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i f 7/I 1 1090 R. F. Ham., J. F. J. I.vmann ani> A. Wyatt 7^1*1 1 9 si Chubb, R. C., and A. K. (Imrchill, IWiJI. I'red|dlaling ulMits vMfialnl with March's rtisnw Vet. Ret. A': 1-7. Churchill, A K., and P. M. 1times, 19*7. Agent of March's disease in Imw culture. Nature don), 215: SM-MO. Churchill, A. K., |o*o. Poultry Industry, lb. Pappcnheimer, A. W., L. C. Dun* and V. Cone, 1929. Studies on tow) paralysis (neurotymphoautosis galHnarum). J. Expt, Med. 49: *.t-*6. Payne, I.. N., and P. M. Ilines, 1947. Studies M March's disease II. Pathogenesis. J. Nall. Can- rrr Inrt .tO: Ml .tOi. _ SeKaal, K., and W. V. lamnrein, I9SI, Control at avian encri'tialnmyrlilft by vaccination. Am. J. Vet. Res. 14: 627-tM.I. Sevoian, M, and I). M. Clumlierlain, I06.S. Avian lymiihomatosia III. Incidence and manifestation in experimentally infected chickens of various ages. Avian Diseases, 7: 07-102. (1969) h of eggs i (ained in dustrial t Lake Mic ratory at PC Iin (B Data o PCBs in t !o determ feed. The Ji Toxicity Studies on Polychlorinated Biphenyls could con in the Chick and that quantities. 1. TOXICITY AND SYMITOMS Studies Hetty M. Rehpkld, R. L. Bradley, Jr. and M. L. Sunde Departments of Food Science and Poultry Science, The University of Wisconsin, Madison 537OS could be from trial: to assess i (Received for publication December 10, 1070) man. INTRODUCTION Subsequent investigation revialed that the Data ob POLYCHLORINATED biphenyls (PCBs) are a series of inert, chemically resistant plasticizers compatible with wide variety of materials, such as paints, amine hardener was the cause and that one of the components, a chlorinated biphenyl a(Aroclor 1242*), seemed to be the problem compound. is of more rect ways animal to mentation. synthetic resins, varnishes and. waxes and levels of 100, 200, 400 and 800 p.p.m. are used to improve such properties as of this compound produced labored respira chemical resistance, water resistance, ad tion, abdominal fluids, hydrojxiricardium, Diets co were prepa hesiveness and flexibility (Monsanto, and swollen kidneys. The mortality was 1248**), v 1968). ' severe and weight gains reduced. the basal c; Since PCBs appear extensively in the en Further work by this group (Flick cl at., solved in p* vironment (Risebrough cl at., 1968) and 1965) suggested that 400 p.p.m. were re small portic because pi their chemical properties and quired to produce serious symptoms. In ad mixing to: widespread use, it appears important to dition to Ihe above symptoms, they ob study their potential effects on biological served cream-colored pancreas, enlarged throughout analyzed ui, systems. ^ adrenal, small spleen, dermatitis and de Drug iWfmii Some work on the effects of PCBs on feathering. Hemoglobin and hematocrit * contain! < chickens has appeared in the literature. were reduced and blood glucose was re i ... 5how on the McCune el at. (1962) observed that an duced slightly. epoxy-resin paint caused symptoms of Koeman ct at. (1969) rejx>rted that j ft PCBs. Ten one-t "chick edema" when chicks were placed in 2000 p.p.m. killed 5 out of 5 Japanese quail (he battery before the paint had hardened. in less than 2 weeks. Amtefson ct at. > ti I he crossing V h.C White I i t .