethylene glycol and sodium hydroxide a t atmospheric p ressu re. In the l a t t e r case, .-a 1 ;eJ = 1) J ind mass y 1975). benzene, denatured 50C), 56; 1 C h ilto n , ion with ' $s te rs and goes subV? iUstyl a t i on ilo produce ited occurs . -7 -1;ft as a byby the )1 and -ol and tte r case, \ | i -I 2,4,5- AMD 2,4,6--TRICHLjOROPHENOLS 251 i f the re a c tio n tem perature exceeds the normal 180C process tem perature, 2 ,3 ,7 ,8 -tetrac h lo ro d ib e n zo -p ara-d io x in is formed by the condensation of two molecules of sodium 2 ,4 ,5 - tric h lo ro phenate under the Influence of the exothermic decomposition of sodium-2-hydroxyethanol (Mllnes, 1971). For additional Inform ation see IARC monograph on ch lo rin ate d dibenzo-paradioxlns (IARC, 1977a). 2 ,4 ,6-Trlchlorophenol From Vindholz (1976), u n less otherw ise s p e c ifie d _ (a) D escrip tio n : Yellow fla k e s (Hawley, 1977) (b) B o ilin g -p o in t: 246C Xc) M elting-point: 69C * i (d) Spectroscopy d a ta : W 296 nm (eJ = 129); 289 nm (eJ = 125) In methanol; in f r a - r e d , n u clear magnetic resonance and mass sp ectral data have been tabulated (G rasselli & Ritchey, 1975). (e) S o lu b ility : g/100 g so lv en t a t 25C: aceto n e, 525; benzene, 113; carbon te tra c h lo rid e , 37; diacetone alcohol, 335; d ieth y l eth er, 354; denatured ethanol, 400; methanol, 525; 7 -- pine o il, 163; Stoddard so lv en t, 16; toluene, 100; turpentine, 37; water, < 0 . (f) V o la tility : Vapour p ressu re I s 1 mm a t 76.5C (Perry & C hilton, 1973). S ta b ility : S table up to i t s m eltin g -p o in t. Heating of the phenate to 280C produced < 0 .1 mg/kg o c ta - and h ep tach lo rin ated dlbenzo-para-dioxins and < 0.02-0."03 mg/kg h exa-, p en ta- and te tra c h lo rln a te d dlbenzo-para-dioxins (Rappe e t a l. , 1978); R e a c tiv ity : Can be converted to sodium s a l t by re a c tio n with sodium carbonate; th e hydroxyl group forms e th e rs , e s te r s and s a lts with m etals and amines; the arom atic po rtio n undergoes . 352 IARC MONOGRAPHS VOLUME 20 su b stitu tio n reactions such as n itra tio n , alkylation, acetyl a tio n and halognation; ch lo rin e atoms can be hydrolysed to produce polyhydroyl benzenes, by reactio n with bases a t elev ated temperatures and p ressu res; oxidative decomposition occurs w ith stro n g o x id izin g agents (Howard & Durkin, 1973). 1.4 Tecb"*^"l products and Im p u rities ' manufactured : with methanol: l-ti Commercir reported In IS company report p. 16) (VS Ini were Imported (VS In tern a tic 2 ,4 ,5 -T ric h lo ro p h en o l i s a v a ila b le in th e VS as a 95Z technic a l grade p ro d u c t. Formulations a v a ila b le In th e VS are concentrated aqueous and non-aqueous s o lu tio n s , co n cen trated s o lid s and em u lslfiab le c o n c e n tra te s. A liq u id form ulation co n ta in s 4 5 .9Z 2 ,4 ,5 -tric h lo ro phenol as th e sodium s a lt . ------2 ,4 ,6 -T ric h lo ro p h en o l a v a ila b le in Japan has a p u rity of 97Z. I t I s a v a ila b le i n th e VS' In aqueous fo rm u latio n s. 2 .3 .7 .8 - T etrach lo ro d lb en zo -p ara-d io x ln was found in 3/6 samples of 2y4^5-trichlorophenol (or I t s sodium s a lt ) i n th e range of 0.07-6.2 mg/kg. .^2,7-D ichloro-, 1 ,3 ,6 ,8 -te tra c h lo ro - and pentachlorodibenzo-pora-dioxlns were found In concentrations of .0.72, 0.30 and 1.5 mg/kg, resp ectiv ely (Firestone e t a t. , 1972). 1 .3 .6 .8 - Tetrachlorodlbenzo-para-dloxln and 2,3,7-trichlorodibenzo- para-dioxin were found in a sample of 2 ,4 ,6 -trichlorophenol a t lev els of 49 and 93 mg/kg, re s p e c tiv e ly . In th e same stu d y , t r i - , t e t r a - and pentachlorodlm ethoxy-dibenzofurans were p resen t In 3/6 samples of 2 , 4 ,5 - . tric h lo ro p h e n o l o r I t s sodium s a l t ; and t e t r a - , p en ta- and hexachloro-..r dibenzofurans were found in one sample of 2 ,4,6-trichlorophenol (Firestone e t a t. , 1972). In a Swedish sample of 2 ,4 ,6 -tric h lo ro p h e n o l, 1.5 mg/kg 2 ,3 ,7 ,8 tetrach lo ro d lb en zo fu ran was found, a s w e ll as 1 7 .5 , 36 and 4.8 mg/kg p e n ta -, h ex a- and heptachlorodibenzofurans; le s s than 3 mg/kg poly c h lo rin ate d dibenzo-para-dioxins w ere.found (Rappe e t a t. , 1979). 2 . Production. Vse, Occurrence and Analysis 2.1 Production and use (a) Production Annual pi M: to be 1-10 mil' 1977a). Japanese Imports since kg annually. 2 .4 .6 -T n 2 .4 .6-T n tlo n of phenol S used in th e V! product o f or\ of phenol. y '< ' ; Commerd reported In IS in which prodi undisclosed at Dismission, IS p rin c ip a l US c ^ 1977b). Ho d a ta c 2.4.6- -since 1965. kg. None was (b ) V s e * {f t . 2,*,5-Tt< 2 .4 .5-Trlchlorophenol 2 .4 .5 - T richlorophenol was f i r s t prepared in 1920 by h eatin g 1 ,2 ,4 ,5 - te trach lo ro b en zen e w ith sodium meth03d.de (R ic h te r, 1944). I t was The major the manufacture (2,4,5-T) (see used in the mai [2 -(2 ,4 ,5 -tric l , acetyllysed to at ^position , 1973). inic a l Jm 'jg j II ivl|lP ft B. s i f i a b l e .*jp .loro- fm 72. i amples o f -6 .2 mg/kg i-dioxins actively 3 m 4i dibenzo- le v e ls of '3, > - --id a ,4,5- achloro- 3,7,8mg/kg poly- ). 2 ,4 ,5 " AND 2,4,6-TRICHLOROPHENOLS Commercial production o f 2 ,4 ,5 -trlc h lo ro p h e n o l in th e US was f i r s t rep o rted In 1950 (US T a r if f Commission, 1951). In 1976, one US company reported production of an undisclosed amount (see preamble, P> 16) (US In te rn a tio n a l Trade Commission, 1977a); 87.2 thousand kg were Imported through th e p rin c ip a l US customs d i s t r i c t s in th a t year (US In te rn a tio n a l Trade Commission, 1977b). Annual production of 2 ,4 ,5-trichlorophenol in A ustria is estimated to be 1-10 m illio n kg. I t was p rev io u sly produced in I ta l y (see IARC, 1977a). Japanese production of' 2 ,4 ,5 -tric h lo ro p h e n o l was stopped In 1971. Imports sin ce th a t year have amounted to approxim ately 10-15 thousand kg annually. 2.4.6- Trichlorophenol - 2 ,4 ,6-T richlorophenol was prepared by Laurent in 1836 by ch lo rin a tio n of phenol (Prager e t a l . , 1923), and th is method i s c u rre n tly used in th e US (Doedens, 1964). In Japan, i t i s produced as a co product of ortho- or para-chlorophenol manufacture by the chlorination of phenol. Commercial production of 2 ,4 ,6 -tric h lo ro p h e n o l In the US was f i r s t rep o rte d In 1950 (US T a r if f Commission, 1951). In 1974, the l a s t year in which production was re p o rte d , one company rep o rted production of an undisclosed amount (see preamble, p . 16) (US In te rn a tio n a l Trade Commission, 1975). In 1976, 1000 kg were imported through the p r in c ip a l US customs d i s t r i c t s (US In te rn a tio n a l Trade Commission, 1977b). - No d a ta on i t s production in Europe were a v a ila b le . 2 .4 .6- Trichlorophenol has been produced commercially in Japan sin ce 1965. In 1977, one company produced an estim ated 120 thousand kg. None was imported or exported. lg 1 ,2 ,4 ,5 - , was / 2 ,4 ,5-Trichlorophenol The major use fo r 2 ,4 ,5 -tric h lo ro p h e n o l i s as an in term ed iate in the manufacture of the herbicide 2,4,5-trlchlorophenoxyacetlc acid (2 ,4 ,5 -T ) (see IARC, 1977b) and i t s e s te r s (Doedens, 1964). I t i s also used in the manufacture of 3 other chemicals used as p e stic id e s: Silvex [2-(2,4,5-trichlorophenoxy)propionic a c id ], Ronnel (O ,0-dim ethyl-0-2,4,5- 1 1 2. KIRK-OTHMER JENCYCLOPEDIA OF CHEMICAL TECHNOLOGY, THIRD EDITION VOLUME 5 CASTOR OIL ---- TO CHLOROSULFURIC ACID A WILEY-INTERSC1ENCE PUBUCATION John Wiley & Sons NEW YORK CHICHESTER BRISBANE TORONTO 7881 f M't/H Copyright 1979 by John W iley & Sons, Inc. All rights reserved. Published simultaneously in Canada. Reproduction or translation of any part of this work beyond that permitted by Sections 107 or 108 of the 1976 United States Copyright Act without the permission of the copyright owner is unlawful. Requests for permission or further information should be addressed to the Permissions Department, John Wiley & Sons, Inc. Library of Congress Cataloging in Publication Data: Main entry under title: Encyclopedia of chemical technology. A t head of title: Kirk-Othmer. " A Wiley-Interscience publication." Includes bibliographies. 1. Chemistry, Technical-- Dictionaries. I. Kirk, Raymond Eller, 1890-1957. I I. Othmer, Donald Frederick, 1904- I I I . Grayson, Martin. IV . Eckroth, David. V. Title: Kirk-Othm er encyclopedia of chemical technology. TP9.E685 1978 660\03 ISBN 0-471-02041-9 77-15820 Printed in the United States of America Casto) Cataly Cataly Cellule 'Cellule Cellule Cellule Cernei Centrii Ceram Cerarci Ceriun Cesiun Chelati Chemii Chemi< Chemk 866 CHLOROPHENOLS Dichiorophenois The main dichlorophenol of commercial interest is 2,4-dichlorophenol. It is used in large volumes in the manufacture of 2,4-dichlorophenoxyacetic acid (2,4-D), and is conveniently manufactured via the chlorination of phenol. In a patented process phenol is dissolved in liquid SO2 and treated with cold gaseous chlorine to give 98% pure 2,4-dichlorophenol (12). If the above procedure is applied to 2-chlorophenol, the product contains 10% 2,6-dichlorophenol. 1,2,3-Trichlorobenzene when sulfonated yields a mixture of 2,3,4-trichlorobenzenesulfonic acid and 2,3,4-trichlorobenzene1,5-disulfonic acid. Treatment of this mixture with alkali hydroxide followed by acid hydrolysis yields 2,3-dichlorophenol (13). 3,4-Dichlorophenol can also be obtained via preparation of the corresponding 3,4-dichlorocumene (propylene plus o-dichlorobenzene) and subsequent oxidation followed by treatment with sulfuric acid (14). Several investigations have been carried out on the hydrolysis of 1,2,4-trichlo robenzene. Treatment with copper, iron, or zinc halides as catalysts has resulted in the formation of 3,5-dichlorophenol (15,16). The hydrogenation of polychlorinated phenols over catalysts consisting of heavy metals, with the addition of FeSCU, CuSO*, NaaS, or sulfur yielded 3-chlorophenol and 3,5-dichlorophenol (17). 3,5-Dichlorophenol has also been prepared via the partial dehalogenation of polychlorophenol using cat alysts prepared from group VIII metals and sulfur or sulfides (18). A wide variety of dichiorophenois have been separated by the use of countercurrent dissociation ex traction (19). Trichlorophenols The most important analogue in the trichlorinated phenol series is 2,4,5-tiichlorophenol. It is used as an intermediate in the manufacture of the herbicides 2.4.5- trichlorophenoxyacetic acid (2,4,5-T) and 2-(2,4,5-trichlorophenoxy)propionic acid (silvex), the germicide hexachlorophene and the insecticides trichloronate and Fenchlorophos. It has also been used in the past as an intermediate in the synthesis of the herbicide erbon (2-(2,4,5-trichlorophenoxy)ethyl 2,2-dichloropropionate). 2.4.5- Trichlorophenol is currently marketed by The Dow Chemical Company and is used as an antifungal agent in several applications (20): in adhesives as a preservative in polyvinyl acetate emulsions; in the automotive industry to preserve rubber gaskets; and in textiles to preserve emulsions used in the rayon industry. 2,4,5-Trichlorophenol is generally applied as a direct additive (ie, directly into molten rubber), or dissolved in the oil phase of emulsions. The sodium salt of 2,4,5-trichlorophenol is also used as a fungicide and bactericide. Uses include (21): adhesives (qv), as with 2,4,5-trichlorophenol; in cooling water as an inhibitor of microbial growth in recirculating water, in foundry core wash, to prevent breakdown of oils and scum formation; in leather dressing and finishes, to prevent the decomposition of nitrogenous compounds; in metal working fluids, to prevent breakdown of oils, emulsifying agents, and other components. An aqueous solution of the sodium salt is normally used in these applications. 2,4,5-Trichlorophenol is manufactured via the chlorination of benzene with 4 moles of chlorine to form 1,2,4,5-tetrachlorobenzene. The tetrachlorobenzene is then hydrolyzed in base to form the desired product. Care needs to be taken to minimize Vol. 5 the formatio rophenol, pr by Dow as a] 2.4.4.6- tetra( (22). In a sii 2.2.4.5.6.6as the main ] di-, and tri-< rachlorophe spraying ch' Tetrachloroi 23,4,6Little ment are present breakdown Thee) catalyst ha tetrachlorc with ion-ej is washed \ Avari to yield in< Pentachlor Penti antimicrol - ---------applicatioi growth in industry, '> blocks; in in the pai: phenol is ; Fungicide The: and has a of adhesi storage a surfaces; in the tie protein-b photogra and presi rot, and during st in indusi primaril- is used j), and process ve 98% 10I, the mated nzeneiy acid tained iichloc acid richloited in inated )uS04, phenol ngcatiety of on ex- ,5-trincides pionic te and thesis >nate). and is vative askets; phenol solved :ricide. iter as revent revent revent olution with 4 >sthen nimize Vol. 5 CHLOROPHENOLS 867 the formation of 2,3,7,8-tetrachlorodibenzo-p-dioxin in this reaction. 2,4,6-Trichlorophenol, produced via the chlorination of phenol, has been manufactured and sold by Dow as a preservative. Chlorination of 2,4,5-trichlorophenol in acidic solution gives 2.4.4.6- tetrachloro-2,5-cyclohexadienone and 2,2,4,5,6,6-hexachloro-3-cyclohexenone (22). In a similar experiment the chlorination of 2,4,6-trichlorophenol also yielded 2.2.4.5.6.6- hexachloro-3-cyclohexen-l-one (23). 2,4,5-Trichlorophenol is obtained as the main product in the chlorination of 3,4-dichlorophenol (24). Mixtures of mono-, di-, and tri-substituted phenols have been obtained via the reduction of*2,3,4,6-tetrachlorophenol (25). 2,4,6-Trichlorophenol har been obtained in yields of 97.5% by spraying chlorine into molten phenol (26). Tetrachlorophenols 2,3,4,6-Tetrachlorophenol is available commercially and is used as a preservative. Little mention is made in the chemical literature of other tetrachlorophenols. They are present in small quantities in pentachlorophenol (27) and are formed during the breakdown of pentachlorophenol in the soil (28). The chlorination of phenol using specifically potassium tellurate, KjTeOa, as dtalyst has been found to give exclusively tetrachlorinated phenols (29). Isomers of tetrachlorophenol and pentachlorophenol have been separated via gradient elution with ion-exchange resins (30). The mixture is adsorbed onto Dowex 2-X8 resin, which is washed with sodium acetate, acetic acid, and methanol. A variety of chlorophenols in aqueous solutions can be degraded with y radiation to yield inorganic chloride and oxalic acid (31). Pentachlorophenol Pentachlorophenol (Penta, PCP) and its sodium salt are used extensively as antimicrobial agents. Pentachlorophenol is used as an antifungal agent in the following applications (32): in the wood industry as a perservative to control termites and fungus growth in building poles, posts, lumber, etc (most extensive use); in the construction industry, to control molds on inert building surfaces such as, tile roofs, and concrete blocks; in the leather industry, to impart mold resistance for upper leather in shoes; in the paint industry, for self protection of protein-based latex paints. Pentachloro phenol is added directly to the formulations or in an appropriate organic solvent (see Fungicide; Industrial antimicrobial agents). The sodium salt of pentachlorophenol is used as an antifungal and antibacterial, and has applications in the following areas (33): adhesives--antimicrobial protection of adhesives based on starch, vegetable, and animal proteins during manufacture, storage and service life; construction materials--control of mold growth on inert surfaces as with pentachlorophenol; leather--prevention of hide deterioration and in the treatment of solutions during tanning; paint--aid in the shelf preservation of protein-based latex paints; petroleum--prevent the growth of bacteria in drilling muds; photographic solutions--control of fungus and slime; pulp and paper--protection and preservation of processing materials, stored pulp and fiberboard against mildew rot, and termites; textiles--protection of finished yarns and cloth against molding during storage; water treatment--control of algal, fungal, and bacterial induced slimes in industrial recirculating water. The sodium salt of pentachlorophenol is utilized primarily in an aqueous solution. 11 3 / *7 3 , 7885 !1T98 Occupational Exposure to H erbicides . . . r A rth u r R. G regory, NIOSH The h e r b ic id e in d u s try in 1979 w ill g ro ss 30 b i l l i o n d o l l a r s . No one would deny T erb icid e s has been found u s e fu l to man. H e rb ic id e s, o r weed k i l l e r s , a re most ^ ften classed as p e s tic id e s . While th is is probably in c o r r e c t, both s tr u c tu r a lly and g e n e r ic a lly , most of us ag ree weeds a r e p e s ts . In 1977, 387,000 to n s w ere aroduced. H erbicides can k i l l weeds on co n tact or can be tra n sp o rte d to o th e r so rtio n s of the p lan t to produce the desired e ffe c t. Dne th in g should be im m ediately e s ta b lis h e d . Most h e r b ic id e s t h a t a r e commonly u sed to d a y aave a low acu te to x ic ity and have caused l i t t l e o r no d i f f i c u l t y in e ith e r m anufac- _ o r a p p lic a tio n . Some good examples o f t h i s a r e Na S u lfam ate d alap o n , p e tro leu m a i l s , Crag h e rb ic id e s and Na B o rate. O ther h e rb ic id e s pose a more s e r io u s problem . M aleic h y d ra z id e and Na C h lo ra te b o th s e v e rly a f f e c t th e CNS. P e n ta ch lo ro p h en o l can cau se d e a th v ia e x c e ss iv e m e ta b o lic stim u la tio n . The p a tie n t a c tu a lly d ie s from s e lf-g e n e ra tio n of h eat (h y p erth erm ia). L e s s e r doses o f p e n ta c h lo ro p h e n o l, e s p e c i a ll y when ab so rb ed th ru th e s k in w i l l p roduce a p e rip h e ra l motor neuropopathy th a t is n early pathognomonic of exposure to th is a e rb ic id e . N itro p h en o lic h e rb ic id e s, such as the d in itro p h en o ls and d in itro c re s o l are a lso h ig h ly to x ic to mammals in c lu d in g humans. They a r e v e i l ab so rb ed th ru th e GI t r a c t t th e sk in as w ell as th e lung. In general they a e ro s o liz e e a s ily . They produce a yv jv s t a i n on th e s k in . They damage th e l i v e r , k id n e y s and CNS by uncoupling o x id a tiv e p h o sp h o ry lizatio n . A granulocytosis and c a ta r a c ts are asso ciated w ith : 7 8 8 6 prolonged contact w ith the n itro p h en o ls. 0000726 DOVV3SG17 - 2- P araq u at and d iq u at a re d ip y rid y ls and have a tremendous a f f i n i t y fo r e p ith e lia l t i s s u e s . As i t c o n c e n tra te s on th e s k in , GI t r a c t o r lu n g s u r f a c e t h i s ty p e o f h e r b ic id e causes inflam m ation and lo c a l n e c ro se s. In se v e re p a ra q u a t p o iso n in g th e m y o c a rd itis and CNS s ig n s a r e m o stly t r a n s i t o r y , b u t th e pulm onary f i b r o s i s which is mild a t f i r s t tends to p ro g ress, and th e p r o lif e r a tio n of th e connective ti s s u e lead s to death' a f te r 1-3 weeks i f the poisoning i s sev ere. I f exposure is m ild e r th e lung f i b r o s i s may develop o n ly over y e a r s . U rea, u r a c il, tr ia z in e , am itro le a n ilid e , a c e ta n ilid e and c a rb a n ila te d e riv a tiv e s have r e c e n tly been developed. W hile they show low sy stem ic to x i c it y in la b o ra to ry a n im a ls , many s u f f e r th e stigm a o f s k in s e n s i t i z a t i o n and have s t r u c t u r a l c h a r a c te r i s t i c s i n common with.known c a rc in o g e n s . Because o f th e p r e s e n t d i f f i c u l t y in g i's tr a t io n and req u irem en t f o r c h ro n ic t o x i c i t y t e s t s , few o f them w i l l ev er re a c h th e m arket. Now to th e goodies - th e chlorophenoxy d e r iv a tiv e s : The s a l t s and e s ta r s of d i and tr ic h lo r o phenoxyacetic a c id a re s im ila r in t o x i c i t y . They a re i r r i t a t i n g to th e s k in , e y e s, r e s p i r a t o r y and GI l i n i n g s . They a r e w e ll absorbed by a l l th r e e r o u te s . The i n t e s t i n e , th e lu n g and th e s k in . None a p p e a r to be s to re d in th e body f o r lo n g . Most a re gone in h o u rs, a l l a f t e r 2 d ay s. Body c le a ra n c e i s e x c e lle n t. T his i s n o t tr u e f o r th e to x ic contam inant T e tra c h lo ro dib.enzo d io x in (TCDDJ, how ever, b u t more on t h a t l a t e r . Most chlorophenoxy e s te rs a re regarded as n o n to x ic, although th e re have been r e p o r te d th r e e c a s e s o f p e r ip h e r a l n eu ro p ath y when exposed to 2-4 D. In 000072?  . - 3- s u s c e p tib le in d iv id u a ls , lo c a l depigm entation has a lso r e s u lte d from prolonged exposure to 245 T. D uring th e 1950's i t became in c re a sin g ly c le a r th a t d if f e r e n t batches of 2,4,5-T had d i f f e r e n t t o x i c i t y and th a t to x i c i t y was due to c o n ta m in a n ts. The m ajor s ig d em o n strated in w orkers were c h lo ra c n e and n e u ro lo g ic d e f i c i e n c i e s . Many o f th e p o s s ib le co n tam in an ts w ere s tu d ie d by Dow ana as a r e s u l t , th e y have a d ju ste d s y n th e t ic m ethods so th a t 2,3,7,8-TCDD th e most p o te n t co n tam in an t was red u ced to 1 ppm. However, d u rin g th e V ie t w ar, Dow was c a lle d on to supply th e ., -- A ^r>> Army w ith la r g e amounts o f th e b u ty l e s t e r o f 245 T and th e s e b a tc h e s (ag en t orange) c o n ta in e d 1-80 ppm c o n ta m in a n ts. /ft *- tr h B ecause o f th e p u b l i c i t y su rro u n d in g t h i s u s e , Dow as w e ll a s o th e r s d id f u r th e r tin g of 23.78 TCDD. The gu in ea p ig was found to be ex trem ely s e n s a tiv e to i t . The LD-50 f o r m ale g u in e a p ig s was l e s s than lpgm./kg PO compared to ov er 100 ug/kg f o r r a b b i t s . Bat% had in te rm e d ia te l e t h a l i t y w ith 45 //gW kg. I n t e r e s t i n g l y IP d osing i n r a b b it s gave an LD 50 o f ap p ro x im ately 400 uETM compared to 100 by mouth and 275 f o r .dermal a b s o rp tio n . M ales a r e more s e n s i t i v e th a n fe m a le s . Dogs w ere le s s se n sa tiv e than ra b b its . The signs of to x ic ity are extrem ely species dependant. A s c ite s i s seen in m ice, p e r i c a r d i a l edema in c h ic k e n s , d e h y d ra tio n in r a t s , a n o re x ia in dogs and h e p a tic n e c r o s is in r a b b i t s . However, a common s ig n in a l l species is loss of w eight. The a c u te s ig n s in w o rk ers exposed to 245 T was c h lo ra c n e in 18% o f 73 p o t e n t i a l l y ex posed. The LD-50 o f 24 D in man i s c lo s e to 500 m g/kg, p er 245 T 100 mg/kg. The o ccu ra n ce o f p o rp h y ria c u tan ea ta rd a in w orkers le a d B le ib e rg to p o s tu la te 2378 TCDD the in g re d ie n t resp o n sib le fo r to x ic ity (1964) * 7888 0000728 2 ,4 ,5 - T was f i r s t r e g is te r e d in 1948. Today some 122 com panies s e l l 245 T in some form in over 400 commercial p ro d u cts. ` Qa , CO CO CD The c h ro n ic t o x i c i t y o f 2378 TCDD i s th e most i n t r i g u i n g . I n monkeys a t v e ry low d o s e s , i t i s d i f f i c u l t to t e l l th e e f f e c ts of low dosing from th e normal aging p ro c e ss. For example, th e signs in clu d e lack of a p p e tite , greying of h a ir , dry s k in , sw ollen e y e lid s , f a llin g h a ir, scaley sk in and lack o f co o rd in atio n in movement. In 197.7, Van M ille r re p o rte d s ig n if ic a n t c a rc in o g e n ic re sp o n se a t 5 p p t in th e .r X , j\jA -) d i e t fo r 78 weeks. There were 6 neoplasms in 5 of te n r a t s tr e a te d . The tumors were ear duct ca, lym phatic leukem ia, adenoca of th e kidney, histocytosarcom a of a p erito n eu m , angiosarcom a of th e sk in and a le y d ig c e l l ademema. However, a t fo u r h ig h e r doses th e re was a la c k of dose response u n t i l th e re was a 1000 fo ld in c r e a s e In d o se . At 5 ppb, th e r e was a 703! in c id e n c e o f neo p lasm s. I n th e US ex p o su re to 2358 TCDD in th e w o rk p lace a p p e a rs to be un d er c o n t r o l , b u t r i n 1971 w a ste o i l c o n ta in in g 11 kg o f TCDD was used to c o n t r o l d u s t on a h o rs e % farm in e a s te r n M is s o u ri. Of th e 86 h o rs e s su b se q u e n tly e x e rc is e d t h e r e , 62 became i l l and 42 d ie d . I f we could have m easured th e d o se in th e h o rs e s t h i s would have b een an LD-50.'. One s ix - y e a r o ld g i r l developed h em orrhagic c y s t i t i s from exposure to th e d u s t. The exact number of dogs, c a t s , and ch ick en s could only be e stim a te d . The ro u te of en try could have been dermal as w ell as in h a la tio n of th e co n tam in a ted d u s t. S o il sam ples tak en 3 months l a t e r re v e a le d 32 mg/Kg TCDD. The h a l f - l i f e o f TCDD in t h i s I n s ta n c e was e s tim a te d a t 1 1 /3 y e a rs (463 d a y s ) . E lg in A ir F orce base where agent orange was te s te d , th e h a l f - l i f e was estim ated at 3 years. 7889 00007:9 OomoMoa - 5- In Seneso, I ta ly in 1976, a chemical p la n t a c c id e n tly exploded and exposed o v er 4 ,0 0 0 p eo p le to TCDD. A book by C a v a lla ro on t h i s e p iso d e o u tl in e s th e a c u te e f f e c t s b u t th e long term e f f e c t s rem ain to be s e e n . No one w i l l deny th e u sefu ln ess of h e rb ic id e s, but en v iro n m en talists argue th a t even though th e 245 T may be r e l a t i v e l y s a f e i f handled p r o p e r ly , th e c o n tam in a tio n d an g ers outw eigh th e b e n e f i t s . T h is may be e s p e c i a ll y t r u e a s u sage c o n tin u e s over the years. The EPA r e c e n tly d ecid ed to ho ld up th e u se o f m ost 245 T and s i lv e x . T h is was based on p o s s ib le te rao lo g y in th e human. While an ep id em ic o lo g ic al stu d y in Oregon shoved an a s s o c ia tio n between 245 T s p ra y in g and in c re a s e d m is c a r r ia g e s , : TCDD was found in any of th e women s u f f e r in g m is c a r r ia g e s . The l i n k o f cau se and e ffe c t is apparent but not estab lish ed . The f i n a l l i n e on TCDD i s s t i l l to be w r itt e n . Van M ille r 1977 o <O . 03SC2I TCDD le v e l 0 1 ppt 5 ppt 50 p p t 500 ppt 1 ppb 5 ppb Incidence of Neoplasms 0 0 5 T o tal Neoplasms 0 0 6 33 4 ...................................... ......... 4 4 5 7 10 Dx -- none none Zymbal Ca Lymph Leuk nephroca h is tio c a angioca le y d ig adenoma rhabdomyosarcoma squamous c e l l tumor astrocytom a fibrom a epltheliosarcom a nephroca seminoma cholangio ca anglo ca 2 histlosarccm as g lio blastem a 4 lung ca 4 hepatomas 2 cholangioca 00C073.1 1 1 *f 7892 126635 J w A REVIEW OF THE GENETIC TOXICOLOGY OF CHLORINATED D IB E N Z O -p -D IO X lN S * J . S. Wassom1, J . E. H u ff2 3 , and N. L o prienou E nvironm ental Mutagen In fo rm atio n Center 2Biom edical Sciences S ection Inform ation Center Com plex/Inform ation D ivision Oak Ridge N a tio n a l L ab o rato ry Oak R id g e, Tennessee 37830 3U n it o f C h em ical C a r c in o g e n e s is In te rn a tio n a l Agency fo r Research on Cancer 69372 Lyon Cedex 2, France ^Laboratory o f Genetics In s titu te o f Anthropology Pisa U n iversity 56100 Pisa, Ita ly C O Work sponsored by the N ational In s titu te o f Environm ental H ealth Sciences under Interagency Agreement 40-247-70 and the Toxicology Inform ation Program /National L ib ra ry o f M edicine under Interagency Agreement 40-274-71 under Union Carbide C orporation co n tract W -7405-eng-26 w ith the U. S. Department o f Energy. *051000 ' O A REVIEW OF THE GENETIC TOXICOLOGY OF CHLORINATED D IB E N Z O -p -D IO X IN S J. S. Wassom, J . E. H u ff, and N. Loprieno SUMMARY (T> <n co & In fo rm a tio n from both published and unpublished sources considered re le v a n t to th understanding of the genetic toxicology of chlorinated dibenzo-?-dioxir,s is summarized in th is review . In te r e s t in w ritin g th is paper was s tim u la te d by the fa c t th a t th is class of compounds, p a rtic u la rly 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n zo - p -d io x in (TCDD), has gained n o to rie ty as an extrem e environm ental and in d u s tria l hazard. Human exposure in th e work p lace occurs when d io x in s are form ed d u rin g the synthesis o f a number o f cornm ercially im portant compounds such as 2 ,4 ,5 - trich lo ro p h en o xyacetic acid , hexachlorophene, and pentachlorophenol. Environ mental contam ination re s u lts from m anufacturing processes and from dioxin contaminants in marketed products. Research on d io x in s as p o te n tia l mutagens was i n i t i a t e d because o f t h e ir s tru c tu ra l s im ila r it y to a c rid in e s , a c la s s o f known in te r c a la tin g ag en ts. To d a te , o n ly fo u r d io x in compounds have- been evalu ated fo r m u ta g e n ic ity : the d i , t e tr a -, and o cta-c h lo rin a te d d e riv a tiv e s and the unsubstituted d ib en zo -p -d io xin . Since o n ly a few o f the many p o ssib le s tru c tu ra l forms o f d io xin s have been te s te d , no conclusions can be made about th e ir p o te n tia l m u ta g e n ic ity o th er than noting th e ir candidacy as in te rc a la tin g agents. F u rth e r, a ll the. p o s itiv e m u ta g e n ic ity and c y to lo g ic a l, e ffe c ts rep o rted thus f a r w ith d io xin s seem to depend on the p o s itio n o f c h lo rin e s u b s titu tio n . The most active~fo rm o f the m olecule is the 2 ,3 ,7 ,8 - d e riv a tiv e (TCDD). 00 D15 0 5 7894 D O W 126687 / D ata a v a ila b le f o r a ssess in g th e m u ta g e n ic p o t e n t ia l o f TCDD a re con f lic t in g and scarce. D iffe re n c e s in te s tin g re s u lts rep o rted in these studies could be a ttr ib u te d to s o lu b ilit y problems w ith th e te s t c h em ical, treatm en t pro to co ls, p u rity o f te s t samples, or to x ic ity . Because there are c o n flic tin g d a ta , a d d itio n a l experim ents are needed b e fo re the m utagenic p o te n tia l o f TCDD and o th e r d io xin s can be determ ined. S tu d ies e x p lo rin g the promoting e ffe c t o f d io x in s on th e m u ta g e n ic ity o f o th e r compounds are als o recommended because e x p e rim e n ts have shown TCDD to be an e x tre m e ly a c t iv e l i v e r enzyme in d u c in g agent th a t enhances the m utag en icity o f c e rta in p o ly c y c lic hydrocarbons such as 3-m ethylcholanthrene. The im portance o f d is c e rn in g th e hazards to human h e a lth from d io x in compounds became ap parent a f t e r an a c c id e n ta l re le a s e o f TCDD from a chem ical p lan t contam inated the Seveso, Ita ly area in July 1976*. This accident revealed th at in s u ffic ie n t data were a v a ila b le to properly evaluate the long term h ealth risks posed by d io xin compounds. Several research p ro jects were th e re fo re in itia te d a fte r the Seveso in c id e n t; i t is hoped th a t many o f the q u e s tio n s co n cern in g th e m u ta g e n ic ity o f TCDD and p o s s ib ly o f o th e r d io x in congeners w ill be answered as a re s u lt o f th is work. * In Jan u ary 1978, a jo in t In te rn a tio n a l Agency fo r Research on Cancer (IA R C )/ US N a tio n a l I n s t i t u t e o f E n viro n m en tal H e a lth S ciences ad hoc W orking Group was convened on the C oo rd in atio n o f E p id em io lo g ical S tudies on the Long-Term Hazards o f C h lo rin ated D ibenzodioxins and C h lo rin ated D ib en zo fu ran s. The p a rtic ip a n ts recommended th a t IARC c o o rd in a te th e fo llo w -u p o f the a c c id e n t-e x p o s e d in d iv id u a ls 0001506fro m f i v e c o u n t r i e s (IA R C I n t e r n a t i o n a l T e c h n ic a l R e p o r t N o. 7 3 / 0 0 1 , 1 9 7 3 ) . 7895 DOW 126688 3 INTRODUCTION C h lo rin ated d ib en zo -p -d io x in s are a group o f chem ical compounds which are among the most to x ic and hazardous -p o llu ta n ts in th e en viro n m en t. These compounds, c o lle c tiv e ly re fe rre d to as d io x in s , are im p u ritie s associated w ith c e rta in end products re s u ltin g from the treatm en t o f c h lo rin a te d benzenes a t elevated tem perature and pressure under a lk a lin e co n d itio n s. The most notable contam inant o f th is group is 2 ,3 ,7 ,8 -te tra c h lo ro d ib e n z o -p -d io x in (TCDD) (see F ig . 1) which may be formed along w ith o th er d io x in compounds during the m anufacture o f several com m ercially im portant products such as the herb icid e 2,4,5-trichlorophenoxyacetic acid (2 ,4 ,5 -T )25, the fungicide pentachlorophenol33, and the germ icide hexachlorophene39. Suspicions o f the possible long-term health hazards o f dioxins arose a fte r i t was found th a t 2 ,4 ,5 -T was te ra to g e n ic in the r a t and mouse1 5 . S h o rtly th e re a fte r i t was discovered th a t the 2 ,4 ,5 -T sample used in th is study con ta in e d ab o u t 30 ppm TCDD1 5 . I t was p r im a r ily th e r e p o r t im p lic a t in g TCDD as a c o n ta m in a n t o f 2 , 4 , 5 - T 1 s t h a t le d to i t s f u r t h e r e v a lu a t io n f o r t e r a t o g e n i c i t y 15 and its eventual te s tin g fo r m u tag en icity2 1 . These and o th er s im ila r rep o rts published during the la te 1950's and e a rly 1970's also stim u lated to x ic o lo g ic a l stu d ies on o th e r d io x in d e riv a tiv e s as w ell as s tu d ie s d e a lin g w ith issues such as environm ental contam ination and movement and a n a ly tic a l d e te c tio n o f these compounds. D eta ile d s ta te -o f-th e a rt reviews summarizing work in these areas have been p u b lish ed 3 5 -3 7 39 or are c u rre n tly being prepared fo r p u b lic a tio n 1 1 *2 0 . Since the question o f p o te n tia l m u tagenicity, carcin o g en icity, and te ra to g e n ic ity o f dioxins has been ra is e d as a re s u lt o f th e in c id e n t in Sevesd, I t a l y * 5 1 *5 9 , we thought i t im portant to re v ie w the in fo rm atio n a v a ila b le on the g en etic to x ic o lo g y o f these compounds. 0001507 7896 Dovtf.26689 t F ig . 1. S tru c tu ra l comparison o f acrid in es and dio xin s 0001508 7 8 $7 ACRIDINES ORNL-OWC 7 7 -2 9 6 9 R H1 HN(CH2)3NCH2CH2CI / och3 \ y 2HCI ICR-191 5 DIOXINS -- TRYPAFLAV1NE DIBENZO-/-DIOXIN 2. 7-DICHLOROOI0ENZOp - DIOXIN 2 .3 ,7 , 8 --TETRACHLORO D IB E N Z O -p -D IO X IN 1,2. 3 .4 .6 . 7.0. 9 OCTACHLORO --DI BENZO L-DIOXIN 6051000 0699ZTMOa CD OO DQW126691 5 The o n ly th eo ry proposed to e x p la in how d io x in s may e x e rt t h e ir m utagenic e f f e c t is th e suggestion t h a t these compounds in t e r c a la t e DMA. T h is mechanism -w as proposed due to the s im ila r ity in s tru c tu re between d io xin s and acrid in es (see F ig . 1 ); th e re fo re , in fo rm a tio n comparing the m u ta g e n ic ity o f d io xin s and a c rid in e s has been included (see "Com parative M u ta g e n ic ity o f D ioxins and A cridines"). The basic d ib en zo -p -d io xin nucleus is n early p lan ar and has e ig h t possible p o in ts o f chem ical s u b s titu tio n . From the m onochloro- to the o c ta c h lo ro d ib e n z o -p -d io x in , a v a r ie t y o f d e r iv a tiv e s a re p o s s ib le and some have proven to be ch em ically p e rs is te n t and b io lo g ic a lly a c tiv e 30. The le th a l dose and to x ic m a n ife s ta tio n s are s tru c tu re and species dependent. O f a ll the p o s s ib le d io xin s tru c tu ra l c o n fig u ra tio n s , TCDD is th e most w id e ly known and te s te d . This d e riv a tiv e has been c a lle d one o f the most p o ten t sm all m olecule to x in s known3 0 . Because o f its extreme to x ic ity and p o te n tia l m u ta g e n ic ity , c a rc in o g e n ic ity * * , and te r a to g e n ic ity 15, la b o ra to ry use should be c a re fu lly c o n tro lle d and a r ig id safety protocol follow ed8. *Seveso, Ita ly (population approxim ately 17,000) is located near the c ity of M ilan. In J u ly 19 7 6 , th e Seveso a re a was a c c id e n t a lly exposed to h ig h le v e ls o f TCDD as a re s u lt o f an explosion in a nearby chemical p la n t. For a comprehensive day-by-day d e s c rip tio n o f th is a c c id e n t, th e read er may w ish to c o n s u lt the book by John 6 . F u l l e r e n t i t l e d The Poison T h a t F e ll f r c i th e S k y , 1 9 7 7 , Random H o u s e , I n c . , Mew York. ^ O * * A t le a s t n in e lo n g -term experim ental c a rc in o g e n ic ity s tu d ie s a re in progress on various ch lo rin ated dioxins (Ghess, M -J ., Bartsch, H ., H u ff, J .E ., and Tom atis, L .t IARC In fo rm a tio n B u lle tin on th e Survey o f Chem icals B eing T ested f o r C a rc in o g e n ic ity , Number 7, Lyon, January 1978, 460 pages). 7899 { / 2 6 9 9 2 T? .6 There are several general a rtic le s which re fe r to the possible m utagenicity o f d i o x i n s 1* " 6 , 6 8 , b u t t h e b u l k o f th e m a t e r i a l r e v ie w e d came fro m a number o f p u b lis h e d - p a p e r s t h a t c o n t a i n e x p e r i m e n t a l r e s u ^ t s 7 1 3 1- 7 , 2 7 ' 2 8 , 3 8 , `* 0 >t' 1 l , 3 , 5 7 , 6 l * d e s c r i b i n g the evalu a tio n o f these compounds fo r mutagenic an d /o r re la te d c y to lo g ic a l e ffe c ts . In fo rm a tio n from unpublished sources is also in clu d ed 9,!*9 . Summaries o f a ll these investig atio n s are presented in the follow ing section. GENETIC TOXICOLOGY TESTING J a c k s o n 1* 0 e v a l u a t e d h i g h l y p u r i f i e d s a m p le s o f 2 , 4 , 5 - T * and TCDD f o r c y to lo g ic a l e ffe c ts in th e A fr ic a n blood l i l y {Hcencnthus kazh zrin as B a k e r). Treatm ents in v o lv in g both compounds in varying pro p o rtio n s were s tu d ie d . In c o n tra s t to the n o -e ffe c t re s u lt w ith a h ig h ly p u rifie d sample o f 2 ,4 ,5 -T , d r a m a t i c i n h i b i t i o n o f m i t o s i s was o b s e rv e d i n c e l l s e x p o s e d e i t h e r t o 1 0 " 1* m o la r 2 ,4 ,5 - T c o n ta in in g 0 .2 pg to 1 .0 pg TCDD p er l i t e r o f w a te r * * o r to a 10"** m olar s o lu tio n o f 2 ,4 ,5 - T c o n ta in in g an unknown le v e l o f TCDD as a co n ta m in a n t. S im ila r r e s u lt s were o b ta in e d when tre a tm e n ts were lim it e d to TCDD alo n e ( 0 .2 pg and 1 .0 pg TCDD p e r l i t e r o f w a t e r * * ) . These tre a tm e n ts also induced form ation o f d ic e n tric bridges and chrom atin fusion w ith form ation o f m u ltinuclei or a single larg e nucleus. Because these effe c ts were not evident in the pure 2 ,4 ,5 -T sample, Jackson concluded th a t the c yto lo g ical e ffe c ts produced w ere due to th e TCDD c o n ta m in a n t. F u rth er inform ation regarding the cytological e ffe c ts produced by 2 ,4 ,5 -T w ill b e a v a i l a b l e soon i n a p a p e r e n t i t l e d "The G e n o t o x ic E f f e c t s o f 2 , 4 , 5 - T " b e in g .. p re p a re d f o r p u b lic a tio n in M u ta tio n Res. by W. F. G ra n t. **J a c k s o n u0 re p o rte d t h a t th e maximum s o lu b ilit y o f TCDD in w a te r was 0 .2 p g / l i t e r and th a t the 1.0 p g /lite r treatm ents were th erefo re probably subjected only to a w ater-saturated solution (0.2 p g /lite r). -A lO CO o c? 7900 E699fcT* DC D a v rin g and Summer18 r e p o r t e d r e s u lt s o b ta in e d w ith a com m ercial sam ple o f 2 ,4 ,5 -T in which d io x in c o n tam in atio n was less than 0.1 ppm. This fo rm u latio n was e v a lu a te d f o r c y t o lo g ic a l e f f e c t s in a w i l d - t y p e D ro s o p h ila m e la n o ja s te r p o p u la tio n by exposing a d u lt f l i e s , 24 hours a f t e r eel os io n , to 250 ppn 2 ,4 ,5 -T in food. Results in d icated th a t th is 2 ,4 ,5 -T form ulation affected e a rly oogenesis and caused s t e r i l i t y . However, i t was not u n e q u iv o c a lly s ta te d th a t the observed s t e r i l i t y was o f g en etic o r ig in . T he fo rm a tio n o f m u ltin u c le a te d c e lls a f t e r tre a tm e n t w ith TCDD has a ls o b e e n o b s e r v e d i n m am m als1 2 2 9 1* 2 . F o r e x a m p le , G r e ig e t a l . 29 t r e a t e d fe m a le Porton ra ts w ith s in g le o ra l doses (5 0 -4 0 0 u g /kg ) o f TCDD d is s o lv e d in d im e th y l- su lfo xid e o r arachis o il. H is to lo g ic a l exam ination o f liv e r c e lls 60 days a fte r tre a tm e n t w ith 100 ug/kg TCDD re v e a le d th a t parenchym al c e ll s tru c tu re s were a lte r e d and many w ere m u ltin u c le a te d . No m itoses w ere observed in any o f these m u ltin u c le a te d c e lls . The only o th e r a b n o rm ality d etected was an occasional pyknotic nucleus. These re s u lts w ere in te r p r e te d as in d ic a tin g th a t TCDD had in te r fe r e d w ith the c a p a c ity o f liv e r c e lls to m aintain th e ir co rrect morphology, thus leading to death and/or s t r u c t u r a l d is o r g a n iz a t io n . B u u -H o i e t a l . 12 have r e p o r te d s i m i l a r o b s e r v a tio n s in liv e r and m yocardial c e lls o f W istar ra ts a fte r sin g le in tra p e rito n e a l i n j e c t i o n s o f TCDD ( 1 0 m g / k g ) . K h e ra and R u d d ic k 1* 1 o b s e r v e d t h a t m i t o t i c fig u re s in m yocardial c e lls were ra re in ra ts tre a te d in utcro during days 6-15 o f g estatio n w ith 250-2000 ug/kg o f 2 ,7-d ich lo ro d ib en zo -p -d io xin per day. K im b ro u g h e t a l . 1*2 h a v e a l s o r e p o r t e d m u l t i n u c l e a t e d c e l l f o r m a t i o n i n th e liv e r s o f New Zealand ra b b its whose ears had been p ain ted d a ily fo r fo u r days w ith 0 .2 ml o f a 20 ug/m l TCDD s o lu t io n . ....... 