MARINE ECOLOGY PROGRESS SERIES Vol. 135: 137-143,1996 Published May 17 Mar Ecol Prog Ser

Intracellular storage of and in different marine vertebrates

Marco ~igro',Claudio ~eonzio~

'Dipartimento di Biomedicina, Universita degli Studi di Pisa, Via Volta, 4, 1-56126 Pisa, Italy 2Dipartimento di Biologia Ambientale, Universita degli Studi di Siena, Via delle Cerchia, 3, 1-53100 Siena, Italy

ABSTRACT: Intracellular storage and levels of mercury and selenium were studied in the livers of top marine predators belong~ngto different vertebrate taxa. Total mercury levels showed very important interspecific variations, ranging from 2.6 pg g.' In tuna and swordfish to several thousand pg g-' (dry weight) in bottle-nosed and Risso's dolphins. However, was less variable, ranging from 1 to 174 pg g.' (dry weight). The ratio between Hg and Se levels was close to equimolarity in marine mammals and cormorants, but a large excess of selenium in relation to mercury was observed in fish. Electron microscopy and x-ray microanalysis revealed mineral granules consisting of clustered crys- talline particles in toothed cetaceans, sea llons and cormorants but not in tuna and swordfish. Granules containing mercury and selenium were malnly located in the cytoplasm of macrophages. These results suggest that the blosynthesis of mineral granules containing mercury and selenium in top marine predators is a common feature among these animals and that the existence of elimination pathways for the excretion of organic mercury might Influence the amount of mercury and selenium stored as min- eral granules in a particular species.

KEYWORDS: Mercury Mercury . Selenium X-ray microanalysis . Marine top predators Mercul-y detoxication

INTRODUCTION reduction in the relative proportion of organic mercury passing from prey to predator has been considered as Due to its long persistence and high mobility in the evidence for a biotransformation process in which marine ecosystem, mercury shows an age-related methylmercury is degraded into less toxic inorganic accumulation and a strong biomagnification in the storage forms (Buhler et al. 1975, Kari & Kauranen food web (Bryan 1979, 1984). Numerous studies have 1978, Smith & Armstrong 1978, Reijnders 1980, Thom- demonstrated that both inorganic and organic mer- son & Furness 1989). cury (mainly methylmercury) occur in marine verte- In the livers of some marine mammals, selenium brates. However, total mercury levels, as well as the and mercury concentrations have often been found to relative proportions of its organic and inorganic be present in a 1:l molar ratio (e.g. Koeman et al. forms, vary largely according to the trophic level, tis- 1973, 1975, Itano et al. 1984, Muir et al. 1988, Leonzio sue analyzed and zoological group (see Thomson et al. 1992). On the basis of this observation, the inter- 1990 for a review). action with selenium has been supposed to account Mercury in fish occurs almost completely in the for the protective effect of this metalloid against mer- organic form (generally >80%) and seldom exceeds cury toxicity (see Pellettier 1986, Cuvin-Aralar & Fur- 1 1-19 g-I (wet weight) (Cappon & Smith 1982). How- ness 1991 for reviews). Although numerous studies ever, in liver tissue of fish-eating marine mammals and have dealt with analytical determinations, data on the seshirds, mrlthylm~rc~~ryusuallv constitutes a very combined intracellular storage of mercury and sele- small proportion (<10%) of the total mercury concen- nium have been obtained only in cetaceans. Martoja trations (e.g. Smith & Armstrong 1978, Gaskin et al. & Viale (1977) first reported the presence of mineral 1979, Falconer et al. 1983. Itano et al. 1984). This granules composed of mercury selenide in the livers

G Inter-Research 1996 Resale offull a]-tide not perrn~tted Mar Ecol Prog Ser 135: 137-143, 1996

