O El Idrissi
email: el-idrissi_o@univ-corse.fr

S Ternengo

B Monnier

G Lepoint

A Aiello

R Bastien

R Lourkisti

M Bonnin

J Santini

V Pasqualini

Idrissi O El

Assessment of trace element contamination and effects on Paracentrotus lividus using several approaches: pollution indices, accumulation factors and biochemical tools

Keywords: Paracentrotus lividus, Asbestos mine, Trace Element Pollution Index, Bioaccumulation, Biomagnification, Oxidative stress

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Introduction

Coastal ecosystems are at the interface between the marine and terrestrial domains and provide many goods and ecosystem services of high value [START_REF] Costanza | Twenty years of ecosystem services: How far have we come and how far do we still need to go?[END_REF]. Coastal marine waters are particularly vulnerable to a wide range of disturbances such as climate change effects and the release into the sea of a wide variety of contaminants (Livingston et al., 1994;[START_REF] Mostofa | Sources, factors, mechanisms and possible solutions to pollutants in marine ecosystems[END_REF]. These habitats are subject to an increase in anthropogenic activities that can lead to high levels of contamination (Islam & Tanaka, 2004). The presence of these contaminants can have a devastating effect on organisms and their ecosystems, making marine pollution a global concern [START_REF] Abdel-Shafy | A review on polycyclic aromatic hydrocarbons: source, environmental impact, effect on human health and remediation[END_REF]Torres et al., 2016).

Among the major pollutants, trace elements are a real ecological issues because of their toxicity, their persistence and their ability to accumulate in marine organisms and even be biomagnified through the trophic chain [START_REF] Rainbow | Metal toxicity, uptake and bioaccumulation in aquatic invertebrates -Modelling zinc in crustaceans[END_REF][START_REF] Bonanno | Trace element compartmentation in the seagrass Posidonia oceanica and biomonitoring applications[END_REF]. The inputs of trace elements in marine environment derived from natural geogenic pollution (geological phenomena) and/or anthropogenic sources (e.g. mining, agriculture, petrochemical industry, aquaculture, sewage waste water) result in increased concentration levels (D [START_REF] Currie | Nomenclature in evaluation of analytical methods including detection and quantification capabilities (IUPAC Recommendations 1995)[END_REF][START_REF] Barhoumi | Assessment of pollution in the Bizerte lagoon (Tunisia) by the combined use of chemical and biochemical markers in mussels, Mytilus galloprovincialis[END_REF]. The trace elements can be classified as essential or nonessential [START_REF] Jiang | Distribution and pollution assessment of heavy metals in surface sediments in the Yellow Sea[END_REF]. Essential elements have a biological function in organisms, such as copper, iron and zinc and non-essential trace elements are not involved in any metabolic mechanism, such as cadmium, mercury and lead [START_REF] Amiard | Les risques chimiques environnementaux : méthodes d'évaluation et impacts sur les organismes[END_REF]. Above a certain threshold, all trace elements present a potential threat for organisms [START_REF] Nordberg | Handbook on the Toxicology of Metals[END_REF]. As a result of the threats caused by trace elements, continuous monitoring of their presence and concentration must be undertaken [START_REF] Richir | A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips[END_REF].

The Mediterranean Sea, which represents 0.82% of the global ocean surface area, is one of the main hotspots of marine biodiversity in the world, with 4-18% of identified marine species [START_REF] Coll | The Biodiversity of the Mediterranean Sea: Estimates, Patterns, and Threats[END_REF]. However, the coastal areas surrounding this semienclosed sea are subjected to the increasing synergistic effects of global changes and intensive anthropogenic activities leading to significant alterations in the structure and functioning of trophic webs and to the degradation of coastal and marine ecosystems [START_REF] Coll | The Mediterranean Sea under siege: spatial overlap between marine biodiversity, cumulative threats and marine reserves[END_REF][START_REF] Ramírez | Spatial congruence between multiple stressors in the Mediterranean Sea may reduce its resilience to climate impacts[END_REF]Stock et al., 2018a). Located in the northwestern Mediterranean Sea, Corsica Island has long been considered as a pristine region with low anthropogenic disturbances resulting in low levels of contamination [START_REF] Gobert | Trace element concentrations in the apex predator swordfish (Xiphias gladius) from a Mediterranean fishery and risk assessment for consumers[END_REF]. However, recent studies have revealed major trace elements contamination near an old asbestos mine in northern Corsica [START_REF] Cary | Etude du fond hydrogéochimique de la Corse. Impacts des activités minières sur la qualité des eaux[END_REF][START_REF] Ternengo | Spatial variations in trace element concentrations of the sea urchin, Paracentrotus lividus, a first reference study in the Mediterranean Sea[END_REF][START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF]. These high levels originate from untreated material directly discharged into the sea during the active period of the asbestos mine between 1948and 1965(BRGM, 2017;;[START_REF] Cary | Etude du fond hydrogéochimique de la Corse. Impacts des activités minières sur la qualité des eaux[END_REF]. The continuous leaching of these mining residues, still present on the sides of the mine, contributes to the dispersal of nickel, chromium and cobalt along the coastline [START_REF] Galgani | Chemical contamination and sediment toxicity along the coast of Corsica[END_REF][START_REF] Kantin | Monitorage de la qualité des eaux et de l'environnement marin -Rapport final -Région Corse. Programme INTERREG IIIA Sardaigne / Corse/ Toscane, MONIQUA N°MCD[END_REF]. Apart from the threat associated with the old asbestos mine, this region is characterized by relatively low levels of anthropogenic pressure (i.e. industrial, agricultural and urban activities; ASR, 2011). Because of these specificities, this sector represents a privileged study area to assess the trace elements contamination from a specific source within the coastal ecosystems.

The bioaccumulation of trace elements by marine organisms can occur through various pathways such as absorption and/or adsorption directly from sediments and interstitial seawater but also through ingestion, which will potentially affect other species along the food chain [START_REF] Adamo | Distribution and partition of heavy metals in surface and sub-surface sediments of Naples city port[END_REF][START_REF] Chen | Distribution and accumulation of heavy metals in the sediments of Kaohsiung Harbor, Taiwan[END_REF]Türkmen et al., 2008). In order to assess bioaccumulation and biomagnification to clarify the toxicity and fate of trace elements in organisms, it is important to determine the concentrations in the different biological and environmental matrices (water column, sediment, plants, animals;Jha, 2004). Echinoderms, and in particular sea urchins, are particularly suitable organisms for use in bioindication studies [START_REF] Parra-Luna | Common sea urchin (Paracentrotus lividus) and sea cucumber of the genus Holothuria as bioindicators of pollution in the study of chemical contaminants in aquatic media. A revision[END_REF]. Along the Mediterranean coast, Paracentrotus lividus (Lamarck, 1816) is frequently considered as a good bioindicator of local pollution because of its wide distribution, abundance, benthic behavior, sedentary habits, rapid response and its recognized sensitivity to a variety of pollutants [START_REF] Sugni | Endocrine disrupting compounds and echinoderms: New ecotoxicological sentinels for the marine ecosystem[END_REF][START_REF] Amri | Seasonal antioxidant responses in the sea urchin Paracentrotus lividus (Lamarck 1816) used as a bioindicator of the environmental contamination in the South-East Mediterranean[END_REF][START_REF] Rouhane-Hacene | Seasonal assessment of biological indices, bioaccumulation, and bioavailability of heavy metals in sea urchins Paracentrotus lividus from Algerian west coast, applied to environmental monitoring[END_REF]. Described as the organs that accumulate the most trace elements, its gonads and gut are of interest in studies assessing contamination levels in coastal marine ecosystems [START_REF] Augier | Teneurs en métaux lourds des oursins comestibles Paracentrotus lividus (Lamarck) prélevés dans quatre secteurs tests du littoral de Marseille (Méditerranée, France)[END_REF][START_REF] Warnau | Distribution of heavy metals in the echinoid Paracentrotus lividus from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations[END_REF][START_REF] Geraci | Nickel, lead, and cadmium induce differential cellular responses in sea urchin embryos by activating the synthesis of different HSP70s[END_REF]. P. lividus is also a species of economic importance due to its valued consumption in several countries (Fernández-Boán et al., 2013;[START_REF] Powell | Extraction and identification of antioxidant poly hydroxynaphthoquinone pigments from the sea urchin, Psammechinus miliaris[END_REF][START_REF] Sun | Use and exploitation of Sea Urchins[END_REF], and ecological significance, controlling the dynamics of seaweeds and seagrass through its grazing activity [START_REF] Lawrence | Effects of feeding on the environment: Echinoidea[END_REF][START_REF] Boudouresque | Chapter 21 -Paracentrotus lividus[END_REF].

