Document aBNOzOVGk0JXaevEKEx8ER57B

Rtprlnitdfrom: PROCEEDINGS OF THE XVth INTERNATIONAL ORNITHOLOGICAL CONGRESS LEIDEN E. J. BRILL 1972 DSW 031225 STLCOPCB4015187 Effects of polychlorinated biphenyls on birds R. G. Heath, J. W. Spann, J. F. Kreitzcr & C. Vance Rurean of Sport Fisheries and Wildlife, Pa/iixenl Wildlife Research denier, FanrH, Maryland, US. A. Jntroducton Polychlorinated biphenyl (PCB) compounds are a group of synthetic chlorinated hydrocarbons that have been used industrially in tank car quantities since the early i93o's (Penning 1930). They are marketed as complex mixtures 1 which function, for example, as plasticizers in diverse materials, as electrical insulators and impregnators, as grinding and cutting oil, hydraulic fluids, and high temperature lubricants, and as heat exchange media 2. 3PCB's are rated in industry as highly toxic compounds and can cause permanent or fatal liver damage (Sax 1963). In 1966, PCB's were identified as environmental contaminants when Jensen (1966) reported residues in Swedish pike, an eagle, and human hair. Identification was con firmed by mass spectrometry. Subsequent reports of residues in numerous terrestrial and aquatic forms have established the global ubiquity of PCB's. They have been reported in British wildlife (Holmes el al. 1967; Prf.stt el a!. 1970), in Scottish and Canadian seals and porpoises (Holden & Marsden 1967), in birds and fish from California and the Pacific (Risebrough el al. 1968), in fish, mussels, and birds of the Netherlands (Koeman et al. 1969), in North American cormorants and pelicans (Anderson et al. 1969), in marine animals of Sweden (Jensen et al. 1969), and in North American Bald Eagles (Bagley et al. 1970). In both environment and laboratory, PCB's behave like DDT and other organochlorine pesticides. Poorly soluble in water but readily so in fats, they concentrate in animal lipids together with pesticidal residues. As with DDT, concentrations tend to increase through trophic levels of food chains (Jensen et al. 1969). PCB's are recovered and detected by the same analytical methods used for organochlorine pesticides, and chemists have necessarily developed procedures to separate them prior to quantification (Koeman et al. 1969; Bagley et al. 1970). Before 1966, PCB's were either misidentified as specific pesticides or viewed as unidentified compounds, possibly pesticidal metabolites (Holmes, et al. 1967); however, detectable quantities have been in the environment at least since 1944 (Jensen 1966). Several routes of entry into the environment have been postulated. For example in dustrial leakage, flushing, or dumping of waste PCB's are conceivable, and refuse burning could release them from plastic materials into the atmosphere. The ecological significance of PCB contamination is virtually uninvestigated. Being toxic compounds they must be classified as potentially hazardous, but toxicological information is limited. Their direct environmental association with several pesticides makes synergistic actions conceivable. Studies to determine effects of PCB's on birds were initiated at the Patuxent Wildlife Research Center in 1968. From these studies we report the following determinations: (1) Median lethal concentrations (I.C60's) of six technical PCB mixtures (Aroclor 12329, 1242, 1 Registered names include Aroclor, Clophni, and Pbcnocblor. 2 Technical Bulletin O/Pl-306, Monsanto Co., St. Louis, Missouri. 