Enantioselective Accumulation ofr-Hexachlorocyclohexane inNorthern Fur Seals andDouble-Crested Cormorants: Effectsof Biological and Ecological Factorsin the Higher Trophic LevelsH I S A T O I W A T A , * , † S H I N S U K E T A N A B E , ‡

T E T S U J I I I D A , ‡ N O R I H I S A B A B A , §

J A M E S P . L U D W I G , | A N DR Y O T A T S U K A W A ⊥

Department of Environmental Veterinary Sciences,Graduate School of Veterinary Medicine, Hokkaido University,N18 W9 North Ward, Sapporo 060, Japan, Department ofEnvironment Conservation, Ehime University, Tarumi 3-5-7,Matsuyama 790, Japan, National Research Institute of FarSeas Fisheries, Orido 5-7-1, Shimizu 424, Japan, The SereGroup Ltd., 138 Road 2 West, Kingsville, Ontario N9Y 2E5,Canada, and Kochi University, Akebono-cho 2-5-1,Kochi 780, Japan

Tissues of northern fur seals (Callorhinus ursinus) fromthe Pacific coast of Japan and double-crested cormorants(Phalacrocorax auritus) from the Great Lakes wereanalyzed in order to explore the enantioselective accumulationof R-hexachlorocyclohexane (HCH). The effects ofbiological and ecological factors such as species, tissue,sex, age, feeding habit, and habitat, which may beattributable to the differences in accumulation betweenenantiomers, were also investigated. The enantiomeric ratios(ERs) of (+)-/(-)-R-HCH in fat tissue of female fur seals,composed of different age groups, collected in 1986 (1.58 (0.25) exhibited greater values than those in abiotic andlower trophic levels previously reported. No age trend ofERs was found in female northern fur seals. Thereappeared to be a temporal transition of ERs in adult femalenorthern fur seals collected in 1971-1988. Regressionanalysis showed a significant relationship between ERs andfeeding habits (p ) 0.003). Analysis of breast muscle ofdouble-crested cormorants exhibited no sex difference inERs. ERs (1.26 ( 0.13) in cormorants from Lake Michiganwere significantly higher than those (1.01 ( 0.18) fromLake Superior (p ) 0.002), suggesting the effects of factorssuch as feeding habit and habitat. Enantiomeric ac-cumulation in the body of double-crested cormorants wastissue-specific. No age trend of ERs was seen in breastmuscle of cormorants. The result implies that sexual maturity,aging and breeding activities are less effective forchanging ERs. The ERs in higher trophic animals couldbe influenced by species-specific metabolism and transportprocess in the body as biological factors and by feedinghabit and habitat as ecological factors.

IntroductionSome persistent and bioaccumulative organochlorines suchas R-hexachlorocyclohexane (HCH), cis-chlordane, trans-chlordane, o,p′-DDT, and some polychlorynated biphenylcongeners are known to have chiral compounds (1-4). Thesecompounds are present as racemic mixtures when synthe-sized. Most of the persistent chiral organochlorines havebeen noted in ubiquitously spreading on global terms (5).Once the chiral organochlorines are released into theenvironment, they may be enantioselectively processed bybiological activities. Numerous reports have shown that inair, water, sediment, soil, and some biological samples thechiral organochlorines are enantioselectively depleted (1-4, 6-10).

The importance to biological and toxicological studiesassociated with enantioselective activity of chiral compoundshas been recently emphasized (11). As is known that the (-)enantiomer of o,p′-DDT is more estrogenic than the (+)enantiomer (12), other environmental chiral contaminantsmay also elicit different toxicological properties. Accurateinformation on stereoselective processes is required whendealing with environmental mixtures of chiral compounds(11).

However, data on enantioselective accumulation of chiralorganochlorines are scarce particularly in higher trophicanimals (8, 13-15). Marine mammals and fish-eatingwaterbirds are severely contaminated by the hazardousorganochlorines. Their reproductive failures, populationdecreases, and mass mortalities have been linked to theexposure (16-18). In addition, little is known for factorsresponsible for the specific accumulation between enanti-omers, as only the limited samples have been analyzed. Evenin the cetaceans there seems to be inter- and intraspeciesvariations in enantiomeric accumulations (15). Nevertheless,due to the small sample size analyzed for each species,reasonable explanations for this variation have not beenestablished. This variation may reflect the divergence inenantioselectivity of some groups to be categorized withdifferent ecological and biological factors such as age andhabitat.