0001512 \ 7901 8 V o s, M o o re , and Z i n k l G7 have s u g g e s te d t h a t TCDD c o u ld be a h e p a to c a rc in o g e n due to its s p e c ific c y to lo g ic a l e ffe c t on th e p r o lif e r a t in g c e lls o f the liv e r . When 5 8 - w e e k - o l d m a le C 5 7 B 1 /6 m ic e vie re t r e a t e d w i t h s i n g l e o r a l d o ses o f TCDD (100, 150-200 p g /k g ), an enlargem ent o f liv e r c e ll n u c le i w as.induced. The LD50 in these s tu d ie s was c a lc u la te d to be 114 p g /k g . In subacute s tu d ie s , 100 four-m onth-old male mice v/ere d iv id e d in to two groups and e ith e r tre a te d fo r two or s ix weeks w ith w eekly TCDD doses o f 0 , 0 .2 , 1 .0 , 5 .0 , o r 25 p g /k g . L iv e r c e lls of mice tre a te d w ith s ix doses o f 25 pg/kg showed p o ly p lo id y , v a c u o liza tio n o f n u c le i, and an increased m ito tic ra te . Green and M oreland27 were the f i r s t to te s t d io xin s (TCDD, 2 ,7 -d ic h lo ro d ib en zo -p -d io xin , and the unsubstituted d ib en zo -p -d io xin ) fo r th e ir a b ility to induce chromosome a b e rra tio n s in mammalian c e lls . In one o f th e ir experim ents27 the d io x in compounds were dissolved in dim ethyl s u lfo x id e and doses o f 10 pg/kg were adm inistered d a ily by in tu b a tio n to male ra ts fo r a fiv e -d a y period. Analyses o f bone marrow preparations from anim als s a c rific e d six hours a fte r the la s t treatm ent were n eg ative. In a second experim ent27, TCDD was d is s o lv e d in an a n is o le /c o r n o il s o lv e n t ( 1 .5 " v / v ) and a d m in is te re d in tr a p e r ito n e a lly to ra ts in doses o f 5, 10, o r 15 pg/kg. Another group o f te s t a n im a ls re c e iv e d 20 p g /kg o f TCDD o r a l l y from th e same t e s t s o lu t io n . A n im als receivin g the higher doses, 15 pg/kg in tra p e rito n e a lly and 20 pg/kg o r a lly , were k ille d 29 days a fte r treatm en t. A gain, bone marrow preparations revealed no evidence o f chromosomal a b e rra tio n s in any o f the an im a ls . As a re s u lt o f t h e s e tw o s t u d i e s 2 7 , t h e a u t h o r s c o n c lu d e d t h a t t h e t e s t compounds (TC D D , 2 , 7 - .. d ic h lo ro d ib e n z o -p -d io x in and d ib e n z o -p -d io x in ) appeared to possess no p o te n tia l fo r producing chromosome a b e rra tio n s in the bone marrow o f m ale ra ts . A group of p o sitive controls treated w ith triethylenem elam ine (0.375 m g/kg), adm inistered . in tra p e rito n e a lly and o r a lly , and used as p o s itiv e c o n tro ls showed a s t a t is t ic a lly *4 o o o o 7902 / Jl q q W 126695 s ig n ific a n t in crease in chromosome a b n o rm alities over u n tre a te d c o n tro ls . In c o n t r a s t w i t h t h e s e e a r l i e r r e s u l t s , G r e e n , M o r e la n d , and S h e y ?-8 , i n a l a t e r s tu d y , found t h a t TCDD s i g n i f i c a n t l y in c re a s e d th e number o f chromosome a b e rra tio n s in r a t bone marrow when a d if f e r e n t e x p e rim e n ta l p ro to co l was used. In t h is e x p e rim e n t2 8 , m ale and fe m a le O sborne-M endel ra ts re c e iv e d TCDD doses o f 0 .2 5 , 0 . 5 , 1 .0 , 2 .0 , o r 4 .0 p g /kg by gavage tw ic e w eekly f o r 13 w eeks. The TCDD samples used were dissolved in a so lven t co n sistin g o f one p a rt acetone to nine p arts corn o i l . Bone marrow from the tre a te d anim als was assayed fo r m ito tic i n h i b i t i o n and chromosome a b e r r a tio n s . No change in th e m it o t ic index was observed a t any dose. C o n versely, a d o s e -re la te d e ffe c t was found when c e lls from both male and fem ale anim als were assayed fo r chromosome breaks. The tre a te d fem ale group showed a s ig n ific a n t in crease a t the 4 pg/kg le v e l as compared w ith the 0 .2 5 pg/kg le v e l (P < 0 .0 1 ) . The tre a te d male group showed a s ig n ific a n t in c re a s e a t th e 2 pg and 4 pg/kg le v e ls as compared w ith the 0 .2 5 pg/kg W e i (P < 0 .0 1 ). The authors cautioned th a t even though the re s u lts were s ig n ific a n t, the observed a c t iv it y should be regarded as only weakly p o sitive. C ze ize l and K ir a ly 17 compared the frequency o f chromosome a b e rra tio n s in the p e rip h e ra l lym phocytes o f 76 workers employed a t a chem ical h erb icid e producing fa c to ry in Budapest, Hungary, along w ith th a t o f 33 control in d iv id u a ls . The m anufacturing process a t th is facto ry favored the form ation of dioxins. Of the workers surveyed, 36 had been exposed to 2,4,5-trich lo ro p h en o xyeth an o l (TCPE) o r K lo rin o l and 26 to B uvinol (a co m bination h e rb ic id e c o n ta in in g TCPE and 2 -c h lo ro -6 -e th y la m in o -4 -is o p ro p y la m in o -l,3 ,5 -tria z in e ). The rem ain in g '14 workers had never been engaged in the production or use o f e ith e r o f these h e rb ic id e s . TCDD c o n ta m in a tio n found in the f in a l h e rb ic id e products has been O 7903 DQV-J126696 10 rep o rted to be less than 0.1 mg/kg and g e n e ra lly not more than 0.0 5 m g/kg6fi. The frequency o f ch ro m atid -typ e and unstable chromosome a b e rra tio n s found by C ze ize l and K ir a ly 17 was h ig h e r (P < 0 .0 0 1 ) in the fa c to r y w orkers than in th e c o n tro ls , regardless o f whether or not they had been d ir e c tly involved in production o f the h e rb icid es. 'However, a b e rra tio n s were more freq u en t in workers p re p a rin g TCPE and B uvinol than in th e o th e r f a c t o r y v/orkers, but the d iffe re n c e was s ig n ific a n t o nly fo r th e c h ro m a tid -ty p e e ffe c t. Chromosome aberrations have also been reported in studies o f Vietnam ese populations exposed to th e h e rb ic id e 2 ,4 ,5 -T in which TCDD was p re s e n t as a co n tam in an t3 2 . In these s tu d ie s , a h ig h er incid en ce o f chromosomal a b n o rm a litie s was reported in in d iv id u a ls brought in co n tact w ith th is h e rb ic id e as a re s u lt o f fo re s t d e fo lia tio n . These stu d ies, however, have been c r itic iz e d as being s ta tis tic a lly unsound (see Hay3132). P re lim in a ry re s u lts o f a cytogenetic in v e s tig a tio n o : TCDD-exposed in d iv id u a ls from the Seveso, It a ly population have been rep o rted by Tenchini e t a 1 . 6 . E xam in atio n s o f th e a ffe c te d p o p u la tio n w ere c o n fin e d to women who became pregnant im m ed iately b e fo re o r a f t e r exposure to TCDD and who had chosen to abort because o f the possible terato g en ic e ffe c ts o f th is compound. Chromosome analyses o f m aternal p e rip h e ra l blood and a b o rtiv e fe ta l tis s u e were scored fo r the presence o f num erical and s tru c tu ra l chromosome v a ria tio n s ; no s ig n ific a n t change was found in the chromosome number o f any sample an alyzed . A lso , no s ig n if ic a n t in c re a s e was observed in th e freq u en cy o f s tru c tu ra l chromosome a b e rra tio n s from m aternal blood sam ples. There was, however, a higher number o f stru ctu ral aberrations in the fe ta l tissues than in maternal blood samples o r fib ro b la s ts from a d u lt tis s u e s , but the frequency o f these in M o 7904 DOVW26697 11 aberrations did not appear to be greater than th a t expected to occur spontan e o u s ly i n c u l t u r e s o f c o m p a ra b le c e l l t y p e s . T e n c h in i e t a l . 6 `( p o in t e d o u t th a t these p re lim in a ry data do not answer the qu estio n o f w hether the higher freq u en cies o f chromosome a b e rra tio n s found in fe ta l tis s u e s were due to chromosome damage caused by TCDD exp o su re. In a n o th e r p r e lim in a r y c y to g e n e tic s t u d y r e l a t e d t o t h e S e v e s o i n c i d e n t 1* 6 , no chrom osom e a b n o r m a l i t i e s w e re fo u n d in the p erip h eral blood c e lls o f 90 workers selected from the chemical plant which was th e source o f th e TCDD co n tam in atio n in Seveso. L ik e w is e , no a b n o rm a litie s were found in chromosomes from p e rip h e ra l blood c e lls o f Seveso r e s i d e n t s l o c a t e d i n t h e a r e a m o s t s e v e r e l y c o n t a m in a t e d w i t h TCDD1*6 . H a y 31 has c ite d re s u lts from a study done a t another in d u s tria l concern regarding the fre q u e n c y o f chromosome a b n o r m a litie s in w o rk e rs p resu m ab ly exposed to TCDD d u rin g the production of 2 ,4 ,5 -tric h lo ro p lie n o l. In th is study, 800 workers with c h lo ra c n e p resu m ab ly caused fro m TCDD exp o su re viere in v e s t ig a t e d and th e fre q u e n c y o f chromosome a b n o rm a litie s d e te rm in e d . The a b n o rm a litie s observed among th ese workers were reported to be not g re a te r than the s ta t is t ic a l norm. TCDD was f i r s t e v a lu a te d f o r m u ta g e n ic ity by H u ssain e t a ! . 38 because o f its s tru c tu ra l s im ila r ity to acrid in es (F ig . 1 ). The assay systems used were (1 ) E scherichia c o li s tra in Sd-4 which measures re v e rs io n from streptom ycin dependence to s tre p to m y c in independence, (2 ) S alm o n ella typkim o'iier. s tr a in s TA1530 and TA1532 which measure reversion from h is tid in e dependence to independence, and (3 ) prophage in d u c tio n in E scherichia c o li K39 c e lls . TCDD was m utagenc in E. c o li S d -4 and in S alm o n ella T A 1 5 3 2 . TCDD had a weak p ro p h ag e-in d u cin g e f f e c t and d id not shew any m utagenic a c t i v i t y in Salm onella TA1530. P a ra lle l studies w ith ac rid in e orange in E. c o li Sd-4 and acrid in e m ustard in Salm onella TA1532 were used as p o s itiv e c o n tro ls . C o ro lla ry re s u lts CD T4 o O o o 7905 869921^00 12 w ith these co n tro l compounds, coupled w ith the well-docum ented knowledge th a t Salm onella s tr a in TA1532 d e te c ts fra in e s h ift m utagens, seem to in d ic a te th a t TCDD was m utagenic v ia in te r c a la tio n w ith DNA. S e i l e r 57 e v a lu a te d TCDD and o c ta c h lo ro d ib e n z o -p -d io x in (OCDD) in S alm onella typhirriuriicTi s t r a in s G 46, T A 1 5 3 0 , T A 1 5 3 1 , T A 1 5 3 2 , and TA1534 and found TCDD to be a strong mutagen in s tra in TA1532, a doubtful mutagen in TA1531 and TA1534, and a nonm utagen i n G46 and T A 1 5 3 0 . OCDD v/as r e p o r t e d as a q u e s t io n a b le m utagen in s tra in s TA1532 and TA1534 and as a nonmutagen in s tra in s G46, TA1530, and T A 1 5 3 1 5 7 . M cC ann1* 9 ( p e r s o n a l c o m m u n ic a t io n ) t e s t e d TCDD i n t h e S a lm o n e lla system using a spot te s t and the standard p la te te s t w ith s tra in s TA1532, TA1535, TA1537, and TA1538, both w ith and w ithout m etabolic a c tiv a tio n . In a ll e x p e r i m e n t s , n e g a t i v e r e s u l t s w e r e o b t a i n e d . T h e r e s u l t s 1* 9 fo u n d w i t h T A 1 5 3 2 a r e in c o n flic t w ith those o b tain ed by S e ile r 57 in th is s tr a in . McCann's n eg ative re s u lts w ith TA1532 cannot be d ir e c tly compared w ith H ussain's e t a l . p o s itiv e s tu d ie s because th e s e a u th o rs 38 used a liq u id in c u b a tio n procedure in th e ir te s t p ro to c o l. The n e g a tiv e response which McCann49 observed w ith the o th e r Salm onella s tra in s (TA 1535, TA1537, and TA1533) adds fu r th e r to the d iffic u lty in understanding the p o ten tial m utagenicity of TCDD, ex p e c ia lly since s tra in s TA1537 and TA1538 are s e n s itiv e to the action o f fram esh ift mutagens. Nebert, T h o rg eirsso n , and F e lto n 53 have a lso rep o rted neg ative fin d in g s w ith s tra in s TA1535 and TA1538. The d iffe re n c e s in re s u lts fo r TA1532 could be due to many f a c t o r s , such as th e s o l u b i l i t y o f th e TCDD s am p le, i t s p u r i t y , tre a tm e n t p ro to c o ls , and the problems involved in handling such a to x ic compound. Table 1 summarizes re s u lts from these stu d ies. 0001517 7S06 13 T a b le 1. M u ta g e n ic ity o f d io x in compounds in S ahr.onella t'j:;him uriur:a DOW Cl G* to D io xin isomer TCDD Strains detecting base-pair substitutions^ S trains detecting frameshi fts^ Referenc' G46 TA1530 TA1535 TA 100 TA1531 TA1532 TA1534 TA1537 TA1532 Mo. 00- 00 0 49 0 0 - 0 0 0 0 0 - 53 0 - 0 0 0 +. 0 0 0 3 8 - - 0 0 ? + ? 0 0 57 OCDD - - 00 ? ? 0 0 57 U n s u b s titu te d d ib e n z o -p d io x in 0 0 . 000 fS e e a ls o T a b le 4 f o r a d e s c rip tio n o f th e Salm onella s tr a in s . D0 -- not te s te d ; ---- negative re s u lts ; + -- p o s itiv e re s u lts ; ? -- doubtful mutagen. R esu lts shown were o b tain ed using d if f e r e n t exp erim en tal p ro to c o ls . 13 0001518 780" 14 DOW Commoner13 te s te d the u n s u b s titu te d d ib e n z o -p -d io x in in fo u r o f th e Ames Salm onella s tr a in s (TA1535, TA1537, TA1533, and TA100) by th e standard p la te DO er> o o -te s t to determ ine the r e lia b ilit y o f the te s t system in distinguishing carginogens from noncarcinogens. Negative re s u lts were reported fo r a ll strain s w ith the c o n c e n tra tio n s te s te d ( 1 .0 , 10, and 100 ug p er p l a t e ) . D im ethyl s u lfo x id e was used as the so lven t in the p rep aratio n o f a ll te s t sam ples.in th is study. F u r t h e r e v id e n c e f o r t h e p o s s i b l e i n t e r c a l a t i n g r e a c t i o n o f TCDD w i t h DNA has been re p o rte d by Kondorosi e t a l . U3. These in v e s tig a to rs checked TCDD and several e le c tro p h ile s (m ethylm ethar.esulfonate, ethylm ethanesulfonate, diepoxy b u tan e, and e th y le n e d ib ro m id e ) f o r t h e i r e f f e c t on th e tr a n s fe c tio n o f Qg Ri!A. The loss o f tra n s fe c tiv ity in th is system in d icates a reactio n w ith s in g le - stranded n u c le ic a c id . In te rc a la tin g agents which do not have a c tiv e side chains ( e .g ., a lk y la tin g groups) would be expected to give negative re s u lts in t h is t e s t because th e y r e a c t p r im a r ily w ith d o u b le -s tra n d e d DMA. A ll th e agents s tu d ie d gave p o s itiv e r e s u lts e xcep t TCDD. U sing TCDD a t c o n c e n tra tio n s r e p o r te d t o be m u tag en ic in E s c h e ric h ia c o li S d -4 and S alm o n ella typkim urium T A 1 5 3 2 3 8 , K o n d o r o s i e t a l . u3 fo u n d no e f f e c t on t h e t r a n s f e c t i o n o f QS RNA. The a u th o rs s ta te d t h a t th e s e r e s u lt s c o n firm th e assum ption t h a t TCDD forms a p h y s i c a l c o m p le x ( i n t e r c a l a t i o n ) w i t h DMA r a t h e r t h a n r e a c t i n g c h e m i c a l l y w ith the nucleic acid. T o x ic it y and dom inant le t h a l s tu d ie s w ith TCDD have been re p o rte d by Khera cr> m -- o and R u d d i c k 1* 1 . G ro u p s o f 2 0 m a le H i s t a r r a t s w e r e d o sed o r a l l y w i t h 4 , 8 , o r 12 ug/kg TCDD per day fo r seven days befo re m ating (s u rv iv a l re s u lts a re shown in T ab le 2 ). S u rvivin g males were caged w ith two u n treated v irg in fem ales fo r f iv e days and t h is regim en was fo llo w e d f o r seven s e q u e n tia l m ating t r i a l s . Fem ales w en 7908 15 T a b le 2 . S u rv iv a l in m ale W is ta r r a t s a f t e r TCDD tre a tm e n t* Dose (ug/kg) Dead anim als Mean su rvival Survivors tim e (days) 12 20 0 17.7 8 11 9 2 0 .1 4 2 18 36.5 *Mo m o rta litie s were observed in the control group. Source: Khera and Ruddick**1 . r 7909 3 0 i.9 Z T M a 16 k ille d nine days a fte r separation from the males and v ia b le embryos, reso rp tio n s i t e s , and c o r p o r a l u t e a w e re c o u n t e d . Mo e v id e n c e was fo u n d f o r t h e i n d u c t i o n o f dominant le th a l m utations during p o st-m eio tic phases o f spermatogenesis although th e in cid en ce o f pregnancies from a ll m ating t r i a l s in each tre a te d group was reduced. H is to lo g ic a l exam ination o f su rvivin g males showed normal te s te s , but the epididym ides were inflam ed w ith sperm granuloma form ation which is analogous to changes seen in the autoimmune re a c tio n fo llo w in g b a c te ria l in fe c tio n or the r e s p o n s e o f t i s s u e s t o f o r e i g n b o d i e s 1* 1 . O th e r ev id e n c e t h a t TCDD produces ad verse e f f e c t s in th e te s te s has been rep o rted by Van M ille r and A lle n 65. In th is stu d y, male Sprague-Dawley rats v/ere fe d d ie ts c o n ta in in g v a rio u s le v e ls o f TCDD f o r 65 w eeks. A nim als re c e iv in g 0 . 0 5 , 0 . 5 , and 1 .0 ppm o f TCDD in t h e i r fo o d d ie d w it h in f o u r w e e k s , and autoposies showed a n o tic e a b le decrease in sperm atogenesis. S e ile r 58 reported th a t a 0 .4 mg/kg dose o f TCDD a d m in is te re d in t r a p e r it o n e a lly to m ale mice p r o d u c e d an a p p r o x im a t e 50% r e d u c t i o n i n t h e r a t e o f t e s t i c u l a r DMA s y n t h e s i s . TCDD and o th e r d io x in s have a ls o been im p lic a te d in causing adverse e ffe c ts in the te s te s o f a number o f o th e r anim als (m ice5 0 , ch icken s5**, guinea p ig s 5 0 , and m o n k e y s 5 **). T h e f a c t t h a t TCDD a p p a r e n t l y r e a c h e s t h e t e s t e s * * 1 5 0 5 **5 8 65 and th e r e p o rt t h a t TCDD has a v/eak chromosome b re a k in g e f f e c t in r a t bone marrow c e l l s 28 s u p p o rt, to some d e g re e , the p o s s ib ilit y th a t TCDD may have th e c a p a c ity C2 VO o o o to act as a weak dominant le th a l agent. F u rth er p re lim in a ry re s u lts reported by Murray e t a l . 52, in a th ree-g en eratio n reproduction study o f Sprague-Dawley r a ts in g e s tin g v a rio u s le v e ls o f TCDD, adds some a d d itio n a l support to th is p o s s ib ility (ta b le 3 ). M u ltig e n e ra tio n stu d ies, however, are not proper in d icato rs o f dominant le t h a lit y , nor is the production o f adverse te s tic u la r e ffe c ts an in d ic a to r o f / m u t a g e n i c i t y . T h e s e d a t a 52 and t h e d a t a c i t e d p r e v i o u s l y 1' 1 5 0 5 **5 S 65 s u g g e s t a need fo r fu rth e r te s tin g . Even w ith the present evidence and sp ecu latio n , i t 7910 Table 3. T h ree-g en eratio n reproduction study o f male and fem ale S prague-D aw ley r a ts in g e s tin g TCDD D aily dose Treatm ent time ingested (ug/kg)a p rio r to m ating (days) 0.1 90 0.01 90 0.001 90 O b s e rv a tio n s Fo F i f2 Decreased f e r t ilit y ; poor su rvival in Fj offspring No e f f e c t on fe rtility ; Ft o ffs p rin g and lit t e r size appeared normal No e f f e c t on f e r tili ty b S ig n ific a n t decrease in f e r t ilit y ; Fz o ffs p rin g had decreased survival ra te and reduced growth ra te ; sm aller lit t e r size No e f f e c t on fe rtility b S ig n ific a n t decrease in f e r t ilit y ; F3 o ffs p rin g had decreased survival ra te and reduced growth ra te ; sm aller lit t e r size No e f f e c t on fe rtility fA n im a ls were m a in ta in e d c o n tin u o u s ly on d ie ts c o n ta in in g s u f f ic ie n t TCDD to p ro v id e th e dose le v e ls shown. ^Due to adverse e ffe c ts observed in F0 p a re n ts , treatm en t was d isco n tin u ed . Compiled from M urray e t al . 5^ . _ _ _ _ _ _ ___ _ _ _ _ _ _ _ _ _ _ _ _ _ _ - -- ------------------------- o o o -- cn 13 ro <X E0L93TM OQ DOW 12670+ I ci is n ot c le a r w heth er th e adverse e ffe c ts produced by TCDD in th e te s te s are the r e s u lt o f d i r e c t TCDD a c t i v i t y o r th e r e s u lt o f some o th e r to x ic response tr ig g e r e d by TCDD. MODIFYING EFFECTS ON MUTAGENESIS The im portance o f metabolism in mutagenesis studies has been considered because many compounds thought to be nonmutagenic are converted to mutagens a fte r m etabolic a c tiv a tio n . P o lycyclic hydrocarbons are a notable example. In d u cib le enzymes in various body organs, p a rtic u la rly the liv e r , are involved in the m etabolic conversion o f promutagens. Agents freq u en tly used to increase these enzyme le v e ls include A ro clo r 1254 (a p o lych lo rin ated b ip h e n y l), p h n o b arb ital, and 3-m eth ylch o lan th ren e. TCDD has been re p o rte d to be a p o te n t s tim u la n t f o r h e p a tic a ry l hydrocarbon hydroylase a c t iv it y in male ra ts (S prague-D aw ley), chick embryos (L eg h o rn ), and fe m a le m ice fro m s e v e r a l in b r e d s t r a in s (C 3H /H eN , C 5 7 B L /6 J , and B A L B /c J )5 5 . When compared w ith re s u lts o b ta in e d in th e r a t w ith 3 -m e th y lc h o la n th re n e , TCDD is 30,000 tim es more potent in inducing aryl hydrocarbon hydroxylase a c t iv it y 55. F e lto n and N e b e rt2* used l i v e r fra c tio n s from C57B1/6N and DBA/2N m ice p re tr e a te d w ith TCDD a t doses ra n g in g from 0 .1 -1 0 0 ug/kg to assay th e in vitro m u t a g e n ic it y o f 3 -m e th y lc h o la n th r e n e and b e n z o [a ]p y r e n e in th e Ames Sabnonella te s t system using s tra in TA1538. A s ig n ific a n t increase in 3-m ethylcholanthrene m utagenesis was observed in te s t samples p re tre a te d w ith liv e r fra c tio n s from TCDD- induced m ice. The m utagenicity of 3-m ethylcholanthrene follow ed q u ite c lo sely the hydroxylase a c tiv ity and cytochrome Pt - 450 form ation. The m utagenicity o f b en zo [a]p yren e was not a ffe c te d when subjected to th is same p ro to c o l. - ........~ C':- c . VI B e rry e t a l . 7 re p o rte d th a t TCDD appears to be an e x c e p tio n a lly p o te n t o 3 and broad-spectrum tran sp lacen tal inducing agent fo r carcinogen-transform ing enzymes found in various tis s u e s . Pregnant Sprague-Dawley ra ts were given s in g le ' 7912 OOW 126705 19 day 17 o f g e s ta tio n . Anim als were k ille d on day 20 o f g e s ta tio n , and homo genates were made o f m aternal liv e r s , lungs, kidneys, ad ren als, and placentas and o f fe ta l liv e r s , kid n eys, and s k in . These homogenates were then tre a te d w ith [ 3H ]b e n zo [a ]p y re n e (BP) and were assayed f o r th e e ffe c ts o f TCDD p re t r e a t m e n t on t h e t is s u e - m e d ia t e d c o v a le n t b in d in g o f BP t o DMA. T is s u e sam ples from th e l iv e r s , lu n g s , and p lacen tas o f T C D D -p retreated dams, as w e ll as samples from liv e r s , lungs, and skin o f t h e ir fe tu s e s , showed an increased c a p a c it y t o c o v a le n t ly b in d BP t o DNA when com pared w it h t is s u e sam p les ta k e n from noninduced control anim als. Berry e t a l . 7 stated th a t "The nature of the TCDD enhancem ent appeared to be t is s u e - s p e c i f i c , and m o re o v e r, th e d a ta from the binding in v itro appeared to c o rre la te w ell w ith the m etabolizing c a p a b ilitie s o f the tis s u e and the m etab o lites formed (B P -7 ,8 -h iy d ro d io l, B P -4 ,5 -d ih y d ro d io l, BP-9 phenol, and BP-3 p henol)". The e f f e c t o f TCDD on th e c o v a le n t b in d in g a c t i v i t y o f b e n z o [a ]p y re n e to DNA and th e m u t a g e n ic it y o f 3 - m e t h y lc h o la n t h r e n e r a is e s th e q u e s tio n o f w h e th e r TCDD can in flu en ce the b io lo g ical a c tiv ity o f o th er p o lycyclic hydrocarbons. The sig n ific a n c e o f th is p o s s ib ility w ill not be fu lly understood u n til ad d itio n al data are availab le. COMPARATIVE M UTAG ENICITY OF D IO X IN S AND A C R ID IN E S The t e s t in g o f TCDD f o r m u ta g e n ic ity and i t s i n i t i a l c l a s s i f i c a t i o n as a mutagen was brought about by it s s tru c tu ra l s im ila r itie s to a c rid in e s (F ig . 1 ). The fo llo w in g sectio n s summarize and compare th e m u ta g e n ic ity o f a c rid in e and d io x in compounds as measured in id e n tic a l te s t systems. These re s u lts n e ith e r prove n o r d is p ro v e th e p o s s ib ilit y th a t TCDD may a c t as an in te r c a la tin g a g e n t.' The e x a c t mode o f a c tio n o f TCDD and o th e r d io x in s w ill be c l a r i f i e d o n ly a f t e r fu rth e r experim entation. oogi;:4 7913 DOW126706 20 B a c te ria Sa Imone l l a tzmhi'Tr.criiva T e s ts w ith s e v e ra l g e n e t ic a lly d e fin e d h is t id in e m utants o f S alm o n ella tvphim uriur, are p resen tly used in a v a rie ty o f ap p licatio n s fo r the evaluation o f chemicals for m u ta g e n ic ity 1-3 (T a b le 4 ) . S alm onella s tr a in s used in th e e v a lu a tio n o f TCDD, OCDD, and the u n su b stitu ted d ib e n zo -p -d io x in are shown in Table 5 , along w ith re s u lts fo r several a c rid in e compounds. E sch erich ia a o li Sd-4 E. c o li Sd-4 is a m utant d e riv a tiv e o f E. c o li B and is used to d e te c t reversions from streptom ycin dependence to streptom ycin independence. Docum entation o f the m o lecu lar mechanism re s u ltin g in th is m u tatio n was not found but rep o rted ly resu lts from a b ase-p air su b stitu tio n m utation10. See T a b le 6 f o r c o m p a ra tiv e r e s u lt s o f TCDD w ith a c r i f l a v i n . Bacteriophages (Prophage Induction) A search o f the Environm ental Mutagen Inform ation Center data base r e v e a le d no in f o r m a t io n on th e a b i l i t y o f a c r id in e s to a c t i v a t e lambda phage in e . c o li K39 c e lls which were used to te s t TCDD33. Reports noting a c tiv a tio n o f lambda phage in a n o th e r E. c o li s t r a i n and n e g a tiv e f in d in g s f o r phage in d u c tio n in S alm o n ella thompson w ere found and a re shown in T a b le 7 . . In an e x te n s iv e review on prophage in d u c tio n in lyso g en ic b a c te ria , Heinem ann33 lis te d a c rid in e orange as having a m oderate p ro p hage-inducing a b ilit y . H u s s a in e t a l . 3S s t a t e d t h a t TCDD had a weal; p r o p h a g e - i n d u c i n g e f f e c t a n d "t h a t such re s u lts agreed w ith data obtained from studies w ith a c rid in e s . The data 0 0 0 1 3 :5 7914 OOW 12670V 21 T a b le 4 . C h a r a c t e r is t ic s o f th e Ames S a trio n e lla t e s t s tr a in s used to e v a lu a te the m u ta g e n ic ity o f TCDD and other dioxins M utation (s ite in S train h is tid in e operon) Other ch aracteristics Type of m utation detected G46 TA1530 TA1531 TA1532 TA1534 TA1535 TA1537 TA1538 TA100 h is G45 h is G45 h is C207 h is C3075 h is D3052 h is G46 h is C3075 h is D3052 h is G46 Normal lipop o lysacch arid e c o a t; DNA e x c is io n r e p a i r present P artial lipopolysaccharide c o a t p r e s e n t; DNA e x c is io n repair deficient P a rtia l lipopolysaccharide c o a t p r e s e n t ; DMA e x c i s i o n repair deficient P artial lipopolysaccharide c o a t p r e s e n t ; DNA e x c is io n repair deficient Normal lip o p o lysacch arid e c o a t ; DNA e x c i s i o n r e p a i r present Lipopolysaccharide d e fic ie n t; DNA e x c is io n r e p a ir d e fe c tiv e Li popolysacchari de d fi ci e n t; DNA e x c is io n r e p a i r d e fe c tiv e Lipopolysaccharide d e fic ie n t; DNA e x c is io n r e p a ir d e fe c tiv e Lipopolysaccharide d e fic ie n t; DMA e x c i s i o n r e p a i r D efective; R factor plasmid present Base s u b s titu tio n Base su b stitu tio n Fram eshift Fram eshift Fram eshift Base su b stitu tio n Fram eshift Fram eshift Base su b stitu tio n Com piled from Ames e t a l . 1 *2 and Simmon-'0 . V-' U 1 \ j ^ 7915 DOW 22 T a b le 5 . M u ta g e n ic ity r e s u lt s w ith S alm o n ella typhirnurium 1 Chemical tested TCDD Strains detecting , base-pair substitutions0 h ic G46 TA1530 TA1535 TA100 00- 0 .0 - 0 0 0` 0 - 0 - - 00 Strains detecting fram eshifts^ Referenc-- TA1531 TA1532 TA1534 TA1537 TA1533 No. 0 - 0 - - 49 0 + 0 0 0 38 0 0 0 0 - 53 ? + ? 0 0 57 OCDD - - 0 0 - ? ? 0 0 57. U n s u b s titu te d d ib e n z o -p d io x in 00 * 0 0 0 13 T' 'a fla v in e - 0 - 0 0 0 0 + + 34,56 Q u in a c rin e - - 0 0 - + - 0 0 11,14 44 P ro fla v in e - 0 0 0 0 0 0 0 + 11,48 IC R -1 7 0 e -/+ 0 0 0 0 + 0 + 0 11,38 45,4 ICR-191 - - 0 0 + + + + + 2,11, 14,4 Acri di ne orange 0 0 0 0 0 0 0 + 0 48 *See T a b le ,4 fo r supporting in fo rm atio n . ~0 -- Mot tested in the papers screened; -- negative re s u lts ; + -- p o sitive re s u lts ; ? -- doubtful m utagen. R esults shown were obtained using d iffe r e n t experim ental p ro to c o ls . ''Acts as both an in te r c a la tin g and an a lk y la tin g agent. s N r\ , UW T. f y D O W 126? 09 23 T a b le 6 . M u ta g e n ic ity r e s u lt s w ith Escherichia c o li S d-4 Chemical tested TCDD A criflavine c+ -- P ositive results. R e s u lts ^ + + R eference Mo. 38 19 j 5 1 5 2 5 7917 d o w i2 67 1 0 24 Table 7. Prophage induction Chemical tested TCDD A cridine orange P ro fla v in e E th a c rid in e la c ta te monom yd rate Host organism E s c h e ric h ia c o li K39 E sch erich ia c o li 18 S a lm o n e lla thompson 19 S a lm o n e lla thompson 19 G+ - P o s i t i v e r e s u l t s ; ---- n e g a t i v e r e s u l t s . R e s u lts 2 + + - Reference No. 38 61 62 62 >. n ~ - ft k.< 1/ i V v 7918 DOW 126711 25 in T ab le 7 in d ic a te th a t a c rid in e s , considered as a group, could be classed as weak when te s te d fo r prophage a c tiv a tio n . P re s e n tly , in s u ffic ie n t data are a v a ila b le a t th is w ritin g fo r a tru e comparison. Mammals Dominant Lethal Studies M a i l i n g and Wassom1* 7 i n t h e i r r e v i e w o f t h e m u ta g e n ic a c t i o n o f c h e m ic a ls rep o rted th a t on ly six a c rid in e compounds have been evalu ated , a t the tim e o f t h e ir re v ie w , in th e dom inant le th a l t e s t . R e s u lts from these te s ts a re shown in Table 8. P o s itiv e re s u lts in the dom inant le th a l te s t are b elieved to be due to chromosome rearrangem ents ra th e r than p o in t m u tatio n s. T h e re fo re , a mutagen t h a t induces p red o m in an tly p o in t m u ta tio n s would go undetected in th is t e s t . Freese26 s ta te d th a t a c rid in e s produce p o in t m utations via in te rc a la tio n by adding o r d e le tin g bases in DNA. Thus, n e g a tiv e re s u lts would be expected i f th e a c t i v i t y o f TCDD resem bled th e in t e r c a la t in g a c tio n o f a c rid in e s as supported by t h e n e g a t i v e r e s u l t s r e p o r t e d b y K h e ra a n d R u d d ic k 1* 1 . H o w e v e r, t h e r e s u l t s o b tain ed by Green and M oreland28 in the r a t bone marrow te s t, coupled w ith the e v i d e n c e t h a t TCDD d o es p ro d u c e a d v e r s e e f f e c t s i n t h e t e s t i s 1*1 ' 5 0 51* * 5 8 ' 6 5 s t i l l le a v e th e u n re s o lv e d q u e s tio n o f w h eth er TCDD can a c t as a weak dom inant le t h a l- in d u c in g ag en t o r cause some o th e r typ e o f damage to th e g e n e tic m a te ria l o f mammalian germ c e lls . Several a c rid in e compounds are known inducers o f chromosome a b e rra tio n s in p la n ts , D rosophil-a, and mammalian c e lls in c u lt u r e 59, b u t no rep o rts on the evalu atio n o f a c rid in e comoounds in the ra t bone marrow te s t were found. R esu lts from o th e r mammalian s tu d ie s w ith a c rid in e s a re shown in 7919 Table 9. 0001520 Z lL 9 Z 1 M O a 26 Table 8. Mammalian dom inant le th a l studies Compound Reported re s u lts 0 TCDD A cridine orange A criflavine 9-Amino a c rid in e IC R -1 70^ Quinacrine hydrochloride T ry p a fla v in e - + - a-- Negative resu lts; + -- p o sitive resu lts. ^Acts as both an in te rc a la tin g and an a lk y la tin g agent. Reference No. 41 47 22 22 63 22 22 7920 31C O r\ V ir LyJ OW126713 27 T a b le 9. Chromosome a b e rra tio n s tu d ie s in c u ltu re d mammalian c e lls Compound TCDD& TCDD5 2,7-D ichlorod ib e n z o -p -d io x in D ibenzo-p-dioxin P roflavine A cridine A crid in e orange C ell type Rat bone marrow c e lls Rat bone marrow c e lls Rat bone marrow c e lls Rat bone marrow c e lls HeLa c e lls Human d ip lo id fib r o b la s ts Human d ip lo id fib r o b la s t s Reported re s u lts ^ + (weak) - - + + + Reference No. 28 27 27 ' 27 59 59 59 a+ - P ositive resu lts; -- negative resu lts. ^ R e s u l t s fr o m t h e s e e x p e r im e n t s 2 7 28 w e re o b t a in e d u s in g d i f f e r e n t e x p e r im e n t a l p ro to co ls, e s p e c ia lly in the dose range te s te d , dosing schedule, and solvents used. 0 VT. \fJ) i ,,TT 7921 28 MUTATION RESEARCH IN PROGRESS The Seveso in cid en t revealed a lack o f inform ation concerning the health - e ffe c ts o f TCDD. As a r e s u lt , a number o f res e a rc h p ro je c ts were in it ia t e d in an a tte m p t t o o b t a in a b e t t e r p e r s p e c tiv e o f th e h a z a rd s posed by TCDD exposure. Most o f these investig atio n s are taking place in laboratories in I t a ly and o th e r European c o u n trie s . In fo rm a tio n is a ls o being c o lle c te d on people o r dom estic an im als exposed to TCDD as a r e s u lt o f th e a c c id e n t a t th e c h e m ic a l p l a n t 1*6 w h ic h c a u s e d t h e c o n t a m i n a t i o n o f t h e S e v e s o a r e a . P r o j e c t s u n d e r way include f e r t i l i t y s tu d ie s , dominant le th a l studies in ra b b its , tran slo catio n tests in m ice, p oint m utation tests using hamster c e lls , cyto lo g ical examinations o f p e rip h e ra l blood from exposed in d iv id a u ls and exposed anim als (c a ttle and r a b b its ), c y to lo g ic a l exam inations o f fe ta l tis s u e from ab o rtu ses, and m ito tic crossing over and recom bination studies in yeast. DISCUSSION AND CONCLUSION O f the many s tru c tu ra lly p o ssib le d io x in c o n fig u ra tio n s , only fo u r have been tested fo r m utagenicity or other re la t-1 e ffe c ts . These are the d i-27, t e t r a - 9 2 7 2 8 3 8 1* 1 * 1* 9 5 3 5 7 , and o c t a - 57 c h l o r i n a t e d d e r i v a t i v e s as w e l l as t h e u n s u b s t i t u t e d d i b e n z o - p - d i o x i n 1 3 2 7 . O f t h e s e , TCDD h a s been tiie m ost fre q u e n tly tested but th e -re s u lts obtained from these stu d ies have not been con c lu s iv e . For exam ple, p o s itiv e re s u lts were re p o rte d when TCDD was te s te d alone f o r c y t o l o g i c a l d i s t u r b a n c e s i n t h e A f r i c a n b lo o d l i l y 1* 0 o r f o r m u t a t io n . i n d u c t i o n i n Salmonella tiipkimuriim*^ >57 and Escherichia e o Z i 3 8 . P o s i t i v e .re s u lts w ere a ls o re p o rte d when 2 ,4 ,5 - T t e s t sam ples c o n ta m in a te d w ith TCDD w ere e v a lu a te d f o r c y t o lo g ic a l e f f e c t s in Drosophilaie and th e A fr ic a n b lo o d l i l y 1' 0 . On t h e o t h e r h a n d , n e g a t i v e r e s u l t s w e r e r e p o r t e d w i t h TCDD i n Salmonella typhinuriur: c o n f l i c t i n g w it h th o s e s tu d ie s r e p o r t i n g p o s it iv e fin d in g s (see T ab le 1 ). The o ctach lo ro d e riv a tiv e (OCDD) was a doubtful 0001533 7922  29 m u ta g e n when e v a l u a t e d i n t h e jo. uhokq t, l a t e s t and t h e u n s u b s t i t u t e d d i b e n z o - p d io x in was nonm utagenic13. C7 o s? H -1 N> cn Mammalian stu d ies w ith d io xin s are scarce. The only data found showed th a t TCDD gave n e g a tiv e re s u lts when te s te d f o r dom inant le th a l e f f e c t s in ra ts '*1 and w eakly p o s itiv e re s u lts when assayed fo r the in d u ctio n o f chromosome aberrations in r a t bone marrow c e lls 28. These data, coupled w ith studies s h o w in g t h a t TCDD d o es r e a c h t h e t e s t e s ' * 1 >5 0 >51*> 5 8 5 5 , i n d i c a t e t h e p o s s i b i l i t y th a t TCDD could a c t as a weak dom inant le th a l inducing agent o r could cause damage to th e g e n e tic m a te ria l w hich m a n ife s ts i t s e l f in some o th e r way. The d ic h lo ro d e r iv a tiv e (DCDD) and th e u n s u b s titu te d d ib e n z o -p -d io x in were both negative in the r a t bone marrow te s t2 7 . T h e c y t o l o g i c a l e f f e c t s p ro d u c e d b y TCDD i n t h e l i v e r o f t r e a t e d a n i m a l s 1*- 2 may be a ttr ib u te d to the to x ic e ffe c ts produced by th e s h o rt-te rm high dose le v e ls . a d m in is te re d in th e s e s tu d ie s and th e f a c t t h a t TCDD tends to c o n c e n tra te in th e l i v e r . P re lim in a ry re s u lts obtained from cyto genetic analyses o f m aternal blood and a b o r tiv e f e t a l tis s u e ta k e n from persons exposed to TCDD d u rin g th e Seveso, I t a l y a c c i d e n t a r e i n c o n c l u s i v e 51* . I n c o n c l u s i v e r e s u l t s h a v e a l s o b een r e p o r t e d 31 fro m s t u d ie s o f p e r ip h e r a l b lo o d c e l l s ta k e n fro m i n d iv id u a ls exposed to TCDD w ith in th e chem ical p la n t w hich caused th e a c c id e n ta l exposure a t Seveso as w ell as from in d iv id u a ls resid in g in h eavily contam inated a r e a s . On th e o t h e r hand, w o rk e rs exposed to th e h e r b ic id e s K lo r in o l and B u v in o l, w hich may have v a ry in g le v e ls o f TCDD c o n ta m in a tio n , had a h ig h e r* incidence o f chrom atid breaks in p erip h eral lymphocytes than did nonexposed in d iv id u a ls 17. Increased freq u en cies in chromosome a b e rra tio n s were also 0001531 reported in Vietnam ese exposed to TCDD-contaminated h erb icid e 2 ,4 ,5 -T , but . i .. j : t . r 7923 30 DOW 126716 The in fo rm a tio n im p lic a tin g TCDD and o th e r d io x in compounds as mutagens is scarce and c o n flic tin g ; th e re fo re , more work is required b efore the m utagenic p o te n tia l o f these compounds can be determ ined. Factors which have co n trib u ted to the d iffic u lty in understanding the experim ental resu lts w ith dioxins, p a r t i c u l a r l y th e TCDD d e r iv a t iv e , in c lu d e th e extrem es in t o x i c i t y o f th ese compounds and th e ir varied s e n s itiv itie s to d iffe r e n t treatm en t p ro to co ls. S o lu b ilit y * seems to be one o f th e more o u tstan d in g problems encountered when w orking w ith d io x in s . To o b ta in com parable d a ta , a ll fu tu re stu d ies should be done under s im ila r treatm en t co n d itio n s whenever p ra c tic a l and w ith samples o f known p u r ity .* * M u ta g e n ic ity assay systems th a t are s e n s itiv e to th e a ctio n o f in te rc a la tin g agents should be considered f i r s t when systems are being selected fo r additional te s ts . For instance, reexam ination in sen sitive s tra in s o f S alm onella, p a r t ic u la r ly TA1532, is needed sin ce th is s tra in is the on ly one which has shown a p o s itiv e response (T ab les 4 and 5 ). Experim ents such as these w ill help resolve the c o n flic tin g re s u lts observed thus fa - in the Ames S alm o n ella t e s t . L ik e w is e , r e t e s t in g f o r dom inant l e t h a l i t y in mammals o r te s tin g in o th e r in vivo mammalian systems which measure damage to the genetic m a te ria l o f germ c e lls should als o be given c o n s id e ra tio n . The te s tin g of d io x in s u sin g th e m u ltip u rp o s e Sacaharcmyces cev e v is ia e s t r a in MP-1 w ould a ls o be o f in te re s t since F ah rig , N ils s o n , and Rappe23 have found th a t the p re d io x in s, In fo r m a tio n on th e s o l u b i l i t y o f TCDD can be found in T a b le 1 o f th e p aper by W.B. Crummett and R .J. S tehl e n title d "D eterm ination o f C h lo rin a te d D ibenzo-pdio xin s and Dibenzofurans in Various M a te ria ls ," Environ. H ealth Perspect. 5 (*1973) 1 5 -2 5 . See a ls o th e p ap er by C. Botre", A. M em oli, and F. A lh a iq u e e n t it le d "TCDD S o lu b iliz a tio n and Photodecom position in Aqueous S o lu tio n s " , E n v iro n : S c i. Technol. 12:335-335,1978. cs o o o **S ee paper by 0. A n ilin e e n title d "P rep aratio n o f C h lo ro d ib en zo -p -d io xin s fo r T o xico lo g ical E valu atio n ," in C hlorodioxins -- O rig in and F ate. Advances in Chemistry S eries No. 120, E.H. B la ir , E d ., American Chemical S o c ie ty , W ashington, D .C ., 7924 f DO 31 4 ,5 ,6 -tric h lo ro -2 -(2 ,4 -d ic h lo ro p h e n o x y )p h e n o l and 4-ch lo ro -2-(2,4-d ich lo ro p h en o xy induce forw ard m utations and m ito tic crossing over in th is yeast s tra in . P en tach lo ro p h en o l (is o m e r p u r ity 99") induced some fo rw ard m u tatio n s and m it o t ic gene co n versio n in th is same y e a s t s t r a in , b u t 2 ,3 ,7 ,8 -te tr a c h lo r o d ib e n z o fu r a n , a compound w hich is s tr u c tu r a lly s im ila r to TCDD, d id not induce any g en etic a c tiv ity in th is organism2 3 . These observations make fu rth e r te s tin g o f dioxins in th is or other yeast strain s which d etect m ito tic crossing-over, gene con versio n , o r forw ard and reverse m utations d e s ira b le (see Zimmermann70} .* Evidence t h a t TCDD is a p o te n t in d u cer o f c e rta in microsomal enzymes p o in ts to the need fo r a d d itio n a l s tu d ie s on the prom oting e f fe c t which d io x in s may have on th e m u ta g e n ic ity o f o th e r compounds. P a r tic u la r a tte n tio n should be given to fo rm ulations in which dioxins are p o te n tia l contam inants. A b e tte r under standing o f the m u tag en icity o f TCDD, and perhaps o th er d io xin s, w ill be obtained once stu d ies stim u la te d by the Seveso tragedy have been p ro p erly e v a lu a te d . A fte r th is m anuscript was w r itte n , B ro n ze tti (p erso n al com m unication)9 sen t the a u th o rs r e s u lt s from some p re lim in a ry exp erim en ts he had done w ith TCDD in th e y e a s t s t r a i n D7 o f Sa.ceh'V'onrjees c s v e v is ia n . T h e g e n e t i c e n d - p o i n t s o f th e s e experim ents were m ito tic cro ssin g -o ver, gene conversion, and reverse m utation. R esults from th ese experim ents, which were done w ith and w ith o u t m etab o lic a c tiv a tio n , re v e a le d th a t TCDD induced a h ig h r a te o f k i l l i n g , and no c ro s s in g - over or gene conversion. A weak reverse m utation inducinq e ffe c t was observed as w e ll as th e presence o f s e v e ra l " p e t it e c o lo n ie s " on th e p la te s screened. The TCDD c o n c e n tra tio n s te s te d in these exp erim en ts w ere 1 , 2 , 4 , 6 , and 8 g/m l. ^^i >.