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Journal of Toxicology and Environmental Health, Part A, 71: 1009–1018, 2008Copyright © Taylor & Francis Group, LLCISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287390801907558

1009

UTEHBiomarker Responses and Decreasing Contaminant Levels in Ringed Seals (Pusa hispida) from Svalbard, Norway

Decreasing Contaminant Levels in Ringed SealHans Wolkers1, Bjørn A. Krafft1, Bert van Bavel2, Lisa B. Helgason1, Christian Lydersen1, and Kit M. Kovacs1

1Norwegian Polar Institute, Tromsø, Norway, and 2MTM Research Center, Örebro University, Örebro, Sweden

Blubber was analyzed for a wide range of contaminants fromfive subadult and eight adult male ringed seals sampled in 2004,namely, for polychlorinated biphenyls (PCBs), hexachloroben-zene (HCB), toxaphenes, chlordanes, dichlorodiphenyldichloroet-hylene (DDE), and polybrominated diphenylethers (PBDEs).Contaminant levels were compared to previously sampled ani-mals from the same area, as well as data from literature for otherarctic wildlife species from a wide variety of locations. Ringedseals sampled in 2004 showed 50–90% lower levels of legacy con-taminants such as PCBs and chlorinated pesticides compared toanimals sampled in 1996 of similar age (14 subadults and 7 adultmales), indicating that the decline of chlorinated contaminantsobserved during the 1990s in a variety of arctic wildlife species iscontinuing into the 21st century. The results also indicated thatPBDE declined in ringed seals; levels in 2004 were about 70–80%lower than in animals sampled in 1998. This is one of the firstobservations of reduced exposure to these compounds and mightbe a first indication that restrictions of production and use ofthese contaminants have resulted in lower exposures in the Arctic.The PCB pattern shifted toward the less chlorinated (i.e., less per-sistent) PCBs, especially in adult ringed seals, possibly as a resultof reduced overall contaminant exposures and a consequentlylower cytochrome P-450 (CYP) induction, which results in aslower metabolism of less persistent PCBs. The overall effectwould be relative increases in the lower chlorinated PCBs and arelative decreases in the higher chlorinated PCB. Possibly due tolow exposure and consequent low induction levels, ethoxyresoru-fin O-deethylation (EROD) activity proved to be a poor biomarkerfor contaminant exposure in ringed seals in the present study. Theclose negative correlation (r2=70.9%)between EROD activity andpercent blubber indicates that CYP might respond to increasedbioavailability of the contaminant mixtures when they are mobi-lized from blubber during periods of reduced food intake.

The Arctic is chronically exposed to a wide variety of con-taminants, most of which originate from lower latitudes

(Pacyna & Oehme, 1988; AMAP, 1998). Monitoring levelsand effects of these pollutants in arctic biota is a political priorityof the circumpolar arctic nations, including Norway. Polychlo-rinated biphenyls (PCBs), chlorinated pesticides like DDT,chlordane and toxaphene, and polybrominated diphenyl ethers(PBDEs) are of special concern because of their persistence andbioaccumulative properties (AMAP, 1998, 2004). These com-pounds are listed by the Stockholm Convention and are includedin the global monitoring program (GMP) that will evaluate theeffectiveness of the convention (www.chem.unep.ch). TheArctic is dominated by the marine environment, and due to thelipophilic nature of these chemicals, they easily enter the foodchain through absorption by lipid-rich arctic plankton. Becausemany of these compounds are persistent they accumulatethrough the food chain, reaching their highest levels in arctictop or apex predators like seals, whales, and polar bears (Ursusmaritimus). In seals, more than 95% of the total amount ofaccumulated contaminants resides in their blubber layer(Stromberg et al., 1990). Besides thermo-insulation properties,blubber lipids also serve as an energy storage; blubber is mobi-lized during the breeding and molting periods as well as othertimes when the animals experience reduced food intake. Dur-ing lipid mobilization accumulated contaminants are releasedinto circulation and reach more sensitive tissues (Lydersen et al.,2002; Jørgensen et al., 2002). Consequently, both tissue con-taminant levels and the response of biomarkers are affected by thedegree of lipid mobilization (Lydersen et al., 2002; Jørgensenet al., 2002). These life-cycle-dependent fluctuations in con-taminant levels and bioavailability pose a considerable chal-lenge to studies dealing with monitoring of contaminant levelsand associated contaminant-induced biological effects.

