CONCENTRATIONS and INTERACTIONS of SELECTED ESSENTIAL and NON-ESSENTIAL ELEMENTS in BOWHEAD and BELUGA WHALES of ARCTIC ALASKA Author(S): Victoria M

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CONCENTRATIONS AND INTERACTIONS OF SELECTED ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES OF ARCTIC ALASKA Author(s): Victoria M. Woshner, Todd M. O'Hara, Gerald R. Bratton, Robert S. Suydam, and Val R. Beasley Source: Journal of Wildlife Diseases, 37(4):693-710. Published By: Wildlife Disease Association DOI: http://dx.doi.org/10.7589/0090-3558-37.4.693 URL: http://www.bioone.org/doi/full/10.7589/0090-3558-37.4.693

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BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries, and research funders in the common goal of maximizing access to critical research. Journal of Wildlife Diseases, 37(4), 2001, pp. 693–710 ᭧ Wildlife Disease Association 2001

CONCENTRATIONS AND INTERACTIONS OF SELECTED ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES OF ARCTIC ALASKA

Victoria M. Woshner,1,2 Todd M. O’Hara,2,4 Gerald R. Bratton,3 Robert S. Suydam,2 and Val R. Beasley1 1 Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802, USA 2 Department of Wildlife Management, North Slope Borough, Barrow, Alaska 99723, USA 3 Department of Veterinary Anatomy and Public Health, Texas A&M University, College Station, Texas 77843, USA 4 Corresponding author (e-mail: [email protected])

ABSTRACT: In this study, we evaluated concentrations of twelve essential and non-essential elements (As, Cd, Co, Cu, Pb, Mg, Mn, Hg, Mo, Se, Ag, and Zn) in tissues of bowhead (Balaena mysticetus) and beluga (Delphinapterus leucas) whales from arctic Alaska (USA) and northwestern Canada. Tissue samples were collected between 1983 and 1997, mostly in 1995–97. The essential elements are reported to develop reference ranges for health status determination, and to help assess known or suspected interactions affecting toxicoses of cadmium (Cd) and mercury (Hg). In some tissues, Cd, Hg, and selenium (Se) were present at concentrations that have been associated with toxicoses in some domestic animals. Nevertheless, tissue levels of all elements were within ranges that have been reported previously in marine mammals. While mean Ag concentrations in beluga whale liver were relatively high (15.91 ␮g/g ww), Ag was not associated with hepatic Se levels or age, contrary to previous findings. Significant associations included: Cd with age, Zn, or Cu; Cu with age, Zn or Ag; and Hg with age, Se, Zn, or Cu. This study found hepatic Hg:Se molar ratios to be consistently lower than unity and different between species. Possible explanations for observed elemental correlations (i.e., interactions) and ancillary mechanisms of Cd and Hg detoxification are discussed. Key words: Balaena mysticetus, Beluga whale, bowhead whale, cadmium, cetaceans, Delphi- napterus leucas, elements, marine mammals, mercury, molar ratio, selenium, silver.

INTRODUCTION mammals off the coast of the Netherlands that were consistent with experimental In general, the Arctic is sparsely popu- monomethylmercury (MHg) toxicosis in lated by humans and has comparatively lit- various laboratory and domestic species. tle industry or agricultural enterprise. Koeman et al. (1973) indicated that prey Consequently, detection of anthropogenic of piscivorous marine mammals contain contaminants in the arctic food chain has approximately 16-fold greater molar Se incited public apprehension, especially than molar Hg, and concluded that the 1:1 among native people who consume marine Hg:Se ratio in liver, kidney and brain, is mammals (Wheatley and Paradis, 1995). established in the marine mammal (Koe- Of particular concern are persistent toxi- man et al., 1975). cants, including some metals, which have Koeman’s theory (Koeman et al., 1975) the potential to accumulate in biotic and of equimolar Hg:Se ratios has been cor- abiotic components of the environment roborated by data on tissues of various ma- (Pacyna and Winchester, 1990). Cadmium rine mammal species, including ringed (Cd) and mercury (Hg) occur naturally; (Phoca hispida) and bearded seals (Erig- however, there is no doubt that recent hu- nathus barbatus) (Smith and Armstrong, man activities have increased the rate at 1978); polar bears (Ursus maritimus) which they and other elements are mobi- (Braune et al., 1991; Dietz et al., 1995); lized to and within the biosphere (Pacyna and harbor porpoises (Phocoena phocoena) and Keeler, 1995). (Paludan-Mu¨ ller et al., 1993) with some Koeman et al. (1973, 1975) examined variation from a 1:1 molar ratio noted (Wa- relationships between levels of Hg and se- gemann et al., 1983; Hansen et al., 1990; lenium (Se) in liver and brain of marine Braune et al., 1991). Departures from uni-

