Marion Pillet
email: marion.pillet@univ-lr.fr

Mathilde Dabrowski

Michel Marengo

Lovina Fullgrabe

Michèle Leduc

Quentin Fontaine

Stéphane Le Floch

Valérie Huet

Carine Churlaud

Pierre Lejeune

Stéphane Le Floch

Hélène Thomas

Preliminary inter-port study of the quality of environments using physiological responses of invertebrates exposed to chronic trace element and organic contamination in Corsica (Mediterranean Sea)

Keywords: pollution mixture, bioindicators, multibiomarkers approach, trace element, organic pollutants, IBR

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Human activities at sea (e.g. fisheries and shipping), on land (e.g. agriculture and industries) or along the coastline (e.g. wastewater treatment plant and massive tourism) cause chronic and diffuse pollution in the marine environment [START_REF] Islam | Impacts of pollution on coastal and marine ecosystems including coastal and marine fisheries and approach for management: a review and synthesis[END_REF]. Urban ports are thus subjected to a myriad of pollutants coming from terrestrial discharges or anthropogenic contaminations [START_REF] Andral | Monitoring chemical contamination levels in the Mediterranean based on the use of mussel caging[END_REF][START_REF] Benali | Integrated use of biomarkers and condition indices in mussels (Mytilus galloprovincialis) for monitoring pollution and development of biomarker index to assess the potential toxic of coastal sites[END_REF][START_REF] Lafabrie | Metal contamination of Posidonia oceanica meadows along the Corsican coastline (Mediterranean)[END_REF]). These pollutants may represent a subsequent risk to human health [START_REF] Fasulo | Metabolomic investigation of Mytilus galloprovincialis (Lamarck 1819) caged in aquatic environments[END_REF][START_REF] Maisano | Effects of petrochemical contamination on caged marine mussels using a multi-biomarker approach: Histological changes, neurotoxicity and hypoxic stress[END_REF]. In these environments, biota is facing diverse stressful conditions and is exposed to a complex mixture of organic and inorganic pollutants. Despite the growing interest in recreational boating and the increasing number of marinas along coastlines all around the world, ecological understanding of these ecosystems is still very scarce [START_REF] Guerra-García | Assessing environmental pollution levels in marinas[END_REF].

In 2008, the Marine Strategy Framework Directive (DCSMM 2008) incited European states to reduce the impact of activity in the marine environment, to achieve or maintain a good environmental status. This status is one of the decisive factors for awarding eco-labels or ecocertifications, such as Blue Flags in marinas [START_REF] Guerra-García | Assessing environmental pollution levels in marinas[END_REF]. It is thus crucial to develop a strong method to assess and monitor contamination in the marine environment, and also to understand the direct or combined effects of pollutants on organisms. Determining how pollutants interact with other chemical stressors to cause additive or synergistic effects on organisms remains one of the greatest challenges in ecotoxicology today. Also, direct chemical analyses of water samples only give a snapshot of the presence of pollutants at a specific date and time. Moreover, many environmental parameters (e.g., temperature, salinity, etc.) affect the bioavailability of pollutants and their toxic effect on organisms could be modified.

Bioindicators are used to consider such influence. Bioindicators are sensitive organisms used to observe induced effects of pollutants and therefore provide information on the impacts to the environment. This evaluation can be measured at several levels, from the molecular and cellular individual levels to the populations up to whole biocenosis level (Bondaruk et al., 2015). The use of these bioindicators is particularly relevant from a biological point of view. The measured concentrations reflect the bioavailable fractions in the marine environment [START_REF] Wang | Chapter 4 -Bioaccumulation and Biomonitoring[END_REF] and confers an integrative measurement of their exposure and effect on ecologically available fractions of environmental pollutants over time [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF].

Bivalves and more precisely mussels received the greatest attention [START_REF] Luna-Acosta | Persistent organic pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic Coast) -Part 2: Potential biological effects[END_REF][START_REF] Rainbow | Cosmopolitan biomonitors of trace metals[END_REF] thanks to their biological characteristics (filter-feeders, global distribution, large abundance, low mobility, easiness to sample, etc.). Mussels are routinely used in regional environmental biomonitoring plans such as OSPAR's Coordinated Environmental Monitoring Programme (CEMP), PNUMA's Program for the Assessment and Control of Marine Pollution within the Mediterranean Action Plan (MED POL agreement of the Barcelona Convention) or the Mussel Watch Project (USA), which was initiated in 1976 and includes the surveillance of coastal regions for their pollutant contents [START_REF] Goldberg | The mussel watch -A first step in global marine monitoring[END_REF].

However, other species (seagrass, fish, marine sponges, holothurians, limpets, etc.) are sometimes better adapted to the environmental conditions of the studied environment and their potential as bioindicators starts to increase [START_REF] Beg | Biomarkers in fish as a measure of the state of marine environment of Kuwait[END_REF][START_REF] Lafabrie | Trace metals assessment in water, sediment, mussel and seagrass species -Validation of the use of Posidonia oceanica as a metal biomonitor[END_REF][START_REF] Mohammadizadeh | Heavy metal accumulation in tissues of two sea cucumbers, Holothuria leucospilota and Holothuria scabra in the northern part of Qeshm Island, Persian Gulf[END_REF][START_REF] Orani | Marine sponges as a powerful tool for trace elements biomonitoring studies in coastal environment[END_REF][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][START_REF] Pérez | Limpets (Patella spp. Mollusca, Gastropoda) as model organisms for biomonitoring environmental quality[END_REF][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 use of native biota as a monitoring tool for bioavailable pollutants in aquatic ecosystems was demonstrated to constitute a highly valuable approach [START_REF] Bertrand | Native crustacean species as a bioindicator of freshwater ecosystem pollution: A multivariate and integrative study of multibiomarker response in active river monitoring[END_REF]).

In the present study, a multibiomarkers approach was chosen to assess the impacts of multiple contaminants. The term biomarker refers to "all the biochemical, cellular, physiological or behavioural parameters which can be measured in the tissues or fluids of an organism or on the whole organism, to highlight the exposure to, or the effects, one or more contaminants" (Garric et al., 2010). Biomarkers were classified into two class: biomarkers of exposure indicate the presence of a contaminant inside an organism due to its interaction with biological molecules, and biomarkers of effect reflect the toxic or sublethal effect of the contaminant on the organism (Luna-Acosta et al. 2017a). In this study, the bioaccumulation of multiple metallic trace elements and organic pollutants represent the biomarkers of exposure. The activity of enzymes involved in several metabolic processes were analysed as biomarkers of effect: antioxidant defences (superoxide dismutase SOD -EC 1.15.1.1 and glutathione-S-transferase GST -EC 2.5.1.18), energetic pathways (lactate dehydrogenase LDH -EC 1.1.1.27, pyruvate kinase PK -EC 2.7.1.40 and phosphoenolpyruvate carboxykinase PEPCK -EC 4.1.1.32), neurotoxicity biomarker (acetylcholinesterase AChE -EC 3.1.1.7) and immune defence mechanisms (laccase type phenoloxidase LAC -EC 1.10.3.2). This multibiomarkers approach was shown to be efficient as it brings a better understanding and an integrated view of the biological responses in environmental risk assessment [START_REF] Cravo | A multibiomarker approach in Mytilus galloprovincialis to assess environmental quality[END_REF][START_REF] Delahaut | A multibiomarker approach for evaluating environmental contamination: common carp (Cyprinus carpio) transplanted along a gradient of metal pollution[END_REF]). However, this approach needs to be coupled with an integrated tool such as the Integrated Biomarker Response (IBR). The IBR summarizes biomarker responses and simplifies their interpretation in biomonitoring programs [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF]. It was described to be useful for evaluating the impact of environmental conditions on the health and physiological status of organisms [START_REF] Serafim | Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems[END_REF]. Compared to individual biomarkers, the IBR was useful to distinguish locations along a stress gradient [START_REF] Bertrand | Native crustacean species as a bioindicator of freshwater ecosystem pollution: A multivariate and integrative study of multibiomarker response in active river monitoring[END_REF]).

In the present study, different sampling techniques (with organisms collected as individual or collected as a pool of individuals) were tested. The objective was to evaluate if analysing the data as a pool, a less expensive and less time-consuming method, would give comparable results to individual analyses. We hypothesised that this "pool" method could be proposed as a simple and effective tool to local authorities for future environmental management.

The secondary objective was to select the best bioindicator for port areas, from several native species. Two molluscs (Mytilus galloprovincialis, Patella sp.), and one echinoderm (Holothuria tubulosa) were selected. Mytilus galloprovincialis was included to obtain a reference species to compare with other studies while the other two species were available all year long in all the sampling locations, easy to sample, and were identified as potential bioindicator species in previous studies [START_REF] Campanella | Trace metals in seagrass, algae and molluscs from an uncontaminated area in the Mediterranean[END_REF][START_REF] Mohammadizadeh | Heavy metal accumulation in tissues of two sea cucumbers, Holothuria leucospilota and Holothuria scabra in the northern part of Qeshm Island, Persian Gulf[END_REF][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][START_REF] Reguera | A review of the empirical literature on the use of limpets Patella spp. (Mollusca: Gastropoda) as bioindicators of environmental quality[END_REF]). These three species allow a global view of the different biological compartments since the mussel is a filter-feeder, the limpet is a grazer, and the sea cucumber is a deposit-feeder. Our hypothesis was that both mussel and limpet would give similar results while sea cucumber would be more contaminated due to its direct presence on the sediment (where contaminants would accumulate).