The ringed seal (Pusa hispida) is an abundant, circumpolararctic species that occupies a central place in the arctic foodweb. Ringed seals feed mostly on pelagic fish and crustaceans.They in turn are preyed on predominantly by polar bears(Ursus maritimus) and constitute an important resource forindigenous people. Due to their high trophic position, this sealspecies is exposed to relatively high levels of halogenated

Received 26 September 2007; accepted 6 December 2007.Address correspondence to Hans Wolkers, Norwegian Polar

Institute, 9296 Tromsø, Norway. E-mail: Hans.Wolkers@npolar.no

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1010 H. WOLKERS ET AL.

organic pollutants. This in combination with their key positionin the arctic food web resulted in them being a species ofchoice for contaminant monitoring studies either to assess con-taminant exposure to arctic top predators (humans and polarbears) through ingestion of ringed seal tissues or to evaluatethe pollution status of the arctic marine environment more gen-erally (AMAP, 1998).

Contaminant exposure is associated with disturbances ofvarious physiological processes such as immunological andendocrine functions (Reijnders, 1986; Brouwer et al., 1989; deSwart et al., 1996; Ross et al., 1996: Troisi et al., 2001: Nealeet al., 2002, 2005a, 2005b). Enzymes of the hepatic cyto-chrome P-450 (CYP) enzyme system, which are involved inbiotransformation of xenobiotics, might also be affected; con-taminant exposure may increase CYP enzyme expression andhence these enzymes become induced and as such may be usedas biomarkers for exposure (Bainy et al., 1999; Wolkers et al.,1996, 1998, 1999a, 2000; Nyman et al., 2003). CYP inductionis also related to adverse effects via its linkage to the formationof biologically active metabolites. The use of these enzymes asfirst-warning indicators for contaminant exposure and contam-inant-induced biological effects may greatly improve monitor-ing efforts by making expensive contaminant analyses morefocused and cost-effective.

This article reports the state of contaminant accumulation inbreeding and molting male ringed seals from Svalbard. Thecomparison of PCBs, toxaphene, and PBDE levels and bodyburdens from samples obtained in the late 1990s (1996 and1998) from the same area (Wolkers et al., 1998, 2004) allowsfor the exploration of temporal trends of these contaminants inSvalbard. Levels and body burdens of a range of pesticideswere also measured in these animals and compared to previ-ously sampled ringed seals. In addition the use of CYP (i.e.,CYP1A activity) as a biomarker was evaluated.

METHODS

Field SamplingFive subadult and eight adult ringed seal males were shot in

Kongsfjorden, Svalbard, during May 2004 as part of a multi-disciplinary scientific research program. Methods for samplecollection and treatment were similar to reference animalssampled in 1996 (14 subadults of 2.4 ± 1.1 (average ± SD) yrold, range 1–4 yr) and 7 adult males of 13.1 ± 6.3 (average ±SD) yr old, range 6–24 yr) (Wolkers et al., 1998). Standardlength (to the nearest cm) and total body mass (to the nearest0.5 kg) were measured. Liver tissue was collected immediatelyafter shooting and frozen in liquid nitrogen. A canine tooth wasextracted for age determination. Aging was performed bycounting annual rings in the cement layer of decalcified stainedlongitudinal sections as described by Lydersen and Gjertz(1987). Animals ≤4 yr were considered sub-adults, whileanimals >4 yr were considered adults. Blubber thickness was

measured to the nearest mm at a point mid-dorsally approxi-mately 60% of the length of the animal from the cranial end,following Ryg et al. (1988). A 10-g blubber column, using thewhole blubber depth, was sampled from this location andstored at –20°C until contaminant analyses. All samples wereanalyzed individually.

ChemicalsAll chemicals were of analytical or specific pesticide grade

and commercially available from Sigma (St. Louis, MO) orMerck (Darmstad, Germany).