>rc i<erH i r nr> o * t h e TCDD s a m o le s . DOW 126118 32 ACKNOWLEDGMENTS The authors are g ratefu l to Dr. H einrich V. M a ilin g , National In s titu te of Environm ental H ealth S ciences, Dr. Sidney G reen, Howard U n iv e rs ity , Dr. W illiam F. G ra n t, M cG ill U n iv e rs ity , and D r. R ichard F. K im b a ll, Oak Ridge N ational L a b o ra to ry , fo r th e ir review and h e lp fu l comments. Thanks are also due D r. Joyce McCann, U n iv e rs ity o f C a lifo r n ia (B e rk e le y ), D r. V in c e n t Simmon, SRI In te rn a tio n a l, and Dr. Georgio B ro n z e tti, N ational In s titu te o f Environmental H ealth Sciences, fo r providing the authors w ith unpublished inform ation. Special acknowledgment is given to the Environm ental Mutagen In fo rm atio n C enter s ta ff fo r review ing the m anuscript and providing a valuable system fo r accessing the c h e m ic a l m u t a g e n e s is l i t e r a t u r e . We a r e e s p e c i a l l y i n d e b t e d t o W ilm a B a r n a r d fo r her p atien ce in typ in g th is m anuscript through it s many revisio n s and updates. o o o i: ; 7 7926 33 REFERENCES 1. Ames, B .N ., E.G. Gurney, J.A . M ille r , and H. B artsch, Carcinogens as frame s h ift mutagens: m etab o lites and d e riv a tiv e s o f 2 -a c e ty l ami n ofluorene and o th e r arom atic amine carcin o g en s, Proc. N a tl. Acad. S c i. (USA), 69 (1972) 3128-3132. 2. Ames, B .N ., F.D . Lee, and W .E. D urston, An improved b a c te ria l te s t system fo r the d e te c tio n and c la s s ific a tio n o f mutagens and carcinogens, Proc. N a tl. Acad. S ci. (USA), 70 (1973) 782-786. 3. Ames, B.N. and C. 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Lid AbMrart - The M tk n l IttJi tncuircd during the production of TCP at Sevno Italy, at pokMbly caused hy an unfotryecahle etothcruiK reaction niih incrcatc of temperature, dour dccompnutnin cf the reaction n u u , formation of p i and me m prcuore The nature of the reaction n Mill uni non and ill be invcvtigatcd further. The detection and mcaMirement of TCDD b> the Company a analyst on material collected at the mic of the accident have punted mcaMict for the protection of the population and the land and for the prevention of further dam aae. Chloracne. the A n letion indtcaiiitg a TCDD etpoaurc. appearing mainly in a emaR eegmem of the population, ha* been generally of mild degree and inclined to rapid and complete healing. Incidence or erveruy of adoictcmi acne and of the current ikin pathology hove not heen affected Hcpatoeounty. ncuroiogMal hndingi or dcranped potphyria m eubahan beta not been ntoerved in the chharacnc f l eet Pregnancy, foetal development, growth of the new born. immunorrtpomc. rate of chromosome aberranom, funciiom of the neurological lyuetn, hacmatological and hepatic condtiionv. muebidity and mortality have been turveyad in the anpoted population. So far the patholoo related to them parametan hai tintaiaed withia the range which a current for the population of ihn region and could not he conetoKd an An ctp o tu it to TCDD. Four per cent of the Aomettic anmtalt living in the comammated aonet AM (pontanaoaely, 9944% of them being wnaH animah. The remaining animate (77,716) ware ilaughterad a t a preventivemeawitt to pentad the food chain. The enalyiB ef TCDD tiaiue tevahravaatod thnt 4 k rather large amount did not naccMarily to rrwponA to eaeinmieal or fonctionnl M ena. The ahminetitir o f TCDO from the body in email a t weR a* large animah indicatMl A n , if TCDD n removed from the Am, all aantieh can he reintinduced into P c feed ehala aft* Ae tegardi the crepe and thr lend. 4 n degeahad chm dcah had praneaRy no effhet an 4k Mora. About W% of 4k TCPO adhered to fohage, grate and crop fur erveeal wuritt until it wee tranahrrad to the euR by 4k ram. Fiald and greenhorn m ah have thawn that TCDD Aeae net hneh 4RR*ew4y heyeirt a depth of 2D in the aeH and that He 90% biaokdewa ie 4k l o n e a a l ta ia t barweaa 9 and 12 wanthe. Only fraettont of the TCDD ia 4k aod can be ftw ad in 4k vegcKtlaa and fruit yaw ing K the contaminnKd an a. Dacontandaatioa eatdd H e haw rigidly aflar ed immeda wlj after the accident either by collecting and dupmmg of i t vngatatdaa or by 4k iprajing of an Mdonor. Today the area winch stall fenced-in could (at made aaaaadblt by rente img the conlaminatad tod K yen and burying them in a concrete P L The natural coune of the bnskdow a would tab* at tenet b -l yr. 1K TRO D U C TIO N O n 10 J uly 1976, a runaway reaction during the production of tricMorophcnol in a subsidiary of the Gtviudan company, the 1CMLSAJ plant in Sevcao, luly, resuhed in the discharge of products oontaining TCDO (2,3.7,t-tetrachlorodibetudtosin) over an area of about 2.1 km1 (TOOacres). This report gives an account of the natura, the estent and (he consequcncas of the accident, as seen 2 yr later. IICM CSA: Industrie G 4a4sla Mede Sue. Aaienerla. J27 ' * . .ci | *t* 000- H4 )1 * k SECTION C. TOXICOLOGY - VOLUME VI C.8 B i n lii C.8.0 Summary of Toxicology Studies with 2,4-D in Birds, Jun<> 1973. O O 509433 C.8.1 The Comparative Toxicity to Birds of Dow Herbicides Containing Chlorophenoxyalkanoic Acids and Derivatives by K. E. Kenaga. Unpublished report GH-R 32 of The Dow Chemical Company, December 29, 1972. C.8.2 The Toxicity of Dow Chlorophenoxyalkanoic Acids and Derivatives when Fed in Diets of White Leghorn Cockerels, r by !:. E. Kenaga. Unpublished report of The Dow Chemical Company, March 9, 1973. C.8.3 The Subacute Toxicity of 2,4-Dichlorophenoxyacetic Acid and 2,4,5-Trichlorophenoxyacetic Acid to Chicks, by c. c. Whitehead and R. J. Pettigrew, Toxicol. Appl. Pharmacol. 21, 348-354, 1972. C.8.4 The Kffect of 2,4-Dichlorophenoxyacetic Acid on Laying Hen.*;, by C. C. Whitehead and R. J. Pettigrew, Br. Poultry Sci. 13' 191-195, 1972. C.8.5 Phenexy-Acid-Induced Renal Changes in the Chicken. I. Ultrastructure, by N.-E. Bjorklund and K. Erne, Acta Vet. Scand. 12, 243-256, 1971. " 79 4 0 000C8C? GCJ'GOS^0 0 -2- C.8.6 The Effect of 2,4-D and Diesel Fuel on Egg Hatchability, r ! by E. D. Kopischke, J. Wildlife Management, 36(4), 1353-1355, 1972. C.8.7 Influence of External Application of Pesticides on Egg Hatchability and Chick Viability, by ,J. D. Somers. Masters Thesis, Guelph University, Guelph, Ontario, Canada, May 1972. C.8.8 (a) Harmful Action of the Herbicide 2,4-D on the Embryonic Development and the Fertility of Game Birds, by Y. Lutz-Ostertag and H. Lutz, Comptes Rendus Acad. Sci. 271, Series D, 2418-2421, December 21, 1970 (in French). (b) Report on Spraying Eggs of Game Birds with 2,4-D. Translation of (a) by M. L. Leng of The Dow Chemical Company, May 18, 1971. (c) Some Recent Findings in the Inhibition of Avian Reproductivety, edited by D. K. Wetherbee, Special Scientific Report -- Wildlife No. 67, September 1962. (Excerpts of chapters by M. J. Landy and R. P. Coppinger.) 7 00028CS 7842 DOW04601U i,______________ ______ ^ ,, r-c?r ! ^ P* ^ r-^ u ^ i . T? 2 ,3-i7 ,S-TETRACHLORODIBENZO-P-DIOXIN: STUDIES ON THE MECHANISM OF ACTION + jALAN POLAND AND EDWARD GLOVER '.cArdle Laboratory fo r Cancer Research, U n iv e rs ity o f W isconsin, Madison, *iscons in 53706 j2 ,3 ,7 ,8 -Tetrach1 orodbenzo-p-doxn (TCDD) , a trace contaminant formed in the commercial synthesis o f 2,A ,5 " tric h lo ro p h e n o l, and one o f the most sotjent small molecule toxins known, is the prototype o f a se rie s o f halogensted aromatic hydrocarbons that a ll a) produce s im ila r patterns o f t o x i c it y , b) are approximate isostereomers, and c) e l i c i t common biochemical responses. Cl n 17 >-- s Cl ry FIGURE 1- 2,3,78 TETRACHL0R0DIBENZ0-P-DIOXIN TOXICOLOGY - The to xico lo g y o f TCDD and related halogenated aromatic hydrocar bons (ch lorin a te d d ib e n zo -p -d io x in s , dibenzofurans, a zo - and azoxybenzenes; and Ii I |_g biphenyls and brominated biphenyls) have been e xte n s ive ly reviewed elsewhere' j! I and' the h istop a th ologic responses are not the subject o f th is review . However II jit ;is essential to b r ie f ly summarize the t o x ic it y produced by TCDD, to appreciate the necessity of reso rting to the in d ire ct approach to the I mechanism o f t o x ic it y presented in th is re p o rt. Supported in part by National In s t it u t e o f Environmental Health Sciences ; Grant R01-ES0-01881, National Cancer In s t it u t e Program P ro je c t Grant 1P01 -CA-22L31*. j R ecipient o f Research Career Development Award K0l-ES-0017. T i O ' . '- ; A b b re via tio n s: TCDD - 2 ,3 ,7 ,8 -tc tra c h lo ro d ib e n z o -p -d io x in ; AI1H - a ry l hydro carbon hydroxylase; MC - 3~mcthylcholanthrene; ALAS - -am inolevul in ic aejid synthetase; ED-- - dose which produces one h a lf the maximal response, In th is case induction of hepatic AHH a c t iv it y ; FL - e q u ilib riu m d is s o c ia tio constant fo r binding; TCAD - 3 , 3 * ,**1-tctrachToroazobcnzene; TCAOB 3,*, 3' ,1 '-tetrachloroazoxybenzene. 0 0 0 7"? j 4 \> w DOW04G017 r6- Species d if f e r both q u a n tita tiv e ly and q u a lita tiv e ly in th e ir response to TCDD and it s congeners 1) L e th a l?t y . The cause of death remains unkno* n . Foljlcwing the adm inistration of a. to x ic dose of TCDD, animals show a slow Lasting syndrome, with a la ten t period before death which is species speci f 1c. Lhere is an order of species s e n s it iv it y to TCDD and related compounds, wl th || guinea pigs and chickens bein^ ve ry s e n s itiv e , rats interm ediate, and mice p e f-g more re s is ta n t. 2) Chloracne and hyperkeratosis. T h is is the most common and -.ch a ra cte ristIc sign o f t o x ic it y observed in humans. Hyperkeratosis aslI been reported in ra b b its, monkeys and h a irle s s m. ice. 3) L iv e r - HepatI o- ce hlu lar necrosis has been reported in rats and rabbits and focal lesions I n mice, but l i t t l e o r no hepatic damage is seen In guinea p ig s . Several species develop disturbances of hepatic porphyrin metabolism. *0 T e ra to g e n lc lty , ! _; emb'ryotoxici ty and fe ta l wastage. TCDD is a potent embryotoxin and teratogen jin mice, ra ts , and chicken embryos. 5) Chick edema. Edema, e s p e c ia lly h yd ro- ^erjicardium , is a c h a ra c te ris tic lesion in young chickens. Edema is also seen | I jin p ic e . 6) In v o lu tio n of lymphoid organs. These compound c h a ra c te ris tic a lly produce thymic atrophy and to a le sse r degree atrophy o f the lymph nodes and il spleen in most species which is accompanied by suppression of the c e llu la r !j immune response in young animals. The e a rly work of Schwetz et a l. 9 and the nore recent report o f McConnell Ie t {a 1. i o indicate that each o f the ch lorin a ted d ib e n zo -p -d.io xi.n s w. hic. h is It o x ic , iwil'l e l i c i t a ll the responses produced by TCDD, i f administered in a s u ffic ie n t dose. The data on halogenated dibenzofuran, a zo- and azoxybenzene and bipnenyl I congeners are more lim ited , but also suggest that those congeners is o s te ric ' i w ith TCDD, produce the same to x ic syndrome. i In s p ite o f the wide spectrum and d is tin c tiv e n e s s o f the histopatholog*/ produced by TCDD, studies on the mechanism of t o x ic it y have been la rg e ly unrewarded. Th is is a ttrib u ta b le to several fa c to rs : 1) The cause of death, Jthe ultim ate "ta rg e t organ" is unknown, making i t d i f f i c u l t to know which tissu e to focus on. 2) I f one studies a p a rtic u la r to x ic e ffe c t c h lo r acne, l i v e r damage, chick edema) as a model, the response is often confined to a p a rtic u la r species; 3) There have been few reports o f to x ic responses t 'i which are d o se -re la te d , e a s ily measured and quant i f inbl e; i|) To date there has .been no rep o rt of TCDD t o x ic it y on any c e ll type in c u ltu re , which lim its in 07 i 1i v it r o s tu d ie s . 5) Studies on the pharmacokinetics and metabolism o f TCDD ti .have provided l i t t l e evidence o f any re a ctive metabolite formation o r cova etit b i nd I ng . Wc have pursued on in d ire c t approach to the mechanism of t o x i c it y , by OGG0733 L. DOW046018 ;.=G!N Hr .-^examining the biochemical e ffe cts produced by TCDD, and then asking how these effects are related to to x ic ity . HOI I f f EKZYMS INDUCTION - TCDD stim ulates the a c t iv it ie s of a number o f enzymes, the i| !moS;t w ell studied o f these being the hepatic microsomal monooxygenases. fo n o - oxygenase induction by TCDD a*) occurs in many itssues and in most species, b) is an extremely s e n s itiv e measure of ch lo rin a ted aromatic hydrocarbon expo sure, and c) gives an e a s ily measured dose-response re la tio n s h ip . The microsomal monooxygenase system is an enzyme complex embedded in the endoplasmic reticulum co n sistin g of NADPH-cytochrome P-hSO reductase and clyto- !chr!ome P-i50, which metabolizes most lip o p h ilic compounds to more polar metabo- !| jlit-e s . In ra t liv e r several d is t in c t types o f cytochrome P-J|50 have been Jid e n tifie d , which d if f e r in the spectrum o f substrates they ca ta lyze and vh ich !are; d if f e r e n t ia lly induced by the adm inistration o f various compounds. A ryl jhydrocarbon hydroxylase (AHH) a c t iv it y (measured as the ra te o f formation of the 3-hydroxy m etabolite o f benzo[a]pyrene) is a measure o f one o r two of jthejse cytochrome P-l50 species, so ca lle d cytochrome P j-il5 0 , induced by TCDD. The' stu dies presented are confined to hepatic AHH a c t i v i t y , but i t should be remembered, th is enzyme a c t iv it y is induced in a number o f tis s u e s . i ;< iI THd'uCTIOM OF HEPATIC AHH ACTIVITY IN THE CHICK EMBRYO - The adm inistra tion o f 1i | TCDD to chicken embryos produces a d o se-rela ted increase in hepatic AHH a c t iv it y measured ?n v i t r o Zk hours la t e r . TCDD produces a 10 to 12 fo ld increase i n AHHj a c t i v i t y ; the dose which produces one h a lf the maximal induction (ED,-q) .is approxim ately 3 ng/egg (0.2 to 0.3 nmol/kg). I n i t i a l l y we tested a ser tes b f jl5 halogenated d ib e n zo -p -d io xin s fo r th e ir potency to induce AHH a c tiv i t v " The- f iv e congeners which induced AHH a c t iv it y had two common p ro p e rtie s : l) ' halogen atoms occupied at i least three, and fo r maximal potency, four of the la te ra l rin g p o sitio n s (p o sitio n s 2,3,7 and 8) and 2) at le a s t one rin g pojsl- tlo n is nonhalogenated. (The o c ta ch lo ro d ib e n zo -p -d io xin is in a c tiv e , but bioth he pta ch lo ro- analogues are a c t iv e ). The same 15 congeners were tested iinn cthe chicken embryo fo r th e ir capacity to induce hepatic -a m in o le v u Iin ic acid synthetase (ALAS) a c t iv it y , the i n i t ia l and r a t e -lim it in g enzyme in heme syn th e sis and an id e n tica l s t r u c t u r e -a c t iv it y re la tio n s h ip was observed, A much la rg e r s c rie s of halogenated d ib e n zo -p -d io xin s and dibenzofurans have .been Subsequently examined for th e ir potency to Induce Ailii a c t i v i t y in the chic c embryo l i v c r ^ ' ^ and in a rat hepatoma c e ll c u ltu r e ^ 1 and confirmed the a aove f incJ i ngs. Cl 00C 0734 L. DOW046019 The most important observation has been that for chlorinated dibenzo-p^dibxir.s there is a n e x c e l l e n t c o r r e l a t i o n b e t w e e n t h eir p o t e n c y to i n d u c e f.HH I^aciiviiy (and A L A S a c t i v i t y ) a n d their toxic potency. T h e m a j o r q u e s t i o n zo be |<aifdresse<i is: H o w d o we exp l a i n this c o r r e l a t i o n ? GENETIC EXPRESSION OF AHH ACTIVITY IN INBRED STRAINS OF MICE The cla s s ic a l inducers or AHH a c t iv it y and cytochrome P^-k50 are the p o ly c y c lic aromatic hydrocarbons, such as 3~nethylcholanthrene (MC). In aj comparison of MC and TCDD fo r th e ir capacity to induce hepatic AHH a c t iv it y in jthe ra t, vie found MC and TCDD produce p a ra lle l dose-response curves, both compounds produce the same maximum response, simultaneous adm inistration o f maximally inducing doses o f MC and TCDD produces no grea ter response than that evoked by e ith e r drug alone. However, TCDD is 30,000 times as potent as KC, and' a sin g le adm inistration produces sustained enzyme induction which last's over 35 d a y s ^ , a re fle c tio n of the long b io lo g ic a l h a l f - l i f e of the compound^ V/hen randomly bred o r ce rta in inbred s tra in s of mice are administered HC,j they respond w ith the induction of hepatic AHH a c t i v i t y ; ce rta in other inbired stra in s when challenged w ith MC f a il to respond17 '13 . The prototyp ical s tra in responsive to MC is C57BL/6J and p ro to typ ica l non-responsive s tra in is i1 DBA/2J. In crosses and backcrosses between C57BL/6J and DBA/2J mice, the jtra it o f responsiveness to aromatic hydrocarbons is inherited in a simple autosopal dominant mode and the genetic locus c o n tro llin g th is t r a i t is ca lle d the Ah locus. The e xtra o rd in a ry potency o f TCDD re la tiv e to MC prompted us to examine it s -e f f e c t s in inbred s tra in s which are responsive and non-responssive to 4C. JCDD induced AHH a c t iv it y in a ll stra in s tested regardless o f th e ir response to ! iQ MC j . In C57BL/6J mice the enzyme a c t iv it y and the sp e ctra l properties of the j! cytpchrome P.-450 that were induced by MC o r TCDD, o r both compunds administerec !I 1 together were found to be s im ila r to those induced in DBA/2J mice by TCDD. i These re su lts suggest that the same gene product(s) is induced in C57BL/6J nice by both compounds and in DBA/2J mice by TCDD. j -Thus, mice which are nonresponsive to MC, do respond to a more potent I stim ulus, TCDD, and th e re fo re these mice do have the s tru c tu ra l and regulatory genes necessary fo r the expression of AHH a c t iv it y . The fa ilu r e o f these mice .to respond to MC suggests that they f a il to recognize MC as a signal for induc- "i . ! \ tio n . V/e postulated that the mutation in nonresponsive mice re s u lts in a . d e fe c tiv e recognition o r receptor s ite for AI!H in du ctio n, a receptor which has i a diminished (or absent) a f f i n i t y for MC. Consider the hypothesis that TCDD ('. I _____ I__ e7h 0000725 D O W 04602U 3Lo..s' HERE 'acts s t the sane receptor as MC, but because o f it s greater potency, and ijence its! presumed greater a f f in it y , i t is able to saturate th is receptor even i^n nonresponsive mice, and in it ia t e the induction of AHH a c t iv it y . T h is hypothesis p re d ic ts inbred mice which are nonresponsive to MC, should respond to TCDD, but should be uniform ly less s e n s itiv e to TCDD, than stra in s v/hich are responsive I to MC. We adm inistered various doses of TCDD to s ix inbred s tra in s o f mice arid hours la t e r measured t h e ir hepatic AHH a c t iv it y '."2"0 . TThLe. dj a__t_a___a__re___p__r_e_s_ enIt. ed F ig . 2 as the log o f the dose versus the fra c tio n a l response (control a c t iv it y = 0, maximal a c t iv it y = 1 .0 ). The three inbred stains which are nonresponsive to MC (DBA/2J, AKR/J, and SJL/J) are less s e n s itiv e to TCDD, tf an arej the responsive s tra in s (C57BL/6J, BALB/cJ and A/J) JL e ^ , the log-dose response curves fo r the nonresponsive s tra in s are sh ifte d to the r ig h t . Tjhe ED^q fo r AHH induction in responsive s tra in s is approximately 1 nmole/kg, i ->uand* fo r nonresponsive stra in s the EDC- is ^ .1 0 nnoles/kg. The data support the hypothesis that TCDD and MC act on the same receptor, and that the Tiut'ation in nonresponsive stra in s is due to a receptor w ith a diminished a ffjin tty fo r inducing compounds. J DOT !O.V- ' ig . 2. Log dose-response curves for the induction o f hepatic AHH a c t iv it y by i- TCDD in s ix inbred s tra in s of mice. The mice were in je cte d in tra p e ri toneaj) ly w ith v a ry in g doses of TCDD dissolved in p-dioxane; 2h hours la te r the animals were k ille d and th e ir liv e rs were assayed fo r AHH a c t i v i t y . The data are E x pressed as fra c tio n a l responses to elim inate s tra in d iffe re n ce s In basal apd 7 maximally induced enzyme a c t iv it ie s (see t e x t ) . Each p o in t represents thej average o f four or fiv e animals. -1 1 J 0) 00726 DOw 04602 \ `.i. CYTOSOLIC BINDING SPECIES 1-- i------------------------------------------------j 'c next wished to search fo r a macromolecular species v/hich had the in v it r o binding pro p e rtie s one would p re d ict fo r the hypothesized receptor based on jthe in v iv o b io lo g y. S p e c ific a lly , one would expect such a moiety to have th^ fo llo w in g in v it r o binding p ro p e rtie s: 1) re v e rs ib ly bind TCDD w ith a high a f f in it y and the binding a f f in it y (K,,) should correspond to the in vljvo potIjency (cDbt;Un) fo r A'riH induction; 2) the Ubinding species from mice which aI|re noriresponsive should have a Tower a f f in it y fo r TCDD than the binding specijes from responsive mice; 3) the rank order o f binding a f f in it ie s o f other h a lo - I genated d ib e n zo -p -d io x ln s , and other ch lo rin a ted aromatic hydrocarbons should correspond to t h e ir rank order o f potencies to induce AHH a c t i v i t y , and k)\ otijer compounds, such as the p o ly c y c lic aromatic hydrocarbons, which induce AHH a c tiv ity , should bind to th is moiety. An I n i t ia l se rie s o f experiments, suggested that there was a moiety which bound ^H-TCDD w ith a high a f f in it y in the l i v e r cytosol (105,000 xg superratant fra c tio n ). The binding o f ^H-TCDD (s p e c ific a c t iv it y 52.5 Ci/mmole) to liv e r cytqsol was' measured by the charcoal-dextran binding assay (fo r d e ta ils see reference 13)j. V/hen hepatic cytosol from C57BL/6J mice was Incubated w ith varying conce nI trations o f T^H-TCDD and T H-TCDD plus a 200-fold excess o f unlabeled 2 ,3 ,7 ,8 " tetrachlorod ib enzofu ran, we observed a small pool o f high a f f i n i t y s ite s (F ig . 3A)j (5 ). In F ig . 3B the same data fo r s p e c ific binding (d isp la ca b le , h1gh a ffiin ity binding) is presented in a Scatchard p lo t . The binding a f f in it y fo r TCD|D, Kp, is 0.27 nil which compares q u ite fa vo ra b ly with the in v iv o potency forj AHH induction in C57BL/6J mice (ED-- = 1 nmole/kg). j The s p e c ific binding of J H-TCDD to liv e r cytosol from C57BL/6J mice was greater at a l l concentrations o f the ra d io lig a n d , than was the s p e c ific bind! to 'liv e r cyto so l from DBA/2J mice. U n fo rtu n a te ly, the lim ited aqueous s o lu b il- ityj of ^H-TCDD prevents us from achieving a s u ffic ie n t concentration to saturate the cyto so l binding spocles from nonrcsponsive mice and estim ating the Kp. ! Another expectation of the cytosol binding species i f i t is the receptor, is !that the in v i t r o binding a f f in i t y o f halogcnated d ib e n zo -p -d io x in congeners '-o t t o :.*. 'should -j j correspond to th e ir in vivo potency ----------------- to induce AHH a c t i v i t y . The bijnd- (*ng a f f in it ie s o f these unlabeled congeners ore measured by th e ir capacity to jI compete v/ith the s p e c ific binding of ^H-TCDD, to mouse l i v e r cytocol and tne Y c s y lt s j n F ig . h are expressed r e la tiv e to the binding a f f i n i t y o f TCDD i (TCDlI D = 100). The b io lo g ic a l potencies of these congeners to Induce hepatic .J 7948 OD 0737 EG!N Ht.r*f s DOW046022 0 10 20 30 40 3H-TCDD (DPMxlO'3) F ig . 3. A) T o ta l, non specific and s p e c ific binding of ( H)-TCDD to hepatic [cytosol from C57BL/6J mice. The c y to s o lic fra c tio n of 1iv e r from C573L/bJ nice 'at |2 mg prgtein/ml in b u ffe r was incubated w ith varying concentrations of (^H )TCDD and ( ^H)-TCDD plus a 200 fo ld excess of 2 ,3 ,7 ,8 -te t rachlorodi benzofuran ,fotj 2 hours at 0. Samples were treated with a suspensi on o f charcoal-de>jt ran to [remove unbound ligand, and the ra d io a c tiv ity q u a n tifi ed. Total binding - n)-TCDD alone (o ); nonspecific or nondisplaceable bind ing - [-W CD D plus 2,3,7,8-tetrachlorodibenzofuran ( & ) ; sp e cific or displa ceable binding (Q ;B) Scatchard p lo t of the s p e c ific binding in A. h 'ji 7' r 'AHH in the chicken embryo (EDC_) are also expressed r e la t iv e to.TCDD. As seen in Figure k, there is a ve ry good correspondence between the rank order of binding a f f i n i t y and b io lo g ic a l potency fo r these compounds. We have observed that other compounds which induce AHH a c t i v i t y , suchj as MC, bcnzo[a]pyrcne and 3-naphthoflavons, compete fo r s p e c ific cytosol binding j s it e s , but drugs which induce other types o f microsomal monooxygenase a c t iv it ie s . ( e . g . phnobarbital, p ,p '-D D T , pregnenolone-16-carbonit r i 1o) and the steroid : hormones and th e ir analogues which have liv e r cytosol binding proteins -- j'all '.'.fail to compete with 3' H-TCDD fo r s p e c ific cytosol bindi.ng s .it e s . ! The c y to s o lic binding species has a ll the pro perties predicted fo r the; ..induction recep tor, most n o ta b ly, s tc re o s p c c ific re co g n itio n o f inducing 1. 7949 00007:8 DOW04G02:) EG IM H I RELATIVE BINDING AFFINITY RELATIVE BIOLOGICAL POTENCY RELATIVE BINDING AFFINITY inoclive (5.4*10'7)* inoclive (27*I0`*1 inoclive (5.4*10'*) Cl Cl inactive (5.4*10"6) inactive (5.4*10-) inactive (2.7x10- ) <4C&Cl Cl inactive (1.1x10 ) RELATIVE BIOLOGICAL POTENCY inactive (9.4*10'*)** inactive (9.4x10-*) inactive (9.4x10-*) inactive (4.7*10-?) inactive (9.4x10-*) inactive (9.410-) inoctive (9.4*10-*) IFI g'. 1|. The cytosol binding a f f in it ie s and b io lo g ica l potencies of dibenzoJa-d'ioxin congeners re la tiv e to TCDD. The binding a f f in it ie s of the dlbenzpp-d.ioxin congeners fo r hepatic cytosol were estimated by the capacity of j these compounds to compete with rH j-TC D D fo r s p e c ific binding s ite s In C57BL/6J 1ivjer c y to s o l. The binding a f f in it ie s are expressed re la tiv e to TCDD whic-h Is assigned a value o f 100. For in a c tiv e analogues, the highest concentration tes'ted that was judged to be soluble is given in parentheses. [The b io lo g ic a l potency (ED__) of each congener was the dose that produced one,' h a lf the maximal induction o f hepatic AHH a c t iv it y in the chicken embryo, and; the potency was expressed re la tiv e to TCDD (TCDD=100). For in a ctive com pounds, the highest dose tested (in moles/kg) is given in parentheses. Thlis assumes the weight o f an average chicken egg Is approximately 50 g. I I I OTTO', a - The absolute value of the Kn fo r TCDD is 0.27 nM. b - The absolute value of the ED-- fo r TCDD induction of hepatic AHH a c t i v i t y in the chicken embryo is 0.31 nmoles/kg. c - The highest concentration tested that was judged soluble in moles/1i te r. d - The highest concentration tested in moles/kg. I .'compounds. Recent experiments both in v i t r o and In v iv o , indicate the cytasol A I, ------------ ^binding species mediates the s p e c ific uptake and binding of 'li-TCDD to hepatic 7950 0000739 4602 L. :--n uic le i, and the experiments suggest that the lig a n d -re ce p to r complex in the cytoI sol translocates to the nucleus * . The Ah locus, which determines the O -o :> o t r a it of responsiveness or nonresponsiveness to p o ly c y c lic aromatic hydrocarbons (orj greater o r le sse r s e n s it iv it y to TCDD, appears to be stru ctu ra l gene ljocus forj the c y to s o lic receptor p ro te in . (The biochemicai and genetic evidencej a il suggests, but does not prove t h is ) . The nutation in nonresponsive mice appears to 'I.re su lt in an a lte re d recep. tor w ith a diminished a f f i n i t y fo r the inducini g I JLJ. compound. COORDINATE GENE EXPRESSION I------- ;-------------------------------------------------------------- The adm inistration o f MC o r TCDD to la b o ra to ry animals stim ulates not only AHH a c t i v i t y , but a number o f other hepatic enzyme a c t iv it ie s . There Is ij jevljdence f o r several of these enzymes, that th is increase in a c t iv it y represents jtrue in d u ctio n , j_.e^ de novo protein syn th e sis. MC and TCDD have been shown to induce the fo llo w in g hepatic enzymes: 8-am inolevul in ic acid syntnetasej, pDPj-glucuronosyi tra n sfe ra se , glutathione-S_-transferase B, aldehyde dehydrjo- jgenase, DT-diaphorase, and o rn ith in e decarboxylase. The induction o f several o f jthese enzymes a c t iv it ie s have been shown to segregate w ith the Ah locus! in ;j I inbred s tra in s of mice, (fo r a more detailed discussion see r e f . 21). I t Would i! appear that the Ah locus co n tro ls not only the expression o f AHH a c t lv it y .j but also the coordinate expression (and perhaps repression) o f a number of other enzymes (F ig . 5 ). i iI1 I STRUCTURE-ACTIVITY RELATIONSHIP j |Considering other classes o f halogenated aromatic compounds, v/e wish to know: l ) i f one can gene ralize about the s tru c tu re necessary to bind to t ie cytosol re c e p to r; and 2) whether there is c o rre la tio n between the potency i f congeners to induce AHH a c t iv it y -- and t h e ir to x ic potency. Dibenzofurans 'thej! s t r u c t u r e -a c t iv it y re la tio n s h ip fo r the ch lo rin a te d dibenzofurans is vIpry sim ila r to that of the dibenzo-p-dioxins fo r binding to the cytosol receptor I ondj inducing AHH a c t iv it y w ith the 2,37 ,8 -tetrachloro~ analogue being the i. i : ' i ! Greenlee, W. F. and A. Poland, nanuscript submitted. In th is re p o rt, we re fe r to the phenotypes o f the Ah locus using the o r i g i nal . term inology: responsive or nonresponsive to p o ly c y c lic aromatic hydror enrbons v/ith the induction o r fa ilu re of induction o f hepatic AHH a c t iv it y . Botn phenotypes respond to TCDD v/ith induction of hepatic AHH a c t iv it y andj d if f e r o n ly in th e ir s e n s it iv it y (responsive m ice, EDr ~ l nmole TCDD/kgJ ` ''nonresponsive" mice ED^^ 10 nmole-; TCDD/kg). Thusf when the nonresponsive phenotype is referred to , i t is nonresponsive to MC and r e la t iv e ly less 1 sensi t i vc to TCDD. I 7{ -000C D O W 046025 I Fig', h. The cytosol binding a f f in it ie s and b io lo g ica l potencies o f dibenzo- p -d .ioxin congeners re la tiv e to TCDD. ihe binding a f f in it ie s o f the dibenzo- Ip-d'io xin congeners fo r hepatic cytosol were estimated by the ca p a city o f | these compounds to compete w ith (^Hj-TCDD fo r s p e c ific binding s ite s In C5:7BL/6J 1iy e r c y to s o l. The binding a f f in it ie s are expressed re la tiv e to TCDD whlcjh is assigned a value of 100. For in a c tiv e analogues, the highest concentration tested that was judged to be soluble is given in parentheses. | [The b io lo g ic a l potency (E D ^ ) of each congener was the dose th a t produced one] h a lf the maximal induction o f hepatic AHH a c t iv it y in the chicken embryo, and; the potency was expressed r e la tiv e to TCDD (TCDD=100). For in a c tiv e com- poupds, the highest dose tested (in moles/kg) is given in parentheses. Th is assumes the weight of an average chicken egg is approximately 50 g. i J l OTTOf / /I f.! i a - The absolute value of the Kn fo r TCDD is 0.27 nM. | b - The absolute value of the ED_n fo r TCDD induction o f hepatic ! AHH a c t iv it y in the chicken embryo is 0.31 nmoles/kg. | c - The highest concentration tested that was judged solu ble in j m olcs/liter. | d - The highest concentration tested in molcs/kg. ..'compounds. Recent experiments both in v it r o and in v iv o . In d ica te the cytosol j binding species mediates the s p e c ific uptake .arid binding o f ^H-TCDD to hepatic 7952 "5000741 DOW04G026 b : g : N n : ni; CYTOPLASM NUCLEUS TCDD<= =TCDD+BP^TCDD-BP ^ *TCDD-BP it AAA5V V VV AHH ACTIVITY A B C (CYTOCHROME 5 -4 5 0 ) ________ V N F ig . 5. Schematic representation o f the model proposed fo r the p le io tro p l c response mediated by the TCDD-receptor complex. BP is the TCDD-binding p-^ otei n or receptor. most a c tiv e . Only th is congener has been tested for t o x i c it y , and I t was found to produce chloracne and edema in chickens 22 Azo and Azoxybenzene 3 ,^ ,3 ' , 4'-Tetrachloroazobenzene (TCAB) and 3,4,3' 4 ' " tetrachloroazoxybenjzene (TCA03) have been reported as contaminants of 3 ,4 -d ic h lo ro a n ? 1ine or h e rb icid e s made from 3 ,4 -d ic h lo ro a n ilin e and are thought to be responsible fo r sev!eral episodes of chloracne in fa c to ry workers 23 ' 24 TCAOB, TCAB, and 3 ,4 ,3 ',4*-tetrachlorohydrazobenzene are a ll approximate stereoisomers o f TjCDD, a ll! bind w ith a high a f f i n i t y to the c y to s o lic binding sp e cies, and are a ll potent inducers o f hepatic AHH in the chicken embryo. Congeners such as ii 3 ,5 ,3 ',5 '" te tra c h lo ro a zo -, 3 ,5 ,3 ',5 '-te tra c h lo ro a z o x y -, and 3,5,3 ,5 -te tr a l chiorohydrazobenzene, azobenzene or azoxybenzene are in a c tiv e as Inducers or com petitive lig a n ds. TCAOB produced acne in h a irle ss mice, but 3 ,5 ,3 ',5 '* jtetrachloroazoxygenzene did not. IHaloqenated Biphenyls ! I Of a se rie s of 16 ch lo rin a te d and brominated biphenyl congeners, only , 0 T 1 O H ' -th ree ( 3 ,4 ,3 ',4 '-t e t r a c h lo r o , 3 ,4 ,5 ,3 ' , 4 ', 5 '-h e x a c h lo ro -, and 3 ,4 ,5 ,3 '4' , 5 '- .1 | # i.jhexabromo-) induced hepatic AHH a c t iv it y and competed fo r the c y to s o lic binding "spe*cies20r . The s tru c tu ra l requirements necessary fo r indu ction o f AHH a c tIiv it y ;v/ere 1) halogen atoms in at least two adjacent la te ra l p o s itio n s on each j! ! -ibenzcne rin g (3 ,4 ,3 ',4 * or 3 ,4 ,5 , 3 ', 4 1, 5 ') ; and 2) the lack o f halogen s u b s ti- 7353 "1 ' ' ..... 00007*2 DOW046027 i.\ Hf !'r. :tuc ions adjacent to the biphenyl bridge {the 2 ,6 ,2 ' or 6' p o s itio n s ) - such subst! tu io ns lead to marked nonplana rity. A more extensive in ve s tig a tio n of microsomal monooxygenase induction by ch lorin a ted biphenyl congeners by Go^ldstein2 et 6a l. supports these conclusions. There are a lim ited number of in v e s tig a tio n s on the t o x ic it y o f ch lo rin a ted biphenyl congeners, but cne refjort by McKinney et_ aj_. 22 is p a r t ic u la r ly in te re s tin g . The authors compared th^ t o x ic it y o f 5 symmetrical hexachlorobiphenyl congeners in chickens. The 3 ,^ ,5 ,3 ' ,4 ' ,5 , - hexachloro-congener had the greatest to x ic potency, and was the o n ly one to produce thymic atrophy and edema in the chickens, to x ic | responses c h a ra c te ris tic of the TCDD-syndrome. We can g en e ra lize about the s t r u c t u r e -a c t iv it y re la tio n s h ip o f each o f these classes o f halogenated aromatic hydrocarbon. Consider a prototype o f each cla ss - TCDD, 2 ,3 ,7 ,8 -te tra ch lo ro d ib e n zo fu ra n , 3 ,4 ,3 ' ,4 '-te tra c h lo rO c z o xybenzene, and 3 ,4 ,3 ',4'-tetrach lorob ip heny1 - depicted in F ig . 6. These compounds are a ll approximate stereoisomers and th e ir molecular structures can I be thought o f as roughly f it t in g into a rectangle 3 x 10A w ith halogen ate ms In the fo u r co rners. P la n a rity or near p la n a rity seems to be e s s e n tia l. In Ij Figure 6 the in v it r o binding a f f in it y fo r the cytosol binding species anc i f ----------------thei potency to induce AHH a c t iv it y in the ch ick fo r each o f these compounds, *i and fo r a number of other tetrahalogenated aromatic compound which are a p p ro xi- I pate stereoisom ers, are compared. The t o x ic it y o f these la t t e r compounds has inotj been te s te d . While many points remain to be answered about the chemical features recog nized by the re ce p to r, the planar rectangular s it e depicted in F ig . 6 seen:s a good f i r s t approxim ation. jA MODEL FOR THE MECHAflISM OF TOXICITY !i j j We have emphasized fo r ch lorinated aromatic hydrocarbon congeners, the jj . .co rre la tio n between th e ir potency to induce AHH a c t iv it y and th e ir to x ic potency. I t is d i f f i c u l t to envision how the induction o f AHH a c t i v i t y , per ;I se.i is in vo lve d in s p e c ific organ t o x ic it ie s such as chloracne o r thymic in vo lu tio n . Th e re fo re , we v/ish to focus on the receptor - the c o rre la tio n between the a f f i n i t y o f congener fo r the receptor and th e ir to x ic potencies. 7O**\w.a / ; ! We p o s t u l a t s t h a t the t o x i c i t y o f t h ese c h l o r i n a t e d a r o m a t i c h y d r o c a r b o n s in m e d i a t a d t h r o u g h the rece p t o r , that is, the initial e v e n t i n t h e i r toxij? action is the stcraospacific recognition a n d binding to the cytosolic binding J s p e p i e s . I t i s s u g g e s t e d that the l i g a n d - r e c e p t o r c o m p l e x c o n t r o l s a b a t t ry \of genes, a n d the ex[)ression (or repression) o f one or more o f these genes ^fvhiph are coordinc.lely expressed results in the observed toxic syndrome.. . J8 5 4 0000743 DOW046028 --r* i v h i 3A I I I * 1 -1 0 A- Flg. 6. Is o s t e r ic tetrahalogenatec aromatic hydrocarbons: Comparison of the!ir potencies to induce AHH a c t iv it y and t h e ir c y to s o lic binding a f f in it ie s . [The b io lo g ic a l potency o f each compound v/as determined from the log doseresponse curve fo r induction of hepatic AHH a c t iv it y in the chicken embryc The; binding a f f i n i t y o f each congener v/as determined by i t s capacity to compete w ith ( H)-TCDD fo r s p e c ific binding s ite s in l i v e r cytosol from jC57(BL/oJ mice. !! I1 I One p re d ic tio n from th is model is immediately obvious. T h e `genetic | evidence suggests that the Ah locus determines the receptor p ro tein and it s binding a f f i n i t y . Thus, i f the t o x ic it y o f TCDD (and re la te d halogenatcd aromatic compounds) is mediated through the recep tor, then inbred stra in s o f j! I n ice w ith a high a f f i n i t y receptor (and which are s e n s itiv e to induction o f >> ! AHH a c t iv it y by TCDD) should be s e n s itiv e to t o x ic it y from TCDD, and nice .w ith a lower a f f i n i t y receptor should be less s e n s itiv e to the t o x ic it y ofj `TCDD. In s h o rt, the t o x ic it y of TCDD should segregate w ith the Ah locus. |To examine t h is , wc chose the in vo lu tio n o f the thymus because 1) we 7355 jfcr/I th is is a " t o x ic response" - (not ju s t a biochemical response) In thajt '000744 I t 'reprosents tissue loss and in young animals i t is associated w ith suppr.essionI o f c e llu la r immunity, and 2) thymic atrophy is dose-related and q u a nI tifi- i a b lIe . C575L/6J, DBA/2J, and hybrid B6D2F./J male mice, were given a single * 4. in tra p e rlto n e a l In je c tio n o f varying dose of TCDD . S ix days la t e r the animals were k ille d , th e ir thymuses dissected out and weighed, and the data expressed as the thymic/body weight ra tio (F ig . 7A) . TCDD produced a l dos.e related decrease in the ra tio o f t'nymus/body w eight. The responsive C57BL/6J mice were more s e n s itiv e than the nonresponsive DBA/2J mice, and 1 the hybrid mice were interm ediate. V/e than bred B6D2F^/J mice (heterozygous at the Ah locus, Aa) w ith DBA/2J mice (homozygous nonresponsive, aa) and the i re su lta n t o ffs p rin g v/ere phenotyped as heterozygous responsive (Aa) or homozygous nonresponsive (a a ). The animals were dosed w ith TCDD as above, k iljle d s ix days la t e r , and th e ir thymus/body weight r a t io determined (F ig . 7B). Since sex influences the thymus/body weight ra tio in co n tro l mice, data from al|es and females are presented separately. As seen in F ig . 7B, fo r both cal!es and females, heterozygous responsive mice (Aa) are more s e n s itiv e to TCDD, than are homozygous nonresponsive (aa) mice. The data in d ic a te that; thymic in vo lu tio n by TCDD segregates with the Ah locus. 1 | Prelim inary experiments in inbred stra in s o f mice, suggest that s e n s itiv ity to the teratogenic e ffe c ts of TCDD (c le f t palate formation) is determined jby 1| jthe Ah locus, j_.. the responsive s tra in s , those with a high a f f i n i t y receptor have a greater incidence o f fetuses with c le f t plates a t a given dose of TCDD. i V/e have reviewed two lin e s of evidence which suggest that t o x ic it y o f I TCDD and related halogenated aromatic compounds is mediated by t h e ir stereo s p e c ific binding to the receptor fo r AHH induction: 1) the c o rre la tio n between the binding a f f in it ie s o f these compounds fo r the c y to s o lic receptor and tjheir .'toxic potencies; and 2) fo r TCDD, the segregation of thymic in v o lu tio n , wljth ths ^Ah locus. Since the Ah locus co n tro ls not only the expression o f AHH a c t iv it y , but several (perhaps many) other genes, i t is tempting to p o stu la te that YCDD jexerts it s t o x ic it y through the expression (or repression) o f one or more of |the genes c o n tro lle d by the recep tor. However, the genetic evidence only permits us to state that t o x ic it y (thymic in vo lu tio n , and probably c le f t C )B 0 ITO '' "vlpalate form ation) segregates w ith the Ah locus. -1 . V/e do not understand why nonhalogenated compounds, which bind to the i'n rVe: duction re ce p to r, such as p o ly c y c lic aromatic hydrocarbons and B-naphthofl'aI vone, ;do not produce the spectrum o f to x ic responses c h a ra c te ris tic o f the chlPrinatec ?95e -+ ;-j Poland, A. and E. C lo ve r, unpublished data. Vs.. i I 000 1C5 rep; OFOgi'OMOd TCDD (m o ln /kg ) F ig . 