Melanie Ourgaud
email: melanie.ourgaud@mio.osupytheas.fr

Sandrine Ruitton

Hugo Bourgogne

Paco Bustamante

Carine Churlaud

Gaël Guillou

Benoit Lebreton

Mireille L Harmelin-Vivien

Mélanie Ourgaud

Benoît Lebreton

Trace elements in a Mediterranean scorpaenid fish: Bioaccumulation processes and spatial variations

Keywords:

come

Introduction

Coastal marine environments represent high value eco-socio-systems, but they are also discharge and accumulation areas of anthropogenic compounds, such as trace elements (TEs) [START_REF] Matthai | Anthropogenic trace metals in sediment and settling particulate matter on a high-energy continental shelf (Sydney, Australia)[END_REF][START_REF] Cobelo-Garcıá | Land inputs of trace metals, major elements, particulate organic carbon and suspended solids to an industrial coastal bay of the NE Atlantic[END_REF]. Such chemical contamination leads to the alteration of marine ecosystems, with an impact at individual, species, population and community levels [START_REF] Fleeger | Indirect effects of contaminants in aquatic ecosystems[END_REF][START_REF] Halpern | A global map of human impact on marine ecosystems[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. The Mediterranean Sea being a semi-enclosed sea, it is predicted to be a particularly sensitive system to any change in its hydrographic conditions due to either climatic or anthropogenic forcings [START_REF] Bethoux | The Mediterranean Sea: a miniature ocean for climatic and environmental studies and a key for the climatic functioning of the North Atlantic[END_REF]The MERMEX Group, 2011). The biogeochemical cycling of TEs at the scale of the Mediterranean Sea is largely governed by atmospheric inputs and air-water exchanges [START_REF] Migon | Transfer of atmospheric matter through the euphotic layer in the northwestern Mediterranean: seasonal pattern and driving forces[END_REF][START_REF] Heimbürger | Trace metal concentrations in the North-western Mediterranean atmospheric aerosol between 1986 and 2008: seasonal patterns and decadal trends[END_REF]The MERMEX Group, 2011). However, in coastal zones and on continental shelves, rivers, such as the Rhône River in the Gulf of Lions, constitute the main input of terrigenous dissolved organic carbon (DOC) [START_REF] Sempéré | Carbon inputs of the Rhône River to the Mediterranean Sea: biogeochemical implications[END_REF] and particulate metals [START_REF] Elbaz-Poulichet | River Inputs of Metals and Arsenic[END_REF][START_REF] Cathalot | The fate of river organic carbon in coastal areas: A study in the Rhône River delta using multiple isotopic (δ 13 C, Δ 14 C) and organic tracers[END_REF]Cossa et al., this issue). In addition, TE input sources include submarine volcanoes, cold seepages and sub-marine freshwater inflows, as well as inputs from large coastal cities, industries, effluents, runoff and marine traffic [START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF].

Certain TEs have the capability to bioaccumulate within aquatic organisms from the environment because of their persistent nature, chemical speciation and bioavailability [START_REF] Macdonald | A new approach suggests that phenomena, such as bioconcentration, biomagnification, and bioaccumulation, result from two fundamental processes[END_REF][START_REF] Castro-González | Heavy metals: implications associated to fish consumption[END_REF]The MERMEX Group, 2011[START_REF] Vieira | Mercury, cadmium, lead and arsenic levels in three pelagic fish species from the Atlantic Ocean: intra-and inter-specific variability and human health risks for consumption[END_REF]. Certain TEs have unknown functions in biological systems and are considered to be potentially toxic to organisms at low concentrations, such as barium (Ba), cadmium (Cd), total mercury (T-Hg) and lead (Pb) [START_REF] Schroeder | Relation of trace metals to human health[END_REF][START_REF] Velusamy | Bioaccumulation of heavy metals in commercially important marine fishes from Mumbai Harbor, India[END_REF]. Other TEs are essential, such as arsenic (As), copper (Cu), chromium (Cr), nickel (Ni), and zinc (Zn), as they play a specific role in metabolism [START_REF] Authman | Metals concentrations in Nile tilapia Oreochromis niloticus (Linnaeus, 1758) from illegal fish farm in Al-Minufiya Province, Egypt, and their effects on some tissues structures[END_REF][START_REF] Bosch | Heavy metals in marine fish meat and consumer health: a review[END_REF] and are required by living organisms in small concentrations to achieve a normal ontogenetic development [START_REF] Merciai | Trace metal concentration and fish size: Variation among fish species in a Mediterranean river[END_REF]. However, essential TEs may also have toxic effects at elevated exposure levels [START_REF] Wood | An introduction to metals in fish physiology and toxicology: Basic principles[END_REF][START_REF] Vrhovnik | Potentially toxic contamination of sediments, water and two animal species in Lake Kalimanci, FYR Macedonia: relevance to human health[END_REF]. The sorption of TEs is affected by chemical (e.g., chemical speciation; [START_REF] Vrhovnik | Potentially toxic contamination of sediments, water and two animal species in Lake Kalimanci, FYR Macedonia: relevance to human health[END_REF], environmental (e.g., pH, temperature and turbidity; Castro-González and Méndez-Armenta, 2008) and biological factors. Contaminants can bioaccumulate during the life of organisms (increases with size or age) and biomagnify with increasing trophic level, but different patterns are observed depending on the contaminant and the organism [START_REF] Harmelin-Vivien | Differential biomagnification of PCB, PBDE, Hg and Radiocesium in the food web of the European hake from the NW Mediterranean[END_REF]. While the contamination of fish by TEs can come directly from the water through respiration or directly through the skin and membranes, the main way of TE inputs into fish is food through the consumption of contaminated prey [START_REF] Hall | Food as the dominant pathway of methylmercury uptake by fish[END_REF]. Bioaccumulation in fish is a complex process that depends on various biological parameters (species, size, age, sex, diet, trophic level and metabolism) [START_REF] Castro-González | Heavy metals: implications associated to fish consumption[END_REF][START_REF] Harmelin-Vivien | Differential biomagnification of PCB, PBDE, Hg and Radiocesium in the food web of the European hake from the NW Mediterranean[END_REF][START_REF] Wood | An introduction to metals in fish physiology and toxicology: Basic principles[END_REF], and differs among tissues due to differences in TE absorption, detoxification and storage mechanisms [START_REF] Has-Schön | Heavy Metal Profile in Five Fish Species Included in Human Diet, Domiciled in the End Flow of River Neretva (Croatia)[END_REF][START_REF] Storelli | Potential human health risks from metals (Hg, Cd, and Pb) and polychlorinated biphenyls (PCBs) via seafood consumption: estimation of target hazard quotients (THQs) and toxic equivalents (TEQs)[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]. Contaminants, such as TEs, emerged as useful ecotracers of trophic patterns and environmental parameters when used in combination with other chemical tracers, such as C and N isotopic compositions [START_REF] Dierking | Spatial patterns in PCBs, pesticides, mercury and cadmium in the common sole in the NW Mediterranean Sea, and a novel use of contaminants as biomarkers[END_REF][START_REF] Chouvelon | Species-and size-related patterns in stable isotopes and mercury concentrations in fish help refine marine ecosystem indicators and provide evidence for distinct management units for hake in the Northeast Atlantic[END_REF]Cresson et al., 2014bCresson et al., , 2015b)), as they provide complementary timeand space-integrated information on bioaccumulation events and trophic positions of fish in the food web.

