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pCB-t Organochlorine Pollutants in Peregrines and Merlins Migrating through Wisconsin Robert W. Risebrough Department of T futritronnl Sciences Institute of Vannc Resources University of C; lifornia Berkeley, Califo nia 94720 ' Gregory I -iorant I.ahoraton Oi nitholocy. f orncll Univcrsi';,, llhaci. New Vork Daniel D. B :rger 1328 N. Jetrerso i Milwaukee, Wistonsin 53202 . Tlie discovery that the Peregrines (Falco peregri/ius) breedit g in the eastern Arctic are laying thin-shelled eg :s and arc showing symptoms of reproductive failure (Berger et al., this issue) suggests -that tl is population is beginning to be affected by th: same extinction process that has resulted in the extirpation of breeding Pere grines in man ' areas of North America and Europe (Hickey,1969; Ratcliffe, 1970). The Peregrines of the eastern United States disap peared before the nature of the extinction pro cess was evident, but subsequent research showed that eggshell thinning had been asso ciated with the population decline (Hickey and Anderson, 1968). In Great Britain there was a considerable amount of evidence that exces sive mortality of adult birds had been a major reason for the rapid decline that began around 1955, coinciding with the introduction of highly toxic insecticides such as dieldrin into agricul tural use (Ratcliffe, 1963; Jefferies and Prestt, 1966). The s> rviving birds, however, exper ienced reproductive failures that were charac terized by shell thinning, egg breakage, and eat ing of the eggs by the adult birds (Ratcliffe, 1967; Ratcliffe, 1970). Although embryonic mortality may be contributing to the decline in reproductive capacity of the Ungava Peregrines (Berger et al., this issue) the pattern of egg breakage associated with shell thinning now observed in this population appears to be iden tical to the symptoms noted previously in south ern Canada (Hall, this issue), the United State; (Hic'ey and Anderson, 1968) and Grett Britain (Ratcliffe, 1970). At the conference convened in Madison. Wisconsin, by J. J. Hickey in 1965 to examine the cruses of disappearance of the Peregrine from the eastern United States, all conceivable factors were considered in detail. Among these, only environmental pollution was consistent with the pattern of extinction observed in Peregrine populations in both Europe ana North Aiuciica (Hickey. 1969). Since repro ductive failure now appears to threaten the survival of the Arctic populations, it has be come imperative to determine more precisely the particular pollutants responsible. In addition to the evidence relating excessive adult mortality to the use of several of the more toxic of the chlorinated hydrocarbon insecti cides, Ratcliffe (1970) has accumulated a con siderable amount of data relating the shell thinning among British birds of prey to the environmental distribution of organochlorine pollutants, including both insecticides with their derivatives and industrial compounds. The latter consist primarily of polychlorinated biphenyls which have become widespread and locally abundant pollutants (Jensen et al., 1969; Kocman et al., 1969; Risebrough et al., 1968). Other pollutants that might be contribu ting to the decline of the North American Peregrines include mercury and lead. Mercury 247 DSW 255255 248 The Canadian Field-Na uralist Voi. 84 is more widespread in the environment than was previously suspected (Fimreite et al,, this issue). Waterfowl may accumulate lead from ingested shotgun pellets (Baglcy and Locke, 1967) and Peregrines preying upon them might acquire dangerous concentrations. There is as yet, however, no evidence that links either metal at environment; I concentrations to the shell thinning phenom mon. Proof is rapidly accumu lating, however, hat one or more of the chlor inated hydrocarbons, particularly the DDT compound DDE. may induce shell thinning under both env ronmcntal and experimental conditions (Hick -y and Anderson, 1968; Porter and Wiemeyer, 1969; Heath et al,, 1969; Anderson et al,, 1969). Very small concentra tions of DDE are associated with significant changes in shell tnickness in eggs of the Brown Pelican (Pelecan ts occidentalis) (Risebrough, Anderson and Schreiber. unpublished observa tions). The role af DDE and of other chlorin ated hydrocarbons found in the environment such as dieldrin and the polychlorinated bi phenyls in produc ing additional sublethal effects that might interfere with reproduction is as yet unclear. Delayed breeding or abnormalities in behaviour could be associated with the high rate of steroid degradation caused by the liver enzymes induced oy these compounds (Jefferies, 1967; Peakall. 