Chemical AnalysesPCBs (IUPAC numbers 28, 52, 74, 99/113, 101, 105, 118,

128, 138, 153, 156, 170/190, and 180), toxaphenes (parlar 26and 50), HCB, p,p-DDE, and chlordanes (cis- and trans-chlordane, cis- and trans-nonachlor, heptachlorepoxide, andoxychlordane), and PBDEs (congeners 28, 47, 66, 85, 99, 100,138, 153, 154, and 183) were measured. About 5 g of blubberwas homogenized in a mortar with sodium sulfate (1:5). About5 g of homogenized tissue was packed in a Suprex standardextraction column (10 ml). An internal standard consisting of amixture of 3 13C-labeled PBDE (BDE 47, 99, 153) and 15PCBs (PCB 28, 52, 70, 101, 105, 118, 138, 153, 156, 170, 180,194, 202, 206, and 209) was added before the solid-phaseextraction. On the top of the sample about 4.5 g basic alumi-num oxide (AlOx) was added as a fat retainer. The extractionswere carried out on a Suprex Autoprep/Accutrap SFE usingCO2 as the supercritical fluid. The chamber temperature was40°C and the pressure was 280 bar during extraction at a flowrate of 2 ml/min for 25 min. All analytes were trapped on aC18 solid sorbent (ODS, Octadecylsilica). The restrictor andtrap temperatures were kept at 45 and 40°C, respectively. Aftercompletion of the extraction the trap was rinsed with 3.5 mlhexane and 3.5 ml methylene chloride at a rate of 2 ml/min.The recovery standard, containing 13C-labeled PCBs 128 and178, was then added and the sample volume was reduced to 30 μltetradecane, producing an extract ready for gas chromatography/mass spectroscopy (GC/MS) analysis. Separate lipid deter-mination was performed by placing ~1 g of the homogenatein a small column and quantitatively extracting it with meth-ylene chloride and hexane (1:1). The weight of the extractedlipids was determined gravimetrically (Manirakiza et al.,2001).

Selected ion recording (SIR) HRGC/LRMS spectra wereacquired using an Agilent 6890 gas chromatograph coupled toa Agilent 5973 mass spectrometer. Chromatographic separa-tion was achieved by splitless injection of 2 μl on a nonpolarDB-5 column (30 m, 0.25 mm ID, 0.25 μm film thickness)using helium as the carrier gas. The GC oven was programmedas follows: 180°C initial hold for 2 min, increase at a rate of15°C/min to 205°C, followed by an increase of 3.7°C/min to300°C, with a final hold at 300°C for 15 min. For PCB and

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DECREASING CONTAMINANT LEVELS IN RINGED SEAL 1011

pesticide analysis the two most abundant ions of the molecu-lar ion cluster were monitored, in addition to masses for the13C-labeled internal standards using electron impact ionization.Quantification was done using a standard containing the inter-nal standard, the recovery standard, and a mixture containingthe different chlorinated pesticides and at least one PCB ateach chlorination level (single level calibration). PBDE andtoxaphene analysis were done using negative chemical ioniza-tion monitoring m/z 79/81 for the PBDEs and the most abun-dant masses of the most abundant fragment for toxaphene.Linearity within the concentration range was routinely checkedusing a five-point calibration curve.

The limit of detection (LOD) was calculated at a signal tonoise ratio of 3 (S/N > 3) and depended on the amount of lipidsextracted. A blank sample was run with each set of samples; noblank levels >10% of the levels measured in the samples wereobserved. Internal standard recoveries varied from 46 and139% depending on the sample and the compounds. In addi-tion, five reference samples (human adipose tissue) were ana-lyzed simultaneously during the project and showed that long-term performance of the method was very good. The laboratorytakes part in international quality assurance/quality control(QA/QC) studies on a regular basis, including a study withinAMAP with good results.

Biochemical AnalysesMicrosomal fractions were prepared from each liver sample

(Rutten et al., 1987) and stored in liquid nitrogen (–198°C) untilanalyses. Microsomal protein concentrations were determined bythe method of Lowry et al. (1951). All CYP-mediated enzymeactivity assays were performed at 37°C in an incubation buffer(50 mM potassium phosphate, pH 7.4) containing an NADPHregenerating system (MgCl2·6H2O 2.5 mM, glucose 6-phosphate5 mM, glucose-6-phosphate dehydrogenase 1 U/ml, and NADP0.5 mM). The rate of ethoxyresorufin O-deethylation (EROD),indicative for CYP1A activity, was determined fluorometrically(Burke & Mayer, 1974) in duplicate in 24-well plates. Measure-ments were performed using a Cytofluor 2300 multiwell fluores-cence plate reader (Millipore Intertech, Marlborough, MA). Theexcitation wavelength was set at 530 nm and the emission wave-length at 590 nm.

