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Reprinted with permission of the Pesticides Monitoring Journal by the Patuxent Wildlife Research Center Bureau of Sport Fisheries and Wildlife U. S. Department of the Interior Laurel, Maryland 20810 Residues of Organochlorine Pesticides, Polychlorinated Biphenyls, and Mercury and A utopsy Data for Bald Eagles, 1969 and 1970* Andre A. Belisle, William L. Relchel, Louis N. Locke, Thair G. Lamont, Bernard M. Mulhem, Richard M. Prouly, Robert B. DeWolf, and Eugene Cromanie ABSTRACT Thirty-nine bald eagles found sick or dead in 13 States during 1969 and 1970 were analyzed for pesticide residues. Residues of DDE, dieldrln, polychlorinated biphenyls (PCB's), and mercury were detected in all bald eagle carcas ses; DDD residues were defected in 38; DDT, heptachlor epoxide, and dichlorobenzophenone (DCBP) were detected less frequently. Six eagles contained possible lethal levels of dieldrin in the brain, and one contained a lethal concentration of DDE (385 ppm) in the brain together with 235 ppm of PCB's. Autopsy revealed that 18 bald eagles were illegally shot; other causes of death were impact injuries, electro cution, emaciation, and infectious diseases. Introduction Reichel et al. (4) presented pesticide residue data from bald eagles (Haliaeetus leucocephalus) collected in 1964 and 1965; a more recent report by Mulhern et al. (2) presented residue and autopsy data on bald eagles for 1966 through 1968. The purpose of this paper is to report the organochlorine pesticides, polychlorinated biphenyls (PCB's), and mercury residues and autopsy data for eagles collected in 1969 and 1970. Sampling and Autopsy Procedures As mentioned in previous report (2,4), a systematic sampling procedure could not be used because of the relatively low populations of these birds and their protected status. Bald eagles found dead or moribund in the field are collected by Federal, State, and private cooperators, packed in dry ice, shipped air express to this laboratory, and stored intact in plastic bags at --25 C. The 13 States from which the 39 samples included in this report were collected are shown in Table 1; 28 birds were collected in 1969 and 11 in 1970, Only field speci mens that were not decomposed were analyzed. Autopsies were performed on all specimens to determine possible causes of death. Tissues for histological study were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with either hematoxylin and eosin, Ziehl-Neelsen acid-fast, periodic-acid Schiff (PAS), Giesmsa, Perl's Prussian blue, or the Van Kossa stain. The entire brain was removed and placed in an acetonerinsed glass jar, and the remaining carcass (except for skin, feet, wings, liver, and gastrointestinal tract) was TABLE 1.--Distribution of eagles collected, by State and year of death, 1969 and 1970 Statv. Florida Illinois Iowa Maine Maryland Michigan Minnesota Missouri North Dakota Ohio South Carolina Wisconsin Virginia Number op Eagles Conecitn 1969 1970 1 21 I 1 1 43 6I 5 3 2 42 1 1 From the Bureau of Sport Fisheries and WUdilfc, Patuxent Wildlife Research Center, Laurel, Md. 20810. Total 28 II Vol. 6, Mo. 3, Decbmbbr 1972 Pages 133-138. Pesticides Monitoring Journal DSW 031195 STLCOPCB4015157 wrapped in aluminum foil. The samples were usually processed for residue analysis within 1 week after dis section. Analytical Procedures The remaining carcass was ground and homogenized in a Hobart food cutter. ORGANOCHLORJNE PESTICIDES The entire brain and a 20-g aliquot of the carcass were then extracted and cleaned up separately, using the procedure described by Mulhern et al. (2). The method consisted of Soxhlet extraction and cleanup by acetoni trile partitioning and Florisil column procedures. The eluate was concentrated and divided in half. One half was analyzed for pesticides, and the other half was set aside for PCB