The use of biomarkers as bioindicators is an important approach in aquatic biomonitoring to assess the relationships between exposure to environmental pollutants and increased effects on individuals and populations [START_REF] Bouzahouane | Multi-biomarkers approach to the assessment of the southeastern Mediterranean Sea health status: Preliminary study on Stramonita haemastoma used as a bioindicator for metal contamination[END_REF]. The effects of pollutants on the marine ecosystem can be expressed in terms of biochemical endpoints [START_REF] Kamel | Effects of increasing temperatures on biomarker responses and accumulation of hazardous substances in rope mussels (Mytilus galloprovincialis) from Bizerte lagoon[END_REF]. The exposure of aquatic organisms to trace elements can lead to the production of reactive oxygen species (ROS; [START_REF] Nieto | Environmental monitoring of Domingo Rubio stream (Huelva estuary, SW Spain) by combining conventional biomarkers and proteomic analysis in Carcinus maenas[END_REF], causing an imbalance between the production of ROS and endogenous antioxidant activity [START_REF] Beyersmann | Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms[END_REF]. Oxidative stress is involved in DNA damage, protein oxidation and lipid peroxidation (Winston & Digiulio, 1991). In view of this, it is relevant to assess the activities of antioxidant enzymes acting against oxidative stress such as catalase (CAT), glutathione peroxidase (GPX), glutathione-s-transferase (GST) and superoxide dismutase (SOD). The content of malondialdehyde (MDA), a by-product of lipid peroxidation, and the level of hydrogen peroxide (H2O2) in the tissues of organisms is also a good indicator of oxidative stress intensity. Therefore, oxidative stress biomarkers are widely used in marine ecotoxicology [START_REF] Kamel | Effects of increasing temperatures on biomarker responses and accumulation of hazardous substances in rope mussels (Mytilus galloprovincialis) from Bizerte lagoon[END_REF][START_REF] Benedetti | Oxidative metabolism of chemical pollutants in marine organisms: molecular and biochemical biomarkers in environmental toxicology[END_REF][START_REF] Ghribi | Trace elements and oxidative stress in the Ark shell Arca noae from a Mediterranean coastal lagoon (Bizerte lagoon, Tunisia): are there health risks associated with their consumption?[END_REF].

The aims of this study are (i) to assess the reliability of different matrices (sea urchin, macroalgae, water column and sediment) to characterize 24 trace elements contamination levels; (ii) to test the effectiveness of several indices used in ecotoxicological studies in order to confirm their use in the assessment of contamination; (iii) to determine the bioaccumulation, biomagnification and biotasediment accumulation factor of trace elements, and (iv) to estimate the effects of trace elements contamination on the oxidative stress of P. lividus.

Material and methods

Study area

The study area is located on the northwest coast of Corsica (NW Mediterranean Sea; Fig. 1A; Fig. 1B). This rocky coast is characterized by the occurrence of several cliffs (40 to 50 m high) formed of ophiolitic rocks (pillow-lavas and prasinites, gabbros, serpentinites and peridotites) and of mineral characteristics of the rubble sand belongs to the serpentine, olivine, pyroxene and amphibole groups [START_REF] Bernier | Coastal progradation and very early diagenesis of ultramafic sands as a result of rubble discharge from asbestos excavations (northern Corsica, western Mediterranean)[END_REF][START_REF] Lafabrie | Metal contamination of Posidonia oceanica meadows along the Corsican coastline (Mediterranean)[END_REF]. In this area, the old asbestos mine of Canari extends along 1 km of rocky coastline (42°49'15''N, 9°19'41'' E; Fig. 1C). After the discovery of the asbestos deposit in 1898, the mine was industrially exploited from 1948 to 1965 (BRGM, 1997). During this active period of exploitation, up to 28,000 tons of asbestos was produced yearly and approximately 4.5 million m 3 of solid mine waste was directly discharged into the Mediterranean Sea (BRGM, 1997;[START_REF] Méria | L'aventure industrielle de l'amiante en Corse[END_REF]. The soils surrounding the old mine are covered by dense scrubland vegetation (upstream of the mine: over 400 m) while the natural revegetation of the exploited areas (downstream of the old mine) remains very reduced (BRGM, 1999).

Although the mine has been closed for over 50 years, the leaching of mining residues, present along the coastline of the mine, still contributes to the dispersal of nickel, chromium and cobalt into the marine environment [START_REF] Galgani | Chemical contamination and sediment toxicity along the coast of Corsica[END_REF][START_REF] Kantin | Monitorage de la qualité des eaux et de l'environnement marin -Rapport final -Région Corse. Programme INTERREG IIIA Sardaigne / Corse/ Toscane, MONIQUA N°MCD[END_REF]. Indeed, in the southern part of the shoreline, there are many beaches (e.g. Albo, Nonza) resulting from the southward transport of discharged rubble (360,000-400,000 m 3 yr -1 ) associated with the old asbestos mine of Canari (Fig. 1C; [START_REF] Bernier | Coastal progradation and very early diagenesis of ultramafic sands as a result of rubble discharge from asbestos excavations (northern Corsica, western Mediterranean)[END_REF][START_REF] Méria | L'aventure industrielle de l'amiante en Corse[END_REF]. The sediment enrichment of these beaches results from two sources: a natural enrichment by coarse sediments from small coastal rivers and an artificial enrichment due to discharged rubbles [START_REF] Pouquet | Le colmatage de la marine d'Albo (Corse) par le stérile de la mine d'amiante[END_REF][START_REF] Bernier | Coastal progradation and very early diagenesis of ultramafic sands as a result of rubble discharge from asbestos excavations (northern Corsica, western Mediterranean)[END_REF]. Greater amounts of sediment are provided by artificial sources (mixture of cobbles and pebbles, sands and silts) continually eroded and winnowed by wave action [START_REF] Bernier | Coastal progradation and very early diagenesis of ultramafic sands as a result of rubble discharge from asbestos excavations (northern Corsica, western Mediterranean)[END_REF]. Currently, the low levels of anthropogenic activity in this area and the local sources of contamination associated with the old mine provide a unique mesocosm to study the contamination in the marine environment (ASR, 2011).

Six sampling sites located along the coast were selected (Fig. 1C): one in front of the old asbestos mine (Old Mine: OM), two to the north (Punta di Canelle: PC; Canelle: CN) and three to the south (Punta Bianca: PB; Albo: AB; Nonza: NZ). Samples were also collected at a reference site (RF), recognized as having a low level of trace elements contamination (Fig. 1D; [START_REF] Gobert | Indices to determine the status of marine coastal water bodies: development, applications and support to management[END_REF][START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF]. These sites were selected to provide a basis for assessing the differences in trace elements concentration in order to establish a potential gradient of contamination and also to better understand the effects of these different concentrations on the organisms.

Collection and processing of biological samples

Individuals (n = 28) of P. lividus sea urchin and macroalgae species observed at each sampling site were collected at two distinct depths for each site (3 m and 6 m depth) in winter (Fig. 1C; Fig. 1D). As indicated by El [START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF], the winter season avoids bias due to the reproductive stage of the urchins. In total, 364 sea urchins of commercial size (~50 mm) were collected and directly transported in a cooler with oxygenated seawater to the laboratory. Among the macroalgae collected at each site, the three most dominant taxa were retained and presented in this study (Ericaria amentacea (C.Agardh) Molinari & Guiry, 2020, Dictyota dichotoma (Hudson) J.V.Lamouroux, 1809 and Ellisolandia elongata (J.Ellis & Solander) K.R. Hind & G.W.Saunders, 2013). Three samples of each macroalgae (≈ 500 g wet weight) were collected at the different sites.

