Tissue-specific congener composition of organohalogen and metabolite contaminants in East Greenland polar bears (Ursus maritimus)

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Tissue-specific congener composition of organohalogen and metabolitecontaminants in East Greenland polar bears (Ursus maritimus)

Wouter A. Gebbink a,b, Christian Sonne c, Rune Dietz c, Maja Kirkegaard c, Frank F. Riget c,Erik W. Born d, Derek C.G. Muir e, Robert J. Letcher a,b,*

a National Wildlife Research Centre, Science and Technology Branch, Environment Canada, Carleton University, Ottawa, Ontario K1S 5B6, Canadab Department of Chemistry, Carleton University, Ottawa, Ontario K1S 5B6, Canada

c Department of Arctic Environment, National Environmental Research Institute, University of Aarhus, Frederiksborgvej 399, DK-4000 Roskilde, Denmarkd Greenland Institute of Natural Resources, P.O. Box 570, DK-3900 Nuuk, Greenland, Denmark

e Water Science and Technology Directorate, Environment Canada, Burlington, Ontario L7R 4A6, Canada

Received 20 February 2007; received in revised form 29 June 2007; accepted 3 July 2007

Tissues-specific (adipose tissue, liver, brain and blood) differences exist for the congener patterns of PCBs, PBDEsand their metabolites/degradation products in East Greenland polar bears.

Abstract

Congener patterns of the major organohalogen contaminant classes of PCBs, PBDEs and their metabolites and/or by-products (OH-PCBs,MeSO2-PCBs, OH-PBDEs and MeO-PBDEs) were examined in adipose tissue, liver, brain and blood of East Greenland polar bears (Ursus mar-itimus). PCB, OH-PCB, MeSO2-PCB and PBDE congener patterns showed significant differences ( p� 0.05) mainly in the liver and the brainrelative to the adipose tissue and the blood. OH-PBDEs and MeO-PBDEs were not detected in the brain and liver, but had different patterns inblood versus the adipose tissue. Novel OH-polybrominated biphenyls (OH-PBBs), one tri- and two tetra-brominated OH-PBBs were detected inall tissues and blood. Congener pattern differences among tissues and blood are likely due to a combination of factors, e.g., biotransformationand retention in the liver, retention in the blood and bloodebrain barrier transport. Our findings suggest that different congener pattern exposuresto these classes of contaminants should be considered with respect to potential target tissue-specific effects in East Greenland polar bears.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Polar bear; Brominated and chlorinated contaminants; Metabolites; Tissue-specific congener composition; East Greenland

1. Introduction

Polar bears (Ursus maritimus) are top predators in the Arcticmarine food web, and adipose tissue contains some of the high-est levels of persistent organohalogen pollutants (POPs) amongArctic species (e.g. Braune et al., 2005; De Wit et al., 2006).These POPs include polychlorinated biphenyls (PCBs),methylsulfone-PCBs (MeSO2-PCBs), various organochlorine

(OC) pesticides including, e.g., DDTs and chlordanes, and con-taminants of recent significance in the Arctic such as polybro-minated diphenyl ether (PBDE) and hexabromocyclododecane(HBCD) flame retardants (Muir et al., 2006; Verreault et al.,2005a). In particular, polar bears from East Greenland havebeen documented to accumulate some of the highest levels ofthese chlorinated and brominated POPs in the adipose and livertissue relative to animals from other circumpolar populations(Bossi et al., 2005; Dietz et al., 2004; Muir et al., 2006; Sandalaet al., 2004; Verreault et al., 2005a).

Monitoring of lipophilic and bioaccumulative POPs inpolar bears has mainly been conducted on adipose tissue andto a lesser extent on whole blood or plasma (Bernhoft et al.,

* Corresponding author. National Wildlife Research Centre, Science and

Technology Branch, Environment Canada, Carleton University, Ottawa,

Ontario K1S 5B6, Canada. Tel.: þ1 613 998 6696; fax: þ1 613 998 0458.

E-mail address: [email protected] (R.J. Letcher).

0269-7491/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.envpol.2007.07.001

Available online at www.sciencedirect.com

Environmental Pollution 152 (2008) 621e629www.elsevier.com/locate/envpol

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1997; Dietz et al., 2004; Muir et al., 2006; Norstrom et al.,1998; Sandau et al., 2000; Sandala et al., 2004; Verreaultet al., 2005a,b, 2006). BDE-47 and -153 were found to bethe dominant congeners in the PBDE pattern in polar bear’sadipose tissue (Dietz et al., 2006; Muir et al., 2006; Sørmoet al., 2006), BDE-47 and -99 were most abundant in wholeblood (Verreault et al., 2005b) while only BDE-47 was de-tected in polar bear’s liver (Kannan et al., 2005). Regardless,limited or no information is available on the congener compo-sition among high lipid content, target tissues such as liver andbrain. Liver and brain tissues are characterized by uniquebiochemical processes that selectively influence the tissue-specific toxicokinetics (levels and congener patterns) of con-taminants. In the liver, enzyme-mediated biotransformationinfluences the fate and molecular form of a compound. Inthe brain the protective bloodebrain barrier (BBB) limits thepassage of foreign chemicals into vital brain areas. For hydro-phobic contaminants (log Kow� 7.5), bioaccumulation amongdifferent tissues is influenced by toxicokinetic factors such asdietary uptake (bioavailability) and lymphatic transport to theliver prior to systemic circulation. Furthermore, the brainblood perfusion is low which, combined with the high affinityof hydrophobic compounds, may result in slower uptake ki-netics for the brain fat compartment (Kelly et al., 2004). Thegeneral dietary uptake route provides a first-pass metabolicclearance in the liver, while slow uptake by the adipose com-partment increases the length of time that a compound remainsin the bloodstream, allowing the liver (and other metabolizingtissues) to influence bioaccumulation.