analysis. The portion to be analyzed for organochlorinc pesticides was streaked on a thin layer (TL) plate, and the pesti cides were separated into four fractions and extracted with hot benzene (f). The pesticides present would be found in the following fractions: Fraction 1--dieldrin, lindane, heptachlor epoxide, endrin, dichlorobenzophenone (DCBP), methoxychlor, and dicofol; Frac tion II--p,p'~ and o,p'-DDD; Fraction III--p,p-' and o,p'-DDT and o,p'-DDE; Fraction IV--p,p'-DDE hep tachlor, aldrin, and mirex. Each fraction was analyzed by gas chromatography (GC) on a 4% SE-30/6% QFI column and confirmed on a 3% QF-1 or a 3% XE60 column; instruments and operating parameters are described in Table 2. TABLE 2.--Gas chromatographic operating parameters using electron capture defection COLUMNS, Glass 6' X ViV O.D. Instrument Detectoi Liquid phase Support Mesh ilit Flow rate Temperature llewletl-Pacliard 5750 Packard nickel 63 tritium 4% SE-30/6% QF-1 3% QF-1 Supelcoport Gas Chrom Q 100/120 100/120 argon/melhane al 60 ml/inlri N> at 40 ml/min WC 175* C Packard trlllum 3% XE-60 Gas Chrom Q 60/80 Ni at 100 ml/min 175 C In addition to the TL zonal separations and dual-column gas chromatographic confirmation, the residues in 10% of the samples were semiquantitatively confirmed by TLC (silica gel plate--2% ethyl ether in hexane develop ing solvent). Positive identification of dieldrin residues in brains of certain eagles was accomplished with the use of an LKB gas chroniatograph-mass spectrometer (GC-MS) equipped with a 2% SE-30, 80/100 mesh column. Operating conditions were: flow rate--35 ml/min of helium; oven temperature--200 C; flash heater--220 C; separator--240 C; and ion source--290 C. The ionization potential was 70 ev,.and accelerating voltage 3.5 kv. The sensitivity was approximately 50 ng. The mass spectra obtained from the samples were compared with spectra obtained from a dieldrin standard in respect to parent peak, Cl" multiplet peaks, base peak, and fragmentation pattern. Polychlorinated biphenyls (PCB's) with GC patterns most closely resembling Aroclor 1254 were found in all samples in Fraction IV and, in smaller amounts, in Fraction III. A previous study (2) showed that PCB compounds (Aroclor 1254 as reference) did not inter fere in the quantitative determination of DDE unless the ratio of PCB's to DDE exceeded 2:1; samples were run on a 3% OV-17 column and quantities were meas ured by a disk integrator. Thus, a separate study was made to determine whether PCB compounds interfered with DDF. when the DDE was quantitated by peak height using the 4% SE-30/6% QF-1 column. Standards of Aroclor 1248, 1254, and 1260 were each mixed with p,p'-DDE to obtain the follwing ratios of PCB's to DDE--1:1, 3:1, 5:1, 10:1, 15:1, and 20:1. The GC instrument parameters were the same as those used to measure DDE in the eagle samples. The results are given in Table 3. JABLE 3.--J he interference from PCB compounds In the determination of p.p -DDE j>,r'-DDE' Ratio or PCB to DDE t:l~[ 3:1 | 5:l^[ lOrl^j 15:1 | 2<L1 AROCLOR 1248 % Recovered % Error 100 102 100 105 105 105 -- 42 - + 3 45 +5 AROCLOR 1254 % Recovered 100 100 100 103 110 122 % Error - - - +3 + 10 +22 ._ ___________ AROCLOR 1260 ----------------------- --j r--------- 1 ---------- --- --- - r--------- 1 .. - - -\ % Recovered 100 100 103 95 92 90 % Error - - 43 --5 --8 -- 10 1 Quantitation by peak height. Using peak height, the amount of DDE recovered re mained within 5% of true value with all three Aroclors up to a 10:1 PCB's to DDE ratio; the error in creased to +10% and --8% at a 15:1 ratio of PCB's to DDE using Aroclor 1254 and 1260, respectively. 