Document JNvNmeqpo3XBjXYE7Qp8GXaM2

9 **|D*<dy and *mto *kaamaco<jocv 17, U0-6M (1*70) \ Comparative Toxicologic Study with Polychlorinatod EMftapftyis ir. Chickons with Special Reference to Porphyria, Edema Formation, Liver Necrosta, and TUK* Residue* J. G. Vo* and J. H. Koeman Awitnu* of I'urrhmry PuiMofy and Ik* lutthtd* of VtKrbwry Pioniuxitety mi foxlroiogy, Unktrtity of Uirtchi, Ulrtthr, Tht Nothttimmk Rtctkti Noctmbtr J, 1969 Compare ive Toxicologic Study with Polychlorinated Biphenyl* in Chicken* with Special Reference to Porphyria, Edema Formation, Liver Necrosi*. atd Ti**ue Residue*. Vo*, J. G. and Koeman, J. H. (1970). Toxleot. A ,>!. Pharmacol. 17, *56-668. In a compar live 60-day oral toxicity tes (400 ppm) in chicken*, three 60%-chlorii ated commercial polychiorinaicd biphenyl (PCB) preparation* were used: compound 1 (PtUnodor HP 6), compound 11 (Clophcn A 60), and compound 111 (Aroclor 1360). Uwnt mortality, mean *urvival tinte, mean weight, and pathological observation; (hydropericardium, abdominal, and utbcuiarvoow edema and cemrolobutor liver necrosi*) a* parameter*, a ngniftcam difference In toxi city u found between the compound*: compound* I and I> (bowed the Mgfcesi, compound III the k>we*t, toxicity. Microscopically oemrotobtrtar liver necrosi: was found in chick* fed compound* 1 and 11. Atrophy of the spleen was found in all test group*. Chemical porphyria wa* found a* a general PCB effect: increased fecal excretion of coproporphyrin and proto porphyrin and fluorescence of tissues occurred in all test group*. Additional experiment* with compound 1 (2000 ppm) in Japanese qiail and rat* con firmed the pxphyrogCnic action. Ga chromatographic analyse* of livtr and brain of dead chick* gave PCB levels that varied between 120 and 2900 ppm. The relationship of hydropericardium (chick edema) and liver oocrodt to the differences in toxicity observed between the technical PCB mixture* is discussed. In recent year* the presence of polychlorinated biphenyls (PCB) ha* been exhibited in fish olid wildlife in many countries, including the United State*. Great Britain, Sweden, and T he Netherlands (Jensen, 1966;'Holmes el el., 1967;Hoklen and Marsden, 1967; Kocirjah if of., 1967; Risebrough et a!., 1968; Koeman et a!., 1969). Co(nfncrc*al 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 media, in protective coatings for wood, metal, and concrete, and for many other application*. Which Of these application* has contributed to the present widespread environmental contt mlnation has not been elucidated. The occupational hazards in the use of PCB have lcga known for years; normal handling of these compounds has not given rise to great dWfitvIUes (Irish, 1963). Am twl experiments with rats and guinea pip have shown that the ingestion of PCB m I 1 f. V... DSW 029271 STLCOPCB4013233 polychlorinated diphenyls toxicity 637 may give rise to liver injury (Miller, 1944). Hydropericardium and abdominal edema were found in chicks (McCunee/o/., 1962; Flick etal., 1965) and Japanese quail (Koeman e/J al., 1969) after PCB ingestion. Risebrough el al. (1968) found that the rate of estradiol metalvolism is markedly increased after PCB treatment in pigeons. This may indicatp that tnesc derivatives may induce hepatic hydroxylases. The data presently available do n >t permit a proper toxicologic evaluation of the amounts detected in wildlife specim ;ns. Kocmnn el al. (1969) found a remarkable resemblance between the gas chrofnutog'ums and mass spectrometer analyses of purified extracts from cider duck issues aid the commercial PCB preparation Phenoclor DP 6. Similar gas chromt togrums and identical mass numbers w*r obtained from other commercial preparations, Clophen A60 and Aroclor 1260. These 3 preparations, which contain on the avc age 6 