7. A) Log dose-response curves fo r the e ffe c t o f TCDD on the ra tio of |thymus/body weight in C57BL/6J, DBA/2J and B6D2Fj/J mice. F ive week oljd male mice were given a sin g le intra pe rito nea l in je c tio n of TCDD dissolved jin p-dioxane o r the solvent alone. S ix days la te r the animals were k ille d , th e ir thymuses weighed, and the data expressed as the ra tio o f the thymus/body w eight. Each point is the naan S .E . of 5 to 8 anim als. The solvent injected (ccJntrol) groups are connected by dashed lines to the re s t o f the dose-response curves. B) ! Log dose-response curve o f TCDD on thymus/body weight ra tio in male and female o ffs p rin g of the B6D2F /J x DBA/2J c ro s s . The o ffs p rin g o f the B6D2F^/J x D3A/2J cross were phenotypea as heterozygous responsive - Aa, o r homozygous, nohresponsive - aa, at the Ah locus. At f iv e and one h a lf weeks o f age th'e animals were dosed w ith TCDD as above and k ille d s ix days la t e r . The data! on pa lies and females are presented sep a ra tely, because the sex o f the mice aflfectec the; ra tio o f thymus/body weight in the solven t injected control groups. E;ach point is the mean S .E . o f 4 to 8 animals. The solvent in jected (control!) groups are connected by dashed lin es to the re s t of the dose-response cu rves. aromatic hydrocarbons. I t is possible that t o x ic it y is related to the i. sustained occupation o f the receptor and the r e la t iv e ly long b io lo g ic a l h a l f - _ lives of the halogenated aromatic compounds versus the ra p id ly metabolized no r w .p o ly c y c lic aromatic hydrocarbons accounts fo r the d iffe re n ce . :.:ii ' The model gives no clue to the ultim ate biochemical lesions responsiblet :J .fo r the t o x ic it y o f these compounds, but merely states that t o x ic it y Is mediatec I !.through binding to the recep tor. For a tis s u e o r c e ll type to develop a toi x ic response to ch lo rin a te d aromatic hydrocarbons the presence o f the receptor! Is 7957 000077 DO W 046031 HF.Rr. :- s s c n ' i a l , but may not be s u f f ic ie n t . * ii iI i' MUTAGENICITY AND CARCINOGENICITY j Recently, several groups have found TCDD to be a potent carcinogen in chronic jfIe- e d in g studies 27-259. Kociba et a l . 27 is a very thorough study maintained ra ts on d ie ts supplying a d a ily dose o f 0 , 1 , 10 and 1 0 0 ng/kg f o r two ye a rs. Female ra ts feed the highest' concentration o f TCDD developed a s ig n ific a n t increase in hepatocellular carcinoma, s t r a t if ie d squamous c e ll carcinoma c f the harJd palate and nasal tu rb in a te and k e ra tin iz in g squamous c e ll carcinoma c f the lung. At a dose o f 100 ng/kg/day nearly one h a lf the female ra ts developed | one o f these neoplasms, suggesting TCDD has a carcinogenic potency comparable iI | ito that o f a fla to x in B ,, which at life tim e dose o f l pg/kg/day has been e s t i - imatied Ij to produce a 50% incidence of hepatocellular carcinoma In the r a t 30 3 . j i The carcinogenic potency o f TCDD is s u rp ris in g in lig h t o f l ) the Tack o f jj convincing evidence that TCDD. is mutagenic in any in v i t r o b a c te ria l te st systems3 3 , and 2) f a ilu r e to demonstrate s ig n ific a n t covalent binding In 1j 3 *7" vivo. We have recently examined the in v iv o binding o f H-TCDD to ra t liv je r pacromolecular fra c tio n s . I f one assumes unextracted r a d io a c t iv it y is equatable With c o va le n tly bound ^H-TCDD, the maximum binding to DMA is 6 pmole o f TCDD !! per mole of nucleotide, 1 to 6 orders o f magnitude lower than that o f most: other chemical carcinogens , and equivalent to one molecule o f TCDD boundI to the1 DMA in every 35 d ip lo id c e lls + . These re s u lts **suggest that i t Is u n likI e ly ji 1 jtha,t the mechanism of oncogenesis is through covalent binding and somatic m u tation. !! j 'Th e mechanlsm(s) by which TCDD produces t o x ic it y and c a rc in o g e n ic ity ajre Unknown. Understanding these processes is important because 1) TCDD Is the ;! .1 prototype o f a large se rie s o f the halogenated aromatic hydrocarbons, manyj o f which are environmental contaminants, 2 ) the studies to date suggest th is l1 se rie s o f compounds acts by ra th er a unique mechanism(s). BO I IO V ACKNOWLEDGEMENTS We would lik e to thank Dr. Andrew Kcnde and his postdoctoral fe llo w s . Drs. James Wade, Mark DeCamp and John A ire y who synthesized a ll the compou ids employed in th is work. We a lso g ra te fu l acknowledge the c o n trib u tio n s of Drs. D ill Greenlee and Joyce Knutson on the b io lo g ica l aspects o f th is v/or ^5$ RPoland, A. and E. G love r, manuscript submitted. in_____ Q j r\ 7 n reogwy^00 : i2 5 * Poland, A. and Clover, fc.. U 9 / / 1 f'.ol. rnarmacot., o , . Izt> Coldstcin, A., Hickman, P. , Bergman, H., McKinney, J.D. and Walker, M.P. (1977) Chem.-Biol. Interactions, 17 69- 8 7 . * 7 : Kociba, R.J., Keyes, P.G., Beyer, J.E., Carreon, R.M., Wade, C.E., Dittenber, D.A., Kalnins, R.P., Frauson, L.E., Park, C.H., Barnard, S.D., Hummel, R.A. and Humiston, C.G. (1973) Toxicol. Appl . Pharmacol., 1(6,' 279-303. 28. Van Miller, J . P . , Lalich, J . J . and Allen, J .R . (1977) Chemosphere, 6, 537-5W. 29.| Moines, P.A., Rust, J.H., Richter, W.R. and Shefner, A.M. (1979) Ann. MY Acad. Sci., in press. 30 J Vogan, G ., Paglialunga, S. and Mewberne, P. (197*0 Food Cosmetic T o x ic o l., 12, 681-685. 31 Hussain, S ., Ehrenberg, L ., L o fro th , G. and G e jv a ll, T . (1972) Ambio, 1, 32-33. 32 S e ile r, J .P . (1973) E xp e rie n tia , 29, 622-623- 33J Wasson, J . S . , H u ff, J .E . and Loprieno, N. (1977/78) Mutat. Res., 1(7, U l-160. 13V Farber, E. (1968) Cancer Res., 28, 1859-1869. 1 ii BOT TOM /i i 71 '1! :i " *95.9 00007^3 nv H i A. /' T626H M Q SECTION II 2,4-D in the Aquatic Environment Mark T. Halter College of Fisheries University of Washington Seattle, Washington 0004196 ZG Z G f l MOQ Summary of Findings on 2,4-D A summary of the content of this review is presented here in terms of specific responses to thirteen relevant questions. 1. What is the purpose of this review? This review attempts to point out the known and potential risks to the aquatic environment associated with the use of the chlorophenoxy herbicide 2,4-D for aquatic plant control in northwest waters through an examination of the relevant literature. This review does not contain information as to whether chemical herbicides are the technique-of-choice for the removal of aquatic plants from those areas where their presence interferes with human activity. 2. Why is the use of 2,4-D as an aquatic herbicide so controversial? The use of 2/4-D in the aquatic environment is controversial due to the chemical structure and properties of the 2/4-D molecule/ coupled with its widespread application in past aquatic plant control programs. 2/4-D is a man-made chlorinated phenyl com pound/ and similar compounds are not produced anywhere in nature. As a chemical group, chlorinated phenyl compounds are generally found to 1) possess varying degrees of biological activity, that is, they may alter the normal functioning of organisms - indeed that is why 2,4-D, for example, kills plants; and 2) be resistant to decomposition by the common chemical and biological mechanisms present in the environment. These two properties, obvious at least to some degree in 2,4-D, have lead to speculations by some scientists and citizens that the extensive use of 2,4-D in the aquatic environment, such as has been witnessed in past plant control programs in other parts of the country, may lead to subtle "poisoning" of the aquatic system, particularly under a regime of annual treatments. 3. Does' 2,4-D exist in different commercial formulations and are some forms preferable to others for application to the acuatxc environment? Yes. 2,4-D is commercially available in both salt and ester formulations and, in the reviewer's opinion, the salt formulations are more suitable for use in the aquatic environment. Common salt 2,4-D formulations include sodium 2,4-D and 2,4-D DMA while . the most widely used ester is 2,4-D BEE. For many years it has been recognized on the basis of laboratory tests that intact (unhydrolysed) 2,4-D esters are more harmful to most aquatic f organisms than are the 2,4-D salt formulations. The use o f esters, especially BEE, in the aquatic environment has not been restricted, however, due to evidence suggesting that p o s t - a p p l i - 0004137 -89- 44 V 6 U 0 cation ester hydrolysis in nature is usually rapid and results in marked detoxification of the parent molecule. However, because rapid hydrolysis of BEE is not yet proven for northwest waters, and because of certain residue problems associated with the past use o f BEE, the reviewer believes the prudent conclusion is that only 2,4-D salts, namely sodium 2,4-D or 2,4-D DMA, should be considered for use in northwest waters. 4. Are the 2,4-D salt products free of harmful or undesirable xmpuritxes? Probably not, although definitive information on this area was not found in the literature. Dated information from various reports suggests small quantities of several impurities are present in 2,4-D. The biological significance of these impurities is not known. The reviewer has inferred, based on recent fishflesh tainting studies conducted with 2,4-D DMA, that currentlymarked 2,4-D DMA formulations contain 2,4-dichlorophenol (2,4-- DCP) in quantities sufficient to cause taste or odor problems in treated waters for an indefinite, although probably limited (days to weeks), period of time. No evidence was found that 2,4-D contains TCDD, the problem contaminant of 2,4,5-T. 5. What techniques do aquatic scientists use to assess the Hazards associated with 2,4-D salts and are these tech niques sufficiently advanced to fully evaluate the potential risks involved? The first tests a chemical such as 2,4-D receives during a testing program are aimed at determining its acute, or short, term, toxicity to several "representative" aquatic life forms. Standardized procedures are available for tests with algae, aquatic invertebrates (bugs), and fish. The test results are expressed in terms of a time-specific LC50 value (e.g. 96 hr LC50), a designation indicating the chemical concentration lethal to 50% of the test organisms in that amount of time. A relatively large number of such tests have been performed with 2,4-D. These tests are not particularly informative except as an index of relative toxicity suitable for a variety of comparative purposes. The next tests often done are subacute exposures in which the concentrations of chemical at which the organisms will survive indefinitely (although the tests are typically only 30-60 days) are estimated. Behavioral, physiological, pathological, or chemical residue tests may be included at this level. In algal tests, oxygen evolution or cell division rate may be measured. If during these tests the chemical exhibits unusual toxicity, or if the chemical is one which will be widely released into the environment, and if sufficient money is available, the full lifecycle chronic toxicity of the chemical may be determined for i - 9 0 - 0004198 j 7963 DOW V49294 select fish.and aquatic invertebrate species. Such technique are currently available only for a handful of fish and invertebrate species. Laboratory chronic tests are often foregone in favor of an application of the chemical at the anticipated usage-rate to one or more outdoor ponds containing "semi-natural" aquatic communities. Such ponds are not totally adequate models of the "real" envi ronment since they lack complexity but they may, in fact, be more sensitive to chemical effects since 1) lack of complexity leads to loss of "stability" and chemical effects may, therefore be more obvious, than in nature and 2) the entire system, rather than just a limited area, receives chemical exposure. However, inter-pond variability in test conditions and results in even between closely-situated ponds, is often a problem with which the investigator must contend. In addition to biological tests, the researcher examines the chemical properties of the test chemical. Water solubility, vapor pressure, and selected partition coefficients often give important clues as to the distribution and fate of the chemical within the environment. Select chemical tests may be performed. These then, are the major techniques presently used to evaluate chemicals prior to their entry into the aquatic environment. Even should a chemical pass through the entire testing protocol and be characterized as environmentally "safe", one cannot help concluding that such a judgement shows extreme optimism on the part of the evaluators. Would the data collected by the procedures outlined above serve as adequate protection for the thousands of fish, aquatic invertebrates, algae and microorganisms that could ultimately be exposed to the chemical through its intended, or sometimes unintended, use? Obviously the answer is "no", and yet we implicitly say "yes" every day as more and more chemicals come on the market. This is a fact of life in modern society but the risks seem managable if we are wise in our use of "approved" chemicals. At this time, the wisest approach seems to be the use of those chemicals that do their job rather selectively and then disappear rapidly. If we compromise and select those chemicals whose properties fall short of these ideals, then the risks should be publicly acknowledged and accepted, which is what this review is all about. 6. What are the chances that 2,4-D salts will harm fish populations in northwest waters? Based on the relevant literature, it is the reviewer's opinion that the risks are low, that 2,4-D salts will cause direct acute or chronic toxicity in northwest fish populations. However, this conclusion involves an extrapolation of data from a few fish shecies ]Lo yet untested species, or of data obtained from ester tests to 2,4-D salt tests, which the reviewer is willing to make eGTi'OOO but that the reader may chose not to assume. For example, the literature reveals that only 13 out of the approximately 36 fish species in Lake Washington have received any type of 2,4-D testing (acute, chronic, field, salt, or ester) and that the chronic toxicity of the 2,4-D salts to any salmonid has never been determined, although limited data collected with ester formulations are available. There is a low risk that fish could be harmed by certain stimu latory effects of 2,4-D salts. There is a high risk that fish populations would at least be altered in composition due to either the indirect effects of 2,4-D, or by the loss of habitat caused by plant removal. The occurrence of such effects would be quite dependent on the total area of plant treatment. There is a high risk that the flesh of fish from the treatment areas would be tainted (flavor impaired) by 2,4-D salt treatments for at least one week after exposure. This perhaps is of more implication to the fisherman than the fish. 7. What are the chances that 2,4-D salts will harm the food webs upon which fish populations depend? The reviewer believes the -literature indicates at least a moderate risk exists that 2,4-D salts may be directly detrimental to the aquatic food chain. This risk is based more on data inadequacies, however, than positive indications of food chain damage. Limited data is available in the effects of 2,4-D salts on aquatic bacteria and fungi, algae, and invertebrates (acute toxicity data only), each isolated under laboratory conditions, but the signifi cance of these findings under complex and inter-related field conditions is not known. The phytoplankton and invertebrate components of the aquatic food chain have been monitored a number of times following past aquatic plant control programs. This body of literature reveals that although the common sense observation can be made that aquatic food chains do not collapse following 2,4-D treatments, there is, in fact, insufficient data to insure that food chains have not undergone subtle alterations due to the direct actions of 2,4-D. The literature reveals that toxicity information of any kind (acute, chronic, field, ester, or salt) is available-only for 6 out of the minimum of 169 species of food chain organisms found in Lake Washington. Food-chain alterations are an almost certainty due to the indirect effects of 2,4-D or to the subsequent alteration in habitat. The exact nature of the alterations, and the subsequent effects on 0004^00 q -92- 0 vv 7 4 9 2 9 6 fish populations, cannot be predicted since the controlling factors, and the ways in which these could be modified due to 2,4-D, are too complex to be subjected to analysis. 8. Will the use of 2,4-D salts result in the accumulation of 2,4-D in the food chain in a manner similar to DDT? No. The literature uniformly documents the absence of 2,4-D accumulation in aquatic food chains. However, there are unresolved questions regarding the length of 2,4-D persistence in some components of the aquatic food chain. The following possibilities have to be acknowledged: 1) The presence of 2,4-D (low ppb) in water for up to six months, possibly longer. 2) The presence of 2,4-D (low ppb) in bottom muds for up to three months. 3) The presence of 2,4-D (mid ppb) in planktonic and benthic invertebrates for three to six months. 4) The presence of 2,4-D or 2,4-DCP (low ppb) in fish for two to four weeks. The biological implications of such low-level 2,4-D residues are not known. 9. Are 2,4-D breakdown products harmful to the aquatic en vironment? Many intermediate breakdown products of 2,4-D have been identified. With the exception of 2,4-DCP, which can be one of the first breakdown products of 2,4-D and may contribute to the tainting problem mentioned earlier, the literature presently suggests these metabolities do not constitute appreciable toxicological risks to aquatic organisms. 10. Will' 2,4-D combine with pollutant chemicals in the water to harm aquatic life"? The answer to this question is not known. The literature contains no environmentally-significant evidence that 2,4-D has combined with other chemicals to bring about observable toxicity in non target organisms. However, the possibility that this could happen is everpresent, and an unquantifiable degree of risk must therefore be assumed. 11. 2 , 4-D salts have been primarily used in parts of the country other than the northwest. Are the results of the scientific studies conducted in those areas valid for the northwest? 0004S01 i -93- 7866 No. Although the general trends provided by the data of these studies are probably valid for the northwest, the literature reveals a less than adequate match-up in biological species between the northwest and other areas. The ongoing studies with 2,4-D by the Canadians in British Columbia may help close this gap but additional small-scale experiences with 2,4-D would be needed prior to more widespread use in the northwest. 12. Is 2,4-D salt the herbicide-of-choice for aquatic plant control in the northwest? No. In the reviewer's opinion, the non-chlorinated herbicide endothall is a better a priori choice for future investigations into chemical methods of plant control in the northwest. 13. Briefly stated, then, what are the risks associated with the use of 2,4-D salts in northwest waters in the absence of additional testing? 1. A low risk that 2,4-D will directly harm fish populations. 2. A very low risk that 2,4-D will harm fish through stimulatory effects. 3. A high risk that the flesh of fish in the treatment area will be temporarily tainted. 4. A high risk that the fish species composition in the treatment will change through indirect effects, or habitat loss. 5. A moderate risk that 2,4-D will directly harm the aquatic food chain. 6. A high risk that aquatic food chains will be altered through the indirect effects of 2,4-D or habitat changes. 7. A very low risk that 2,4-D breakdown products will harm aquatic life. 8. A very low risk that 2,4-D will combine with pollutant chemicals to induce harm in aquatic life. r* 9. Noncharacterizable risks associated with the presence of impurities in the commercial formulation of 2,4-D and with the persistence of 2,4-D in the environment at low levels. 10. In addition, the fact that most of the species in northwest waters have net been involved in past 2,4-D evaluation tests must be acknowledged. 0004202 -94- TABLE OF CONTENTS P6Z6W VK SUBJECT PAGE Title Page 88- Summary of Findings on 2,4-D 89 Table of Contents 95 Introduction 97 Toxicity of 2,4-D to Aquatic Organisms 102 Phytoplankton Conclusions 102 102 Invertebrates and Amphibians Conclusions Zooplankton Benthos Field Studies Amphibians 106 106 107 107 108 110 Fish Conclusions 2 f4-D Acid or Sodium Salt 2.4- D DMA 2.4- D BEE Water Quality *.nd 2,4-D Toxicity to Fish 110 110 112 114 116 119 Waterfowl Conclusions 120 120 Nontarget Aquatic Plants 121 Additional .Nonquantifiable or Potential Effects of 2,4-D on the AquaticSystem 121 Potential Ecosystem Stimulation by2,4-D 122 Effects of 2,4-D on Aquatic Bacteria and Fungi 122 Tainting of Water and Fish-Flesh by2,4-D 123 Avoida'nce of 2,4-D by Aquatic Organisms 124 Acquired Resistance to 2,4-D 125 0004S03 7368 D O W 7492yy SUBJECT Synergistic Toxicities of 2,4-D with Other Chemicals Concern Regarding Carcinogenicity of 2,4-D to Aquatic Organisms Indirect Effects of 2,4-D Use Species Comparisons Between Literature Toxicity Data and Northwest Waters Relationship Between 2,4-D Concentrations Known to be Toxic to Aquatic Organisms and Those Likely to Occur During Herbicide Treatments Persistence Conclusions Persistence in Water Persistence in Hydrosol (Sediment) Persistence in Invertebrates Persistence in Fish Persistence in Plants 2,4-D Breakdown Products of Metabolites Persistence of Metabolites or Breakdown Products Acute and Chronic Effects of 2,4-D Metabolites and Breakdown Products Purity of Technical 2,4-D Formulations Effects and Persistence of 2,4-D Impurities Possible Accidents During Herbicide Treatment Literature Cited Appendix A The Questions Upon Which the Union Bay Study Herbicide Literature Search is Based r*Appendix B Species List for Lake Washington, Exclusive of Vascular Aquatic Plants, Waterfowl, and Mammals - -96- PAGE 126 126 127 127 128 131 131 135 140 142 143 146 146 147 146 148 149 150 152 170 172 C004S04 INTRODUCTION O O f G f ' ', This review provides an assessment of the potential ecological implications attending use of the chlorophenoxy herbicide 2,4-D for aquatic plant control in northwest waters. In particular, this report responds to a series of questions about 2,4-D (Appendix A) developed toy the Union Bay Study Technical Review Committee during discussions on possible control strategies for the aquatic plant Myriophyllum spicatum in Lake Washington. Only aquatic ecological effects are considered here; potential human health implications are discussed in a companion report by Shearer (1979). The use of 2,4-D, both for aquatic and terrestrial plant control',' has become controversial in recent years due to the apprehensions of some scientists and citizens (see Warnock and Lewis, 1978) regarding the possible consequences to human and environmental health of long term low, and sometimes periodically high, doses of 2,4-D. This con cern intitially focused on the 2,4-D horaologue 2,4,5-T, for which U.S. registration was recently suspended by the U.S. Environmental Protection Agency (EPA), but has now increasingly shifted to 2,4-D based on the continued widespread use of this herbicide in agri- and silviculture, urban horticulture, and, to a lesser extent, aquatic plant control. In its chemical structure (Fig. 1), 2,4-D shares the chlorinated aromatic moiety common to many of the toxic chemicals recognized to be persistent in the environment, such.as DDT, dieldrin, toxaphene, polychlorinated biphenyls (PCB), pentachlorophenols (PC? and dioxins. Unlike these more highly chlorinated compounds, however, the 2,4-D molecule contains only two chlorine atoms,per substituted aromatic nucleus, a structure more susceptible to breakdown by biolo gical organisms and thus less likely to be persistent in the environ ment. In this labile structure, together with an exceptionally large body of broadly-based research worldwide (see review by the National Research Council of Canada, 1978), is the basis-for past and con tinuing statements regarding the general "safety" of 2,4-D and, hence, its widespread use. For example, in a 1972 report on the degradation of synthetic organic molecules in the biosphere prepared by the National Academy of sciences, Dr. Stanley Dagley wrote of 2,4-D "Being wise after the event, we can say that 2,4-D is just the sort of her bicide we would have designed, had we been guided by our knowledge of microbial metabolism." (p. 345). However, with growth and development in (1) the ability of chemists to detect ever smaller amounts of her bicide residues in the environment; (2) the overall knowledge of biologists regarding the structure and function of biological and eco logical systems; and (3) the environmental awareness of the general public, the meaning of the "safety" of widely distributed synthetic chemicals has become ill-defined (see How Safe is Safe? by the National Academy of Sciences, 1974) and the willingness of the public to assume "safety" as determined by public or private scientists has somewhat diminished. Moreover, such re-evaluations and incredulities ;eera justified. It is perhaps frustrating but true that regardless of the specific chemical of concern and the amount of research available on that chemical, particularly if the chemical is a synthetic 7870 -97- CCG'iSOS DOW 749301 Name: 2,4-D acid Molecular weight: 221 grams/mole Water Solubility: 900 mg/1 (ppm) pKa: '"'-'2.9 ' Name: 2,4-D Dimethylamine (DMA) Molecular weight: 266.1 grams/mole Water solubility: 3000 grams/liter o - ch2cooch2ch2och2ch2ch2ch3 Cl Name: 2,4-D Butoxyethanol ester (BEE) Molecular weight: 321.2 grams/mole Water solubility: 12 ppm Figure 1. Common forms of 2,4-dichlorophenoxyacetic acid and some chemical properties (from the Herbicide Handbook of the Week Science Society of America, 1975, or Zepp et al. 1975) 000406 -98- kh r > n "pollutant," knowledge and methodology are insufficiently advanced to determine at what concentration that substance will ultimately prove "harmless" to the total environment. In consequence, for the fore seeable future, risk must attend the intentional or unintentional application of. any synthetic chemical to the environment. At best, the results of research and subsequent regulation on any specific** chemical can serve only to prevent most of the gross, and some of the more subtle chronic, effects on a limited number of life form's. This perspective should be maintained when evaluating the following infor mation on 2,4-D in the aquatic environment (also see Hirst and Bank, 1971) In the past, the aquatic effects of chemicals have been assessed under a host of different techniques and conditions, as will be evi dent in this report for the specific case of 2,4-D. -Such diversity in experimental approach, although considerably less diverse than nature itself, has historically yielded a fragmentary array of data which, when assembled for use by a regulatory agency, has resulted in water quality standards or decision-making policies, that have unfortunately been based on non-uniform, and sometimes poorly rationalized, criteria (see Sprague, 1976). In an attempt to improve upon this situation, the field of aquatic toxicology in liaison with the Environmental Protection Agency has over the last five to ten years been actively involved in the development of progressively standardized test protocols for use in the assessment of chemical hazards to the aquatic environment. For example, algal tests have recently been standardized around a single species, Selenastrum capricornutum (Miller, et al. 1978). Fish and invertebrate tests have been .standardized around a few select species for several years (EPA, 1978a, 1975) with daphnia and the fathead min now likely to remain the most commonly tested representatives of each group for some time to come (Maki, 1979). Standardized model eco- systems, such as those developed by Metcalf and co-workers (1971) or Taub (1969) will' probably be used extensively in the future for hazard prediction (Witherspoon, et al. 1976). The primary objective of these development efforts has been the provision of a systematic framework of data collection which will permit the rational entry of new chemi cals into the environment as well as the elimination of some of the old. On the one hand, there is no question that these standar dizations represent progress towards a vitally needed data base for use in aquatic toxicology and the water quality decision-making pro cess. On the other hand, there is a potential danger in that if these protocols serve to concentrate relatively scarce research monies on a few selected approaches, the possibility exists that some of the more sensitive components of the environment could be systematically ignored during the chemical screening process, thus actually de creasing overall environmental protection. If without other merit, the piecemeal approach of the past was, on occasion, able to uncover some rather curious effects. Hopefully, however, at least two factors will work to prevent future oversights. First, those developing stan dard methods, as well as aquatic toxicologists as a whole, are aware _QQ_ that the number of species now available for testing in the laboratory is inadequate and in need of expansion (Buikema and Benfield, 1979; Sprague# 1976; EPA# 1975). Standardization around the presently used species has only come about due to successful past adaptations of these particular animals to laboratory conditions. Additional species c a n be incorporated into test protocols as the appropriate rearing information becomes available. The same is true for completely novel test approaches. Secondly# the ultimate test in most existing or pro posed chemical development protocols in one or more field evaluations in the geographic area of interest (EPA# 1978b; Cairns# et al.# 1978; Committee on Water Criteria# 1973; Sprague# 1971). Properly conducted field tests have historically provided the most integrative, and pro bably the best# approach to the determination of the environmental significance of a chemical. Given# then# the combination of a battery o standardized laboratory tests and final field trials# the develop m e n t of chemicals such as herbicides# as now being pursued# seems m a r k e d l y superior to the procedures of the past (an account of the development of aquatic plant control chemicals as of 30 years ago is provided in Goudey (1946)# a report which if nothing else# attests to the true resiliency of the aquatic environment). If initially more expensive than past procedures# the long-term costs of present deve lopmental methodologies# whether to industry or to society as a whole# ar e likely to be considerably lower if new chemicals are in fact more environmentally acceptable and thereby less subject to public controversy. The following review is concerned with identifying the aquatic risk potential associated with 2,4-D specifically. No information on the chlorophenoxy herbicides in general# or 2#4#5-T and dioxins in particular# is included (but these are covered in the recent review by the National Research Council of Canada, 1978). Similarly# only the 2. 4 - D formulations likely to be applied to# or used to assess the effects on, aquatic systems are considered, these being either pure 2 . 4 - D acid# the sodium salt# the dimethylamine salt (hereafter referred to as DMA), or the butoxyethanol ester (hgereafter referred to as B E E ) . The chemical structures of these forms are shown in Figure 1. Some data derived from different 2#4-D formulations have b e e n included in several sections if particularly relevant to the discussion. Likewise# monitoring studies documenting the presence of 2 . 4 - D residues in flowing waters have been included despite lack of knowledge regarding the source and original formulation of the 2#4-D. T h i s review is also almost completely concerned with the freshwater environment although some studies with anadromous# and a few with marine or estuarine, species have been cited. The international literature has been included to the extent allowed by the constraints of time and translation. t Others have reviewed 2#4-D in the aquatic environment including the National Research Council of Canada (1978), Gangstad (1978)#. Warnock and Lewis (1978)# Dost (1978)# Redden (1978), Brown (1978)# , 1 I 1 r 1 t , ' i -1 0 0 - O Miller (1977), Morley and Reid (1977) , Council for Agriculture S c i e n c e ^ and Technology (1975), Allebone, et al. (1975), Schultz and Harmon (1974), Pimentel (1971) and Daly (1971). 749304 Finally, the concluding introductory remarks of Dr. Stuart Hurlbert in his review on the secondary effects of pesticides on -* aquatic ecosystems seem particularly worth of repetition here: In this review, the writer has generally refrained from criti cizing the designs, or lack thereof, of the studies cited and the consequent statistical weakness of their conclusions; it is enough to say that the majority of the studies are defective in these respects. If a cited result is of great importance or interest to the reader, he will have to consult the original article to assess its validity. Despite this unfortunate but hardly unique state of affairs, a review of this literature should be .useful since biologists can often extract useful infor mation from data that high-church statisticians would regard as hopeless, biology having advanced considerably before statistics emerged as its auxiliary discipline. 1 Nearly all of ti\e studies cited in this review have been collected, and are on file at the Metro library. -101- *1 t TOXICITY OF 2,4-D TO AQUATIC ORGANISMS 3YTOPLANKTON inclusions: t^baub . Under optimized laboratory conditions, 2,4-D acid or sodium salt produces moderate to low toxicity (>50 ppm) in many algal spe cies. BEE has been tested less often but may be relatively more toxic (4 p p m ) . DMA has not been tested in the laboratory. Non-toxic concentrations of 2,4-D directly stimulate the growth of several algal species in t h e .laboratory. Under field conditions, the effects of various 2,4-D formulations on algae have not been well documented. The available evidence, including the not unusual presence of algal blooms (whether of direct or indirect origin) following 2,4-D applications, suggests permanent toxic effects, to at least some algal species, do not occur. , Laboratory evidence suggests algae do not concentrate 2,4-D anion through sorption mechanisms. Similar tests with BEE, which might be expected to show positive results, have not been conducted. Meager evidence suggests algae can metabolize 2,4-D but this area has received little attention. Meaninglful. assessments of the effects of chemicals on algal rowth and production cure made with difficulty since upon chemical sposure thse functions may be either stimulated or inhibited, and, : inhibited, the effect may be either algicidal (cell death) or Igistatic (cessation of cell division). (Miller, et al., 1978). Lth 2,4-D, algal toxicity has primarily been examined in the laboraory, although some field studies have included qualitative attempts 3 determine effects on natural populations. Under conditions of laboratory bioassay, algal survival, growth, ad production seem generally tolerant to moderately high con- antrations of 2,4-D acid or sodium salt (Table 1). In general, the aitiation of toxic effects requires exposure to more than 50 ppm 2,4acid or sodium salt, in most of the species or strains tested (Das id Singh, 1977; Singh, 1974; Morre, 1974; Voight and Lunch, 1974; ertagnolli and Nadakavukaren, 1974; Poorman, 1973; Venkataraman and ajyalakshmi, 1972; Bingham, 1972; and Vance and Smith, 1969). More usceptible species do exist however, as seen with Nosto sp. by ankataraman and Rajyalakshmi (1972) and Scenedesmus sp. by Bingham ; j.972) and Stadnyk, et al. (1971) , where at least temporary effects ar*- apparent at 1 to 25 ppm 2,4-D. That an algae (Euglena g r a c i l i s ) a ie to recover from 2,4-D induced cell pathology once herbicide posure is discontinued was shown by Poorman (1973). 0004210 1 7S 76 Direct stimulation of algal growth by non-toxic amounts of 2,4-D, which could be considered an undesirable effect under certain con ditions, has been shown in at least three species (Das and Singh, 1977; Bertagnolli and Nadakavukaren, 1974? and Poorman, 1973). Whether such stimulation would occur at 2,4-D levels likely to result from actual herbicide treatments (see later section) is questionable. Bertagnolli and Nadakavukaren (1974) measured stimulation in Chlorella pyrenoidosa at about 122 ppm but not at lower concentrations. In the other two studies, the growth of Anabaenopsis raciborskii and Euglena gracilis was stimulated at the lowest concentration tested (10 ppm in each case), so the potential for stimulating effects at yet lower 2,4-D concentrations remains. Only a few laboratory tests have been done with 2,4-D BEE, but algae appear to be more susceptible to this formulation than to the acid or sodium salt. Using 36 algal isolates, Butler, et al. (1975) noted mild growth inhibition at 4.0 ppm BEE, although 0.001 to 1.0 ppm produced no measurable effects. Interspecific patterns of suscep tibility were not determined. These authors cited two earlier reports on BEE done with saltwater algae (Butler, 1965 and Walsh, 1972) in support of their own (somewhat optimistic) conclusion that the available data suggest "the use of butoxy ethanol ester of 2,4-D in nature will not cause any large decrease in algal growth." Butler (1977) recently presented a thorough, non-critical review of pesticide effects on algae. The herbicide section of this review includes most of the papers.in Table 1 of this report, plus a number of additional papers not found in Table 1 since they were published in a foreign language or otherwise generally unavailable. However, on the basis of the summaries provided by Butler, none of these papers present data inconsistent with the trends evident from the work included in Table 1. As seen in Table 1, laboratoryalgal assays vary in detail and length but overall represent a rather homogeneous approach to hazard assessment since the assays are uniformly conducted under optimal con ditions of nutrients, light, and temperature. To what degree are results determined under these conditions applicable to natural systems of variable and sub-optimal characteristics? This question cannot be answered at the present time but is being actively researched as demonstrated by a recent symposium (International Association for Theoretical and Applied Limnology, 1978). Miller, et al. (1978) warned that "caution should be observed in interpreting results" of algal assays in which growth has been inhibited and Rubinstein, et al. (1975), after a review of algal test methodology, cbncluded that "acute phytotoxicity tests, using algae, can be completed in relative short time periods, ranging from a few hours to a few days. Methods of determining chronic effects are, however, poorly developed for general application and the interpretation of results is, at this time, questionable." That 2,4-D is generally of low laboratory toxicity to single algal species suggests that the acute hazards of 2,4-D to algae are minimal. However, it should be 0004^1^ 1 T a b la 1. S u m m a rie s o f a tu d ia a on th a f f a c t 'o f 2 ,4 -D on p h y to p la n k to n . im m iii________ * S co tt a t 1. 1910 **- r<Mi.t<> OKA o b ln a o n * na c it a d by i i> * <11 i id SHA lin ib y at *1. 1177 tw M H tm -----------------H U ll MO M 3 M M *v o U v a t i' De M d l l a f h 1977 MA. a c t iv a l c td la a t 14*C C b v ll aodluo v it o tro c o a la o a a ta ocha and M u tila r Orookar l i a b i and C o la c id y a V o l.hc |4 lyn ck im m 197 1971 OKAi d a la f o * lo o apo lla d M I 99 a c tiv a 1971 oes 1971 Ac Id 197 A<d M C M 3C 3C ll C |4*C *COoaooatv H 7*0 7 *lla U 4 ( 4 a .il ( , ea a .ta l M li u i M il r a - 1 4 l. t a a m 4 1 i H K l d a te odlu 'yoad ootoc* r la to l'a o o lo tio * * - 1 1 ediica M orra* 41*9* l a r t a ^ n o l l l nd Madakaukran tooraan b b ltM y at a i. V nnkatactaan and ajyalabafuai 197 1974 1974 * H | M * M tl(* H IM Ih I O odiu, 71 a c tiv o iH tM Ia a l OOdltM 31C )1C )0C 'ir a U a a d iu t* Cbo-19 o o d ita i O old'a Oacal a 4 iic 197) M I . )0 t a c tiv a W lM Iu l 197) 1973 U l i 301 a c t iv o *9 ta d la t A id 31C O ifo o Buriana aadiua > < -)% )0C C u rrlto c A Bound* docto C a ro lin a d itro o o ft-fro tu e rta n ! a o lo tlo n co CO S> DOW *749307 iim matti Hnasal I l M t4 ( fo 4 li 4 U IN w ild t r f * P w , 4 IM t 4 w ild 1 f f * 4 *!# h i l l lt 4 v M 1*4-# U M U M t *1 H IM . iilllt l ItH lH 9 C M M tlU tp 1444 l 4 I4 m ( t o d . f la b p ra e a a t f l U l H v lU 1 |p * 1,4-0 la d M n v - - )4 b M i pbataaydfchaale *4 p ir a t ic a u U * m ( i m #4 la a i i l t l o a . Itp a a a ia aa#a. wafa I I I I I * 4 4 ppd 1 .4 -0 . C d lM illt h i i m U m i Cdlaraoaecua ap. Lpaabya ap. daadaaaa v a r ia b ili ia d aaaav l i ia y a l u t * 4 14-1144 ppa 1 ,4 -0 . daadaaaapaia a lb a re lli O aaptiaa tu fy la le b a ce ia cy. l l< a M a aa a c. I I ppd 1 ,4 - 0 . O a td le lia a p ca ya i aa w ater a i alraaadaaha. C d ia ro p b y ll a aaaewcoi, alp fw a lU e tIv a ly aaapl#4 td ca w fb a vt M y . dad aaaay - 14 4 y . 4 .441 - 4 ppa 1 .4 -0 la a ta i. Bapaaai a lfa la 4.41 ppa 1 .4 -0 i t Iv a c a la Idea a a a lv a a i la 1 .4 -0 laaa la Ida a a ila a . e c c i t a l a u k a a aa l i i l . t a d aaaay 1-14 ia y a t a a t a i l i t e i 114 ppa 9 paci a f d a a id la ila ia a a faw ai 14 a ic a ia c a f V fa If 4 a ira la c a f d ita a c d la ap. iaa P a iIa a tru e #p. O iaaaiaaava ap. Aakl#lra4#aw a #p. lim i 1 ppa ONA I k i m i H a l f a l p c e iu a t ia , I ppo 414 M t , Water i f f ( M *4t4. A lfa l (oath i t l M l i t M lik M a l l f a i l M U llty Moi4 baevy i l | t l d lo fl* la t M k 4u i ( m i | r l * i ( . K M f f M i a i l a m m I p o o l, do f f a c t a m i m 4 m m | a f Id a I n i t 4 fu a a tla a a . I t ppa - a l a l ravtb a tla u la tia l i t ppa aa a f la a i da flo w 'd r a ta d a l Oaataaaa la c a ll auadaf 444 M - la d ld llla a a f a itra p a a f la e t io a 144# ppa - a a a p la ta a v id la d ld l ll a a O a ta c a la a i aa *a a c a a v la tla a fa c ia * a f 1. 