M Bouchoucha
email: marc.bouchoucha@ifremer.fr

Trace element contamination in fish impacted by bauxite red mud disposal in the Cassidaigne canyon (NW French Mediterranean)

Keywords: Trace element, bioaccumulation, arsenic speciation, ICP-MS, fish, industrial discharge

From 1966 to 2015, the Gardanne alumina refinery discharged some 20 million tons of bauxite residue (called red mud) into the Cassidaigne Canyon (northwest French Mediterranean) with impacts on local ecosystem functioning. Although these red muds contained high levels of trace elements (TE), in particular titanium (Ti), vanadium (V), aluminium (Al) and arsenic (As), surprisingly, their impacts on fish contamination levels and the risk related to fish consumption have been little studied until now. Here, 11 trace elements (Al, As, Cd, Cr, Co, Cr, Mn, Ni, Pb, Ti and V) were analyzed in muscle and, when possible, liver, from 1 308 fish of 26 species from an impacted zone in the vicinity of the Cassidaigne Canyon and a reference zone, unaffected by red mud disposals. Moreover, 66 arsenic speciation analyses were performed. Although the impact of human activities on the levels of fish contamination by trace elements is generally not easy to assess in situ because it is blurred by interaction with biological effects, we highlighted significant contamination of the fish species collected from the Cassidaigne Canyon, especially by the main trace elements attributable to the discharges of the Gardanne alumina refinery, namely Al, V and Ti. Moreover, inorganic toxic As concentrations were higher in the impacted zone. The results of this baseline research also confirmed the concern previously raised regarding Hg in Mediterranean organisms and that trace element contamination levels in fish are generally negatively related to fish length for all TE except Hg.

Introduction

Hazards to human health stemming from exposure to individual contaminants or groups of contaminants have emerged as a major concern in recent years (e.g. [START_REF] Copat | Heavy metals concentrations in fish and shellfish from eastern Mediterranean Sea: Consumption advisories[END_REF][START_REF] Damiano | Accumulation of heavy metals to assess the health status of swordfish in a comparative analysis of Mediterranean and Atlantic areas[END_REF][START_REF] Miniero | Persistent toxic substances in Mediterranean aquatic species[END_REF][START_REF] Percin | Some trace elements in front and rear dorsal ordinary muscles of wild and farmed bluefin tuna (Thunnus thynnus L. 1758) in the Turkish part of the eastern Mediterranean Sea[END_REF][START_REF] Suarez-Serrano | Procambarus clarkii as a bioindicator of heavy metal pollution sources in the lower Ebro River and Delta[END_REF][START_REF] Turkmen | Determination of metals in fish species from Aegean and Mediterranean seas[END_REF].

Among the wide range of toxic substances contaminating the marine environment, specific focus has been given to trace elements [START_REF] Castro-Gonzalez | Heavy metals: Implications associated to fish consumption[END_REF]. Naturally present in the Earth's crust, trace elements (TE) are also found in the marine environment [START_REF] Bryan | The effects of heavy metals (other than mercury) on marine and estuarine organisms[END_REF][START_REF] Mason | Trace metals in aquatic systems[END_REF]. However, human activities have considerably increased their spread into aquatic ecosystems [START_REF] Asante | Trace elements and stable isotope ratios (delta C-13 and delta N-15) in fish from deep-waters of the Sulu Sea and the Celebes Sea[END_REF][START_REF] Nriagu | Quantitative assessment of worldwide contamination of air, water and soils by trace-metals[END_REF][START_REF] Squadrone | Human exposure to metals due to consumption of fish from an artificial lake basin close to an active mining area in Katanga (DR Congo)[END_REF], especially those in marine coastal areas [START_REF] Ourgaud | Trace elements in a Mediterranean scorpaenid fish: Bioaccumulation processes and spatial variations[END_REF]. TE are generally classified into two categories on the basis of their metabolic role and their regulation by organisms. Essential TE (e.g. iron, copper and zinc) are involved in metabolic processes, including functional and structural ones [START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF]. They are generally efficiently regulated by marine animals, and present a narrow range of variation in their tissues, except when ambient concentrations in seawater or food reach very high concentrations [START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF][START_REF] Chapman | Water quality assessments: a guide to the use of biota, sediments, and water in environmental monitoring: E & Fn Spon London[END_REF][START_REF] Vallee | Zinc coordination, function, and structure of zinc enzymes and other proteins[END_REF], or when they are involved in a specific metabolic activity at a specific life stage (e.g. juvenile growth; [START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF]. On the contrary, some TE (e.g. cadmium, lead and mercury) are not essential for marine life and their concentrations in organisms depend mainly on environmental levels [START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF]. Whether essential or nonessential, TE become toxic above a certain threshold [START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF].

From 1966 to 2015, the Gardanne alumina refinery discharged bauxite residue (called red mud) through a pipe at 320 meters depth in the Cassidaigne Canyon in the northwest French Mediterranean. This mud then spread along the canyon axis and on its lateral flanks to great depth (>2000 m). A centimetric red layer of bauxite residue can now be observed along the canyon axis, more than 50 km away from the pipe. The red mud contained high levels of TE, in particular titanium (Ti), vanadium (V), aluminium (Al), and to a lesser extent arsenic (As) [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF][START_REF] Fontanier | Deep-sea foraminifera from the Cassidaigne Canyon (NW Mediterranean): Assessing the environmental impact of bauxite red mud disposal[END_REF]. These elements are present in several mineral oxi-hydroxide phases (hematite, goethite, ilmenite, rutile, anatase, brookite and kaolinite) that are not dissolved by the industrial process used to extract alumina from bauxite ore at the Gardanne alumina refinery [START_REF] Fontanier | Deep-sea foraminifera from the Cassidaigne Canyon (NW Mediterranean): Assessing the environmental impact of bauxite red mud disposal[END_REF]. Red mud disposal into the Cassidaigne Canyon was expected to lower the biodiversity and abundance of marine communities and to increase the potential uptake of bioavailable TE into the tissues of marine organisms, their bioaccumulation through food webs and ultimately into human fishconsuming communities [START_REF] Ellis | Submarine tailings disposal (STD) for mines: An introduction[END_REF][START_REF] Gray | Mercury and methylmercury contents in minewaste calcine, water, and sediment collected from the Palawan Quicksilver Mine, Philippines[END_REF][START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF][START_REF] Swales | Biological monitoring of the impacts of the Ok Tedi copper mine on fish populations in the Fly River system, Papua New Guinea[END_REF][START_REF] Williams | Assessment of mercury contamination and human exposure associated with coastal disposal of waste from a cinnabar mining operation, Palawan, Philippines[END_REF].

Since the 1960s, several environmental impact studies of the Gardanne alumina production activity have been performed to document the effects of these disposals on biodiversity [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF] but, surprisingly, up to now the impact of red mud residues in the Cassidaigne Canyon on TE contamination of fish and the risk related to fish consumption have been studied only twice. In 2004, 9 TE were analyzed in 28 samples from 12 fish species and in 2013, 27 TE were analyzed in 11 samples from 8 fish species [START_REF] Anses | Note d'appui scientifique et technique de l'Agence nationale de sécurité sanitaire de l'alimentation , de l'environnement et du travail relatif à l'impact potentiel sur la santé humaine du rejet en Méditerranée d'effluents issus des activités de transformation de minerai de bauxite[END_REF][START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF]. These studies did not identify any cumulated risk linked with fish consumption [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF]. However, their conclusions were limited:

(i) because the number of fish analyzed per species was very low; and (ii) because thresholds were not compared with baseline values measured in a control zone, precluding any clear conclusion on actual contamination. Indeed, for elements without regulatory thresholds such as Ti and V, a robust impact assessment should include a comparison of trace metal concentrations in fish from the impacted area and from an non-impacted area chosen as control. Moreover, As is one of the elements present in the red muds discharged in the Cassidaigne Canyon. Arsenic can biomagnify in food webs [START_REF] Francesconi | Arsenic species in seafood: Origin and human health implications[END_REF][START_REF] Rahman | Bioaccumulation, biotransformation and trophic transfer of arsenic in the aquatic food chain[END_REF], but this element exists in a number of chemical forms in nature, all with very different toxicity characteristics, with inorganic species being the most hazardous [START_REF] Saha | A review of arsenic poisoning and its effects on human health[END_REF]. Therefore, the determination of the total As content is certainly not sufficient for estimating its potential toxic impact and its speciation must be studied. Indeed, very few data are available on As speciation in the Mediterranean context (see [START_REF] Fattorini | Chemical special-lion of arsenic in different marine organisms: Importance in monitoring studies[END_REF][START_REF] Storelli | Mercury and arsenic speciation in the muscle tissue of Scyliorhinus canicula from the Mediterranean sea[END_REF][START_REF] Storelli | Organic and inorganic arsenic and lead in fish from the South Adriatic Sea, Italy[END_REF]. To our knowledge, As speciation in fish collected close to the Cassidaigne Canyon has never been studied.

