www.elsevier.com/locate/scitotenv

Science of the Total Environm

Histology of selected immunological organs in polar bear

(Ursus maritimus) from East Greenland in relation to

concentrations of organohalogen contaminants

M. Kirkegaarda,b,*, C. Sonnea,c, P.S. Leifssonb, R. Dietza,

E.W. Bornd, D.C.G. Muire, R.J. Letcherf

aDepartment of Arctic Environment, National Environmental Research Institute, Frederiksborgvej 399, 4000 DenmarkbDepartment of Pharmacology and Pathobiology, The Royal Veterinary and Agricultural University, Bulowsvej 17, 1870 Denmark

cDepartment of Anatomy and Physiology, The Royal Veterinary and Agricultural University, Bulowsvej 17, 1870 DenmarkdGreenland Institute of Natural Resources, P.O. Box 570, DK-3900 Nuuk, Greenland, DenmarkeNational Water Research Institute, Environment Canada, Burlington, ON Canada L7R 4A6

fGreat Lakes Institute for Environmental Research, University of Windsor, Windsor ON Canada N9B 3P4

Received 20 May 2004; accepted 22 September 2004

Abstract

Samples of lymph nodes (axillary, n=54 and inguinal, n=45), spleen (n=60), thymus (n=11) and thyroid tissue (n=5) from a

total of 82 polar bears (Ursus maritimus) collected in East Greenland 1999–2002 were examined histologically. The purpose

was to relate histology to concentrations of organohalogen contaminants (OHCs) [i.e., sum (A)PCBs, ADDTs, AHCHs, ACHLs,HCB, Dieldrin and APBDEs] determined in adipose tissue, as studies on polar bears have indicated that some OHCs act as

immunosuppressive agents. Secondary follicle counts were evaluated in spleen and lymph nodes, and semiquantitively divided

into four groups (0: few/absent to 3: high). In the spleen, a high secondary follicle count was found in 21% of the cases (12/60),

and this was significantly higher in subadults ( pb0.01) compared to adults of both sexes. Also in the lymph nodes a high

secondary follicle count was found in 20% of the cases (20/99), and in the axillary lymph nodes changes were significantly

higher in subadults ( pb0.05) compared to adults of both sexes. Significantly weak correlations between concentrations of

OHCs and the amount of secondary follicles in lymph nodes was found, but probably occurred as a consequence of other

multiple stress factor(s) (all: pb0.05) and also one significant, but modest positive correlation was found between APBDEconcentrations and secondary follicle counts ( pb0.01; r=0.41). In spleen, a significant relation between low concentrations of

OHCs in adipose tissue and few/absent secondary follicles was found with respect to ACHLs, AHCHs, HCB and Dieldrin. No

histopathological observations (e.g., neoplasia) were found in spleen or lymph nodes, nor in thymus or thyroid. In conclusion,

the present data suggest that the exposure concentrations of OHCs to polar bears are unlikely to have resulted in adverse effects

0048-9697/$ - s

doi:10.1016/j.sc

* Correspondi

Roskilde, Denm

E-mail addr

ent 341 (2005) 119–132

ee front matter D 2004 Elsevier B.V. All rights reserved.

itotenv.2004.09.034

ng author. National Environmental Research Institute, Department of Arctic Environment, Frederiksborgvej 399, DK-4000

ark. Tel.: +45 46 30 19 53; fax: +45 46 30 19 53.

ess: mki@dmu.dk (M. Kirkegaard).

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132120

on the tissues in question, although ACHLs, AHCHs, HCB and Dieldrin were related to increased secondary follicle counts in

the spleen.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Polar bear; Ursus maritimus; OHCs; Lymph node; Spleen; Thyroid; Thymus

1. Introduction

Generally, organohalogen contaminant (OHC) and

especially polychlorinated biphenyl (PCB) exposure

is believed to negatively affect reproduction and

survival in marine mammals, e.g., in seals from the

Baltic Sea (Bergman and Olsson, 1985; Bergman,

1999) and in polar bears (Ursus maritimus) from

Canadian and Svalbard populations (Letcher et al.,

1996; Wiig et al., 1998; Bernhoft et al., 2000; Lie et

al., 2004a,in press; Skaare et al., 2001a; Haave et al.,

2003; Oskam et al., 2003). Immunosuppressive

effects of OHC exposure may be an environmental

issue in marine species (Vos and Luster, 1989), e.g.,

such effects were found to correlate with concen-

trations of aryl hydrocarbon receptor (AhR)-active

OHCs including non-ortho PCBs [e.g., 3,3V,4,4V-tetrachlorobiphenyl (CB-77) and 3,3V,4,4V,5-penta-chlorobiphenyl (CB-126)] and polychlorinated

dibenzo-p-dioxins/furans (PCDDs/Fs) in harbour

seals (Phoca vitulina) fed Baltic fish under semifield

experiments (Swart et al., 1994; Ross et al., 1995,

1996). Non-ortho PCBs and PCDDs/Fs are also

suspected to cause an increasing prevalence of

intestinal ulcers in Baltic grey seals (Halichoerus

grypus) (Bergmann et al., 1996; Bergman, 1999).

In wildlife, histopathological changes in kidneys,

adrenals, thyroids a.o. organs have been correlated to

concentrations of OHCs in several marine mammals

such as harbour seal, Greg seal, ringed seal (Phoca

hispida botnica) and harbour porpoise (Phocoena

phocoena) (Bergman and Olsson, 1985; Schumacher

et al., 1993; Bergman et al., 2001, Siebert et al., 2002) as

well as in fish (bream, Abramis brama) (Koponen et al.,

2001). In harbour porpoise some OHCs have direct

affect on the thymus, causing atrophy, and some affect

the immune system by increasing lymphocyte depletion

from lymphatic organs (Siebert et al., 2002). Immuno-

suppressive effects aremeasured by biomarkers, such as

reduced antibody production when exposed to a foreign

antigen, changes in T-cell populations, decreased

delayed-type hypersensitivity, decreased resistance to

virus infections and decreased natural killer cell activity

(Wong et al., 1992; Tryphonas, 1994).

In polar bear studies from Canada and Svalbard in

1998 and 1999, results show that organochlorines

(OHCs) in combination with specific biological factors

(sex, age, condition, body mass) significantly influence

serum IgG concentrations and antibody production

after immunisation with influenza and reo viruses and

tetanus toxoid. In addition, the results demonstrate in

particular, a negative association between APCBs andthe serum IgG concentrations or production of anti-

bodies to influenza and reo viruses (Lie et al., 2004a).

For lymphocyte proliferation, the results of the

statistical analyses show that high concentrations of

PCBs and/or pesticides were found to decrease specific

lymphocyte function and thus may produce impaired

resistance against infections (Lie et al., in press). The

study demonstrates that high concentrations of APCBsinfluence parts of the humoral immunity in polar bears,

as the ability to produce antibodies following immu-

nisation was impaired (Lie et al., 2004a).