The major objective of this study is to provide more dataon enantioselective accumulation of a chiral organochlorinein the higher trophic level animals for which biological andecological factors are well-known. The present study focuseson R-HCH as a model compound. Northern fur seals(Callorhinus ursinus) from the Pacific coast of Japan anddouble-crested cormorants (Phalacrocorax auritus) from theGreat Lakes were selected as the higher trophic animals. Theseanimals are the top predators in these ecosystems and areknown to accumulate organochlorines in high concentrations(16, 19). The present study also attempts to clarify factorsresponsible for the differences in accumulation betweenenantiomers. The effects of biological and ecological factorssuch as species, tissue, sex, age, feeding habit, and habitatare discussed.

Materials and MethodsSample Collection and Preparation. The northern fur sealswere almost annually collected off Sanriku, Japan, and from

* Corresponding author telephone: +81-11-706-5102; Fax: +81-11-717-7569; E-mail: iwatah@vetmed.hokudai.ac.jp.

† Hokkaido University.‡ Ehime University.§ National Research Institute of Far Seas Fisheries.|| The Sere Group Ltd.⊥ Kochi University.

Environ. Sci. Technol. 1998, 32, 2244-2249

2244 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 15, 1998 S0013-936X(98)00115-1 CCC: $15.00 1998 American Chemical SocietyPublished on Web 06/24/1998

the Okhotsuk Sea during winter-spring seasons in 1971-1988, under the license from the Ministry of Agriculture,Forestry and Fisheries of Japan (Table 1). Adult female furseals more than 20 years old were selected to examinetemporal trend of enantiomeric accumulation of R-HCH. Toelucidate the age trend of enantioselectivity, the female furseals collected in 1986 and of various ages were used. Thefat tissues around the mammary gland were analyzed. Dataon age, body weight, and length were available for all animals(19).

Double-crested cormorants were collected from LakeSuperior (Tahquamenon Island) and Green Bay (Little GullIsland and Gravelly Island) of Lake Michigan (Table 1). Thecormorants were banded immediately after hatching andwere recovered from May to August in 1990, and the ages ofcormorants were determined by the recoveries of bandedanimals. These cormorants were dissected after removingthe feathers and separated in breast muscle, liver, bonemarrow, skin, and the others (carcass). Breast muscles of allthe cormorants from the lakes were used to investigatewhether there are age-, sex-, and habitat-specific variationsin the enantiomeric accumulation. Six Lake Michigancormorant liver, bone marrow, skin, and carcass sampleswere also analyzed to examine the tissue-specific enantio-selectivity. These samples were stored at -20 °C untilanalysis.

Chemical Analysis. The analysis of R-, â-, and γ-HCHisomers was performed following the procedure in previouspapers (15, 19).

The enantiomeric composition of R-HCH in animal tissueswas determined by separation using a 30 m cyclodex-Bcolumn (0.25 mm i.d. and 0.25 µm film thickness; J&WScientific USA). According to R-HCH concentrations insamples, 5-20 µL of sample eluates was injected into high-resolution gas chromatograph (Hewlett-Packard 5890 seriesII) with 63 Ni electron capture detector with a moving needletype injection system (splitless and solvent cut mode,Shimadzu Co. Ltd., Japan). The column is coated withpermethylated â-cyclodextrin in 14% cyanopropylphenyl-84% methyl polysiloxane. The oven temperature of gaschromatograph was programmed from an initial temperatureof 80 °C (3 min hold) to a final temperature of 230 °C (30 minhold) at a rate of 2 °C/min. Injector and detector temper-atures were set at 200 and 250 °C, respectively. Carrier andmakeup gases were helium (flow rate 1.5 mL/min) andnitrogen (flow rate 60 mL/min), respectively.

HCH isomers were quantified from individually resolvedpeak heights with corresponding peak heights of the authenticstandard. Recoveries of HCH isomers through the wholeanalytical procedures by spiking 50 ng of individual isomersto corn oil were more than 90% for all the isomers (n ) 3).Reported concentrations were not corrected for the recoverypercentage.

