Document NNmwgkZL9aKZoVqZepbojBJyw

TOXICOLOGY AND APPLIED PHARMACOLOGY 17, 656-668 11910} Comparative Toxicologic Study with Polychlorinated Biphenyls in Chickens with Special Reference to Porphyria, Edema Formation, Liver Necrosis, and Tissue Residues J. G. Vos AND J. H. Koeman Institute of Veterinary Pathology and the Institute of Veterinary Pharnnn ,,/,>?* and Toxicology, University of Utrecht. Utrecht. The Netherlands Received November j, 1969 Comparative Toxicologic Study with Polychlorinated Biphenyls in Chickens with Special Reference to Porphyria, Edema Formation, Liver Necrosis, and Tissue Residues. Vos, J. G. and Koeman, J. H. (1970V Toxicol. Appl. Pharmacol. 17, 656-668. In a comparative 60-day ora] toxicity test (400 ppm) in chickens, three GO"(.-chlorinated commercial polychlorinated biphenyl (PCB) preparations were used: compound I (Phenoclor DP 6), compound II (Clophen A60). and compound III (Aroclor 1260). Using mortality, mean survival time, mean weight, and pathological observations (hydropericardium, abdominal, and subcutaneous edema and centrolobular liver necrosis) as parameters, a significant difference in toxi city was found between the compounds-: compounds I and II showed the highest, compound III the lowest, toxicity. Microscopically centrolobular liver necrosis was found in chicks fed compounds I and If. Atrophy of the spleen was found in all test groups. Chemical porphyria was found as a general PCB effect: increased fecal excretion of coproporphyrin and proto porphyrin and fluorescence of tissues occurred in alt test groups. Additional experiments with compound I (2000 ppm>in Japanese quail and rats con firmed the porphyrogenic action. Gas chromatographic, analyses of liver and brain of dead chicks gave PCB levels that varied between 120 and 2900 ppm. The relationship of hyctropericardium (chick edema.) and. liver necrosis to the differences in toxicity observed between the technical PCB mixtures is discussed. In recent years the presence of polychlorinated biphenyls (PCB) has been exhibited :a fish and wildlife in many countries, including the United States, Great Britain, Sweden, and The Netherlands (Jensen, 1966; Holmes et aL, 1967; Holden and Marsden, 1967; Koeman et al., 1967; Risebrough et al., 1968; Koeman et at., 1969). Commercial PCB preparations are oily fluids consisting of a mixture of different chlorinated biphenyls. These chemicals are extremely stable with very low aqueous solubility. They are used as lubricants, as fluids for heat transfer and dielectric medix. in protective coatings for wood, metal, and concrete, and for many other applications. Which of these applications has contributed to the present widespread environmental contamination has not been elucidated. The occupational hazards in the use of PC3 have been known for years; normal handling of these compounds has not given rise to great difficulties (Irish, 1963). Animal experiments with rats and guinea pigs have shown that the ingestion of PC3 656 DSW 255379 POLYCHLORINATED BIPHENYLS TOXICITY 657 may give rise to liver injury (Miller, 1944). Hydropericardium and abdominal edema were found in chicks (McCune et a!., 1962; Flick, et al., 1965) and Japanese quail (Koejnan et al., 1969) after PCB ingestion. Risebrough et al. (1968) found that the rate of estradiol metabolism is markedly increased after PCB treatment in pigeons. This may indicate that these derivatives may induce hepatic hydroxylases. The data presently available do not permit a proper toxicologic evaluation of the amounts detected in vildkfe specimens. Koeman et al. (1969) found a remarkable resemblance between the evs chromatograms and mass spectrometer analyses of purified extracts from eider duck (issues and the commercial PCB preparation Phenoclor DP 6. Similar gas chromatograms and identical mass numbers were obtained from other commercial preparations, Clophen A60 and Aroclor 1260. These 3 preparations, which contain on the average 6 chlorine atoms per molecule, were used in the present comparative toxi cologic study, chicks, quail, and rats being used as the experimental animals. METHODS The PCB preparations were obtained from Prodelec in France (compound I, Phenocor DP 6), Bayer in Germany (compound II, Clophen A60 Lot No. 912434), and Mon santo in the United States (compound III, Aroclor 1260 Lot No. AK.