Finally, the levels of contamination and the subsequent pollutant-induced stress syndrome were compared in several North Corsica port areas, never studied before. We hypothesised that the level of contamination would be quite low due to the restricted size of the considered port areas. E 9° 17' 54.373''),Île Rousse (N 42° 33' 59.116'';E 8° 56' 7.778'') and Calvi (N 42° 33' 59.116'';E 8° 45' 29.094'') ports were chosen as exposed locations (Figure 1). These three ports are small marinas with low hydrodynamism. St-Florent, Île-Rousse and Calvi ports can respectively accommodate 950, 250 and 500 boats. All are located close to boat rental agencies and oil stations; St-Florent and Calvi are also subjected to the impact of their fairing area; and St-Florent is located at the mouth of the Aliso River. The STARESO station in the Revellata bay (N 42° 34' 49.584''; E 8° 43' 31.511'') was chosen as the reference location (Figure 1). It is distant from any city and is used for only 2 boats. One water sample was taken from each location to evaluate the environmental trace element contamination.

Material and methods

St

Fig. 1 Map of the experimental locations in North Corsica (France) and anthropogenic pressures observed next to the different ports Three species (Mytilus galloprovincialis, Patella sp. and Holothuria tubulosa) were sampled in January 2020 in four different locations of North Corsica (France) (Figure 1). The objective was to collect 20 mussel and limpet individuals (10 for bioaccumulation analyses and 10 for other analyses) and 10 sea cucumber individuals (both kind of analyses being possible on one individual thanks to the large size of sea cucumber). The final number of individuals was dependent of the available wild organisms on each site. Mussels (n=17; length = 76.75 ± 9.03 mm) were found only in St-Florent port. Limpets (n = 13-14 per location; length = 35.24 ± 5.36 mm) were found everywhere except in St-Florent port. Sea cucumbers (n = 5-7 per location; length = 154.1± 43.77 mm) were collected in the four locations.

During the sampling, additional organisms were collected in one location to test the "pool" sampling techniques: 13 mussels (length = 68.56 ± 8.84 mm) and four sea cucumbers (length = 193.25 ± 34.67 mm) were collected in in Île Rousse. Tissues were dissected, pooled and processed following the same method than for the "individual" sampling technique (detailed below for all the analyses). After sampling, individuals were immediately placed in dry ice and shipped to La Rochelle (France) within 48 h in order to be analysed. In the lab, half (n = ~7) of the mussels, limpets and sea cucumbers tissues were kept at -20°C for bioaccumulation analyses. The other half was kept at -80°C for all the other analyses.

A. Biomarkers of exposure (bioaccumulation) a) Trace element assessment

The soft tissues of mussel and limpet were hand grinded with a ceramic mortar and pestle. The body wall of sea cucumber was chopped into small pieces. The samples were freeze dried for 48-72 h. Then, the samples and the two reference materials (DOLT-5, dogfish liver, and TORT-3, lobster hepatopancreas, National Research Council Canada) were weighted around 0.25 mg and digested by addition of 6:2 (v/v) 67-70% HNO3/34-37% HCl mixture (Fisher, trace metal quality). The digestion process was carried out overnight at room temperature followed by a microwave digestion (30 min with constantly increasing temperature up to 120 °C, then 15 min at this temperature). At the end of these steps, the volume of the samples was completed with ultrapure water until 50 mL. Silver (Ag), barium (Ba), cadmium (Cd), cobalt (Co), chromium (Cr), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), antimony (Sb), selenium (Se), tin (Sn) and vanadium (V) contents were analysed using an Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) Vista-Pro (Varian Inc., Palo Alto, California, USA). Aluminium (Al), arsenic (As), copper (Cu), iron (Fe) and zinc (Zn) were analysed using an Inductively Coupled Plasma Mass Spectrometry (ICP-MS) XSeries 2 (ThermoFisher Scientific, Waltham, Massachusetts, USA).

Mean recovery rates were 76% (Al / DOLT-5), 55% (Ag / DOLT-5), 93% (As / DOLT-5) and 100% (As/ TORT-3), 90% (Cd / DOLT-5) and 95% (Cd / TORT-3), 82% (Co / DOLT-5) and 98% (Co / TORT-3), 97% (Cr / TORT-3), 97% (Cu / DOLT-5) and 95 (Cu / TORT-3), 105% (Fe / DOLT-5) and 98% (Fe / TORT-3), 92% (Mn / TORT-3), 87% (Mo / DOLT5) and 85% (Mo / TORT-3), 83% (Ni), 103% (Pb / DOLT-5) and 79% (Pb / TORT-3), 93% (Se / DOLT-5) and 91% (Se / TORT-3), 92% (V / TORT-3) and 95% (Zn / DOLT-5) and 101% (Zn / TORT-3). Limits of quantification were 19.28 (Al), 0.01 (Ag), 0.1 (As), 0.1 (Ba), 0.01 (Cd), 0.01 (Co), 0.1 (Cr), 0.96 (Cu), 3.86 (Fe), 0.01 (Mn), 0.1 (Mo), 0.04 (Ni), 0.01 (Pb), 0.04 (Sb), 0.39 (Se), 0.02 (Sn), 0.96 (V) and 3.86 (Zn) μg.g -1 of dry weight.

The trace element contamination of the water samples was also analysed using ICP-OES (dilution to 1/5 for water samples and seawater standards, in 2 % nitric acid) and ICP-MS (dilution to 1/20 for water samples and seawater standards, in 2 % nitric acid). b) Organic pollutants assessment Tissues of organisms (soft tissues of mussel and limpet and body wall of sea cucumber, measured as a pool of seven individuals) were analysed for organic pollutants contents. 22 polycyclic aromatic hydrocarbons (PAHs), including the 16 Environmental Protection Agency (EPA) PAHs, 14 polychlorinated biphenyls (PCBs) and seven pesticides were analysed in tissues by stir bar sorptive extraction-thermal desorption-gas chromatographytandem mass spectrometry (SBSEGC-MS/MS) following [START_REF] Lacroix | Development of an innovative and "green" stir bar sorptive extraction-thermal desorption-gas chromatography-tandem mass spectrometry method for quantification of polycyclic aromatic hydrocarbons in marine biota[END_REF] method. Tissues were briefly digested by saponification and analytes were extracted for 16 h at 700 rpm using polydimethylsiloxane stir-bars (Twister 20 mm × 0.5 mm, Gerstel). Bars were subsequently analysed using a gas chromatography system Agilent 7890A coupled to an Agilent 7000 triple quadripole mass spectrometer (Agilent Technologies) and equipped with a Thermal Desorption Unit combined with a Cooled Injection System (Gerstel). The GC column was a Restek Rxi-5 ms (30 m, 0.25 mm, 0.25 μm). Analytes were quantified relatively to deuterated compounds using a calibration curve ranging from 0.01 ng to 30 ng per bar. Results are expressed as ng.g -1 dry weight. Limits of quantification (LOQ) were calculated by the calibration curve method [START_REF] Shrivastava | Methods for the determination of limit of detection and limit of quantitation of the analytical methods[END_REF]

B. Biomarkers of effect analyses a) Enzymatic biomarkers

Individuals were dissected to keep the digestive gland for mussel, the whole soft tissues for limpets and the body wall for sea cucumber. The tissues were weighted and grounded in liquid nitrogen using a MM400 Retsch © (GmbH, Éragny, Luxemburg) mixer mill. Then they were hand homogenized on ice in one volume of phosphate buffered saline (PBS) solution (100 mM, pH 7.5) containing 0.1 % Triton X-100 and one mM ethylenediaminetetraacetic acid (EDTA) to avoid protein degradation. The samples were centrifuged 15 min at 12500 g at 4°C (Sorvall Legeng Micro 17R, ThermoFisher Scientific, Waltham, Massachusetts, USA). The supernatant was collected, divided into aliquots and used for the subsequent biomarkers of effect and protein analyses.

The capacity to respond to oxidative stress was evaluated using SOD. SOD activity protocol is based on the inhibition of a superoxide-driven NADH oxidation and was measured following the rate of NADH oxidation at 340 nm [START_REF] Paoletti | A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts[END_REF]). The GST is an enzyme involved in detoxification process. It was determined by measuring the production of GS-DNB at 340 nm [START_REF] Habig | Glutathione S-transferases. The first enzymatic step in mercapturic acid formation[END_REF]. The activities of the indices of glycolysis and gluconeogenesis pathways (PK, PEPCK, and LDH) were determined by measuring the NADH consumption at 340 nm.

The method used for PK and LDH followed the [START_REF] Childress | Depth-related enzymic activities in muscle, brain and heart of deep-living pelagic marine teleosts[END_REF] protocol modified by [START_REF] Bailey | High swimming and metabolic activity in the deep-sea eel Synaphobranchus kaupii revealed by integrated in situ and in vitro measurements[END_REF]. For PEPCK, the [START_REF] Petrescu | Determination of phosphoenolpyruvate carboxykinase activity with deoxyguanosine 5′-diphosphate as nucleotide substrate[END_REF] method, adapted by [START_REF] Jamieson | Interactions between oestradiol and glucocorticoid regulatory effects on liver-specific glucocorticoid-inducible genes: possible evidence for a role of hepatic 11â-hydroxysteroid dehydrogenase type 1[END_REF] was used. However, LDH and PEPCK activities were below the detection levels in mussel and sea cucumber respectively. AChE is widely used to estimate neurotoxic impacts of pollutants, especially organophosphates, carbamates, several heavy metals and surfactants. Its activity was assayed by recording the production of 5-thio-2-nitrobenzoate at 405 nm [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF].

Finally, the LAC enzyme is involved in immune, antioxidant and detoxification processes (Luna-Acosta et al. 2017a). Its activity was measured according to [START_REF] Luna-Acosta | First evidence of laccase activity in the Pacific oyster Crassostrea gigas[END_REF], following the oxidation of p-phenylenediamine at 420 nm.