Recent studies on polar bears, limited to adipose tissue andblood, have revealed a number of novel, persistent and meta-bolically associated organohalogen contaminants. One suchclass is broadly defined as halogenated phenolic contaminants(HPCs) and is comprised by a growing number of chlorinatedand brominated substances. Information is lacking about thepresence, accumulation and congener patterns of HPCs amonghigh lipid tissues in polar bears. For example, Sandala et al.(2004) reported OH-PCBs (at levels exceeding PCBs), 4-OH-HpCS (4-hydroxy-heptachlorsytrene) and PCP (penta-chlorophenol) in blood of East Greenland polar bears, andshowed that 4-OH-CB146, 4-OH-CB187, 4-OH-CB193 and4,40-diOH-CB202 dominated the OH-PCB congener pattern.OH-PCBs are metabolic products of PCB biotransformation(Letcher et al., 2000). Although OH-PCBs have not been re-ported to accumulate in adipose tissue of polar bears or anyother wildlife species, OH-PCBs have been measured inSwedish human adipose tissue at low levels relative to blood(Guvenius et al., 2002). Verreault et al. (2005b) reported sev-eral OH-PBDE congeners at low to non-detectable concentra-tions in blood plasma of Svalbard polar bears. OH-PBDEs,possible metabolites of PBDEs and related MeO-PBDEswere detected at low concentrations in Svalbard polar bearplasma, with 40-OH-BDE49 and 6-MeO-BDE47 as major con-geners. The high levels of chlorinated phenolics in polar bearblood appear to be related to retention by competitive bindingto TTR (transthyretin) which is a thyroid hormone transportprotein (Palha, 2002). Several OH-PCBs and 4-OH-HpCS

have up to 6.6 times higher affinity for the natural TTR ligand,thyroxine (T4) (Malmberg, 2004; Sandau et al., 2000). A classof PCB metabolites, the MeSO2-PCBs, has been reported inthe adipose tissue of circumpolar polar bears, including thosefrom East Greenland (Sandala et al., 2004; Verreault et al.,2005a). The bioaccumulation pattern of MeSO2-PCBs (and3-MeSO2-p,p0-DDE) is similar to that of PCBs (Letcheret al., 1998). Letcher et al. (1995) and Sandala et al. (2004)demonstrated that 40-MeSO2-CB87, 30-MeSO2-CB101 and40-MeSO2-CB101 were the dominant congeners in polar bearadipose tissue and blood plasma.

Little attention has been given to brominated and chlori-nated chemical residues in polar bears, and especially thestudy of physicalechemical properties of these contaminants,which can influence toxicokinetics and thereby tissue-specificcomposition of accumulated congeners. Depending on physi-calechemical properties, which are a function of moleculargroup substitutions and structure, the congener-specific con-taminant toxicokinetics can be altered due to, e.g., the typeof halogenation and particularly the presence or absence of ad-ditional phenyl groups (e.g., MeO and OH groups). Such fun-damental characteristics of organohalogens will influence theaffinity to specific tissues and macromolecules (Hakk andLetcher, 2003; Letcher et al., 2000). The objective of the pres-ent study is to identify and examine the tissue-specific conge-ner patterns of different chlorinated and brominated POPs andtheir by-products in the liver, adipose tissue, blood and brainof East Greenland polar bears.

2. Experimental section

2.1. Sample collection and age estimation

Adipose, brain and liver tissue, and whole blood samples were collected

from adult male (n¼ 10) and female (n¼ 10) polar bears in the Ittoqqortoor-

miit/Scoresby Sound area in central East Greenland between 69�000N and

74�000N in 1999e2001 (Dietz et al., 2004, 2006; Sandala et al., 2004). Brain

sub-samples (ca. 5 g) were taken from medulla oblongata (brain stem) and

pons via the skull foramen magnum and wrapped in aluminum foil. Liver sam-

ples were collected randomly from the right lobes and frozen in polyethylene

(PE) plastic bags. Further liver sub-sampling (ca. 5 g) was done in the labora-

tory where the sample core was cut clean and wrapped in aluminum foil.

Whole blood samples were transferred into vacutainers (lithium heparin,

Greiner�). All sample were taken <1 h post mortem and kept frozen at tem-

peratures of �5 to �20 �C prior to sample extraction. The ages were estimated

at 5.5 to 25 years by counting the annual growth layers in the cement of an I3

tooth after decalcification, thin sectioning (14 mm) and staining with toluidine

blue (Dietz et al., 2004; Sandala et al., 2004).