693 694 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001 ty have been interpreted as evidence that differ with age, tissue type, gender, spe- the mechanism for Hg detoxification has cies, etc.; (3) evaluate elemental interac- been overwhelmed or is not Se-depen- tions; and (4) report ‘‘normal ranges’’ of dent. Arima and Gakura (1979) found a essential elements. linear increase in muscle total Hg with age MATERIALS AND METHODS in odontocetes, in conjunction with a Hg: Se molar ratio of approximately 1.5:1, Sample collection and processing which they construed as an excess of Hg. From 1983 through 1997, tissues were col- Zeisler et al. (1993) noted a positive cor- lected from bowhead and beluga whales relation of Se with both silver (Ag) and to- through the cooperation of Alaskan Eskimo (Inuit) subsistence hunters. Forty-one bowhead tal Hg (THg) in liver of beluga whales whales were sampled from 1983 through 1990 (Delphinapterus leucas). Becker et al. near Barrow (71.33ЊN, 156.00ЊW) (n ϭ 31), (1995) further investigated relationships Kaktovik (70.10ЊN, 143.23ЊW) (n ϭ 3), and among Ag, Se, and Hg in beluga whales. Wainwright (70.55ЊN, 159.95ЊW) (n ϭ 7), Alas- Hepatic total Hg concentrations were sim- ka. From 1995–97, 21 bowhead whales were sampled near Barrow, Alaska. Between 1992 ilar between Alaskan belugas and Atlantic and 1996 beluga whales were sampled in late pilot whales. In contrast, hepatic Ag con- June/early July at Pt Lay, Alaska (69.66ЊN, centrations in belugas were one to three 162.83ЊW) (1992, n ϭ 9; 1995, n ϭ 10; 1996, orders of magnitude greater than in pilot n ϭ 10); in spring (May, 1997) at Point Hope Њ Њ ϭ whales (5.93–107.4 vs. 0.13–0.33 ␮g/g (68.30 N, 166.63 W), Alaska (n 9); in August of 1996 (n ϭ 1) and July of 1997 (n ϭ 3) at ww). Becker et al. (1995) further specu- Barrow, Alaska; summer of 1993 at the North- lated that Ag at these concentrations might west Territories (Inuvik) of Canada (68.32ЊN, adversely affect the Se status of Alaskan 133.50ЊW) (n ϭ 7); and in July at Kaktovik, belugas through competitive binding, ren- Alaska (1997, n ϭ 1). Tissues collected included dering them more vulnerable to Hg toxi- liver, kidney, muscle, blubber and epidermis. Tissues were either frozen or chilled and sub- cosis. sequently processed under clean conditions fol- Major concerns regarding element lev- lowing a strict protocol (Becker, 1995) to min- els in these species include potential toxic imize contamination from handling. This in- effects to wildlife and human consumers. cluded rinsing tissue with HPLC grade water We present what may be considered ‘‘nor- (Sigma Chemical Co., St. Louis, Missouri, USA) prior to sub-sampling and cutting with mal’’ or reference ranges of essential ele- titanium knives on a clean, Teflon-covered sur- ments in apparently healthy (as deter- face. Subsamples of tissues were placed in acid- mined by gross and histologic examination; washed scintillation vials (Fisher Scientific, Woshner et al., 2000b) subsistence-har- Pittsburgh, Pennsylvania, USA) in preparation vested bowhead and beluga whales that for heavy metals analysis. All tissues were frozen (Ϫ20 C) immediately after processing. Fro- may be used when a mineral deficiency is zen samples were express delivered (24–48 hr) suspected, for comparison to other spe- to College Station, Texas (USA) for archiving cies, to identify interactions that may and analyses under appropriate National Ma- counter toxicoses (i.e., Se and Hg), or to rine Fisheries Service permits. Teeth were col- establish guidelines for human consump- lected from belugas for age determination using counts of dentinal growth layer groups tion. A better understanding of essential (GLGs), which have been estimated to deposit elements in arctic marine mammals will at a rate of two GLGs annually (Goren et al., help to improve health assessments and 1987). Total GLGs were used in all analyses. disease investigations. Body length was used as a surrogate for age in The objectives of this study were to (1) bowhead whales (George et al., 1999). evaluate concentrations of selected essen- Metals analysis tial and non-essential elements in tissues In preparation for elemental analysis, tissue of bowhead and beluga whales harvested samples were weighed (wet weight or ww), by Alaskan Native subsistence hunters; (2) dried to constant weight, and digested in a wet to determine whether these element levels ash procedure using concentrated (70%) nitric WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 695 acid and microwave processing at 120 C for 60 Washington, USA; Bloom and Crecelius, 1983; min. All metals, except Cd and zinc (Zn), were Bloom and Fitzgerald, 1988). The detection analyzed using a Perkin-Elmer (Norwalk, Con- limits were 1.1 ng/g and 6.6 ng/g ww for MHg necticut, USA) model SIMAA 6000 graphite and Hg(II), respectively. The second total Hg furnace atomic absorption spectrophotometry value given in the following results was calcu- (GFAAS) instrument, equipped with an AS-60 lated by summing the MHg and Hg(II) values; autosampler and Zeeman background correc- SHg [SHg ϭ sum of Hg(II) and MHg]. All Hg tion (Perkin-Elmer); Cd and Zn were deter- data were reported on a wet weight (ww) basis. mined using a Perkin-Elmer instrument model Due to financial constraints and known low lev- 306 flame AAS. Detailed methodology followed els of Hg in bowhead tissues, inorganic that described by Smoley (1992) and modified [Hg(II)] and organic (MHg) Hg were deter- by Bratton et al. (1997). For bowhead whale mined only in beluga liver, kidney, muscle, and samples collected prior to 1995, tissues were epidermis. dry-ashed at 480 F and analyzed by AAS with Smith-Hieftje background correction, using bo- Statistical analysis vine liver standard #1577 (National Bureau of Student’s t-test was used to compare elemen- Standards, USA) as reference material (Bratton tal concentrations between genders within spe- et al., 1993, 1997). All concentrations were re- cies. With few exceptions (i.e., beluga whale ported on a ppm wet weight (ww) basis. The epidermal Hg(II), renal Se, and renal and he- detection limit for all elements was approxi- patic As), significant differences were not ob- mately 0.01 ␮g/g (ppm). served between sexes, so data were pooled for further analyses. One-way Analysis of Variance Mercury analysis (ANOVA) with Tukey’s post-hoc test tested for differences in elemental concentrations be- In preparation for total Hg analysis at the tween tissues within species, and between spe- Texas Veterinary Diagnostic Laboratory (Ama- cies within tissue, with cetaceans in this study rillo, Texas, USA) wet tissue samples were analyzed in conjunction with ringed seals and weighed and transferred to 250 ml quartz vol- polar bears (Woshner et al., 2001). Following umetric digestion tubes. Sample digestion and Conover (1971), the values for each element analysis followed Korsrud et al. (1985) with mi- were converted to ranks and Pearson correla- nor modifications. Briefly, 15 ml of a tertiary tions calculated between elemental concentra- acid mixture, 15 N nitric acid, 36 N sulfuric tions within a tissue type, and between ele- acid, and 70% perchloric acid (5:1:1) (Sigma mental concentrations and age indices. Linear Chemical Co., St. Louis, Missouri, USA) was regression was used to examine associations be- added to each tube. After 4 hr at room tem- tween selected elements within tissue and spe- perature, samples were heated in a five-step se- cies. All statistical analyses were done using quence over a period of 6 hr to a final temper- Systat (SPSS, Inc., Chicago, Illinois, USA; ature of 200 C, where it was maintained for 2 1998) computer software with a P-value of hr. After cooling, digests were transferred to Ͻ0.05 considered significant. For purposes of volumetric flasks and brought to 25 ml with de- calculating summary statistics (computed only ionized water. Samples were placed in 150 ml when Ͼ50% of samples had detectable levels reaction vessels, each containing 20 ml of 1 N for a given element), values below the mini- HCl. Each analytical run included blank di- mum detection limit (MDL) were expressed as gests, working standards, and one standard ref- one-half the MDL, in accordance with accept- erence material (DOLT-2; National Research ed statistical practice (Gilbert, 1987). Council, Ottawa, Ontario, Canada). Direct determination of total Hg (THg) concentrations RESULTS was via cold vapor AAS using a Thermo-Jarrell Ash (Franklin, Massachusetts, USA) model S- Bowhead whales 11 AAS with a Thermo-Jarrell Ash AVA-440 Total body length of bowhead whales atomic vapor accessory. Mercury vapor was generated through the automated addition of ranged from 4.0 (a fetus) to 17.7 m. Mean 2.5 ml of 5% stannous chloride to the sealed concentrations of elements in bowhead reaction vessel. The detection limit for THg whale tissues are presented in Table 1. was approximately 1 ng/g (ppb ww). Mean concentration of Cd was highest in For determination of divalent Hg [Hg(II)] kidney (20.02 ␮g/g), with a range of 0.04 and monomethyl Hg (MHg), wet tissue sam- to 64.0 ␮g/g ww. Mean hepatic Cd was ples were digested (Bloom, 1992), and analyzed ␮ by cold vapor atomic fluorescence spectrome- 9.63 g/g ww and significantly lower than try (CVAFS; Frontier Geosciences, Seattle, in kidney, while levels in muscle, blubber 696 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001

TABLE 1. Concentrations (␮g/g ww) of selected elements in tissues of Alaskan bowhead whales.