After sampling, macroalgae were rinsed with ultrapure MilliQ™ water and placed at -20 °C until chemical analysis. The specimens of P. lividus were measured, weighed and dissected. The sex ratio has been respected to avoid bias. Gonads and guts collected from sea urchins (n = 16) from each station were immediately weighed and stored at -20 °C. Frozen samples were lyophilized (CHRIST LCG Lyochamber Guard 121550 PMMA/Alpha 1-4 LD plus) and ground with an agate mortar.

Approximately 0.2 g of each dried material was mineralized in Teflon digestion vessels, in a closed microwave digestion labstation (Ethos D, Milestone Inc. Sorisole, Italy), using nitric acid (HNO3, 60%) and hydrogen peroxide (H2O2, 30%) as reagents (Suprapur grade, Merck, Darmstadt, Germany).

Collection and processing of sediment samples

Sediment samples were collected at each station using a 0.1 m 2 Van Veen grab from the bottom (top 20 cm). After collection, the sediments were stored at -20 °C until chemical analysis. Frozen sediments were lyophilized (BenchTop 3L, VirTis Company Inc.) and were eluted for 4 h at room temperature with 30 mL of HCl 1N (Suprapure grade, Merck, Darmstadt, Germany), according to [START_REF] Townsend | Trace metal characterisation of marine sediment reference materials MESS-3 and PACS-2 in dilute HCl extracts[END_REF]. A 4-h extraction time in HCl 1N ensures the removal of the available precipitated trace elements while not favouring the extraction of natural geogenic metals [START_REF] Snape | Characterisation of the dilute HCl extraction method for the identification of metal contamination in Antarctic marine sediments[END_REF]. Eluates were then diluted to an appropriate volume of 50 mL, centrifuged for 10 min at 2000 rotations per minute and separated from their remaining culot prior to being analyzed.

Trace element concentrations

Bioavailable concentrations in the seawater column

The concentration of trace elements in the seawater column at each site was measured using diffusive gradients in thin films (DGTs) devices (DGT Research Ltd., UK). DGT units, fixed with a nylon fishing line to a plastic stage buried in the sediment, floated freely in the seawater column for one week. After recovery, DGTs were rinsed with ultrapure MilliQ™ water and stored at 4 °C until analysis. As trace metal diffusion coefficients through the diffusive layer depend on water temperature, average temperatures were recorded using HOBO TidbiT® v2 loggers (accuracy: ± 0.21 °C).

Trace element analysis

The concentration of 24 trace elements (Ag, Al, As, Ba, Be, Bi, Cd, Co, Cr, Cu, Fe, Hg, Li, Mn, Mo, Ni, Pb, Sb, Se, Sn, Sr, U, V, Zn) were determined by Inductively Coupled Plasma Mass Spectrometry using Dynamic Reaction Cell technology (ICP-MS ELAN DRC II, Perkin Elmer), according to the method described by [START_REF] Richir | A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips[END_REF]. In order to check the purity of the chemicals used, analytical blanks were analyzed similarly to the samples and were performed every 40 samples.

Analytical accuracy was checked by analyzing Certified Reference Materials (CRM):

DORM-4 (fish protein), NIST1566b (oyster), NIST2976 (mussel tissue), BCR-60 (Lagarosiphon major), BCR-661 (Platyhypnidium riparioides) and GBW07603 (bush twigs and leaves). For each TE, detection limit (LD) and quantification limit (LQ) were calculated, depending on their specific blank distribution (Grinzaid et al. 1977;[START_REF] Currie | Nomenclature in evaluation of analytical methods including detection and quantification capabilities (IUPAC Recommendations 1995)[END_REF]. The total Hg (THg) content of biological samples was determined using atomic absorption spectrometry at 254 nm, in a Direct Mercury Analyser (DMA 80 153 Milestone, Minnesota, USA). Quality assessment was operated using replicates, standards (THg 100 ng g-1 ), blanks (HCl 1%) and CRM at the beginning and the end of each series. The results are expressed in milligrams of element per kilogram of dry weight (mg kg -1 DW) for tissue and sediment, and in milligrams of element per litre (mg L -1 ). TEs with values below the detection limit were removed from the database.

For the others, concentrations below the LD were replaced with a value of LD/2, as reported by [START_REF] Skrbić | Principal component analysis of indicator PCB profiles in breast milk from Poland[END_REF].

Histological study

The gonads of six sea urchins from different stations were fixed in formaldehyde in order to perform a histological study and determine the reproductive stage. This enables us to determine whether trace elements levels are influenced by the reproductive stage in this study. Gonadal tissues were dehydrated using ethanol (from 70 to 100%) then placed in Neo-Clear™, xylene substitute, before being embedded in paraffin. After cooling the blocks, two duplicate sections (thickness: 5 μm) were taken using a microtome, mounted on slides, and air-dried for the deparaffinization of the tissue in preparation for rehydration. After that, the sections were stained with Masson's Trichrome and observed under a light microscope to determine the stage of maturity based on the stages described by Byrne (1990): stage 1, recovery; stage 2, growing; stage 3, premature; stage 4, mature; stage 5, partly spawned; stage 6, spent.

Biochemical analyses

For the oxidative stress study, the gonads of six sea urchins per station were fixed in liquid nitrogen before storage at -80 °C. Samples were homogenized using a Potter-Elvehjem homogenizer in chilled phosphate buffer (100 mM, pH 7.4; 25 mg w/w per mL of buffer) containing 20% glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a serine protease inhibitor. The homogenates were centrifuged at 15,000 × g for 30 min at 4 °C and the supernatant was used for biochemical assays. Protein concentration was measured as described in Bradford (1976) and was used to normalize the final unit for biomarker responses. Biomarkers including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione-Stransferase (GST) and determination of malondialdéhyde (MDA) content were assessed as described by [START_REF] Greani | Effect of chronic arsenic exposure under environmental conditions on bioaccumulation, oxidative stress, and antioxidant enzymatic defenses in wild trout Salmo trutta (Pisces, Teleostei)[END_REF]. The hydrogen peroxide (H2O2) concentration was determined using a PeroxiDetect Kit (Sigma, Aldrich, St. Louis, MO, United States) as described by [START_REF] Lourkisti | Triploid citrus genotypes have a better tolerance to natural chilling conditions of photosynthetic capacities and specific leaf volatile organic compounds[END_REF].

Elemental and isotopic analyses

Gut content of P. lividus (n = 12 per site) and food sources (D. dichotoma, E.

elongata and E. amentacea) were analyzed through elemental and isotopic analyses.

The sea urchins and macroalgae samples were dried at 60 °C for 48-96 h and successively ground to a homogenous powder with agate mortal. As acidification is known to alter N stable isotope ratios [START_REF] Mateo | Effects of sample preparation on stable isotope ratios of carbon and nitrogen in marine invertebrates: implications for food web studies using stable isotopes[END_REF], acidified samples (E. elongata) were analyzed twice: once for stable C isotopic ratios, using decarbonated material, and once for N isotopic ratios, using native material. Samples of food source (1-5 mg) and gut content (2-3 mg) were subsequently loaded into tin capsules (8 x 5 mm, Elemental Microanalysis). Stable isotope ratio measurements were performed via continuous flow-elemental analysis-isotope-ratio mass spectrometry (CF-EA-IRMS) at University of Liège, using a Vario Micro Cube elemental analyser (Elementar Analysensysteme GmBH, Hanau, Germany) coupled to an Isoprime 100 mass spectrometer (Isoprime, Cheadle, UK). Isotopic ratios of C and N were expressed conventionally [START_REF] Coplen | Guidelines and recommended terms for expression of stableisotope-ratio and gas-ratio measurement results[END_REF], using standard delta (δ) notation relative to their respective international standards, Vienna-Pee Dee Belemnite (VPDB) and atmospheric N2. Certified reference materials (CRM) of sucrose (IAEA-C6, δ 13 C = -10.8 ± 0.5‰) and ammonium sulphate (IAEA-N2, δ 15 N = 20.3 ± 0.2‰) obtained from the International Atomic Energy Agency (IAEA, Vienna, Austria), were used for the measurement of isotopic ratios. These CRM, procedural blanks and internal replicates (i.e., glycine and in-house seagrass reference materials) were used to assess the analytical precision. Standard deviations on multi-batch replicate measurements were 0.1‰ for δ 13 C and 0.2‰ for δ 15 N.

