Arno Bringer
email: arno.bringer1@univ-lr.fr

Jérôme Cachot

Emmanuel Dubillot

Grégoire Prunier

Valérie Huet

Christelle Clérandeau

Louise Evin

Hélène Thomas

Intergenerational effects of environmentally-aged microplastics on the Crassostrea gigas

Keywords: Oyster, microplastics, reproduction, larval behaviour, development and growth, intergenerational effects, Crassostrea gigas

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The growing use of plastics dramatically increases their production in Europe (57.9 million tons, PlasticsEurope, 2020) and in the World (368 million tons). Whether from land, atmosphere or rivers, anthropogenic pollutants, including plastics, end up in the marine environment [START_REF] Halpern | A global map of human impact on marine ecosystems[END_REF].

In recent years, microplastics (MPs) has drawn the public's attention. MPs are defined as plastic particles between 1 µm and 5 mm [START_REF] Cole | Microplastics as contaminants in the marine environment: a review[END_REF][START_REF] Wagner | Microplastics in freshwater ecosystems: what we know and what we need to know[END_REF], while nanoplastics (NPs) are smaller than 100 nm [START_REF] Lambert | Characterisation of nanoplastics during the degradation of polystyrene[END_REF][START_REF] Gigault | Marine plastic litter: the unanalyzed nanofraction[END_REF]. MPs are spread in all oceans [START_REF] Barnes | Accumulation and fragmentation of plastic debris in global environments[END_REF], including in the Arctic Ocean [START_REF] Zarfl | Are marine plastic particles transport vectors for organic pollutants to the Arctic?[END_REF]. The MP size range distribution in the environment depends on hydrodynamic conditions, and degradation processes [START_REF] Enders | Abundance, size and polymer composition of marine microplastics > 10 µm m in the Atlantic Ocean and their modelled vertical distribution[END_REF].

MPs occurrence in ocean is variable over time and space. Oceanic gyres and coastal areas as hot spots of MP accumulation [START_REF] Law | Plastic accumulation in the North Atlantic subtropical gyre[END_REF]. In Europe, 73 % of the plastic production resorts to 6 polymers (PlasticsEurope, 2020): polypropylene (PP), high and low-density polyethylene (HDPE and LDPE), polyvinyl chloride (PVC), polyurethane (PUR), polyethylene terephthalate (PET) and polystyrene (PS).

Once ingested, MPs cause abrasions, obstructions and alterations of physiological structures, affecting the survival of individuals [START_REF] Wright | The physical impacts of microplastics on marine organisms: A review[END_REF]. MPs can also generate chemical damage, as they contained a large variety of additives [START_REF] Pérez-Albaladejo | Plastics and plastic additives as inducers of oxidative stress[END_REF][START_REF] Hermabessiere | Occurrence and effects of plastic additives on marine environments and organisms: A review[END_REF] and can sorbed persistent organic pollutants and heavy metals [START_REF] Rochman | Policy: classify plastic waste as hazardous[END_REF][START_REF] Bakir | Transport of persistent organic pollutants by microplastics in estuarine conditions[END_REF][START_REF] Gorman | Organic contamination of beached plastic pellets in the South Atlantic: Risk assessments can benefit by considering spatial gradients[END_REF]Naik et al., 2019). Recent studies have documented the potential effects of MPs on marine organisms and in particular bivalves [START_REF] Tallec | Nanoplastics impaired oyster free living stages, gametes and embryos[END_REF][START_REF] Sussarellu | Oyster reproduction is affected by exposure to polystyrene microplastics[END_REF][START_REF] Revel | Realistic environmental exposure to microplastics does not induce biological effects in the Pacific oyster Crassostrea gigas[END_REF]. However, most of the MPs studied were of commercial origin and very few studies focused on the impacts of environmental MPs [START_REF] Pannetier | Environmental samples of microplastics induce significant toxic effects in fish larvae[END_REF][START_REF] Cormier | Chemicals sorbed to environmental microplastics are toxic to early life stages of aquatic organisms[END_REF][START_REF] Gardon | Toxic effects of leachates from plastic pearl-farming gear on embryo-larval development in the pearl oyster Pinctada margaritifera[END_REF].