In this context, the specific aim of this study was to assess, for the first time, the direct impact of red mud disposal in the Cassidaigne Canyon on the TE concentrations, including As species, of several common fish species of commercial interest. The hypothesis tested here was that the burden for fish captured close to the Cassidaigne Canyon would be higher for red mud-related trace elements, in particular in Al, Ti and V, than those of fish from other non-impacted areas.

Materials and methods

Study area

The Cassidaigne Canyon is a short canyon (<50 km) located in the northern part of the western Mediterranean Sea, east of the Gulf of Lions (Fig. 1). Its head borders Cassis Bay, only 8 km from the coast, and presents in its deeper part two narrow passages located 3 km and 17 km, respectively, from the shelf break (170 m depth). This canyon has been considerably affected by the massive disposal of bauxite residues [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF] that lasted almost 50 years and is estimated at 20 million tons [START_REF] Anses | Note d'appui scientifique et technique de l'Agence nationale de sécurité sanitaire de l'alimentation , de l'environnement et du travail relatif à l'impact potentiel sur la santé humaine du rejet en Méditerranée d'effluents issus des activités de transformation de minerai de bauxite[END_REF]. Below 350 m, the entire seabed along the canyon axis was covered by the red mud flow which spread more than 60 km from the pipe, with a significant proportion of the mud being propagated at depths >2000 m [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF]. Today, this red mud also covers steep inclined rock and is found underneath overhangs [START_REF] Fabri | Megafauna of vulnerable marine ecosystems in French mediterranean submarine canyons: Spatial distribution and anthropogenic impacts[END_REF]. During strong north-eastern Mistral wind events, especially during winter, quantities of this red mud could be transported northward, from the canyon to the continental shelf [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF].

To assess the impacts of red mud disposal on fish contamination, two zones were considered (Fig. 1).

The first was adjacent to the Cassidaigne canyon head (called 'impacted zone', IZ), where detectable impact was most likely to occur (see [START_REF] Dauvin | Towards an impact assessment of bauxite red mud waste on the knowledge of the structure and functions of bathyal ecosystems: The example of the Cassidaigne canyon (north-western Mediterranean Sea)[END_REF][START_REF] Fontanier | Deep-sea foraminifera from the Cassidaigne Canyon (NW Mediterranean): Assessing the environmental impact of bauxite red mud disposal[END_REF]. The second zone (called 'reference zone', RZ) was in the vicinity of the Stoechades Canyon. As water masses in this part of the Mediterranean flow westward along the continental margin [START_REF] Gatti | The Rhone river dilution zone present in the northeastern shelf of the Gulf of Lion in December 2003[END_REF], the Stoechades Canyon is far from the influence of industrial discharge and thus not impacted by bauxite residues (MC. Fabri, pers. com.). The head of the Stoechades Canyon is located in the Gulf of Hyères, less than 5 km from the coast, providing similar bathymetry and physico-chemical conditions that allow comparing the two canyons. (Fig. 1).

Fish sampling

Twenty-six commercial species were selected in this study with regard to their importance in the communities of both systems and in the diet of the local population. In addition, these species encompass a wide range of feeding habits and living environments. The sampling can thus be considered representative of all fish functional groups occurring in the area. The aim of the sampling program was to obtain similar species from both the impacted and the reference zone. Fish were collected during 40 fishing operations (16 in the impacted zone, 24 in the reference zone) conducted between June 15 th 2015 and July 27 th 2015 by professional fishermen using different fishing gears such as bottom and pelagic trawls, trammel nets, hooks, etc. The geographical coordinates of the fishing operations were systematically collected by an on-board scientific observer.

Individual fish were stored on board and transferred to the laboratory in ice boxes where all fish were measured (total length to the nearest mm) and weighed (total mass to the nearest 0.1 g) before dissection. Specific measurements were performed in the muscle, the main edible tissue in fish). White dorsal muscle was sampled, individually placed in polyethylene bags and kept frozen at -20°C until chemical analysis. For larger species, namely Conger conger, Merluccius merluccius, Scyliorhinus canicula, Galeus melastomus and Raja clavata, livers were also collected when possible, as liver is the organ mostly involved in detoxication processes.

Determination of trace element concentrations

For each sample, concentrations in 11 TE (Al, As, Cd, Co, Cr, Hg, Mn, Ni, Pb, Ti and V) were analyzed.

The analyses of total TE were carried out using a 7700x ICP-MS (Agilent Technologies, Courtaboeuf, France), equipped with a third-generation octopole reaction system (ORS3) using helium as the collision gas. Further details of the instrument settings and performance criteria of this method, validated in-house and accredited by the French Committee of Accreditation (COFRAC), were described in [START_REF] Chevallier | Simultaneous determination of 31 elements in foodstuffs by ICP-MS after closed-vessel microwave digestion: Method validation based on the accuracy profile[END_REF]. Briefly, 0.2 to 2 g of sample was weighed precisely in a quartz digestion vessel and then wet-oxidised with a mixture of 3 mL of ultra-pure water and 3 mL of ultrapure HNO3 (67% v/v) in a Multiwave 3000 closed microwave digestion system (Anton-Paar, Courtaboeuf, France). After cooling to room temperature, the digested samples were transferred into 50 mL polyethylene tubes into which a solution of a mixture of internal standards (yttrium, scandium and rhenium) at 2 µg L -1 and ultrapure water was added to the final volume before analysis using ICP-MS. One randomly selected vessel was filled with reagents only and taken through the entire procedure as a blank. The limits of detection (LOD) were estimated at 0.042, 0.001, 0.0003, 0.0005, 0.005, 0.004, 0.0025, 0.025, 0.0012, 0.025, 0.0005 mg.kg -1 wet mass (mg.kg -1 wm) whereas the limits of quantification were estimated at 0.083, 0.002, 0.0005, 0.001, 0.010, 0.008, 0.005, 0.050, 0.0025, 0.050, 0.0001 mg.kg -1 wm for Al, As, Cd, Cr, Hg, Mn, Ni, Pb, Ti and V respectively. Chemical speciation measurements were carried out on the seafood species in which total arsenic exposure was most prevalent: sea breams (gilthead sea bream Sparus aurata, black spot sea bream Pagellus bogaraveo, common two-banded sea bream Diplodus vulgaris, white sea bream Diplodus sargus and red sea bream Pagellus erythrinus), redfish and rays. These species contribute more than 65% of total arsenic exposure for human consumers. For each species, between 2 and 8 composite samples per zone were analyzed. Each composite sample consisted of 3 to 5 sub-samples. Arsenic speciation was performed by HPLC/ICP-MS (HPLC Ultimate 300, Dionex, Voisins le bretonneux, France; ICP-MS X-Series II , Thermo Scientifc, Courtaboeuf, France) coupling in standard mode after microwave-assisted extraction (Multiwave 3000, Anton-Paar). This method was validated in-house, accredited by COFRAC, and described by [START_REF] Leufroy | Determination of seven arsenic species in seafood by ion exchange chromatography coupled to inductively coupled plasma-mass spectrometry following microwave assisted extraction: method validation and occurrence data[END_REF]. The following arsenic species were analyzed: As(III), As(V), monomethylarsonic acid (MA), dimethylarsinic acid (DMA), and arsenobetaine (AsB). For all these species, an LOD/LOQ of 0.002/0.005 mg As kg -1 wm was assessed under robust conditions. These speciation analyses were used to determine the levels of inorganic arsenic (sum of As(III) and As(V)). In parallel, the total arsenic concentration in the composite samples was also measured by the ICP-MS method described above for total TE analysis.

Several internal quality controls (IQCs) and associated criteria were used to ensure the reliability of the results. Each run included a standard calibration, blanks, several certified reference materials (CRMs): TORT-2 lobster hepatopancreas, ERM-CE278k mussel tissue and SRM 1548a typical diet, chosen to cover the 11 elements of interest (for total TE analysis) and BCR-627 tuna fish tissue (for As speciation analysis), two different spiked sample solutions, several fish samples including two samples analyzed in duplicate and a mid-range standard analyzed for every eight samples and at the end of the sequence. The set criteria were as follows: calibration (r² > 0.995), blanks (values < limit of quantification (LOQ)), internal standards (values within 70 and 130% of the target value), mid-range standards (values within 80 and 120% of the target value), spiked standard solutions (spike recovery within 70 and 130% of the theoretical spiked standard value), CRMs (Z-score < ± 2) and duplicates (acceptable if the relative standard deviation (RSD) ≤ 20% when the mean value ≥ 5 x LOQ or RSD ≤ 40% when the mean value ≥ LOQ and < 5 x LOQ). When the acceptance criteria were not met, the results were discarded and the samples were re-analyzed [START_REF] Millour | Internal quality controls applied in inductively coupled plasma mass spectrometry multi-elemental analysis in the second French Total Diet Study[END_REF].

