Environ. Sci. Technol. 1998, 32, 2244-2249
Enantioselective Accumulation of r-Hexachlorocyclohexane in Northern Fur Seals and Double-Crested Cormorants: Effects of Biological and Ecological Factors in the Higher Trophic Levels H I S A T O I W A T A , * ,† S H I N S U K E T A N A B E , ‡ TETSUJI IIDA,‡ NORIHISA BABA,§ JAMES P. LUDWIG,| AND RYO TATSUKAWA⊥ Department of Environmental Veterinary Sciences, Graduate School of Veterinary Medicine, Hokkaido University, N18 W9 North Ward, Sapporo 060, Japan, Department of Environment Conservation, Ehime University, Tarumi 3-5-7, Matsuyama 790, Japan, National Research Institute of Far Seas Fisheries, Orido 5-7-1, Shimizu 424, Japan, The Sere Group 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) from the Pacific coast of Japan and double-crested cormorants (Phalacrocorax auritus) from the Great Lakes were analyzed in order to explore the enantioselective accumulation of R-hexachlorocyclohexane (HCH). The effects of biological and ecological factors such as species, tissue, sex, age, feeding habit, and habitat, which may be attributable to the differences in accumulation between enantiomers, 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 and lower trophic levels previously reported. No age trend of ERs was found in female northern fur seals. There appeared to be a temporal transition of ERs in adult female northern fur seals collected in 1971-1988. Regression analysis showed a significant relationship between ERs and feeding habits (p ) 0.003). Analysis of breast muscle of double-crested cormorants exhibited no sex difference in ERs. ERs (1.26 ( 0.13) in cormorants from Lake Michigan were significantly higher than those (1.01 ( 0.18) from Lake Superior (p ) 0.002), suggesting the effects of factors such as feeding habit and habitat. Enantiomeric accumulation in the body of double-crested cormorants was tissue-specific. No age trend of ERs was seen in breast muscle of cormorants. The result implies that sexual maturity, aging and breeding activities are less effective for changing ERs. The ERs in higher trophic animals could be influenced by species-specific metabolism and transport process in the body as biological factors and by feeding habit and habitat as ecological factors.
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Introduction Some persistent and bioaccumulative organochlorines such as R-hexachlorocyclohexane (HCH), cis-chlordane, transchlordane, o,p′-DDT, and some polychlorynated biphenyl congeners are known to have chiral compounds (1-4). These compounds are present as racemic mixtures when synthesized. Most of the persistent chiral organochlorines have been noted in ubiquitously spreading on global terms (5). Once the chiral organochlorines are released into the environment, they may be enantioselectively processed by biological activities. Numerous reports have shown that in air, water, sediment, soil, and some biological samples the chiral organochlorines are enantioselectively depleted (14, 6-10). The importance to biological and toxicological studies associated with enantioselective activity of chiral compounds has been recently emphasized (11). As is known that the (-) enantiomer of o,p′-DDT is more estrogenic than the (+) enantiomer (12), other environmental chiral contaminants may also elicit different toxicological properties. Accurate information on stereoselective processes is required when dealing with environmental mixtures of chiral compounds (11). However, data on enantioselective accumulation of chiral organochlorines are scarce particularly in higher trophic animals (8, 13-15). Marine mammals and fish-eating waterbirds are severely contaminated by the hazardous organochlorines. Their reproductive failures, population decreases, and mass mortalities have been linked to the exposure (16-18). In addition, little is known for factors responsible for the specific accumulation between enantiomers, as only the limited samples have been analyzed. Even in the cetaceans there seems to be inter- and intraspecies variations in enantiomeric accumulations (15). Nevertheless, due to the small sample size analyzed for each species, reasonable explanations for this variation have not been established. This variation may reflect the divergence in enantioselectivity of some groups to be categorized with different ecological and biological factors such as age and habitat. The major objective of this study is to provide more data on enantioselective accumulation of a chiral organochlorine in the higher trophic level animals for which biological and ecological factors are well-known. The present study focuses on R-HCH as a model compound. Northern fur seals (Callorhinus ursinus) from the Pacific coast of Japan and double-crested cormorants (Phalacrocorax auritus) from the Great Lakes were selected as the higher trophic animals. These animals are the top predators in these ecosystems and are known to accumulate organochlorines in high concentrations (16, 19). The present study also attempts to clarify factors responsible for the differences in accumulation between enantiomers. The effects of biological and ecological factors such as species, tissue, sex, age, feeding habit, and habitat are discussed.