3 I.ast two digits of Aroclor numbers denote chlorine percentage. DSW 031226 STLCOPCB4015188 476 1IF.ATH ET AI,,: SYMPOSIUM ON CHKMICAE POI.UJDON 1248, 1254, 1260, and 1262) in diets of 2-week-old Mallards Anas platyrbyncbos, Pheasants Phasianus cotchicus, Dobwhitc Colinns virginianns, and Japanese Quail Cotnrnix colurnix. (2) Joint actions of three ratios of Aroclor 1254 and DDE combined in diets of Japanese Quail for 5 days. (3) Reproductive effects of sublcthal dietary levels of Aroclor 1254 on Mallards and Bobwhite. Dietary Toxicitiks of PCB's Toxicitif.s of individuae Aroclors Dietary LCjo's were derived to assess the relative toxicities of the different Aroclors and to compare the toxicities with those of several common organochlorine pesticides. The LC5o's also serve to rank the sensitivities of the four species to each chemical. An LC50 is defined here as ppm of a chemical in an ad libitum diet expected to produce 50 percent mortality among 2-weck-old birds in 8 days, 5 days of toxic diet followed by 3 days of untreated diet (the latter included to detect mortality induced beyond the dosage period). The test protocol was that described by Hradi & L. Sdckf.i. (1965). Briefly, chemical was introduced as a dietary component because ingestion is seemingly the principal route of exposure in the wild. Aroclors and pesticides were dissolved in corn oil and then mixed thoroughly with dry feed in a ratio of 2 parts of solution to 98 parts of feed. An equal amount of pure corn oil was added to control diets. To standardize experimental conditions, all Aroclors were tested simultane ously on a given species in a completely randomized design. Each LC50 was derived from a group of 60 birds placed in six pens of 10 each, One of six dietary levels was assigned to each pen. Levels were spaced geometrically over a range expected to produce from 10 to 90 percent mortality. Pens of control birds were used to adjust LC5o's for extraneous mortality. LCjo's were computed by met hods of probit analysis (Finney, 1952). Table 1 presents LCjo's, with 95 percent confidence limits, of Aroclors, DDE, DDT, dieldrin, and endrin for the four test species. These determinations show that dietary toxicities of Aroclors were, with few exceptions, less than those of the four organochlorine pesticides. Endrin was approximately 100 times, and dieldrin 10 times, as toxic as any Aroclor; and DDT was as much as 4 times as toxic. Aroclors were somewhat more toxic to Mallards than DDE, as were the highlychlorinated Aroclors to Bobwhite; otherwise, DDE1 was more toxic than PCB's. Presst^ */. (1970) found Aroclor 1254 to have 1/13 the lethal toxicity of DDT to adult Bengalese Finches Loucbura striata in a 56-day feeding study. There was a positive relationship between the percentage of chlorine in a technical Aroclor and its toxicity. The relationship held true for those containing less than 60 percent chlorine, Aroclors 1260 and 1262 deviating slightly. Mallards were relatively less responsive to chlorine content than the three gallinaceous species. DSW 031227 STLCOPCB4015189 11 HATH ET AT.! SYMPOSIUM ON CHEMICAL POLLUTION 477 Table i Toxicity comparisons of Aroclors (polychlorinated biphenyls) and four organoCHLORINE PESTICIDES IN DIETS OF 2-WEF.K-OLD BIRDS Chemical LC50V, with 95% confidence limits parenthesized Mallard Pheasant Bobwhite Japanese Quail Aroclor 1232s Aroclor 1242 Aroclor 1248 Aroclor 1254 Aroclor 1260 Aroclor 1262 DDE DDT dieldrin endrin -- 3180 (2610-3880) 2795 (2265-3420) 2700 (2160-3310) 1975 (1365-2750) 3QIO (2460-3635) 357 (2810-4670) 1870 (1500-2370) 200 (160-240) 22 (i7-3i) 0 1 -u. 3150 (2625-3950) 2080 (1840-2350) I3IO (r165-1480) 1090 (965-1230) 1260 1*35 (1085-1405) 840 (730-970) 310 (25 5-375) 55 (49-61) (m-5-17-5) 3000 (2575-3500) 2100 (1705-2610) 1175 (965-1440) 605 (410-840) 745 (575-935) 870 (700-1070) 825 (695-980) 610 (515-725) 39 (33-45) 15 (10.5-24) >5000 >5000 4845 (435 5-5410) 2900 (2600-3240) 2185 (1915-2480) 2290 (2040-2575) 1355 (1110-1650) 57 (470-690) 56 (51-61) 15 (12.5-17) 1 LCS0: ppm chemical in ad libitum diet expected to produce 50% mortality in 8 days, j days of toxic diet followed by 3 days of untreated diet. Each determination based on 60 birds. * Last two digits of Aroclor numbers denote chlorine percentage by weight. There were consistent species differences in sensitivity to