2.2. Contaminant extraction

The extraction and clean up of the adipose, brain, liver tissue and blood for

PCBs, PBDEs and MeSO2- and OH-containing compounds have been previ-

ously described in detail, although modifications were necessary for halogenated

phenolic compounds in adipose and brain tissues (Chu et al., 2003; Dietz et al.,

2004; McKinney et al., 2006; Muir et al., 2006; Sandala et al., 2004; Verreault

et al., 2005a,b). Briefly, approximately 0.5 g of adipose tissue was homogenized

with Na2SO4, spiked with the internal standards [six 13C-labeled PCBs (CB-28, -

52, -118, -153, -180 and -194), two PBDEs (BDE-30 and -71), 3-MeSO2-2-CH3-

2030405050-pentachlorobiphenyl, four 13C-labeled OH-PCBs (40-OH-CB120,

40-OH-CB159, 40-OH-CB172, 40-OH-CB187) and 20-OH-BDE28] and extracted

622 W.A. Gebbink et al. / Environmental Pollution 152 (2008) 621e629

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with DCM/n-hexanes (1:1). Percent lipid was determined gravimetrically, which

was same for liver and brain samples. Lipids were degraded/removed by the ad-

dition of concentrated H2SO4, and protonated MeSO2-PCBs/-DDEs (in H2SO4)

were separated from all other neutral contaminants (in organic phase). Bulk lipid

removal could not be effectively accomplished by gel permeation chromatogra-

phy (GPC) as all HPCs were poorly recovered. This is in contrast to numerous

previous reports that incorporated GPC in the isolation of less polar and non-

OH-containing organochlorines such as PCBs, but did not include HPCs (Dietz

et al., 2004; McKinney et al., 2006; Muir et al., 2006; Sandala et al., 2004;

Verreault et al., 2005a,b). The aqueous acid phase was diluted with H2O (1:1)

and the MeSO2-PCBs were extracted with n-hexanes and eluted through a basic

silica column (2 g, 33% 1 M KOH by w/w) with 20 ml DCM. The neutral con-

taminants were separated from HPCs by aqueous KOH partitioning and com-

bined with the MeSO2-PCB fraction before being eluted through a Florisil

column (8.0 g, 1.2% H2O deactivated by w/w). Fraction 1, containing PCBs,

PBDEs and MeO-PBDEs, was concentrated and solvent exchanged to 1 ml

TMP in preparation for GCeMSD analysis. Fraction 2, containing MeSO2-

PCBs, was concentrated to w0.5 ml and loaded on a basic alumina column

(3 g, 2.3% H2O deactivated by w/w) and eluted with 50 ml DCM/n-hexanes

(1:1) in preparation for GCeMSD analysis. The aqueous KOH fraction, contain-

ing HPCs, was then acidified with concentrated H2SO4 to wpH 2. The reproto-

nated HPCs were extracted from the acidified fraction with MtBE/n-hexanes and

derivatized to their methoxy analogues using diazomethane. The methoxylated

HPCs were purified on a silica column (3 g, 22% H2SO4 deactivated by w/w) and

eluted with 15% DCM in n-hexanes. The extract, containing OH-PCBs, OH-

PBDEs and possibly as yet unidentified HPCs (methylated to their MeO-

analogues), was concentrated 100 ml in preparation for GCeMSD analysis.

Approximately 2 g of brain tissue was homogenized with Na2SO4, trans-

ferred into a pre-cleaned extraction thimble and spiked with the internal stan-

dards and Soxhlet extracted for 4 h with 150 ml acetone/n-hexanes (1:1).

HPCs and neutrals were cleaned up and isolated in an identical fashion as de-

scribed for polar bear adipose in preparation for GCeMSD analysis. Approxi-

mately 0.5 g of liver tissue was homogenized with Na2SO4, spiked with the

internal standards and extracted with 200 ml DCM/n-hexanes (1:1). Further

cleaned up and isolated HPCs and neutrals were in an identical fashion as de-

scribed for polar bear adipose in preparation for GCeMSD analysis. Approxi-

mately 2 g of whole blood was spiked with the internal standards followed by

the addition of 1 ml 6 M HCl and 3 ml 2-propanol. All contaminants were ex-

tracted with MtBE/n-hexanes (1:1), and by KOH partitioning the HPCs were

separated from the neutral contaminants. Further clean up and isolation of

HPCs and neutrals was conducted in an identical fashion as described for polar

bear adipose in preparation for GCeMSD analysis. The percent lipid was de-

termined by a sulfo-phospho-vanilin reaction using an olive oil derived cali-

bration curve (Sandau, 2000).

2.3. Quantification of contaminants

PCBs analyses were carried out on an Agilent 6890 GCe5973 MSD, fitted

with a DB-5 column (30 m, 0.25 mm ID, 0.25 mm film thickness, J&W Scien-

tific). The GC oven temperature program for PCB was as follows: 100 �C(3 min), 20 �C/min to 180 �C, 2.5 �C/min to 300 �C. The MS was set in EI

ionization mode, with the ionization voltage set at 70 eV. The source and quad-

rupole temperature were 230 �C and 150 �C, respectively. In SIM, the [M]þ

and [Mþ 2]þ were monitored for 51 PCB congeners (see Table 1). For

MeSO2-PCBs the oven temperature program was 100 �C (3 min), 20 �C/min

to 220 �C (1 min), 3 �C/min to 280 �C (8 min). The MS was set in the

ECNI mode with an ionization voltage of 70 eV. The source and quadrupole

temperature were 180 �C and 150 �C, respectively. Methane was used as col-

lision gas. Using SIM the [M]� and [Mþ 2]� ions were monitored for each

chlorinated homolog group, and 24 congeners were monitored (see Table 1).

