P Bustamante
email: paco.bustamante@univ-lr.fr

S Grigioni

R Boucher-Rodoni

F Caurant

P Miramand

Bioaccumulation of 12 trace elements in the tissues of the nautilus Nautilus macromphalus from New-Caledonia

Keywords: New Caledonia, trace metals, cephalopods, digestive gland, nautilus

Soils in New Caledonia are particularly rich in metals among which Fe and Ni are intensively exploited. Due to important natural erosion in tropical latitudes and to mining activities, coastal waters are enriched in Co, Cr, Fe and Ni. In deeper waters lives a cephalopod species which is considered as a living fossil, the nautilus Nautilus macromphalus. In this study, 12 traces elements were analysed in the tissues of 4 nautilus specimens. Results showed high metal concentrations compared to data available for cephalopod from temperate waters. These concentrations were often in the same order than those encountered in bivalves or gastropods from contaminated areas. Relatively high concentrations of Ni and Cr in the hemolymph strongly suggest a high exposure of N.

macromphalus to these metals. Among the tissues, the digestive gland have the highest concentrations of Cd, Co, Fe, V and Zn while for Ag, Al, As, Cr and Ni, renal and pericardial appendages exhibited the highest values. Despite this, the digestive gland contained the largest quantities of all metals with the exception of As and Mn which were mainly found in the body 2 muscular remains. These results highlighted the major role of digestive gland and excreting organs in the metabolism of metals in these cephalopod species.

Introduction

Cephalopods play a key role in many marine ecosystems [START_REF] Amaratunga | Advances in assessment of world cephalopod resources[END_REF][START_REF] Rodhouse | Antarctic cephalopods -a living marine resource ?[END_REF]). Trace elements have been investigated extensively in many marine organisms but rarely in cephalopods. Some elements are essential, others toxic, and for a number of them the role is still not known. The studies concerning trace elements in cephalopods molluscs are limited to species targeted by commercial fisheries such as the cuttlefish Sepia officinalis [START_REF] Decleir | Determination of protein-bound copper and zinc in some organs of the cuttlefish Sepia officinalis L[END_REF][START_REF] Schipp | Distribution of copper and iron in some central organs of Sepia officinalis (Cephalopoda). A comparative study by flamless atomic absorption and electron microscopy[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et Terres Rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], the octopuses Octopus vulgaris and Eledone cirrhosa [START_REF] Ghiretti-Magaldi | Pathways of terminal respiration in marine invertebrates. I. The respiratory system in cephalopods[END_REF][START_REF] Rocca | Copper distribution in Octopus vulgaris Lam. hepatopancreas[END_REF][START_REF] Renzoni | Mercury concentration in the water, sediments and fauna of an area of the Tyrrhenian coast[END_REF][START_REF] Froesch | Octopus chromatophores accumulate nickel[END_REF], Ueda et al. 1979[START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Barghigiani | Interaction between selenium and cadmium in Eledone cirrhosa of the Northern Tyrrhenian Sea[END_REF][START_REF] Rossi | Mercury in Eledone cirrhosa from the Northern Tyrrhenian Sea: contents and relation with life cycle[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et Terres Rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], some Ommastrephid squids [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF], Ueda et al. 1979[START_REF] Smith | Radio-nuclides and heavy metals in Nototodarus gouldi[END_REF][START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Yamada | Organotin compounds and polychlorinated biphenyls of livers in squid collected from coastal waters and open ocean[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et Terres Rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF]) and some Loliginid squids [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Yamada | Organotin compounds and polychlorinated biphenyls of livers in squid collected from coastal waters and open ocean[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et Terres Rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF]). Among these species, high levels of metals have generally been recorded, the digestive gland of Coleidae concentrating many trace elements, such as cadmium, copper, silver or zinc [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF][START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Finger | Molecular association of Cu, Zn, Cd and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF][START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et Terres Rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF], Bustamante et al 1998a). The most striking feature is that the digestive gland stores most of the total cadmium, reaching up 98% in some species [START_REF] Bustamante | Etude des processus de bioaccumulation et de détoxication d'éléments traces (métaux lourds et Terres Rares) chez les mollusques céphalopodes et bivalves pectinidés. Implication de leur biodisponibilité pour le transfert vers les prédateurs[END_REF].

Present cephalopods comprise two subclasses: the Coleoidae, with ca 650 species and the Nautiloidae with a single genus (Nautilus) and an as yet undetermined number of species (3 to 7 according to the authors). Nautilus is the last representative of the ectocochleate cephalopods and is considered as a living fossil. This taxon shares a number of common anatomical structures with present cephalopods, but some organs are specific to Nautilus.

Among the nautilus species, Nautilus macromphalus is endemic to the New Caledonian waters. In this area, natural erosion and mining activity have provoked an enrichment of several metals, mainly Co, Cr, Fe and Ni, of the New Caledonian waters and consequently in the coral reef food webs [START_REF] Monniot | Cellular sites of iron and nickel accumulation in ascidians related to the naturally and anthropic enriched New Caledonian environment[END_REF]). These authors have described the metal enrichment in several species of filter-feeding ascidians from the shallow waters. But no data are available on species living off the New Caledonia coast.

For these reasons, levels of trace elements in the tissues of Nautilus macromphalus from New Caledonia were investigated here and compared to previous results reported for Coleoid species.

Materials & Methods

Sampling and sample preparation

2 males and 2 females Nautilus macromphalus were taken in baited traps from the barrier reef off Nouméa (New Caledonia). Samples were frozen upon arrival at the Nouméa Aquarium in individual plastic bags. Each individual was weighed and sexed. The digestive gland, gills, renal and pericardial appendages, digestive tract and genital tract were totally removed. In addition, pieces of muscle and crop were sampled to determine metal concentrations. The remains of the animals were thus composed of arms, the rest of the muscles and the rest of the crop. The stomach was emptied before metal analysis.

Analytical procedure

Tissue samples were dried for several days at 80°C to constant weight. Two aliquots of approx. 300 mg of each homogenised dry sample were digested with 4 ml of 65 % HNO 3 and 1 ml of 70 % HClO 4 at 80°C until the solution was clear. After evaporation, the residues were dissolved in 0.3 N nitric acid.

Cadmium, Copper and Zinc were determined both by atomic absorption spectrophotometry (AAS) and by induced coupled plasma mass spectrophotometry (ICP-MS). Other elements (Ag, As, Al, Co, Cr, Fe, Mn, Ni and V) were analysed only by ICP-MS.

Appliances used for metals determination were a Varian spectrophotometer Vectra 250 Plus with Deuterium background correction and a Varian ICP-MS Ultra Mass 700. Reference tissues, dogfish liver DOLT-2 (NRCC), Orchard-Leaves (NBS) and MA-A-2 fish-flesh standard (IAEA), were treated and analysed in the same way as the samples. The results for the standard reference materials (Table 1) are in good agreement with certified values. The detection limits were (µg/g dry wt): 0.002 (Ag), 0.005 (Al), 0.15 (As), 0.005 (Cd), 0.005 (Co), 0.005 (Cr), 0.027 (Cu), 0.15 (Fe), 0.017 (Mn), 0.002 (Ni), 0.005 (V) and 0.15 (Zn). Metal concentrations in tissues are given relatively to the dry weight (µg.g -1 dry wt) while the distribution percentages were calculated for wet weight.

Results

Metal concentration in the organs of nautilus are reported in Figure 1. The percentage of heavy metals in each tissue are shown in Figure 2.

Mean metal concentrations in whole Nautilus macromphalus were 2.4 µg Ag.g -1 , 19.1 µg Al.g -1 , 186 µg As.g -1 , 16.0 µg Cd.g -1 , 2.7 µg Co.g -1 , 2.6 µg Cr.g -1 , 73 µg Cu.g -1 , 258 µg Fe.g -1 , 17µg Mn.g -1 , 6.7 µg Ni.g -1 , 3.2 µg V.g -1 and 260 µg Zn.g -1 (Figure 1). Variation coefficients ranged from 10% for As to 45% for Co and Ni.

Metal levels in soft tissues

In the four sampled nautilus, the digestive gland was the major site of concentration for Cd, Co, Fe, V and Zn with 28.2-60.5 µg Cd.g -1 , 4.4-12.5 µg Co.g -1 , 477-953 µg Fe.g -1 , 6.4-10.6 µg V.g -1 and 515-963 µg Zn.g -1 (Figure 1). The digestive gland also concentrated Ag, As, Cr, Cu and Ni at levels closed to the highest concentrations recorded in the other tissues with 1.1-6.8 µg Ag.g -1 , 133-194 µg As.g -1 , 3.4-5.5 µg Cr.g -1 78-174 µg Cu.g -1 and 9.0-26.3 µg Ni.g -1 (Figure 1). Ag, Al, As, Cr and Ni concentrations were also remarkable in excreting tissues, i.e. renal and pericardial appendages, which exhibited the highest concentrations for these metals. In fact, Ni concentrations ranged from 5.6 to 40.0 µg.g -1 in renal appendages. Pericardial appendages concentrated Ag from 13.8 to 28.0 µg.g -1 , Al from 45 to 305 µg.g -1 , As from 155 to 412 µg.g -1 and Cr from 6.0 to 9.4 µg.g -1 (Figure 1).

Muscles exhibited generally the lowest concentration for all metals except for As which exhibited the highest concentration in this tissue (203-354 µg As.g -1 dry weight ; Figure 1).

Mn was present in high concentration in the gills and the renal appendages (i.e. 42-151 µg.g -1 and 16-152 µg.g -1 , respectively) while most tissues exhibited concentrations lower than 20 µg.g -1 . The highest Cu concentration was found in the hemolymph, with mean values of 1055 ± 12 µg.g -1 . Thus, it is not surprising to find relatively high Cu concentrations in the gills (i.e. 127 ± 38 µg.g -1 ).

Percentage distribution of metals in soft tissues

With the exception of As and Mn which were mainly found in the body muscular remains (50 ± 12% and 42 ± 16%, respectively), the digestive gland contained the largest quantities of all metals : 75 ± 3% of Ag, 62 ± 11% of Al, 96 ± 3% of Cd, 95 ± 2% of Co, 57 ± 16% of Cu, 65 ± 5 % of Cr, 87 ± 4% of Fe, 84 ± 8 of Ni, 95 ± 3% of V and 88 ± 3% of Zn (Figure 2).

Although the concentration of some metals was high in the renal and pericardial appendages, these tissues contain in fact low amounts of metals because of their small mass (Figure 2).

Discussion

The variability of metal concentration was relatively small among the four studied individuals.

Low variation coefficients for toxic metals such as Ag, Cd or V (26%, 41% and 34%, respectively) are noteworthy. Nevertheless, coefficients of variation for Cd are similar to those found in two octopus species from the Southern Indian Ocean, Benthoctopus thielei and Graneledone, reported to have very high Cd levels in their tissues (Bustamante et al. 1998a).