1. M M llU r lu a la 4-4 dawa. T d la was aa*a l4 c a la da * l v . * C b lo ra o d y ll a d lfd a la ic a a ta a a l ate Idaa a a a tc a l # tr a * . P a a a ld la o tla u la tla a a . O laloa ia c a it y vaa d lfd la K a a la a a l c i a a . 4.441 - 1.4 ppa aa a ffa c i# aa pcawtb 4*4 p p a i 14 la c # # l a awad a f a w liw a a ll# d o w la f paa fc a w td . d a p a M la f aa Ida a lf a l tra la J4 -4 M a f Ida 1 .4-0 m i ap p a ra a tly a a i d a ll# 4 la aaa -2,4-4 aoapowai# la 1 weak#. Ocawid i r a a t lc a lly a t d atd tc a ita a a t la v a la la a l l j p a ci. , ta d aaaayi 1-1# ia y a la a f a la a 14C -2.4-0 a p ia d a by a a l l# a a a a r# i. T a a ta i 9 t a I 444 OP* l#4-0 ta d a a a ayi 1 -4 vaake. T a a ta i wp l a 114 ppo 1 .4 - 0 ta d aaaayi 4-1 4 4y. TaalaO 144-1144 ppa 1 .4 - 0 t a d a a a ayi 14 doura le a p . A i i i t l o c a l dlaa d a a lo a l a a i ra a a ira tla n ta a ta ia a lu iv i l a a t a i 1.4 4 ppd l a 1144 ppa. t a aaaayi 1 ia ya la a f. T a a ta i l- l# # m 1,4-0 f i a l i a d a arva clla aa. la y aaa ayi I t ia y a la a f T ta 4 | t a 144 fpa 1,4-0 C a ala lw a a ia apay 4fa a i a a a a c w a la a ld a fla w a ta a a y a tla a lia la ia rra lia m i c m c i m 19 fcaadw ala a i a a r Ia a 4d a l la a t a i, C y liavaporava ap. O d ia ta li pyraaaldo gw fla p r a c llla "P ia c ile * 21 a t r a ia a o f d iu o -fra a a a lfa C. alcrop aw a i O aoi rowed t 1-14 ppa. aaaa a t 44-144 ppa. a ia a id a r la u ti Pale aa atd a t 14-44 ppa. paa a t 41-14 ppa. A. a iiu la M i O a a i f ia w id a t 14-114 ppa paa a t 144-144 ppa. a a rc e # *a i O o o i owed #1 14-144 p p a . poor I 444-1444 ppa. O p ta la a f 14C -1.4-0 fa v a i ta da ` M i * 141 r a iw c lla a ia r e a liv e a d a a rv a i f l a i a a id f a lfa l a a lla w ild v a ia r. da f f a a t a aa a w c v lv a l ac f r a v t b 1 a a a c a a ira ila a a up la 144 ppa. Oa a f f a a t # aa a l f a l p r e v ia a t 144-144 ppa. fra fr a a a lv a i o ra ta a t 444-1144 ppa. C a ll ilv ls la a y a c d ra a lra lla , c a ll v a lfd t, ai e v i l ila a a ia r w a a ffa c ta i a t a l l eaaeavia la v a la , la a p lc a lla a a a i p d o la a ya td a a la la d ld l t a i dova 1 ppa. da a ffa c i aa la a f ia a u tc la a i u p ta k e *1 144 ppa. do aiw araa a f fa c i aa proved a t 14*14 pp, a t i a u l a l l o a a t |4 p p a . C ra v td l a d l d l t a i by 11 a t i y 7 dp 144 ppa . C a ll p a ld a la p y a l a i 1 144 ppa d u i t u r b a i f a c a ti a ll | ia y a ia ala w a te r. da iif f a c a a c a a a a r a a u ic o f d a e d ie iia ic a a ia a a l, d a ta i oa I f p it a llo law . 4<wm l i a i t a i bava 1 pp ia 1 a tc a ia a f d oa too a * . ir o w ih l i a i t a i dova 1# pa ia I t i r a l a o f d o a io c a p .j prow td l i a i t a i aba* loo 1 17 a l f a l o t f # l a e i f io w t d l i a i t a i adov 44 pp ia 2 a lp a i a t c a lM . able 1 (Continued) Author() u Val---- tnd ftinqh-- OM 1173 1*74 3,4-0 ftinwUtia XX: Matar 31% oroUbbriaBtOolditUd iwanldiuiolutioi Siadnyk, r t 1 MajtalU, t *1 Vano and btich piare 1*73 M3 u n Ob 34-41 lb fatc*tiv--a--Uq--dU-- UC QW-10 Ndlui d tk aknM trlM U->% TV* m wvoiw IX Jttld *tacbni--l pMi* 3K M m U --dii IM I 34*3^; toil m IMO 10-17% r- co ]> DOW? 49308 1ewuLbsisssi L* MMyi H I ln| Tnti tWMMlWi 1.4- JetcUi O U tftlU n r -- M itra rbloyrtmn coi y rilU Ut VI 114% lmp I4C n l t o toga 0*0 41 teoHAa. Ml 4 . i. ivo a.- mnplmtuUomitwodioni1oo*rCt oftif anmlioirtofUlnyoUUi w>nlti<otliO I t a oooayi 4 * * lo o p IIlnofcrotrpoMUploomtUaratoPidt.taMn*a ocWIo mpiotUat m i o ta nt a. 2k l i tu*. I n oa IM I. 12 fb lo j'ilr o n w i p i i i i i I n p td rim attortalo pironiira n2ofiu.ra plora* odoro rm lw I . in c u l i v n w c * oc lim o * i t 1M Rp C- P srn ro ldC M m ia lit o t o * H o * t t ppo C- n M tfd tU ito c fc I M ia l it o H ( p i t . p u o lflro n d o p ia g li 0 p o o r oc 2S ppu O f li Ip U wfmcim o * lr I . n d r io a ta a o rtm l 2 .1 -0 , < l toU y t p ii 4 .1 . U n ta r U lu i oc II* * . aydroiylotta wmtvWrm Im A. n o tfiU o tifM --olili ym ot. UP* t % il m a lto Il M badi lo o orn il0i t atar Im an U. il-- H11 Wc <* oc ky 141 a 4 y U , 1 C m M l i U o n u oc ito c ia d . 4/1 2 p a n n o r a t pr ooowt 24 ta u ro p o o l t io t o M to t ta ta M 2 ta o . 4 /1 1 poroso eoo h ir to t, M 24 ta o . C b lo ra p ly lU y n t lr a t a lo t U ta o . bue ta d if f a m i u ta o . 14 c lA o r io r 2 n j b u t o f >p-- rlrw ta lo l^ U C ie o n o o ano o r ip i* , f t o t a i ' n t i ln r l 2 ,4 -0 no m a ta fa c to m a l o t m n ro n rn rlio o ip to Hartr y il ar a tr o t o * x * r llr if p o n d 00 tom " a ita * fe llro L a p t a n n a * b u i o n o ro * ly 1 m ta o . a y Im o boo 0 o ta p llo p r tifo c t. a adirato nta co pytyton. 00042.13 DOW 749303 - ^cognized that these same tests provide no information about multi. ^gal species interactions, algal-invertebrate interactions, and the associated area of nutrient dynamics (Dr. Freida Taub, University of Washington, personal communication) that are so important in overall aquatic productivity. ** A number of monitoring studies subsequent to field applications of 2,4-D have included at least some attempt to assess effects'on algal populations (Table 1). None of these are free from inadequacies in sampling, representative control areas, or pre-treatment natural history information but together seem to confirm the laboratory fin dings of little toxicity to most algae at the 2,4-D levels used for plant control (Brooker, 1976? Whitney, et al., 1973; Wojtalik, et al., 1971; Pierce, 1961). Several field reports document the occurrence of growth stimulation of some species, but whether through direct or indirect mechanisms is unclear. (Scott, et al., 1978? Stallings and Huckins, 1978? Robinson as cited in Capp (1978); Brooker, 1976; Paris and Lewis, 1973). Two field studies suggest temporary reductions may occur in some phytoplankton species.immediately following treatment but no sub sequent long-term effects. In Wojtalik, et al. (1971).6 of 12 algal species collected in pre-treatment tows were not present in samples taken 24 hours after 2,4-D DMA treatments but did reappear after two weeks. At the same time, 4 of 12 species were more abundant 24 hours p t treatment and at two weeks. However, due to the non-rigorous nature of the sampling and the questionable relationship between the control and treatment areas of this study, the statistical and lim nological accuracy of these findings is doubtful. Similarly, Pierce (1961) stated the phytoplankton appeared to be "sick" after a pond was treated with 2.6-3.6 ppm 2,4-D (formulation not given) but returned to health after several weeks. According to the author, the "sickness" may have been a result of 2,4-D pellets collected and temporarily stored in the same containers as the algae during transport to the laboratory, again a confounding detail A detailed quadruplicate study on the effects of 2,4-D DMA on warmwater pond limnology and geochemical cycling is presently in progreess at the Denver field station of the U.S. Fish and Wildlife Service. Only preliminary details have been released (Scott et al, 1978) but it appears that any algal effects produced by 2,4-D levels up to 2 ppm are more likely to be stimulatory than toxic. Because algae can represent a very large physical surface area within the water column, particularly under bloom conditions, several laboratory investigations have concerned the sorption potential of 2,4-D onto algae under various conditions. Voight and Lynch (1974) j found very low sorption of 2,4-D acid by Coelastrum microporum unless the pH was adjusted to 5 and then the sorption was so weak that a ten fc' reduction in residues could be produced by simply washing the tr-uted cells with water. Boehm and Mueller (1976) measured about a two fold concentration of 2,4-D (formulation not oiven) over the 11 ** I^ 0 0 0 4 ti4 H/ ppb exposure concentration by Scenedesmus acutus at pH 7, equilibiruro occurring in 4-8 hours. Valentine and Bingham (1974), who were investigating the potential use of algae for 2,4-D residue removal from water, showed that only Scenedesmus quadricaudata, of the four algal species tested, sorbed appreciable quantities of 2,4-0 (formu lation not given), but only at pH 4.7. Sorption equilibrium was reached in about one hour, as cell density and temperature dependent, and independent of 2,4-D concentration in the exposure medium between levels of 0.01 to 0.50 ppm. Taken together, these studies suggest algal sorption of 2,4-D under natural conditions is not likely to serve as a concentrating mechanism of 2,4-D residues. All this work was presumably done with 2,4-D anion; had 2,4-D BEE been tested, significant sorption of this more lipid soluble form might be antici^ pated. However, Paris, et al. (1975), in testing the sorption of a O number of pesticides on algae, bacteria, and fungi, concluded that sorption tests with BEE were not necessary due to the rapid hydrolysis Q of BEE to 2,4-D under natural conditions. The results of the field study by Wojtalik, et al. (1971) poten tially conflict with the above conclusion in that "plankton" samples collected from the Guntersville Reservoir after 2,4-D DMA treatment contained up to 3.6 ppm 2,4-D thirty days later. In fact, the authors concluded that "removal of liquid DMA 2,4-D from water by plankters that adsorb or absorb the herbicide is an important form of loss of the applied material." Their table 8 indicates 100% of the 2,4-D in surface water samples was sequestered in the plankton fraction of the sample at 24 hours post treatment and that more than 1 ppm 2,4-D was retained in the plankton for at least 3 months. These findings are difficult to interpret since "plankton" was not defined by the authors as to the relative composition of zooplankton vs. phytoplankton. Furthermore, after three months in a reservoir, the plankton popula tions present at the time of treatment would likely be gone either through natural mortality or down-current movement, or even if still present, would probably have eliminated any 2,4-D residues either through active (see below) or passive mechanisms. If so, presence of 2.4- D residues at 3 months post treatment defies explanation. Possibly the "2,4-D" represents sample contamination or an interfering residue unique to plankton. Since no other study to date confirms this finding, it would seem worthwhile for future monitoring studies to investigage 2,4-D residue persistence in phytoplankton. Two studies were found on 2,4-D metabolism by algae. Valentine and Bingham (1974) incubated Scendesmus quadricauda in radio-labelled 2.4- D for 24 hours, then used thin layer chromatography to show that two hydroxylated and two unidentified 2,4-D metabolites were formed arter one day. Butler, et al. (1975) exposed 21 algal strains to 0.01 ppm 2,4-D BEE for two weeks then analyzed for the loss of herbicides. Under these conditions, 2,4-D losses ranged from 13 to 64% depending on the particular algal species. This limited evidence suggests -algae are capable of metabolizing 2,4-D but the area is in need for further investigation. -105- 0004215 T T E G f 'lMO<:i INVERTEBRATES AND AMPHIBIANS Conclusions: 1. Under laboratory conditions, 2,4-D acid or sodium salt produces moderate to very low acute toxicity (generally > 100 ppm) to-the relatively few zooplankton representatives that have been tested.. 2. Under laboratory conditions, 2,4-D DMA and BEE produce moderate to moderately high acute toxicity (1 to 10 ppm) to at least two common zooplankters. 3. Under laboratory conditions, 2,4-D BEE produces moderately high acute toxicity (0.5 to 2 ppm) in the benthic invertebrate repre sentatives that have been tested. 2,4-D DMA produces generally' very'low toxicity ( > 100 ppm), to these same species. 4. No laboratory, total life-cycle chronic toxicity tests for inver tebrates were found but some acute tests with various 2,4-D for mulations have been done with eggs or early life stages. Only moderate toxicity ( > 10 ppm) was found in these tests. . 5. A recent test by Zimakowsky-Gnoinska (1977) showed that a sample population of aquatic sowbugs exposed to 41 ppm of 2,4-D sodium salt or more, for 24 hours were reduced through mortality by 90% over a several month period when held in clean water. This study clouds the meaning of results obtained in invertebrate acute toxicity tests. Field studies, however, though less than conclu sive, do not support the idea that similar delayed mortality occurs under natural conditions. 6. The many field studies available suggest that 2,4-D in any form does not cause direct permanent reductions in aquatic inver tebrate populations. In the majority of these studies, however, the data, as presented in published form, are too weak to permit absolute conclusions. 7. Limited evidence suggests amphibians are quite resistant to the effects of 2,4-D. Buikema and Benfield (1979) recently stated, after a thorough review of current invertebrate toxicity test methodology, that inver tebrate "toxicity research has evolved considerably in the last ten years but one cannot avoid concluding that the 'state of the art' is still in a primary stage of development" and, therefore, "Unless we pursue research in this area, we will never be able to claim that we are protecting, or will protect, the aquatic biota." As outlined by these authors, past laboratory invertebrate tests have typically bean performed without regard to the physiological, nutritional, beha ve oraly and environmental requirements of the particular test species, J thus could presumably yield misleading information. Although, as indicated earlier, it would seem that field trials subsequent to t 7982 -106- 0 G Q 4 2 ri6 laboratory tests would, at least in the case of 2,4-D, compensate for many of the compromises and shortcomings present in laboratory work, these criticisms of Buikema and Benfield remain relevant to the following material on 2,4-D, much of which was developed in the laboratory. ZooDlankton In laboratory tests, several zooplankton species have con sistently been shown to be relatively insensitive to the 2,4-D acid a nd sodium salt formulations {Table 2). Daphnia magna were not killed by 100 ppm 2,4-D in 26 hours (rosby and Tucker, 1966) or in 48 hours (Sanders, 1970). The 48 hour LC50 for Simocephalus vetulus was 310 p p m (Klekowski and Zvirgzds, 1971). Four Cyclops sp. required expo sure to at least 620 ppm sodium 2`,4-D before toxic effects were signi ficant (Wierzbicka, 1974a) and 0 to 4 hours old Cyclops vernalis nauplii, presumably a very sensitive life stage, required 37 ppm 2,4-D acid to produce a 48 hour LC50 and 8.7 ppm for a 96 hour LC50 (Robertson and Bunting, 1976). Korde et al. (1976) exposed the eggs and early instars of Daphnia magna to either 1658 or 3315 ppm sodium 2.4- D during development and found no effect on egg respiration but depressed juvenile respiration at these very high levels. When 2,4-D BEE and DMA were tested against two zooplankton spe cies, both were about equally toxic to Daphnia magna 95.6 and 4.0 ppm, respectively, in 48 hours), while BEE was slightly more toxic than DMA to the seed shrimp Cypridopsis vidua (1.8 versus 8.0 ppm in 48 -hours) (Sanders, 1970). These few data suggest both the BEE and DMA forms of 2.4- D are more acutely toxic than the acid or sodium salt to at least some invertebrate species. B e n th o s As with zooplankton, only a few benthic invertebrates have been tested in the laboratory but the results uniformly suggest that 2,4-D B E E is realtively more toxic to the Benthos than DMA (Table 2). For example, the 48 hour LC50 of DMA for scud (Gammarus fasciatus) , the aquatic sowbug, and the glass shrimp was greater than 100 ppm for each species, while it was 5.9, 3.2 and 1.4 ppm, respectively, for BEE (Sanders, 1970). Similarly, the comparative 96 hour LC50 values of D M A and BEE for Gammarus lacustris were 100 ppm and 0.44 ppm, respec tively (Sanders, 1969). Sanders and cope (1968), testing an unspecified (but presumably salt) formulation of 2,4-D and BEE, found 96 hour LC50 values for the stonefly Pteronarcys californica of 15 and 1.6 ppm, respectively. . In a saltwater study of interest, Davis and Hidu (1969) used unspecified "salt" and "ester" formulations of 2,4-D in determining 14 day LC50 levels of 64.3 and 0.74 ppm, respectively, with larval oysters (Crassostrea virqinica). Butler (1965) exposed adult oysters of this same species to 0.1 ppm BEE for one week and observed no mortality. -107- 0004217 ^ I 1 i3 li-3 I r * I US 3 . 3 I! . i i I i 5 f i I iI <t ; I] as 2 sl * iS i i ! 3 4 Z ai 5 3 ti i CM f-f(r-OO4- 1 3 J l i 35 f a 1 i 1 I Table 2 (Continued) \iLhoct>) ota dutnev, t 1. 1S71 COOU> l*?l ?,4-o ronaiiackm *r**uUr * rw . H-K^C % Matar Currituck to a d . torti Q m ilM AcU 17-20* MS & * m ard : M t Itti UMuxtad al tvli^lAi Itti Juli*. *t 1. 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U bwkiif I P in * -- - i llacsta ly aurf< 1W ` I -- n r m i l -- 4 fee-- p i-- i ri I r r t e n l i U -- 4 -- illy. 4*14-- f l -- -- <*a. 14 |ta W l-- " atmaHy" "tayfly" dui t m l u 9 m ite r 4 Urinerm tp*, 1 ( my b-- , e-- ap. c i-- p lm otte y d PaailicrU trte ria c a I --fc-olri Uripeiac I ttmaps4*1 *M9uMrI.) UaloUy--oldUl Caseari fa r 14 day, ari la r-- ri. I sa vir luios Plalri -- lo * tasplaa te ta a4 1. II. -- 12a-- t a M K t a t a -- , la b i m m ritte a n a jilta jwytolaa p ia tr lm n ilfi m yflU a lar vas ta le a afeita atItaaeap.oa1aacuvur--aad*aa.ri--aMyaal--tuoriiiwyte a44pI.t1paanybBB Plaid f f l io llo ta f 1.4 -0 tra -- oca Som -- 1*41 rpuiasaasmfaumsavIrtist aIaioi d tuaiittaa 'ioarl-- ' paorliadta a wlcypr'i* a**ifta4 0 0 0 4 2 *1 3 IO GO S> * v ii* --V cfonpa In d n t e n n post t m s -- c moapc duruvi are la p U N p o rtu l ta rn 4-- U ly n p f b i teta m a rita . *to rivaraa affacta --nari ari a y 2.4-S) la v a i. Ma d m t e y antal ahn-- a l i t i -- tatari. c ita ta is s i * -- lourd. Sfao rm piemsttaa tIaI m4mrtac U m tyK J(2m21U1 %bue se i (k u obmUm iJ. IdmJ 4dfu M j M n*um C44bchyfrW. LnOa*taMcei loh *a vunl Ub4 appwmd by i than 1)44 pp- Hmfaotacery--iv--lo--mf.roIPfal--Ulirft--ta--ta--lint,yomum. rimitpa--aaltilrlia)tnrteaa--y.t--t--aM.wtetirle--uscy--tatnleuupttonseln--<l--2a,4u-m0tUead ta-tMlc M IN n teli hours. M ten. M IM pf. I.J4 4.44 44 f11..44 ' fi*. Ms fiacca ori 104 pf 2.4*0 on pan--acia, ns otacas affacts m aoto m l--tte ttti--p i aUcv#ae. i -- braco pap-- ferita riuo ta habitat ctev-- M oyator a o r ta lity -- ari. ! d days a bue e rta --lity a o lia in a tta r affocta bKyX1 rieayoaa tatari. iala etaiari com1e1anra..vite fM tib la t-- r u y rad u n iv i noeta in ocplfteto. efface r o tt a on banthoa. -- fe b iw ia , c a ^ c ila a . ^ t a .w atraacm nt U fo m ailm atuilaM . *ajorono affaci M . .1**.441-0uuuatmt niHo ii.rfe ro y affan .-tn DOW 7 4 9 3 1 5 Sigmon (1979) reared the larvae of a midge (Chironomus sp.) through pupation and adult emergence in 1 to 3 ppm BEE/ noting some reductions in survival at each life stage. These findings seemingly could be of environmental signifiance; however/ the author concluded based on arguments of likely exposure concentrations at periods (see later section), that athe effect is not likely important in nature.* Crayfish (Orconectes nais) exposed for 48 hours to 100 ppm BEE and DMA were not affiecteci by either formulation (Sanders, 1970). The acid form of 2,4-D has not often been used for benthos testing. A single report by Sanders (1970) lists a surprisingly low 48 hour LC50 value of .3.2 ppm 2,4-D acid for Gammarus fasciatus. The significance of the above short-term toxicity tests has recently been clouded by the publication of a paper in Europe by Zimakowska-Gnoinska (1977). In this work, groups of the aquatic sowbug Asellus aquaticus were exposed to from 41 to 1830 ppm sodium 2,4-D for 24 hours, then transferred to clean water holding aquaria for 150 days. During this post-exposure period, mortality was greater than 90% in all experimental groups, while less than 10% in the control group. A prolonged period of delayed mortality was thus evi dent. Although (1) the exposure concentrations in this test were quite high; (2) the test wasdone only.with a single species; and (3) similar effects have notbeen documented in the field for other invertebrate population (as discussed below), this test must be of concern until the area is further explored. It is interesting that, in the U.S., Hansen and Kawatski (1976) recently called for a 24 hour post-exposure observation period for invertebrate tests but in light of the above results, this would seem less than adequate. Field Studies While laboratory studies suggest invertebrates could be adversely affected by 2,4-D, particularly when the treatment formulation is BEE, field trials and investigations provide little, if any, evidence that direct permanent effects to invertebrates have resulted from such treatments. This conclusion cannot be satisfactorily supported by any one particular study, since, when isolated and judged by present stan dards, most show certain deficiencies in design, scope, or reporting. However, given the absence of effects generally noted, the number of studies reported, and the time period encompassed by data collection (20 years), the assumption that any measurable direct effects on invertebrate populations would have been detected by this time seems reasonable. Two studies have directly focused on aquatic invertebrates i following treatments of their habitat with 2,4-D. In perhaps the most thorough invertebrate field study on 2,4-D available, Brooker (1974) jonitored the invertebrate populations of an English drainage, channel for six months after the application of a mixture of 2,4-D DMA and (i CGG4220 .< f| b V i< / A !U U dalapon to emergent and ditchbank vegetation. That an herbicide mixcure was used is not a complication, since no undesirable effects were noted. The control for this study was a nearby channel of similar characteristics receiving the traditional method of plant removal, manual cutting. Replicated samples of both sediment cores and pond net sweeps were collected at two week intervals in each stream. The^ 2.4- 0 treatment of the brook, which produced maximum 2,4-D concen tration in water of 29 ppb, was in May, and in June, the normal-summer booms in the invertebrate populations took place. Density fluctua tions in the five major toxnomic family groups present were considered normal. Jaccard species diversity indices'as calculated both before and after treatment were the same. The appendix of the paper, which provides an overall summary of the data, documents no change in the status of 49 species, increases in 7 species, and the serendipitous appearance of 29 species in either the control or treated stream, in the months following herbicide treatment. Marshall and Rutschky (1974) conducted a similar, but more limited, study in a cove of a small lake in Pennsylvania treated with 2,4-0 BEE granules. Twentyone invertebrate taxonomic families were sampled at 1, 3, and 5 weeks post treatment. While no change in the overall species diversity index was present at 5 weeks, the species composition had changed. Odonates and Baetids had decreased in number, possibly through emergence (although this could not be verified since the necessary pre-treatment natural history information was not available), while tendipeds and oligochaetes increased in.number. This study was omplicated by the loss of oxygen in the hypolimnion for a week or more following herbicide treatment. Thus the observed invertebrate shifts could not-be distinctly categorized as either direct or in direct effects of 2,4-D treatment. That invertebrate populations are not permanently damaged by direct toxicity due to 2,4-0 treatments has been the conclusion offered by most reports describing past large-scale plant control programs (Gangstad, 1978? Whitney et al, 1973? Wojtalik et al., 1971? Smith and Isom, 1967). While this assessment may not be inaccurate, the amount of data collected to support this contention seems, in every case, disproportionately low in relation to the total scope of the particular project. As one example, the study by Smith and Isom (1967) is the single published account of the treatment of some 8000 acres of water milfoil in TVA reservoirs with over 350,000 pounds of 2.4- D BEE. Given a project of this magnitude, it is somewhat sur prising that the report devotes only three rather general paragraphs to a discussion of invertebrate studies, these being limited to a few benthic species, and supportive data are completely absent.. While it is clear that these authors, as well as others cited above, were of the opinion that 2,4-D did not adversely influence the invertebrate community, the data as presented provide less than adequate support for this assertion. The unique reports of Pierce (1960, 1961), although written in a _ atber informal narrative style, suggest not only the effects of a scientist deeply involved in her work, but describe two of the very^ D JC ujU lA / irtv l& lx ^ rrv (jL /N d d b u /M tl/ was available prior to the addition of herbicide. Pierce had per sonally studied the treatment pond for several years prior to the 2,4D studies and thus, when after two seasons of work, the conclusion was reached (although informally documented) that 2,4-D had no effect on the benthos or zooplankton, the reader is inclined to believe that this was probably so. Q Lim (1978) reported a simulated field-use of 2,4-D B E E `"in a l a r g ^ indoor model ecosystem in which both plants and gastropods were pre sent. The snail population was apparently immediately reduced by the 2.4- D treatment but 12 months later appeared to have returned to normal. T R B t - / Ml As discussed earlier in the phytoplankton section, Wojtal'ik et al. (1971) documented the persistence of 2,4-D residues in two com ponents of the invertebrate biota in TVA reservoirs, plankton and mussels. These residues were apparently not acutely toxic but of otherwise unknown consequence. Future programs should include speci fic studies on both these invertebrate groups^ Amphibians Only two laboratory studies were located on the toxicity of 2,4-D to amphibians, both conducted with tadpoles. Sanders (1970) noted no effects of 100 ppm 2,4-D DMA to Pseudacris triseriata in 0.0008 to 50 ppm 2,4-D acid for 48 hours, than in clean water for five days. No mortality or adverse developmental effects were evident. In a third study, Lhoste and Roth (1946, as cited in Pimentel, 1971) reported that the development of frog (Rana temporaria) eggs incubated in a 0. 5. colution of 2,4-D (5000 ppm) was inhibited. In the only field studies which specifically mention frogs, Pierce (1960, 196-1) reported no effects on frog abundance as a result of partial treatment of Long Pond, New York, with "granular" 2,4-D. FISH Conclusions: 2.4- D Acid.or Sodium Salt 1. Under laboratory conditions, 2,4-D acid or sodium salt produces moderate to low toxicity ( > 50 ppm) in the warmwater fish spe cies that have been tested. 2. No chronic toxicity studies with 2,4-D acid or sodium salt were found but a number of studies have shown only moderate toxicity of these forms to the developing eggs and fry of several species of warmwater fish. 3. Under laboratory conditions, 2,4-D acid or sodium salt.produces moderate acute toxicity (10 to 50 ppm) in salmonid fry and fingerlings. ( 0004333 4. Salmonid chronic toxicity has not been determined using 2,4-D acid or sodium salt. 5. Field studies have not been conducted in the U.S. with 2,4-D acid or sodium salt. 2.4- D DMA 1. Under laboratory conditions, 2,4-D DMA produces low acute toxi city (> 100 ppm) in the warmwater fish species that have been tested. 2. Under laboratory conditions, 2,4-D DMA produces low acute toxi city to the eggs and fry of several warmwater fish species. 3. Laboratory chronic toxicity tests at DMA concentrations up to 2.0 ppm have not affected the survival, growth, or reproduction of the fathead minnow. However, several sublethal physiological parameters of the fathead minnow, i.e. metabolic rate, collagen synthesis, timing of reproduction, were altered by these same concentrations. 4. Past and ongoing field studies reveal no adverse effects of 2,4-D DMA on warmwater fish, however, additional quantitative data would be desirable. 5. Under laboratory conditions, 2,4-D DMA produces low acute toxi city ( > 100 ppm) in the three salmonid species that have been tested. 6. Under laboratory conditions, the ability of coho salmon smolts to survive saltwater challenge tests has twice been shown to be unaffected by 2,4-D DMA. * 7. No salmonid field or reproductive studies were found for 2,4-D DMA. 2.4- D BEE 1. Under laboratory conditions, 2,4-D BEE produces high acute toxi city (0.5 - 2.0 ppm) in the warmwater fish species that have been tested.. 2. Under laboratory conditions, a chronic toxicity test conducted with the fathead minnow showed 0.3 ppm BEE or less did not alter ** the survival, growth and reproduction of this species under con tinuous exposure conditions. ' 3. A field study done with PGBE, an ester 2,4-D formulation similar in toxicity to BEE, showed single applications of 1 ppm of PGBE or les produced no effects on bluegill reproduction. Single -1 1 1 - o applications of 5 to 10 prm PGBE, killed some adult fish, a e l a y e d O spawning, but did not affect number of fry produced. 4 . Despite marked laboratory toxicity, field studies with BEE have not recorded adverse effects to warmwater fish under natural con ditions. However, additional quantitative data would be desirable. 749319 5. Under laboratory conditions, 2,4-D BEE produces high acute toxi city (0.5 - 2.0 ppm) in at least four salmonid species. 6. Under laboratory conditions, the ability of sockeye salmon smolts and pink salmon fry to survive saltwater challenge tests has not been affected by a prior 24 hour exposure to 1 ppm BEE. 7. Under laboratory conditions, chronic egg and fry toxicity tests conducted with two salmonid species with two 2,4-D ester for mulations other than BEE (BE and PGBEE) indicate that water resi dues should not exceed approximately 20 to 35 ppb BE and 30 to 55 ppb PGBEE under chronic exposure conditions, or 100 ppb under single exposure conditions, if salmonids are to be protected. 8. Field studies with BEE in large coldwater lakes have not been reported in the past but are presently ongoing in the Okanogan Basin of Canada. As true for most other chemicals, considerably more information is available regarding the effects of 2,4-D on fish than for the other aquatic biota, since fish are the component of the freshwater system most frequently collected for human food and, therefore, most likely to be of concern in those aquatic chemical research programs influ enced by economic, legal, or political considerations. For coherent presentation, the effects of 2,4-D in fish have been divided into three parts based on the chemical formulation of 2,4-D used in each study, i.e., 2,4-D acid or sodium salt; 2,4-D DMA, and 2,4-D BEE. Within each section, information on warmwater species precedes that on salmonid species. 2,4-D Acid or Sodium Salt The acute toxicity of 2,4-D acid or sodium salt to warmwater fish as measured in the laboratory is moderate to low ( > 10 ppm), as seen in Table 3 (Rehwoldt et al i977? McCorkle et al, 1977; Kamler, 1974; Sergeant et al, 1971; Butler, 1965; King and Penfound, 1946, Harrison and Rees, 1946). As acute toxicity bioassays, done under static water conditions with arbitrary endpoints, these tests merely provide a gross indication of the toxicity of this form of 2,4-D relative (to other formulations or other chemicals. In the study by Rehwoldt et al (1977),<vhi,ch provides LC50 values at 24, 48, and 96 hours for seven fish species> mortality had stabilized at 96 hours in 3/7 species, appeared to be gradually decreasing in 3/7 species and was much lower at 96 hours compared to 48 hours in one species (Fundulus diaphanus). CG04S24 79Q The authors further stated that in separate tests no chronic toxicity was evident, but no details or data were offered in support of this statement. No long-term reproduction studies were found for 2,4-D acid or sodium salt but several European workers have done tests with the developing eggs and fry of three warmwater fish species. While these tests provide information about a particularly sensitive life stage, ^ they do not assess equally important questions of whether the repro; ductive physiology or behavior of adult fish are adversely affected. Kamler (1972) and Matlak (1972), in companion studies, examined the same group of carp (Cvprinus carpio) eggs reared in 0, 5, or 50 ppm solutions of 2,4-D from two hours post fertilization to day 10 of develooment. The high 2,4-D concentration induced various pathologies and metabolic effects throughout the- study and killed all of the fry at about 200 hours. The low 2,4-D concentration (5 ppm) did not pro duce pronounced adverse effects except that hatching was delayed by about 14 hours compared to the control eggs and slight reductions in metabolic rate were measured. The no-observable-effect level of 2,4-D sodium salt would therefore be somewhat below 5 ppm. Kamler et al. (1974) essentially repeated and confirmed these results but also per formed 48 hour acute toxicity tests with'eggs and larvae. In the latter tests very high 2,4-D concentrations (3200 ppm) were needed to produce effects within 48 hours, leading the authors to conclude that short term egg-fry toxicity tests were the "least sensitive of all acute tests," at least of the types used in their experiments. After exposing the eggs of the European loach (Misgurnus fossilis) to a single very high concentration (3315 ppm) of sodium 2,4-D, Klekowski et al. (1977) found some effects on oxygen consumption, in agreement with earlier findings by Korde and Zbirgzos (1971). Both of these studies were done for theoretical reasons and appear to provide no practical information except possibly as demonstration of the low acute toxicity of sodium 2,4-D when exposure period is short. Biro (1979) reared the eggs of the European bleak (Alburnus alburnus) in 25 to 3200 ppm sodium 2,4-D and found effects at all concentrations. At 300 ppm or higher, eggs were killed within 24 hours. At more than 50 ppm, malformations were seen in hatching embryos and overall mortality was approximately 75%. At the lowest 2,4-D level, 25 ppm, egg and larvae mortality were slightly higher than the controls but the larvae at hatch were normal in appearance. Following a discussion of these results and a short literature review, the author concluded that maxi mum tolerable sodium 2,4-D level during the developmental period of this species might be about 0.5 to 1.0 ppm. Salmonids also appear to be generally resistant to acute effects a moderate levels of 2,4-D acid or sodium salt although some inter specific variability is evident. The Fish-Pesticide-Research Laboratory (unpublished) reported 96 hour LC50 values of 64 and 45 ppm 2,4-D acid to Salmo clarkii and Salvelinus namavcush respectively. Meehan et al (1975).determined the 96 hour acute toxicity of 2,4-D acid to the fry and fingerlings of six salmonid species. Cono and chum salmon fry, coho fingerlings, and rainbow and Dolly Varaen trout 1 1 j t 1 ' i v -113- 0 0 0 4 2 .2 5 - D O W 7 4 9 3 2 1T ib * 3. S u a u r U t o f s t u d l a a on th* a f f a c t a o f 2,4-D on f i a h . \ IU * 4 d h aim _ P tf l)l l.t-P fagaw ddon ______________ S H U llC in * M ini H* < -- U* IIM yi Mwi>n< af a la rva# la f a ta l aka a a a ta la la f l . t - k m U i Im W I I i t )IM im ||a fraaa) im IHIt*U-*) ** iai*c i*ie Caaylata 4 a t d a d a a l * t M w *( la b l M t i t n . i m M . f f l i 1.4 "I I f l a k / l l l b ta a t. % I m I i IM N> a lly . M h u t M llv ilr a ta tw tl. !* watar a k *lla * a a* fa(f*aa4. I . l t U IM M * U tltl. Caltaa kaha waa a tli M 99a U b * a ta d a kiaaaaaya t i l l aflaaaaaat. U I ta a ta . Vaata4 M l i t l . l r t f M m 414 a M lw tla i a M lla a fa taat. MCkM Wee*#* m | Mayar l i l i l ! aa4 Iw atlaa akwaiai at a l. 111! M l *A*waklaaa* M U 9wty| aatar, la w v ty l ata. w t t f l a t t lyaal ka tyj t i U i aata 11C h i *c M il ink m t i l l M 14 aa bm M U ka!4 M -ll*C MC allvatac. p l i l . l # ka4i M l eaeeatit*e4 aata a4J*at4 ta t a d aaa f t t*4 taraaa taval taa# a ta tla klaaaaay. U llU ta /ta a k . iaa# a ta tla klaaaaaya. Alaa aaaa aaraala t a * |* a lty ta a ta MU ( la k I t m I l t | k a (a i kabaa ta M aaya fa a t katak. Alaa 414 l i M l i t a l I la l 4 aaa klaaaaay. Itallwataf i * 4.4 0wt4aa< a la a d a aaal. M M M taca. 4lala ittU tb lia t al 1 tl> M i M M t t f a r 44 4ara. 4 aaaM. a ltk M l altkaat IM A. acAl I I n a i M * l k M fa# M 1* U b . a ta tla klaaaaay. M U ta taata. I flak/taa k U b . a ta d a kiaaaaaya. Aaaa akcaaia wacfc, 4 a ta lla aat M -aaatk t i f t i w a ta | . l * l# t-# . a*yf at *1 tlakaw tal at at* Halaa |aa*ct aalyl fa ta a latkaa m 4 tack /iM yrve. ' M frr m 4 k a rk lt I t b t iiM l aalyl M il M il M il M14 MU ltwibtyai tckaatacyl MU MC U.C u*e alatlaatM n iiM ilin n U b . a tc a d a taa. I 4 t l i H a t a*faa4 i t l . l u i . i tfm ana la M laya. U b tta tla b itM ttf IM ta |M Iaawa*a4 la a a t f i 41a t la alta I a t H l n ac I . i a | ) ) U fgm M * , l a t r l i a i i t a M a lta a4. U b aaaay. C t ll M H calakaw u a a ft t n i la 14 M M 99a i I h . U b *af. b fM M ta V*a*aa. | , t - k aatat < t ila | la ta aaa al4a. C a n la tiiM t i M i t t la la k atabla taaba. ta a akcaaia m l i U y ta a t. Iw *iai# raw *4 Caffa4witlaa a a a lta c a i. ____ l i i i ln . IhyM lw i k|i U k .U h H at . ti* *4 lir u n ii1. Vty I I l ea*av*4a*v. lM<rM * * f l l i l i M llkM *ff*< Itf |IH ll* H f IkM I i t tM M iM 4 Iry I i m m ifN M * M t IM * 1M f f * va# I I i l B M lk *14 M a M U lt ln M f ir! U n i f la I U l* M |l* b , HW4U II Bm AT I*** affa#. M !<# M IM U ll | M I. t0e. 1. M|mMOiUImihUM M a *i# t | IM f tlllM I I k iN lrt Iti* ilM k ll | f f l t Mbf< M * M I M* M4 lr M ll 1. M i k l l M li la m i 1.1 pfm M la I I I m k U l M f l U k . la a tta a ia i i m la a ta , a l l a r a li* **< * I l l l M a l ! ! * l . f f * . la ta * i t a v i . u M i # * * ! Iva h I m Im U 14 M f U l l t i f a 4.1 fa . a a a t t a l l l f la ay i a tla m c m i M a i aa*aaatalaaa laaa Uaa . l U f a a w t la I m fa r 14 I m i * * * * * *+ ia t lH a t t i l l a # * U * l 4 M ae* Ila * a 4 l*a l 1 wtaafaaa M M a ia la m I I m I m . ICS * Im 1 a a a llla a ila l n i la lla r u i m i la * te la ta * tra v i t f e f l l * *4 #*! I t i l i * U t N i r l aaiat a a i aaa ialv i t a l a i 44 a, M a i a tta a ia aafaavtaa, a tta u m 14 la 11 a l M t M *aa t a l a la i ta H a t aa aaa aaa taa 14 la 11 f f * M * Im i I N f i a l i aaa la a ia l* 4 i a t a l a(ala f f l t * a a ila a **e a l4 aa fa4*a v a lle a # la aaaaa a l 144 f i fa k iU k l l i m m i I m . la i ta a * aa f * 414 aa affa aaaia la a la U y * l l l l M l f aa M I l a a l t l l y u l a* # aaaialacaa* aa B M U IU y a ata< va ti a ffa a ia aaa4. Ol co > Maia* aaa aillu # M aiala U fkM M Latala lttaaa ttaaatatiialia ul ai uitawla fy w lN atti it la ia a vMalvw t iM f* lN r**eal*a al*fuiM ( a iu ta I yai #14 I I a* 1 aa a a t t a iliy la | le tijm l . fitta# ft* M fltW M t . vaattM a C. a * t la l. (lla a ttN H itt* 144.1 1114..14 1T.| in .# iri .. 141.1 11.1 m44..1i 144.1 *4a1..1v M iH* M14..4? 44.4 M I. M*a..?r BaM a tta a ia ffa a M fa v a i la M - 4 te M m i I *m U w r t i a t m u r n m m i u i i m , a# .a V4ft*1*.*4*j*n* .y.......I..m...i.h...h akaMllaN.a la v a la m V ii m 4 larva# i f m i ara U n n i la al# ve. u aaa- f a tt a ! * * l . * t . a a a ffa a ia a t a a f l t a il a * . i f H I ara f a t i l l l a a l 4 la a e ta ia la , -# , u a a fita ila a aaa 4a#taaaa4. * alea !< **< I alea fa i 4 ** l alea M M fN lO P fH lM a lt** * * ll Uaa 4f?*4 * f f * ! M *J a *l ll* W 3 1 1 .4 -f. a lla tta a lia rv tlIfU i aa*l4la *1fa1vaetaataUlvai1M w a4tMa ll i y w i t a l l i aaa la a ra a ta i fr S ^ 3 * * u T S ."J [:` " **4 uu M ** -- !*#!!* Mlenv alla a4f 11 1 |gl | m ] , . M I ala aaa W i i i * - * * * ** i*. a l a l i . <? GGG4 2 2 & f ? J r ( 7094 fingerlings experienced no mortality at 10 ppm, or, in the trout species, at 50 ppra. Partial mortalities did occur in chum and coho salmon fry, and some groups of coho fingerlings, at 50 ppm. Sockeye salmon smolts experienced up to 20 per cent mortality at 50 ppm, but no mortality at lower concentrations. Pink salmon fry were the most sensitive salmonid group, with individual groups experiencing 0 to~*20 per cent mortality at 1 to 5 ppm 2,4-D and 0 to 100 percent mortality at 10 to 50 ppm. C izr.nbt Davis et al. (1972) conducted an n vitro study to determine whether 2,4-D acid could disrupt the activity of the rainbow trout gill APT-ase enzyme complex, but found no effect at an exposure level of about 22 ppra. No studies were found on salmonid chronic toxicity or reproduc tive system effects using sodium 2,4-D or acid. The sodium salt of 2,4-D has apparently not been widely used for plant control in the U.S., although at least one sodium 2,4-D product is marketed in the U.S., including Washington State (Aguacide Pellets, Aquacide Co., St. Paul, Minn.). No field studies of sodium 2,4-D or acid were located. Sowever, the field studies reported in the next section on 2,4-D DMA should provide equivalent information since, upon dissociation, the sodium and DMA salts of 2,4-D yield the identical 2,4-D anion. 