All concentrations are given in mg.kg -1 wm. When data were available in both muscle and liver, the liver to muscle concentration ratios were calculated.

Statistical analyses

The statistical analysis of the results obtained on both matrices (muscle and liver) of fish was performed only for species presenting a minimum sample size of 15 individuals per zone (IZ and RZ).

For these species, Pearson correlation tests were used to investigate the relationship between the TE levels and fish length. Moreover, spatial variations in TE concentrations were analyzed according to the zone for each matrix separately. To do this, PERMANOVAs were performed. They allow handling complex, unbalanced multiple-factor designs and do not assume normal error distributions [START_REF] Anderson | Permutation tests for univariate or multivariate analysis of variance and regression[END_REF]. Euclidean distance similarity matrices were generated and the factor 'sampling area' was treated as fixed. Since it can affect contamination levels, fish length was considered as a covariate. Data were log(X+1) transformed prior to statistical analyses. In each case, p-values were calculated by 999 random permutations of residuals [START_REF] Anderson | Permutation tests for univariate or multivariate analysis of variance and regression[END_REF]. Concentrations below LOD were set to 0 whereas concentrations below LOQ were set to LOD. Due to the large number of species and TE analyzed, a large number of statistical analyses were performed simultaneously in this study, thereby increasing the proportion of false positives. Therefore, a multiple testing correction procedure was conducted to control the risk of reporting too many false positives and we used a false discovery rate (FDR) method to adjust for the inflation of p-values [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]. All data processing and statistical analyses were performed using R software (R Core Team, 2014) and PRIMER 6 software with the PERMANOVA add-on [START_REF] Clarke | Change in marine communities: an approach to statistical analysis and interpretation[END_REF]. The significance level for the tests was consistently set at α = 0.05.

Results

A total of 1 308 fish of 26 species were collected during this study (769 individuals and 23 species at IZ; 539 individuals and 21 species at RZ, with 18 species collected in both zones) (Table 1,2).

Considering the two matrices (muscle and liver), 1 597 analyses of 11 trace elements were performed (928 for IZ and 669 for RZ). Among the 26 species sampled, only 9 (Conger conger, Coris julis, Diplodus vulgaris, Helicolenus dactylopterus, Merluccius merluccius, Mullus surmuletus, Pagellus bogaraveo, Scorpaena porcus and Scyliorhinus canicula) reached the minimum sampling size required to investigate spatial variations and the relationship between TE and fish length. These species represented more than 67% of the total chemical analyses performed. With regards to As speciation, only 66 analyses were performed due to financial constraints (Table 3). This number was too small to correctly explore differences between the two sampling areas and test the effect of fish size on contamination but these results nonetheless provide preliminary information on the burdens of this little investigated TE.

The trace element concentrations in the muscle and the liver of different fish species collected from the two sampling areas are given in Table 2. In muscle, the distribution patterns in the TE concentrations were as follows: As > Al > Hg > Mn > Ti > Pb > Cr > V > Ni > Cd > Co for IZ and As > Al > Hg > Mn > Ti > Pb > Cr > V > Ni > Co > Cd for RZ. In the liver, the sequence was:

As > Al > Mn > Hg > Cd > Ti > Pb > Co > V > Cr > Ni for IZ and As > Al > Mn > Hg > Cd > V > Pb > Co > Ti > Cr > Ni for RZ.
Liver to muscle concentration ratios were consistently higher than 1 for Al, Cd, Co, Cr, Mn, Pb and V and reached up to 360 for Cd in M. merluccius from RZ. Ratios were lower than 1 (i.e. higher concentrations in muscle than in liver) for As and Hg. Due to the high number of values under LOD, it was generally not possible to calculate those ratios for Ni. Finally, the liver to muscle concentration ratios for Ti were below 1 for C. conger and S. canicula but over 1 for M. merluccius and Raja clavata.

Contamination levels in muscle differed significantly between species for all TE except Ni (Table 1), and no general specific trend could be observed. Nevertheless, the highest concentrations of As were found in the shark species S. canicula (p < 0.001) and the highest concentrations of Hg were found in H. dactylopterus and S. canicula (p < 0.001). Considering livers, species-dependent patterns in TE concentrations were less blurred and quite similar between sampling zones, with S. canicula presenting the highest Al, As, Cd, Co and Hg concentrations, M. merluccius the highest Mn concentrations and C. conger the highest Cr, Pb and V concentrations (Table 2).

The only compound of inorganic As (Asi) found during this study was AS(III) while As(V) concentrations were systematically below LOD. Inorganic arsenic concentrations in fish muscle were not correlated with total As (AsT) concentrations (Table 3), and the average percentage of Asi (relative to AsT) ranged between 0.01 and 2.69% for all the species considered and in both sampling zones. This percentage of Asi was systematically higher in individuals from IZ than those from RZ.

The relationships between fish length and concentrations differed between trace elements, species and tissues (Table 4). The vast majority of relationships, whether significant or not, were nonetheless negative, even for high concentrations of TE in the disposal. On the contrary, Hg was the only TE with a positive relationship for all species and tissues. Finally, As exhibited an intermediate pattern as positive relationships were observed for 8 species × tissue cases studied, 5 of which were significant (muscle of C. conger, S. canicula and P. bogaraveo, liver of S. canicula and C. conger).

Regarding the spatial trends, TE concentrations varied according to the sampling zone but the nature of the variation differed according to the species and the tissue considered (Table1, 2). Indeed, 36 out of the 132 comparisons of TE concentrations between sampling areas remained significant after adjusting for FDR (muscle and liver grouped), with 23 and 13 comparisons displaying significantly higher concentrations in IZ and RZ, respectively. For the main TE from the Gardanne alumina refinery discharges, namely Al, V and Ti, when spatial variations existed, the highest concentrations in fish muscle were systematically observed in IZ. By contrast, for As, Co, Hg, Mn and Pb, no clear pattern was shown with concentrations higher in IZ for some species and higher in RZ for others. Finally, for Cd, Cr and Ni, no difference in TE concentrations were observed between the two zones whatever couple of species × tissue was considered.

Discussion

Very few studies have assessed the impact of the red mud disposal in the Cassidaigne Canyon on contamination levels in fish. To our knowledge, this is the first time that this has been investigated so thoroughly, with the analysis of 11 TE for a large number of individuals (n=1308) from 26 different fish species. In addition, two tissues (muscle and liver) were taken into account, comparing, when possible, contamination levels between the impacted zone and a reference one. Moreover, although several studies have already been conducted on TE in Mediterranean fish (e.g. [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] Iamiceli | Metals in Mediterranean aquatic species[END_REF][START_REF] Mille | Trace metal concentrations in the muscle of seven marine species: Comparison between the Gulf of Lions (North-West Mediterranean Sea) and the Bay of Biscay (North-East Atlantic Ocean)[END_REF], some elements such as Al, Ti and V have rarely been investigated [START_REF] Eisler | Chapter 3 -Fishes[END_REF]. Lastly, the present study provides a baseline dataset for various Mediterranean fish species and allows a preliminary investigation of certain factors affecting contamination.

A large number of the fish analyzed in this study presented concentration levels higher than the limits defined by the European Commission in fish muscle [START_REF] Ec | Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs[END_REF] T. trachurus and Z. faber) had Hg concentration kevels above the European regulatory limit (0.5 mg.kg -1 wm in teleosts, 1 mg.kg -1 wm in sharks), confirming the concern raised previously regarding Hg in Mediterranean organisms [START_REF] Cossa | Influences of bioavailability, trophic position, and growth on methylmercury in hakes (Merluccius merluccius) from northwestern Mediterranean and northeastern Atlantic[END_REF][START_REF] Harmelin-Vivien | Difference of mercury bioaccumulation in red mullets from the north-western Mediterranean and Black seas[END_REF][START_REF] Koenig | New insights into mercury bioaccumulation in deepsea organisms from the NW Mediterranean and their human health implications[END_REF].

The straightforward pattern observed for Hg in the present study may be largely driven by biological and biogeochemical mechanisms at play at the scale of the whole NE Mediterranean [START_REF] Cossa | The Mediterranean mercury anomaly, a geochemical or a biological issue[END_REF] rather than from the local influence of red mud disposals. For Pb and Cd, the number of individuals with concentrations above the EU regulatory limits in fish muscle (0.3 mg.kg -1 wm and 0.05 mg.kg -1 wm for Pb and Cd, respectively), were lower, 8 and 19, respectively, which represent between 0.6 and 1.5% of the total number of fish analyzed. Between 75 and 90% of these threshold overruns concerned IZ, confirming the conclusions of [START_REF] Ourgaud | Trace elements in a Mediterranean scorpaenid fish: Bioaccumulation processes and spatial variations[END_REF], who found that these TE were more concentrated in S. porcus collected in the bay of Marseille (close to the Cassidaigne Canyon) than in those from the Gulf of Hyères. There is no regulatory limit for total As in fish muscle, but arsenic exposure is a significant worldwide environmental health concern. Overall, the data we obtained here appear of great concern with regard to total As concentrations. However, the risk of potential As poisoning through fish consumption should be further assessed through the specific determination of the inorganic As content in fish tissues. Most of the As in the tissues of marine animals is bound in organoarsenic forms [START_REF] Neff | Ecotoxicology of arsenic in the marine environment[END_REF] and considered of low toxicity [START_REF] Hindmarsh | Arsenic, its clinical and environmental significance[END_REF]. In this study, the main form of As found in fish was arsenobetaine which accounts for up to 100% of As in certain species (Table 3). This form is not toxic to humans [START_REF] Neff | Ecotoxicology of arsenic in the marine environment[END_REF].