The total protein content was measured with the bicinchoninic acid protein assay kit (Sigma-Aldrich®) that is similar to the [START_REF] Lowry | Protein measurment with the Folin phenol reagent[END_REF] procedure.

All activities measurements were done on a SpectroStar Nano spectrophotometer (BMG labtech, Champigny-sur-Marne, France).

All chemicals were obtained from Sigma-Aldrich®. All analyses were performed in technical duplicates for each sample, using standard methods adapted for a microplate reader. For all enzymatic analysis, homogenates were diluted to obtain linear reaction slopes for a minimum of five minutes at 25 °C. Total and specific enzymatic activities were measured and expressed as U (μmol.min -1 ).g -1 of wet tissue and U.mg -1 of protein, respectively. b) Glycogen and total lipids Tissues were grounded in liquid nitrogen using a MM400 Retsch © (GmbH, Éragny, Luxemburg) mixer mill and then hand homogenized on ice in one mL of citrate buffer (0.1 M, pH 5). Half of the sample was placed immediately at -80 °C until the total lipid analyses. The other half was incubated for four min in boiling water and then placed at -80 °C.

The determination of the total lipid concentration is based on the improved sulfo-phosphovanillin reaction from [START_REF] Frings | Improved Determination of Total Serum Lipids by the Sulfo-Phospho-Vanillin Reaction[END_REF]. For the glycogen, the sample was incubated with amyloglucosidase resulting in the complete hydrolysis of glycogen to glucose as described in [START_REF] Carr | Quantitative semi-automated enzymatic assay for tissue glycogen[END_REF].

C. Statistics analyses a) Test on the "pool" technique

To test the "pool" technique, the mean of the "individual" samples was compared to with the one of the "pooled" samples. The percentage of difference between these two values was compared for biomarkers of effect and exposure.

b) Biomarkers

Normality and homogeneity of variances were verified by Shapiro and Levene tests. Data were log10 transformed to avoid heteroscedasticity when possible.

Pearson's and Spearman's (in case of heteroscedasticity) correlations between total and specific enzymatic activity responses were verified for all enzymes.

In limpet and sea cucumber, one-way ANOVAs (or non-parametric Kruskal-Wallis tests in case of heteroscedasticity) were used to test the effects of location on the biomarkers of effect and trace element bioaccumulation, followed by respectively Tukey or Mann-Whitney Wilcoxon post-hoc test when a significant effect was found (P < 0.05). Due to many values below the limit of quantification (<LOQ), testing the difference between location was not possible for the organic pollutants' bioaccumulation data. To determine the most important parameters in our multi-parameter analysis, principal component analysis (PCA) was performed on trace element and organic contaminants concentration in tissue of limpet and sea cucumber grouped per location.

Finally, for limpet and sea cucumber, the "Integrated Biomarker Response" (IBR, [START_REF] Devin | The integrated biomarker response revisited: optimization to avoid misuse[END_REF])) was calculated using the response of biomarkers of effect (results from the "individual" sampling technique). On R software, the functions from the package IBRtools were used [START_REF] Devin | The integrated biomarker response revisited: optimization to avoid misuse[END_REF] based on [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF]).

Results

A. Test on the "pool" technique

For all the biomarkers, the difference between the value obtained for "pooled" samples and the average of the values obtained for each "individual" samples were classified into three categories: < 10% of difference, 10-30% of difference and > 30% of difference (Figure 2). The difference between the "pool" and the "individual" samples varied according to the species and class of biomarkers. In mussels, the difference between "pool" and "individual" techniques was less than 30% in 6.25, 7.22 and four times out of 10 for the biomarkers of effect, the metallic trace elements and the organic pollutants bioaccumulation respectively. For limpet, the same was observed 7.8, 9.4 and five times out of 10 and for sea cucumber, it was 5.7, 5.5 and 0 times out of ten. On the contrary to the other biomarker classes, the difference was superior or equal to 30% in all species for the organic pollutants.

B. Trace element contamination of water

Among the 18 metallic trace elements measured in water samples (Table 1), only five (Co, Cu, Mn, Mo and Pb) were above the limit of quantification. The highest values for Cu and Mn were observed in St-Florent and the one of Mo was detected in Calvi. The higher number of metallic trace elements was detected in Île Rousse. No trace element contamination was observed at STARESO.

C. Biomarkers of exposure

For the trace element contamination (Table 2), differences between location were statistically tested only for limpet and sea cucumber as wild mussel was found only in St-Florent. While the difference between location were found only for Mn (P < 0.001) regarding sea cucumber, differences were observed for Al, As, Ba, Co, Cr, Cu, Fe, Mn, Ni, Pb, Se, V and Zn in limpet.

The contamination trend in limpet differed according to the trace element: for Al, Cu, Fe, Mn, Se (P < 0.001) and Co (P < 0.01), higher levels were found in Calvi and Île Rousse compared to STARESO; for Ba (P < 0.05) and Pb (P < 0.001), the contamination level is lower in STARESO compared to those in Île Rousse; for Zn and V (P < 0.001), Calvi was the most contaminated location; for Cr and Ni (P < 0.001), STARESO and Calvi contamination was higher than Île Rousse; for As (P < 0.001), Calvi presented a lower contamination than Île

Rousse, and STARESO presented intermediate levels. For all the other metallic trace elements (Ag, As, Cd, Mo, Sb, Sn), the contamination registered in organisms was similar among locations. Organic pollutants bioaccumulation (Table 3) was only detected in St-Florent for mussel and sea cucumber and in Île Rousse for limpet. Mussels accumulated more organic pollutants ( 14HAPs, eight PCBs and one pesticide) than limpets (8 HAPs) or sea cucumbers (5 HAPs).

Concerning the bioaccumulation of the different organic pollutant categories, mussel was the only organism to accumulate PCBs and pesticides. Specific and total activities measured in mussel, limpet and sea cucumber were significantly correlated (R between 0.89 and one depending of the enzyme, P < 0,001). Total activities and specific activities can be found in Supplementary Tables 1.

The response of the antioxidant system varied according to the species (Figure 4). In limpet the SOD and GST activities were much higher than those detected in mussel (and in sea cucumber for GST). The effect of location was significant for sea cucumber SOD activity. Its SOD activity was 1.2 times higher (P < 0.01) in individuals collected in STARESO and Île Rousse than in those from St-Florent.

Fig. 4

Total enzymatic activity of superoxide dismutase (U/g of wet tissue) and glutathione Stransferase (mU/g of wet tissue) in digestive gland of mussel, soft tissue of limpet and body wall of sea cucumber collected in North Corsica in January (mean ± standard deviation).

Different letters indicate significant difference between the location, within the same species (P < 0.05). Circles indicate the value for the pooled data collected in the same location.

LDH, PK and PEPCK (Figure 5) are respectively involved in fermentation, glycolysis and neoglycolysis. Significant variations of their activities were only observed for limpets. In mussel and sea cucumber, the activity of these three enzymes was similar. In limpet collected in STARESO, the PK activity was significantly higher than in Calvi and Île Rousse, and the PEPCK activity was significantly higher than in Calvi. The activity of AChE (Figure 6), used as an indicator of neurotoxicity, was similar according to the location for all three organisms. The same was noted for the activity of LAC, enzyme involved in immunity processes. The lipid and glycogen levels (Figure 7) presented approximately the same levels in mussel and in limpet. For limpet, no differences between locations were noted either for the lipids or glycogen contents. In sea cucumber, the glycogen content was similar among location while the lipids content was significantly lower in Calvi compared to the other locations. The IBR calculated using the biomarkers of effect response gave variable results according to the organism (Figure 8). In limpet, the IBR value was the highest in Calvi, the lowest in STARESO and intermediate in Île Rousse. In sea cucumber, the lowest IBR values were found in STARESO and Calvi, the greatest values were found in Île Rousse. They were analysed considering a multibiomarkers approach (indicators of stress, energetic metabolism, neurotoxicity, immunity, metallic trace elements/organic pollutants bioaccumulation).

In accordance with our first hypothesis, the results of this study validate the use of the "pool" sampling technique. This work therefore confirms the conclusion of several other studies focusing on the analyses of organic pollutant in herring gulls (Larus arentatus) [START_REF] Turle | Validation of the use of pooled samples for monitoring of contaminants in wildlife[END_REF] or on the analyses of metallic trace elements and biomarker of effect on black scallop (Mimachlamys varia) collected in port areas [START_REF] Breitwieser | Assessment of the biological quality of port areas: A case study on the three harbours of La Rochelle: The marina, the fishing harbour and the seaport[END_REF]. Both authors showed the reliability of the "pool" sampling technique, as the means of individual analyses and those of pooled samples was lower than the normal analytical variation. A validation was however necessary in the present study because the results might differ according to the studied organisms and the considered biomarkers. Here, the "pool" technique was especially reliable for Al, Cd, Co, Cu, Mo, Ni, Pb, Se and Zn accumulations in limpet; for LAC activities, Cd, Co, Cr and Se accumulations in mussel; and for AChE and LAC activities, Ag, Cr, Mo, Ni, Se and Zn accumulations in sea cucumber. The reliability of the "pool" technique for metallic trace element accumulation was especially observed in limpet. However, the "pool" sampling techniques is not adapted to the organic pollutant bioaccumulation analyses in our study. This is mainly because most of the analysed organic pollutants are close to or below the detection limit.