of Cuvier's dolphins. Successively, similar granules Light and electron microscopy. Samples were pre- were described in the striped dolphin and the role of served in buffered formalin, post-fixed in osmium macrophages in mercury selenide (HgSe) biomineral- tetroxide, dehydrated in an ascending ethanol series, ization hypothesized (Nigro 1994). More recently, exchanged in propylene oxide and embedded in Epon- HgSe was also reported in the respiratory system of Araldite. Liver samples were cut on a LKB ultratome I11 bottle-nosed dol.phins and short-flnned pilot whales, ultramicrotome, using glass or diamond knives. Semi- where it wa.s associated with soot particles (Rawson et thin sections (1 pm thick) were stained with toluidine al. 1995). blue and used for light microscope observations. The The aim of this paper is to improve the knowledge relative abundance of mercury selenide in the liver of of the intracellular storage of mercury and selenium the species studied was qualitatively estimated in in marine vertebrates and to verify if the biomineral- semi-thin sections observed with the lOOx oblective. ization is a characteristic of toothed cetaceans ex- Thin sections were mounted on carbon-formvar coated clusively or shared with other species subjected to a copper grids and double stained with saturated aque- high Hg dietary intake. To achieve this goal, a com- ous uranyl acetate for ultrastructural examinations. parative study using transmission electron micro- Some unosmicated liver sections underwent a proteo- scopy and x-ray microanalysis was done on the livers lytic treatment with 0.5% aqueous pronase solution at of top predators belonging to different taxa of marine 37°C for 3, 6 and 9 h before staining. Post fixation and vertebrates. staining were omitted for x-ray and electron diffraction analysis. All samples were examined with a Philips M-400 MATERIALS AND METHODS electron microscope equipped with an EDAXx-ray de- tector and multichannel analyzer, operating at 120 kV. Samplings. No animal was deliberately killed for this Quantitative x-ray microanalysis was done compar- research, all liver samples were obtained from mam- ing the relative intensity of the Se Kaand HgLa peaks mals and birds that died of natural causes or from (peak - background) in the sample spectra with that fish that were caught commercially. Liver samples of obtained from a standard made of finely powdered Risso's Grampus griseus and bottle-nosed dolphin Tur- commercial mercury selenide (Strem chemicals) dis- siops truncatus were obtained from cetaceans stranded persed onto a copper specimen grid (see 'Results'). along Italian coasts (in the framework of the activity of the Italian Center for the Study of Cetaceans). Sea lion Otaria byronia livers were obtained from the colony of RESULTS Mar del Plata, Argentina, in the framework of a EU research cooperation between Italy and Argentina. The concentrations of total mercury, methylmercury Cormorant Phalacrocorax carbo liver samples were and, selenium in the liver samples are reported in provided by the National Institute for Wildlife Blology, Table 1. Total Hg level varied widely among the spe- Bologna, Italy, and were collected in the PO delta, Italy. cies investigated; the highest value (in toothed ceta- Swordfish Xiphias gladius and tuna Thunnus thynnus ceans) being 4 orders of magnitude higher than the liver samples were obtained from fishermen of Ustica lowest one (fish) (Table 1). Methylmercury varied and Favignana islands (southern Tyrrhenian Sea) re- between 1 and 174 pg g-l (Table 1).Mercury and sele- spectively. Chemical analysis. Liver samples Table 1 Total mercury, methylmercury and selenium levels (pg g-' dry weight) were put in plastic bags and stored at and selenium:mercury molar ratio in the liver of specimens studied by electron -20°C prior to processing for chemical microscopy analysis. Total mercury was assayed in 0.1 g aliquots of freeze-dried ti.ssue Species Total mercury Methyl- Selenium Molar ratio sa.mples after digestion in a. Teflon mercury Se:Hg bomb for 9 h at llO°C with 2.0 m1 of Mammals nitric acid (Stoeppler & Backaus 1978). Bottle-nosed dolphin 13270 174 4330 0.83 The solution was analyzed by a cold Risso's dolphin 3828 53 1413 0.93 vapor atomic absorption spectrophoto- Sea lion 89 1 20 324 0.92 meter with a Perkin-Elmer Model 2280 Birds Cormorant 131 18 55 1.06 apparatus. Fish Methylrnercury was analyzed by gas Tuna 2.6 1 10 97.3 chromatography following the method Swordfish 3.2 1 19 15.03 of Horvat et al. (1990). P P - Nigro & Leomo: Hg and Se ~nlracellularstorage In marlne vertebrates