As Cd Co Cu Pb Mg Mn THg Se Zn

Livera Mean 0.201 9.631 0.021 11.111 0.031 294.71 2.271 0.0601 1.611 34.941 SDb 0.16 10.43 0.01 21.48 0.02 46.5 0.92 0.073 0.81 16.16 n 54 55 20 55 55 20 20 55 55 55 Kidney Mean 0.091 20.022 0.021 3.282 0.021,2 322.51 0.532 0.0381,2 1.581 27.722 SD 0.09 17.29 0.01 1.41 0.02 102.7 0.18 0.033 0.42 11.31 n 48 48 20 48 48 20 20 47 48 48 Muscle Mean 0.081 0.053 NDc 1.502 0.021,2 509.82 0.152 0.0172,3 0.353 38.941 SD 0.13 0.07 0.87 0.02 198.1 0.07 0.011 0.33 17.14 n 35 36 14 43 36 21 14 35 42 43 Blubber Mean 0.582 0.023 ND 0.602 0.022 49.93 0.032 0.0022,3 0.063 2.583 SD 0.68 0.02 0.52 0.01 22.1 0.02 0.007 0.03 2.08 n 38 34 11 41 34 11 11 30 38 34 Mean 0.081 ND ND 0.532 ND 521.02 0.062 0.0062,3 0.752 15.173 SD 0.05 0.10 40.6 0.02 0.004 0.36 2.14 n 15 8 8 15 8 15 8 8 15 15 a Liver Mo and Ag were 0.38 and 0.34 ␮g/g ww, respectively; not detected in other tissues. b Standard deviation. c ND ϭ not detected in greater than 50% of the samples. The same superscript number (1,2,3) indicates mean level for that element is not significantly different among those tissue(s) as determined by one-way ANOVA with Tukey’s post-hoc test. and epidermis were significantly lower ble 3) and the associated r2 was only 0.19 than liver and kidney Cd levels. Mean con- (Table 3). Linear regression analysis of centration of As was highest in blubber body length versus Se, Ag, or Cd in liver, (0.58 ␮g/g). Levels of lead (Pb) and THg as well as Cd in kidney, were all significant were consistently low in all tissues; the (Table 3). Among these regressions the highest mean concentrations were found closest associations were between body in liver 0.03 ␮g/g (Pb) and 0.06 ␮g/g ww length and Cd in kidney (r2 ϭ 0.40) and (THg). Mean Cu, Mn, Ag, and Zn levels liver (r2 ϭ 0.38). were significantly greater in liver than in Beluga whales kidney; and As, Co, Pb, Mg, THg, and Se were not significantly different between Mean concentrations of elements in tis- liver and kidney. Mean Mg concentrations sues of beluga whales are presented in Ta- were highest in muscle and epidermis. ble 4. Highest mean tissue concentration Significant correlations (P Ͻ 0.05) be- of As was in blubber (1.41 ␮g/g), which tween variables within tissue are given in exceeded mean levels in other tissues by Table 2. A number of elements were sig- at least four-fold (Table 4). Mean Cd was nificantly correlated with body length, in- highest in kidney (12.15 ␮g/g ww) followed dicating that their tissue concentrations ei- by liver (3.81 ␮g/g ww) and was lowest in ther increase or decrease with age. He- muscle, blubber, and epidermis. Mean Ag patic Cu, Ag and Mn concentrations was highest in liver (15.91 ␮g/g ww). Ap- showed significant negative correlations preciable mean Se concentrations were with body length. Linear regression anal- observed in liver (41.41 ␮g/g ww), kidney yses of Se versus THg was conducted (Ta- (6.27 ␮g/g ww) and epidermis (9.56 ␮g/g WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 697

TABLE 2. Pearson’s correlation coefficients between centrations were highest in liver, but var- selected significantly correlated variablesa (P Ͻ 0.05) in Alaskan bowhead whales. ied depending upon analytical method employed. As previously noted, calculated to- rPntal Hg (SHg) was determined through summation of Hg(II) and MHg. In gen- Liver eral, SHg tended to be lower than total Hg Cd vsb Lgthc 0.70 Ͻ0.001 54 Cd vs As 0.32 0.019 54 determined directly (THg) by approxi- Cu vs Lgth Ϫ0.39 0.003 54 mately two-fold, although not necessarily THg vs As 0.32 0.018 54 on a case-by-case basis. Mean hepatic SHg THg vs Cd 0.47 Ͻ0.001 54 was 12.42 ␮g/g ww as opposed to mean Se vs Lgth 0.33 0.016 54 THg of 23.29 ␮g/g ww (Table 4). This dis- Se vs Cd 0.49 Ͻ0.001 54 Se vs THg 0.40 0.003 54 crepancy between total Hg values attrib- Ag vs Lgth Ϫ0.65 0.002 20 utable to analytic methodology was not Ag vs Cd Ϫ0.60 0.005 20 nearly as pronounced in other tissues, Ag vs Cu 0.78 Ͻ0.001 20 which had lower total Hg levels, as well as Ϫ Ag vs Se 0.57 0.009 20 (in the case of muscle and epidermis) a Zn vs As Ϫ0.27 0.047 54 Zn vs Se 0.37 0.006 54 greater proportion of MHg. When calculated as a percentage of SHg, MHg oc- Blubber curred at a mean level of 14.2% in liver, Zn vs Lgth Ϫ0.547 0.002 30 12.8% in kidney, 96.0% in muscle, and Kidney 97.1% in epidermis. In contrast, when cal- Ͻ Cd vs Lgth 0.74 0.001 48 culated as percentages of THg, MHg was Cd vs Zn 0.32 0.026 48 THg vs Cd 0.46 0.001 47 7.4% in liver, 11.8% in kidney, 94.0% in Se vs Cu Ϫ0.31 0.033 48 muscle, and 113.0% in epidermis. Regard- Zn vs Cu Ϫ0.38 0.008 48 less of calculation method, it is evident Zn vs THg 0.35 0.017 47 that virtually all Hg in muscle and epider- Zn vs Se 0.31 0.031 48 mis is in the methylated form while ap- Muscle proximately 90% of mean total Hg present Cd vs Lgth 0.75 Ͻ0.001 35 in liver and kidney is inorganic [Hg(II)]. THg vs Lgth 0.42 0.012 35 In general, tissue elemental concentra- Se vs Cu 0.63 Ͻ0.001 42 Zn vs Cu Ϫ0.63 Ͻ0.001 42 tions did not appear to differ significantly Zn vs Se Ϫ0.68 Ͻ0.001 42 between males and females, but we noted Epidermis a few exceptions. Mean epidermal Hg(II) ␮ Se vs Lgth 0.797 Ͻ0.001 15 was higher in males (0.03 g/g ww) than Se vs As 0.735 0.002 15 in females (0.02 ␮g/g ww) but the biolog- Zn vs As 0.698 0.004 15 ical significance of a 0.01 ␮g/g difference Zn vs Se 0.571 0.026 15 is difficult to interpret. Among animals a Data were ranked prior to analysis. from which kidney and liver were ob- b vs ϭ versus. tained, males were significantly older than c ϭ Lgth body length. females. Mean Se level in kidney was greater in males (29.72 ␮g/g ww) than in ww), with Se in liver being significantly females (18.81 ␮g/g ww). Males had sig- higher than in the other four tissues (Table nificantly higher concentrations of As than 4). Levels of Cu, Pb, Mn, THg, Mo, Se, females in kidney (0.30 ␮g/g ww in males Ag, Hg (II), MHg, and SHg were signifi- vs. 0.20 ␮g/g ww in females) and liver cantly higher in liver versus kidney (Table (0.16 vs. 0.12 ␮g/g ww in males and fe- 4). Magnesium was highest in muscle. males, respectively). Mean Cd and Co levels were higher in Significant correlations between vari- kidney than liver (Table 4). ables within tissue are given in Tables 5 Among beluga whale tissues, Hg con- and 6. A number of elements correlated 698 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001

TABLE 3. Linear regression parameters for THg versus Se (␮mol/Kga) in liver, and selected elements (␮g/g wwb) versus body length (Lgth) in Alaskan bowhead whales (P Ͻ 0.05 only).