Such results suggest efficient regulation processes of toxic metals in Nautilidae even Cd levels were lower in Nautilus macromphalus than in these octopuses. Some other toxic elements, Ag, Co, Cr, Ni and V, were more concentrated in nautilus than in temperate cephalopods such as the cuttlefish Sepia officinalis and the octopuses Eledone cirrhosa and Octopus vulgaris [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF]. This might be due to accumulation during nautilus life span (10-15 years), much longer than Coleoid's (1-3 years). Moreover, several essential metals, such as Fe, Mn and Zn were also highly concentrated in N.

macromphalus, but Cu concentrations almost the same as in Coleoidae species [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF], Bustamante et al. 1998a). High metal concentrations might reflect the ambient life conditions of nautilus. Mineral extraction activity, mainly Ni, is important in New Caledonia : Ni enrichment in the waters and food webs might thus account for high levels of Ni in N. macromphalus. Although the dissolved Ni have not been measured, large amounts in coastal sediments suggests that abnormal concentration may occur in sea water [START_REF] Bryan | Heavy metal contamination in the sea[END_REF][START_REF] Monniot | Cellular sites of iron and nickel accumulation in ascidians related to the naturally and anthropic enriched New Caledonian environment[END_REF]. Indeed, [START_REF] Monniot | Cellular sites of iron and nickel accumulation in ascidians related to the naturally and anthropic enriched New Caledonian environment[END_REF] reported very high Ni concentrations in the body and the tunic of Ascidia sydneiensis from coastal waters, reaching 80.4 and 119.5 µg/g dry wt, respectively. Compared to other molluscs species, Ni concentrations in N. macromphalus were in the same order of magnitude than in several deposit feeder species such as Scrobicularia plana or Macoma balthica but are higher than in filter feeder species such as Pecten maximus or Mytilus edulis (Table 2). It was also the case for Co and Cr. High levels of Ni, Co and Cr in deposit feeder bivalves were related to direct exposure through contaminated sediments [START_REF] Bryan | Indicators of heavy metal contamination in the Looe estuary (Cornwall) with particular regard to silver and lead[END_REF]. This strongly suggest that contamination is not only located on the New Caledonian coast but reach deeper waters where nautilus lives. This hypothesis is reinforced by the relatively high concentrations for these metals encountered in the hemolymph. Indeed, in circulatory fluid as hemolymph, turnover of metals is supposed to be rapid and by the way, metal concentrations relatively low, with the exception of Cu which is a main component of hemocyanin. It is the case for most of the trace elements analysed but not for Ni and Cr (Figure 1). This supposed a high exposure of nautilus to these metals.

Concerning Fe, N. macromphalus exhibit higher concentrations than in cephalopods from temperate waters that could also indicated contamination (Table 2). Nevertheless, Fe concentrations are lower than in bivalve and gastropod species from contaminated areas and were relatively low in hemolymph (Table 2).

Table 3 compares the trace element concentrations in the digestive gland of Nautilus macromphalus with those reported for other cephalopod species. The relative weight of the digestive gland in our samples is higher than in most of Coleoidae species. Indeed, the digestive gland consists of 20±5% of the fresh weight of the soft tissues of N. macromphalus while in Coleoidae, it represent 6 to 10% of the total body weight. Thus, when metals were highly concentrated in the digestive gland (e.g. Cd), metal concentrations in the whole individuals were proportionally higher than in other cephalopod species.

Ag, Cd, Co, Cr, Ni and V levels in the digestive gland of N. macromphalus are higher than those reported for Eledone cirrhosa and Sepia officinalis from the English Channel.

Nevertheless, several squid species, i.e. Loligo opalescens, Ommastrephes bartrami and Sthenoteuthis oualaniensis from the Pacific Ocean, exhibit higher concentrations of Ag in their digestive gland [START_REF] Martin | High copper concentrations in squid livers in association with elevated levels of silver, cadmium, and zinc[END_REF]. These authors explain these very high Ag concentrations by the release of the metal in the Californian waters. This suppose very high ability of cephalopods to concentrate Ag. For Cd, these squids and the octopuses from Kerguelen Islands (i.e. Graneledone sp. and Benthoctopus thielei) have far higher concentrations in the digestive gland than nautilus (Bustamante et al. 1998a). Although Cd concentrations in N. macromphalus were higher than in squids and cuttlefish from temperate waters (Table 3, Bustamante et al. 1998b), it doesn't indicate a contamination as they are in the same order of magnitude than in octopus species from these areas. Concentrations of Cu, Fe, Mn and Zn in the digestive gland of N. macromphalus, all essential elements, are in the same order of magnitude as those of other cephalopod species. Thus, these elements are properly regulated in the digestive gland of N. macromphalus as in the other cephalopod species. Results for As appear to be the first reported for the cephalopod digestive gland. For Al, concentrations can only be compared with those in the squid Nototodarus gouldi [START_REF] Smith | Concentrations of the elements Ag, Al, Ca, Cd, Cu, Fe, Mg, Pb and Zn, and the radionuclides 210 Pb and 210 Po in the digestive gland of the squid Nototodarus gouldi[END_REF] which are ranged from 1.5 to 20 µg/g (Table 3).

Globally, concentrations of the toxic elements in the digestive gland of Nautilus macromphalus are generally higher than those reported for cephalopods from the French coasts, i.e. Sepia officinalis, Eledone cirrhosa and Octopus vulgaris (Table 3).

In the Coleoidae species, branchial hearts have been reported to concentrate essential elements such as Fe and Cu [START_REF] Fox | Adenochrome a glandular pigment in the branchial hearts of the octopus[END_REF][START_REF] Ghiretti-Magaldi | Pathways of terminal respiration in marine invertebrates. I. The respiratory system in cephalopods[END_REF][START_REF] Nardi | Isolation and distribution of adenochrome(s) in Octopus vulgaris[END_REF][START_REF] Schipp | Distribution of copper and iron in some central organs of Sepia officinalis (Cephalopoda). A comparative study by flamless atomic absorption and electron microscopy[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF]. These organs also concentrate toxic and radioactive elements such as 241 Am, Co, Ni, 239-240 Pu and V [START_REF] Ueda | Amounts of trace elements in marine cephalopods[END_REF], Nakahara et al. 1979[START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Guary | High concentrations of transuranic and natural radioactive elements in the branchial hearts of the cephalopods Octopus vulgaris[END_REF][START_REF] Guary | Experimental studies on the biokinetics of plutonium and americium in the cephalopod Octopus vulgaris[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF]. Moreover, kidneys of cephalopods have been shown to store Cd, Cu, Fe, Mn, Ni and Pb [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF][START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF].

High concentrations of trace elements (Ag, Al, As, Cr, Fe, Ni and Mn) were found in organs concerned with excretion, i.e. the pericardial and renal appendages (Figure 1). These results, and those concerning the digestive gland, suggest that these metals could follow another way of detoxification in nautilus than in the few Coleoidae species studied. The digestive gland would assume mainly the detoxification of metals such as Cd, V or Zn. The hypothesis is strengthened by the trace element levels in the circulatory fluid. Indeed, as the food can be considered as the main source of metals in cephalopods since they are carnivorous, the hemolymph would be a major vector for metal distribution among the different tissues. Metal levels in the hemolymph of Nautilus macromphalus showed relatively low concentrations for most elements, except for Cu which is present in hemocyanin. This is particularly clear for Cd, V and Zn which exhibited the lowest concentrations in the hemolymph (Figure 1).

The other tissues store trace element in much lower amounts than the digestive gland, the renal and the pericardial appendages. Nevertheless, muscles which generally exhibit the lowest metal concentrations, showed the highest As concentrations. This could be due to high consumption of crustaceans which are particularly rich in As.

Investigations on trace element concentrations in the tissues have highlighted the key role of the digestive gland of nautilus in the metabolism of metals, as is also the case in Coleoidae. This organ is indeed the major site of concentration of Cd, Co, Fe, V and Zn and, compared to other tissues, it shows also high levels of Ag, As, Cr, Cu and Ni (Figure 1). The digestive gland contains the highest percentage of metals, with the exception of As and Mn (Figure 2). [START_REF] Miramand | Concentration and distribution of heavy metals in tissues of two cephalopods, Eledone cirrhosa and Sepia officinalis, from the French coast of the English Channel[END_REF] have classified some trace elements based on the ratio between the concentration in the digestive gland and in the muscle. Thus, in the digestive gland, three groups of elements can be evidenced in Nautilus macromphalus : poorly concentrated elements, i.e. Al, As, Cr, Cu and Mn (ratio <10), moderately concentrated elements, i.e. Fe, Ni and Zn (ratio >10 to <50) and highly concentrated elements, i.e. Ag, Cd, Co and V (ratio > 50). Similar ratios were found in Coleoidae (i.e. Sepia officinalis and Eledone cirrhosa) for Cr, Mn, Fe, Cd and Ag. On the contrary, V and Co are highly concentrated in the digestive gland of Nautilus whereas they are respectively poorly and moderately concentrated in Coleoidae. In the same way, Ni and Zn are moderately concentrated in the digestive gland of Nautilus but only poorly concentrated in S. officinalis and E. cirrhosa. High levels and storage of toxic metals in the digestive gland suggests efficient detoxification processes in this organ. Moreover, these high levels of toxic elements do not apparently disturb essential elements metabolism. Thus, it would be of particular interest to study detoxification processes in the digestive gland of nautilus.

Conclusion

Previous studies have demonstrated the ability of cephalopods to concentrate many trace elements. Data from nautilus species were particularly interesting on an evolutionary point of view. Indeed, as several elements are considered to be toxic for biota, marine animals would evolve to counteract their toxicity. It seems to be the case in cephalopods as they are able to growth and reproduce with very high metal concentrations. In their case, detoxification strategy involve storage mechanisms of these elements. This strategy appear to be efficient, and probably applied to minimise energetic cost, and is common among Nautilidae and Coleoidae cephalopods.

Enrichment of metals in New Caledonian waters appears to not be limited to coastal waters. Indeed, contamination by Co, Cr and Ni is supported 1) by high levels in whole animals which are in the same order of magnitude than in bivalves and gastropods from contaminated areas 2) by relatively high levels in excretory organs and in hemolymph that indicate a high exposure to these metals 3) by higher ratios between concentrations in the muscle and in the digestive gland that show an enrichment in this organ. Further studies are needed to evaluate the scale of the contamination by these metals and possible effects on the ecosystems. [START_REF] Miramand | High concentrations of some heavy metals in tissues of the Mediterranean octopus[END_REF]

  Figure 1. Trace elements concentrations (µg/g dry wt) in the tissues of Nautilus macromphalus from New Caledonia. Scale bars represent 1 standard deviation. DG: digestive gland; DT: digestive tract; GT: genital tract; Hly: hemolymph; Mus: muscle; Papp: pericardial appendages; Rapp: renal appendages; Rem: remainders; WI: whole individual.

Table 1 .