In both sampling zones, our results clearly showed significant differences in the bioaccumulation of a range of TE between the 26 selected species, suggesting a diversity response among fish (Table 1, 2). Indeed, the accumulation of TE in fish does not directly reflect their simple concentration in the ambient environment as it is affected by environmental factors, species biology and the physicochemical properties of the contaminant [START_REF] Cossa | Influences of bioavailability, trophic position, and growth on methylmercury in hakes (Merluccius merluccius) from northwestern Mediterranean and northeastern Atlantic[END_REF][START_REF] Cresson | Are red mullet efficient as bio-indicators of mercury contamination? A case study from the French Mediterranean[END_REF]. Thus, assessing a site-specific contamination pattern requires good understanding of all these parameters although the interpretation of the results remains complex because of the existence of interactions between them. Notwithstanding some general trends could still be discerned. Some TE such as Hg are wellknown for their biomagnification property, namely the trend for increasing concentrations along food webs [START_REF] Bryan | Bioaccumulation of marine pollutants[END_REF]. This process in particular is responsible for the high Hg concentrations generally observed in high trophic level species such as marine apex predators [START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF] like S. canicula, M. merluccius, G melastomus, Z. faber, etc. In this study, Hg concentrations in fish muscle from IZ varied between 0.014 mg.kg -1 in L. aurata and 1.613 m. kg -1 in S. canicula (Table 1). The interspecific variation in mean trophic levels, ranging from 2.8 for L. aurata to 3.8 S. canicula (Cresson et al., 2014b;Froese and Pauly, 2014;[START_REF] Stergiou | Feeding habits and trophic levels of Mediterranean fish[END_REF], could partially explain this differences in the TE concentrations between these species. Moreover, an increasing number of studies have documented relatively high levels of TE contamination in shelf-edge and deep species (Cresson et al., 2014a;[START_REF] Hornung | Trace-metam content in deep-water sharks from the eastern Mediterranean-Sea[END_REF], especially when compared to coastal species [START_REF] Chouvelon | Enhanced bioaccumulation of mercury in deep-sea fauna from the Bay of Biscay (north-east Atlantic) in relation to trophic positions identified by analysis of carbon and nitrogen stable isotopes[END_REF]. The 26 species selected here live at drastically different depths, from the surface down to a few meters depth for C. julis, L. ramada and L. aurata, to several hundreds of meters for G. melastomus, H. dactylopterus and P. bogaraveo (Froese and Pauly, 2014). In this study, C. julis, L. ramada and L. aurata were captured between 5 and 35 m depth whereas G. melastomus, H. dactylopterus and P. bogaraveo were caught between 141 and 391m depth. This may explain a part of the inter-specific variations in TE contamination and the lower TE concentrations generally observed in L. ramada, L. aurata and C. julis (Table 1), Finally, inter-specific differences in TE concentrations may also be driven by contrasting life-history and behavior. Factors such as preferred habitat (e.g. benthic and pelagic), longevity, diet and foraging behavior (e. g. vertical migrations and type of prey consumed), metabolic rates and biochemical response [START_REF] Burger | A framework and methods for incorporating gender-related issues in wildlife risk assessment: Gender-related differences in metal levels and other contaminants as a case study[END_REF][START_REF] Zhao | Role of living environments in the accumulation characteristics of heavy metals in fishes and crabs in the Yangtze River Estuary, China[END_REF] have been demonstrated to affect rates of TE uptake and excretion from both food and water pathways. Previous works showed that species that are continuously in contact with contaminants in sediment have developed higher detoxication abilities, resulting in lower body burdens while being exposed to the same contaminant load as pelagic species [START_REF] Sole | Esterase activities and lipid peroxidation levels in offshore commercial species of the NW Mediterranean Sea[END_REF]. This may explain why TE concentrations in the benthic ray R. clavata are genarally lower than those o the demersal S. canicula (Table1, 2), despite similar trophic levels (Froese and Pauly, 2014;[START_REF] Stergiou | Feeding habits and trophic levels of Mediterranean fish[END_REF] and living depth (both species were captured between 100 and 380m depth).

Fish length is often recognized to be of importance in determining the rate of physiological processes that influence uptake, distribution and elimination of TE [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]. However, except for Hg, our analysis revealed no conclusive evidence that mean TE concentrations were systematically linked to fish length, which is in good agreement with the results reported in the literature. Indeed, although it is generally accepted that Hg load in fish body is strongly affected by size it is still not clear if the other TE accumulations are size-dependent. This is supported by the fact that most TE (with the exception of As and Hg) do not biomagnify along aquatic food webs [START_REF] Fey | Does trophic level drive organic and metallic contamination in coral reef organisms?[END_REF][START_REF] Xu | Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus[END_REF]. The available literature on the correlation between TE concentrations in muscle tissue and fish length and weight have shown that a dependency exists for some species (e.g. [START_REF] Gobert | Trace element concentrations in the apex predator swordfish (Xiphias gladius) from a Mediterranean fishery and risk assessment for consumers[END_REF], while for other species, no dependency is found (e.g. [START_REF] Mille | Trace metal concentrations in the muscle of seven marine species: Comparison between the Gulf of Lions (North-West Mediterranean Sea) and the Bay of Biscay (North-East Atlantic Ocean)[END_REF]. The absence of relationship likely indicates that fish regulate some TE at a certain concentration required for efficient metabolic activities [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] Hornung | Trace-metam content in deep-water sharks from the eastern Mediterranean-Sea[END_REF]. Moreover, when significant, the relationships between TE (other than Hg) and fish lengths are generally negative [START_REF] Endo | Hg, Zn and Cu levels in the muscle and liver of tiger sharks (Galeocerdo cuvier) from the coast of Ishigaki Island, Japan: Relationship between metal concentrations and body length[END_REF]. This may result from a higher metabolic activity in fast-growing young individuals, requiring higher amounts of these elements than older individuals or through a higher excretion rate and/or dilution of metal burden with growth, and lower metabolism [START_REF] Eisler | Chapter 3 -Fishes[END_REF]. In addition, length is commonly used as a proxy of age and chronic exposure to contaminants, as length is quickly measured, while accurate age determination requires complex and time-consuming procedures (e.g. otolith or vertebrae reading for teleost and chondrichtyans, respectively). This pattern is nonetheless blurred by the fact that growth rate affects the age -length relationship. As a result, individuals of the same length but from zones with different nutritional inputs (and thus different growth rates) may have different age and may have been thus exposed to contaminant during different periods of time.