Bivalves are commonly used in studies to assess water quality and habitat. These filter feeders, in particular Crassostrea gigas oysters, are sensitive to alterations in water quality, which makes them good bioindicators and bioaccumulators for the study of pollutants [START_REF] O'connor | National distribution of chemical concentrations in mussels and oysters in the USA[END_REF], and in particular of MPs [START_REF] Ward | Selective ingestion and egestion of plastic particles by the blue mussel (Mytilus edulis) and eastern oyster (Crassostrea virginica): implications for using bivalves as bioindicators of microplastic pollution[END_REF][START_REF] Bonanno | Perspectives on using marine species as bioindicators of plastic pollution[END_REF]. In fact, oysters (C. gigas) are considered sentinel species of anthropized coastal ecosystems, due to their wide distribution, their sedentary lifestyle, and their sensitivity to a large range of pollutants [START_REF] Conger | Shell growth of unfed oysters in the laboratory: a sublethal bioassay system for pollutants[END_REF][START_REF] His | A comparison between oyster (Crassostrea gigas) and sea urchin (Paracentrotus lividus) larval bioassays for toxicological studies[END_REF][START_REF] Quiniou | Marine water quality assessment using transplanted oyster larvae[END_REF]. Found in the bivalve tissues, MPs appear as fibres, fragments or granules [START_REF] Cho | Abundance and characteristics of microplastics in market bivalves from South Korea[END_REF][START_REF] Jing-Xi | Separation and identification of microplastics in digestive system of bivalves[END_REF]. MP size and shape vary according to the tissue and species [START_REF] Li | Microplastics in commercial bivalves from China[END_REF]. Phuong et al. (2018a) highlighted that MP contamination fluctuated according to seasons, species and their living conditions (wild or farmed). Several previous studies have shown the presence of MPs in the tissues of oysters living on the French Atlantic coasts (Van Cauwenbergh & Janssen, 2014;[START_REF] Phuong | Factors influencing the microplastic contamination of bivalves from the French Atlantic coast: Location, season and/or mode of life?[END_REF]. Sussarelu et al. (2016) demonstrated that the PS-MPs ingestion altered energy flows, metabolism and homeostasis in oysters, resulting in a loss of energy dedicated to their reproductive function. They also noted impacts on the gametogenesis, with a decrease in quantity and quality of the gametes produced. Several studies underlined MPs effects on adult bivalves -including on their gametogenesis mechanism, and early larval stages. [START_REF] Cole | Ingestion of nanoplastics and microplastics by pacific oyster larvae[END_REF] demonstrated that oyster larvae ingested PS-MPs, although their study differed in terms of larval stages and particle sizes. [START_REF] Tallec | Nanoplastics impaired oyster free living stages, gametes and embryos[END_REF] reported a significant increase in malformation and developmental arrest (early mortality) frequencies in oyster larvae exposed to PS-NPs. [START_REF] Bringer | Experimental ingestion of fluorescent microplastics by pacific oysters, Crassostrea gigas, and their effects on the behaviour and development at early stages[END_REF] studied the ingestion and effects of fluorescent MPs (mixed polymers) on oyster larvae. They documented that 72h-old D-larvae ingested MPs, which were detected in their digestive tract. This study also highlighted that MPs are able to stick to the locomotor cilia of D-larvae, influencing their swimming behaviour at 24-h of development. In addition, Bringer et al. (2020a) demonstrated that an exposure to 4-13 µm HDPE-MPs, affected the oyster maximum swimming speed and trajectories of oyster larvae. A recent study focused on effects environmental MPs on the early stages of the pearl oyster, Pinctada margaritifera [START_REF] Gardon | Toxic effects of leachates from plastic pearl-farming gear on embryo-larval development in the pearl oyster Pinctada margaritifera[END_REF]. This work documented the impacts of aged plastics on the embryo-larval and larval stages, leading to malformations and mortalities depending on the tested concentrations [START_REF] Gardon | Toxic effects of leachates from plastic pearl-farming gear on embryo-larval development in the pearl oyster Pinctada margaritifera[END_REF].