Environmental monitoring programs for coastal ecosystems are developing in the Mediterranean Sea for providing scientific knowledge in the assessment of the health and sustainability of ecosystems [START_REF] Tomasello | Biochemical and bioaccumulation approaches for investigating marine pollution using Mediterranean rainbow wrasse, Coris julis (Linneaus 1798)[END_REF][START_REF] Copat | Heavy metals concentrations in fish and shellfish from eastern Mediterranean Sea: Consumption advisories[END_REF][START_REF] Miniero | Persistent toxic substances in Mediterranean aquatic species[END_REF][START_REF] Personnic | An ecosystem-based approach to assess the status of a Mediterranean ecosystem, the Posidonia oceanica seagrass meadow[END_REF][START_REF] Naccari | Toxic metals in pelagic, benthic and demersal fish species from Mediterranean FAO Zone 37[END_REF][START_REF] Bonito | Evaluation of the global impacts of mitigation on persistent, bioaccumulative and toxic pollutants in marine fish[END_REF]. Studies carried out on TE bioconcentration and/or biomagnification in Mediterranean species investigated magnoliophytes [START_REF] Pergent-Martini | Posidonia oceanica: a biological indicator of past and present mercury contamination in the Mediterranean Sea[END_REF][START_REF] Pergent | Mercury levels and fluxes in Posidonia oceanica meadows[END_REF][START_REF] Lafabrie | Trace metals assessment in water, sediment, mussel and seagrass speciesvalidation of the use of Posidonia oceanica as a metal biomonitor[END_REF][START_REF] Lafabrie | Utilization of the seagrass Posidonia oceanica to evaluate the spatial dispersion of metal contamination[END_REF], invertebrates [START_REF] Perez | Effects of 12 years' operation of a sewage treatment plant on trace metal occurrence within a Mediterranean commercial sponge (Spongia officinalis, Demospongiae)[END_REF][START_REF] Lafabrie | Trace metals assessment in water, sediment, mussel and seagrass speciesvalidation of the use of Posidonia oceanica as a metal biomonitor[END_REF][START_REF] Angeletti | Cadmium bioaccumulation in Mediterranean spider crab (Maya squinado): Human consumption andhealth implications for exposure in Italian population[END_REF], and fish [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF][START_REF] Copat | Evaluation of a temporal trend heavy metals contamination in Posidonia oceanica (L.) Delile, (1813) along the western coastline of Sicily (Italy)[END_REF][START_REF] Copat | Heavy metals concentrations in fish and shellfish from eastern Mediterranean Sea: Consumption advisories[END_REF]. However, most studies on fish concern continental shelf species and are focused on Hg contamination [START_REF] Harmelin-Vivien | Difference of mercury bioaccumulation in red mullets from the northwestern Mediterranean and Black seas[END_REF][START_REF] Wood | An introduction to metals in fish physiology and toxicology: Basic principles[END_REF][START_REF] Cossa | Influences of bioavailability, trophic position, and growth on methylmercury in hakes (Merluccius merluccius) from Northwestern Mediterranean and Northeastern Atlantic[END_REF]Cresson et al., 2014aCresson et al., , 2015aCresson et al., , 2015b)). Thus, there is a lack of knowledge on TE concentrations in fish species living in coastal waters of the northwestern Mediterranean Sea [START_REF] Uluozlu | Trace metal content in nine species of fish from the Black and Aegean Seas, Turkey[END_REF].

In France, the annual per capita fish consumption ranges from 30 to 60 kg (FAO, 2012;[START_REF] Vieira | Fish consumption recommendations to conform to current advice in regard to mercury intake[END_REF] and seafood has always been an important part of the diet of Mediterranean populations [START_REF] Faget | Le milieu marin méditerranéen: conflits, usages et représentations: le cas du golfe de Marseille (début XVIIIe-début XXe siècles)[END_REF].

Given the potential deleterious effects to human health related to the consumption of contaminated fish [START_REF] Domingo | Benefits and risks of fish consumption: Part I. A quantitative analysis of the intake of omega-3 fatty acids and chemical contaminants[END_REF][START_REF] Martorell | Human exposure to arsenic, cadmium, mercury, and lead from foods in Catalonia, Spain: temporal trend[END_REF][START_REF] Vieira | Mercury, cadmium, lead and arsenic levels in three pelagic fish species from the Atlantic Ocean: intra-and inter-specific variability and human health risks for consumption[END_REF], it is crucial to better understand the processes of TE accumulation in Mediterranean coastal fishes and to evaluate the subsequent risk due to fish consumption. The black scorpionfish, Scorpaena porcus Linnaeus, 1758, while not the most consumed species, is largely used in traditional recipes (fish soup, fried scorpionfish fillets and the famous "bouillabaisse") and is a common component of local fisheries all year round [START_REF] Leleu | Métiers, effort and catches of a Mediterranean small-scale coastal fishery: the case of the Côte Bleue Marine Park[END_REF]. This species is common in all of the Mediterranean Sea between 5 and 30 m depth, but may be occasionally recorded deeper. This a sedentary macrocarnivore living on rocky substrates and seagrass beds [START_REF] Harmelin-Vivien | Food partitioning among scorpaenid fishes in Mediterranean seagrass beds[END_REF]. It plays an important role in the trophic functioning of these ecosystems, being one of the most common predators and accounting for 18-35% of the total fish biomass [START_REF] Bell | Fish fauna of French Mediterranean Posidonia oceanica seagrass meadows. I: Community structure[END_REF][START_REF] Ourgaud | Response of a seagrass fish assemblage to improved wastewater treatment[END_REF]. For these characteristics, S. porcus could be used as an indicator of TE contamination in Mediterranean coastal fish species. Thus, the aim of the present study was to provide some baseline information on the concentration levels of different TEs (As, Ba, Cd, Cr, Cu, Ni, T-Hg, Pb, and Zn) susceptible to revealing anthropic influences in coastal marine fishes or being a threat to health security. For this purpose, S. porcus was collected from different locations subjected to different degrees of anthropic inputs. We used a multi-tracer approach (TEs, C and N stable isotopes, stomach contents and otolith analyses) and analyzed muscles that are the main portion of the fish eaten by consumers. The main questions addressed are (1) Did TE concentrations differ among sampling sites and were they related to differences in the biological parameters of fish or characteristics of the environment? (2) Were these differences similar for all TEs analyzed? (3) Could bioaccumulation and/or biomagnification processes be evident for some TEs? (4) Are TE concentrations recorded below European regulations for fish flesh commercialization?

Materials and methods

Study area and sample preparation

This study was carried out in Posidonia oceanica seagrass meadows in three sampling sites of the Bay of Marseille, in the vicinity of one of the largest cities on the Mediterranean Sea, and in one site of the Gulf of Hyères (HY), a less urbanized or industrialized area, considered as a reference site (Fig. 1). The three Marseille sites were subjected to different anthropic and freshwater inputs: Côte Bleue (CB), located in the northern area of the bay and not directly influenced by Marseille urban sewage, but sporadically exposed to the influence of Rhône River waters [START_REF] Petrenko | Circulation in a stratified and wind-forced Gulfof Lions, NW Mediterranean Sea: in situ and modeling data[END_REF]; Prado Bay (PB), subjected to episodic inputs from the small coastal Huveaune River and to constant urban and harbor inputs; and Plateau des Chèvres (PC), located in the southern area of the bay close to the outlet of the Cortiou wastewater treatment plant, and thus directly exposed to nutrient-rich urban sewage [START_REF] Bellan | Pollution by sewage in Marseille[END_REF][START_REF] Arfi | Cortiou: évolution d'un site marin soumis à un rejet urbain[END_REF].