Many studies have shown that TE accumulate in various organs of fish at different levels [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] Dural | Bioaccumulation of some heavy metals in different tissues of Dicentrarchus labrax L, 1758, Sparus aurata L, 1758 and Mugil cephalus L, 1758 from the Camlik lagoon of the eastern cost of Mediterranean (Turkey)[END_REF][START_REF] Fernandes | Bioaccumulation of heavy metals in Liza saliens from the Esmoriz-Paramos coastal lagoon, Portugal[END_REF][START_REF] Long | Metallothionein induction and bioaccumulation kinetics of Cd and Ag in the marine fish Terapon jarbua challenged with dietary or waterborne Ag and Cu[END_REF][START_REF] Mormede | Heavy metal concentrations in commercial deep-sea fish from the Rockall Trough[END_REF][START_REF] Uluturhan | Heavy metal contaminants in Red Pandora (Pagellus erythrinus) tissues from the Eastern Aegean Sea, Turkey[END_REF]. With the exception of Hg, As and Ti, the liver to muscle concentration ratios calculated in this study were generally over 1 and reached up to 360. This is not surprising because, in general, TE are preferentially accumulated in the liver of aquatic organisms [START_REF] Henry | Heavy metals in four fish species from the French coast of the eastern English Channel and Southern Bight of the North Sea[END_REF], which likely results from detoxification processes [START_REF] Eisler | Chapter 3 -Fishes[END_REF]. For Hg, higher concentrations are commonly found in fish muscle. Indeed, methylmercury is the predominant form of Hg in fish, ranging from 80% to 100% [START_REF] Bloom | On the chemical form of mercury in edible fish and marine invertebrate tissue[END_REF][START_REF] Chouvelon | Oligotrophy as a major driver of mercury bioaccumulation in medium-to high-trophic level consumers: A marine ecosystem-comparative study[END_REF][START_REF] Magalhaes | Intra-and inter-specific variability in total and methylmercury bioaccumulation by eight marine fish species from the Azores[END_REF] and is mainly accumulated in fish muscle due to its affinity to protein [START_REF] Amlund | Accumulation and elimination of methylmercury in Atlantic cod (Gadus morhua L.) following dietary exposure[END_REF][START_REF] Harris | The chemical form of mercury in fish[END_REF]. This difference in chemical affinity explains why the liver to muscle concentration ratios for Hg ranged here from 0.47 to 0.97. Considering As, Eisler (2010) stated that hepatic concentrations are usually higher than those found in muscle tissues, a pattern not observed in the present study as the liver to muscle concentration ratios ranged between 0.34 and 0.90. Such ratios below 1 have already been reported in benthic (Pteromylaeus bovinus and Myliobatis aquila) and pelagic (Pteroplatytrygon violacea) rays from the northern Adriatic Sea [START_REF] Slejkovec | Bioaccumulation of Arsenic Species in Rays from the Northern Adriatic Sea[END_REF], in S. canicula from the North Sea [START_REF] De Gieter | Total and toxic arsenic levels in North Sea fish[END_REF], in 22 fish species from the New Caledonia lagoon [START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF], and in 32 teleost species from the island of Guam (Pacific Ocean) [START_REF] Denton | Trace metals in marine organisms from four harbours in Guam[END_REF]. Therefore, the As partition between muscle and liver seems to be speciesdependent (e.g. [START_REF] Hellou | Distribution of elements in tissues of yellowtail flounder Pleuronectes ferruginea[END_REF][START_REF] Raimundo | Trace-element concentrations in muscle and liver of 11 commercial fish species from Condor Seamount, Azores Archipelago (Portugal)[END_REF][START_REF] Raimundo | Elemental composition of two ecologically contrasting seamount fishes, the bluemouth (Helicolenus dactylopterus) and blackspot seabream (Pagellus bogaraveo)[END_REF] and possibly related to differences in the bioavailability of forms of this element [START_REF] Francesconi | Arsenic species in seafood: Origin and human health implications[END_REF]. Finally, very few data exist on Ti concentrations in fish and, to our knowledge, the accumulation of this element in different organs has never been studied. The distinct accumulation patterns observed here may result from differences in the physiological response of the species, as well as differences in lifehistory and behavior of fish.

As confirmed by sediment analysis [START_REF] Fontanier | Deep-sea foraminifera from the Cassidaigne Canyon (NW Mediterranean): Assessing the environmental impact of bauxite red mud disposal[END_REF], the Cassidaigne Canyon has been subjected to considerable TE inputs, mainly in Al, Ti, V from the alumina refinery's by-products. In general, these TE are considered non-essential for marine life and their concentrations in organisms mainly depend on their environmental levels [START_REF] Amiard | Comparative study of the patterns of bioaccumulation of essential (Cu, Zn) and non-essential (Cd, Pb) trace metals in various estuarine and coastal organisms[END_REF]. Therefore, it was expected that fish from IZ would contain consistently higher concentrations of red mud-related TE than those from RZ. Among all the species, Al was consistently more accumulated in fish from IZ, likely resulting from industrial releases. Aluminum is also the TE most concentrated in red mud. Laboratory experiments showed that the bauxite from the Gardanne plant releases substantial quantities of Al in seawater [START_REF] Pagano | Toxicity of bauxite manufacturing by-products in sea urchin embryos[END_REF]. In the marine environment (pH 8.0-8.3, salinity 35), this Al is theoretically mostly present in two forms: the aluminate anion Al(OH)4 -and to a lesser extent neutral aluminum hydroxide Al(OH)3 - [START_REF] Millero | Effect of Ocean Acidification on the Speciation of Metals in Seawater[END_REF]. There are very few studies on the transfer of aluminate anions and aluminum hydroxide to aquatic organisms and even fewer focusing on fish species. Our results suggested that fish are able to at least partially accumulate these Al forms. This observation can be extended to other living organisms as Al bioaccumulation has also been reported in marine plants collected nearby a coastal bauxite sludge disposal site [START_REF] Malea | Concentration of Aluminium in Halophila stipulacea (Forsk.) archers. and the substrate of the Antikyra Gulf, Greece[END_REF]. For the other TE attributable to the Gardanne alumina refinery discharges (Ti and V), we found that when spatial variations existed, concentrations in fish muscle were systematically higher in IZ than in RZ, but this difference concerned a limited number of species. It could be hypothesized that they are not readily bioavailable for species and/or they are poorly transferred through the food chain up to apex predators. This result is similar to those of [START_REF] Bourcier | Ecoulement des boues rouges dans le canyon de la Cassidaigne (décembre 1968)[END_REF], [START_REF] Bourcier | Les «boues rouges» déversées dans le canyon de la Cassidaigne (région de Marseille)[END_REF] and [START_REF] Fontanier | Deep-sea foraminifera from the Cassidaigne Canyon (NW Mediterranean): Assessing the environmental impact of bauxite red mud disposal[END_REF], who observed that there was no chemical impact of red muds on macrobenthic fauna and the structure of deep-sea foraminifera communities in the Cassidaigne Canyon. They suggested that most of the toxic TE in the red mud impacting the sites are not bioavailable but simply locked in solid phases.

Conclusion

The impact of the human activities on the levels of fish contamination by TE is not easy to assess in situ because it is blurred by the interaction of biological effects (e.g. [START_REF] Bouchoucha | Growth, condition and metal concentration in juveniles of two Diplodus species in ports[END_REF]Cresson et al., 2014a;[START_REF] Metian | Trace element bioaccumulation in reef fish from New Caledonia: Influence of trophic groups and risk assessment for consumers[END_REF]. A thorough sampling campaign and numerous analyses allowed highlighting the significant contamination of fish species collected from the Cassidaigne Canyon area, especially for Al and to a lesser extent for Ti and V. Although not statistically proved, it was also shown that fish collected close to the alumina refinery pipe had higher Asi concentrations than those from the reference area. In the next step, it would be interesting to assess human dietary exposure to these metals through the consumption of fish from the Cassidaigne Canyon zone. This would allow better evaluation of the health risk for humans consuming these fish. Moreover, the solubility of many TE is highly redox-sensitive, thus the elemental concentrations in seawater change accordingly (e.g. [START_REF] Morford | The geochemistry of redox sensitive trace metals in sediments[END_REF]. Consequently, if the redox conditions change in the sites contaminated by red mud, this would influence the solubility of TE, making them bioavailable to marine organisms. Therefore, it seems essential to continue regular and long-term monitoring of marine organisms from this area to prevent any further environmental deterioration and to assess human exposure.

Figures

  Figures

Figure 1 :

 1 Figure 1: Location of the sampling station and sampling zones. The star represents the Gardanne alumina refinery pipeline outlet.

  . A total of 444 individuals from 15 different species(C. conger, C. julis, D. sargus, D. vulgaris, G. melastomus, H. dactylopterus,

M. merluccius, M. surmuletus, M. barbatus, P. bogaraveo, R. clavata, S. canicula, T. mediterraneus,

Table 1 :

 1 Mean TE concentrations ± standard deviation (in mg.kg -1 wm) measured in fish muscle from the red mud impacted (IZ) and the reference (RZ) 658 zones. Minimum and maximum values are given in brackets. 659 660

