Tissue distribution of metals in striped dolphins (Stenella coeruleoalba) from the Apulian coasts, Southern Italy

Marine Environmental Research 49 (2000) 55±66 www.elsevier.com/locate/marenvrev

Tissue distribution of metals in striped dolphins (Stenella coeruleoalba) from the Apulian coasts, Southern Italy N. Cardellicchio a,*, S. Giandomenico a, P. Ragone a, A. Di Leo a a

CNR - Istituto Sperimentale Talassogra®co, via Roma 3, 74100 Taranto, Italy

Received 20 December 1995; received in revised form 10 February 1999; accepted 10 May 1999

Abstract Tissue distributions of metals (mercury, lead, cadmium, zinc, copper, iron, manganese) were determined in six specimens of striped dolphins (Stenella coeruleoalba, Meyen) stranded on the Apulian coasts (Southern Italy) between February and June 1987. Methyl mercury and selenium were also determined in the liver samples. The liver accumulated the highest concentrations of metals, except for cadmium and chromium. Metal levels were higher than those found in dolphins living in the Atlantic, but lower than those recorded in the same species from the French Mediterranean coasts. Necroscopic surveys found that all specimens were a€ected by haemorrhagic gastritis, but the cause was not clear. While it was not possible to related the death of dolphins to a speci®c cause, or to contaminants, the accumulation of metals is likely to contribute to the health of the organism and represents a risk factor for dolphins. # 2000 Elsevier Science Ltd. All rights reserved.

1. Introduction Many studies have been carried out concerning metal accumulation in dolphins from di€erent areas of the world, for example, the Japanese coasts (Honda, Tatsukava & Fujiyama, 1982; Honda, Tatsukava, Itano, Miyazaki & Fujiyama, 1983), the Paci®c coasts (AndreÂ, Ribeyre & Boudou, 1990; Itano, Kawai, Miyazaki, Tatsukawa & Fujiyama, 1984a), the Atlantic coasts (Gaskin et al., 1974; Law et al., 1991; * Corresponding author. 0141-1136/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S0141-1136(99)00048-3

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Marcovecchio, Moreno, Bastida, Grepe & Rodriguez, 1990). In the Mediterranean sea, the studies concern chie¯y dolphins stranded along the French and Italian coasts (AndreÂ, Boudou, Ribeyre & Bernhard, 1991; Augier, Park & Ronneau, 1993; Capelli, De Pellegrini & Minganti, 1989; Carlini & Fabbri, 1989; Leonzio, Focardi & Fossi, 1992; Martoja & Viale, 1977; Thibaud, 1978; Viale, 1978). The study of heavy metals accumulation is important because of the toxic e€ects that these pollutants can cause, especially in organisms at the top of the marine foodchain. Between 14 February and 30 June 1987, 40 dolphins were found dead along the coasts of Apulia (Southern Italy): 31 specimens of Stenella coeruleoalba, two of Grampus griseus, two of Tursiops truncatus and ®ve specimens of cetaceans (probably Stenella coeruleoalba) which could not be identi®ed since they were badly preserved. This phenomenon represented an exceptional event for the Italian coasts. In fact, dolphins stranded in Apulia amounted to 40% of the cetaceans stranded throughout 1987 (Cagnolaro & Notarbartolo di Sciara, 1992; Cardellicchio et al., 1987; Centro Studi Cetacei, 1988; Notarbartolo di Sciara, 1990). However, it was not a single massive stranding, but rather the stranding of single dolphins over a relatively short period of time and within quite a restricted coastal area. Some of the well-conserved specimens were subjected to autopsy in order to ascertain the causes of death, at the same time chemical analyses were carried out to determine the level of contaminants. In this paper, the concentrations of metals (mercury, Hg; cadmium, Cd; lead, Pb; zinc, Zn; copper, Cu; iron, Fe; chromium, Cr; manganese, Mn) in tissues and organs of six specimens of S. coeruleoalba (four males and two females) are presented. Metal levels have also been compared with those found in dolphins from other geographical areas, in order to show a possible relationship between the accumulation and the contamination of the marine environment. 2. Materials and methods Organ and tissue samples (liver, brain, kidney, lung, muscle, blubber and melon) of six dolphins were collected during autopsy. Fig. 1 shows the coastal area where the dead cetaceans were found. It was observed that ®ve dolphins were adult (three male and two females): their length and weight ranged from 190 to 208 cm and 57 to 92 kg, respectively. Unfortunately, their ages were not estimated from analysis of their teeth, but ®ve of the dolphins were certainly older than 10 years, and one male specimen was a young suckling individual (length 107 cm, weight 14 kg). From observations on S. coeruleoalba from the Paci®c, these cetaceans are about 100 cm long at birth (Miyazaki, 1977; Miyazaki, Fujise & Fujiyama, 1981). Six months later, at the end of the suckling period, they become 150 cm long, reaching the maximum length (220±230 cm) at the age of 11±12 years. In the Mediterranean S. coeruleoalba is usually 10% shorter than the specimen from the Paci®c (Andre et al., 1991). Samples were collected only from well-preserved animals (n ˆ 6) to avoid putrefaction processes a€ecting the analytical results. Samples were transferred to

