Environ. Sci. Technol. 2006, 40, 5668-5674
Xenoendocrine Pollutants May Reduce Size of Sexual Organs in East Greenland Polar Bears (Ursus maritimus) C H R I S T I A N S O N N E , * ,† P A L L S . LEIFSSON,‡ RUNE DIETZ,† ERIK W. BORN,§ ROBERT J. LETCHER,| LARS HYLDSTRUP,⊥ FRANK F. RIGET,† MAJA KIRKEGAARD,† AND DEREK C. G. MUIR# Department of Arctic Environment, National Environmental Research Institute, Frederiksborgvej 399, P.O. Box 358, DK-4000 Roskilde, Denmark, Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Bu ¨ lowsvej 17, DK-1870 Frederiksberg, Denmark, Greenland Institute of Natural Resources, P.O. Box 570, DK-3900 Nuuk, Greenland, Denmark, National Wildlife Research Centre, Canadian Wildlife Service, Environment Canada, and Carleton University, Raven Road, Ottawa, Ontario K1A 0H3, Canada, University Hospital of Hvidovre, Kettegaards Alle´ 30, DK-2650 Hvidovre, Denmark, and National Water Research Institute, Environment Canada, Burlington, Ontario L7R 4A6, Canada
Reproductive organs from 55 male and 44 female East Greenland polar bears were examined to investigate the potential negative impact from organohalogen pollutants (OHCs). Multiple regressions normalizing for age showed a significant inverse relationship between OHCs and testis length and baculum length and weight, respectively, and was found in both subadults (dichlorodiphenyl trichloroethanes, dieldrin, chlordanes, hexacyclohexanes, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs)) and adults (hexachlorobenzene [HCB]) (all p < 0.05). Baculum bone mineral densities decreased with increasing chlordanes, DDTs, and HCB in subadults and adults, respectively (all p < 0.05). In females, a significant inverse relationship was found between ovary length and ΣPCB (p ) 0.03) and ΣCHL (p < 0.01), respectively, and between ovary weight and ΣPBDE (p < 0.01) and uterine horn length and HCB (p ) 0.02). The study suggests that there is an impact from xenoendocrine pollutants on the size of East Greenland polar bear genitalia. This may pose a risk to this polar bear subpopulation in the future because of reduced sperm and egg quality/ quantity and uterus and penis size/robustness.
Introduction One key factor for the population size of polar bears (Ursus maritimus) and mammals in general is the functionality of * Corresponding author phone: +45-46-30-19-54; fax: +45-4630-19-14; e-mail:
[email protected]. † National Environmental Research Institute. ‡ The Royal Veterinary and Agricultural University. § Greenland Institute of Natural Resources. | National Wildlife Research Centre, Canadian Wildlife Service, Environment Canada, and Carleton University. ⊥ University Hospital of Hvidovre. # National Water Research Institute, Environment Canada. 5668
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the reproductive organs and the ability to produce surviving cubs. The life-span and adult survival of polar bears are among the highest for mammals in general, while the reproductive rates is among the lowest for terrestrial mammals (1-3). Polar bears are seasonal breeders, exhibit delayed implantation, and usually give birth to two cubs (4-8). Mating takes place in April-June, and implantation occurs in late September, resulting in birth in late December (4-8). Reproductive success is susceptible to extrinsic environmental factors such as food access, climatic oscillations, and anthropogenic xenoendocrine disrupting chemicals (9-11). The reproductive organs are susceptible to changes in homeostasis induced by organochlorines through the hypothalamus-pituitary-target organ axis (12, 13). The functioning of reproductive organs and their ability to reproduce is a complex interaction and timing between endocrine (hormonal) and immune parameters (12, 13). Impaired fertility has been associated with a negative impact from environmental anthropogenic organohalogen compounds (organochlorines and brominated flame retardants) in both male and female mammals, resulting in testicular dysfunction (e.g., low sperm count and altered spermatozoa morphology) and pathology (e.g., endometriosis, leiomyomas, occlusions, and stenosis) in the female reproductive tract, hence leading to a reduction in the number of surviving offspring (13-17). Polar bears from East Greenland, Svalbard, and the Western Russian Arctic are the most OHC-polluted species in the Arctic as they rely on blubber from mainly ringed seals (Phoca hispida) and bearded seals (Erignathus barbatus) (2, 9). The seal blubber contains significant amounts of OHCs due to their lipophilic nature. Several correlative studies on polar bears have indicated that OHCs may have an impact on steroid/peptide hormones and vitamins (18-22), the immune system (23-25), internal organs (26-28), and the skeletal system (29). Furthermore, a suppression of the cellular immunity and hypospadias was found in Greenland sledge dogs dietary-exposed to similar OHC levels via OCpolluted minke whale (Balaenoptera acutorostrata) and ringed seal blubber (30, 31). However, nothing is known about the impact on polar bear reproductive organs from OHCs. Studies of Svalbard subpopulations have indicated a negative impact from OHCs on the population size, although results were not conclusive (32, 33). Studies of pseudohermaphroditism (abnormal enlarged clitoris) and the potential relationship to pollution in polar bears are not conclusive either (34-36). To determine if OHCs have a similar negative impact on East Greenland polar bear genitalia, levels of OHCs were compared with macro- and microscopic morphology of reproductive organs in 99 individuals sampled during 1999-2002. These new correlative results are intended to fill part of the existing knowledge gap in understanding the significance, nature, and effects of chronic environmental OHC exposure.
