Environ. Sci. Technol. 2000, 34, 2668-2674
The Enantioselective Bioaccumulation of Chiral Chlordane and r-HCH Contaminants in the Polar Bear Food Chain K A R I N W I B E R G , * ,† R O B E R T J . L E T C H E R , ‡,@ COURTNEY D. SANDAU,‡ R O S S J . N O R S T R O M , ‡,§ MATS TYSKLIND,† AND TERRY F. BIDLEMAN# Department of Chemistry, Environmental Chemistry, Umeå University, SE-901 87 Umeå, Sweden, Centre for Analytical and Environmental Chemistry, Department of Chemistry, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario, K1S 5B6 Canada, Environment Canada, National Wildlife Research Center, Hull, Quebec, K1A 0H3 Canada, and Canadian Meteorological Service, 4905 Dufferin Street, Downsview, Ontario, M3H 5T4 Canada
The enantiomer ratios (ERs) of R-HCH and chlordane related compounds (CHLs) were examined in the polar bear food chain (arctic cod-ringed seal-polar bear), using chiral gas chromatography-mass spectrometry (GCMS). The cod showed near-racemic mixtures (ER ) 1) for most of the compounds. In contrast, ERs in ringed seal and polar bear were frequently nonracemic (ER * 1), due to enantiomer-specific biotransformation. As (+)-R-HCH was transferred up the food chain, it became more abundant relative to (-)-R-HCH. For the CHLs, there was no uniform trend for the ER changes and the increasing trophic level. Apparent chiral biomagnification factors (BFs) were calculated and up to a 20-fold difference in the BF between enantiomers was found. Analysis of chiral BFs relative to CB-153 indicated that oxychlordane was formed by ringed seal and metabolized by polar bears. However, the ERs did not change significantly as a result of these biotransformations. Multivariate statistical methods revealed the clustering of sample categories and were used to investigate the relationships between the ERs, chemical residue concentrations, and biological data. ERs were important variables for the sample groupings and for the class separation of male/female seals and fat/ liver tissues. The variance in the cytochrome P450 CYP2B protein content of bear liver could be explained by the variances in chemical residue data. In this analysis the ERs were of secondary importance. The ERs of some highly recalcitrant CHLs in polar bear adipose showed linear relationships with the age of the bears.
Introduction Persistent lipophilic organochlorine compounds (OCs) biomagnify in the marine ecosystem, and OCs are therefore found at high concentrations at the top of the food chains. The arctic marine food chain is simple due to the limited biodiversity in the arctic marine environment. The polar bear (Ursus maritimus) is the top predator, eating mostly blubber 2668
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of ringed seal (Phoca hispida), which in turn consumes largely arctic cod (Boreogadus saida) and amphipods (1). Several OCs pesticides as well as their metabolites are chiral and exist as two enantiomers. Some common chiral OCs are R-hexachlorocyclohexane (R-HCH), o,p′-DDT and components of technical chlordane, and toxaphene. Chlordanes are a group of cyclodiene compounds, which contain many chiral compounds (2) with high biomagnification potential (3-5). The main constituents of technical chlordane (cis- and trans-chlordane and heptachlor ) tCHL, cCHL, and HEPT) are chiral as well as many of the bioaccumulating minor components, e.g. MC5, MC6, MC7, and U82 (2, 6). The CHL metabolites, oxychlordane (OXY) and heptachlorexo-epoxide (HEPX), are also chiral and are persistent in biota (7). High levels of Chlordane related compounds (CHLs), mainly OXY, together with polychlorinated biphenyls (PCBs) and PCB methyl sulfones (MeSO2-PCBs), are known to be the most persistent compound classes in polar bear tissues (8). Technical HCH consists of several isomers, of which the most abundant, R-HCH (∼70%), is the only chiral compound. In general, HCHs as a group are found to exhibit low biomagnification factors (BFs) in the polar bear food chain (3). Enantiomers have identical lipophilicity, vapor pressure, polarity, and other physical and chemical properties important to their environmental fate. However, the relative abundance of the enantiomers may be changed after being subjected to biotic processes. Enantiomer ratios (ERs) in technical products are racemic (ER ) 1), whereas nonracemic ERs are frequently found in the environment. It is of importance to determine the level