Aquaculture professionals regularly use plastic tools [START_REF] Lusher | Microplastics in fisheries and aquaculture: status of knowledge on their occurrence and implications for aquatic organisms and food safety[END_REF]. Following extreme hydroclimatic events (storms, winds or strong currents, etc.), professional materials become detached and drift towards beaches and coasts during high tides. Plastics, used in French oyster farming, could deteriorate, fragment and become toxic when broken down into particles of MPs/NPs. In addition, the additives released could potentially be more harmful to the marine environment than the polymer itself (phthalates, PAHs, metals ;[START_REF] Bringer | Coastal ecosystem inventory with characterization and identification of plastic contamination and additives from aquaculture materials[END_REF]. The French shellfish market is important. The sustainability of shellfish companies depends on the quality of the water.

This study aimed at assessing effects of environmentally-aged MPs (from shellfish farming) on adult oysters (C. gigas) and their offspring. The MPs used (138.6 ± 2.3 µm) were a cocktail of three different aged-polymers: oyster crops (HDPE), collector cups (PP) and collector tubes (PVC). The data obtained included: (i) MP exposure of two-month-old adult oysters, with a follow-up on survival, biometrics, potential ingestion/excretion (tissues/biodeposits) and biomarker responses; (ii) larvae from exposed parents, with a 72-hour follow-up on swimming behaviour, development (malformations and developmental arrest) and length growth.

Materials and Method

Preparation and analysis of environmentally-aged cocktail of MPs

Plastic macrodebris were recovered on the French Atlantic beach (South-West, France), more precisely in the Angoulins-sur-Mer municipality [START_REF] Bringer | Coastal ecosystem inventory with characterization and identification of plastic contamination and additives from aquaculture materials[END_REF]. These were sorted to identify plastics from the oyster farming industry. HDPE oyster culture crops, PP cup collectors and PVC tube collectors, were selected. A GC-MS pyrolysis analysis (700 °C) of the three plastic materials selected (manual cutting of 130-200 μg samples) allowed the identification of the main polymer. After a rough cut (3-4 cm), the plastics were crushed using a stationary metal hammer, crushing the plastics several times.

Then, the plastics were sieved using 5 mm, 1 mm and 100 μm sieves. The grinding was carried out at room temperature so as not to influence the nature of the polymers materials. MPs cocktail of 138.6 ± 2.3 μm were recovered (three analytical replicates in laser granulometry, Malverne ®). MPs cocktail was composed of 28 % HDPE, 40 % PP and 32 % PVC [START_REF] Andrady | Microplastics in the marine environment[END_REF]. Each solution was prepared in pure Milli-Q-water (Millipore ®) and kept in the dark at 4 °C (Figure 1). To carry out a quantitative analysis of MPs in experimental seawater, each cocktail solution (0.1, and 10 mg. L -1 MP) was tested through a flow cytometer (Attune Acoustic Focusing Cytometer ®) according to the protocole described previously (Bringer et al., 2020a ;[START_REF] Bringer | Experimental ingestion of fluorescent microplastics by pacific oysters, Crassostrea gigas, and their effects on the behaviour and development at early stages[END_REF]. Two-millilitre samples from each MP exposition solution were vortexed (StarLab Vortex IR ®, 12,000 rpm for 20 sec) to homogenise the solutions and then 300 μL were taken for flow cytometry analysis. A calibration was carried out, thanks to previous studies (Bringer et al., 2020a) with commercial MPs, to achieve an analysis rate of 500 μL.min -1 and a saturation of 10,000 particles maximum detected. From the control conditions (10 μm filtered seawater), control analytical samples (n=12) were created. The blank obtained enabled to remove background particles (natural present in seawater). Using seawater filtered at 0.2 μm, a first calibration was conducted to select the < 200-time detected particles. MPs measurements in seawater were assessed at T1 (D17), T2 (D35) and T3 (D56) for all exposure conditions.