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Fig. 1. Coastal area in the Mediterranean sea where dolphins (Stenella coeruleoalba) were found.

PTFE-containers and frozen at ÿ20 C. Before the analysis, samples were homogenized in a te¯on Ultra-Turrax T25 homogenizer (Janke and Kunkel, Staufen, Germany); for Cd, Pb, Zn, Cu, and Fe analysis, 0.5 g homogenates were digested under pressure in a te¯on vessel with 10 ml Ultrex-grade HNO3 (J.T. Baker, Phillipsburg, NJ, USA) for 4 h at 160 C. Cd, Pb, Cr and Mn were then determined by graphite furnace atomic absorption spectrophotometry (GF-AAS) using a Perkin Elmer (Norwalk, CT, USA) Zeeman 3030 spectrophotometer. Cu, Zn and Fe, instead, were determined by ¯ame atomic absorption spectrophotometry. For total Hg determination, samples were digested under pressure with 10 ml Ultrex-grade H2SO4-HNO3 (J.T. Baker) mixture (1:1) for 4 h at 160 C. Hg concentrations were determined by cold vapour atomic absorption spectrophotometry using a Perkin Elmer 1100 B spectrophotometer. A 1000 mg/l stock solution of the various metals was used for preparing standard solutions. Diluted standard solutions were prepared through the serial dilution of stock solutions with ultra-pure deionized water (<0.1 mS at 25 C) obtained using a Milli-Q system (Millipore, Milford, MA, USA). The detection limits for the Cd, Cr, Pb and Hg were lower than 10 ng gÿ1 wet weight (<0.010 mg/l). In the liver samples methyl mercury and selenium were also determined. After an acid digestion of the sample with 10 ml Ultrex-grade HNO3 (4 h at 160 C), selenium was determined by GF-AAS. Methyl mercury was determined by gas chromatography: according to Westoo (1968), 1 g of homogenate was treated with 1.5 ml of concentrated HCl and 8 ml of toluene in order to extract methyl mercury chloride.

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The methyl mercury chloride was, therefore, converted into an aqueous complex with cystein hydrochloride and the toluene phase was discarded. The complex was broken with HCl and methyl mercury was back-extracted as the chloride into a new portion of toluene. The toluene was concentrated to 100 ml and 1 ml was injected into a gas chromatograph (Carlo Erba 5600, Milan, Italy) equipped with a Ni63 electron capture detector and a silica column WCOT (25 m length, 0.22 mm i.d.) packed with Carbowax 20M. The known addition method was used to quantify all metals and methyl mercury. The validity of analytical methods was con®rmed with certi®ed Standard Reference Materials (DOLT-2: dog®sh liver) obtained from the National Research Council of Canada. Precision and accuracy are reported in Table 1. 3. Results Table 2 shows mean, standard deviation and range of metal concentrations in the di€erent tissues and organs examined. The highest concentrations of total mercury (Hgtot) were found in the liver, followed by the lung, brain, muscle and kidneys. The lowest Hg concentration was in the blubber. Compared with the Hgtot, the percentage methyl mercury in adult specimens ranged from 2.1 to 6.8%. In the suckling specimen, the methyl mercury percentage was higher (55%). Concerning other toxic metals, Cd accumulated primarily in the kidneys; lower concentrations are observed in the muscle, blubber and brain. Pb also accumulated in the liver; the lowest levels were found in the blubber, brain and melon. There was no preferential accumulation of Cr in any organ; the highest concentrations were found mainly in the blubber and in the lung. Zn, Cu, Fe and Mn have been identi®ed as essential metals in enzymatic reactions; these metals are toxic only at high levels. Fe was accumulated in the liver

