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Enantioselective analysis of organochlorine pesticides in herring and seal from the Swedish marine environment
Malfne Pollution Bulletin, Vol. 36, No. 5. pp. 345-353, 1998
Pergamon PII: S0025-326X(97)00187-2
© 1998 Elsevier Science Ltd. All rights reserved Printed in (;real Britain (1(125-326X/98$19.1)0+lL(~)
Enantioselective Analysis of Organochlorine Pesticides in Herring and Seal from the Swedish Marine Environment KARIN WIBERG*§, MICHAEL OEHMEt, PETER HAGLUND*, HEIDI KARLSSONt, MATS OLSSON~: and CHRISTOFFER RAPPE*
*Institute of Environmental Chemistry, Ume~ University, S-901 87 Ume~, Sweden tlnstitute for Organic Chemistry, University of Basel, St Johanns-Ring 19, CH-4056 Basel, Switzerland ~;Contamination Research Group, Swedish Museum q# Natural History, Box 50007, S-10405 Stockholm, Sweden
Factors influencing the changes of enantiomeric ratios (ERs) in herring (Clupea harengus), grey seal (Halichoerus grypus), harbour seal (Phoca vitulina) and ringed seal (Phoca hispida) collected along the Swedish coastline were studied by enantioselective high-resolution gas chromatography (HRGC). Three-year-old male and female herring individuals from different sampling sites were selected, whereas seal blubber and liver samples represented different species, sexes and geographical locations. Enantioselective analysis of ~-hexachlorocyclohexane (~-HCH), chlordanes and chlordane metabolites were performed. In general, herring showed similar ERs within the Baltic Sea. Results indicate that species specific differences are important factors controlling the ERs of Baltic seals. Inverse ERs in seal blubber and liver, and a low deviation from the racemic ratio of some components in the liver, indicate that not only enantioselective degradation but also stereoselectivity of other processes are responsible for the changes of ERs in biota. © 1998 Elsevier Science Ltd. All rights reserved Keywords: pesticides; bioaccumulation; degradation; enantiomers; Baltic seals; herring.
The Baltic Sea is known to be highly polluted by organochlorines. Many of them are bioaccumulated and biomagnified and thus found at very high concentrations in high trophy living organisms. The elevated levels of organochlorines in the marine environment are suspected to be associated with reproductive §Corresponding author. Tel.: +46-90-786-56-72; + 4 6 - 9 0 - 1 8 - 6 1 - 5 5 ; e-mail: k w g ~ c h e m . u m u . s e .
fax:
impairment and a high incidence of pathological changes observed in all of the three Baltic seal species: grey seal (Halichoerus grypus), harbour seal (Phoca vitulina) and ringed seal (Phoca hispida) (Olsson et al., 1994). Some of the organochlorines are chiral and thus exist as enantiomers. The pesticide chlordane, which consists of more than 140 components, includes many chiral components. Not only the main constituents, cis-chlordane, trans-chlordane and heptachlor, but also many of the bioaccumulating minor components, i.e. the MC4, MC5, MC6, MC7 (Miyazaki compounds, Miyazaki et al., 1985) and U82 (Dearth and Hites, 1991a,b), are chiral. Heptachlor is rapidly degraded in the environment, whereas the main metabolic product, heptachlor-exo-epoxide, is a persistent chiral compound. Similarly, cis-chlordane, trans-chlordane and the nonachlors are mainly degraded to the persistent chiral metabolite oxychlordane (Nomeir and Hajjar, 1987). These degradation processes are slow and thus the precursors as well as the metabolites are detected in biota. Technical chlordane was used widely on farmlands and for termite control in the 195(1s through to the 1980s, but is now banned or severely restricted in most countries. Due to atmospheric long-range transport, chlordane has been dispersed world-wide and is now found in remote areas such as the Arctic or in regions where it not has been in use. Another persistent ubiquitous pesticide is hexachlorocyclohexane (HCH). It was introduced as a pesticide during the Second World War and is now the predominant organochlorine detected in the ocean environment (lwata et al., 1993). The technical mixture consists of several isomers, of which the most abundant, ~-HCH 345
Marine Pollution Bulletin (80%), is chiral. The use of technical HCH was stopped in most countries, but continues to be used, mainly in India (Li et al., 1996). The enantiomers in the technical pesticide products are present as racemates. However, most biological processes show enantiomeric specificity and once the chiral components are spread in the environment, the enantiomer ratios (ERs) are changed due to selective metabolism or membrane transport. Many authors have found species specificity of enantioselective accumulation/metabolization of a-HCH and chlordanerelated compounds in biota (Buser et al., 1992; Buser and Mfiller, 1992, 1993, 1994; Faller et al., 1991; Mfiller et al., 1992; Oehmc et al., 1994; Pfaffenberger et al., 1992; M6ssner et al., 1992; Tanabe et al., 1996). However, so far, very little is known about the factors influencing the enantioselectivity of accumulation/ degradation mechanisms. For a better understanding, the objective of this work was to study the influence of various factors, such as species, sex and geographical location, on the enantiomeric composition found in a number of selected herring (Clupea harengus) and seal samples collected along the Swedish coastline.
Materials and Methods Chemicals" All the solvents, except tetrahydrofuran, were of high purity (glass distilled) and obtained from Burdick and Jackson (Muskegon, MI, USA). The tetrahydrofuran (unstabilized) was of HPLC grade from Labscan Ltd (Dublin, Ireland). The reference compounds, ~-HCH, trans-chlordane, cis-chlordane, oxychlordane and heptachlor-exo-epoxide, were obtained as crystalline reference compounds of > 95% purity (Dr Ehrenstorfer, Augsburg, Germany) and the enantiomer enriched reference standards of ~-HCH, heptachlorexo-epoxide and oxychlordane originated from Axact Standards Inc. For other pure or enriched reference standards see Oehme et al., 1997. The technical chlordane was obtained from Alltech, Deerfield, IL, USA. Solutions of 1.0 ng ~tl-1 were made with iso-octane as solvent. The Florisil had a particle size of 0.1500.250ram and originated from Merck (Darmstadt, Germany, cat. no. 12518.0100). Samples Herring and seal samples were collected from different locations along the Swedish coastline (Fig. 1). Three-year-old male and female herring individuals were selected for analysis (Table 1). Their weight varied between 24 and 39 g. The seal samples represented different species, sexes, ages and geographical locations (Table 2). For each seal individual, both adipose tissue and liver samples were analysed. The samples were deep frozen (-18°C) and kept in polyethylene bags or pre-cleaned glass jars until analysis. 346
Clean-up The whole herring samples (viscera excluded), 1.1 g of seal blubber or 11.5 g of seal liver were homogenized with 500 g water-free NazSO4 (we-treated for 6 h at 600°C). After dehydration overnight, the samples were packed into glass extraction columns. Extraction was performed with 150 or 300ml acetone/hexane 12.5:1) and 150 or 300ml hexane/diethyl ether (9:1), the higher volumes for the herring samples. The lipid contents, 3.7-14% in herring, 93-100% in seal blubber and 3.0-5.9% in seal liver, were determined gravimetrically on 10% of the extracts. After solvent volume reduction to 5-10 ml, the major amount of lipids was removed by polyethylene film dialysis and gel permeation chromatography (Zook et al., 1992). The samples were transferred to dialysis tubes of 350 mm length and 26 mm diameter. They were dialysed for 72 h in glass columns filled with 150 ml cyclopentane. After 16 and 48h the solvent was drained and the column was refilled with fresh cyclopentane. The portions of cyclopentane were combined and the solvent volume was reduced to 1-3 ml by slow evaporation in a fume-hood. For the gel permeation chromatography, two columns of 300 mm length and 7.5 mm i.d. packed with 5 lain PL-Gel (styrene-divinylbenzene copolymer, pore size 5 nm, Hewlett-Packard) were connected in series. The seal sample extracts were split into three portions of about 1 ml, which were injected separately. The pesticide fraction was collected between 15.4 and 16.8 ml using tetrahydrofuran at a flow rate of 0.7 ml rainThe three fractions were pooled after volume reduction to 1 ml, separation into three fractions was carried out on a deactivated (1.2% w/w H20) column (3011 mm long by 10 mm i.d.) containing 8.0 g Florisil. The first fraction (20 ml hexane) was discarded, c~-HCH and most of the chlordanes of interest were collected in fraction 2 (38 ml dichloromethane/hexane 15:85) and heptachlor-exo-epoxide was collected in fraction 3 (25 ml dichloromethane/hexane 50:50). Chromatographic separation and instrumentation The enantiomer specific analysis of the chiral pesticides was carried out by gas chromatography (GC) using capillaries coated with mixtures of modified !3-cyclodextrins and polysiloxanes. The reasons for using various capillary columns, or column combinations, were the inability to resolve all chiral pesticides on one single column, as well as the accessibility of the columns. It is known that the elution orders of the enantiomers can vary among columns with similar stationary phases (Oehme et al., 1997). Therefore, the enantiomer elution sequences of all columns were tested with pure enantiomers or (+)-enantiomer enriched reference standards and are given in Table 3. The determination of the ERs was carried out by low resolution mass spectrometry using the selected ion monitoring mode. For each compound, the two most intense ions above m/z 181) were recorded. Compound
Volume 36/Number 5/May 1998
identification was made by retention time and abundance ratio of the two selected ions. Compounds where no reference standards were available, such as U82, MC5, MC6 and MC7, were identified according to the procedure of Oehme et al. (1994). It was assumed that the chiral compound X~ was similar to that reported by Buser et al. (1992) by comparing relative retention times of technical chlordane and environmental samples.
