Environ. Sci. Technol. 2000, 34, 2126-2130
Enantiomer Ratios of Chlordane Congeners Are Gender Specific in Cod (Gadus morhua) from the Barents Sea H E I D I K A R L S S O N , † M I C H A E L O E H M E , * ,† SONJA SKOPP,† AND IVAN C. BURKOW‡ Organic Analytical Chemistry, University of Basel, Neuhausstrasse 31, CH-4057 Basel, Switzerland, and Norwegian Institute for Air Research, The Polar Environmental Centre, N-9296 Tromsø, Norway
The enantiomer ratios of some chlordane congeners were found to be gender-specific in cod from the Barents Sea. Concentrations of U81, U82, trans-chlordane, MC5, cischlordane, MC7, trans-nonachlor, MC6, and cis-nonachlor as well as the metabolites cis-heptachlorepoxide and oxychlordane were determined. Female cod preferentially accumulated (-)-cis-chlordane and (-)-trans-chlordane, while male cod had an excess of (+)-cis-chlordane and (+)trans-chlordane. A significant difference was also observed for MC6. Only chlordane congeners without a chlorine atom at the C3 position showed gender-specific enantiomer ratios. No exception was found within the sample set of 16 individuals. Furthermore, the isomer and enantiomer patterns were very similar in muscle tissue, gonads, and liver.
Introduction The pesticide chlordane consists of at least 147 compounds (1). Some are chiral and are present as racemates in the technical product. Although chlordane was banned in the U.S.A. in 1987, it is still found in the environment. Chlordane accumulates in the marine food web (2, 3). Particularly in the Arctic, chlordane belongs to the most abundant environmental pollutants. Levels in polar bears and Inuits are only exceeded by those of toxaphene and some polychlorinated biphenyl congeners (2, 4). Little is known about differences in biological effects between enantiomers of chlorinated pesticides such as R-HCH, chlordane, o,p′-DDT, and toxaphene. Toxicity studies of heptachlor revealed that the metabolite (-)-cis-heptachlorepoxide was more active than (+)- and (-)-heptachlor or their racemate (5, 6). Moreover, only the (+)-enantiomer of chlordene showed a pesticidal activity due to bioactivation by metabolization to (-)-cis-chlordene epoxide (7). (+)Chlordene, (-)-chlordene epoxide, and (+)-cis-heptachlor epoxide are both more active than their antipods and have the same absolute stereochemical configuration (5). Another study showed that the enantiomers of o,p′-DDT have different estrogenic activities in immature female rats. The (-)enantiomer is more potent (8). The enantiomer ratios of chlordane congeners and metabolites have been studied in various marine biota such * Corresponding author phone: +41 61 639 23 00; fax: +41 61 639 23 01; e-mail:
[email protected]. † Organic Analytical Chemistry, University of Basel. ‡ Norwegian Institute for Air Research. 2126
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 34, NO. 11, 2000
FIGURE 1. Structures of the analyzed chiral chlordane congeners. Note that the structure of U81 is unknown. as herring, cod, salmon, seals, and polar bears (9-11). However, an investigation of the influence of biological parameters such as age and gender on the observed enantiomer ratios require the analysis of a sufficient number of well-characterized animals which was not the case in the cited studies. In the presented work, levels and enantiomer ratios of chlordane congeners were determined in cod from the Barents Sea. The following compounds were selected: U81, U82, trans-chlordane, MC5, cis-chlordane, MC7, transnonachlor, MC6, and cis-nonachlor as well as the metabolites cis-heptachlorepoxide and oxychlordane. Structures of the determined compounds are given in Figure 1 and their IUPAC names in Table 1 (for further details about nomenclature, consult refs 1, 12, and 13). Sixteen cod liver samples from biological well-characterized individuals as well as selected samples of muscle and gonads were analyzed in order to find possible relations between biological parameters and observed enantiomer ratios.
Experimental Section Samples and Sample Preparation. Cod moving from the Barents Sea to the Lofot Islands for spawning was caught outside Kvaløya, Norway (70°N,17°E) in January 1995. It belonged to the Norwegian-Russian population which moves annually from the Barents Sea to the Lofot Islands during the period end of December to February for spawning in March. Sixteen individuals were selected, and age, sex, weight, and other parameters were determined (see Table 2). Age was determined by counting the annual rings of the otoliths. Details of the sample clean up are given in ref 14. 10.1021/es991288z CCC: $19.00
2000 American Chemical Society Published on Web 04/29/2000
TABLE 1. IUPAC and Common Names for Environmentally Important Octachlorinated Chlordanes and for the Internal Standard MC8 IUPAC name
common namesa
1-exo-2-exo-4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane 1-exo-2-endo-4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane structure not identified 1-exo-2-endo-3-exo-4,5,6,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane 1-exo-2-endo-3-exo-4,5,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane 1-exo-2,2,4,5,6,7,8,8-nonachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane 1-exo-2-exo-3-endo-4,5,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane 1-exo-2-exo-3-exo-4,5,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindane
cis-chlordane, no. 62 (1) trans-chlordane, no. 22 (1) U81 (1) U82 (1, 13), no. 49 MC5 (12), no. 59 (1) MC6 (12), no. 64 (1), nonachlor III (2) MC7 (12), no. 68 (1) MC8 (12), no. 78 (1)
a Common names are in accordance to Miyazaki et al. (12), Dearth et al. (1), and Muir et al. (2). Additional information regarding nomenclature can be obtained in ref 14.
