The first part of the present study evaluated tissue concentrations of twelve essential and non-essential elements in four arctic marine mammal species important as subsistence resources to indigenous Alaskans. Species sampled included: bowhead whales, beluga whales, ringed seals, and polar bears. Concentrations of As, Cd, Co, Cu, Pb, Mg, Mn, Hg, Mo, Se, Ag, and Zn, were analyzed in liver, kidney, muscle, blubber, and epidermis (the latter in cetaceans only). Elements that were identified as having tissue concentrations, which in domesticated species would have been considered higher than normal and/or even toxic, were Cd, Hg, Ag, and Se. However, the concentrations of these elements were consistent with previous reports for arctic marine mammals. Remaining elements were at concentrations within normal ranges for domesticated species, although Cu was found frequently at concentrations that would be considered marginal or deficient in terrestrial domesticated animals. Across-species comparisons revealed that Cd was highest in kidney, followed by liver in all four species. Its concentrations were frequently correlated with Cu, Zn, Hg, and Se. Cadmium accumulated with age in bowhead and beluga whales, especially in liver and kidney. The relationships between Cd and Hg, and between Cd and Se were believed to be due to mutual accretion with age, although direct interactions could not be ruled out, especially with respect to Cd and Se. Associations between Cd and Cu, and Cd and Zn were potentially attributable to mutual binding with the inducible protein, metallothionein. This assumption was supported by the observation that Cd:Zn ratios in liver and kidney displayed a significant linear relationship to age and that this ratio either increased slightly (in kidney and liver of bowheads) or remained constant (in kidney and liver of belugas) with age. In general, Se was highest in liver and kidney of all four species, where it was frequently at concentrations that would have been deemed elevated or toxic for domesticated species, although within ranges previously reported for arctic marine mammals. Selenium increased with age indices, and was highly correlated with Hg, and often with Cd as well. Mercury also increased with age, and liver contained the highest tissue concentration in the cetacean and pinniped species. The pattern of Se accumulation in polar bears differed, with highest concentrations found in kidney, which suggested that this tissue may be the primary site for Hg detoxification in this species, as is the case for terrestrial mammals. Compared to the other three species, bowhead whales had very low Hg concentrations in all tissues. The highly significant linear relationship between Hg and Se noted in various tissues (particularly liver) of all four species was presumed due to binding of these two elements to each other following demethylation of MHg. This assumption was supported by the observations that while Se and Hg both accumulated with age, the fraction of total Hg that was composed of MHg decreased with age. The quantity that represented the difference between total Hg measured directly and calculated total Hg [i.e., SHg = Hg(II) + MHg], also increased with age in beluga liver. This connoted that a portion of the total Hg present was in an organic form other than MHg, and that this form accumulated with age. Alternatively, this portion, which was apparently not measured by either the Hg(II) or MHg procedures, may have been lost during extraction. Species in this study had mean hepatic Hg:Se molar ratios that were below unity. This implies that Hg concentrations may have been below some threshold level, after which subsequent accumulation proceeds in a 1:1 molar ratio fashion with Se. Alternatively, it might suggest that a 1:1 Hg:Se molar ratio is not a prerequisite for protection from Hg toxcosis among marine mammals, because none of the animals in the present study exhibited lesions typically associated with Hg toxicosis. In beluga liver, concentrations of Ag were elevated when compared to domesticated species. The only element that showed a significant linear association to Ag was Cu—a relationship that was observed in all four species. This suggested that Ag and Cu may be associated through a common ligand, possibly metallothionein. The association between Ag and Se in beluga liver was less strong than that between Hg and Se; moreover, Ag did not increase with age. These findings indicate that Ag probably does not compete with Hg for Se binding, and therefore is unlikely to substantially inhibit detoxification of Hg in beluga whales. In the second portion of this research, tissues from bowhead whales, beluga whales and ringed seals were examined at both the gross and light microscopic level. The purpose of this evaluation was three-fold: to describe the normal histologic appearance of tissues; to perform a routine histologic survey of tissues that would contribute to a general health assessment, and; to scrutinize tissues for lesions that might support a diagnosis of toxicosis caused by Cd, Hg, Ag, or Se. Tissues examined were chosen on the basis of their propensity to be targets for toxicologic injury from the specified elements (with the exception of brain) and included, but were not limited to, the tissues analyzed chemically. Special stains were used to identify particular pigments or tissue components. Overall, the bowhead whales evaluated appeared healthy and had low parasite burdens. The most common lesion, which was observed in all bowheads, was a non-inflammatory chronic renal periglomerular and interstitial fibrosis. This lesion was not typical of Cd-induced nephropathy, and it did not appear to be associated with renal Cd burdens. Nevertheless, thresholds of Cd-induced renal injury are not known for cetacean species, and more whales need to be examined