*rn diets co Published with the approval of the Director of * Registered trademark for a compound con i " PCBs. Ail e the Wisconsin Agricultural Experiment Station taining 42% chlorine. Sold by Monsanto, St. Louis, College of Agricultural and Lilt Sciences, Madison. Mo. ** Obtained I i 332632 It STLCOPCB4079208 Toxicity ok I'ni.vniLomNATKo Biimiknvui 1091 (1fr9) have rc|xtrU'(l on the PCI) content Tahw. I. -Hoiat dM of eggs of |H'lionns and cormorants ob tained in remote areas ns well ns war in dustrial arcits. Fat extracted from fish in Lake Michigan was examined in this labo ratory and contained up to 120 p.p.m. l'CUs (Bradley, 1970). Data on the effect of various levels of PCBs in the diet of the chick could be used to determine safe levels of l'CUs in chick Orntind yellow corn Wheal multilines Alfnlfa meal (220 I.U. A/am) Meat scrap (50% protein) Sovlx-an meal (44% protein) Fish meal (60% protein) Indiarri salt Chick nice oyster ahcll Granite grit . fm./kilo gram 525 too 50 so 200 50 5 10 to feed. The possibility exists that ingredients could contain significant levels of PCBs mg./kilo gram and that poultry could ingest significant MnSO, quantities. Studies on PCB toxicity in the chick Vitamin Hit (.152 mgs./gm.) Riboflavin (44.5 mga./gm.) Penicillin (44.5 mgn./gm.) could be used in combination with data Vitamin A (10000/gm.) ii from trials on other experimental animals Vitamin Da (<W5fl/gm.) I l to assess the potential hazard of PCI) to i man. 156 K4 55 99 221 99 Data obtained by direct experimentation . electrically heated batteries with raised is of more value than that obtained in indi wire floors. Feed and water were provided rect ways and the young chick is an easy ad libitum. Chicks were weighed weekly, animal to control for this type of experi deaths and toxicity symptoms were re mentation. corded daily. Seven birds that died on PCB EXPERIMENTAL Diets containing various levels of PCBs ' were prepared by adding a PCI) (Aroclor 1248**), which contains 48% chlorine, to the basal diet in Table 1. PCBs were dis solved in petroleum ether and mixed into a small portion of the basal diet prior to final mixing to facilitate even distribution throughout the mixture. The basal diet was analyzed using the method of the Food and Drug Administration (1968) and found to diets were examined by the Wisconsin State Animal Health Laboratory in Madison for gross pathology and indications of toxicity. The investigators also autopsied many of the dead birds. Some of the birds were killed, their livers removed, weighed and reported as a percentage of body weight. . Experiments were terminated at 4) to 5 weeks. In reporting the results, some of the data were combined and are indicated jn the Results and Discussion section. contain < 1 p.p.m. of compounds which RESULTS AND DISCUSSION show on the chromatograph to be similar to Survivors and average weights of groups PCBs. of 10 chicks each fed diets containing 0, 10, I Ten one-day-old chicks, resulting from 50, 100, and ISO p.p.m. PCBs were plotted the crossing of New Hampshire males and in Figure 1. A depressed growth rate was S.C. White Leghorn females, were placed observed with dietary levels of 50 p.p.m. on diets containing various amounts of PCBs and greater. When 100 p.p.m. and PCBs. All experiments were conducted in ISO p.p.m. l'CUs were added to the diet, toxicity dill not increase proportionately. ** Obtained from Momaato, Si. Louts, Mo. This has also been reported in rats by \ 1 i I I I | i ! [ ii i i f i. I DSW 332633 STLCOPCB4079209 " ri - 1092 II. M. RkiikkU), K. L. Hkaim.kv, Jb. anii M. L. Suniik would nffi'Cl growth, symptoms and toxicity diet gainr in the chick. Average weights of chicks fed ned 79 diets containing levels of I'CHs from 10 Sixteen < 50 p.p.m. are shown in Figure 2. Three ex P-P-m. I'C/ periments have lccn averaged together in out of 20 (h i this figure. In one of the three experiments the 20 and 40 p.pjn. groups were not in out of 30 i Levels of m cluded in the experimental design, Growth growth depi depression resulted from PCBs even at lev and continu els where no deaths occurred. There was the experim. i \ considerable variation in how individual birds react on the same diet. Two birds on Mortality with 