Aroclors. Bobwhite were most sensitive, followed in turn by Pheasants, Mallards, and Japanese Quail. LCjo's for Japanese Quail (the most tolerant species) were from 3 to 4 times as high as for Bobwhite. Mallards were about twice as tolerant as Pheasants, which were nearly as sensitive as Bobwhite. The order of species sensitivity to the organochlorine pesticides was somewhat different; Mallards were from 2 to 4 times as tolerant as the gallinaceous species, the sensitivities of the latter being relatively similar. During PCB poisoning, birds became lethargic and tended to assume a crou ching position within the final 24 hours of life. They displayed mild tremors during the final hour, tremors being less violent than those associated with DDT poisoning. Autopsies of eight Bobwhite chicks poisoned by Aroclor 1254 revealed a DSw 031228 STLCOPCB4015190 478 HEATH ET AL..' SYMPOSIUM ON CHEMICAL POLLUTION general decline in physical condition accompanied by a complete absence of fat, and there was excessive fluid in the pericardial sac. Similar effects from Aroclor 1242 were reported in Vantress-White Rock, cross-bred chicks (McCune et al. 1962). Vos & Koeman (1970) describe detailed pathological changes in Ilubbart cockerels fed diets with 400 ppm of one of three 60 percent-chlorinated commer cial PCB preparations. Joint toxicity of Aroclor 1254 and DDE Using the above 8-day protocol, Japanese Quail were fed diets containing combinations of Aroclor 1254 and DDE to test the possibility of synergistic action. Three ratios of the chemicals were tested, one combining 50 percent of the known effective dosages of both chemicals, one combining 90 percent of an effec tive dosage of Aroclor with 10 percent of that of DDE, and one combining 10 percent of an effective dosage of Aroclor with 90 percent of that of DDE. Both chemicals were also administered separately as is necessary in tests of joint action. Analysis was by the method of Wadlf.y (1945). All joint actions proved to be essentially additive. In terms of effective dosages the 50:50 percent mixture was about 91 percent as toxic as the individual chemi cals, the 90 percent Aroclor: 10 percent DDE was 98 percent as toxic, and the 10 percent Aroclor 190 percent DDE was 95 percent as toxic. Clearly there was no suggestion of synergism. Reproductive Effects of PCB's Two-year studies to determine effects of Aroclor 1254 on Mallard and Bobwhite reproduction began at the Center in 1968. DDE was included as a com parative treatment because of its close environmental association with PCB's and because of its capacity to impair Mallard reproduction (Hf.ath et al. 1969). A test for synergistic effects on reproduction using a mixture of Aroclor 1254 and DDE was necessarily confined to the Bobwhite study. Chemicals were administered in the diet. Mallard treatments included dietary concentrations of Aroclor 1254 at 25 ppm and DDF1 at 10 ppm, Bobwhite received Aroclor 1254 at 50 ppm, DDE at 30 ppm, and a mixture of 25 ppm Aroclor 1254 plus 15 ppm DDE. Diets were prepared by dissolving chemicals in corn oil and mixing one part oil solution with 99 parts of dry commercial feed. Test birds had been hatched in incubators, the progeny of captive breeders phenotypically indistinguishable from wild birds. Mallards were housed in wooden pens constructed on concrete slabs; pens were spread with straw and supplied with troughs of running water. Bobwhite were maintained outdoors in wire-floored cages. A completely randomized design was used throughout both studies. Five hens osw 031229 STLCOPCB4015191 HEATH F,T ALT SYMPOSIUM ON CHEMICAL POLLUTION 479 and two drakes were allotted to each Mallard pen, and each treatment was replicated in four pens. Bobwhite cages were assigned three hens and two cocks; each treatment was replicated in six cages. Mallards received treated diet ad libitum from about u weeks before their first laying