	Species	zon e	Tissue	N	Total length (mm)	Total mass (g)	Al	As	Cd	Co	Cr	Hg
	Chelon labrosus	IZ	muscle	1	406	860.3	0.320	1.659	0.000	0.002	0.021	0.128
	Conger conger	IZ	muscle		710 ± 171 (416 -1119)	799.5 ± 643.2 (94.5 -2894.4)	0.062 ± 0.159 (0.000 -0.858)	22.115 ± 11.886 (5.308 -53.193)	0.003 ± 0.007 (0.000 -0.038)	0.002 ± 0.003 (0.000 -0.015)	0.021 ± 0.029 (0.000 -0.109)	0.38 ± 0.339 (0.07 -1.872)
	Conger conger	RZ	muscle		716 ± 203 (334 -1035)	817.5 ± 672.6 (50.8-2341.7)	0.032 ± 0.102 (0.000 -0.465)	26.867 ± 19.009 (8.613 -86.156)	0.003 ± 0.008 (0.000 -0.041)	0.001 ± 0.001 (0.000 -0.003)	0.017 ± 0.023 (0.000 -0.085)	0.345 ± 0.218 (0.066 -1.003)
	Coris julis	IZ	muscle		128 ± 1.9 (103 -212)	20.9 ± 12.5 (8.6 -83.5)	0.106 ± 0.112 (0.000 -0.389)	4.213 ± 2.266 (1.08 -10.100)	0.001 ± 0.001 (0.000 -0.002)	0.007 ± 0.003 (0.000 -0.017)	0.031 ± 0.035 (0.000 -0.145)	0.148 ± 0.105 (0.095 -0.700)
	Coris julis	RZ	muscle		125 ± 14 (86 -147)	17.0 ± 5.3 (5.1 -29.4)	0.149 ± 0.237 (0.000 -1.11)	7.633 ± 7.786 (0.586 -33.938)	0.004 ± 0.015 (0.000 -0.085)	0.009 ± 0.004 (0.000 -0.019)	0.014 ± 0.019 (0.000 -0.065)	0.174 ± 0.245 (0.035 -1.369)
	Dicentrarchus labrax	RZ	muscle	1	349	346.6	0.000	0.675	0.000	0.002	0.027	0.222
	Diplodus sargus	IZ	muscle	4	269 ± 21 (235 -289)	357.2 ± 86.9 (214.6 -433.3)	0.026 ± 0.051 (0.000 -0.102)	5.28 ± 2.908 (2.203 -9.221)	0.001 ± 0.000 (0.001 -0.001)	0.004 ± 0.001 (0.003 -0.004)	0.016 ± 0.012 (0.000 -0.028)	0.756 ± 0.364 (0.429 -1.264)
	Diplodus sargus	RZ	muscle		245 ± 45 (173 -375)	308.7 ± 189.1 (101 -969.4)	0.385 ± 1.784 (0.000 -11.076)	11.911 ± 6.165 (1.218 -24.651)	0.001 ± 0.001 (0.000 -0.005)	0.007 ± 0.008 (0.000 -0.047)	0.009 ± 0.016 (0.000 -0.073)	0.429 ± 0.296 (0.084 -1.369)
	Diplodus vulgaris	IZ	muscle		226 ± 20 (189-279)	203.5 ± 59.0 (83.6 -359.4)	0.262 ± 0.154 (0.000 -0.683)	4.346 ± 1.188 (1.585 -7.514)	0.001 ± 0.005 (0.000 -0.024)	0.003 ± 0.002 (0.000 -0.010)	0.016 ± 0.045 (0.000 -0.228)	0.442 ± 0.176 (0.124 -0.786)
	Diplodus vulgaris	RZ	muscle		213 ± 24 (149 -249)	164.0 ± 56.8 (46.9 -283.9)	0.052 ± 0.083 (0.000 -0.304)	6.919 ± 3.132 (3.209 -15.573)	0.001 ± 0.001 (0.000 -0.003)	0.005 ± 0.002 (0.000 -0.011)	0.016 ± 0.019 (0.000 -0.096)	0.389 ± 0.159 (0.082 -0.798)
	Engraulis encrasicolus	IZ	muscle		139 ± 8 (121-157)	16.8 ± 2.9 (10.2 -25.5)	0.030 ± 0.074 (0.000 -0.363)	3.804 ± 0.759 (1.688 -5.900)	0.005 ± 0.002 (0.002 -0.009)	0.01 ± 0.002 (0.006 -0.017)	0.019 ± 0.018 (0.000 -0.059)	0.119 ± 0.046 (0.035 -0.247)
	Engraulis encrasicolus	RZ	muscle	6	118 ± 11 (100 -129)	10.6 ± 2.7 (5.5 -12.5)	1.769 ± 2.503 (0.160 -5.711)	4.664 ± 0.846 (3.830 -6.252)	0.005 ± 0.002 (0.000 -0.007)	0.016 ± 0.004 (0.011 -0.022)	0.035 ± 0.019 (0.017 -0.070)	0.084 ± 0.02 (0.065 -0.118)
	Galeus melastomus	IZ	muscle		459 ± 81 (323 -578)	283.4 ± 147.3 (81.8 -545.7)	0.574 ± 0.583 (0.000 -3.090)	33.122 ± 7.735 (22.517 -55.173)	0.001 ± 0.002 (0.000 -0.006)	0.001 ± 0.002 (0.000 -0.007)	0.019 ± 0.017 (0.000 -0.074)	1.47 ± 0.842 (0.553 -3.833)
	Galeus melastomus	RZ	muscle	4	327 ± 31 (277 -354)	89.8 ± 23.1 (51.2 -110.0)	1.923 ± 1.016 (0.812 -2.829)	28.503 ± 9.637 (17.587 -41.050)	0.001 ± 0.000 (0.001 -0.001)	0.003 ± 0.001 (0.002 -0.005)	0.038 ± 0.005 (0.031 -0.042)	0.908 ± 0.139 (0.799 -1.089)
	Helicolenus dactylopterus	IZ	muscle		187 ± 31 (114 -248)	109.6 ± 51.2 (22.2 -264.9)	0.694 ± 0.683 (0.137-3.364)	5.281 ± 2.442 (2.111 -12.100)	0.001 ± 0.001 (0.000 -0.006)	0.001 ± 0.002 (0.000 -0.005)	0.029 ± 0.086 (0.000 -0.525)	1.045 ± 0.456 (0.273 -2.730)
	Helicolenus dactylopterus	RZ	muscle		194 ± 33 (139-291)	130.7 ± 72.2 (37.6 -402)	0.217 ± 0.301 (0.000 -1.839)	10.166 ± 5.484 (2.690 -25.008)	0.001 ± 0.001 (0.000 -0.003)	0.003 ± 0.002 (0.000 -0.010)	0.013 ± 0.024 (0.000 -0.137)	1.099 ± 0.368 (0.382 -2.059)
	Liza aurata	IZ	muscle		281 ± 24 (241-330)	193.3 ± 71.1 (111.7 -388.8)	0.064 ± 0.084 (0.000 -0.217)	0.561 ± 0.253 (0.285 -0.960)	0.000 ± 0.000 (0.000 -0.000)	0.01 ± 0.002 (0.006 -0.013)	0.027 ± 0.025 (0.000 -0.076)	0.014 ± 0.01 (0.000 -0.028)
	Liza aurata	RZ	muscle	7	328 ± 25 (282 -359)	307.3 ± 65.6 (212.6 -385.9)	0.050 ± 0.089 (0.000 -0.218)	1.302 ± 0.477 (0.832 -2.230)	0.000 ± 0.000 (0.000 -0.000)	0.011 ± 0.006 (0.000 -0.018)	0.024 ± 0.036 (0.000 -0.088)	0.021 ± 0.012 (0.013 -0.047)
	Liza ramada	IZ	muscle		319 ± 50 (239 -389)	288.7 ± 144.8 (105.7 -506.2)	0.591 ± 0.447 (0.101 -1.770)	0.992 ± 0.589 (0.243 -2.091)	0.000 ± 0.000 (0.000 -0.000)	0.006 ± 0.003 (0.002 -0.010)	0.019 ± 0.019 (0.000 -0.06)	0.094 ± 0.095 (0.007 -0.321)
	Merluccius merluccius	IZ	muscle		354 ± 132 (233 -795)	439.1 ± 518.4 (89.1 -2305.2)	0.460 ± 0.474 (0.000 -2.139)	6.512 ± 2.707 (3.353 -18.323)	0.002 ± 0.007 (0.000 -0.043)	0.001 ± 0.005 (0.000 -0.021)	0.005 ± 0.011 (0.000 -0.041)	0.454 ± 0.524 (0.08 -3.073)

Table 1 (continued):

 1 Mean TE concentrations ± standard deviation (in mg.kg -1 wm) measured in fish muscle from the zone (IZ) impacted by red mud and the reference (RZ) zones. Minimum and maximum values are given in brackets.