Experimental exposure of adult oysters and larvae

In June 2019, adult oysters Crassostrea gigas [START_REF] Bayne | A scientific name for Pacific oysters[END_REF][START_REF] Bayne | The proposed dropping of the genus Crassostrea for all pacific cupped oysters and its replacement by a new genus Magallana: a dissenting view[END_REF] purchased from a commercial hatchery (24 mo, 15.75 ± 1.5 g) were transferred to our laboratory. After a 7-day acclimatisation period, the oysters were placed in 9 experimental tanks of 37.5 L (n=30 oysters per tank, with three replicates tanks per condition) with 3 large buffer tanks (one buffer tank per condition). Tanks were supplied with filtered (10 µm) UV-treated (78.3 L.h -1 ) running seawater (filtered seawater, FSW) at a temperature of 18.5 ± 0.1 °C, salinity of 35.3 ± 0.06 and natural photoperiod. During the experiment, the tank temperature was gradually increased to faithfully recreate the environmental conditions of spring on the French Atlantic coast [START_REF] Zapata-Restrepo | Effects of the interaction between temperature and steroid hormones on gametogenesis and sex ratio in the European flat oyster (Ostrea edulis)[END_REF]. Seawater came from the Atlantic Ocean (South-West, France) in the Pertuis Breton and more precisely it was pumped in the Baie de l'Aiguillon (La Rochelle, France). Once a day, oysters were fed with a mixed diet (T.Isochrysis galbana,

Experimental exposure

Oyster farm next to the collect area Pavlova, Tetraselmis, Thalassiosira and Nanno, Shelfish diet, VariconAqua). Tanks (n=3/condition) containing the exposed oysters, were connected to a buffer tank, equipped with an air bubbling system in order to aerate FSW, and carry out a continuous water renewal in a closed system. The buffer tanks had the same physicochemical parameters as the exposure tanks. Every two days, water was renewed to prevent bacterial growth and provide good conditions for oysters. Three conditions were set up: control condition (without MPs), 0.1 mg. L -1 MP, and 10 mg. L -1 MP (Figure 2). In each buffer tank, a pumping system was set up to homogenise MPs in water. After a two-month exposure, oysters were mating to obtain embryos. Then, oyster larvae were grown in filtered seawater (0.2 µm) at 24 °C (optimal temperature for development, [START_REF] Robinson | Gonadal cycle of Crassostrea gigas kumamoto (Thunberg) in Yaquina Bay, Oregon and optimum conditions for broodstock oysters and larval culture[END_REF] and potential effects of MPs on their swimming behaviour and development were recorded at 24-, 48-and 72-hours (Figure 2). After fertilisation, behaviour, development, and growth analyses were carried out on D-larvae.

Biometric analysis and survival

A length growth monitoring was performed on adult oysters exposed to MPs for two months. A followup was carried out at D0 (T0), D17 (T1), D35 (T2) and D56 (T3). Oysters (n=30/condition) were measured (length, width and thickness) with a digital calliper and weighted (whole weight, wet flesh and empty shell). A condition index was calculated in order to compare the growth of exposed oysters to control ones. [START_REF] Bodoy | Etude comparative de différents indices de condition chez l'huître creuse (Crassostrea gigas)[END_REF] defined the index condition by the ratio of wet flesh to whole weight minus empty shell. The delta condition index was the difference between the beginning and end of the exposure period. A survival monitoring was carried out, removing dead oysters every two days and counting the total dead oysters for each condition over the two-month period.

Microplastic analysis in adult oysters tissues and faeces

Methodological details have been placed in the section Supplementary data (S1).