␤ c 2 Tissue Variables n 1-Coef r P (regression)

Liver THga vsd Sea 55 0.02* 0.190 0.001 Seb vs Lgth (m) 55 0.088 0.146 0.004 Agb vs Lgth (m) 20 Ϫ0.067 0.199 0.049 Cdb vs Lgth (m) 55 1.802 0.378 Ͻ0.001 Kidney Cdb vs Lgth (m) 48 3.328 0.396 Ͻ0.001 a Expressed on a molar basis (␮mol/Kg). b Expressed on mass basis (ppm ww). c ␤ Ͻ 1 coefﬁcient. For THg vs Se, a * denotes that the slope was signiﬁcantly (P 0.05) different from one. d vs ϭ versus.

(both positively and negatively) with age MHg exhibited a significant positive linear (GLGs). Both organic and inorganic forms relationship with age, as did Se and Cd of Hg accumulated with age in all tissues (Table 7). except blubber. Levels of Cu declined as Species comparison a function of age in liver, muscle, and epidermis. Linear regression parameters of Bowhead whales had greater mean he- Se versus various forms of Hg; and of Se patic Cd levels than beluga whales (P Ͻ versus the summed molar quantity of total 0.05), while the reverse was true for Hg, Hg and Ag are shown in Table 6. There Se and Ag. Zinc levels were lower in kid- was a significant linear relationship be- ney of bowhead whale, while the mean tween total Hg and Se (on a molar basis) Cd:Zn molar ratio was significantly great- in liver and kidney, with r2 values of 0.61 er for bowhead whale (mean ϮSD Cd:Zn and 0.55, respectively; but no such rela- ϭ 0.44 Ϯ 0.37) as compared to the beluga tionships between Se and Hg or Ag were whale (mean ϮSD Cd:Zn ϭ 0.19 Ϯ 0.08). evident in muscle, blubber, or epidermis. Mean Hg levels in muscle were signifi- Liver and kidney were the only tissues cantly higher in beluga whale, and the with detectable concentrations of Ag; sig- mean hepatic THg:Se molar ratio was significant linear relationships between Se nificantly greater in beluga whale as com- and Hg ϩ Ag were observed, with r2 val- pared to bowhead whale [0.279 Ϯ 0.169 ues of 0.59 in liver and 0.55 in kidney (Ta- (SD) vs. 0.016 Ϯ 0.020 (SD), respective- ble 7). Pearson’s correlation coefficient for ly]. the relationship between liver Ag and Se DISCUSSION was not statistically significant, while the correlation between THg and Se was high- The two cetacean species sampled for ly significant (Table 5). Both total Hg and this study, bowhead and beluga whales, are Se showed significant accumulation with important subsistence resources to Alaskan age in the liver (Table 5). Natives, and may be good indicators of Among linear regression analyses for se- some arctic ecosystem contamination. lected elements versus age (GLGs) (Table These species are year-round inhabitants 7), hepatic Se, Cd, THg, and MHg all in- of the Arctic and Subarctic (Stewart and creased significantly with age. In liver, the Stewart, 1989; Moore and Reeves, 1993; proportion of total mercury present as Waller, 1996), and occupy different tro- MHg (%MHg) decreased with age (Table phic levels. Biomagnification of toxic met- 7). In contrast, the difference (HgD) be- als in beluga whales is likely, as they con- tween THg and SHg increased with age. sume a variety of prey, including fish [i.e., In the kidney, THg, SHg, Hg(II), and Arctic cod (Boreogadus saida), Arctic char WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 699 . 1 2 2 2 c 24 23 24 15 3.09 0.58 0.471 4.58 1.16 0.70 10.32 — — 12.42 NA 1 2 3 2,3 c 24 23 24 15 0.88 0.35 0.54 — — 0.464 1.72 0.59 1.12 0.67 NA 1 2 2,3 2 c 24 23 24 15 2.80 0.05 0.014 4.02 0.05 0.02 10.05 — — 10.68 NA 1 1,2 2 3 1 50 45 24 11 17 6.96 8.97 1.04 4.02 10.59 16.81 40.36 36.92 30.87 47.19 1 2 2 b b 24 24 11 11 17 0.03 0.002 0.06 0.01 10.20 15.91 ND ND 1 2 2 2 2 50 45 24 16 17 3.24 0.06 0.36 7.47 6.27 0.28 0.58 9.56 32.67 41.41 1 2 b b 24 24 11 11 11 0.18 0.01 0.01 ND ND — 0.77 1 2 2 2 2 24 11 11 11 24 3.880 0.829 0.035 0.317 4.97 1.19 0.03 0.60 25.93 23.29 1 2 3 2 3 24 24 11 11 11 0.90 0.69 0.10 0.32 0.07 3.99 1.51 1.65 0.39 0.44 4 1 1,2 3 2,3 24 24 11 17 24 69.36 88.08 11.15 83.52 44.91 107.45 261.8 322.7 410.3 386.4 1 2 b b 50 45 11 11 11 0.01 0.03 0.01 ND ND — 0.02 1 2 2 2 2 45 24 16 17 50 0.42 0.41 0.35 0.18 1.94 0.92 0.52 0.50 23.76 20.91 1 2 1,2 1 24 24 11 11 11 0.01 0.03 0.01 0.01 0.02 0.04 0.02 0.01 — ) indicates mean level for that element is not significantly different among those tissue(s) as determined by one-way ANOVA with Tukey’s post-hoc test g/g ww) of selected elements in Alaskan beluga whales. ␮ 1,2,3 1 2 3 b 50 45 11 11 11 1.76 6.02 0.01 3.81 0.02 12.15 ND — 1 1 1 2 1 As Cd Co Cu Pb Mg Mn THg Mo Se Ag Zn HgII MHg SHg 50 45 11 16 17 0.06 0.17 0.06 0.91 0.31 0.14 0.27 0.10 1.41 0.30 4. Concentrations ( a a a a a Mean SD Mean SD Mean SD Mean SD Mean SD n n n n n ABLE Not detected in greater than 50% of the samples. SD is standard deviation. Not analyzed. Mean GLGs were 26 for liver, 25 for kidney, and 22 for muscle, blubber, and epidermis. The same superscript number ( T Liver a b c Kidney Muscle Blubber Epidermis 700 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001

TABLE 5. Pearson’s correlation coefﬁcients between selected signiﬁcantly correlated (P Ͻ 0.05) variablesa in liver and kidney of Alaskan beluga whales.