Results and Discussion The ERs presented in Tables 1 and 2 were calculated as the area ratio of the ( + ) / ( - ) enantio-
mers or, in the case of an unknown elution order, as the area of first to second eluting enantiomer. For a positive identification the abundance ratio of the two ions monitored had to be within _+ 10% compared to a reference standard. The ERs in the herring studied arc listed in Table 1. Most of the compounds showed deviations from the originally racemic ratio. They were largest for transchlordane and heptachlor-exo-epoxidc (ER on average 0.37 and 0.56 respectively) and smallest for U82 and MC7 (ER on average 1.1 and 0.96 respectively). Comparing the ERs and the ER profiles (the relative magnitude of the ERs within one sample) among
5
Skagerrak . ,,, N ~ J I ~~~.,~ H(1-°) 6Y BalticSea
Fig. I Map of the sampling sites of herring and seal. The numbers of the herring samples (see Table I) are given in parentheses with an H in front. Seal samples are marked with plain numbers according to Table 2.
347
Marine Pollution Bulletin TABLE l
Data for sample characterization and ERs of selected organochlorines in Baltic herring. Herring sample #
Sex
Age (yr)
Sampling site
Year of collection
'~-HCH +/-
U82 E~/E2
tr-CHL +/-
cs-CHL +/-
MC5 El/E2
MC7 EJE_,
H EP-exo +/-
OXY +/-
1 2 3 4 5 6 7 8 9
F M M M M F F F F
3 3 3 3 3 3 3 3 3
Utklippan Utklippan Utklippan Landsort Landsort Landsort Harufjarden Harufjarden Harufjarden
1993 1993 1993 1993 1993 1993 1993 1993 1993
0.79 n.d. n.d. 0.81 n.d. 0.81 0.83 n.d. n.d.
1.1 1.2 1.2 1.1 1.2 1.0 1.0 1.1 1.0
0.48 0.2(I 0.27 0.58 0.47 0.36 0.35 0.27 0.32
1.1 2.1 1.2 1.3 1.5 1.2 1.2 1.6 1.6
1.5 1.4 1.6 1.5 1.4 1.6 1.2 1.2 1.2
1.1 11.9 1.0 1.(I 1.(I (1.9 11.9 i i
0.58 0.56 0.51 (I.58 0.49 0.55 0.4(I 0.65 0.68
1.4 1.4 1.4 1.4 I. 1 1.5 1.4 1.4 1.5
M, male; F, female; yr, year; i, interference; n.d., not detected. For calculations and other abbreviations see Fig. 2.
o f t h e E R s f o u n d by B u s e r et al. (1992) fall in the E R r a n g e p r e s e n t e d in this study. T h e o n l y e x c e p t i o n was M C 7 , w h i c h B u s e r et al. (1992) f o u n d to b e slightly l o w e r ( E R = 0.83). T h e E R s f o u n d in t h e seal s a m p l e s r a n g e d f r o m v a l u e s close to r a c e m i c ratios to a l m o s t total d e p l e t i o n o f o n e e n a n t i o m e r . A s can b e s e e n f r o m T a b l e 2, m a n y s i m i l a r i t i e s exist b e t w e e n t h e a n a l y s e d s a m p l e s , but s t r o n g d e v i a t i o n s w e r e also f o u n d . H o w e v e r , d u e to t h e l i m i t e d n u m b e r o f s a m p l e s , only t e n t a t i v e c o n c l u s i o n s could be drawn. As expected, the ERs of the three
individuals, n o o b v i o u s d i f f e r e n c e s c o u l d b e f o u n d . T h i s i n d i c a t e s t h a t t h e E R v a r i a t i o n a m o n g Baltic h e r r i n g is small. N e i t h e r s a m p l i n g site w i t h i n t h e Baltic S e a n o r sex s e e m s to h a v e a s u b s t a n t i a l i n f l u e n c e o n t h e E R (Fig. 2a,b). B u s e r et al. (1992) e x a m i n e d t h e E R s in Baltic herring. Assuming a reversed enantiomer elution s e q u e n c e o f M C 5 o n t h e p e r m e t h y l a t e d 13-cyclodextrin ( P M C D ) - b a s e d c a p i l l a r y c o l u m n u s e d by B u s e r et al. (1992) c o m p a r e d to t h e tert.-butyldimethylsilyl 13-cyclodextrin ( B S C D ) c o l u m n u s e d in this study, t h e m a j o r i t y
TABLE 2
Data for sample characterization and ERs of selected organochlorines in blubber and liver of seals sampled along the Swedish coastline. Sample # Species, Sex l_x~cation, Year of collection
Age (yr) Health status Condition
1 GS, M Gavlebukten, 1989 2 GS, F Gotland, n.a. 3 GS, F Eggegrund, 1989 4 GS, F Eggegrund, 1989 5 GS, F IJ3vstabukten, 1989 6 RS, M Kalmarsund, n.a. 7 RS, F Ronnebyfjorden, 1993 8 HS, F Kalmarsund, 1983 9 HS, M Skiinekusten, n.a. 10 HS, M West Tj6rn, 1988
1 Healthy Not starved 38 Unhealthy Starved 0 Healthy Not starved 1 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved
~-HCH +/-
Xi EI/Ez
U82 EJE2
tr-CHL +/-
cs-CHL +/-
MC5 El/E2
MC7 Et/E2
MC6 El/E2
HEP-exo +/-
OXY +/-
blubber liver
1.1 n.d.
i n.d.
4.7 2.4
0.71 0.91
1.8 0.26
i 3.5
i I).39
0.90 11.54
0.27 <11.5
1.4 11.97
blubber liver
1.7 3.6
0.76 0.94
0.77 0.62
0.52 0.58
i i
i i
11.20 0.15
11.12 n.d.
1.1 0.86
blubber liver
1.6 2.7
0.94 0.45
5.2 4.8
I).19 0.40
0.64 0.27
6.0 10
0.58 0.32
0.75 0.46
11.55 <11.5
1.4 0.78
blubber liver
2.2 n.d.
1.0 0.42
8.4 5.7
0.21 0.25
0.47 0.49
4.9 7.7
0.68 11.33
11,58 0,39
0.50 < 0.5
1.4 1.11
blubber liver
1.0 n.d.
0.92 0.37
5.1 2.6
0.74 0.94
0.48 0.24
3.7 6.3
0.98 0.41
0.66 11.44
0.25 <0.7
1.2 11.71
blubber liver
1.1 1.4
1.1 0.38
7.1 3.8
0.39 0.75
0.28 0.72
3.2 0.58
2.0 11.40
0.16 0.62
0.30 <0.7
11.92 11.56
blubber liver
1.0 n.d.
1.1 n.d.
4.1 1.5
0.21 0.22
2.9 4.6
2.2 2.1
0.30 0.08
0.25 0.13
0.41 < 11.7
1.3 i
blubber liver
2.0 n.d.
1.3 0.27
18 13
2.6 0.42
0.14 i
i i
i i
1.3 0.74
0.12 < 0.7
0.77 0.5 I
blubber liver
3.4 n.d.
1.3 0.41
22 21
3.1 11.56
0.21 i
4.1 19
1.9 i
1.3 1.1
11.11 <0.3
I).62 0.44
blubber liver
3.6 n.d.
0.94 n.d.