Materials and Methods Sample Collection and Preparation. The northern fur seals were almost annually collected off Sanriku, Japan, and from * Corresponding author telephone: +81-11-706-5102; Fax: +8111-717-7569; E-mail:
[email protected]. † Hokkaido University. ‡ Ehime University. § National Research Institute of Far Seas Fisheries. || The Sere Group Ltd. ⊥ Kochi University. S0013-936X(98)00115-1 CCC: $15.00
1998 American Chemical Society Published on Web 06/24/1998
a All the ratios and concentrations are expressed as average ( standard deviation. ER, enantiomeric ratio of (+)-R-HCH to (-)-R-HCH. HCH isomer concentration, ng/g on fat weight basis. R/γ, ratio of R-HCH to γ-HCH.
16.5 ( 12.0 4.0 ( 0.43 2.5 ( 0.46 3.6 ( 0.78 4.2 ( 0.52 3.9 ( 1.3 7.7 ( 5.1 36 ( 39 58 ( 53 23 ( 25 27 ( 23 46 ( 49 164 ( 159 140 ( 135 138 ( 118 72 ( 65 103 ( 77 126 ( 106 1990 Lake Michigan
male/female
0.025-6
brain bone breast muscle liver skin carcass
6 6 6 6 6 6
>3.6 1.25 ( 0.19 1.24 ( 0.14 1.08 ( 0.20 1.08 ( 0.13 1.08 ( 0.17
45 ( 79 278 ( 388 159 ( 217 151 ( 208 193 ( 254 228 ( 303
2.6 ( 0.49 2.2 ( 0.42 61 ( 36 72 ( 63 158 ( 99 141 ( 104 1990 Lake Michigan
male female
0.025-6 0.025-3
breast muscle breast muscle
7 3
1.27 ( 0.15 1.25 ( 0.11
196 ( 183 62 ( 50
7.3 ( 0.53 8.0 ( 2.4 58 ( 26 60 ( 44 420 ( 157 420 ( 250 breast muscle breast muscle 2-5 0.12-4 1990 Lake Superior double-crested cormorant
male female
fat fat 3-23 20-23 female female Pacific coast of Japan northern fur seal
1986 1971-1988
4 7
1.03 ( 0.10 1.00 ( 0.22
147 ( 156 99 ( 135
4.3 ( 0.84 3.3 ( 1.0 38 ( 11 67 ( 24 159 ( 24 208 ( 66 51 53
1.58 ( 0.25 1.56 ( 0.23
376 ( 152 467 ( 178
r/γ γ-HCH β-HCH r-HCH ER no. of samples organ age (year) sex collection year location species
TABLE 1. Enantiomeric Ratios of r-HCH and Concentrations of HCH Isomers in the Tissues of Northern Fur Seals and Double-Crested Cormorantsa
the Okhotsuk Sea during winter-spring seasons in 19711988, under the license from the Ministry of Agriculture, Forestry and Fisheries of Japan (Table 1). Adult female fur seals more than 20 years old were selected to examine temporal trend of enantiomeric accumulation of R-HCH. To elucidate the age trend of enantioselectivity, the female fur seals collected in 1986 and of various ages were used. The fat tissues around the mammary gland were analyzed. Data on age, body weight, and length were available for all animals (19). Double-crested cormorants were collected from Lake Superior (Tahquamenon Island) and Green Bay (Little Gull Island and Gravelly Island) of Lake Michigan (Table 1). The cormorants were banded immediately after hatching and were recovered from May to August in 1990, and the ages of cormorants were determined by the recoveries of banded animals. These cormorants were dissected after removing the feathers and separated in breast muscle, liver, bone marrow, skin, and the others (carcass). Breast muscles of all the cormorants from the lakes were used to investigate whether there are age-, sex-, and habitat-specific variations in the enantiomeric accumulation. Six Lake Michigan cormorant liver, bone marrow, skin, and carcass samples were also analyzed to examine the tissue-specific enantioselectivity. These samples were stored at -20 °C until analysis. Chemical Analysis. The analysis of R-, β-, and γ-HCH isomers was performed following the procedure in previous papers (15, 19). The enantiomeric composition of R-HCH in animal tissues was determined by separation using a 30 m cyclodex-B column (0.25 mm i.d. and 0.25 µm film thickness; J&W Scientific USA). According to R-HCH concentrations in samples, 5-20 µL of sample