Fish were sampled by fishermen operating a small beam trawl (1.5 × 0.5 m, 8 mm stretched mesh size) [START_REF] Harmelin-Vivien | Description d'un petit chalut à perche pour récolter la faune vagile des herbiers de posidonies[END_REF] during both day and night as diel differences occur in the activity rhythms, feeding behavior and position of fish associated with P. oceanica seagrass meadows [START_REF] Harmelin-Vivien | Ichtyofaune des herbiers de posidonies du Parc national de Port-Cros: 1. Composition et variations spatio-temporelles[END_REF]. Five trawls of 10 min duration at a constant towing speed of 2.8 km h -1 (combined surface area covered of 3500 m 2 ) were carried out between 8 and 18 m depth. A total of 588 individuals of S. porcus were collected. Once caught, fish were immediately stored on ice in polyethylene bags. In the laboratory, fish were measured to the nearest mm (total length, TL) before dissection and sex determination. Dissections were performed on an acid (10% HCl) cleaned glass worktop with ceramic scalpels and forceps, wearing powder free single use nitrile gloves. All instruments were cleaned with 10% HCl, rinsed with Milli-Q water between each sample and dried at 100 °C for 2 h. Dorsal white muscle samples of fish without skin were individually put into polyethylene bags and kept frozen at -20 °C until analysis. The number of fish processed per type of analysis is given in Table 1.

Stomach content analysis

Stomach contents were removed and stored in 95% ethanol before analysis. As ontogenic variations of diet are high in S. porcus [START_REF] Harmelin-Vivien | Food partitioning among scorpaenid fishes in Mediterranean seagrass beds[END_REF], individuals were separated into three size/age classes (see Section 2.1). Prey items in stomachs were sorted under a stereo microscope to the lowest possible taxonomic group and counted. The different prey categories were grouped according to fish size class, sampling site and period (day or night). Prey items were dried for 24 h at 50 °C and weighed ( ± 0.01 mg dry weight, dw). The importance of prey categories in the S. porcus diet was assessed by their percentage by number (%N), percentage by weight based on digested non-reconstituted prey weight (%W), percentage of occurrence (%O) and index of relative importance (%IR; [START_REF] Pinkas | Food habits of albacore, bluefin tuna, and bonito in California waters[END_REF], calculated as % IRI = (IRI/Σ IRI) × 100, where IRI = %O × (%N + %W).

Otolith analysis

The age (in years) of the individuals was estimated by the otolith analysis of individuals covering all size/age classes. Sagittal otoliths were removed, cleaned, weighed ( ± 0.1 mg) and their length ( ± 0.1 mm) was measured. As weight and length did not differ between right and left otoliths (Wilcoxon tests, p > .05), the left otolith was arbitrarily chosen for age reading. Sagittae were immersed in distilled water and observed by transparency using a stereo microscope under reflected light without any additional preparation (Fabien Morat, pers. comm.). Reading was done from the nucleus to the anterior extremity of the rostrum by counting annual annuli. One annulus was the combination of an opaque mark and a hyaline mark, as already observed in other Scorpaenidae [START_REF] Massutí | Growth variability of blue-mouth Helicolenus dactylopterus (Osteichthyes: Scorpaenidae) in two western Mediterranean areas: Alboran Sea and Balearic Sea[END_REF][START_REF] Mesa | Age and growth of Madeira scorpionfish, Scorpaena maderensis Valenciennes, 1833, in the central Mediterranean[END_REF][START_REF] Demirhan | Age, growth and food composition of Scorpaena porcus (Linnaeus, 1758) in the southeastern Black Sea[END_REF][START_REF] Scarcella | Comparison of growth rates estimated by otolith reading of Scorpaena porcus and Scorpaena notata caught on artificial and natural reefs of the northern Adriatic sea[END_REF]. Each otolith was analyzed by three different observers to obtain the fairest estimate of age. Correlation was established between the total otolith length (T o L, mm) and the age of the individuals (years).

Isotopic and trace element analyses

Samples of dorsal white muscle were freeze-dried and ground to a fine powder before analysis [START_REF] Pinnegar | Differential fractionation of δ 13 C and δ 15 N among fish tissues: implications for the study of trophic interactions[END_REF]. Measurements of C and N isotopic compositions were performed at the individual level with an elemental analyzer (Flash EA 1112, Thermo Scientific, Milan, Italy) coupled with an isotope ratio mass spectrometer (Delta V Advantage with a Conflo IV interface, Thermo Scientific, Bremen, Germany) at the LIENSs stable isotope facility of the University of La Rochelle, France. Briefly, samples of 0.40 ± 0.01 mg of freezedried powder were weighed and sealed in tin capsules for analysis. The results are expressed following the delta (δ) notation, as deviations from standards (Vienna Pee Dee Belemnite for δ 13 C, N 2 in air for δ 15 N):

δX (‰) = [(R sample /R standard ) -1] × 10 3 ,
where X is 13 C or 15 N and R is 13 C/ 12 C or 15 N/ 14 N, respectively. Analytical precision, based on the analyses of acetanilide (Thermo Scientific), used as laboratory internal standard was < 0.15‰ for carbon and nitrogen. Calibration was done using reference materials (USGS-24, IAEA-CH6, -600 for carbon; and IAEA-N2, -NO-3, -600 for nitrogen). The C/N ratio provided by the elemental analyzer was used as a proxy of the lipid content. A value of the C/N ratio lower than 3.7 indicates that the lipid content is low and does not affect the isotopic composition of carbon [START_REF] Sweeting | Effects of chemical lipid extraction and arithmetic lipid correction on stable isotope ratios of fish tissues[END_REF]. The δ 15 N value of individuals was used as a proxy of their trophic position, as commonly done [START_REF] Newsome | A niche for isotopic ecology[END_REF][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] Sebastiano | High levels of mercury and low levels of persistent organic pollutants in a tropical seabird in French Guiana, the Magnificent frigatebird, Fregata magnificens[END_REF].

The analysis of As, Ba, Cd, Cr, Cu, T-Hg, Ni, Pb and Zn in fish was performed at the individual level for the biggest fish and on pools of individuals (from the same site, size/age class and sex) for the smallest ones. The required mineralization of samples was performed on aliquots of ∼250 mg digested in 3 mL (for masses lower than 100 mg) or 5 mL (for masses higher than 100 mg) of a 3:1 (v:v) mixture of 65% HNO 3 (6 mL) and 37% HCl (2 mL) (Merck Suprapur® quality). Acid mineralization was performed overnight, at room temperature, and then in a microwave for 30 min with increasing temperature up to 120 °C, and 15 min at 120 °C (1200 W). After the mineralization process, each sample was diluted to 50 mL with Milli-Q water, according to the volume of acid added to the mineralization. Elements were analyzed using an ICP-OES (Vista-Pro, Varian, Mulgrave, Victoria Australia) or an ICP-MS (X SeriesII, Thermo Fisher Scientific, Bremen, Germany). T-Hg does not require a mineralization step and so it was directly analyzed from the dried samples by atomic absorption spectrometry with an Advanced Mercury Analyzer (AMA 254, Altec, Prague, Czech Republic) as described by [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North Eastern Atlantic waters: influence of geographical origin and feeding ecology[END_REF].

Precision and repeatability of TE analyses were assessed with certified reference materials (dogfish liver DOLT-4 and lobster hepatopancreas TORT-2; NRCC), treated and analyzed in the same way as the samples. The results were in good agreement with the certified values, and the relative standard deviations were always below 15%, proving good repeatability of the method. Blanks were tested in each analytical batch. All element concentrations were given on a dry weight basis (µg g -1 dw). The detection limits (µg g -1 ) were 0.0008 (T-Hg), 0.003 (Cd and Pb), 0.01 (Cr, Cu, and Ni), 0.03 (As and Ba), and 0.52 (Zn).