There were no standards available for 3-MeSO2-CB64, 30-MeSO2-CB87, 3-

MeSO2-CB91, 4-MeSO2-CB91, 30-MeSO2-CB141, 40-MeSO2-CB141 and 4-

MeSO2-CB149. Relative response factors (RRF) of other congeners with the

same number of chlorination were used. For OH-PCBs the oven ramping pro-

gram was 80 �C (1 min), 10 �C/min to 250 �C (5 min), 5 �C/min to 300 �C(5 min). The MS was set in the ECNI mode with an ionization voltage of

70 eV. The source and quadrupole temperature were 200 �C and 150 �C, re-

spectively. Methane was used as collision gas. In SIM the [M]�, [Mþ 2]�

and [M� 15]� {[M�CH3]�} ions of the MeO-containing derivatives of all

OH-PCBs were monitored for 33 OH-PCBs (see Table 1). The PBDEs and

MeO-PBDEs were determined by GCeMS-ECNI fitted with a DB-5 column

(15 m, 0.25 mm ID, 0.25 mm film thickness, J&W Scientific). The temperature

program was 90 �C, 20 �C/min to 310 �C (15 min). The MS was set in ECNI

mode, with an ionization voltage of 70 eV. The source and quadrupole temper-

ature were 150 �C and 106 �C, respectively. Methane was used as collision

gas. Using SIM, the isotopic bromine anions (m/z 79 and 81) were monitored.

Thirteen PBDEs and 15 MeO-PBDE congeners were monitored (see Table 1).

Table 1

The arithmetic mean (�SE) of concentrations (ng/g wet wt) of classes of neutral and phenolic organohalogen compounds in the tissues of female and male polar

bears from East Greenland (n¼ 20)

Analyte Adipose Blood Brain Liver

Mean� SE Range Mean� SE Range Mean� SE Range Mean� SE Range

Lipid (%) 90� 4 28e120 1.3� 0.1 0.88e1.9 21� 1 13e29 11� 1 5.1e19P

-PCBa 5387� 606 887e12262 40� 9 7e204 148� 23 31e459 3125� 372 1412e7443P

-MeSO2-PCBb 455� 123 61e2648 7� 2 1e52 12� 1 9e23 225� 61 31e1268P-OH-PCBc 60� 8 20e173 1020� 132 385e2888 18� 3 4.8e51 355� 36 176e714

P-PBDEd 83� 19 24e422 1.2� 0.1 0.5e1.9 2.9� 0.4 <0.5e7.6 40� 4 14e103

P-OH-PBDEe 0.9� 0.5 <0.3e10 2.9� 1.0 <0.5e13 n.d. <0.2 n.d. <0.5P-MeO-PBDEf 4.3� 1.7 <0.3e25 0.16� 0.06 <0.5e0.78 n.d. <0.5 n.d. <0.5

n.d. e Not detected.a P-PCB: CB-28/-31, -42, -44, -49, -52, -60, -64/-71, -66/-95, -70, -74, -84/-101, -87, -97, -99, -105, -110, -118, -128, -129/-178, -138, -141, -146, -149, -151,

-153, -156/-171/-202, -158, -170/-190, -172, -174, -177, -179, -180, -182/-187, -183, -194, -195, -196/-203, -200, -201 and -206.b P-MeSO2-PCB: 30/40-MeSO2-CB49, 3/4-MeSO2-CB52, 3/4-MeSO2-CB64, 3/4-MeSO2-CB70, 30/40-MeSO2-CB87, 3/4-MeSO2-CB91, 30/40-MeSO2-CB101,

3/4-MeSO2-CB110, 30/40-MeSO2-CB132, 30/40-MeSO2-CB141, 3/4-MeSO2-CB149 and 3/4-MeSO2-CB174.c P-OH-PCB: 40-OH-CB79, 4-OH-CB97, 40-OH-CB101/4-OH-CB134, 4-OH-CB107/40-OH-CB108, 20-OH-CB114, 3-OH-CB118, 40-OH-CB120, 40-OH-

CB127, 40-OH-CB130, 30-OH-CB138, 4-OH-CB146, 40-OH-CB159, 4-OH-CB162, 4-OH-CB163, 40-OH-CB172, 40-OH-CB177, 4-OH-CB178, 30-OH-CB180,

30-OH-CB182, 30-OH-CB183, 30-OH-CB184, 4-OH-CB187, 4-OH-CB193, 40-OH-CB199, 40-OH-CB200, 40-OH-CB201, 40-OH-CB202, 4,40-diOH-CB202,

30-OH-CB203/40-OH-CB198 and 40-OH-CB208.d P-PBDE: BDE-17, -28, -47, -66, -85, -99, -100, -138, -153, -154, -183, -190 and -209.e P-OH-PBDE: 60-OH-BDE17, 60-OH-BDE17, 60-OH-BDE49, 20-OH-BDE68, 6-OH-BDE47, 3-OH-BDE47, 5-OH-BDE47, 40-OH-BDE49, 4-OH-BDE42,

6-OH-BDE90, 6-OH-BDE99, 2-OH-BDE123, 6-OH-BDE85 and 6-OH-BDE137.f P-MeO-PBDE: 40-MeO-BDE17, 60-MeO-BDE17, 20-MeO-BDE28, 4-MeO-BDE42, 3-MeO-BDE47, 5-MeO-BDE47, 6-MeO-BDE47, 40-MeO-BDE49,

60-MeO-BDE49, 20-MeO-BDE68, 6-MeO-BDE85, 6-MeO-BDE90, 6-MeO-BDE99, 2-MeO-BDE123 and 6-MeO-BDE137.