chlorine atoms per molecule, were used in the present comparative toxicologic study, chicks, quail, and rats being used as the experimental animals. METHODS The PCB preparations were obtained from Prodelec in France (compound f, Pheno clor DP 6), Bayer in Germany (compound II, Clophen AC ) Lot No. 912434), and Mon santo ir| the United States (compound 111, Aroclor 1260 Lot No. AK-3). One-day-old cockerels (Hubl art laying breed) were used in these studies. The chicks were kept under a continuously operating heating lamp throughout the experiment. On the second day they wcjre weighed, individually wing-banded, and distributed randomly into 4 groups. The birds received diets containing PCB as follows: group I, 400 ppm compound 1; group 1 ,400 ppm compound II; group Ilf, 400 ppm compound III; group IV served as control. Thcyjwcrc led and watered ad libitum and were weighed twice weekly. The feed con sisted of u common type 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 into the ration. 1 he chicks were kept on these rations for 60 days and killed. These birds and the animals that died during the experiment were necropsied. Two chicks from group l( died during the first 3 days. At necropsy it appeared that they were not killed by the PjCB 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 needle on a2-ml syringe into a small slit in the pericardium. All the pericardial fluid was removeej from the anterior part of the heart sac by intermittent aspiration. The volume of Sie fluid removed was measured to the nearest 0.05 ml. The incidence of abdominal and subcutaneous edema and the weights of the liver and spleen were recorded. The liver, spjeen, and kidneys were fixed in 10% buffered formalin and processed in a con ventional manner for histologic examination. Sections (7 p) were stained with hematoxylin-eosjn and Perls's iron stain. Moreover, selected sections were stained for amy loid willy methyl violet according to Lillie (1965), with Congo red, with PAS, with PASAlcian blue, and with Fettrot for lipids. Prompted by experience obtained in parallel studies vyith hexachlorobcnzene, observations were made in ultraviolet light using red fluorescence as an indicator for the presence of excess quantities of porphyrins. Fluor escence of liver, bile (after opening of the gall bladder), small intestine, and bone was I1 DSW 029272 STLCOPCB4013234 I 658 VOS AND KOEMAN recorded. Fcca! coproporphyrin and protoporphyrin were determined by the method described by Rimington (1961), using a Beckman spectrophotometer. jPCB levels in livers and brains of selected birds were determined by gas chromato graphy. The tissues were extracted with petroleum ether in a Soxhlct extraction appara tus after drying with anhydrous sodium sulfate. Cleanup of the samples was carried oijt by liquid-liquid partition with dimethyl formamide a id column chromatography using activated Florisil. The PCB preparations in the final extract were measured by gas-liquid chromatography (204-1 B Vuriun Aerograph witn electron capture detection), The Pyrcxglasscolumn(5fect x-J inch), filled with 10% DC200onCaschromQ(80-l00 m;*h), wus opcruied m 200C with nitrogen a* the carrier ga (about 50 nil/mln). 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 not necessarily identical with the PCB compound used in the treatment. For quantification, I |icak. considered to be representative, was selected. This was the peak with r.v ~ 1.45 rc alive to dicldrin. The approximate amount of total PCB residue was calculated from th: height of this peak with compound I as the standard. In a number if tissue extracts the amounts of PCB wer; measured also by a Dohrm inn C 250 / niicrocoulonietric detection system (wi h a T-300-S-halogcn cell), coupled to a Microtek MT-220 gas chromatograph. The