Liver rPn Kidney rPn

As vsb GLGsc 0.54 Ͻ0.001 46 Cd vs GLGs 0.61 Ͻ0.001 41 Cd vs GLGs 0.54 Ͻ0.001 46 THg vs GLGs 0.59 0.007 20 Cd vs As 0.33 0.018 50 MHg vs GLGs 0.84 Ͻ0.001 20 Cu vs GLGs Ϫ0.58 Ͻ0.001 46 THg vs Cd 0.52 0.009 24 THg vs GLGs 0.68 0.001 20 Hg(II) vs GLGs 0.59 0.006 20 THg vs Cd 0.49 0.014 24 Hg(II) vs Cd 0.44 0.034 23 THg vs SHg 0.79 Ͻ0.001 24 Hg(II) vs THg 0.86 Ͻ0.001 23 Hg(II) vs GLGs 0.65 0.002 20 SHg vs GLGs 0.64 0.003 20 Hg(II) vs Cd 0.45 0.028 24 SHg vs Cd 0.46 0.027 23 Hg(II) vs Cu Ϫ0.51 0.012 24 SHg vs THg 0.86 Ͻ0.001 23 Hg(II) vs THg 0.77 Ͻ0.001 24 SHg vs Hg(II) 0.99 Ͻ0.001 23 MHg vs GLGs 0.78 Ͻ0.001 20 SHg vs MHg 0.85 Ͻ0.001 23 MHg vs Cd 0.54 0.007 24 Se vs GLGs 0.60 Ͻ0.001 41 MHg vs THg 0.71 Ͻ0.001 24 Se vs Cd 0.65 Ͻ0.001 45 MHg vs Hg(II) 0.62 0.001 24 Se vs THg 0.73 Ͻ0.001 24 SHg vs GLGs 0.71 0.001 20 Se vs Hg(II) 0.67 Ͻ0.001 23 Se vs GLGs 0.75 Ͻ0.001 46 Se vs MHg 0.67 0.001 23 Se vs As 0.43 0.002 50 Se vs SHg 0.68 Ͻ0.001 23 Se vs Cd 0.62 Ͻ0.001 50 Ag vs GLGs 0.48 0.032 24 Se vs THg 0.75 Ͻ0.001 24 Ag vs THg 0.69 Ͻ0.001 24 Se vs Hg(II) 0.62 0.001 24 Ag vs Hg(II) 0.87 Ͻ0.001 23 Se vs MHg 0.65 0.001 24 Ag vs MHg 0.65 Ͻ0.001 23 Se vs SHg 0.64 0.001 24 Ag vs SHg 0.84 Ͻ0.001 23 Ag vs Cu 0.59 0.002 24 Ag vs Se 0.49 0.014 24 Zn vs Cd 0.57 Ͻ0.001 50 Zn vs Cd 0.49 0.001 45 Zn vs Se 0.35 0.017 45 a Data were ranked prior to analysis. b vs ϭ versus. c Growth layer groups (GLGs), GLGs/2 approximates age in years (Goren et al., 1987).

(Salvelinus alpinus), whitefish (Coregonus to accumulate non-essential elements sp.), and Pacific herring (Clupea haren- might be considered less than that of polar gus)], as well as invertebrates such as bears, seals, or toothed whales. However, shrimp (Argus sp., Crangon sp., and Eu- age-dependent accumulation in particular alus sp.), and squid (Octopus sp.) (Seaman tissues, coupled with a long bowhead et al., 1982). Seaman et al. (1982) recount- whale lifespan (Ն100 yr) (George et al., ed evidence that large belugas (generally 1999) increase the probability that some older males) consumed larger fish than did metals amass. the smaller whales, as the diet of adult be- While the current study relies heavily on luga males apparently contained a substan- correlation analysis, many correlations tially greater amount of larger fish species, among our data have associated P-values such as cods (Gadus sp.) than did the diet of Ͻ0.01 or Ͻ0.001, which are not liable of adult females or immature animals of to be due to chance. Moreover, we scru- either gender. Bowhead whales (Balaena tinized data for common patterns that mysticetus) feed near the base of the food would implicate elemental interactions chain upon small invertebrates collectively across tissues and species. Also, it is im- known as krill, which is comprised of a va- portant to recognize that relationships be- riety of euphausiid, copepod, mysid, and tween individual elements may be due to amphipod species (Lowry, 1993). As lower age (thus mutually associated) or to a di- trophic level consumers, their opportunity rect interaction. WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 701

TABLE 6. Pearson’s correlation coefﬁcients between selected signiﬁcantly correlated variablesa (P Ͻ 0.05) in muscle, epidermis, and blubber of Alaskan beluga whales.

Muscle rPnEpidermis rPn

Cu vsb GLGsc Ϫ0.73 0.01 11 Cu vs GLGs Ϫ0.59 0.032 13 Hg(II) vs GLGs 0.82 0.002 11 THg vs GLGs 0.62 0.043 11 Hg(II) vs As 0.66 0.028 11 THg vs Cu Ϫ0.63 0.038 11 Hg(II) vs Cu Ϫ0.64 0.001 11 Hg(II) vs GLGs 0.77 0.004 12 Hg(II) vs THg 0.65 0.032 11 Hg(II) vs As 0.52 0.045 15 MHg vs GLGs 0.73 0.011 11 Hg(II) vs THg 0.93 Ͻ0.001 11 MHg vs As 0.68 0.022 11 MHg vs GLGs 0.81 0.001 12 MHg vs Cu Ϫ0.61 0.002 24 MHg vs Cu Ϫ0.74 0.002 15 MHg vs THg 0.84 0.001 11 MHg vs THg 0.76 0.006 11 MHg vs Hg(II) 0.83 Ͻ0.001 24 MHg vs Hg(II) 0.60 0.018 15 SHg vs GLGs 0.73 0.011 11 SHg vs GLGs 0.81 0.001 12 SHg vs As 0.68 0.022 11 SHg vs As 0.61 0.017 17 SHg vs Cu Ϫ0.63 0.001 24 SHg vs Cu Ϫ0.74 0.001 15 SHg vs THg 0.84 0.001 11 SHg vs THg 0.77 0.006 11 SHg vs Hg(II) 0.84 Ͻ0.001 24 SHg vs Hg(II) 0.61 0.017 15 SHg vs MHg 1.0 Ͻ0.001 24 SHg vs MHg 1.0 Ͻ0.001 15 Se vs Cu Ϫ0.64 0.001 24 Se vs As Ϫ0.65 0.005 17 Se vs Hg(II) 0.49 0.015 24 Se vs THg Ϫ0.70 0.016 11 Zn vs Cu 0.42 0.042 24 Zn vs THg Ϫ0.71 0.015 11 Zn vs Se Ϫ0.68 Ͻ0.001 24 Zn vs MHg Ϫ0.60 0.019 15 Ϫ Blubber Zn vs SHg 0.59 0.02 17 Se vs Cu 0.72 0.002 12 Zn vs Cu 0.79 0.004 11 a Data were ranked prior to analysis. b vs ϭ versus. c Growth layer groups (GLGs), GLGs/2 approximates age in years (Goren et al., 1987).

Arsenic tramethylarsonium cation, which was de- Arsenic was relatively low in most tis- tected in pinniped, but not cetacean liver sues of both cetacean species, with highest (Goessler et al., 1998). These organic ar- As levels in blubber, which exceeded con- senic compounds are considered relatively centrations in liver about ten-fold. Similar non-toxic to mammals and are apparently findings have been reported for Canadian excreted unchanged (Vahter et al., 1983; narwhal, in which As concentrations were Sabbioni et al., 1991; Neff, 1997). also highest in blubber (Wagemann et al., Previous research has found no accu- 1983). The finding of highest As levels in mulation of hepatic As with age, nor any blubber indicates that it is probably in or- association of As with other trace elements ganic form and thus lipid soluble. Bratton (Becker et al., 1997). In the present study, et al. (1997) speciated hepatic and renal As in beluga liver showed a highly signifi- As in a single bowhead whale via high-per- cant positive correlation with age, which formance liquid chromatography (HPLC) may implicate accrual of As over time. Al- and ascertained that 98% of As was in the ternatively, higher levels of As in larger form of arsenobetaine, which is the pre- (i.e., older) as compared to smaller whales dominant form of As in marine animals may reflect different environmental expo- (Neff, 1997). Several other organic As sures, prey selection, and/or qualitative compounds have been identified in a va- differences in blubber constituents. Larg- riety of marine organisms (including ma- er, older whales may consume prey higher rine mammals): arsenocholine, dimethylar- in As than that typically taken by younger, sinic acid, methylarsonic acid and the te- smaller animals. Or, the higher As levels in 702 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001

TABLE 7. Linear regression parameters for Sea versus various forms of Hga or Hg ϩ Aga in tissues of Alaskan beluga whales, and selected elements (ppm ww) versus GLGsb (age).