The OHCs classes PCBs, dichlorodiphenyltri-

chloroethanes (DDTs), hexachlorocyclohexanes

(HCHs), chlordanes (CHLs) and PCDDs/Fs that are

present in the tissues of polar bears, have shown to be

transferred transplacentally from mother to foetus in

utero and to cubs via mother’s milk (Bernhoft et al.,

1997; Polischuk et al., 2002). Many of these com-

pounds have demonstrated or have the potential to

function as agonists and/or antagonists with respect to

hormone-dependent processes in endocrine target

organs and tissues (Colborn et al., 1993; De March et

al., 1998; Damstra et al., 2002; AMAP, 2004). OHCs in

exposed organisms can interfere with the main endo-

crine pathway, which may result in disruption of

different endocrine target organs like adrenals, thyroid,

parathyroid, thymus, testicles and ovaries.Mechanisms

resulting in the disruption of endocrine homeostasis

can occur due to modulation/interference of the

concentrations of corticosterone, thyroid hormones

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132 121

[thyronine (T3) and thyroxine (T4)] and other sex

hormones (Colborn et al., 1993; Feldman, 1995; De

March et al., 1998; Bergman, 1999; Damstra et al.,

2002; AMAP, 2004). OHC exposure can induce

hypothalamus-mediated stress resulting in hyperadre-

nocorticism (adrenocortical hyperplasia), which is a

clinical condition in mammals caused by high concen-

trations of cortisol (glucocorticoids). High glucocorti-

coid concentrations interfere with metabolism in

relation to the immune system leading to anti-inflam-

matory and immune-suppressive responses. In the

Baltic ringed seal and grey seal, hyperadrenocorticism

has been correlated to high concentrations of PCBs and

DDTs (Bergman and Olsson, 1985; Feldman, 1995; De

March et al., 1998; Bergman, 1999; AMAP, 2004).

In order to assess whether relatively high concen-

trations of OHCs in tissues may be linked to

deleterious effects on health of polar bears in East

Greenland, samples of lymph nodes, spleen, thyroid

and thymus tissues were collected 1999–2002 from a

total of 82 polar bears and analysed histologically in

relation to and compared to the OHC residue

concentrations in adipose tissue of these bears.

2. Materials and methods

2.1. Sampling

Fat samples from polar bears were collected by

local subsistence hunters in the Ittoqqortoormiit/Score-

sby Sound area in central East Greenland between

69800VN and 74800VN in 1999–2001. All tissue

samples were taken as soon as possible postmortem

and stored in separate polyethylene (PE) Whirlpak

bags. All samples were kept at outdoor temperature

(�5 to �20 8C) until transferred to a freezer (�10 to

�20 8C). Samples were shipped frozen from Scoresby

Sound to Roskilde, where the portion of fat that had

been in contact with the PE was trimmed off and the

remaining part was transferred to precleaned glass

containers with cleaned aluminium foil in between the

lid and the glass container. Further storage was at�20

8C. Samples from spleen (n=60), lymph nodes

[axillary (n=54) and inguinal (n=45)], thyroid (n=5)

and thymus (n=11) were stored in a solution of

phosphate buffered one part formaldehyde (5% sol-

ution) and nine parts ethanol (96% solution) to prevent

freezing damage. The thymus undergoes involution

after maturation (Dellman and Eurell, 1998), after

which it becomes small, similar in size to the thyroid.

This relatively small lobe size in adults made it

difficult for the Inuit hunters to locate; therefore, only

a few samples of thyroid and thymus were obtained.

2.2. Age determination

The age determination was carried out by counting

the cementum Growth Layer Groups (GLG) of the I3(or P1) tooth using the methods described by Hensel

and Sorensen (1980) and Dietz et al. (1991). Based on

the assigned ages the animals were grouped into adult

females (z5 years), adult males (z6 years) and the

remaining as subadults (Norstrom et al., 1998;

Henriksen et al., 2001).

2.3. Histology

All tissue samples were trimmed, processed con-

ventionally, embedded in paraffin (Sakura Tissue

TekRVIP, Sakura Finetek, USA), and cut into 2–4

Am slices. Sections were stained with haematoxylin-

eosin (HE) for histological examination. All slides

were examined with a Leica DMLB microscope with

50, 100, 200, 400 and 1000� magnification (pictures

were taken and stored by a Leica DC300 digital

camera). Histological findings (secondary follicles)

were assessed semiquantitatively and assigned to four

groups (index) as few/absent (group 0), low (group 1),

moderate (group 2), high (group 3). The index was

calculated as an average of secondary follicles from

repeated counts in four randomly selected low power

fields (50� magnification). In lymph nodes group 0

represented 0–3 secondary follicles, group 1: 4–5

secondary follicles, group 2: 6–8 secondary follicles

and group 3: 9–18 secondary follicles. In spleen

groups were; 0: 0–1 secondary follicles, group 1: 2

secondary follicles, group 2:3 secondary follicles and

group 3: 4–8 secondary follicles.

2.4. Contaminant analyses

2.4.1. PCBs and OHCs

Polar bear blubber samples (n=94)were analysed for

polychlorinatedbiphenyls (PCBs), dichlorodiphenyltri-

chloroethanes (DDTs), hexacyclohexanes (HCHs),

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132122

CHLordanes (CHLs), hexachlorobenzene (HCB) and

dieldrin according to Sandala et al. (2004) and Dietz et

al. (2004) at theGreat Lakes Institute for Environmental

Research (GLIER), University ofWindsor, Canada. An

external standard quantification approach used for

PCBs and OHCs in the adipose tissues was based on

peak area of the GC-AECD response, which is

described in detail in Dietz et al. (2004). Briefly,

APCBs is the sum (A) of the concentrations of the 51individual or coeluting congeners (if detected): CB #

31/28, 52, 49, 44, 42, 64/71, 74, 70, 66/95, 60, 101/84,

99, 97, 87, 110, 151, 149, 118, 146, 153, 105, 141, 179,

138, 158, 129/178, 182/187, 183, 128, 174, 177, 171/

202/156, 200, 172, 180, 170/190, 201, 203/196, 195,

194, 206. CB congeners are numbered according to the

corrected IUPAC numbering scheme as described by

Guitart et al. (1993). ADDTs is the sum of 4,4V-DDT,4,4V-DDD and 4,4V-DDE. AHCHs is the sum of the a-,

h- and g-hexachlorocyclohexane.ACHLs is the sum of

oxychlordane, trans-chlordane, cis-chlordane, trans-

nonachlor, cis-nonachlor and heptachlor epoxide.

Contaminant fractions were subsequently sent to the

National Water Research Institute [Environment Can-

ada, Burlington, Ontario, Canada L7R 4A6 (NWRI)]

for determination of brominated diphenyl ether (PBDE)

flame retardants.

2.4.2. PBDEs

PBDEs (n=93) were determined by electron

capture negative ion (low resolution) MS using an

external standard. Briefly, APBDEs is the sum (A) ofthe concentrations of the 35 individual or coeluting

IUPAC congeners (if detected): BDE# 10, 7, 11, 8, 12/

13, 15, 30, 32, 28/33, 35, 37, 75, 71, 66, 47, 49, 77,

Table 1

Percentual distribution of lymphocytic accumulation (secondary follicles) i

of splenic tissue in subadult and adult East Greenland polar bears sample

Groups Tissue

Axillary lymph nodes Inguinal lym

Secondary follicle

frequency

Subadults Adults All Subadults

(0) Few/absent 10% (2) 30% (10) 22% (12) 18% (4)

(1) Low 10% (2) 32% (11) 24% (13) 27.5% (6)

(2) Moderate 35% (7) 23.5% (8) 28% (15) 36.5% (8)

(3) High 45% (9) 14.5% (5) 26% (14) 18% (4)

Sum 100% (20) 100% (34) 100% (54) 100% (22)

Numbers (n) are given in brackets.