-3). One-day-old cockerels (Hubbart laying breed) were used in these studies. The chicks were kept under 5.continuously operating heating lamp throughout the experiment. On the second day coey were weighed, individually wing-banded, and distributed randomly into 4 groups. Tan birds received diets containing PCB as follow-s: group I, 400 ppm compound I; group 11.400 ppm compound II; group HI, 400 ppm compound III; group IV served as control. They were fed and watered ad libitum and were weighed twice weekly. The feed con sisted of a common ty pe of starter ration. The PCB was dissolved in acetone and mixed carefully into a premix. After this the acetone was evaporated and the premix was mixed cue uic ration. The chicks were kept on these rations for 60 days and killed. These birds *nd the animals that died during the experiment were necropsied. Two chicks from sroup 11 died during the first 3 days. At necropsy it appeared that they were not killed by die PCB treatment (no liver necrosis, no edema, no tissue fluorescence). They were discarded and not included in the calculations. At necropsy hydropericardium volume was determined by inserting a blunt-tipped "cedle on a 2-ml syringe into a small slit in the pericardium. All the pericardial fluid was removed from the anterior part of the heart sac by intermittent aspiration. The volume -rliie fluid removed was measured to the nearest 0.05 ml. The incidence of abdominal *"41 subcutaneous edema and the weights of the liver and spleen were recorded. The l'yer. spleen, and kidneys were fixed in 10% buffered formalin and processed in a con ditional manner for histologic examination. Sections (7 fx) w'ere stained with hematmvuu-eosin and Perk's iron stain. Moreover, selected sections were stained foramy- ^ nil methyl violet according to Lillie (1965), with Congo red, with PAS, with PAS"''Cian blue, and with Fettrct for lipids. Prompted by experience obtained in parallel -coics with hexachlorobeiuene, observations were made in ultraviolet light using red -ortscence as an indicator for the presence of excess quantities of porphyrins. FluorN;nce ol liver, bile (after opening of the gall bladder), small intestine, and bone w;as DSW 255380 STLCOPCB4061071 658 VOS AND KOEMAN recorded. Fecal coproporphyrin and protoporphyrin were determined by the method described by Rimington (1961), using a Beckman spectrophotometer. PCB levels in livers and brains of selected birds were determined by gas chromato graphy. The tissues were extracted with petroleum ether in a Soxhlet extraction appara tus after drying with anhydrous sodium sulfate. Cleanup of the samples was carried out by liquid-liquid partition with dimethyl formamide and column chromatograph-,, using activated Florisil. The PCB preparations in the final extract were measured by gas-liquid chromatography (204-1 B Varian Aerograph with electron capture detection v. The Pyrex glass column (5 feet x inch), filled with 10 % DC 200 on Gaschrorn Q (80-1 O') mesh), was operated at 200C with nitrogen as the carrier gas (about 50 ml/rnin). Only an approximate quantitative determination of the residue from the chromato gram of the tissue extracts could be made, since the composition of the residue was necessarily identical with the PCB compound used in the treatment. For quantification1 peak, considered to be representative, was selected. This was the peak with rx = 1.45 relative to dieldrin. The approximate amount of total PCB-residue was calculated from the height of this peak with compound I as the standard. In a number of tissue extracts the amounts of PCB were measured also by a Dobrmann C 250 A microcoulometric detection system (with a T-300-S-halogen ceil;., coupled to a Microtek MT-220 gas chromatograph. The chromatograph was equipped with a Pyrex column (6 feet x inch) filled with a mixed-bed column packing (QF ! plus DC 200) described by Burke and Holswade (1966). With this system 0.6 /zg of Phenoclor DP6 gave about a half-scale response on a 1-mV recorder (microcoulomens: range: 200 ohms). By this method the content of chlorine in the extract is determined, by addition of the chlorine contents of the different PCB peaks. Since the average chlorine content of our compounds was 60%, the total residue could be calculated. Here again, the result is an approximation since the residue may have a different chlorine contend Additional experiments with special reference to porphyrin biosynthesis were made with compound I in 5 adult female Japanese quail weighing 112-130 gandS adult female Wistar rats weighing 173-212 g at a dose level of 2000 ppm in the dry food. Monaiity of the animals in both tests was scored, and