In this study, the enantiomer eluting first from theâ-cyclodextrin column was regarded as (+). This elutionpattern has already been assigned using the â-cyclodextrinby other authors (1). Enantiomeric ratio (ER) was definedas the ratio of peak areas of (+)-R-HCH to (-)-R-HCH. Forthe confirmation of accuracy of peak areas, ERs calculatedwith peak areas of both enantiomers in northern fur sealsamples randomly chosen were compared to those with peakheights. The average difference between the two calculationmethods of ERs was 3.3 ( 2.2% (n ) 8), showing the smallvariations. The ER in R-HCH standard solution was deter-mined to be 0.984 ( 0.007 (n ) 6) by injections of 50 pg ofR-HCH. The value was almost close to 1.0 as a racemic ratioand was found to be reproducible with a standard deviationof (0.71%. This reproducibility agreed well with other studiesso far reported (8, 9). Since the average resolution limit, TA

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VOL. 32, NO. 15, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2245

defined as total peak height/unresolved peak height, was 7.2for the standard, 3.6 was determined as the resolution limitof samples for acceptable ER values. In addition, differ-ence in retention time (RT) of both enantiomers was alsorecorded to check RT lag by the appearance of any inter-ferring peaks. The results of RT differences by the injectionof 50 pg of standard solution was in the range of 0.150-0.154with an average of 0.152 (n ) 6). The standard solution ofR-HCH was injected in every set of sample analysis, andchanges in RT of enantiomers and deterioration in qualityof the â-cyclodextrin column were monitored. Only datawithin 0.150-0.154 of RT lag were used for further consid-eration.

Statistical Analysis. Statistical analyses were performedusing StatView (version 4.0, Abacus Concepts, Inc.). Regres-sion analysis was applied to examine the relationship of ERsand feeding habit. Correlations between ERs and age wereexamined by Spearman’s rank correlation test. Student’st-test was conducted to verify the gender difference in HCHresidue levels and ERs. Mann-Whitney’s U-tests were usedto detect habitat differences in HCH concentrations and ERs.ANOVA and Fisher’s protected least significant differencepost-hoc test (Fisher’s test) were conducted for the detectionof tissue-specific and age-dependent (growth stage) ac-cumulation of HCH isomers and enantiomers.

Results and DiscussionNorthern Fur Seal. Concentrations of HCH isomers andERs of R-isomer in the fat tissue of female northern fur sealsare given in Table 1.

The average concentrations of R-, â-, and γ-HCH isomersin fur seals collected in 1986 were 159 ( 24, 376 ( 152, and38 ( 11 ng/g on fat weight basis, respectively, showing thatthe â-isomer was the most abundant. The ratio of R- toγ-HCH isomer (R/γ) was 4.3 ( 0.84 on average, whichexhibited a comparable value to that in the technical HCHmixture largely used in Asian countries (5). Average HCHisomer concentrations in adult fur seal groups of the 1971-1988 collection were slightly higher within a factor of 2 thanthose in fur seals with various ages collected in 1986. Thehigher average in the 1971-1988 group may be arising fromthe inclusion of relatively higher residue levels in the1970ssamples, while no consistent age trend in the 1986 collectionwas observed. The age and temporal trends of HCH residuelevels have already been discussed elsewhere (19).

ERs in fat tissues of female northern fur seals with differentage, which were collected in 1986, were in the range of 1.08-2.07 (average 1.58 ( 0.25). There was no significant cor-relation (Spearman’s rank correlation test, r ) 0.36, p > 0.01)between ER and age (Figure 1). Considering that the maturityage of female northern fur seal is known to be 4-7 years, thisweak relationship implies that enantioselective transfer anddegradation are unlikely to show a direct association withsexual maturity, aging, and breeding activities such as deliveryand lactation.

As the second factor to be concerned, sample collectionyear was inspected in relation to ER (Figure 2). In 53 fattissue selections of northern fur seals older than 20 years,ERs were measured. The average ratio of this group was1.56 ( 0.23 and quite similar to those of the different agegroup (1.58 ( 0.25). Linear regression analysis did not allowprediction of ERs in fat tissues of adult female northern furseals from the sample collection year. ERs preferably seemedto undergo a transition depending on the sampling period;the average ratio was 1.69 ( 0.11 in 1971-1975, transferredto a lower value (1.39 ( 0.14) until 1983, and subsequentlytended to increase again (1.69 ( 0.29) in 1985-1987 (Figure2). No correlation between ERs and HCH isomer residuelevels was found. To account for the temporal transition of