Experimental Section From the subsistence hunting regulated by the Home Rule of Greenland (Nuuk), testicles and/or penises and bacula (n ) 55) and ovaries and uteruses (n ) 44) were obtained from East Greenland polar bears in the Scoresby Sound area (69°00′ N to 74°00′ N) during 1999-2002. All organs were fixed in a formaldehyde/alcohol solution (3.5% formaldehyde, 86% EtOH, and 10.5% H2O, phosphate buffered). Subcutaneous adipose tissue was sampled from 82 of the bears for OHC analyses and stored frozen in separate poly(ethylene) plastic bags. Further procedures are described in Sonne et al. (27). 10.1021/es060836n CCC: $33.50
2006 American Chemical Society Published on Web 08/16/2006
FIGURE 1. Baculum DXA scanning image of bone mineral density in (A) a 28-year-old and (B) a 7-year-old East Greenland polar bear. Note the high-density areas of cortical bone tissue (H) and the lower density areas (L) of trabecular bone tissue. Age Determination. The age determination was carried out by counting the cementum growth layer groups (GLGs) of the lower I3 tooth after decalcification, thin sectioning (14 µm), and staining (toluidine blue), using the method described by Dietz et al. (37). When necessary, the individuals were categorized as subadults, adult males, and adult females by the criteria: adult males g6 years, adult females g5 years, and the remaining as subadults (8). Gross Anatomy, Histology, and Bone Mineral Density. Length and weight was recorded as an average of left and right testes before the tissue was trimmed, processed conventionally, embedded in paraffin, sectioned at about 4 µm and stained with haematoxylin (Al-haematein)-eosin (HE) for routine diagnostics (38). In males, all slides were examinated consecutively from the seminiferous tubules to the caput of the epididymis. The maximal diameters (µm) of 5 randomly chosen seminiferous and epididymal tubules were only measured in 11 and 26 individuals respectively, due to immature closure and/or suboptimal fixation. The presence of histopathology (fibrosis, atrophy, hyalinisation and inflammation) of seminiferous tubules and interstitium was recorded (thirteen individuals were excluded from these histological examinations due to suboptimal fixation.) Penises and bacula were investigated grossly before recording baculum weight, length, and bone mineral density (BMD). Dual X-ray absorptiometry (DXA) was applied to all bacula for detecting osteoporosis by use of a Norland XR 26 X-ray bone densitometer (Norland Corporation, Wisconsin, USA). The X-ray osteodensitometry determined BMD (grams of calcium phosphate (hydroxyapatite) per square centimeter) as described by Sonne et al. (29). A software-generated image of baculum BMD is given in Figure 1. In females, dimensions of the uterine corpus (diameter) and each uterine horn (diameter at midpoint and length) and ovaries (length, weight) were measured. Data are given as the average values of the measurement of the left and right organs. (The individual with the enlarged clitoris reported by Sonne et al. (35) was not included in the present investigation.) Analyses of OHCs. Subcutaneous samples of polar bear adipose tissue (n ) 81) were analyzed for polychlorinated biphenyls (PCBs), chlordanes (CHLs), dichlorodiphenyl trichloroethanes (DDT), dieldrin, hexacyclohexanes (HCHs), hexahlorobenzene (HCB), and polybrominated diphenyl ethers (PBDEs) according to Dietz et al. (40) and Muir et al. (41). Statistics. The statistical analyses were performed with the SAS statistical software package (SAS 9.1 and enterprise guide 3.0; SAS Institute Inc., Cary, NC), and the significance level was set to p ) 0.05, while levels at 0.05 < p e 0.10 were considered a trend. The OHC data were log-transformed (base e) prior to the analyses to meet the assumption of normality and homogeneity of the variance. Differences in seasonal testis/baculum/ovary/uterus size (weight, length and diameter) were tested within analyses of covariance with weight and length, respectively, as dependent