1970). The volume that emerged from the 1965 conference in Madison (Hickey, 1969) concludes with the statement by Hickey and Roelle "How much industrial pollutants like the polychlorinated biphenyls have also contri buted to some of these phenomena promises to be an absorbing chapter in the research of the immediate future" (Hickey and Roelle, 1969). The present paper reports on the polychlorina ted biphenyls and other chlorinated hydro carbons in fat biopsy samples obtained from ten Peregrines and seven Merlins (Pigeon Hawk. Falco columbarius) trapped at the Cedar Grove Ornithological Station on the western shore of Lake Michigan during the fall migrations of 1968 and 1969. Materials and Methods All of the Peregrines were immature birds of the year and therefore approximately three months old. Seven were biopsied in 1968 and three in 1969. All of the Merlin samples were obtained in 1969. Three were adult males, one an adult female, and the remaining three were immatu, cs of the year. The biopsy technique has been described previously (Endcrson and Berger, 1968). There have been no indications that this procedure harms tie birds in any way. Biopsy samples have b :cn obtained from Harriers (Circus cyaneus) that have been observed for many months afterwards (Frances Hamcrstrom, per sonal communication). The adipose tissue biopsy samples were frozen, cither in acetone-washed glass vials capped with aluminum foil, or in sterile packets lined internally with aluminum foil which are used in medicine for antiseptic gauzes. The samples were weighed in the laboratory to ob tain wet weights and then ground in a Waring blendor with anhydrous sodium sulfate. Lipids were obtained by soxhlet extraction for at least six hour, with a hot mixture of two parts of hexane ;.nd one part of acetone. The organic solvents were removed by evaporation. When small amounts of sodium sulfate were carried over into the extract, the lipid was dissolved in petroleum ether and washed with water. The biopsy SuiVtpiwS VvcTC aiJ'icUi. i tiC Pcicgrine samples averaged 0.49 grams, with a range from 0.0496 to 0.983 gm, and the Merlins averaged 0.162 gm, with a range from 0.0497 to 0.5608 gm. Some samples were found to consist mainly of connective tissue rather than lipid. The Peregrine samples consisted of an average of 69% by weight of extractable lipid, with a range from 31% to 91%. One Merlin sample consisted of only 5% lipid, the average percent lipid among the Merlin samples was 35%. and the highest value was 68%. The lipid extracts were dried at 65 for 8-12 hours, weighed in the beakers, dissolved in hexane and divided into one or more aliquots. The aliquot analysed for the DDT and PCB compounds was passed through a modified Davidow column (Stanley and LeFavourc, 1965). This column, consisting of celite, sul furic acid and fuming sulfuric acid, permits rapid cleanup with quantitative recov- DSW 255256 STLCOPCB4060947 1970 Risebrough et. al.: Pollutants in Peregrines and Merlins 249 erics of the DDT compounds and PCB. Although not an important factor for the present study, it permits much larger amounts of lipid material to be treated at one time than docs the florsil method. This procedure, how ever destroys dicldrin and to determine this compound another method must be employed. Another aliq.iot of the lipid extract, in hexane, was partitioned twice with acetonitrile, the combined acetonitrile fractions were evaporated to dryness, ^dissolved in petroleum ether, and applied to a litirisii column. The column was rinsed first with 200 ml of petroleum ether and 200 ml of 6% ethyl ether in petroleum ether. Dicldrin was eluted by passing 200 ml of 15% ethyl ether in petroleum ether through the col umn. Control.; had indicated that 90% or more of the dieldrir applied to a florisil column eluted in this fraction. Blank samples consisting of redistilled petro leum ether analysed through the entire proce dure showed 10 substances that might interfere with the deter nination of any of the chlorinated hydrocarbons reported here. The extracts were analysed with a Microtek gas chromatograph equipped with a tritium