chromatograph was equipped with a Pyrcx column (6 feet x \ inch) filled with a mixed-bed column packing (QF I pljjs DC 200) described by Burke and Holswade (1966). With this system 0.6 /xg of Phcnoclor DP6guvc about a half-scale response on a I-mV recorder (microcoulomcter ra lge: 200 ohms). By this method the content of chlorine in the extract is determined by addition of the chlorine contents of the dillerent 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 content. Additional experiments with special reference to porphyrin biosynthesis were made wi^h compound I in 5 adult female Japanese quail weighing 112-130 g and 8 adult female wjstar rats weighing 173-212 g at a dose level of 2000 ppm in the dry food. Mortality ofjthe 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 Wilcoxon test for 2 unrelated samples (van der Waerden, 1957). Experiments with Chickens RESULTS All chicks from groups f and II died during the experiment, while the mortality in group III birds was only 15% (Table I). Death was accompanied by ruffled feathers and ataxia as the main signs; loss of feathers was often seen. Hydropericordium (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 f and 11 only, with the highest incidence in group II (Table 1). ------------------------- --- DSW 029273 STLCOPCB4013235 S D " CL 3 3 J ?? n o cl? 3 c; ? s v 5 ^ o. v ? cl POLYCHLORINATED BIPHENYLS TOXICITY STLCOPCB4013236 TABLE 1 Mortality, Mean Survival Time and Pathologic Observations or Chick* Fed 400 ppm PCB for 60 Days and of Control Birds Groups Number of N deaths Mean survival* time (days) Number of birds with edema Hydropericardium* Abdominal Subcutaneous Number of birds with liver necrosis I (Phenoclor) II (Clophcn) 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 0 0 * The ranee of values b ihown in parentheses. * 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 ate, with interpolation for inicrmediateajcs. O in O N) lO IV a *>J I 660 VOS AND KOEMAN Macroscopic red fluorescence, under ultraviolet light indicating porphyrin*, was detected in all birds of the 3 test groups; fluorescence of liver, bile, small intestine, and 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 I and II) showed the highest incidence of fluorescence in the bile and small intestine followed by the liver and bone (Fig. I). Only bone fluorescence was pronounced in the killed birds from group HI (Fig. I). This finding is in agreement with theexcrction ofcoproporphyrin and proto porphyrin it. 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. In the mean . time, large amounts of porphyrin had been deposited in the bones. <D r* \ ( rut [' "] LIVER pm SMALL INTESTINE BILE BONE Fig. I, 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 front group III were significantly reduced (Table 3). Liver necrosis was observed in 9 birds from groups I and 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 029275 i STLCOPCB4013237 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 mescjichymul cells between the necrotic liver parenchymal cells could be observed. Fatty degeneration could be scon also (positive for Fettrott). Protein droplets of different tize were sometimes present in the sinusoids near damaged cells as well as in tome central veins. Ljiver parenchymal cells with pycnotic nuclei coul< be observed in liver sections from all 3 experimental groups, A small amyloid dcpos'i was seen in liver sections of I bird each from groups I and II. TABLE 2 COPROP^lRRHYRIN AND PROTOPORPHYRIN CONTENTS Oig/g DRY WEIGHT) OF FECES OF CHICKS Fed PCB for 60 Days and of Control Birds Coproporphyrin Days after start of feeding 1 Croups 11 m IV Protoporphyrin Groups I II lti IV 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 8.3 15.2 4.5 0.8 19.4 20.0 -- 13.: 11.5 8.9 -- 0.6 25.C 16.1 25.2 -- 9.7 11.5* 14.2 1.4 26.'