12 19
1.8 0.76
0.18 i
4.9 1.9
1.5 i
1.0 0.84
i i
1.0 i
19 15
GS, grey seal; RS, ringed seal; HS, harbour seal; M, male; F, female; n.a., not available. For calculations and other explanations see Table 1 and Fig. 2 348
Volume 36/Number 5/May 1998
juvenile female grey seals were similar, indicating that individuals of the same sex and species resemble each other in the ER profiles. No obvious influence of sampling sites or sex was observed. The fcmale harbour seal from Kalmarsund, Baltic Proper (seal #8) and the two male harbour seals from the coast of Sk~,ne, Baltic Proper (seal #9) and West Tj6rn, Skagerrak (seal #1(I) exhibited similar ER profiles (Fig. 3a). This is interesting since the chlordane body burden of juvenile harbour seals was found to be lower in the Skagerrak (sum of chlordancs 0.5(I-0.63 I-~gg ~) compared to the Baltic, Kalmarsund (1.5-1.8 l,tg g - ~) (Andersson and Wartanian, 1992, Table 1). In the case of ringed seal and grey seal some gender-specific differences were indicated, mostly due to the ERs of cis-chlordanc and MC5. Further studies are needed to find out whether these differences are statistically significant. The evidence of species-specific differences of the ER profiles was strong. In Fig. 3b the ERs in blubber from juvenile males can be seen (seal #1 vs 6 vs average 9-1(I). As shown, all species show distinct enantiomeric profiles and the most remarkable differ-
ences were the large deviations of the ERs of U82, heptachlor-exo-epoxide and zt-HCH in harbour seal, the strong dominance of the last cluting MC6-enantiomer in ringed seal and inverse ER of trans- and c/s-chlordane in harbour seal and grey seal respectivcly. As seen from Table 2, the corresponding juvenile females compare similarly (seal # 3 - 5 vs 7 vs 8), but here the ringed seal had inverse ER of cis-chlordanc and the at-HCH of harbour seal did not show a large deviation compared to the other two species. Explanations for these findings may be different feeding habits and variations in the enantioselectivity of uptake, cell membrane transport or degradation. Species-specific ER profiles of chlordane-related compounds have also been found by Buscr et al. (1992), Buser and Miiller (1992), Buser and Miiller (1993) and Oehme et al. (1994). Several authors have reported various ERs of zt-HCH in a number of species (Faller et al., 1991; Miiller et al., 1992; Pfaffenberger et al., 1992; M6ssncr et al., 1992; Tanabe et al., 1996). Tanabe et al. (1996) suggested that the ER differences
TABLE 3 Chromatographic separation conditions and instrumentation. Component 7-HCH
HEP-exo
Elution order
GC
LRMS
Matrix
+/-
Fison GC8000
Fison MD8()0, El, 70 eV, 250°C
Herring
+/
CP Chirasil DEX CB column (PMCD), 25 m × (I.25 ram, He 45 cm s hzlet: 250°C splitless I rain Oven: 160°C(6)-3°C rain 1-250°C(15) Fison Gcg()(I()
Fison MDg()(), El, 70 eV, 250°C
Herring
HP 5897. interface HP5988, ECNI, CH~, 0.5 torr, 200°C
Herring
tr-CHL, cs-CHL, MC5, MC7, U82, OXY
+/ +/ unknown +/
X], HEP-exo, OXY
unknown +/+/-
tr-CHL, cs-CHL, MC5, MC7, U82, MC6
tr-CHL cs-CHL, MC5, MC7, U82, MC6
+/+/-
unknown unknown
+/-/+
unknown unknown
10% BSCD/PS(}86, 18 m x (1.3 mm, He 45cms hlh't: 25(1°C, splitless 1 min Oven: 16(1°C(6)-3°C min 1-225°C(15) HP 589(I Tandem column: RT~2330+30% BSCD/PS(}86 (glass), 24 m x {).25 ram+ 18 m x (/.3 mm, He 35-45 cm s htlet: 251)°C, splitless 2 rain Oven: 60°C(2)-25°C min 1-185°C-3°C rain i_ 20(}°C(30) HP 5890 10¢4 BSCD/PS086 18m×0.3mm, He45ems i hth't: 25(1°C, on-column Oven: 811°C(3)-25°C rain 1-185°C(25)
HP 5890
Tandem column: RTx2330+ 10% BSCD/PS086 (glass), 25 m × 0.25 r a m + Ig m × (l.3 ram, He
Finnigan 4500, ECNI, CH4,
Seal
Seal
0.4 torr, 200°C
HP 5989, ECNI, CH4, ().5 torr, 200°C
Seal
35-45 cm s t 25 m ×0.25 ram+25 m ×0.25 ram, He or35-45 cm s Tandem column: RTx2330+ 10% BSCD/PS086
(fused silica), Oven: 60°C(2)-25°C rain 1_185oc_3oc rain - i_ 200°C(30) Inh't: 250°C, splitless 2 rain
PMCD, permethylated [~-cyclodextrin; BSCD, tert.-butyldimethylisilyl-13-cyclodextrin:
HP, Hewlett Packard. For other abbreviations see Fig. 2.
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Marine Pollution Bulletin
could be due to metabolization of the enantiomers by different enzymes and that the distortion of the ER might be due to unbalanced degradation processes. Furthermore, they suggested that one is less likely to find large deviation from the racemic ER in animals with low metabolic capacity such as molluscs and fish and this was illustrated by a number of ~-HCH ERs in various species (see Fig. 3 in Tanabe et al., 1996). The ERs for chlordane and a-HCH presented in this study support this hypothesis. The ER range is, in general,
smaller in herring (ER range > 0.2 to < 1.6) compared to seal (ER range >0.1 to 22). Further supportive data were reported by Buser et al. (1992) (see Table II therein), who found ER ranges for Me4, MC5, MC7, trans- and cis-chlordane from 0.38 to 1.35 in Baltic herring and Baltic salmon, whereas Baltic seal ranged from 0.24 to 2.7. Further, the data for U82 in Baltic seal, reported by Buser and Miiller (1993), showed considerable changes of the ERs compared to Baltic herring and Baltic salmon, which were close to
(a) 2.00
, ----T-r~ ,
t.00
~
J
.
0.50
0.33
[] Harufj~irden
0.25
[] Landsort [] Utklippan
0.20
a-HCH
U82
tr-CHL
cs-CHL
Me5
MC7
HEP-exo
OXY
(b) 2.00
1.00
T
= W
I
I
0.50
0.33
[] Males
0.25
[] Females 0.20
a-HCH
U82
tr-CHL
cs-CHL
MC5
MC7
HEP-exo
Fig. 2 ERs in Baltic herring, a-HCH = ~-HCH; U82, XI = octachloro chlordane congener of unknown structures; cs-, tr- and O X Y =cis-, trans- and oxy-chlordane; M e 5 , M C 7 = o c t a chloro chlordane congeners; M e 6 = nonachloro chlordane congener; HEP-exo = heptachlor-exo-epoxide; P M C D = permethylated I~-cyclodextrin; BSCD = tert.-butyldimethylisilylf3-cyclodextrin. For components with known elution order (HCH, tr-CHL, cs-CHL, OXY and HEP-exo) ER is calculated as the area ratio of (+)-/( - ) - e n a n t i o m e r , and otherwise as the area ratio of the first to second eluting enantiomer. (a) Average, high and low values of herring from various sampling sites in the Baltic sea. (b) Average, high and low values of males vs females.
350
OXY
Volume 36/Number 5/May 1998
26 T (a) [] Female HS, Baltic Sea: blubber #8
21 [] Male HS, Baltic Sea: blubber #9 16 [] Male HS, Skagerack: blubber #10
6.0
ii:t U82
21
xl
tr-CHL cs-CHL MC5
MC7
MC6
OXY
HEP- a-HCH exo
'(b) [ ] Male GS: b l u b b e r #1
[] Male RS: b l u b b e r #6
16
[] Male HS: average b l u b b e r #9-10 11
6.0 1.0
i
r'i~z4,
'"I_I
'
__,~
~:
0,17
0.09
U82
Xl
tr-CHL cs-CHL
MC5
MC7
MC6
OXY
H E P - a-HCH exo
11
(c)
9.0
O J u v e n i l e female GS: average b l u b b e r #3-5
7.0
[ ] J u v e n i l e female GS: average liver #3-5
S.O 3.0 1.0 0.33 0.20 0.14
t
U82
Xt
tr-CHL cs-CHL
MC5
MC7
MC6
OXY
a-HCH
Fig. 3 ERs in seals sampled along the Swedish coastline. For calculations and abbreviations see Fig. 2. (a) Female and male harbour seal blubber; various sampling sites (seal #8-10). (b) Male seal blubber; grey seal vs ringed seal vs harbour seal (seal #1 vs #6 vs average of #9-10). (c) Juvenile female grey seal; blubber vs liver (average, high and low values of seal #3-5 blubber vs liver).