eluates was injected into highresolution gas chromatograph (Hewlett-Packard 5890 series II) with 63 Ni electron capture detector with a moving needle type injection system (splitless and solvent cut mode, Shimadzu Co. Ltd., Japan). The column is coated with permethylated β-cyclodextrin in 14% cyanopropylphenyl -84% methyl polysiloxane. The oven temperature of gas chromatograph was programmed from an initial temperature of 80 °C (3 min hold) to a final temperature of 230 °C (30 min hold) at a rate of 2 °C/min. Injector and detector temperatures were set at 200 and 250 °C, respectively. Carrier and makeup gases were helium (flow rate 1.5 mL/min) and nitrogen (flow rate 60 mL/min), respectively. HCH isomers were quantified from individually resolved peak heights with corresponding peak heights of the authentic standard. Recoveries of HCH isomers through the whole analytical procedures by spiking 50 ng of individual isomers to corn oil were more than 90% for all the isomers (n ) 3). Reported concentrations were not corrected for the recovery percentage. In this study, the enantiomer eluting first from the β-cyclodextrin column was regarded as (+). This elution pattern has already been assigned using the β-cyclodextrin by other authors (1). Enantiomeric ratio (ER) was defined as the ratio of peak areas of (+)-R-HCH to (-)-R-HCH. For the confirmation of accuracy of peak areas, ERs calculated with peak areas of both enantiomers in northern fur seal samples randomly chosen were compared to those with peak heights. The average difference between the two calculation methods of ERs was 3.3 ( 2.2% (n ) 8), showing the small variations. The ER in R-HCH standard solution was determined to be 0.984 ( 0.007 (n ) 6) by injections of 50 pg of R-HCH. The value was almost close to 1.0 as a racemic ratio and was found to be reproducible with a standard deviation of (0.71%. This reproducibility agreed well with other studies so far reported (8, 9). Since the average resolution limit,
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defined as total peak height/unresolved peak height, was 7.2 for the standard, 3.6 was determined as the resolution limit of samples for acceptable ER values. In addition, difference in retention time (RT) of both enantiomers was also recorded to check RT lag by the appearance of any interferring peaks. The results of RT differences by the injection of 50 pg of standard solution was in the range of 0.150-0.154 with an average of 0.152 (n ) 6). The standard solution of R-HCH was injected in every set of sample analysis, and changes in RT of enantiomers and deterioration in quality of the β-cyclodextrin column were monitored. Only data within 0.150-0.154 of RT lag were used for further consideration. Statistical Analysis. Statistical analyses were performed using StatView (version 4.0, Abacus Concepts, Inc.). Regression analysis was applied to examine the relationship of ERs and feeding habit. Correlations between ERs and age were examined by Spearman’s rank correlation test. Student’s t-test was conducted to verify the gender difference in HCH residue levels and ERs. Mann-Whitney’s U-tests were used to detect habitat differences in HCH concentrations and ERs. ANOVA and Fisher’s protected least significant difference post-hoc test (Fisher’s test) were conducted for the detection of tissue-specific and age-dependent (growth stage) accumulation of HCH isomers and enantiomers.
FIGURE 1. Age trend of ERs of r-HCH in fat tissues of female northern fur seals. No significant correlation between ER and age was found (Spearman’s rank correlation test, r ) 0.36, p > 0.01).