Statistical analyses

Spatial differences in diet composition were tested using Spearman's rank correlations coefficient on the%IRI of prey categories. Correlations between size and carbon and nitrogen isotopic compositions in fish muscle were assessed using generalized linear models (GLMs; [START_REF] Legendre | Numerical Ecology[END_REF]. GLMs were also used to analyze the correlation between fish size/age classes δ 15 N values or TE concentrations.

The variability in size, age, isotopic compositions and TE levels were analyzed independently using a univariate PERMANOVA [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. The PERMANOVA routine was performed for testing the simultaneous responses of one (δ 13 C, δ 15 N, and each TE) or more (nine TEs) variables to all factors (site, class and sex) in an analysis of variance (ANOVA and MANOVA) based on an experimental design of any resemblance measure using permutation methods [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF][START_REF] Mcardle | Fitting multivariate models to community data: a comment on distance-based redundancy analysis[END_REF][START_REF] Anderson | PERMANOVA+ for PRIMER: Guide to Software and Statistical Methods[END_REF]. The variables were normalized prior to analyses. Euclidean distance similarity matrices were generated. This method is equivalent to a parametric MANOVA, but free from the assumptions of normality and the homoscedasticity of residuals [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. P-values were obtained using 9999 random unrestricted permutations of raw data and Type I sums of squares. Monte Carlo P-values were considered when there were not enough possible permutations (< 2000). Significance was set at P-value less than 0.05 ( * = 0.05, * * = 0.01, and * * * = 0.001). The differences in mean TE concentrations between site, class and sex were described by a non-metric Multi-Dimensional Scaling ordination (nMDS; Clarke 1993; Clarke and Gorley 2006) calculated by the dissimilarity matrix among samples on the basis of the binomial dissimilarity measure [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF]. If the stress levels from 2-dimensional ordination plots were lower than 0.2, the analyses were validated [START_REF] Clarke | Non-parametric multivariate analyses of changes in community structure[END_REF]. Then, correlation vectors were calculated with the Spearman rank correlation index. Lastly, a multivariate PERMANOVA [START_REF] Anderson | A new method for non-parametric multivariate analysis of variance[END_REF] was performed, based on the binomial deviance (scaled) dissimilarity measures. Analyses were performed using PRIMER 6 software (6.1.16) and the PERMANOVA add-on (1.06) (Plymouth Marine Laboratory, Plymouth, United Kingdom) (Clarke and Warwick, 2001).

Results

Size, age and sex ratio

A total of 588 individuals were sampled (78 at CB, 110 at PB, 209 at PC, and 191 at HY). Fish were classified into three classes corresponding to three age and size classes: (C1) juveniles (aged 0+, < 70 mm TL); (C2) sub-adults (1-3 years, 71-130 mm TL); and (C3) adults (3-6 years, > 131 mm TL) (Table 2). The average total length for all individuals was 109 mm ( ± 29 mm) for a mean age of 2 years ( ± 1 year). A significant difference among sampling sites was observed, with larger and older individuals at HY than at the three Marseille (i.e., PB, CB, PC) sites (PERMANOVA, F = 14.19, p < .001, HY > PB = CB = PC, for TL; and PERMANOVA, F = 17.25, p < .001, HY > PB = PC = CB, for age). The sex ratio was well balanced (51% females; 49% males) and independent of sampling site (PERMANOVA, F = 1.28, p > .05). Fish with indeterminate sex represented 20% of samples, with the predominance of juvenile individuals in the smallest size/age class.

Diet

Out of the 588 individuals analyzed, 103 had empty stomachs (27%) and 18 contained prey items at an advanced state of decomposition (3%) that did not allow correct taxonomic identification. The results were therefore obtained from the analysis of 467 stomach contents. Diet composition differed significantly between size/age classes (Spearman rS, all p < .05), indicating a gradual change in S. porcus diet with the growth of individuals and an increase in mean prey size (Table 3). Juveniles preyed mainly on small crustaceans, such as small Caridea, isopods and amphipods, while subadults and adults consumed more diversified and larger prey, such as Brachyura and Teleostei. However, Caridea remained the principal prey in all size/age classes. Subadults consumed less Isopoda and Amphipoda and more Brachyoura, Galatheidae and Teleostei than juveniles. The diet of adults mainly consisted of Caridea, Teleostei and Brachyura. If the prey categories consumed by S. porcus were the same at all sampling sites, with Caridea always preferentially consumed, their relative proportions in the fish diet differed significantly among sites, with individuals from PB and PC differing from those of CB and HY. S. porcus from PB and PC presented similar diets (rS = 0.79, p > .05), with higher proportions of Caridea (65.0 and 81.7%IRI, respectively) and Amphipoda (14.0 and 10.0% IRI, respectively) consumed, than at CB and HY (Caridea: 33.2 and 57.4%IRI, respectively; and Amphipoda: < 3% IRI). In contrast, the diet of S. porcus was similar at CB and HY (rS = 0.71, p > .05), where the species ingested higher proportions of Isopoda (15.5% and 25.9% IRI, respectively), Galathea (> 3.0%IRI) and Teleostei (8.7 and 3.0% IRI, respectively), than at PB and PC (Isopoda: < 2.0%IRI; Galathea: < 1.0%IRI; and Teleostei: < 2.0%IRI).

Stable isotope compositions

In this study, the mean C/N ratio of S. porcus muscle was 3.2 ± 0.1 (Table 4), so no correction was applied on δ 13 C values for lipids. A wide range of δ 13 C values was observed among all S. porcus analyzed (from -18.8 to -16.5‰, Δ δ 13 C = 2.3‰) and δ 15 N values (from 6.4 to 12.0‰, Δ δ 15 N = 5.6‰). Such variations were related to differences among both sites and size/age classes (Fig. 2). Carbon isotopic compositions were significantly less enriched for individuals collected at PC and PB than at CB and HY (PERMANOVA, F = 37.43, for δ 13 C; p < .001), and δ 15 N values were significantly lower at HY than at CB, PB and PC (PERMANOVA, F = 57.74, for δ 15 N, p < .001) (Table 4). In addition, juveniles exhibited significantly lower δ 13 C and δ 15 N values than adults, regardless of the site (PERMANOVA, F = 4.79, p < .01, for δ 13 C; F = 21.06, p < .001 for δ 15 N). Significant correlations between length and δ 15 N values were observed at CB (R 2 = 0.55, p < .001), PB (R 2 = 0.34, p < .05), PC (R 2 = 0.64, p < .01) and HY (R 2 = 0.69, p < .001), while all correlations between TL and δ 13 C values were not significant (p > .05 at all sampling sites) (Fig. 3).

TE concentrations

All TEs analyzed were found in varying concentrations (Table 5). Ranges of detected concentrations for the nine elements quantified in S. 4a andb).