of each enantiomer in environmental samples, since enantiomers may exhibit different biological and toxicological activity (9, 10). For example, (+)-R-HCH was more effective than (-)-R-HCH in cytotoxic and growth stimulation bioassays of primary rat hepatocytes (11), the insecticidal toxicity of several cyclodienes was found to differ significantly between enantiomers as well as between enantiomers and racemates (10), and finally, enantiomers of atropisomeric PCBs showed different potencies toward the induction of several xenobioticmetabolizing enzymes or the accumulation of uroporphyrin (9). Investigations of R-HCH have shown that the ERs in species belonging to the lower part of the marine food chain (e.g. mussels) essentially reflect the ERs in the surrounding water (12). A drastic ER change in the ascension of trophic levels in a food chain was demonstrated by Hu ¨ hnerfuss et al. (13), who compared the ERs of R-HCH in eider duck and mussel. While the mussel had a slight excess of the (-)enantiomer, a large change in the ER was found in the duck, with the (+)-enantiomer dominating. In general, species at high trophic levels have been found to exhibit significantly nonracemic mixtures of R-HCH (14) and CHLs (7, 15). ER changes have not been rigorously defined in any food chain. Moreover, only a few attempts have been made to systematically examine the factors affecting the ER changes in biota (15-17). The objectives of the present study were to (1) examine changes in ERs of chiral chlordane compounds and R-HCH as a consequence of enantioselective biochemical processes * Corresponding author phone: +46-90 786 5672; fax: +46-90 12 81 33; e-mail:
[email protected]. † Umeå University. ‡Carleton University. §Environment Canada, National Wildlife Research Center. #Canadian Meteorological Service. @ Present address: Research Institute of Toxicology, Utrecht University, P.O. Box 80.176, NL-3508 TD Utrecht, The Netherlands. 10.1021/es990740b CCC: $19.00
2000 American Chemical Society Published on Web 05/25/2000
TABLE 1. Mean Concentrations (ng/g lipid) ( Standard Deviation (SD) of Selected Organochlorines (OCs) in the Polar Bear Food Chain objects
abbreviations lipid percentage (%) variables (+)-trans-chlordane (-)-trans-chlordane (+)-cis-chlordane (-)-cis-chlordane MC5-E1 MC5-E2 MC6-E1 MC6-E2 MC7-E1 MC7-E2 trans-nonachlor cis-nonachlor MC2 compound C C-5 U-2 U-3 U-4 (+)-heptachlor-exo-epoxide (-)-heptachlor-exo-epoxide (+)-oxychlordane (-)-oxychlordane (+)-R-hexachlorocyclohexane (-)-R-hexachlorocyclohexane β-hexachlorocyclohexane γ-hexachlorocyclohexane PCB-153 age (yr)
arctic cod two pools of nine cod each 6.5/7.0
Chemical Residues (ng/g lipid) tCHL+ 7.5/13 tCHL7.7/13 cCHL+ 20/19 cCHL21/20 MC5-1 2.0/4.3a MC5-2 2.4/4.7a MC6-1 N/A MC6-2 N/A MC7-1 0.67/1.4a MC7-2 0.74/1.4a tN 34/31 cN n.d/13 MC2 N/A C N/A C5 N/A U2 N/A U3 N/A U4 N/A HEPX+ 7.9/9.1 HEPX5.6/7.4 OXY+ 1.1/1.6 OXY0.8/1.3 RHCH+ 150/150 RHCH160/160 βHCH 13/5 γHCH 28/29 CB-153 1.9/0.7 Biological Data N/A
CYP2B isozyme protein content (pmol/mg)
N/A
ringed seal blubber n ) 11
polar bear fat n)7
polar bear liver n ) 13
89 ( 6
71 ( 16
6.9 ( 0.9
12 ( 5 22 ( 9 16 ( 6 5.3 ( 2.1 110 ( 70a 300 ( 200a 6.1 ( 4.1a 23 ( 12a 5.1 ( 3.4a 24 ( 12a 170 ( 70 110 ( 50 N/A N/A N/A N/A N/A N/A 41 ( 14 60 ( 19 210 ( 80 150 ( 70 290 ( 170 270 ( 150 99 ( 47 40 ( 54 61 ( 19
0.20 ( 0.20a 0.02 ( 0.01a 0.07 ( 0.08a 0.02 ( 0.03a 3.6 ( 3.8 29 ( 32 130 ( 30 370 ( 70 i i 300 ( 100 N/A 53 ( 50 43 ( 34 33 ( 36 40 ( 46 100 ( 110 7 ( 16 300 ( 50 140 ( 20 1500 ( 300 920 ( 160 400 ( 50 190 ( 70 130 ( 60 N/A 2700 ( 600
7.3 ( 5.4a 2.3 ( 2.0a 2.5 ( 2.3a 2.0 ( 2.6a 18 ( 9 190 ( 120 320 ( 220 2000 ( 1200 i i 250 ( 90 N/A 120 ( 80 950 ( 660 230 ( 380 245 ( 110 28 ( 37 500 ( 400 2200 ( 1800 660 ( 540 11 000 ( 12 000 8 400 ( 8100 440 ( 200 130 ( 50 69 ( 34 N/A 7700 ( 4000
N/A
median: 8.5 range: 3-13 N/A
median: 9.0 range: 3-22 20 ( 7§
N/A
a
Semiquantitative data (see Material and Methods); § (22); N/A: not analyzed; i: interference; chlordane nomenclature is used as follows: MC# and compound C: technical chlordane compounds identified by Miyazaki (33), MC5 and MC7: octachloro-, and MC6: nonachloro-chlordane compounds; C5: (5+3) technical chlordane component (Environ. Can., unpublished data); U2, U3, U4: unidentified chlordane related compounds, probably decomposition compounds or metabolites ((3, 8, 19) and Environ. Can., unpublished data).