at necropsy, observations were made under ultraviolet light; occasionally cryostat sections of liver and kidneys were examined in a fluorescence microscope (Zeiss, exciting filters BG 38/2.5 and BG 12/4, barrier filters 53 and 33, Osram super pressure lamp HBO 200 W). Statistical analyses, on a one-tail significance level, were done according the Wilcoxnn test for 2 unrelated samples (van der Waerden, 1957). RESULTS Experiments with Chickens All chicks from groups I and II died during the experiment, while the mortality in group III birds was only 15% (Table 1). Death was accompanied by ruffled feathers and ataxia as the main signs; loss of feathers was often seen. Hydropericardium (Flick et al, 1965) with clear and straw-colored pericardial fluid, was a common finding in groups I and II and was rare (not statistically significant) in group III compared with the control group. Abdominal and subcutaneous edema was observed in groups I and II only, with the highest incidence in group II (Table I). DSW 255381 STLCOPCB4061072 TABLE 1 Mortality, Mean Survival Time and Pathologic Observations o>- Chicks Fed 400 ppm PCB eor 60 Days and of Control Birds Groups Number of .V deaths Mean survival0 time (days) Number of birds with edema Hydropericardium1' Abdominal Subcutaneous Number of bircs with liver necrosis I (Phenoclor) 11 (Clophen) III (Aroclor) IV (Control) 24 24 22 22 20 3 20 0 24.3 (12-58) 20.5 (13-29) -- -- 18 20 3 0 85 97 00 00 9 9 Au 0 " The range of values is shown in parentheses. 6 Positive hydropericardium response was considered to be over 0.2 ml pericardial fluid in chicks 20 days of age and 0.4 ml in chicks 60 days of age. with interpolation for intermediate ages. DSW 255382 STLCOPCB4061073 660 VOS AND KOr.MAN' Macroscopic red fluorescence, under ultraviolet light indicating porphyrins, was detected in all birds of the 3 test groups: fluorescence of liver, bile, small intestine. ;.nij bone were used as parameters. There was a remarkable difference in the location of the porphyrins. The chicks that died during the experiment (chiefly groups [ and II) showed the highest incidence of fluorescence in the bile and small intestine followed bv the lig and bone (Fig. 1). Only bone fluorescence was pronounced in the killed birds from sroco HI (Fig. 1). This finding is in agreement with theexcretion ofcoproporphyrtn ami prot, ,, porphyrin in the feces (Table 2). The excretion in group III reached a maximum at about 4 weeks followed by a gradual decrease to the level of the control birds. Tn the mean time, large amounts of porphyrin had been deposited in the bones. LIVER SMALL INTESTINE 1 | BILE H|[ BONE Fig. 1. Percentage of chicks showing fluorescence under ultraviolet light in liver, bile, small intestine, and bone. A: Chicks that died during the experiment; B: chicks killed at the end of the experiment. Mean liver weight and relative liver weight of the killed birds from group III was sig nificantly increased compared with group IV, mean body weight and mean spleen weight of chicks from group III were significantly reduced (Table 3). Liver necrosis was observed in 9 birds from groups land II (Table 1). It varied between slight (some lobules) and severe (nearly all lobules). The occurrence of liver necrosis was independent of survival time. The foci of centrolobular necrosis extended in many DSW 255383 STLCOPCB4061074 POLYCHLORINATED BIPHENYLS TOXICITY 661 cases from one lobule to another lobule, resulting in a microscopic picture with the por tal triad as the center of the viable tissue (Fig. 2). In some of these sections proliferation of mesenchymal cells between the necrotic liver parenchymal cells could be observed. Fatty degeneration could be seen also (positive for Fettrott). Protein droplets of different ait were sometimes present in the sinusoids near damaged cells as well as in some central veins. Liver parenchymal cells with pycnotic nuclei could be observed in liver sections from all 3 experimental groups. A small amyloid deposit was seen in liver sections of i bird each from groups I and IL - TABLE 2 CbPROPORHIiYRIN AND PROTOPORPHYRIN CONTENTS (fig/g DRY WEIGHT) OF FeCES OF CHICKS v Fed PCB for 60 Days and of Control Birds laT Coproporphyrin . Days after start of feeding I Groups II III IV Protoporphyrin Groups I II III IV ^ 7 -- H 14 19 26 36 58 4.2 3.2 7.1 9.4 12.6 13.4* -- 4.1 3.7 6.0 9.8 36.7-- -- 2.7 2.2 -- 4.4 13.6 10.0 2.5 0.8 0.8 -- 0.6 -- 1.4 2.4 -One bird still alive at the moment of sampling. `.Three birds still alive at the moment of sampling. 