ERs, an attempt was made in examining the association withthe feeding habit. Using the recorded data on the stomachcontents of northern fur seals collected during the period ofJanuary-March after 1980 (20-22), ERs were plotted againstannual ratios (v/v %) of fish content to total stomach content,and a regression analysis was conducted (Figure 3). Ac-cordingly, a significant relationship (r2 ) 0.40, p ) 0.0029)was noted between ER and feeding habit. In the beginningof 1980s, 70-80% of total stomach content was fish, and theremainder was squid. In the following years, stomach contentwas mostly fish. Although no ER data are available forstomach contents, a difference of ERs between fish and squidis expected, and it could be reflected in the seals. The

FIGURE 1. Age trend of ERs of r-HCH in fat tissues of female northernfur seals. No significant correlation between ER and age was found(Spearman’s rank correlation test, r ) 0.36, p > 0.01).

FIGURE 2. Temporal trend of ERs of r-HCH in fat tissues of adultfemale northern fur seals.

FIGURE 3. Relationship between r-HCH ERs in fat tissues of adultfemale northern fur seals and their feeding habit. The percentageof fish content is an average value calculated from total stomachcontent in all the fur seals collected in each year.

2246 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 32, NO. 15, 1998

variation of feeding habit might be a potential factor forexplaining the temporal transition of fur seal ERs.

Mossner et al. (13) have measured ERs (1.20-1.88) in theblubber of three neonatal northern fur seals of different healthstatus. These values were in the range of those in this study,supporting our result that there was no correlation betweenER and age. Other chiral analyses (8) have also recordedthat blubber of harbor seals (Phoca vitulina) from the GermanNorth Sea coast had an excess of (+)-R-HCH, with ERs in theseals (2.17 and 2.33, n ) 2) that were somewhat higher thanthose in fur seals. Our previous study (15) also noted thatERs found in the blubber of 10 species of adult male cetaceansfrom the North Pacific and Indian Oceans ranged from 1.6to 2.8. In addition, Hummert et al. (14) have reported thatenrichment of the (+) enantiomer was found in most ofmarine mammals from the Northern Hemisphere. Accordingto another study (23), most of ERs in lower trophic animalssuch as blue mussel and flounder were almost less than 1.0with small variations and close to those in the surroundingwater. The comparison of ERs among the environmentalcompartments implies that stereoselectivity is pronouncedin higher trophic levels, preferentially depleting the (-)enantiomer through the food chain in a marine ecosystem.However, some exceptions have been found in other mammaltissues. In roe-deer liver, sheep liver, and hooded sealblubber, an excess of the (-) enantiomer has been reported(14, 24, 25). Although the reasonable explanation of suchreversals in enantioselectivity in those animals has not beenconfirmed, specific enzymatic processes and/or preferentialaccumulation of the (-) enantiomer by selective permeationthrough membranes might elicit this effect (13, 24). Analternative explanation is the effect of an enantioselectivesource in their habitat. If there is a significant source of the(-) enantiomer that can be incorporated into these animals,ERs in their tissues, to some extent, may reflect those in thesource. Some authors have reported geographical variationsin R-HCH enantioselectivity of seawater; an excess of (+)-R-HCH was found in the Bering and Chukchi Seas, andinversely (-)-R-HCH was enriched in the Canada Basin ofthe Arctic Ocean and the Greenland Sea (7). The similargeographical variations have also been seen in seawaterwithin the North Sea (1).

Double-Crested Cormorant. Residue levels of HCHisomers and ERs of the R-isomer in double-crested cormo-rants are also listed in Table 1.

