Jessica Kojadinovic
email: jessica.kojadinovic@univ-nantes.fr

Paco Bustamante

Matthieu Le Corre

Richard P Cosson

Trace elements in three marine birds breeding on Reunion Island (Western Indian Ocean) Part 2 : Factors influencing their detoxification

Keywords: Metals, Metallothioneins, Sub-cellular distribution, Homeostasis, Regulation, Tropical seabirds

Seabird tissues collected between 2002 and 2004 from Barau's Petrel (Pterodroma baraui), Audubon's Shearwater (Puffinus lherminieri bailloni) and White-tailed Tropicbird (Phaethon lepturus) colonies on Reunion Island were analyzed for metallothioneins (MTs) and trace element content. The subcellular distribution between soluble and insoluble fractions of Cd, Cu, Fe, Mn, Se and Zn was determined in liver and kidney tissues. In both tissues, the soluble fraction of the cell concentrated most of the Cd and Se, whereas Fe, Mn and Zn were preferentially accumulated in the insoluble fraction. The distribution of these elements varied with the tissue, the age of the bird and the species. Furthermore, the distributions of Fe and Mn were somewhat influenced by the bird's physical condition. Metallothionein levels were measured in the soluble fraction after heat denaturation. The levels of these proteins varied from 5.5 ± 2.7 mg.g -1 d.w. to 11.4 ± 6.2 mg.g -1 d.w., depending on the species and the tissue considered. Metallothionein levels were significantly different between liver and kidney only in the White-tailed Tropicbird. In the three species, MT levels in kidney were significantly higher in adults than in juveniles. The bird's weight also had an influence on hepatic and renal MT levels, but not the gender nor the reproductive status. The implication of MTs in Cu and Zn homeostasis, and Cd and Hg detoxification are discussed. In addition, clues on Hg regulation by Se were found, especially in Barau's Petrel, where the levels of these two elements were very significantly correlated.

Introduction

Marine birds accumulate high levels of trace elements due to their position in marine food webs and their long life span. Cadmium (Cd) and mercury (Hg), which are often naturally present in the environment, are toxic to marine biota. Essential elements, such as copper (Cu), iron (Fe), manganese (Mn), selenium (Se) and zinc (Zn), are necessary for the metabolism but can however cause adverse effects when their concentrations in the organism becomes excessive. Quantification of trace element levels in different tissues of the organism is an indicator of the bioavailable fraction of the element in the environment [START_REF] Hogstrand | Metallothionein as an indicator of heavy metal exposure in two subtropical fish species[END_REF]. Nevertheless, these measures do not indicate whether the organism is stressed or affected by the contaminant. The physico-chemical form of storage of the element may however inform on the tissue(s) where detoxification takes place.

In marine vertebrates, detoxification is the result of many processes. One of the most commonly studied is the binding of metals to metallothioneins [START_REF] Mason | Metal speciation and bioavailability in aquatic systems[END_REF]. Metallothioneins (MTs) constitute a class of soluble low molecular weight metallo-proteins characterized by their heat stability, their high cystein content and a lack of aromatic amino acid [START_REF] Hamer | Metallothionein[END_REF]. These proteins play an important role in the transport and storage of trace elements. They also provide protection against the toxic effects of certain metals by sequestrating and reducing the amount of free metal ions and acting as a "rescue" function for structures impaired by inappropriate metal-binding [START_REF] Hamilton | Evaluation of metallothionein measurement as a biological indicator of stress for cadmium in brook trout[END_REF][START_REF] Vallee | The function of metallothionein[END_REF][START_REF] Roesijadi | Metallothionein and its role in toxic metal regulation[END_REF]. Since their discovery, in 1957, the existence of MTs was firmly established in a large number of animals [START_REF] Hamza-Chaffai | Physico-chemical forms of storage of metals (Cd, Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from Tunisian coast: ecotoxicological consequences[END_REF]. MTs have been shown to occur in liver and/or in kidneys of various species of seabirds, such as Leach's Storm Petrel (Oceanodroma leucorhoa), the Atlantic Puffin (Fratercula arctica), the Doublecrested Cormorant (Phalacrocorax auritus), the Herring Gull (Larus argentatus), the Blackheaded Gull (Larus ridibundus), the Northern Fulmar (Fulmarus glacialis), the Guillemot (Uria aalge), the Kittiwake (Rissa tridactyla) and the Common Scoter (Melanitta nigra) [START_REF] Osborn | Naturally occuring cadmium and zinc binding protein from liver and kidney of Fulmarus glacialis, a pelagic North Atlantic seabird[END_REF][START_REF] Elliott | Heavy metal and metallothionein concentrations in Atlantic Canadian seabirds[END_REF][START_REF] Wenzel | The suitability of oiled Guillemots (Uria aalge) as monitoring organisms for geographical comparisons of trace element contaminants[END_REF]. Measurements of MT levels and their relationships with element levels constitute an attractive insight to trace element behavior in the organism.

Another common detoxification process relies on the insolubilization of metals as mineral concretions. A typical example is the formation of insoluble tiemannite granules after the binding of Hg by Se in the liver of many marine mammals and certain birds [START_REF] Koeman | Mercury-selenium correlations in marine mammals[END_REF][START_REF] Martoja | Identification of tiemannite as a probable product of demethylation of mercury by selenium in cetaceans. A complement to the scheme of the biological cycle of mercury[END_REF][START_REF] Nigro | Mercury exposure and elimination rates in captive bottlenose dolphins[END_REF][START_REF] Decataldo | Association of metals (mercury, cadmium and zinc) with metallothionein-like proteins in storage organs of stranded dolphins from the Mediterranean sea (Southern Italy)[END_REF][START_REF] Ikemoto | Detoxification mechanism of heavy metals in marine mammals and seabirds: Interaction of selenium with mercury, silver, copper, zinc, and cadmium in liver[END_REF]. Enriched Cd granules are also known to exist in the White-sided dolphin (Lagenorhyncus acutus) kidneys [START_REF] Gallien | Cadmium-containing granules in kidney tissue of the Atlantic white-sided dolphin (Lagenorhyncus acutus) off the Faroe Islands[END_REF].

The nature of the detoxification process adopted by an organism gives an insight on its degree of exposure to toxic elements. For example, MTs tend to take part in the detoxification of elements to which the exposure is low or sudden [START_REF] Chan | Nephrotoxicity in rats following liver transplantation from cadmium-exposed rats[END_REF], whereas granules attest the exposure to higher levels over a longer period of time [START_REF] Palmisano | Speciation of mercury in dolphin liver: a two stage mechanism for the demethylation accumulation process and role of selenium[END_REF].

Our work focuses on three seabird species breeding on Reunion Island which differ in their dietary ecology and in their contamination by elements as discussed in the first part of this study (Kojadinovic et al., submitted for this volume). Barau's Petrel (Pterodroma baraui) is an oceanic bird which feeds almost exclusively on cephalopods. Juveniles accumulate particularly high levels of Hg and Cd, and adults mainly have high Hg, Fe and Se levels. The White-tailed Tropicbird (Phaethon lepturus) also forages over oceanic water but closer to Reunion Island, and is less specialized on squid. In comparison to the other two species, high levels of Cu, Fe, Mn and Zn were observed in juveniles and Cu and Mn in adults. Finally, Aububon's Shearwater (Puffinus lherminieri bailloni) forages closest to the island, feeds as much on cephalopods as it does on fish [START_REF] Bailey | The pelagic distribution of seabirds in the Western Indian Ocean[END_REF][START_REF] Jaquemet | Seabird community structure in a coastal tropical environment: importance of natural factors and fish aggregating devices (FADs)[END_REF]) and shows the lowest Hg levels (Kojadinovic et al., submitted for this volume). Information on species-specific differences in the sensitivity to trace elements would be important in evaluating the adverse effects of elements on wild birds. Thus, in this second part of our work, we examine trace element regulation in Reunion seabirds by studying the subcellular distribution of trace elements and MT levels in liver and kidney as well as factors influencing MT levels. The interaction between MTs and Cd, Cu, Hg, Zn, and the relationships between elements will be discussed in order to understand the potential detoxification strategies used by these birds.

Materials and methods

Study site and species

Barau's Petrel, Aububon's Shearwater and the White-tailed Tropicbird used in this study originated from colonies established on Reunion Island (21 • 7'S, 55 • 33'E) in the Western Indian Ocean. As noted earlier, these seabird species have contrasting dietary ecologies during their breeding season (approximately 3 months) during which they feed more or less in proximity to the island. Outside their breeding period, individuals scatter in the waters of the Indian Ocean (Barré et al. 1996, Stahl and[START_REF] Stahl | Distribution, abundance and aspects of the pelagic ecology of Barau's Petrel (Pterodroma baraui) in the south-west Indian Ocean[END_REF]. During that time, dietary habits of the three species remain unknown.

Bird sampling

Fifty two Barau's Petrels, 61 Audubon's Shearwaters and 49 White-tailed Tropicbirds were sampled (Kojadinovic et al., submitted for this volume). Since these birds were found before or shortly after their accidental death, we are confident that the conditions in which the birds died had no effect on their elemental and MT levels. Each bird was measured, weighed and aged. Adults were sexed and their reproductive status noted. The liver, kidneys and pectoral muscles were removed during the dissection, weighed and refrozen prior to analysis. At this stage, the muscular and body conditions were evaluated by MC and BC indices respectively (Kojadinovic et al., submitted for this volume).

Sample preparation

To prepare for elemental and MT determination, livers and kidneys were blended, lyophilized and ground to a fine powder. One hundred mg aliquots of tissue were homogenized on ice in 6 ml of 100 mM Tris buffer with β-mercaptoethanol at pH = 8.1, then centrifuged. Metal content was determined in the pellet (C1). The obtained supernatant (S1) was divided for metal analysis (5 ml) and MT analysis (1 ml). Prior to MT dosage, the supernatant aliquot was submitted to heat denaturation and centrifugated to separate the heat-stable proteins from the denatured proteins (Figure 1). The supernatant S2 stemming from the second centrifugation was frozen (-80 • C) until MT quantification. The analysis of Cd, Cu, Fe, Mn, Se and Zn conducted in S1 and C1 called for an extra step in the preparation protocol. Both fractions were completely dried and digested with 1 ml of 15 N nitric acid at 60 • C for 48 h before being diluted in 10 ml of deionized water.

Metallothioneins were not analyzed in muscle tissue. Muscle samples were thus dried at 55 • C in an oven before being ground. Two hundred mg aliquots were digested with 3.5 ml of 15 N nitric acid and diluted in 10 ml of deionized water.

Accuracy and reproducibility of the preparation were tested by preparing 10 replicates of reference standards (National Research Council, Canada) and 11 blanks along with each set of samples. The sample preparation was done in "metal-free" conditions (Kojadinovic et al., submitted for this volume).