F~gs.1 to 3 Ultrastructure of mammal livers. F* Granipusgriseus (Risso's dolphin). Liver (HgSe) granules (arrows) In a macrophage adjacent to a cap~llarylumen (C) E.erythrocytes; scale bar = 2 5 pm Fig. 2 Turs~opstruncatus (bottle-nosed dol- ph~n).Liver granules composed of tlny spherical part~cles(arrows) and larger polygonal crystals (arrow heads) clustered together, scale bar = 0 1 pm FI~3. Otana byl-on~a(sea lion). Liver granules composed exclusively of spherical particles; scale bar = 0 1 pm nium occurred in a 1-1 molar ratio in marine mammals morants (Figs. 1 to 3), but not in tuna and swordfish. and in cormorants, whereas a large excess of selenium Granules were very abundant in both odontocetes and was observed in tuna and swordfish (Table 1). much less numerous in sea lions and cormorants The ultrastructural investigations done on liver sec- (Table 2).Granules were most frequently observed dis- tions revealed irregularly shaped dense granules In tributed along the sinusoids or in the connective tissue bottle-nosed and R~sso'sdolphins, sea lions and cor- surrounding blood vessels (Fig. 1). In high magnification

Table 2 Relative abundance (arbitrary unlts) and quantitat~vex-ray microanalysls of liver granules In marlne verteblates SeK/HgL mean latio (* SD) of SeKu and HgLu peak lntensit~es(peak - background) 111 the granules Se/Hg atomlc rat10 of selen~umand mercury In the granule n number of granules analyzed by x-ray m~croanalysis

Standal d Bottle-nosed Rlsso's Sea lion Cormorant Cormordnt HgSe dolph~n dolph~n pronascl Mar Ecol Prog Ser 135: 137-143, 1996

electronmicrographs, delphinid granules were seen to HgLn 1 SeKtr be composed of tightly clustered spherical particles mea- suring approximately 15 nm in d~ameterand larger polygonal particles (of 70 to 80 nm) w~thwell-defined shapes (Fig. 2). In sea lions and cormorants (Fig. 3),gran- ules consisted only of spherical particles of about 15 nm. Liver granules produced electron diffraction pat- terns consisting of continuous rings and superimposed discrete spots in the bottle-nosed and Risso's dolphins (Fig. 4). This finding, along with the above-reported ultrastructural data, suggests that 2 types of crystalline particles occurred in the liver granules of toothed cetaceans, namely the t~nyspherical ones and the larger euhedral crystals. On the other hand, diffraction patterns consisting only of continuous rings were observed in sea lions and cormorants (data not shown), suggesting a structu.re made of only random1.y orlented micro crystallites. In all the species investigated, the diffraction parameters were similar t.o those of mercury selenide (tiemannite). Mercury and selenium were the only elements detected in granules by the x-ray microanalysis in the transmission electron microscope (Fig. 5). The relative SeKa intensities of the HgLa and SeKa peaks (as peak areas) in the spectra of liver granules were calculated and compared with values obtained from the HgSe standard. Since the proportion of mercury and sele- nium atoms in the standard was 1:l (stoichiometric value), the Hg:Se ratio in the liver granules was calcu- lated according to the following proportion: Fig 5 Part of the x-ray spectrum of the liver granules in Se:Hg = different marine vertebrates showing the HgL and SeK (SeKa:HgLa in the sample):(SeK:aHgLa m the standard). peaks. (A] Bottle-nosed dolphin; (B) sea lion; (C) cormorant; (D) HgSe standard. Vertical scale: x-ray counts; horizontal The results (Table 2) indicate that the Se:Hg ratios in scale: x-ray energy the granules of Risso's dolphin (0.98 + 0.06), bottle- nosed dolph~n(1.04 + 0.04), and sea lion (1.13 + 0.17) were very close to the stoichiometric value. The Se:Hg ratio obtained for the cormorant (0.6 * 0.06) was significantly lower, indicating an excess of Hg with respect to Se (Table 2). As a working hypothesis, we consider that the excess Hg may be bonded to sulfur substituting for selenium in the mlneral structure of the granule, or to any SH-rich proteln associated with the granules. Sulfur was not detected by the x-ray micro- analysis (resolution 150 eV) due to overlapping of the SK peak (2.3 keV) with the HgM peak (2.2keV) (not shown in Fig. 5). To verify the presence of proteins associated with the granules, some sections underwent proteolytic treatment. After incubation with aqueous pronase solution no change in fine morphology (data not shown) or quantitative composition of the granules (Table 2) was observed. These results suggest that the excess mercury in the cormorant granules was proba- bly bonded to inorganic sulfur as part of their crys- Fig. 4. Grampus griseus (Risso's dolphin). Electron diffraction patterns produced by liver granul.es, show~ngcontinuous talline structure and isomorphically substituting for rings and superimposed dlscrete spots selenium, rather than to SH-groups of proteins. Nlgro & Leonzlo: Hg and Se intracellu1a1-storage In marine vertebrates 141