* -- 13.7 2.4 -- -- 5.8 2.8 4.5 -- 2.3 -- 6.1 8.1 One bird dill alive at tlK moment of sampling. * Throe birds still alive at the moment of sampling. The spleens of all birds in the test groups were small, described already by Flick ct a!. (1965), Compared with the controls. In general, in the spleens of all chicks of groups I, If, and 111 which died during the experiment a reduction in the amount of the red pulp, and atrophy of the white pulp and lymphoid foci (Fig. 3), caused by a suppressive or destructive action, could be observed, resulting in a small spleen with a wrinkled capsule.j In 2 t>irds from group III we found necrosis in the center of a lymphoid focus; in some bij'dsfrom groups 1 and II eosinophilicdcposits were seen in the center of the white pulp (Fjg. 3). These deposits were purple with methyl violet, red with PAS. red purple with PAS-Alcian blue, and light brown with the Congo red slain. With Congo red stain they showed some green birefringence under polarized light, although fluorescence under tlje UV microscope was yellow instead of pink. These data are strong indications of amyloid. Tubutar dilatation in the kidneys, described already by McCune el al. (1962) was also common, usually to a slight degree, in most chicks from groups I and II, but rare in group l|l. This dilatation was sometimes accompanied by the presence of leukocytes and red! blood cells in the lumen of the tubules. Pcrls'p iron stain gave the following results: the presence of iron in liver KupfTer cells test birds seemed to be about twice as much as that in control birds. In some birds If Ka> VOS AND KOEMAN STLCOPCB4013239 TABLE 3 Organ Weights of Killed Chicks Fed PCB (Comfouno III, Aroclor) tor 60 Days and of Control Birds* Groups . Ill (Aroclor) IV (Control) Body Liver Liver Spken Spleen N (b) - (g) (g/1001 body weight) <S) (g/100 g body weight) 17 703 (514-335)* 29.7 (22.6-35.9)* 4.31 (3.47-5.91 Y 20 921 (820-1010) 24.6 (19.5-28.4) 2.67 (2.17-2.97) 0.90 (0.40-1.50)* 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. P < 0.025. * P < 0.01. 4 P <0.005. o (S) O M sO (V *sj sJ POLYCHLORINATED BIPHENYLS TOXICITY 663 i I i iI t i Fig. :. Liver of a croup | chick with severe ccmroiobular necrosis, resulting in a reversed picture with the portal triad becoming the center (arrow). Note the strong eosinophilic parenchymal cells at the mar gin of necrotic and vital tissue. Hematoxylin and eosin. xl25. 't Fie. Spleen of a group 1 chick with atrophy of a lymphoid focus and atrophy of the white pulp. The wh tc pulp has been replaced by homogeneous eosinophilic material, probably amyloid (arrow). Hematoxylin and eosin. x500. DSW 029278 I Ii i STLCOPCB4013240 664 VOS AND KOCMAN from oil test groups we found iron in the cytoplasm of liver parenchymal cells, but this wasj 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, rcsjvctivcly, in the cytoplasm of kidney tubular cells. Approximate liver residues of PCB in 25 selected birds we c determined by gaschromajography. Thi? selection included birds which died in the first part of the test, birds which died later and birds with liver necrosis. Brain analysts were made in 12 chicks. 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 fhe 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 maeroscopically at necropsy. The bird that died last showed a strong fluorescence ofliver, kidney, small intestine, and muscles. Cryostat sections examined by fluorescent micros' opy revealed an intensely red fluorescence i f 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. Pro iferation of probably RES cells was a common finding. Peris's iron stain was positive for both Kupffer and parenchyma! cells. 