Metal analysis

Cadmium, Cu, Fe, Mn, Se and Zn were analyzed in S1 and C1 by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES Varian Vista Pro CCD). Total levels were obtained by adding the elemental masses found in S1 and C1 and dividing by the aliquot mass. Total Hg analyzes were carried out with an Advanced Mercury Analyzer (ALTEC AMA 254). Detection limits and recovery rates are given in Kojadinovic et al. (submitted for this volume).

Metallothionein analysis

Among the 162 individuals, 70 were analyzed for their hepatic and renal metallothionein levels. Differential pulse polarographic analysis (DPP) was used in order to determine the amount of MTs in the heat-denatured soluble fraction (S2). DPP is a technique based on -SH compounds determination according to Brdička reaction [START_REF] Brdička | Polarographic studies with the dropping mercury method. A new test for proteins in the presence of cobalt salts in ammoniacal solution of ammonium chloride[END_REF] as described by [START_REF] Thompson | An improved electrochemical method for the quantification of metallothionein in marine organisms[END_REF]. Model 303A static mercury drop electrode (SMDE) was used. Certified rabbit liver MTs (Sigma Chemical Co., St. Louis, MO) was used to carry out the calibration according to the method of standard additions. The system consisted of a beveled capillary, Hg working electrode, a platinum counter electrode and an Ag/AgCl reference electrode. Results are expressed in mg of MTs per g of dry homogenized tissue. For comparison with concentrations given in relation to wet weight, refer to the mean moisture contents given hereafter: from 66 to 72% in liver, from 73 to 78% in kidney and from 65 to 71% in muscle.

Statistical analysis

Statistical analyses were performed using the GNU R statistical system (R Development Core Team 2005). Data were first checked for normality by means of Shapiro-Wilk tests. In cases of non-departure from normality, parametric tests were used in the subsequent analyses. When Shapiro-Wilk test p-values were less than 0.05, non-parametric analogues were used. Prior to the use of ANOVA for independent samples, in addition to normality, the homogeneity of the variances of the tested samples was checked by means of Bartlett tests. In case of departure from normality or non-homogeneity of the variances, Kruskal-Wallis tests were applied instead.

The significance of differences of MT levels between liver and kidney was tested by means of t-tests or Wilcoxon (W) tests according to normality. The influence of species and MC on MT content was tested by means of ANOVA or Kruskal-Wallis tests followed by Tukey's HSD tests. The influence of age, sex and reproductive status was tested by means of t-tests or Wilcoxon tests. For each species, dependencies between trace element and MT levels were studied by means of Pearson's linear correlation coefficient. In order to study the factors influencing the distribution of elemental levels between the soluble and insoluble fractions, the latter tests were applied to the ratio between the mass of an element in the soluble fraction and the total mass of this element in both fractions.

Levels of significance of the null hypotheses associated with these tests will be divided into classes of p-values represented by the following codes : NS ≥ 0.05 ; * < 0.05 ; * * < 0.01 ; * * * < 0.001. SD shall stand for standard deviation and CV for coefficient of variation.

3 Results

Sub-cellular elemental distribution

The subcellular distribution of analyzed elements is given in Table 3.1 as the percentage of the element recovered in the soluble fraction versus the total amount of metal in the given tissue. It is assumed that, after a centrifugation at 1 800 g for 60 min, the nucleus and debris of various origins have sedimented to constitute C1, whereas S1 corresponds to the soluble fraction which contains the MTs. Figures 2 and3 illustrate the levels of these elements in the soluble fraction in relation to the whole tissue levels. The distribution pattern of trace elements between both fractions was similar in the three species. Cadmium, Se and hepatic Cu were more present in S1 whereas Fe, Mn, and renal Cu and Zn were in a larger part found in C1. In most cases, the proportion of trace elements in the soluble fraction was higher in liver than in kidney. Furthermore, the concentrations of each element in S1 increased with the total cellular elemental concentrations (Figures 2 and3). Moreover, the linear correlations between levels in the soluble fraction and in the whole tissue were generally better in liver than in kidney. They were particularly good for Cd, Cu, Se and Zn (Figures 2 and3).

In spite of the general similarity of the results in the 3 species, some differences were observed between the Petrel, the Shearwater and the Tropicbird. The distribution of hepatic Cd and Cu was significantly different between Audubon's Shearwater and the White-tailed Tropicbird (P T ukey : * * * ). In kidney, the two Procellariiforms differed in the distribution of Cu (P T ukey : * ) and Fe (P T ukey : * ), the Shearwater differed from the Tropicbird in the distribution of Cu and Zn (P T ukey : * * * ) and Barau's Petrel differed from the Tropicbird in the distribution of Mn (P T ukey : * * ).

The only significant influence of age on trace element distribution, all species combined, was found for Fe (P t-test : * ), Mn (P t-test : * * ) and Se (P W ilcoxon : * * * ) in liver. The accumulation of these elements in the soluble fraction was higher in adults than in juveniles.

Sex, across all, did not have an impact on the distribution of trace elements, except for hepatic Zn (P t-test : * * ) which was found to accumulate significantly more in the soluble fraction in females than in males. In the same way, the reproductive status of adults did not influence the distribution of trace elements in the majority of cases. It was however observed that renal Mn and Zn levels were higher (resp. P W ilcoxon : * * and * ) in the insoluble fraction of incubating birds.

The influence of the muscular condition index (MC) on the distribution of Cd, Cu, Fe, Mn, Se and Zn was tested. Hepatic Mn, and hepatic and renal Fe, were proportionally more present in the soluble fraction than in the insoluble fraction in birds with good muscular condition (resp. P Kruskal-W allis : * * , P AN OV A : * and P AN OV A : * ). Although less obvious, the same trend was observed for Fe and Mn with the body condition index (BC). In the Tropicbird, hepatic and renal Fe and liver Mn were preferentially accumulated in S1 in larger birds. Indeed, significant positive correlations were found between the bird's body weight and hepatic levels of Fe in S1 (r = 0.723 * * * , n = 51), the bird's weight and kidney Fe (r = 0.347 * , n = 51), and the bird's weight and liver Mn (r = 0.651 * * * , n = 51). The opposite tendency was noticed for kidney and liver Zn in Barau's Petrel (resp. r = -0.971 * * , r = -0.93 * , n = 5) but may result from the low number of samples.

Metallothioneins

Metallothionein levels found in liver and kidney of Barau's Petrel, Audubon's Shearwater and the White-tailed Tropicbird are presented in Table 2. The linear correlations between MT and elemental levels are given in Table 3. Positive correlations between total Cd, Cu, Hg and Zn levels and MT levels in liver and kidney indicate that MT content of these organs increases with metal levels. At the species level, the highest MT concentrations were found in Barau's Petrel (Table 2) which exhibited the highest Hg levels of the three species (Kojadinovic et al., submitted for this volume).

Metallothionein levels were significantly different between liver and kidney in the Whitetailed Tropicbird (P t-test : * * * ). However this was not the case for the two Procellariiform species. Besides, MT levels were well correlated between both tissues in the three bird species (Table 3).

In the three species combined, renal MT levels were significantly higher in adults than in juveniles (P W ilcoxon : * * * ), while there were no significant differences in liver.

As it was observed for trace element levels (Kojadinovic et al., submitted for this volume), the gender nor the reproductive status of the adults of each species had any influence on the MT levels.

Body condition (BC) did not seem to have any influence on MT levels of each species (Table 3). However, the muscular condition (MC) had an impact on MT concentrations in liver and kidney, the lower MT levels being found in birds with the best MC. Furthermore, significant negative correlations were found between the total weight of the bird and MT levels, especially in liver.

Table 3: Correlations between MT levels and total metal levels, MT levels and the bird's body condition (BC) and, MT levels and the bird's weight. The letters ".L", ".K" or ".M" placed after the abbreviation of an element indicate the tissues (liver, kidney and muscle, respectively) in which the element is considered. PB: Barau's Petrel ; PLB: Audubon's Shearwater ; PQ: White-tailed Topicbird.

Metallothioneins can bound atoms of Cd, Cu, Hg and Zn [START_REF] Hamer | Metallothionein[END_REF]). The number of atoms of Cd, Cu and Zn was calculated per gram of dry tissue and summed (denoted by molCdCuHgZn in Table 3), in order to examine the potential linkage of MTs with the 4 atoms simultaneously. Although the degree of correlation varied from one species to the other, the same general pairs of well correlated variables appeared, such as MT-Zn in liver and kidney. The summed Cd, Cu, Hg and Zn atoms were also well correlated with MT levels in the corresponding tissue in all three species. Positive correlations were also found between MT levels in a tissue and metal levels in the other. For example, hepatic MT levels were correlated to muscular Cu and Zn levels in all species, and renal MT levels were correlated to Cd levels in muscle, liver and kidney.

Discussion

In addition to its concentration in a tissue, the toxicity of a an element is subject to a number of factors such as the interaction of this element with others, its chemical speciation and its availability, which is dictated by its location in a tissue as well as at a cellular level. The study of elemental subcellular distribution, along with the investigation of potential detoxification processes, is of evident necessity in understanding the path followed by each element in the birds' organism.

The most evident subcellular distribution trend was that, in all three species, the proportion of each element in the soluble fraction was higher in the liver than in the kidney (Table 3.1). The function of each organ probably explains this distribution pattern. On one hand, the liver is an organ of great metabolic activity and a reservoir for essential elements which must be available, and are thus located in the soluble fraction. On the other hand, the kidney is an organ of which the principal function is the excretion of the organism's waste material, but it is also a sink for elements that can not be excreted.

In the three species Fe, Mn and renal Cu, were in a larger part found in the insoluble fraction (Table 3.1). The trends observed for these essential metals were similar to those observed in various seabirds and other marine animals [START_REF] Anan | Subcellular distribution of trace elements in the liver of sea turtles[END_REF][START_REF] Ikemoto | Detoxification mechanism of heavy metals in marine mammals and seabirds: Interaction of selenium with mercury, silver, copper, zinc, and cadmium in liver[END_REF][START_REF] Anan | Multielemental accumulation and its intracellular distribution in tissues of some aquatic birds[END_REF]) and confirm that these elements are part of the composition of organelle and membrane debris that sedimented during centrifugation. Copper concretions may also account for the renal Cu found in C1 [START_REF] Bremner | Assay of extracellular metallothionein[END_REF]. The muscular condition index had an impact on the distribution of hepatic Mn, and hepatic and renal Fe which were proportionally more present in the soluble fraction than in the insoluble fraction in birds with good muscular condition. Given the importance of pectoral muscles in flight, it can be suposed that when they are well developed and actively used by the bird it enhances metabolic activity in the organism. An active metabolism implies a great demand in energy. It is thus not surprising to find, in these healthy birds, essential metals present in the soluble fraction, where they are more available. Hepatic Cu was mainly present in S1 where its levels were very significantly correlated to the MT level in the White-tailed Tropicbird. In this bird, the levels as well as the proportion of hepatic Cu in S1 were the highest of the three studied species. The correlation between Cu and MTs observed in liver may be the sign of the regulation of this element in the Tropicbird. In contrast, regulation in the other two species may be less efficient. Further studies are needed to verify this last hypothesis.

