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Exposure to persistent organic pollutants reduces testosterone concentrations and affects sperm viability and morphology during the mating peak-period in a controlled experiment on farmed Arctic foxes (Vulpes lagopus) Christian Sonne, Peter A. Torjesen, Eva Fuglei, Derek C.G. Muir, Bjorn M. Jenssen, Even Jørgensen, Rune Dietz, and Øystein Ahlstrøm Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b00289 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 19, 2017

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Environmental Science & Technology

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Exposure to persistent organic pollutants reduces testosterone concentrations and

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affects sperm viability and morphology during the mating peak-period in a controlled

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experiment on farmed Arctic foxes (Vulpes lagopus)

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Christian Sonne1,*, Peter A. Torjesen2, Eva Fuglei3, Derek C. G. Muir4, Bjørn Munro

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Jenssen1,5,6, Even Jørgensen7, Rune Dietz1 and Øystein Ahlstrøm8

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1

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and Technology, Frederiksborgvej 399, POBox 358, DK-4000 Roskilde, Denmark (Sonne:

Department of Bioscience, Arctic Research Centre (ARC), Aarhus University, Faculty of Science

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[email protected]; R. Dietz: [email protected])

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2

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Norway (mailto:[email protected])

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Norwegian Polar Institute, Fram Centre, NO-9296 Tromsø, Norway ([email protected])

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Aquatic Contaminants Research Division, Environment and Climate Change Canada, Burlington ON

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Canada L7S 1A1 ([email protected])

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5

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Norway ([email protected])

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6

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Longyearbyen

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7

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Tromsø, Norway ([email protected])

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8

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Ås, Norway ([email protected])

Department of Endocrinology, Hormone Laboratory, Oslo University Hospital, NO-0514 Oslo,

Department of Biology, Norwegian University of Science and Technology, NO-7491 Trondheim,

Department of Arctic Technology, The University Centre in Svarbard, POBox 156, NO-9171

Department of Arctic and Marine Biology, UiT the Arctic University of Norway, N, NO-9037

Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, NO-1433

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*Corresponding author: Professor Christian Sonne, DScVetMed, PhD, DVM, Dipl. ECZM

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(Wildlife Health), Aarhus University, Faculty of Science and Technology, Department of

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Bioscience, Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark. Tel. +45 3078

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3172; fax: +45 8715 5015; Email address: [email protected] (C. Sonne).

1 ACS Paragon Plus Environment


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Abstract

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We investigated testosterone production and semen parameters in farmed Arctic foxes by

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dietary exposure to persistent organic pollutants (POPs) for 22 months. Eight male foxes were

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given a diet of POP-contaminated minke whale blubber whereas their eight male siblings

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were fed a control diet containing pig fat as main fat source. The minke whale-based feed

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contained a ∑POPs concentration of 802 ng/g ww whereas the pig-based feed contained

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∑POPs of 24 ng/g ww. At the end of the experiment, ∑POP concentrations in adipose tissue

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were 8856±2535 ng/g ww in the exposed foxes and 1264±539 ng/g ww in the control foxes.

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The exposed group had 45-64% significantly lower testosterone concentrations during their

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peak mating season compared to the controls (p ≤ 0.05), while the number of dead and defect

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sperm cells was 27% (p = 0.07) and 15% (p = 0.33) higher in the exposed group. Similar

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effects during the mating season in wild Arctic foxes may affect mating behavior and

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reproductive success. Based on these results we recommend testosterone as a sensitive

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biomarker of POP exposure and that seasonal patterns are investigated when interpreting

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putative endocrine disruption in Arctic wildlife with potential population-level effects.

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Keywords: Arctic, Endocrine disruption, Health effects, Pollution, Reproductive organs, Top

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predator.