623W.A. Gebbink et al. / Environmental Pollution 152 (2008) 621e629

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For the OH-PBDE and OH-PBB analysis, the chromatographic and mass spec-

tral parameters were the same as described for OH-PCBs. Using SIM, the iso-

topic bromine anions (m/z 79 and 81) were monitored for 14 OH-PBDE

congeners (see Table 1). Given that there were no commercially available au-

thentic standards for OH-PBB congeners (i.e., their MeO-PBB analogues), the

homolog structures of the apparent MeO-PBBs were confirmed using GCe

high resolution MS (GCeHRMS). GCeHRMS analyses were preformed on

a HewlettePackard 5890 Series GC connected to a VG Autospec, double fo-

cusing-magnetic sector mass spectrometer. The GC was equipped with a fused

silica DB-5 column (30 m, 0.25 mm ID, 0.25 mm film thickness, J&W Scien-

tific), and the temperature ramping program started at 100 �C (2 min), 20 �C/

min to 180 �C, 5 �C/min to 300 �C (6 min). Ionization was preformed by elec-

tron ionization (EI) with an electron voltage of 32 eV, and the source tem-

perature was 280 �C. The mass spectrometer was operated at full scan mode

(75e550 amu) at 1500 mass units resolution.

2.4. Quality control

The analytes were identified by comparison of their relative chromato-

graphic retention time to authentic reference standards. The mean recoveries

were based on the internal standards, and recoveries of all contaminants were

on average 79� 30% in the adipose, 74� 33% in the blood, 51� 64% in the

brain and 77� 37% in the liver. PCBs were calculated using an external standard

approach and were only recovery corrected when recovery was <80%. Recov-

eries for all other contaminants were inherently recovery corrected using the in-

ternal standard quantification method. Method blank samples were analyzed to

monitor for background interferences and contamination. Traces of BDE-47, -99

and -100 were found systematically in the blanks during the PBDE analysis, be-

tween <1% and 5% of the analytes in the sample, and thus background sub-

tracted per block of samples. The method limits of quantification (MLOQs)

for PCBs and PBDEs, based on a signal to noise (S/N ) ratio of 10, were around

0.1 ng/g wet wt for all the tissues, for MeSO2-PCBs, OH-PCB and OH-PBDE the

MLOQs were around 0.05 ng/g wet wt for all the tissues.P

-PCB were within

5% of the consensus values of SRM 1945 pilot whale blubber homogenate

(NIST; Gaithersburg, MD, USA) reported by Schantz et al. (1995),P

-PBDE

were within 13% of the values reported by Kucklick et al. (2004) on SRM 1945.

2.5. Data analysis

Prior to data handling, organohalogen concentrations were log10-trans-

formed for optimal normality of the data distribution (Zar, 1984). The organo-

halogen pattern differences between polar bear liver, blood, brain and adipose

samples were investigated using the relative proportions (wet weight basis) of

individual congener concentration to the total organohalogen concentrations.

Individual organohalogen compounds were included in sums only if they

were detected in 50% or more of the samples in a given matrix. For these com-

pounds, the samples with concentrations below the MLOQs were assigned

a randomly generated value between zero and the compound-specific

MLOQ. The differences between the congener patterns (log10-transformed)

and tissues were investigated by an analysis of variance (ANOVA), followed

by the Fisher post-hoc test. Statistica� (StatSoft, Tulsa, OK, USA) was used

as statistical software and a was set to p� 0.05.

3. Results and discussion

3.1. PCB and PCB metabolites

Based on the percent of PCB congener toP

-PCB concen-trations, the PCB congener patterns were similar in the three

4’-OH-CB2024’-OH-CB172

Adipose

Blood

Brain

Liver

CB99 CB138 CB153 CB170/190 CB180

Mean [CBx]/ -PCB] concentration ratio

Other PCBs

0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

0.0 0.2 0.4 0.6 0.8 1.00.0 0.2 0.4 0.6 0.8 1.0

Adipose

Blood

Brain

Liver

BDE47 BDE99 BDE100 BDE153 BDE154/BB153

Mean [BDEx]/ -PBDE] concentration ratio

Other PBDEs

Adipose

Blood

Brain

Liver

Mean [MeSO2-CBx]/ -MeSO2-PCB] concentration ratio

MeSO2-CB49

MeSO2-CB64

3’ 4’

MeSO2-CB70MeSO2-CB87

MeSO2-CB101

MeSO2-CB110

3 43 4 3’ 4’ 3’ 4’ 3’ 4’4

MeSO2-CB141

Other MeSO2-PCBs

Adipose

Blood

Brain

Liver

Mean [OH-CBx]/ -OH-PCB] concentration ratio

4-OH-CB146 4-OH-CB193

4’-OH-CB199

4,4’-diOH-CB202

4’-OH-CB2084-OH-CB1874-OH-CB163Other OH-PCBs

A

DC

B

Fig. 1. The mean concentration ratio (relative to the sum concentrations of each class) of major congeners of (A) PCBs, (B) OH-PCBs, (C) MeSO2-PCBs and (D)

PBDEs in adipose, brain and liver tissue, and whole blood of polar bears (n¼ 20) from East Greenland.