Considering this, the "pool" technique allows researchers to obtain reliable results at lower costs [START_REF] Breitwieser | Assessment of the biological quality of port areas: A case study on the three harbours of La Rochelle: The marina, the fishing harbour and the seaport[END_REF]). This can be advantageous when the analyses are quite expensive (trace element or organic pollutants analyses for example), when the population is composed of a limited number of individuals, to gain time in laboratory analyses or to reduce the number of organisms (3 R's: Reduce, Reuse, Recycle). This technique has however some disadvantages as it does not allow to precisely assess the impact of a mix of contaminants nor to detect extreme or abnormal values as with inter-subject studies. Also, it may have negative consequences when the biological variation is important or not known [START_REF] Bignert | The need for adequate biological sampling in ecotoxicological investigations: a retrospective study of twenty years pollution monitoring[END_REF][START_REF] Bignert | Biological variation an important factor to consider in ecotoxicological studies based on environmental samples[END_REF].

Our second hypothesis supposed that both mussel and limpet would give similar results while sea cucumber would be more contaminated. This was shown to be wrong by our results. In fact, biomarkers gave more responses in limpet than in sea cucumber. They allowed statistical discrimination of the studied locations. Mussel had a similar response as limpet, however, mussel are only found in St-Florent, where the primary production is enhanced by freshwater inputs coming from the Aliso River. In the other locations, the oligotrophic conditions are not suitable for the installation of mussel. In mussel, it is then difficult to affirm that the biomarker response reflects the pollution on the locations rather than the starvation effect. Considering this and despite the bioindicative capacity of mussel shown in many studies [START_REF] Caricato | Seasonal variation of biomarkers in Mytilus galloprovincialis sampled inside and outside Mar Piccolo of Taranto (Italy)[END_REF][START_REF] Lehtonen | Mussel Caging and the Weight of Evidence Approach in the Assessment of Chemical Contamination in Coastal Waters of Finland (Baltic Sea)[END_REF][START_REF] Uluturhan | Seasonal variations of multi-biomarker responses to metals and pesticides pollution in M. galloprovincialis and T. decussatus from Homa Lagoon, Eastern Aegean Sea[END_REF], mussel is not adapted to the environmental conditions of North Corsica port areas. Mussel should only be used in complementarity with other organisms. In sea cucumber, the only effect of location observed on the biomarkers of effect was for the lipids content and SOD activity. Compared to the other locations, lipid contents were lower in Calvi. This might reflect a lower food supply at this location. The SOD activity was higher in STARESO and Île Rousse compared to the one in St-Florent. SOD is involved in antioxidative defences, and this response could reflect more stress in STARESO and Île Rousse. However, the significant difference in SOD activity was only detected in total activity and not in specific activity, meaning there is no significant difference at a protein level.

The variation in SOD total activity is not confirmed by the trace element bioaccumulation in sea cucumber: most metallic trace elements (except Mn) have similar levels among location.

Mn bioaccumulation varied across sites and St-Florent seems to be the most polluted one. The higher metallic contamination in St-Florent compared to the other locations is also confirmed by the multivariate analysis that includes all contaminants. The biomarkers of exposure and biomarkers of effect gave contradictory responses in St-Florent. This could be explained by the presence of some HAPs detected or even of other pollutants not analysed in the present study (e.g., emerging contaminants). Considering these results, the sea cucumber might not be an efficient bioindicator species in North Corsica. However, previous studies demonstrated the reliability of sea cucumber as bioindicator [START_REF] Aydin | Effects of anthropological factors on the metal accumulation profiles of sea cucumbers in near industrial and residential coastlines of İzmir, Turkey[END_REF][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] Our results confirm the bioaccumulative capacities of sea cucumber, but the lack of correlation with the biomarker of effects should be considered when using this species as bioindicator. The tissue analysed in the present study (body wall of sea cucumber), could also impacted our results. The sensibility of body wall might be lower compared to the one of other tissues such as respiratory tree or intestine [START_REF] Aydin | Effects of anthropological factors on the metal accumulation profiles of sea cucumbers in near industrial and residential coastlines of İzmir, Turkey[END_REF].

The limpet seems to be the most promising organism to monitor the effects of pollutants in North Corsica port areas. In our study, it bioaccumulates more metallic trace elements in Calvi and Île Rousse (significant difference between location for Al, As, Ba, Co, Cu, Cr, Fe, Mn, Pb, Se, V and Zn) and some HAPs (benzothiophene, biphenyl, fluorene, anthracene, 2methylfluoranthene, chrysene, benzo(e)pyrene and benzo(g,h,i)perylene) in Île Rousse.

Limpets also seem more impacted in Calvi and Île Rousse than in STARESO. For example, PK activity is lower in Calvi and Île Rousse, and PEPCK activity is lower in Calvi, compared to those in STARESO. The biomarkers of exposure response are correlated with the biomarkers of effect response (for both total and specific activities). Our results confirm the reliability of limpets as a bioindicator, their sensibility to detect contamination difference among locations and the possibility to use them as a good substitute or complement to mussels for biomonitoring marine pollution. Our results verified the bioaccumulative capacities of limpets for various contaminants (but especially for Cd, Pb, Cu & Zn) demonstrated by several authors [START_REF] Conti | Baseline trace metals in Patella caerulea in a central Tyrrhenian ecosystem (Pontine Islands archipelago and Lazio region coastal sites, Italy)[END_REF][START_REF] Mbandzi | Temporal and spatial variation of heavy metal concentration in four limpet species along the southeast coast of South Africa[END_REF][START_REF] Pérez | Limpets (Patella spp. Mollusca, Gastropoda) as model organisms for biomonitoring environmental quality[END_REF]. From an ecological point of view, limpet is as interesting as a bioindicator species as mussel: its biomarkers of effects responses are linked to those of the biomarker of exposure [START_REF] Gharred | Effect of multiple pollution on the cholinesterase activity, morphometry and reproduction performance of Patella caerulea collected from eastern Tunisian coasts[END_REF][START_REF] Gouveia | Can shell alterations in limpets be used as alternative biomarkers of coastal contamination?[END_REF][START_REF] Pérez | Limpets (Patella spp. Mollusca, Gastropoda) as model organisms for biomonitoring environmental quality[END_REF][START_REF] Silva | Using biomarkers to address the impacts of pollution on limpets (Patella depressa) and their mechanisms to cope with stress[END_REF].

Considering the complexity of biochemical responses to chronic pollution, the use of a multibiomarkers approach is strongly recommended [START_REF] Caricato | Seasonal variation of biomarkers in Mytilus galloprovincialis sampled inside and outside Mar Piccolo of Taranto (Italy)[END_REF]). The IBR is a method combining several biomarker responses of effect into a general "stress index". In the present study, it was calculated for limpets and sea cucumbers from the different locations. It allowed for the identification of the most impacted location. Firstly, for both species, the IBR was the lowest in STARESO. This underscores the absence of trace element or organic pollutants contamination and confirms the use of STARESO as a reference location. The highest IBR values were not located in the same port for limpet and sea cucumber. The IBR calculated with the limpet responses shows that Calvi seems the most impacted location while the one calculated with the sea cucumber responses shows higher values for Île Rousse and St-Florent.

Previous studies used the IBR to provide indication of the environmental stress caused by environmental [START_REF] Serafim | Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems[END_REF] or anthropogenic [START_REF] Cao | Seawater acidification increases copper toxicity: A multi-biomarker approach with a key marine invertebrate, the Pacific Oyster Crassostrea gigas[END_REF][START_REF] Ji | Using multi-integrated biomarker indexes approach to assess marine quality and health status of marine organism: a case study of Ruditapes philippinarum in Laizhou Bay, China[END_REF]Luna-Acosta et al. 2017b;[START_REF] Silva | Using biomarkers to address the impacts of pollution on limpets (Patella depressa) and their mechanisms to cope with stress[END_REF] factors. However, the biological responses must be interpreted cautiously and in combination with chemical data to avoid misinterpretation [START_REF] Cravo | A multibiomarker approach in the clam Ruditapes decussatus to assess the impact of pollution in the Ria Formosa lagoon, South Coast of Portugal[END_REF]. The calculation of the IBR is highly dependent on a number of a priori choices [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF]. It will not explain the biological meaning, but will rather provide the mathematical relations between the biological data [START_REF] Ji | Using multi-integrated biomarker indexes approach to assess marine quality and health status of marine organism: a case study of Ruditapes philippinarum in Laizhou Bay, China[END_REF]). In our study, the IBR gives us a first integrated view of the situation in the North Corsica port. As already shown in previous studies, the IBR is a promising tool for scientists and managers to classify the toxic potential of coastal areas [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF][START_REF] Benali | Integrated use of biomarkers and condition indices in mussels (Mytilus galloprovincialis) for monitoring pollution and development of biomarker index to assess the potential toxic of coastal sites[END_REF].

Finally, in accordance with our last hypothesis, the pollution on the considered areas is still low compared to other bigger port areas in the Mediterranean Sea [START_REF] Galgani | Chemical contamination and sediment toxicity along the coast of Corsica[END_REF]. This is mainly due to the small size of the ports (between 250 and 900 boats) considered in the present study. Despite some anthropic pressures registered close to the ports (such as boats rental agencies), the chemical contamination was still below the reglementary threshold values.

For future research, it will be necessary to obtain more data on environmental parameters and on the port contamination. This could be done by measuring environmental conditions (temperature, salinity, oxygen or chlorophyll-a), by collecting more water samples and by adding sediments analyses. Furthermore, it will be necessary to integrate more biomarker data (for example other contaminants such as tributyltin) or condition index to consider the intraspecific variability of wild organisms and how their feeding mode influence their response to chemical exposure. Longer biomonitoring is also needed to improve the evaluation of the contaminated locations. Finally, to more precisely test the bioindicative capacities of species, some experimental studies with controlled conditions would be essential.