Renal Zn was found essentially in the insoluble fraction of the cell in the three seabirds, whereas hepatic Zn's distribution differed among species. Audubon's Shearwater accumulated hepatic Zn preferentially in the insoluble fraction whereas in Barau's petrel and the White-tailed Tropicbird the accumulation was slightly oriented towards the soluble fraction. Audubon's Shearwater was characterized by the lowest levels of MTs and of most elements. Further investigations are needed in order to understand whether this difference in the accumulation pattern in Audubon's Shearwater is the result of differences in its storage capacities (e.g. the presence of a smaller amount of ligands in the soluble fraction) or whether these differences result from its diet which is much richer in fish than the other two species. As a general rule, the subcellular distribution of Zn in the three Reunion species (71 to 81% of the total Zn in the subcellular fraction, Table 3.1) seems to be less oriented towards the soluble fraction than in other aquatic birds [START_REF] Anan | Multielemental accumulation and its intracellular distribution in tissues of some aquatic birds[END_REF]. Furthermore, renal Mn and Zn levels were higher in the insoluble fraction of incubating birds in relation to birds presenting no incubation patch. These differences in Mn and Zn accumulation may be due to the particular physiological state of incubating birds, such as temporarily modified nutritional patterns for example.

Cadmium, Hg and Zn were found to be positively correlated in the seabirds from Reunion Island (data not shown) and had a tendency of increasing in the soluble fraction with the increase of their whole tissue levels (Figures 2 and3). Although Zn is considered as the main inducer of MT synthesis, MTs have greater affinities for Cd, Cu and Hg than for Zn. As such, Cd, Cu and Hg ions are expected to displace Zn ions from Zn-binding sites through a metal-metal exchange reaction [START_REF] Eaton | Dose-response effects of various metal ions on rat liver metallothionein, glutathion, heme oxygenase, and cytochrome P-450[END_REF]. The displaced Zn is then available for initiating MT induction [START_REF] Roesijadi | Metallothionein and its role in toxic metal regulation[END_REF].

The induction of MT synthesis is known to be associated with an increase of the metal resistance of animals (Eaton et al. 1980, Duncan and[START_REF] Duncan | Tolerance and resistance to cadmium in white suckers (Castostmus commersoni) previously exposed to cadmium, mercury, zinc or selenium, Can[END_REF]. Indeed, MTs main known functions are (i) the homeostatic regulation of intracellular metals by the binding of excess metals that enter the cell and (ii) a "rescue" function for structures impaired by inappropriate metal-binding [START_REF] Hamilton | Evaluation of metallothionein measurement as a biological indicator of stress for cadmium in brook trout[END_REF]. The correlations observed between MTs and metal levels in these species suggest that MTs could act as a reservoir for toxic Cd and Hg or for the excess Cu and Zn. Correlation between MT levels and molCdCuHgZn, and between MT and Zn levels, were systematically stronger (higher r values) than correlations between levels of MTs and Cd, Cu or Hg considered separately (Table 3). Consequently, we may conclude that MTs seem to be most often bound (i) exclusively to one or several Zn ion(s), or (ii) simultaneously to Cd, Cu, Hg and Zn. Furthermore, not only was Cd positively correlated with MT levels in the three species but it was mostly present in the soluble fraction of the cell, where the large majority of the MT burden is found (Table 3.1). Cd-induced renal toxicity is thought to be associated with Cd not bound to MTs [START_REF] Chan | Metal accumulation and detoxification in humans, in Metal metabolism in aquatic environments[END_REF]. In this study, the sampled birds died from poaching, collisions or other types of accidents, not indicating fatal intoxication. It can thus be suspected that most of the Cd in S1 was bound to MTs, supporting the hypothesis of a MT-mediated detoxification of Cd. However, MT-Cd compounds seemed unequally distributed between the liver and the kidneys. As noted earlier, MT levels were higher in adults than in juveniles in kidney but not in liver. Interestingly, this does not correspond to Cd trends where this non-essential metal was more concentrated in adults than in juveniles in kidney as well as in liver (Kojadinovic et al., submitted for this volume). Cadmium found in the liver may be proportionally less bound to MTs in comparison with renal Cd. Cadmium can exit from the liver in the form of Cd-MT through the blood stream and accumulate in the kidney [START_REF] Chan | Nephrotoxicity in rats following liver transplantation from cadmium-exposed rats[END_REF]) which leads us to perceive the liver as a "transition organ" for Cd-MT, and the kidney as a "storage organ" for Cd-MT.

Mercury levels in the storage organs of adult Barau's Petrels (the most Hg-enriched of the 3 species) were very high. However, histological examinations of liver and kidney conducted in seabirds showed Hg levels similar to those found in Barau's Petrel (21 ± 28 µg.g -1 d.w. vs 24 ± 14 µg.g -1 d.w.) and failed to reveal indications of tissue damage associated with these Hg levels [START_REF] Elliott | Heavy metal and metallothionein concentrations in Atlantic Canadian seabirds[END_REF]). This might be related to efficient Hg detoxification processes in the studied seabirds. Besides the detoxification of Cd, MTs could also be suspected to play a part in the detoxification of Hg. This is, however, difficult to prove since Hg subcellular distribution was not determined, and MT-Hg correlation in Barau's Petrel was weak. Moreover, the interaction between Hg and Se is considered as the main protective process against Hg toxicity [START_REF] Magos | Comparison of the protection given by selenite, selenomethionine and biological selenium against the renotoxicity of mercury[END_REF][START_REF] Elliott | Heavy metal and metallothionein concentrations in Atlantic Canadian seabirds[END_REF], Kim et al. 1996a). Indeed, Hg is demethylated in the presence of Se and stored as non-toxic and non-biodegradable, insoluble tiemannite granules in the liver of certain marine mammals and birds [START_REF] Koeman | Mercury-selenium correlations in marine mammals[END_REF][START_REF] Nigro | Mercury exposure and elimination rates in captive bottlenose dolphins[END_REF][START_REF] Ikemoto | Detoxification mechanism of heavy metals in marine mammals and seabirds: Interaction of selenium with mercury, silver, copper, zinc, and cadmium in liver[END_REF]. A 1:1 molar ratio between Hg and Se in the liver of seabirds strongly suggests the existence of mercuric selenide (HgSe) [START_REF] Martoja | Identification of tiemannite as a probable product of demethylation of mercury by selenium in cetaceans. A complement to the scheme of the biological cycle of mercury[END_REF], Kim et al. 1996a[START_REF] Decataldo | Association of metals (mercury, cadmium and zinc) with metallothionein-like proteins in storage organs of stranded dolphins from the Mediterranean sea (Southern Italy)[END_REF]). Such ratios were not found in the Reunion seabirds. Relatively low exposure to Hg may be the reason for the apparent absence of HgSe granules in the insoluble fraction, which was, in addition, confirmed by little Se accumulation in the insoluble fraction. A threshold level of 100 µg.g -1 d.w. of Hg was noted in the Black-footed Albatross (Diomedea nigripes), level above which the detoxification of Hg by Se was triggered (Kim et al. 1996a). Mercury burdens in Reunion adult seabirds were well under this threshold level with hepatic concentrations of 24.31 µg.g -1 d.w. in Petrels, 1.72 µg.g -1 d.w. in Shearwaters and 1.89 µg.g -1 d.w. in the White-tailed Tropicbird (Kojadinovic et al., submitted for this volume). In these birds, Se was chiefly compartmentalized in the soluble fraction of the cells (Table 3.1), and Se levels in S1 increased with levels in the whole liver tissue (Figure 2). Although Hg subcellular distribution was not studied, significant correlations between Hg and Se suggest that it has a similar distribution pattern. [START_REF] Anan | Multielemental accumulation and its intracellular distribution in tissues of some aquatic birds[END_REF] interpreted such results as an indication of the formation of Hg-Se complexes bound to thiol-containing glutathione (GSH) molecules [START_REF] Zilmer | The Glutathione system as an attractive therapeutic target[END_REF] in the soluble fraction. These compounds would act as the precursor of the mineralization of HgSe in lysosomes. Further investigations on total Hg and methylmercury (MeHg) distribution in the subcellular fractions should be carried out in liver and kidney of these three species in order to substantiate these hypothesis.

Conclusion

Many free-living seabirds exhibit relatively high concentrations of trace elements, Cd and Hg in particular, with apparently little or no evident ill effects. This is the case of the birds considered during our study, especially Barau's Petrel. In the three seabirds, a MTmediated Cd detoxification process seems to take place. The involvement of MTs in Cu and Zn homeostasis was also confirmed. Metallothionein synthesis can be induced by a variety of factors such as various conditions of physical and physiological stress, including starvation [START_REF] Nordberg | Metallothioneins: historical review and state of knowledge[END_REF], Debacker et al. 2001b). The study of the muscular condition and the body weight of these seabirds confirmed this statement. They should thus be taken into account when comparing MT data. Furthermore, it appears as though a Se-Hg detoxificating interaction exists in the two Procellariiform species.

As a whole, this work showed that trace element availability and detoxification were mainly influenced by the same factors that had an important impact on trace element levels in these birds (Kojadinovic et al., submitted for this volume): age, diet and phylogeny. Trace element levels and MT levels in the three species decreased in the following order: Barau's Petrel, White-tailed Tropicbird and Audubon's Shearwater. This order follows a gradient of (i) weight of the bird (mean weights of adults were resp. 306 g, 276 g and 197 g), (ii) percentage of cephalopods in their diet (respectively, 98%, 80% and 50%) and distance of their fishing zones from the coast during the breeding period. However the White-tailed Tropicbird stood out in various ways, such as, the strategies developed by the fledgling to cope with fasting (Kojadinovic et al., submitted for this volume), higher percentages of every element in the soluble fraction of its cells, its efficient Cu regulation, the apparent absence of Se-Hg detoxificating interaction in this bird and the potential excretion of Se through eggs in female Tropicbirds (Kojadinovic et al., submitted for this volume). In the light of these results, the Tropicbird's particularities may be linked to the phylogenetic distance which separates this Phaethontidae from the two Procellariidae species.

Figure 1 :

 1 Figure 1: Sample preparation for trace element and MT analysis in seabird tissues.

Figure 2 :

 2 Figure 2: Elemental levels in the soluble fraction in relation to their levels in the whole liver of Barau's Petrel, Audubon's Shearwater and the White-tailed Tropicbird.

Figure 3 :

 3 Figure 3: Elemental levels in the soluble fraction in relation to their levels in the whole kidney of Barau's Petrel, Audubon's Shearwater and the White-tailed Tropicbird.