All lymph nodes (axillary and inguinal) in group 3 (high)=20% (20 out o

100, 119, 99, 116, 85, 155/126, 105, 154, 153, 140,

138, 166, 183, 181, 190 (Muir et al., in preparation).

Gas chromatographic conditions for the PBDEs were

described by Luross et al. (2002).

2.5. Statistics

The statistical analyses were performed with the

SAS statistical software package (SAS V8) and a

significance concentration of p=0.05 was used (except

if stated otherwise). Contaminant data was log-trans-

formed (base e) prior to the analyses in order to better

meet the assumption of normality and homogeneity of

the variance. To test for differences in frequency of

secondary follicles among age and sex groups the

non-parametric Kruskall–Wallis test was applied. To

test for significant differences in mean concentrations

of organohalogens among groups of secondary

follicles, a single factor one-way analysis of variance

(ANOVA) was employed. When significant differ-

ences were found these were tested among each other

by the Tukey post hoc test. Finally, the non-parametric

Spearmann Rank test was used to test for significant

correlations between concentrations of OHCs and

number of secondary follicles.

3. Results

3.1. Lymph nodes

Fifty-four samples from axillae (ALN) and 45

samples from inguinal lymph nodes (ILN) were

examined and evaluated histologically. Of these, 42

n 54 axillary lymph nodes, 45 inguinal lymph nodes and 60 samples

d 1999–2002

ph nodes Spleen tissue

Adults All Subadults Adults All

22% (5) 20% (9) 0% (0) 11% (4) 7% (4)

30% (7) 29% (13) 25% (6) 45% (16) 37% (22)

40% (9) 38% (17) 37.5% (9) 36% (13) 37% (22)

8% (2) 13% (6) 37.5% (9) 8% (3) 21% (12)

100% (23) 100% (45) 100% (24) 100% (36) 100% (60)

f 99).

Fig. 1. Histology of a lymph node from a normal 5-year-old East

Greenland male polar bear sampled 1999 (note secondary lymphatic

follicles in the cortex). Trabeculum (T), Mantle zone (M) and

germinal centre (G). (HE, 100�.).

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132 123

(ALN: 20; ILN; 22) were from subadults, 37 (ALN: 19;

ILN; 18) from adult males and 20 (ALN: 15; ILN; 5)

from adult females (Table 1). Microscopically the

appearance of polar bear lymph nodes resembled that

of the ringed seal (P. hispida) (Dyce et al., 1987; Habel,

1992; Welsch et al., 1997). The nodes were surrounded

Fig. 2. Lymphocytic histological findings in the four distinguished groups

follicles). (B) Group 1 (4–5 secondary follicles). (C) Group 2 (6–8 seconda

germ center. HE (100�: panels B and C), (50�: panels A and D).

by a fibrous capsule from which fibrous trabeculae

(bTQ; Fig. 1) extended into the node to form a

supporting framework, carrying blood vessels and

nerves. The surface of the lymph nodes was penetrated

by a number of afferent lymphatic vessels, which

opened into a subcapsular sinus, forming networks

extending into the hilus. The lymph cortex consisted of

primary and secondary lymphatic follicles separated by

diffuse lymphatic tissue (Dellman and Eurell, 1998).

The primary follicles consisted of naive B cells and

some memory T cells and the secondary lymphoid

follicles consisted mainly of B cells in various stages in

the secondary follicle (bGQ Fig. 1), characterised by

open nuclei and more cytoplasm. The B-cells were also

less densely packed than the smaller naive B cells and

the few Tcells that formed the darker mantle zone (bMQFig. 1) of the follicle (Stevens and Lowe, 1997).

3.1.1. Histology

None of the changes found in the examination of

the lymph nodes could be categorised as severe

histopathological observations (as neoplasia or fib-

rosis). Secondary follicles were found and distributed

as shown in Table 1 and Fig. 2. For group 3, a

. Arrows indicate secondary follicles. (A) Group 0 (0–3 secondary

ry follicles). (D) Group 3 (9–18 secondary follicles). Arrows indicate

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132124

higher count of secondary follicles in the axillary

lymph nodes was found for subadults compared to

adult males and females ( pb0.05). No similar

significant differences in the inguinal lymph node

were found.

3.1.2. OHCs

Concentrations of tissue residues (blubber) of

OHCs (ng/g lw) of PCBs (51 congeners), DDTs

(4,4V-DDD, 4,4V-DDE, 4,4V-DDT), HCHs (a-HCH, h-HCH, g-HCH), CHLs (oxychlordane, trans-chlor-

dane, cis-chlordane), HCB, PBDEs (35 congeners)

and dieldrin are described in Dietz et al. (2004).

APCBs and ACHLs constituted the highest concen-

trations followed by ADDTs (4,4V-DDE constituted

the most), dieldrin and AHCHs, while HCB and

APBDEs carried the lowest concentrations. AMeSO2-

PCB and 3-MeSO2-4,4V-DDE concentrations were not

included in these comparisons due to covariance, i.e.,

A-MeSO2-PCB to A-PCBs and 3-MeSO2-4,4V-DDEto 4,4V-DDE ratios for the present Greenland bears

Table 2

Mean values of APCBs, ADDTs, AHCHs, ACHLs, HCB, Dieldrin and APlymph nodes and spleen in East Greenland polar bears (1999–2002) anal

Tissue Compound

APCBs ADDTs AHCH

Axillary lymph nodes

Mean (ng/g lw), all groups 6060 n.s. 456 n.s. 195 n

0 (Few/absent) 7184 566 219

1 (Low) 6608 361 238

2 (Moderate) 5353 480 166

3 (High) 5477 442 165

Inguinal lymph nodes

Mean (ng/g lw), all groups 6586 n.s. 492 n.s. 191 n

0 (Few/absent) 8107 526 267

1 (Low) 7406 523 187

2 (Moderate) 5159 452 159

3 (High) 5605 405 136

Spleen

Mean (ng/g lw), all groups 6252 n.s. 1098 n.s. 185*

0 (Few/absent) 3443 429 112

1 (Low) 6421 503 189

2 (Moderate) 6043 387 188

3 (High) 6979 487 194

Significance concentrations from the single factor one-way ANOVA ( F-tes

significance: n.s..

* Significance at pb0.05 level.

** Significance at pb0.01 level.

were relatively constant values of 0.08F0.03 and

0.06F0.05, respectively (Sandala et al., 2004). Addi-

tional information on OHC concentrations and infor-

mation on sex, season differences and time trends in

the East Greenland polar bear are available in Dietz et

al. (2004) and Sandala et al. (2004).