histologically in conjunction with analysis of tissue Cd residues. Acute myodegeneration was observed in cardiac and/or skeletal muscle of a few bowheads, and was presumed to reflect a hunting-induced exertional myopathy. The beluga whales examined were generally in good body condition and appeared healthy grossly, but they had much higher parasite burdens than bowhead whales. In particular, prevalence in belugas of pulmonary nematodiasis was high, being especially common among whales obtained from Pt. Hope compared to those from Pt. Lay. Grossly, firm, caseous nodules were associated with lungworms, while histologically, the associated pulmonary changes ranged from mild chronic inflammation and focal granuloma formation to catarrhal granulomatous and eosinophilic verminous bronchopneumonia. Another change observed in some belugas and believed to be associated with lungworm infection, was multifocal pulmonary arterial medial hypertrophy and degeneration. Beluga whales harvested at Pt. Lay (summer) frequently showed evidence of hepatic and pancreatic atrophy, while whales taken at Pt. Hope (spring) did not. This was believed to result from anorexia during migration—a supposition corroborated by the lack of stomach contents among Pt. Lay whales. Another prominent histologic finding among belugas was hepatic telangectasia, which occurred with significantly greater frequency and severity in Pt. Hope belugas than in those from Pt. Lay. The etiology and significance of this lesion could be not be ascertained, although it was not believed to be associated with any of the elements analyzed in this study. Mild thickening of Bowman’s capsule was seen frequently in belugas. However, this lesion was not typical of Hg or Cd-induced nephropathies, and did not appear correlated with kidney concentrations of these metals. This lesion was believed to be a normal consequence of aging in belugas, although a metal etiology for it could not be excluded irrefutably. In general, ringed seals were in good body condition and appeared healthy on gross examination. Among seals evaluated histologically, the most common finding was a mild, chronic, focal or periportal hepatitis, with focal hepatocellular necrosis sometimes apparent. Although a metal etiology for this lesion could not be definitively ruled out, in the absence of other lesions that would support a diagnosis of metal toxicosis, an infectious etiology was considered more credible. Two out of sixteen seals had embryologic remnants (an epidermoid cyst and an ultimobranchial cyst)—lesions that are usually considered incidental. While no toxic (metal or otherwise) etiology could be ascertained for these lesions, the incidence of retained embryologic remnants seemed high. A number of xenobiotics are known to be endocrine-disruptors, and the potential for such an etiology among these seals should be examined further. Lipofuscin deposition was ubiquitous among all three species examined histologically. Lipofuscin was most prevalent in hepatocytes, but also commonly was observed in various other tissue and cell types, especially in cardiac and skeletal myocytes, and in uriniferous tubular epithelial cells. The third portion of this study employed autometallographic (AMG) development of light microscopic tissue sections to amplify and localize deposition of inorganic Hg in liver and kidney of beluga and bowhead whales. No staining occurred among bowhead tissues, confirming the extremely low concentration of Hg determined through chemical analyses. In beluga kidney sections, AMG granules were seen throughout the uriniferous tubular epithelium, showing that Hg deposits throughout the nephric tubule, and not solely in the proximal tubular epithelium. In liver tissue, AMG granules were deposited primarily in periportal regions among whales with lower hepatic Hg burdens. In addition to periportal deposition, AMG granules were observed in pericentral and mid-zonal regions in the belugas sampled that had higher liver Hg concentrations (generally older animals). Granules were densely concentrated in stellate macrophages, especially near portal triads. Granules also were distributed in hepatocellular cytoplasm, generally concentrated toward the bile cannalicular domain of the cell. Granules were discrete, potentially indicating that Hg was confined within lysosomes. These observations suggested that inorganic Hg deposits initially in periportal regions of young animals, with subsequent accumulation occurring pericentrally, and finally, midzonally as the whales age. Computer-assisted densitometric analysis was used for semi-quantitative evaluation of AMG staining intensities. These AMG staining intensities were well correlated with concentrations of Hg determined via chemical analysis. Areas with AMG-staining were identified and compared with location of lipofuscin in the same field, visualized with fluorescent microscopy. While AMG granules and lipofuscin deposits sometimes were co-localized, they more often were not. In addition, abundant lipofuscin deposition was seen in livers of younger belugas with little to no Hg-catalyzed AMG staining. Also, lipofuscin concentrated predominantly in pericentral regions. These observations suggested that in the healthy marine mammals of this study, marked hepatic lipofuscin deposition most often occurred independently of Hg accumulation. Consequently, hepatic lipofuscin is likely to be a poor indicator of Hg-induced damage in belugas. The abundant lipofuscin deposition in livers of marine mammals was interpreted as most likely denoting a heightened exposure to oxidative stress that is probably inherent to a marine mammalian existence. These oxidative stressors may include a diet high in polyunsaturated fatty acids (PUFAs), alternating hypoxia and abundant oxygenation, and periodic bouts of anorexia associated with migration.