Table 1 :

 1 Subcellular distribution of hepatic and renal elemental burdens in adult Barau's Petrels, Audubon's Shearwaters and White-tailed Tropicbirds, expressed in percentage of the element recovered in the soluble fraction versus the total amount of metal in the given tissue (Mean ± SD).

		Barau's Petrel Audubon's Shearwater White-tailed Tropicbird
		(n=6)	(n=16)	(n=21)
	Liver
	Cd	81 ± 19	74 ± 8	84 ± 4
	Cu	61 ± 11	55 ± 9	70 ± 7
	Fe	45 ± 14	33 ± 14	41 ± 19
	Mn	36 ± 12	27 ± 10	33 ± 15
	Se	79 ± 3	76 ± 10	81 ± 8
	Zn	58 ± 10	46 ± 15	60 ± 8
	Kidney
	Cd	65 ± 15	56 ± 10	58 ± 16
	Cu	43 ± 17	25 ± 12	46 ± 15
	Fe	42 ± 9	26 ± 12	32 ± 11
	Mn	14 ± 5	18 ± 9	28 ± 7
	Se	78 ± 12	72 ± 3	66 ± 13
	Zn	21 ± 19	19 ± 11	35 ± 11

Table 2 :

 2 Metallothionein concentrations (Mean ± SD, mg.g -1 d.w.) in liver and kidney of seabirds from Reunion Island. For both tissues, the significance of the level differences among the three species is given in the last column. The letters a and b were used to indicate which species differ in terms of MT levels: species sharing the same letter do not differ significantly.

		Barau's Petrel Audubon's Shearwater White-tailed Tropicbird
	n	16	21	33
	Liver
	Mean ± SD 11.44 ± 6.22 a	6.01± 4.87 b	9.22 ± 5.86 ab	ANOVA: *
	CV%	8	7	9
	Kidney
	Mean ± SD 10.39 ± 6.02 a	6.64 ± 2.94 b	5.45 ± 2.67 b	ANOVA: * * *
	CV%	8	7	7

Acknowledgments

This research was supported by the Conseil Général de La Réunion. J.K. also benefited from support of the Conseil Régional de La Réunion and the European Social Fund through a PhD grant. The authors would like to thank E. Robert, M. Rouquette, P. Grondin and N. Ghanem in La Réunion for their help in the tissue preparation and C. Churlaud, Centre Commun d'Analyse (La Rochelle), for her assistance with the analyzes on the ICP-AES. We would also like to thank the numerous volunteers who actively participate in the SEOR Petrel rescue program. We are also grateful towards the referees for their helpful comments on the manuscript.


Jessica Kojadinovic
email: jessica.kojadinovic@univ-nantes.fr

Michel Potier

Matthieu Le Corre

Richard P Cosson

Paco Bustamante

Bioaccumulation of trace elements in pelagic fish from the Western Indian Ocean

Keywords: Metals, Fish, Contamination, Detoxification, Guidelines

Trace elements were analyzed in fish of commercial interest to determine their importance in marine systems of the Western Indian Ocean and their bioaccumulation patterns. The results are equivalent or lower than levels reported in ichthyofauna worldwide. Certain values of muscular Cd, Hg, Pb and Zn were however above thresholds for human consumption. Levels varied among tissues, species and with fish length, but were seldom influenced by the nutritional condition of the fish, its gender and its reproductive status. Correlations between hepatic Hg and Se levels in Swordfish (r 2 = 0.747) and Yellowfin Tunas (r 2 = 0.226), and among metallothionein linking metals imply the existence of detoxification processes in these species. Differences in levels between fish from the Mozambique Channel and Reunion Island probably reflect differences of diets rather than differences of elemental availability in both environments.

Capsule : Metal bioaccumulation was quantified in four species of pelagic fish.

Introduction

Metals and metalloids, whether of natural or anthropogenic origin, are present in all ecosystems throughout the world. The occurrence of unnaturally high levels of metals in regions distant from anthropogenic activities, such as the Arctic and the Antarctic, is of concern, and provides an incentive to study metals in food webs of other isolated areas. Tropical waters are less monitored than marine environments from temperate and polar regions, more particularly southern tropical oceans which are often considered as less contaminated than the northern ones. Accordingly, the tropical zone of the Indian Ocean has, up to the present day, received little attention from researchers with reference to levels of trace elements in marine organisms [START_REF] Kureishy | Total mercury content in some marine fish from the Indian Ocean[END_REF][START_REF] Matthews | Mercury content of commercial important fish of the Seychelles, and hair mercury levels of a selected part of the population[END_REF][START_REF] Mwashote | Levels of cadmium and lead in water, sediments and selected fish species in Mombasa, Kenya[END_REF][START_REF] Robinson | Observations on the levels of total mercury (Hg) and selenium (Se) in species common to the artisanal fisheries of Seychelles[END_REF][START_REF] Kojadinovic | Trace elements in three marine birds breeding on Reunion Island (Western Indian Ocean) Part 1: Factors influencing their bioaccumulation[END_REF].

Large fish such as Swordfish (Xiphias gladius), Yellowfin Tunas (Tunnus albacares), Skipjacks (Katsuwonus pelamis) and Common Dolfinfish (Coryphaena hippurus), which are at the top of marine food webs, are particularly exposed to high levels of trace elements through their food [START_REF] Bryan | Bioaccumulation of marine pollutants[END_REF]. In addition, these pelagic organisms are high performance fish with very high metabolic rates, and consequently high food intake rates, a property that accentuates the exposure to trace elements. Because of their trophic position and bioaccumulation capacities, these fish will be used as bio-indicators of elemental levels to verify the pristine character of the tropical Western Indian Ocean waters.

Through the study of trace element levels in these four commercially important pelagic fish caught in the Mozambique Channel and in waters surrounding Reunion Island, this paper presents novel data on elemental impregnation of marine fish in the western part of the tropical Indian Ocean. Trace elements assimilated from food are transported in the blood, deposited in various tissues and excreted or stored. This paper aims to determine whether the global patterns of trace element accumulation and excretion are similar among species and between study sites. To address these points, three non-essential elements (cadmium (Cd), mercury (Hg) and lead (Pb)) and five essential elements (copper (Cu), iron (Fe), manganese (Mn), selenium (Se) and zinc (Zn)) were analyzed in the liver, kidney and muscle tissue.

The differences in elemental bioaccumulation among tissues were tested. In addition, the potential influence of factors such as physical condition, size, gender and reproductive status on elemental levels were investigated. Furthermore, species' elemental impregnations were compared within study zones, as well as between them. Because the levels of certain elements were dependent of the size of the fish, length-adjusted levels were used to compare their levels in each species found in Reunion Island waters and in the north of the Mozam-bique Channel. The relationships between elements were investigated through the study of the correlations existing among them. Furthermore, considering the nutritional value of fish and the large quantities consumed in this part of the world, our results are briefly discussed in the light of European guidelines for human consumption.

2 Materials and methods The teleost species studied during this investigation were sampled in 2004 from two geographically distant sites in the Western Indian Ocean. The first site corresponds to waters surrounding Reunion Island, a 2 512 km 2 French territory located 700 km east of Madagascar (21 • 7'S ; 55 • 33'E). The second sampling zone was situated in the northern part of the Mozambique Channel, delimited by the following coordinates : 10 • 34'S and 17 • 07'S, and 41 • 11'E and 47 • 19'E (Figure 1). These two sites correspond to two major fishing (purse seine and longline) areas of the Western Indian Ocean [START_REF] Weenarain | Impact économique des activités thonières industrielles[END_REF].

Study site and species

Four pelagic marine fish (the Swordfish, the Yellowfin Tuna, the Skipjack and the Common Dolfinfish) were chosen for this study with respect to their bioaccumulaion capacities and their importance in human consumption.

Fish sampling and preparation

Table 1 Sample information on pelagic fish from the tropical Western Indian Ocean.

A total of 72 fish were sampled in the northern part of the Mozambique Channel and 115 in waters surrounding Reunion Island. Sample information is given in Table 1. Each fish was measured, and weighed when possible. The LJFL (from the tip of the lower jaw to the fork of the caudal fin) was measured on Swordfish whereas the FL (from the tip of the snout to the fork of the caudal fin) was noted for the others species. Individuals were sexed during dissection by the examination of the gonads. The nutritional and reproductive states of each animal were assessed as they may affect the elemental levels. Consequently, gonads were weighed in order to calculate the gonadosomatic index (GSI) as it is generally considered a good measure of gonad maturation and spawning readiness. The GSI is based on the broad assumption that proportionally larger gonads indicate greater development [START_REF] West | Methods of assessing ovarian development in fishes -a review[END_REF]. It was calculated as follows:

GSI = Gonad weight
Body weight × 100 Furthermore, the liver was weighed and used to calculate a hepatosomatic index (HSI) which serves as an indicator of the body condition of the individual.

HSI = Liver weight

Body weight × 100

Due to the limited data on the body weight of the sampled fish, this parameter was extrapolated from the length using weight-length relationships presented in Table 2.

Table 2 Relationships between weight and length for 4 tropical pelagic fish: data from studies in the Indian Ocean. These relationships are presented as exponential regressions on the following model: W=a.L b where W is the fish weight and L the length.

The liver, kidneys and digestive system were removed and placed in separate plastic bags. White muscle was sampled for analysis in the abdominal area above the vent of the fish. Samples were transported to the laboratory in coolers and frozen at -20 • C. To prepare for elemental determination livers, kidneys and muscles were partially thawed out, blended, dried and ground to a fine powder. Muscles were dried in an oven at 50 • C to constant mass for 72 h whereas liver and kidney samples were freeze dried.

Metal analysis

The analysis of Cd, Cu, Fe, Mn, Pb, Se and Zn calls for an extra step in the preparation protocol. Two aliquots of 0.2 to 0.4 g of each sample were digested with 3.5 ml of 15 N nitric acid at 60 • C for 48 h before being diluted to 10 ml with deionized water. To avoid metal contamination, all glass and plastic utensils were washed with detergent, plunged in a bath of mixed nitric (35 ml.l -1 ) and chlorhydric (50 ml.l -1 ) acids for a minimum of 24 h, rinsed 3 times in deionized (Milli-Q quality) water and dried in an oven at 50 • C before use.

Cadmium, Cu, Fe, Mn, Pb, Se and Zn were analyzed by Inductive Coupled Plasma Atomic Emission Spectrometry (ICP-AES Varian Vista Pro CCD). Total mercury (Hg) analysis was carried out with an Advanced Mercury Analyzer (ALTEC AMA 254) on aliquots ranging from 7 to 24 mg of dry sample weighed to the nearest 0.01 mg.

Accuracy and reproducibility of the preparation were tested by preparing replicates of lobster hepatopancreas (TORT-2), dogfish liver (DOLT-3) and dogfish muscle (DORM-2) reference standards (National Research Council, Canada) and blanks along with each set of samples. Detection limits and recovery rates are presented in Table 3. Results under the detection limit were considered as null values. Elemental levels are expressed in µg.g -1 of dry weight (d.w.). The mean moisture content in each species went from 67 to 73% in liver, from 65 to 77% in kidney and from 67 to 82% in muscle. They were used for the conversion of our results to wet weight basis (w.w.) for comparison with other studies.