variables, age as a covariable and season (spring, JanuaryJuly, vs fall, August-December) as a class variable (season according to Roosing-Asvid et al. (8), including their firstorder interaction link (age × season). This was conducted on individuals up to 10 years of age as it was reasonable to believe that the age versus testis/ovary size was following a linear relationship within this age span, after which an asymptotic weight/length was reached. After a successive reduction of nonsignificant interaction, judged from the typeIII sum of squares (p > 0.05), the significance of each of the remaining factors was evaluated from the final model leastsquare mean. The relationship between macroscopic testis/ ovary/uterus measurements (weight, length, and diameter) and histology (size of the seminifery and epididymidy lumen) was tested by Pearsons’ linear correlation analysis for normality data. In the evaluation of histology seasonal and age patterns, a Chi-square and a one-way analysis of variation (ANOVA) were employed, respectively. Finally, the relationship between age and testis/ovary/uterus length and weight was investigated using the Gompertz growth model (y ) k1 exp(k2 exp(k3x))), and the same was used for age versus baculum weight, length, and BMD, respectively. Then, a one-way ANOVA was performed to test for differences in mean concentrations of each group of OHCs (PCBs, DDTs, CHLs, dieldrin, HCHs, HCB, and PBDEs) between subadults and adults. The results were finally evaluated from Tukey’s post hoc test. To test the relationship between concentrations of OHCs and age, a linear regression model was employed for subadults and adults, respectively. Differences in OHC levels between individuals with and without testis histological changes were tested with analyses of covariance with OHC group as dependent variables, age as a covariable, and the presence of histopathology (1 vs 0) as a class variable, including their first-order interaction link (age × presence of histopathology). After a successive reduction of nonsignificant interaction, judged from the typeIII sum of squares (p > 0.05), the significance of each of the remaining factors was evaluated from the final model leastsquare mean. The same model was used to test for seasonal variation in OHC concentrations with season (spring vs fall) as a class variable. When analyzing the relationship between OHC group and testis/baculum/ovary/uterus size, Pearsons’ linear correlation analysis was used for the initial data exploration, while multiple regression analyses (size and BMD as dependent variables; age and OHC group as explanatory variables) controlling for age were used to test for statistically significant relationships. The relationships between OHC group and size were finally evaluated from the parameter estimates, r2 and p-value. On the basis of Gompertz growth models, the analyses were employed on subadult and adult males, respectively.
Results Testis and Penis Morphology. Testicles and exact ages were available from 51, and penises/bacula from 52 individuals, VOL. 40, NO. 18, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Basic Statistics (Mean ( Standard Deviation (n)) of the Reproductive Organs from 55 Male and 44 Female East Greenland Polar Bears Sampled During 1999-2002 variable
subadults
adults
age testis weight (g) testis length (mm) baculum weight (g) baculum length (mm) baculum BMD (g/cm2) uterine corpus diameter (mm) uterine corpus length (mm) uterine horn diameter (mm) uterine horn length (mm) ovary length (mm) ovary weight (g)
2.95 ( 1 (49) 14.04 ( 10.40 (23) 51.59 ( 9.14 (22) 4.35 ( 2.53 (23) 117.17 ( 28.99 (23) 0.1710 ( 0.0520 (23) 5.58 ( 1.39 (19) 56 ( 25.9 (8) 4.41 ( 0.98 (22) 91.88 ( 16.13 (20) 22.87 ( 4.93 (23) 2.43 ( 0.88 (22)
10.23 ( 6.45 (49) 53.35 ( 21.93 (26) 74.04 ( 9.59 (24) 13.58 ( 5.27 (27) 172.11 ( 17.51 (27) 0.3065 ( 0.0603 (26) 7.5 ( 2.38 (18) 84.89 ( 19.76 (9) 6.69 ( 1.66 (21) 118.9 ( 20.86 (21) 22.83 ( 4.54 (21) 3.05 ( 1.06 (16)