(e.g. biotransformation) in the polar bear food chain, (2) study the biomagnification of CHLs and R-HCH enantiomers, and (3) evaluate the relative modeling power of ERs and CHL and HCH residue concentrations in multivariate statistical approaches in the comparison of residue pattern differences among species, tissues, and sexes and in describing enzyme induction in polar bears.
Materials and Methods Tissue Sampling, Extraction, and Cleanup. Polar bear fat (n ) 7) and liver samples (n ) 13) were collected from male polar bears, 3-22 years of age. Subcutaneous adipose tissue from the base of the tail and liver samples from the distal portion of the right lobe of freshly killed polar bears were taken as part of the Inuit hunt in the Resolute Bay area of Canadian Arctic (74°N, 95°W). Bears A-D were collected in 1992 and bears E-M in 1993. The samples were taken within 3 days of each other at the height of hyperphagia in the end of April, when they were actively feeding on ringed seals. Fat and liver samples for chemical analysis were stored at -40 °C, and the liver samples for enzymatic and immunochemical analysis were stored at -80 °C until further use. Blubber (n ) 11, five males and six females) and liver (n ) 7, four males and three females) samples of ringed seals and two pools of nine whole arctic cod were collected from the same area and approximately in the same time period as for polar bear and
stored at -20 °C or lower until further use. The ringed seals were all adults (5-8 years of age) except for two juvenile females ( 1 indicates formation (1). Because of the relatively large range in concentrations, the semiquantitative nature of some of the data, and low numbers of samples (Table 1), BF values are likely to have relatively large uncertainty. However, normalizing the data to CB-153 should remove much of the variance associated with differences in concentration among individuals, because residue patterns are much more similar among individuals than concentrations (18). BF′ values are therefore a more reliable indication of biomagnification potential than BF values. For comparison, CB-153 is also included in Table 3. From cod to seal, the BF′ values for the enantiomers of tCHL, cCHL, HEPX, and R-HCH were between 0.005 and 2672
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0.16, indicating that they were all metabolized by the seal considerably faster than CB-153 (Table 3). The BF values of these compounds ranged between 0.3 and 9. The BF and BF′ values differed 2-3 times between enantiomers of tCHL, cCHL, and HEPX, resulting in ER changes from cod to seal (Figure 1). Selective formation of HEPX could occur, but heptachlor, its only precursor (31), was not found in cod. The enantiomers of R-HCH appear to be eliminated from seals at similar rates, with a slight preference for accumulation of RHCH+ (Figure 1a). For enantiomers of the three MC compounds, BFs were higher (up to 85) and BF′ was in the 0.1-1.3 range, indicating slow clearance similar to or somewhat faster than that of CB-153. However, there were relatively great differences in bioaccumulation between enantiomers, especially for MC7 and MC6 (Figure 1c). The second eluting enantiomer of MC5, MC6, and MC7 had 3-5 times higher biomagnification values (BF and BF′) than the first enantiomer (Table 3). OXY was clearly formed by seals (BF′ ≈ 2), presumably by metabolism of chlordane and nonachlor isomers taken up via cod consumption. Consequently, OXY had the highest BF value of the analyzed compounds (≈150). It is interesting to note that this did not change the ER from cod to seal (Figure 1d). The biomagnification values were in general lower from seal to bear than from cod to seal. BFs were lower than 1 for all compounds except the metabolites and MC6. This is in large part due to the greater biotransformation capacity of polar bear. The BF′ values of enantiomers of tCHL, cCHL, and MC5 were
