Arch Environ Contam Toxicol (2010) 59:652–660 DOI 10.1007/s00244-010-9510-9

Polychlorinated Biphenyls and Reproductive Performance in Otters From the Norwegian Coast Hanne Christensen • Thrine Moen Heggberget Arno C. Gutleb

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Received: 3 October 2009 / Accepted: 22 March 2010 / Published online: 11 April 2010 Ó Springer Science+Business Media, LLC 2010

Abstract Eurasian otter (Lutra lutra) has shown decreasing population trends in most areas of Europe until recently, when populations in some areas started to recover. For Norway it was postulated that PCB concentrations in the south would be high and that levels in otters will show a geographic pattern that can be related to female otter reproductive health. Concentrations of PCBs (measured as the sum of 30 congeners ranging from 0.58 to 29 mg/kg lipid weight [geometric mean 6.18 mg/kg]) were lower than those found in otters from most other European countries. PCB concentrations did not decrease in otters collected during the period from 1979 to 1990. However, a south-to-north gradient of increasing PCB concentrations in otter livers was found along the Norwegian coast. Actual PCB concentrations had not affected the reproductive health of female otters (implanted embryos, implantation sites, regressive structures). This provides valuable information on PCB concentrations tolerated by Eurasian otters at the population level.

Eurasian otter (Lutra lutra) populations showed a substantial decline across much of their European range during the period from 1960 until approximately a decade ago

H. Christensen
T. M. Heggberget Norwegian Institute for Nature Research, 7004 Trondheim, Norway A. C. Gutleb (&) Department Environment and Agro-Biotechnologies, Centre de Recherche Public–Gabriel Lippmann, 4422 Belvaux, Grand-Duchy of Luxembourg, France e-mail: [email protected]

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despite the fact that otter hunting, which was the main cause of declining populations in the decades before 1960, was banned throughout almost all of Europe (Mason and Macdonald 1986). In the last 10–15 years, populations have started to recover in many of the areas that had previously experienced declining populations (Christensen 2007; Kranz et al. 2007; Ruiz-Olmo 2007). This dramatic decline of the otter is generally attributed to contaminants accumulating in the aquatic food chain. However, although pollution is generally agreed on as the factor to have caused the final decline of already dwindling populations in Europe, the actual causative agent is still controversial. Three groups of compounds are possibly responsible: polychlorinated biphenyls (PCBs) (Sandegren et al. 1980; Olsson et al. 1981; Broekhuizen 1989; Leonards et al. 1996a; Smit et al. 1998; Gutleb 2002; Gutleb and Jefferies 2007), dieldrin (Jefferies and Hanson 2002), and mercury (Gutleb et al. 1998b; Mason 1989; Kruuk 1995). With the exception of negative correlation of PCBs with retinoid levels and health status of otters (Leonards et al. 1996a; Simpson et al. 2000; Gutleb and Murk 2002) virtually no biologic data to support or reject the role of these three groups in otter declines has been published. The current study was originally designed to test the hypothesis that otters along the southern coast of Norway have disappeared as a result of high PCB levels and consequent reproductive failure and is part of a thesis (Christensen 1995) that has never been published in peerreviewed form. At this stage of discussion, which compounds were responsible for the decline of otters is still ongoing. Therefore, data that can support either hypothesis are of importance because otter populations are extending into areas that still have high contaminant levels.

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Animals, Materials, and Methods Collection of Otters Between 1972 and 1992, [1000 otter carcasses were collected along the coast of Norway and sent to the Norwegian Institute for Nature Research (NINA). The majority of these animals were found between 61°N and 71°N. Approximately 80% of these animals were killed in road accidents or drowned in fishnets. Of all these animals, only 135 female otters were in good enough condition that detailed investigation of the reproductive tract could be done. It has been well established that the majority of otters found dead are male (Kruuk 1995; Gutleb et al. 1998a, b). One hundred ten animals of both sexes and all age categories were selected for analyses of PCB concentrations. For each female otter chosen, a male otter of similar age and from the closest possible location was selected.

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In the present study, the following coastal definitions were used: southern coast region 58°N, 59°40 N, western coast region 59°41 N, 63°25 N, and northern coast region: 63°26 N, 71°N (Fig. 1). Animals were assigned to five coastal sections with increasing distance from the southeastern base point, i.e., the coastal border between Norway and Sweden. The median distance of the five sections to the coastal base point was as follows: section 1 = 859 km, section 2 = 1193 km, section 3 = 1495, section 4 = 1800 km, and section 5 = 2185 km. Potential Confounding Factors Temporal- and age-related trends in PCB concentrations may influence results when analysing geographic differences, and this was considered in the analyses. Temporal trends in PCB concentrations from 1978 to 1990 were analysed by selecting juvenile otters (\1 year) of both