It was tested whether concentrations of APCB,ADDTs, AHCHs, ACHLs, HCB, Dieldrin and

APBDEs could explain the difference in distribution

of secondary follicles. No significant difference was

found between any of the seven groups of contami-

nants and the frequency of secondary follicles in

axillary and inguinal lymph nodes (ANOVA test,

Table 2). On the other hand, significant correlations

between contaminant concentrations and follicle score

(not grouped) were found in three cases (Table 3).

These were APBDEs and the mean number of

secondary follicles/focus in the inguinal lymph nodes

( pb0.01; r=0.41), a negative correlation between

APCBs and follicle score in inguinal lymph nodes

( pb0.05; r=�0.35) and a negative correlation

BDEs (ng/g lw) divided on score groups within axillary and inguinal

ysed in the present study

s ACHLs HCB Dieldrin APBDEs

.s. 1233 n.s. 119 n.s. 186 n.s. 57 n.s.

999 89 172 55

1280 103 180 57

1300 138 196 60

1282 134 190 54

.s. 1309 n.s. 133 n.s. 193 n.s. 60 n.s.

1197 127 184 85

1695 172 229 62

938 99 168 46

1412 111 146 39

1248* 130** 187** 56 n.s.

478 53 81 31

1152 105 176 56

1176 120 183 53

1679 199 236 68

t) are presented between counts of secondary follicles and OHCs. No

Table 3

Spearmann Rank correlation coefficients between concentrations of APCBs, ADDTs, AHCHs, ACHLs, HCB, Dieldrin and APBDEs in adipose

tissue and number of secondary follicles/focus in lymph nodes and splenic tissue from East Greenland polar bears (1999–2002)

Tissue Compounds

APCB ADDT AHCH ACHL HCB Dieldrin APBDEs

Axillary

Lymph nodes (r) �0.18 n.s. �0.002 n.s. 0.26 n.s. 0.24 n.s. �0.35* 0.25 n.s. 0.28 n.s.

Inguinal

Lymph nodes (r) �0.35* 0.03 n.s. �0.03 n.s. 0.04 n.s. 0.15 n.s. 0.02 n.s. 0.41**

Spleen (r)

0.17 n.s. �0.15 n.s. 0.26 n.s. 0.24 n.s. �0.35* �0.16 n.s. �0.15 n.s.

P-values: nonsignificant: n.s..

* Significant at pb0.05 level.

** Significant at pb0.01 level.

Fig. 3. Histology of spleen tissue from a normal 8-year-old male Eas

Greenland polar bear. (A) Lymphatic nodule (N) with artery (A). (B

Lymphatic nodule (N), capsule (C) and trabecula (T). (HE, 50�.).

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132 125

between HCB and the number of secondary follicles/

focus in axillary lymph nodes ( pb0.05; r=�0.35).

3.2. Spleen

Spleen samples from 24 subadults, 20 adult males

and 16 adult females were examined and evaluated

histologically. Microscopically the appearance of

polar bear spleen tissue was composed of capsule

(bCQ, Fig. 3), trabeculae (bTQ, Fig. 3), white pulp (5–

20%: rich in lymphocytes and active in immune

responses), red pulp (antigen trapping and storage of

red blood cells), and it had a marginal zone between

the white and red pulp (Dellman and Eurell, 1998).

The white pulp consisted of two types of tissues

comprising B-or T-cells and was similar to that of the

paracortex and superficial cortex of the lymph nodes.

B cells formed germinal centers in the lymphatic

nodules (bNQ, Fig. 3), usually located near an arteriole

(bAQ, Fig. 3), where irregular masses around the

central arteries were formed by white pulp T cells.

3.2.1. Histology

No essential histopathological observations were

found. The histological differences were categorised

into four groups depending on the number of

secondary follicles (Fig. 4 and Table 1).

3.2.2. OHCs

It was tested whether concentrations of APCBs,ADDTs, AHCHs, ACHLs, HCB, Dieldrin and

APBDEs could explain the difference in distribution

of secondary follicles. A significant difference in

mean values of ACHLs and AHCHs (both: pb0.05)

and HCB and Dieldrin (both: pb0.01) was found for

t

)

Fig. 4. Histological findings in spleen tissue in the four distinguished groups. (A) Group 0 (0–1 germ centers). (B) Group 1 (2 germ centers).

(C) Group 2 (3 germ centers). (D) Group 3 (4–8 germ centers), -more germ centers are visible in the actual focus of the microscope. Arrows

indicate germ center. (HE, 50�.).

Fig. 5. Thyroid tissue from a normal 4-year-old East Greenland

male polar bear. Thyroid follicle (F) and lumen filled with colloid

(C). (HE, 50�.).

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132126

all groups of follicle score (Table 2). The Tukey post

hoc test of these four contaminants showed that group

0 had significantly lower contaminant concentrations

compared to groups 1–3 ( pb0.05) (exception was

HCB, where group 0 was significantly different only

from group 3). No significant correlations were found

between APCBs, ADDTs, AHCHs, ACHLs, Dieldrinand APBDEs, and secondary follicle score; however,

a negative correlation was found between HCB and

follicle score (Table 3).

3.3. Thyroid and thymus

The thyroid gland of the polar bear was divided

into irregular lobes and each thyroid follicle (bFQ, Fig.5) comprised an epithelium composed of secretory

thyroid follicular cells secreting the stained colloid

(bCQ, Fig. 5) rich in thyroglubulin (storage form of

thyroxine) (Dellman and Eurell, 1998). The thymus

was a lympheaepithelial organ, composed of thymic

lobules (bLQ, Fig. 6). Controlled T-cell production was

located in the cortex (bCQ, Fig. 6) and maturation of

T-cells were indicated in the medulla (bMQ, Fig. 6).All the examined samples of the thyroid (n=5) and

thymus (n=11) tissue were found to be similar to

normal mammalian tissues and no histopathological

changes were detected (Figs. 5 and 6). The contam-

inant concentrations of these few animals for the

thyroid (ng/g lw) were APCBs=2341–8124 (n=5),

ADDTs=86–205 (n=5) and APBDEs=26–69 (n=3);

for the thymus, APCBs=2466–8372 (n=8),

ADDTs=101–249 (n=8) and APBDEs=25–36 (n=3).

Fig. 6. Normal thymus tissue from a 4-year-old East Greenland

female polar bear. Thymic lobules (L), medulla (M), and cortex.(C).

(HE, 50�.).

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132 127

3.4. Hydroxylated (HO) metabolites

Modulation effects on endocrine systems in wild-

life may also occur as a result of PBDE and PCB

exposure and or subsequent hydroxylated (HO) PBDE

and HO-PCB, respectively, metabolite formation

(Letcher et al., 2002; Darnerud, 2003; Hakk and

Letcher, 2003; Legler and Brouwer, 2003). For

example, HO-PBDE metabolites have been shown

in vitro to competitively interact with human trans-

thyretin (TTR, thyroid hormone transport protein)

(Meerts et al., 2000). HO-PCBs have also demon-

strated thyroid-related activity in humans and wildlife,

including possible activity in Canadian polar bear

(Brouwer et al., 1998; Sandau et al., 2000b; Fisk et al.,

submitted for publication). The present East Green-

land polar bears have been found to contain among

the highest blood-plasma concentrations of HO-PCB

than that of any other species or population (Sandala

et al., 2004).