␤ c 2 Tissue Variables n 1-Coef r P (regression)

Liver Sea vsd THga 24 1.90* 0.614 Ͻ0.001 Sea vs THga ϩ Aga 24 1.51 0.594 Ͻ0.001 Sea vs Hg(II)a 24 2.95 0.222 0.020 Se (ppm) vs HgDe (ppm) 24 0.61* 0.568 Ͻ0.001 Kidney Sea vs THga 24 0.90 0.525 Ͻ0.001 Sea vs THga ϩ Aga 24 0.89 0.551 Ͻ0.001 Liver THg (ppm) vs GLGsb 20 1.34 0.353 0.006 Se (ppm) vs GLGs 46 1.56 0.408 Ͻ0.001 Cd (ppm) vs GLGs 46 0.07 0.279 Ͻ0.001 MHg (ppm) vs GLGs 20 0.05 0.503 Ͻ0.001 %MHg vs GLGs 20 Ϫ0.76 0.295 0.013 HgDe vs GLGs 20 0.95 0.297 0.013 Kidney THg (ppm) vs GLGs 20 0.20 0.354 0.006 Se (ppm) vs GLGs 41 0.15 0.346 Ͻ0.001 Cd (ppm) vs GLGs 41 0.27 0.346 Ͻ0.001 HgII (ppm) vs GLGs 20 0.13 0.278 0.017 MHg (ppm) vs GLGs 20 0.02 0.641 Ͻ0.001 SHg (ppm) vs GLGs 20 0.15 0.331 0.008 a Expressed on a molar basis (␮mol/Kg). b Growth layer groups (GLGs), GLGs/2 approximates age in years (Goren et al., 1987). c ␤ ϩ 1 coefﬁcient. For Se vs various forms of Hg or THg Ag in liver and kidney, a * denotes that the slope was signiﬁcantly (P Ͻ .05) different from one. d vs ϭ versus. e Mercury level difference, HgD ϭ total Hg (THg) Ϫ sum Hg (SHg) (all in ppm).