1 he feeding of compound 1 (2000 ppm) to 8 white rats caused a marked loss of weight as cjompared 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 unqcr UV light, lire 2 rats that died next had fluorescence of the incisors and the small fstine, respectively, while the 2 rats that died last showed fluorescence both of incisors the small intestine. An important finding was the presence of an enlarged liver, as ; also found in Japanese quail fed compound I (2000 ppm), and a small spleen. Sec- \s of the liver revealed a centrolobular degeneration (vacuolated, foamy cells with pycnotic nuclei near the central veins). In some sections focal necrosis could be seen. The disappearance of structure in the white pulp of the spleens, together with a reduction in the red pulp and the presence of green-brown pigment in macrophages was notable; Perls's stain for iron was strongly positive. Therefore sidcrosiscould bediagnosed in the lessen together with its appearance in Kupffer and parenchymal cells of the liver and tubular cells of the kidney. i I 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 long list ofcompounds that produce chemical porphyria. Granick (1965, 1966) has presented evidence that the chemical pophyrin is the result of the induction of 8-aminolcvulinic oci^J synthetase in liver mitochondria in the ease of 3,5-dicthoxycarbonyl-l,4-dihydro2,4.6-trimethylpyridinc and allylisopropylacetaniide. It is of interest to note that Risebrough et at. (1968) have demonstrated that PCB also induces steroid hydroxylases in I the1 liver of birds. I DSW 029279 STLCOPCB4013241 POLYCHLORINATED BIPHENYLS TOXICITY STLCOPCB4013242 TABLE 4 Chemical Analyses of Liver and Brain in Chicks Fed PCB for 60 Days* Group I (Pftcnodor) History Liver Brain Group 11 (Oophen) History Liver Brain Group 111 (Aroclor) History Liver Brain Died on: Died on: Died on: 12th day 2200 -- 13th day 800 -- 16th day 950 420 16th day 1300 ' 490 14th day 1100 -- 1100" -- 1900" -- 17th day* 410 -- 17th day 680 -- 21st day* 120 _ !7thday 1600 320 31st day 2400 270 22nd day* 210 70 19th day* 190 120 Killed on: 220" -- 20th day 610 ~ 60th day 250 40 22nd day* 310 210 22nd day 420 380 340" -- 24th day* 210" 180 -- -- 22nd day 420 -- 23rd day 860 -- 60th day 60th day 220 210 -- -- 150" -- 26th day 390 320 28th day 1400 -- 1400" -- 31st day* 2100 380 58th day 2900 700 o <A s: * The approximate contents are Riven in parts per million. * Chicks with liver necrosis. . o * Results obtained by microcoulomctric detection; all other figures are results obtained by electron capture detection. Nj vO l\J cc o V* I 666, VOS AND KOCMAN Ip our experiments with compound III in the chicks it was noted that the excretion of coproporphyrin and protoporphyrin decreased to nearly normal values after a maxi mum effect at 26 days of treatment. Whether this is due to an adaptation which enables thepnima! to eliminate these compounds more efficiently, to an age-dependent change in elimination capacity or susceptibility, is not known. The high incidence of tissue fluor escence in the dead chicks from groups I and II is remarkab c. The excretion of copro porphyrin and protoporphyrin is also high at that time. Tfe possibility has to be con sidered that the presence of large amounts of porphyrins could have a toxic effect in its owl), and may have contributed to the death of these birds. T*'hc earlier manifestation of porphyria in chicken and quail, compared with the rat, resembles the difference found in hexacblorobenzenc-induccd porphyria: early nianifeslntion of porphyria in the Japanese quail, late manifestation in the rat (Vos et at., 1968). Flick et at. (1965) observed that chicks fed PCB had lower hemoglobin levels than controls. This findingcombined 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 of increased erythrocyte breakdown. Besides the po phyria induced