Table 1 Precision and accuracy of analytical methods obtained using a certi®ed dog®sh liver (DOLT-2)a Metals

Cadmium Chromium Copper Iron Lead Manganese Total mercury Selenium Zinc HgMe (as Hg) a b

DOLT-2 Certi®ed

Foundb

20.8‹0.5 0.37‹0.08 25.80‹1.1 1103‹47 0.22‹0.02 6.88‹0.56 1.99‹0.10 6.06‹0.49 85.8‹2.5 0.693‹0.053

20.1‹0.90 0.42‹0.10 26.10‹0.80 1165‹36 0.18‹0.08 6.96‹0.60 1.75‹0.20 6.27‹0.75 79.2‹4.6 0.750‹0.100

The concentration are given in mg gÿ1 dry weight. Number of replicates is 5.

Table 2 Tissue

Cd

Cr

Fe

Pb

Cu

Mn

Zn

Hg

HgMe

Se

Brain

0.03 (0.03) 0.02±0.07

0.03 (0.01) 0.02±0.06

113 (15) 96±136

0.33 (0.31) 0.01±0.82

1.13 (0.23) 0.82±1.33

0.47 (0.20) 0.30±0.81

9.04 (5.96) 2.18±18.61

13.90 (5.97) 6.86±22.03

NA

NA

Liver

1.75 (1.27) 0.11±3.27

0.03 (0.02) 0.02±0.05

307 (93) 233±464

1.05 (0.72) 0.44±2.19

7.73 (2.22) 4.96±10.00

3.19 (1.48) 1.48±5.42

24.65 (7.32) 16.71±33.34

189.16 (28.55) 156.23±216.70

8.82 (3.70) 3.37±12.19

80.25 (12.71) 65.84±92.40

Blubber

0.05 (0.02) 0.04±0.05

0.10 (0.07) 0.02±0.19

94 (54) 29±173

0.37 (0.23) 0.21±0.78

0.63 (0.42) 0.27±1.33

0.52 (0.68) 0.08±1.70

5.09 (3.57) 2.70±11.85

1.38 (0.91) 0.62±2.92

NA

NA

Melon

0.04 (0.02) 0.02±0.07

0.08 (0.04) 0.04±0.11

97 (29) 47±120

0.21 (0.13) 0.01±0.30

0.30 (0.27) 0.02±0.62

0.45 (0.23) 0.23±0.84

2.50 (1.85) 0.48±5.47

1.96 (1.22) 1.27±4.14

NA

NA

Muscle

0.04 (0.02) 0.04±0.05

0.03 (0.02) 0.02±0.05

293 (32) 252±339

0.41 (0.36) 0.02±0.98

0.85 (0.32) 0.50±1.24

0.27 (0.10) 0.18±0.41

7.15 (3.10) 1.77±11.45

10.87 (2.47) 8.13±14.37

NA

NA

Lung

0.13 (0.11) 0.02±0.31

0.12 (0.04) 0.07±0.15

244 (36) 200±283

0.64 (0.49) 0.19±0.87

0.64 (0.37) 0.39±1.28

0.45 (0.24) 0.25±0.80

22.65 (21.92) 5.62±60.20

28.68 (14.23) 15.49±51.59

NA

NA

Kidney

7.02 (4.08) 1.83±12.82

0.05 (0.02) 0.02±0.10

190 (34) 160±248

0.44 (0.55) 0.02±1.31

1.45 (0.51) 0.80±2.08

0.63 (0.03) 0.60±0.66

15.22 (7.70) 4.44±24.39

10.30 (2.16) 7.57±13.05

NA

NA

NA 2.06 3.32 0.28 1.21 0.28 NA

NA 5.00 0.04 0.04 0.40 0.48 NA

NA 16.99 3.34 1.31 4.48 24.64 NA

NA 1.96 0.29 0.14 0.52 0.29 NA

NA 1.08 NA NA NA NA NA

NA 4.30 NA NA NA NA NA

B. Metal concentrations (g/g wet wt.) in the suckling specimen Brain Liver Blubber Melon Muscle Lung Kidney a

NA 0.53 0.02 <0.01 <0.01 0.04 NA

NA 0.02 <0.01 <0.01 0.02 <0.01 NA

NA 172 135 68 148 201 NA

NA 0.21 0.28 0.32 0.12 0.14 NA

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A. Average concentration (g/g wet wt.), standard deviation in parentheses and range of metals in adult specimens of Stenella coeruleoalbaa

NA, not analysed. 59