As, Ba, Cd, T-Hg, Pb, and Zn concentrations displayed significant differences among the four sampling sites (Table 5, Fig. 5). Pair-wise tests showed that the highest TE concentrations were not always measured in the same site. As concentration was significantly higher at PC than at the three other sites. Higher Ba concentrations were found at CB and PB than PC and HY, while the highest T-Hg concentrations were recorded in the Bay of Marseille at PB and PC. Concentrations of Cd, Pb and Zn were all higher at the three Marseille sites than at HY. Cr, Cu and Ni were the only elements that did not statistically differ in concentration with site. TE concentrations also displayed significant differences among fish size classes, except for Cr, Ni and Zn (PERMA-NOVA, F = 16.73, p < .05 for As; F = 103.82, p < .01 for Ba; F = 5.85, p < .01 for Cd; F = 26.62, p < .05 for Cu; F = 2115.00, p < .01 for T-Hg; and F = 1408.40, p < .01 for Pb), while sex had no effect on TE concentrations (results not shown, PERMANOVA, F = 5.46, p > .05). Pair-wise tests revealed that adults always presented the highest concentrations in As and T-Hg (Fig. 4). In contrast, Ba, Cd, Cu and Pb concentrations were significantly higher in either juveniles or in sub-adults. The potential trophic transfer of TEs was estimated using the relationships between trace element concentrations and δ 15 N values of individuals. Generally, a significant positive correlation between them indicates that the element is biomagnified through the food chain, whereas a significant negative correlation suggests that biodiminution has occurred. In the present study, for all individuals combined, significant correlations between age, size and δ 15 N values were observed for T-Hg (r = 0.88, p < .001, r = 0.84, p < .001, and r = 0.68, p < .01, respectively), but not for the eight other TEs analyzed. Positive, but not significant, correlations (p > .05) were observed for As and negative, but not significant, correlations (p > .05) for the other TEs.

Discussion

This study provided baseline information on the concentration status of nine TEs in the black scorpionfish Scorpaena porcus, a common sedentary fish representative of the spatial variation of coastal environments in the northwestern Mediterranean Sea. Three elements (Cd, Hg and Pb) have been more studied as they are subjected to international regulation [START_REF] Ec | portant fixation de teneurs maximales pour certains contaminants dans les denrées alimentaires[END_REF], while the others, (As, Cr, Cu, Ba, Ni, and Zn) have rarely been previously investigated in this region.

Influence of site

Contamination patterns are driven by local contamination sources, environmental factors and the biology of the species [START_REF] Cossa | Influences of bioavailability, trophic position, and growth on methylmercury in hakes (Merluccius merluccius) from Northwestern Mediterranean and Northeastern Atlantic[END_REF]Cresson et al., 2015a), and assessing a site-specific contamination pattern requires a good understanding of all these parameters. The quantification of TE concentrations in the muscle of S. porcus collected in seagrass beds, combined with the measurement of C and N isotopic compositions, allowed the discrimination of local populations from four coastal sites investigated in the northwestern Mediterranean Sea. It was also demonstrated that spatial patterns of TE concentrations differed among trace elements, but As and Zn were always the most concentrated TEs, and Ba, Pb, and Cd the least concentrated ones. Cr, Cu, and Ni were the only three TEs that displayed similar concentrations in S. porcus muscle at all sites. This could likely be related to the similar concentrations of these elements in the main crustacean prey species ingested by S. porcus at these sites (Ourgaud, unpublished data), as food is the main route of TE inputs in fish [START_REF] Hall | Food as the dominant pathway of methylmercury uptake by fish[END_REF]. On the reverse, the other TEs analyzed exhibited significant spatial variations. The highest As concentration was observed at PC, while higher T-Hg levels were recorded at PB and PC, and higher Ba levels at CB and PC. Cd, Pb and Zn were all observed in higher concentrations at the three sites of the Bay of Marseille compared to the site located at HY. These results reflected a higher anthropic influence in Marseille Bay than at Hyères, due to the size and scale of the activity of this large city, as TEs are commonly used in many industrial, harbor and urban activities (The MERMEX Group, 2011;[START_REF] Bonnard | Zinc et composés minéraux[END_REF][START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF][START_REF] Falcy | Mercure et composés minéraux[END_REF]. Moreover, the Bay of Marseille is subjected also to additional TE inputs related to the sporadic intrusions of Rhône River waters in the north of the bay [START_REF] Sempéré | Carbon inputs of the Rhône River to the Mediterranean Sea: biogeochemical implications[END_REF][START_REF] Fraysse | Intrusion of Rhône River diluted water into the Bay of Marseille: generation processes and impacts on ecosystem functioning[END_REF]. High concentrations of As and Zn are often recorded in marine fish and shellfish (Kim et al., 2007a;[START_REF] Copat | Heavy metals concentrations in fish and shellfish from eastern Mediterranean Sea: Consumption advisories[END_REF][START_REF] Vieira | Fish consumption recommendations to conform to current advice in regard to mercury intake[END_REF]. The high As concentrations observed in S. porcus at PC could be related to As inputs in the marine environment by Cortiou sewage waters [START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF] but also by high industrial effluents dumped into the Cassidaigne canyon located eastward, not far from PC and upstream from the main current [START_REF] Millot | Circulation in the Western Mediterranean Sea[END_REF]. The higher concentrations of Zn compared to those of other essential elements, partly resulted from its crucial importance for living organisms, which is well studied in fish [START_REF] Watanabe | Trace minerals in fish nutrition[END_REF][START_REF] Bury | Nutritive metal uptake in teleost fish[END_REF][START_REF] Pouil | Dietary Zn and the subsequent organotropism in fish: no influence of food quality, frequency of feeding and environmental conditions (pH and temperature)[END_REF]. However, the high concentrations of Zn recorded in S. porcus in Marseille sites could also be attributed to its high level in the local environment. Zn is considered as an anthropogenic component, used, for example, in Zn based sacrificial anodes of boat traffic and in other industrial activities [START_REF] Matthiessen | Sources and Potential Effects of Copper and Zinc Concentrations in the Estuarine Waters of Essex and Suffolk, United Kingdom[END_REF][START_REF] Hu | Assessing heavy metal pollution in the surface soils of a region that had undergone three decades of intense industrialization and urbanization[END_REF]. As with most contaminants, As and Zn are predominantly assimilated and bioaccumulated from dietary sources and surrounding waters [START_REF] Xu | Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus[END_REF][START_REF] Bury | Nutritive metal uptake in teleost fish[END_REF][START_REF] Mathews | Dominance of dietary intake of metals in marine elasmobranch and teleost fish[END_REF].