Analyses of biochemical biomarkers in adult oysters

Individual sample processing

Adult oysters were removed from their experimental tank, dissected to extract shell tissue, and submerged in liquid nitrogen and stored at -80 °C for analysis. For each condition and each sampling date (T1, T2 and T3), digestive glands (n=5/conditions) were homogenised in ice-cold phosphate buffer (100 mM, pH 7.2, 1100 mOsm). The homogenates were then centrifuged for 15 min at 12,500 g at 4 °C and the Final Fractions (FF), corresponding to the supernatant, were used for biochemical assays. FF was stored at -80 °C for analyzes. Analytical protocols for each biomarker have been placed in the Supplementary data section (S2).

Reproduction of adult oysters after a two-month exposure

Mature oysters were cleaned to avoid external contaminations and remove stuck microorganisms. Male and female oysters were induced to spawn by thermal stimulation (alternating immersion in FSW at 18 °C and 28 °C for 30 min) or by stripping the gonad (when thermal stimulation was ineffective) [START_REF] Mai | Environmental concentrations of irgarol, diuron and S-metolachlor induce deleterious effects on gametes and embryos of the Pacific oyster, Crassostrea gigas[END_REF][START_REF] Gamain | Combined effects of temperature and copper and S-metolachlor on embryo-larval development of the Pacific oyster, Crassostrea gigas[END_REF]Bringer et al., 2020a). Males and females were individually isolated in beakers (500 mL of FSW) at their spawning temperatures [START_REF] Parker | The effect of ocean acidification and temperature on the fertilization and embryonic development of the Sydney rock oyster Saccostrea glomerata (Gould 1850)[END_REF] for 10 minutes, before being removed. Eggs and sperm from two individuals were selected to create a single pairing. To eliminate debris and faeces, both were sieved separately using 50 μm (sperm) and 100 μm (eggs) meshes (Sefar Nitex ®). Sperm mobility was assessed and eggs counted under a microscope (Motic ®) at 100x magnification. Eggs were fertilised with sperm at a ratio of 1:10 (egg:sperm). Fertilisation success was studied under a microscope at 100x magnification, 1 hour after the start of the fertilisation. Embryos were then counted and transferred to a 24-well microplate (Greiner Bio-One, Cellstar without detectable DNase, RNase, human DNA and pyrogens ®) used in embryotoxicity according to previously published protocols [START_REF] His | A comparison between oyster (Crassostrea gigas) and sea urchin (Paracentrotus lividus) larval bioassays for toxicological studies[END_REF][START_REF] Quiniou | Bio-indicateur de la toxicité potentielle de milieux aqueux : bio-essai "développement embryo-larvaire de bivalve[END_REF] and the AFNOR standard procedure (NF ISO 17244, 2015). The fertilized eggs (approximately 300-350 eggs) were placed in wells containing 2 mL of filtered seawater (0.2 µm) and incubated in a dark climatic chamber at 24 °C for 24-, 48-and 72-hour.

Larval behaviour, development and growth

After a 24-, 48-and 72-hour incubation, the swimming behaviour was recorded under a microscope (ZEISS Axio Observer Z1 ®) at 100x magnification in a naturally-lit air-conditioned room, set at 24 °C. Two-minute videos were recorded. For the three replicates (corresponds to three different couples with fertilisation success), results are expressed in mean ± SEM, with 6 videos by couple, i.e., n=18 videos/condition. A freeware application (VirtualDub, Windows ®) was used to subsample videos to 4 fps (frame per second) and convert them to AVI format [START_REF] Gamain | Assessment of swimming behavior of the Pacific oyster D-larvae (Crassostrea gigas) following exposure to model pollutants[END_REF]Bringer et al., 2020a,b).