Results and Discussion Northern Fur Seal. Concentrations of HCH isomers and ERs of R-isomer in the fat tissue of female northern fur seals are given in Table 1. The average concentrations of R-, β-, and γ-HCH isomers in fur seals collected in 1986 were 159 ( 24, 376 ( 152, and 38 ( 11 ng/g on fat weight basis, respectively, showing that the β-isomer was the most abundant. The ratio of R- to γ-HCH isomer (R/γ) was 4.3 ( 0.84 on average, which exhibited a comparable value to that in the technical HCH mixture largely used in Asian countries (5). Average HCH isomer concentrations in adult fur seal groups of the 19711988 collection were slightly higher within a factor of 2 than those in fur seals with various ages collected in 1986. The higher average in the 1971-1988 group may be arising from the inclusion of relatively higher residue levels in the1970s samples, while no consistent age trend in the 1986 collection was observed. The age and temporal trends of HCH residue levels have already been discussed elsewhere (19). ERs in fat tissues of female northern fur seals with different age, which were collected in 1986, were in the range of 1.082.07 (average 1.58 ( 0.25). There was no significant correlation (Spearman’s rank correlation test, r ) 0.36, p > 0.01) between ER and age (Figure 1). Considering that the maturity age of female northern fur seal is known to be 4-7 years, this weak relationship implies that enantioselective transfer and degradation are unlikely to show a direct association with sexual maturity, aging, and breeding activities such as delivery and lactation. As the second factor to be concerned, sample collection year was inspected in relation to ER (Figure 2). In 53 fat tissue selections of northern fur seals older than 20 years, ERs were measured. The average ratio of this group was 1.56 ( 0.23 and quite similar to those of the different age group (1.58 ( 0.25). Linear regression analysis did not allow prediction of ERs in fat tissues of adult female northern fur seals from the sample collection year. ERs preferably seemed to undergo a transition depending on the sampling period; the average ratio was 1.69 ( 0.11 in 1971-1975, transferred to a lower value (1.39 ( 0.14) until 1983, and subsequently tended to increase again (1.69 ( 0.29) in 1985-1987 (Figure 2). No correlation between ERs and HCH isomer residue levels was found. To account for the temporal transition of 2246
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FIGURE 2. Temporal trend of ERs of r-HCH in fat tissues of adult female northern fur seals.
FIGURE 3. Relationship between r-HCH ERs in fat tissues of adult female northern fur seals and their feeding habit. The percentage of fish content is an average value calculated from total stomach content in all the fur seals collected in each year. ERs, an attempt was made in examining the association with the feeding habit. Using the recorded data on the stomach contents of northern fur seals collected during the period of January-March after 1980 (20-22), ERs were plotted against annual ratios (v/v %) of fish content to total stomach content, and a regression analysis was conducted (Figure 3). Accordingly, a significant relationship (r2 ) 0.40, p ) 0.0029) was noted between ER and feeding habit. In the beginning of 1980s, 70-80% of total stomach content was fish, and the remainder was squid. In the following years, stomach content was mostly fish. Although no ER data are available for stomach contents, a difference of ERs between fish and squid is expected, and it could be reflected in the seals. The
variation of feeding habit might be a potential factor for explaining the temporal transition of fur seal ERs. Mo¨ssner et al. (13) have measured ERs (1.20-1.88) in the blubber of three neonatal northern fur seals of different health status. These values were in the range of those in this study, supporting our result that there was no correlation between ER and age. Other chiral analyses (8) have also recorded that blubber of harbor seals (Phoca vitulina) from the German North Sea coast had an excess of (+)-R-HCH, with ERs in the seals (2.17 and 2.33, n ) 2) that were somewhat higher than those in fur seals. Our previous study (15) also noted that ERs found in the blubber of 10 species of adult male cetaceans from the North Pacific and Indian Oceans ranged from 1.6 to 2.8. In addition, Hummert et al. (14) have reported that enrichment of the (+) enantiomer was found in most of marine mammals from the Northern Hemisphere. According to another study (23), most of ERs in lower trophic animals such as blue mussel and flounder were almost less than 1.0 with small variations and close to those in the surrounding water. The comparison of ERs among the environmental compartments implies that stereoselectivity is pronounced in higher trophic levels, preferentially depleting the (-) enantiomer through the food chain in a marine ecosystem. However, some exceptions have been found in other mammal tissues. In roe-deer liver, sheep liver, and hooded seal blubber, an