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Introduction

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Arctic top predators biomagnify high concentrations of lipophilic persistent organic pollutants

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(POPs) due to their trophic position and the lipid rich Arctic food chains.1-4 The group of

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POPs includes persistent environmental contaminants such as polychlorinated biphenyls

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(PCBs) known to have endocrine disruptive and immune toxic effects in Arctic wildlife such

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as polar bears (Ursus maritimus) and Arctic foxes (Vulpes lagopus).1,5,6 Previous controlled

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exposure studies have focused on using carnivorous model species to investigate effects of

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POPs in Arctic predatory species since studies of wild living animals often only allow

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correlative comparison of exposure and biomarker endpoints. Such model species have

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included the Greenland sledge dog (Canis familiaris) and farmed Arctic foxes since these are

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phylogenetically close to e.g. polar bears and wild Arctic foxes.1 Such an experimental

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approach allows investigating multigenerational effects of POPs on e.g. immune and

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endocrine endpoints that are relevant for wildlife toxicology.1,7-9 It also allows mimicking the

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seasonal variability in food availability and emaciation, which is a characteristic trait in

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endemic Arctic wildlife.1 For example, polar bears may starve for extended periods during

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gestation, denning and ice-free summer periods.10,11

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Wild Arctic foxes mate from the end of February until mid-April.12 They are

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monogamous and pairs are territorial in the breeding season from end of February until late

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August where the pups leave the den. The gestation period is 52-54 days and the foxes deliver

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around mid-May. Their body fat is naturally depleting during spring to reach the lowest in

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summer (June-July) when it constitutes only 6% of the body mass (June-July).13 In addition,

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they may experience periods during winter where they do not find food and starve for days or

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weeks.13,14 During periods when stored body lipids are mobilized, lipophilic POPs are

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released from the adipose tissue and become bioavailable and redistributed to sensitive organs

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such as e.g. thyroid glands, gonads and central nervous system.15,16 Since the endocrine



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glands and the central nervous system are vital for reproduction, the starvation-related peaks

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in POP exposure during the reproductive season are of concern for Arctic wildlife.1,2

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Effects on reproductive organs are important to assess when studying POP exposure in

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wildlife key species as it affects the demographics and population size via the effect on

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survival and recruitment.17-19 Investigating the relationship between POPs and sex steroids

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gives an indication of endocrine disruption and adverse reproductive health effects on e.g.

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semen quality and testicular dysgenesis syndrome (TDS), which includes development of

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hypospadias, testicular malign neoplasms and cryptorchidism.20 To date, it has not been

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possible to investigate the relationship between POP exposure and semen quality in any

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Arctic wildlife species while several human studies show that exposure to POPs affects semen

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quality and decrease testosterone concentrations.21,22

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To investigate the effects of POPs on the seasonal pattern of testosterone

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concentrations and semen parameters in Arctic top predators, we conducted a 2-year

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exposure-control trial. Eight brother-pairs of farmed Arctic fox were exposed to either a diet

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containing naturally POP-contaminated minke whale blubber (Balaenoptera acutorostrata) or

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to a control diet containing lard from domesticated pigs (Sus scrofa) as fat source. From

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weaning to 2-years of age, foxes were exposed to these respective diets. After 17 months of

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exposure (August 2003 to January 2005) we obtained weekly blood samples during the period

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January to April 2005 while semen was sampled in early April. The foxes were euthanized in

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June 2005 and abdominal adipose tissue collected to study the bioaccumulation of POPs. Here

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we report on the differences in testosterone concentrations and semen quality during the

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spring peak of mating among the POP exposed foxes and their controls.

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Material and Methods

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Housing and feeding


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Eight newly weaned brother-pairs of male foxes (54 days old) were separated into two

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groups, one POP exposed group (n=8) and one control group (n=8) (Table 1). The groups

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were balanced with respect to body mass and genotype (brother pairs) and all foxes were

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individually housed in semi-outdoor cages (1.5×1.2×1.0 m) exposed to natural photoperiod

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and ambient temperature at the Norwegian University of Life Sciences, Ås, Norway. The

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exposed group received wet feed with minke whale blubber as main fat source, whereas the

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control group received lard from pigs as main fat source. The whale-based feed had ∑POPs

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concentrations of 802 ng/g ww while the lard-based feed had ∑POPs concentrations of 24

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ng/g ww. Further information on the composition of the two diets with respect to various

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ingredients and POP concentrations and compositions are published elsewhere.15,23 To

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simulate the changes in annual feeding and body fat content of free-ranging Arctic foxes, both

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groups were given high-energy feed from 13 Aug 2003 to 4 Jan 2004 and again from 8 Aug

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2004 to 28 Nov 2004. During the period Jan 2004 to Aug 2004 and again from Nov 2004 to

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June 2005, the foxes were all fed low energy feed. This is a recommended feeding regime that

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simulates the natural cycling in food access during autumn when animals deposit body fat

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which they rely on as energy source during winter/spring when food resources are scarce or

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absent. The two groups received identical rations of feed throughout the study. Feed was

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given once a day, and water was supplied ad libitum. The experiment was carried out at the

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research farm at the Norwegian University of Life Sciences, Ås, Norway and performed on a

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licence granted by the Norwegian Animal Research Committee (www.fdu.no). All

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experimental procedures followed Norwegian protocols for ethical standards for the use of

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live animals and the experiments were treated according to national and international

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guidelines for animal research.