season through their second year of laying. A similar schedule was initiated with Bobwhite; however, heavy mortality prior to the second breeding season limited the study to a single year. All eggs were collected daily for 8 weeks each season and stored at i6C and j 5 percent relative humidity. At intervals of 2 weeks they were examined for cracked shells, incubated, and candled after 2 and 3 weeks of incubation to measure embryonation and embryo survival. Hatchlings were leg-banded for identification Table 2 Reproductive success among penned Mallards maintained for two seasons1 on feeds containing Aroclor I 25 4 OR DDK season chemical added to feed (ppm) control Aroclor 1254 <*J> DDE (10) Pens of birds (5 99, 2 $$ each) Hcn-scasons (8 weeks each) Total eggs laid Eggs laid per hen-seasons Eggs cracked (%) Eggs embryonated of eggs set (%) Embryos alive at 3 weeks (%) Normal hatchlings of j-week embryos (%) Normal hatchlings alive at 14 days (%) 14-day ducklings of embryonated eggs (%) 14-day ducklings per hcn-scasons 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 1969 1970 4 4 20 20 590 630 29.5 }'-5 4 9 86 88 98 9* 70 59 94 93 64 5i 13.0 11.6 4 4 20 19 453 468 21.7 24.6 6 9 94 90 96 92 <>3 64 91 96 55 57 8.8 10.4 4 4 19.1 18 628 632 32.8 35-J 20* 28s 80 68 94 88 47* 53 97 92 45* 43s 7.8 6.7 1 Dosage started December 19, 1968; eggs collected from March 12 through May 6, 1969, and from March 18 through May 12, 1970. 8 Differences from controls statistically significant (P < o,05). OSw 031230 STLCOPCB4015192 480 HEATH ET AL.: SYMPOSIUM ON CHEMICAL POLLUTION Table 3 Reproductive success among penned bobwihte fed diets containing Aroclor 1234, DDE, OR A MIXTURE OP BOTH CHEMICALS1 chemical added to feed (ppm) control Aroclor DDE Aroclor 1254 -f DDE (50) (30) (25) 4-(15) Pens of birds (3 ?? 2 <?<? each) Hen-seasons (8 weeks each) Total eggs laid Eggs laid per hen-scason Eggs cracked (%) Eggs embryonated of eggs set (%) Embryos alive at 3 weeks (%) Normal hatchlings of 3-weck embryos (%) Normal hatchlings alive after 14 days (%) 14-day chicks of embryonated eggs (%) 14-day chicks per hen-season 6 16.6 347 20.9 10 88 97 96 78 73 11.5 O00 66 16.4 15-3 4*5 22.4 27,1 75 90 93 97 98 91 96 72 7 64 66 11.5 13.1 ,3 14.9 386 26.0 7 93 97 92 7i 63 13.5 1 Dosage started April 3, 1968; eggs collected from April 27 through June 21, 1968. None of the above differences V'as significant at the p/ percent level of confidence. with parental pen and observed on diet free of chemical for 14 days. Data were recorded for each pen for every 2-week set of eggs, From these data we calculated the seasonal parameters of reproductive success presented in Tables 2 and 3. Measurements were analyzed by weighted analysis of variance, pen statistics being weighted according to the number of eggs or hatchlings on which they were based. Angular transformations were applied to percentages for analysis, and means were separated by methods of Duncan (1955) and Kramer (1956). Prior to incubation eggs with sound shells from alternate 2 - week sets were sampled to measure shell thickness. Mean thicknesses of dry shell, with membrane, are presented in Tables 4 and j. Effects on Mallard reproduction Aroclor 1254 did not produce statistically significant effects on mallard repro duction at the 95 percent level of confidence (Table 2). The apparent reduction in egg production, and subsequently in ducklings per hen, resulted from one pen of DSM 031231 STLCOPCB4015193 HEA TH ET AL. I SYMPOSIUM ON CHEMICAL POLLUTION 481 Table 4 Mean thickness op eggshells op penned Mai,lards maintained through two seasons on FEED CONTAINING ArOCLOR I254 OR DDE chemical added to feed (ppm) season none Aroclor (*S) DDE (io) No. of eggs Shell thickness (mm) 1969 1970 1969 1970 28 32 0-375 0.365 2? 