Conclusion

This study is the first to give information on the ecological quality of the port areas in North

Corsica. The biomonitoring carried out using three bioindicator species (M. galloprovincialis, Patella sp. and H. tubulosa) demonstrated that the choice of a species adapted to the local environment is of primary importance for efficient biomonitoring. In North Corsica, the limpet is present in most locations in comparison to the mussel, and it responds better than the sea cucumber. For future biomonitoring, it would be very interesting to use the limpet in combination with the mussel to have a global view of the ecological quality of the water in the area. The combination between limpet and mussel will also allow the comparison with worldwide biomonitoring surveys that mainly use Mytilus sp.

The results of the present study show that the water contamination in North Corsica port areas is relatively low compared to other ports areas in the Mediterranean Sea. For future research, it is necessary to continue this monitoring over time and at different seasons to obtain more reliable data.

Finally, the validation of the sampling techniques as "pool" is important for management purpose. Even if the "pooled" data limits the analyses for fundamental research, this method gives reliable results to help the local managers and authorities for planning integrated environmental management of the port area.

  and were 25 (naphthalene, phenanthrene and PCB 138), five (dibenzothiophene, fluoranthene, 2-methylfluoranthene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, perylene, indeno(1,2,3-cd)pyrene, dibenzo(a,h)anthracene, benzo(g,h,i)perylene, PCB 153 and 4-4-DDE), 2.5 (benzothiophene, biphenyl, acenaphthylene, acenaphthene, fluorene, anthracene, pyrene, benzo(a)anthracene, PCB 7, PCB 28, PCB 52, PCB 35, PCB 101, PCB 77, PCB 135, PCB 118, PCB 156, PCB 180, PCB 169, aldrine, isodrine, dieldrine and endrine) and 0.5 (PCB 105, hexachlorobenzene and 2-4-DDE) ng.g -1 dry weight. Analytical quality control was done using the Standard Reference Materials 1974c "Organics in Mussel Tissue (Mytilus edulis)" provided by the National Institute of Standards (SRM) and Technology (NIST, Gaithersburg, USA).

Fig. 2

 2 Fig. 2 Occurrence of the different class of difference (in %) between « pool » and

  When analysing the results of all contaminants (trace element and organic contaminants) in sea cucumber tissues, the PCA principal component (Dim1) explained 36.9% while the second principal component (Dim2) explained 18.7 % of the total variation. It was difficult to determine the most important factors for the model because of the high number of parameters (Figure3A). Contaminant concentrations in tissue from St-Florent seemed to be more explained by Dim1. Dim1 was related to concentrations of Cd, Co and Mn. In the other locations, contaminant concentrations in tissues seemed to be better explained by Dim2. Dim2 is mainly related to Mo, Sb, Ag, Ba and Ni (Figure3A, supplementary material 1). In limpet, the PCA principal component (Dim1) explained 30.7% while the second principal component (Dim2) explained 17.7 % of the total variation. While contaminants concentration in tissue from Île Rousse seemed to be explained by both the Dim1 and Dim2, contaminants concentration in tissue from STARESO and Calvi seemed to be more explained by Dim1.

Fig. 3

 3 Fig.3PCA results for trace element and organic contaminants in (A) sea cucumber and (B)

Fig. 5

 5 Fig. 5 Total enzymatic activity of lactate dehydrogenase, pyruvate kinase and

Fig. 6

 6 Fig. 6 Total enzymatic activity of acetylcholinesterase and laccase (mU/g of wet tissue) in

Fig. 7

 7 Fig. 7 Total lipids and glycogen concentrations (mg/g of wet tissues) in digestive gland of

Fig. 8

 8 Fig. 8 Integrated Biomarker Responses (IBR, mean) calculated for limpet and sea cucumber

Fig. 3 Fig. 4 Fig. 4 Fig. 5 Fig. 6

 34456 Fig. 3 Map of the experimental locations in North Corsica (France) and anthropic pressures

Fig. 7 Fig. 8

 78 Fig. 7 Total lipids and glycogen concentrations (mg/g of wet tissues) in digestive gland of

Table 1 :

 1 Trace element concentration (µg/L) in water sample collected in the different locations in January. Grey cells indicate values higher than the limit of detection.

		STARESO	Calvi	Île Rousse St-Florent
	Ag	< 10	< 10	< 10	< 10
	Al	< 200	< 200	< 200	< 200
	As	< 10	< 10	< 10	< 10
	Ba	< 10	< 10	< 10	< 10
	Cd	< 2	< 2	< 2	< 2
	Co	< 1	1,0	< 1	< 1
	Cr	< 10	< 10	< 10	< 10
	Cu	< 2	2,6	5,5	12,3
	Fe	< 2500	< 2500	< 2500	< 2500
	Mn	< 1	< 1	< 1	18,94
	Mo	< 10	12,7	11,0	< 10
	Ni	< 100	< 100	< 100	< 100
	Pb	< 1	< 1	1,9	< 1
	Sb	< 1	< 1	< 1	< 1
	Se	< 40	< 40	< 40	< 40
	Sn	< 2	< 2	< 2	< 2
	V	< 100	< 100	< 100	< 100
	Zn	< 2500	< 2500	< 2500	< 2500

Table 2 :

 2 Trace element concentration (µg/g of wet tissue) in soft tissue of mussel and limpet and body wall of sea cucumber collected in North Corsica in January 2020 (mean ± standard deviation, N=7). Letters indicate significant difference between the locations (P < 0.05) within the same species.

	mussel	limpet

Table 3 :

 3 Organic pollutant concentration (µg/g of wet tissue) in soft tissue of mussel and limpet and body wall of sea cucumber collected in North 361No organic pollutants were detected in limpet collected in STARESO and Calvi or in sea cucumber collected in STARESO, Calvi and Île Rousse. However, organic pollutants concentrations in tissues of organisms were below the Environmental Quality Standards (EQS) defined by the 2013/39/EU directive for biota.

	362	Corsica in January (mean ± standard deviation, N=1-7). <LD = below the detection limit.
			mussel				limpet				sea cucumber
			St Florent (individual)	St Florent (pool)	STARESO Calvi	Île Rousse (individual)	Île Rousse (pool)	STARESO Calvi	Île Rousse	St Florent (individual)	St Florent (pool)
		naphthalene	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		benzothiophene	<LD	<LD	<LD	<LD	4.92 ± 0.72	<LD	<LD	<LD	<LD	<LD	<LD
		biphenyl	<LD	<LD	<LD	<LD	4.61 ± 2.03	3.02	<LD	<LD	<LD	5.52	<LD
		acenaphthylene	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		acenaphthene	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		fluorene	<LD	<LD	<LD	<LD	3.89	2.98	<LD	<LD	<LD	4.15	<LD
		dibenzothiophene	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		phenanthrene	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		anthracene	3.63	<LD	<LD	<LD	3.18	<LD	<LD	<LD	<LD	<LD	<LD
		fluoranthene	6.58	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		2-methylfluoranthene	9.80 ± 0.85	8.47	<LD	<LD 11.51 ± 3.07	14.47	<LD	<LD	<LD	<LD	<LD
		pyrene	4.06	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		benzo(a)anthracene	10.36 ± 9.38	3.64	<LD	<LD	<LD	2.69	<LD	<LD	<LD	<LD	<LD
		chrysene	18.35 ± 15.55	8.12	<LD	<LD	7.72 ± 1.59	12.39	<LD	<LD	<LD	<LD	<LD
		benzo(b)fluoranthene	16.98	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		benzo(k)fluoranthene	18.71	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		benzo(e)pyrene	10.23 ± 6.73	<LD	<LD	<LD	5.62 ± 0.64	10.88	<LD	<LD	<LD	<LD	<LD
		benzo(a)pyrene	9.41	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		perylene	9.24 ± 4.35	5.36	<LD	<LD	<LD	<LD	<LD	<LD	<LD	8.28 ± 2.19	5.30
		indeno(1,2,3-cd)pyrene	5.75	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD
		dibenzo(a,h)anthracene	12.65	<LD	<LD	<LD	<LD	<LD	<LD	<LD	<LD	8.52	<LD
		benzo(g,h,i)perylene	12.67 ± 1.84	9.88	<LD	<LD	7.89 ± 3.98	7.05	<LD	<LD	<LD	9.17 ± 3.29	<LD

  ; Turk Culha et al. 2016), especially for bioaccumulation analyses. For example, Turk Culha et al.(2016) detected average levels of metallic trace elements equal to 0.18 µg/g for Cd, 2.43 µg/g for Cu, 2.09 µg/g for Pb, 14.58 µg/g for Ni, 16.86 µg/g for Zn, and 73.46 µg/g for Fe in sea cucumber (H. tubulosa) sampled in the Dardanelles Strait (Turkey). In our study, for the similar metallic trace elements, the levels were lower for Cd, Pb and Ni (respectively 0.05; 1.55 and 2.52 µg/g) and higher for Cu,Zn and Fe (respectively, 4.8; 41.67 and 126.45 µg/g) in the same species.

Mr. B. Pantalacci, manager of the shellfish farm SARL Etang de Diane, and his employees who provided mussels. This work was supported by the "QUAlity of the marine environment in the Mediterranean POrt areas" (QUAMPO) project, funded by the "Fonds Européen pour les Affaires Maritimes et la Pêche" (FEAMP) -European Maritime and Fisheries Fund (EMFF) [grant number PFEA800219DM0940001, 2019].

Funding

This work was supported by the "QUAlity of the marine environment in the Mediterranean POrt areas" (QUAMPO) project, funded by the "Fonds Européen pour les Affaires Maritimes et la Pêche" (FEAMP) -European Maritime and Fisheries Fund (EMFF) [grant number PFEA800219DM0940001, 2019].