TABLE 2. Age, Gender, and Weight of the Analyzed Cod Individuals sample no.
age (years)
gender
weight (kg)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
6 12 11 8 8 7 7 7 7 9 6 6 7 6 6 6
female female female female female female female female female male male male male male male male
1.9 9.6 6.9 3.4 4.7 3.9 2.0 3.0 1.8 4.7 1.0 1.3 2.4 1.5 1.6 3.0
Briefly, about 1 g of liver, 20-50 g of muscle, or 20 g of gonads were homogenized with a 10-15-fold excess of water-free sodium sulfate. MC8 and/or 13C12 PCB-118 were added as internal standards prior to column extraction of lipids with ethyl acetate/cyclohexane (1+1). MC8 is an octachlorinated chlordane that is structurally related to the target compounds but not 13C12 PCB-118 (see Figure 1). Since the use of MC8 as internal standard is relatively new, the well-tested 13C12 PCB-118 was added to the samples as well. The use of two internal standards ensured the compatibility between samples prepared before and after MC8 was obtained. Further details about the isolation of MC8 are given in refs 13 and 14. Lipids were removed by gel permeation chromatography on Biobeads SX3 with ethyl acetate/cyclohexane (1+1) as mobile phase. The samples were fractionated on aluminum oxide deactivated with 5% water (w/w). Mixtures of n-hexane/tertbutyldimethyl ether were employed to elute the target compounds. Before quantification 1,2,3,4-tetrachloronaphthalene was added as recovery standard, also called performance standard. Isomer Specific Analysis. Analysis was carried out on a HP 5890 Series II gas chromatograph (GC) connected to a HP 5989B mass spectrometer (MS) operating in negative ion chemical ionization (NICI) mode with methane as reagent gas. Detailed information about MS parameters is given in ref 13. Isomer specific analysis of heptachlor and octa- and nonachloro congeners of chlordane was carried out on a capillary column of 22 m length and 0.25 mm i.d. coated with 0.14 µm of polymethylsiloxane (Ultra1, Hewlett-Packard). The following temperature program was applied: 60 °C for 2 min, then 20 °C/min to 120 °C, followed by 4 °C/min to 250 °C kept for 2 min. Quantification ions for heptachlor (m/z 299.8, m/z 301.8) and the recovery standard 1,2,3,4tetrachloronaphthalene (m/z 263.8, m/z 265.8) were recorded within a retention time window of 6.0-19.5 min and a dwell
time of 100 ms. A dwell time of 80 ms was applied to the following ions monitored from 19.6 to 39.5 min: internal standard, 13C12-labeled PCB118, m/z 377.8 and m/z 339.8; octachloro congeners including the internal standard MC8, m/z 407.8 and m/z 409.8; and nonachloro congeners, m/z 441.8 and m/z 443.8. The Ultra 1 capillary column used previously did not separate cis- and trans-heptachlorepoxide, which made it unsuitable for quantification of the chlordane metabolites (14). Therefore, cis- and trans-heptachlorepoxide as well as oxychlordane were separated on a 30 m long column of 0.25 mm i.d. coated with 0.1 µm of 90%-biscyanopropyl-10%phenylcyanopropyl polysiloxane (Rtx-2330, Restek, PA). The separation was performed as follows: 70 °C for 2 min, then 15 °C/min to 190 °C followed by 1.5 °C/min to 220 °C, and finally 15 °C/min to 240 °C, isothermal for 10 min. The quantification ions for heptachlor (m/z 299.8, m/z 301.8), oxychlordane (m/z 421.8, 423.8), and the recovery standard 1,2,3,4-tetrachloronaphthalene (m/z 263.8, 265.8) were monitored from 7 to 17.5 min with a dwell time of 100 ms. From 17.5 to 41.3 min ions for cis-heptachlor epoxide (m/z 315.8, m/z 317.8), trans-heptachlor epoxide (m/z 351.8, m/z 353.8), and 13C12-labeled PCB 118 (m/z 337.8, m/z 339.8) as well as octa- (m/z 407.8, 409.8) and nonachloro congeners (m/z 441.8, m/z 443.8) were recorded with a dwell time of 80 ms. Helium of 