Species sampled: bowhead whale (Balaena mysticetus), beluga whale (Delphinapterus leucas), ringed seal (Phoca hispida) and polar bear (Ursus maritimus). Tissues sampled: Liver, kidney, blubber, muscle and epidermis (the last from whales only) for toxicology; Multiple tissues for histology.
Some tissues also banked with Alaska Marine Mammal Tissue Archival Program (AMMTAP)
From 1995 through 1997, tissues were collected from four species of arctic marine mammals through the cooperation of Eskimo hunters during subsistence food gathering activities. The species sampled were bowhead whales (Balaena mysticetus), beluga whales (Delphinapterus leucas), ringed seals (Phoca hispida) and polar bears (Ursus maritimus). Bowhead whales (n = 20), polar bears (n = 24), and ringed seals (n = 19) were obtained near Barrow, Alaska. Bowheads were sampled during annual spring and fall hunts, that usually take place from mid-April to the end of May, and from mid-September to the end of October. Data from bowheads harvested 1995-97 were combined with data collected previously (1983-90; Bratton et al., 1997). These previous data were from forty-one bowhead whales obtained near Barrow (n = 29), Wainwright (n = 7), Kaktovik (n = 4), and Savoonga (n = 1). Seals were collected during the summer (June/July), while polar bears generally were obtained during the winter (December – February). Beluga whales were collected associated with several villages, including: Pt. Lay (n = 10), Pt. Hope (n = 9), Barrow (n = 4), and Kaktovik (n = 1). At Pt. Lay, beluga samples were collected during an annual hunt that takes place during June/July. At other villages, belugas were generally harvested opportunistically during Spring bowhead whale hunts (May). Data from belugas harvested during 1996-97 were combined with data obtained previously (1992-95; Tarpley et al., 1995). These included data from belugas harvested during the annual summer hunt at Pt. Lay in 1992 (n = 9) and 1995 (n = 10), as well as seven belugas sampled in July, 1993, from the Northwest Territories, Canada. Tissues collected included liver, kidney, muscle, blubber and epidermis, the latter being obtained from the two cetacean species only. To facilitate comparative discussion, blubber was defined as the subcuticular fat of polar bears and seals, as well as the true blubber of cetaceans. Tissues were either frozen or chilled and subsequently processed under clean conditions following a strict protocol (Becker et al., 1999) to minimize contamination from handling. This included rinsing tissue with HPLC grade water (Sigma Chemical, St. Louis, Missouri, USA) prior to sub-sampling and cutting with titanium knives on a clean, Teflon-covered surface. Subsamples of tissues were placed in acid-washed scintillation vials (Fisher Scientific, Pittsburgh, PA) in preparation for heavy metals analysis. All tissues were frozen (-20C) immediately after processing. Frozen tissues were packed in insulated coolers on ice and shipped via 2-day air express to College Station, Texas (College of Veterinary Medicine) for elemental analysis. Teeth were collected from belugas for age determination using dentinal growth layer groups (GLGs), which have been estimated to deposit at a rate of two GLGs annually (Goren et al., 1987). Body length (tip of rostrum to fork) was used as a surrogate indicator of age in bowhead whales (Schell and Saupe, 1993; George et al., 1999). Metals Analysis In preparation for elemental analysis, tissue samples were weighed [wet weight (ww)], dried to constant weight, and digested in a wet ash procedure using concentrated (70%) nitric acid with microwave processing at 120°C for 60 minutes. All metals except Cd, zinc (Zn), and Hg were analyzed using a Perkin-Elmer (Norwalk, Connecticut, USA) model SIMAA 6000 graphite furnace atomic absorption spectrophotometer (GFAAS) instrument, equipped with an AS-60 autosampler and Zeeman background correction (Perkin-Elmer). Cadmium and Zn were determined by flame atomic absorption spectrophotometry (AAS), using a Perkin-Elmer instrument model 306. Methodology followed USEPA recommendations as authored by Smoley (1992), described in detail by Bratton et al. (1997), with the modification of a microwave wet ash digestion, rather than a previously used dry ash procedure. All concentrations were reported on a wet weight basis (in mg/g ww). Each analytical run, with duplicates of each of the following, included: samples, blanks, reference materials, spiked references, and spiked samples. Percent recovery was calculated as follows: % spike recovery = {[(Cs x Vs) – (Cu x Vu)] / (Ct x Vt)} x 100 where: Cs = concentration in spiked sample Vs = total volume of spiked sample (i.e., vol. of sample + vol. of spike) Cu = concentration in unspiked sample Vu = total volume of unspiked sample Ct = concentration of spiking solution Vt = volume