DISCUSSION short-finned pilot whale and reported HgSe in the lung and hilar lymph nodes to be associated with soot par- Since all the species investigated lived far from focal ticles. Martoja & Berry (1980) supposed that tiemannite man-made sources of pollution (with the exception of was the product of a process of biosynthesis and repre- the cormorant), their mercury and selenium levels sented the final stage of methylmercury degradation. should be natural. Total mercury concentration in the They also suggested that selenium was directly in- liver varied widely, the highest values (measured in volved in the demethylation process and concluded odontocetes) being up to 4 orders of magnitude higher that the synthesis of HgSe could be expected in species than the lowest ones (~npelagic predatory fish). Simi- showing a 1:lmolar ratio of mercury and selenium. larly, the proportion of methylmercury was highly vari- The results of the present study support the conclu- able among species investigated, being 1 to 2'+1of total slons of Martoja & Berry (1980).In fact, the occurrence Hg in marine mammals, about 13'i/;, In cormorants and of mineral granules containing Hg and Se was ob- more than 30'5 in tuna and swordfish. Differences in served in 2 species of Dolphinidae, namely the bottle- total mercury concentrations and the relative propor- nosed dolphin and Risso's dolphin. However, it was not tions of organic and inorganic forms in the livers of limited to toothed cetaceans only, as it was also marine vertebrates are considered to be the conse- observed in sea lions and cormorants. In all these spe- quence of some interacting factors, namely mercury cies, the presence of mineral granules was accompa- dietary intake, elimination and detoxication/storage nied by a Hg:Se molar ratio close to 1 and a low per- (Thornson & Furness 1989, Andre et al. 1991). All the centage of organic mercury. study species occupy a terminal posltlon in the marine As an alternative to biosynthesis, mineral granules food web, t:hus they may be reasonably considered containing mercury and selenium may form as a result similar with reference to their methylmercury dietary of the high affinity of these 2 elements, without any intake. Differences in methylmercury elimination biochemical processes. Thi.s hypothesis 1s not sup- capacity and/or age related storage of detoxified forms ported by the finding of relatively h~ghmercury and may therefore account for the observed interspecific selenium concentrations in baleen whales stranded variability in Hg concentrations. along the Mediterranean coasts. In these whales in Demethylation, namely the transformation of organic fact, no trace of mineralized forms of mercury or sele- mercury into the less toxic inorganic form, is believed nium storage could be found (M. Nigro unpubl. obs.), to occur in those marine mammals and birds which suggesting the occurrence of other unknown forms of tend to accumulate high levels of prevalently inorganic detoxication/storage as an alternative to biomineral- mercury (e.g. Smith & Armstrong 1978, Gaskin et al. ization. 1979, Falconer et al. 1983, Itano et al. 1984). The very Although the paucity of data does not permit wide high mercury concentration in some f~sh-eatingmarine generalizations, th.e differences in the abundance of vertebrates raises the question of how they avoid toxic mercury selenide granules observed In the species effects. In many papers the chelnical interaction investigated appeared to be mainly related to mercury between mercury and selen~umhas been indicated as excretion capacity and detoxication/storage. In dolphins, one of the possible mechanisms leading to mercury mineral granules were very abundant and the half-life of detoxication (see Pellettier 1986, Cuvin-Aralar & Fur- mercury was estimated to be 1000 d, suggesting a very ness 1991 for reviews). This conclusion was based on limited excretion capacity (Itano & Kawai 1986).More- the observation that mercury and selenium levels are over, the mercury dietary intake for dolphins living in the often correlated in an equimolar ratio, in the liver of Mediterranean Sea was estimated at about 1 mg d-' marine mammals (e.g. Koeman et al. 1973, 1975, ltano (Nigro & Leonzio 1993). Such a high mercury intake et al. 1984, Hansen 1990, Muir al. 1988, Leonzio et al. accompanied by negligible excretion might have forced 1992). However, the only dlrect evidence of the occur- toothed cetaceans to develop, over an evolutionary time rence of a non-toxic product of mercury and selenium scale, a very efficient process of mercury detoxication ~nteraction,has concerned toothed cetaceans. In these and storage. Similarly, dolphin lymphocytes have been organisms, insoluble mineral granules composed of demonstrated to be much more resistant to both the mercury selenide (tiemannlte) were first described cytotoxic and genotoxic effects of methylmercury than by Martoja & Viale (1977) in the liver of the Cuvier rat or human cells after in vitro exposure to the toxic dolphin. Successively, Nigro (1994) reported similar pollutant (Betti & Nigro 1996). granules in the striped dolphin and hypothesized Pinnipeds, like dolphins, can degrade methylmer- that mercury se!enide preripitation mainly occurs in cury, accumulating mainly inorganic Hg in the liver macrophage eterophagosomes. More recently Rawson (Buhler et al. 1975, Kari & Kauranen 1978, Smith & et al. (1995) found HgSe in both the liver and in the Armstrong 1978, Reijnders 1980). Our data show that respiratory system of the bottle-nosed dolphin and at least part of this form of mercury is stored as mer- 142 Mar Ecol Prog Ser 135 137-143, 1996