Spatial variations of TE concentrations in S. porcus could be related, in part, to differences in their isotopic compositions, themselves linked to differences in the isotopic values of organic matter (OM) sources among sites. First, the lower δ 13 C values found in S. porcus individuals at PC and PB in Marseille Bay could be explained by a higher influence of riverine OM inputs at these sites, as continental OM is known to be depleted in 13 C compared to marine OM [START_REF] Riera | Temporal variation of δ 13 C in particulate organic matter and oyster Crassostrea gigas in Marennes-Oléron Bay (France): effect of freshwater inflow[END_REF]Kim et al., 2007aKim et al., , 2007b;;[START_REF] Harmelin-Vivien | Comparison of C and N stable isotope ratios between surface particulate organic matter and microphytoplankton in the Gulf of Lions (NW Mediterranean)[END_REF]. Second, the higher δ 15 N values of fish from the three Marseille sites reflected an organic enrichment of the environment due to higher anthropogenic inputs in this area [START_REF] Darnaude | Trophodynamic linkage between river runoff and coastal fishery yield elucidated by stable isotope data in the Gulf of Lions (NW Mediterranean)[END_REF][START_REF] Cresson | Spatio-temporal variation of suspended and sedimentary organic matter quality in the Bay of Marseilles (NW Mediterranean) assessed by biochemical and isotopic analyses[END_REF]. The similar lower δ 13 C values of S. porcus at PC and PB could be related to local inputs of freshwater from the Cortiou sewage treatment plant and the Huveaune River, as already measured in coastal OM and phytoplankton at these sites by [START_REF] Rastorgueff | Feeding strategies and resource partitioning mitigate the effects of oligotrophy for marine cave mysids[END_REF] and [START_REF] Cresson | Spatio-temporal variation of suspended and sedimentary organic matter quality in the Bay of Marseilles (NW Mediterranean) assessed by biochemical and isotopic analyses[END_REF]. Fish from PC were exposed daily to the sewage waters of Cortiou, composed of the treated effluent from the Marseille conurbation and the waters of the Huveaune, Prado, and Jarret rivers. During heavy rains, the Huveaune River waters were directly discharged into Prado Bay and had an impact on fish from PB seagrass meadows [START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF]. At these sites, S. porcus presented particularly high concentrations of As, Pb, T-Hg and Zn, all TEs mainly related to anthropic activities but also Ba, which traces from a continental influence. In the area, the Rhône River is the most important source of Ba [START_REF] Salminen | Geochemical Atlas of Europe. Part I: Background Information, Methodology and Maps[END_REF], which may explain the highest Ba concentration in S. porcus recorded at CB, a site more subjected to the sporadic influence of this river than PB and PC [START_REF] Fraysse | Intrusion of Rhône River diluted water into the Bay of Marseille: generation processes and impacts on ecosystem functioning[END_REF]. Thus, differences in the proportions of terrestrial and marine organic matter inputs at the base of seagrass food webs between Marseille Bay and Hyères were reflected in the differences of isotopic compositions observed in S. porcus from the two areas (i.e., lower δ 13 C values and higher δ 15 N values at Marseille, higher δ 13 C values and lower δ 15 N values at Hyères), and in their concentrations of some trace elements (generally higher As, Ba, Cd, T-Hg, Pb, and Zn at Marseille linked to higher human and industrial pressures).

Influence of diet and trophic level

The dietary uptake is the dominant entry pathway of contaminants in most organisms [START_REF] Zhao | Biomagnification of radiocesium in a marine piscivorous fish[END_REF] and the main route for TE bioaccumulation [START_REF] Willis | Relative contributions of food and water in the accumulation of zinc by two species of marine fish[END_REF][START_REF] Hall | Food as the dominant pathway of methylmercury uptake by fish[END_REF][START_REF] Xu | Exposure and potential food chain transfer factor of Cd, Se and Zn in marine fish Lutjanus argentimaculatus[END_REF]. Contaminant concentration in organisms depends on many factors: trophic group, biological characteristics (e.g., species, swimming behavior, metabolic activities), uptake and elimination kinetics, chemical speciation and bioavailability of contaminants, local environmental conditions (salinity, temperature, pH, alkalinity, etc.), concentrations in water and sediments [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]. Bioavailability of TEs from ingested food is strongly influenced by the nature of the food and varies considerably among species (e.g., [START_REF] Luoma | Biological processes[END_REF]. As S. porcus is a sedentary species [START_REF] Bell | Fish fauna of French Mediterranean Posidonia oceanica seagrass meadows. II: Feeding habits[END_REF], isotopic compositions and TE concentrations measured in its muscles reflect its local feeding in Posidonia oceanica seagrass meadows. Its diet is mainly composed of Decapoda crustaceans and Teleostei. Spatial variability in fish diet could also be linked to spatial differences (nature and frequency) of organic matter inputs, and contributed to explain spatial differences in TE concentrations. The high similarity of fish diet at PC and PB could be related to the chronic inputs of terrestrial and sewage water POM [START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF], which favored the populations of detritivorous benthic invertebrates (Amphipoda, Caridea, Brachyoura, Anomura) [START_REF] Bellan | Mise en évidence de modèles éco-biologiques dans des zones soumises à pertubations par matières organiques[END_REF], found in large numbers in the stomach contents of S. porcus at these sites. The higher consumption of these detritivorous prey at PC and PB contributed in turn to a higher concentrations in some trace elements, such as Hg, in fish from these sites.

A significant increase of δ 15 N values with size and age was observed in this species at all sampling sites, while δ 13 C values did not differ. The trophic level increase (estimated by δ 15 N) in larger and older individuals was linked to ontogenetic modifications of their diet, with the consumption of prey of increasing size and trophic level when growing (Harmelin-Vivien et al., 1989, this paper), along with morphological, physiological and growth rate differences between juvenile and adult fish [START_REF] Perga | Laboratory measures of isotope discrimination factors: comments on Caut, Angulo and Courchamp[END_REF]. The increasing size of the mouth allows a broadening of the captured prey spectrum, resulting in the consumption of larger and more diverse prey, such as larger Caridea, Brachyoura and Teleostei [START_REF] Bell | Fish fauna of French Mediterranean Posidonia oceanica seagrass meadows. II: Feeding habits[END_REF][START_REF] Stergiou | Feeding habits and trophic levels of Mediterranean fish[END_REF][START_REF] Chouvelon | Species-and size-related patterns in stable isotopes and mercury concentrations in fish help refine marine ecosystem indicators and provide evidence for distinct management units for hake in the Northeast Atlantic[END_REF]Cresson et al., 2014b) in order to satisfy the increasing energy needs of the individuals [START_REF] Harmelin-Vivien | Food partitioning among scorpaenid fishes in Mediterranean seagrass beds[END_REF][START_REF] Karpouzi | The relationships between mouth size and shape and body length for 18 species of marine fishes and their trophic implications[END_REF]. The present study revealed major differences in TE concentrations with fish size/age class for most elements analyzed, while sex had no influence. Higher concentrations of As and T-Hg were recorded in adults, and higher concentrations of Ba, Cd, Cu, and Pb in juveniles and sub-adults, while no effect of fish size was observed for Ni, and Zn. However, significant correlations between TE concentrations and size, age and δ 15 N values of individuals were evident only for T-Hg, indicating that bioaccumulation during the life of S. porcus and biomagnification with its trophic level occurred only for T-Hg. No significant correlations with size, age or trophic level was observed for all the other elements.

Our results on T-Hg bioaccumulation and biomagnification were consistent with many studies that report positive correlations between δ 15 N values or trophic level, and total or organic Hg in various marine organisms and food webs [START_REF] Campbell | Mercury and other trace elements in a pelagic Arctic marine food web (Northwater Polynya, Baffin Bay)[END_REF][START_REF] Magalhães | Intra-and inter-specific variability in total and methylmercury bioaccumulation by eight marine fish species from the Azores[END_REF][START_REF] Ikemoto | Biomagnification of trace elements in the aquatic food web in the Mekong Delta, South Vietnam using stable carbon and nitrogen isotope analysis[END_REF][START_REF] Dierking | Spatial patterns in PCBs, pesticides, mercury and cadmium in the common sole in the NW Mediterranean Sea, and a novel use of contaminants as biomarkers[END_REF][START_REF] Nfon | Trophodynamics of mercury and other trace elements in a pelagic food chain from the Baltic Sea[END_REF][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 | Differential biomagnification of PCB, PBDE, Hg and Radiocesium in the food web of the European hake from the NW Mediterranean[END_REF][START_REF] Hao | Trace elements in fish from Taihu Lake, China: Levels, associated risks, and trophic transfer[END_REF][START_REF] Chouvelon | Species-and size-related patterns in stable isotopes and mercury concentrations in fish help refine marine ecosystem indicators and provide evidence for distinct management units for hake in the Northeast Atlantic[END_REF]. The absence of correlation between the concentrations of the other TEs and S. porcus descriptors (δ 15 N values, TL, age) indicated that they were neither biomagnified nor biodiminished during the fish ontogenetic development or through the food web. Similar results are reported in several studies [START_REF] Ikemoto | Biomagnification of trace elements in the aquatic food web in the Mekong Delta, South Vietnam using stable carbon and nitrogen isotope analysis[END_REF][START_REF] Zhang | Large-scale spatial and interspecies differences in trace elements and stable isotopes in marine wild fish from Chinese waters[END_REF][START_REF] Hao | Trace elements in fish from Taihu Lake, China: Levels, associated risks, and trophic transfer[END_REF], while other ones indicate a biodiminution of As, Cd and Pb in freshwater and marine ecosystems [START_REF] Asante | Trace elements and stable isotopes (δ 13 C and δ 15 N) in shallow and deep-water organisms from the East China Sea[END_REF]. Some laboratory studies suggest that low assimilation efficiencies and high efflux rates of Cd and Pb in marine organisms decrease their biomagnification potentials in food webs [START_REF] Wang | Interactions of trace metals and different marine food chains[END_REF], while others find evidence of Cd biomagnification in food webs [START_REF] Dietz | Comparison of contaminants from different trophic levels and ecosystems[END_REF]. The same controversy is observed for the relationship of As concentration with δ 15 N values in the North Pacific Ocean [START_REF] Asante | Trace elements and stable isotopes (δ 13 C and δ 15 N) in shallow and deep-water organisms from the East China Sea[END_REF]: biodiminution, versus Asante et al., 2010: biomagnification).