351
Marine Pollution Bulletin
racemic. The findings of smaller deviations from racemic values in fish and other animals with low metabolic capacity compared to their predators indicate that the influence of food is of minor importance. However, the assumption that enzymatic degradation is the most important process for changes in ER is contradictory to the quite different ER profiles in ringed seal and harbour seal. They both belong to the genus Phoca and there should not be much difference in their enzyme systems. in the liver, the metabolic capacity is higher than in other organs. Therefore, one would expect larger deviations from the racemic ER in the liver compared to the blubber. As can be seen from Table 2 and in Fig. 3c, this is the case for most of the compounds. The same trend was also observed by M6ssner et al. (1992), who reported significantly larger deviations for the ERs of a-HCH in fur seal liver than in blubber. Component U82 and trans-chlordane do not follow this trend, which indicates that the mechanisms leading to changes of ERs are not only attributed to enantioselective degradation, but also to uptake and/or transport across cell membranes. A change of the ER of a-HCH due to the transport across the 'blood-brain barrier' has been reported for harbour seals (M6ssner et al., 1992) and for double-crested cormorant (see Fig. 3 in Tanabe et al., 1996). Buser and Mi.iller (1994) have shown that the degradation of (+)- and (-)-tram-chlordane form (+)- and (-)-oxychlordane respectively. Therefore, the enantioselective formation of metabolites is an important process which change ERs in addition to the mechanisms mentioned above. Interestingly, in many samples (seal #1-3, 5, 6, 8-10) the ERs were inverse in the liver and the corresponding blubber extract. The reason for this could be that several enantioselective processes (degradation, uptake, cell membrane transport) are involved in the change of ER. The old and starved female grey seal (seal #2) suffered from a disease complex interpreted as hyperadrenocortisism, which includes uterine occlusions. Substantially increased ERs of U82, trans-ch[ordane, MC6 and heptachlor-exo-epoxide were observed in the blubber and liver samples of this female in comparison to the juvenile healthy grey seal females (seal #3-5). Further studies are under way to find out whether age, health and/or feeding status have an influence on the ERs. In summary, herring had, in general, similar a-HCH and chlordane ERs within the Baltic Sea and thus no spatial south-north trends could be indicated. In addition, no differences between male and female herring were observed. Results indicate that speciesspecific differences are important factors controlling the ER of Baltic seals, whereas the sampling site along the Swedish coastline seems to play a minor role. Further investigations are needed to find out whether indicated differences between individuals of different 352
sex, age and health status exist. Inverse ER in blubber and liver and a low deviation from the racemic ratio of some components in the liver indicate that not only enantioselective degradation but also the stereoselectivity of other processes such as enantioselective uptake, cell membrane transport and metabolite formation are responsible for the changes of ER in biota. We wish to thank Dr Terry Bidleman, Atmospheric Environment Service, Downsview, Canada, for kindly providing the (+)-enantiomer-enriched reference standards of ~-HCH, heptachlorexo-epoxide and oxychlordane. This work was supported by the Swedish Environmental Protection Agency 'Persistent Organic Pollutants' scientific program under the project number 30415A and by the Swiss Science Foundation under the project number 21 I)0-(/43399.95. Andersson, O. and Wartanian, A. Levels of polychlorinated camphenes (toxaphene), chlordane compounds and polybrominated diphenyl ethers in seals from Swedish waters. Amhio, 1992, 8, 550-552. Buser, H.-R. and M~ller, M. D. Enantiomer separation of chlordane components and metabolites using chiral high resolution gas chromatography and detection by mass spectrometric techniques. Analytical Chemistry, 1992, 64, 3168-3175, Buser, H.-R. and MiJller, M. D. Enantioselective dctermination of chlordane components, metabolites and photoconversion products in environmental samples using chiral high resolution gas chromatography and mass spectrometry. Environmental Science and Technology, 1993, 27, 1211-1220. Buser, H.-R. and Mfiller, M. D. Identification of the (+)- and (-)-enantiomers of chiral chlordane compounds using chiral highperformance liquid chromatography/chiroptical detection and chiral high resolution gas chromatography/mass spectrometry. Analytical Chemistry, 1994, 66, 2155-2162. Buser, H.-R., MiJller, M. D. and Rappe, C. Enantioselective determination of chlordane components using high resolution gas chromatography-mass spectrometry with application to environmental samples. Environmental Science and Technology, 1992, 26, 1533-1540. Dearth, M. A. and Hites, R, A. Complete analysis of technical chlordane using negative ionisation mass spectrometry. Environmental Science and Technology, 1991a, 25, 245-254. Dearth, M. A. and Hites, R. A. Chlordane accumulation in people. Environmental Science and Technology, 1991b, 25, 1279-1285. Failer, J., Hfihnerfuss, H., K6nig, W. A. and Ludwig, P. Gas chromatographic separation of the enantiomers of marine organic pollutants. Distribution of u-HCH enantiomers in the North Sea. Marine Pollution Bulletin, 1991, 22, 82-86. lwata, H., Tanabe, S., Sakai, N. and Tatsukawa, R. Distribution of persistent organochlorines in oceanic air and surface seawater and role of ocean on their global transport and fate. Environmental Science anti Technology, 1993, 27, 1080-1098. Li, Y.-F., Mcmillan, A. and Scholtz, M. T. Global HCH usage with l°x 1° longitude/latitude resolution. Environmental Science and Technology, 1996, 30, 3525-3533. Miyazaki, T., Yagmagishi, T. and Matsumoto, M. Isolation and structure elucidation of some components in technical grade chlordane. Archives of Environmental Contamination and Toxicology, 1985, 14, 475-483. M6ssner, S., Sparker, T. R., Becket, P. R. and Ballschmiter, K. Ratios of enantiomers of alpha-HCH and determination of alpha, beta- and gamma-HCH isomers in brain and other tissues of neonatal northern fur seals (Callorhinus ulwinus). Chemosphere, 1992, 24, 1171-1180. Miiller, M. D., Schlabach, M. and Oehme, M. Fast and precise determination of ~-hexachlorocyclohexane enantiomer in environmental samples using high-resolution gas chromatography. Environmental Science and Technolo,w, 1992, 26, 566-569. Nomeir, A. A. and Hajjar, N. P. Metabolism of chlordane in mammals. Reviews q]"Envil~mmental Contamination attd Toxicology, 1987, 100, 1-22. Oehme, M., Kallenborn, R., Wiberg, K. and Rappe, C. Simultaneous enantioselective separation of chlordanes, nonachlor compounds
Volume 36/Number 5/May 1998 and o,p'-DDT in environmental samples using tandem capillary columns. Journal q[ High Resohttion Chromatography, 1994, 17. 583-588. Oehme, M., Miiller, L. and Karlsson, H. (1997) High-resolution gas chromatographic test for the characterisation of enantioselective separations of organoehlorine compounds. Application to tert.butyldimethylsilyl 13-cyelodextrin. JotoTtal of" Chromatography, A, 775, 275-285 Olsson, M., Karlsson, B. and Ahnland, E. Diseases and environmental contaminants in seals from the Baltic and the Swedish west coast. The Science of the Total Em,iro/lment, 1994, 154, 217-227.
Pfaffenberger, B., H0hnerfuss, H., Kallenborn, R., G0nther, A. G., K6nig, W. A. and KrOner, G. Chromatographic separation of the enantiomers of marine pollutants. Part 6. Comparison of the enantioselective degradation of ~¢-hexachlorocyclohexane in marine biota and water. Chemosphere, 1992, 25, 719-725. Tanabe, S., Kumaran, P., lwata, H., Tatsukawa, R. and Miyazaki, N. Enantiomeric ratios of ~-hexachlorocyclohexane in blubber of small cetaceans. Marine Pollution Bulletin, 1996, 32, 27-31. Zook, D. R., Buser, H.-R., Bergqvist, P.-A., Rappe, C. and Olsson, M. Detection of tris(chlorophenyl)methane and tris(4chlorophenyl)methanol in ringed seal (Phoca hispMa) from the Baltic Sea. Ambio, 1992, 21,557-560.