cury selenide granules. However, unlike toothed ceta- In conclusion, we can say that ultimate marine con- ceans, pinnipeds possess efficient methylmercury ex- sumers, particularly fish-eating animals, have the com- cretlon capacity through the fur (Wenzel et al. 1993) by mon task of reducing methylmercury toxicity as a con- virtue of the strong binding between organic mercury sequence of their high dietary intake. The processes and sulfhydryl groups of keratins (Crewther et al. are fundamentally direct elimination of methylmer- 1965) In addition, seals are known to detoxify mercury cury and/or biotransformation and bioaccumulation by binding it to metallothioneins (Olafson & Thomson of a non-toxic product. The mechanisms of the latter 1974, Hamanaka et al. 1982, Mochizuchi et al. 1985, process are largely unknown, occurring mainly In feral Tohyama et al. 1986). All these features might account animals, such as dolphins, which can be investigated for the lower Hg level usually found in pinnipeds using exclusively non-destructive approaches. More with respect to dolphins (see Thomson 1990) and the studies are needed to clarify both the physiological scarcity of liver granules found in the present study. mechanisms of mercury and selenium biomineraliza- Birds eliminate methylmercury via the feathers (e.g. tion and the factors influencing its occurrence among Honda et al. 1986, Furness 1993). This has been marine organisms. demonstrated to be an important mercury excretion pathway in these organisms (Braune & Gaskin 1987). Seabirds with slow moult cycles, such as albatrosses, Acknocvledgements The authors are grateful to Dr U. Agrimi and Dr A. Massi for furnishing respectively toothed cetaceans have a restricted elimination capacity (Thomson & and cormorant llver samples, Prof. M. Mellini for hls hclp in Furness 1989). According to Thomson & Furness interpretation of mineraloyic data and Prof. S. Bonotto tor (1989) these birds solve the problem by converting instructive comments on the manuscript. Thls is publication a proportion of organic mercury into an inorganic no. 53 of the Italian 'Centro Studi Cetacei' storage, performing a toxicologically less demanding alternative to the accumulation of methylmercury. The LITERATURE CITED specimens of cormorant studied showed high total mercury levels and a low ratio of organic to inorganic Andre JM, Boudou A, &beyre F (1991) mercuryaccumulation in delphinidae. Wat Air Soil Pollut 56:187-203 mercury, which are typical features of birds with lim- Betti C. Nigro M (1996) The comet assay for the evaluation of ited mercury excretion capacity. The fact that liver the genetic hazard of pol.lutants In cetaceans. Prel~minary granules containing mercury and selenium were found results on the genotoxic effects of methyl mercury on the in the study species demonstrates that the biomineral- bottle-nosed dolphin (Tursiops truncatus) lyrnphocytes in ization, as a non-toxic