17 0.360 0.368 27 26 0.3391 0,328' 1 Differences from controls statistically significant (P < 0.05). Table 5 Mean thickness op eggshells of penned Bobwihte maintained through one season on PEED CONTAINING AROCLOR 12)4, DDK, OR A MIXTURE OF BOTH CHEMICALS chemical added to feed (ppm) none Aioclot (5) DDE (3) Aroclor 1254 4 DDE (25H-O5) No. of eggs Shell thickness (mm) 14 0.207 17 0.208 20 0.205 18 0.215 None of the above differences was significant at the 9; percent level of confidence. breeders failing to lay normally; however, egg production in the remaining Aroclor pens did not differ markedly from that in control pens. Other reproductive determinations differed little between control and Aroclor treatments. In particular, Aroclor caused no detectable increase in eggshell cracking or reduction in shell thickness; DDE induced significant shell thinning and cracking and a reduction in hatching success, as was reported in earlier work (Heath etal. 1969). Effects on Bobwhite reproduction Bobwhite reproduction after 1 to 3 months of dosage was not measurably affected by either Aroclor 1254 or DDE, administered singly or in combination (Table 3). Numerically, mean egg production was lowest, and percentages of cracked eggs highest, in control birds, although differences were not substantiated at the 5 percent level of significance. The suggested decrease in chick survival in all experimental groups was not significant. DSW 031232 * STLCOPCB4015194 482 HEATH ET AT.: SYMPOSIUM ON CHEMICAL POLLUTION Effects on eggshell thickness A preliminary investigation of PCB effects on mallard eggshell thickness was conducted in 1968. Single groups of year-old birds comprised of eight hens and one drake were fed diets containing o, 10, or 500 ppm of Aroclor 1254 from March 21 to April 29. biggs were collected from March 22 to April 11 (21 days) and again from April 25 to 29 (5 days). Eggs were opened and shells dried with forced air for 16 hours at z-jC. Shell thickness was measured at the waist with a Starrett dial gage graduated in 0,01 mm units. Average shell thicknesses did not differ significantly among treatments during the first collection period. Mean thicknesses from the o, 10, and 500 ppm Aroclor treatments were 0.357, 0.348, 0.354 mm in samples of 41, 36, and 27 eggs, respec tively. During the second period mean thicknesses were 0.357, 0.370, and 0.378 mm in 18, 30, and 17 eggs. It can not be determined if the 6 percent increases in mean shell thickness in both Aroclor 1254 treatments are real, since eggs could not be identified with individual hens, and pen replication was lacking. Certainly the Aroclor 1254 treatments did not induce shell thinning. Analysis of shell thickness measurements from the 2-year Mallard study, in which biweekly pen averages served as sampling units in a split-plot design, failed to indicate thickness changes from Aroclor 1254 at 25 ppm (Table 4). DDE at 10 ppm induced significant shell thinning both years (P < 0.05). The numerical increase in Aroclor 1254 shells the second year relative to decreases in control and DDE shells (years x treatments interaction) was not significant. Analysis of variance of Bobwhite eggshell data revealed no measurable differ ences in mean shell thickness among treatments (Table 5). Of particular note is the fact that DDE induced eggshell thinning in Mallards but not in Bobwhite. PCB residues in Mallard eggs Two second-year Mallard eggs from the Aroclor 1254 treatment contained 56 ppm and 33 ppm of PCB, wet weight. Measurement was by thin layer chromato- Figure i. Gas chromatograms of technical Aroclor 1254 (solid line) and Aroclor 1254 resi dues from a Mallard egg (dotted line) of a hen maintained on feed containing 25 ppm. DSW 031233 STLCOPCB4015195 HEATH ET AL.: SYMPOSIUM ON CHEMICAL POLLUTION 483 graphy (B. M. Mulhern et al. 1971). Methods of cleanup were essentially those described by Reichkl et al. (1969). The egg residues were also analyzed by gas-liquid chromatography (GLC) using an OV-17 column. Comparison