The relative contribution of macroalgae to the diet of the sea urchin P. lividus was estimated with a Bayesian isotopic mixing model (MixSIAR, [START_REF] Stock | MixSIAR GUI User Manual[END_REF]Stock et al., 2018b). The application of the mixing model provided accurate information concerning the contribution of macroalgae species to the sea urchin tissues and recognized the main components of the diet under different conditions [START_REF] Peterson | Stable isotopes as tracers of organic matter input and transfer in benthic food webs: a review[END_REF][START_REF] Fry | Stable isotope ecology[END_REF][START_REF] Wing | Plasticity in the diet of two echinoderm species across an ecotone: microbial recycling of forest litter and bottomup forcing of populations structure[END_REF][START_REF] Cabanillas-Terán | Trophic ecology of sea urchins in coral-rocky reef systems, Ecuador[END_REF]. The model runs included the isotopic signatures of each individual, isotopic compositions of food sources (mean ± SD) and trophic enrichment factors (TEFs; mean ± SD) corresponding to the isotopic composition difference of consumer tissues and the potential food sources [START_REF] Mascart | Seasonal dependence on seagrass detritus and trophic niche partitioning in four copepod ecomorphotypes[END_REF]. Though the use of suitable TEFs is required to run mixing model, there are no specific TEF for the taxa studied here, and we used widely applicable values (i.e., 0.4 ± 1.2‰ for C, 2.3 ± 1.6‰ for N) from [START_REF] Mccutchan | Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur[END_REF]. The model was run using the MixSIAR 3.1.12 package in R 4.1. The results of the mixing model showing the calculated sea urchin dietary proportions were represented using mean ± SD and 95% credible intervals (CI95) indicating the intervals of probability density function distributions.

Numerical procedures

In order to compare the contamination levels at the different sites, the Trace Element Pollution Index (TEPI) was calculated for each site. The TEPI allows a reliable comparison of study sites, regardless of trace elements or the biological model used [START_REF] Richir | A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips[END_REF]. The index was calculated with concentrations measured in gonads, digestive tract and macroalgae to confirm its robustness. The data were standardized by mean normalization as recommended for the calculation by [START_REF] Richir | A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips[END_REF]. TEPI values were calculated using the following formula:

TEPI = (Cf1 * Cf2 * … * Cfn) 1/n
where Cfn is the mean normalized concentration of the trace element at each site and n is the number of trace elements examined. The higher the TEPI value, the more contaminated the site is. In order to classify and compare the trace elements according to their spatial variability on all the studied sites, the Trace Element Spatial Variation Index (TESVI) was determined for each element according to [START_REF] Richir | A reassessment of the use of Posidonia oceanica and Mytilus galloprovincialis to biomonitor the coastal pollution of trace elements: New tools and tips[END_REF]. For each trace element, TESVI was calculated as follows:

TESVI = [(xmax / xmin)/(∑(xmax / xi) / n)] * SD
where xmax and xmin are the maximum and minimum mean concentrations recorded among the n sites, xi are the mean concentrations recorded at each of the n sites, and SD is the standard deviation of the mean ratio ∑(xmax / xi) / n. For a given trace element, the higher the value of the TESVI, the more its environmental levels will vary overall across the study area. Therefore, the higher the TESVI, the more representative it is of a site.

Bioaccumulation is generally referred to as a process in which the chemical concentration in an organism achieves a level that exceeds that in the environment, the diet, or both [START_REF] Gobas | Revisiting bioaccumulation criteria for POPs and PBT assessments[END_REF]. The extent to which chemicals bioaccumulate is expressed by several values, including bioaccumulation factor (BAF), biomagnification factor (BMF) and biota-sediment accumulation factor (BSAF). BAF is ratio of the steady chemical concentrations in an aquatic water-respiring organism (CB, g kg -1 WW) and the water (CW, g L -1 ) determined from field data in which sampled organisms are exposed to a chemical in the water and in their diet. BAF is calculated using the following formula: CB /CW. BFM is the ratio of the steady state chemical concentrations in an organism (CB, g kg -1 WW) and the diet of the organism (CD, g kg -1 WW) determined from field data. Lastly, BSAF is the ratio of the chemical concentrations in an organism (CB, g kg -1 DW) and the sediment (CS, g kg -1 DW).

Data were expressed as mean ± standard error (SE) and analyzed using XLSTAT software. The data were transformed in order to meet the conditions of application of the parametric tests and to reduce the effect of outliers skewing the data distribution.

Analyses of variance (ANOVA) followed by post-hoc Tukey's honestly significant difference (HSD) tests were performed. The relationship between enzymatic activities and trace elements was measured by Pearson correlation coefficient. A significant difference was considered a p-value less than 0.05.

Results

Distribution of trace element content

The levels in Be and Bi levels were below the detection limit in all matrices analyzed, so they were not considered in the statistical analyses of the results. In addition, Hg concentrations were also below the detection limit in the sediments and the seawater column. For the sake of clarity, only 14 representative examples chosen among the 24 studied trace elements are graphically illustrated throughout the paper, and discussion mainly revolves around these selected examples. The mean trace element concentrations measured in the different matrices (sea urchins, macroalgae, sediment and seawater column) are presented in Fig. 2. Se> Pb> Mo> Cd> Ag in sediment (Fig. 2).

Spatial variations of trace elements

There is no significant difference in trace elements concentrations between the samples collected at 3 m depth and those collected at 6 m depth. The TEPI was calculated with concentrations in sea urchin gonads, sea urchin guts and macroalgae (Fig. 3). In gonads and guts, a gradient of contamination clearly appeared, with higher contamination to the south of the old asbestos mine, particularly at Albo (gonads: 1.367, guts: 1.172) and Nonza (gonads: 1.161, guts: 1.201; Fig. 3A; Fig. 3B). In contrast, the reference site is the least contaminated, with a TEPI of 0.456 for the gonads and 0.529 for guts (Fig. 3A; Fig. 3B).

The TEPI calculated with macroalgae showed a more contrasting gradient with the Canelle site, located north of the mine, similarly contaminated to Nonza (0.902 and 0.907, respectively; Fig. 3C). The four trace elements with the highest TESVI in gonads, guts and macroalgae are Co, Cr, Fe and Ni (Table 1). The highest concentrations were noted at Albo for the four trace elements (Table 1). Therefore, Co concentrations are 10 to 20-fold higher at Albo than at the reference site, Cr 96 to 172-fold, Fe 2 to 9-fold and Ni approximately 38-fold.

Bioaccumulation, biomagnification and biota-sediment accumulation factors

The BAF and BSAF were calculated with the concentrations in the gonads and guts of the sea urchin and with concentrations measured in macroalgae (Table 2). Most trace elements were most bioaccumulated in macroalgae then the guts and finally the gonads (Al, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V; Table 2). Only Zn had a higher accumulation in the gonads in comparison to the guts and macroalgae (Table 2).

Among the macroalgae studied, the isotopic analysis highlighted that sea urchins collected fed mainly on E. elongata (96.6 ± 2.3%; CI95: 90.8-99.6%) and very little on D. dichotoma (2.5 ± 2.2%; CI95: 0.1-8.4%) and E. amentacea (0.9 ± 0.9%; CI95: 0-3.2%; Fig. 4). In order to calculate the BMF, a weighted average of macroalgae was used for accuracy. Therefore, the formula used in this study was:

CB/(CD1 * 0.966 + CD2 * 0.025 + CD3 * 0.009)
where, CB is the concentration measured in guts, CD1 the concentration in E. elongata, CD2 in D. dichotoma and CD3 in E. amentacea. The highest BMFs were obtained for Ag, Cd and Mo, elements for which the guts accumulate more than the other matrices (Table 2).

Biotic factors of the sea urchin

The histological analysis highlighted that sea urchins were mostly at stage 4 (mature) and stage 5 (partly spawned; Fig. 5). However, variations were observed at Punta Bianca where ~30% of the sea urchins had already spawned and at the reference site where 50% of the sea urchins had reached the premature stage (Fig. 5).