624 W.A. Gebbink et al. / Environmental Pollution 152 (2008) 621e629

Author's personal copy

tissues and blood, and the pattern was dominated by five con-geners: CB-99, -138, -153, -170/-190 and -180, which madeup 79e95% of

P-PCB (Fig. 1A). However, there were signif-

icant differences in the relative composition of PCB congenersamong tissues. In the liver, e.g., CB-99/

P-PCB was signifi-

cantly lower relative to the adipose tissue and brain( p< 0.007), while CB-138/

P-PCB was significantly higher

( p< 0.005) relative to the other tissues. The CB-153/P-PCB and CB-180/

P-PCB were significantly higher in

the brain relative to the other tissues ( p< 0.05). The differ-ence in PCB congener pattern in brain and liver could be a re-sult of biochemical processes, biotransformation in the liverand the congener-specific discrimination by the BBB. Regard-less, the PCB congener patterns were consistent with previousreports for polar bear adipose, blood and liver (Kannan et al.,2005; Letcher et al., 1998; Muir et al., 1988; Norstrom et al.,1998; Sandala et al., 2004; Verreault et al., 2006). In the pres-ent East Greenland bears, the trend for

P-PCB concentrations

was adipose tissue> liver> brain> blood (Table 1; Gebbinket al., submitted for publication).

OH-PCB congeners were detected in all three tissues andblood. OH-PCBs have been shown to be oxidative and biolog-ically active metabolites of PCBs (Letcher et al., 2000), andhave been shown to be more concentrated than PCBs in bloodor plasma of East Greenland and Canadian bears (Sandalaet al., 2004; Sandau et al., 2000). The mean percent compositionof congeners was dominated by nine congeners in all tissues,and comprising 73e98% of

P-OH-PCB concentration

(Fig. 1B, Table 1). The detected OH-PCBs were all meta- orpara-OH substituted and were pentachloro- to nonachloro-substituted. These congeners were also shown to dominate theOH-PCB congener pattern in the blood of Canadian and EastGreenland polar bears (Sandala et al., 2004; Sandau et al.,2000). Other quantitatively abundant congeners were 40-OH-CB107/40-OH-CB108, 30-OH-CB138, 4-OH-CB178 and 30-OH-CB180. There was a significant difference in the relativecomposition of OH-PCB congeners among tissues ( p< 0.05).Especially in the brain, the 4-OH-CB146, 4-OH-CB163, 40-OH-CB172, 4-OH-CB193 and 4,40-diOH-CB202 to

P-OH-

PCB ratio showed a significant difference ( p< 0.04) comparedto the other tissues. In the adipose tissue, the 4-OH-CB146 and40-OH-CB208 to

P-OH-PCB concentration ratios showed sig-

nificant differences relative to the other tissues ( p< 0.04),while the 4,40-diOH-CB202/

P-OH-PCB in all the tissues and

the blood was significantly different from each other( p< 0.04). The trend for mean

P-OH-PCB concentrations fol-

lows blood [ liver> adipose tissue> brain (Table 1), whichmeans that OH-PCBs appear to be more protein associatedrather than lipid associated as is the case with POPs in general(Gebbink et al., submitted for publication; Verreault et al.,2005b). This is exemplified by the fact that environmentally rel-evant OH-PCB congeners have been shown to competitivelybind to the T4 blood transport protein TTR (Malmberg, 2004).

MeSO2-PCBs have been shown to be persistent and bioac-cumulate in the adipose tissue of polar bears from circumpolarpopulation, including those from East Greenland (Letcheret al., 1998; Verreault et al., 2005a). In the present study, a total

of 23 MeSO2-PCB congeners were detected in all polar beartissues. The mean percent composition of 13 congenersmade up 60e86% of

P-MeSO2-PCB concentration

(Fig. 1C). Several more minor congeners in all the tissueswere 3-/4-MeSO2-CB52, 3-/4-MeSO2-CB91, 30-/40-MeSO2-CB132, 3-/4-MeSO2-CB149 and 3-/4-MeSO2-CB174. Therewas a significant difference in the relative composition ofMeSO2-PCB congeners among tissues ( p< 0.05). In the liver,the ratio of all para-substituted MeSO2-PCBs, 3-MeSO2-CB64and 30-MeSO2-CB87 was significantly different relative to theother tissues ( p< 0.04). In all tissues and blood the proportionof 40-MeSO2-CB101 and 4-MeSO2-CB110 was significantlydifferent ( p< 0.04). Also, the proportion of 3/4-MeSO2-CB70 and 40-MeSO2-CB87 in the brain was significantlygreater relative to the other tissues ( p< 0.03). The differentMeSO2-PCB congener pattern in the liver relative to the othertissues may be associated with protein binding as MeSO2-PCBs have been found to bind to the fatty acid binding protein(FABP) in the liver of rats and chicken (Haraguchi et al., 1997;Larsen et al., 1992; Letcher et al., 2000). ComparableMeSO2-PCB congener patterns were found in the adiposetissue from Canadian polar bears (Letcher et al., 1995),and in the adipose tissue and blood from East Greenlandpolar bears (Sandala et al., 2004). On a wet weight basis thetrend for mean

P-MeSO2-PCB concentrations follows

adipose> liver> brain> blood (Gebbink et al., submittedfor publication).

3.2. PBDE, OH-PBDEs and MeO-PBDEs

A total of 13 BDE congeners were detected in the polarbear tissues (Fig. 1D). The total percent composition amongthe tissues of the congeners BDE-47, -99, -100, -153, and -154 was 86e91% of

P-PBDE concentration (Table 1).