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and muscle. In general, liver was the accumulation organ for Cu, Zn and Mn; the lowest concentrations were in the blubber and melon. 4. Discussion 4.1. Mercury distribution Table 3 shows a comparison among metal concentrations found in dolphins from di€erent marine areas. The results indicate that Hg levels in dolphins from the Mediterranean are generally higher than those found in the same species from the Atlantic (Andre et al., 1991). Considering the high mobility of cetaceans, these levels re¯ect the general contamination of the broad and poorly de®ned area in which the cetaceans live. In the Mediterranean the highest metal concentrations are found in dolphins from the French and Tyrrhenian coasts, though the literature data are not sucient to provide a general view of the whole basin. The presence of natural Hg sources in the Mediterranean sea and Hg rich inputs via rivers ¯owing through the Mount Amiata area (one of the richest natural reserves of cinnabar; Bacci, 1989), results in very high Hg concentrations in ®sh tissues from the Tyrrhenian area (north-western Mediterranean; Bernhard, 1988). Hg levels in dolphins stranded in Apulia in 1987 (this study) are lower than those found in dolphins from the French Mediterranean and Northern Tyrrhenian coasts (Andre et al., 1991; Augier et al., 1993; Leonzio, Focardi & Fossi, 1992). Hg concentrations, however, were comparable to those found in S. coeruleoalba from the Japanese coasts (Honda et al., 1983; Itano et al., 1984a), even though in this case animals had been captured. In this study, Hg distribution is in accordance with other observations (Augier et al., 1993; Honda et al., 1983; Itano, Kawai, Miyazaki, Tatsukawa & Fujiyama, 1984b). Wagemann and Muir (1984) have considered the limit of Hg tolerance for the mammal's liver to be in the range of 100±400 mg gÿ1 wet weight; Table 2 concentrations fall within this range. The dolphin accumulates Hg mainly as methyl mercury through the diet; a methyl mercury biological half-life of about 1000 days has been estimated for S. coeruleoalba by Itano and Kawai, (1981). In agreement with other observations (Viale, 1978), the proportion of HgMe in the liver is less than 10% in adult specimens. This con®rms a demethylation process in the liver, whose mechanism has not been explained yet. Demethylation is probably activated when HgMe concentration exceeds a threshold value (Palmisano, Cardellicchio & Zambonin, 1995). It has been proposed that at high HgMe concentrations the liver accumulates Se, which is somehow involved in the detoxication process. According to Koeman, Peeters, Koudstaal-Hol, Tjioe and de Goeij (1973) the molar ratio Hg0 /Se (Hg0 =HgtotÿHgMe) in the liver of dolphins (Delphinus delphis) was approximately 1. In this study the average value of this ratio was 1.12 in adult specimens. Such observations led to the hypothesis that the ®nal compound of HgMe demethylation could be HgSe (tiemannite), an insoluble and little toxic compound. The presence of HgMe in the cytoplasm of dolphin hepatic cells has been

Table 3 Metal concentrations (mg gÿ1 wet wt.) in dolphins (compiled from the sources indicated) Locality