Significant differences were noted between the concentrations measured in the male and female gonads. Females exhibited higher concentrations of Cd, Mn, Se, and Zn while high Mo, Pb, and V levels were found in males (p-value < 0.05). In contrast, no significant differences were found for biomarkers of stress. In the guts, the only significant difference between males and females is measured for Zn with a concentration 1.5-fold higher in females (p-value < 0.001).

Biochemical analyses

There was no significant difference between sites regarding MDA content and specific activities of SOD and GPX (Fig. 6). At the same time, the specific activity of CAT and the H2O2 levels were higher in sea urchins collected at Albo, Nonza and at the reference site (Fig. 6). Finally, the highest specific activity of GST was measured in sea urchins at Albo and three sites (Reference site, Nonza and Old Mine; Fig. 6). Specific activities of CAT and GST as well as H2O2 levels in sea urchin gonads showed significant positive correlation (p-value < 0.001, Table A.1). In addition, there were significant positive correlations for several trace elements with CAT and H2O2 (Table A .1). Accordingly, increased concentrations of Al, Cr, Cu, Fe, Mn, Pb, and Se resulted in higher specific CAT activity and H2O2 levels in the gonads (p-value < 0.05).

Discussion

The study of environmental contamination requires a deep knowledge of the distribution of pollutants and their concentrations in biotopes and organisms [START_REF] Lagadic | Utilisation de biomarqueurs pour la surveillance de la qualité de l'environnement[END_REF][START_REF] Ramade | Introduction à l'écotoxicologie : Fondements et applications[END_REF]. For this purpose, trace element concentrations were analyzed in sea urchins (gonads and guts), three macroalgae species, the seawater column and sediment. The trace elements follow an almost similar sequence regardless of the environmental matrices studied, although there are some notable differences. Fe is the predominant trace element resulting from its essential character [START_REF] Phillips | Strategies of trace metal sequestration in aquatic organisms[END_REF]Lohan & Tagliabue, 2018) or from the contamination of the ecosystem as described in the literature (Brik et al., 2018). Fe is an essential trace element playing a major role for many species due to its role in various physiological processes (e.g. photosynthesis, enzymatic activity, reproduction, etc.) and it is also included in geochemical processes [START_REF] Sunda | Bioavailability and bioaccumulation of iron in the Sea[END_REF][START_REF] Thuróczy | Observation of consistent trends in the organic complexation of dissolved iron in the Atlantic sector of the Southern Ocean[END_REF]. However, in contrast to the literature, Fe concentrations exceeded Zn concentrations in the gonads [START_REF] Strogyloudi | Metal and metallothionein concentrations in Paracentrotus lividus from Amvrakikos gulf (Ionian Sea-Greece)[END_REF][START_REF] Ternengo | Spatial variations in trace element concentrations of the sea urchin, Paracentrotus lividus, a first reference study in the Mediterranean Sea[END_REF][START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF]. This is due to the remarkably high concentrations of Fe, 2 to 8fold higher than in many studies [START_REF] Warnau | Distribution of heavy metals in the echinoid Paracentrotus lividus from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations[END_REF][START_REF] Storelli | Heavy metals in the aquatic environment of the Southern Adriatic Sea, Italy: macroalgae, sediments and benthic species[END_REF][START_REF] Soualili | Assessment of seawater pollution by heavy metals in the neighbourhood of Algiers: use of the sea urchin, Paracentrotus lividus, as a bioindicator[END_REF][START_REF] Strogyloudi | Metal and metallothionein concentrations in Paracentrotus lividus from Amvrakikos gulf (Ionian Sea-Greece)[END_REF]. According to [START_REF] Blum | Identification des zones à risque de fond géochimique élevé en éléments traces dans les cours d'eau et les eaux souterraines du basin Rhône-Méditerrannée-Corse[END_REF], the serpentinites of Cap Corse could generate significant Fe content explaining these high contents at the studied sites. Although Fe is an essential requirement for marine organisms, an excessive concentration could be harmful to their health and must therefore be monitored [START_REF] Fosmire | Zinc toxicity[END_REF]. Zn is the second element measured in high concentration in the gonads. It is the only trace element with a higher accumulation factor in the gonads than in the guts and macroalgae. This accumulation could be due to its intervention in gametogenesis and in particular in ovogenesis explaining its high content in females compared to males [START_REF] Unuma | Quantitative changes in yolk protein and other components in the ovary and testis of the sea urchin Pseudocentrotus depressus[END_REF][START_REF] Unuma | Zinc-binding property of the major yolk protein in the sea urchin -implications of its role as a zinc transporter for gametogenesis[END_REF]. In contrast to the sediment, macroalgae, gonads and guts of sea urchins where Pb is among those with the lowest concentrations, this trace element is the second most abundant in the distribution sequence of the seawater column. It is one of the most abundant and common non-essential trace elements in the environment [START_REF] Mishra | Lead detoxification by coontail (Ceratophyllum demersum L.) involves induction of phytochelatins and antioxidant system in response to its accumulation[END_REF]. Its concentration in the seawater column (2.17 ± 0.83 mg L -1 ) is considerably higher than previously reported in the literature [START_REF] Lin | Lead in the southern East China Sea[END_REF]Bruland & Lohan, 2003). This is may be due to point contamination of the water column during the period of this study or contamination of DGTs during material handling. Seawater column analysis is reported to be less relevant in the literature for trace element monitoring due to its high fluctuations caused by several factors such as hydrodynamic energy [START_REF] Jahan | Assessment of trace elements pollution in sea ports of New South Wales (NSW), Australia using macrophytobenthic plant Ecklonia radiata as a bio-indicator[END_REF]. Moreover, it is not always possible to analyze all the contaminants in this biotope, the concentration levels being too low and the analytical techniques not being sensitive enough in this study for Hg.

Calculation of the TEPI through measurement of trace element contents in the gonads and guts of the sea urchin enabled us to determine a gradient of contamination with a higher concentration to the south of the old asbestos mine, in particular at Albo and Nonza. The occurrence of a similar north-south gradient within this area has already been demonstrated in other matrices such as mussels [START_REF] Kantin | Étude de la contamination par les éléments traces en Méditerranée à l'aide d'organismes bio-intégrateurs[END_REF]. In the literature, it has been established that asbestos wastes were accumulated to the south of the old asbestos mine (BRGM, 1997;[START_REF] Cary | Etude du fond hydrogéochimique de la Corse. Impacts des activités minières sur la qualité des eaux[END_REF].

These materials migrated along the coastline due to the effect of swell and prevailing marine currents (BRGM, 1997). As a result, the wastes expanded the coastline southward for 5 km from the old mine, progressively filling the beach at Albo and creating the beach at Nonza (about 1 500 m long; BRGM, 1997). A total of one million tons of excavated material was transported to the artificial beaches during the 35 years of operation of the site [START_REF] Boulmier | Evaluation des coûts de réhabilitation de l'ancienne exploitation d'amiante de Canari en Haute-Corse[END_REF]Hervé et al., 1997;[START_REF] Cary | Etude du fond hydrogéochimique de la Corse. Impacts des activités minières sur la qualité des eaux[END_REF]. This observation highlights the important role that the marine environment plays in the diffusion and distribution of contaminants in the environment. Hence, it is necessary to take care during the selection process of the sampling site for ecotoxicological studies and to consider which can contribute to the variability of contaminants at local scale. Moreover, sites at 3 and 6 m depth are similarly impacted by diffusion, indicating that the contamination affects the whole of the studied bathymetric range.