50 p.p the diet containing .30 p.p.m. PCBs each 3. At 34 da) weighed 208 g. at 3 weeks of age. At 4 on the diet c < weeks of age one had gained 14 g. and the mortality cu other 105 g. The 40 p.p.m. growth curve 15 days but wns almost identical with the 30 p.p.m. the birds inc curve. One bird on the diet containing 50 General cd p.p.m. PCBs lost 52 g. between 3 and 4 diets contain weeks of age (206-154). One bird on this edema was y developed bcl Fic. 1. Survival and average weight* from 10 chicks fed various levels of P(JBi the wattles in of age (Figur been fed diet- Tucker and Crabtree (1970), Mortality was as severe with 50 p.p.m. as with 3 times as much PCRs in the diet. The survi vors are shown directly on Figure 1. All 10 p.p.m. PCBs the report f: Health Labo generalised ed> survived unless indicated. Flick ct al. (1965) reported no deaths when 24 Single Comb White Leghorn cockerels were fed diets containing 200 p.p.m. PCBs (Aroclor 1242) and.only 4 deaths in 24 chicks on a ( c c diet containing 400 p.p.m. PCBs. Aroclor 1242 is a.PCR which contains 6% less chlorine than Aroclcr 1248 and presumably this accounts for somewhat lower toxicity. Total mortality with Japanese quail was observed between 6 and 55 days after feed ing a diet of 2,000 p.p.m. PCRs (Koeman (l al., 1969). The 10 p.p.m. level resulted f, C; u C; uQ,; S^ ti Zi in small insignificant increase in weight gain especially during the last } week. The next experiment was designed to as certain how levels below 50 p.pjn. PCBs Kms. 2. Average weights of chkks ted vsriiss levels of PCBs. (20 chicks started in etch iW except for the 20 and 40 p.p.m. where only 10 chicks were started.) Fio. J, Surviv i DSW 332634 i STLCOPCB4079210 Toxicity ok I'oi.vciimihinatkii Hipiiknyi.h iom y dk't Kninc<l 10 g. (21.1-22.1) ami one d gained 79 g. (208 -287). i- Sixteen out of .10 chicks died when 50 p.p.m. PCBs were fed, yet only 4 deaths In out of 20 occurred at 40 p.p.m. and 1 death out of .10 at 50 p.p.m. to 5 weeks of age. Levels of more than 10 p.p.m. resulted in a growth depression starting at 1 week of age and continuing through the fifth week when IS the experiment was terminated. _ al Mortality results nf 5 separate studies in with 50 p.p.m. PCBa are shown in Figure :h 3. At 34 days, 50r/o of the chicks had died Fio. 4. Typical swellings in chicks with PCB 4 on the diet containing 50 p.p.m. PCBs. The induced edema. Note: the swelling lielow the beak ic mortality curve was near normal the first and adjacent to the wattles. i-e 15 days but losses became more severe as n. the birds increased ini age. effusion into the pericardial sacs, muscles, ;o General edema was observed in birds fed subcutaneous tissues and abdominal cavity 4 diets containing > 20 p.p.m. PCBs. The were observed. Apparently, the permeabil lis edema was severe enough so that swelling ity of chick tissues increases in the pres developed below the beaks and adjacent to ence of PCBs. Others have observed edema the wattles in most chicks at 2) to 3 weeks due to feeding PCBs but not the double of age (Figure 4). When chicks which had chin effect shown here. McCune ct al. been fed diets containing 50, 100 and 150 (1962) and Flick ct al. (1965) reported p.p.m. rCBs were examined postmortem, that edema was observed in chicks fed the report from the Wisconsin Animal 400 p.p.m. PCBs in the diet, however, ' Health Laboratory indicated extensive ederpa was virtually absent from 24 chicks generalized edema. Large amounts of serous fed 200 p.p.m. PCBs. Species, sex and 6% Fkj. J. Survival of chkka on diets containing SO p.p.m. PCBs. (S4 chicks started in S studies) DSW 332635 I 1 STLCOPCB4079211 . " I i 4 .r$m ; * , j 3 .' 