 1 Comparison of trace elements concentrations (µg/g dry wt) of dogfish liver DOLT-2 (NRCC), Orchard Leaves SRM 1571 (NBS) and fish-flesh homogenates MAA2 (IAEA) determined in the present study with certified values.

	Standard	Ag	Al	As	Cd	Co	Cr	Cu	Fe	Mn	Ni	V	Zn
	DOLT-2
	Present study	0.556±0.041	26.9±3.5	16.1±0.2	20.4±0.27	0.22±0.01	0.39±0.09	26.8±0.3	1072±17	6.26±0.11	0.24±0.04	-	86.3±1.2
	Certified values	0.608±0.032	25.2±2.4	16.1± 1.1	20.8±0.05	0.24±0.05	0.37±0.08	25.8±1.1	1103±47	6.88±0.56	0.20±0.02	-	85.8±2.5
	Orchard-Leaves
	Present study	-	-	-	0.13±0.02	(0.15)	(2.3)	12±1	290±15	85±6	1.5±0.1	(0.5)	25±1
	Certified values	-	-	-	0.11±0.02	(0.2)	(2.3)	12±1	300±20	91±4	1.3±0.2	(0.6)	25±3
	MA-A-2
	Present study	0.09±0.03	-	-	0.063±0.002	0.06±0.01	1.1±0.1	3.7±0.2	51±2	0.69±0.09	1.2±0.1	-	32±3
	Certified values	0.10±0.01	-	-	0.066±0.004	0.08±0.01	1.3±0.1	4.0±0.1	54±1	0.81±0.04	1.1±0.2	-	33±1

Table 3 .

 3 Trace element concentrations (µg/g dry wt) determined in the digestive gland of cephalopods from this study and from the literature.

	Species	Ag	Al	As	Cd	Co	Cr	Cu	Fe	Mn	Ni	V	Zn	Authors
	Nautilus macromphalus	4.45±2.68	32.1±16.3	166±26	45.1±13.2	7.8±3.6	4.4±1.1	106±46	666±231	10.1±1.7	16.3±7.8	8.8±2.0	672±208	Present study
	Sepia officinalis	6.15±1.75			12.67±0.35 3.27±0.6	1.1±0.1	315±3	244±28	3.3±0.1	1.3±0.4	5.0±1.3	571±47	Miramand & Bentley (1992)
	Loligo opalescens	251.1±12.6			85.0±51.6			5350±3210	111±73				247±131	Martin & Flegal (1975)
	L. opalescens	45.9±19.0			121.5±57.9			8370±3130	87±49				449±201	"
	Nototodarus gouldi				33±30			363±238					830±355	Finger & Smith (1987)
	N. gouldi	3.3±1.4	7.7±4.0		50±25			246±298	745±440	4.2±1.1			696±295	Smith et al. (1984)
	Ommastrephes bartrami	12.1±8.6			287±202			195±212	399±204				163±55	Martin & Flegal (1975)
	Stenoteuthis oualaniensis	24.1±10.9			782±255			1720±151	319±67				513±288	"
	Eledone cirrhosa	3.20±1.74			24.00±1.75 2.06±0.0	0.8±0.1	456 ±11	287±13	4.2±1.6	2.5±0.1	3.3±0.5	646±86	Miramand & Bentley (1992)
	Benthoctopus thielei				215			42					416	Bustamante et al. (1998a)
	Graneledone sp.				369			1092					102	Bustamante et al. (1998a)
	Octopus vulgaris							2550	1920					Ghiretti-Magaldi et al. (1958)
	O. vulgaris				50±10			2500±700	700±130	7.0±0.5		4.5±1.0	1450±400

Acknowledgements

We thank Pascale Joannot, director of the Nouméa Aquarium, the ORSTOM Center of Nouméa and the crew of the O.V. Dawa for providing nautilus specimen. This work was supported financially by the "Conseil Régional Poitou-Charentes" and Rhodia (Rhône-Poulenc).


R Bazinette

J Paillol

C O Laux

D Bessière

F Massines

Excitation of dielectric barrier discharge using sinusoidal and nanosecond waveform: application to silicon solar cell

Keywords: DBD, homogeneous, glow, radiofrequency, nanopulse, silicon nitride

Different diffuse dielectric barrier discharges (DBDs) are studied in the same configuration for different applied voltage waveforms: Glow, Townsend-like and radiofrequency (RF) DBD's for a sinusoidal excitation ranging from 50 kHz to 9 MHz and the nano-second repetitive pulsed DBD (NRP-DBD). The power of the RF and the NRP-DBD is 10 to 30 times larger than that of the low frequency DBD's but according to emission spectroscopy the electron energy and thus the energy transfer to the gas are quite different. The consequence on the chemical kinetic is illustrated by the difference in the conditions leading to a SiN:H having the same refractive index.

Introduction

Atmospheric pressure diffuse or homogeneous discharges are streamer free discharges. Low frequency diffuse dielectric barrier discharges (DBDs) have been abundantly studied [1]. Recently efforts have been made to increase the power of these discharges, in order to increase the efficiency of processes such as thin film growth. In the present work two solutions are compared: (i) the increase of the frequency excitation i.e., of the discharge repetition rate up to the point where the physics of the discharge change, adding wave riding to the ionization mechanisms, and (ii) the increase of the voltage up to a value largely higher than the breakdown voltage so rapidly that localized streamers are avoided thanks to a nanopulsed power supply. The aim of this work is to determine how the physics of homogeneous discharges change with the voltage waveform, and how this affects the induced chemistry through the analysis of thin film properties. The gas mixture is NH 3 diluted in Ar to have the benefit of NH 3 Penning ionization by Ar metastables to obtain a diffuse discharge when the excitation frequency and the voltage are low. SiH 4 is added to get SiNx:H thin films useful as antireflective and passivating thin films of p type silicon solar cells.

Experimantal set-up

Fig. 1 is a schematic view of the experimental reactor with two plasma areas defined by the two high-voltage electrodes metalized on an internal face of alumina square tubes. The ground electrode is on the other side of the 1 mm gap. It is also covered with an alumina plate. The metallization area is twice 14×50 mm 2 for high voltage (HV) electrodes and 50×50 mm 2 for the low-voltage one. A silicon wafer (CZ (100)) is used as a substrate to produce SiN coating at 400 °C. In this case, the substrate holder moves below the HV electrodes to cover the entire surface. The discharge cell is in an airtight reactor. The ambient air is removed with a primary vacuum, and the reactor is filled with argon up to a pressure of 760 Torr. The Ar flow rate is set to 3 l /min and the sum of NH 3 and SiH 4 is equal to 200 ppm. The waveforms used are: (i) a pulsed voltage generated by FID III (FPG20-30MkS50) generator to make pulses between 5 and 50 ns with a repetitive rate between 1 and 30 kHz. The high voltage electrode is always anode side. (ii) Sinusoidal voltage generated by waveform generator connected to an amplifier. Since the voltage at the output of the amplifier is not sufficient to ignite a discharge, the amplifier output is fed into a high-voltage transformer. Up to 60 kHz, an audio amplifier (Crest Audio CC4000) coupled to a high-voltage transformer with a large bandwidth is used. The maximum available voltage is 3 kV. The circuitry allows voltage and frequency to be independently adjusted. For higher frequency, we use the frequency resonance of the RLC circuit defined by the discharge cell capacitor and the inductance of a homemade transformer. The power injected is maximized and the power reflected is minimized. Different homemade transformers allow to work between 140 kHz and 9.6 MHz up to 1 kV. The amplifier is a PRANA GN500.

P-III-6-2

The discharge current is measured with a current probe (Lilco LTD 13W5000 400Hz-100MHz), and the voltage applied to the electrodes is measured with a high voltage probe (Tektronic P6015A 75 MHz). They are connected to a digital oscilloscope Tektronix DPO4104 (1 GHz). The average discharge power (P) is determined from the measurements of voltage (V(t)) and current (I(t)) over one cycle (T):

(1) Plasma photographs are taken with an intensified CCD camera PI-MAX II (Princeton Instruments) with a camera lens working from 200 to 1100 nm. Exposure time can be as short as 5 ns.

Optical emission spectroscopy (OES) with long exposure time (100 ms) is made with a Maya2000Pro spectrometer which has a 14 µm square pixel size, slit of 25µm and an optic fiber of 600 µm with a collimator optic.

Results

Discharge modes

Fig. 2 presents the maximum value of the diffuse DBD power and the breakdown voltage as a function of the frequency between 1 kHz to 7.5 MHz for nanosecond repetitive pulsed and sinusoidal waveforms. The FID generator works between 1 and 30 kHz. The pulse energy does not depend on the frequency and can reach 1.6 mJ for 50 ns. The power increases linearly with the repetitive frequency up to 25 W cm -3 but a filamentary discharge is observed above 17 W cm -3 . As the gas and the surface do not recover their initial properties however, the voltage increase is so high that the breakdown voltage is not affected. Its value is constant around 1.3 kV. Fast imaging shows that maximum plasma emission is close to the anode with weak emission close to the cathode (Fig. 3). This could be explained by emission from the cathode and the higher electron density close to the anode [START_REF] Takana | Proc. 21 st Int. Symp. on Plasma Chemistry -ISPC 2013[END_REF] associated to a rather uniform electric field. 3).

The power of this diffuse DBD is limited at around 1 W cm -3 . The breakdown voltage is almost constant at around 850 V because the lower excitation frequency (50 kHz) is high enough to enhance memory effects. Breakdown is controlled by ions drift (mainly NH 3 + ), metastable diffusion and UV photons propagation inducing secondary emission from the cathode.

Between 270 and 520 kHz, the ion time transit from anode to cathode is longer than the voltage polarity inversion and according to the light intensity from the cathode to the anode, the cathode fall do not form (Fig. 3). In this range, the discharge is considered as a Townsend like DBD (TDBD) associated to a higher power because electrons have a larger energy than in the GDBD as the electric field is large whatever the position in the gap. The maximum power is twice that of a GDBD. It is associated with a decrease of the breakdown voltage due to ion accumulation in the gap which offers the possibility to drift to the cathode before breakdown-generating electrons. When the frequency further increases, ion trapping in the gap is more efficient. It enhances the positive space charge induced by electron displacement. The higher the frequency, the lower the breakdown voltage (150 V at 8.4 MHz). Around 1 MHz, there is a transition domain between TDBD and RFDBD where the power is lower than for other frequencies because the discharge is increasingly weakened by the gap.

Above 3 MHz, the amplitude of electron motion becomes smaller than the gap value limiting electron losses at the anode. Consequently, the discharge begins to be controlled by electron losses. The power density increases by a factor of 30 until 36 W cm -3 . This is not the maximum value, as the voltage amplitude was limited by the power supply. In this range, the radiofrequency DBD (RFDBD) operates in alpha mode. The maximum ionization rate takes place in the plasma bulk at the sheath border due to sheath oscillation and in the bulk because of the rather high ion-electron recombination rate at atmospheric pressure.