4. Discussion

Studies on IgG concentrations have indicated that

some OHCs function as immunosuppressive sub-

stances in polar bears (Bernhoft et al., 2000; Skaare

et al., 2001b; Lie et al., 2002, 2004a,in press; Fisk

et al., submitted for publication). In a normal

functioning immune system, a suppression of

immunity can lead to decreased host resistance to

infection. This could possibly be seen as increased

infection rates, expressed by high secondary follicle

counts in lymph nodes and spleen, although this has

not been proved. In a suppressed immune system,

decreased activity in the immune organs could be

expected, but this depends on the mechanism of the

xenobiotic on the immunity, and for many of the

OHCs this is not clear and suppression is dose/

exposure dependent. Furthermore, the effects of

synergy among OHCs and the lack of controlled

studies evaluating histopathological changes in

relation to OHCs in wild animals, makes the

evaluation about cause and effect problematic.

Currently, reference material on histopathological

effects of contaminants in wild animals is sparse

compared to controlled laboratory studies, and more

research on the issue is needed.

4.1. Lymph nodes

Our present results showed significant correla-

tions between the frequency of secondary follicles in

the lymph nodes and concentrations of OHCs in

adipose tissue, but as these correlations showed both

weak negative and positive relationships, OHC

exposure is not likely linked to modulation of the

frequency of secondary follicles, and other possible

multiple stress factors probably explain any modu-

lation effects. However, a modest, positive correla-

tion was found in relation to APBDE concentrations.

Evidence is accumulating that exposure to very high

concentrations of PBDEs causes immunomodulatory

effects in laboratory animals (McDonald, 2002;

Darnerud, 2003; AMAP, 2004). Exposure of mice

to BDE-47 or Bromkal 70-5DE suppressed the

proliferation of lymphocytes and the production of

antibodies (Darnerud and Thuvander, 1998;

Thuvander and Darnerud, 1999). Thymotoxic effects

occurred in mice exposed to BDE-71 (Fowles et al.,

1994). High secondary follicle counts (group 3) were

found in 20% of all lymph nodes and this

accumulation of secondary follicles indicated a

response to antigens. The significantly higher num-

ber of secondary follicles in the axillary lymph

nodes of subadults compared to adults showed a

higher immune response among the young animals,

similar to the inguinal lymph nodes. Germinal

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132128

centers in secondary follicles are not present at birth

(Nielsen, 1977), so the difference was evaluated to

possibly be a natural age related immune adaptation/

maturation to antigens in subadult polar bears

(Grundmann and Vollmer, 1990).

4.2. Spleen

In 20% of the spleen samples, the frequencies of

secondary follicles were categorised as high (group

3). Again a significantly higher number of secondary

follicles in the spleen of subadults (37.5%) indicated

an activity in immune response not found in adults

(8%), as is described in humans by Stevens and

Lowe (1997). ACHLs, AHCHs, HCB and Dieldrin

showed a significant difference in mean values

among groups 0–3 in the spleen and the post hoc

analysis showed that only the group with few or

absent secondary follicles (group 0) was significantly

lower in contaminant concentrations compared to

groups 1–3. This could possibly have indicated the

immunological reaction against foreign antigens

circulating in the blood and thereby the possible

increase of the IgG response. Siebert et al. (2002)

found a relation between elevated concentrations of

DDE and spleen depletion in harbour porpoise (P.

phocoena), although these concentrations (adults

from North Sea, Baltic Sea, Norway and Island)

were much higher (range: 62–14,000 ng/g lw, n=30)

than found for all polar bears in the present study

(range: 74–1152 ng/g lw, n=92).

4.3. Thyroid and thymus

In general, no severe histopathological observa-

tions (atrophy, fibrosis and neoplasia) could be found

in either of the investigated tissues. In laboratory

studies of effects of PCB on the thyroid gland, in

utero, dietary and/or intra peritoneal (ip) exposure to

rats have shown histopathological changes like

decreases in serum T4, increased follicular cell height

and decreased colloid area (Ness et al., 1993; Saeed

and Hansen, 1997; Hood et al., 1999; Khan et al.,

2002). In Table 4, the concentrations used in the

investigations of relations between organohalogens

and thyroid changes in laboratory mammals (rats),

harbour seals, grey seals and harbour porpoise are

compared to the concentrations found in the adipose

tissue (blubber), as well as the estimated daily intake

by the East Greenland polar bears in the present

study.

According to Born and Bocher (2001), an baverageQpolar bear (weight: 200 kg) consumes app. 1000 kg

blubber per year. Based on concentrations in ringed

seal blubber from East Greenland sampled 1999–2002

(Riget, unpublished data) the average intake of a 200

kg baverageQ polar bear of APCBs is 13 Ag/kg APCBs/day as seen in Table 4. This is, of course, a rough

estimate but makes it comparable with concentrations

used in other investigations. Based on Riget et al.

(2003), the concentrations in East Greenland are a

factor 2–4 higher compared to West Greenland. These

data were used in evaluating the intake in East

Greenland polar bears (Table 4). It is seen that the

concentrations found and the daily intake calculated

are significantly lower in the bears compared to the

laboratory mammals (rats) which, as well as a low

sample size, could explain that we did not find a

relation between thyroid histology (atrophy and

fibrosis) and concentrations of PCBs.

4.4. Wildlife

Earlier studies of wildlife have shown that histo-

pathological changes in kidney, adrenals, uterus,

thyroids a.o. were related to concentrations of organo-

halogens in marine mammals such as harbour seal (P.

vitulina), grey seal (H. grypus), ringed seal (P. hispida

botnica) and harbour porpoise (P. phocoena) (Table

4) (Bergman and Olsson, 1985; Schumacher et al.,

1993; Bergman et al., 2001; Siebert et al., 2002).

Siebert et al. (2002) found a significant relation

between the degree of depletion of lymphoid organs

of harbour porpoise (P. phocoena) and elevated

concentrations of PBDEs and PCBs. In the present

study of polar bears, all thyroid and thymus samples

examined showed normal tissue and also much lower

concentrations of PBDE than that of the harbour

porpoises (P. phocoena) investigated by Siebert et al.

(2002) (Table 4).

Bernhoft et al. (2000) investigated possible

immunotoxic effects of OHCs in polar bears from

Svalbard sampled during 1991–1994, and found

blood plasma concentrations of IgG negatively

correlated with APCBs (mean ranges in different

age groups: 11200–21,700 ng/g lw, Bernhoft et al.,

Table 4

Range in the concentrations of organohalogens (ng/g w.w.) in wildlife species and laboratory studies where a link between contaminants and

pathology has been postulated, compared to concentrations in adipose tissue or daily intake in the bears of the present study