Statistical AnalysesFour groups of PCB congeners, with different resistances

toward metabolic breakdown, were distinguished based on thechlorine substitution pattern on the ortho (o), meta (m), andpara (p) positions (Bruhn et al., 1995). Group I (PCBs 153 and180), congeners with no vicinal H atoms in o,m or m,p posi-tions, are considered persistent. Group II (PCBs 52, 101), con-geners with vicinal m,p H atoms, are metabolized by CYP1Aand 2B/3A enzymes. Group IIIa (PCBs 28, 74, 105, 118, 156),congeners with vicinal H atoms at o,m positions, and a maxi-mum of one o-Cl, are metabolized by CYP1A. Group IIIb

(PCBs 99/113, 128, 138, 170/190), congeners with vicinal H-atoms at o,m positions, and two or more o-Cls, are consideredpersistent.

The total blubber (as a percent of body weight) was calcu-lated as described by Ryg et al. (1990) and the total blubbercontaminant body burden, expressed as micrograms contami-nant per kilogram body mass was calculated (Wolkers et al.,1998). Geometric means and 95% confidence intervals werecalculated for concentrations of individual contaminants aswell as contaminant groups.

Differences between adults and subadults as well as betweenanimals from 1996 and 2004 were assessed using Student’s t-testafter logarithmic (ln) transformation of individual data. Signifi-cance was preset at p < .05. Multiple regressions were carriedout for all animals, with contaminant concentrations as explana-tory variables, to assess the effect of contaminants on ERODactivity using R2.0.1 (http://www.r-project.org, Denmark, 2005),after ln transformation of the individual data. Age and percentfat were included as covariates. Regression analyses were alsoused to directly assess the relationship between CYP and percentof blubber.

RESULTS

Body MeasurementsThe adult male ringed seals were on average 11.3 ± 6.2 yr

old (range 6–22 yr), while the subadult males were 2.8 ± 1.1 yr ofage (range 2–4 yr). Adult males had significantly higher aver-age body mass than subadults (74.8 ± 11.8 vs. 49.6 ± 7.9 kg),while percent of total blubber did not differ markedly betweenadults (27.6 ± 4.4%) and subadults (31.7 ± 3.8%).

Contaminant Concentrations and Burdens 1: 2004 DataGeometric mean total PCB levels were 448 ng/g lipid (95%

CI 295–678 ng/g lipid) for adults and 198 ng/g lipid (95% CI:127–305 ng/g lipid) (Figure 1, a and b). Adults had signifi-cantly higher levels for all PCBs and PCB groups than sub-adults, except PCB 128 (Figure 1, a and b). PCB body burdensfor adults were significantly higher for PCB groups II and IIIaand total PCBs than for subadults (Figure 1c).

Pesticide levels were relatively low in all animals studied.HCB was ~6 ng/g lipid for both adults and subadults, whileΣ chlordanes was about 150 and 90 ng/g lipid for adults andsubadults respectively (Table 1). Toxaphene congener 26and 50 levels were below 5 ng/g lipid in both subadults andadults (Figure 1d). Pesticide levels did not differ signifi-cantly between adults and subadults, except for p,p-DDE,which showed significantly higher levels in adults (about300 ng/g lipid compared to 135 ng/g lipid) (Figure 1d andTable 1).

PBDE concentrations were generally low, with BDE conge-ners 66, 138, and 183 below detection. Generally, levels weresignificantly higher in adults than in subadults (Table 2);

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1012 H. WOLKERS ET AL.

FIG. 1. (a) Congener-specific mean PCB concentrations (ng/g lipid) and 95% confidence intervals in blubber of ringed seals from Svalbard sampled in 1996and 2004. Asterisk indicates significant differences between juvenile and adult seals sampled in 2004 at p < .05. (b) Concentrations of different metabolic groupsof PCBs (ng/g lipid) and 95% confidence intervals in blubber of ringed seals from Svalbard sampled in 1996 and 2004. Asterisk indicates significant differencesbetween juvenile and adult seals sampled in 2004 at p < .05. Group I: persistent congeners with no vicinal H atoms in o,m or m,p positions. Group II: congenerswith vicinal m,p H atoms and 0–3 o-Cls, considered to be metabolized by CYP 2B/3A and to a lesser extent CYP1A enzymes. Group IIIa: congeners with novicinal H atoms at m,p positions, and a maximum of 1 o-Cl, considered to be metabolized by CYP1A. Group IIIb: persistent congeners with no vicinal H atoms atm,p positions, and 2 or more o-Cls. (c) PCB body burdens (μg/kg body weight) and 95% confidence interval, according to metabolic group, in ringed seals fromSvalbard sampled in 1996 and 2004. Asterisk indicates significant differences between juvenile and adult seals sampled in 2004 at p < .05. Group I: persistentcongeners with no vicinal H atoms in o,m or m,p positions; group II: congeners with vicinal m,p H atoms and 0–3 o-Cls, considered to be metabolized by CYP 2B/3A and to a lesser extent CYP1A enzymes; group IIIa: congeners with no vicinal H atoms at m,p positions, and a maximum of 1 o-Cl, considered to bemetabolized by CYP1A; group IIIb: persistent congeners with no vicinal H atoms at m,p positions, and 2 or more o-Cls. (d) Congener-specific toxapheneconcentrations (ng/g lipid) in blubber of ringed seals from Svalbard, sampled in 1996 and 2004. Asterisk indicates significant differences between juvenile andadult seals sampled in 2004 at p < .05.