134 Pesticides Monitoring Journal QSW 031196 STLCOPCB4015158 To determine the efficiency of the analytical procedure, a pesticide recovery study was run with duck wings; 20-g aliquots of duck wing homogenates, containing trace quantities of pesticides, were spiked to contain the fol lowing: 5.0 ppm DDE, 0.2 ppm DDD, O.i ppm DDT, 0.2 ppm dieldrin, and 5.0 ppm Aroclor 1254. The average recoveries from three determinations were 77% DDE, 95% DDD, 79% DDT. 85% dieldrin, and 90% PCB's. In reporting residues in eagle samples, no corrections were made for recoveries. The lower limit of detection was arbitrarily set at 0.05 ppm; quantities <0.05 ppm were reported as trace. PCB's The second half of the eluate was analyzed for PCB's using the semiquantitative determination by thin layer chromatography (TLC) as described by Mulhern et at. (3) with the following modification: the sample eluate was evaporated to dryness, followed by the addition of 2 ml of oxidizing agent (1.5 g of CrOi in 59 ml of HOAc and 1 ml of H>0). This agent was prepared by saturat ing 59 ml HOAc with a portion of the 1.5 g CrO; the remaining CrO crystals were dissolved with 1 ml H*0. The lower detection limit was set at 1.0 ppm; a trace value for PCB's was considered to be <1.0 ppm. TOTAL MERCURY Total mercury was determined by cold vapor atomic absorption. A separate 2-g portion of the homogenized carcass was digested by refluxing with sulfuric and nitric acids and then oxidized with hydrogen peroxide. Hydroxylamine hydrochloride and stannous chloride were added to the digest to reduce mercury (II) ions to mercury metal. The sample was aerated, and the mercury in the air stream passing through a gas cell was measured by atomic absorption. The lower limit of detection was approximately 0.02 ppm in a 2-g sample. uene (4:1) were added. The mixture was shaken thor oughly, centrifuged, and chilled until the water layer was frozen. The organic layer was then decanted into a tube for injection into a MicroTek MT220 gas chroma tograph equipped with a Ni" detector on pulsed voltage operation. When chopped mallard muscle tissues spiked with 2 or 6 /u.g of mercury as methyl mercury' hydroxide were analyzed by this procedure, 81% recoveries were obtained. Two column packings were used; one con sisted of 15% Carbowax 20 M on 60/80 mcsh-Chromosorb G, acid washed, DCMS treated, at 185 C. Under these conditions injection of 1 ng of Hg as methyl mercury bromide produced a peak height of 10% of full-scale deflection. Some fouling of the detector was encountered with this column, and the degree of sen sitivity was only fair. Thus, a second column packing, 5% Hi-Eff-lOB (phenyl diethanolamine succinate) on 60/80 mesh Gas Chront Q, was used. Operating tem peratures of this column ranged front 145 to 160 C. Injection of approximately 0.4 ng Hg as methyl mercury bromide produced a peak height of 10% of full-scale deflection. This was considered to be the lower limit of reliable detection for the instrument and these operat ing conditions. This column packing apparently did not foul the detector, although continual clogging of the long exit tube on the detector was observed. This prob lem was partially alleviated by wrapping the exit tube with heating tape. Results and Discussion RESIDUES The data for chlorinated pesticides, PCB's and total mercury residues found in bald eagles collected in 1969 and 1970 are summarized in Table 4. Results for eagle carcasses and brains are reported on a wet-weight basis. Medians rather than arithmetic means were used be cause the residue levels were asymmetrically distributed. All 39 carcass samples contained DDE, dieldrin, PCB's, and mercury; 38 contained p,p'-DDD. Methyl mercuric hydroxide and mercuric chloride were added to eight 2-g portions of an eagle carcass, and from 90% to 107% of the mercury was recovered. METHYL MERCURY Methyl mercury in ground bald eagle carcasses was determined in duplicate by the method of Jensen (Naturvardsverkels Specialanalytiska Laboratorium Lanktbrukshogskolan Uppsala, Sweden. Unpublished method sent to Organization for