2,4-D DMA The acute toxicity of 2,4-D DMA to warmwater fish as determined by laboratory bioassay is low (Table 3), all short-term LC50 deter minations being in excess of 100 ppm or, not uncommonly, 200 ppm (Fish-Pesticide Research Laboratory, unpublished; McCorckle, et al., 1977; Schultz, 1973; Butler, 1965; Hughes and Davis, 1965; Hughes and Davis, 1963; Davis and Hardcastle, 1959). A limited number of laboratory chronic toxicity and fish repro duction studies have also been done with DMA. Mayer and Mehrle (1974), for which only the abstract has been published, as verified by Dr. F. Mayer, Fish-Pesticide Research Laboratory, personal communi cation) exposed adult fathead minnows to continuously renewed con centrations of 0.12 to 2.0 ppm 2,4-D DMA from two months prior to spawning through reproduction. No effects on adult growth and sur vival or egg production and fry survival were found except that spawning activity, across all exposure levels, was initiated one month sooner than observed in control fish. This apparent prematurity in spawning, which corresponded to a generalized increase in metabolic rate as determined by separate radio-iodine uptake tests in the same fish, suggests that 2,4-D is physiologically active in fish, although not in a toxic manner. This is further indicated by the results of. collagen level tests performed in fathead minnows chronically exposed, to 0.2 to 2.0 ppm DMA in the laboratory (Mayer et al, 1977). After two months, although growth was not affected, bone collagen levels -114- 0 G0 & 2 9 r -pyo were significantly reduced at 1 and 2 ppm DMA. Reduced collagen in fish is of unknown biological significance at present but presumably is indicative of at least some degree of chronic stress. It should be remembered that these tests indicating sub-lethal physiological acti vity were performed under chronic exposure conditions, and similar exposure regimes under natural conditions are not likely (see section on persistence). Hiltibran (1967) exposed the eggs and fry of four common warmwater fish to 25 ppm 2,4-D DMA for eight days but noted no adverse effects. Similarly, the 96 hour LC50 of DMA to fathead minnor eggs was 1400 ppm whereas fry and fingerling values ranged from 320 to 360 ppm (Fish-Pesiticde Research Lab, unpublished). Several field studies have confirmed the low toxicity of DMA as measured in the laboratory. Wojtalik et al (1971) reported no fish mortalities following treatment of TVA reservoirs with DMA. They further stated that the treatment year, "1969, was one of the best fishing years ever for crappie, redear sunfish, bluegill, and largemouth bass in Guntersville Reservoir. . ... In addition to sport fish ing, thousands of pounds of commercial fish (channel catfish, smallmouth buffalo, carp, and drum) were harvested by commercial fishermen; herbicide treatment did not affect either their catch or market acceptance." Information on the following year's catch, that is, one reproduction cycle post treatment, would be of interest but was not provided in this study. Schultz and Whitney (1974) similarly noted no fish mortalities following treatment of a canal in the Florida Loxahatchee Wildlife Refuge with a combination of DMA and another 2,4-D salt. Schultz (1973) exposed bluegills, largemouth bass, and channel catfish in outdoor plastic pool model ecosystems to one-time doses of 0.5, 1.0, and 2.0 ppm 2,4-D DMA for 84 days and observed no adverse effects on the fish. Similarly, no fish died when Stalling and Huckings (1978) used the same protocol to study DMA dyna mics in bluegill sunfish. Schultz and Harmon (1974) although pri marily concerned with herbicide residues in fish, reported no fish mortality but successful bluegill reproduction in ponds treated with DMA. According to Scott et al (1978), bluegills in ponds treated with 2 ppm DMA showed no toxic effects but did grow faster than fish in control ponds. Whether this was believed to be an indirect or direct effect of 2,4-D was not stated. Three studies have tested the toxicity of 2,4-D DMA to salmonids. The fry of Chinook salmon and rainbow trout were both unaffected by exposure to 100 ppm DMA for 96 hours under laboratory conditions (Fish-Pesticide Research Laboratory, unpublished) while coho salmon sffiolts were not killed when exposed to DMA at concentrations up to 200 ppm for six days (Lorz, et al., in press). The latter study further showed no effect of DMA on either the gill ATP-ase enzyme complex or the ability of the -smolts to successfully pass a seawater challenge test. Likewise, Bouck and Johnson (1979) recently showed no signifi cant effects on short-term fresh- or sea-water survival of coho salmon -115- 0004250 V'-- C A. ; l V V * ! V / U / . U smolts treated with 30 ppm 2,4-D DMA for 1 hour (that DMA was the for mulation used in this test was confirmed by L. Marking, LaCrosse National Fish Research Laboratory, personal communication). No studies were found on the effects of DMA on either salmonid reproduction or on salmonids under natural conditions. 2,4-D BEE Under conditions of laboratory bioassay, 2,4-D BEE shows marked acute toxicity to warmwater fish species, being, in general, some 10 to 50 times more toxic than 2,4-D acid or sodium salt and at least 100 times more toxic than 2,4-D DMA (Table 3). As seen in Inglis and Davis (1972), Sergeant et al. (1971), Alabaster (1969), Mount and Stephan (1967), Hughes and Davis (1965), Hughes and Davis (1963), and the Fish-Pesticide Research.Laboratory (unpublished), the acute toxi city is between 1 and 5 ppm for most of the six species tested. That granular BEE is less toxic than a liquid formulation to bluegills was shown to Hughes and Davis (1965); this was apparently a function of the slower release of active ingredient from the pelleted form. Rodgers and Stalling (1972) exposed bluegills and channel catfish to a single dose of 1.0 ppm BEE for 120 hours in the laboratory and observed no mortality or histopathological effects. A complete, life-cycle laboratory chronic toxicity test on the fathead minnow was conducted with 2,4-D BEE by Mount and Stephan `(1967). Nominal BEE concentrations were set at 0.07 to 1.8 ppm, but, due to rapid hyprolysis of BEE to 2,4-D within the dilution apparatus and test aquaria, actual BEE concentrations were from 0.01 to 0.31 ppm BEE and 0.01 to 0.80 PPM 2,4-D. No adult fish mortalities, histopathologies, or effect on sexual condition were noted at any of these concentrations. Eggs and fry were also unaffected except when eggs from control fish were incubated directly under the contaminated water flowing into the highest exposure level tank, which reportedly resulted in exposure levels of about 1.5 ppm BEE. These eggs were killed within 48 hours. The authors concluded that 0.3 ppm BEE was a "safe" level in natural waters. A field study with 2,4-D involving bluegill reproduction was done by Cope et al. (1970). The 2,4-D formulation in this work was not BEE, rather 2,4-D propylene glycol butyl ester (PGBE), but the findings are reported here since both compounds are esters of very similar acute toxicity to bluegills (Hughes and Davis, 1973; Fish-Pesticide Research Laboratory, unpublished) and they provide information about the poorly known effects of 2,4-D on fish reproduction. In this study, five small ponds, each stocked with 600 bluegills, were treated with a single application of either 0.1, 0.5, 1.0, 5.0, or 10.0 ppm PGBE, 3.0 ppm being the PGBE concentration normally applied for the control of aquatic plants, according to the authors. In the ponds treated with O.l, 0.5 and 1.0 ppm PGBE, no effects on bluegill survival, growth, or reproduction resulted from the treatment, despite the presence of some histopathology in the fish dur-ing the first two weeks post treatment. f -1K_ Fish nesting began 11 days after treatment and spawning occurred over the subsequent three weeks. The number of fry produced in the treat ment ponds was reportedly the same in control ponds but no supportive data was presented. The authors further indicated that leaks in the plastic barriers separating adjacent ponds may have confounded the overall reproduction analyses. At the 5 and 10 ppm PGBE treatment levels, fish kills were noted immediately after treatment, two fish at 5 ppm, and 112 fish (20% of population) at 10 ppm. Well-described, gross histopathology was evident in moribund fish, but, in surviving fish, the lesions eventually dissipated with time. The seasonal growth of surviving fish at these higher treatment levels was actually greater than at the lower treatment levels and control. The authors attributed this to (1) increased food availability (the more complete plant kills at the higher treatment levels left less protection for aquatic insects), and (2) lower fish density, thus, less competition. Spawning was delayed by about two weeks at the higher PGBE treatment levels but did occur and egg and fry survival were apparently normal. Overall, this study suggests that even when the toxic ester formula tions of 2,4-D are misused, i.e., at three times the normal appli cation rate or five times the laboratory derived LC50 value, permanent reductions in bluegill populations are not likely to occur. Studies monitoring field applications of 2,4-D BEE have been unable to document and direct adverse effects on fish populations as a result of BEE treatments. Such studies have generally consisted of holding caged fish in the treatment areas, plus systematic or random net-capture surveys of fish population at various time periods post treatment. The reports by Smith and Isom (1967) on the TVA reser voirs, Whitney et al (1973) on Currituck Sound, Gangstad (1978) on the southern waters of the U.S. generally, Pierce (1960, 1961) in the northeast (formulation assumed to be BEE) and the British Columbia Water Investigations Branch in the northwest (Lira and Lozoway, 1978) are uniform in their appraisal of no observable direct effects on fish populations as a result of BEE treatments. The toxic potential of BEE as measured in the laboratory is apparently not realized under the BEE concentrations and environmental conditions present during actual field use. The fairly rapid hyprolysis of BEE to 2,4-D in nature is probably the key factor responsible for this generally observed lack of environmental toxicity, as will be discussed later in the section on persistence. The acute toxicity of 2,4-D BEE to salmonids in the laboratory equals or slightly exceeds the high toxicity measured in warmwater species-. Inglis and Davis (1972) determined a 96 hour LC50 for BEE liquid to rainbow trout fry of about 1.5 ppm. A very similar figure wa& determined for granular BEE with coho salmon fry by AmChem Products, Inc., as part of their confidential registration information submitted to the EPA. Rodgers and Stalling (1972) exposed rainbow trout fingerlings to single doses of 0.3 and 1.0 ppm BEE for seven days in the laboratory, without finding mortality or histopatho'logical effects. 0G04232 -117- Preliminary results from chronic toxicity studies being conducted with several salmonid species by the International Pacific Salmon Fisheries Commission of New Westminster, British Columbia were pro vided by that organization for the purpose of this review. The tests consisted of short and long term laboratory bioassays of the eggs, alevins and fry of sockeye, pink, and coho salmon, plus rainbow treut. Nominal BEE test concentrations ranged from 0.05 to 1.0 ppm, although hydrolysis reduced these levels to presently unknown levels of* BEE and 2,4-0, much as found by Mount and Stephan (1967). In seven day bioassays, 1.0 ppm BEE killed all fish of each species in each test. At 0.7 ppm BEE, the following mortality was noted: Salmonid SDecies Life Stage 1 Killed Sockeye Sockeye Coho Pink Rainbow Trout Fingerling Smolt Fry Fry Fry 32.5 0 30 ' 20 100 No fish were killed at concentrations below 0.3 ppm BEE. Long term bioassays included: Species Life Stage Bioassay Length Pink Salmon Coho Salmon Rainbow Trout Rainbow Trout Alevin to fry Egg to fry Egg to fry Alevin to fry 34 days 102 days 85 days 17 days In these tests, complete mortality occurred in all tests at 1.0 ppm and 0.7 ppm BEE. At 0.3 ppm only the rainbow trout exposed from egg to fry showed significant mortality (58%). In all tests, surviving fry at 0.3 ppm were significantly shorter than their respec tive controls, while at less* than 0.3 ppm no adverse effects were evi dent. Sockeye salmon smolts and pink salmon fry exposed to 1.0 ppm BEE for 24 hours then challenged by immersion in 28% seawater for four days showed no abnormal response. Woodward and Mayer (1978) recently reported the results of salmo nid tests conducted with several 2,4-0 esters, but not BEE, which are discussed here under the same rationale as provided for the inclusion of Cope et al (1970) in the warmwater section. In these tests, the fry and eggs of cutthroat and lake trout were exposed to the butyl (BE) , isooctyl (IOE), and propylene glycol butyl ether esters (PGBEE) of 2,4-D under a variety of water quality conditions. To determine short term toxicity, the fry of each species were bioassayed under static conditions in the laboratory, yielding 96 our LC50 values ranging from 0.49 to 1.22 ppm BE or PGBEE depending on the particular test conditions. IOE was not toxic at 60 ppm. Chronic toxicities of 3E and PGBEE were evaluated by incubating the eggs and resulting fry of each fish species in continuously-renewed 2,4-D solutions from 0004233 -1 1 8 - shortly after fertilization to 50 days post hatching. Based on a number of egg and fry growth parameters, the authors concluded that more than 24 and 33 ppb 2,4-D BE, and more than 31 and 52 ppb PGBEE, could adversely affect cutthroat and lake trout, respectively, under conditions of chronic exposure. To simulate the type of exposure which might typically occur in salmonid streams after 2,4-D use in a . forested watershed, the authors exposed just-hatched cutthroat alevins to either 0, 25, 50, 100, or 500 ppb 2,4-D BE and PGBEE for seven days under static conditions, then transferred the fry to clean, flowing water for 60 days. The highest concentration of each herbicide (500 ppb) killed all of the fry in each test but none of the lower con centrations produced mortality. The authors concluded that after a single application of BE or PGBEE to a watershed, residues in natural waters should not exceed 100 ppb if salmonids are to be protected. The many field studies cited previously in the warmwater section provide essentially no information on the field safety of BEE to salmonids. In the past, intentional applications of 2,4-D for plant control in salmonid habitats have been few, or at least have gone unreported. The more usual problem in Northwest watersheds has been the presence of 2,4-D residues in streams draining large, managed forests, a subject area beyond the scope of this review. Stream con tamination by 2,4-D has been a primary concern of Dr. Logan Norris of the U.S. Forest Service, who has discussed this problem in the litera ture (Logan 1971). However, it is obvious that forest streams differ markedly from the large, relatively cold-water lakes in which aquatic 'plants are likely to be of problem in the Northwest, and studies such as those now being conducted in the Okanogan basin of British Columbia (Lim and Lozoway, 1978) could provide much needed information, although to date no information on salmonids has been released. Water Quality and 2,4-D Toxicity to Fish Water quality encompasses many parameters, but the toxicity of chemicals to fish is most often influenced by water hardness, pH, and the inter-related factors of temperature and dissolved oxygen. With 2.4- D, at least three studies have reported that acute toxicity is not affected by water hardness (Woodward and Mayer, 1978; Inglis and Davis, 1972; Fish-Pesticide Research Laboratory, unpublished). Low hydrogen ion concentrations (below 5) could theoretically increase the toxicity of 2,4-D, since it is a weak acid and therefore subject to ionization effects (see Woodward and Mayer, 1978), but this is not likely to be of significance under normal environmental conditions. However-; pH does influence the rate of chemical hydrolysis of BEE to 2.4- D (as will be discussed later) and at higher pH BEE would be detoxified much more readily than at lower pH. Conditions of low oxy gen or high temperature would be expected to accelerate the rate of death of fish at higher toxic concentrations of 2,4-D but not necessarily change the toxic level, in other words, the fish would simply die more quickly but at the same 2,4-D concentrations (Brown, 1973). Under conditions of marginal oxygen, temperature, and 2,4-D levels, the acute or chronic toxicity of 2,4-D cannot be predicted. 0 0 0 4 2 ,3 4 The interaction of all three parameters would have to be determined empirically through experimentation. WATERFOWL Conclusions: o t4 U 0 J U 1. The toxicity of 2,4-D has been studied in only two waterfowl spe cies, mallards and Canadian geese. 2. 2,4-D is essentially non-toxic to mallards when admsinistered in the diet for short periods. 3. A single dose of 1500 ppm 2,4-D acid in female mallards produced temporary (2 day) egg-shell thinning, but this was attributed to anorexia rather than a physiological effect. 4. Canadian geese fed 1000 ppm 2,4-D (unspecified formulation) for periods up to 230 days appeared outwardly normal but had lowered weight gain and some internal histopathology. 5. Field studies with waterfowl at aquatic plant control sites have not been reported but also do not appear to be necessary. A variety of waterfowl are known to include the aquatic plant Myriophyllum spicatum in their diet (Florschutz, 1973). Following mllfoil*control programs, therefore, waterfowl would likely be tem porarily exposed to herbicide residues through the ingestion of mori bund or decaying plant material or possibly through the direct consumption of intact herbicide pellets, if a pelleted formulation was used. The oral toxicity of herbicides, specifically 2,4-0, to waterfowl is thus of interest. Mallards have been the primary species used in 2,4-D toxicity tests. Hill et al. (1975) reported that five day feedings of 5000 ppm 2.4- D DMA or BEE to three week old mallard ducklings produced no mor tality. DeWitt et al. (1963, 1962) determined LD50 values of 2500 and 5000 ppm for DMA and BEE, respectively, in feeding trials that took between 10.and 100 days. DeWitt et al. (1964) fed 200 ppm 2,4-D BEE to adult mallards for periods up to 126 days but found no mortality. Tucker and Crabtree (1970) report the oral toxicity of several 2,4-D formulations, all producing LD50 values greater than 1000 ppm. All of the doses in the above studies refer to ppm 2,4-D in the diet. Haegele and Tucker .(1974) found that a single dose of 1500 ppm 2.4- D acid in the diet of mallards produced temporary (2 day) egshell thinning. The thinning was attributed to a temporary reduction in food intake following herbicide treatment, this, rather than a direct physiological 2,4-D effect, leading to eggs of poor shell quality.' S/helajon et al. (1964) fed Canadian geese a diet containing 1000 ppm 2,4-D for periods up to 230 days. While these birds outwardly GGG4 ^ ^ (' 8 0 0 1 appeared to be normal, their overall weight gain was reduced by about 30% compared to the controls and some internal organ histopathology was present. Two geese returned to uncontaminated food appeared to have regained complete health in about 7 months. Only a single report was located on the residues of 2,4-D in waterfowl resulting from aquatic plant control programs. Schultz and Whitney (1974) found that a combined sample of three gallinule ducks (Gallinula chloropus) collected in a Florida wildlife refuge treated with 2,4-D contained 0.3 and 0.675 ppm 2,4-D in the breast muscle and liver, respectively, one day after the treatment. Additional samples collected over the following week contained no residues. NONTARGET AQUATIC PLANTS The selectivity of 2,4-D for water milfoil even in the presence of other plant species is a primary reason 2,4-D is currently the herbicide-of-choice for milfoil control. This selectivity has been adequately documented in the literature (Gangstad, 1977; Gangstad et al, 1976; Wojtalik et al, 1971). Little can actually be said regard ing 2,4-D effects on nontarget plant species, except that non-target plants are afforded a margin of protection only by virtue of the sen sitivity of milfoil to 2,4-D. Tripling, or in some cases only doubling, the 2,4-D application rate used, for milfoil control will result in the death of most nontargent plants. . Given the stimulatory effects of sublethal 2,4-D concentration noted in the following section of this report, it would not be sur prising to learn that the growth of milfoil or nontarget plants not killed by the herbicide treatment could be enhanced. ADDITIONAL NONQUANTIFIABLE OR POTENTIAL EFFECTS OF 2,4-D ON THE AQUATIC SYSTEM Based on the overall information on 2,4-D and herbicides in gen eral, the following non-quantifiable or potential effects of 2,4-D seem to merit discussion: 1. Potential aquatic ecosystem stimulation by 2,4-D. 2. Effects of 2,4-D on aquatic bacteria and fungi. 3. Tainting of water and fish-flesh by 2,4-D or breakdown products. 4. ..Avoidance of 2,4-D by aquatic organisms. 5. Acquired resistance to 2,4-D by plants or organisms. 6. Synergistic toxicities of other chemicals with 2,4-D. 7. Concern regarding carcinogenicity of 2,4-D to aquatic organisms. 8. Indirect effects of herbicides, including 2,4-D. i -1 2 1 - POTENTIAL ECOSYSTEM STIMULATION BY 2,4-D aa yeenTy > Several components of the aquatic ecosystem are known to be sti mulated by the presence of 2,4-D at subtoxic levels. As described earlier in this, report, algal production can be directly enhanced by 2,4-D (Das and Singh, 1977; Bertagnolli and Nadakavnkaren, 1974: " Poorman, 1973), as can fathead minnow metabolism, as well as reproduc tive timing (Mayer and Merhle, 1974). An in vitro laboratory study by Weiss (1976) showed that the mitotic activity and overall synthesis of cultured rainbow trout cells were stimulated by sublethal amounts (but 50 ppm) of 2,4-D. Petruk (1969) believed that 2,4-D induced a drama tic temporary increase in the bacterial population of a treated pond, although the possibility that this was a secondary effect cannot be eliminated. The ongoing research with 2,4-D on pond communities out lined in Scott et al. (1978) suggests several levels of stimulation within warmwater ponds but these have yet to be fully analyzed and validated. None of these effects are presently known to occur under the 2,4-D concentrations and exposure durations associated with aquatic plant control treatments, with the possible exception of algal * or bacterial stimulation. Stimulation has been primarily evident under closely monitored indoor or outdoor laboratory conditions and may well be of no ultimate environmental significance. The principal concern surrounding stimulatory effects, particularly if evident only in select components of the aquatic system, is that they could serve to "unbalance" what are often well coordinated ecological cycles. As simply one hypothetical example from many that could be chosen, it is not difficult to imagine a situation under which the spring 2,4-D treatment of aquatic plants could result in the reproductive stimula tion of a particular fish species in such a manner that fry hatch pre cedes the normally simultaneous appearance of a critical food item or particular temperature regime, thus adversely affecting, in a very subtle way, the annual recruitment to that fish population. Again, such an effect is hypothetical and, even if present, might be mini mized by factors such as immigration into the treated area of normally-superfluous fish production from non-treatment areas, or growth compensation. Nonetheless, it would seem prudent for investi gators involved in future 2,4-D field studies to be mindful of possi ble stimulatory effects. EFFECTS OF 2,4-D ON AQUATIC BACTERIA AND FUNGI Natural bacterial and fungal populations of the aquatic system have been largely neglected by North American aquatic researchers, particularly aquatic toxicologists. Consequently, little information on the effects of 2,4-D on these organisms is available, which is unfortunate since microorganisms are of critical importance in both the cycling of nutrients and energy in the aquatic system, as well as in the ultimate breakdown of the 2,4-D molecule itself. The Russian paper by Petruk (1964) cited earlier found that, at the leyel applied for aquatic plant control, 2,4-D may be stimulatory to at least some bacteria, an interesting finding since it suggests CGQ4237 ' 8003 2.4- D may promote its own breakdown once applied to water. However, a more recent study by Golovleva et al. (1977) failed to find such sti mulation within small "microplots" isolated in natural waters follow ing the addition of 200 ppm 2,4-D. This was attirbuted to lack of certain organic substances which serve as cosubstrates in bacterial growth and 2,4-D breakdown. These authors also tried the interesting experiment of spiking the microplots with high concentrations of laboratory-reared herbicide-degrading microorganisms in order to hasten the breakdown of applied herbicide. These experiments also proved unsuccessful, since the added bacteria died within 24 hours. Apparently either the biotic or abiotic factors were not adequate to sustin the high bacterial populations. Other studies have determined the levels at which 2,4-D is harm ful to bacteria. For example, Walker and Newman (1956) noted that the laboratory growth of a Mycoplana bacterium was not reduced until expo sure concentrations reached 5000 ppm 2,4-D, while Worth and McCabe (1948) found that 200 to 20,000 ppm 2,4-D was required to inhibit the growth of several aerobic bacteria. In a study of primarily academic interest, Wedemeyer (1966) found that the "nonmetabolic" uptake of 2.4- D into the bacterium Pseudomonas flourescens was a two step pro cess involving an initial sorption to the cell wall and then passive diffusion into the cell. Experiments on the effects of 2,4-D DMA on benthic microorganisms are ongoing at the Fish-Pesticide Research Laboratory in Denver, Colorado, but the results are not yet available (Scott et al., 1978). In a short note, Mayer (1976) reported that 2,4-D DMA markedly reduced the incidence of infections by the common aquatic fungus Saprolegnia on fathead minnow eggs during chronic toxicity tests uti lizing DMA concentrations up to 2 ppm. This finding suggests DMA may have fungistatic or fungicidal properties which could be of signifi cance to natural fungal populations. The many studies presented in later sections on 2,4-D persistence and metabolism show that a variety of bacteria are known to be capable of metabolizing or cometabolizing 2,4-D under laboratory conditions, and these further serve as demonstration that the past emphasis with respect to 2,4-D and bacteria has been the effect of the bacteria on 2.4-D. The converse effect, namely that of 2,4-D on bacteria, has not been well studied, particularly in situ, and the area is in need of attention (see the review by Pfister, 1974). TAINTING OF WATER AND FISH-FLESH - BY 2,4-D i The first breakdown product, and frequent impurity, of 2,4-D is 2.4- D dichlorophenol (2,4-DCP), a fairly potent water and fish-fleSh tainting agent. A good deal of concern was expressed over this ' problem during the early 1960's (Aly and Faust, 1963 and 1965; McKee and Wolfe, 1963) but, oddly enough, the area was almost completely -123- 0004S3S ignored, at least in the literature, in the intervening years until only recently. Folmar (1979) has just addressed the problem of fishflesh tainting in a paper now being reviewed for publication. Other than Folmar's work, only two studies were located in which the problem was even mentioned, Mount and Stephan (1967) determined the levels at which 2,4-D did not affect the survival, growth and reproduction of the fathead minnow but stated only that these limits did "not apply to acceptable concentrations for preventing tainting of fish flesh." Wojtalik et al. (1971) reported that "at no time were unusual tastes or odors reported in fish" and further that the commercial market acceptance was not affected following 2,4-D treatments in TVA reser voirs, but these comments appear to represent the authors' collective impressions rather than hard evidence. Folmar (1979) examined 2,4-D tainting in the flesh of rainbow trout. Three groups of trout in raceways were exposed to 0.05, 0.09, or 0.15 ppm 2,4-D DMA for four hours, then held in clean water for 1, 4, or 7 days post exposure. On each sampling day, the fish were filleted and one fillet was prepared for*2,4-D residue analysis, while the other was retained for taste evaluation by a panel of 12 people at Colorado State University. In these taste tests, the panel judged the taste of the fish from the highest 2,4-D exposure level (0.15 ppm) to be inferior to that of the control fish on each of the four post exposure sampling dates. The taste of the fish exposed to lower con- ntrations was judged to be acceptable throughout the study, although still not quite as good as the control fish. These results suggest that whenever DMA concentrations greater than 0.15 ppm result from aquatic plant control programs, fish flesh may be tainted for a mini mum of one week. The source of the off-flavor in Folmar's study is of interest. The residue analysis on the fish fillets showed that very little or no 2,4-D remained in fish collected in the post exposure period. Folmar suggested tainting may have been due to 2,4-D metabo lites, or an herbicide-induced accumulation of natural body products in the fish. Given, however, that fish are unable to metabolize 2,4-D, as will be shown in a later section, and that the raceway resi dence time of the applied herbicide was so short as to preclude micro bial breakdown or photodecomposition, then it seems likely that the taining may have resulted from 2,4-DCP impurities in the liquid 2,4-D DMA used for the study. Unfortunately, since the 2,4-DCP content of currently marketed 2,4-DMA is not publicly known, this possibility cannot be verified. Tests on flesh tainting with other fish species would be of value, particularly if the test fish were collected during actual field treatments with 2,4-D. Information on the degree to which currently marketed 2,4-D formulations taint water would also be desirable. '< AVOIDANCE OF 2,4-D BY AQUATIC ORGANISMS The single report by Smith and Ison (1967) that the application of 2,4-D to TVA reservoirs "appeared to result in some movement of 0004239 lake fish o.ut of the treated area" has been repeatedly cited as evi dence that fish may avoid 2,4-D treated areas and thus reduce the potential for incurring adverse effects. No other field study located, during this literature search confirms fish avoidance under field con ditions. However, a number of laboratory studies, all done with a Ymaze, all of questionable environmental significance, have tried to determine specific levels at which several aquatic organisms might avoid 2,4-D. Folmar (1978) found that mayfly nymphs did not avoid 2,4-D concentrations up to 100 ppm, even though under mortal stress at 100 ppm. Folmar (1976) noted that rainbow trout fry did avoid 2,4-D DMA concentrations of 1 ppm or higher but concluded that avoidance would be unlikely under field conditions. Glass shrimp, an estuarine invertebrate, avoided 2,4-D BEE levels of 1 ppm of higher and consis tently selected the lower of two 2,4-D concentrations when such a choice was offered (Hansen et al., 1973). Gambusia and Sheepshead" minnows also avoided 1 ppm 2,4-D but not 0.01 ppm (Hansen et al., 1972; Hansen, 1969). None of these studies were controlled for possible 2,4-DCP impurities in the 2,4-D formulations. It is not inconceivable then, that some organisms might avoid an area treated with 2,4-D. The duration of the avoidance is unknown. At any rate, avoidance is a behavior pattern which obviously favors the more mobile inhabitants of an area. ACQUIRED RESISTANCE TO 2,4-D * Just as it is the case that the more often 2,4-D is added to a particular system, the more rapid will be the rate of its subsequent decomposition, it is also true that the more often 2,4-D is applied, the more rapid will be the induction of resistance to the toxic effects of 2,4-D in all aquatic biota. Patterns of resistance in the biota are not easily predicted; resistance is a complex phenomenon mediated by a variety of behavioral, anatomical, and-biochemical fac tors, all ultimately a reflection of the genetic composition of the particular life form. Acquired resistance to 2,4-D might be expected to take place rather slowly since applications are usually made only once a year. Certainly aquatic plants are subject to the greatest selective pressure as a result of herbicide treatments. Their rate of resistance.development might be predictable based on the terrestrial plant control experience but the location of such information was beyond the scope of this review. Among" the aquatic animal forms, the insects and fish with short life cycles might be expected to show the higher rates of resistance development. At the present time, only one aquatic -animal species, the fish Gambusia affinis, is known to have a resistance to 2,4-D, and this apparently represents a cross-resistance induced by chlorinated hydrocarbon pesticides other than 2,4-D (Chambers et al, 1977; Fabacher and Chambers, 1974). In principle, resistance could ultimately render 2,4-D useless as an aquatic herbicide or protect aquatic animals from adverse effects of 2,4-D. In practice, resistance would probably not appear for years, if ever. 0004^40 -125- o SYNERGISTIC TOXICITIES OF 2,4-D WITH OTHER CHEMICALS C * * no The potential for synergisms (supra-additive toxicities) between 2.4- D and other chemicals has been poorly explored in aquatic animais and is likely to remain so for an indefinite future period. The num ber of chemical combinations is, in a practical sense, inexhaustible and, at the exposure concentrations necessary to mimic environmental levels, the likelihood of positive results is too low to justify any presently known type of systematic screening. Potential synergisms are therefore always one of the given risks associated with the use of 2.4- D, or any other chemical, in the environment. At present, there is no evidence that 2,4-D has combined with other chemicals to produce synergistic toxicity to non-target organisms in the aquatic environ ment. The occasionally-cited studies on the synergism between 2,4-D butyl ester and carbaryl (Statham and Lech, 1975, 1976) done with rainbow trout were conducted for academic purposes at high exposure concentrations and do not reflect any known or probable field situa tion. Scott et al. (1978) summarized experiments conducted at the National Fisheries Research Laboratory in Lacrosse, Wisconsin', in which rainbow trout pretreated with polychlorinated biphenyl (PCB) were found to be "more sensitive than the controls" to acutely-toxic concentrations of 2,4-D DMA in short term tests. Presumably this refers to exposures of more than 100 ppm 2,4-D. These experiments seem to point to partially-additive acute toxicity between PCB abd 2.4- D, but not supra-additive toxicity. Likely candidates for com bination tests with 2,4-D include other herbicides or carriers that might be mixed with 2,4-D prior to application, or possibly other che micals known to be present as pollutants in specific treatment areas. For example, Brooker (1976) field tested the combination of 2,4-D and dalapon in an English stream (without finding effects, synergistic or otherwise) since these two chemicals are apparently often used .to gether in Great Britain. The Washington State Department of Fisheries (Holland et al. I960) tested the combination of 2,4-D PGBE and 2,4,5-T PGBE to coho salmon in trying to determine whether a reported fish kill was due to the runoff of a particular brand of commercial fer tilizer. Matida et al. (1976) tested a similar combination commonly used in Japanese agriculture. Pierce (1968) used a combination of 2.4- D and fenac, as well as 2,4-D plus Banvel-D in Nobska Pond, Massachusetts. None of these combinations in any of these studies produced supra-additive toxicities. v ^ CONCERN REGARDING CARCINOGENICITY OF. 2,4-D TO AQUATIC ORGANISMS No information was found regarding the possible carcinogenicity, of 2,4-D to aquatic animals, which is not surprising, since such tests are not routinely performed. Similarly, data on mutagenicity or tera togenicity in aquatic animals are lacking. Interest in the area of ;uatic animal carcinogens is developing, as evidenced by the recent symposium proceedings on aquatic -p1o2l6l-utants as carcinogens in both ^ 0 humann and aquatic organisms presented by Kraybill et al. (1977). OQQ^ Whether concerns in this area will ultimately be translated into a requirement for the inclusion of the appropriate tests on aquatic ani mals into chemical registration protocols is not known at this time. INDIRECT EFFECTS OF 2,4-D USE As stated by Robson and Barrett (1977) in their review on the ecological effects of aquatic herbicides, "Even if a herbicide could be developed which was specific to one species of weed, with no toxic effects on other weeds or animals, it would still produce an effect on the ecosystem." Such effects are generally termed indirect or secon dary actions of herbicides, and these have been well discussed in the literature (Robson and Barrett, 1977; Newbold, 1977, Strange et al., 1975; Newbold, 1975, Brooker and Edwards, 1975; Hurlbert, 1975; Morley and Reid, 1975; Frank, 1972; Walker, 1971; Mullison, 1970). The reader interested in secondary effects is referred particularly to Hurlbert (1975) and Newbold (1975) for excellent, well-exampled dis cussions. Briefly listed, the principal areas of concern are the consequences of oxygen loss, nutrient releases, .detritus build-up, aquatic community shifts, habitat loss, and food web (energy flow) alterations subsequent to herbicide treatments. It should also be n$ted that indirect effects are not always detrimental to aquatic populations, and may, on occasion, be somewhat beneficial. SPECIES COMPARISONS BETWEEN LITERATURE TOXICITY DATA .. AND NORTHWEST WATERS - A lake Washington Species list (Appendix B), exclusive of vascu lar plants, waterfowl, and mammals, was compiled from the reports of White (M?5), the Washington Cooperative Fishery Unit (1974), Wydoski (1972), Thut (1969), Comita and Anderson (1959) Scheffer and Robinson (1939), and Edmondson (personal communication). This list contains 205 taxa and yet is probably incomplete, particularly in the inverte brates. It seems reasonable to assume this list reflects the species composition of most Northwest freshwaters. A comparison (Appendix B) of the organisms for which at least some 2,4 - D .toxicity data is available with those found in Lake Washington shows that at the genera level 13/71 alga, 2/20 protozoans, 6/10 zooplankton, 22/68 benthic invertebrates, and 13/36 fish taxa (or 56/205 taxa overall) have been used or monitored in one or more 2,4-D evaluations. At the species level, only 19/205 taxa are represented, of which 13/19 are fish species. At the non-fish species level, therefore, the match-up is only 6/169 taxa, a frequency much lower tha might be considered desirable. Moreover, a simple match-up of species provides no information on the quantity or quality of datai available for that species. Some match-ups at the species and genera level simply reflect the appearance of that organism during a single field study. From these match-ups, it is clear that any contentions that 2,4-D will not harm the.biota of Lake Washington must be based more on^faith than fact, including the generalizations made in the -127- 0004242 0 q \H 7 4 9 3 3 8 next section of this report. Many believe that the amount of 2,4-D data presently available for salmonids does not allow for the adequat protection of this taxonomic group. However, it is obvious from Appendix B that salmonids in fact represent the best studied group, with respect to 2,4-D, in Northwest waters. Unfortunately, this reflects not an overabundance of information on salmonids, but rather a paucity of data on the other biota. At very minimum, a thorough small-scale field trial with 2,4-D in Lake Washington should precede any broader application for water milfoil control. The physical conditions in Lake Washington differ from those in the available studies primarily in the presence of cold deep waters in proximity to some of the proposed treatment areas. As discussed in a later section, movement of 2,4-D into these waters could promote the persistence of residue, although at extremely low levels. Lake Washington bays themselves are not characterized by physical con ditions markedly different from those present in several 2,4-D field studies. Again, the primary physical factor of concern is the pattern of water movement within each bay, in this case in order to allow con trol of plant kill patterns as well as to minimize 2,4-D residues in non-treatment areas. RELATIONSHIP BETWEEN 2,4-D CONCENTRATIONS KNOWN TO BE TOXIC TO AQUATIC ORGANISMS AND THOSE LIKELY TO OCCUR DURING HERBICIDE TREATMENTS L Given 2,4-D DMA as the selected herbicide: The target concentration of DMA for milfoil control in Northwest waters would probably not exceed 2.0 ppm. At or below such a concen tration, the available lab and field data indicate, as discussed in the previous sections, that-algal populations would not experience mortality but could be stimulated, zooplankton could experience par tial temporary population reductions, and benthic invertebrates and fish would experience no observable effects. These effects would not be expected to be much worse even if the target concentration was exceeded by several-fold. Given BEE as the selected herbicide: Granular 3EE would be applied to Northwest waters at no greater than the rate of 100 lbs. pellets/acre, or the equivalent of 29 lbs. BEE/acre or 20 lbs. 2,4-D/acre. The 2,4-D concentrations in water would depend entirely on the depth at the application site and some representative figures are provided in Table 4. These figures correspond to the maximum theoretical concentrations possible, and assume immediate release, no dilution, and, in the case of 3EE, no hydrolysis. Actual concentrations in the field would be lower than those presented in Table 4, but not by any predictable numerical fac tor. Depth stratified gradients of 2,4-D could occur. At the^water depth of typical water milfoil habitat, 5 to 15 feet, 2,4-D BEE i t j n t; G 0 0 4 4 3 -128- DOW ? 