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Statements and declarations

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

The authors have no competing interests to declare that are relevant to the content of this article.

Ethical Approval

This study does not involve human participants or vertebrate animals.

Consent to Participate

Yes

Consent to Publish

Yes

List of captions:


Marion Pillet
email: marion.pillet@univ-lr.fr

K Garrett Evensen

Michel Marengo

Pierre Lejeune

Helen C Poynton

Hélène Thomas

First insight into the development of a new transcriptomic tool in French Corsica harbors

Keywords: chronic pollution, emergent contaminant, C-BED, biomonitoring, M. galloprovincialis, multibiomarkers

Coastal harbor areas are subjected to a myriad of pollution sources with largely unknown effects. Such complex chemical mixtures are difficult to monitor but transcriptomics is a promising approach for such biomonitoring. The present study was designed to verify the use of the Coastal Biosensor for Endocrine Disruption (C-BED) assay, previously developed to detect emerging contaminants and their effects on Mytilus edulis, on another mussel species, Mytilus galloprovincialis. Mussels were caged on St-Florent harbor (contaminated) and on Revellata Bay (reference) for three months. A classical multibiomarkers approach was coupled to the C-BED assay. The results of both approaches were analysed using the Integrated Biomarkers Responses (IBR) and compared to each other. Both approaches demonstrated a higher contamination and probable endocrine disruption of mussels in St-Florent, compared to the reference station. These results confirm that the C-BED assay provides an innovative method to expands our ability to detect emerging contaminants.

Introduction

Coastal harbor areas are susceptible chronic and diffuse marine pollution due to contaminant run-off from natural and anthropogenic sources (e.g. terrestrial discharges, anthropogenic contaminations) and estuarine characteristics (e.g., low hydrodynamism) [START_REF] Islam | Impacts of pollution on coastal and marine ecosystems including coastal and marine fisheries and approach for management: a review and synthesis[END_REF]. This pollution is a serious threat to the marine environment and recreational activities and could pose a subsequent risk to human health [START_REF] Maisano | Effects of petrochemical contamination on caged marine mussels using a multi-biomarker approach: Histological changes, neurotoxicity and hypoxic stress[END_REF]. Recently, the presence of legacy pollutants (trace elements, PAHs, PCBs) has declined within urban coastal systems, but emerging contaminants (e.g. pharmaceuticals, perfluorinated alkylated substances, nanomaterials) are becoming more prominent even has their impacts are not fully understood [START_REF] Maruya | The Mussel Watch California pilot study on contaminants of emerging concern (CECs): Synthesis and next steps[END_REF][START_REF] Richardson | Water Analysis: Emerging Contaminants and Current Issues[END_REF]. The concern about these compounds has increased due to endocrine disruptive effect and their potential to cause population level effects in wildlife at low exposure levels [START_REF] Blalock | Assessing legacy and endocrine disrupting pollutants in Boston Harbor with transcriptomic biomarkers[END_REF]. In addition, these chemicals are frequently found together in complex mixtures. Determining the impacts of emerging contaminants and how they interact with other chemical stressors to cause additive or synergistic effects is one of the major challenges in ecotoxicology today [START_REF] Van Den Brink | Toward sustainable environmental quality: Priority research questions for Europe[END_REF].

In 2008, the European Commission approved the Marine Strategy Framework Directive (DCSMM, 2008), a legislation aimed at achieving or maintaining "Good Environmental Status" in EU waters. In North Corsica, to our knowledge, no study has been done within its harbor areas, although it is important to evaluate and monitor the quality of the waters and the ecosystem.

Marine biomonitoring consists in observing and assessing the natural or induced alterations of marine ecosystem and this evaluation can be measured at several levels, from the molecular, cellular, and individual levels to the populations up to whole biocenosis level [START_REF] Bondaruk | Identification of hazards for water environment in the upper Silesian Coal Basin caused by the discharge of salt mine water containing particularly harmful substances and radionuclides[END_REF]. Bivalves and especially mussels have been used as bioindicators for decades to investigate the toxicity of pollutants. The classical example of biomonitoring is the Mussel Watch concept, which was initiated in 1976, consisting in the surveillance of United States coastal regions for their pollutant contents [START_REF] Farrington | Goldberg's proposal of "the Mussel Watch": Reflections after 40 years[END_REF]. The use of these bioindicators is particularly relevant from a biological point of view as the directly measured concentrations reflect the bioavailable fractions in the marine environment [START_REF] Wang | Chapter 4 -Bioaccumulation and Biomonitoring[END_REF]. Mussels are ideal for biomonitoring studies because they are filter feeders, are distributed worldwide, and have capacity to bioaccumulate diverse contaminants [START_REF] Banni | Combined effects of n-TiO2 and 2,3,7,8-TCDD in Mytilus galloprovincialis digestive gland: A transcriptomic and immunohistochemical study[END_REF][START_REF] Canesi | Short-term effects of environmentally relevant concentrations of EDC mixtures on Mytilus galloprovincialis digestive gland[END_REF][START_REF] Ozkan | Multi-biomarker responses after exposure to pollution in the Mediterranean mussels (Mytilus galloprovincialis L.) in the Aegean coast of Turkey[END_REF][START_REF] Strehse | Marine bivalves as bioindicators for environmental pollutants with focus on dumped munitions in the sea: A review[END_REF]. They also provide several biomarker responses, such as decreased antioxidant defences in the presence of pollution [START_REF] Beyer | Blue mussels (Mytilus edulis spp.) as sentinel organisms in coastal pollution monitoring: A review[END_REF].

Despite the sensitivity of conventional biomarkers for biomonitoring and risk assessment [START_REF] Handy | Ecotoxicity test methods for engineered nanomaterials: Practical experiences and recommendations from the bench[END_REF], these indicators may not respond to low level, chronic exposures to pollutants or exposure to emergent pollutants. More integrative tools, are needed that offer greater sensitivity and specificity across diverse pollutant classes [START_REF] Gottschalk | Engineered nanomaterials in water and soils: A risk quantification based on probabilistic exposure and effect modeling[END_REF]. Transcriptomic and other High Throughput Screening (HTS) approaches, are of great interest because they allow the screening of a large set of genes and the impact of pollutants on their expression [START_REF] Châtel | Transcriptomic approach: A promising tool for rapid screening nanomaterial-mediated toxicity in the marine bivalve Mytilus edulis -Application to copper oxide nanoparticles[END_REF][START_REF] Poynton | Ecotoxicogenomics: Emerging Technologies for Emerging Contaminants[END_REF].

In the present study, a classic multi-biomarker approach was coupled to an innovative transcriptomic approach that uses the biological responses of blue mussels to detect the additive impacts of complex contaminant mixtures found in urban coastal areas. The Coastal Biosensor for Endocrine Disruption (C-BED), was designed based the responses of Mytilus edulis exposed to estrogenic compounds in the lab, and was successfully validated by detecting endocrine disrupting compounds (EDCs) in field collected mussels in Northeastern U.S.A. [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF][START_REF] Blalock | Assessing legacy and endocrine disrupting pollutants in Boston Harbor with transcriptomic biomarkers[END_REF]. The assay uses the transcriptomic responses of a specific set of biomarker genes chosen because of their roles in endocrine pathways and sexual development, and their robust responses to endocrine disrupting stressors.

The goal of this paper is to translate this innovative method to a different mussel species, M. galloprovincialis, and to test the incorporation of a molecular tool in a classic biomonitoring in Mediterranean harbors of North Corsica.

Material and methods

Caged mussels (M. galloprovincialis) were deployed in St. Florent harbor (exposed site,North Corsica,N 42° 40' 50.362'';E 9° 17' 54.373'') and in the Revellata Bay (control site, N 42° 34' 49.584''; E 8° 43' 31.511'') for three months before sampling in September 2020 and January 2021. At the end of the exposure, 20 mussels were collected for subsequent classic biomarkers (n = 10) and molecular (C-BED, n = 10) analyses.

A. Classic biomarker approach

Individuals collected for the classic biomarkers were immediately placed in dry ice, shipped to the laboratory within 48 h and kept at -80°C until enzymatic analyses.

The digestive gland of mussel was dissected and ground in liquid nitrogen using a MM400 Retsch© (GmbH, Éragny, Luxemburg) mixer mill and homogenized on ice in phosphate buffered saline (PBS) solution (100 mM, pH 7.5) containing 0.1 % Triton X-100 and 1 mM methylene diamine tetra-acetic acid (EDTA) to avoid protein degradation. The samples were centrifuged, and supernatant was collected for the subsequent biomarkers of effect and protein analyses.

A multibiomarkers approach was used by testing superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), pyruvate kinase (PK), acetylcholinesterase (AChE) and laccase-type phenoloxidase (LAC). SOD activity protocol is based on the inhibition of a superoxide-driven NADH oxidation and was measured following the rate of NADH oxidation at 340 nm [START_REF] Paoletti | A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts[END_REF]. CAT activity was determined according to [START_REF] Aebi | Catalase in vitro[END_REF] by measuring the rate of decomposition of hydrogen peroxide at 240 nm. GPx activity was assayed according to [START_REF] Paglia | Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase[END_REF] as modified by [START_REF] Janssens | Reduced enzymatic antioxidative defense in deep-sea fish[END_REF] by monitoring the consumption of NADPH at 340 nm. GST activity was determined by measuring the production of GS-DNB at 340 nm [START_REF] Habig | Glutathione S-transferases. The first enzymatic step in mercapturic acid formation[END_REF]. The activity of PK was determined by measuring the NADH consumption at 340 nm, according to [START_REF] Childress | Depth-related enzymic activities in muscle, brain and heart of deep-living pelagic marine teleosts[END_REF] as modified by [START_REF] Bailey | High swimming and metabolic activity in the deepsea eel Synaphobranchus kaupii revealed by integrated in situ and in vitro measurements[END_REF]. AChE activity was assayed by recording the production of 5-thio-2-nitrobenzoate at 405 nm [START_REF] Ellman | A new and rapid colorimetric determination of acetylcholinesterase activity[END_REF]. Finally, the LAC activity was measured according to [START_REF] Luna-Acosta | First evidence of laccase activity in the Pacific oyster Crassostrea gigas[END_REF], following the oxidation of phenylenediamine at 420 nm. The total protein content was measured with the bicinchoninic acid protein assay kit (Sigma-Aldrich®) that is similar to the [START_REF] Lowry | Protein measurment with the Folin phenol reagent[END_REF] procedure.