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180170/19015615313812811810510199/113745228

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*

153, 180153, 180 28, 74, 105, 118, 15628, 74, 105, 118, 156 99, 128, 138, 17099, 128, 138, 17052, 10152, 101

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DECREASING CONTAMINANT LEVELS IN RINGED SEAL 1013

however, BDE 47 was the predominant PBDE in both adultsand subadults and made up about 85% of all PBDEs measured.

Contaminant Concentrations and Burdens 2: Temporal TrendsPCBs

Substantially lower concentrations of all PCB congenersmeasured were found for both subadult and adult animals sampled

in 2004 as compared to those sampled in 1996 (Wolkers et al.,1998) (Figure 1, a and b), with the largest differences beingrecorded in adult animals. Subadults sampled in 2004 had ~3-fold lower levels of the different PCB groups compared to sub-adults sampled in 1996 (Figure 1b). In adult animals, the morepersistent PCB from groups I and IIIb showed 6- and 4.4-foldlower levels, respectively, in 2004 as compared to 1996, whilegroup IIIa was less than 3-fold lower in the 2004 as comparedto the 1996 adults (Figure 1b). The decrease of the persistent

FIG. 1. (Continued)

28, 74, 105, 118, 15652, 101153, 180

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153, 180 52, 101 28, 74, 105, 118, 156 99, 128, 138, 17099, 128, 138, 170

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1014 H. WOLKERS ET AL.

PCBs (group I and IIIb) was also less in subadults than inadults (Figure 1b). In addition, the PCB pattern, expressed asthe percent of an individual PCB to total PCBs, changedbetween 1996 and 2004, particularly in adults. Generally, non-persistent PCB increased, with the most pronounced change forPCB 28, which rose from 0.5 to 1.4%; PCB 105 increased from1.9 to 4.1%, and PCB 118 rose from 4.7 to 10.1% of ΣPCBs.The persistent PCB decreased: PCB 128 from 1.1 to 0.5%;PCB 153 from 34.9 to 26.7%; PCB 170/190 from 3.4 to 2.9%;and PCB 180 from 9.7 to 7.5% of ΣPCB.

Contaminant body burdens also showed a marked differencebetween animals sampled in 1996 and 2004 (Figure 1c). Burdensfor the different PCB groups in subadults were 3- to 4-fold

lower in 2004 animals as compared to 1996 animals. Similar tothe trends for concentrations, the adults showed an even morepronounced difference in body burden between the 1996 and2004 animals than for the subadults. PCB from groups I, II,and IIIb showed 6-fold lower burdens in the 2004 adult ani-mals as compared to a 3- to 4-fold lower burden for PCB fromgroup IIIa, while subadults showed ~3- to 4-fold lower burdensfor all PCB groups (Figure 1c)

Pesticides and PBDESimilar to PCB, toxaphene congeners 26 and 50 also

showed a marked difference between 1996 and 2004 animals,

TABLE 1 Geometric Mean Concentrations of Chlorinated Pesticides in Blubber of Male Ringed Seals From Svalbard

HCB p,p-DDE cis-chl trans-chl cis-nona trans-nona hepta oxy Σ chl

Subadult malesGM 5.4 135.4 4.8 2.2 1.8 41.0 1.5 41.8 93.595% low 4.2 104.1 4.0 1.7 1.3 28.9 1.0 29.4 65.895% high 7.0 176.2 5.7 2.8 2.5 58.2 2.4 59.3 132.7

Adult malesGM 7.1 300* 6.3 2.9 2.0 57.5 2.7 75.0 14895% low 6.2 185 4.8 2.0 1.3 40.7 1.8 53.0 10495% high 8.2 487 8.3 4.1 3.1 81.3 4.0 106 209

Malesa

GM 22.0 1040.0 — — — 173.0 — — —

Note. HCB = hexachlorobenzene; DDE = dichlorodiphenyldichloroethylene; chl = chlordane; nona = nonachlor; hepta = heptachlor epoxide;oxy = oxychlordane; Σ chl = total chlordane. Asterisk indicates significant difference between subadults and adults at p < .05.

aData (adults and subadults sampled in 1995 in Svalbard) from Muir et al. (2000).