by the PCB preparations, here is a significant differencj: in their toxicity, in spite of the resemblance in the gas chromatograms and spcctro- metric analyses (Koeman et at., 1969). The high incidence of ;dema and liver necrosis in thc'Clophen and the Phenoclor group, compared with t he Aroclor-fcd chicks, is striding. In the cose of our chicks fed Aroclor 1260 we found a lower incidence of edema com pared with the ch cks of McCunc ct at. (1962) and Flick et at. (1965). This could be explained by a diffciencc in the PCB preparation (they used Aroclor 1242, which contains 42% 6fchlorine) or by differences in diet (Flick et at., 1963) or strain of animals. The relationship between the toxic nature of PCB and the chick edema factor (Anonymous, 1968) have already been discussed by Flick et at. (1965). Gas chromatograms of PCB fractions (Aroclor) obtained by thin-layer chromatography showed peaks with about the same retention times as reference chick edema factor samples (Huang ct a!., I961?). One of the toxic substances of the chick edema factor has been isolated and characterized (hexachlorodibenzo-p-dioxin) by Cantrell et at. (Anonymous, 1968). Recently Higginbotham et at. (1968) found the presence of hexachlorodibenzo-pdioldn in pyrolyzntcs of tetrachlorophcnol. The related compound tctrachlorodibcnzo- p-dioxin, which may be found under abnormal conditions in the synthesis of trichlorophejrtOl, is known to cause a very potent "chloracne"' and liver necrosis (Bauer el at., 1961; Linn Jones and Krizck, 1962). The edema incidence and liver necrosis found with Clonhen and Phenoclor, together with chloracne-like lesions observed in the experi ments of von Wcdel et at. (1943) with dermal application of a technical mixture of PCB and chlorinated naphthalene, suggests the hypothesis that in technical PCB mixtures a hydropericardium, liver necrosis, and ''chloracne"-causing factor or factors arc pre sent. Also environmental contamination with this possible factor must be considered. Work is in progress to determine the compound(s) responsible for the severe edema formation and liver necrosis. The residues of PCB in liver and in brain are high. Figures found with thecoulomelric detection system are in reasonable agreement with figures found with the electron cap ture system. Chromatograms of both systems arc nearly identical. The approximate j , ' f i DSW 029281 STLCOPCB4013243 on of maxiablet tgein (loort>proiconin iu e rat, nonlrl of., levels aeroquail, vn. liflersetrostiiin kt, U com ae exitains aonyms of ; with tt a!., J and 1968), ttO-penzoiloroel a!., 1 with xperi- fpcb ures a > preicrcd, dema nctric ] capimate POLYCHLORINATED DIPHENYLS TOXICITY 667 K'B residues in liver do not seem to be correlated with the duration of survival. Liver I residues seem to be particularly low in animals which died with liver necrosis. Brain residues are lower than corresponding liver residues. The variation in the residue levels of prude PCB in both liver and brain is high. For the time being this limits the usefulness of |hcse figures for the evaluation of crude PCB residue levels in tissues of wildlife speci men found in the field. ACKNOWLEDGMENTS Wc wish 1 > thank Mr. H. L. van dcr Maas for his help with the chemistry of the PCB prepara tions and Mr. R, H. de Vos (Central Institute for Nutrition and Food Research, T.N.O,, Zcisl) ! for the micracoulometric analyses of the samples. We acknowledge the contributions of Mr. A. Muscb, Mr. E. W. A. Kamperdi)k and the technical assistance of Mr. J. H. Pennings. REFERENCES i An jn'Vmous (1968). The chick edema factor. Nutr. Rev. 26, 28-30. Bauer, H., Schulz. K. H., and SpitOELiERO, U. (1961). Bcrufiiche Vcrgiftungen bei dcr Her- sjcllung v >n Chlorphcnol-Vcrbindongen. Arch. Gewerhepathoi. Gewvrhehyg. 