Data analysis

Statistical analyses were performed using the GNU R statistical system (R Development Core Team, 2005). All statistical samples submitted to tests were first checked for normality and homogeneity of the variances by means of Shapiro-Wilk and Bartlett tests respectively. In the case of non-departure from normality, parametric tests were used in the subsequent analysis, otherwise, non-parametric analogues were used.

The significance of differences of trace element levels among tissues was tested by one way repeated measures analysis of variance (ANOVA) or Friedman (F) tests and followed by multiple pairwise comparison t-tests (t) or Wilcoxon (W) tests for paired samples using Bonferroni's p-value correction. The influence of species on elemental levels was tested by Kruskal-Wallis (KW) tests followed by Wilcoxon tests for independent samples using Bonferroni's p-value correction. The influence of sex and sampling location on trace element levels were tested by means of t-tests or Wilcoxon tests. Trace element content in fish tissues may be influenced by the size of the individual (e.g. [START_REF] Jaffar | Selected trace metal concentrations in different tissues of fish from coastal waters of Pakistan (Arabian Sea)[END_REF][START_REF] Monteiro | Mercury content of Swordfish, Xiphias gladius, in relation to length, weight, age and sex[END_REF][START_REF] Bloom | A survey of size-specific mercury concentrations in game fish from Maryland fresh and estuarine waters[END_REF][START_REF] Mormede | Trace elements in deep-water fish species from the Rockall Trough[END_REF][START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF][START_REF] Agusa | Concentrations of trace elements in marine fish and its risk assessment in Malaysia[END_REF]. The difference in the average length of fish is thus a bias when comparing subsamples using comparison tests such as those described above. In order to truly measure the effect of the sampling location, tested subgroups should contain fish of equal mean size. An analysis of covariance (ANCOVA) was thus performed for each species, in order to adjust the elemental levels in both sampling sites (Mozambique Channel and Reunion Island) using fish length as a covariate. The ANCOVAs were realized on log metal values and log lengths. Residuals were checked for normality by means of Shapiro tests, and for homocedaticity by plotting fitted values vs. residuals [START_REF] Venables | Statistics and computing[END_REF][START_REF] Faraway | Evaluation of ecotoxicological effects of endocrine disrupters during a four-year survey of the Mediterranean population of swordfish (Xiphias gladius)[END_REF]. Furthermore, the potential influence of the reproductive status (GSI) and body condition (HSI) on elemental burdens were investigated, for each species (data from both areas combined), by means of Pearson's linear correlation coefficient. This method was also applied to detect dependencies among elemental levels. The resulting correlation matrices were represented by dendrograms built using the average linkage as aggregation criterion.

Levels of significance of the null hypotheses associated with these tests will be divided into classes of p-values represented by the following codes : NS ≥ 0.05 ; * < 0.05 ; * * < 0.01 ; * * * < 0.001. SD will stand for standard deviation and CV for coefficient of variation.

3 Results

Trace element levels

Levels of Cd, Cu, Fe, Hg, Mn, Pb, Se and Zn in liver, muscle and kidney of Swordfish, Yellowfin Tunas, Skipjack Tunas and Common Dolphinfish caught in the North of the Mozambique Channel and in Reunion Island waters are presented in Table 4, and their dispersion illustrated in Figures 2 to 4.

The level sequence of the tested elements were, in most cases, equal in both areas, and very similar among species (Table 4). Trends were alike in liver and kidney, as they generally adopted the following sequence: Fe > Zn > Cd > Cu ⋍ Se > Hg ⋍ Mn > Pb and Fe > Zn > Se ⋍ Cd ⋍ Cu > Hg ⋍ Mn > Pb respectively (where ⋍ indicates that the two adjacent elements exchange places in the sequence according to the species and the location). In muscle, Zn levels were always higher than Fe levels contrarily to what was noted above (Zn > Fe > Se > Cu ⋍ Hg ⋍ Cd ⋍ Mn > Pb). Furthermore, the sequence of non-essential metal is noteworthy since, in all species-location subgroups but Mozambique Channel Common Dolfinfish, it was as follows: Cd > Hg > Pb in liver and kidney, and Hg > Cd > Pb in muscle. Lead levels were low in all subgroups, for the 3 tissues, with a certain number of values under the detection limit and 50% under the quantification limit (QL) of the ICP-AES (Table 3). The reader should keep in mind that, consequently, the precision of these values is not maximal. Concerning the other elements, all levels were above the QL except for 12 values of Cu in Skipjack muscle. Contrarily, hepatic Cu levels were exceptionally high in Yellowfin Tunas sampled in Reunion waters.

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Factors influencing trace element levels

Tissue related differences

The two major vectors of trace element assimilation in fish are water, through the gills, and more importantly food [START_REF] Dallinger | Contaminated food and uptake of heavy metals by fish: a review and a proposal for further research[END_REF][START_REF] Reinfelder | Trace element trophic transfer in aquatic organisms: A critique of the kinetic model approach[END_REF][START_REF] Smith | Uptake and elimination of radiocaesium in fish and the "size effect[END_REF]. Metals and selenium circulate through the body via blood and are accumulated in various tissues. Statistical tests applied on all the sampled individuals showed that the accumulation of elements varied between liver, kidney and muscle. Lead was the exception since Pb levels did not differ significantly between tissues for Skipjacks (p F riedman = 0.15), Swordfish (p F riedman = 0.184 ; p F riedman = 0.05) and Yellowfin Tunas (p F riedman = 0.683 ; p F riedman = 0.076) fished in the Mozambique Channel and Reunion waters respectively. In Common Dolfinfish, levels of Pb were however higher in kidney than in the other two tissues (p < 0.015; Table 4). The general accumulation pattern of Cd, Cu, Mn showed that liver (followed by kidney, then muscle) was a privileged tissue in all four species. In Swordfish and Skipjacks, liver was also the target tissue for Fe, Hg, Se (except in Swordfish from Reunion) and Zn.

Conversely, the allocations of Fe, Se and Zn in Yellowfin Tunas and Common Dolfinfish were oriented towards kidney (followed by liver and muscle), with the exception of Zn in Common Dolfinfish from the Mozambique Channel. Mercury levels in Mozambique Channel Common Dolfinfish were highest in muscle (followed by liver and kidney), while they were not significantly different in Common Dolfinfish from Reunion waters (p F riedman = 0.572). Mercury accumulation in Yellowfin Tunas was also different between sites. In Yellowfin Tunas from the Mozambique Channel Hg accumulated mostly in kidney (followed by liver and muscle), while in individuals caught in Reunion waters Hg was mainly found in liver (followed by kidney, then muscle).

Influence of size

Hepatic, renal and muscular Hg levels were indeed correlated with the length of the fish in the four species (considering results of both sites combined). Cadmium levels in Skipjacks and Swordfish were also positively correlated to fish length in all three tissues. Selenium levels were influenced by size in all Skipjack tissues and in either liver or kidney of the other species. On the contrary, the low Pb levels did not show any correlation with fish length. Although essential elements are regulated by the organism, some were nevertheless correlated to fish length (Table 5).

Table 5 Correlations between elemental levels and fish length, expressed by determination coefficients (r 2 ) and p-values. Negative correlations are marked by (-).

Influence of sex, reproduction status and body condition

In the previous sections, males and females were pooled since, in most cases, no influence of sex was revealed within the species-location subgroups which were tested (all but Mozambique Common Dolfinfish and Reunion Yellowfin Tunas). Certain exceptions were however noted. In Skipjacks, muscular Fe and Se, and hepatic Zn were significantly higher in males than in females (p t-test = 0.029, p W ilcoxon = 0.005 and p t-test = 0.001 respectively). Conversely, hepatic Cu levels were higher in females than in males (p t-test < 0.001). In Common Dolfinfish, hepatic Fe, Mn and Zn levels were higher in females than in males (p t-test = 0.04, 0.047 and 0.019 respectively). These results must be regarded cautiously since the effect of inter-sex fish length differences was not tested because of a lack of data.

In the light of the results of correlations between elemental levels, and the GSI and the HSI, the influence of both factors appeared to be species dependent. A GSI value of 0.25%±0.13 and a HSI mean value of 1.06%±0.35 were calculated for all Swordfish combined. The reproduction status was positively correlated only to hepatic Cu (r 2 = 0.42, p = 0.031). Negative correlations were observed between HSI and renal (r 2 = 0.304, p = 0.004), muscular (r 2 = 0.24, p = 0.009) and hepatic (r 2 = 0.134, p = 0.028) Hg, and hep-atic Cd (r 2 = 0.164, p = 0.014) and Se (r 2 = 0.141, p = 0.024). Positive correlations were noted for renal and muscular Mn (r 2 = 0.188, p = 0.034 ; r 2 = 0.139, p = 0.042 respectively).

In Yellowfin Tunas, the absence of dependencies between elemental levels and GSI (0.28% ±0.21) attests that there was no influence of the reproduction status on metal levels. Renal Pb was positively correlated to the fish's body condition (HSI = 0.65%±0.24; r 2 = 0.211, p = 0.011). Negative correlations were noted between HSI and Zn in liver (r 2 = 0.185, p = 0.007) and muscle (r 2 = 0.126, p = 0.031), and Hg in muscle (r 2 = 0.147, p = 0.021).

The GSI in Skipjacks (2.03%±0.90) was correlated to the largest number of elements: hepatic (r 2 = 0.16, p = 0.021) and muscular (r 2 = (-) 0.146, p = 0.034) Mn, hepatic Se (r 2 = 0.159, p = 0.021), renal Fe (r 2 = 0.156, p = 0.046) and hepatic Cd (r 2 = 0.149, p = 0.026). Furthermore, hepatic (r 2 = 0.292, p = 0.001) and muscular (r 2 = 0.18, p = 0.022) Cd, and muscular Fe (r 2 = 0.137, p = 0.048) were negatively correlated to the HSI (0.80%±0.31).

The Common Dolfinfish was the only species for which there was no relation between the HSI (1.15%±0.84) and elemental burdens. Nevertheless, the GSI (1.28%±1.21) was positively correlated to renal (r 2 = 0.224, p = 0.005) and muscular (r 2 = 0.169, p = 0.017) Hg and hepatic Mn (r 2 = 0.206, p = 0.013).

Geographical differences

The comparison of the two study areas in term of fish elemental impregnation is presented in Figures 2 to 4. The significance of the level difference, which is indicated for each species below the boxplots, corresponds to the result of a ttest or a Wilcoxon test when the given element was not correlated to the length of the fish considered. Otherwise (Table 5), ANCOVAs were applied in order to eliminate the bias of having different fish sizes between sampling areas. In this case, the result which is given corresponds to the level of significance of the null hypotheses associated to the regression slope of the elemental level against fish length.