There were no sex differences in HCH residue levels ofthe cormorant breast muscles both in Lake Superior andLake Michigan (Student’s t-test; p > 0.05). No significantdifferences were observed in the residue levels of â- andγ-HCH including male and female samples between the twolakes (Mann-Whitney’s U-test; â-HCH, p ) 0.62; γ-HCH, p) 0.70). R-HCH residues (420 ( 212 ng/g on fat weight basis,n ) 11) in Lake Superior were significantly higher than those(153 ( 95 ng/g, n ) 10) in Lake Michigan (p ) 0.0011). Asa result, in Lake Superior cormorants the R-isomer wasdominant, although Lake Michigan cormorants as well asnorthern fur seal fat had the â-isomer at the highestproportion. The difference of R-HCH residue levels incormorant breast muscles between the two lakes resulted inthe variation (Mann-Whitney’s U-test, p ) 0.0001) in R/γratios (Lake Superior, 7.8 ( 1.9; Lake Michigan, 2.5 ( 0.50).The similar regional pattern of R/γ ratios has also beenreported by McConnell et al. (26), where R/γ ratios (7.4) inwater from Lake Superior were found to be higher than those(3.3) in the Green Bay water. These observations may reflectthe effects of a fresh input via atmosphere of R-HCHoriginated from technical HCH for Lake Superior and a localpoint source of lindane (γ-HCH) lowering the R/γ ratio inGreen Bay (26). A comparison of individual HCH isomerresidues among tissues using a Fisher’s test along with two-

factor factorial ANOVA showed that no significant differenceswere detected for tissue-specific accumulation of each isomer(R-HCH, p ) 0.788; â-HCH, p ) 0.659; γ-HCH, p ) 0.241),although average concentrations of â- and γ-HCH wereapparently lower in the brain than in the other tissues. Thiswas due to the large variations of concentrations. Ratherthan being tissue-specific, age was a more effective factor foraccumulation of all the HCH isomers. HCH concentrationsin the age class of 0.025-0.063-years-old cormorant chicks(n ) 3) were significantly lower for all the isomers (Fisher’stest, p < 0.0001) than those in more than 1-year-old animals(n ) 3). As for the comparison of R/γ ratios in tissues, brainsamples revealed significantly larger ratios (16.5 ( 12.0, n )6) than the other tissues (Fisher’s test, p < 0.0001). Thepreferential accumulation of R-HCH in brain has also beenfound in marine mammals such as striped dolphin andnorthern fur seal (13, 27). A specific transport processassociated with the blood-brain barrier and lipid composi-tion consisting of polar phospholipids and cholesterols inbrain have been proposed as causative factors. Interestingly,brains from more than 1-year-old cormorants significantly(Fisher’s test, p < 0.0001) increased in R/γ ratios (27.1 ( 4.1,n ) 3) as compared to the younger cormorants (5.8 ( 1.1,n ) 3), while the other tissues showed no significant agetrends (p > 0.1). These results suggest that the blood-brainbarrier is relatively leaky to γ-HCH in newborn cormorantsand subsequently becomes tighter in adults.

Factors on sex and habitat for cormorant ER were initiallyexamined (Table 1). Average ERs of breast muscles ofcormorants from Lake Superior were 1.03 ( 0.10 (n ) 4) formale and 1.00 ( 0.22 (n ) 7) for female, while those of maleand female cormorants from Lake Michigan were 1.27 (0.15 (n ) 7) and 1.25 ( 0.11 (n ) 3), respectively. There wasno significant sex difference in ERs of cormorant breastmuscles from the two lakes (Lake Superior, p ) 0.846; LakeMichigan, p ) 0.806). This may indicate that sex-steroidmetabolizing enzymes are not involved for depleting enan-tiomers. Following this result, the gender factor was notconsidered further.

ERs were significantly different (Mann-Whitney’s U-test;p ) 0.0022) between animals from Lake Michigan (1.26 (0.13, n ) 10) and those from Lake Superior (1.01 ( 0.18, n) 11), suggesting that habitat is likely to be a critical factor.The following three parameters in association with habitatmay play a role in regional variations of the ER. First, thedifferences between the two habitats may be attributable tocormorant diet. As composition of the diets is not the samein the two lakes (28), the difference in ER in their diet wouldaffect to the ratio in the predator. Another parameter maybe contaminant level. If the enantiomers are metabolizedby xenobiotic compound-inducible enzymes such as cyto-chrome P450, cormorant ERs would also be changed ac-cording to the different contaminant levels to which cor-morants are exposed. In fact, it is well-known that LakeMichigan (Green Bay) is much more contaminated bypolychlorinated biphenyls and dioxins than Lake Superior(16, 29). The other parameter concerning habitat may beascribed to fresh input of R-HCH. Considering the fact thatin cormorant breast muscles from Lake Superior R-HCH level(420 ( 212 ng/g) was significantly higher than those fromLake Michigan and ER (1.01 ( 0.18) was inversely lower,R-isomer freshly input and incorporated into the highertrophic level prior to being subjected to a marked enantio-selective degradation might reduce ER up to almost 1.0 inLake Superior cormorants. In contrast, in the Lake Michiganecosystem, recycling and enantioselective processes on along-term basis of R-HCH previously used might have raisedthe ER. In the present study, it was difficult to separate themost likely source of variation from diet, contaminant level,and fresh input of R-HCH. Further studies are needed to

VOL. 32, NO. 15, 1998 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 9 2247

determine the most influential parameter to have causedthe variation of ERs found in two habitats of cormorants.