Fig. 1 Map of Norway showing coastal regions. Shaded areas indicate the coastal interval where the otter carcasses were collected and the median distance from the southeastern base point (*). I and II = location of sampling areas for fish

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sexes. Age-related concentrations were analysed separately for sex. Because data on physiologic status and its seasonal variation in otters were insufficient, any influences from such factors were not considered in the analysis. Analyses of PCBs Fat samples from the dorso-caudal region of the 110 otters were collected during necropsies at NINA and stored at -20 °C until further analyses were performed at the Centre for Industrial Research (SINTEF), Norway. Homogenization, extraction, cleanup, and analyses of the samples were performed in 1992 and 1993 with methods that were at state-of-the art at that time (Skaare et al. 1988). The samples were analysed according to SINTEF’s quality accredited standard method, and measured values of quality assurance samples were all within the acceptable range. Thirty individual PCB congeners [PCBs 56, 95, 99, 101, 105, 110, 114, 118, 128, 137, 138, 146, 153, 156, 167, 170, 173, 180, 183, 186, 187, 189, 193, 194, 195, 201, 203, 205, 206, and 209 (Ballschmitter and Zell 1980)] were quantified and summarized and are reported here as RPCBs so that the results of this study can be compared with those published during that time period. Age Estimation Three age categories of otter (first year, second year, and older) were determined from cranial development (Stubbe 1969; Heggberget 1984, 1988) by comparison with skulls of known age. The age of older animals was estimated by counting cementum layers in the apical part of the root of a sectioned canine tooth (Heggberget 1984). Analyses of Reproduction On the whole, the method described in Heggberget and Christensen (1994) and Heggberget (1998) was used in this study. The uteri were searched for implanted embryos, implantation sites, regressive structures (resorptions and mummified foetuses), and visible pathologic changes (e.g., occlusions and tumours). The implantation sites were classified according to regeneration stage of the uterus wall (Martin et al. 1976). Wounds and distensions of the uterine wall at the implantation sites showed recent parturition. The second stage was characterized by dark brown or black pigmentation of the placental scars at the sites, and implantation sites in the final stage were indicated by white scars, often containing small amounts of bright orange pigment. The presence, developmental stage, and number of corpora lutea were classified (Langvatn 1992). When comparing the frequency of different reproductive stages in mature female otters in the western and the

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northern region, all stages of development were included when analysing the mean litter size at birth, unlike the earlier study (Heggberget and Christensen 1994) in which weak black-and-white placental scars were excluded. Estimates, including all developmental stages, will give a lower calculation of litter size because the weak scars are old and some may no longer be visible. However, we decided to include all stages because only a few animals showed recent parturition as placental wounds or clear, black placental scars. The study was carried out in agreement with all provisions enforced by the National Animal Research Authority. Statistical Analysis All data in the tables are reported as means and ranges (minimum to maximum). Statistical evaluation was made by one-way analysis of variance (ANOVA) after testing the data for normality, and pairwise comparisons of means within significant treatments were made using Bonferroni’s test, which controlled type I errors where appropriate. Mann–Whitney U-test was used to test for statistical significant differences in concentrations of PCBs in otters where appropriate. Differences in the proportion of female otters with signs of ovulation were tested with v2 contingency analysis. Time series were tested by applying simple regression analysis. The acceptance level for all analyses was set at P \ 0.05. Analyses were performed using SPSS/ PC?, version 6.0 (SPSS, Chicago, IL). Regression analysis was performed using GraphPad Prism 4 (San Diego, CA).

Results Because age estimation became less accurate with increasing age because animals [5 years often had several cementum layers in the sectioned tooth that were difficult to distinguish clearly, all results for animals [5 years were pooled.

PCB Concentrations in Otters PCBs were detectable in the dorso-caudal fat of all animals (n = 110), and RPCBs ranged from 0.58 to 29 mg/kg lipid weight (overall arithmetic mean of 7.41 mg/kg [SD 4.71] and overall geometric mean of 6.18 mg/kg). No significant time trends in PCB concentrations in juvenile coastal otters (\ 1 year) were found for the period 1978 to 1990 (simple regression of log concentrations; slope = –0.0001, r2 = 0.0005, P = 0.89, n = 41; Fig. 2). For these calculations older animals were excluded to avoid any confounding effects of age.