353
Pergamon PII: S0025-326X(97)00187-2
© 1998 Elsevier Science Ltd. All rights reserved Printed in (;real Britain (1(125-326X/98$19.1)0+lL(~)
Enantioselective Analysis of Organochlorine Pesticides in Herring and Seal from the Swedish Marine Environment KARIN WIBERG*§, MICHAEL OEHMEt, PETER HAGLUND*, HEIDI KARLSSONt, MATS OLSSON~: and CHRISTOFFER RAPPE*
*Institute of Environmental Chemistry, Ume~ University, S-901 87 Ume~, Sweden tlnstitute for Organic Chemistry, University of Basel, St Johanns-Ring 19, CH-4056 Basel, Switzerland ~;Contamination Research Group, Swedish Museum q# Natural History, Box 50007, S-10405 Stockholm, Sweden
Factors influencing the changes of enantiomeric ratios (ERs) in herring (Clupea harengus), grey seal (Halichoerus grypus), harbour seal (Phoca vitulina) and ringed seal (Phoca hispida) collected along the Swedish coastline were studied by enantioselective high-resolution gas chromatography (HRGC). Three-year-old male and female herring individuals from different sampling sites were selected, whereas seal blubber and liver samples represented different species, sexes and geographical locations. Enantioselective analysis of ~-hexachlorocyclohexane (~-HCH), chlordanes and chlordane metabolites were performed. In general, herring showed similar ERs within the Baltic Sea. Results indicate that species specific differences are important factors controlling the ERs of Baltic seals. Inverse ERs in seal blubber and liver, and a low deviation from the racemic ratio of some components in the liver, indicate that not only enantioselective degradation but also stereoselectivity of other processes are responsible for the changes of ERs in biota. © 1998 Elsevier Science Ltd. All rights reserved Keywords: pesticides; bioaccumulation; degradation; enantiomers; Baltic seals; herring.
The Baltic Sea is known to be highly polluted by organochlorines. Many of them are bioaccumulated and biomagnified and thus found at very high concentrations in high trophy living organisms. The elevated levels of organochlorines in the marine environment are suspected to be associated with reproductive §Corresponding author. Tel.: +46-90-786-56-72; + 4 6 - 9 0 - 1 8 - 6 1 - 5 5 ; e-mail: k w g ~ c h e m . u m u . s e .
fax:
impairment and a high incidence of pathological changes observed in all of the three Baltic seal species: grey seal (Halichoerus grypus), harbour seal (Phoca vitulina) and ringed seal (Phoca hispida) (Olsson et al., 1994). Some of the organochlorines are chiral and thus exist as enantiomers. The pesticide chlordane, which consists of more than 140 components, includes many chiral components. Not only the main constituents, cis-chlordane, trans-chlordane and heptachlor, but also many of the bioaccumulating minor components, i.e. the MC4, MC5, MC6, MC7 (Miyazaki compounds, Miyazaki et al., 1985) and U82 (Dearth and Hites, 1991a,b), are chiral. Heptachlor is rapidly degraded in the environment, whereas the main metabolic product, heptachlor-exo-epoxide, is a persistent chiral compound. Similarly, cis-chlordane, trans-chlordane and the nonachlors are mainly degraded to the persistent chiral metabolite oxychlordane (Nomeir and Hajjar, 1987). These degradation processes are slow and thus the precursors as well as the metabolites are detected in biota. Technical chlordane was used widely on farmlands and for termite control in the 195(1s through to the 1980s, but is now banned or severely restricted in most countries. Due to atmospheric long-range transport, chlordane has been dispersed world-wide and is now found in remote areas such as the Arctic or in regions where it not has been in use. Another persistent ubiquitous pesticide is hexachlorocyclohexane (HCH). It was introduced as a pesticide during the Second World War and is now the predominant organochlorine detected in the ocean environment (lwata et al., 1993). The technical mixture consists of several isomers, of which the most abundant, ~-HCH 345
Marine Pollution Bulletin (80%), is chiral. The use of technical HCH was stopped in most countries, but continues to be used, mainly in India (Li et al., 1996). The enantiomers in the technical pesticide products are present as racemates. However, most biological processes show enantiomeric specificity and once the chiral components are spread in the environment, the enantiomer ratios (ERs) are changed due to selective metabolism or membrane transport. Many authors have found species specificity of enantioselective accumulation/metabolization of a-HCH and chlordanerelated compounds in biota (Buser et al., 1992; Buser and Mfiller, 1992, 1993, 1994; Faller et al., 1991; Mfiller et al., 1992; Oehmc et al., 1994; Pfaffenberger et al., 1992; M6ssner et al., 1992; Tanabe et al., 1996). However, so far, very little is known about the factors influencing the enantioselectivity of accumulation/ degradation mechanisms. For a better understanding, the objective of this work was to study the influence of various factors, such as species, sex and geographical location, on the enantiomeric composition found in a number of selected herring (Clupea harengus) and seal samples collected along the Swedish coastline.
Materials and Methods Chemicals" All the solvents, except tetrahydrofuran, were of high purity (glass distilled) and obtained from Burdick and Jackson (Muskegon, MI, USA). The tetrahydrofuran (unstabilized) was of HPLC grade from Labscan Ltd (Dublin, Ireland). The reference compounds, ~-HCH, trans-chlordane, cis-chlordane, oxychlordane and heptachlor-exo-epoxide, were obtained as crystalline reference compounds of > 95% purity (Dr Ehrenstorfer, Augsburg, Germany) and the enantiomer enriched reference standards of ~-HCH, heptachlorexo-epoxide and oxychlordane originated from Axact Standards Inc. For other pure or enriched reference standards see Oehme et al., 1997. The technical chlordane was obtained from Alltech, Deerfield, IL, USA. Solutions of 1.0 ng ~tl-1 were made with iso-octane as solvent. The Florisil had a particle size of 0.1500.250ram and originated from Merck (Darmstadt, Germany, cat. no. 12518.0100). Samples Herring and seal samples were collected from different locations along the Swedish coastline (Fig. 1). Three-year-old male and female herring individuals were selected for analysis (Table 1). Their weight varied between 24 and 39 g. The seal samples represented different species, sexes, ages and geographical locations (Table 2). For each seal individual, both adipose tissue and liver samples were analysed. The samples were deep frozen (-18°C) and kept in polyethylene bags or pre-cleaned glass jars until analysis. 346
Clean-up The whole herring samples (viscera excluded), 1.1 g of seal blubber or 11.5 g of seal liver were homogenized with 500 g water-free NazSO4 (we-treated for 6 h at 600°C). After dehydration overnight, the samples were packed into glass extraction columns. Extraction was performed with 150 or 300ml acetone/hexane 12.5:1) and 150 or 300ml hexane/diethyl ether (9:1), the higher volumes for the herring samples. The lipid contents, 3.7-14% in herring, 93-100% in seal blubber and 3.0-5.9% in seal liver, were determined gravimetrically on 10% of the extracts. After solvent volume reduction to 5-10 ml, the major amount of lipids was removed by polyethylene film dialysis and gel permeation chromatography (Zook et al., 1992). The samples were transferred to dialysis tubes of 350 mm length and 26 mm diameter. They were dialysed for 72 h in glass columns filled with 150 ml cyclopentane. After 16 and 48h the solvent was drained and the column was refilled with fresh cyclopentane. The portions of cyclopentane were combined and the solvent volume was reduced to 1-3 ml by slow evaporation in a fume-hood. For the gel permeation chromatography, two columns of 300 mm length and 7.5 mm i.d. packed with 5 lain PL-Gel (styrene-divinylbenzene copolymer, pore size 5 nm, Hewlett-Packard) were connected in series. The seal sample extracts were split into three portions of about 1 ml, which were injected separately. The pesticide fraction was collected between 15.4 and 16.8 ml using tetrahydrofuran at a flow rate of 0.7 ml rainThe three fractions were pooled after volume reduction to 1 ml, separation into three fractions was carried out on a deactivated (1.2% w/w H20) column (3011 mm long by 10 mm i.d.) containing 8.0 g Florisil. The first fraction (20 ml hexane) was discarded, c~-HCH and most of the chlordanes of interest were collected in fraction 2 (38 ml dichloromethane/hexane 15:85) and heptachlor-exo-epoxide was collected in fraction 3 (25 ml dichloromethane/hexane 50:50). Chromatographic separation and instrumentation The enantiomer specific analysis of the chiral pesticides was carried out by gas chromatography (GC) using capillaries coated with mixtures of modified !3-cyclodextrins and polysiloxanes. The reasons for using various capillary columns, or column combinations, were the inability to resolve all chiral pesticides on one single column, as well as the accessibility of the columns. It is known that the elution orders of the enantiomers can vary among columns with similar stationary phases (Oehme et al., 1997). Therefore, the enantiomer elution sequences of all columns were tested with pure enantiomers or (+)-enantiomer enriched reference standards and are given in Table 3. The determination of the ERs was carried out by low resolution mass spectrometry using the selected ion monitoring mode. For each compound, the two most intense ions above m/z 181) were recorded. Compound
Volume 36/Number 5/May 1998
identification was made by retention time and abundance ratio of the two selected ions. Compounds where no reference standards were available, such as U82, MC5, MC6 and MC7, were identified according to the procedure of Oehme et al. (1994). It was assumed that the chiral compound X~ was similar to that reported by Buser et al. (1992) by comparing relative retention times of technical chlordane and environmental samples.