All activities measurements were done on a SpectroStar Nano spectrophotometer (BMG labtech, Champigny-sur-Marne, France). Chemicals were obtained from Sigma-Aldrich®. All analyses were performed in duplicate using standard methods adapted for a microplate reader.

For all enzymatic analysis, homogenates were diluted to obtain linear reaction slopes for a minimum of five minutes at 25 °C. Specific enzymatic activities were measured and expressed as U (μmol.min-1).mg-1 of protein.

B. Transcriptomic approach

For the C-BED assay, mussel (n = 10 for each period) were immediately dissected and the digestive gland was kept in RNAlater. Total RNA was extracted using TriReagent (Molecular Research Center, Cincinnati, OH), according to the manufacturer's protocol.

Samples were then treated with DNase (New England Biolabs, Ipswich, MA) to remove any residual DNA. Samples were cleaned up using a NEB Monarch RNA Cleanup Kit (New England Biolabs) to purify the RNA samples. RNA was reverse transcribed to cDNA using EpiScript Reverse Transcriptase (Lucigen, Middleton, WI) [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF][START_REF] Blalock | Assessing legacy and endocrine disrupting pollutants in Boston Harbor with transcriptomic biomarkers[END_REF][START_REF] Hines | Comparison of histological, genetic, metabolomics, and lipid-based methods for sex determination in marine mussels[END_REF].

The C-BED assay uses real time quantitative PCR (RT-qPCR) and consists of eleven genes: two sex marker genes (for sex identification: VERL, VCL), eight biomarker genes (Cav-1, StAR, MBTP, BCAS3, SR, ADX, GCL32E, Mat89bb), and one reference gene (18S) [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF][START_REF] Blalock | Assessing legacy and endocrine disrupting pollutants in Boston Harbor with transcriptomic biomarkers[END_REF]. M. edulis primers sequences from [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF] were tested on the M. galloprovincialis samples and verified on an agarose gel. All primers, except VERL, produced a product. New primers for M. galloprovincialis VERL (GenBank: VDI63802.1) were designed using Primer3 (v4.1.0) (F: GCT GTT GCG TTG CTA TCA CT R: TGC AGA TGA CGC CTT TAT GC) and were tested and verified using qPCR.

All qPCR reactions were performed as described previously [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF] using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, Waltham, MA). Standard curves for all primers, including the newly designed M. g.,VERL primers, used had an r 2 of 0.99 and a primer efficiency between 95-105% [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF]. The difference in expression in VERL and VCL was used for sex identification of M. galloprovincialis mussels [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF][START_REF] Hines | Comparison of histological, genetic, metabolomics, and lipid-based methods for sex determination in marine mussels[END_REF]. A difference in Ct greater than 2.5 indicates the mussel is a male, less than -2.5 is a female, and between -2.5 and 2.5 is unknown (i.e., sex cannot be determined) [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF][START_REF] Hines | Comparison of histological, genetic, metabolomics, and lipid-based methods for sex determination in marine mussels[END_REF]. Expression of the eight biomarker genes were normalized to 18S expression. The C-BED assay was performed on all mussels including those identified as male, female, or undetermined. This is in contrast with our previous study using the C-BED assay, which limited assay responses to male mussels only [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF]. We have decided to extend the assay to all mussels because (1) there was limited difference between the IBR score of males vs females in this study (see supplement) and (2) in the presence of endocrine disrupting compounds, male mussels may become phenotypically female and be identified as female mussels (Evensen in prep). Therefore, focusing only on mussels identified as male following an exposure is not including the responses of all genetically male mussels.

C. Statistics

Data were compiled into the Integrated Biomarker Response (IBR). IBR was developed by [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF]. The IBR was described to be useful in environmental quality assessment to evaluate the impact of environmental conditions on the health of organisms and their physiological status [START_REF] Serafim | Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems[END_REF] and was shown to be useful to distinguish among sites located along a stress gradient, while individual biomarkers were not able to do so [START_REF] Bertrand | Native crustacean species as a bioindicator of freshwater ecosystem pollution: A multivariate and integrative study of multibiomarker response in active river monitoring[END_REF]. [START_REF] Devin | The integrated biomarker response revisited: optimization to avoid misuse[END_REF] adjusted the IBR calculation by determining every permutation of the IBR based on different positions of the biomarkers and calculating the median IBR value. The median IBR value was calculated for both sites at both time points. On these data, normality and homogeneity of variances were verified by Shapiro and Levene tests and data. As these conditions were not found and no data transformations was possible, nonparametric Kruskall-Wallis tests, were performed to analyse the effect of treatment, followed by Mann-Whitney Wilcoxon post-hoc test when a significant effect was found.

Results

Mussel sex identification was performed to evaluate for skewed sex ratios. There was no significantly skewed sex ratio at Revellata Bay at either time points or at St. Florent January 2021. There was a significantly skew sex ratio (p<0.05) at St. Florent in September 2021 (Figure 1). At St. Florent in September, there was only one male out of the ten sampled mussels; the remaining mussels were all female. The classical biomarkers showed higher IBR values (figure 2) at St-Florent (median = 5.37 in September and 4.62 in January) compared to Revellata Bay (median = 0.105 in September and 2.11 in January), and a greater difference in the summer.

The relative importance of each biomarker varies according to season and site: in St-Florent in September, all enzymes present relatively low coefficients, while the higher coefficients are observed for LAC, GPx, GST and PK in St-Florent in January and for CAT, ACHE and SOD in Revellata Bay at both seasons. Florent compared with Revellata Bay (Figure 3). At both time points, the IBR values at St. Florent were higher than Revellata Bay. In September 2020 and January 2021, Revellata Bay had IBR values less than 1, while St-Florent had IBR values greater than 1, reaching 8.76 in September 2020 (Figure 3). At both sites, the IBR values for January were lower than those for September (Figure 3). Based on the IBR values, we ranked the sites and time points based on risk of endocrine disruption: Revellata Bay January 2021 < Revellata Bay September 2020 < St. Florent January 2021 < St. Florent September 2020. in the water, causing a stress response in the St. Florent mussels. These results confirm observations described in previous environmental biomonitoring studies. St-Florent is usually described as under moderate anthropogenic pressure [START_REF] Gobert | Assessment of the ecological status of Mediterranean French coastal waters as required by the Water Framework Directive using the Posidonia oceanica Rapid Easy Index: PREI[END_REF] or even as a highly contaminated site [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]. The contamination is usually ascribed to high levels of metallic trace elements such as Co, Cr, Fe or Ni, linked to waste from the former Canari asbestos mine, closed in 1965, located at the northwest of the Gulf of St-Florent [START_REF] Andral | Monitoring chemical contamination levels in the Mediterranean based on the use of mussel caging[END_REF][START_REF] Luy | Chemical contamination along the Mediterranean French coast using Posidonia oceanica (L.) Delile above-ground tissues: a multiple trace element study[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]. However, there is little data available on pollution levels in this region, especially for organic contaminants.

C-BED assay is translatable across Mytilus species

Here, we adapted the C-BED assay, which was originally developed in M. edulis [START_REF] Blalock | Transcriptomic and Network Analyses Reveal Mechanistic-Based Biomarkers of Endocrine Disruption in the Marine Mussel, Mytilus edulis[END_REF], to the closely related M. galloprovincialis. This assay is used as an early indicator for possible developmental and reproductive impairments. There was a higher median IBR value at both time points at St. Florent (Sept: 8.79, Jan: 1.49) than at Revellata Bay (Figure 3).

The results of the C-BED assay indicate that mussels at St. Florent are responding to EDCs. Furthermore, the significantly feminized sex ratio (p<0.05) at Revellata Bay in September (Figure 3C), during the same sampling period as the highest IBR values, strongly suggests that endocrine disruption is occurring in these mussels. In addition, the difference in IBR values at the impacted and reference site show that this assay can be applied to M. galloprovincialis. The Mytilus species complex are found globally, from South America to the North Atlantic to the Pacific [START_REF] Oyarzún | Blue mussels of the Mytilus edulis species complex from South America: The application of species delimitation models to DNA sequence variation[END_REF][START_REF] Wilson | New diagnostic SNP molecular markers for the Mytilus species complex[END_REF]. With the success of application of the C-BED assay to M. galloprovincialis, this assay can be utilized in other Mytilus species.