TABLE 2 Geometric Mean Concentrations of Selected Polybrominated Diphenyl Ethers in Blubber of Male Ringed Seals From Svalbard

BDE 28 BDE 47 BDE 85 BDE 99 BDE 100 BDE 153 BDE 154 Σ BDE

SubadultsGM 0.43 6.0 0.05 0.16 0.30 0.04 0.09 7.195% low 0.30 4.6 0.04 0.10 0.23 0.03 0.07 6.095% high 0.60 7.8 0.07 0.28 0.39 0.04 0.12 8.5

AdultsGM 0.81* 13.7* 0.12* 0.25 0.71* 0.13* 0.26* 16.1*95% low 0.61 11.2 0.09 0.17 0.50 0.09 0.15 12.295% high 1.1 16.9 0.17 0.38 1.0 0.20 0.45 21.3

Subadultsa

GM 0.13 16.8 nd 0.66 0.39 — nd 18.395% low 0.10 12.1 — 0.42 0.27 — — 14.195% high 0.15 23.3 — 1.04 0.58 — — 23.7

Note. BDE = brominated diphenyl ether. Asterisk indicates significant difference between subadults and adults at p < .05.aData (subadults sampled in 1998 in Svalbard) from Wolkers et al. (2004).

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DECREASING CONTAMINANT LEVELS IN RINGED SEAL 1015

with levels in subadults and adults from 2004 more than 2-foldlower compared to those in 1996 (Figure 1d). Other pesticidesas well as PBDE concentrations showed marked differencesbetween animals sampled in Svalbard in 2004 and animalssampled in 1995 (Muir et al., 2000) and 1998 (Wolkers et al.,2004) (Tables 1 and 2). Levels of HCB and trans-nonachlorfell by about 75%, while DDE declined by almost 90% (Table 1).PBDEs were also ~70–80% lower in 2004 as compared to1998 (Table 2).

CYP Activities in Relation to Contaminants and Blubber Content

Average EROD activities were 102.7 ± 68.3 pmol/min/mgprotein in adults and 59.8 ± 46.9 pmol/min/mg protein insubadults, but the difference was not statistically significant.Linear multiple regression analyses did not reveal any signifi-cant relationships between EROD and contaminants, althoughthere was a significant negative correlation (r2=70.9%)between EROD and percent of blubber (i.e., the condition ofthe animal) that could be established (Figure 2).

DISCUSSIONRinged seals may be considered model candidates for moni-

toring contaminant accumulation and contaminant-inducedbiological effects because of their high abundance and circum-polar distribution as well as their central place in the arctic eco-system. They are the dominant species in the diet of polar bearsthroughout the Arctic, and also play an important part of Inuitdiets in many coastal communities, linking them directly tohuman health issues. In contrast to the polar bear’s extremelyefficient metabolism of many contaminants, the ringed seal’smoderate ability to metabolize contaminants results in moredifferent compounds in higher concentrations accumulating in

their tissues, making them more suitable for monitoring pur-poses (Wolkers et al., 2004). The advantage of using maleseals in monitoring is that, unlike females, they don’t excretepart of their contaminant burden in the milk, and as such, malesreflect the total exposure over their entire life span.

Information on temporal trends in contaminant concentra-tions is relatively scarce for the European Arctic during the lastdecade. To our knowledge, this is the first study reporting on adeclining PBDE trend in marine mammals in the Arctic. Lev-els declined by about 70% between 1998 and 2004. Thepresent study also showed a substantial decline in PCBs, and awide variety of chlorinated pesticides between 1996 and 2004in ringed seal from Svalbard. The animals from 1996 were ofsimilar age, sampled in the same season using similar samplingmethods.