of the GLC chromatograms of the mallard eggs with an Aroclor 1254 standard reveals that early peaks from the egg are greatly diminished (Fig. 1). A similar result was reported in brains and livers of Japanese Quail (Koeman et al. 1969). The differences may be due to differential absorption of PCB's in the system or to metabolism of low-chlorine molecules. A positive relationship has been shown between the number of chlorine atoms per PCB molecule and GLC retention time (Koeman et al., op. cit.; Bagley et al. 1970). Chronic effects of PCB's on breeders There were no deaths among 19 hen and 8 drake Mallards fed diets with 25 ppm Aroclor 1254 from December 19, 1968, to May 6, 1970I There was complete survival among control birds during this period, although two of 20 hens and one of 8 drakes on 10 ppm DDE treatment died prior to the second breeding season. As mentioned earlier, Bobwhite suffered heavy mortality prior to the second breeding season, and the study was terminated. Reproductive effects of PCB's in chickens In a study conducted for the Monsanto Company 1 significant reproductive impairment in White Leghorn chickens Galius gallus fed Aroclor 1242 at 10 or 100 ppm or Aroclor 1254 at 100 ppm was demonstrated. The dosages reduced egg production and hatchability and caused thin eggshells. There were no measurable effects from Aroclor 1242 at 1 ppm, from Aroclor 1254 at 1 or 10 ppm, or from Aroclor 1260 at 1, 10, or 100 ppm. Discussion It is encouraging that the lethal toxicity of PCB compounds proved to be less than that of DDT to the species investigated. However, the four-fold difference in sensitivity to PCB's observed between two gallinaceous species indicates that there may be species much more sensitive than those tested. Aroclor 1254 did not induce measurable changes in eggshells or in reproductive success of Mallards at 25 ppm or Bobwhite at 50 ppm; however the material did induce such changes in White Leghorns at 100 ppm (a severe dosage) but not at 10 ppm. Unfortunately the findings do not permit a valid comparison of sensitivity to Aroclor 1254 between White Leghorns and Mallards or Bobwhite, The fact that Aroclor 1242 affected reproduction at 10 ppm, while Aroclor 1254 did not, suggests effects from specific PCB isomers not necessarily related to chlorine 1 Industrial Bio-Test Laboratories, Inc., Report No. J 7300, June 4, 1970. To be published. OSW 031234 STLCOPCB4015196 484 HEATH ET AI,. : SYMPOSIUM ON CHEMICAL POLLUTION content. Eggshell thinning from PCB's has been suggested by other investigators (Andf.rson et at. 1969; Pkakall 1970). There are, of course, many aspects of PCB toxicity not investigated in this study. Extensive, integrated research will be required to assess fully the effects of polychlorinated biphenyls on birds. Summary Toxicides of six technical polychlorinated biphenyl (PCB) compounds (Arocior 1232, 1242, 1248, 1254, 1260, and 1262) were studied on penned Mallards Anasplafyrhjncbos, Pheasants Phasianus cokbicns, Bobwhite Col'tnus virginiams, and Japanese Quail Coturnix coturnix at the Patuxent Wildlife Research Center, Laurel, Maryland. LCM's expressed as ppm of PCB in dry feed, were determined for 2-wcck-old birds fed treated diets for 5 days. Arocior toxicity, generally less than that of DDT, was found to be positively correlated with chlorine percentage (last two digits of Arocior number). The joint toxicity of Arocior 1234 and DDE on Japanese Quail was additive, not synergistic. Low dietary levels (25 and 50 ppm) of Arocior 1254 produced no measurable repro ductive effects in Mallards and Bobwhite. Acknowledgements We wish to thank the Monsanto Company for permission to cite unpublished research findings, Dr, L. N. I.ockf. for autopsies and evaluations of gross pathology, Messrs. W. L. Rfjchf.l and B. M. Mulhf.rn for chemical analyses, and Mesdames H. M. Nelson and H. L. Young for statistical