Species

and study

Tissue

affected

Organohalogen

compound

Concentration ng/g

(blubber) or

mg/kg/day (food)

n Concentration (blubber (ng/g) or

estimated daily intake mg/kg/day)

for East Greenland polar bears

n

Wildlife

Harbour seala,b,c Several ADDTs 2000–13,000 38 74–1113 92

Harbour seala,b,c Several APCBs 6000–110,000 37 898–20,407 92

Grey seala Several ADDTs 11,000–1 600,000 38 74–1113 92

Grey seala Several APCBs 32,000–5,300,000 37 898–20,407 92

Harbour seald,e Thyroid APCBs 21,240–140,830 32 2341–8124 5

Harbour porpoisef Thyroid PCB 153 380–2500 50 756–2624 5

Harbour porpoisef Thyroid ADDTs 760–2330 50 86–205 5

Harbour porpoisef Thyroid APBDEs 17– 4900 71 26–69 5

Harbour porpoisef Thymus APBDEs 17–4900 71 25–36 3

Harbour porpoisef Spleen DDE 160–14,000 98 85–982 49

Laboratory study

Ratg Thyroid PCB-28, 118,153 Diet: 8–64 mg/kg/day 14 0.013 mg/kg/day APCBs(ring seal diet estimate)

92

Rath Thyroid Aroclor 1242 Diet: 24–60 mg/kg

for 2–7 days

19 0.013 mg/kg/day APCBs(ring seal diet estimate)

92

Rati Thyroid Aroclor 1254 Diet: 25–200 ppm

ad libitum for 7 days

30 0.013 mg/kg/day APCBs(ring seal diet estimate)

92

Ratj Thyroid PCB-95, 101 ip: 4–32 mg/kg/day

for 2 days

33 0.013 mg/kg/day APCBs(ring seal diet estimate)

92

a Wildlife data from Blomkvist et al. (1992).b Wildlife data from Mortensen et al. (1992).c Wildlife data from Schandorff (1997).d Wildlife data from Lucas et al. (1990).e Wildlife data from Schumacher (1993).f Wildlife data from Siebert et al. (2002).g Laboratory studies data from Ness et al. (1993).h Laboratory studies data from Saeed and Hansen (1997).i Laboratory studies data from Hood et al. (1999).j Laboratory studies data from Khan et al. (2002).

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132 129

1997). Polar bear studies from Canada and Svalbard

in 1998 and 1999 also showed that OHCs were

negatively correlated to serum IgG concentrations

and antibody production, after immunisation with

influenza and reo viruses and tetanus toxoid. Like

earlier studies, the results also demonstrated in

particular, a negative association between APCBsand the serum IgG concentrations or production of

antibody concentration to influenza and reo viruses

(Lie et al., 2004a).

Highest of the APCBs in the polar bears of this

study from East Greenland (collected from 1999 to

2002) was around 18,100 ng/g lw (mean: 6140 for

all ages). Recent findings have shown that a

substantial decrease (29.9–81.2%) in the tissue

concentrations, dependent of the investigated com-

pound analysed, have taken place over the last

decade (Dietz et al., 2004). The lower burden of

contaminants could hence explain the lack of

obvious histopathological observations. In rodents,

it is known that organ weights and histopathology

are not the most sensitive endpoints for detecting

alterations in immune functions (Luster et al., 1988),

and although the lower burden of contaminants is a

possible explanation, another possibility is that

histopathology is not a sensitive enough method of

detecting immune suppression in polar bears exposed

to OHCs.

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132130

5. Conclusion

It was investigated whether OHCs have contrib-

uted to histopathological observations in lymph

nodes, spleen, thymus and thyroid glands of the

Greenland Sea polar bear collected 1999–2002. No

essential histopathological observations were found in

any of the tissues examined. No significant relations

between concentrations of organohalogens and the

amount of secondary follicles were found in lymph

nodes although significant correlations were found for

APBDEs (positive), APCBs (negative) and HCB

(negative) and follicle score. Significantly lower

concentrations of organohalogens in spleen samples

categorized in group 0 (few/absent secondary fol-

licles) were found, which indicated a positive relation-

ship between the OHCs, ACHLs, AHCHs, HCB and

Dieldrin, and the number of secondary follicles in the

spleen. The histopathological evaluation of the few

samples of thymus and thyroid revealed no change

either. In conclusion the data suggest that the exposure

concentrations of contaminants to polar bears in East

Greenland today are unlikely to result in adverse

effects on tissues of lymph nodes, spleen, thyroid and

thymus (histopathology) although an effect, described

as increased secondary follicle count, was observed in

splenic tissue for the contaminants ACHLs, AHCHs,HCB and Dieldrin.

Acknowledgements

Danish Cooperation for Environment in the Arctic

and The Commission for Scientific Research in

Greenland are acknowledged for financial support,

Jonas Brbnlund who gathered the samples through

local hunters, Hanne Tuborg Sandell and Birger

Sandell who helped with local contacts to hunters.

Steen Andersen (Foxtrot) made the instruction video

for the polar bear hunters, for which Lars 2by also

supplied footage. The laboratory technicians at

National Water Research Institute and Great Lakes

Institute for Environmental Research (Mr. Greg Sandla

and Ms. Rodica Lazar) are acknowledged for con-

ducting the chemical analysis. The laboratory techni-

cians at the Laboratory of Pathology for conducting

the histology slides, and finally the reviewers are

acknowledged for valuable comments to this article.

References

AMAP. Amap Assessment 2002: Persistent organic pollutants in the

arctic. Oslo (Norway)7 Arctic Monitoring and Assessment

Programme (AMAP); 2004. xvi+310 pp.

Bergman A. Health condition of the Baltic grey seal (Halichoerus

grypus) during two decades. Apmis 1999;107:270–82.

Bergman A, Olsson M. Pathology of Baltic grey seal and ringed seal

females with special reference to adrenocortical hyperplasia: is

environmental pollution the cause of a widely distributed

disease syndrome? Finn Game Res 1985;44:47–62.

Bergman A, Bergstrand A, Bignert A. Renal lesions in Baltic grey

seals (Halichoerus grypus) and ringed seals (Phoca hispida

botnica). Ambio 2001;30(7):397–409.

Bergmann A, Bignert A, Helander B, Olsson M. Miljfgifta och

marina toppkonsumenter. Ostersjf ’95: 2rsrapport fr3n den

marina miljfovervakningen, Stockholms Marina Forskningscen-

trum. Sweden7 Stockholm University; 1996. p. 43–8.

Bernhoft A, Wiig O, Skaare JU. Organohalogens in polar bears

(Ursus maritimus) at Svalbard. Environ Pollut 1997;96:159–75.

Bernhoft A, Skaare JU, Wiig O, Derocher AE, Larsen HJS. Possible

immunotoxic effects of organochlorines in polar bears (Ursus

maritimus) at Svalbard. J Toxicol Environ Health A 2000;

59(7):561–74.

Blomkvist G, Roos A, Jensen S, Bignert A, Olsson M. Concen-

trations of sDDT and PCB in seals from Swedish and Scottish

waters. AMBIO 1992;21(8):539–45.

Born EW, Bfcher J. The ecology of Greenland. Nuuk7 Ministry of

Environment and Natural Resources; 2001.

Brouwer A, Morse DC, Lans MC, Schuur AG, Murk AJ, Klassen-

Wehler E, et al. Interactions of persistent environmental

organohalogens with the thyroid hormone system: mechanism

and possible consequences for animal. J Toxicol Ind Health

1998;14:59–84.

Colborn T, Vom Saal FS, Soto AM. Developmental effects of

endocrine-disrupting chemicals in wildlife and humans. Environ

Health Perspect 1993;101:378–84.