1. s ) ' i 2Qu2n2* 25 i 1004 II, M. Kkmkki.ii, K. L. Hmadi.kv, Jh. anii M. I* Nundk Fig. 5. Hyckoperica'dium in chick fed a diet containing 50 p.p.m. PCB*. lower chlorine content of PCBs used in their experiments might account for no or slight edema at 100 PCBs. . An enlarged heart sac is a characteristic symptom of PCB toxicity (McCune ct al., 1962; Flick et al., 1965; Koeman ct al., 1969). A typical example is shown in Fig ure 5. In postmortem examination of five 5- week old birds on a diet containing 50 p.p.m. PCBs we removed 10 ml. of fluid from 3 heart sacs, 2 ml. from one, while on the last the heart sac appeared normal. Flick ct al. (1965) found only 1 ml. (aver age) when 400 p.p.m. were fed for 3 weeks. A gasping condition was also observed with high levels of PCBs in the diet. This could be the result of an enlarged heart sac exerting pressure which prevents normal heart action. This would reduce cardiovas cular capacity. Moreover, normal lung function would be impaired by the serous fluid and hemorrhages which were observed in postmortem examination. A noticeable decrease in the development of comb and wattles was observed with chicks fed diets containing 4Q and 50 |i4.in. Wits, l4"is ric|irrs*l<iii at aertnaiaty wKiuil rliiirnrlrilnflw wu* ulnrrvrd at M) |i*p.m. and no imllrcnlili1 ikirmw* wiih oh nerved wIhmi chicks were fed 10 nod 20 iquu. I'CIts. This effec t Inis ihiI Invii rr- INirleil j>revhni.`ily for chickens. Kisehrongh ft al. (1968) has rejmrlcd that IK'Its in jected intramuscularly in pigeons resulted in an increase in estradiol mctaltolitrs in the liver 7 days after injection. DDT which is structurally similar to PCBs exhibited hormonal degradation in studies using tes tosterone and progesterone (Peakall, 1967). Degradation of steroid hormones by hepatic enzymes which were induced by PCBs could account for the depression of secondary sexual characteristics which were observed. In some birds fed a diet containing 50, 100 or 150 p.p.m. PCBs, a postmortem ex amination revealed that the proventriculus was atypically solid and firm to the touch. Full and distended crops were observed in some birds fed 50 p.p.m. PCBs in the diet. In one experimental group, 6 out of 10 birds had abnormally distended crops at 2 weeks of age. This condition lasted only 2 or 3 days and was not observed at the lower levels of dietary PCBs. This indi cates a temporary stasis of the digestive tract. . Three deaths which the investigators ob served were preceded by a convulsion. Other chlorinated hydrocarbons are known to interfere with the nervous system. DDT attacks the nervous system of animals and disrupts the ion-transport mechanism (Matsumura, 1969). This disruption can result in respiratory failure or secondary poisoning by neurotoxins produced in the animal during DDT poisoning. Liver as a percentage of body weight in creased as PCBs in the diet increased (Ta ble 2). This increase in liver as percentage of body weight was also observed when diets contained chick edema factor (CEF) I | \I 1 I were fed man, lor, that min affected, by tin- w< IVialim muscular which ha or 150 i j rous fluir hearts : Bleeding many chi periment Many as edenu integrity utilizatio mins. Sympt are simi containir dude: edema, . cell wall liver as stituent contain (Yartzof ilarity t tion of one mig tion is si Other Talz 2. PCB* ad to die p.p.m 0 0 50 150 150 150 DSW 332636 STLCOPCB4079212 I i Toxicity or Polychlokinatkd Hiphknyls 1095 were fed to chicks (Campbell and Fried Flick ct al., 1965) with chickens and man, 1966). The data do show, however, Tucker and Crabtree (1970) with ducks that actual liver weights |ier bird are not reported that higher levels are required for affected. The percentage change is caused toxic symptoms to develop than were by the weight reduction. " ' needed