excess of the (-) enantiomer has been reported (14, 24, 25). Although the reasonable explanation of such reversals in enantioselectivity in those animals has not been confirmed, specific enzymatic processes and/or preferential accumulation of the (-) enantiomer by selective permeation through membranes might elicit this effect (13, 24). An alternative explanation is the effect of an enantioselective source 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 the source. Some authors have reported geographical variations in R-HCH enantioselectivity of seawater; an excess of (+)R-HCH was found in the Bering and Chukchi Seas, and inversely (-)-R-HCH was enriched in the Canada Basin of the Arctic Ocean and the Greenland Sea (7). The similar geographical variations have also been seen in seawater within the North Sea (1). Double-Crested Cormorant. Residue levels of HCH isomers and ERs of the R-isomer in double-crested cormorants are also listed in Table 1. There were no sex differences in HCH residue levels of the cormorant breast muscles both in Lake Superior and Lake Michigan (Student’s t-test; p > 0.05). No significant differences were observed in the residue levels of β- and γ-HCH including male and female samples between the two lakes (Mann-Whitney’s U-test; β-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). As a result, in Lake Superior cormorants the R-isomer was dominant, although Lake Michigan cormorants as well as northern fur seal fat had the β-isomer at the highest proportion. The difference of R-HCH residue levels in cormorant breast muscles between the two lakes resulted in the variation (Mann-Whitney’s U-test, p ) 0.0001) in R/γ ratios (Lake Superior, 7.8 ( 1.9; Lake Michigan, 2.5 ( 0.50). The similar regional pattern of R/γ ratios has also been reported by McConnell et al. (26), where R/γ ratios (7.4) in water from Lake Superior were found to be higher than those (3.3) in the Green Bay water. These observations may reflect the effects of a fresh input via atmosphere of R-HCH originated from technical HCH for Lake Superior and a local point source of lindane (γ-HCH) lowering the R/γ ratio in Green Bay (26). A comparison of individual HCH isomer residues among tissues using a Fisher’s test along with two-
factor factorial ANOVA showed that no significant differences were detected for tissue-specific accumulation of each isomer (R-HCH, p ) 0.788; β-HCH, p ) 0.659; γ-HCH, p ) 0.241), although average concentrations of β- and γ-HCH were apparently lower in the brain than in the other tissues. This was due to the large variations of concentrations. Rather than being tissue-specific, age was a more effective factor for accumulation of all the HCH isomers. HCH concentrations in the age class of 0.025-0.063-years-old cormorant chicks (n ) 3) were significantly lower for all the isomers (Fisher’s test, p < 0.0001) than those in more than 1-year-old animals (n ) 3). As for the comparison of R/γ ratios in tissues, brain samples revealed significantly larger ratios (16.5 ( 12.0, n ) 6) than the other tissues (Fisher’s test, p < 0.0001). The preferential accumulation of R-HCH in brain has also been found in marine mammals such as striped dolphin and northern fur seal (13, 27). A specific transport process associated with the blood-brain barrier and lipid composition consisting of polar phospholipids and cholesterols in brain 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 age trends (p > 0.1). These results suggest that the blood-brain barrier is relatively leaky to γ-HCH in newborn cormorants and subsequently becomes tighter in adults. Factors on sex and habitat for cormorant ER were initially examined (Table 1). Average ERs of breast muscles of cormorants from Lake Superior were 1.03 ( 0.10 (n ) 4) for male and 1.00 ( 0.22 (n ) 7) for female, while those of male and female cormorants from Lake Michigan were 1.27 ( 0.15 (n ) 7) and 1.25 ( 0.11 (n ) 3), respectively. There was no significant sex difference in ERs of cormorant breast muscles from the two lakes (Lake Superior, p ) 0.846; Lake Michigan, p ) 0.806). This may indicate that sex-steroid metabolizing enzymes are not involved for depleting enantiomers. Following this result, the gender factor was not considered 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 habitat may play a role in regional variations of the ER. First, the differences between the two habitats may be attributable to cormorant diet. As composition of the diets is not the same in the two lakes (28), the difference in ER in their diet would affect to the ratio in the predator. Another parameter may be contaminant level. If the enantiomers are metabolized by xenobiotic compound-inducible enzymes such as cytochrome P450, cormorant ERs would also be changed according to the different contaminant levels to which cormorants are exposed. In fact, it is well-known that Lake Michigan (Green Bay) is much more contaminated by polychlorinated biphenyls and dioxins than Lake Superior (16, 29). The other parameter concerning habitat may be ascribed to fresh input of R-HCH. Considering the fact that in cormorant breast muscles from Lake Superior R-HCH level (420 ( 212 ng/g) was significantly higher than those from Lake Michigan and ER (1.01 ( 0.18) was inversely lower, R-isomer freshly input and incorporated into the higher trophic level prior to being subjected to a marked enantioselective degradation might reduce ER up to almost 1.0 in Lake Superior cormorants. In contrast, in the Lake Michigan ecosystem, recycling and enantioselective processes on a long-term basis of R-HCH previously used might have raised the ER. In the present study, it was difficult to separate the most 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