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Fig. 2 Temporal trend in the concentration of PCBs (mg/kg lipid weight) in fat from juvenile coastal otters (\1 year old) during the period from 1978 to 1990 (simple regression of log concentrations; slope = -0.0001, r2 = 0.0005, P = 0.89)

When subdividing the otter samples with regard to sex and age, mature male otters ([ 3 years) had significant higher PCB levels (geometric mean 8.37 mg/kg) than mature female otters (geometric mean 5.43 mg/kg) (oneway ANOVA, P = 0.03). PCB levels in juvenile male and female otters (\1 year) did not show any significant differences (geometric mean 3.89 mg/kg to 5.40 mg/kg) (oneway ANOVA, P = 0.09). Because there were significant differences for mature animals, geographic trends were analysed separately for the two sexes. A significant increase in PCB concentrations with age (r = 0.58; P \ 0.01) and distance from the southeastern base point (r = 0.44; P \ 0.01) was found for male otters (Fig. 3 [top and bottom panels]). Mean age of the male otters increased significantly northward along the coast. Age accounted for 26% of the total variation in PCB concentrations (variance analyses with partial correlation, r = 0.51, P \ 0.0001), whereas the south–north gradient accounted for 10% of total variation (variance analyses with partial correlation, r = 0.32, P \ 0.02). In female otters, no significant relations between PCB concentrations and age were found (r = –0.44; P [ 0.05) (Fig. 4 [top panel]). However, as with male otters, a significant relation between PCB concentrations and distance from the southeastern base point were observed (r = 0.232; P \ 0.01) (Fig. 4 [bottom panel]). When corrected for age, 5% of the total variation of the PCB concentrations was accounted for by the south–north gradient (variance analyses with partial correlation, r = 0.22, P = 0.05).

Reproductive Performance: Implantation Sites No significant differences were found in the proportion of female otters with visible implantation sites (fresh and old) between the western (56% [n = 34]) and northern coastal regions (58% [n = 111]) (v2 = 0.004; df = 1; P = 0.95). No significant differences for the total number of implantations in female otters were found between the western and the northern coastal regions (Mann–Whitney U-test, P = 0.622), nor was there a significant difference in number of implantations in implanted female otters between the western (2.2 [range 1 to 3]) and northern (2.4 [range 1 to 4]) coastal populations (Mann–Whitney U-test, P = 0.44). Ovulation No significant differences in the proportion of female otters with signs of ovulation were observed between the northern (25% of 102 female otters with signs of ovulation) and the western coastal regions (33% of 33 females with signs of ovulation) (v2 = 0.59; df = 1; P = 0.44). None of the western female otters had a corpus luteum verum (CLV) because they did not have any foetuses. Of the pregnant female otters with intact ovaries in the northern region, all had CLV. Regressing ovulation structures (corpus luteum spureum [CLS]) and resorptions were seldom found, and differences between the two regions were not significant (Mann– Whitney U-test, P = 0.68). In the 33 female otters from the western coastal region, CLS were observed in 3 otters, and

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Fig. 3 (Top panel) Age-related and (bottom panel) geographic trends in concentrations of PCBs (mg/kg lipid weight) in fat of male coastal otters from Norway. Concentrations are presented as percentiles in box plots, and numbers of otters are presented in parentheses

5 CLS were observed in the 102 female otters from the Northern coast region. Two female otters in the northern coastal region had signs of resorptions in the uterus. In one female otter from the north, a leiomyoma was observed in the uterus. Postpartum regressive stages of CLV, i.e., corpus albicans (CA), were common and often numerous in the ovaries of female otters from both regions, and because they may

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have resulted from consecutive and therefore different pregnancies, they were not included in the analysis.

Discussion In general, in otters from the Norwegian coast collected before 1990, PCB levels were low to moderate compared

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Fig. 4 (Top panel) Age-related and (bottom panel) geographic trends in concentrations of PCBs (mg/kg lipid weight) in fat of female coastal otters from Norway. Concentrations are presented as percentiles in box plots, and number of otters are presented in parentheses

with other regions in Europe. A significant trend for a concentration gradient northward along the coast was observed. No significant differences in reproductive histories of western and northern female otters were found. Results indicate that coastal otters in Norway were likely not exposed to critical levels of PCBs, at least not in those areas at the time when the animals were collected. We cannot exclude the possibility that PCBs contributed to the

disappearance of otters along the southern coast of Norway, but no samples are available from the region to support or refute this. Temporal and Geographic Trends PCB concentrations in the carcasses of animals \ 1 year old collected from 1978 to 1990 did not show a decreasing