Tissue

Various dolphins

Mediterranean sea

Liver 14±604 Kidney 1.20±2.20 Various tissue

Delphinus delphis

New Zealand

Liver

35.0±72.0

0.21±1.55

Stenella Japanese coasts coeruleoalba

Liver Muscle Kidney

20.5 7 8.7

6.3

Stenella Japanese coasts coeruleoalba

Liver Kidney

205 14.7

Tursiops gephyreus

Liver Kidney

86 13.4

Stenella French Atlantic coeruleoalba coasts

Liver

1.2±87

Stenella British Isles coeruleoalba

Liver

11

0.7

9.7

Stenella Italian MediterLiver coeruleoalba ranean coasts (Tyr- Muscle rhenian coasts) Kidney Brain

324.4a 36.8a 64.7a 15a

0.05a 0.66a 0.1a 0.1a

7.33a 0.18a 44.8a 0.11a

Stenella French Medicoeruleoalba terranean coasts

668.4 87.2 33.3 0.5±146.5

Tursiops truncatus a

Argentina

South Carolina coasts

Liver Kidney Brain Liver

Pb

0.1±10

Cd 1.20±2.22

Cr

0.1±2.5

Fe

Cu

Se

80±380 95±669

Source Viale et al., 1978

8.1 2.0 3.1

25

Zn

30.0±40.0 9.3±24.0

Koeman et al., 1972

44.5 11.4 30.1

Honda et al., 1983

Itano et al., 1984a 0.8 28.4

77.7 29.5

196.2 93.6

Marcovecchio et al., 1990 Andre et al., 1991

0.5

11

56 225a 46.2a 114.1a 66.9a

Law et al., 1991 106a 10.5a 49.8a 9.2a

Leonzio et al., 1992

Andre et al., 1991 0.006±0.272

1.17±78.98

8.8±271.1 0.18±47.20

Beck et al., 1997

61

Value expressed as mg gÿ1 dry weight.

Hg

N. Cardellicchio et al. / Marine Environmental Research 49 (2000) 55±66

Species

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con®rmed by X-ray spectrography in Ziphius cavirostris (Martoja & Berry, 1980) and in S. coeruleoalba (Nigro, 1994). In the suckling specimen (Table 2B) the HgMe percentage is high (55%) and Hg0 /Se ratio is 13.7. In this young dolphin the HgMe demethylation has probably not been activated yet. 4.2. Distribution of other metals Due to the limited number of references, it is dicult to compare the results obtained in this study. The only data concerning dolphins from the Mediterranean are those given by Viale (1978) and Leonzio et al. (1992). According to Viale (1978), Cr does not have a preferential accumulation organ in dolphins from Corsican coasts: the maximum levels were reached in the brain. However, for evaluation of Cr toxic e€ects, it would be necessary to establish the oxidation state of the metal (speciation). For Fe, the highest concentrations were recorded by Viale in the liver and in the kidneys. Cd, Pb, Cu and Zn concentrations in this study can be critically evaluated with those levels recorded by Law et al. (1991) in specimens from the British coasts and by Honda et al. (1983) in dolphins from the Japanese coasts. Cd, Cu and Zn levels in the Mediterranean dolphins were similar to those found by these authors. Cd accumulated preferentially in the kidneys; Fujise, Honda, Tatsukawa and Mishima (1988) indicated that renal dysfunction attributable to Cd can occur in marine mammals with Cd liver concentrations greater than 20 mg gÿ1 wet weight. None of the dolphins analysed in our study exhibited these concentrations. The essential metals concentrations were not high and their variation was dependent on the physiology and the metabolism of the various specimens; the Cu and Zn concentrations seem to be homeostatically controlled (Law et al., 1991, 1992). Law et al. (1991) suggested that it is possible to hypothesize a range of liver concentrations within which this regulation is active, for example, approximately 3±30 mg gÿ1 for Cu and 20±200 mg gÿ1 for Zn. They further postulate that animals outside this range are those whose regulating mechanism may be impaired. In marine mammals homeostatic control of Cu and Zn may be mediated by metallothionein (MT; Muir et al, 1988). The function of MTs in the control of metal availability has been outlined in Roesijadi (1992). Di€erent Mts able to bind Cd, Zn, Hg and Cu have been identi®ed in marine mammals (Mochizuki, Suzuki, Sunaga, Kobayashi & Doi, 1985; Olafson and Tompson, 1974; Tohyama, Himeno, Watanabe, Suzuki & Morita, 1986). Different biological processes can occur in marine organisms to reduce metal toxic e€ects; among these, the induction of MT synthesis is very important in metal detoxi®cation (Viarengo & Nott, 1993). The combinations of metals with MTs reduce metal `mobility' in the organism and represent an important `mechanism of protection' in mammals. Metal distribution in di€erent tissues is related to proteins able to bind the metal with sulphidrilic and hydroxylic groups. There is a low anity between metals and lipids. Dolphins assimilate metals both through water and food. Metal assimilation from water occurs mainly through passive di€usion of the metal as soluble compounds. Gaskin (1986) studied water assimilation in small `odontoceti' (Cetacea); they assimilate a water quantity equal to 77 ml/kg of body weight per