computations. Appendix The chemical names of compounds mentioned in this paper are: DDE i,i-dichloro-2,2-bis (p-chlorophenyl) ethylene DDT i,i,i-trichloro-z,2-bis (p-chlorophcnyl) ethane Dieldrln i,2,3,4,io,io-hcxachloro-6,7-epoxy-i,4,4a,5,6,7,8,8a-octahydro-i,4,endo-exo-5,8- dimcthanonaphthalenc Endrln i^^^.ro.to-hcxachloro-fij-epoxy-i^^a.j.fi^.S.Sa-octahydro-i^-endo-endo-j.S- dimethanonnphthalcne PCB Polychlorinated biphenyl References Anderson D. W., Hickey J. J., Riserrougii R. W. .Hughes D. F. & Christensen R. E. (1969) Significance of chlorinated hydrocarbon residues to breeding pelicans and cormorants. The Canadian Field Naturalist 83, 91-112. Bagley G. E., Rf.iciihi. W. L. & Cromartik E. (1970) Identification of polychlorinated bi phenyls in two bald eagles by combined gas-liquid chromatography-mass spectrometry. Journal of the Association of Official Analytical Chemists 53, 251-26U Duncan D. B. (1955) Multiple range and multiple F tests. Biometrics 11, 1-42. Finney D. J. (1952) Probit Analysis, 2nd Ed., Cambridge University Press. 318 p. Heath R. G., Spann J. W. & Kreitzkr. J. F. (1969) Marked DDE impairment of mallard reproduction in controlled studies, Nature 224, 47-48. OSW 031235 STLCOPCB4015197 HI'.ATH ET AL.: SYMPOSIUM ON CHEMICAL POLLUTION 485 IIpatu R. G. & Stickkl L. F. (1965) Protocol for testing the acute and relative toxicity of pesticides to penned birds. F. 18-21 in U.S. Fish and Wildlife Circular 226. Holden A. V. & Marsdf.n K. (1967) Organochlotine pesticides in seals and porpoises. Nature 216, 1274-1276. Holmes D. C., Simmons J, H. & Tatton J, O'G. (1967) Chlorinated hydrocarbons in British wildlife. Nature 216, 227-229. Jensen S. (1966) Report of a new chemical hazard. New Scientist 32, 612. Jensen S., JoiiNin.s A. G., Olsson M. 6c Oiterlind G. (1969) DDT and PCB in marine animals from Swedish waters. Nature 224, 247-230. Kop.man J. H., Ten Nof.vhr df. Brauw M. C. 6c de Vos R. H. (1969) Chlorinated biphenyls in fish, mussels and birds from the River Rhine and the Netherlands coastal area. Nature 221, 1126-1128. Kramer C. Y. (1956) Extension of multiple range tests to group means with unequal numbers of replication. Biometrics 12, 307-310. McCune E. I.., Savage J. E. 6c O'Dell B. L. (1962) Hydropericardium and ascites in chicks fed a chlorinated hydrocarbon. Poultry Science 41, 295-299. Mulhf.rn B. M., Cromartie E., Rf.ichkl W. L. & Bkmsle A. A. (1971) Semi-quantitative determination of polychlorinated biphenyls in tissue samples by thin layer chromatography. Journal of the Association of Official Analytical Chemists. (In Press). Peakall D. B. (1970) Pesticides and the reproduction of birds. Scientific American 222, 72-78. Penning C. FI. (1930) Physical characteristics and commercial possibilities of chlotinated di phenyl, Industrial and Engineering Chemistry 22, 1180-1182. Prf.sst I., Jefferies D. J. & Moore N. W. (1970) Polychlorinated biphenyls in wild birds in Britain and their avian toxicity. Environmental Pollution J, 3-26. Rr.icnr.i. W. L., Lamont T. G, & Cromartie E. (1969) Residues in two bald eagles suspected of pesticide poisoning. Bulletin of Environmental Contamination and Toxicology 4, 24-30. Rishrrough R. W., Peakall D. B., Herman S. G., Kirven M. N. & Rif.che P. (1968) Poly chlorinated biphenyls in the global ecosystem. Nature 220, 1098-1102. Sax N. I, (1963) Dangerous properties of industrial chemicals, 2nd Ed. Reinhold, New York, N.Y. 1343 p. ..... Vos J. G. & Kop.man J. H. (1970) Comparative toxicologic study with polychlorinated bi phenyls in chickens with special reference to porphyria, edema formation, liver necrosis, and tissue residues. Toxicology and Applied Pharmacology 17, 656-668. Waolfy F. M. (1945) The evidence requited to show synergistic action of insecticides and a short cut in analysis. United States Bureau of Entomology and Plant Quarantine, ET-223. 6 p. OSW 031236 STLCOPCB4015198