and a nickel-63 electron capture detector. Glass columns were used containing 3% QF-1, or a mixture of 3% QF-1 and 10% DC-200, on Chromosorb W, 80-100 mesh, acid washed and HMDS treated. Other laboratories (Reynolds, 1969; Armour and Burke, 1970) have developed methods for separating the polychlorinated biphenyls from some or all of the chlorinated hydrocarbons of insecticide origin. The polychlorinated biphenyl compounds emerge at different times from the gas chromatograph and occasionally a peak may coincide or interfere with the peak produced by one of the insecticides. We have found it possible, however, to quantify PCB. DDE, p,p'-DDD, p,p'-DDT, dieldrin and endrin on the basis of the procedure outlined above, with out any attempt at separating the PCB. Dieldrin and DDE emerge at exactly the same timte on the DC-200 column, but on the QF-1 column there is no significant interference between dieldrin and DDE, or between dieldrin and any of the polychlorinated biphenyls encountered in environmental samples. DDE frequently con stitutes 90% or more of the DDT compounds present in wildlife. The electron capture re sponse of DDE is considerably greater than that of PCB, and although one of the PCB compounds emerges at approximately the same time as DDE, the error introduced in the DOE determination is less than experimental cr or even when the total PCB concentration is up to five times that of the DDE. Vermeer and Ri ynolds (1970) have commented upon the diificulty in separating the DDT compounds p. >'DDD and p.p'-DDT from PCB. On the DC-2 X) column, both DDT compounds emerge at approximately the same time as a PCB pc ik (Anderson et al., 1969). On the QF-1 colunr n, however, there is no interference between p,;/DDT and any of the polychlorinated biphen Is usually encountered in environmental sample . Although many different chlorinated bipheryl compounds arc released into the environmei t, relatively few persist in the higher levels of fo<d chains. Moreover, these are found in appro; imately the same proportions in environmental samples over wide areas of the world. The chromatograms of the PCB compounds in the Peregrines analysed in the present study are therefore similar to the chromatograms of Pacgrine extracts from Mexico, California, and Amchitka Island (Risebrough, unpublished re sults). Moreover, the chromatograms are similar to those obtained from extracts of Brown Pelican eggs from several areas in both North and South America (Risebrough, unpublished results). It is frequently possible, therefore, to treat the PCB mixture as a single compound and to obtain accurate relative concentrations of PCB using any one of the several peaks. The peak emerging at the same time as p,p'-DDD on the QF-1 column is almost always of the same height as two of the other peaks. It is therefore possible to obtain a crude estimate of the DDD present without initial separation of the PCB. This assumption may be tested and the identification of p,p'-DDT and p.p'-DDD con firmed by saponification (Anderson et al., 1969; Risebrough er al., 1969). An aliquot of the DSW 255257 250 The Canadian Field-Naturalist Vol. 84 extract, in hexane or petroleum ether, is re fluxed in 5% KOH in ethyl alcohol. The hexane is then separated from the alcohol and potassium hydroxide by the addition of water and a sample injected into the gas chromatograph. The pro cedure converts both DDT and DDD to their respective ethylene derivatives, DDE and DDMU, but leaves the PCB profile of the chromatograms unchanged. Since the profile of the PCB peaks was similar, but not identical to the profile observed in chromatograms of the commercial prepara tion Aroclor 1254, PCB was quantified by comparing the total area of the PCB peaks emerging after DDE with the areas of the same peaks in chromatograms of standard Aroclor 1254. The procedure is therefore arbitrary, but permits the determination of relative, amounts of PCB within tht correct order of magnitude of absolute amounts. Moreover, the values ob tained can be readjusted as methodology is improved. Results and Discussion Enderson and Berger (1968) have previously reported on the chlorinated hydrocarbon resi dues found in biopsy fat samples from immature Peregrines that were also trapped during fall migration at Cedar Grove, Wisconsin. These samples, analysed by the Wisconsin Alumni Research Foundation in Madison, Wisconsin, contained an