Among the BDE-47, -99, -100, -153 and -154 congeners thetrend showed a significant brain localization preference forBDE-47 ( p< 0.03), a significant preference of BDE-99 to ac-cumulate in the liver ( p< 0.001), and a significant accumula-tion preference of BDE-153 in the subcutaneous adipose tissue( p< 0.001). Our findings are consistent with other polar bearstudies where BDE-47 and -153 were the dominant congenersand BDE-154 a minor in adipose tissue samples (Dietz et al.,2006; Muir et al., 2006; Sørmo et al., 2006). In blood plasmaBDE-154 was more dominant than BDE-153 (Verreault et al.,2005b) while Kannan et al. (2005) only detected BDE-47 inthe liver of Alaskan polar bears. The trend for

P-PBDE con-

centrations follows adipose> liver> blood> brain (Table 1;Gebbink et al., submitted for publication).

Among the 14 OH-PBDE congeners monitored, only twocongeners were detected. However, there were tissue-specificdifferences for the minimal number of OH-PBDE congenersthat were quantifiable. The 6-OH-BDE47 congener was foundonly in the adipose tissue, while 3-OH-BDE47 was foundmainly in the blood but also in adipose tissue (Fig. 2). Ver-reault et al. (2005b) found OH-PBDEs in polar bear plasmafrom Svalbard, but not 3-OH-BDE47 and 6-OH-BDE47. Wequantified OH-PBDEs at or near quantification limit levels

625W.A. Gebbink et al. / Environmental Pollution 152 (2008) 621e629

Author's personal copy

in all tissues (Table 1). The 3-OH-BDE47 and 6-OH-BDE47congeners have been reported to form metabolically in ratsafter dietary exposure of BDE-47 (Marsh et al., 2006). How-ever, 6-OH-BDE47 has also been identified as a natural prod-uct, produced by Dysidea herbacea (Carte and Faulkner,1981). MeO-PBDEs were presently detected in the adiposetissue and blood but not in the liver and brain (Table 1). We de-tected a total of three MeO-PBDE congeners, which differedamong the tissues (Fig. 2). Besides being possible PBDE me-tabolites, MeO-PBDEs could also be of a natural origin. SeveralMeO-PBDE congeners are known to be produced by sponges,and have been found to accumulate in marine mammals (Teutenet al., 2005). The ortho-MeO-substituted congeners seem to fa-vour accumulation in the adipose tissue and blood, rather thanaccumulation in the liver and brain. Previously, one of the MeO-PBDE congeners found in the present study was detected in theplasma of Svalbard polar bears (Verreault et al., 2005b).

3.3. OH-PBBs

We unexpectedly detected three unknown, brominated phe-nolic contaminants in the present bears, which we identified asOH-PBBs. In determinations of MeO-PBDEs (methylated de-rivatives of OH-PBDEs) by GCeMS(ECNI) monitoring of theisotopic 79Br� and 81Br� anions, three unknown and highly re-sponsive peaks were detected in the methylated, HPC fractionfrom polar bear adipose tissue (Fig. 3a). All the three me-thoxylated, brominated unknowns were eluted at the same re-tention time as tri- and tetra-brominated MeO-PBDEs, whichsuggested they were MeO-containing compounds that were ei-ther tri- or tetra-brominated, based on comparable tempera-ture-dependent volatilities that influence GC elution times.As exemplified by unknown 2 (Fig. 3b), GCeMS(ECNI) fullscan mass spectra (m/z 50e550 amu) showed the same isoto-pic ion clusters for Br� (m/z 79) and Br2

� (m/z 161) for allthree unknowns. The mass spectrum of unknown 1 showed ad-ditional negative ion clusters at m/z 247, 325 and 405 (notshown). A hypothetical structure for unknown 1 would betri-brominated MeO-PBB with m/z 405 being the [M� 15]�

ion. The [M� 15]� ion is a common fragment ion for MeO-PCBs (Sandau, 2000). Unknown 2, with ion clusters centered

around m/z 403 and 483 (Fig. 3b), is consistent with tetra-brominated MeO-PBB, and unknown 3 with ion cluster cen-tered around m/z 325, 405 and 481 is also consistent witha tetra-brominated MeO-PBB.

GCeHRMS(EI) was utilized to confirm the methylatedOH-PBBs in the HPC fraction from polar bear adipose. Infull scan mode (m/z 75e550 amu, 1500 mass units resolution),the isotopic ion clusters [M� CH3]þ and [M� 2Br� CH3]þ

were detected for unknown 2 (Fig. 3c). Coincidental ionsfrom hydrocarbon contamination in the solvent (TMP) co-eluted with unknown 1, and made it impossible to detected un-known 1 by GCeHRMS(EI). Unknown 3 was detected in thefull scan, but the EI sensitivity was not sufficient to determinethe exact molecular mass. Regardless, the m/z ratios of the[M� CH3þ 2]þ, [M� CH3þ 4]þ, [M� CH3þ 6]þ ions inthe [M� CH3]þ isotopic ion cluster of unknown 2 were allwithin 0.04 amu of the theoretical mass. In addition, furtherstructural confirmation was possible as the m/z ratiosof [M� 2Br�CH3]þ, [M� 2Br� CH3þ 2]þ, [M� 2Br�CH3þ 4]þ ions in the [M� 2Br� CH3]þ isotopic ion clusterof unknown 2 were all within 0.03 amu of the theoreticalmass. Although GCeHRMS(EI) confirmation was only possi-ble for unknown 2, it is highly probable that unknowns 1 and 3are also MeO-PBBs due to the similar mass spectra obtainedby GCeMS(ECNI). The origin of the detected OH-PBBs isunclear. It is possible that OH-PBBs are formed via biotrans-formation of PBBs (Gardner et al., 1979; Kohli et al., 1978;Koss et al., 1994). Isnansetyo and Kamei (2003) identified2,20-diOH-BB80 as a natural compound produced by the ma-rine sponge Pseudoalteromonas phenolica.