	Species	zone	tissue	N	Total length (mm)	Total mass (g)	Mn	Ni	Pb	Ti	V
	Chelon labrosus	IZ	muscle	1	406	860.3	0.069	0.000	0.005	0.001	0.001
	Conger conger	IZ	muscle 31	710 ± 171 (416 -1119)	799.5 ± 643.2 (94.5 -2894.4)	0.346 ± 0.260 (0.07 -1.035)	0.000 ± 0.000 (0.000 -0.000)	0.007 ± 0.007 (0.000 -0.031)	0.067 ± 0.114 (0.000 -0.54)	0.006 ± 0.0140 (0.000 -0.077)
	Conger conger	RZ	muscle 23	716 ± 203 (334 -1035)	817.5 ± 672.6 (50.8-2341.7)	0.515 ± 0.383 (0.058 -1.273)	0.000 ± 0.000 (0.000 -0.000)	0.006 ± 0.008 (0.000 -0.031)	0.070 ± 0.115 (0.000 -0.466)	0.004 ± 0.004 (0.000 -0.017)
	Coris julis	IZ	muscle 32	128 ± 1.9 (103 -212)	20.9 ± 12.5 (8.6 -83.5)	0.182 ± 0.058 (0.097 -0.304)	0.007 ± 0.024 (0.000 -0.109)	0.025 ± 0.019 (0.000 -0.076)	0.059 ± 0.06 (0.000 -0.254)	0.030 ± 0.017 (0.000 -0.090)
	Coris julis	RZ	muscle 31	125 ± 14 (86 -147)	17.0 ± 5.3 (5.1 -29.4)	0.196 ± 0.074 (0.107 -0.415)	0.003 ± 0.011 (0.000 -0.051)	0.014 ± 0.012 (0.000 -0.043)	0.114 ± 0.127 (0.000 -0.513)	0.048 ± 0.043 (0.007 -0.191)
	Dicentrarchus labrax	RZ	muscle	1	349	346.6	0.151	0.000	0.000	0.000	0.000
	Diplodus sargus	IZ	muscle	4	269 ± 21 (235 -289)	357.2 ± 86.9 (214.6 -433.3)	0.064 ± 0.01 (0.056 -0.076)	0.000 ± 0.000 (0.000 -0.000)	0.005 ± 0.007 (0.000 -0.015)	0.000 ± 0.000 (0.000 -0.000)	0.007 ± 0.003 (0.003 -0.011)
	Diplodus sargus	RZ	muscle 39	245 ± 45 (173 -375)	308.7 ± 189.1 (101 -969.4)	0.061 ± 0.032 (0.035 -0.177)	0.015 ± 0.065 (0.000 -0.382)	0.023 ± 0.121 (0.000 -0.757)	0.044 ± 0.086 (0.000 -0.509)	0.004 ± 0.004 (0.000 -0.020)
	Diplodus vulgaris	IZ	muscle 28	226 ± 20 (189-279)	203.5 ± 59.0 (83.6 -359.4)	0.115 ± 0.051 (0.067 -0.266)	0.017 ± 0.068 (0.000 -0.334)	0.024 ± 0.070 (0.000 -0.368)	0.019 ± 0.038 (0.000 -0.131)	0.016 ± 0.023 (0.000 -0.13)
	Diplodus vulgaris	RZ	muscle 53	213 ± 24 (149 -249)	164.0 ± 56.8 (46.9 -283.9)	0.102 ± 0.082 (0.032 -0.475)	0.000 ± 0.000 (0.000 -0.000)	0.025 ± 0.029 (0.000 -0.125)	0.019 ± 0.036 (0.000 -0.197)	0.019 ± 0.0260 (0.000 -0.154)
	Engraulis encrasicolus	IZ	muscle 60	139 ± 8 (121-157)	16.8 ± 2.9 (10.2 -25.5)	0.345 ± 0.091 (0.039 -0.533)	0.006 ± 0.025 (0.000 -0.157)	0.004 ± 0.002 (0.000 -0.012)	0.045 ± 0.051 (0.000 -0.287)	0.021 ± 0.012 (0.002 -0.073)
	Engraulis encrasicolus	RZ	muscle	6	118 ± 11 (100 -129)	10.6 ± 2.7 (5.5 -12.5)	0.478 ± 0.210 (0.262 -0.879)	0.028 ± 0.023 (0.000 -0.051)	0.072 ± 0.100 (0.000 -0.216)	0.115 ± 0.105 (0.039 -0.257)	0.032 ± 0.021 (0.018 -0.072)
	Galeus melastomus	IZ	muscle 32	459 ± 81 (323 -578)	283.4 ± 147.3 (81.8 -545.7)	0.118 ± 0.024 (0.081 -0.198)	0.007 ± 0.036 (0.000 -0.201)	0.053 ± 0.068 (0.005 -0.305)	0.191 ± 0.147 (0.054 -0.754)	0.003 ± 0.004 (0.000 -0.019)
	Galeus melastomus	RZ	muscle	4	327 ± 31 (277 -354)	89.8 ± 23.1 (51.2 -110.0)	0.210 ± 0.098 (0.140 -0.354)	0.000 ± 0.000 (0.000 -0.000)	0.141 ± 0.104 (0.035 -0.280)	0.218 ± 0.03 (0.182 -0.247)	0.005 ± 0.002 (0.002 -0.006)
	Helicolenus dactylopterus	IZ	muscle 37	187 ± 31 (114 -248)	109.6 ± 51.2 (22.2 -264.9)	0.096 ± 0.037 (0.055 -0.276)	0.006 ± 0.036 (0.000 -0.221)	0.086 ± 0.100 (0.006 -0.435)	0.127 ± 0.111 (0.000 -0.647)	0.004 ± 0.005 (0.000 -0.026)
	Helicolenus dactylopterus	RZ	muscle 46	194 ± 33 (139-291)	130.7 ± 72.2 (37.6 -402)	0.086 ± 0.025 (0.051 -0.167)	0.004 ± 0.019 (0.000 -0.103)	0.003 ± 0.003 (0.000 -0.012)	0.038 ± 0.031 (0.000 -0.105)	0.002 ± 0.002 (0.000 -0.009)
	Liza aurata	IZ	muscle 12	281 ± 24 (241-330)	193.3 ± 71.1 (111.7 -388.8)	0.094 ± 0.027 (0.064 -0.155)	0.010 ± 0.022 (0.000 -0.060)	0.001 ± 0.003 (0.000 -0.008)	0.021 ± 0.036 (0.000 -0.114)	0.003 ± 0.003 (0.000 -0.011)
	Liza aurata	RZ	muscle	7	328 ± 25 (282 -359)	307.3 ± 65.6 (212.6 -385.9)	0.093 ± 0.054 (0.057 -0.196)	0.018 ± 0.025 (0.000 -0.053)	0.008 ± 0.01 (0.000 -0.029)	0.010 ± 0.017 (0.000 -0.036)	0.007 ± 0.005 (0.000 -0.013)
	Merluccius merluccius	IZ	liver	48	364 ± 135 (233 -795)	471.6 ± 534.3 (97.7 -2305.2)	1.69 ± 0.545 (0.081 -2.748)	0.008 ± 0.027 (0.000 -0.147)	0.015 ± 0.019 (0.000 -0.107)	0.130 ± 0.229 (0.000 -1.27)	0.034 ± 0.030 (0.000 -0.135)
	Merluccius merluccius	IZ	muscle 53	354 ± 132 (233 -795)	439.1 ± 518.4 (89.1 -2305.2)	0.145 ± 0.194 (0.067 -1.151)	0.031 ± 0.186 (0.000 -1.314)	0.01 ± 0.044 (0.000 -0.308)	0.018 ± 0.042 (0.000 -0.229)	0.002 ± 0.003 (0.000 -0.022)

Table 2 :

 2 Mean TE concentrations ± standard deviation (in mg.kg -1 wm) measured in fish liver from the zone (IZ) impacted by red mud and the reference (RZ) zones. Minimum and maximum values are given in brackets.

	Species	zon e	Tissue	N	Total length (mm)	Total mass (g)	Al	As	Cd	Co	Cr	Hg
	Conger conger	IZ	liver	30	696 ± 157 (416 -1020)	729.7 ± 525.3 (94.5 -2161.8)	0.173 ± 0.131 (0.000 -0.535)	19.809 ± 9.831 (5.278 -48.487)	0.117 ± 0.082 (0.000 -0.263)	0.021 ± 0.007 (0.000 -0.032)	0.07 ± 0.084 (0.000 -0.377)	0.331 ± 0.203 (0.042 -1.033)
	Conger conger	RZ	liver	21	691 ± 195 (334 -1010)	817.5 ± 672.6 (50.8-2341.7)	0.334 ± 0.458 (0.000 -1.931)	24.035 ± 14.403 (6.931 -59.475)	0.117 ± 0.127 (0.001 -0.503)	0.019 ± 0.009 (0.000 -0.038)	0.073 ± 0.208 (0.000 -0.950)	0.286 ± 0.211 (0.055 -0.867)
	Merluccius merluccius	IZ	liver	48	364 ± 135 (233 -795)	471.6 ± 534.3 (97.7 -2305.2)	1.963 ± 2.563 (0.000 -14.479)	5.194 ± 1.859 (2.803 -12.569)	0.071 ± 0.101 (0.000 -0.514)	0.033 ± 0.016 (0.000 -0.072)	0.028 ± 0.044 (0.000 -0.256)	0.442 ± 0.729 (0.06 -4.644)
	Merluccius merluccius	RZ	liver	23	388 ± 51 (284 -476)	469.7 ± 178.9 (147.3 -858.4)	0.922 ± 0.699 (0.000 -3.13)	3.961 ± 1.217 (2.413 -8.010)	0.06 ± 0.049 (0.012 -0.208)	0.025 ± 0.012 (0.006 -0.053)	0.026 ± 0.054 (0.000 -0.165)	0.261 ± 0.219 (0.064 -0.972)
	Raja clavata	IZ	liver	21	605 ± 116 (440 -826)	1493.4 ± 940.0 (431.4 -3820.1)	0.819 ± 0.712 (0.000 -2.885)	33.368 ± 20.832 (13.88 -81.629)	0.291 ± 0.278 (0.001 -1.255)	0.04 ± 0.023 (0.000 -0.090)	0.005 ± 0.008 (0.000 -0.029)	0.666 ± 0.613 (0.261 -2.476)
	Raja clavata	RZ	liver	5	749 ± 68 (669 -849)	2293.3 ± 650.1 (1567.7 -3181.4)	1.539 ± 1.146 (0.463 -3.349)	30.499 ± 4.233 (26.315 -36.691)	0.346 ± 0.230 (0.130 -0.653)	0.078 ± 0.025 (0.046 -0.104)	0.006 ± 0.013 (0.000 -0.028)	0.669 ± 0.521 (0.237 -1.425)
	Scyliorhinus canicula	IZ	liver	60	417 ± 58 (244 -509)	244.1 ± 91.2 (39.4 -400.4)	1.873 ± 1.727 (0.238 -7.033)	29.800 ± 16.703 (3.827 -81.501)	0.202 ± 0.220 (0.000 -0.971)	0.059 ± 0.042 (0.003 -0.186)	0.036 ± 0.064 (0.000 -0.393)	1.129 ± 1.191 (0.121 -6.146)
	Scyliorhinus canicula	RZ	liver	81	370 ± 62 (244 -500)	182.7 ± 104.2 (41.3 -485.4)	2.594 ± 2.362 (0.466 -14.108)	27.772 ± 12.341 (2.900 -63.125)	0.167 ± 0.149 (0.001 -0.857)	0.068 ± 0.034 (0.026 -0.168)	0.036 ± 0.037 (0.000 -0.169)	0.496 ± 0.383 (0.077 -2.182)