of spike placed into sample Reference materials obtained from the National Bureau of Standards (NBS, USA) or the National Research Council of Canada (NRC, Canada) included: dogfish muscle (DORM), dogfish liver (DOLT), oyster tissue, and bovine liver. Reproducibility of results was assured by calculating the relative standard deviation for results from a minimum of ten replications of each standard along with comparisons of relative differences between duplicate tissue sample digests. Targeted within and between run variability for duplicated samples and standard digests was less than 10% for tissues or reference standards with higher metal concentrations, or less than 20% for tissues or reference standards containing lower levels of metals (Bratton et al., 1997). The detection limit for all elements was approximately 0.01 mg/g (ppm). Quality assurance/quality control information is summarized in Tables 1.1 through 1.4. Slight differences from the above methods were employed for bowhead whale samples collected prior to 1995 (Bratton et al., 1993; 1997). For these samples, tissues were dry-ashed at 480°F and analyzed by AAS with Smith-Hieftje background correction, using bovine liver standard #1577 (NBS, USA) as reference material. Mercury Analysis In preparation for total Hg analysis at the Texas Veterinary Diagnostic Laboratory (Amarillo, Texas USA) through direct detection, wet tissue samples were weighed, dried, reweighed, placed in 250 ml quartz volumetric digestion tubes, and digested in a mixture of 5 parts 15 N nitric acid and 1 part each of 36 N sulfuric acid and 70% perchloric acid. After four hours at room temperature, the samples underwent a heating phase in which the temperature was raised in a five-step sequence over a period of six hours to a final temperature of 200°C, where it was maintained for two hours. After cooling, digests were transferred to volumetric flasks and brought to 25 ml with deionized water. Samples were placed in reaction vessels to which 20 ml of 1 N HCl were added. Each analytical run included blank digests, working standards, and one standard reference material (DOLT-2; NRC, Canada). Direct determination of total Hg (THg) concentrations was via cold vapor AAS (AAS-CVG) at a wavelength of 254 nm using a Thermo-Jarrell Ash (Franklin, Massachusetts, USA) model S-11 AAS with a Thermo-Jarrell Ash AVA-440 atomic vapor accessory. The detection limit for THg was approximately 1 ng/g ww. For determination of divalent Hg [Hg(II)] and monomethyl Hg (MHg), 0.5 g. tissue samples were digested in 25% KOH in methanol for 2 hours at 90°C (Bloom, 1992) and brought to 40 ml with methanol. Each batch of 20 samples included 3 digestion blanks, 2 standard reference materials (DORM-2 and DOLT-2), a duplicate digestion and a matrix spike duplicate digestion. Analysis for MHg was performed by aqueous phase ethylation of digest aliquots at pH 5. Aqueous phase ethylation uses aqueous tetraethylborate anion to convert HgX2 species to methylethylmercury, and CH3HgX species to methylethylmercury (Bloom, 1989). Non-ionic Hg species [i.e., elemental Hg (Hg0) and dimethylmercury] do not react with the tetraethylborate anion (Bloom, 1989). Ethylation of labile Hg compounds is followed by purging and trapping of the volatile ethyl mercury analogs on Carbotrap (Supleco, Bellefonte, Pennsylvania, USA; Bloom and Fitzgerald, 1988). The Carbotraps were then thermally desorbed into a packed GC column (15% OV-3 on Chromasorb-WAW DCMS; Supleco, Bellefonte, Pennsylvania, USA) held at a constant 100 C. The ethyl analogs were separated by the GC column and the carrier gas then pyrolized at 800 C to break all compounds down to Hg0 prior to detection by cold vapor atomic fluorescence spectrometry (CVAFS)(Frontier Geosciences, Seattle, WA). The detection limit calculated from 27 blanks was 1.1 ng/g ww for this study. Hg(II) was analyzed by SnCl2 reduction of digest aliquots at pH 1, followed by purging and trapping of the elemental Hg (Hg0) on gold-coated quartz sand (Bloom and Crecelius, 1983). The trapped Hg was then thermally desorbed into the carrier gas leading to the cell of the CVAFS detector. The detection limit of Hg(II) for 27 blanks in this study was 6.6 ng/g. Recoveries of spikes for both MHg and Hg(II) were 100 ± 13 %. The second Hg value given in the following results, summed Hg (SHg), was calculated by adding the MHg and Hg(II) values. As for the other elements, all Hg data were reported on a wet weight basis. Due to financial constraints, determination of inorganic and organic Hg [Hg(II), MHg, and SHg] was done only on selected tissue samples, including beluga whale liver, kidney, muscle, and epidermis; ringed seal liver and kidney; and polar bear liver and muscle.
See above (methods)
Radionuclide contaminant burdens in arctic marine mammals harvested during subsistence hunting