As long as the discharge breakdown and development are controlled by the electron emission from the cathode, the power of the diffuse DBD is limited, but when the cathode does not largely participate in the ionization the power can be increased while still maintaining the diffuse mode.

The power increase induces a gas temperature increase (170 °C for 11 W cm -3 ) i.e., a gas density decrease but this is not at the origin of the discharge stabilization. The temperature increase is much smaller in the case of NRP-DBD at least partly because of the small duty cycle. This is of interest to treat thermosensitive surfaces such as polymers.

Emission spectroscopy

The irradiance of G-, RF-, NRP-DBD is shown in Fig. 4. The main emissions lines depend on the discharge mode. We observe theA 3 Π-X 3 Σ -NH molecular band system at 330 and 336 nm and Ar* emission corresponding to transitions between different levels of 3p 5 4p (≈ 13 eV) and the four levels of 3p 5 4s (from 11.55 to 11.83 eV). In GDBD, NH emission at 342 nm dominates while at higher frequency (RFDBD) it is NH at 336nm and for the NRP-DBD it is Ar emissions. Ar emission is related to high energy electrons so the emission spectrum is a signature of electrons of higher energy than in the case of sinusoidal waveform.

For a sinusoidal voltage, the increase of the excitation frequency tends to decrease argon emission which is in agreement with a lower applied voltage and thus a lower electric field applied on the electrons. Moreover in RFDBD, a broad emission continuum in the UV/Vis range appears whereas argon emission is very weak. This continuum has been attributed to low energy electrons slowed down by the interaction with Ar atoms (Bremsstrahlung). This is observed in low pressure RF discharges [START_REF] Park | [END_REF] and is in agreement with the effect of the parameters: voltage and frequency increase the gas ionization and thus the electron density, N e , whereas a temperature increase or pressure decrease reduces the density of Ar atoms, N gas . At 9.6 MHz, the continuum is rather well fitted according to Eq. 2 assuming a Maxwellian distribution function, a mean electron energy of 1 eV and an electron density is between 10 11 and 10 12 per cm 3 because of a dual distribution function due to cold electrons is the bulk and high energy electrons coming from oscillations of the sheaths.

(2) dσ(E)/dν is the Bremsstrahlung cross section calculated from e-Ar elastic cross-section modulated by photon emission.

The higher the continuum amplitude, the lower the argon emission and the higher the NH (336 nm) emission which is the one requiring the lowest electron energy. We conclude that, in RF-DBD, the electron energy is low compared to G-DBD and the density of electrons is higher. This is in agreement with the higher power and a lower breakdown voltage.

In conclusion, according to emission spectra and to the electrical behavior, the electron energy increases from RFDBD to GDBD and from GDBD to NRP-DBD.

Thin films

To illustrate the consequences of the DBD physics on chemical kinetics, the influence of the ratio [NH 3 ]/[SiH 4 ] on SiN:H refractive index has been studied (Fig. 5). One advantage of SiN:H, is that its refractive index is easily tuned. Its value is directly correlated to the Si/N ratio in the film which is straightforwardly related to the ratio R = [NH 3 ]/[SiH 4 ] [START_REF] Lecouvreur | Proc. 23 rd Eur[END_REF]. Whatever the DBD mode, thin films made at 400 °C are dense, without porosity and it is always possible to optimize R to have coating properties compatible with the application. The optimum value for antireflective coating of crystalline silicon solar cells is 2.09 [START_REF] Quoizola | Proc. 25 th Eur. Photovoltaic Solar Energy Conf[END_REF]. In GBDB, the optimum ratio is R = 1.75. In RFDBD, the NH 3 concentration must be increased and in NRP-DBD it has to be decreased.

This can be related to the difference in NH 3 + flow to the surface . NH 3 + is the dominant ion because Penning ionization is important, i.e., if the density of metastable integrated other time is high compared to that of electrons. And, moreover, charge transfer between Ar 2 + and NH 3 is effective. In RFDBD, the density of metastable should be low, moreover ions are confined in the gas bulk and their contribution to the surface chemistry is low compared to the case of GDBD where they actively participate in the discharge development. The NRP-DBD is unipolar and the substrate was on the

λ λ ν ν σ ν ν ν λ λ d dE E f d d d E d v h N N d Q h e gas         = ∫ ∞ ) ( ) ( P-III-6-2
cathode, so the flux of NH 3 + should be rather high. This is confirmed by the increase of the refractive index, for the same NH 3 /SiH 4 value, when the substrate is on the anode. Fig. 6 presents the growth rate as a function of NH 3 +SiH 4 obtained with the different DBD modes. For the GDBD, the growth rate saturates around 30 nm/min when the precursor concentration increases to 200 ppm. In that case, the growth rate is limited by the energetic species density, i.e., by the discharge power. The RFDBD which operated at higher power reaches 80 nm/min. The growth rate increases with precursor concentration showing that it is the precursor concentration that controls the growth rate. For the same power, similar growth rates are obtained with the NRP-DBD. If the power of that discharge is lower than that of GDBD, the growth rate is also lower. So we can conclude that more than the energy of electrons it is the power and the precursor concentration that control the growth rate. Growth rate as a function of precursor concentration for the different waveforms.

Conclusions

The influence of the excitation waveforms on the behavior of a homogeneous DBD was studied. For sinusoidal excitation, three different discharge regimes are identified [START_REF] Bazinette | [END_REF]. When the frequency increases, the discharge physics transitions from glow DBD to Townsend DBD and then to radiofrequency DBD. The transition from GDBD to TDBD is related to ion trapping in the gas gap. This is true until electrons begin to be trapped and ionization begins to be controlled by sheath oscillations. For GDBD in the 50 -520 kHz range, the power varies markedly with the discharge regime: a factor of 2 was observed between GDBD and TDBD. The transition from TDBD to RFDBD is associated with a drastic increase of the power, related to higher electron density.

The RFDBD reaches 35 W cm -3 for the highest frequency tested. This large power increase is associated with a decrease in the breakdown voltage by a factor of 3, reflecting the variation of the dominant ionization mechanisms and electric field distribution over the gas gap. The electron density decreases between GDBD and TDBD, whereas the electron energy increases because of the field uniformity. In contrast, in the RFDBD the electron density is expected to be markedly higher, and with a lower electron energy associated with a Bremsstrahlung emission.

The NRP-DBD allows reaching much power than low frequency sinusoidal DBD with higher average electron energy. These different voltage waveforms allow to change the electron energy distribution function. Whatever the waveform, it is possible to obtain a dense layer of silicon nitride useful as antireflective coating for silicon solar cell but the optimized conditions depend on the plasma excitation. RFDBD and NRP-DBD allow to reach high growth rate.

Fig. 1 .

 1 Fig. 1. Discharge cell.

Fig. 2 .

 2 Fig. 2. Breakdown voltage (full line) and maximum power (dash line) of diffuse DBD as a function of voltage frequency for NRP and sinusoidal waveforms.

Fig. 3 .

 3 Fig. 3. Typical light profile from the anode (on the left) to the cathode (on the right) when the light is maximum for the different DBD modes. For sinusoidal waveforms, the curves of Fig. 2 can be separated into four arrays corresponding to each discharge regime: glow, Townsend-like, transition and radiofrequency [Error! Bookmark not defined.]. At 50, 140 and 200 kHz the discharge operates in glow DBD (GDBD) mode characterized by a higher light emission close to the cathode (Fig.3).The power of this diffuse DBD is limited at around 1 W cm -3 . The breakdown voltage is almost constant at around 850 V because the lower excitation frequency (50 kHz) is high enough to enhance memory effects. Breakdown is controlled by ions drift (mainly NH 3

Fig. 4 .

 4 Fig. 4. Plasma irradiance: a) 50 kHz (GBDB), b) 9.6 MHz (RFDBD), and c) NRP-DBD (5 ns, 1 kHz).

Fig. 5 .

 5 Fig. 5. Rrefractive index as a function of [NH 3 ]/[SiH 4 ] for the different waveforms at 400 °C.

Fig. 6 .

 6 Fig.6.Growth rate as a function of precursor concentration for the different waveforms.

Acknowledgements

It is a pleasure to acknowledge the "BATIR" FUI Project (F11211007U) financed by OSEO.


Dr Paco Bustamante

P Bocher

Y Chérel

P Miramand

F Caurant

Distribution of trace elements in the tissues of benthic and pelagic fish from the Kerguelen Islands

Keywords: Antarctic, Heavy metals, Cd-enrichment, Myctophidae

New information on the concentrations of Cd, Cu, Hg and Zn in the liver, kidney and muscles of 8 marine benthic and pelagic sub-Antarctic fish species are presented to determine the importance of these metals in the marine systems of the Kerguelen Islands.

Compared to the reported metal concentrations in other Antarctic fish species, the present results are globally within the same range of concentrations, although Cd displayed a very high interspecific variability in liver and kidney. Indeed, the highest Cd concentrations in liver, ranging from 10.0 to 52.1 µg.g -1 dry wt but also the lowest Cd concentrations in muscles (< 0.030 µg.g -1 dry wt) have been displayed by the pelagic Myctophidae Gymnoscopelus piabilis. Metal concentrations differences might be related to diet and feeding habits of benthic and pelagic fish species. However, Cd and Hg concentrations in the edible muscle are lower than the French limit values (≤ 0.155 µg Cd g -1 dry wt and ≤ 1.51 µg Hg g -1 dry wt) for these toxic metals as well as for edible and non-commercially interesting fish species. Results for Cd in fish tissues are consistent with the hypothesis of Cd-enrichment in the polar food webs typically explained by essential elements depletion. In fact, Zn concentrations in fish from the Kerguelen Islands are comparable to those of other areas but low Cu concentrations 2 in fish livers, ranging from 0.9 to 24.7 µg.g -1 dry wt, might indicate low availability of this essential element in these sub-Antarctic waters.

INTRODUCTION

Heavy metal concentrations in the organisms from the Austral Ocean have been globally poorly investigated. However, all the studies report high toxic metal concentrations, especially for Cd and Hg, in comparison with those from northern temperate waters. Such metals enrichment have been reported for polar areas (AMAP 1998[START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF][START_REF] Sanchez-Hernandez | Trace element contamination in Antarctic ecosystems[END_REF] but in the Antarctic and sub-Antarctic food webs, it remains unclear even if the extreme environmental conditions (e.g. temperature, seasonal alternation, essential elements availability) might play a key role on the processes of uptake, storage and elimination of the metals by organisms. Moreover, local environmental factors such as volcanism or upwellings could increase metal concentrations in the marine environment.

Specifically, in the Kerguelen Islands environment, high concentrations of Cd have been found to occur in 2 species of benthic octopuses [START_REF] Bustamante | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF]. Generally, cephalopods are known to strongly accumulate Cd in their digestive gland but the concentrations reported for the Kerguelen octopuses were particularly high. Such high Cd levels have also been reported for some Antarctic zooplankton species as a likely result of very low essential elements availability to these organisms [START_REF] Rainbow | Copper, cadmium and zinc concentrations in oceanic amphipod and euphausiid crustaceans, as a source of heavy metals to pelagic seabirds[END_REF][START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF]. The Cd enrichment in the zooplankton cannot be fully considered as the direct result of anthropogenic contamination owing to the very low levels reported for other zooplankton species living in the same waters (see the data compiled by [START_REF] Sanchez-Hernandez | Trace element contamination in Antarctic ecosystems[END_REF].