99.996% purity was used as carrier gas, and the column head pressure was 1.0 bar for both isomer selective columns. Enantiomer Selective Separation. Analysis was performed with the same GC-MS system and NICI-MS conditions as for the isomer selective analysis. A tandem column system was employed for U81, U82, cis- and trans-chlordane, MC5, and MC6. It consisted of a capillary in front (30 m length, 0.25 mm i.d.) coated with 0.1 µm of Rtx-2330 connected to an enantioselective capillary (23 m length, 0.25 mm i.d.) coated with a 0.14 µm thick film of heptakis(2,3,6-tri-O-tertbutyldimethylsilyl)-β-cyclodextrin diluted in PS086. The coupling of the two columns enabled separation of all cisand trans-chlordane enantiomers without coelution as well as from trans-nonachlor (for further information, see ref 15). The temperature program was as follows: 90 °C for 2 min, then with 15 °C/min to 180 °C, isothermal for 44 min, followed by 2 °C/min to 230 °C, isothermal for 2 min. A column head pressure of 1.7 bar was applied. MC7, cis-heptachlor epoxide, and oxychlordane were separated into enantiomers on a capillary of 30 m length and 0.25 i.d. coated with 0.25 µm of heptakis (2,3,6-tri-O-tert-butyldimethyl-silyl)-β-cyclodextrin in BGB25 (BGB-172, BGB, Switzerland). The following temperature program was applied: 60 °C for 2 min, then 15 °C/min to 160 °C, followed by 7 °C/min to 220 °C, isothermal for 15 min. The carrier gas pressure was 1.0 bar. For all separations the injector temperature was set to 240 °C and the transfer line to 250 °C. Injections of 1 µL were performed splitless with the split closed for 2 min. Quantification was carried out with the internal standard method VOL. 34, NO. 11, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 3. Average Concentrations (Wet Weight Basis), Standard Deviations, and Enantiomer Ratios (ERs) of Chlordane Congeners and Metabolites in 16 Livers from Male and Female Coda concentration (ng/g) compound
male
U81 U82 trans-chlordane MC5 cis-chlordane MC7 trans-nonachlor MC6 cis-nonachlor oxychlordane cis-heptachlor epoxidec
enantiomer ratios (ERs)
female
nab
nab
5.3 ( 2.1 7.7 ( 5.3 9.2 ( 4.2 53 ( 24 2.1 ( 0.87 88 ( 42 2.6 ( 1.9 36 ( 19 12 ( 4.2 4.8 ( 2.2
6.2 ( 1.5 6.6 ( 6.7 11 ( 3.4 48 ( 15 2.99 ( 0.68 108 ( 18 0.93 ( 0.31 46 ( 20 14 ( 7.0 5.8 ( 4.1
male
female
0.91 ( 0.06 0.85 ( 0.04 1.33 ( 0.39 0.80 ( 0.04 1.19 ( 0.09 0.89 ( 0.04 not chiral 0.92 ( 0.12 not chiral 1.04 ( 0.21 1.52 ( 0.13
0.86 ( 0.08 0.81 ( 0.08 0.63 ( 0.16 0.79 ( 0.07 0.81 ( 0.07 0.95 ( 0.04 not chiral 0.20 ( 0.08 not chiral 1.26 ( 0.41 1.81 ( 0.79
a Enantiomer ratios are given as concentration ratios of (+)- to (-)-enantiomers for cis- and trans-chlordane and first to second eluting enantiomer for all other compounds. Gender specific differences which are significant at p < 0.05, are marked in bold. b Not analyzed. c Partly nondetectable in males (n ) 5).
TABLE 4. Concentrations on Lipid Weight Basis of Chlordane Compounds in Muscle, Gonads, and Liver from Five Cod Individualsa sample no.
U82 (ng/g)
tr-CD (ng/g)
MC5 (ng/g)
cis-CD (ng/g)
MC7 (ng/g)
tr-Nona (ng/g)
cis-Nona (ng/g)
muscle F1 gonad F1 liver F1 muscle F3 gonad F3 liver F3 muscle F4 gonad F4 liver F4 muscle M10 gonad M10 liver M10 muscle M11 gonad M11 liver M11
16 8.5 53 4.1 2.8 11 3.3 3.6 6.8 5.4 0.8 9.6 5.0 0.4 4.8
24 113 53 0.7 2.1 57 3.5 4.9 5.8 6.6 1.5 12 4.6 0.9 6.7
29 15 106 11 4.5 31 5.0 6.9 17 4.8 1.2 19 4.0 0.6 8.6
179 105 462 58 34 109 125 113 114 55 16 144 34 7.5 58
10 5.4 21 4.7 1.6 5.9 1.7 2.0 2.8 1.7 0.5 4.7 1.6 0.3 2.4
292 132 686 113 47 251 55 65 133 97 21 291 71 9.0 106
121 73 352 53 31 119 26 41 75 37 12 125 25 4.7 50
OXY (ng/g) 82 10 Int 28. 11 Int 15 16 18 20 4.4 27 35 2.7 18
cis-HEP (ng/g)
sum (ng/g)
lipid c (%)
10 1.7 14 8.0