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day. 18% (14 ml) is drunk directly, 51% (39 ml) is assimilated with food and the remaining 31% (24 ml) is assimilated through the skin. Cetacean skin is sensitive and vulnerable because it is not protected by mucous or scales, or any keratin layer as in other mammals (Viale, 1978). Food, however, is the main source of contamination: the S. coeruleoalba feeds on mesopelagic ®sh (sardines) and squids (about 5 kg a day). The metal accumulation is also in¯uenced by age, length, weight, sex and the sea area where dolphins live (Andre et al., 1990, 1991; Itano et al., 1984b). The length of S. coeruleoalba increases until the age of 12 years and then remains constant. After this age, metals accumulate in a constant body volume (Andre et al., 1991). For the six specimens examined, it has not been possible to establish a correlation between metal concentrations and length or weight. The number of specimens was rather small and the adult specimens had approximately the same length; furthermore, their weight had probably been in¯uenced by stress factors before death. For most of the dolphins, the thickness of subcutaneous adipose tissue was considerably reduced; before dying the dolphins had lost much of their fat reserves. The controversial question of sex in¯uence has been discussed by various authors (Gaskin, Stone®el, Suda & Frank, 1979; Honda et al., 1983; Wageman, Snow, Lutz & Scott, 1983). Usually females are smaller than males; the di€erent physiology can in¯uence metal assimilation or elimination. For example, females can eliminate Hg during pregnancy (Jernelov, 1986) or suckling (WHO, 1976). Methyl mercury can overcome the placental barrier reaching high blood levels in the fetus. Such transfer represents a risk to the o€spring. In Table 3, it is apparent that the geographical area in¯uences metal contaminant levels, but there is no clear correlation between metal accumulation and mortality. While the synergic e€ects of the various pollutants are not well understood, the accumulation of toxic compounds could lead to a stress condition in an organism. For example, it has been supposed that the accumulation of pollutants like polychlorinated biphenyls (PCBs), could impair immunologic defences of an individual making cetaceans more exposed to infections, especially viral ones (Borrel & Aguilar, 1991). It has been suggested that PCB compounds could cause such e€ects and high concentrations of these compounds have been reported for dolphins from Italian waters (Corsolini et al., 1995). In this work, however, the PCB levels in the blubber of dolphins examined were lower than those found in the other Mediterranean areas (Cardellicchio, 1995). Necroscopic surveys carried out in this study, showed that all specimens were a€ected by hemorrhagic gastritis in the glandular stomach and in the ®rst section of the intestine (Cardellicchio et al., 1987). In some cases a duodenal ulcer was found. The cause of these pathologies has not been explained. The causes of death have been investigated by means of a post-mortem study of the animals. Traumas, su€ocation, infective diseases or parasitic infections cannot be considered as causes of death on the basis of necroscopic and microbiologic surveys, even if some specimens were a€ected by parasitic cysts in the abdominal muscles. The remains of cephalopods have been found in the stomach of the suckling dolphin; this dolphin probably lost its mother and attempted to feed. Although dolphins may be able to tolerate