Economic Cooperation and Development members, Sept. 1968). In this proce dure the lipids were extracted from a 1-g sample, wet tissue, by shaking with 30 ml methanol:ethyl ether, 3:1 mixture. After centrifuging and decanting, the remain ing tissue was dried in vacuo, and 1 ml each of 0.5 M HBr and 0.5 M CuBri followed by 10 ml benzene:tol- The median value of DDE in the 1970 carcass samples was 2.6 times larger than in 1969, but both values were close to or within the median range of 4.9-16.6 ppm found for 1964-1968 (2,4). For 1970, the median concentration of DDE in the brain was over 14 times greater than any median value observed since 1964; however, no trend can be established from these data because of the small number of samples collected, the wide range in DDE levels, and the absence of a sys tematic sampling procedure. Only two States, Min nesota and Wisconsin, were represented continuously from 1964-1970, often by one or two birds. Significant correlations between residues of PCB's and DDE in the brain were obtained for both 1969 and 1970 samples; the correlations indicate that in the 28 Vol. 6, No. 3, December 1972 135 DSW 031197 STLCOPCB4015159 samples statistically analyzed, there was an association between the levels of PCB's and DDE in the brain (Table 5). A more general inference may become pos sible with the analysis of future samples. The correla tion between residues of PCB's and DDE in the brain could not be attributed to interference of PCB's with GC quantitation of DDE since the study of PCB inter ference in DDE quantitation showed only a 5% error in the amount of DDE recovered at the 10:1 level of PCB's to DDE and only one sample had a PCB's to DDE ratio greater than 10:1 (viz 11:1). The median value of PCB's to DDE in brains was 3.1 in 1969 and 2.2 in 1970. The data for methyl mercury residues in 29 of the 39 carcasses are presented in Table 6; the 10 carcasses containing <1.0 ppm total mercury were not analyzed. Seven samples, which were reanalyzed in duplicate, con tained less than 65% methyl mercury, presumably due to the presence of significant amounts of inorganic mercury in the soft tissues or in the bone fragments mixed with the ground carcass. The equivalent ppm mercury present as methyl" mercury ranged from 0.38 to 44.56 ppm Hg. Lethal levels of methyl mercury for bald eagles have not been established. One finding of prime interest was that pesticide poison ing possibly accounted for the death of seven bald eagles. Residues in these birds were semiquantitatively con firmed by TLC. In addition, dieldrin residues in four of these birds were analyzed and confirmed by GC-MS. Six of the eagles contained possible lethal levels of diel drin in the brain (Table 7); two of these birds were collected in 1969 and four, in 1970. The effects of the COMTOUHD p,r`- DDE p,p'-I>DD p.p'-DDT Dkldrin Heptachlor epoxide DicMorobe ntophe none PCB Compounds Mercury TABLE 4.--Pesticide residues in bald eagles, 1969 and 1970 IT = <0.05 ppm]1 Yea* Median Carcass Ranoe Residues in PPM 1 N* Median 1969 1910 1969 1910 1969 1910 1969 1970 1969 1970 1969 1970 1969 1910 1969 1970 6.9 tB 1.0 1.5 0.22 0.10 0.41 0.74 0.06 0.10 0,42 T 10 20 1.5 2.5 0.16-30 1.5-78 0.06-0.27 0.28-11 0.07-0.75 0.09-1. i T-6.5 <0.10-18 T-0.35 T-0.41 0.080.71 T T-150 4-200 0.52-43 0.47-9.4 28 11 27 11 12 6 28 11 22 9 7 1 28 II 28 11 0.68 26 0.20 1.5 0.06 0.34 0.27 2.0 0.04 0.36 0.31 0.30 2.5 46 ............................ ' ........ Brain Range N* T-62 0.22-385 T-2.7 0.05-7.2 0.06 0.23-0.69 T-8.0 0.23-11 0.003-0.06 0.06-1.0 0.07-0.81 T-l.t T-65 T-230- 28 11 18 11 1 4 18 10 6 7 4 4 28 11 NOTE: A tout of 28 birds were collected In 1969 end 11 birds in 1910. 1 Calculated on a wet-weight basis. * Number of specimens that contained residues: the median is based on this number. TABLE 5.