Cadmium levels in the kidney of Reunion Yellowfin Tunas were above those found in the Mozambique Channel. In contrast, Cd levels in Swordfish muscle and Common Dolfinfish liver were highest in the Mozambique Channel. For Cu, muscular and hepatic levels were not different from one zone to the other, contrarily to renal levels which were higher in Reunion for Common Dolfinfish and Yellowfin Tunas and in the Mozambique Channel for Swordfish. Similar muscular and hepatic Fe burdens were found in Swordfish and Yellowfin Tunas from both areas. However, in Common Dolfinfish, Fe levels were significantly different in all tissues. Dolphinfish Mn and Pb levels were also significantly different between areas in all tissues, with higher Pb burdens in Mozambique Channel fish. The results obtained for the other species were variable. Mercury and selenium followed the same trends in the three tissues. Whether significantly or not higher levels were found in Swordfish from Reunion waters and in Common Dolfinfish from the Mozambique Channel. In the Yellowfin Tuna, Hg and Se burdens were similar between areas of origin. For Swordfish, Yellowfin Tunas and Common Dolfinfish, Zn levels were not significantly different between areas regardless of the tissue, except for Yellowfin Tuna kidney and muscle.

It is interesting to note that within a species the level of an element was, in some cases, higher in Mozambique Channel fish for one tissue and higher in Reunion fish for another tissue (e.g. Fe and Mn in Dolfinfish and Zn in Yellowfins).

Inter-specific differences

Detailed results on inter-specific differences are presented in Table 4. Generally the ranking of the four species with respect to their elemental burdens was equivalent in both study zones. Several general trends can be highlighted, as for Se, and Cu, Fe and Zn in liver, which levels were highest in Yellowfin Tunas and lowest in Common Dolfinfish (Swordfish and Skipjacks showing intermediate values). Cadmium and Hg also shared common patterns: Swordfish being the most impregnated and Common Dolfinfish the least. It is however noteworthy that, in spite of its small size and short lifespan, Skipjacks had Cd levels equivalent to those found in the Swordfish. Hardly any differences existed among species in terms of Pb burdens. Furthermore, the Common Dolfinfish, which is the smallest species in terms of weight, had the highest levels of renal Fe and Mn (in all tissues for fish caught around Reunion and in liver for Mozambique Channel fish).

Relationships between trace element levels

Accumulation behaviors of trace elements in the organism result in part from their interaction. The correlation matrices linking trace element levels in the various tissues are represented, for each species, by the dendrograms of common to the four species: Fe, Se and Zn in muscle, and Cu, Cd and Se in liver (limited to Cu and Se in Skipjacks). Relationships among elements were more species-specific in kidneys with evident correlations among Cu, Fe and Se in Swordfish ; Fe, Mn and Zn in Yellowfin Tunas ; Fe and Se, and Cu and Zn in Skipjacks ; and Cu, Fe and Mn in Common Dolfinfish. Moreover, the correlation between Hg and Se in liver increased with the Hg impregnation among species (Figures 5678): Common Dolfinfish (r 2 = 0.007 ; p > 0.05), Skipjacks (r 2 = 0.063 ; p > 0.05), Yellowfin Tunas (r 2 = 0.226 ; p = 0.001) and Swordfish (r 2 = 0.747 ; p < 0.001).

Discussion

Element levels varied among tissues and species, and in many cases, with fish length. Individual patterns of elemental bioaccumulation will thus be discussed according to the tissue, the fish's physiology and detoxification processes. Furthermore, the differences in levels observed between fish of the Mozambique Channel and Reunion Island will be interpreted and levels compared with other oceans and health guidelines.

Individual patterns of elemental bioaccumulation

Elemental distribution among tissues

The general accumulation pattern of Cd, Cu, Mn (liver>kidney>muscle) confirmed what has previously been observed by most authors (e.g. [START_REF] Mackay | Selenium and heavy metals in black marlin[END_REF][START_REF] Jaffar | Selected trace metal concentrations in different tissues of fish from coastal waters of Pakistan (Arabian Sea)[END_REF][START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF][START_REF] Bustamante | Distribution of trace elements in tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF]Storelli et al., 2005;[START_REF] Licata | Heavy metals in liver and muscle of Bluefin Tuna (Thunnus thynnus) caught in the straits of Messina (Sicily, Italy)[END_REF]. Interestingly the accumulation pattern of Cd in fish differs from that of other vertebrates such as birds, mammals and turtles in which kidney is the target tissue for Cd (e.g. [START_REF] Furness | Heavy metals in the marine environment[END_REF][START_REF] Furness | Birds as Monitors of Environmental Changes[END_REF][START_REF] Furness | Cadmium in birds[END_REF][START_REF] Dietz | Lead, cadmium, mercury and selenium in Greenland marine animals[END_REF]Storelli et al., 2005b) .

Literature on the distribution of Fe, Se and Zn shows that there exists a certain variation in accumulation patterns among tissues according to the species [START_REF] Mackay | Selenium and heavy metals in black marlin[END_REF][START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF][START_REF] Deheyn | Evidence for enhanced bioavailability of trace elements in the marine ecosystem of Deception Island, a volcano in Antarctica[END_REF]. These observations corroborate our results since the main target tissue of these elements was different in Swordfish and Skipjacks vs. Yellowfin Tunas and Common Dolfinfish.

Hg in fish tissues usually follows the liver>muscle order in terms of accumulation levels [START_REF] Mackay | Selenium and heavy metals in black marlin[END_REF]Storelli et al., 2005). Yet, dominating Hg levels in muscle, as it was noted for Common Dolfinfish of the Mozambique Channel, have also been observed in Bluefin Tunas fished in the Messina straits [START_REF] Licata | Heavy metals in liver and muscle of Bluefin Tuna (Thunnus thynnus) caught in the straits of Messina (Sicily, Italy)[END_REF], and in benthic fish caught around the Kerguelen Islands [START_REF] Bustamante | Distribution of trace elements in tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF]. Moreover, elemental turnover rates are, as a general rule, higher in liver and kidney than in muscle. Hence, elemental levels in the muscle reflect the elemental intake on a longer time frame than the other tissues.

Since Common Dolfinfish are susceptible to cover large distances, it can be suggested that their muscle impregnation is the result of the accumulation of trace elements from preys living in other environments than where the fish were caught and which contained higher Hg levels than those consumed by Common Dolfinfish in the northern Mozambique Channel.

The only organotropism of Pb was noted in Common Dolfinfish where levels were highest in kidney. It is difficult to compare this result to others since renal concentrations are rarely investigated in fish. Most studies in fish include liver and muscle and conclude that Pb levels are higher in the former than in the latter [START_REF] Mackay | Selenium and heavy metals in black marlin[END_REF][START_REF] Mormede | Trace elements in deep-water fish species from the Rockall Trough[END_REF][START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF]Storelli et al., 2005). The absence of organotropism in the other species may be attributed to the low Pb levels found in these fish.

Physiology dependent element accumulation

Metabolic activity in young individuals is usually higher than in older fish [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF]. Since the contaminant uptake rate is positively linked to the metabolic rate in marine animals, it can be supposed that metal accumulation would be highest in young fish. Furthermore a dilution effect of growth on elemental levels may also explain the negative correlation observed in this study with muscular Cu and Mn in Swordfish. The variability of the relationships observed for the other essential elements (positive or no correlation) may simply be attributed to their contrasted elemental regulation in relation with the environmental conditions, and by consequent physiological needs, at a given period. As for Cd, Hg and Se, their constant accumulation over the fish's lifetime and the absence (or limited) excretion of these elements results in their positive correlation with fish length. Added to the large range of sizes of the fish in each subgroup (Table 1), the bioaccumulation of Cd, Hg and Se with size is most probably the cause of the large individual variations in levels observed within species-location subgroups (high CVs in Table 4). Furthermore, in Yellowfin Tunas and Common Dolfinfish, for which Cd burden was not correlated with fish length, it is possible that both phenomena described above counteracted leading to a stabilization of Cd levels. This hypothesis could also be made for Pb which levels do not correlate with the age/size of animals [START_REF] Mackay | Selenium and heavy metals in black marlin[END_REF][START_REF] Dietz | Lead, cadmium, mercury and selenium in Greenland marine animals[END_REF].

In most cases, elemental levels measured in male and female fish were not different in spite of physiological specificities of each gender, such as higher growth rates in female Swordfish. This observation was substantiated by the relatively small impact that the physiological changes implicated in reproduction had on trace element levels in these marine fish. The correlations between GSI and elemental levels, mostly essential elements, were different among the four species. The accumulation of essential elements during a period such as reproduction seems natural to account for the increased activity of the metabolism. The decrease of Mn levels with GSI in Skipjacks muscle indicates that this element is probably mobilized from muscle during the period of gonad development to be used elsewhere.

The decrease of some trace element levels with the increase of the HSI, or body condition, in Swordfish, Yellowfin Tunas and Skipjacks is probably due to a differential increase of the liver and the body weight during fish growth.

Detoxification processes

The basis of metal toxicity is the inhibition of enzymatic systems of cells, resulting from substitution of other metal ions (mainly Cu 2+ , Zn 2+ and Ca 2+ ) [START_REF] Jackson | Mercury in aquatic ecosystems[END_REF]. One of the most common detoxification strategies observed in marine vertebrates is the binding of metals to metallothioneins [START_REF] Mason | Metal detoxification in aquatic organisms[END_REF]. Metallothioneins (MTs) provide protection against toxic effects of certain metals, Cd in particular, by sequestrating and reducing the amount of free metal ions and acting as a "rescue" function for structures impaired by inappropriate metal-binding [START_REF] Hamilton | Evaluation of metallothionein measurement as a biological indicator of stress for cadmium in brook trout[END_REF][START_REF] Vallee | The function of metallothionein[END_REF][START_REF] Roesijadi | Metallothionein and its role in toxic metal regulation[END_REF]. Since their discovery, in 1957, the existence of MTs was firmly established in a large number of animals including fish, in which they occur in liver, kidney, gill and muscle [START_REF] Hogstrand | Binding and detoxification of heavy metals in lower vertebrates with reference to metallothionein[END_REF][START_REF] Hamza-Chaffai | Physico-chemical forms of storage of metals (Cd, Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from Tunisian coast: ecotoxicological consequences[END_REF][START_REF] Olsson | Influence of seasonal changes in water temperature on cadmium inducibility of hepatic and renal metallothionein in rainbow trout[END_REF][START_REF] De Boeck | Differential metallothionein induction patterns in three freshwater fish during sublethal copper exposure[END_REF][START_REF] Scudiero | Fish and mammalian metallothioneins: a comparative study[END_REF]. Besides Cd, MTs also have high affinities to Ag, Cu, Hg and Zn. These ions may be simultaneously linked to this protein. Hence, although MTs were not investigated in the present study, the correlations between Cd, Cu, Hg and Zn may provide clues to whether MTs play a detoxifying role in the studied fish. The comparatively very low Cd levels in the muscle of all fish (Table 4) suppose effective processes of Cd sequestration in liver and kidney. Moreover, positive correlations were noted between levels of Cd and levels of Cu, Hg and Zn in liver and kidney in all four species (Table 6 andFigures 5-8). It is thus possible that renal and hepatic MTs could act as a sink for toxic Cd and Hg or for the excess Cu and Zn in these species. This hypothesis should, of course, be substantiated by MTs analysis.