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Sampling



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During Jan-April 2005, blood was regularly collected once a week to explore the seasonal

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variation of plasma testosterone in relation to their mating season in March/April, while

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semen was collected in the first week of April 2005. Plasma was separated by centrifugation

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at 1613 G (9 cm diameter, 4000 rpm) for 15 minutes and transferred to Eppendorf tubes, and

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kept frozen at -20°C pending analysis. For semen collection, each male was brought indoor

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one by one and placed in a purpose made box for semen collection. The fox was fixated with

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a neck lock at the end of the box. Testicles were palpated and graded for firmness and size.

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Ejaculates for quality assessment were obtained by masturbation. It was possible to collect

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ejaculates of semen and prostate secretion from seven controls and eight exposed foxes. These

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samples were taken directly in a plastic semen glass for further quality assessment of volume,

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density, prostate fluid, live/dead cells, normal/defect cells and motility (Table 1).

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On 16 June 2005, the foxes (24 months old) were fasted for 24 h and sacrificed using electrocution.

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Testosterone analysis and semen data

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Concentrations of testosterone (T) in plasma were determined using a RIA kit from Orion

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Diagnostica, Turku, Finland after ether extraction. The intra-assay and total assay CV were

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7% and 9%, respectively. Data are given in nmol/L. Further details on the analyses are found

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in Grindflek et al. (24) and Hallanger et al. (8). The quality assessment of the semen included

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volume (measured directly from the collection glass; mL), density (subjectively graded

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visually from 0-4), prostate fluid (graded visually 0 or 1), % sperm cell alive (subjectively

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graded by microscopy ×200 of a native unstained semen smear), motility (subjectively graded

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by microscopy from 0-3), % normal and testes weight (graded by palpation 0-5).

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Persistent organic pollutants (POPs) analysis


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Analyses were conducted using methods described in Johansen et al. (25). In brief, samples

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were homogenized and Soxhlet extracted with dichloromethane. PCB/OCPs were isolated

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from lipid co-extractives by gel permeation chromatography followed by fractionation on a

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silica gel column. Extracts were analyzed for 104 PCB congeners and 35 OCPs and

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chlorinated by-products using gas chromatography with electron capture detection. The

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compounds presented used in the present investigation included ∑PCB (polychlorinated

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biphenyls: CB-1, 3, 4-10, 7-9, 6, 8-5, 19, 12-13, 18, 15-17, 24-27, 16-32, 54-29, 26, 25, 31-

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28, 50, 33-20, 53, 51, 22, 45, 46, 52, 43, 49, 47-48, 44, 59, 42, 71-41-64, 40, 100, 63, 74, 70-

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76-98, 66, 95, 91, 55, 56-60, 92, 84, 101, 99, 119, 82, 97, 81-87, 136, 110, 82, 151, 135-144,

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147, 107, 149, 118, 133, 114-134, 131, 146, 153, 132, 105, 141, 179, 137, 176, 130, 163-138,

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158, 129, 178, 175, 182-187, 183, 128, 167, 185, 174, 177, 202-171, 156, 173, 157-200, 172,

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197, 180, 193, 191, 199, 170-190, 198, 201, 203-196, 189, 208-195, 207, 194, 205, 206, 209),

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∑PCB10 (CB-180, CB-156, CB-163/138, CB-105, CB-153, CB-118, CB-101, CB-52, CB-

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31/28), ∑DDT (dichlorodiphenyltrichloroethane: o,p-DDD, p,p-DDD, o,p-DDE, p,p-DDE,

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o,p-DDT, p,p-DDT), ∑CHL (chlordanes: γ-Chlordane, Heptachlor Epoxide, Oxychlordane, α-

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Chlordane, transnonachlor, cis-nonachlor, Heptachlor), ∑HCH (hexachlorohexane: α-HCH, β-

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HCH, γ-HCH), ∑CBZ (1245-TTCB, PECB, HCB) and ∑POPs (Table 1). Certified reference

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materials from the National Institute of Standards and Testing (NIST 1774b mussel, NIST

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1588a cod liver oil), and laboratory blanks consisting of all reagents, were analyzed with each

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batch of samples. All concentrations are given as ng/g ww.