average concentration of 19.4 ppm of DDE on a lipid basis. DDE concentra tions in the fat of females at the Arctic breeding grounds are much higher, in the order of 300 ppm or higher (Berger et al., this issue). The ten Peregrine samples analysed in the present study contained 17.9 12.3 ppm DDE, 0.23 0.34 ppm p,p'-DDD, 0.71 0.68 ppm p,p'-DDT, 18.8 it 12.5 ppm total DDT (DDE + p,p'-DDD + p,p'-DDT), and 52.2 32.3 ppm PCB. Seven samples were analysed for dieldrin, which averaged 0.40 0.64 ppm. All residues are expressed on an extractable lipid basis, with the 95% confidence limits of the standard error of the mean. The values for p,p'-DDD and p,p'-DDT are lower than those previously reported by WARF, Inc., presum ably a result of interference by PCB in the earlier determinations (Anderson et al., 1969). The dieldrin values arc equivalent to those previous'y reported and there is no significant difference in the DDE concentrations. On a lipid basis, the average values reported by WARF, Inc. weie 19.3 ppm DDE, 0.8 ppm DDD, 1.2 ppm p,p'-DDT and 0.3 ppm dieldrin (Enderson and Berber, 1968). First year immature Peregrines migrating from the Arctic have therefore low residues of DDE. Unfortunately little is known about the relations lips among dietary levels of DDE and PCB, cqiilibrium concentrations of these com pounds i i various tissues, and the times required to achieve an equilibrium concentration. The low con :cntrations found in these Peregrines presuma nly reflect therefore both the age of the birds, which possibly has not permitted them to accumul ite the high concentrations found in adults, aid lower levels of contamination found in some of the prey species. An understanding of the pi armacodynamics of DDE and PCB in birds is me of the conspicuous deficiencies in our knowledge in the field of pollution ecology. Within several ecosystems so far studied, the concentrations of DDE in birds tends to parallel the concentrations of PCB. The ratio may be approximately one to one, as in San Francisco Bay (Risebrough et al., 1968). DDE is several times more abundant than PCB in several Pacific marine ecosystems (Risebrough et al., 1968), but in Florida, PCB is approximately three times as abundant as DDE in the Brown Pelicans (Risebrough, Gress, Anderson and Schreiber, unpublished manuscript). The cor relation coefficient between the DDE and PCB concentrations is frequently highly significant. These observations have suggested that the ratios observed reflect the local fallout pattern of the DDT and PCB compounds, and that the DDE and PCB show similar patterns of accu mulation, concentration and excretion in the birds and possibly other members of the ecosys tem. Among the 10 Peregrine samples analysed, PCB was more abundant than DDE by ap proximately three times. The correlation co- DSW^2Sa STLCOPCB4060949 1970 Risebrough et. al.: Pollutants in Peregrines and Merlins 25 i efficient is 0.8897, which is significant at the 0.001 level. Although it is not known from which area of the Arctic these birds have come, the results suggest that over a considerable area of the tundra ecosystems occupied by the Pere grines. this ratio reflects the local fallout pattern of DDE and PCB. The four adult Merlins averaged 302 ppm DDE (139-704) in the lipid fraction. This value may be compared with an average concentra tion of 392 ppm in the lipid of 9 breeding Peregrines from the Mackenzie River region (Enderson and Berger, 1968), 725 ppm in the fat of four adult Peregrines from Alaska (Cade et al., 1968) and 334 ppm in the lipid of 9 adult Peregrines from Ungava (Berger et al.. this issue). Merlins therefore may possess al most the Sami level of contamination as the Peregrines and might therefore be expected to show symptoms of shell thinning. The concen trations of total DDT in the adult Merlins averaged 314 ppm. 96% of the DDT residues in these Merlins therefore consisted of DDE. The PCB concentrations averaged 196 ppm (44-467). The samples were not analysed for dieldrin. In the three immatures, DDE averaged 49.3 ppm (10.8, 18.6 and 119.3), total DDT aver aged 50.3 ppm, and PCB averaged 28.6 ppm (5.7, 29.0 and 51.2). With the exception of one immature Merlin that contained 18.6 ppm DDE and 51.2 ppm PCB, a ratio similar to that found in the Peregrines, all other Merlins contained more DDE than PCB. With the one immature excepted, PCB was significantly correlated with DDE (r = 0.9820, p less than 0.001, four degrees of freedom). Most of the Merlins, therefore, appear to be coming from a region of the boreal forest where the pollutant fallout pattern is different from that in the tundra ecosystem occupied by the Peregrine. When PCB was found to be present in Pere grines, occasionally in high concentrations, it seemed likely that these pollutants were con tributing to the reproductive failures associated with population decline (Risebrough et al., 1968). The dominant symptom of reproductive failures of the Peregrines in Britain has been shell thinning associated with egg breakage, disappearance of the eggs, and eating of the eggs by the adult birds (Ratcliffc, 1970). In the Ungava population of Peregrines in the eastern Arctic, shell thinning and egg breakage arc nov contributing to reproductive failures (Berger e' al., this issue). The evidence available to date suggests, however, that PCB is not a cause or the si ell thinning observed in many population:, of biids. No correlation between thinning am PCB was found in the eggs of the White Pelican (Peleranus erythrorhynchos) (Anderson et al., 1969 or in eggs of the Great Blue Heron (Ard>a herodias) (Vermeer and Reynolds. 1970 . An analysis of approximately 200 eggs of th - Brown Pelican from both eastern anti weste rn North America has shown that DDE rathei than PCB is associated with the she!. thinn:ng (Risebrough. Grcss, Anderson and Schre ber, unpublished ms.) Whenever corre lations between PCB and shell thinning have been observed, as in Double-Crested Cormor ants >Phalacrocorax auritus), PCB is also cor related with DDE (Anderson et al.. 1969). Pe;.kall (1970) has reported that p.p'-DDT delays egg laying in the Ringdove (Streptopelia risoria) and that the delay is associated wit! lower concentrations of estradiol in the blooa early in the breeding cycie. Peakall had earlier reported (in Risebrough et al., 1968) that technical DDT, p.p'-DDE, and PCB induce the synthesis of liver enzymes that degrade estradiol, and that PCB is a more potent enzyme inducer thanns DDE-. Delayed breeding may therefore be one of the environmental effects of PCB, an effect that could be critical to Arctic-breeding birds which must complete their reproductive cycle within a restricted period of time. A recent investigation in Japan of the "Yusho" or Rice Oil Disease has traced the cause to rice oil contaminated by chlorinated biphenyls (Katsuki, 1969). Both the contamin ated rice oil and the Japanese commercial PCB produce symptoms when fed to chicks similar to those observed in the "chick edema" syn drome (Goto et al., 1969). This disease of chicks was first observed in the United States in 1957, and among the symptoms were accu- i dSVM 255259 STLCOPCB4060950 252 The Canadian Field-Naturalist Voi. 8- mutation of fluid in the pericardial sac and in the abdominal cavity, subcutaneous edema, and liver and-kidney damage (Verrett, 1970). Chlorinated dibenzo-p-dioxins have been shown to be one of the groups of chlorinated synthetic compounds that produce this disease (Higgin botham et. al., 196S). These compounds have also been shown to be present in the herbicide 2,4,5-T and induce both mortality and embry onic deformities in chicks at very low concen trations (Verr-.u, 1970). A similar group of compounds, the chlorinated dibenzofurans, are almost as toxic (Schulz, 1970). These resemble the chlorinated biphenyls in structure and might be derived from them. Whether they are now present in the environment and pose a threat to wildlife promises to be "an absorbing chapter in the research of the immediate future" (Hickey and I'.oelle, 1969). Acknowledgements The research was supported by the National Science Foundation grants GB 6362 and GB 11649. , Literature Cited Anderson, D. \V., Hickey, J. J., Risebrough, R. W., Hughes, D. F., and Christensen. R. E. 1969. Sig nificance of chlorinated hydrocarbon residues to breeding pelicans and cut inuiams. Canadian FieldNaturalist 83(2): 91-112. Armour, J. A. ard Uurkc, J. A. 1970. Method for separating polychlorinated biphenyls from DDT and its analogs. Journal of the Association of Official Analytical Chemists 53: 761-768. Bagley, G. E. and Locke, L. N. 1967. The occur rence of lead in tissues of wild birds. Bulletin of Environmental Contamination and Toxicology 2: 297-305. Berger, D. D.t Anderson, D. 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