Several studies report metal concentrations in the tissues of 13 fish species of various Antarctic areas [START_REF] Honda | Organ and tissue distribution of heavy metals, and their growth-related changes in Antarctic fish, Pagothenia borchgrevinki[END_REF][START_REF] Lenihan | Intense and localised benthic marine pollution around McMurdo Station, Antarctica[END_REF][START_REF] Capelli | Mercury and selenium in Adamussium colbecki and Pagothenia bernacchii from the Ross Sea (Antarctica) collected during Italian expeditions 1988-89[END_REF][START_REF] Szefer | Mercury and major essential elements in seals, penguins, and other representative fauna of the Antarctic[END_REF], Minganti et al. 1994[START_REF] Cshpf | Plomb, cadmium et mercure dans l'alimentation : évaluation et gestion du risque[END_REF][START_REF] Bargagli | Elevated cadmium accumulation in marine organisms from Terra Nova Bay (Antarctica)[END_REF], 1998ab, de Moreno et al 1997[START_REF] Marquez | Metal (Fe, Zn, Mn and Cu) levels in the Antarctic fish Notothenia coriiceps[END_REF]). However, no data for the fish from the Kerguelen Islands are available to date. This sub-Antarctic Archipelago is of a great ecological interest since millions of seabirds and numerous marine mammals breed there every year [START_REF] Guinet | Consumption of marine resources by seabirds and seals in Crozet and Kerguelen waters: changes in relation to consumer biomass 1962-85[END_REF]. Furthermore, as in many sub-Antarctic areas, commercial fisheries developped at the end of the 60s, targetting fish of the families Nototheniidae and Channichthyidae [START_REF] Duhamel | La situation de la pêche aux Iles Kerguelen en 1981[END_REF]. Thus, baseline information on heavy metal concentrations in the tissue of fish are needed in order to evaluate the fish quality for human consumption as well as for piscivorous predators.

For these reasons, selected heavy metals have been analysed in the liver, kidney and muscles of several fish species, including benthic (neritic) and pelagic (oceanic) ones. Thus, 35 specimens representing 8 different species from the Kerguelen Island waters have been individually analysed for each Cd, Cu, Hg and Zn contents. Metal concentrations and tissue distribution, are compared between species from different Antarctic areas, and influence of the diet of benthic and pelagic species on the accumulation are discussed.

MATERIALS AND METHODS

Sampling and sample preparation

Pelagic fish were collected on cruises of the RV "La Curieuse" during austral summer. (Table 1).

The fish were separated by species and stored on board at -20°C in plastic bags prior to analysis. Subsequently, the length, weight and sex of fish were determined. Moreover, otoliths of Myctophidae species were taken out to ensure identification of the species. Then, specimens were dissected and liver, kidney and muscle tissues were treated separately. The remainders of each dissected individual were also analysed individually in view of determining the percentage distribution of the metals. Characteristics of the samples (i.e. family, species, length, weight and sex) are shown in Table 1.

Analytical procedure

Separated tissue samples were dried to a constant weight for several days at 60°C and then homogenised. Whenever possible, 2 aliquots of approx. 300 mg of each homogenised dry sample were digested with 5 ml of 65% HNO 3 and 0.3 ml of 70% HClO 4 at 80°C for 24 h.

The residues obtained after evaporation of the acids were dissolved in 0.3 N nitric acid. Cd, Cu and Zn were assayed using flame and graphite furnace atomic absorption spectrophotometer Varian 250 Plus with deuterium background correction.

For Hg, aliquots ranging from 10 to 50 mg of dried material have been analysed directly in a Advanced Mercury Analyser spectrophotometer, Altec AMA 254. Hg determination involved evaporation of Hg by progressive heating until 800°C under oxygen atmosphere for 3 min and subsequent amalgamation on a Au-net. Afterwards, the net was heated to liberate the collected mercury and subsequently measured by UV atomic absorption spectrophotometry. However, Hg analysis were not performed in the liver and kidney of the pelagic fish nor for Lepidonotothen squamifrons.

Quality assurance was assessed using dogfish liver DOLT-2 (NRCC) and dogfish muscle DORM-2 (NRCC) as reference materials. These standards were treated and analysed under the same conditions as the fish samples, and recoveries of the metals ranged from 92 to 105%.

Detection limits, calculated as 3 SD of the mean of eight blanks, were 0.004 for Cd, 0.5 for Cu, 3 for Zn and 0.005 for Hg (µg.g -1 dry wt). All metal concentrations in fish tissues are also reported in µg.g -1 dry wt. Water contents allowing recalculations of the metal concentrations from dry wt to wet wt are given in Table 1.

Statistical procedures

Statistically analysis of results used commercially available packages. As concentration of some elements did not follow a normal distribution, non parametric analysis using Kruskall-Wallis test for multiple comparisons and Mann-Whitney U-test provided by Minitab 13.1 for Windows. A statistically significant difference was considered to exist whenever the probability is lower than P ≤ 0.05.

RESULTS

Results on heavy metal concentrations in the tissues of fish from the Kerguelen Islands are compiled in Tables 2, 3 and 4 for liver, kidney and muscle, respectively.

In liver, metal concentrations exhibited a large variability among species. For Hg, concentrations differ in 2 orders of magnitude, i.e. between 0.042 and 1.51 µg.g -1 dry wt. The Myctophidae Gymnoscopelus piabilis and the Notothenidae Lepidonotothen squamifrons showed significantly higher Cd and Zn concentrations in liver. For Cu and Zn, Paranotothenia magellanica also exhibited the highest concentrations. Among the 3 organs considered, liver displays the highest Cd and Cu concentrations. Compared to muscle, hepatic Cd concentrations are 2 to 4 orders of magnitude higher whereas Cu concentrations are only 2 to 10 times higher (Table 2).

Despite the heterogeneity of metal concentrations in liver, no clear segregation between pelagic and benthic fish could be done. This was also the case for kidney. Compared to muscle, kidney shows elevated concentrations of Cd and Cu. Among the 3 tissues, kidney had the highest Zn but the lowest Hg concentrations. As in liver, the Myctophidae Gymnoscopelus piabilis and the Notothenidae Lepidonotothen squamifrons exhibited significantly higher renal Cd concentrations compared to the other fish species. Renal Cu concentrations were significantly elevated only for G. piabilis (Table 3).

Compared to the results for liver and kidney, Cd, Cu and Zn concentrations appeared to be low in fish muscles although the important variability among species is remarkable (Table 4).

Muscular Cd and Zn concentrations were generally lower in pelagic fish than in benthic ones.

Similarly, mean Hg concentrations varied on 3 orders of magnitude in the muscle (from 0.044 to 1.19 µg.g -1 dry wt) in both benthic and pelagic fish. Nevertheless, the Channichthydae Channichthys rhinoceratus exhibited clearly the highest Hg concentrations (Table 4).

Complete dissection of fish allowed to calculate the distribution of metals between liver, kidney (representing less than 5% and than 1% of the whole body mass) and the remaining tissue including muscle. The percentage of each metal contained in these compartments is shown in Table 5.

Cd and Cu concentrations displayed the same distribution pattern in the tissues of benthic and pelagic fish: liver > kidney > muscle. Despite several orders of magnitude between Cd concentrations in muscle and liver, the latter generally contained less than 50% of the total body burden of Cd. Thus, most of this Cd was muscular except for the Channichthydae Channichthys rhinoceratus (87% of Cd located in liver; Table 5). Owing to the small differences between muscle and liver Cu concentrations and to the respective proportions of these tissues in the fresh weight of fish, less than 20% of the metal was stored in the hepatic compartment (Table 5).

Zn concentrations showed the following sequence : kidney > liver > muscle but the two first organs contained less than 15% of the total body burden of Zn.

Although no clear differences of concentrations between the three tissues appears, the muscular parts of fish contained up to 90% of the total body burden of Hg due to its significance (Table 5).

DISCUSSION

Levels of trace metals

Despite the past and present human fishing pressure on some of the Austral Ocean areas and their major role in the ecosystems [START_REF] Guinet | Consumption of marine resources by seabirds and seals in Crozet and Kerguelen waters: changes in relation to consumer biomass 1962-85[END_REF][START_REF] Cherel | Food and feeding ecology of the neritic-slope forager black-browed albatross and its relationships with commercial fisheries in Kerguelen waters[END_REF], data on metal levels in the fish still lacks. Such assessment is particularly true for the sub-antarctic zone while Antarctic Ocean is more documented. Thus, this study represent the first investigation concerning the distribution of trace elements in the tissue of 8 fish species from the sub-Antarctic Kerguelen Islands. A recent review of trace element contamination around Antarctica [START_REF] Sanchez-Hernandez | Trace element contamination in Antarctic ecosystems[END_REF] allows to compare the present results with those of several fish species from the Antarctic Ocean.

Overall, the present results about metal concentrations in the tissues of benthic and pelagic fish from the Kerguelen Islands waters fall within the range for the other fish species from the Antarctic Ocean (data compiled in Table 678). Reported data for fish liver (Table 6) range from 0.86 to 21.6 µg Cd g -1 , from 3.3 to 92 µg Cu g -1 , from 0.02 to 0.82 µg Hg g -1 and from 87.7 to 106 µg Zn g -1 (values are expressed in dry wt after conversion from wet wt for some data using a 4.0 correction factor). Generally, the heavy metal levels in fish liver from the Kerguelen Islands tended to be slightly higher than those reported in the current litterature (Table 6), except for Hg which is closely in the same concentration range.

Concerning kidney, data for Antarctic fish are rarely reported (Table 7). They range from 0.17 to 10.1 µg Cd g -1 , and from 0.09 to 2.60 µg Hg g -1 (dry wt) while the only values reported for Cu and Zn concern two benthic species, i.e. Notothenia coriiceps [START_REF] Marquez | Metal (Fe, Zn, Mn and Cu) levels in the Antarctic fish Notothenia coriiceps[END_REF] and

Trematomus bernacchii (Bargagli et al. 1998b). Despite such few data, it also appears that several fish species from the Kerguelen Islands show higher heavy metal concentrations in the kidney compared to other Antarctic fish species. More fish muscle values from the Antarctic Ocean are available (Table 7). Thus, our results are closely within the same range of the reported data: from 0.01 to 1.0 µg Cd g -1 , from 0.2 to 2.5 µg Cu g -1 , from 0.01 to 1.79 µg Hg g -1 , and from 2.0 to 125 µg Zn g -1 (values are expressed in dry wt after conversion from wet wt for some data using a 5.0 correction factor). Considering the French limit value for human consumption of toxic metals, i.e. 0.5 µg.g -1 for Cd and 2.5 µg.g -1 for Hg [START_REF] Cshpf | Plomb, cadmium et mercure dans l'alimentation : évaluation et gestion du risque[END_REF], metal concentrations in fish muscles appear to be low, as well for the commercially fished species like Channichthys rhinoceratus, Champsocephalus gunnari and Notothenia rossii, than for the other species presented here. Heavy metals investigations in fish mainly consider concentrations in muscle and/or in liver, in detriment of gills or kidney as well. Furthermore, little information concerning metal distribution has been reported especially for the Antarctic fish. For this reason, comparison with fish outside the Austral Ocean was necessary and revealed that metal distribution in the tissues of Austral Ocean fish is very similar to that of fish from the temperate waters [START_REF] Miramand | Reproductive cycle and heavy metals in the organs of red mullet, Mullus barbatus (L.), from the northwestern Mediterranean[END_REF].