The higher PCB and PBDE concentrations in adult thansubadult ringed seals found in this study is in agreement withprevious studies (Cameron et al., 1997; Wolkers et al., 1998;AMAP, 2004) and can be explained simply by a time-associatedincrease in contaminant exposure and accumulation thatexceeds biotransformation capacity. In contrast, pesticides didnot show an age-related effect, suggesting that most pesticidesare not as persistent and bioaccumulative as PCB and PBDE inseals, and are biotransformed. The relatively nonpersistentnature of many pesticides in seals was supported by resultsfrom a previous study on harp seals (Phoca groenlandica),where the persistence of pesticides, expressed as biomagnifica-tions factor and metabolic index (Bruhn et al., 1995), was sub-stantially lower than that of the PCB (Wolkers et al., 1999b).

Declines in PCBs and chlorinated pesticides have beenobserved in a variety of wildlife species in both the Canadianand the European Arctic over the last decades (AMAP, 2004).The results from the present study indicate a continuing declinein PCBs and pesticides between 1996 and 2004 in ringed sealmales (50–90%). Since the early 1970s PCBs and DDT have

FIG. 2. Relationship between EROD (pmol/min/mg protein) and percent of blubber in male ringed seals from Svalbard.

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declined in the Western Canadian Arctic (AMAP, 1998, 2004).Between 1975 and 2000 ringed seals from this area showed a2- to 5-fold decline for PCB and DDT, reflecting lower levelsin source regions (Muir et al., 1988, 1999; Muir, 1996; Weis &Muir, 1997: Addison & Smith, 1998; Hickie et al., 2005). Similarto ringed seals, polar bears from the Canadian Arctic showed adecline in PCB levels through the 1990s; however, in contrastto the ringed seals from the present study, chlordanes and DDElevels remained largely unchanged, possibly due to a continu-ous input of these compounds into the Canadian Arctic(Norstrom, 2001). In fact, in some areas within the CanadianArctic chlordane levels actually increased slightly between1990 and 2001 (AMAP, 2004). For example, beluga whales(Delphinapterus leucas) from the Canadian Arctic showed aclear decline in PCB, while they displayed a slight increase inchlordane concentrations between 1982 and 1997. No changein DDT or toxaphenes was observed in these animals (Stern,1999; Stern & Addison, 1999).

Similar to the Canadian Arctic, declining contaminant lev-els have been observed in Greenland as well as the EuropeanArctic over the past decades. Similar to the present ringed sealstudy, Dietz et al. (2004) found a significant reduction in PCBsas well as chlorinated pesticides between 1990 and 2000 inpolar bears from Eastern Greenland; PCBs and chlordanesdeclined by 75 and 31–75%, respectively. Henriksen et al.(2001) observed decreasing levels of PCB 153 in polar bearsfrom Svalbard during the 1990–1998 period. A similar declinewas observed in harp seal pups from the White Sea; i.e., PCBs,DDT, HCB, and HCH declined 50–70%, between 1992 and1998 (Muir et al., 2001). However, after 1998 the levelsseemed to stabilize in bears (Henriksen et al., 2001; AMAP,2004), possibly due to a steady state between exposure andexcretion. Nevertheless, decreasing contaminant levels inringed seals from the present study indicate that PCBs contin-ued to decline after 1998. During an 8-yr time span, levels ofPCB as well as pesticides dropped by as much as 50–80% inboth subadults and adults.

The overall PCB pattern showed a shift toward the lowerchlorinated PCBs in adult ringed seals from 1996 to 2004. Asimilar shift, where the fraction of more highly chlorinated,more persistent PCBs, declined, while the less chlorinatedPCBs increased relative to total PCBs, was also observed inpolar bears from the Canadian Arctic throughout the 1990s(Norstrom, 2001). Such changes in PCB patterns are mostlikely due to interactions between environmental contaminantdynamics and toxicokinetics in the animals, such as changes incontaminant exposure and contaminant metabolism in the ani-mal. However, this shift toward the lower chlorinated PCBseems counterintuitive: reduced exposure and continued con-taminant metabolism might result in a selective decline of lesspersistent, generally less chlorinated, PCBs in the body. How-ever, data suggest the opposite, i.e., relatively higher presenceof the less chlorinated PCBs. This might be the result of thelower overall exposure in the animals, resulting in a lower

CYP induction and a consequent slower metabolism of lesspersistent PCBs. The overall effect of this reduced biotransfor-mation would be a relative increase in the lower chlorinated,less persistent PCBs, as observed in the present study.