49339 P Table 4. Nominal Concentrations of 2,4-D in Hater of Various Depths Following Application of 100 Pounds/Acre BEE Pellets, Assuming Immediate Release and No Dilution VOLUME Water Depth Cu/Ft/Acre Liters/Acre BEE (ppm)a 2# 4-D (ppm) 1 43560 1232748 10.68 7.31 2 87120 2465496 5.30 3.68 3 130680 3698244 3.57 2.46 4 174240 4930992 2.67 1.84 5 217800 6163740 2.14 1.47 6 261360 7396988 1.78 1.22 7 304920 8629236 1.52 1.05 8 348480 9861984 1.33 9 392040 11094732 1.19 0.92 0.82 10 435600 12327480 1.07 0.74 15 653400 18491220 0.71 aBEE pellets contain 29% BEE. BEE concentrations assume no hydrolysis. S 0.50 bBEE pellets correspond to 20% 2#4-D. 2,4-D concentrations assume immediate hydrolysis. CT1 sta so o o OOV* 7 4 9 3 4 0 centrations would not exceed 2.14 to 0.7 ppm. At these levels, safet to aquatic organisms is completely dependent on the rapid hydrolysis of BEE to 2,4-D. If hydrolysis was delayed, and there was no dilu tion, invertebrate and fish skills, and temporary declines in algal production, would be expected. However, available evidence suggests that the hydrolysis of BEE to 2,4-D is relatively rapid under natural conditions, that dilutions and pellet release rate do serve to..keep residues at low levels, and that biota toxicity, therefore, is not an anticipated outcome of a single BEE application. -130- PERSISTENCE Conclusions: Free anion is the molecular form in which 2,4-D is likely to per sist in the environment. Salt formulations of 2,4-D dissociate to y/ld anion, while ester formulations hydrolyze, usually within 24 hours according to limited available information, to the anion molecufe. Persistence in water 1. Under laboratory conditions, 2,4-D has repeatedly been shown to persist unchanged in water for up to six months in the absence of the proper enzyme systems (2,4-D degrading microorganisms) or other energy inputs. 2. Under laboratory conditions 2,4-D has repeatedly been shown to be decomposed in water in periods of hours to days when the proper enzyme systems (2,4-D degrading microorganisms) were present. 3. Under some warmwater field conditions, 2,4-D has repeatedly been shown to be reduced to non-detectable levels in water of closed water bodies (pools or ponds) in approximately one month. 4. Under some warmwater field conditions, 2,4-D has occasionally been shown to persist for about 3 to 6 months in the water of closed water bodies. 5. No reports on the persistence of 2,4-D in coldwater pools or ponds were located. 6. The persistence of 2,4-D in the water of large lake and reser voirs can be correlated to the water flow through the treatment area. Persistence in high flow areas is shorter than in low flow areas, however residues at the low ppb level may be present for up to several months post treatment in either situation. 7. Persistence of 2,4-D at extremely low levels may be encouraged by water movements in lakes, reservoirs, and streams. 3. The available evidence suggests 2,4-D is not sorbed to particles or environmental surfaces. 9. The available evidence suggests 2,4-D. in the formulations used for aquatic plant treatments, does not volatilize from water sur faces. 10. Under laboratory conditions, 2,4-D is readily degraded by photo- decomposition. Under field conditions, the contribution of 0G0424G , so o * photodecomposition to 2,4-D breakdown is unknown. Theoretical calculations suggest a moderate contribution, at best. wU CC CO Persistence in Hydrosol ^ tc 1. Onder warmwater field conditions, 2,4-D applied as DMA appears in the sediment at the ppb level and persists for about one.month. 2. Under warmwater field conditions, 2,4-D applied as BEE has shown erratic patterns of hydrosol persistence. The study by Smith and Isom (1967) documented the persistence of 58.8 ppm 2,4-D in hydrosol for at least 10 months. Several other studies conducted with BEE have reported only short to moderate periods of persistence. Persistence in Invertebrates 1. Persistence of 2,4-D in invertebrate species have been primarily documented in warmwater bivalve molluses. 2. Bivalve molluses generally accumulate 2,4-D in the low ppm (1 to 10) ppm range and then slowly eliminate the residues over the subsequent 2 to 6 months. 3. Zooplankton may retain 2,4-D residues for extended periods (3 to 6 months) but this finding needs to be confirmed. Persistence in Fish 1. Under laboratory conditions, warm and coldwater fish species rapidly accumulate, hydrolyze, and eliminate 2,4-D BEE? 2,4-D acid is not accumulated under similar conditions. 2. Warmwater fish apparently cannot metabolize 2,4-D. However, warmwater fish can accumulate and use the bacterial breakdown products of 2,4-D for tissue elaboration and growth. 3. Field.studies uniformly document the general absence of 2,4-D accumulation and persistence in warmwater fish. 4. No studies were located regarding the persistence of 2,4-D in salmonids under field conditions. Persistence in Plants 1. Plants accumulate high concentrations (5 to 80 ppm) of 2,4-D. 2. The persistence of 2,4-D in dead plant material has not been well documented. 3. ! Plants not killed by 2,4-D may require several months to elimi nate 2,4-D residues. r n_ CG04247 ' 8 0 1 3 -132- This section deals with the persistence of the 2/4-D anion mole cule, once a particular 2,4-D formulation has been applied to an aquatic system. The anion is the molecular form of interest since upon application, the salt formulations of 2,4-D, e.g., sodium or DMA, dissociate to the free anion in water,, while the ester formulations, e.g., BEE or PGBE, eventually hydrolyze to yield 2,4-D anion. The rate of hydrolysis of the ester formulations is of critical importance to their safe use since, as documented earlier, the toxicity of 2,4-D esters to the aquatic biota is markedly higher than for the free anion. That ester hydrolysis usually occurs within hours after intro duction into either warm or cold water was demonstrated in the BEE toxicity tests by Mount and Stephan (1967) and Martens (1979), wherein nominal BEE test concentrations could not be maintained in the test chambers (due to rapid hydrolysis) even under toxicant renewal con ditions. These authors attributed hydrolysis to microbial action (enzymatic hydrolysis) but chemical hydrolysis may also occur under certain conditions. Zepp, et al. (1975), in measuring the rate of chemical hydrolysis of BEE under various laboratory conditions, showed that the half-life (time to 50% hydrolysis) of the BEE molecule could be predicted by the equation: T (1/2) * -0.692 Kb (OH) * Where: T (1/2) * BEE half-life in water Kb * rate constant of hydrolysis (OH) hydroxyl ion concentration of the water This equation clearly demonstrates the importance of water.pH (a measure of acidity reflecting both the (H) and (OH) levels in water) in determining the rate of chemical hydrolysis of BEE. The higher the pH (and water in aquatic plant beds often has high pH), the more rapid will be the transformation of BEE to 2,4-D. For example, Zepp et al. calculated the T(%) for BEE at pH 9 to be 0.6 hours while at pH 6, it would be 26 days. The parameter Kb in the equation is directly tem perature dependent, so that lowqer temperatures, as characteristic in some northwest waters, would be expected to retard hydrolysis. Unfortunately, because the lowest temperature tested by Zepp was 28Cr a rather high environmental water temperature, their equation will not provide an accurate prediction of the BEE chemical hydrolysis rate in colder waters without some modification in the Kb parameter. Daly (1971) also determined the time required for BEE hydrolysis in the laboratory using a variety of waters and conditions, although no attempt was made to differentiate between enzymatic and chemical 0GQ4243 6^ M o a hydrolysis. In distilled water at pH 6.5 under various lighting regi mes, the conversion of BEE to 2,4-D ranged from 53 5o 67% in ten days at 29C while only 35% in ten days at 2C with exposure to light. Such test conditions would seem to promote primarily chemical hydroly sis although some microbial or photochemical conversion may have occured. When-the tests were repeated using pond or "polluted" (high bacterial content) water, transformation from BEE to 2,4-D was com plete within 24 hours in each case. Rodgers and Stalling (1972), working in the laboratory, found that hydrolysis of BEE to 2,4-D took place within 24 hours if fish were present in the water, but was delayed to 90 hours (90% hydrolysis) in the absence of fish. The water for these tests came from a deep well and was therefore probably of low microorganism content. Given low microorganism content, coupled with pH of 7, it is not surprising that hydrolysis was delayed in the absence of fish, since conditions were favorable for neither enzymatic nor chemical mechanisms. Paris, et al. (1975), demonstrated that a bacteria and fungus isolated from natural waters could enzyma tically hydrolyze 50 to 95% of 2,4-D enters in as little as 15 minu tes . In summary, it is clear that in cold, sterile waters of neutral pH, the hydrolysis rate of the toxic BEE to 2,4-D can be markedly pro longed. However, under almost all environmental conditions, particu larly in habitats where aquatic plants are abundant, the available laboratory evidence suggests that the chemical and enzymatic hydroly sis of BEE to 2,4-D occurs rapdily, probably within 24 hours. This tcan be the only explanation for the lack of biota toxicity and 2,4-D accumulation noted in field monitoring studies following BEE applica tions. Rapid hydrolysis has yet to be proven in the laboratory under the type of conditions more characteristic to the northwest but at least one study suggests that coldwater enzymatic hydrolysis does not proceed significantly slower than in warmwater (Martens, 1979). Much information was found regarding the persistence of 2,4D anion in the biota and waters of ponds, lakes and streams. These studies are presented in condensed form in Table 5. Most, if not all., of this work supports the conclusion that environmentasl applications of 2,4-D ultimately dissipate to undetectable levels, though the exact fate of the 2,4-D has never been determined under field conditions. Several fates can be surmised from laboratory findings. The dissipa tion time course tends to be variable and not subject to accurate pre diction. On occasion, residues in select components of the environ ment have shown atypical patterns of persistence; such instances have been emphasized in the following sections. The overall information has been organized under these general headings: water, hydrosol, fish, invertebrates, and plants. The reader is reminded that the per sistence of chemicals such as 2,4-D at low levels in the aquatic* environment is a problem of completely unknown consequence. Some researchers regard persistence as unacceptable, others are little con cerned (see Kahn, 1977). At the present time, 2,4-D residues in'the low ppb range pose no know threat (as measurable by currently available techniques) to aquatic environmental health. ) t ) {_ t i r 0G 04S43 -134- Persistence In Water Upon application to water, 2,4-D anion could theoretically, depending on existing conditions, (1) persist unchanged at either detectable or undetectable levels, depending on the dynamics of the water body; (2) undergo uptake and metabolism by plant, animal, or microorganism cells; (3) undergo photodecoroposition; (4) undergo volatilization; or (5) undergo sorption to physical surfaces. There is little doubt that the 2,4-D anion can be characterized as a stable chemical structure in water in the absence of enzyme systems or various energy inputs (Paris, et al., 1973; Frank, 1972; Faust and Suffett, 1966). This was convincingly demonstrated by DeMarco, et al. (1967), who showed that concentrations of 50 ppb 2,4-D in sterilized, light-shielded waters were unchanged after 100 days in a simulated impoundment. Even in non-sterile water, if the proper microorganisms are not present, 2,4-D will persist for extended per iods. Aly and Faust (1964) found no breakdown of 2,4-D in lighted laboratory flasks containing 3 ppm 2,4-D in aerobic "lake water" after a 120 day incubation period. Schwartz (1967) failed to isolate the proper bacteria from a number of polluted natural sources and found "In spite of excellent conditions for biological activity, at least 60% of the 2,4-D persisted for 3 to 6 months. . . On this basis it ma y be concluded that 2,4-D would not be degraded materially by the microorganisms present in a natural water supply." Daly (1971) `repeatedly found no breakdown of 2,4-D anion in a variety of 14 day laboratory flask tests. Watson (1977) reported that 2,4-D anion was stable in river water-mud solutions for periods up to six months, par ticularly during times when the river water contained low levels of bacteria. i In contrast to these findings, several laboratory findings have shown that 2,4-D degradation in water can occur relatively rapidly when certain microorganisms are present; in fact, under enrichedculture techniques, high concentrations of 2,4-D can be decomposed in a period of hours (Houston, 1975; Loos, et al., 1967; Okey and Bogan, 1965; Aly and Faust, 1964). Heromet and Faust (1969) showed that the rate of 2,4-D breakdown in aqueous solution depended on (1) micro organism concentration, (2) 2,4-D concentration, and the relative ratios between the two. Decomposition proceded more rapidly when both herbicide and microorganism concentrations were higher, and also when the microorganisms were first acclimated to 2,4-D as a carbon source..- The authors concluded that although 2,4-D could be degraded readily in the laboratory, the aquatic environment would not provide conditions favor-able to the rapid breakdown of. 2,4-D. DeMarco, et al. (1967) demon-strated that in natural river water, SO ppb solution of 2,4-D were decomposed within 10 days whether the water temperature was warm (22-26C) or cold (10-13C), as long as the water was kept aerobic. Okey and Bogan (1965) found that sewage sludge organisms in water could completely degrade a 600 ppm Solution of 2,4-D in as little as 6 hours. Robson (1966) noted that 2,4-D added to 0004S5G T a b l e 5. 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M i l 1914 i*n > - fu r m MOlto * t A l, 1914) 1919 (m Q e l. i t a l. 1914 to * r i t t a d C a*tad 1914 I M " M f M r lt t. 194?, 19491 T ri/\l* r ni M ttot* 1974 1971 1914 'toto'Uv*.*1. 14TT 2.4-0 fo m iU tio n (9 * O O W 749348 Q p e a itm lm o f to w n t *vU d la v a re tura 2 4 o r 4 lid ie n fH y 1 en |e n i| -ii c t pprm . 9.0)2 to tft 4.4 nta s w lo u t nC d h l^ n 091 4.44 1V ia , too i*-n e t 1.12 I h im , it t 11.1 IV m n todita 11400Wpp11*t%t4ar, SOi pF" 23*C 29*^tilUt 4 "r Un lu U li rd d tlM Un a d toaaaf 1911 1914 1914 19T4 Un fe ta l 4B M id lo * UMlSo4 KX I n io/* et) ln?r41ntt 2.0 Pto NS 20 lto/ert eti* ta fra lla * loo ito IJ -lA . P rotocol M 4 m kM i , I . ) . 1 14. , N , to . l i , n i | l t i yo I ter .m i a i o fo n i In F io rii , O n lU , ni H m m . a to ra d S p H lr tti ta to o 1,4-0 W la by bactnrU ani h |l m ia r lin t if c y o o n d ltlm a . Ufe w r / i oa ro ta * oC ( f e r in i bydm lyeU o R feo 1,4*0, p em ly tl , m i U t ilit y . 1,4-5 im lO t M o p U i In Ornadtm r ii 1,4-0raaidmo maitarai In ha mear o a r l M bcum, foolo, lofooao, r pana In nettata Chitad ftofeoo M loriia plant ^rayfefi. lapU a parara11? tafean ac I, , K a r i n dapa 1,4*0 fa ta ta s i to r itp w l In t o t Poataplm nU lU io o m r a Uaaa pam p a rla i. 1,4*0 n i lila im m uri I In oom ral H O q m 1* * (topo tillo A ife an m m a riC U i 1,4-0 tn a n a ik . ( d a iln y ta to m fear* in i a io ooU oetai fn a m rlo m fe o m U a ria m o , tacar an m itad v itti l,4 -o *tta n I m i t a i In ita d o rt, t a i mo om ito v itti a a o la tin i e 3,4-0^ tona In ta b a ta i. d o w ip n lM topO a i tapfe U nf 1,4-0 M looU feai fim i natu ra i m aom ta a ^ h U feoraw y rrletaanfe t to n lfm , O aa* ita la m a tom i m a i to Ungulata tof a m lo f o m t aro ani o o ilm n to v lto iln natom i m ta r la dina oontalnlnv 1,4-0 ItoJoaa m i a ita r o rfa n i* M u d i, it a a to iq u o tt U a M o a 1,4-0 v ltM n Ita t o t o f l nonicorad. 1,4-0 m a tta * a n lte ra i In aaUefe otomna (M a mofean* Patata m a* a io -ym r parlad. 1.4-0 rm ltaa n a d tu a l lo tta otar o lappa Indoor nudai M itia ii t e t a m il u n ta . 1,4*0 rm tom a u n ito rta In fid i rii ta tm a i a 1 4 0 0 -llta r W iloar M a l eooayttuaa t e 14 day, `d i ' fita , tta o tta r td to n it fir n . 1,4-0 tenlduaa nonifeerad In m t piota froa tta nur th are o f (a taanaaan, t.C . Tabi 5 (Oontinued P arala ta w ca _________________________ Im Ii*O W ri F io rii* - t . l M 1.1 fp i In t ir fa r 14 in v i, than t n o n ^ku cu b le O M ifl* 4 .0 | to t.F u n ln t M fa r U l y t, tta n a t ron-<H(jctM jt* lv^U f in In m to r fa r d t ff, tta n t n m ^ ttK U b l ln r l i M t iM fi afe lew to *U < fffe I v o li m feo Si daya thm f m . ili acuri ponda panamily hom i i l a r it rfea o f 4 1 n l| U , m tN aolt p a ri, hut i l n neolvod (O la tiw ly h lp ta r I n it ia l dona a feo a p p lica tio n rro r. A ll ta c e v i! r i A f f i ra p id i? hyicolyaad K t to 3,4-o. ani? a lm tn in ma eaam . frmm doonrfaticn o M 4 oeeurrad In ta a in c ^ n t tafea o f ( M a l ta rtro ly a l* o f V X la appaimi an pif. R lfta r tta pii, tta ifilito ta hpholy i li . Ffeofeolyoio oscura v ltM n houn tr ia r Ufe c a rii eIura bat aay M n m olto m ia r n a te a l co n d itima I f l t oscura i t 11. v to la tllltv o f om o U or? Irar ta ro ! en tta o ra tle a l calcula t i ara. tfe U tillty ta lf - ltf a lo m ioyo fa r B . Ito feoafe m iar m i fto a o f 1,4-0 m ld m by dar , to a n r , In 10/1$ ferala caaidmo ta i m ta dly ia e lln a i, 4/1$ o rn i tooufe C!u ama la m i , m i 1/1$ h o m i tnem mad la m io afe ia y . fe oo rra U tio n e tri* hom i 1,4-0 B o a lp a tlo i hut eonralafeai ritti mfear dapcn, m ta r im para aura, a r i poot 1,4*0 fa m i afe l i feo V ii ppb In U / l) mfear la p U a . M qtaat la m ia a n fe to i In tram a a la lif arfeon araaa. 1,4-0 rm lduao In f id i m a roporta dly C.S feo l . f ppa 1,4-0 h it pattern b f m id a n y hom baan dm to v a n tia lla l tacMcfeda uaa top p riv a ta parti . tom e tafear o lln e ta i O v in i Ita auaaer-aubmi p a rla i i l i not tr a ililm i 1 ,4 -0 .In 4 a ito . !ttm tafear eoltactod d to ln f tta v tn e ir p a ric i dld toatoi cM i l,4 *o In 1-4 a n ta In 1 eaam, tu t d ld ro t In 1 o tta r eaam. tota jm m ta ly U n ta M i 1,4-0 In iapa tu t aB om lm a lly to c* <0 dopa ffUTTwwfan laag 4 M to tta X U n p io to * fo lla i to pernota tta ra p ii I U m * M w o f 1,4-0 and. In Cmfe, tta a k im p n lm O d ra t a o r in . sta ta r, 1,4-0 w o n e tm a lly brotan io m rm iU y In r m adempio* i a tfln f M a u n a , auto a p re p lo ie tn , te tta r le tam ata tha rata o f tonatalo a i,' O tta r t a n pauau tta tta f r a t t i o f ra -3 ,4 -o m ta te U s lta wlrm if f U no to tta a d lliy to 1,4*0 ta aahfex i. .4-0 ta la tt to Ufe $M /lIto U t a fa ly fto . ife tt vara In tta la to '*-- ---- " t o bat a a m i* 1.4 ppb. t e tta flre fe J m a ta , rn tm 1,4-0 la m i m a ta f r a N to n o m b. aIdrna ta to a t lll datactdbla a t 4 aentta but fo ia by Pi auntha. tal tor n itim v it t i f it a , 3 py* 1,4*0 m (ducato t l . l n U Uya but tta n femad afe O la la m i t e tta ra a J iriq e i tta SI daya. In acoayatea vttheu t fit a , 1,4-0 lo rlv V e lin a ri to atanr la lly toatactabla la m i bp day 14. la t i ta , 1,4-Q i l i nefe a x m iU ta bufe tta faraatoom producta m M c u a tlitto t a m a o tta cortan ( ra ye n ta am ptovth aarartituanta. r. ,.M m UM In n *. but vara ty p M y 1 feo )$ ppb __ . 4 laV w m ter, -tnM_i,__J_,__-_o__n_m__lA_j_-- paafcod a t $00 ppb, but u n t? .1* ^ Ito t a m a ln IT dava. In e la , 1,4-0 rm lduaa a n about $00 p |b afe 41 tova bufe am fe day l$ 4 . In pLerta, 1,4-0 rm lduaa vara * M ln lt la lly h it daellnad to ).$ m i t I mtota i mea p ia t 34 0004S53 aa nn water in flasks with mud on the bottom could degrade 2,4-D within 12 days after a single application, and within 7 days if a second appli cation was made one month later. Using a very similar water-mud flask system, Sharpee (1973) also found that high concentrations of 2,4-D could be broken down by microorganisms within 10 days. These widely divergent laboratory findings on the persistence of"* 2,4-D in water suggest.that the results of field studies might also be somewhat less than uniform. This was confirmed by the located reports. In the following pages, the studies conducted in closed water bodies, i.e., pools or ponds, are presented first, since simple dilution as a mode of dissipation is not a factor, and those studies conducted in open water bodies, i.e., partially-treated large lakes or reservoirs, are considered subsequentlv. Several studies, all conducted in warm to temperate-water ponds, suggest that 2,4-D persists about 30-40 days or less following her bicide treatments. Schultz and Harmon (1974) sprayed DMA 2,4-D at various rates on nine ponds in Florida, Georgia and Misouri; noting that 2,4-D persisted for 14 days in the Florida and Georgia ponds and 28 days in Missouri ponds. Persistence was independent of initial spray rates and water 2,4-D concentrations. Sampling continued for about 5 month post treatment but no 2,4-D was found after one month. Frank and Comes (1967) applied BEE 2,4-D at a rate of 1.33 ppm to a small pond near Denver, but noted maximum water 2,4-D concentrations ->f only 0.067 ppm, apparently as a result of poorly dissolving pellets. By day 24, 0.019 ppm 2,4-D was still present in the water but none was detected at day 36. Cope et al. (1970) found that 2,4-D, as measured by a cucumber seed bioassay technique, was essentially gone after about 35 days from warmwater ponds treated with 0.1 to 10 ppm 2,4-D PGBE. Again, dissipation was independent of initial con centration applied. Schultz (1973), in a triplicated experiment per formed in outdoor plastic pools, showed that 2,4-D disappeared from water in about 35 days, regardless of the original amount applied (0.5-2.0 ppm). His analyses continued for 84 days post treatment. In all of the above tests, the patterns of 2,4-D persistence prior to disappearance is often different than might be anticipated. It would seem logical for the residues to be maximal immediately after treat ment then steadily decrease until gone. In actuality, 2,4-D residues frequently tend to gradually rise above initial levels from about day 3 to day 20 and are sometimes at maximum just before disappearance. Such patterns may reflect 2,4-D released from dying aquatic plants but a definitive explanation was not found in the located literature. Two small-scale experiments of simulated 2,4-D use have documented rather lengthy persistence of 2,4-D in water. Dim (1978) added 2,4-D to large indoor plastic cylinders containing water, mud, plants, and some organisms. Water 2,4-D concentrations were initially 0.028 ppm, gradually rose to 0.26 ppm at'about 30 days, plateaued at this level cor an additional 30 days, then gradually dropped and were nondetect.ble at about 6 months. Stalling and Huckins (1978) added 2 ppm 2,4-D to outdoor plastic pools with and without fish. In the pool -136- C G C `^ 3 -02Q without fish, 2,4-D residues gradually declined and were essentially gone after 84 days. In the pool with fish, 2,4-D declined by about 50% within 8 days, then remained essentially constant at this level (1 ppm) over the remainder of the test. Several types of analysis con firmed the absence of 2,4-D metabolites in this pool. Finally, Averitt and Gangstad (1976) reported mixed patterns of persistence following treatment of 15 pools, ponds and lagoons with 2.4- D. Ten of 15 cases showed marked reductions, but not disappear ance, of 2,4-D residues in 28 days; 4/15 showed approximately no change in residues between days 1 and 28; and 1/15 showed higher levels on day 28 compared to day 1. Given these data, an appropriate generalization concerning the persistence of 2,4-D in the water of warmwater ponds is not obvious. A reasonable statement might be that 2,4-D applied to warmwater ponds probably will be near undetectable levels at about one month post treatment, but this is in no way certain. Persistence of 2,4-D resi dues at the low ppb or high pptr level for three to six months post treatment might well be anticipated. Patterns of residue persistence prior to dissipation, especially during the first three weeks post treatment, are not subject to accurate prediction. No studies on the persistence of 2,4-D in cold water ponds were located. * The most extensive report on the persistence of 2,4-D in the waters of a large, dynamic aquatic system is that of Wojtalik, et al. (1971). These authors documented 2,4-D concentrations in waters of four treatment areas and a number of water treatment plants along the TVA Guntersville reservoir following large-scale sprayings of 2,4-D for water milfoil control. As might be anticipated, 2,4-D persistence patterns in water were largely correlated with*the mass water move ments through the treatment areas. In higher water exchange areas (Comer Bridge and Ossa-win-tha), 2,4-D concentrations in water were about 0.5 to 1.5 ppm 8 hours after treatment, 0.05 to 0.5 ppm 24 hours after treatment and then gernarally at trace or undetectable levels at two weeks and thereafter over the 6 month monitoring period. In the North Sauty section, which received moderate water exchange, residues followed about the same patterns as above except that at two weeks 0.2 to 0.7 ppm 2,4-D was present in the water, then subsequently undetec table. In the Jagger Branch, which received only low water exchange, 2.4-D was reportedly in excess of 5 ppm for five days and above 1 ppm for an additional three days. At two weeks, the concentration had dropped to about 0.6 ppm and was undetectable at four weeks. However, a t ^ w o and three months post treatment, residues again rose to 0.02 to 0.04 ppm before returning to non-detectable amounts at six months. The water entering drinking water treatment plants during the herbi cide application periods generally contained between 2 and 100 ppg 2.4- D. However, the North Marshall plant, presumably on the Jagger 3ranch, pumped water containing in excess of 1 to 5 ppm 2,4-D for over CG04255 0 O V>f 7 4 9 3 5 1 a one week period. 2,4-D levels gradually declined in the low ppb level in the.subsequent three weeks. In a study conducted in a northwest watershed, Lira and Lozoway (1978) applied 2,4-D pellets to test plots in the north arm of Lake Okanogan, British Columbia, to control water milfoil. The treatment plots were known to have underwater lateral current movements. The maximum 2,4-D residues measured after these treatments was 0.14*`ppra, while more typical residues ranged from 0.02 to 0.05 ppm before declining to non-detecta'ole levels three to eight days post treatment. Very low (0.3 to 34 ppb) 2,4-D levels were found in waters outside the treatment area for three days after treatment but not at subsequent sampling intervals. Other less complete reports on 2,4-D persistence in open water bodies were also found. Daly (1971) found 2,4-D at 2.4, 3.5 and 5.0 ppm at 1, 3, and 7 days following treatment of a ten acre plot on Lake Seminole, Ga., with 100 lb. BEE/acre. Unfortunately, no further sam ples were collected. Smith and Isom (1967) reported that 2,4-D ranged from nondetectable to 157 ppb in the waters of two TVA reservoirs following treatments with 40 to 100 lbs. 2,4-D BEE pellets/acre. Schultz and Whitney (1974) found only 1 to 16 ppb 2,4-D in the water of a Florida canal immediately after spray treatments of water hyacinth; the hyacinth had supposedly absorbed most of the applied herbicide. Water 2,4-D residues subsequently disappeared and reap peared at the 1 to 4 ppb range during the 57 day post treatment s.ampling period. Finally, Whitney et al. (1973) detected no 2,4-D on the water of Currituck Sound, North Carolina, after treatments of milfoil beds with 3EE pellets. This unusual finding was reportedly due to either the observed flooding of the treatment area shortly after the herbicide application or possibly 2,4-D degradation during shipment of the water samples to the laboratory. Based on these studies, the application of 2,4-D to open water bodies does not necessarily insure that 2,4-D persistence patterns in water will be much different than observed in smaller ponds with no source of diluent water. Certainly, the application of 2,4-D to large lakes requires no fewer precautions, if the persistence of residues is of concern. Clearly, 2,4-D was abused in the Jagger 3ranch of the Guntersville reservoir, just as it was probably quite safely applied to Lake Okanagan in Canada. If the water body is characterized by significant water movements, then persistence of 2,4-D will probably be short due to simple dilution to undetectable levels. If the water body is more stagnant, persistence patterns will be quite similar to those observed in closed water bodies and the same generalizations as stated earlier are relevant here. Knowledge of the patterns of water movement in a large water body should be considered a prerequisite for any herbicide treatment, if the fate of the applied material is of concern. Whether water movements such as found in lakes, reservoirs, and -138- 0G04Z56 'V streams influence the true persistence of 2,4-D, that is, decomposi tion vs. simple dilution, is a question which merits discussion. In addition to the low 2,4-D residues sometimes found for prolonged periods after the types of treatments described above, 2,4-D is also found at ppb levels in rivers and streams throughout the U.S. and Canada apparently as a result of herbicide run-off from agricultural and urban areas (See Table 5: Gummer, 1978; Truhler and Reed, 1976; Lee Choi et al., 1976; Morris, 1975; Schulze et al., 1973; Frank et a l . , 1970; Bartley and Eattrup, 1970; Manigold and Shulze, 1969). No information was found regarding the status of such residues, particu larly as to whether they are subject to degradation while in transit. If the factors encouraging microbial 2,4-D breakdown in laboratory are warm, nutrient-rich, 2,4-D rich conditions, then it is reasonable to assume that cool, nutrient-poor conditions present in moving waters, particularly in the Northwest, do not promote the degradation of extremely dilute concentrations of 2,4-D. Water movements, which pro bably serve to reduce the formation of bacterial complexes as well as being the mechanism of dilution, must therefore be considered a factor which favors the persistence of 2,4-D in water, albeit at very low levels. Whether 2,4-D can undergo sorption to the variety of particles and surfaces found in natural waters has.been investigated by a number of workers. DeMarco et al. (1967) determined that only extremely .small quantities of 2,4-D sorbed to the sides of large metal impound ments, or the suspended sediment in the water of the impoundments, following 100 day tests. The algal sorption studies cited in an earlier section of this report were generally negative. Work by Aly and Faust (1964) and Hague et al. (1968) revealed that .pure clays sorb only very small amounts of 2,4-D. For example, to remove 1 mg. of 2.4- D anion from a liter of water requires 26 grams of kaolinite clay (Aly and Faust, 1964). Several compounds are known to be relatively efficient sorbers of 2,4-D, however. As reported by Aly and Faust (1965) and Weber and Gould (1966), powdered activated charcoal will sorb about 80 mg. 2,4-D/gram. More recently, Miller and Faust (1972a, 1972b), Faust and Miller (1973), and Khan (1974) discovered that both synthetic and natural organo-clay complexes will sorb appreciable quantities of 2,4-D, but neither type of complex is known to be widespread.in the environment. Khan (1973) further showed that humic acid, a natural organic compound often found in water, does physically sorb 2,4-D. While this suggests humic acids could serve as natural 2.4- D scavengers in water, the subsequent section on the persistence of 2,4-D insediments, which generally have high concentrations of humic materials, shows that sediments typically do not have high 2,4-D residues, and in the few instances where high residues have been observed, the most likely explanation is undissolved pellets rather than sorption. In the monitoring study discussed earlier, Wojtalik et al. (1971) believed that "plankton" sorbed significant quantities of 2.4- D but this determination waits confirmation. Finally, Wedemeyer (1966) found that sorption of 2,4-D to bacterial cell walls was the first step in the uptake of 2,4-D by bacteria. At the present time, it does not appear that sorotion is a sianificant factor in the r 8023 CG0457 cnonTW M O ^ distribution and fate of 2/4-D in the aquatic environment. It would not be surprising, however/ for a future investigation to demonstrate that 2/4-D residues at the ppb or pptr levels actually persist in association with some type of organic complex. Volatilization of 2/4-D from water surfaces apparently has no^ role in reducing the persistence of 2/4-D in water. The salt formu lations of 2/4-D are considered to be almost completely nonvol-atile (Grover, 1976? QueHee and Sutherland, 1974). In the unlikely circum stances that the BEE formulation of 2,4-D would persist in water, Zepp et al. (1975) calculated that the vaporization half-life in a pond one meter deep would be 895 days (2.5 years). The period would be even longer for deeper waters. Photodecomposition is a frequently, and often improperly, cited mode of dissipation for 2,4-D in water. The complexities attending the study of the photolysis-of chemicals in natural waters were recently reviewed by Zepp and Baughman (1978). Crosby (1972) conclu ded a review of photodecomposition by stating "But does photodecom position of pesticides really take place in the aquatic world? Are light-energized transformations of pesticides environmentally impor tant? Unfortunately, almost no incontrovertible evidence has been reported. . . final proof of its extent and significance must await either more sophisticated methods for detecting and measuring tran sient chemical species or the actual application of photochemical principles to the practical scale removal of pesticides from water." A number of studies document the photodegradation of 2,4-D by either artificial light or sunlight in the laboratory (Zepp et al., 1975? Boval and Smith, 1973; Croby and Tutass, 1966; Aly and Faust, 1964? Bell, 1956). The paper by Aly and Faust (1964) has been often cited by others as evidence that 2,4-D is broken down by sunlight even though the authors concluded "ultraviolet radiation from the sun is expected to be an insignificant factor in the decomposition of 2,4-D compounds in surface waters." If laboratory results are ultimately confirmed under field conditions, the calculations of Zepp et al. (1975) provide an indication of the potential contribution of photode composition to 2,4-D breakdown. The figures show that the estimated half-life of 2,4-D in a one meter deep pond of pure water at 25C in the southern U.S. would be about 20 days. This would, of course, represent a very minimum value for the deeper, less-sunlit waters of the Northwest. Persistence in Hydrosol (Sediment) The application of 2,4-D DMA liquid for aquatic plant control has not resulted in high hydrosol 2,4-D concentrations or unusual patterns of persistence within the hydrosol. Schultz sprayed three treatment levels of DMA (0.15 to 2.0 ppm) on nine plastic pool model ecosystems. During days 1 through 9 post treatment, hydrosol 2,4-D levels were roughly stable at about 6.02 to 0.20 depending on the particular pond, and by day 2^1 were generally non-detectable over the remainder of the 34 day test period. After one day, Schultz and Harmon (1974) found -140- ( COG4u3 2,4-D residues between 0 and 170 ppb in nine ponds treated at three levels (three each in Florida, Georgia, and Missouri) of DMA. The hydrosol 2,4-D level was directly related to the treatment levels, the most residue occurring in the ponds receiving the highest treatments. In the southern ponds, 2,4-D residues were always undetectable by 14 days post treatment. In the Missouri ponds, residues remained present at day 28 (17 to 150 ppb), but were gone by day 56. Following 2,4-D sprayings of thick mats of water hyacinth in a Florida canal, Schultz and Whitney (1974) could detect no 2,4-D residues in bottom muds during a 113 day post treatment monitoring period. Wojtalik et al. (1971) found 100 to 450 ppb 2,4-D for up to three months after heavy treatments of the Jagger branch of the TVA Guntersville reservoir with DMA. No 2,4-D was detected in the sediments at 6 months post treat ment. While 2,4-D DMA sprayings have produced uniformly low hydrosol 2,4-D residues, the use of 2,4-D BEE pellets has produced erratic hydrosol 2,4-D levels, ranging from very high to low, with quite unde sirable patterns of persistence in some instances. The worst case is the TVA experience reported by Smith and Isom (1967). In the Watts Bar Reservoir, the few sediment samples analyzed showed 2,4-D residues ranging from 0.95 to 56 ppm four days post treatment, 0.15 to 35 ppm 24 days post treatment, and 0.24 to 58.8 ppm 10 months post treatment. In the Guntersville reservoir, 2,4-D residues in the hydrosol were generally in the mid-ppb range over the 9 month monitoring period except one sample at 42 days contained 33.6 ppm. These are unaccep table patterns of persistence in the reviewer's opinion. Frank and Comes (1967) attempted to apply 1.33 ppm 2,4-D to the water of a small pond using BEE pellets, but achieved a water 2,4-D concentration of only 0.024 ppm at day 1. with a hydrosol concentration of about 5 ppm. These sediment residues decreased by about 50% after one week, were still detectable (0.10 ppm) at 56 days, and gone at 85 days. Both of these studies suggest treatments conducted with poorly formulated 2,4- D pellets. Wilkinson (1964) demonstrated that the rate of release of BEE from pellets was influenced by such factors as temperature, pellet size, and to a lesser extent, bottom sediment. However, while the variability produced by such factors is sufficient to account for the results of Frank and Comes (1967), they do not exaplin those of Smith ande Isom (1967). Several studies conducted with BEE pellets more recently than the 1960's have produced more acceptable 2,4-D sediment concentrations. Whitney et al. (1973), monitoring 2,4-D concentra tions in the sediment at four stations in Currituck Sound, North Carolina, following BEE treatments, found 200 to 650 ppb at all sta tions and these were approximately the same at the end of the three week monitoring period. Daly (1974) measured about 100 ppb 2,4-D in the^sediroents of a treatment plot on Lake Seminole, Georgia, for 12. days post treatment, then the residues declined to nondetectable levels. Finally, Lira and Lozoway (1978) measured 50 to 460 ppb in the sediments of Lake Okanogan, B. C. immediately following BEE pellet treatments. Residues remained present at day 8 but had disappeared by day 17. 