Interferences on routes of metal uptake

The very low Cd concentrations in the muscle of most of the fish species investigated (including Myctophidae) suppose effective processes of Cd sequestration in liver and kidney and demonstrate that dietary uptake of Cd is likely to be the most important route of assumption in Kerguelen Islands fish. Enrichment of cadmium in liver and kidney with respect to muscles is particularly evident for zooplankton-eating fish, such as Myctophidae [START_REF] Hulley | The Fishes of the Southern Ocean[END_REF]). Thus, these species from the Kerguelen Islands displayed the highest Cd concentrations in liver and kidney, i.e. from 10.0 to 52.1 µg.g -1 dwt in Gymnoscopelus piabilis compared to 0.86 to 21.6 µg.g -1 dwt in the liver of the species from other areas (Table 6). The diet of Gymnoscopelus consists of crustaceans plankton including euphausiids and euphausiid larvae, hyperiids, and mysids [START_REF] Hulley | The Fishes of the Southern Ocean[END_REF]). Some of these planktonic species from the Austral Ocean (e.g. euphausiids) exhibit relatively high levels of Cd for crustaceans, with values ranging from 0.15 to 3.4 µg.g -1 dwt in the krill Euphausia superba [START_REF] Rainbow | Copper, cadmium and zinc concentrations in oceanic amphipod and euphausiid crustaceans, as a source of heavy metals to pelagic seabirds[END_REF][START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF]). However, some other planktonic species could reach extremely high levels of Cd like the hyperiid amphipod Themisto gaudichaudii, which concentrate the metal from 8.0 to 118 µg.g -1 dwt [START_REF] Hennig | Metal concentrations in Antarctic zooplankton species[END_REF][START_REF] Rainbow | Copper, cadmium and zinc concentrations in oceanic amphipod and euphausiid crustaceans, as a source of heavy metals to pelagic seabirds[END_REF]). Similarly to those reported results, T. gaudichaudii show very high Cd concentrations ranging from 21.2 to 81.7 µg.g -1 dwt in the Kerguelen Island waters (Bustamante, unpublished data). Thus, the high Cd levels recorded in the kidney and liver of G. piabilis would be a direct result of a Cd-rich diet. On the contrary, fish species having the lowest Cd concentrations in their tissues, e.g. Notothenia rossii or Paranotothenia magellanica, are benthic feeders, and include algae, polychaetes, crustaceans, gastropods and fish in their diet [START_REF] Gon | Fishes of the Southern Ocean[END_REF]. However, the Trematomus fish which are typical benthic feeders of the Antarctic Ocean, exhibit relatively high values reaching 21.6 and 10.1 µg Cd g -1 in their liver and kidney, respectively (Tables 6 &7). This difference can be due to a substantial heterogeneity of age. Thus, these benthic species, as N. rossii and P. magellanica used in our study were juveniles (less than 4 years old) while Trematomus were supposed to be adults after length measurements as suggested by [START_REF] Gon | Fishes of the Southern Ocean[END_REF].

Interference on the origin of the metals

Elevated Cd concentrations in the biota from the Austral Ocean were surprinsingly high for this area remote from human activities. However, except in some localised spots, anthropogenic Cd contamination does not occur in the Austral Ocean, suggesting that the high concentrations found in the examined marine species are essentially due to natural conditions. Thus, the elevated concentrations of this toxic element in both benthic and pelagic invertebrates seem to correpond to Cd-abnormalities in polar waters as inferred for crustaceans and molluscs [START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF][START_REF] Bargagli | Elevated cadmium accumulation in marine organisms from Terra Nova Bay (Antarctica)[END_REF][START_REF] De Moreno | Heavy metals in Antarctic organisms[END_REF][START_REF] Ritterhoff | Trace metals in field samples of zooplankton from the Fram Strait and the Greenland sea[END_REF][START_REF] Bustamante | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF][START_REF] Sanchez-Hernandez | Trace element contamination in Antarctic ecosystems[END_REF]. Indeed, the low Cd concentrations in fish muscle indicate low artificial contamination but the elevated Cd concentrations in both liver and kidney highlight exposure of the fish to the metal. Thus, the high Cd levels in liver and kidney of several Antarctic fish species is a matter of concern.

As it occurs for the Arctic fish [START_REF] Macdonald | Cadmium in marine invertebrates and arctic cod in the Canadian Arctic. Distribution and ecological implications[END_REF][START_REF] Hellou | Heavy metals and other elements in three tissues of cod Gadus morhua from the Northwest Atlantic[END_REF][START_REF] Zauke | Heavy metals in fish from the Barents Sea (summer 1994)[END_REF], bioaccumulation of Cd in the fish from the Austral Ocean is difficult to explain since the Cd concentrations in the sea water from polar regions are low. Nevertheless, the occurrence of upwelling of deep waters in the Kerguelen region [START_REF] Plancke | Phytoplankton biomass and productivity in the Subtropical Convergence area and shelves of the western Indian subantarctic Islands. In : Llano GA (ed) Adaptations within Antarctic ecosystems[END_REF] should bring an enrichment of Cd in the surface waters. The increase of Cd bioavailability in the Kerguelen Island waters by upwelled deep waters might be at the origin of an increase of Cd concentrations in the marine biota on a local scale, as discussed for benthic octopus from this area [START_REF] Bustamante | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF]). On the other hand, [START_REF] Bucciarelli | Iron and manganese in the wake of the Kerguelen Islands (Southern Ocean)[END_REF] found an iron enrichment of the coastal waters of the Kerguelen Islands and explained it in terms of direct inputs of terrestrial material, as a consequence of riverine discharge, soil leaching by rainwater and aeolian input by strong winds, but also by inputs from the sediments due to effluxes from the sediment at the water interface and resuspension from the sediment. However, our results for Cd but also for Cu, Hg and Zn in the tissue of Kerguelen Islands fish do not show an enrichment following the same processes as Fe. Indeed, Cd concentrations in both liver and kidney of fish caught in close coastal waters (i.e. Notothenia rossii and Paranotothenia magellanica) are the lowest among the 8 studied species. Similarly to fish, metal concentrations in crustaceans also distinguish the coastal samples from the continentalshelf ones. For example, the hyperiid amphipod Themisto gaudichaudii display Cd concentrations from 21.2 to 27.3 µg.g -1 dwt in the coastal waters (Morbihan Gulf) while 68.2 to 80.7 µg Cd.g -1 dwt were found in the individuals from the shelf (unpublished data). These new findings point out the necessity to investigate carefully the metal concentrations, particularly for Cd, in the waters surrounding Kerguelen Islands, to determine the sources to biota in this area.

Cd enrichment in marine animals from the Austral Ocean was also proposed to occur for organisms suffering essential elements deficiency, which have evolved very efficient mechanisms of elemental uptake [START_REF] Petri | Trace metal in the crustaceans in the Antarctic Ocean[END_REF]). However, these mechanisms are probably non specific to the essential metal and so, Cd might be absorbed by the same pathways than elements like Cu or Zn. Zn deficiency is not evident in the Kerguelen Island waters as its concentrations in fish tissues from this area are of the same order than concentrations reported for fish from various areas [START_REF] Hellou | Heavy metals and other elements in three tissues of cod Gadus morhua from the Northwest Atlantic[END_REF][START_REF] Roméo | Heavy metal distribution in different fish species from the Mauritanian coast[END_REF][START_REF] Zauke | Heavy metals in fish from the Barents Sea (summer 1994)[END_REF]). Similarly to fish, Zn concentrations found in the digestive gland of octopus from the Kerguelen Islands are of the same order compared to other cephalopod species [START_REF] Bustamante | Cadmium, copper and zinc in octopuses from Kerguelen Islands, Southern Indian Ocean[END_REF]. As for Cd, Zn could also be carried by upwelled waters, becoming more available in this area than in open Ocean where upwellings do not occur.

Contrarily to Zn, low concentrations of Cu in the liver of fish from the Kerguelen Islands might be due to a low availability of this element. Indeed, Cu concentrations appear to be under close physiological regulation in most species [START_REF] Thompson | Metal levels in marine invertebrates[END_REF]). In the same way, low Cu concentrations were also found in octopus from Kerguelen Islands compared to data reported for other cephalopods.

CONCLUSION

The present work provides new information on the distribution of heavy metals in fish from the Kerguelen Islands. Considering the muscle, concentrations of Cd and Hg in both benthic and pelagic species are below the values fixed as a limit by the CSHPF (1995). On the other hand, liver and kidney display very high Cd consequently to high exposure through diet, but very low hepatic Cu concentrations. This is in accordance with the hypothesis of Cd enrichment related to Cu deficiency. In this context, studies on the detoxification and storage processes of Cd in the liver and kidney of Kerguelen Islands fish should be thoroughly carried out.

  Myctophidae and Gempylidae were caught in the eastern part of the peri-insular shelf in February 1998, using a IYGPT trawl (International Young Gadoid Pelagic Trawl, opening 12 X 7 m) with 10 mm mesh size in the cone. Benthic fish were captured either by net fishing overnight in the Morbihan Bay (Notothenia rossii and Paranotothenia magellanica) or by commercial trawling on the Kerguelen shelf (Channichthys rhinoceratus, Chamsocephalus gunnari and Lepidonotothen squamifrons)

Table 1 .

 1 Characteristics of the fish samples, together with the water content in the tissues and organs allowing conversion of dry wt to wet wt metal concentrations.