Another prevalent detoxification process relies on the insolubilization of metals as mineral concretions. A typical example is the formation of non-toxic insoluble tiemannite granules after the mobilization and demethylization of Hg by Se in the liver of many marine mammals, as well as certain birds and fish [START_REF] Koeman | Mercury-selenium correlations in marine mammals[END_REF][START_REF] Mackay | Selenium and heavy metals in black marlin[END_REF][START_REF] Martoja | Identification of tiemannite as a probable prod-uct of demethylation of mercury by selenium in cetaceans. A complement to the scheme of the biological cycle of mercury[END_REF][START_REF] Nigro | Mercury exposure and elimination rates in captive bottlenose dolphins[END_REF][START_REF] Decataldo | Association of metals (mercury, cadmium and zinc) with metallothionein-like proteins in storage organs of stranded dolphins from the Mediterranean sea (Southern Italy)[END_REF][START_REF] Ikemoto | Detoxification mechanism of heavy metals in marine mammals and seabirds: Interaction of selenium with mercury, silver, copper, zinc, and cadmium in liver[END_REF]). Among the four species considered here, the correlation between Hg and Se in liver increased with the Hg impregnation of the given species: Common Dolfinfish (r 2 = 0.007 ; p > 0.05), Skipjacks (r 2 = 0.063 ; p > 0.05), Yellowfin Tunas (r 2 = 0.226 ; p = 0.001) and Swordfish (r 2 = 0.747 ; p < 0.001). Although the mean molar ratios of Se:Hg in liver were different from 1:1 in Swordfish (57:1) and in Yellowfin Tunas (483:1), the significant correlations between these two elements provides an argument for the existence of Se mediated Hg detoxification in both species. Indeed, it is possible that the presence of large quantities of Se in the liver conceal Se:Hg granules. Further investigation is needed to confirm the presence of tiemmanite granules in large fish such as Swordfish.

Trace element levels in fish of the Western Indian Ocean

The pelagic fish considered in this investigation are ideal bioaccumulators as they are characterized by metabolic rates approximately three folds higher than most active fish species [START_REF] Korsmeyer | Tuna metabolism and energetics[END_REF], as well as digestion and growth rates two to five times higher than those of other species of equal body size (Storelli et al., 2005). However, these four fish cover a large ranges of sizes, habitats (in terms of depth), and differ somewhat in terms of their dietary preferences. These are potential factors explaining the differences in their trace element burdens. Swordfish, for example, were the most impregnated in Hg and Cd. These fish stand out by their very large size and their long lifespan that allow long-term bioaccumulation. Furthermore, Swordfish feed on larger preys than Yellowfin Tunas and Common Dolfinfish, especially concerning cephalopods. Moreover the high levels of Cd may be related to the high proportion of cephalopods in the diet of the sampled Swordfish, in comparison to the other three species which mostly preyed on fish (Potier Unpubl. data). Indeed, cephalopods have been reported as a vector of the transfer of cadmium to top marine predators [START_REF] Bustamante | Cephalopods as a vector of the transfer of cadmium to top marine predators in the North-East Atlantic Ocean[END_REF]. Swordfish feed in epipelagic and mesopelagic waters. Mesopelagic waters, that are poorly oxygenated, house the process of Hg methylation, transformation of Hg from an inorganic to an organic form more easily absorbed by living organisms. Mesopelagic animals are thus more susceptible of having high Hg burdens and transfering them to organisms of higher trophic levels [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF][START_REF] Thompson | Seabirds as biomonitors of mercury inputs to epipelagic and mesopelagic marine food chains[END_REF]. Among the four species, Swordfish is the most susceptible of integrating Hg through such a pathway since Yellowfin Tunas, Skipjacks and Common Dolfinfish feed almost exclusively on epipelagic preys [START_REF] Quéro | R: A language and environment for statistical computing[END_REF][START_REF] Opic | Poissons commerciaux du sud-ouest de l'Océan Indien[END_REF][START_REF] Potier | Feeding partitioning among tuna taken in surface and mid-water layers: The case of yellowfin (Thunnus albacares) and bigeye (T. obesus) in the Western Tropical Indian Ocean[END_REF]. Yellowfin Tunas, for their part, had highest levels of most essential elements. This latter species is phyllogenetically close to the Skipjack [START_REF] Collette | Systematics of tunas and mackerels (Scombridae). In: Tuna physiology, ecology, and evolution[END_REF] which presented similar trace element values in many cases. Furthermore, with the exception of Mn, elemental levels were lowest in the Common Dolfinfish. This probably results from the Dolfinfish's small size, short lifespan (4 years) and very high growth rate [START_REF] Massuti | Reproductive biology of dolphin-fish (Coryphaena hippurus l.) of the island of Majorca (western Mediterranean)[END_REF]NOAA fisheries, 2005).

Non-essential metals do not present any function for the fish's metabolism and are by consequent not regulated by the metabolism. The amount of Cd, Hg and Pb in fish organisms can thus serve as an indication of environmental levels of these metals. For example, although not always in significant proportions, Pb mean levels were highest in tissues of all species caught in the Mozambique Channel. Hence, it could be assumed that Pb levels are higher in prey species inhabiting the north of the Mozambique Channel than in those found in waters surrounding Reunion Island. However, biological and ecological characteristics of each teleost species vary from one area to the other and may also affect metal levels. This was most likely the case for levels of Cd and Hg which predominance area was not always the same for the three species. These differences may be explained by shifts in the species' dietary prey composition and/or trophic position between areas, rather than differences in environmental levels. More specifically, the higher Cd levels in Swordfish and Common Dolfinfish sampled in the Mozambique Channel than in Reunion waters can be explained by a larger amount of cephalopods in their diet (50% and 38% resp. in the Mozambique Channel vs 31% and 2% resp. in Reunion, determined by stomac content analysis, Potier Unpubl. data). These conclusions are in accordance with the results obtained for Hg in Swordfish. Mercury levels were highest in Reunion waters where Swordfish feed mainly on fish (68% with respect to 44% in the Mozambique Channel) which are regarded as Hg bioaccumulating prey [START_REF] Arcos | Mercury levels in seabirds and their fish prey at the Ebro Delta (NW Mediterranean): the role of trawler discard as a source of contamination[END_REF]. A larger dataset of Common Dolfinfish from the Mozambique Channel (> 6 fish) is needed to confirm that these fish are more impregnated in Cd, Hg and Pb than those collected in Reunion waters, irrespectively of their size. In Yellowfin Tunas, the higher Cd levels in Reunion were not in accordance with their piscivorous diet (99% fish in Reunion and 86% in the Mozambique Channel, Potier Unpubl. data) as most fish species generally contain relatively low Cd levels [START_REF] Bustamante | Distribution of trace elements in tissues of benthic and pelagic fish from the Kerguelen Islands[END_REF]. In the light of these results, it seems likely that the diet of Yellowfin Tunas from Reunion waters changes during the year and includes a significant proportion of cephalopods, which increases their exposure to Cd [START_REF] Bustamante | Cephalopods as a vector of the transfer of cadmium to top marine predators in the North-East Atlantic Ocean[END_REF] ; changes in diet which may be linked to changes in habitat.

Comparison with other oceans and guidelines

Despite the human fishing pressure on Swordfish, tunas and, to a lesser extent, Dolphinfish from the Indian Ocean [START_REF] Pianet | Etat des stocks de thonidés dans l'Océan Indien[END_REF], there is a great lack of knowledge on trace element levels in these fish. A compilation of data on trace element values from various studies (Tables 7 &8) allows to compare the present results to those of phylogenetically related, or similar size species from the Indian Ocean and other locations in the world.

Few guidelines for effects of metals in fish have been established for the protection of the fish. Guidance values in muscle are however recommended in some countries, such as 20 µg.g -1 w.w. for Cu and 50 µg.g -1 w.w. for Zn (MAFF, 1995). None of the muscle samples reached the limits set for Cu. For Zn, although all mean values were inferior to 50 µg.g -1 w.w., some of the most Zn-enriched Yellowfin Tuna and Skipjack muscle samples were superior to the guidance level.

Mercury. A great deal of publications on marine fish report only Hg levels because this metal is of particular concern for human health (e.g. [START_REF] Carrington | An exposure assessment for methylmercury from seafood for consumers in the United States[END_REF][START_REF] Marcotrigiano | Heavy metal, polychlorinated biphenyl and organochlorine pesticide residues in marine organisms: Risk evaluation for consumers[END_REF]Burger andGochfeld, 2004, 2005;Burger et al., 2005;[START_REF] Rasmussen | A review of mercury in seafood: Special focus on tuna[END_REF]. Inorganic mercury occurring naturally or from pollution is converted to methyl-mercury (MeHg) by microorganisms and is biomagnified up the food chain. Consequently, a large percentage of Hg is present as toxic MeHg in the edible portions of fish consumed by man [START_REF] Cappon | Mercury and selenium content and chemical form in fish muscle[END_REF][START_REF] Bloom | A survey of size-specific mercury concentrations in game fish from Maryland fresh and estuarine waters[END_REF][START_REF] Wagemann | Percent methylmercury and organic mercury in tissues of marine mammals and fish using different experimental and calculation methods[END_REF]. The most widely established guideline value for Hg levels in marine predatory fish is 1 µg.g -1 w.w. (IPCS, 1987;[START_REF] Epa | Guidance for assessing chemical contaminant data for use in fish advisories. Volume II. Risk assessment and fish consumption limits[END_REF]. In the Mozambique Channel and Reunion Island, 3% and 43% of the Swordfish respectively, had muscular Hg levels exceeding the authorized limits (Figure 2). This last result on Reunion Swordfish should however be taken very cautiously and not generalized owing to the very small sample size (7 fish). In Swordfish liver, 40% of Hg values were above the permissible level in Mozambique Channel Trace element levels (Mean ± SD, µg.g -1 w.w.) in the muscle of large pelagic fish.