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high metal concentrations, there is no information on the long-term or sublethal impact of contaminants. Concentrations of metals in the livers of these animals were low, and therefore trace metal contaminants were unlikely to have contributed to mortality. However, the study of the distribution and metal accumulation in stranded dolphins may contribute to a better understanding of the stranding phenomena and the observed pathologies. References AndreÂ, J. M., Ribeyre, F., & Boudou, A. (1990). Mercury contamination levels and distribution in tissues and organs of Delphinids (Stenella attenuata) from the eastern tropical Paci®c in relation to biological and ecological factors. Mar. Environ. Res., 30, 43±72. AndreÂ, J., Boudou, A., Ribeyre, F., & Bernhard, M. (1991). Comparative study of mercury accumulation in dolphins (Stenella coeruleoalba) from French Atlantic and Mediterranean coasts. Sci. Total Environ., 104, 191±209. Augier, H., Park, W. K., & Ronneau, C. (1993). Mercury contamination of the striped dolphin Stenella coeruleoalba Meyen from French Mediterranean coasts. Mar. Pollut. Bull., 26(6), 306±311. Bacci, E. (1989). Mercury in the Mediterranean. Mar. Pollut. Bull., 20(2), 59±63. Beck, K. M., Fair, P., McFee, W., & Wolf, D. (1993). Heavy metals in livers of Bottlenose Dolphins stranded along the South Carolina Coast. Mar. Pollut. Bull., 34(9), 734±739. Bernhard, M. (1988). Mercury in the Mediterranean. UNEP Reg. Seas Rep. Studies, No. 98. (141 pp.). Athens: UNEP. Borrell, A., & Aguilar, A. (1991). Were PCB levels abnormally high in striped dolphins a€ected by the Western Mediterranean die-o€? European Research on Cetaceans, 5, 88±90. Cagnolaro, L., & Notarbartolo di Sciara, G. (l992). Research activities and conservation status of cetaceans in Italy. In Protezione della fauna marina e introduzione di specie alloctone. Boll. dei Musei e degli Ist. Biol., Genova, 56±57, 69±85. Capelli, R., De Pellegrini, R., & Minganti, V. (1989). Preliminary results on the presence of inorganic, organic mercury and selenium in dolphins (Stenella coeruleoalba) from the Ligurian sea (pp. 19±24). European Cetacean Society ed., Proceedings of the 3rd Annual Conference (pp. 19±24), La Rochelle, France, 24±26 Feb., 1989. Cardellicchio, N. (1995). Persistent contaminants in dolphins: an indication of chemical pollution in the Mediterranean sea. Water Science and Technology, 32(9-10), 331±340. Cardellicchio, N., Cozzi, B., Notarbartolo di Sciara, G., Pantarotto, C., Pastore, M., & Sebastio, C. (1987). Relazione della commissione peritale circa le cause della morte degli organismi marini sul litorale pugliese da febbraio a giugno 1987. Pretura di Otranto ed., (p. 209). Otranto, Italy. Carlini, R. & Fabbri, F. (1989). Mercury, methyl mercury and selenium in Italian stranded Cetaceans (pp. 25±31). European Cetacean Society ed., Proceedings of the 3rd Annual Conference (pp. 25±31), La Rochelle, France, 24±26 Feb., 1989. Centro Studi Cetacei (1988). Cetacei spiaggiati lungo le coste italiane. Rendiconto 1988. Atti Soc. Ital. Sci. Nat. Museo Civ. Ist. Nat. Milano, 129(4), 411±432. Corsolini, S., Focardi, S., Kannan, K., Tanabe, S., Borrel, A., & Tatsukawa, R. (1995). Congener pro®le and toxicity assessment of polychlorinated biphenyls in dolphins, sharks and tuna collected from Italian coastal waters. Mar. Environ. Res., 40, 33±53. Fujise, Y., Honda, K., Tatsukawa, R., & Mishima, S. (1988). Tissue distribution of heavy metals in Dall's porpoise in the northwestern Paci®c. Mar. Pollut. Bull., 19, 226±230. Gaskin, D. E. (1986). Kidney and water metabolism. In M. M. Bryden & R. Harrison, Research on dolphins (pp. 129±148). Oxford: Clarendon Press. Gaskin, D. E., Smith, D. J. D., Arnold, P. W., Louisy, M. V., Frank, R., Holdrinet, M., & McWade, J. W. (1974). Mercury, DDT, dieldrin and PCB in two species of Odontoceti (Cetacea) from St. Lucia, Lesser Antilles. J. Fish. Res. Board Can., 31, 1235±1239.

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