	Family	Localisation	Sample	Length	Fresh	Sex		Water content
			size		weight			(%)
	Species			(mm)	(g)		Liver	Kidney Muscle
	Pelagic fish
	Gempylidae
	Paradiplospinus gracilis	Oceanic zone	1	370	67	♂	55	78	71
	Myctophidae
	Gymnoscopelus nicholsi	Oceanic zone	4	144 ± 15	31 ± 8	4 ♀	42 ± 6 45 ± 11 62 ± 2
	Gymnoscopelus piabilis	Oceanic zone	5	151 ± 11	37 ± 6	5 ♀	56 ± 8 48 ± 15 76 ± 3
	Benthic fish
	Channichthyidae
	Champsocephalus gunnari	Kerguelen shelf	5	314 ± 13 202 ± 34 1 ♂, 4 ♀ 73 ± 2	73 ± 5	76 ± 2
	Channichthys rhinoceratus	Kerguelen shelf	5	344 ± 52 435 ± 150 2 ♂, 3 ♀ 75 ± 4	83 ± 2	81 ± 3
	Nototheniidae
	Paranotothenia magellanica	Morbihan bay	5	157 ± 8	92 ± 17 2 ♂, 3 ♀ 77 ± 2	81 ± 2	78 ± 0
	Notothenia rossii	Morbihan bay	5	237 ± 41 300 ± 135 2 ♂, 3 ♀ 73 ± 4	81 ± 1	80 ± 1
	Lepidonotothen squamifrons Kerguelen shelf	5	284 ± 29 279 ± 90 3 ♂, 2 ♀ 71 ± 8	78 ± 4	76 ± 2

Table 2 .

 2 Mean ± SD and range of the metal concentrations (µg.g -1 dry wt.) in the liver of benthic and pelagic fish from the Kerguelen Island waters.

	Metal	Species	N	Mean ± SD	Range	Group
						1 2 3
		Paradiplospinus gracilis	1	-	0.94
	Pelagic	Gymnoscopelus nicholsi	4	4.23 ± 0.34	3.90-4.66	|
		Gymnoscopelus piabilis	5	28.5 ± 16.9	10.0-52.1	|
	Cd	Notothenia rossii	5	2.82 ± 1.60	0.82-4.26	|
		Paranotothenia magellanica	5	4.15 ± 2.19	2.03-7.35	|
	Benthic	Channichthys rhinoceratus	5	4.37 ± 2.00	2.73-6.78	|
		Champsocephalus gunnari	5	5.52 ± 4.48	1.04-10.6	| |
		Lepidonotothen squamifrons	5	10.8 ± 4.59	5.42-15.4	| |
		Paradiplospinus gracilis	1	-	14.1
	Pelagic	Gymnoscopelus nicholsi	4	5.8 ± 1.3	5.1-7.5	|
		Gymnoscopelus piabilis	5	10.2 ± 3.5	6.3-14.8	|
	Cu	Notothenia rossii	5	4.8 ± 1.6	3.2-7.3	|
		Paranotothenia magellanica	5	16.8 ± 4.5	13.7-24.7	|
	Benthic	Channichthys rhinoceratus	5	4.0 ± 1.2	3.2-6.1	|
		Champsocephalus gunnari	5	3.1 ± 2.5	0.9-7.1	|
		Lepidonotothen squamifrons	5	3.8 ± 1.1	2.5-5.6	|
		Paradiplospinus gracilis	1	NA	-
	Pelagic	Gymnoscopelus nicholsi	4	NA	-
		Gymnoscopelus piabilis	5	NA	-
	Hg	Notothenia rossii	5	0.513 ± 0.208 0.219-0.743	|
		Paranotothenia magellanica	5	0.370 ± 0.114 0.245-0.524	|
	Benthic	Channichthys rhinoceratus	5	0.686 ± 0.510	0.160-1.51	|
		Champsocephalus gunnari	5	0.047 ± 0.006 0.042-0.055 |
		Lepidonotothen squamifrons	5	0.078 ± 0.032 0.052-0.133 |
		Paradiplospinus gracilis	1	-	112
	Pelagic	Gymnoscopelus nicholsi	4	92.9 ± 18.2	70.8-113	| |
		Gymnoscopelus piabilis	5	142 ± 30.6	108-184	|
	Zn	Notothenia rossii	5	99.0 ± 18.9	75.6-119	|
		Paranotothenia magellanica	5	143 ± 16.2	123-165	|
	Benthic	Channichthys rhinoceratus	5	70.4 ± 13.8	61.4-93.0	|
		Champsocephalus gunnari	5	62.7 ± 32.0	28.8-100	|
		Lepidonotothen squamifrons	5	110 ± 32.3	63.5-149	| |
	N, independent samples. NA : not analysed. Bars (|) indicates groups identified by non parametric tests.

Table 3 .

 3 Mean ± SD and range of the metal concentrations (µg.g -1 dry wt.) in the kidney of benthic and pelagic fish from the Kerguelen Island waters.

	Metal	Species	N	Mean ± SD	Range	Group
						1 2 3 4 5
		Paradiplospinus gracilis	1	-	1.69
	Pelagic	Gymnoscopelus nicholsi	4	2.66 ± 1.03	2.07-4.19	|
		Gymnoscopelus piabilis	5	15.7 ± 8.10	5.92-28.5	|
	Cd	Notothenia rossii	5	0.13 ± 0.09	0.04-0.27	|
		Paranotothenia magellanica	5	0.28 ± 0.20	0.10-0.59	| |
	Benthic	Channichthys rhinoceratus	5	0.31 ± 0.10	0.20-0.43	|
		Champsocephalus gunnari	4	2.57 ± 0.78	1.58-3.31	|
		Lepidonotothen squamifrons	5	4.28 ± 1.42	1.99-5.89	|
		Paradiplospinus gracilis	1	-	7.2
	Pelagic	Gymnoscopelus nicholsi	4	4.8 ± 1.3	3.5-6.4	| |
		Gymnoscopelus piabilis	5	10.7 ± 5.4	6.1-19.5	|
	Cu	Notothenia rossii	5	3.7 ± 0.7	3.0-4.9	|
		Paranotothenia magellanica	5	4.4 ± 0.8	3.7-5.7	|
	Benthic	Channichthys rhinoceratus	5	3.2 ± 0.7	2.1-3.8	|
		Champsocephalus gunnari	4	2.2 ± 0.7	1.2-2.7	|
		Lepidonotothen squamifrons	5	4.0 ± 1.0	2.9-4.8	|
		Paradiplospinus gracilis	1	NA	-
	Pelagic	Gymnoscopelus nicholsi	4	NA	-
		Gymnoscopelus piabilis	5	NA	-
	Hg	Lepidonotothen squamifrons	5	NA	-
		Notothenia rossii	5	0.470 ± 0.252 0.134-0.721	|
	Benthic	Paranotothenia magellanica	5	0.102 ± 0.010 0.092-0.119	|
		Channichthys rhinoceratus	5	0.533 ± 0.327	0.172-1.05	|
		Champsocephalus gunnari	4	0.030 ± 0.006 0.024-0.038 |
		Paradiplospinus gracilis	1	-	110
	Pelagic	Gymnoscopelus nicholsi	4	86.2 ± 6.8	77.4-93.5	| |
		Gymnoscopelus piabilis	5	113 ± 23.1	86.1-146	| |
	Zn	Notothenia rossii	5	84.1 ± 8.8	70.5-92.3	|
		Paranotothenia magellanica	5	138 ± 15.9	122-161	|
	Benthic	Channichthys rhinoceratus	5	143 ± 38.3	102-202	|
		Champsocephalus gunnari	4	85.7 ± 15.2	69.4-106	| |
		Lepidonotothen squamifrons	5	181 ± 66.4	113-264	|
	N, independent samples. NA : not analysed. Bars (|) indicates groups identified by non parametric tests.

Table 4 .

 4 Mean ± SD and range of the metal concentrations (µg.g -1 dry wt.) in the muscle of benthic and pelagic fish from the Kerguelen Island waters.

	Metal	Species	Mean ± SD	Range	N	Group
						1 2 3 4
		Paradiplospinus gracilis	-	0.006	1
	Pelagic	Gymnoscopelus nicholsi	0.010 ± 0.008 0.004-0.021	4	|
		Gymnoscopelus piabilis	0.016 ± 0.009 0.006-0.029	5	|
	Cd	Notothenia rossii	0.049 ± 0.011 0.034-0.064	5	|
		Paranotothenia magellanica	0.035 ± 0.021 0.014-0.063	5	| |
	Benthic	Channichthys rhinoceratus	0.051 ± 0.025 0.024-0.090	5	|
		Champsocephalus gunnari	0.086 ± 0.052 0.034-0.155	5	|
		Lepidonotothen squamifrons	0.053 ± 0.026 0.026-0.085	5	|
		Paradiplospinus gracilis	-	0.4	1
	Pelagic	Gymnoscopelus nicholsi	2.5 ± 0.7	1.9-3.4	4	|
		Gymnoscopelus piabilis	1.2 ± 0.4	0.8-1.7	5	|
	Cu	Notothenia rossii	0.7 ± 0.1	0.6-0.9	5	| |
		Paranotothenia magellanica	0.9 ± 0.0	0.9-1.0	5	|
	Benthic	Channichthys rhinoceratus	0.5 ± 0.2	0.2-0.8	5	|
		Champsocephalus gunnari	0.6 ± 0.1	0.4-0.8	5	|
		Lepidonotothen squamifrons	1.0 ± 0.3	0.7-1.4	5	|
	Pelagic	Paradiplospinus gracilis	-	0.251	1
		Gymnoscopelus nicholsi	0.205 ± 0.126 0.157-0.297	4	| |
	Hg	Gymnoscopelus piabilis	0.310 ± 0.126 0.177-0.475	5	|
		Notothenia rossii	0.255 ± 0.059 0.192-0.344	5	|
	Benthic	Paranotothenia magellanica	0.140 ± 0.037 0.097-0.191	5	|
		Channichthys rhinoceratus	1.19 ± 0.367	0.606-1.51	5	|
		Champsocephalus gunnari	0.044 ± 0.012 0.034-0.065	5	|
		Lepidonotothen squamifrons	0.126 ± 0.033 0.094-0.180	5	|
		Paradiplospinus gracilis	-	10.0	1
	Pelagic	Gymnoscopelus nicholsi	9.2 ± 4.0	6.6-15.0	4	|
		Gymnoscopelus piabilis	9.9 ± 1.2	8.4-11.3	5	|
	Zn	Notothenia rossii	19.6 ± 1.1	18.6-21.0	5	|
		Paranotothenia magellanica	22.0 ± 1.6	20.2-23.7	5	|
	Benthic	Channichthys rhinoceratus	33.2 ± 7.4	23.6-40.7	5	|
		Champsocephalus gunnari	29.1 ± 1.9	27.1-31.1	5	|
		Lepidonotothen squamifrons	19.9 ± 2.6	16.7-22.9	5	| |
	N, independent samples. Bars (|) indicates groups identified by non parametric tests.

Table 5 .

 5 Distribution of the metals in benthic and pelagic fish from the Kerguelen Islands waters. Mean % ± SD referred to the fresh weight of organs and tissues.