The Albo and Nonza beaches are characterized by black pebbles constituted by serpentine (BRGM, 1997). The serpentinites are characterized by high levels of Fe and Mg [START_REF] Morrison | A regional-scale study of chromium and nickel in soils of northern California, USA[END_REF] which may explain the high Fe concentrations found at Albo with a high TESVI whatever the matrices studied. Co, Cr, and Ni are also present in high concentrations at Albo with significant concentration differences compared to the reference site. Previous works have also highlighted a high contamination of Ni, Cr and Co in marine sediments in the area adjacent (15 km of coastline) to the old asbestos mine at Canari [START_REF] Andral | Evaluation de la qualité des eaux basée sur l'utilisation de stations artificielles de moules en Méditerranée : résultats de la campagne[END_REF][START_REF] Kantin | Monitorage de la qualité des eaux et de l'environnement marin -Rapport final -Région Corse. Programme INTERREG IIIA Sardaigne / Corse/ Toscane, MONIQUA N°MCD[END_REF] and an accumulation of these elements in several marine organisms (Bouchoucha & Andral, 2010;[START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF]. According to several authors, in addition to Fe, serpentinites are naturally enriched in other trace elements such as Cr, Ni and Co [START_REF] Morrison | A regional-scale study of chromium and nickel in soils of northern California, USA[END_REF][START_REF] Siebecker | Nickel speciation in serpentine soils using synchrotron radiation techniques[END_REF]Tashakor et al., 2011), explaining these high concentration levels in the region and particularly to the south of the old mine. The general formula of serpentine is Mg5 {Si2O4} (OH)4 and substitution of Mg by Fe (II), Fe (III), Cr, Al, Ni and Mn may occur [START_REF] Mével | Serpentinization of abyssal peridotites at mid-ocean ridges[END_REF]. In the same way, large amounts of Mg and/or Fe are reported, disproportionate richness in Ni, Cr and Co and poorness in Ca in the serpentinite [START_REF] Lee | Pedogenesis in a wetland meadow and surrounding serpentinitic landslide terrain, northern California, USA[END_REF][START_REF] Pal | Biosorption of cobalt by fungi from serpentine soil of Andaman[END_REF]. Thus, even though mine drainage was the main cause of these contamination variations, there is a general sensitivity related to a particularly concentrated geochemical background. The assessment of ecosystem quality requires good knowledge of the natural geochemical context in order to distinguish the trace elements naturally present in the environment from those resulting from anthropogenic activities.

Serpentine soils are considered a source of geogenic pollution by many researchers because of the trace elements they contain and their potential deleterious effect at high concentrations in the environment [START_REF] Caillaud | Trace metals distribution from a serpentinite weathering at the scales of the weathering profile and its related weathering microsystems and clay minerals[END_REF]Tashakor et al., 2011).

In this regard, recent experiments have been performed to determine the impacts of concentrations measured in this area on Paracentrotus lividus but no significant effect were highlighted on the larvae, even in the presence of chronic contamination (El Idrissi et al., 2022a, 2022b).

The TEPI of macroalgae also indicated strong contamination at Albo but no northsouth contamination gradient has been highlighted. Indeed, the macroalgae located in the northern part of the mine exhibited contamination levels similar to those located in the south, resulting in a gradient around the mine rather than north-south. This result may be related to a reversal phenomenon of the littoral drift (from south to north) during a strong storm in 1973 causing a transport of sediment from the rubble discharge towards the north [START_REF] Bernier | Coastal progradation and very early diagenesis of ultramafic sands as a result of rubble discharge from asbestos excavations (northern Corsica, western Mediterranean)[END_REF].

Macroalgae may be better able to accumulate than sea urchins as indicated by the different accumulation factors calculated in this study. Thus, the macroalgae can be particularly useful to determine the presence of a contaminant at the specific location.

However, it is necessary to be careful when interpreting contamination levels at the risk of overestimating them (Richir, 2013). Moreover, if the aim of the study is to identify the magnitude of well-integrated contamination, the sea urchin may be a better tool for assessing a contamination gradient.

It is commonly recognized that the accumulation of contaminants in marine organisms occurs through three compartments: the seawater column, food and sediments [START_REF] Bouzahouane | Multi-biomarkers approach to the assessment of the southeastern Mediterranean Sea health status: Preliminary study on Stramonita haemastoma used as a bioindicator for metal contamination[END_REF]. Accumulation can lead to high internal concentrations resulting in toxicity, even when external concentrations are low (ECHA, 2017). According to some authors, sediment often plays the role of a local sink for contaminants, which can increase the concentrations of benthic organisms that indiscriminately ingest sediment particles while feeding [START_REF] Li | Source apportionment and source-to-sink transport of major and trace elements in coastal sediments: Combining positive matrix factorization and sediment trend analysis[END_REF][START_REF] Jacobs | Biota sediment accumulation factors for invertebrates: review and recommendations for the oak ridge reservation[END_REF]. In the present study, the BSAFs are mostly low (less than 1) indicating low bioaccumulation [START_REF] Warnau | Distribution of heavy metals in the echinoid Paracentrotus lividus from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations[END_REF]. These low values are notably related to high trace element concentrations in the sediment. Only Ag, Cd, Mo, Se and Zn have BSAFs higher than 1 with concentrations in organisms equivalent to those recorded in the literature (e.g. Guendouzi et al., 2017;Rouane-Hacene et al., 2017;[START_REF] Warnau | Distribution of heavy metals in the echinoid Paracentrotus lividus from the Mediterranean Posidonia oceanica ecosystem: seasonal and geographical variations[END_REF]. A high BSAF does not necessarily indicate a risk and rather suggests high concentrations in the organism due to its essential role in physiological processes and low concentrations in sediments, as with Zn for instance. Therefore, the efficiency of trace element absorption from different sources may vary according to ecological needs, organism metabolism and concentrations in different compartments [START_REF] Bouzahouane | Multi-biomarkers approach to the assessment of the southeastern Mediterranean Sea health status: Preliminary study on Stramonita haemastoma used as a bioindicator for metal contamination[END_REF]. Variations in trace elements within the same species can therefore have different origins. The reproductive stage of the sea urchin is known as a parameter influencing trace element concentrations [START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF]. In the present study, the sea urchins are almost all at the same stage of reproduction except at Punta Bianca where about 30% of the sea urchins have already spawned (stage 5) and at the reference site where 50% of the gonads are premature (stage 3). This information probably indicates a low overestimation of gonadal contamination levels at Punta Bianca and the reference site compared to the other investigated sites where a dilution effect occurs [START_REF] El Idrissi | Seasonal change in trace element concentrations of Paracentrotus lividus: Its use as a bioindicator[END_REF]. In this study, BAFs indicate that the majority of trace elements are most bioaccumulated in macroalgae then the gut and later the gonads. Therefore, the use of guts might be more interesting than gonads in studies assessing local variations in trace element contamination. Indeed, this compartment of the sea urchin bioaccumulates more making concentrations higher and therefore the analytical analysis more accurate. In addition, in contrast to the gonads, the majority of the trace elements do not vary according to the sex. Investigations should be conducted to assess whether reproductive stages and the source of food have an impact on the concentrations in the guts. In the present study, we estimated BMF using the guts and macroalgae that the urchin could potentially feed on. Although in some conditions, P. lividus can be omnivorous [START_REF] Wangensteen | A wolf in sheep's clothing: carnivory in dominant sea urchins in the 50 Mediterranean[END_REF], it remains mainly herbivorous [START_REF] Agnetta | Functional traits of two co-occurring sea urchins across a barren/forest patch system[END_REF][START_REF] Boudouresque | Chapter 21 -Paracentrotus lividus[END_REF]. Several factors such as seasonality [START_REF] Verlaque | Relations entre Paracentrotus lividus (Lamarck) et le phytobenthos de Méditerranée occidentale[END_REF], abiotic parameters or resources available within the biotope [START_REF] Paine | The effects of grazing by sea urchins Stongylocentrotus spp. on benthic algal populations[END_REF][START_REF] Frantzis | Relations trophiques entre les oursins Arbacia lixula et Paracentrotus lividus (Echinoidea Regularia) et le phytobenthos infralittoral superficiel dans la baie de Port-Cros (Var, France)[END_REF] can influence its choice. Therefore, for this work, three species of macroalgae were collected because of the presence of P. lividus in their beds and their high abundance in the sites. Isotopic analyses identified E. elongata as the main food source compared to D. dichotoma and E. amentacea. Frantzis & Grémare (1992) have already demonstrated a preference of P. lividus for this macroalgae which is consistent with our results. The most biomagnified trace elements (Ag, Cd and Mo) are those measured at low concentrations and are among the last elements in the distribution regardless of the matrices studied. Luy et al. (2011) have also studied these elements and, according to them, the levels of Ag and Mo measured in seagrass reflect the background level of agriculture in this area.