In addition to legacy contaminants like PCBs and chlori-nated pesticides, the results from the present study indicatethat PBDEs are also declining. Ringed seals from Svalbardsampled in 1998 showed about 3-fold higher levels (Wolkerset al., 2004) than in the animals from the current study. Tem-poral trend data for PBDE in the Arctic are relatively scarce.Previous publications reported substantial increases of thesecompounds between the early 1980s and the mid 1990s inbeluga whales from the Canadian Arctic (Stern & Ikonomou,2000, 2001) and a 9-fold increase between the early 1980sand 2000 in Canadian ringed seals (Ikonomou et al., 2002).PBDE 47 in ringed seals in eastern Greenland, sampled in2001, was still about 3- to 4-fold higher compared to theseals from the present study (Vorkamp et al., 2004). Also,PBDE levels in Californian harbor seals are still increasing(Neale et al., 2005). The observed PBDE decline in ringedseals from the present study is most likely due to strict regu-lations concerning production and use of these compounds inEurope. Recently, Europe has taken initiatives to phase outthe use of all PBDEs. In August 2004 two commercial mix-tures of PBDEs, penta- and octa-BDE, were banned (Cox &Efthymiou, 2003), while a full ban of deca-BDE is consid-ered by 2008. The decline of these compounds in ringedseals from Svalbard, before the official ban in 2004, mightbe explained by voluntarily phasing out some of thesecompounds by the chemical industries in anticipation of theofficial ban.

CYP 1A activity, measured as EROD activity, has beenwidely used a marker of contaminant exposure (Letcher et al.,1996; Wolkers et al., 1998; Bainy et al., 1999; Nyman, 2003).Correlation between EROD activity and PCB and DDTconcen-trations could be as high as 60% in polluted animals (Nymanet al., 2003). However, correlations between CYP and contam-inants do not necessarily indicate a causal relationship, andcompounds co-accumulating may actually produce theobserved induction. In addition, differences in contaminantbioavailability due to physiological factors, such as changes inlipid cycles, may lead to different results when relating con-taminants to biomarkers such as CYP. In other words, thedetection of biomarker responses as well as contaminant-induced biological effects may vary with changes in contami-nant bioavailability related to lipid dynamics. Ringed sealsbreed and molt from approximately April to June, and foodintake is dramatically reduced, particularly in males, whileblubber is mobilized and metabolized to cover energydemands. During this period, animals will exhibit varyingdegrees of lipid mobilization, resulting in varying degrees inincrease in contaminant concentrations in their tissues as wellas elevated contaminant bioavailability (Lydersen et al., 2002,Jørgensen et al., 2002). This might result in biomarker

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responses of varying degrees, for example, from cytochromeP-450 (CYP) enzyme activities (Jørgensen et al., 2002). Themale ringed seals from the present study were in a state of neg-ative energy balance due to breeding and molting. During thisperiod, they are probably most sensitive to the effects of accu-mulated contaminants. Thus, the absence of a direct relation-ship between contaminants and EROD was somewhatsurprising. Nyman et al. (2003) found a close relationshipbetween contaminants and EROD activity in seals from theBaltic. In the current study EROD activity was related to con-taminants accumulated in blubber. Although blubber pollutantsreflect the overall exposure over time, the circulating levels inthe seals during sampling may simply have been too low toelicit an EROD response. The seals from the Nyman et al.(2003) study were heavily polluted and possibly above a cer-tain threshold for a clear EROD response. In addition, it is pos-sible that some key compounds might have been lacking in thechemical analyses and the correlation calculations in our study.This possibility was supported by the negative associationbetween the percent of total blubber and EROD, suggesting aCYP induction possibly associated with the mobilization andconsequent increased bioavailability of the accumulated con-taminant mixture, including compounds that were not includedin the chemical analyses.

CONCLUSIONThe current study showed that between 1996 and 2004

levels of legacy contaminants, like PCBs and chlorinatedpesticides, decreased by 50–80% in ringed seals from Sval-bard. The PCB pattern in males shifted to the less persistentPCBs, possibly due to a reduced exposure and consequentreduction in contaminant metabolism, resulting in anincreased relative presence of the less chlorinated PCBs. Inaddition, most likely due to strict regulations concerningPBDEs, these compounds showed a decrease of about 70%,which is one of the first documented cases of decreasingPBDEs in the Arctic. Although EROD was a poor biomarkerfor blubber contaminants in the ringed seals in the presentstudy, possibly due to low exposure and consequent lowinduction levels, the association between EROD and percentof blubber suggests that it may respond to increased bio-availability of the contaminant mixture released into bloodwhen blubber is mobilized.

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