form of Hg and Se storage, is not vitro. Mar Pollut Bull (in press) Braune BM, Gaskin DE (1987) 4Iercury levels in Bonaparte's restricted to marine mammals but also occurs in gull (Larus ph~ladelphia)during autumn moult in the seabirds. It could be hypothesized that mineral gran- Quoddy region, New Brunswick, Canada. Arch Environ ules are formed if elimination through the feathers Contam Toxicol 16:539-549 cannot match the dietary intake of mercury. Bryan GW (1979) Bioaccumulation of marine pollutants. Phi1 Trans R Soc Lond B 286:483-505 Tuna and swordfish occupy a similar trophic level to Bryan GW (1984) Pollution due to heavy metals and thew dolphins and consequently have a similar mercury compounds. In: Kinne 0 (ed) Marine ecology. LYiley, dietary intake. Nevertheless they have the lowest total Chichester, p 1289-1431 mercury concentrations, th.e highest proportion of Buhler DR, Claeys RR, Mate BR (1975) Heavy metals and organic mercury and lack mineral granules. In chlorinated hydrocarbon residues in Californian sea lions (Zalophus cdfifornldnus). J Fish Res Bd Can 32.2391-2397 practice, the biomagnification factor with respect to Cappon CJ, Smith JC (1982) Chemical form and distribution mercury levels in prey is 500 for delphinids and 30 for of mercury and selenium in edible seafood. Bull Environ predatory pelagic fish (Nigro & Leonzio 1993). Physio- Contam Toxicol 29:285-289 logical differences between fish and marine mammals Copin-Monteyut G, Courau PH, Laumond F (1984) Occur- rrnce of mercury In the dtrnospherc and waters of the may partly explaln their different capacity to store Mediterranean. FAO Fisheries Report KO. 325 Supple- mercury. For instance, fish have gills that malntaln a ment:5 1-57 constant and direct contact between the blood and sea Crewther WG, Fraser RDB, Lennox FG. Lindlcy H (1965) The water When large quantities of methylmercury are chemistry of kcratins. Adv Prot Chem 20.191-346 ingested with the diet, as occurs in large pelagic Cuvin-Aralar MLA, Furncss R (1991) hl~rci~ryand sclcnium interdctions: d rcvlcw. Ecotoxicol Environ Saf 21:348-364 fish, the load may reach concentrations of more than Falconer RC, Davies IM, Topping G (1983) Trac~metals in thc 100 pg ml-' whereas mercury levels In th.e water range common porpoise Phocena phocena. hIar Environ Rcs 8: from 5 to 25 X 10-' pg ml-' (Copin-Montegut et al. 119-127 1984) This enormous difference suggests that mercury Furness RW (1993) Birds as monitor of pollutants. In: Furness RW, Greenwood JJD (eds) l3lrds as monltors of environ- exchange wou.ld easily occur through the gradient rnentdl change Chi~pmanarid Hall, London, p 86-143 blood-seawater, giving fish a further efficient elimina- Gaskin DE, Stonef~eldKI, Suda P, Frank R (19791 Changes In tion system, bcside renal a.nd bile excretion. mercury levels In harbor porpoises (Phocrnd phocrncll Nigro & Leonzio: Hg and Se intracellular storage in marine vertebrates 143