Damstra T, Barlow S, Bergman A, Kavlock R, Kraak GVD. Global

assessment of the state-of-the-science of endocrine disruptors-

WHO;2002. 180 pp.

Darnerud PO. Toxic effects of brominated flame retardants in man

and in wildlife. Environ Int 2003;29:841–51.

Darnerud PO, Thuvander A. Studies on immunological effects of

polybrominated diphenyl ethers (PBDE) and polychlorinated

biphenyls (PCB) exposure in rats and mice. Organohalog

Compd 1998;35:415–8.

Dellman HD, Eurell J, 1998. Textbook of veterinary histology, fifth

ed. Oslo (Norway)7 Lippincott Williams and Wilkins;1998. p.

128–303.

de March BGE, de Wit C, Muir DGC, Braune B, Gregor DJ,

Norstrom RJ, et al. Chapter 6: persistent organic pollutants.

AMAP assessment report: arctic pollution issues. Oslo

(Norway)7 Arctic Monitoring and Assessment Programme;

1998. p. 183–372.

Dietz R, Heide-Jbrgensen MP, H7rkfnen T, Teilmann J, Valentin N.

Age determination of European harbour seal (Phoca vitulina L).

Sarsia 1991;76:17–21.

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132 131

Dietz R, Riget F, Sonne-Hansen C, Letcher R, Born EW, Muir

DCG. Polychlorinated biphenyls and organochlorine pesticides

in East Greenland polar bears (Ursus maritimus), 1990–2001.

Sci Total Environ 2004;331:107–24.

Dyce KM, Sack WO, Wensing CJG, 1995. Veterinary anatomy, 3rd

ed. Saunders;1987. p. 417–35.

Feldman EC. Hyperadrenocorticism. In: Ettinger SJ, Feldman EC,

editors. Textbook of veterinary internal medicine, vol. II.

Philadelphia7 W.B. Saunders; 1995. p. 1538–78.

Fisk AT, de Wit C, Wayland M, Kuzyk ZZ, Burgess N, Letcher R,

et al. An assessment of the toxicological significance of

anthropogenic contaminants in Canadian arctic wildlife. Sci

Total Environ;2004 [submitted for publication].

Fowles JR, Fairbrother A, Baecher-Steppan L, Kerkvliet N.

Immunologic and endocrine effects of the flame-retardant

pentabromondiphenyl ether (DE-71) in C57BL/6J mice. Tox-

icology 1994;86:49–61.

Grundmann E, Vollmer E. Cell types and functions. Reaction

patterns of the lymph nodeCurrent topics in pathology, vol. 84.

Berlin7 Springer; 1990. p. 288 part 1.

Guitart R, Puig P, Gomeezcacatalan J. Requirements for a stand-

ardidized nomenclature criterium for PCBs-computer-assisted

assignment of correct congener denomination and numbering.

Chemosphere 1993;27(8):1451–9.

Haave M, Ropstad E, Derocher AE, Lie E, Dahl E, Wiig a., et al.Polychlorinated biphenyls and reproductive hormones in female

polar bears at Svalbard. Environ Health Perspect 2003;111(4):

431–6.

Habel RE. Splanchnologia. In: Schaller Oscar, editor. Illustrated

veterinary anatomical nomenclature. Stuttgard (Germany)7 Enke

Verlag; 1992. p. 194–7.

Hakk H, Letcher RJ. Metabolism in the toxicokinetics and fate of

brominated flame retardants—a review. Environ Int 2003;29:

829–39.

Henriksen EO, Wiig O, Skaare JU, Gabrielsen GW, Derocher AE.

Monitoring PCBs in polar bears: lessons learned from Svalbard.

J Environ Monit 2001;3:493–8.

Hensel RJ, Sorensen FE. Age determination of live polar bears. Int

Conf Bear Res Manage 1980;4:95–100.

Hood A, Hashmi R, Klaassen CD. Effects of microsomal enzyme

inducers on the thyroid-follicular cell proliferation, hyper-

plasia, and hypertrophy. Toxicol Appl Pharmacol 1999;160(2):

163–70.

Khan MA, Lichtensteiger CA, Faroon O, Mumtaz M, Schaeffer DJ,

Hansen LG, 2002. Toxicol Sci 2002;65(1):52–61.

Koponen K, Myers MS, Ritola O, Huuskonen SE, Seppa-Lindstrom

P. Histopathology of feral fish from a PCB-contaminated

freshwater lake. Ambio 2001;30(3):122–6.

Legler J, Brouwer A. Are brominated flame retardants endocrine

disruptors? Environ Int 2003;29:879–85.

Letcher RJ, Nordstrom RJ, Lin S, Ramsay MA, Bandiera SM.

Immunoquantitation and microsomal monooxygenase activities

of hepatic cytochromes P450 1A and P450 2B and chlorinated

hydrocarbon contaminant levels in polar bear (Ursus mariti-

mus). Toxicol Appl Pharmacol 1996;137:127–40.

Letcher RJ, Lemmen JG, van der Burg B, Brouwer A, Bergman A,

Giesy JP, et al. In vitro antiestrogenic effects of aryl methyl

sulfone metabolites of polychlorinated biphenyls and 2,2-bis(4-

chlorophenyl)-1,1-dichloroethene on 17 beta-estradiol-induced

gene expression in several bioassay systems. Toxicol Sci 2002;

69(2):362–72.

Lie E, Larsen HJS, Derocher AE, Lunn N, Norstrom R,Wiig O, et al.

Do POPs impair the infection resistance of polar bears (Ursus

maritimus). AMAP conference abstract; 2002.

Lie E, Jbrgen H, Larsen S, Johansen GM, Derocher AE, Lunn NJ,

et al. Does high organochlorine (OC) exposure impair the

resistance to infection in polar bears (Ursus maritimus)?: Part I

Effect of OCs on the humoral immunity. J Toxicol Environ

Health 2004a;67:555–82.

Lie E, Jbrgen H, Larsen S, Johansen GM, Derocher AE, Lunn NJ,

et al. Does high organochlorine (OC) exposure impair the

resistance to infection in polar bears (Ursus maritimus)?: Part I

Effect of OCs on mitogen and antigen induced lymphocyte

profileration. J Toxicol Environ Health 2004b [in press].

Lucas B, Vetler W, Fischer P, Heidemann G, Plotz J. Characteristic

chlorinated-hydrocarbon patterns in the blubber of seals from

different marine regions. Chemosphere 1990;21(1–2):13–9.

Luross JM, Alaee M, Sergeant DB, Cannon CM, Whittle DM,

Solomon KR, et al. Spatial distribution of polybrominated

diphenyl ethers and polybrominated biphenyls in lake trout from

the Laurentian Great Lakes. Chemosphere 2002;46:665–72.

Luster MI, Munson AE, Thomas PT, Holsapple MP, Fenters JD,

White Jr KL, et al. Development of a testing battery to assess

chemical-induced immunotoxicity: National Toxicology Pro-

gram’s guidelines for immunotoxicity evaluation in mice.

Fundam Appl Toxicol 1988;10:2–19.

McDonald TA. A perspective on the potential health risks of

PBDEs. Chemosphere 2002;46:745–55.