0004253 025 -141- O O W 74U35 In summary, the available information on the persistence of 2,4-D in hydrosol indicates that high concentrations of 2,4-D do not accumu late and persist in the hydrosol under usual circumstances. If, how ever, high concentrations of 2,4-D are deposited into the hydrosol due to either the use of poorly formulated pellets or excessive applica tion rates, there is evidence high concentrations of 2,4-D may persist in the hydrosol for extended periods of time. Persistence in Invertebrates cr. Only a few studies have reported the persistence of 2,4-D in invertebrates, and most of these concern the bivalve molluses. Butler (1965) determined that, eastern oysters exposed to continuously-renewed solutions, of 0.1 ppm 2,4-D BEE for seven days accumulated 18 ppm 2,4-D acid but then totally eliminated these residues when placed into clean water for seven days. Coakley et al. (1964) briefly reported findings of 3.5 to 3.8 ppm of 2,4-D as BEE in oyster and clam samples collected three days after BEE treatments of test plots in Chesapeake Bay. Additional samples at later times were apprently not collected. In the TVA Watts Bar reservoir, two mussel samples collected four days after BEE treatments contained about 0.4 and 0.7 ppm BEE, while in the Guntersville reservoir mussels collected at 1, 3, 6, and 42 days post treatment contained 0.24 to 1.12, 0.18 to 1.0, less than detectable quantities (<0.14), and nondetectable to 0.20 ppm, respectively 'raith and Isom, 1967). Wojtalik et al. (1971) reported 2,4-D per sistence in mussels in TVA reservoirs following DMA treatments but the results are confounded by the presence of significant amounts of 2,4-D in the ore-treatment samples. Samples collected a month before treat ment generally contained 0.07 to 2.7 ppm 2,4-D while those collected during the month after treatment contained 0.05 to 0.97 ppm 2,4-D. By five months post treatment, 2,4-D residues in mussels were often below detectable limits but occasionally as high as 0.26 ppm. This is also the study discussed earlier that found extended persistence in "plank ton" 2,4-D residues. Residues peaked at 3.6 ppm 2,4-D in "plankton" at 30 days post treatment and at six months were still 0.37 ppm. Since laboratory studies show little sorption of 2,4-D by algae, it is assumed by the reviewer that the invertebrate fraction of the "plank ton" account for these residues. As discussed earlier, the persis tence of 2,4-D residues in transient zooplankton populations is diffi cult to explain. It is further difficult to determine how much cre dence is due a single sample of 0.71 grams of zooplankton (as was the 6 month sample) taken from a multi-thousand acre reservoir. This paucity of data, coupled with the apparently uncontrolled use of 2,4-D in the reservoir as evidenced by the 2,4-D found in pretreatment sam ples, suggests that a good case has not been made for the long-term persistence of 2,4-D in plankton. < Three mussel samples collected 42 days post treatment by Lim and T-ozoway (1978) as part of the Lake Okanogan BEE studies contained 2,4- r'esidues of 0.52, 0.40 and 0.12 ppm. No residues were present in a mussel sample collected on day 156. Finally, Whitney et al. (1973) found 0.13 to 0.23 ppm 2,4-D in composite samples of grass shrimp, -142- CG04260 S02S damselflies, and amphipods at 24 hours post treatment and these declined to nondetectable levels within three weeks post treatment. In conclusion, mussels generally appear to accumulate 2,4-D to the low ppm level immediately following treatments, then slowly elimi nate it in the following months. Residues in the ppb range might still be evident at six months. Zooplankton may retain 2,4-D residues for a period of months but this is in need of confirmation. Persistence in Fish Next to determinations of the acute toxicity of 2,4-D to fish, research on the patterns of uptake and elimination of 2,4-D in fish under both laboratory and field conditions represents the area for which presently available information is possibly most complete. The only additional information desirable would be a field study on the persistence of 2,4-D in salmonids under natural conditions. Rodgers and Stalling (1972) monitored the accumulation and per sistence of 2,4-D BEE in eight tissues of three fish species, either fasted or fed, at two exposure concentrations under laboratory con ditions. Due to the amount of data generated by this number of com binations, only the results with muscle tissue will be considered here, since muscle is the portion of the fish normally consumed in the O^S. Visceral tissues generally showed the same persistence patterns as did muscle except that initial concentrations were usually higher. Tn rainbow trout muscle, 2,4-D residues were 1.2 to 4.4 ppm at 3 hours, declined to 0.1 ppm at 24 hours, and were at trace ( < 0.05 ppm) or nondetectable levels after 48 hours. In bluegill muscle, 2,4- D residues peaked at 1.7 to 46.6 ppm at 2 hours, declined to 0.1 to 3.5 ppm at 24 hours, were detectable at 0.1 to 0.2 ppm at 48 hours, but were nondetectable at 120 hours (5 days). Fasted fish uniformly accumulated higher 2,4-D levels than fed fish, for unknown reasons. BEE was hydrolyzed to 2,4-D within fish bodies, presumably by either plasma or liver ester-ase enzymes, so that only 2,4-D was present in the various non-liver tissues during the persistence period. Trout accumulated the least 2,4-D, catfish were intermediate, and bluegills accumulated most. The amounts of 2,4-D accumulated by the fish in these experiments are higher than would be expected under natural con ditions, since BEE would normally be hydrolyzed to 2,4-D by a number of non-fish mechanisms (e.g. microbial enzymatic action or chemical hydrolysis) which were not present under the laboratory conditions of this study. Because such hydrolysis did not occur in this study, BEE, a more l'ipid-soluble molecule than 2,4-D, was accumulated to relati vely high levels. However, when the authors exposed channel catfish and bluegills to 1 ppm 2,4-D acid for 120 hours,.residues in fish were at non-detectable levels ( < 0.01 ppm 2,4-D) over the entire test period. ,^ \ Schultz (1973) measured the persistence of 2,4-D in tissues of channel catfish, largemouth bass, and bluegills over an 84 day period following the application of DMA at either 0.5, 1.0, or 2.0 ppm to -143- 0G04LG1 outdoor plastic pool model ecosystems. Additional laboratory tests measured the persistence of 2,4-D in fish exposed to 1 ppm for two weeks then placed into clean water for four weeks. The effect of tem perature and water pH on 2,4-D accumulation was also determined. In the outdoor pool studies, 2,4-D residues in the various tissues of each fish species appeared to slowly rise from less than 1 ppm at 1~* week to from 5 to 100 ppm at 12 weeks, based on radiometric analyses. However, gas and thin-layer chromatographic tests revealed that* these residues did not reflect intact 2,4-D but rather unidentified C-14 fragments of the originally applied 2,4-D molecules. Similar results were found in the laboratory tests; unidentified metabolites per sisted, but not 2,4-D. Temperature (uptake of C-14 2,4-D at 17 vs. 25C) did not influence uptake but uptake at pH 6 was slightly greater than at pH 9. In summary, at no time was intact 2,4-D found above trace quantities ( < 5 ppb) in the muscle tissues of the three fish species, although unidentified 2,4-D metabolites, usually in increas ing quantities, were present throughout the studies. Stalling and Huckins (1978) extended Schultz's work using a series of more exten sive and sophisticated chemical procedures. Bluegills were exposed to a single application of radio-labeled 2,4-D DMA using two outdoor model systems similar to those employed by Schultz. One system was sprayed with 2 ppm DMA, the other was left as the control. After 34 days, again the radiometric residues had risen steadily over the test period and again it was demonstrated that the residues were not intact 2,4-D. Rather, the C-14 was found to have been incorporated into amino acids and glycogen; lipids, free fatty acids and triglycerides; and unidentified but water soluble residues. No 2,4-D intermediate metabolites were found. The 2,4-D had therefore been rapidly broken down to hydrocarbon fragments subsequently utilized by the fish for the synthesis of normal body tissues. However, using two separate techniques, the authors were unable to demonstrate the ability of fish to metabolize 2,4-D, and thus concluded that 2,4-D was probably de graded by microorganisms in the model ecosystems, with the resultant carbon fragments being accumulated by the fish. c 3 a c< O C Sikka, et al. (1977, same data as presented in Sikka, 1977) also studied the uptake and metabolism of 2,4-D DMA in bluegills and chan nel catfish. Fish were exposed to 2 ppm solution of DMA for up to 168 hours under laboratory conditions. Under these conditions, bluegills accumulated about 0.9 ppm 2,4-D within 24 hours and residues remained constant at this level over 168 hours. Catfish accumulated 0.20 ppm by 24 hours and again this level remained constant for the remainder of th test-period. Residue in edible tissues corresponded to only about 5 and 10% of the total body residues in bluegill and catfish, respectively. No metabolites were present. Since micro-organisms were apparently absent from the exposure systems, and the fish them selves were unable to metabolize 2,4-D, the constant 2,4-D tissue levels noted in the fish during the entire exposure period probably represent a simple equilibrium between fish and.water that could have persisted indefinitely, or at least until water quality was njade more suitable for microbial growth due to the presence of fish. The authors also deterarined the fate of 2,4-D injected intraperitoneally -144- GG0462 ( 3028 into bluegills. Approximately 90% of injected 2,4-D was excreted by the fish withih 6 hours. Again, no evidence of metabolism was found. A research group at the Mount Desert Island Biological Laboratory has recently determined the fate of 2,4-D injected into two marine forms, the dogfish and the winter flounder (Pritchard and James, 1978; Guarinao, James and Bond, 1977; James and Bend, 1976). These studies show, as is the case in freshwater species, that both species can excrete at least 50% of injected 2,4-D within 2 days, and in the case of dogfish, 90% within 6 days. Moreover, in both species a taurine conjugate of 2,4-D is formed prior to excretion so that, on occasion, less than 10% of the 2,4-D is excreted in free form. This represents the only known metabolism of 2,4-D in fish. Based on laboratory evidence, the accumulation and persistence of 2.4- D in fish as a result of aquatic plant control programs does not appear likely and this has been confirmed under field conditions. In general, 2,4-D persistence in fish is somewhat difficult to determine under field conditions since the exact extent and timing of the 2,4-D exposure regime cannot be exactly known for a mobile population. Thus, fish with low 2,4-D residues may represent recent immigrants from non-treated areas, while fish groups exhibiting apparent extended persistence may in fact reflect some condition such as continued dietary exposure. All of the field data on fish tissue persistence are based on whole body analyses, except for those studies by Schultz and Whitney (1974) and Toetz (1976), which reflect fillet analysis. After treatment of TVA reservoirs with BEE pellets, Smith and Isom (1967) reported only one fish sample out of eleven to have 2,4-D residues in excess of the 0.14 ppm detectability limit and that sample contained 0.15 ppi. That sample was collected 50 days post treatment, however. Wojtalik et al. (1971) found 2,4-D residues in 2 of 23 post treatment samples of eight fish species of the Guntersville TVA reser voir following DMA treatments. Gizzard shad, a planktivorous species, had 0.34 and 0.`22 ppm at 1 and 3 months, respectively, but undetec table quantities at 2 and 6 months. Residues were found in no otherspecies although sampling was not initiated until 4 weeks post treat ment. Also, one pre-treatment sample of largemouth bass contained 0.15 ppm 2,4-D. Whitney et al. (1973) measured 2,4-D residues in 13 of 38 fish samples of six fish species collected 1 to 21 days follow ing treatments of Currituck Sound with BEE pellets. The highest con centration found was 0.24 ppm in one largemouth bass sample taken 8 days post treatment. Of eight samples analyzed 2 and 3 weeks post treatmertt, only one contained 2,4-D residues (0.17 ppm). Schultz and Whitney (1974) found 2,4-D in 19 to 53 post treatment fish samples from a Florida canal treated with 2,4-D salts. The highest concent tration was 0.16 ppm in one sample of bluegill sunfish. Eight sam ples contained residues less than 10 ppb, seven contained between 11 and 50 ppb 2,4-D. Schultz and Harmon (1974) treated fish ponds, in Florida, Georgia, and Missouri with 2,4-D DMA. forty-five of 307 fish samples contained 2,4-D residues. Only 2 to 307 samples contained 2.4-D residues after 14 days post treatment, and these amounted to 5 -id;- and 10 ppb in largemouth bass samples collected from a Georgia pond on day 28 post treatment. The high concentration of the study was 1.08 ppm in a catfish sample collected in Florida, one day after treatment. Most other residues were 50 ppb or less. This study provides perhaps the strongest field data in support of the non-persistence of 2,4-D *n fish. Toetz (1976) reported residues of 2,4-D in fish from Lake Fort Cobb, Oklahoma, but the information provided is badly confounded by the presence of high 2,4-D levels in the "control" samples. w C < Based on the above laboratory and field data, ample evidence suggests 2,4-D is neither accumulated nor persistent in fish tissue. This has been demonstrated in salmonids, under laboratory conditions (Rodgers and Stalling, 1972) but, for the purpose of decision-making regarding Northwest waters, a verification under field conditions would be desirable. Persistence in Plants Little information was located regarding the persistence of 2,4-D in aquatic plants, although the recent study by Lim and Lozoway (1978) provides a good indication of what is probably the typical persistence of 2,4-D in water milfoil in Northwest waters. Plants contained very high 2,4-D levels (60 to 80 ppm) immediately after BEE treatments, but residues declined to 1 to 3 ppm by one week and persisted at these levels for about 5 weeks post treatment. Residues averaged 0.8 and 0l5 ppm at 72 days post treatment but had declined to non-detectable l'evels at day 156. Daly (1971) reported that 2,4-D persisted in the dead milfoil of a warm Georgia lake at 4 ppm for 14 days, at which time the plants decomposed. Smith and Isom (1967) found that three samples of water milfoil collected 24 hours after BEE treatments con tained less than 0.14, 3.36, and 8.36 ppm 2,4-D but no additional samples were analyzed. It is hardly surprising that 2,4-D reaches high concentrations in aquatic plants, since they are the target species. The subsequent persistence depends on whether the plant is killed by the accumulated herbicide. If death occurs, persistence is not dependent on plant action but either the rate at which the plant tissue is decayed by microorganisms or the rate at which the residual 2,4-D diffuses out of the dead plants. This area has apparently not been studied. If the plant is not killed, metabolism of the 2,4-D could probably occur by any of the mechanisms known for terrestrial plants, as outlined in Kearney and Kaufman (1975). Information may exist regarding the meta bolism of 2,4-D by aquatic plants, but this was not actively sought during the literature search. 2,4-D BREAKDOWN PRODUCTS OR METABOLITES Freshwater fish are not known to metabolize. 2,4-D (Stalling and duckins, 1978? Sikka et al., 1977). Whether invertebrates can metabo lize 2,4-D has not been studied. Phytoplankton are known to at least hydroxylate 2,4-D (Valentine and Bingham, 1974). Plant and microbial CG04SS4 2.4- D metabolism have been rather extensively studied in terrestrial species (Loos, 1975)/ but less so in aquatic species (Paris and Lewis, 1973), and while it would seem reasonable to presume that terrestrial organism pathways could be transferred to the aquatic system, Sanborn (1974) was not able to do so in his model ecosystem study. In that test, six known 2,4-D metabolites did not correspond to any of the seven unidentified metabolites isolated during the study* The author labeled these findings as "disturbing." Photodecomposition may also play a minor role in the breakdown of 2,4-D in water, as discussed earlier. The known metabolites of 2,4-D produced by microorganisms (Paris and Lewis, 1973) include: DOW749360 6-bydroxy-2,4-dichlorophenoxy acetic acid 2.4- dichlorophenol 4-chlorocatechol 3.5- dichlorocatechol cis,cis-3-chloromuconic acid cis,cis-2,4-dichloromuconic acid 4-carbxymethylene but-2-enolide maleyl acetic acid chloro maleylacetic acid 3-ketoadipic acid 2-chloro-4-ketoadipic acid chlorosuccinic acid * succinic acid chloride carbon dioxide water Plant 2,4-D metabolism products include many of the above plus a number of additional hydroxylated and conjugated forms (Loos, 1975). Under the presently proposed photodecomposition scheme (Crosby, 1972; Crosby and Tutass, 1966) 2,4-D forms one of three phenolic compounds which in turn are readily hydroxylated to form humic substances, com mon natural organic compounds. .PERSISTENCE OF METABOLITES OR BREAKDOWN PRODUCTS Most of the metabolites listed in the preceding section are intra-cellular intermediates whose exact persistence is not known. 2.4- D molecules enter the cell and, sometime later, carbon dioxide, water, .and chloride are produced. If the intermediates never leave the cell, they can be of no environmental consequence. However, a nulfiber of examples of only partial 2,4-D metabolism have been cited in the reviews by Loss (1975) and Allebone et al. (1975).. Presumably, aquatic plant and bacterial cells that are unable to fully metabolize 2.4- D would excrete the metabolites into the ambient water. The fate of such molecules is unknown. In the only report located of direct aquatic relevance, Sharpee (1973) found that during a laboratory microbial degradation of 50 ppm 2,4-D in water and mud requiring eight -147- QGQ4t>L< f 8031 (JV V days for completion, 0.1 ppm 2,4-dichlorophenol appeared in the water for two days and then disappeared over an additional two days, all before the initial 2,4-D was completely degraded. Photodecomposition of 2,4-D in the laboratory has been shown to release 2,4-dichlorophenol into the water (Aly and faust, 1964; CrtSsby and Tutass, 1966; Boval and Smith, 1973; Zepp et al., 1975), however, Aly and Faust (1964) also showed that the subsequent photodecomposi tion rate an environmental pH values of the formed 2,4-dichlorophenol is 10 to 20 times faster than that of 2,4-D. Furthermore, Boval and Smith (1973) demonstrated in the laboratory that the presence of 2,4dichlorophenol catalyzed the more rapid photodecomposition of the parent 2,4-D molecule. It would seem, then, that the persistence of 2,4-dsichlorophenol at the levels formed by biotic or abiotic factors, is not likely to be a problem in the aquatic environment. More will be said below regarding 2,4-dichlorophenol as an impurity in 2,4-D technical formulations. c C c * ACUTE AND CHRONIC EFFECTS OF 2,4-D METABOLITES AND BREAKDOWN PRODUCTS With the exception of 2,4-dichlorophenol, the toxicological effects of the intermiatiary metabolites of 2,4-D are unknown. Of the many papers and reviews on 2,4-D metabolism, only two mention concerns about the toxicological impact of metabolites. Bollag et al. (1968), In reference to 2,4-D metabolites, state that "the toxicity of these products to higher plants or to other organisms is unknown. Inasmuch as they are found in cultures of a microorganism obtained from soil,, they may accumulate during the decomposition of phenoxy herbicides. In this regard, it is of interest that compounds containing unsatur ated lactone rings commonly possess some form of physiological activity. Sharpee et al. (1973) repeat this warning about the unsaturated lac tone ring, specifically, chlorobutenolide. The ultimate 2,4-D breakdown products, either humic acids, suc cinic acid, carbon dioxide, chloride, or water, are all natural, completely-acceptable compounds or elements. Furthermore, that such end products, or even perhaps intermediary metabolites, may be used in the growth process of aquatic animals, with no discernable adverse effects, was elegantly demonstrated by Stalling and Huckings (1978). PURITY OF TECHNICAL 2,4-D FORMULATIONS Since inquiries by the reviewer to chemical manufacturers were not answered, the purity of technical 2,4-D formulations had to be determined from the generally inadequate information available in the published literature. All of the major reviews on the safety of phenoxy herbicides National Research Council of Canada, 1978; Ramel, 1978; Council for agricultural Science and Technoogy, 1975; Hazardous Materials Advisory Committee* .1974)^have stated either directly or indirectly that 2,4-D -148- CG04oo 8032 does not contain 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD). Pre sumably, some of these committees have received proprietary informa tion to this effect from chemical companies, since published data in support of this contention are usually not offered (see Johnson, 1971). The presence of TCDD in 2,4-D is not expected based on the raw chemi cals and techniques used in the production process, and Wollson et al. (1972) found none in 28 samples of 2,4-D analyzed. Collins et al. (1971) and Khera and McKinley (1972) found no TCDD in the 2,4-D they used for mammalian toxicological studies. The following impurities have been reported in 2,4-D by various investigators: 2,4-dichlorophenol (Faust and Aly, 1963) 2,6-dichlorophenoxyacetic acid (Hazardous Materials Advisory Committee, 1974) Bis-(2,6-dichlorophenoxy) methane (Huston, 1972) 2,2',4,6'-tetrachloro-diphenoxy-methane (Huston, 1972) B i s - (2,4-dichlorophenoxy) methane (Huston, 1972) Hexachlorodibenzo-para-dioxin (Woolson, 1972) Most of these reports are relatively dated; the degree to which they reflect either the qualitative or quantitative chemical impuri-. ties associated with current 2,4-D production techniques is not known. In the reviewer's opinion, the chemical impurity of the 2,4-D formulalions used for aquatic plant control should be regarded as an unknown, 'unless specific information is provided by the manufacturer. EFFECTS AND PERSISTENCE OF 2,4-D IMPURITIES Of the impurities listed in the previous section, toxicological and persistence data are available only for 2,4-dichlorophenol. In the only report located on the toxicity of 2,4-dichlorophenol, Holcombe and Phipps (1979, abstract only) found that 3.11 ppm 2,4-DCP reduced the growth of fathead minnow fry in 32 day exposure tests. The "safe" concentration for fathead minnow eggs and fry was estimated to be between 2 and 3 ppm 2,4-DCP. Faust and Aly (1963) determined the degree of 2,4-DCP contamina tion of several 2,4-D formulations and conducted a series of labora tory tests on the persistence of 2,4-DCP in water under various conditions. The granular BEE formulation tested contained 560 ppm 2,4-DCP, an amount found empirically to contaminate a 1 ppm applica tion of BEE with about 10 ppb 2,4-DCP. This level was above reported taste and odor detectability limits. Under clean water conditions, this level of 2,4-DCP was stable in water for a period of 218 days, much as 2/4-D itself is stable under similar conditions (see earlier section on persistence in water). The persistence time could -be shor tened if sewage or ethanol was added to the test flasks and in one -149- s ( test with aerobic, buffered lake water, the half-lives of 500 and 100 ppb solutions of 2,4-DCP were found to be only six days in each case. Again, it is unknown whether the current 2,4-D formulations are contaminated with 2,4-DCP to the degree noted in this 16 year old study. Whether they are or not, it appears that 2,4-DCP breakdown is subject to the same constraints as noted earlier for 2,4-D. Thus, if the proper conditions are not present 2,4-DCP will persist; if they are present, breakdown can be rather rapid. The 2,4-DCP concentra tions likely to result from the use of contaminated 2,4-D would not be acutely toxic to the fathead minnow. The effect on all other organ isms is unknown. As discussed earlier, the potential water and fishflesh tainting problem with 2,4-DCP has not been adequately resolved. POSSIBLE ACCIDENTS DRING HERBICIDE TREATMENTS The sinking of an application boat would not be a particularly desirable event within an herbicide treatment program but the sequelae could be minimized if the herbicide was kept containerized at all times during the application process. No reports regarding any type of unusual precaution necessary for the safe application of 2,4-D were located Of the ^ : control, the ester formulations appear to present greater inherent risks since n acute toxic effects are imminent if their hydrolysis is delayed in nature; 2) their rate of hydrolysis in Northwest waters has not yet been positively determined, and 3) past studies in other areas of the country have demonstrated some highly undesirable patterns of per sistence associated with the use of ester formulations. These inher ent risks are eliminated by the selection of 2,4-D formulations other than esters for use in any further investigations or applications. It is the reviewer's strong recommendation, then, that should 2,4-D be seriously considered for organized aquatic plant control in Washington State, that only non-ester 2,4-D formulation (sodium 2,4-D, 2,4-D DMA) be considered for use. Additional research should precede any extensive use of 2,4-D salts in Washington. The critical information lacking at the present time are the results of a field study conducted with 2,4-D in the Lake Washington area. Such a study, if thoroughly done, might answer the following questions; 1. Are the generalizations based on the overall literature regarding the toxicity of 2,4-D to algae, invertebrates, and fish, valid for Lake Washington species? 2. Are the patterns of 2,4-D persistence in Northwest waters accep table, particularly with respect to residues in algae, inverte brates (especially zooplankton and mussels), and fish (especially salmonids)? 0004263 -1 =;n_ 3. Is there any evidence of ecosystem stimulation by 2f4-D in Northwest waters? 4. What are the secondary effects of 2,4-D in Northwest waters? Also necessary is: 1. More information on 2,4-D purity. 2. Further testing on organisms of the type done by zimakowskaGnoinska (1977), i.e., long term effects of a single high dose. 3. More information regarding the current status of the tainting problem. 4. At least a partial chronic toxicity test with all life stages of a salmonid species. 5. Some information on whether 2,4-D is carcinogenic in fish. An interesting set of data would be the results of a thorough present-day ecological survey of one of the branches of the TVA reser voirs that were heavily treated with 2,4-D ten years ago in comparison to a similar study of an appropriate control area. Could or would such a survey detect any long-term effects ao*tributable to the 2,4-D treatments? Should milfoil control with 2,4-D be contemplated in the absence of local testing, special attention should be given to the ongoing experiments on chemical weed control being conducted by the Canadian government in the Okanagan basin. CC04263 -151- (. 8 0 3 5 D O W 749365 LITERATURE CITED ALABASTER, J.S. 1969. Survival of fish in 164 herbicides, insecti cides, wetting agents, and miscellaneous substances. Internatl. Pesticide Control 11(2): 29-35. ALLEBONE, J.E., R.J. HAMILTON AND B. RAVENSCROFT. 1975. Environmental organic chemistry of 2,4-dichorophenoxyacetic acid. Environ. Chem. 1: 160-190. ALY, O.M. AND S.D. FAUST. 1964. Studies on the fate of 2,4-D and ester derivatives in natural surface waters. J. Agr. Food Chem. 12(b): 541-546. ALY, O.M. AND S.D. FAUST. 1965. Removal of 2,4-dichlorophenoxyacetic acid derivitives from natural waters. J. American Water Works Assoc. 56 (2): 221-230. AMCHEM PRODUCTS, INC. 1978. The acute toxicity of 2,4-D equivalent aqua-kleen, 20% by weight lot #A10884-1 to the coho salmon Onchorhycus kistutch (Walbaum). ANONYMOUS. 1978. Aquatic hazard evaluation of chemical pesticides. Bioscience 28(9): 600-601. 1" & V ER ITT, W.K. AND E.O. GANGSTAD. 1976. Dissipation of residues of 2.4- D in static waters. J. Environ. Qual. 5(2): 145-149. BARTLEY, T.R., JR., AND A.R. HATTRUP, JR. 1970. 2,4-D contamination and persistence in irrigation water. Proc. West. Soc. Weed Sci. 23: 10-33. BERTAGNOLLI, 3.L. AND M.J. NADAKAVUKAREN. 1974. Physiological responses of Chlorella pyrenoidosa to 2,4-dichlorophenoxyacetic acid. J. Ex. Bot. 25: 180-188. BINGHAM, S.W. 1972. Improving water quality by removal of pesticide pollutants with aquatic plants. Project Completion Rept. OWRR A033-VA, Nat. Tech. Infor. Serv. No. PB-213-495. 70 pp. BIRO, P. 1979. Acute effects of the sodium salt of 2,4-D on the early developmental stages of bleak, Alburnus alburnus. J. Fish Biol. 14: 101-109. BOEHM, H.H. 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Dynamics of an ester of 2,4-D in organs of three fish species. Weed Sci. 20(1): 101-105. .-RUBINSTEIN, R., E. CUIRLE AND H. COLE. 1975. Test methods for * assessing the effects of chemicals on plants. EPA, Office of Toxic Substances, Washington, D.C. EPA 560/5-75-008. SANBORN, J.R. 1974. The fate of select pesticides in the aquatic environment. U.S. NTIS JPB-239-749, 83 pp. SANDERS, H.O. 1970a. Pesticide toxicities to tadpoles of the western chorus frog, Pseudacris triseriata and Fowler's toad, Bufo woodhousii fowlerii. Copela 1970(2): 246-251. SANDERS, H.O. 1970b. Toxicities of some herbicides to six species of freshwater crustaceans. J, Water Poll. Cont. Fed. 8: 1544-1550. SANDERS, H.O. 1969. Toxicity of pesticides to the crustacean Gammarus lacmstris, U.S. Dept. Interior, Fish and Wildlife Serv. Tech. Paper 25. 18 pp. SANDERS, H.O. AND O.B. COPE. 1968. The relative toxicities of several pesticides to naiads of three species of stonefies. Limnol. Oceanoq. 13(1): 112-117. SCHAFFER. 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Monogr. 39(1): 79-100. . -* T O E T Z , D. 1976- Residues of 2,4-D in flesh of selected fish species in Lake Fort Cobb as a result of herbicide use on Eurasian milfoil, Myriophyllum spicatum. Unpublished report, Bur. Reclamation, U.S. Dept. Interior, Amarillo, Texas. TRUHLAR, J.F. AND L.A. REED. 1976. Occurrence of pesticide residues in four streams draining different land use areas in Pennsylvania, 1969-1971. Pest. Monit. J. 19(3): 101-110. TUCKER. R.K. AND D.G. CRABTREE. 1970. Handbook of toxicity of pesti cides to wildlife. U.S. Bur. Sport Fish, and Wildlife Resource Publ. No. 84. 131 pp. VALENTINE, J.P. AND S.W. BINGHAM. 1974. Influence of several algae on 2,4-D residues in water. Weed Sci. 22: 358-363. VANCE, B.D. AND D.L. SMITH. 1969. The effects of five herbicides on three green algae. Texas J. Sci. 20: 329-337. VOIGHT, R.A. AND D.L. LYNCH. 1974. Effects of 2,4-D and DMSO on pro caryotic and eucaryotic cells. Bull. Environ. Contam. Toxicol. 12(4): 400-405. VENKATARAMAN, G.S. AND B. RAJYALAKSHMI. 1972. Relative tolerance of nitrogen-fixing blue-green algae to pesticides. Indian J. Aqri. Sci. 42(2): 119-121. WALKER, C.R. 1971. Herbicide chemicals and their effect on the aquatic environment. 24th Ann. Meet. So. Weed Sci. Society, Unpublished Manuscript. WABKER, R.L. AND A.S. NEWMAN. 1956. Microbial decomposition of 2,4dichlorophenoxyacetic acid. Appl. Microbiol. : 201-206. WALSH, G.E. 1972. Effects of herbicides on photosynthesis and growth of marine unicellular algae. Hyacinth Contr. J. 10: 45-48.. -167- OGG&S5 T v V {)J WARNOCK, J.W. AND J. LEWIS. 1978. The other face of 2,4-D: A Citizen's Report. South Okanogan Environmental Coalition, Pentictin, Brit., Col. Penticton Commercial Printing, Ltd. WASHINGTON COOPERATIVE FISHERY UNIT. 1974. A study of the aquatic life of the University of Washington Canoe House area. Unpublished MS. 25 pp. WATSON, J.R. 1977. Seasonal variation in the biodegradation of 2,4-D in river water. Water Res. 11(2): 153-157. WEBER, W.J., JR. AND J.P. GOULD. 1966. Sorption of organic pesti cides from aqueous solution. pp 280-304. In: Rosen, A.A. and H.F. Kraybill (Eds.) Organic pesticides in the environment. Advan. Chem. Ser. GO. 309 pp. WEDEMEYER, G. 1966. Microorganisms and pesticides at Seattle. In: Progress in Sport Fishery Research, 1965. Bur. Sport. Fish, and Wildl. Res. Publ. 17: 54-55. WEED SCIENCE SOCIETY OF AMERICA. 1974. Herbicide Handbook. Third edition. WEISS, J.S.E.V. 1976. The morphological changes and survival effects observed among monkey kidney cells, girardi heart cells, and *. rainbow trout gonad cells, in vitro, in response to exposure to * various herbicides. Piss. Abstracts Int. 36(8): 3736B. WHITE, S.T. 1975. The influence of piers and bulkheads on the aquatic organisms in Lake Washington. M.S. thesis, College of Fisheries, Univ. of Washington. WHITNEY, E.W., A.B. MONTGOMERY, C.E. MARTIN, AND E.O. GANGSTAD. 1973. The effects of a 2,4-D application on the biota and water quality in Currituck Sound, North Carolina. Hyacinth Contr. J. 11: 1317. WIERZBICKA, M. 1974. Hemol-ymph concentration in Cyclopoida copepodids during active and resting stage and the efect of 2,4-D sodium salt. Pol. Arch. Hydrobiol. 21 (2): 269-273 WIERZBICKA, M. 1974. Influence of 2,4-D sodium salt on the survival of some copepoda species. Pol. Arch. Hydrobiol. 21(2): 275-282. WITHERSPOON, J.P., E.A. BONDIETTI, S. DRAGGAN, F.P. TAUB, N. PEARSON, AND J.R. TRABALKA. 1976. State-of-the-art and proposed testing for environmental transport of toxic substances. Environ. Sci. Div. Publ. No. 893. Oak Ridge Natl. Lab., ORNL/EPA-1, 165 pp. WILKINSON, R.E. 1964. Subaqueous release of herbicides from granules. Weeds 12: 69-76. 042SG -168- "OJTALIK, T.A., T.F. HALL, AND L.O. HILL. 1971. Monitoring ecologi cal conditions associated with wide scale applications of DMA 2,4-D to aquatic environments. Pest. Monit. J. 4(4): 184-203. 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Production of chemical transfor mation of pollutants in the aquatic environment, pp. 237-263. In: Hutzinger, 0., L. H. Van Lelyveld, and B.C.J. Zoetaman (Eds.) Aquatic Pollutants: Transformation and Biological Effects ZIMAKOWSKA, D. 1973. Effects of 2,4-D sodium of various con centrations on respiration of an aquatic crustacean Asellus acuaticus. Pol. Arch. Hydrobiol. 20 (3): 469-473. ZIMAKOWSKA-GNIONSKA, D. 1977. Toxicological and physiological aspects of the action of herbicide, sodium salt of 2,4-D, on Asellus aquaticus. L. (Isopoda). Pol. Arch. Hvdrobiol. 24 (3): 389-411. 0004237 E DO W 327J78 sud. uy v.fL. owe K . Olsen C. O'Hare l'ilo ill_>a AJ U . C . J \J\S\J\JJ. y K No. 66681-2 Charge 102-0007360-A THE EFFECTS OF REPEATED APPLICATIONS OF SYMMETRICAL TETRACHLORODIBENZODIOXIN ON GUINEA PIG EARS Problem To determine whether folliculitis is produced in the guinea pig ear following repeated applications of symmetrical tetrachlorodibenzo dioxin in benzene. M a te r ia ls Used 0.01# and O.OOI56Symmetrical tetrachlorodibenzodioxin isolated from caustic insoluble oil. Vehicle - benzene. Experimental Procedure 0.1 cc/ear/day of each solution described above was applied to the outer surface of guinea pig ears. Five animals were used with each solution strength. Three animals were employed as benzene controls. The total animals used numbered thirteen. Results and Conclusions All test animals rapidly lost weight and became quite ill. Three of the animals exposed to the O.OI56 concentration died after four, five and six applications respectively. All control animals gained weight and appeared outwardly quite Healthy. Gross autopsies on the test pigs revealed no severe liver or kidney damage. Results strongly suggest that the guinea pig absorbs symmetrical tetrachlorodibenzodioxin systemically and that six ear applications of the O.OI56solution in benzene comprise a lethal dose. Under the conditions of the experiment folliculitis was not produced in the guinea pig ear following six applications of O.OI56 symmetrical tetrachlorodibenzodioxin in benzene. C. P. O'Hare Biochemical Research Laboratory 1701 Building CPCisjl / 8S5 0000524 l )V/027179 Table THE EFFECTS OF REPEATED APPLICATIONS OF SYMMETRICAL TETRACHLORODIBENZODIOXIN ON GUINEA PIG EARS W eig ht Animal Number Of S o lu tio n Days On S t a r t Of When Number A p p lic a tio n s S tre n g th Exposure Exposure Autopslec L iv e r Autopsy Kidney Lungs 1 5 O .O l# 7 401 226 Cong. + L ig h t in color + Cong. + 2 5 O .O l# 7 497 314 O.K. "" Cong. + Cong. ++ 3 5 0 .0 1 # 6 440 337 Died a f t e r fiv e a p p lic a tio n s . 4 6 0 .0 1 # 7 471 318 Died a f t e r s ix a p p lic a tio n s . 5 4 0 .0 1 # 6 510 319 Died a f t e r fo u r a p p lic a t io n s . ~6 5 0 .0 0 1 # 7 452 312 O.K. Cong. + 7 5 0 .0 0 1 # 7 442 324 O.K. Cong.? 8 6 0 .0 0 1 # 7 469 350 O.K. O.K. 9 6 0 .0 0 1 # 7 471 340 O.K. ? 10 6 0 .0 0 1 # 7 489 382 O.K. Cong. + O.K. O.K. Cong. + Cong. + O.K. 11 5 Benzene 7 329 351 O.K. Cong. +++ Cong. ++ Pneumonia ++ 12 o Benzene 7 389 409 O.K. ? 13 6 Benzene 7 464 475 O.K. ? O.K. Pneumonia 0000525 Qj Q> 114 I ~14 _ C f) total, viral -ill 1A as hu ey^Jas;;!. TLj abundant nuclear, viral RN/ csqassca at 10 h'byo iraxckdbiJ from a contiguous region, 65% of the genorm in length. In som s cxss,.viral.RNA sequences complementary to mRN/ sequences are abundant in the nnnlp.s VJhm r^rtnUc^a ~-'1 -- *-- rr) -)JL sas &stracts il uu3 (S No o 4 '^Fcaisaceffl'uiina' tbs, late stage of infection./r *' o *NJ Vrt ro 00 o - ! ^ 0mKS ^ ^ .iK' /:;,. J E N S S ^ ,:D A G ^ ;a n d :LAKS RENBER#G .'(D iv. Genet., Enviror Toxicol. Unit, Wallenberg Lab., Lilia Frcscati, 104 05 Stockholm 50, Swed. .... EbSributccn aad;cy0cg2aeak:QC`5t o f 2,4-IB and 2,4,5 -T pbenoxyacetic acids i ; tSsai1 ijte s a . C H E ^ -B IO L INTERACT 14(3/4): 291-299. 1 9 7 6 - The phenoxyacetic acids 2 ,4 - 0 and 2,4,5-T , extensively used as herbicide were tested for cytogenetic effects by means o f induced micronuclei i erythrocytes of mouse bone marrow. Because of the high experimenta resolution power, this is a particularly suitable test system for the detection o weak chromosome breaking activity in mammals. The cytogenetic tests wer supplemented with chemical analyses of the concentration of the tes substances reaching the target cells. The cytogenetic tests 24 h and 7 day after the treatment with 2 ,4 -D or 4,5-T . showed no detectable increase o micronuclei in the erythrocytes as compared to the controls. A weak toxi CGfM C'3 3 f 11 [April 1977] effect o f 2 ,4 -D and 2,4,5-T on the mitotic activity was indicated, as judged by the decrease of the percentage of polychromatic erythrocytes. Gas chromatographic analysis of plasma and cell fractions from blood and bone marrow showed that less than 5% o f the test substances present in the plasma appeared in the cells. The lack o f penetration of the phenoxyacetic acids in the cells is in accordance with the rapid excretion of them known to occur in the mammalian body. Although this experiment does not constitute a reliable measure of the mutagenic potential of 2 ,4 -D and 2,4,5-T , in practice the lack of penetration o f these substances into the cells indicates that they do not constitute cytogenetic hazard to man. O <5 CD o iSD i. t' lestca zT 'r.n ir i -. k* IV. ' J.S i f . ; v jy -Y IJ ur. ^ .s - i ^ , | :^h'17v-..'- tir ~s. f ."th"*?- '' -id :,: e'.'v*-<jh.'ivTjjc'.r-rtcir.. ', _>jrli i i t -ri W. )ir . rr-.-up S ' lits ;.i .-s k : ' s- t .* . * i p r i '-'r.' C sifcia- is h c ris i z-.rj . Jtion.", sj:v :i :*'* a-. :hf }c-, s .a ..vu: Trnrt-i- * to , '- f f t i i t sport :. . :j.e . . . in u s env: r.-ssvsi p r're p ris Jo rin c ( ?1* ne..:n: jr.i t ' tt. *t-.: Jacted f o r ' j1 document ' >d by th e many y i n th e io s t fYJW-.' I5*V*\n.:;;i.,- p;.1 te- - *.yy- O -- :---- =S1- ro Coi" o. 20 y o a rn , b7 th e UK* ior' ... . 1 A ... ^ .. (o. accession to t ro ; rec ./. . . d . une Foc A j & v ' $ 7 7 ;/<2t and . and y.od. Vrf. f[/0 </ * Roc th e EEC, w ith th e r e s u lt in g need to ' ice w ith basis p ro v is io n s o f th e A ct0 , ar/1 by th*: s u b s ta n tia l amendment n r ; r o th e r l e g is l a t i o n . The l a t t e r changes .'. > c o n fu s io n ; Anyone w is h in g t o suggest r England and Valeo should w r ite to the. r i c u lt u r o , F is h e rie s and F o o l, Boon f Road London. SWiP 2AE. In S co tla n d reuses are Foods 2 ra n ch , C c n ttic h Rone . lrew s Rouse, E dinburgh, EDI JDS, and " ' : artm ent o f H e a lth and S o c ia l S e rv ic e s , "'JSB. 2 ,4 - 2 A2ID 2 ,4 ,5 -T RSVIE-JED K - W ? 2 - O 0 O) K-VS6Q-Oeo) " A r e p o r t'e n title d "The S a fe ty o f the H erbicides 2 ,4 -2 and 2 ,4 ,5 -T ", by . 2 . J . l 'u rn e r , has been iB sued os F o re s try C o m is s io n B u lle t in n o .57 (EMSO, London, 1977\ p p . 5 6 , 1 .2 0 ) . I t re v ie w s th e p r o p e r tie s , m a n u fa c tu re , mode o f a c tio n and unes o f 2 ,4 -2 and 2 ,4 ,5 -T , t h e i r o ffo c ts on dom estic liv e s to c k and man, e f f e c t s on o th e r la n d organism s and p e rs is te n c e in t e r r e s t r i a l -. environm ents, and a c t i v i t y and p e rs is te n c e in a q u a tic environm ents. Hie r e p o r t. in g e n e ra l p re s e n ts a re a s s u rin g p ic tu r e o f th e tw o h e rb ic id e o , now th a t 2 ,4 ,5 -T is produced e s s e n tia lly fre e o f 2 .3 ,7 ,8 -to tra c h lo ro d lb o n z o - -d lo x ln , . a lth o u g h i t acknowledges th e p o s s ib ilit y o f in d ir e c t o ffo c ts on anim al com m unities as a r e s u l t o f rem oval o f t h o i r p la n t fo o d s and s h e lt e r . The hazard t o man fro m th e d i l u t e s o lu tio n s th a t are row uaed f o r moat weedc o n tro l treatm ents is regarded as low , even i f contam ination o f food c r -w ater s u p p lie s should o c c u r. o O o o IO rc . HEALTH Aim SA5ETI CDIBAiJCE KOTES : The T e c h n ic a l B a ta IloteB p r e v io u s ly is s u e d by EM F a c to ry In s p e c to ra te , and th e C S lA 's Dotes o f Guidance produced by th e Employment M edical A d viso ry S e rv ic e , are being p ro g re s s iv e ly replaced by Guidance IJctes from the H e a lth and S a fe ty E x e c u tiv e . These a re p u b lis h e d i n f i v e s e rie s , d e s i& ia te d M e d ic a l, E n v iro n m e n ta l H yg ie n e , C hem ical S a fe ty , P la n t and M a c h in e ry , and G e n e ra l, and a re a v a ila b le fro m EK30 a t a p r ic e o f 5Qp each. I n th e m e d ic a l s e r ie s , G uid.incc ilo to o MS 1-9 d e a l r e s p e c tiv e ly w it h le a d -- a e -lis a l s u rv e illa n c e ,, a n th ra x , s k in te s ts in d e rm a titis and o ccu p a tio n a l chest .d ise a se , organic duct surveys;, lung fu n c tio n , chest X -raya in dust diseases, c o lo u r v is io n and is o c y a n a te s -- m e dical s u r v e illa n c e . In th e e n v iro n m e n ta l lyric.** s e r ie c , h e a lth o n i s a fe ty p re c a u tio n s f o r cadmium, chromium, a n ilin e , .ir s e n lc , a r s in e , n t ib in e .md t e i y l l i i a a r t th v s u b je c ts o f EH 1, 2 , 4 , 9 , 11, 12 and 1 3 r e s p e c t iv e ly , w h ile Eli 5 d e a ls w ith tr ic h lo r o e t h y le n e and EH 10 i s ?n hygiene s ta r.j-.ru r. f.-.r asbestos and mc-usurc-ac-nt o f. i t s c o n c e n tra tio n s i n a irfccm e d u s t. F -rs ig a tio n u s in g m ethyl b r '- a iie and guidsmee on a r t ic le s and substances f o r ;;so a t work a re the. s u b je c ts o f nos 1 and *i, r e s p e c t iv e ly , in the general s e rie s , the l a t t e r d e a lin g v/.cn c e rta in p ro v is io n s o f the H ealth and S a fe ty a t Work e t c . A c t . 1974. IIABIOACTIVITY 2ECLIKES FUHTKSl. ` F u rth s r evidence o f the c o n tin u in g d e c lin e in radioactive- f . illc u t v s u ( j , $0 ; \ .1 1` ; S *:,*.*. 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