kidney and liver of males could result from ship between As and any other element gender-related prey preferences, blubber across tissues or species. constituents or physiology. Because As in these whales is probably Lead in organic form and lipid-soluble, it can Caution must be exercised in interpre- likely traverse the placenta and mammary tation of Pb levels in animals taken using gland, so that reproductively active fe- lead ammunition as was the case for be- males might transfer As to their offspring. luga whales. Higher Pb levels were ob- While inorganic As is known to cross the served in liver as compared to kidney in placental barrier (Lindgren et al., 1984), both cetacean species; nevertheless, all tis- studies on placental transfer of the organic sues had very low Pb levels, which were As compounds found in seafood are lack- well below concentrations likely to be as- ing. However, it is not unreasonable to associated with toxic effects (Puls, 1994). sume it could cross the placenta, because the predominant form of organic As in fish Cadmium, copper, zinc and crustaceans, arsenobetaine, is distrib- In bowhead and beluga whales mean uted widely throughout the body, is not kidney Cd concentrations were two- to metabolized, and appears to be excreted four-fold higher than in liver. Cadmium in rapidly in the urine (Vahter et al., 1983). these two tissues was elevated in both spe- Although there were statistically significant cies compared to what are considered nor- correlations between As and various other mal levels in domestic species such as dogs elements, we found no consistent relation- and cattle (Puls, 1994). However, the Cd WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 703 concentrations found in this study are well characteristic of chronic Cd toxicosis were within ranges previously reported for arc- not observed (Woshner, 2000). tic marine mammals (Hansen et al., 1990; In bowhead and beluga whales, Cd ac- Wagemann et al., 1996; Dietz et al., 1996, cumulated with age in both liver and kid- 1998). While the kidney Cd concentrations ney, corroborating a well-documented observed in this study are not unusual for phenomenon (Wagemann et al., 1983, some marine mammals, the threshold for 1989, 1990; Dietz et al., 1996; Becker et Cd-induced chronic renal toxicosis is not al., 1997; Krone et al., 1999); Cd also ac- known for these species. Among terrestrial cumulated with age in muscle of bowhead mammals, including humans, a renal cor- whales. Cadmium demonstrated a signifi- tical concentration of 200 ␮g/g ww has cant positive correlation with Se in liver of been acceded as the critical threshold for both whale species, as well as in kidney of chronic Cd toxicosis, seen clinically as a belugas. While Se has been shown to ame- low molecular weight proteinuria (WHO, liorate toxic effects of Cd in laboratory an- 1992). Our results, as well as most previ- imals (Ridlington and Whanger, 1981; ous studies, have documented Cd concen- Wahba et al., 1993; Rana and Verma, trations in renal tissue of marine mammals 1996), its association with Cd in the two from entire renicules, including medulla, whale species studied could have resulted and thus underestimate cortical Cd con- from coincidental accumulation of these centrations by about 25% (Dietz et al., two elements with age. If Se were actively 1998). For example, Cd has been reported and proportionately assimilated as a con- at levels in excess of 50 ␮g/g ww in the sequence of Cd exposure in cetaceans, intact bowhead renicules, which includes bowhead whales would have exhibited medullary and cortical tissue (Bratton et higher Se levels than were observed in this al., 1997). Since Cd concentrates in prox- study. Additionally, Cd was positively cor- imal tubular cells of the cortex, which av- related with Hg in liver and kidney of both erages 0.22 (22%) the thickness of the en- species, probably as a consequence of mu- tire renicule (Haldiman and Tarpley, tual accretion with age. Because of these 1993), it might be assumed that cortical findings, it seems doubtful that Se plays a Cd levels are proportionately greater. major role in Cd detoxification in these Thus, Cd cortical levels may reach con- species; nevertheless, the possibility exists centrations approximating 200 ␮g/g, the that Se may, at least to some degree, form accepted threshold for renal toxicity temporary or stable complexes with Cd (WHO, 1992). Very few studies of marine similar to the tiemannite (Hg-Se) com- mammals have included histopathological plexes previously noted in liver of marine examination of kidney in conjunction with mammals (Martoja and Berry, 1980). chemical analyses. One exception was a While simple evaluation of tissue residues study of ringed seals divided into three cannot adequately address this issue, pre- groups on the basis of kidney Cd levels. sent and future studies, especially using Concentrations in the five seals with the cell culture systems, may help elucidate highest renal Cd burden averaged 399 ␮g/g the processes of Cd metabolism in marine ww and the five with the lowest having a mammal species. range of only 1.63 to 5.19 ␮g/g ww. No Cadmium correlated positively with Zn lesions typical of chronic renal Cd toxicosis in kidney of both species, as well as in liver were observed among initially frozen renal of belugas. Copper also correlated posi- tissue sections examined microscopically tively with Ag in liver of both species. In- (Dietz et al., 1998). Histopathologic ex- tercorrelations among various combina- aminations of these beluga and bowhead tions of the elements Cd, Cu, Hg, Ag and whales in the present study would appear Zn were common. It has been shown re- to support this assertion, because lesions peatedly that both Cd and Hg accumulate 704 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001 with age (Norstrom et al., 1986; Braune et kidney. Wagemann et al. (1996) also found al., 1991; Muir et al., 1992; Wagemann et mean Zn levels to be similar across these al., 1996). Cadmium binds primarily to latter three tissues within species (nar- metallothionein (MTH), an inducible, whals, belugas, and ringed seals), and Zn small molecular weight metal-binding pro- (and Cu) levels tended to track those of tein important for both homeostasis and Cd. While we observed similar associations detoxification of various metals, especially among Zn, Cd, and Cu within species, dif- Cd, Zn and Cu. Although we did not eval- ferences in mean Zn levels among tissues uate MTH’s role in the current study, the were more marked. This is most likely be- association between Cd, Zn and Cu is cause Alaskan animals have a lower Cd most likely due to common binding with burden compared to the Canadian ani- MTH, various isoforms of which are pre- mals, mirroring the geologic gradient of sent in a wide array of tissue types. While Cd, which decreases from east to west the correlation of Cd with Hg is likely due across North America (Wagemann et al., to mutual accumulation over time, MTH 1996). is also capable of binding Hg and Ag, al- A significant negative correlation was beit with lesser affinity than for Cd, Zn, or seen between Cu and age in liver, muscle Cu (Kaim and Schwederski, 1991). Various and epidermis of belugas as well as in liver metals are capable of inducing MTH syn- of bowheads, confirming a phenomenon thesis, in turn increasing the opportunity previously reported in marine mammals for binding of Cd and other metals to it (Wagemann et al., 1983, 1989). This de- (Piotrowski et al., 1974; Sugawara and Su- cline in tissue Cu could result from loss of gawara, 1984). Consequently, intercorre- Cu over time, dilution of Cu levels by in- lations of the aforementioned metals could creased tissue mass with age, or decreased result both from mutual binding to induc- tissue level requirements for Cu (i.e., met- ible MTH, as well as coincidental accu- abolic regulation). mulation with age via non-MTH process- Silver es. Previous studies in marine mammals have shown that most Hg is in the ‘‘insol- Belugas had mean hepatic Ag levels that uble’’ rather than the ‘‘soluble’’ cellular were markedly greater than those ob- fraction, and thus not MTH-associated served in bowhead whales (this study), (Lee et al., 1977; Wagemann et al., 1984). ringed seals or polar bears (Woshner et al., Whales in the present study had mean 2001), corroborating a previously reported hepatic Cu concentrations that would be phenomenon for this species (Becker et considered below the normal range for al., 1995). The mean hepatic Ag concen- dogs or cattle (Puls, 1994). Nevertheless, tration for beluga whales was approximate- levels in this study were in agreement with ly 300 times greater than levels in kidney. previously published data in cetacean spe- Other tissues of beluga (muscle, blubber cies (Wagemann et al., 1996; Krone et al., and epidermis) exhibited uniformly low Ag 1999). The levels of essential elements, levels, as did all tissues of bowheads. Beck- like Cu, are reported here for apparently er et al. (1995) noted that in beluga liver, healthy animals and may be useful in cases both Ag and Hg were correlated positively where a deficiency is suspected. with Se, although Ag was more strongly In belugas, the highest mean Zn con- correlated with Se than was Hg. In addi- centration occurred in the epidermis. Wa- tion, there was a trend toward increasing gemann et al. (1996) noted a similar ob- concentrations of all three elements with servation in Canadian belugas and nar- age. Similar positive correlations for he- whals; muktuk (epidermis and blubber patic Ag with Se or age were not evident combined) contained approximately two to in the present research. In kidney of be- three times more Zn than muscle, liver or luga and liver of bowhead whales Ag was WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 705 positively correlated with Se, Cd, and var- compensate for the Se-rich fish diet con- ious forms of Hg. A positive correlation sumed by toothed cetaceans. Beluga between hepatic Ag and Cu was observed whales molt annually, possibly reducing Se in both species examined and might con- burdens through this mechanism. In bow- note binding by a common ligand. head whales, mean Se levels in liver and The elevated levels of hepatic Ag in be- kidney were similar, and were significantly lugas remain a conundrum. This phenom- lower than in liver and kidney of belugas. enon may be a consequence of some dis- In general, Se increased with age indi- tinctive alimentary predilection, or may re- ces in liver, kidney and epidermis of bow- flect a novel physiologic function for this head whales, as well as in liver and kidney metal. The fact that hepatic Cu is low of belugas, which echoed prior research among belugas in comparison to