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Statistical analyses

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All data were analyzed prior to statistical analyses for meeting the criteria of normal

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distribution, equal variance and homoscedasticity. A two-sided, two-sample student t-test was

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applied to test for differences between control and exposed groups. All statistical analysis was



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conducted using SAS 9.3 for Windows. The statistical significance was set to α=0.05 while

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0.05 < p < 0.1 was considered a trend.

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Results

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A summary of the results of the testosterone, semen and POP analyses is given in Table 1.

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Plasma testosterone concentrations were analyzed in all samples taken weekly during the

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period from January to April 2005, semen variables the first week of April 2005 and POP

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concentration at euthanization 16 June 2005. POP concentrations were significantly highest in

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the exposed group for all compounds (t-test: all p > 0.01). According to Figure 1,

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concentrations of especially PCBs, DDTs and Chlordanes were several folds higher in the

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exposed group.

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When comparing the average testosterone concentrations of all pooled data of the

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entire study period no significant difference was found between the two group of control and

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exposed foxes (t-test: p = 0.17; Table 1). Therefore, the data was fitted to a 3rd order

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polynomial model showing that the spring peak of testosterone was around 1 April after

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which there was a decline in the testosterone concentrations in both groups (Figure 2).

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Concentrations of testosterone on 22 March, 11 April and 21 April were significantly lower

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(45-64%) in the exposed group of foxes as compared to controls (t-test: all p ≤ 0.05). Thus, a

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clear and significant difference between the two groups was evident in the peak-period of

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testosterone production.

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The analysed semen variables did not differ between the two groups of exposed and

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control foxes except for the number of live sperm cells (Table 1). The number of dead sperm

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cells was 27% higher in the exposed foxes as compared to the control foxes (Figure 3) and

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that difference was close to being statistically significant (t-test: p=0.07). Similar, the number

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of defect sperm cells was 15% higher in the exposed group (t-test: p = 0.33).

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Discussion

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Testosterone

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The Arctic fox is a seasonal breeder and farmed Arctic foxes have their mating season

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approximately from middle of March to the end of April. The timing of the breeding period is

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governed by the change in day-length, which triggers and regulates hormone production that

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affects reproductive organs. Adult males go through the same annual cycle of testicular

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development or “puberty” as young males does their first year of mating.26 Preparations for

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spermatogenic activity in the testes starts as early as in October/November, and further

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testicular development with increased weight and volume take place until the mating period

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starts in March. In July, testicular regression is completed and baseline testosterone levels are

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reached.26 In farm conditions, one male is normally used to fertilize 6-8 females during

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natural mating, but far more with artificial insemination.

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To our knowledge, no information is available in the international scientific literature

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on semen quality and testosterone concentrations in wild Arctic foxes. Thus, the weekly

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fluctuations during spring peak are not described and therefore the data in the control group is

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the best we have in order to describe the inhibition of POP exposure on testosterone

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concentrations and semen quality. Extrapolating from other arctic top predators such as polar

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bears or even other fox species is not advisable due to species-specific differences. Overall,

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the plasma testosterone concentrations in the present study were similar to those levels

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previously reported in farmed foxes during January-May.27,28 The testosterone concentrations

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that we observed during the reproductive spring peak period were significantly lower in the

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exposed foxes as compared to in the control foxes. Previous results from the study of farmed

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Arctic foxes exposed to POPs at the research facilities at Ås have been reported elsewhere

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and are summarized in Table 2.8,9,15,23,29,30 Hallanger et al. (2012) also reported significant, 4-

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fold lower plasma testosterone concentrations in juvenile POP exposed Arctic foxes

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compared to control foxes. In the study by Rogstad et al. (9) no reduction in testosterone was 9 ACS Paragon Plus Environment


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found in the POP exposed adult arctic foxes; however, since these animals were sampled in

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mid-June the seasonal pattern around spring peak was not taken into account, which may

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explain the results. The results by Hallanger et al. (8) indicate that the exposed juvenile foxes

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had a delayed pubertal reproductive development based on a single endpoint after 4 month of

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POP exposure. In the present study, there were no between-group differences in the