ERs in several tissues of cormorants from Lake Michiganwere compared (Table 1). The result showed that ERs ofbrain samples (>3.6) were apparently higher than those ofthe other tissues. Regarding the other tissues, ERs in bone(1.25 ( 0.19) and breast muscle (1.24 ( 0.14) on averagewere slightly higher than those in liver (1.08 ( 0.20), skin(1.08 ( 0.13) and carcass (1.08 ( 0.17). However, nosignificant difference among these tissues except brain wasdetected by one-factor ANOVA (p ) 0.21). Higher ERs onlyin the brain samples suggest that tissue-specific metabolismand/or transport process in the body associated with a chiralselective protein are also factors to be related. Our result inextreme enrichment of the (+) enantiomer in brain was inaccordance with the case of northern fur seal brain previouslyreported, in which ERs of more than 30 (n ) 2) were recorded(13). The excess of the (+) enantiomer in brain has beenexplained in terms of an enantioselective transport processconcerning a specific blood-brain barrier (13). On the otherhand, our study showed that the depletion of (-)-R-HCHenantiomer in the brain of cormorants with various ages isunlikely to be linked with the poor penetration of theγ-isomer. While the latter phenomenon is age dependent(growth stage) as observed, the former is not. Theseobservations indicate that independent processes may beresponsible for the prevention from intrusion of (-)-R-HCHand γ-isomer into the brain.

Age variation of the enantioselectivity was also examinedusing the cormorant muscle samples (Figure 4). Similar tothe result from northern fur seals, no age trend of ERs wasseen in the breast muscle of cormorants. No significantcorrelations between ER and age in the two lakes (LakeMichigan, r ) 0.009, p ) 0.98; Lake Superior, r ) 0.45, p )0.16) were found by Spearman’s rank correlation test. Thisagain supports that factors on aging and breeding activitiesare less effective.

The data comparable to the cormorant ERs were givenfrom the enantiomer analyses of common eider duck liverby Pfaffenberger et al. (23). An apparent depletion of (-)-R-HCH with the large variation has been observed in theliver samples from two sites in the German Bight (ER ) 1.76- ∞, n ) 8). In the case of common eider duck, the abilityto degrade (-)-R-HCH in the liver appeared to be dependenton the physical condition of the animals; ducks sufferingfrom a parasitic disease showed relatively lower ERs, whileanimals in a healthy condition exhibited greater ERs.Considering that all the cormorants used in this studyappeared to be healthy, the differences in ERs between thetwo bird species might have arisen from the species-specificaccumulation processes rather than the other factors, whichmay be interpreted by more efficient degradation of (-)-R-HCH in common eider duck than cormorant.

The present study reveals that the ERs in these highertrophic level animals might be influenced by species-specificmetabolism and transport process in the body as biologicalfactors and by feeding habit and habitat as ecological factors.The other biological factors such as sex and age were lesseffective for changing ERs. This study also showed that theenantioselectivity is temporally and regionally varied, evenwhen the same species and tissues are monitored. Con-sidering all these facts, enantiomeric analysis of chiralcompounds could be a useful tool for elucidating a transientecological and ecotoxicological states in higher trophicanimals of the ecosystem.

Acknowledgments

This study was supported by grants-in-aid for the ScientificResearch from the Ministry of Education, Science and Cultureof Japan to H.I. (Grants 09306021 and 09876084).

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FIGURE 4. Age trend of ERs of r-HCH in breast muscles of cormorantsfrom Lake Michigan. No significant correlation between ER andage was found (Spearman’s rank correlation test, r ) 0.009, p )0.98).

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Received for review February 4, 1998. Revised manuscriptreceived May 1, 1998. Accepted May 13, 1998.

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