Several studies suggest that exposure to trace element contamination can induce a cascade of events such as ROS production [START_REF] Farombi | Biomarkers of oxidative stress and heavy metal levels as indicators of environmental pollution in African cat fish (Clarias gariepinus) from Nigeria Ogun River[END_REF][START_REF] Nieto | Environmental monitoring of Domingo Rubio stream (Huelva estuary, SW Spain) by combining conventional biomarkers and proteomic analysis in Carcinus maenas[END_REF]. Thus, antioxidant defense mechanisms are crucial to maintain the redox balance between pro-oxidants and antioxidants in aerobic organisms [START_REF] Limon-Pacheco | The role of antioxidants and antioxidantrelated enzymes in protective responses to environmentally induced oxidative stress[END_REF][START_REF] Chan | Biomarker responses in oysters Crassostrea hongkongensis in relation to metal contamination patterns in the Pearl River Estuary, southern Chinazstar[END_REF]. In order to estimate the effects of trace element contamination on the oxidative stress of P. lividus, analyses were performed on the gonads of sea urchins collected at different locations of the contamination gradient (north to south). The highest specific activity of CAT, GST and H2O2 was reported in the south of the old asbestos mine where the contamination is the highest. A positive correlation between the specific activity of CAT, H2O2 and some trace elements is noted confirming an effect of contaminants on the oxidative stress of sea urchin and on the reliability of biomarkers used in this study. In the mitochondrial respiratory chain, H2O2 can react directly with metal ions such as Fe or Cu, by the Fenton reaction, and form the hydroxyl radical which is a powerful oxidant [START_REF] Regoli | Oxidative pathways of chemical toxicity and oxidative stress biomarkers in marine organisms[END_REF][START_REF] Mejdoub | Oxidative stress responses of the mussel Mytilus galloprovincialis exposed to emissary's pollution in coastal areas of Casablanca[END_REF]. Consequently, the elimination of H2O2 is a key strategy of organisms against oxidative stress (Regoli et al., 2002a;[START_REF] Mejdoub | Oxidative stress responses of the mussel Mytilus galloprovincialis exposed to emissary's pollution in coastal areas of Casablanca[END_REF]. According to [START_REF] Giarratano | Assessment of antioxidant responses and trace metal accumulation by digestive gland of ribbed mussel Aulacomya atra from northern Patagonia[END_REF], the simultaneous induction of GST and CAT activities suggests a similar pattern for hydrogen peroxide removal.

Therefore, the increase in these enzymes suggests an activation of detoxification processes, probably reflecting high stress (Louiz et al., 2016). A large number of other studies have shown higher activity of CAT and GST in organisms collected from contaminated sites [START_REF] Bougherira | Assessment of contaminant migration in groundwater from an industrial development area, Annaba District NE Algeria[END_REF][START_REF] Keblouti | The use of weather radar for rainfallrunoff modeling, case of Seybouse watershed (Algeria)[END_REF]Bouzenda et al., 2017). Regoli et al. (2002b) consider CAT a sensitive and important biomarker of oxidative stress superior to SOD, explaining the invariance of specific SOD activity in this study. These high specific activities of CAT and GST as well as this high H2O2 content were also measured at the reference site which however has a low concentration of trace elements. The main difficulty in using biomarkers in the natural environment is the interference with other environmental factors [START_REF] Lagadic | Utilisation de biomarqueurs pour la surveillance de la qualité de l'environnement[END_REF]. In contrast to experiments under controlled conditions, various factors can lead to responses of biochemical parameters in the natural environment [START_REF] Lagadic | Utilisation de biomarqueurs pour la surveillance de la qualité de l'environnement[END_REF][START_REF] Ramade | Introduction à l'écotoxicologie : Fondements et applications[END_REF]. As a result, many factors such as meteorological conditions, interactions with other contaminants than those studied, or interspecific relationships can complexify the interpretation of oxidative stress responses [START_REF] Lagadic | Utilisation de biomarqueurs pour la surveillance de la qualité de l'environnement[END_REF]. In this case, the same habitat at the reference site was chosen as for the sites near the old asbestos mine in order to minimize variation; it thus appears that other factors led to these high values. Further study would be of interest to determine the source of this significant oxidative stress response. Finally, despite the higher levels of H2O2 in sea urchin gonads collected to the south of the mine and at the reference site, the level of MDA, a by-product of lipid peroxidation, did not significantly vary, suggesting that the antioxidant enzyme system of P. lividus prevented oxidative damage from occurring [START_REF] Amri | Seasonal antioxidant responses in the sea urchin Paracentrotus lividus (Lamarck 1816) used as a bioindicator of the environmental contamination in the South-East Mediterranean[END_REF][START_REF] Ding | Accumulation, tissue distribution, and biochemical effects of polystyrene microplastics in the freshwater fish red tilapia (Oreochromis niloticus)[END_REF].

Conclusion

This study assessed the reliability of sea urchins and macroalgae in the assessment of contamination in coastal ecosystems. The sea urchin gut appears to be a good bioindicator tool for assessing trace element levels. In this context, it would be interesting to perform complementary studies in order to understand the influence of several parameters such as the season and the food sources of the sea urchin. TEPI allowed identification of the different levels of contamination by highlighting the gradients of contamination in the old asbestos mine using sea urchins and macroalgae. TESVI enabled us to determine with efficiency the trace elements characteristic (Co, Cr, Fe, Ni) of the Albo site. The high levels of these trace elements in the different matrices are due to the discharges of the old mine swept along by the sea current and the geology of the region composed by serpentinites.

The contamination generated by the old asbestos mine causes oxidative stress very well regulated by the antioxidant mechanisms of the sea urchin. This study also highlighted the need for caution when interpreting biomarkers of stress under environmental conditions due to the different factors involved. Finally, this research shows that the effects of the old asbestos mine are still present and in order to prevent them, it is essential to stabilize the surrounding environment and soils through revegetation action and to maintain monitoring of the marine environment.

Currently, a large-scale project to remediate and to rehabilitate the area is underway.

A study of the effects of this project on the surrounding marine ecosystems would be relevant at short-term, beacause of the increased risk of contamination from this rehabilitation work, and in the long-term to monitor potential beneficial effects. Canelle; OM: Old Mine; PB: Punta Bianca; AB: Albo; NZ: Nonza; RF: Reference site).

The higher the TESVI value, the greater the spatial variation of that element among the sampling locations. In dark grey, the higher values. Dissimilar letters denote significant differences between groups (p-value < 0.05).

  Bouzenda, R., Soltani, N., Khebbeb, M.E.H., 2017. Assessment of pollution in the Gulf of Annaba (Algeria) by monthly measurements of two biomarkers in a fish species Liza aurata. Journal of Entomology and Zoology Studies 5(1), 366-372. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72(1-2),248-254. https://doi.org/10.1016/0003-2697(76)90527-3 Brik, B., Aydi, A., Riahi, C., Sdiri, A., Regaya, K., 2018. Contamination levels and vertical distribution of trace metals with application of geochemical indices in the sediment cores of the Bizerte Lagoon-Ichkeul lake complex in northeastern Tunisia. Arabian Journal of Geosciences 11, 23-15. https://doi.org/10.1007/s12517-017-3382-5 BRGM, 1997. Mine de Canari -Rejet des stériles en mer et évolution du trait de côte. Rapport BRGM/ R39277, 114p BRGM, 1999. Evaluation des coûts de réhabilitation de l'ancienne exploitation d'amiante de Canari en Haute-Corse. Rapport BRGM/ R39159, 127p Bruland, K. W., Lohan, M. C., 2003. Controls of Trace Metals in Seawater. Oceans and Marine Geochemistry. H. D. Holland and K. K. Turekian (Eds). Elsevier Ltd. 6. 23-47. Byrne, M., 1990. Annual reproductive cycles of the commercial sea urchin Paracentrotus lividus from an exposed intertidal and a sheltered subtidal habitat

Figure 1 .Figure 2 .

 12 Figure 1. Location of the study area in Corsica Island (A, B) and the sampling sites (C, D). PC: Punta di Canelle; CN: Canelle; OM: Old Mine; PB: Punta Bianca; AB: Albo; NZ: Nonza; RF: Reference site

Figure 3 .