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testosterone concentrations in January after 17 month of POP exposure, which indicate that

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there is no effect on the testosterone concentrations outside the reproductive period.28 It was

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only during the reproductive period March-April with elevated testosterone concentrations

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that there were clear and significant differences between exposed and control foxes with

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exposed foxes having the lowest levels. The increase in testosterone production during the

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reproductive period is important for semen production and reproductive health. Therefore, an

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attenuated testosterone production due to POP exposure is worrying and may influence the

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breeding outcome of wild Arctic foxes in their natural habitats.2,3

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Of relevance to the Arctic, testosterone and testes size has been measured in Svalbard

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and East Greenland polar bears as well as captive sledge dogs and Arctic foxes showing that

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POPs and especially PCBs have an effect on e.g. testosterone concentrations and testes

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size.7,8,31,32,33 Studies of male polar bears from Svalbard also show that testosterone

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concentrations were negatively correlated with ΣPCB.32 A controlled study of West

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Greenland sledge dogs similar to the present also showed that plasma testosterone levels were

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lowest in the exposed group following 12 month of POP exposure.31 These two studies

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support the finding in the present study; that POPs have the potential of depressing plasma

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testosterone concentrations in Arctic male mammals. In both the sledge dog and farmed

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Arctic fox study, higher hepatic Cyp-450 activity was found in the exposed group and that

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may lead to an increased testosterone metabolism reducing the blood concentrations.15,34

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Similar relationships between internal POP concentrations and testosterone concentrations

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have been shown for humans in multiple studies. For example, Schell et al. (21) and Vitku et 10 ACS Paragon Plus Environment

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al. (22) showed that POP exposure was inversely correlated to testosterone concentrations

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likely due to an influence of steroidogenesis via up- and downregulation of enzymes in the

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downstream biotransformation and steroid production.7,35

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Semen parameters

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Semen quality evaluation by microscopy is based on subjectively estimation of motility,

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viability and morphology.36 Farmed blue fox semen normally contains 80-90 % viable

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spermatozoa and 80% with normal progressive motility while motility rates less than 65%

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indicate subnormal fertility.36 Abnormal spermatozoa rate is rarely higher than 5% and levels

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higher than 20% is usually associated with low fertility.36 In the present study, there was a

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clear trend towards lower rates of sperm viability and higher rates of abnormal spermatozoa in

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the exposed group of foxes compared to the controls. In fact, the prevalence of defect sperm

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cells was around 25% in the POP exposed group of foxes, which indicate that the fertility was

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reduced. POP exposure has been reported to cause low quality semen and testicular

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dysgenesis syndrome (TDS) that includes development of hypospadias, testicular malign

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neoplasm (cancer) and cryptorcism.20,22 The difference in sperm cell mortality and

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morphology in the present study was not significant; however, the results point towards that

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POPs could have an effect that is in accordance with other studies of both wild and laboratory

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animals, as well as in humans.22,37-39

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Thyroid hormones, vitamins and Cyp-450

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There are several mechanistic and physiological explanations for the effects of POP exposure

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on the testosterone concentrations during the peak reproductive period in farmed Arctic foxes.

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One is, as previously mentioned, that PCBs may alter the testicular steroidogenesis of

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testosterone formation and also the liver metabolism via increased hepatic Cyp-450

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activity.7,15 Another, and likely very important, explanation may be the indirect effects on 11 ACS Paragon Plus Environment


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testosterone homeostasis from the endocrine disruption of thyroid hormones. In previous

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studies of POP exposed farmed Arctic male foxes at Ås and in POP exposed Greenland

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sledge dogs and polar bears, decreases in FT4:FT3 ratio has been reported.8,9,40-42 Such

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reductions in thyroid hormones are important, as there are mechanisms of crosstalk between

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endocrine systems.43 The crosstalk between hypothalamic-pituitary-thyroid (HPT) and

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hypothalamic-pituitary-gonadal (HPG) axis regulates metabolism and there is increasing

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evidence that THs are also involved in gonadal differentiation and reproductive function as

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well as production of steroids.43 Thyroid hormones seem to affect the synthesis of

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testosterone, which is important for growth and development as well as reproductive function.