8.3 19.4 13.2 25.0 9.7 26.3" -- 15.2 20.0 11.5 16.1 11.5" -- -- 4.5 8.9 -- 25.2 14.2 13.7 5.8 2.8 4.5 -- 2.3 -- 6.1 8.1 'The spleens of all birds in the test groups were small, described already by Flick et al. compared with the controls. In general, in the spleens of all chicks of groups I, H : d rn which died during the experiment a reduction in the amount of the red pulp, ana atrophy of the white pulp and lymphoid foci (Fig. 3), caused by a suppressive nr destructive action, could be observed, resulting in a small spleen with a wrinkled capsule. 1'.' 2 birds from group Ell we found necrosis in the center of a lymphoid focus; in Tsac- birds from groups I and II eosinophilic deposits were seen in the center of the white "-lp (Fig. 31. These deposits were purple with methyl violet, red with PAS, red purple PAS-Alcian blue, and light brown with the Congo red stain. With Congo red stain `Try showed some green birefringence under polarized light, although fluorescence the UV microscope was yellow instead of pink. These data are strong indications amyloid. _* Tubular dilatation in the kidneys, described already by McCune et al. (1962) was also ~3iv,on. usually to a slight degree, in most chicks from groups I and II, but rare in Jtaup in | (ds dilatation was sometimes accompanied by the presence of leukocytes red blood cells in the lumen of the tubules. Perk s iron stain gave the follow ing results: the presence of iron in liver Kupfler ceils lest birds seemed to be about tw ice as much as that in control birds. In some birds STLCOPCB40I TABLE 3 Organ Weights of Killed Chicks Fed PCB (Compound III, Aroclor) for 60 Days and of Control Birds3 Groups Body V (g) Liver (g) Liver (g/100 g body weight) Spleen (g) Spleen (g/100 g body weight) III (Aroclor) IV (Control) 17 703 (514-835)' 29.7 (22.6-35.9)" 4.31 (3.47-5.91)' 20 921 (820-1010) 24.6 (19.5-28.4) 2.67 (2.17-2.97) 0.90 (0.40-1.50)3 1.35 (0.95-1.82) 0.136 (0.048-0.288) 0.146 (0.113-0.185) "The range of values is shown in parentheses. 3 P< 0.0:5. : CP <0.01. J P < 0.005. STLCOPCB4061076 POLYCHLORINATED BIPHENYLS TOXICITY 663 ^lc- 2. Liver of a group I chick with severe cenirolobular necrosis, resulting in a reversed picture with `Portal triad becoming the center (arrow). Note the strong eosinophilic parenchymal cells at the mar- v* necronc and vital tissue. Hematoxylin and eosin. 125. _ P>G. 3. Spleen of a group I chick with atrophy of a lymphoid focus and atrophy of the white pulp. `Awhile pulp has been replaced by homogeneous eosinophilic material, probably amyloid (arrow), ttaatow lin and eosin. . 500. DSW 255386 STLCOPCB4061077 664 VOS AND KOEMAN from all test groups we found iron in the cytoplasm of liver parenchymal cells, but this was absent in control livers. In 8,7,4, and 0 birds from groups I, II, III, and IV, respec tively, iron was demonstrated in macrophages in the spleen and in 7,2, 2, and 0 birds, respectively, in the cytoplasm of kidney tubular cells. Approximate liver residues of PCB in 25 selected birds were determined by gas chro matography. This selection included birds which died in the first part of the test, bads which died later and birds with liver necrosis. Brain analyses were made in 12 In 7 liver extracts the approximate residue was measured by the microcoulometric detection system. The results are given in Table 4. Experiments with Japanese Quail and White Rats The feeding of compound I (2000 ppm) in 5 Japanese quail resulted in mortality be tween 5 and 13 days, associated with the following signs: slight tremor, ataxia, and ruffled feathers. Four quail showed positive fluorescence macroscopically at necropsy. The bird that died last showed a strong fluorescence of liver, kidney, small intestine, and muscles. Cryostat sections examined by fluorescent microscopy revealed an. intensely red fluorescence of all liver parenchymal cells. Examination of the kidneys showed a strong fluorescence of tubular cells, especially in the cortex. Hydropericardium was observed in 3 birds. Sections of the enlarged livers showed lysis, pycnosis, and necrosis of liver cells. Proliferation of probably RES cells was a common finding. Perls's urea stain was positive for both Kupffer and parenchymal cells. ' The feeding of compound I (2000 ppm) to 8 white rats caused a marked loss ofweigjkt as compared with control animals. The rats died between 12 and 56 days after the start, of the experiment. At necropsy 4 rats that died within 3 weeks showed no fluorescence under UV light, the 2 rats that died next had fluorescence of the incisors and the small intestine, respectively, while the 2 rats that died last showed fluorescence both ofinasora and the small intestine. An important finding was the presence of an enlarged liver. 