Sanitary risk assumption

Among the analyzed TEs, the European Commission only has set a limit for Cd, T-Hg and Pb [START_REF] Ec | portant fixation de teneurs maximales pour certains contaminants dans les denrées alimentaires[END_REF] in seafood (Cd: 0.05 µg g -1 wet weight (ww); Hg: 0.5 µg g -1 ww; and Pb: 0.3 µg g -1 ww). T-Hg is of priority concern because of its neurotoxic, immunotoxic and teratogenic effects in vertebrates [START_REF] Schlacher-Hoenlinger | Accumulation, contamination, and seasonal variability of trace metals in the coastal zone-patterns in a seagrass meadow from the Mediterranean[END_REF][START_REF] Tartu | To breed or not to breed: endocrine response to mercury contamination by an Arctic seabird[END_REF]. The values found in S. porcus from northwestern coastal sites in the present study for Cd (0.135 ± 0.092 µg g -1 ww), Hg (0.004 ± 0.001 µg g -1 ww) and Pb (0.005 ± 0.003 µg g -1 ww) are lower than the limits defined by the European Commission in fish muscle [START_REF] Ec | portant fixation de teneurs maximales pour certains contaminants dans les denrées alimentaires[END_REF].

Comparison with data available in the literature indicates that TE concentrations found in the muscle of S. porcus at Marseille Bay and Hyères seagrass meadows were lower than or similar to the values reported for this species by Ulzuolu et al. (2007) in the Aegean Sea or other marine fish (predators of invertebrates and small fish) in the Mediterranean Sea [START_REF] Harmelin-Vivien | Difference of mercury bioaccumulation in red mullets from the northwestern Mediterranean and Black seas[END_REF][START_REF] Ennouri | Metallic contamination of Red Mullet (Mullus barbatus) and Round Sardinella (Sardinella aurita) from the Gulf of Tunis/La contamination metallique du rouget de vase (Mullus barbatus) et de la sardinelle (Sardinella aurita) du golfe de tunis[END_REF]Cresson et al., 2014a) and other regions (e.g., [START_REF] Monteiro | Mercury content in relation to sex, size, age and growth in two scorpionfish (Helicolenus dactylopterus and Pontinus kuhlii) from Azorean waters[END_REF][START_REF] Miao | Comparative concentrations of metals in marine species from french frigate shoals, North Pacific Ocean[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] Briand | Spatial variability of metallic and organic contamination of anguilliform fish in New Caledonia[END_REF]. Concentrations in the muscle of S. porcus were not alarming. However, in order to assess the level of contamination for this species and the associated risk of consumption, additional analyses, i.e. speciation analyses of As and Hg, and TEs in different parts of fish (e.g., liver), are required.

Conclusion

The analysis of the concentrations of nine trace elements (TEs), combined with the determination of diet, age, isotopic compositions and trophic level of the black scorpionfish Scorpaena porcus enabled the separation of local populations at four sampling sites submitted to different level of anthropic pressures in the northwestern Mediterranean Sea. Thus, S. porcus appears to be a good candidate for the monitoring of environmental contamination in coastal waters, given its sedentarity, predatory feeding behavior, high trophic level and its high abundance in Mediterranean coastal habitats. The risk of potential adverse effects through the consumption of S. porcus in the south of France appears to be limited, because the concentrations recorded are lower than the toxicological reference values (Cd, Pb and Hg). However, significant bioaccumulation and bioamplification processes were observed for T-Hg and were nearly significant for As but not for the other TEs analyzed. Large-scale monitoring studies of S. porcus could thus represent an appropriate tool to assess the contamination status of the environment by these two elements (Hg and As), as well as their speciation, which present particular sanitary risks to human health associated with the consumption of seafood [START_REF] Nadal | Exposure to metals through the consumption of fish and seafood by the population living near the Ebro River in Catalonia, Spain: health risks[END_REF][START_REF] Lloret | Challenging the links between seafood and human health in the context of global change[END_REF].

Fig. 1 .

 1 Fig. 1. Location of the sampling sites along the northwestern Mediterranean Sea (gray points) (CB: Côte Bleue; PB: Prado Bay; PC: Plateau des Chèvres; and HY: Hyères). Cortiou: location of Marseille sewage output. Bathymetric data from SHOM, 2010.

Fig. 3 .

 3 Fig. 3. Linear between total length (TL, mm), and C and N isotopic compositions (‰) of Scorpaena porcus at the four sampling sites (white circle: CB; light gray square: PB; gray diamond: PC; and black triangle: HY). *** : p < .001; ** : p < .01; * : p < .05; and ns (nonsignificant): p > .05.

Fig. 4 .

 4 Fig. 4. (A) Two-dimensional nMDS ordination plot of trace element concentrations in the muscle of Scorpaena porcus according to sampling site (CB: Côte Bleue; PB: Prado Bay; PC: Plateau des Chèvres; and HY: Hyères); and (B) correlation vectors (binomial deviance resemblance) and 2D stress (0.09; good as < .2). Polygon ellipse lines are drawn for each size/age-class (white: juveniles; light gray: sub-adults; and dark gray: adults). Trace elements are indicated by sampling site symbols (white circle: CB; light gray square: PB; gray diamond: PC; and black triangle: HY).

Fig. 5 .

 5 Fig.5. Concentrations (Mean ± SD) of the nine trace elements (TEs, µg g -1 dw) in the muscle of Scorpaena porcus in the four sampling sites (in white, CB: Côte Bleue; in light gray, PB: Prado Bay; in dark gray, PC: Plateau de Chèvres; and in black, HY: Hyères). For convenience, TEs are ranked according to concentration ranges. Significant differences among sites are indicated by different letters (ns: not-significant, p > .05).