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The indirect effects from POPs on vitamins and thyroid hormones may therefore be important

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mechanism for endocrine disruption and testosterone concentrations during mating season in

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male foxes.31,43

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Reproduction

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The PCB concentrations in the exposed farmed foxes was 10-100 folds lower compared to

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those reported in wild Arctic foxes in Svalbard and Iceland but similar to those from other

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Arctic regions.3,44-46 Such geographic differences are related to the diet, since foxes feeding

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from the marine ecosystem have higher levels of PCB compared to foxes feeding a terrestrial

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diet.44,45 Based on the present results, the effects from POP exposure may influence the

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mating season of wild Arctic foxes. Testosterone plays an important role in physiological and

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behavioral traits related to reproduction.47,48 A reduction of testosterone levels due to high

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POP exposure at the time of mating likely have negative effects on the reproductive outcome

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as reported in dogs with reduced testicular function.49 There are no studies that in a simple

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way directly link testosterone concentrations, semen quality and fertility, which disables an

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evaluation of the 45-65% testosterone reduction on reproductive outcome in the present study.

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Males with low testosterone levels may however not be that attracted to mating, pair bonding, 12 ACS Paragon Plus Environment

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or in aggression towards other males. One hypothesis could be that these males could be less

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competitive compared to less contaminated foxes. If large parts of the population suffer from

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high POP levels, this may have a significant negative effect on the population level due to

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possible changes in mating behavior and reproductive success. Arctic adapted species are

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specifically vulnerable to POP exposure because they experience seasonal periods where

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almost all adipose tissue depots are mobilized and the accumulated POPs become

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bioavailable.13,14,44 Such periods are stressful for both sexes and may have a potential negative

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effect on the population level, since reproduction and winter survival are vital demographic

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parameters for the Arctic fox. According to Pedersen et al. (3), Arctic foxes in Svalbard are at

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risk of POP induced effects, however, such a link has not been proven by field studies yet.

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Pharmacokinetic modelling of polar bears and sledge dogs suggest that the Arctic foxes in

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Svalbard may be in risk of reproductive effects caused by exposure to endocrine disrupting

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POPs.50,51

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In summary, concentrations of ∑POPs in adipose tissue of exposed foxes were

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significantly higher than in the control foxes while blood concentrations of testosterone

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followed a seasonal pattern with the lowest concentrations measured in January and the

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highest in mid-March to mid-April, which is the normal mating season of Arctic foxes.

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Testosterone concentrations were significantly lower in the exposed group of foxes during

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their peak reproductive period while semen parameters did not differ much between the two

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groups; however, there was a trend toward a higher number of dead and defect sperm cells in

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the exposed foxes. A POP-induced depression of testosterone levels in the breeding season of

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wild Arctic foxes with very high POP body burden may affect the mating behavior and

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reproductive success, with a potential negative effect on the population level. Based on the

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present results from farmed foxes we conclude that POPs may have negative on reproductive

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performance in wild living Arctic foxes males by reducing testosterone levels and by



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negatively affecting semen quality. We also recommend that the seasonal patterns needs to be

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investigated when interpreting the endocrine disruption in Arctic wildlife.


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Acknowledgements

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The Norwegian Research Council (project no. 153484/S30) is acknowledged for funding the

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study. Furthermore, we thank the staff at the research farm in Ås for proper care of the foxes,

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Karoline Sivertsen, and Ingeborg G. Hallanger for laboratory assistance. Kjell Andersen Berg

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(† 8 September 2006) at Department of Reproduction and Forensic Medicine at Norwegian

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School of Veterinary Science Oslo Norway is acknowledged for conducting the semen

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analyses. Mary Williamson, Ed Sverko and Enzo Barresi (Environment Canada Burlington

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ON) are acknowledged for conducting the analysis of POPs. The authors declare no

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competing financial interest.



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References

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1. Sonne, C. Health effects from long-range transported contaminants in Arctic top predators:

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An integrated review based on studies of polar bears and relevant model species. Environ. Int.