--Association between PCB and DDE residues in bald eagles, 1969 and 1970 Yeas Carcass PCB/DDF. Median Ranoe N> Correlation Coefficient Brain PCB/DDE Median Ranoe 1969 1.5 0.4-10 26 0.287 3.1 0.9-11 1910 1.3 0.5-3.3 11 0.451 2.2 0.6-7.5 ___________ _______ t Number of specimens that contained more than trace amounts of both PCB compounds and DDE. r - <o.oi. r = <o.o5. CoaSEtATION Coefficient *0.908 0.152 136 Pesticides Monitoring Journal DSW 031X98 STLCOPCB4015160 high levels of dieldrin in the brains of the (wo eagles that drowned may have caused the birds to fall into the water and drown. The dieldrin levels ranged from 4.6 to 11 ppm, wet-weight basis. Experimental and field data have shown that the lowest lethal brain residue for dieldrin is about 4 or 5 ppm (5). The seventh eagle, an adult female from Michigan, contained 385 ppnt of DDE and 6 ppm DDD in the brain, together with 235 ppnt of PCB's. The level of DDE alone is well within the lethal range (6), but the high level of PCB's suggests that these compounds may have also contributed to death. In this study and previous studies (2,4) since 1964, with the exception of one eagle of unknown sex, all bald eagles found to be possibly poisoned by dieldrin or DDT metabolites have been females. Of the 153 eagles analyzed during 1964-1970, 15 (9.8%) possibly died of dieldrin poisoning. AUTOPSY DATA The autopsy results for the 39 bald eagles are sum marized in Table 8. Illegal shooting remained (he most frequent single cause of death among the bald eagles TABLE 6.--Mercury found in 29 bald eagle carcasses, 1969 and 1970 Total ppm Hg < 1.01 1.02 1.12 1.13 1.17 1.26 1.31 1.42 1.49 1.53 1.53 1.70 1.72 1.17 2.07 2.18 2.20 2.50 2.74 2.96 3.01 3.58 4.47 5.65 5.83 8.32 9.40 11.00 43.00 Methyl Mercury Equivalent ppm Hg 0.68 0.96 1.01 1,09 0.98 0.76 0.90 0.92 1.28 1.41 0.38 0.80 1.36 1.75 1.74 1.45 1.88 1.99 2.44 0.48 2.85 1.56 2.05 4.68 3.98 6.71 2.72 7.60 44.56 % or Total Hg Found as Methyl Mercury 67.3 94.1 90.2 96.5 83.8 60.3 68.7 64.8 85.9 92.2 24.8 47.0 79.1 98.9 84.0 66.5 85.4 79.6 89.0 16.2 94.7 43.6 46.4 82.8 68.3 80.6 28.9 69.1 103.6 MEDIAN 2.07 1.45 79.6 1 Of ihc 39 samples collected, 10 coniHlned <1.0 ppm total mercury end, thus, were no! analyzed for metltyl mercury. Vol. 6, No. 3, December 1972 examined in this laboratory with 46% of the birds col lected during 1969-1970 having been shot. The four eagles dying from impact injuries, most fre quently as the result of hitting a power line or tower, included one eagle from Virginia which had been struck by a private jet aircraft. Three eagles were extremely emaciated; two of these were later found to have possible lethal levels of dieldrin in (he brain (Table 7), and the third, a bird from Wis consin, had multiple injuries from porcupine quills. Two quills were still embedded in the back of the eagle's oral cavity, suggesting that this eagle died from the effects of starvation and secondary bacterial infection (sec below). TABLE 7.--Data on six suspected cases of possible dieldrin poisoning and one suspected case of DDE poisoning. 1969 and 1970 State Year Missouri Wisconsin Illinois Michigan Minnesota Maryland 1969 1969 1970 1970 1970 1970 Agi: 1 ................. RF51DUE Sex IN tJRAtN (PPM ) .. DIE1.DRIN Autopsy Findings ......... Ad F *4.6 Drowning, emaciation Ad F * 6.5 Open a F 4.6 Open <Ad F *4.8 Drowning 1m F * 5.9 Emaciated; shotgun pellets around tail Ad F II Open DDE/PCB Michigan 1970 Ad 385/235 Parasitic enteritis * Ad ~ adult, 1m -- immature. s Dieldrin analyzed and confirmed by gas chromatography-mass spec trometry. * Open no diagnosis could be made on the basis of autopsy finding*. TABLF 8.