Results and Discussion The ERs presented in Tables 1 and 2 were calculated as the area ratio of the ( + ) / ( - ) enantio-
mers or, in the case of an unknown elution order, as the area of first to second eluting enantiomer. For a positive identification the abundance ratio of the two ions monitored had to be within _+ 10% compared to a reference standard. The ERs in the herring studied arc listed in Table 1. Most of the compounds showed deviations from the originally racemic ratio. They were largest for transchlordane and heptachlor-exo-epoxidc (ER on average 0.37 and 0.56 respectively) and smallest for U82 and MC7 (ER on average 1.1 and 0.96 respectively). Comparing the ERs and the ER profiles (the relative magnitude of the ERs within one sample) among
5
Skagerrak . ,,, N ~ J I ~~~.,~ H(1-°) 6Y BalticSea
Fig. I Map of the sampling sites of herring and seal. The numbers of the herring samples (see Table I) are given in parentheses with an H in front. Seal samples are marked with plain numbers according to Table 2.
347
Marine Pollution Bulletin TABLE l
Data for sample characterization and ERs of selected organochlorines in Baltic herring. Herring sample #
Sex
Age (yr)
Sampling site
Year of collection
'~-HCH +/-
U82 E~/E2
tr-CHL +/-
cs-CHL +/-
MC5 El/E2
MC7 EJE_,
H EP-exo +/-
OXY +/-
1 2 3 4 5 6 7 8 9
F M M M M F F F F
3 3 3 3 3 3 3 3 3
Utklippan Utklippan Utklippan Landsort Landsort Landsort Harufjarden Harufjarden Harufjarden
1993 1993 1993 1993 1993 1993 1993 1993 1993
0.79 n.d. n.d. 0.81 n.d. 0.81 0.83 n.d. n.d.
1.1 1.2 1.2 1.1 1.2 1.0 1.0 1.1 1.0
0.48 0.2(I 0.27 0.58 0.47 0.36 0.35 0.27 0.32
1.1 2.1 1.2 1.3 1.5 1.2 1.2 1.6 1.6
1.5 1.4 1.6 1.5 1.4 1.6 1.2 1.2 1.2
1.1 11.9 1.0 1.(I 1.(I (1.9 11.9 i i
0.58 0.56 0.51 (I.58 0.49 0.55 0.4(I 0.65 0.68
1.4 1.4 1.4 1.4 I. 1 1.5 1.4 1.4 1.5
M, male; F, female; yr, year; i, interference; n.d., not detected. For calculations and other abbreviations see Fig. 2.
o f t h e E R s f o u n d by B u s e r et al. (1992) fall in the E R r a n g e p r e s e n t e d in this study. T h e o n l y e x c e p t i o n was M C 7 , w h i c h B u s e r et al. (1992) f o u n d to b e slightly l o w e r ( E R = 0.83). T h e E R s f o u n d in t h e seal s a m p l e s r a n g e d f r o m v a l u e s close to r a c e m i c ratios to a l m o s t total d e p l e t i o n o f o n e e n a n t i o m e r . A s can b e s e e n f r o m T a b l e 2, m a n y s i m i l a r i t i e s exist b e t w e e n t h e a n a l y s e d s a m p l e s , but s t r o n g d e v i a t i o n s w e r e also f o u n d . H o w e v e r , d u e to t h e l i m i t e d n u m b e r o f s a m p l e s , only t e n t a t i v e c o n c l u s i o n s could be drawn. As expected, the ERs of the three
individuals, n o o b v i o u s d i f f e r e n c e s c o u l d b e f o u n d . T h i s i n d i c a t e s t h a t t h e E R v a r i a t i o n a m o n g Baltic h e r r i n g is small. N e i t h e r s a m p l i n g site w i t h i n t h e Baltic S e a n o r sex s e e m s to h a v e a s u b s t a n t i a l i n f l u e n c e o n t h e E R (Fig. 2a,b). B u s e r et al. (1992) e x a m i n e d t h e E R s in Baltic herring. Assuming a reversed enantiomer elution s e q u e n c e o f M C 5 o n t h e p e r m e t h y l a t e d 13-cyclodextrin ( P M C D ) - b a s e d c a p i l l a r y c o l u m n u s e d by B u s e r et al. (1992) c o m p a r e d to t h e tert.-butyldimethylsilyl 13-cyclodextrin ( B S C D ) c o l u m n u s e d in this study, t h e m a j o r i t y
TABLE 2
Data for sample characterization and ERs of selected organochlorines in blubber and liver of seals sampled along the Swedish coastline. Sample # Species, Sex l_x~cation, Year of collection
Age (yr) Health status Condition
1 GS, M Gavlebukten, 1989 2 GS, F Gotland, n.a. 3 GS, F Eggegrund, 1989 4 GS, F Eggegrund, 1989 5 GS, F IJ3vstabukten, 1989 6 RS, M Kalmarsund, n.a. 7 RS, F Ronnebyfjorden, 1993 8 HS, F Kalmarsund, 1983 9 HS, M Skiinekusten, n.a. 10 HS, M West Tj6rn, 1988
1 Healthy Not starved 38 Unhealthy Starved 0 Healthy Not starved 1 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved 0 Healthy Not starved
~-HCH +/-
Xi EI/Ez
U82 EJE2
tr-CHL +/-
cs-CHL +/-
MC5 El/E2
MC7 Et/E2
MC6 El/E2
HEP-exo +/-
OXY +/-
blubber liver
1.1 n.d.
i n.d.
4.7 2.4
0.71 0.91
1.8 0.26
i 3.5
i I).39
0.90 11.54
0.27 <11.5
1.4 11.97
blubber liver
1.7 3.6
0.76 0.94
0.77 0.62
0.52 0.58
i i
i i
11.20 0.15
11.12 n.d.
1.1 0.86
blubber liver
1.6 2.7
0.94 0.45
5.2 4.8
I).19 0.40
0.64 0.27
6.0 10
0.58 0.32
0.75 0.46
11.55 <11.5
1.4 0.78
blubber liver
2.2 n.d.
1.0 0.42
8.4 5.7
0.21 0.25
0.47 0.49
4.9 7.7
0.68 11.33
11,58 0,39
0.50 < 0.5
1.4 1.11
blubber liver
1.0 n.d.
0.92 0.37
5.1 2.6
0.74 0.94
0.48 0.24
3.7 6.3
0.98 0.41
0.66 11.44
0.25 <0.7
1.2 11.71
blubber liver
1.1 1.4
1.1 0.38
7.1 3.8
0.39 0.75
0.28 0.72
3.2 0.58
2.0 11.40
0.16 0.62
0.30 <0.7
11.92 11.56
blubber liver
1.0 n.d.
1.1 n.d.
4.1 1.5
0.21 0.22
2.9 4.6
2.2 2.1
0.30 0.08
0.25 0.13
0.41 < 11.7
1.3 i
blubber liver
2.0 n.d.
1.3 0.27
18 13
2.6 0.42
0.14 i
i i
i i
1.3 0.74
0.12 < 0.7
0.77 0.5 I
blubber liver
3.4 n.d.
1.3 0.41
22 21
3.1 11.56
0.21 i
4.1 19
1.9 i
1.3 1.1
11.11 <0.3
I).62 0.44
blubber liver
3.6 n.d.
0.94 n.d.