respectively. Basic statistics for all testes and bacula measurements are given in Table 1. No obvious gross pathology was found in testes or bacula. Testis weight was highly correlated with testis length (p < 0.0001; r > 0.93, n ) 47), and testis weight was significantly higher during sexual activity (January-July) than during resting (August-December) (p < 0.05). No seasonal difference was found for testis length, baculum size, or BMD (all p > 0.24). Testis Histology. Histology was conducted on testis tissue from all 55 individuals. Twenty-two of the bears were caught in spring (January-July), 17 of the bears were caught during fall (August-December), and 16 were of unknown sampling date. The diameters of the seminiferous and epididymal tubules were highly correlated with the weights and lengths of the testes (all p < 0.0001; r2 > 0.77; n ) 25). Multifocal fibrosis, atrophy, hyalinization of basement membranes, and/or inflammation was recorded in the testes of 20 bears (Figure 2). Five of the “spring” bears (23%) and five of the “autumn” bears (29%) exhibited these lesions, and hence no seasonal difference was found (p ) 0.64). Furthermore, the mean age of individuals with and without lesions did not differ (p ) 0.38). Organohalogen and Age in Males. Adipose tissue for OHC analyses was available from 43 male polar bears (Table 2). The relationships between concentrations of each OHC group and the two age categories were tested (Table 2). The analyses showed that only mean ΣPCB levels were significantly higher in adults when compared to subadults (p < 0.05) and that chlordanes and dieldrin decreased significantly with age for the entire data set (p < 0.05). There were no significant differences between spring and fall in mean concentrations
TABLE 2. OHC Concentrations (Mean ( Standard Deviation, ng/g Lipid Weight (lw)) in Subcutaneous Adipose Tissue of 43 Male and 39 Female East Greenland Polar Bears Sampled During 1999-2001 OHCs
subadult males (n ) 21)
adult males (n ) 22)
subadult females (n ) 18)
adult females (n ) 21)
∑PCB 5519 ( 2341 7237 ( 2503a 7115 ( 3131 6046 ( 3952 ∑CHLd 1197 ( 684 1020 ( 432 1999 ( 934b 1619 ( 1447 ∑DDT 401 ( 243 463 ( 326 457 ( 230 339 ( 210 dieldrinc 196 ( 108 199 ( 170 219 ( 99 175 ( 69 ∑HCHe 174 ( 53 180 ( 59 199 ( 77 175 ( 159 HCB 112 ( 107 109 ( 177 114 ( 77 74 ( 76 ∑PBDE 53 ( 33 59 ( 40 56 ( 34 40 ( 23 a Significantly higher in adults when compared to subadults at p e 0.05. b Significantly higher in subadults when compared to adults at p e 0.05. c Significantly negative relationship to age in males at p e 0.05 (r2 ) 0.12). d Significantly negative relationship to age in males at p e 0.05 (r2 ) 0.10). e Significantly negative relationship with age at p e 0.05 (r2 ) 0.46) for adult females.
of any of the OHC groups (all p > 0.05) (Table 2). Due to the significant age dependency of OHC concentrations the further statistical analyses were conducted on subadults and adults separately. Organohalogens and Testis/Baculum Size. To remove the influence from spring vs fall fluctuations, only testis length was used in the multiple regression analyses of OHC relationships to testis size. Testis length decreased significantly with ΣPBDE, ΣHCH, ΣDDT, and chlordanes in subadults (all p < 0.05), while a decreasing trend was found
FIGURE 2. Left panel: Accumulation of spermatozoa and seminiferous epithelial cells in normal epididymal lumen of a 15-year-old East Greenland bear caught March 24, 2000 (HE, 10×, bar, 100 µm). Right panel: Testis histopathology in a 5-year-old East Greenland polar bear caught January 29, 2001. Tubular atrophy, hyalinization of basement membranes, interstitial fibrosis, and mononuclear cell infiltrations (inflammation) (HE, 5×, bar, 20 µm). 5670
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TABLE 3. Significant Contaminant Results from the Multiple Regression Analyses of Testis Size (Weight and Length) versus Age and Contaminant Concentrations in East Greenland Male Polar Bears Sampled During 1999-2001a age group subadults
adults
pcont
parameter equation
page
r2
n
length ) 5.27 × age - 6.61[ln(∑PBDE)] + 63.42 length ) 3.94 × age - 13.71[ln(∑HCH)] + 112.13 length ) 4.83 × age - 5.85[ln(∑DDT)] + 73.32 length ) 4.84 × age - 8.19[ln(∑CHL)] + 95.98
0.040 0.031 0.019 0.018
0.002 0.022 0.004 0.004
0.49 0.51 0.53 0.54
20 20 20 20
length ) 1.63 × age - 4.32[ln(HCB)] + 46.37 weight ) 5.36 × age - 9.70[ln(∑HCH)] - 23.94 weight ) 2.59 × age - 16.52[ln(dieldrin)] + 115.88
0.090 0.033 0.082
0.079