in these experiments. Generally, Postmortem examinations indicated the other workers have not worked with I muscular hemorrhaging in some birds levels below 100 p.p.m. This could account which had died on diets containing 50, 100 for the differences in the results. The or 150 p.p.m. PCBs. Lungs contained se best possibility at this time for the differ rous fluid and some hemorrhaging. Some ences is the relative toxicity of PCBs based hearts showed petechial hemorrhages. on the chlorine content. Only the com Bleeding from the mouth was observed in pound containing 48% chlorine was tested many chicks which had died during the ex in these experiments but chickens certainly i periments reported here. . are affected by relatively low levels of this Many of the symptoms of toxicity such compound. as edema, hemorrhaging and lack of tissue integrity suggest that PCBs interfere with SUMMARY utilization or absorption of fat soluble vita Maximum tolerance of polychlorinated mins. biphenyls containing 48% chlorine in the Symptoms observed in chicks fed PCBs chick is approximately 50 p.pjn. Symptoms are similar to those reported when , diets observed in the chick fed polychlorinated containing CEF were fed. Symptoms in biphenyls included 1) depressed weight clude: 1) depressed body weight, 2) gain, 2) edema, 3) gasping for breath, 4) edema, 3) pericardial fluid, 4) increased hyperpericardial fluid, 5) internal hemor cell wall permeability and 5) an increase id rhaging, 6) depression of secondary sexual liver as percentage body weight. The con characteristics and 7) an increase in liver stituent responsible for CEF is known to as percentage of body weight. Several of contain a high proportion of chlorine these symptoms were present when levels of (Yartzoff ct al., 1961). Because of the sim more than 20 p.p.m. of PCBs were fed. ilarity to symptoms and the high propor tion of chlorine iVn both PCBs and CEF,9 one might postulate that their mode of ac tion is similar. Other workers (McCune et al., 1962; REFERENCES Anderson, D. W., J. J. Hickey, R. W. Risebrough, D. F. Hughes and R. E. Christensen, 1969. Sig nificance of chlorinated hydrocarbon residues to breeding pelicans and cormorants. Can. Field Table 2.--Liver at % body weiuhl on toxic Unit #/ PCPs with 2\ week-eld chicks Natur. S3: 91-112. Bradley, R. L., Jr., 1970. Personal communica tion. PCBs added to diet p.p.m. 0 0 Weight of birds i 218 195 Weight Liver as of liver . % body wt. 85.8 2.7 6.7 3.7 Campbell, T. C., and L. Friedman, 1966. Chick edema factor: Some tissue distribution data and toxicologic effects in the rat and chick. Proc. Soc. Exp. Biol. Medf 121: 1283-1287. Flick, D. F., R. G. ODell and V. A. Childs, 1965. Studies of the chick edema disease, 3. Similar ' 50 170 5.8 3.4 ity of symptoms produced by feeding chlori- ( nated biphenyl. Poultry Sci. 44: 1460-1465. 150 150 106 139 4.8 6.2 4.5 4.5 Food and Drug Administration, H. E. W., 1968. 150 151 6.8 4.5 Pesticide Analytical Manual. I. Methods Which Detect Multiple Residues. Washington, D.C. I * .> '1 ; k i r [ DSV\J 332637 ( I STLCOPCB4079213 10*>6 n. M. Rkhkki.o, R. L. Bradley, Jr. and M. L. Sunuk Kfx-mnn, J. H.| M. C. Tfn Nnevcr Da llrnux nnd R. H. De Vo% 1060. C'ldorinnlnl liiplirnyk In imiwh aiul Wiril* from flip River klilnc and ihr XrtherUrwU r,,xl,,l area Nxlwr, Ml! 1126 1126. MaiMimiii*. y., 1'itrt, hll'l', nniviil iln'iih o ini iu mriltiiniMii n( VhIi* Ni. Mur., Nov, 1060, |). 