	Metal Habitat	Species	N	Liver	Kidney	Muscular parts
						and remainders
		Paradiplospinus gracilis	1	11.7	1.5	86.8
	Pelagic	Gymnoscopelus nicholsi	4	36.1 ± 13.1	4.0 ± 1.7	59.9 ± 13.9
		Gymnoscopelus piabilis	5	47.8 ± 15.4	5.7 ± 2.5	46.5 ± 17.6
	Cd	Champsocephalus gunnari	5	13.8 ± 12.0	0.6 ± 0.6	85.6 ± 12.3
		Channichthys rhinoceratus	5	87.1 ± 7.5	0.6 ± 0.4	12.4 ± 7.2
	Benthic	Paranotothenia magellanica	5	21.3 ± 5.2	0.6 ± 0.3	78.1 ± 5.7
		Notothenia rossii	5	41.2 ± 14.2	0.5 ± 0.3	58.3 ± 15.0
		Lepidonotothen squamifrons	5	28.7 ± 11.2	2.1 ± 1.1	69.2 ± 12.7
		Paradiplospinus gracilis	1	19.6	0.7	79.7
	Pelagic	Gymnoscopelus nicholsi	4	6.2 ± 2.5	1.0 ± 0.6	92.4 ± 5.7
		Gymnoscopelus piabilis	5	11.3 ± 3.0	2.3 ± 0.8	86.5 ± 3.3
	Cu	Champsocephalus gunnari	5	5.1 ± 5.0	0.3 ± 0.3	94.5 ± 5.3
		Channichthys rhinoceratus	5	7.5 ± 1.1	0.5 ± 0.2	92.0 ± 0.9
	Benthic	Paranotothenia magellanica	5	10.9 ± 2.7	1.1 ± 0.1	87.9 ± 3.9
		Notothenia rossii	5	6.8 ± 2.8	1.4 ± 0.3	91.8 ± 2.6
		Lepidonotothen squamifrons	5	6.5 ± 5.2	0.9 ± 0.1	92.6 ± 8.8
		Champsocephalus gunnari	5	2.8 ± 0.8	0.1 ± 0.1	97.1 ± 1.8
		Channichthys rhinoceratus	5	4.3 ± 1.6	0.4 ± 0.3	95.4 ± 1.2
	Hg Benthic	Paranotothenia magellanica	5	5.6 ± 3.0	0.6 ± 0.3	93.8 ± 4.2
		Notothenia rossii	5	7.1 ± 2.6	1.7 ± 0.6	91.2 ± 2.9
		Lepidonotothen squamifrons	5	1.5 ± 1.0	ND	98.5 ± 1.5-
		Paradiplospinus gracilis	1	8.9	0.6	81.5
	Pelagic	Gymnoscopelus nicholsi	4	10.7 ± 2.0	1.9 ± 0.9	87.3 ± 3.9
		Gymnoscopelus piabilis	5	2.3 ± 5.2	1.9 ± 0.3	95.9 ± 5.2
	Zn	Champsocephalus gunnari	5	2.0 ± 1.1	0.2 ± 0.2	97.8 ± 1.4
		Channichthys rhinoceratus	5	3.1 ± 0.8	0.7 ± 0.7	96.3 ± 1.7
	Benthic	Paranotothenia magellanica	5	3.7 ± 0.5	1.4 ± 0.2	95.0 ± 1.3
		Notothenia rossii	5	6.0 ± 1.6	1.4 ± 0.4	92.6 ± 2.1
		Lepidonotothen squamifrons	5	4.7 ± 2.7	1.2 ± 0.2	94.1 ± 5.0
	ND : not determined.

Table 6 .

 6 Metal concentrations in the liver of fish from the Antarctic Ocean. Values represent the ranges or mean ± SD, expressed as µg.g -1 dwt or wwt.

	Species		Sample area	N	Cd	Cu	Hg	Zn	Basis	Reference
	Chaenocephalus aceratus	Antarctic peninsula	4	1.01-1.25	3.3-5.4	-	87.7-106.0	dwt	Szefer et al. (1993)
	Chionodraco hamatus	Terra Nova Bay	20	1.02-14.53	-	-	-	dwt	Bargagli et al. (1996)
	"		"	18	-	-	0.02-0.44	-	dwt	Bargagli et al. (1998)
	"		"	16	0.99 ± 0.97	0.85 ± 0.70	-	18.70 ± 5.13	dwt	Santovito et al. (2000)
	Cryodraco antarcticus	"	6	0.86-4.25	-	-	-	dwt	Bargagli et al. (1996)
	Notothenia coriiceps (male) South Shetland Islands	9	-	2.46 ± 0.84	-	29.55 ± 8.64	wwt	Marquez et al (1998)
	"	(female)	"	10	-	1.58 ± 0.38	-	23.62 ± 2.20	wwt	Marquez et al (1998)
	Pagothenia borchgrevinski	Syowa Station	18	0.30-2.46	0.92-5.88	0.005-0.026	22.4-34.2	wwt	Honda et al. (1983)
	Trematomus bernacchii	Terra Nova Bay	18	3.36-21.60	-	-	-	dwt	Bargagli et al. (1996)
	"		"	11	-	-	0.10-0.82	-	dwt	Bargagli et al. (1998)
	"		"	15	7.30 ± 5.61	5.19 ± 3.03	-	44.35 ± 24.72 dwt	Santovito et al. (2000)
	Trematomus hansoni	"	18	5.09-16.42	-	-	-	dwt	Bargagli et al. (1996)
	"		"	18	-	-	0.08-0.69	-	dwt	Bargagli et al. (1998)
	Trematomus newnesi	"	8	0.98-5.75	-	-	-	dwt	Bargagli et al. (1996)
	"		"	7	-	-	0.09-0.28	-	dwt	Bargagli et al. (1998)
	Trematomus sp.		Winter Quarters Bay	-	5.0-21.0	5.0-23	-	110-140	wwt	Lenihan et al. (1990)
	"		Cinder cones	-	12 ± 4	14 ± 2	-	127 ± 25	wwt	"

Table 7 .

 7 Metal concentrations in the kidney of fish from the Antarctic Ocean. Values represent the ranges or mean expressed as µg.g -1 dwt or wwt.

	Species		Sample area	N	Cd	Cu	Hg	Zn	Basis	Reference
	Chionodraco hamatus	Terra Nova Bay	20	0.26-0.87	-	-	-	dwt	Bargagli et al. (1996)
	"		"	20	-	-	0.10-0.85	-	dwt	Bargagli et al. (1998a)
	Cryodraco antarcticus	"	6	0.18-0.62	-	-	-	dwt	Bargagli et al. (1996)
	Notothenia coriiceps (male) South Shetland Islands 10	-	1.56 ± 0.43	-	23.30 ± 6.63	wwt	Marquez et al (1998)
	"	(female)	"	10	-	1.72 ± 0.56	-	23.69 ± 10.16 wwt	Marquez et al (1998)
	Pagothenia bernacchii	Terra Nova Bay	16	-	-	0.227-2.50	-	dwt	Capelli et al. (1991)
	Trematomus bernacchii	Terra Nova Bay	18	2.05-10.11	-	-	-	dwt	Bargagli et al. (1996)
	"		"	10	-	-	0.32-2.60	-	dwt	Bargagli et al. (1998a)
	"		"	27	1.8-3.0	5.1-5.7	0.47-0.83	110-116	dwt	Bargagli et al. (1998b)
	Trematomus hansoni	"	18	1.60-3.29	-	-	-	dwt	Bargagli et al. (1996)
	"		"	13	-	-	0.10-1.75	-	dwt	Bargagli et al. (1998a)
	Trematomus newnesi	"	8	0.17-3.91	-	-	-	dwt	Bargagli et al. (1996)
	"		"	8	-	-	0.09-0.82	-	dwt	Bargagli et al. (1998a)

Table 8 .

 8 Metal concentrations in the muscle of fish from the Antarctic Ocean. Values represent the ranges or mean expressed as µg.g -1 dwt or wwt.

	Species	Sample area	N	Cd	Cu	Hg	Zn	Basis	Reference
	Chionodraco hamatus	Terra Nova Bay	20	0.01-0.03	-	-	-	dwt	Bargagli et al. (1996)
	"	"	7	-	-	0.01-0.92	-	dwt	Bargagli et al. (1998a)
	"	"	10	0.97 ± 0.47	0.70 ± 0.67	-	6.27 ± 3.73	dwt	Santovito et al. (2000)
	Chaenocephalus aceratus	Antarctic peninsula	3	0.05-0.13	1.0-2.0	-	28.6-35.5	dwt	Szefer et al. (1993)
	Cryodraco antarcticus	Terra Nova Bay	6	0.01-0.06	-	-	-	dwt	Bargagli et al. (1996)
	Notothenia coriiceps (male) South Shetland Islands 10	-	1.00 ± 0.34	-	4.77 ± 1.08	wwt	Marquez et al (1998)
	"	South Orkney Islands	11	< 0.05	0.04-0.50	0.01-0.10	1.00-6.70	wwt	de Moreno et al (1997)
	" (female)	South Shetland Islands 10	-	4.79 ± 0.81	-	1.01 ± 0.62	wwt	Marquez et al (1998)
	"	South Orkney Islands	17	< 0.05	0.05-0.40	0.01-0.09	2.00-5.40	wwt	de Moreno et al (1997)
	Notothenia gibberifrons	Antarctica	3	0.02-0.04	0.71-0.98	-	20.7-24.2	dwt	Szefer et al. (1993)
	Pagothenia borchgrevinski	Syowa Station	22	0.01-0.04	0.17-1.39	0.002-0.009	4.27-8.15	wwt	Honda et al. (1983)
	Pagothenia bernacchii	Terra Nova Bay	18	-	-	0.230-0.990	-	dwt	Minganti et al (1994)
	Trematomus bernacchii	Terra Nova Bay	18	0.01-0.08	-	-	-	dwt	Bargagli et al. (1996)
	"	"	12	-	-	0.17-1.79	-	dwt	Bargagli et al. (1998a)
	"	"	27	0.03-0.05	1.9-2.5	0.49-0.74	22.8-23.6	dwt	Bargagli et al. (1998b)
	"	"	10	0.74 ± 0.48	0.32 ± 0.23	-	2.88 ± 1.89	dwt	Santovito et al. (2000)
	Trematomus hansoni	"	18	0.01-0.05	-	-	-	dwt	Bargagli et al. (1996)
	"	"	15	-	-	0.11-1.08	-	dwt	Bargagli et al. (1998a)
	Trematomus newnesi	"	8	0.02-0.05	-	-	-	dwt	Bargagli et al. (1996)
	"	"	7	-	-	0.09-0.82	-	dwt	Bargagli et al. (1998a)
	Trematomus sp.	Winter Quarters Bay	-	0.1-0.2	0.1-0.3	-	0.4-25	wwt	Lenihan et al. (1990)
	"	Cinder cones	-	0.2	0.9 ± 0.8	-	41 ± 13	wwt	"

Acknowledgements. We thank Guy Duhamel and the crew of the RV "La Curieuse" for their help in the collection of the fish specimens. This work was financially supported by the "Conseils Généraux des Deux-Sévres et de la Charente Maritime", and by the Institut Français pour la Recherche et la Technologie Polaires and the Terres Australes et Antarctiques Françaises.