Meerts IATM, van Zanden JJ, Luijks EA, van Leeuwen-Bol I,

Marsh G, Jakobsson E, et al. Potent competitive interactions of

some brominated flame retardants and related compounds with

human transthyretin in vitro. Toxicol Sci 2000;56:95–104.

Mortensen P, Bergmann A, Bignert A, Hansen H, Harkonen T,

Olsson M. Prevalence of skull lesions in harbour seals (Phoca

vitulina) in Swedish and Danish museum collections 1835–

1988. AMBIO 1992;21(8):520–4.

Muir, DCG, Dietz, R, Riget, FF, Sonne, C, Letcher, RJ, Born, EW,

Polybrominated diphenylethers in East Greenland polar bears

(Ursus maritimus) 1990–2001. [in preparation].

Ness DK, Schantz SL, Moshtaghian J, Hansen LG. Effects of

perinatal exposure to specific PCB congeners on the thyroid-

hormone concentrations and thyroid histology in rat. Toxicol

Lett 1993;68(3):311–23.

Nielsen EH, 1977. Kompendium i speciel histologi, 3.ed. Denmark7

F.A.D.L.s Forlag; 1977. p. 11–162.

Norstrom RJ, Belikov SE, Born EW, Garner GW, Malone B,

Olpinski S, et al. Chlorinated hydrocarbon contaminants in polar

bears from eastern Russia, North America, Greenland, and

Svalbard: biomonitoring of arctic pollution. Arch Environ

Contam Toxicol 1998;35:354–67.

Oskam IC, Ropstad E, Dahl E, Lie E, Derocher AE, Wiig a, et al.Organochlorines affect the major androgenic hormone, testos-

terone, in male polar bears (Ursus maritimus) at Svalbard. J

Toxicol Environ Health A 2003;66(22):2119–39.

M. Kirkegaard et al. / Science of the Total Environment 341 (2005) 119–132132

Polischuk S, Ramsay M, Norstrom RJ. Body burdens and tissue

concentrations of organohalogens in polar bears (Ursus mariti-

mus) vary during seasonal fasts. Environ Pollut 2002;118:29–39.

Riget FF, Johansen P, Glasius M, Vorkamp K, Dahlgaard H, Muir D,

et al. Chapter 4 Marine environment. In: Riget FF, Christensen J,

Johansen P, editors. AMAP Greenland and the Faroe Islands

1997–2001. Denmark7 Ministry of Environment; 2003.

Riget, FF. Senior scientist, National Environmental Research

Institute, PO Box 358, Frederiksborgvej 399, DK-4000 Ros-

kilde, Denmark; Unpublished data. Ph: (+45) 46301948; e-mail:

ffr@dmu.dk.

Ross PS, de Swart RL, Reijnders PJH, Van Loveren H, Vos JGV,

Osterhaus ADME. Contaminant related suppression of delayed-

type hypersensitivity and antibody response in harbour seals fed

herring from the Baltic Sea. Environ Health Perspect 1995;103:

162–7.

Ross PS, de Swart RL, Timmermann HH, Reijnders PJH, Vos JGV,

Van Loveren H, et al. Suppression of natural killer cell activity

in harbour seals (Phoca vitulina) fed Baltic Sea herring. Aquat

Toxicol 1996;34:71–84.

Saeed A, Hansen LG. Morphometric changes in the prepubertal

femal rat thyroid gland following acute exposure to 2,2V44V-tetrachlorobiphenyl and aroclor 1242. J Toxicol Environ Health

1997;51(5):503–13.

Sandala GM, Sonne-Hansen C, Dietz R, Muir DCG, Valters K,

Bennett ER, et al. Methyl sulfone and hydroxylated PCB

metabolites in adipose and whole blood of polar bear (Ursus

maritimus) from Scoresby Sound, Greenland. Sci Total Environ

2004;331:125–41.

Sandau CD, Meerts IATM, Letcher RJ, McAlees AJ, Chittim B,

Brouwer A, et al. Identification of 4-hydroxyheptachlorostyrene

in polar bear plasma and its binding affinity to transthyretin: a

metabolite of octachlorostyrene? Environ Sci Technol 2000;34:

3871–7.

Schandorff S. Developmental stability and skull lesions in the

harbour seal (Phoca vitulina) in the 19th and 20th centuries. Ann

Zool Fennici 1997;34:151–66.

Schumacher U, Zahler S, Horny HP, Heidemann G, Skirnisson K,

Welsch U. Histological investigations on the thyroids of marine

mammals (Phoca vitulina , Phocoena phocoena) and the

possible implications of marine pollution. J Wildl Dis 1993;

29(1):103–8.

Siebert U, Vossen A, Baumg7rtner W, Mqller G, Beineke A,

McLachlan M, et al. Investigations of the influence of Pollutants

on the endocrinium and immune system of harbour porpoise in

the German North and Baltic Seas. UBA F+E Vorhaben FKZ 2

99 65 211/01 2002:1–308.

Skaare JU, Bernhoft A,Wiiga., NorumKR, Haug E, Eide DM, et al.

Relationship between plasma levels of organochlorines, retinol

and thyroid hormones from polar bears (Ursus maritimus) at

Svalbard. J Toxicol Environ Health A 2001;62:227–41.

Skaare JU, Hansen HJ, Lie E, Ropstad E, Bernhoft A, Derocher AE,

et al. Relationships between high loads of organic contaminants

and health effects in arctic mammals—immune response and

chlorinated environmental pollutants in polar bear. Pharmacol

Toxicol 2001;88(Suppl. 1):14.

Stevens A, Lowe J, 1994. Human histology, 2nd ed. New

York7 Mosby;1997. p. 117–258.

Swart RD, Ross PS, Vedder LJ, Timmerman HH, Heisterkamp S,

Loveren HV, et al. Impairment of immune function in harbour

seals (Phoca vitulina) feeding on fish from polluted waters.

Ambio 1994;23:155–9.

Thuvander A, Darnerud PO. Effects of polybrominated diphenyl

ether (PBDE) and polychlorinated biphenyl (PCB) on some

immunological parameters after oral exposure in rats and mice.

Toxicol Environ Chem 1999;70:229–42.

Tryphonas H. Immunotoxicity of polychlorinated biphenyls: present

status and further considerations. Exp Clin Immunogenet 1994;

11:149–62.

Vos JG, Luster MI. Immune alterations. In: Kimborough RD, Jensen

S, editors. Halogenated biphenyls, terphenyls, naphthalenes,

dibenzodioxins and related products. New York7 Elseviere

Science Publishers; 1989. p. 295–322.

Welsch U, Schwertfirm S, Skirnisson K, Shumacher U. Histolog-

ical, histochemical and fine structural observations on the lymph

node of the common seal (Phoca vitulina) and the grey seal

(Halichoerus grypus). Anat Rec 1997;247:225–42.

Wiig a, Derocher AE, Cronin MM, Skaare JU. Female pseudo-

hermaphrodite polar bears at Svalbard. J Wildl Dis 1998;34(4):

792–6.

Wong S, Fournier M, Coderre D, Banska W, Krzystyniak K.

Environmental immunotoxicology. In: Peakall DB, editor.

Animal biomarkers as pollution indicators. London7 Chapman

and Hall; 1992. p. 8–189.