terrestrial findings in these and other marine mam- species suggests that Cu may be limiting mal species (Dietz et al., 1996; Bratton et in this species or not essential at the levels al., 1997), as did the close association be- typically noted in terrestrial species. Since tween Hg and Se. While Hg-Se complexes Ag is chemically similar to Cu (in terms of have been demonstrated in liver tissue of its valence shell configuration) and rela- marine mammals, the precise structure, tively benign except at high concentra- function, and distribution of these com- tions, it may function in homeostasis of Cu plexes is not known (Martoja and Berry, or other essential metals in belugas by way 1980; Pelletier, 1985; Nigro and Leonzio, of MTH, but this is speculative. Australian 1996), and presence of them in other tis- dugongs were found to have hepatic Ag sues has not been addressed. In controlled concentrations comparable to those of be- experiments, Se has been proven to miti- lugas in our research (Denton et al., 1980). gate toxic effects of Hg (Stillings et al., This finding, in conjunction with an age- 1974; Ridlington and Whanger, 1981; Cu- related decline in liver Cu led Denton et vin-Aralar and Furness, 1991), Cd (Rid- al. (1980) to suggest that aged dugongs lington and Whanger, 1981; Rana and Ver- may become Cu deficient, as Ag may oc- ma, 1996) and Ag (Ridlington and Whan- cupy sites vacated by Cu. ger, 1981; Wagner et al., 1975). It has been proposed that the ameliorative action of Se Selenium and mercury towards these three metals occurs via an- Mean Se concentrations in liver and kid- tioxidant activity because other antioxi- ney tissue of belugas were consistent with dants such as reduced glutathione (GSH) levels associated with toxicosis in domestic and vitamin E offer similar protection species such as cattle and dogs (Puls, (Rana and Verma, 1996), or because these 1994), although well within ranges previ- metals deplete the Se-dependent enzyme ously reported for this species (Wagemann glutathione peroxidase (GSH-Px; Wagner et al., 1996). In belugas, Se concentrations et al., 1975; Sidhu et al., 1993). Evidence in liver were approximately 6-fold higher also exists for Se acting as an antioxidant than levels in kidney. Belugas had higher through formation of metal selenide com- Se concentrations in epidermis than bow- plexes, in particular for Ag (Aaseth et al., head whales. High concentrations of Se 1981) and Hg (Bjo¨rkman et al., 1995). The have been reported in epidermis of harbor strength of association between Cd and Se porpoises from Greenlandic waters (Dietz in the present study often rivaled that of et al., 1990; Paludan-Mu¨ ller et al., 1993). Hg and Se. While this could be due mere- Selenium in epidermis at concentrations ly to independent, concurrent increase approximately 100 times those of Hg led with age, an interaction between Cd and Paludan-Mu¨ ller et al. (1993) to speculate Se cannot be ruled out. that epidermal tissue may serve as a stor- Total Hg was present in liver and kidney age and/or excretory mechanism for Se to of beluga at concentrations that would be 706 JOURNAL OF WILDLIFE DISEASES, VOL. 37, NO. 4, OCTOBER 2001 considered high or even toxic among do- mals was not borne out in the present re- mestic species (Puls, 1994), but within search. Regression analyses of hepatic Se ranges that have been reported for marine versus THg (or THg vs. Se) expressed in mammals (Koeman et al., 1973, 1975; molar quantities for both species evaluated Dietz et al., 1990; Braune et al., 1991; Wa- revealed slopes significantly different from gemann et al., 1996). As previously men- one. While the slope for the linear regres- tioned, total Hg concentrations in beluga sion of Se versus THg ϩ Ag (expressed as whale tissues varied depending upon ana- molar quantities) did not differ significant- lytical method employed, such that SHg ly from one, the lack of a significant cor- was lower than THg, especially in liver. relation between THg and Ag in liver, in This discrepancy could be due to interla- conjunction with the age associations for boratory variation; alternatively, it might these elements noted earlier, indicates that suggest that a substantial portion of the Hg the accumulation of Ag is not responsible in the organic fraction exists in a form oth- for disrupting a 1:1 Hg:Se ratio, as it does er than MHg (Wagemann et al., 1997). not appear to compete with Hg for Se. The mean difference in the fraction of Various researchers have presented infor- MHg obtained as percentage of THg ver- mation supporting and refuting the consis- sus SHg in the liver was approximately 7%. tency of a 1:1 ratio in tissues of marine Nevertheless, whether computed as a per- mammals. Some authors have proposed a centage of THg or SHg, the majority of hypothesis of a threshold concentration of Hg in the liver (ϳ90%) of beluga whales Se equivalent to the physiologically essen- was inorganic [Hg(II)]. Conversely, virtu- tial level of Se plus a reservoir of this met- ally all (Ͼ90%) of the Hg in beluga muscle alloid (Hansen et al., 1990; Krone et al., and epidermis was in the form of MHg. 1999). These workers suggested that Hg In beluga liver, Hg(II), MHg and total creates a far greater binding capacity for Hg (either THg or SHg) increased signif- Se, which eventuates the accumulation of icantly with age (GLGs). Of all forms of the two elements in a 1:1 molar ratio fash- Hg, MHg had the lowest rate of accumu- ion. If this is the case, it is possible that lation (0.054 ␮g/g MHg per GLG) but the hepatic Hg concentrations encountered in most linear relationship to age, with a re- this study did not consume the Se reser- gression coefficient of 0.50. While absolute voir and thus did not stimulate markedly concentrations of hepatic MHg increased increased Se uptake and binding. Regard- with age, the percentage of hepatic total less, it is important to recognize that orig- Hg in the form of MHg declined signifi- inally, Koeman et al. (1973, 1975) arrived cantly with age. In beluga epidermis and at this 1:1 ratio by averaging regression muscle (where virtually all Hg is in the slopes of Hg versus Se for 56 animals of form of MHg), SHg, Hg(II) and MHg nine different marine mammal species, as were positively correlated with age well as detecting a 1:1 Hg:Se molar ratio (GLGs), and there was a positive correla- in brain, kidney and liver from a single tion between THg and age in epidermis. seal. The tendency for Hg:Se molar con- No significant correlation was observed centrations to converge on a 1:1 ratio was between Hg and Se in epidermis, while in offered as evidence that Se complexation muscle, Se was positively correlated with with Hg was the probable mode of detox- Hg(II), but not with any other form of Hg. ification in marine mammals, occurring The MHg form was negatively correlated through an active process rather than by with Cu in both epidermis and muscle and passive coaccumulation. While this per- with Zn in epidermis. ception was profound, subsequent re- The 1:1 molar relationship purported to searchers have suggested that deviations be characteristic of the association be- from this 1:1 molar status indicate abnor- tween hepatic Hg and Se in marine mam- mality, or even toxicity unaccompanied by WOSHNER ET AL.—ESSENTIAL AND NON-ESSENTIAL ELEMENTS IN BOWHEAD AND BELUGA WHALES 707 detected evidence of compromised health Japanese Society of Scientific Fisheries 45: 623– (Arima and Gakura, 1979; Caurant et al., 626. BECKER, P. R., E. A. MACKEY,R.DEMIRALP,M.M. 1996). Findings of the present study do SCHANTZ,B.J.KOSTER, AND S. A. WISE. 1997. not counter the recognized relationship of Concentrations of chlorinated hydrocarbons and Hg with Se, or the premise that this as- trace elements in marine mammal tissues ar- sociation could be the primary mode of Hg chived in the U.S. National Biomonitoring Spec- detoxification in marine mammals. Nev- imen Bank. Chemosphere 34: 2067–2098. , , ,R.SUYDAM,G.EARLY,B. ertheless, it may not be necessary to KOSTER, AND S. A. WISE. 1995. Relationship of achieve a 1:1 relationship even in tissues silver with selenium and mercury in the liver of that accumulate high levels of Hg (liver of two species of toothed whales (Odonotocetes). belugas) for the animals to be protected Marine Pollution Bulletin 30: 262–271. from Hg toxicosis. The current research BJO¨ RKMAN, L., K. MOTTET,M.NYLANDER,M.VAHT- ER,B.LIND, AND L. FRIBERG. 1995. Selenium also suggests the existence of ancillary de- concentrations in brain after exposure to meth- toxification mechanisms for Hg in these ylmercury: Relations between the inorganic mer- species, especially in tissues other than liv- cury fraction and selenium. Archives of Toxicol- er. ogy 69: 228–234. Results of the present study indicate a BLOOM,N.,AND W. F. F ITZGERALD. 1988. Deter- mination of volatile mercury species at the pi- strong need for more comprehensive re- cogram level by low-temperature gas chromatog- search of mercury speciation, metal local- raphy with cold-vapor atomic fluorescence de- ization at the cellular level, coordinated as- tection. Analytica Chemica Acta 208: 151–161. sessment of health of marine mammals by . 1992. On the chemical form of mercury in gross and histological examination, and edible fish and marine invertebrate tissue. Ca- nadian Journal of Fisheries and Aquatic Sciences physiologic/behavioral assessments. These 49: 1010–1017. issues form the focus of our on-going re- , AND E. A. CRECELIUS. 1983. Determination search and present us with great challeng- of mercury in seawater at sub-nanogram per liter es in understanding the toxicodisposition, levels. Marine Chemistry 14: 49–59. interactions, essentially and potential ad- BRATTON, G. R., W. FLORY,C.B.SPAINHOUR, AND E. M. HAUBOLD. 1997. Assessment of selected verse effects of these elements in arctic heavy metals in liver, kidney, muscle, blubber, marine mammals. and visceral fat of Eskimo harvested bowhead whales Balaena mysticetus from Alaska’s north ACKNOWLEDGMENTS coast. Final report submitted to the North Slope We are very grateful to the many subsistence Borough Dept. of Wildlife Management, Barrow, hunters who allowed us to sample their animals Alaska, 233 pp. for this and other studies particularly those ,C.B.SPAINHOUR,W.FLORY,M.REED, AND hunters from Point Hope, Point Lay, and Bar- K. JAYKO. 1993. Presence and potential effects row (Alaska). This project was primarily funded of contaminants. In The bowhead whale, Special by a grant from the Office of Naval Research Publication Number 2 of The Society for Marine (grant number N00014-1-0797) to the North Mammalogy, J. J. Burns, J. J. Montague, and C. Slope Borough; additional support was provid- J. Cowles (eds.). 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