 3 Figure 3. Trace Element Pollution Index (TEPI) determined using sea urchins (gonads: A, orange points, guts: B, purple points) and macroalgae (C, green points), of the seven sites (PC: Punta di Canelle; CN: Canelle; OM: Old Mine; PB: Punta Bianca; AB: Albo; NZ: Nonza; RF: Reference site). No macroalgae samples were collected at RF.

Figure 4 .

 4 Figure 4. Relative contributions of three food sources (Dictyota dichotoma, Ellisolandia elongata and Ericaria amentacea) to the diet of P. lividus individuals sampled near the old asbestos mine at Canari (Corsica, NW Mediterranean).

Figure 5 .

 5 Figure 5. Maturity stage of sea urchin gonads (%) at each site (PC: Punta di Canelle; CN: Canelle; OM: Old Mine; PB: Punta Bianca; AB: Albo; NZ: Nonza; RF: Reference site).

Figure 6 .

 6 Figure 6. Changes in specific activities of (A) superoxyde dismutase (SOD), (B) catalase (CAT), (C) glutathione peroxidase (GPX), (D) glutathione-S-transferase (GST) and changes in (E) hydrogen peroxide (H2O2) and (F) malondialdehyde (MDA) contents in gonads of P. lividus collected at seven sites (PC: Punta di Canelle; CN: Canelle; OM: Old Mine; PB: Punta Bianca; AB: Albo; NZ: Nonza; RF: Reference site).

Figure 6 .

 6 Figure 6.

  Trace element concentrations follow the sequence: Fe> Zn> Al> Ni> Cr> Mn> Cu> V> Se> Co> Mo> Cd> Ag> Pb in sea urchin gonads; Fe> Al> Cr >Ni> Zn> Mn> Cu> V> Se> Co> Cd> Mo> Ag> Pb in sea urchin guts; Fe> Al> Ni> Cr> Mn> Cu> V> Zn> Co> Pb> Mo> Cd> Ag in macroalgae; Fe> Pb> Al> Ni> V> Zn> Se> Mn> Cr> Cu> Co> Mo> Cd> Ag in seawater column and Fe > Al> Ni> Cr> Mn> V> Co> Cu> Zn>

Table Captions Table 1 .

 Captions1 Trace Element Spatial Variation Index (TESVI) of 22 trace elements examined in sea urchins (gonads and guts) and macroalgae from six sites near the old asbestos mine (Corsica, NW Mediterranean Sea; PC: Punta di Canelle; CN:

Table 2 .

 2 Bioaccumulation (BAF), biomagnification (BMF) and biota-sediment accumulation (BSAF) factor of 14 trace elements. TE: Trace elements; SW:

	Seawater.

Table 1 .

 1

	1144
	1145
				Sea urchins
						Macroalgae
		Gonads		Guts
	Trace elements
			Site
		TESVI		TESVI	Site xmax TESVI	Site xmax
			xmax
	Ag	1.622	NZ	3.964	CN	0.216	PC
	Al	4.896	AB	1.321	AB	0.776	AB
	As	0.704	AB	0.962	OM	2.357	AB
	Ba	0.710	PC	0.182	PB	n.a	n.a
	Cd	1.354	PC	0.370	CN	0.639	PC
	Co	8.243	AB	31.789	AB	10.124	AB
	Cr	194.926	AB	406.518	AB	13.335	AB
	Cu	0.589	NZ	0.750	NZ	4.422	AB
	Fe	16.854	AB	15.238	AB	10.913	AB
	Hg	1.703	AB	0.576	RF	n.a	n.a
	Li	0.160	AB	0.247	PB	n.a	n.a
	Mn	3.078	AB	6.650	AB	3.897	AB
	Mo	0.454	PC	0.652	RF	1.871	AB
	Ni	56.107	AB	50.454	AB	17.229	AB
	Pb	0.296	RF	0.488	RF	0.422	CN
	Sb	0.794	CN	0.304	RF	0.185	AB
	Se	0.790	NZ	3.457	NZ	3.451	AB
	Sn	1.002	AB	2.190	RF	2.893	AB
	Sr	0.960	AB	0.520	PB	0.237	PC
	U	3.293	CN	0.120	PB	n.a	n.a

Table 2 .

 2

			BAF		BMF		BSAF
	TE	gonads/	guts/	macroalgae/	guts/	gonads/	guts/	macroalgae
		SW	SW	SW	macroalgae	sediment	sediment	/sediment
	Ag	50.3526 163.6319	29.8274	5.5936	38.7261 167.7989	22.9402
	Al	11.2258	46.8450	117.4314	0.1401	0.0038	0.0210	0.0395
	Cd	33.4045 181.6255	13.1774	6.9715	23.6158 171.2038	9.3159
	Co	5.5608	18.1816	41.7896	0.1351	0.0205	0.0894	0.1541
	Cr	7.5379	45.2430	98.6556	0.1514	0.0086	0.0691	0.1131
	Cu	5.0336	14.3122	21.8451	0.2092	0.2221	0.8419	0.9638
	Fe	13.1917	59.3457	148.6595	0.1393	0.0086	0.0516	0.0970
	Mn	3.7815	18.2950	36.8864	0.1556	0.0109	0.0702	0.1061
	Mo	3.1471	13.0912	2.7865	3.2869	2.5737	14.2747	2.2788
	Ni	2.2670	11.1937	32.2017	0.1214	0.0081	0.0532	0.1149
	Pb	0.0097	0.0361	0.1022	0.1169	0.0794	0.3938	0.8352
	Se 170.7810 502.6702	958.6628	0.1535	1.5232	5.9777	8.5503
	V	0.7858	1.8492	4.5681	0.1535	0.0720	0.2259	0.4185
	Zn	38.8205	10.4672	5.4270	0.8050	8.7230	3.1360	1.2195

Acknowledgments

This work was supported by the University of Corsica (France) and the University of Liege (Belgium). This paper has the MARE number MARExxx. This study would not have been possible without the financial support of the French Government, the Collectivity of Corsica and the MISTRALS (Mediterranean Integrated STudies at Regional And Local Scales) research program, for whose cooperation the authors are grateful. This work is part of the STARECAPMED (STAtion of Reference and

Grinzaid, E.L., Zil'bershtein, K.I., Nadezhina, L.S., Yufa, B.Y., 1977. Terms and methods of estimating detection limits invarious analytical methods. Journal of Analytical Chemistry USSR 32, 1678-1684. Guendouzi, Y., Soualili, D.L., Boulahdid, M., Boudjenoun, M., Mezali, K., 2017. Seasonal variation in bioavailability of trace metals in the echinoid Paracentrotus lividus (Lamarck, 1816) from Algerian coastal waters: effect of physiological indices.

Regional

Studies in Marine Science 14, 112-117. https://doi.org/10.1016/j.rsma.2017.05.010 Hervé, J.P., Le Bars, P., Juncy, G., Lamy, N., Cosqueric, M., 1997. Mine de Canari (Haute-Corse). Rejet des stériles en mer et évolution du trait de côte. BRGM/RR-39277-FR. Islam, M.S., Tanaka, M., 2004. Impacts of pollution on coastal and marine ecosystems including coastal and marine fisheries and approach for management: a review and synthesis. Marine Pollution Livingston, D.R., Förlin, L., George, S.G., 1994. Molecular biomarkers and toxic consequences of impact by organic pollution in aquatic organisms. In: Sutcliffe, D.W. (ed) Water quality & stress indicators in marine and freshwater systems: linking levels of organisation. Ambleside, UK, Freshwater Biological Association, p154-171 Lohan, M.C., Tagliabue, A., 2018. Oceanic Micronutrients: Trace Metals that are Essential for Marine Life. Elements 14(6), 385-390. https://doi.org/10.2138/gselements.14.6.385 Louiz, I., Ben Hassine, O.K., Palluel, O., Ben-Attia, M., Aït-Aïssa, S., 2016. Spatial and temporal variation of biochemical biomarkers in Gobius niger (Gobiidae) from a southern Mediterranean lagoon (Bizerta lagoon, Tunisia): influence of biotic and abiotic factors.