Table 8 Trace element levels (Mean ± SD or range, µg.g -1 w.w.) in the liver (a) and kidney (b) of large pelagic fish.

samples, and 64% in Reunion samples (Figure 3). In kidney, the percentage of exceeding values was 29% in the Mozambique Channel samples, and 31% in Reunion samples (Figure 4). The Hg guideline was also reach in 5%, 14% and 18% of muscles, livers and kidneys of Yellowfin Tunas caught in Reunion Island waters. Overall, the Hg levels detected during this study were nevertheless congruent with, or lower than, Hg level found in the literature. For example, Hg levels in Yellowfin Tunas from Taiwan waters were 23 and 10 times superior to levels in Yellowfin Tunas from the Mozambique Channel and Reunion waters respectively. Furthermore, Hg values in muscle of Common Dolfinfish from Reunion waters were five times lower than those measured in Common Dolfinfish of equal mean length fished in the Atlantic. The comparison of Hg values in marlins with those found in the present species is also noteworthy as the former are so much superior to the latter (Tables 7 &8). In this section, hepatic and renal levels are compared to thresholds established for muscle. However there are no health standards for liver and kidney because these organs are seldom, if ever, consumed or used for oil production, therefore concentrations exceeding the guideline values are unlikely to be a health hazard.

Cadmium. The accumulation of Cd is proportionally less oriented towards the muscle than Hg. Cadmium is however highly toxic (e.g. [START_REF] Järup | Hazards of heavy metal contamination[END_REF].

The threshold concentration of this metal in fish muscle destine to human consumption set by the European Commission is 0.1 µg.g -1 w.w., thus 10 times lower than that set for Hg. Muscular levels of Cd exceeded the legal limit in 43%, 18%, 81% and 5% of cases for Swordfish, Yellowfin Tunas, Skipjacks and Common Dolfinfish caught in Reunion waters respectively. In fish from the Mozambique Channel, percentages were as follows: 76%, 0% and 0% in Swordfish, Yellowfin Tunas and Common Dolfinfish respectively. Hepatic and renal values of Cd were higher than authorized limits in all fish. However, the reader should bear in mind that these standards have been established for muscle with a margin of safety. It is difficult to compare the Cd levels of Swordfish from this study to those from the Mediterranean (Tables 7 &8) because of the propensity of Cd to accumulate with length in this species and the large size difference between Swordfish sampled in both basins (the mean length being approximately twice shorter in the Mediterranean sample). For Yellowfin Tunas, in which Cd was not correlated to length, muscular Cd levels are in line with that of other Tunas, although slightly higher than Cd values in Yellowfin Tunas from Taiwan waters and lower than those in Bluefin Tuna from the Black Sea.

Lead. Fish caught in the northern Mozambique Channel and around Reunion Island presented lower Pb levels than equivalent species fished in the Mediterranean or in Taiwan waters. For comparison sake, the present Pb values in muscle are similar to those found in small sculpins and halibut from Greenland [START_REF] Dietz | Lead, cadmium, mercury and selenium in Greenland marine animals[END_REF]. Lead found in wild fish can however reach very high levels such as in the Streamlined spinefoot, Siganus oramin, from Pearl River Estuary, in South China, in which Pb values were 33 times higher than the maximum level recorded during this study (30.7 vs. 0.93 µg.g -1 w.w.) [START_REF] Ip | Heavy metal and Pb isotopic compositions of aquatic organisms in the Pearl River Estuary, South China[END_REF]. Volcanic activity is one of the largest natural input of Pb into the atmosphere [START_REF] Ramade | Précis d'Ecotoxicologie[END_REF]. In the light of the low Pb levels in Reunion fish, it appears that the volcanic activity in Reunion Island has no measurable affect on the surrounding marine ecosystem Pb levels. Furthermore, the isolation of the Western Indian Ocean with respect to large anthropogenic activity centers, added to the short half-life of Pb in water (25 days), and the absence of biomagnification proprieties for this metal, have apparently preserved the fish living in this region from anthropogenic Pb (mostly originating from antiknock additive in petroleum, wast incineration, metallurgy, paints, etc). The guideline value adopted the European Commission for Pb in marine fish muscle 0.4 µg.g -1 w.w.. Levels in fish sampled during this study were all under this limit except one Swordfish caught in the Mozambique Channel and one Skipjack.

This study of pelagic fish shows that the tropical waters of the Western Indian Ocean have low levels of non essential metals. Monitoring studies should however be set up as some fish contain values of muscular Cd, Hg and Zn superior to thresholds for human consumption, particularly Swordfish caught in waters surrounding Reunion Island.

Fig. 1 .

 1 Fig. 1. Map of the study area with sampling zones materialized by shaded circles.

  Fig.2. Comparison of elemental levels (µg.g -1 d.w.) in muscle of fish caught in the Mozambique Channel (MOZ) and Reunion Island waters (RUN). For each species, the significances of the level differences between both study sites are indicated below the boxplots. Horizontal dotted lines mark the European guidance levels for human consumption. COR: Common Dolphinfish; SKJ: Skipjack Tunas; SWO: Swordfish; YFT: Yellowfin Tunas; W: Wilcoxon test; AOCV: analysis of covariance; NS: Non significative.

  Fig.4. Comparison of elemental levels (µg.g -1 d.w.) in kidney of fish caught in the Mozambique Channel (MOZ) and Reunion Island waters (RUN). For each species, the significances of the level differences between both study sites are indicated below the boxplots. Horizontal dotted lines mark the European guidance levels for human consumption. COR: Common Dolphinfish; SKJ: Skipjack Tunas; SWO: Swordfish; YFT: Yellowfin Tunas; W: Wilcoxon test; t: t-test; AOCV: analysis of covariance; NS: Non significative.

Fig. 5 .Fig. 6 .Fig. 7 .

 567 Fig.5. Dendrogram illustrating linear correlations between trace elements in Swordfish. The minimum levels of significance of the linear correlation coefficients among groups of elemental levels are encoded in the dendrogram. NS: non significant. The letters ".L", ".K", ".M" and placed after the abbreviation of an element indicate the tissues (resp. liver, kidney and muscle) in which the element is considered.

Fig. 8 .

 8 Fig.8. Dendrogram illustrating linear correlations between trace elements in the Common Dolphinfish. The minimum levels of significance of the linear correlation coefficients among groups of elemental levels are encoded in the dendrogram. NS: non significant. The letters ".L", ".K", ".M" and placed after the abbreviation of an element indicate the tissues (resp. liver, kidney and muscle) in which the element is considered.

Table 3

 3 Detection limits (DL), quantification limits (QL) and recovery rates.

  Comparison of elemental levels (µg.g -1 d.w.) in muscle of fish caught in the Mozambique Channel (MOZ) and Reunion Island waters (RUN). For each species, the significances of the level differences between both study sites are indicated below the boxplots. Horizontal dotted lines mark the European guidance levels for human consumption.

	MOZ	RUN RUN MOZ	RUN	MOZ	RUN	MOZ	RUN RUN MOZ	RUN	MOZ	RUN
		Fe					Hg
	MOZ	RUN RUN MOZ	RUN	MOZ	RUN	MOZ	RUN RUN MOZ	RUN	MOZ	RUN
		Mn					Pb
	MOZ	RUN RUN MOZ	RUN	MOZ	RUN	MOZ	RUN RUN MOZ	RUN	MOZ	RUN
		Se					Zn
						MOZ	RUN RUN MOZ	RUN	MOZ	RUN

COR: Common Dolphinfish; SKJ: Skipjack Tunas; SWO: Swordfish; YFT: Yellowfin Tunas; W: Wilcoxon test; AOCV: analysis of covariance; NS: Non significative.

  Fig.3. Comparison of elemental levels (µg.g -1 d.w.) in liver of fish caught in the Mozambique Channel (MOZ) and Reunion Island waters (RUN). For each species, the significances of the level differences between both study sites are indicated below the boxplots. Horizontal dotted lines mark the European guidance levels for human consumption. COR: Common Dolphinfish; SKJ: Skipjack Tunas; SWO: Swordfish; YFT: Yellowfin Tunas; W: Wilcoxon test; t: t-test; AOCV: analysis of covariance; NS: Non significative.

							Non-outlier Max.
							Non-outlier Min.
							3rd quartile
							Mean
							1st quartile
							Outlier
				Cd						Cu
	Levels (µg.g-1 d.w.)	0.3					67
		MOZ RUN	RUN	MOZ	RUN MOZ	RUN	MOZ RUN	RUN	MOZ	RUN MOZ	RUN
		W: *		AOCV: NS	AOCV: NS	W: NS		AOCV: NS	W: NS
				Fe						Hg
	Levels (µg.g-1 d.w.)
							3.3
		MOZ RUN	RUN	MOZ	RUN MOZ	RUN	MOZ RUN	RUN	MOZ	RUN MOZ	RUN
		t: **		W: NS	W: NS	AOCV: *		AOCV: **	AOCV: NS
				Mn						Pb
	Levels (µg.g-1 d.w.)
							0
		MOZ RUN	RUN	MOZ	RUN MOZ	RUN	MOZ RUN	RUN	MOZ	RUN MOZ	RUN
		AOCV: **		AOCV: NS	W: NS	W: ***		W: NS	W: *
				Se						Zn
		250
	Levels (µg.g-1 d.w.)	200 150 100					166 0
		MOZ RUN	RUN	MOZ	RUN MOZ	RUN	MOZ RUN	RUN	MOZ	RUN MOZ	RUN
		}	}	}	}
			SKJ	SWO
		W: NS		AOCV: *	AOCV: NS	t: NS		AOCV: NS	AOCV: NS
							10

  Comparison of elemental levels (µg.g -1 d.w.) in kidney of fish caught in the

	Mozambique Channel (MOZ) and Reunion Island waters (RUN). For each species,
	the significances of the level differences between both study sites are indicated below
	the boxplots. Horizontal dotted lines mark the European guidance levels for human
	consumption. COR: Common Dolphinfish; SKJ: Skipjack Tunas; SWO: Swordfish;
	YFT: Yellowfin Tunas; W: Wilcoxon test; t: t-test; AOCV: analysis of covariance;
	NS: Non significative.

Table 6

 6 Correlations between Cd and Cu, Hg and Zn levels, expressed by determination coefficients (r 2 ) and p-values.

  Although they lack uniformity, guideline values are however available for fish consumers, both human and wildlife. The European Union has set regulations for Cd, Hg and Pb in muscle (legislations 466/2001 and 221/2002). Legal thresholds are inexistent for essential elements in Europe.

Table 7

 7

Acknowledgments

This research was supported by the Conseil Général de La Réunion. J.K. also benefited from support of the Conseil Régional de La Réunion and the European Social Fund through a PhD grant. The authors would like to thank J. Bourjea and M. Taquet (Ifremer -La Réunion), as well as Maeva Pêche au Gros, Réunion Fishing Club, L. & P. Berthier, and other anonymous Reunionese fishermen for their assistance in the sampling effort. We would also like thank E. Robert, M. Rouquette, P. Grondin, N. Ghanem (University of La Réunion) and C. Churlaud, (Centre Commun d'Analyse -La Rochelle), for their help in the sample preparation and analysis. We also acknowledge the referees for their helpful comments on the manuscript.