-- Probable causes of bald eagle mortality, 1969 and ]970 ' Cause or Death Number of Eaglfs Shot Dieldrin 1 DDE 1 Impact Electrocution Enunciation Nephrosis Streptococcal infection Avian cholera Trapping injuries Open Total 18 6 1 4 2 1 I I 1 1 3 39 1 See Tabic 7 for details. DSW 031199 137 STLCOPCB4015161 One eagle from Michigan had an old trapping injury which had become secondarily infected and resulted in a fibrinous pericarditis. A pure culture of a gram-positive rod, subsequently identified as a Lactobacillus, was isolated from the pericardium. This isolate of Lactobacil lus was noninfectious to laboratory mice and domestic pigeons and probably should be regarded as a post mortem contaminant. Although Pseudomonas aeruginosa was isolated from the kidney and from an old infected bullet leg wound of an eagle from Wisconsin, the cause of death was a more recent gunshot wound which produced massive hemorrhage into the pericardial sac. Pasteurella multocida, the causative organism of avian chlorea, was isolated from three eagles. The first isola tion was from an adult male from Ohio and apparently represents a frank case of avian chlorea. The second isolation of P. multocida was from the heart of the emaciated eagle which had been injured by porcupine quills; the third isolation was from the liver of an eagle shot in Florida. Several parasitic conditions were observed in these 39 eagles. Schizonts of Leucocytozoon were found in the hearts of an eagle from Maryland and one from Illinois; both eagles had possible lethal levels of dicldrin in the brain (Tabic 7). Another eagle from Michigan, subse quently found to have high levels of DDE and PCB's in the brain, had a severe enteritis caused by a large number of as yet unidentified flukes; although the enteritis was severe, it was not regarded as the cause of the eagle's death. Conclusion Since the bald eagle is located at the top of food chains and is the final recipient of environmental pollutants, the presence of PCB's, DDE, dieldrin, and mercury in all 39 samples from 13 States, demonstrates continued widespread environmental contamination by these com pounds. Acknowledgment Appreciation is extended to J. O. Knislcy and P. Jones who assisted in the autopsies and dissections of eagles, to L. T. Young who prepared sections for histological examination, and to the cooperators from many States and organizations who submitted eagles for analysis. Special acknowledgment is due to Dr. R. Shillinger and his staff (Animal Health Laboratories, Maryland Depart ment of Agriculture, College Park, Md.) for the identi fication of the bacterial isolates and to the National Animal Disease Laboratory, USDA, Ames, Iowa, for identification of the Lactobacillus isolate. Sec Appendix for chemical names of compounds discussed in this paper. LITERATURE CITED (1) Mulhcrn, B. M. 1968. An improved method for the separation and removal of organochlorine insecticides from thin layer plates. 1. Chromatogr. 34:556-558, (2) Mulhern, B. M.. W. L. Reichel, L. N. Locke, T. G. Larnont, A. Belisle, E. Cromartie, G. E. Baglcy, and R. M Prouty. 1970. Organochlorine residues and autopsy data from bald eagles 1966-68. Pestic. Monit. J. 4(3):141-144. (3) Mulhern, B. M., E. Cromartie, H'. L. Reichel, and A. A. Belisle. 1971. Scmiquantilalive determination of poly chlorinated biphenyls in tissue samples by thin layer chromatography. J. Assoc. Off. Anal. Chem. 54(3): 548-550. (4) Reichel, W. L,, E. Cromartie, T. G. Lamont, B. M. Mul hern, and R. M. Prouty. 1969. Pesticide residues in eagles. Pestic. Monit. J, 3(3): 142-144. (5) Stickel, W. //., L. F. Stickel, and 1. W. Spann. 1969. Tissue residues of dieldrin in relation to mortality in birds and mammals, p. 174-204. In M. W. Miller and G. C. Berg fed.] Chemical Fallout, Current Research on Persistent Pesticides. Chas. C. Thomas, Springfield, III. (6) Stickel, W. H,, L. F. Stickel, and F. B. Coon. 1970. DDF and DDD residues correlated with mortality of experi mental birds, p. 287-294. In W. B. Deichmann fed.] Pesticides Symposia. Halos and Assoc., Miami, Fla. DSW 031200 138 Pesticides Monitoring Journai JNT : 3 73 STLCOPCB4015162