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The present study reveals that the ERs in these higher trophic level animals might be influenced by species-specific metabolism and transport process in the body as biological factors and by feeding habit and habitat as ecological factors. The other biological factors such as sex and age were less effective for changing ERs. This study also showed that the enantioselectivity is temporally and regionally varied, even when the same species and tissues are monitored. Considering all these facts, enantiomeric analysis of chiral compounds could be a useful tool for elucidating a transient ecological and ecotoxicological states in higher trophic animals of the ecosystem. FIGURE 4. Age trend of ERs of r-HCH in breast muscles of cormorants from Lake Michigan. No significant correlation between ER and age was found (Spearman’s rank correlation test, r ) 0.009, p ) 0.98). determine the most influential parameter to have caused the variation of ERs found in two habitats of cormorants. ERs in several tissues of cormorants from Lake Michigan were compared (Table 1). The result showed that ERs of brain samples (>3.6) were apparently higher than those of the other tissues. Regarding the other tissues, ERs in bone (1.25 ( 0.19) and breast muscle (1.24 ( 0.14) on average were slightly higher than those in liver (1.08 ( 0.20), skin (1.08 ( 0.13) and carcass (1.08 ( 0.17). However, no significant difference among these tissues except brain was detected by one-factor ANOVA (p ) 0.21). Higher ERs only in the brain samples suggest that tissue-specific metabolism and/or transport process in the body associated with a chiral selective protein are also factors to be related. Our result in extreme enrichment of the (+) enantiomer in brain was in accordance with the case of northern fur seal brain previously reported, in which ERs of more than 30 (n ) 2) were recorded (13). The excess of the (+) enantiomer in brain has been explained in terms of an enantioselective transport process concerning a specific blood-brain barrier (13). On the other hand, our study showed that the depletion of (-)-R-HCH enantiomer in the brain of cormorants with various ages is unlikely to be linked with the poor penetration of the γ-isomer. While the latter phenomenon is age dependent (growth stage) as observed, the former is not. These observations indicate that independent processes may be responsible for the prevention from intrusion of (-)-R-HCH and γ-isomer into the brain. Age variation of the enantioselectivity was also examined using the cormorant muscle samples (Figure 4). Similar to the result from northern fur seals, no age trend of ERs was seen in the breast muscle of cormorants. No significant correlations between ER and age in the two lakes (Lake Michigan, r ) 0.009, p ) 0.98; Lake Superior, r ) 0.45, p ) 0.16) were found by Spearman’s rank correlation test. This again supports that factors on aging and breeding activities are less effective. The data comparable to the cormorant ERs were given from the enantiomer analyses of common eider duck liver by Pfaffenberger et al. (23). An apparent depletion of (-)R-HCH with the large variation has been observed in the liver samples from two sites in the German Bight (ER ) 1.76 - ∞, n ) 8). In the case of common eider duck, the ability to degrade (-)-R-HCH in the liver appeared to be dependent on the physical condition of the animals; ducks suffering from a parasitic disease showed relatively lower ERs, while animals in a healthy condition exhibited greater ERs. Considering that all the cormorants used in this study appeared to be healthy, the differences in ERs between the two bird species might have arisen from the species-specific accumulation processes rather than the other factors, which may be interpreted by more efficient degradation of (-)R-HCH in common eider duck than cormorant. 2248
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Acknowledgments This study was supported by grants-in-aid for the Scientific Research from the Ministry of Education, Science and Culture of Japan to H.I. (Grants 09306021 and 09876084).
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Received for review February 4, 1998. Revised manuscript received May 1, 1998. Accepted May 13, 1998. ES980115R
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