ImageJ (1,52a software) was used to analyse the videos. These (AVI format) were first converted to grayscale image, before being transformed into a stack of binary images. Swimming parameters of numbered D-larvae, including their maximum speed (pixel/s) and trajectories, were calculated using the wrMTrck plugin. Consequently, each larva was assigned an identification number reported in the result file. Using the protocol published by [START_REF] Gamain | Assessment of swimming behavior of the Pacific oyster D-larvae (Crassostrea gigas) following exposure to model pollutants[END_REF] and improved by Bringer et al., 2020a, three different types of larval path were noted: (1) rectilinear; (2) circular and (3) motionless. A graphic tablet (Wacom Bamboo Pen&Touch ®) and image processing software (Photos, Windows 10) enabled to quantify and characterise the swimming trajectories. Once processed with a plugin, the videos were analysed to ensure each larva was only detected once. Then, 25 μL of 1 % buffered formaldehyde (Sigma-Aldrich Chemical ®, St. Quentin Fallavier, France) were added to each well. The percentage of abnormal D-larvae (developmental abnormalities) and developmental arrests were recorded [START_REF] His | A comparison between oyster (Crassostrea gigas) and sea urchin (Paracentrotus lividus) larval bioassays for toxicological studies[END_REF][START_REF] Quiniou | Bio-indicateur de la toxicité potentielle de milieux aqueux : bio-essai "développement embryo-larvaire de bivalve[END_REF] according to the standardised AFNOR procedure (NF ISO 17244, 2015).

To validate the bioassays, the control condition should have contained less than 20 % of abnormal larvae (NF ISO 17244, 2015). For the three replicates (three couples of oyster genitors), results are expressed in mean ± SEM. In each condition, twelve wells of approximately 200 D-larvae were analysed, i.e., n=2400 D-larvae counted/condition.

A length growth analysis using the ImageJ software was carried out, studying larvae exposed for 24-, 48-and 72-hours (n=20/condition with three replicates, i.e., a total of n=60/condition). Each larva were measured lengthwise [START_REF] Talmage | The effects of elevated carbon dioxide concentrations on the metamorphosis, size, and survival of larval hard clams (Mercenaria mercenaria), bay scallops (Argopecten irradians), and Eastern oysters (Crassostrea virginica)[END_REF][START_REF] Helm | Hatchery operation: culture of larvae basic methodology, feeding and nutrition, factors influencing growth and sur-vival, and settlement and metamorphosis. Hatchery Culture of Bivalves: A Practical Manual[END_REF]Bringer et al., 2021a). Exposed larvae were then visualised (ZEISS Axio Observer Z1, x20 objective) and photographed. Body length was determined from photographs. Results are expressed in mean ± SEM.

Statistics

Data are expressed in mean ± SEM. A logrank test, using the Kaplan-Meier method, was carried out to analyse the survival of adult oysters. In case of abnormal distribution, data were transformed using the following formula: P'=arcsin√r; P corresponds to raw data (frequency of larvae abnormalities) specified in P values from 0 to 1 [START_REF] Legendre | Transformation:1-4[END_REF]. Homogeneity of variance (Levene's test) and normality of distribution (Shapiro-Wilk) were assessed. In order to compare the various treatments, an analysis of variance (ANOVA) using Tukey's post-hoc test was performed. For data not entering in the scope of a parametric test, a statistical analysis was carried out through the Kruskal-Wallis Test.

Differences in tested concentration means were then assessed using the Kruskal Nemenyi Post-hoc test with the PMCMR package (the Tukey test equivalent for non-parametric data).

Significance difference was accepted when p-value < 0.05. Statistical analysis was completed using R and graphs from Microsoft Excel ®. Through flow cytometry, the different concentrations of the MPs cocktail were measured in the experimental waters used for the exposure of adult oysters (Table 1) at different sampling times (T1, T2 and T3). At 0.1 mg. L -1 MP, very little variation was noted between the first two sampling times (T1 and T2), ranging from 655 ± 53.0 to 677 ± 67.6 MP.mL -1 . At T3, the concentrations of MPs increased slightly, with 943 ± 60.7 MP.mL -1 . At 10 mg. L -1 MP, values at the three sampling times (T1, T2 and T3) were stable. However, the difference in concentration factor between the two conditions of exposure to MPs seems to be lower than theory.