from the Bay of Fundy (Canada) and ddjdcent waters dur- in two species of seals by high performance liquid chro- ing 1969-1977. Arch Envil-on Contam Toxicol 8:733-762 matography-atomic absol-ption spectrophotometry. Comp Halnanaka T, Itoo T, Mishimcl S (1982) Age-related change Blochem Physiol 82(C).248-254 and distribution of cadmium and zinc concentrat~onin Mu~rDCJ, Wagemann R, Grift NP, Norstrom RJ, Simon M, stellar lion (Eumetopasjubata) from the coast of Hokaido. L~enJ (1988) Organochlorine chemical and heavy metal Japan. Mar Pollut Bull 1357-61 contaminants in white-beaked dolphins (Lagenorynchus Hanscn JC (1990) Human exposure to metals through con- albirostris) and pilot whales (Globicephala n~elaena)from sumption of marine foods: a case study of exceptionally the coast of Newfoundland. Canada. Arch Environ Toxicol high Intake among Greenlanders. In: Furness RW, Rain- 17 613-628 bow PS (eds) Hedvy metals in the marine environment. Nigl-o M (1994) Mercury and selenium localization in macro- CRC Press, Inc, Boca Raton, p 227-243 phages of the striped dolphin, Stenella coeruleoalba. Honda K, Nasu T, Tatsukava R (1986) Seasonal changes in J Mar Biol Ass UK74:975-978 mercury accumulat~onin the black-eared klte, Milvus Nlgro M, Leonzio C (1993) Mercury selen~deaccumulation migrans Ijneatus. Environ Pollut 42:3325-334 in dolphins. In: Evans PH (ed) Procc.c,dings 7th Annual Horvat M, Byrne AR, \lay K (1990) A modified method for the Conference of the European Cetacean Society, Inverness, determination of methylmercury by gaschromatography. Scotland, February 1993 Cambridge, p 212-215 Talanta 37.207-212 Olafson RW, Thomson JAJ (1974) Isolation of heavy lnetal Itano K,Kawai S (1986) Changes of mercury content and bio- binding proteins from marine vertebrates Mar Biol 28: logical half-life of mercury In the str~peddolphins. In: 83-86 Fujiyama T (ed) Studies on the levels of organochlorine Pellettier E (1986) Mercury-selenium interactions in aquatic compounds and heavy metals in marine organisms. Uni- organisms. a review. Mar Environ Res 18:lll-132 versity of Ryukyus, p 49-73 Rawson AJ, Bradley JP, Teetsov A, Rice SB. Haller EM, Itano K, Kawai S, Miyazaki N, Tatzukawa R, Fujiyama T Patton GW (1995) A role for airborne particulate in high (1984) Mercury and selenium levels in striped dolphins mercury levels of some cetaceans. Ecotoxicol Environ caught off the Pacific coasts of Japan. Agric B101 Chem 48: Saf 30:309-314 1109-1116 Reilndei-s PJH (1980) 0rganoch.lorine and heavy metal Kari T, Kauranen P (1978) Mercury and selenium contents in residues in harbor seals from the Wadden Sea and their seals from fresh and brackish waters in Finland. Bull possible effects on reproduction. Neth J Sea Res 14:30-65 Environ Contam Toxicol 19:273-280 Smith TG, Armstrong FXJ (1978) Mercury and selenium in Koeman JH, Peeters VHM, Koudstaal-Hol CHM, Tjioe PS, De ringed and bearded seal tissues from Arctic Canada. Goeu JJh4 (1973) Mercury selenium correlation in marine Arctic 31:75-84 mammals. Nature 246:385-386 Stoeppler M, Backaus F. (1978) Pretreatment studles with bio- Koeman JH, Van de Ven WSM, De Goeu JJM, Tjioe PS, van logical and environmental materials, I. System of pressur- Haaften JL (1975) Mercury and selenium in marine ized multisample decomposition. Fresenius Z Anal Chem mammals and birds. Sci Total Environ 3:279-287 231:116-200 Leonzio C. Focardi S, Fossi C (1992) Heavy metals and sele- Thomson DR (1990) Metal levels in marine vertebrates. In: nium in stranded dolphins of the northern Tyrrhenian Furness RW, Rainbow PS (eds) Heavy metals in the marine (NW Mediterranean). Sci Total Environ 119:77-84 environment. CRC Press, Inc. Boca Raton, p 143-182 Martoja R, Berry JP (1980) Identification of tiemanmte as a Thomson DR, Furness RW (1989) The chemical form of probable product of demethylation of mercury by sele- mercury stored in south Atlantic seabirds. Environ Pollut nium in cetaceans. Vie Milleu 30;7-10 60:305-317 Martoja R, Viale D (1977) Accumulation de granules de Tohyama C, Himeno SJ. Watanabe C, Suzuki T, hlorita M seleniure mercurique dans le foie d'odontocetes (Mammi- (1986) The relationship of the increased level of metollo- feres, Cetaces): un mecanisme possible de detoxication du thioneins with heavy metals in the tissues of the harbor methylmercure par le selenium. C r Hebd Seanc 11cad Sci, seal (Foca vitulina). Ecotoxicol Envlron Saf 12:85-94 Paris D 285 109-112 Wenzel C, Adelung D. Kruse H, Wassermann 0 (1993) Trace Mochizuch~Y, Suzuki KT, Sunaga H, Kobaydshi T, Doi R metal accumulation in hair and skin of the harbour seal, (1985) Separation and characterization of metallothionein Phoca vitulina. Mar Pollut Bull 26:152-155

This article was submitted to the edltor A4anuscrlpt fjrst received: Septeniber 22, 1995 Revised version accepted: Ucccniber 11, 1995