6 0, McCunc, K. J. B. tvvnuc and It. L. O'Dell, Pen kail, 1). II,, 1067, Pesticide-induced enzyme lirrnkdown nf slefolris in lilrds. Nature, II6j 505 406. MManfirti, It W,i< II lit Iih H It I'h.i-ilhh ft 11'iiiii`l nnl <4 ft In in,, /<,,y /'/// flilhill Ifl,"( lllliln ilyl- III Hit Ith/lHl Hi/ 'J >1111 hi tine, )m nun no/. Tucker, K. K., ami It. (i. t'mill i re, l`i/0. Ilmnl- Imnk of Toxicity of I'esliddes to Wildlife. U. S. MATI.iu si* bhli lui.n inn hth' i f pi pa ,,l, 4 I'li liiiiinary | wood shavings I, weeks in order 1062. Hy,lro|>cricAr(lium nnd ascilci in chicka frd a chlorinated hydrocnrlron. Poultry Sci. 41: 205-200. Monsanto, 196$. Aroclof Plasticizers. Tech. Hull. 0/PL-306. Monsanto Organic Chemistry Divi sion, St. Louis, Mo. l)e|l. of Interior, liurcau of S|iorl Kislieries and Wildlife Resources Publication No. 84. Yartzoff, A., I). Fireslonc, I). Hanes, W. Horwiir, L. Friedman and S. Ncslieim, 1961. Studies of the chick edema factor. II. Isolation of toxic substance. J. Amer. Oil Chem. Soc. 31: 60-62. reuse. The reuse,, was being used /. each succeeding t; use. Thus, the rc the sixth trial ha, its 7th use) and ti addition, received Fumigation and Reuse of Broiler Litter* i Pens were rand, I to each treatment The pens assigned Y.Ernest Ross and Allen Miyaiiara* of necessity, remai Il Prfiarfment of Animal Sciencti, Univtruly oj 'Hawaii, Honolulu, Hawaii 96822 (Received for publication December 11. 1970) f 1\I the experiment, wh |)cns were reassign. klW*,*-- ' THE periodic scarcity of conventional thyl bromide. In a controlled laboratory litter materials is a matter of increas study and under "semi-field conditions" ing concern to broiler producers in matnhyey reported that methyl bromide killed One hundred ai chicks were started trials, they were pi parts of the country. In areas where the Aspergillus Jumigains on agar, Ascaridia \ A top of the litter i1<I problem has been especially acute some poultrymen have resorted to reusing the lit ter for 2 or more broods. galli eggs and Eimcria tenella oocysts in a small amount of water when used at the rate of 1 cc. per quart of space or 1 pound Jr aluminum or card inches high. In the were placed direct); dart of each trial, Although Kennard and Chamberlin per 100 square feet of floor space. Irrors nnd one chic ij 1 (1947, 1948a, b, 1949, 1951) have demon strated the effectiveness of built-up litter pathologists and poultrymen are still con Klepscr cl al. (1962) fumigated two broiler houses under field conditions at the rate of 1/2 and 1 lb. of methyl bromide |xr 4 1Il4 ti-ed |>er pen. After ; were expanded and cerned about the possibility of perpetuating 100 square feet of floor space. Test dishes disease organisms to succeeding broods. distributed in the litter showed good con l it automatic water Brooding was done m brooders, at the rat The use of a soil fumigant, methyl bro trol of E. neealrix and variable results with t. l*rn. 1 I mide, by Andrews cf al. (1943) and Boney Ascaris suutn ova. They also rejjorted no (1948) for control of coccidia and interme significant mortality among chicks placed { (i Ul chicks used w, bred broiler chicks oh l i diate hosts of some internal parasites sug on the fumigated litter within a few hours gested to Edgar and King (1955) (he pps- following termination of the fumigation pe He, tell commercial t ftau',1 regular broi sibility of sterilizing poultry litter with me- riod. Their preliminary report, however, did not contnin data on the subsequent per commercial fe, "nc trial all chick 'Journal Series No. 1278 of the Hawaii Agri formance of these chicks. f t,|"ry un(| n,, rcct cultural Kx|>crlmcnl Station. ,1 The puqiosc of this study, therefore, w 'Stale Department of Agriculture, Honolulu, . to determine the effect of methyl bromlile Hawaii 96814. Present address: Department of feed. Trials 1 ` trials 4 an, Animal Sciences, University of Hawaii, Honolulu, fumigation of reused litter on chick growth, "rtt the chicks were Hawaii 96822. feed conversion and mortality. -1 * 6 when they were DSW 332638 STLCOPCB4079214