12 19
1.8 0.76
0.18 i
4.9 1.9
1.5 i
1.0 0.84
i i
1.0 i
19 15
GS, grey seal; RS, ringed seal; HS, harbour seal; M, male; F, female; n.a., not available. For calculations and other explanations see Table 1 and Fig. 2 348
Volume 36/Number 5/May 1998
juvenile female grey seals were similar, indicating that individuals of the same sex and species resemble each other in the ER profiles. No obvious influence of sampling sites or sex was observed. The fcmale harbour seal from Kalmarsund, Baltic Proper (seal #8) and the two male harbour seals from the coast of Sk~,ne, Baltic Proper (seal #9) and West Tj6rn, Skagerrak (seal #1(I) exhibited similar ER profiles (Fig. 3a). This is interesting since the chlordane body burden of juvenile harbour seals was found to be lower in the Skagerrak (sum of chlordancs 0.5(I-0.63 I-~gg ~) compared to the Baltic, Kalmarsund (1.5-1.8 l,tg g - ~) (Andersson and Wartanian, 1992, Table 1). In the case of ringed seal and grey seal some gender-specific differences were indicated, mostly due to the ERs of cis-chlordanc and MC5. Further studies are needed to find out whether these differences are statistically significant. The evidence of species-specific differences of the ER profiles was strong. In Fig. 3b the ERs in blubber from juvenile males can be seen (seal #1 vs 6 vs average 9-1(I). As shown, all species show distinct enantiomeric profiles and the most remarkable differ-
ences were the large deviations of the ERs of U82, heptachlor-exo-epoxide and zt-HCH in harbour seal, the strong dominance of the last cluting MC6-enantiomer in ringed seal and inverse ER of trans- and c/s-chlordane in harbour seal and grey seal respectivcly. As seen from Table 2, the corresponding juvenile females compare similarly (seal # 3 - 5 vs 7 vs 8), but here the ringed seal had inverse ER of cis-chlordanc and the at-HCH of harbour seal did not show a large deviation compared to the other two species. Explanations for these findings may be different feeding habits and variations in the enantioselectivity of uptake, cell membrane transport or degradation. Species-specific ER profiles of chlordane-related compounds have also been found by Buscr et al. (1992), Buser and Miiller (1992), Buser and Miiller (1993) and Oehme et al. (1994). Several authors have reported various ERs of zt-HCH in a number of species (Faller et al., 1991; Miiller et al., 1992; Pfaffenberger et al., 1992; M6ssncr et al., 1992; Tanabe et al., 1996). Tanabe et al. (1996) suggested that the ER differences
TABLE 3 Chromatographic separation conditions and instrumentation. Component 7-HCH
HEP-exo
Elution order
GC
LRMS
Matrix
+/-
Fison GC8000
Fison MD8()0, El, 70 eV, 250°C
Herring
+/
CP Chirasil DEX CB column (PMCD), 25 m × (I.25 ram, He 45 cm s hzlet: 250°C splitless I rain Oven: 160°C(6)-3°C rain 1-250°C(15) Fison Gcg()(I()
Fison MDg()(), El, 70 eV, 250°C
Herring
HP 5897. interface HP5988, ECNI, CH~, 0.5 torr, 200°C
Herring
tr-CHL, cs-CHL, MC5, MC7, U82, OXY
+/ +/ unknown +/
X], HEP-exo, OXY
unknown +/+/-
tr-CHL, cs-CHL, MC5, MC7, U82, MC6
tr-CHL cs-CHL, MC5, MC7, U82, MC6
+/+/-
unknown unknown
+/-/+
unknown unknown
10% BSCD/PS(}86, 18 m x (1.3 mm, He 45cms hlh't: 25(1°C, splitless 1 min Oven: 16(1°C(6)-3°C min 1-225°C(15) HP 589(I Tandem column: RT~2330+30% BSCD/PS(}86 (glass), 24 m x {).25 ram+ 18 m x (/.3 mm, He 35-45 cm s htlet: 251)°C, splitless 2 rain Oven: 60°C(2)-25°C min 1-185°C-3°C rain i_ 20(}°C(30) HP 5890 10¢4 BSCD/PS086 18m×0.3mm, He45ems i hth't: 25(1°C, on-column Oven: 811°C(3)-25°C rain 1-185°C(25)
HP 5890
Tandem column: RTx2330+ 10% BSCD/PS086 (glass), 25 m × 0.25 r a m + Ig m × (l.3 ram, He
Finnigan 4500, ECNI, CH4,
Seal
Seal
0.4 torr, 200°C
HP 5989, ECNI, CH4, ().5 torr, 200°C
Seal
35-45 cm s t 25 m ×0.25 ram+25 m ×0.25 ram, He or35-45 cm s Tandem column: RTx2330+ 10% BSCD/PS086
(fused silica), Oven: 60°C(2)-25°C rain 1_185oc_3oc rain - i_ 200°C(30) Inh't: 250°C, splitless 2 rain
PMCD, permethylated [~-cyclodextrin; BSCD, tert.-butyldimethylisilyl-13-cyclodextrin:
HP, Hewlett Packard. For other abbreviations see Fig. 2.
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Marine Pollution Bulletin
could be due to metabolization of the enantiomers by different enzymes and that the distortion of the ER might be due to unbalanced degradation processes. Furthermore, they suggested that one is less likely to find large deviation from the racemic ER in animals with low metabolic capacity such as molluscs and fish and this was illustrated by a number of ~-HCH ERs in various species (see Fig. 3 in Tanabe et al., 1996). The ERs for chlordane and a-HCH presented in this study support this hypothesis. The ER range is, in general,
smaller in herring (ER range > 0.2 to < 1.6) compared to seal (ER range >0.1 to 22). Further supportive data were reported by Buser et al. (1992) (see Table II therein), who found ER ranges for Me4, MC5, MC7, trans- and cis-chlordane from 0.38 to 1.35 in Baltic herring and Baltic salmon, whereas Baltic seal ranged from 0.24 to 2.7. Further, the data for U82 in Baltic seal, reported by Buser and Miiller (1993), showed considerable changes of the ERs compared to Baltic herring and Baltic salmon, which were close to
(a) 2.00
, ----T-r~ ,
t.00
~
J
.
0.50
0.33
[] Harufj~irden
0.25
[] Landsort [] Utklippan
0.20
a-HCH
U82
tr-CHL
cs-CHL
Me5
MC7
HEP-exo
OXY
(b) 2.00
1.00
T
= W
I
I
0.50
0.33
[] Males
0.25
[] Females 0.20
a-HCH
U82
tr-CHL
cs-CHL
MC5
MC7
HEP-exo
Fig. 2 ERs in Baltic herring, a-HCH = ~-HCH; U82, XI = octachloro chlordane congener of unknown structures; cs-, tr- and O X Y =cis-, trans- and oxy-chlordane; M e 5 , M C 7 = o c t a chloro chlordane congeners; M e 6 = nonachloro chlordane congener; HEP-exo = heptachlor-exo-epoxide; P M C D = permethylated I~-cyclodextrin; BSCD = tert.-butyldimethylisilylf3-cyclodextrin. For components with known elution order (HCH, tr-CHL, cs-CHL, OXY and HEP-exo) ER is calculated as the area ratio of (+)-/( - ) - e n a n t i o m e r , and otherwise as the area ratio of the first to second eluting enantiomer. (a) Average, high and low values of herring from various sampling sites in the Baltic sea. (b) Average, high and low values of males vs females.
350
OXY
Volume 36/Number 5/May 1998
26 T (a) [] Female HS, Baltic Sea: blubber #8
21 [] Male HS, Baltic Sea: blubber #9 16 [] Male HS, Skagerack: blubber #10
6.0
ii:t U82
21
xl
tr-CHL cs-CHL MC5
MC7
MC6
OXY
HEP- a-HCH exo
'(b) [ ] Male GS: b l u b b e r #1
[] Male RS: b l u b b e r #6
16
[] Male HS: average b l u b b e r #9-10 11
6.0 1.0
i
r'i~z4,
'"I_I
'
__,~
~:
0,17
0.09
U82
Xl
tr-CHL cs-CHL
MC5
MC7
MC6
OXY
H E P - a-HCH exo
11
(c)
9.0
O J u v e n i l e female GS: average b l u b b e r #3-5
7.0
[ ] J u v e n i l e female GS: average liver #3-5
S.O 3.0 1.0 0.33 0.20 0.14
t
U82
Xt
tr-CHL cs-CHL
MC5
MC7
MC6
OXY
a-HCH
Fig. 3 ERs in seals sampled along the Swedish coastline. For calculations and abbreviations see Fig. 2. (a) Female and male harbour seal blubber; various sampling sites (seal #8-10). (b) Male seal blubber; grey seal vs ringed seal vs harbour seal (seal #1 vs #6 vs average of #9-10). (c) Juvenile female grey seal; blubber vs liver (average, high and low values of seal #3-5 blubber vs liver).