Table 1

 1 Number of individuals of the black scorpionfish Scorpaena porcus by sampling site (CB:

	Côte Bleue; PB: Prado Bay; PC: Plateau des Chèvres; and HY: Hyères), size/age class (C1:
	juveniles, < 70 mm total length (TL), aged 0+; C2: sub-adults 71-130 mm TL, 1-3 years;
	C3: and adults, > 131 mm TL, 3-6 years), and sex (U: undetermined; F: female; and M:
	male) for analyses of stomach contents, isotopic compositions (C and N) and trace ele-
	ments (TEs).
		Site				Class		Sex
		CB PB	PC	HY	C1 C2	C3 U	F	M
	Stomach contents 78	110 209 191 45 458 85 128 234 226
	Stable isotopes	18	16	13	21	17 34	17 34	18	16
	Trace elements	7	16	19	77	40 72	7	64	30	25

Table 2

 2 Mean ( ± SD) size and age of Scorpaena porcus according to sampling site and size/age class.

	Site	Class	Total length (mm)	Age (year)
	Côte Bleue (CB)	Juveniles	54 ± 12	0 +
		Sub-adults	105 ± 19	1.58 ± 0.79
		Adults	160 ± 27	3.67 ± 1.15
	Prado Bay (PB)	Juveniles	41 ± 5	0 +
		Sub-adults	86 ± 20	1.14 ± 0.69
		Adults	161 ± 8	3.75 ± 0.50
	Plateau des Chèvres (PC)	Juveniles	51 ± 10	0 +
		Sub-adults	100 ± 14	1.40 ± 0.55
		Adults	159 ± 21	4.00 ± 0.82
	Hyères (HY)	Juveniles	67 ± 2	0.20 ± 0.45
		Sub-adults	101 ± 10	1.50 ± 0.53
		Adults	152 ± 25	3.67 ± 1.03

Table 3

 3 Diet composition of Scorpaena porcus according to size/age classes. Dietary indices:% frequency of occurrence (%FO),% by number (%N),% by dry weight (%W) and% index of relative importance (%IRI) of each prey category. Others: Stomatopoda, Mollusca, crustacean remains and particulate organic matter. Numbers of stomachs with prey are indicated in brackets.Table 4C and N isotopic compositions and C/N (Mean ± SD) of Scorpaena porcus muscles according to sampling site and class.

	Prey items	Juveniles (43)			Sub-adults (3 6 3)			Adults (79)
		%FO	%N	%W	%IRI	%FO	%N	%W	%IRI	%FO	%N	%W	%IRI
	Crustacea
	Amphipoda	25.0	22.2	5.1	15.6	9.5	7.5	1.4	3.6	0.6	0.5	< 0.1	< 0.1
	Isopoda	26.3	22.8	25.4	24.5	12.9	10.3	4.9	7.3	7.6	7.8	2.8	4.2
	Caridea	40.8	48.7	65.1	57.6	35.4	46.8	42.2	56.6	31.3	38.4	26.4	40.9
	Brachyura	-	-	-	-	8.8	8.2	19.3	9.4	19.8	16.5	31.7	17.8
	Galathea	1.2	0.7	2.5	0.5	8.1	6.0	13.0	7.4	7.9	7.2	3.6	4.0
	Annelida	1.6	1.6	0.8	0.5	1.9	2.3	0.5	0.3	0.6	0.5	< 0.1	< 0.1
	Teleostei	-	-	-	-	4.2	2.8	11.0	4.8	10.8	10.3	29.5	19.2
	Others	5.0	4.0	1.0	1.4	19.3	16.0	7.8	10.7	21.9	19.0	6.0	13.7

Fig. 2. C and N isotopic compositions (‰) in the muscle of Scorpaena porcus according to sampling site (○ CB: Côte Bleue; □ PB: Prado Bay; ♢ PC: Plateau des Chèvres; and Δ HY: Hyères) and size/age classes of individuals (white: juveniles < 70 mm total length, aged 0+; gray: sub-adults 71-130 mm, 1-3 years; and black: adults > 131 mm, 3-6 years).

Table 5

 5 Comparison of trace element concentrations (Mean ± SD, µg•g -1 dw) in the muscle of Scorpaena porcus among sampling sites. Comparisons were performed by PERMANOVA and the Pair-wise column refers to the sites compared. *** : p < .001; ** : p < .01; * : p < .05; and ns (non-significant): p > .05.

		Côte Bleue (CB)	Prado Bay (PB)	Plateau des Chèvres (PC)	Hyères (HY)	PERMANOVA	p	Pair-wise test (site)
	As	25.63 ± 14.19	31.27 ± 23.77	89.53 ± 48.16	45.59 ± 11.72	F = 15.99	*	PC > HY -PB -CB
		(17.44-42.01)	(11.15-57.50)	(54.72-144.5)	(26.37-66.30)
	Ba	0.30 ± 0.09	0.26 ± 0.10	0.23 ± 0.06	0.22 ± 0.01	F = 84.23	**	CB -PB > PC -HY
		(0.19-0.35)	(0.19-0.37)	(0.18-0.30)	(0.20-0.23)
	Cd	0.029 ± 0.010	0.025 ± 0.012	0.021 ± 0.014	< 0.020	F = 4.97	**	CB -PB -PC > HY
		(0.02-0.04)	(0.02-0.04)	(0.02-0.03)	-
	Cr	0.64 ± 0.28	2.72 ± 4.16	1.75 ± 0.94	2.78 ± 3.32	F = 0.93	ns
		(0.48-0.96)	(0.18-7.53)	(0.83-2.70)	(0.15-8.73)
	Cu	0.58 ± 0.01	0.50 ± 0.15	0.60 ± 0.10	0.56 ± 0.16	F = 3.44	ns
		(0.57-0.58)	(0.35-0.65)	(0.49-0.68)	(0.38-1.04)
	Hg	0.70 ± 0.47	1.03 ± 1.01	0.93 ± 0.86	0.48 ± 0.27	F = 196.07	**	PB -PC > CB -HY
		(0.42-1.24)	(0.44-2.19)	(0.30-1.91)	(0.24-1.35)
	Ni	0.33 ± 0.05	0.36 ± 0.39	0.52 ± 0.32	1.33 ± 1.87	F = 0.38	ns
		(0.28-0.36)	(0.10-0.81)	(0.23-0.87)	(0.10-4.69)
	Pb	0.029 ± 0.010	0.025 ± 0.012	0.028 ± 0.019	0.023 ± 0.005	F = 1090.70	**	PC -CB -PB > HY
		(0.02-0.04)	(0.02-0.04)	(0.02-0.07)	(0.02-0.03)
	Zn	48.66 ± 5.45	61.62 ± 12.95	46.89 ± 15.85	15.57 ± 1.92	F = 619.20	***	PB -CB -PC > HY
		(45.52-54.95)	(53.96-76.56)	(29.48-60.50)	(13.01-19.07)

Acknowledgments

This study was carried out as a part of WP2 MERMEX-MISTRALS and is a contribution to the international IMBER and LOICZ projects. We are grateful to R. Sempéré, MERMEX co-PI, and to F. Carlotti and F. Van Wambeke, co-leaders of WP2 MERMEX, for helpful suggestions on the manuscript. This study forms part of the CONTAM project of M I O. This work was funded by the OHM "Littoral méditerranéen" project of the CNRS/AMU -DRIIHM LabEx. M. O. acknowledges financial support from the Provence-Alpes-Côte-d'Azur Region (PhD grant), the city of Marseille, and the Rhône-Mediterranean and Corsica Water Agency. Thanks are express to the Calanques National Park, Côte Bleue MPA and the fishermen of Hyères. The authors are grateful to the crews of the R/ V Antedon II and L'armandia and SAM-M I O platform for their operation at sea. We thank M. Brault-Fravou from the platform Analyses Elémentaires of LIENSs for her support in trace element analysis. The LIENSs laboratory acknowledges the CPER (Contrat de Projet Etat-Région) for funding the IR-MS, the ICPs and the AMA. The IUF (Institut Universitaire de France) is acknowledged for its support to PB. Finally, the authors are indebted to three anonymous reviewers for their detailed comments, improvements and helpful suggestions on a previous version of the manuscript.