Table 2 (

 2 continued): Mean TE concentrations ± standard deviation (in mg.kg -1 wm) measured in fish liver from the zone (IZ) impacted by red mud and the reference (RZ) zones. Minimum and maximum values are given in brackets.

	Species	zone	tissue	N	Total length (mm)	Total mass (g)	Mn	Ni	Pb	Ti	V
	Conger conger	IZ	liver	30	696 ± 157 (416 -1020)	729.7 ± 525.3 (94.5 -2161.8)	0.934 ± 0.214 (0.280 -1.361)	0.022 ± 0.045 (0.000 -0.186)	0.069 ± 0.044 (0.000 -0.169)	0.021 ± 0.043 (0.000 -0.151)	0.096 ± 0.064 (0.003 -0.331)
	Conger conger	RZ	liver	21	691 ± 195 (334 -1010)	817.5 ± 672.6 (50.8-2341.7)	1.119 ± 0.213 (0.768 -1.553)	0.000 ± 0.000 (0.000 -0.000)	0.102 ± 0.150 (0.008 -0.657)	0.055 ± 0.079 (0.000 -0.208)	0.131 ± 0.096 (0.006 -0.368)
	Merluccius merluccius	IZ	liver	48	364 ± 135 (233 -795)	471.6 ± 534.3 (97.7 -2305.2)	1.69 ± 0.545 (0.081 -2.748)	0.008 ± 0.027 (0.000 -0.147)	0.015 ± 0.019 (0.000 -0.107)	0.130 ± 0.229 (0.000 -1.27)	0.034 ± 0.030 (0.000 -0.135)
	Merluccius merluccius	RZ	liver	23	388 ± 51 (284 -476)	469.7 ± 178.9 (147.3 -858.4)	1.444 ± 0.383 (0.924 -2.629)	0.014 ± 0.037 (0.000 -0.113)	0.015 ± 0.025 (0.000 -0.117)	0.022 ± 0.035 (0.000 -0.095)	0.017 ± 0.012 (0.002 -0.042)
	Raja clavata	IZ	liver	21	605 ± 116 (440 -826)	1493.4 ± 940.0 (431.4 -3820.1)	0.798 ± 0.336 (0.129 -1.506)	0.003 ± 0.014 (0.000 -0.066)	0.015 ± 0.012 (0.000 -0.049)	0.043 ± 0.062 (0.000 -0.255)	0.051 ± 0.049 (0.000 -0.231)
	Raja clavata	RZ	liver	5	749 ± 68 (669 -849)	2293.3 ± 650.1 (1567.7 -3181.4)	0.859 ± 0.185 (0.571 -1.023)	0.013 ± 0.028 (0.000 -0.063)	0.019 ± 0.008 (0.008 -0.027)	0.036 ± 0.056 (0.000 -0.128)	0.081 ± 0.044 (0.04 -0.151)
	Scyliorhinus canicula	IZ	liver	60	417 ± 58 (244 -509)	244.1 ± 91.2 (39.4 -400.4)	0.873 ± 0.435 (0.208 -2.165)	0.012 ± 0.042 (0.000 -0.211)	0.026 ± 0.017 (0.003 -0.079)	0.078 ± 0.084 (0.000 -0.343)	0.038 ± 0.034 (0.002 -0.168)
	Scyliorhinus canicula	RZ	liver	81	370 ± 62 (244 -500)	182.7 ± 104.2 (41.3 -485.4)	1.147 ± 0.437 (0.488 -3.143)	0.011 ± 0.026 (0.000 -0.112)	0.029 ± 0.02 (0.007 -0.151)	0.074 ± 0.084 (0.000 -0.442)	0.129 ± 0.332 (0.007 -2.421)

Table 4 :

 4 Pearson correlation coefficients between TE concentrations and length in the muscle and the liver of fish collected in this study. Significant 698 differences are highlighted by bold characters ( * p < 0.05, ** p < 0.01, *** p < 0.001) 699 700

	Species	Tissue N IZ N RZ	Al	As	Cd	Co	Cr	Hg	Mn	Ni	Pb	Ti	V
	Conger conger	muscle	31	23	-0.057	0.56 ***	-0.036	-0.058	-0.337 *	0.535 ***	-0.293 *		-0.42 **	-0.469 ***	-0.290 *
	Coris julis	muscle	32	31	-0.072	-0.258 *	-0.044	-0.347 **	-0.067	0.07	-0.252*	-0.080	-0.163	-0.042	-0.458 ***
	Diplodus vulgaris	muscle	28	53	0.096	0.075	0.116	-0.162	-0.041	0.738 ***	-0.116	0.308 **	-0.342 **	-0.344 **	-0.125
	Helicolenus dactylopterus muscle	37	46	-0.251 *	-0.049	-0.101	-0.193	0.007	0.529 ***	-0.577 ***	0.021	-0.289 **	0.133	-0.241 *
	Merluccius merluccius	muscle	53	24	-0.320 **	-0.084	0.027	0.041	-0.016	0.851 ***	-0.043	-0.038	0.272 *	0.147	-0.077
	Mullus surmuletus	muscle	68	72	-0.167 *	0.136	0.077	0.181 *	0.13	0.470 ***	-0.004	0.052	-0.034	-0.043	0.122
	Pagellus bogaraveo	muscle	27	30	0.004	0.270 *	0.091	-0.274 *	-0.043	0.226	0.045	0.081	0.087	0.152	-0.033
	Scorpaena porcus	muscle	39	71	-0.363 ***	0.151	-0.171	-0.167	-0.046	0.338 ***	-0.163	-0.112	-0.311 **	-0.357 ***	-0.382 ***
	Scyliorhinus canicula	muscle	63	82	-0.120	0.250 **	0.206	0.119	-0.352 ***	0.467 ***	0.166 *	-0.167 *	-0.004	-0.343 ***	0.092
	Conger conger	liver	30	21	-0.197	0.472 ***	0.158	0.525 ***	-0.496 ***	0.444 **	0.151	-0.214	-0.270	-0.248	0.323 *
	Merluccius merluccius	liver	48	23	0.402 **	-0.186	0.583 ***	0.254*	-0.173	0.748 ***	-0.449 ***	-0.170	0.276 *	0.472 ***	0.555 ***
	Scyliorhinus canicula	liver	60	81	-0.182 *	0.370 ***	0.337 ***	0.299 ***	-0.309 ***	0.434 ***	-0.260 **	-0.211 *	0.1	-0.314 ***	0.177

* 701

Merluccius

Acknowledgements

This work received financial support from the Environment and Sustainable Development Ministry.

We are grateful to F. Ruchon and local fishermen for their help in fish sampling. Thanks are also due to C. Tomasino, D. Louedoc, C. Ravel, A. Vaugier and F. Chavanon for their assistance during the laboratory work.

 (243 -280) 264.7 ± 99.5 (194.4 -335.1) 12.62 ± 10.02 (5.53 -19.7)0.032 ± 0.002 (0.031 -0.033) 0.000 ± 0.000 (0.000 -0.000) 0.004 ± 0.005 (0.000 -0.007) 13.1 ± 10. 1 (5.99 -20.3)