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Marine Pollution Bulletin
racemic. The findings of smaller deviations from racemic values in fish and other animals with low metabolic capacity compared to their predators indicate that the influence of food is of minor importance. However, the assumption that enzymatic degradation is the most important process for changes in ER is contradictory to the quite different ER profiles in ringed seal and harbour seal. They both belong to the genus Phoca and there should not be much difference in their enzyme systems. in the liver, the metabolic capacity is higher than in other organs. Therefore, one would expect larger deviations from the racemic ER in the liver compared to the blubber. As can be seen from Table 2 and in Fig. 3c, this is the case for most of the compounds. The same trend was also observed by M6ssner et al. (1992), who reported significantly larger deviations for the ERs of a-HCH in fur seal liver than in blubber. Component U82 and trans-chlordane do not follow this trend, which indicates that the mechanisms leading to changes of ERs are not only attributed to enantioselective degradation, but also to uptake and/or transport across cell membranes. A change of the ER of a-HCH due to the transport across the 'blood-brain barrier' has been reported for harbour seals (M6ssner et al., 1992) and for double-crested cormorant (see Fig. 3 in Tanabe et al., 1996). Buser and Mi.iller (1994) have shown that the degradation of (+)- and (-)-tram-chlordane form (+)- and (-)-oxychlordane respectively. Therefore, the enantioselective formation of metabolites is an important process which change ERs in addition to the mechanisms mentioned above. Interestingly, in many samples (seal #1-3, 5, 6, 8-10) the ERs were inverse in the liver and the corresponding blubber extract. The reason for this could be that several enantioselective processes (degradation, uptake, cell membrane transport) are involved in the change of ER. The old and starved female grey seal (seal #2) suffered from a disease complex interpreted as hyperadrenocortisism, which includes uterine occlusions. Substantially increased ERs of U82, trans-ch[ordane, MC6 and heptachlor-exo-epoxide were observed in the blubber and liver samples of this female in comparison to the juvenile healthy grey seal females (seal #3-5). Further studies are under way to find out whether age, health and/or feeding status have an influence on the ERs. In summary, herring had, in general, similar a-HCH and chlordane ERs within the Baltic Sea and thus no spatial south-north trends could be indicated. In addition, no differences between male and female herring were observed. Results indicate that speciesspecific differences are important factors controlling the ER of Baltic seals, whereas the sampling site along the Swedish coastline seems to play a minor role. Further investigations are needed to find out whether indicated differences between individuals of different 352
sex, age and health status exist. Inverse ER in blubber and liver and a low deviation from the racemic ratio of some components in the liver indicate that not only enantioselective degradation but also the stereoselectivity of other processes such as enantioselective uptake, cell membrane transport and metabolite formation are responsible for the changes of ER in biota. We wish to thank Dr Terry Bidleman, Atmospheric Environment Service, Downsview, Canada, for kindly providing the (+)-enantiomer-enriched reference standards of ~-HCH, heptachlorexo-epoxide and oxychlordane. This work was supported by the Swedish Environmental Protection Agency 'Persistent Organic Pollutants' scientific program under the project number 30415A and by the Swiss Science Foundation under the project number 21 I)0-(/43399.95. Andersson, O. and Wartanian, A. Levels of polychlorinated camphenes (toxaphene), chlordane compounds and polybrominated diphenyl ethers in seals from Swedish waters. Amhio, 1992, 8, 550-552. Buser, H.-R. and M~ller, M. D. Enantiomer separation of chlordane components and metabolites using chiral high resolution gas chromatography and detection by mass spectrometric techniques. Analytical Chemistry, 1992, 64, 3168-3175, Buser, H.-R. and MiJller, M. D. Enantioselective dctermination of chlordane components, metabolites and photoconversion products in environmental samples using chiral high resolution gas chromatography and mass spectrometry. Environmental Science and Technology, 1993, 27, 1211-1220. Buser, H.-R. and Mfiller, M. D. Identification of the (+)- and (-)-enantiomers of chiral chlordane compounds using chiral highperformance liquid chromatography/chiroptical detection and chiral high resolution gas chromatography/mass spectrometry. Analytical Chemistry, 1994, 66, 2155-2162. Buser, H.-R., MiJller, M. D. and Rappe, C. Enantioselective determination of chlordane components using high resolution gas chromatography-mass spectrometry with application to environmental samples. Environmental Science and Technology, 1992, 26, 1533-1540. Dearth, M. A. and Hites, R, A. Complete analysis of technical chlordane using negative ionisation mass spectrometry. Environmental Science and Technology, 1991a, 25, 245-254. Dearth, M. A. and Hites, R. A. Chlordane accumulation in people. Environmental Science and Technology, 1991b, 25, 1279-1285. Failer, J., Hfihnerfuss, H., K6nig, W. A. and Ludwig, P. Gas chromatographic separation of the enantiomers of marine organic pollutants. Distribution of u-HCH enantiomers in the North Sea. Marine Pollution Bulletin, 1991, 22, 82-86. lwata, H., Tanabe, S., Sakai, N. and Tatsukawa, R. Distribution of persistent organochlorines in oceanic air and surface seawater and role of ocean on their global transport and fate. Environmental Science anti Technology, 1993, 27, 1080-1098. Li, Y.-F., Mcmillan, A. and Scholtz, M. T. Global HCH usage with l°x 1° longitude/latitude resolution. Environmental Science and Technology, 1996, 30, 3525-3533. Miyazaki, T., Yagmagishi, T. and Matsumoto, M. Isolation and structure elucidation of some components in technical grade chlordane. Archives of Environmental Contamination and Toxicology, 1985, 14, 475-483. M6ssner, S., Sparker, T. R., Becket, P. R. and Ballschmiter, K. Ratios of enantiomers of alpha-HCH and determination of alpha, beta- and gamma-HCH isomers in brain and other tissues of neonatal northern fur seals (Callorhinus ulwinus). Chemosphere, 1992, 24, 1171-1180. Miiller, M. D., Schlabach, M. and Oehme, M. Fast and precise determination of ~-hexachlorocyclohexane enantiomer in environmental samples using high-resolution gas chromatography. Environmental Science and Technolo,w, 1992, 26, 566-569. Nomeir, A. A. and Hajjar, N. P. Metabolism of chlordane in mammals. Reviews q]"Envil~mmental Contamination attd Toxicology, 1987, 100, 1-22. Oehme, M., Kallenborn, R., Wiberg, K. and Rappe, C. Simultaneous enantioselective separation of chlordanes, nonachlor compounds
Volume 36/Number 5/May 1998 and o,p'-DDT in environmental samples using tandem capillary columns. Journal q[ High Resohttion Chromatography, 1994, 17. 583-588. Oehme, M., Miiller, L. and Karlsson, H. (1997) High-resolution gas chromatographic test for the characterisation of enantioselective separations of organoehlorine compounds. Application to tert.butyldimethylsilyl 13-cyelodextrin. JotoTtal of" Chromatography, A, 775, 275-285 Olsson, M., Karlsson, B. and Ahnland, E. Diseases and environmental contaminants in seals from the Baltic and the Swedish west coast. The Science of the Total Em,iro/lment, 1994, 154, 217-227.
Pfaffenberger, B., H0hnerfuss, H., Kallenborn, R., G0nther, A. G., K6nig, W. A. and KrOner, G. Chromatographic separation of the enantiomers of marine pollutants. Part 6. Comparison of the enantioselective degradation of ~¢-hexachlorocyclohexane in marine biota and water. Chemosphere, 1992, 25, 719-725. Tanabe, S., Kumaran, P., lwata, H., Tatsukawa, R. and Miyazaki, N. Enantiomeric ratios of ~-hexachlorocyclohexane in blubber of small cetaceans. Marine Pollution Bulletin, 1996, 32, 27-31. Zook, D. R., Buser, H.-R., Bergqvist, P.-A., Rappe, C. and Olsson, M. Detection of tris(chlorophenyl)methane and tris(4chlorophenyl)methanol in ringed seal (Phoca hispMa) from the Baltic Sea. Ambio, 1992, 21,557-560.
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