2* was also found in Japanese quail fed compound I (2000 ppm), and a small spleen-Sec tions of the liver revealed a centrolobular degeneration (vacuolated, foamy cells with pycnotic nuclei near the central veins). In some secti ons focal necrosis could be seen.The disappearance of structure in the white pulp of the spleens, together with a reduction ia the red pulp and the presence of green-brown pigment in macrophages was notable: Perls's stain for iron was strongly positive. Therefore siderosis could be diagnosed La tbe Iespen together with its appearance in Kupffer and parenchymal cells of the liver tubular cells of the kidney. _ DISCUSSION From the results it can be concluded that porphyria is a common finding in animals treated with PCB, so commercial polychlorinated biphenyls extend the already Jong list ofcompounds that produce chemical porphyria. Gtfick( 1965,1966) has presented evidence that the chemical pophyrft is the result of the induction of 8-aminolevulinic acid synthetase in liver mitochondria in the case of 3,5-diethoxycarbonyl-l,4-dihydro2,4,6-trimethylpyridine and allylisopropylacetamide. It is of interest to note that Risebrough et al. (1968) have demonstrated that PCB also induces steroid hydroxylases in the liver of birds. DSW 255387 STLCOPCB4061078 rXEi 4 Chemical Analyses of Liver and Brain in Chicks Fed PCB for 60 Days' Group I (Phenoclof) History Liver Braid Group It (Clophen) History Livef Braid Group III (Aroclor) History Liver Brain Died on: 12th day 16th day 21st day4 22nd day4 22nd day4 24th day4 28th day 31st day4 58th day 2200 1300 1900c 120 210 220c 310 210C 180 150c 1400 1400c 2100 2900 . -- 490 -- -- 70 -- 210 -- -- -- -- -- 380 700 Died on: 13th day 14th day 17th day4 17th day 19th day4 20th day 22nd day 22nd day 23rd day 26th day 800 1100 410 1600 190 610 420 420 860 390 -- -- -- 320 120 -- 380 -- -- 320 Died on: 16th day 17th day 31st day Killed on: 60th day 60th day 60th day 950 1100 680 2400 250 340* 220 210 420 -- -- 270 40 -- -- -- * The approximate contents are given in parts per million. * Chicks with liver necrosis. e Results obtained by microcoulometric detection; ail other figures are results obtained by electron capture detection. DSW 255388 STLCOPCB4061079 666 VOS AND KOEMAN In our experiments with compound III in the chicks it was noted that the excretion m coproporphyrin and protoporphyrin decreased to nearly normal values after a max*, mum effect at 26 days of treatment. Whether this is due to an adaptation which enable* the animal to eliminate these compounds more efficiently, to an age-dependent changes elimination capacity or susceptibility, is not known. The high incidence of tissue fluor escence in the dead chicks from groups I and II is remarkable. The excretion of copt^ porphyrin and protoporphyrin is also high at that time. The possibility has to be con sidered that the presence of large amounts of porphyrins could have a toxic effect in as own, and may have contributed to the death of these birds. The earlier manifestation of porphyria in chicken and quail, compared with the ru, resembles the difference found in hexachlorobenzene-induced porphyria: early ma*. festation of porphyria in the Japanese quail, late manifestation in the rat (Vos et at. 1968). Flick et al. (1965) observed that chicks fed PCB had lower hemoglobin level* than controls. This finding combined with the increased iron-positive staining of macro phages in the spleen, liver, and kidney parenchymal cells of chicks, Japanese quail, and rats found in our experiments are indicative ofincreased erythrocyte breakdown. Besides the porphyria induced by the PCB preparations, there is s significant differ ence in their toxicity, in spite of the resemblance in the gas chromatograms and spectra- metric analyses (Koeman et al., 1969). The high incidence of edema, and liver necrosasi the Clophen and the Phenoclor group, compared with the Arodor-fed chicks, it striking. . In the case of our chicks fed Aroclor 1260 we found a lower incidence ofedema cant- pared with the chicks of McCune et al. (1962) and Flick et aL (1965). This couki beca ptained by a difference in the PCB preparation (they used Aroclor T242, which coats** 42% of chlorine) or by differences in