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511 512 513 514 515


TABLES Table 1. Data on biometrics, testosterone, semen and POPs analysed in the group of control (n=8) and exposed (n=8) farmed Arctic foxes dietary exposed to persistent organic pollutants for 22 months. Data given as mean±SD. Biometrics Body weight (kg) Testes weight (0-5) Testosterone (nmol/L) 07-Jan 14-Jan 21-Jan 28-Jan 04-Feb 09-Feb 15-Feb 25-Feb 07-Mar 18-Mar 22-Mar 01-Apr 11-Apr 21-Apr Mean Jan-Apr Semen Volume (mL) Density (0-4) Prostate fluid (0-1) Alive sperm cells (%) Dead sperm cells (%) Motility (0-3) Normal sperm cells (%) Defect sperm cells (%) POPs (ng/g ww) ∑PCB ∑PCB10 ∑DDT ∑CHL ∑HCH ∑CBZ ∑POPs

516 517

Control

Exposed

5.54±0.50 4.57±1.13

5.39±0.44 4.43±0.79

0.89±0.77 1.99±1.81 1.26±0.95 1.13±0.87 2.15±1.31 2.15±1.28 3.10±1.86 4.16±1.86 3.27±1.04 6.73±1.62 6.85±3.64 8.29±3.07 4.19±2.63 4.26±1.87 3.49±2.84

1.33±0.90 1.11±1.01 1.48±1.27 1.77±1.29 2.02±1.20 2.02±1.23 1.92±1.07 4.49±2.60 2.84±1.76 5.10±3.60 3.49±2.38* 6.13±3.47 1.52±0.77* 2.4±1.83* 2.68±2.37

0.44±0.42 1.86±0.90 0.71±0.49 82.50±31.81 17.50±6.89 2.00±1.00 90±5.48 10±5.48

0.53±0.35 2.29±1.38 0.57±0.53 55.71±32.71*tr 44.29±32.71*tr 2.00±1.29 75.71±33.72 24.29±33.72

443±193 290±132 3±1 73±31 2±0.3 5±1 816±3

2771±798* 1638±1071* 362±684* 1041±733* 25±7 21±15 5859±1984*

*: significant difference between control and exposed group at p < 0.05. *tr: non-significant trend (p = 0.07).



518 519 520

Table 2. Changes in thyroid hormones (TT3, TT4, T4, T3, FT3, FT4), testosterone (T) and vitamin parameters of farmed Arctic foxes experimentally exposed to POPs for 4-22 months. ↓: decrease; ↑: increase; : No change.

TT3

FT3

TT4

FT4

TT3: FT3

a

a

a

a

b

521

Page 24 of 29

a

T4: T3 ↓c b

TT4: FT4 ↑c

TT4: TT3 c

c

FT4: FT3 ,↓c

T ,↓a,b d

Vit A Liver

Vit A Blood

a

a

Sources: Hallanger et al. (8); Rogstad et al. (9); Sonne (1), Helgason et al. (15).


Vit E Liver ↓b

Vit E Blood ↓a

Cyp450 ↑d

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522 523 524 525 526 527 528 529 530 531 532 533 534 535 536


FIGURE LEGENDS Figure 1. Concentrations of POPs (ng/g ww) in abdominal adipose tissue from the control and exposed group of farmed Arctic foxes fed a diet containing lard and minke whale lipids, respectively, for 2 years. Data is given as mean values while error bars indicate SD. *: statistical significant difference (p < 0.01). Figure 2. Concentrations of testosterone (nmol/L) for each of the 14 weeks (7 Jan-21 Apr 2005) that testosterone was measured in blood of each farmed Arctic fox of the control and exposed group included in the study. Mean data fitted a 3rd order polynomial model. Figure 3. Semen data for exposed and control group of farmed Arctic foxes kept in the controlled experiment for 2 years. Data is given as mean values while error bars indicate SD. *tr: trend (p = 0.07). See Table 1 for more information.



537 538

Page 26 of 29

FIGURES 9000

Control

8000

*

Exposed

7000

ng/g ww

6000 5000 4000

* *

3000

*

2000

*

1000 0

539 540

TOTAL PCB

sPCB10

sDDT

SCHL

SHCH

FIGURE 1

26

ACS Paragon Plus Environment

SCBZ

Sum POPs

Page 27 of 29


12

* 10

nmol/L

8

* 6

*

4

2

0 24-Dec

13-Jan

2-Feb Controls

541 542

22-Feb Exposed

13-Mar Poly. (Controls)

FIGURE 2

27


2-Apr Poly. (Exposed)

22-Apr

12-May


*tr

100 90 80 70 60 50 40 30 20 10 0

Controls Exposed

Volume (mL)

543 544

Page 28 of 29

Density (0-4)

Prostate fluid (0-1) Alive sperm cells (%)

Motility (0-3)

FIGURE 3

28


Normal sperm cells Testis weight (0-5) (%)

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254x190mm (96 x 96 DPI)


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