Benefits of a multi-biomarker approach

Using IBR values from both approaches indicates mussels are exposed to a variety of contaminants including contaminants that elicit classic stress responses and those that cause endocrine disruption. Interestingly, both approaches indicated that contamination levels and effects are more severe during the summer. These effects might be due to the higher touristic activity in summer or to the elevated temperature for example. A single compound at a low concentration may not produce a detectable adverse effect in an organism. However, many contaminants at low concentrations may cause additive impacts, resulting in a detectable effect [START_REF] Giuliani | Transcriptional and catalytic responses of antioxidant and biotransformation pathways in mussels, Mytilus galloprovincialis, exposed to chemical mixtures[END_REF][START_REF] Strömgren | The combined effect of copper and hydrocarbons on the length growth of Mytilus edulis[END_REF][START_REF] Widdows | Mussels and environmental contaminants : bioaccumulation and physiological aspects[END_REF]. Adding a transcriptomic assay to biomonitoring can provide vital information because gene expression changes are highly responsive and sensitive [START_REF] Aymoz | Timing of gene expression in a cell-fate decision system[END_REF][START_REF] Bendjilali | Time-Course Analysis of Gene Expression During the Saccharomyces cerevisiae Hypoxic Response[END_REF]. Furthermore, utilizing multiple assays and IBR values provides greater insight to possible exposures and stress responses organisms may be experiencing [START_REF] Beliaeff | Integrated biomarker response: A useful tool for ecological risk assessment[END_REF][START_REF] Devin | The integrated biomarker response revisited: optimization to avoid misuse[END_REF][START_REF] Giuliani | Transcriptional and catalytic responses of antioxidant and biotransformation pathways in mussels, Mytilus galloprovincialis, exposed to chemical mixtures[END_REF][START_REF] Kim | Integration of multi-level biomarker responses to cadmium and benzo[k]fluoranthene in the pale chub (Zacco platypus)[END_REF].

Conclusion

Here we demonstrated that the C-BED assay is translatable to a new Mytilus species, broadening its application and that its inclusion within a multi-biomarker monitoring program provides insight into the chemicals present and causing biological effects within urban harbors.

Determining the impacts of emerging contaminants and how they interact with other chemical stressors to cause additive or synergistic effects is one of the major challenges in ecotoxicology today. The global application of the C-BED assay addresses this challenge and will significantly enhance our ability to predict environmental impacts of contamination found in our urban coastal ecosystems and harbors. Through this, we anticipate the development of a transformative tool for coastal environmental assessment into management decisions for coastal areas.

Figure 1 :

 1 Figure 1: Sex identification results at Revellata Bay and St. Florent. Bars greater than 2.5 are

Figure 2 :

 2 Figure 2: Median IBR values calculated using data from the classical biomonitoring data

Figure 3 :

 3 Figure 3: Median IBR values for Revellata Bay (September 2020, January 2021) and St Florent

Acknowledgment

The authors are grateful to Mr. B. Pantalacci, manager of the shellfish farm SARL Etang de Diane, and his employees who provided mussels.

Funding

This work was supported by the "QUAlity of the marine environment in the Mediterranean POrt areas" (QUAMPO) project, funded by the "Fonds Européen pour les Affaires Maritimes et la Pêche" (FEAMP) -European Maritime and Fisheries Fund (EMFF) [grant number PFEA800219DM0940001, 2019].

Authors Contributions

Marion Pillet: conceptualization, methodology, validation, data collection, formal analyses, investigation, writing. K. Garrett Evensen: methodology, validation, data collection, formal analyses, investigation, writing.

Michel Marengo: conceptualization, methodology, supervision, project administration, funding acquisition.

Pierre Lejeune: conceptualization, funding acquisition.

Helen C. Poynton: conceptualization, supervision, funding acquisition.

Hélène Thomas: supervision, project administration, funding acquisition.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

The authors have no competing interests to declare that are relevant to the content of this article.

Ethical Approval

This study does not involve human participants or vertebrate animals.

List of captions:


Mathéo Richard

Dany Jaffuel

Jean-Pierre Mallet

Raphael Gilson

Maxime Patout

Emeline Fresnel

Marius Lebret
email: mariuslebret@gmail.com

General nomenclature of air leak patterns: A proposal

A B S T R A C T

Leakage is a common issue with PAP therapy and may contribute to short-term noncompliance with treatment. PAP manufacturers use different methods to estimate and report air leaks (median, mean, p90, p95, % of important air leak, time spent on major leaks), which makes the analysis and comparisons challenging. Leak intensity is a well-known parameter of major importance as it can significantly affect the performance of PAP devices. However, there is a lack of knowledge regarding another leakage parameter, namely the leakage pattern. As of now, there is no standard classification for leak patterns in real-life settings. Our objective was to develop and propose a comprehensive nomenclature for leakage patterns observed during nasal auto-PAP therapy.

We examined 50 ventilatory polygraphy recordings conducted with auto-PAP, based on data from a prior study in which the unintentional leak flow was precisely measured over time. We have categorized leaks into two main types: continuous leaks, which appear and persist for an extended period, and discontinuous leaks, which vanish more rapidly. Continuous leaks can have both abrupt or gradual apparitions and terminations. Their "shape" can be either interrupted or constant. Discontinuous leaks can have both abrupt or gradual apparitions and terminations. Due to the relatively short observation scale (less than 5 min minutes), this type of leakage is not characterized by a specific shape between apparition and termination. This nomenclature could be a valuable tool that facilitates comparative bench tests or clinical studies. This tool could support manufacturers in developing precise algorithms for leak compensation, residual AHI estimation and the accuracy of CPAP monitoring data. Lastly, establishing a standardized approach for describing and categorizing air leakage patterns could assist clinicians in identifying device-related issues, such as patient discomfort arising from specific types of leakage.

Introduction to the case

Positive Airway Pressure (PAP) is considered the first-line treatment for moderate to severe Obstructive Sleep Apnea (OSA) syndrome [START_REF] Lévy | Obstructive sleep apnoea syndrome[END_REF]. While the causes of PAP cessation are multifactorial [START_REF] Gagnadoux | Influence of marital status and employment status on long-term adherence with continuous positive airway pressure in sleep apnea patients[END_REF] unintentional leakage is a common issue with PAP therapy and may contribute to short-term non-compliance with treatment [START_REF] Valentin | Air leak is associated with poor adherence to autopap therapy[END_REF]. PAP manufacturers use different methods to estimate and report air leaks (median, mean, p90, p95, % of important air leak, time spent on major leaks), which makes the analysis and comparisons of studies more challenging [START_REF] Schwab | An official American thoracic society statement: continuous positive airway pressure adherence tracking systems. The optimal monitoring strategies and outcome measures in adults[END_REF]. Leak intensity is a well-known parameter of major importance as it can significantly affect the performance of PAP devices [START_REF] Lebret | Responses of bilevel ventilators to unintentional leak: a bench study[END_REF]. However, there is a lack of knowledge regarding another leakage parameter, namely the leakage pattern. As of now, there is no standard classification for leak patterns in real-life settings, and it remains unknown whether the leakage pattern has a greater impact on the patient compared to the leakage intensity. It is crucial to note that there is no correlation between the patient's clinical assessment of leakage and the measurement of leakage by the PAP device [START_REF] Rotty | Mask sideeffects in long-term CPAP-patients impact adherence and sleepiness: the InterfaceVent reallife study[END_REF].

Image analysis

Our primary objective was to establish and develop a comprehensive nomenclature for leakage patterns observed during nasal auto-PAP therapy. Following this, we intend to simulate these leakage patterns using a realistic bench model. We examined 50 ventilatory polygraphy recordings conducted with auto-PAP, totalling over 12,000 min of treatment time. Our analysis was based on data from a prior study [START_REF] Lebret | Feasibility of type 3 polygraphy for evaluating leak determinants in CPAP-treated OSA patients: a step toward personalized leak management[END_REF], in which the unintentional leak flow was precisely measured over time for each patient's recording. However, in that previous study [START_REF] Lebret | Feasibility of type 3 polygraphy for evaluating leak determinants in CPAP-treated OSA patients: a step toward personalized leak management[END_REF], we did not evaluate the effects of leak patterns or magnitudes on clinical outcomes, leaving this area unexplored. The nomenclature we derived from this analysis is summarized in Figs. 1 and2.

Discussion

As per Baltzan et al. [START_REF] Baltzan | Leak profile inspection during nasal continuous positive airway pressure[END_REF], we have categorized leaks into two main types: continuous leaks, which appear and persist for an extended period, and discontinuous leaks, which vanish more rapidly. The presence of intermittent, non-continuous leaks with irregular patterns may have different effects on the PAP device and/or the patient compared to leaks with more consistent and stable characteristics over time (i.e., continuous leaks). Moreover, our proposal introduces: i) a novel approach by identifying two major patterns of leak onset and termination, irrespective of whether it is a discontinuous or continuous leakage pattern; ii) two major leak patterns based on the shape of continuous leakage.

As CPAP telemonitoring-guided interventions continues to expand in the field of sleep apnea, it becomes essential to validate the quality of data generated by CPAP monitoring systems before incorporating them into the decision-making process. So far, sleep medicine professional societies have been unable to persuade manufacturers to standardize the way they report leakages [START_REF] Schwab | An official American thoracic society statement: continuous positive airway pressure adherence tracking systems. The optimal monitoring strategies and outcome measures in adults[END_REF]. With this general nomenclature for air leakage patterns, we aim to provide a valuable tool that facilitates comparative bench tests and clinical studies. This tool can support manufacturers in developing precise algorithms for leak compensation, residual AHI estimation and the accuracy of CPAP monitoring data. Lastly, establishing a standardized approach for describing and categorizing air leakage patterns could assist clinicians in identifying device-related issues, such as patient discomfort arising from specific types of leakage.

CRediT authorship contribution statement

Mathéo Richard and. Dany Jaffuel and. Jean-Pierre Mallet and Marius Lebret: manuscript preparation and review manuscript. Raphael Gilson and. Maxime Patout and. Emeline Fresnel: Literature search, data collection, study design, analysis of data and manuscript preparation.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Lebret Marius reports a relationship with Air Liquide Medical Systems and KerNel Biomedical that includes: employment.