diet (Flick etaL, 1963) or strain ofanimals.' The relationship between the toxic nature of PCB and the chick edemaJactor (Anony mous, 1968) have already been discussed by Flicker al. (1965). Gas chromatograms cf PCB fractions (Aroclor) obtained by thin-layer chromatography showed peaks wk about the same retention times as reference chick edema factor amples (Huang ef at, 1967). One of the toxic substances of the chick edema factor has been isolated sad characterized (hexachlorodibenzo-p-dioxin) by Cantrell et al. (Anonymous, I9SH- Recently Higginbotham et al. (1968) found the presence of hexachkjrodibenzD-f- dioxin in pyrolyzates of tetrachlorophenol. The related compound tetrachlorocGbasac*- />-dioxin, which may be found under abnormal conditions in the synthesis of tricM*^ phenol, is known to cause a very potent "chloracne" and liver necrosis (Bauer et d. 1961; Linn Jones and Krizek, 1962). The edema incidence and liver necrosis found Clophen and Phenoclor, together with chloracne-like lesions observed m the expo ments of von Wedel et al. (1943) with dermal application of a technical mixture oTPCl and chlorinated naphthalene, suggests the hypothesis that in technical PCB mixnno* hydropericardium, liver necrosis, and "chloracne"-causing factor or factors are pt* sent. Also environmental contamination with this possible factor must be consderei Work is in progress to determine the compound(s) responsible for the severe edc** formation and liver necrosis. - The residues of PCB in liver and in brain are high. Figures found with the coulomb** detection system are in reasonable agreement with figures found with the electrons*" ture system. Chromatograms of both systems are nearly identical. The approxua* OS\N 255389 STLCOPCB4061080 POLYCHLORINATED BIPHENYLS TOXICITY 667 PCB residues in liver do not seem to be correlated with the duration of survival. Liver residues seem to be particularly low in animals which died with liver necrosis. Brain residue are lower than corresponding liver residues. The variation in the residue levels ofcrude PCB in both liver and brain is high. For the time being this limits the usefulness ofthese figures for the evaluation of crude PCB residue levels in tissues of wildlife speci men found in the field. ACKNOWLEDGMENTS We wish to thank Mr. H. L. van der Maas for his help with the chemistry ofthe PCB prepara tions and Mr. R. H. de Vos (Central Institute for Nutrition and Food Research, T.N.O., Zeist) for the microcoulometric analyses of the samples. We acknowledge the contributions of Mr. A. Musch, Mr. E. W. A. Kamperdijk and the whnirat assistance of Mr. J. H. Pcnnings. :f REFERENCES Anonymous (1968). The chick edema factor. Nutr. Rev. 26, 28-30. Bauer, H., Schulz, K. H., and Spiegelberg, U. (1961). Berufliche Vergiftungen bei der Her- stellung von Chlorphenol-Verbindungen. Arch. Gewerbepathol. Gewerbehyg. 18, 538-555. Burke, J. A., and Holswade, W. (1966). A gas chromatographic column for pesticide residue analysis: Retention times and response data. J. Ass. Offic. Anal. Chem. 49, 347-385. Fuck, D. F., Douglass, C. D., and Gallo, L. (1963). Studies of the chick edema disease. 1. Body water distribution and effect of diet. Potitry Sci. 42, 855-862. Fuck, D. F., O'Dell, R. G., and Childs, V. A. (1965). Studies of the chick edema disease. 3. Similarity ofsymptoms produced by feeding chlorinated biphenyl. Poultry Sci. 44,1460 1465. Granick, S. (1965). Hepatic porphyria and drug-induced or chemical porphyria. Ann. N. Y. Acad. 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(1963). Industrial Hygiene and Toxicology (F. A. Patty, ed.), 2nd ed., Vol. 2, pp. 1340-1343. Wiley (Interscience), New York. IptSEN, S. (1966). Report of a new chemical hazard. New Sci. 32, 612. Koeman, J. H., Oskamp, A. A. G., Veen, J., Brouwer,., Rooth, J., Zwart, P., v.d. Broek, E-, and van Genderen, H. (1967). Insecticides as a factor in the mortality of the sandwich tern (Sterna sandvicensis). Meded. Rijks/ac. Landbouwwetensch. Gent 32, 841-854. Koeman, J. H., ten Noever de Brauw, M. C., and de Vos, R. H. (1969). Chlorinated biphenyls in fish, mussels and birds from the river Rhine and the Netherlands coastal area. Nature (London) 221, 1126-1128. ^Uje, R. D. (1965). Histopathologic Technic and Practical Histochemistry, pp. 518-519. . McGraw-Hill, New York. Uss Jones, E., and Krizek, H. (1962). A technic for testing acnegenic potency in rabbits, applied to the potent acnegen, 2,3,7,8-tetrachlorodibenzo-p-dioxin. J. Invest. Dermatol. 511-517.