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trend, unlike what was reported for otters of all age classes collected during the same time period in Europe (Roos et al. 2001; Simpson et al. 2000). However, the overall mean concentration in this study of 7.4 mg/kg is considerably lower than the 17 mg/kg reported for otter samples collected earlier in Norway (Sandegren et al. 1980). The age of male otters increased significantly northward along the coast, and this contributes to the increasing levels of PCBs recorded with increasing distance from the southeastern base point. This increase in tissue concentrations in male otters from south to north fits well with the expected cold condensation pattern of volatile compounds (Wania and Mackay 1993). PCB concentrations in otters from the Norwegian coast were generally low to moderate compared with otters from other areas of Europe (Mason and Macdonald 1986; Mason and Madsen 1993; Mason and O’Sullivan 1992; Mason and Ratford 1994; Kruuk 1995; Kruuk and Conroy 1996; Leonards et al. 1996a; Gutleb and Kranz 1998; Roos et al. 2001; Gutleb and Jefferies 2007) or comparable with those found in Orkney (Mason and Reynolds 1988) or Western Scotland (Kruuk and Conroy 1996). Reproduction-, Sex-, and Age-Related RPCBs in Eurasian Otters The observed increase in PCB concentrations in male otters with age can be explained by the continuous accumulation by way of food in both sexes as well as the lack of an efficient excretion mechanism in male otters as lactation is for female otters (Mason and Madsen 1993; Leonards et al. 1996b). Seals can excrete up to 15% of body burden through lactation (Addison and Brodie 1977), and PCBs are also transferred to the foetus by way of the placenta before birth (Bleavins et al. 1980, 1982). No sex differences are observable in otters \1 year old in which milk is the main route of exposure, and excretion is low. Pups start to take solid food at the age of 2 months and follow their mother on hunting excursions at the age of 4 months (Mason and Macdonald 1986). The relatively high PCB concentrations found in animals \1 year reflect the fact that PCBs are transferred by way of milk, and similar agerelated concentrations of PCBs were found in Denmark (Mason and Madsen 1993). The reproductive history of female coastal otters at the southernmost limit of the present range did not differ from the northern population for any of the parameters investigated (proportion of female otters with visible implantation sites, total number of implantation sites, signs of ovulation, etc.). This can probably be explained by the overall low PCB concentrations found in this study. Although preovulatory stages have been affected in rodents (Ørberg and Kihlstrøm 1973; Brezner et al. 1984) and monkeys

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(Barsotti et al. 1976; van den Berg et al. 1988), no such effects were observed in mink, which are closely related to otters (Kihlstrøm et al. 1992). In examinations of Baltic grey and ringed seals with sterility that was most probably PCB induced (both uterine horns occluded), CA were found in the ovaries, indicating that this part of the reproductive cycle was more or less physiologically normal (Bergman and Olsson 1985). If the findings from these species also apply to otters, only abortions, resorptions, pathologic changes in the uterus, and reduced litter size should be expected among reproductive effects. In the ovulation stage, the regressive structure CLS should occur, and, indeed, resorptions and CLS were observed in a few animals, although the observed effects were not statistically significant. For otters, a proposed effect level of 50 mg/kg fat weight was used to evaluate or predict potential negative effects of PCB exposure on populations (Jensen et al. 1977). This effect level was solely based on data from mink, but in a later study, Bleavins et al. (1980) described large differences in sensitivities between different other species in the mustelid family. In contrast with other areas of Europe, not a single otter measured in Norway exceeded this value, which has been predicted to potentially impair reproduction (Mason and Macdonald 1986; Lafontaine et al. 1990; Mason and Madsen 1993; Mason and O’Sullivan 1992; Mason and Ratford 1994; Kruuk and Conroy 1996; Gutleb and Kranz 1998; Roos et al. 2001). A strong negative correlation was observed between hepatic vitamin A levels, PCB concentrations expressed as TCDD-equivalents, and health status in Eurasian (Leonards et al. 1996a; Murk et al. 1998) and southern sea otters (Enhydra lutris nereis) (Kannan et al. 2007). This is also supported by reports of an increase in vitamin A correlating with decreasing PCB concentrations in Eurasian otter carcasses (Simpson et al. 2000).

Conclusion The present study shows that PCB concentrations present in otters collected before 1990 along the Norwegian coast were lower than concentrations in otters from most other European countries. No correlations of PCB concentrations with effects of female reproductive tissues were observed. Based on these results, however, the hypothesis that previous high PCB concentrations contributed to the disappearance of otters along the southern coast cannot be rejected. No otter carcasses were available from these areas, and sample collection simply may have been too late to pick up the effect. We therefore consider that this data set, which represents the only available data on PCB concentrations in

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Eurasian otters with parameters related to reproductive health, provides important evidence that PCB levels found in the otters from this study are not impairing the reproductive parameters presented. Furthermore, because otters are or will very likely recolonize areas that still have high concentrations of PCBs (Boscher et al. 2010), the data also have a potential value for future risk assessments for otter populations. Acknowledgments The Directorate for Nature Management, the Norwegian Research Council, and the Norwegian Institute for Nature Research supported the doctoral study for which these data were originally produced. Lesley Wright did a wonderful job in reading the ‘‘continental’’ English. Thanks to two anonymous reviewers for their comments, which greatly improved the original version.

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