18, 538-555. fiUKKf,J. A., and Holswade, W. (1966). A gas chromate -raphic column for pesticide residue analysis: Retention times and response data. J. 0,'ic. Ami. Chan. 49, 347-385. Fujrx, D. F., Douglass, C. D., and Gallo, L. (1963). Studies of the chick edema disease. 1. Body wat'-r distribution and effect of diet. Poultry Sci. 42, 855-862. Fu|:k, D. F , ODell, R. G., and Childs, V. A. (1965). Studies of the chick edema disease. 3. Similarity of symptoms produced by feeding chlorinated biphenyl. Poultry Set. 44, 1460 1465. t GrAnick, S. (1965). Hepatic porphyria and drug-induced or chemical porphyria. Ann. N. Y. Acad. Sci. 123, 188-197. GrAnjck.S. 11966). The induction in vitro of the synthesis of 5-aminolevulinic acid synthetase ip cltcmic; I porphyria: A response to certain drugs, sex hormones and foreign chemicals J. Biol. Chem. 241. 1359-1375. Higcingbotham, G. R., Huang, A., Firestone, D,, Verrft, J,, Ress, J.,and Campbell, A. D. (1968). Chemical and toxicological evaluations of isolated and synthetic chlorodcrivativcs oj' dibenz%v>-dioxin. Nature (London) 220, 802-703. Holden, A, V.. and Marsden, K. (1967). Organochlorine pesticides in seals and porpoises. Nature (London) 216, 2174-1276. Holmes, D. C,, Simmons, J. H., and Tat7on, J. O. G. (1967). Chlorinated hydrocarbons in British wildlife. Nature (London) 216, 227-229. Huang, A,, Firestone, D.,and Campbell, A. D. (1967). Detection of chick edema factor and other chlorinated hydrocarbons in fats and oils by electron capture gas chromatography: uic of TLC as a preliminary cleanup procedure. J. Ass. Offic. Anal. Chan. 50. 16-21. i Irish, O, D. (1963). industrial Hygiene and Toxicology (F. A. Patty, ed.), 2nd ed., Vol. 2, pp. 1340-1343. Wiley (Intcrscience), New York. Jensen, S. (1966). Report of a new chemical hazard. New Sci. 32, 612. Kocman, J. H.. Oskamp, A. A. G.. Veen, J.. Brouwer, E.. Rooth, J.. Zwart, P,, v.d. Broek, Ej, and van Genderen, H. (1967). Insecticides as a factor in the mortality of the sandw ich tern (Sterna sandviccnsis). Mcdcd. Rijksfac. Uwdhoawwctensch, 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, 1126-1! 28. LtLtlE, R. D. (1965). Histopathologic Teclmic and Practical Histochemistry, pp. 518-519. McGraw-Hill, New York. Lnn Jones, E., and Krizek, H. (1962). A technic for testing acnegcnic potency in rabbits, applied to the potent acnegen, 2,3,7,8-tetrachlorodibcnzo-^-dioxin. J. Invest. Dermatol. 39, $11-517. DSW 029282 ! i t i i I i i i i i I i i i i STLCOPCB4013244 668 VOS AND KOCMAN McCone, E. L., Savage, J. E,, and O'Deil, B. L. (1962). Hydropericardium and a sc'- chicks fed a chlorinateJ hydrocarbon. Poultry Set. 41. 295-299. i(.lea, i. W. (1944). Pathologic changes in snimals exposed to a commercial chlorinatcu Tdiphenyl. Pith. Health Pep. 59. 1085-1093. Rimington, C. (1961). Quantitative determination of porphobilinogen and porphyrins in urine and faeces. Broadsheet No. 36 of the Association of Clinical Pathologists. Copies may be obtained from Dr. R. B. H. Tierney, Pathology Labor; lory, 75, Boutporl Street, Barn staple. Devon. Riti tiROucit. R. W,, Ricche. P.. Peakali, D. B., Herman, S. G., and Kirven. M. N. (1968). Polychlorinated biphenyls in the global ecosystem. Nature (London) 220, 1098-1102. van r>t r Wai ri>i n, B. L. (1957). Mathvmatisehe Statistik, pp. 269-284. Springer, Berlin, yus wuhl, H., Holla, w. A,, anu e^enton. 3, {1947), OH.enotipntin ilia toxic ctVecu result ing from exposure to chlorinated naphthalene and chlorinated phenyls with suggestions for prevention. Rubber Age (New York) 53, 419-426. Yos, 5. G., Breeman, H. A., and Benschop, H. (1968). The occurrence of the fungicide hexaChlorobcnrene in wild birds and its toxicological importance. Meded. Rijksfac. Landbouwweiensch. Cent. 33, 1263-1269. i 4 DSW 029283 STLCOPCB4013245