4. Conclusions

To our knowledge, this is the first comparative report ofPCB, PBDE, OH-PCB, MeSO2-PCB, OH-PBDE and MeO-PBDE metabolite/degradation product, congener patternsamong polar bear tissues, and specifically including the brain.Regardless of the overall tissue and blood concentrations, con-gener patterns of these contaminants showed a significant dif-ference especially in the liver and brain for several congeners.Biotransformation and protein binding in the liver and the

0

0.4

0.8

1.2

1.6

3-OH-BDE47 6-OH-BDE46 6'-MeO-BDE17 6-MeO-BDE47 6-MeO-BDE85

0.4

0.8

1.2

1.6M

ean

[OH

-BD

Ex]

/[Σ-

OH

-PB

DE

] ra

tioM

ean [MeO

-BD

Ex ]/[Σ

-MeO

-PBD

E] ratio

Adipose Blood

Fig. 2. The mean concentration ratio� SE of OH-PBDE and MeO-PBDE congeners (relative to theP

-OH-PBDEs andP

-MeO-PBDEs) in the adipose tissue

(black bars) and whole blood (grey bars) of East Greenland polar bears.

626 W.A. Gebbink et al. / Environmental Pollution 152 (2008) 621e629

Author's personal copy

BBB in the spinal stem region of the brain could be biochem-ical processes which might influence the congener patterns.Three OH-PBB congeners were detected in all the polarbear tissues, although the source is unclear, and we are un-aware of any previous reports of OH-PBBs in any aquatic orterrestrial vertebrate (Table 2).

Our findings show that tissue composition of congenerpattern varies as a function of the multiple congener class inquestion. The pattern composition differences for PCBs,

OH-PCBs, MeSO2-PCBs, PBDEs and OH-PBBs indicatethat there are congener-specific mechanisms into or out ofthe brain, liver, adipose tissue and blood. Clearly, our findingssuggest that exposures with respect to congener patterns mayelicit target tissue-specific effects in East Greenland polarbears. In a separate report, we provide an in-depth examinationof the tissue composition and body burdens of the individualor sum concentrations of classes with multiple congeners, ofa wider variety of chlorinated and brominated contaminants

10 12 1614 18 20 22 24 2826 30 32 3634Time

100000

Abu

ndan

ce

200000

300000

400000

500000

2’-OH-BDE28 (IS)

OH-Br3-BB (1)

OH-Br4-BB (2)

OH-Br4-BB (3)

323.9159

325.9137

327.9093

481.7717

485.7566

489.7615

[M-2Br-CH3]+ [M-CH3]+

A

C

300 320 360340 380 400 420 440 460 480 500 520m/z

20000

40000

60000

80000

100000

120000

140000

160000

0

Abu

ndan

ce

350300100 150 200 250 400 450 500

390 400 420410 430 460450440 490480470

79

161403

483

m/z

403

483

[M-Br-CH3]-

[M-CH3]-

B

Fig. 3. GCeMSD (ECNI (m/z 79/81)) mass chromatogram of the brominated phenolic compound fraction from polar bear adipose tissue (derivatized to their cor-

responding MeO-compounds) (A). Full scan mass spectra of OH-Br4-BB (2) by GCeECNI-MS (B) (50e550 amu) and GCeEI-HRMS (C) (75e550 amu, 1500

units mass resolution).

Table 2

The arithmetic mean (�SE) concentrations (ng/g wet wt) of apparent OH-PBB congeners (Fig. 3) in the tissues of polar bear from East Greenland (n¼ 20)

Adipose Blood Brain Liver

Mean� SE Range Mean� SE Range Mean� SE Range Mean� SE Range

OH-Br3-BB (1) 7.4� 3.6 <0.2e65 0.13� 0.11 <0.04e2.2 5.3� 2.6 <0.05e48 0.85� 0.35 <0.08e5.6

OH-Br4-BB (2) 1.5� 1.5 <0.2e31 n.d. <0.04 n.d. <0.05 n.d. <0.08

OH-Br4-BB (3) 5.3� 5.2 <0.2e104 0.13� 0.13 <0.04e2.6 4.3� 1.9 <0.05e30 2.4� 1.2 <0.08e24

n.d. e Not detected, MLOQ 0.04e0.2 ng/g wet wt.

627W.A. Gebbink et al. / Environmental Pollution 152 (2008) 621e629

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of contrasting physico-chemical properties (e.g., polarity andlipophilicity) (Gebbink et al., submitted for publication).

Acknowledgements

Funding for this project is provided by the Natural Sciencesand Engineering Research Council of Canada and Canada Re-search Chairs Program (to R.J.L.). Supplemental support wasalso provided by the Molson Foundation. Danish Cooperationfor Environment in the Arctic and The Commission for Scien-tific Research in Greenland are thanked for facilitating the col-lection of polar bear tissue samples. We wish to thankDr. Goran Marsh and Prof. Dr. Ake Bergman (Departmentof Environmental Chemistry, Stockholm University, Sweden)for generously providing authentic MeO- and OH-PBDEsand MeSO2-PCB standards. We also thank Francois Cyr atNWRC for performing the GCeHRMS(EI) analysis, andMs. Melissa McKinney for helpful suggestions in this study.

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