Results

Analysis of MP concentrations

State of health and reproductive effort in adult oysters

To monitor growth and shape, a condition index was built from biometric measurements on adult oysters. However, there was no significant difference in the condition index between the control and exposed oysters (0.1 and 10 mg. L -1 MP) with respectively 0.36 ± 0.03; 0.30 ± 0.02 and 0.31 ± 0.04.

Following a two-month exposure, oyster mortality rate differed significantly between the three exposure conditions (0, 0.1 and 10 mg. L -1 MP). The control condition exhibited the lowest mortality rate of 2.2 % (n=90/condition). At 0.1 mg. L -1 MP, a cumulative mortality of 13.3 % was quantified. At 10 mg. L - After two months exposure under controlled laboratory conditions, some adult oysters reached sexual maturity and developed mature gonads, allowing reproduction (n=10/conditions). Six oysters from the control condition emitted gametes. Seven oysters from the 0.1 mg MP. L -1 condition and 8 oysters from the 10 mg. L -1 MP condition were mature and ejected gametes through thermal shock (Table S3). In terms of sex determination, oysters in the control condition were predominantly maturing females (4:2, female: male) while oysters exposed to MPs were mostly males with mature gametes (2:5 for 0.1 mg.

L -1 MP and 3:5 for 10 mg. L -1 MP, Table S1). The quality of gametes (well-formed oocytes and motile spermatozoa) and was observed under a microscope (Motic). In the control condition, 100 % of the gametes ejected being of good quality (Figure S4). 57 % and 50 % of the gametes from oysters exposed to 0.1 and 10 mg. L -1 MP were of good quality (Table S3). When bivalves expelled poor-quality gametes, they were not selected to proceed to in vitro fertilisation. Three couples for each condition (i.e. three replicates) were formed to obtain embryos, then becoming D-larvae.

MP analysis in adult oyster tissues and biodeposits

MPs were quantified in digested tissues and biodeposits from exposed adult oysters (Table 2). In the control oysters, there were no microplastics in the tissues for the three sampling times. In exposed oysters, MPs were detected in the tissues at all sampling times (T1, T2 and T3). At T1, a greater quantity (increase factor of 5.4) of MPs is noted in the tissues of oysters exposed to 10 mg. L -1 MP compared to the 0.1 mg. L -1 MP condition. Same observation at T2 and T3, with increase factors of 3.7 and 4

respectively. The two exposure conditions noted a decrease in the MPs present in the tissues at T3, compared to T2. Under the control conditions, there were no MPs in the biodeposits. In exposed oysters, MPs at T1 were quantified as follows: 0.084 ± 0.02 at 0.1 mg of MP .L -1 and 6.4 ± 2.2 MPs.mg -1 at 10 mg. L -1 MP. At T2 and T3, the MPs in the biodeposits were markedly increased compared to T1.

Analysis of biochemical biomarkers in the adult oyster digestive gland

Figure 4 presents biomarker results on adult oysters exposed to environmental MPs (cocktail of HDPE, PVC and PP from oyster farming). SOD and GST activities are both induce under oxidative stress. The MDA activity is defined as lipid peroxidation and the LAC activity as environmental stress.

Superoxide Dismutase (SOD) and Glutathione S-transferase (GST) assays: oxidative stress

The SOD activity in oysters from the three conditions tested (control, 0.1 and 10 mg. L -1 MP) decreased significantly at T3 (respectively 34.3 ± 3.3, 32.6 ± 2.5 and 30.0 ± 3.3 U/mg of protein), in comparison with T1 (respectively 51.4 ± 5.4, 60.9 ± 7.0 and 49.7 ± 8.3 U/mg of protein) and T2 (respectively 60.5 ± 13.4, 72.8 ± 7.2 and 73.5 ± 20.5 U/mg of protein; ANOVA, p<0.01). Both the control and exposed oysters followed similar trends. It cannot be concluded that there is a significant difference in SOD

HDPE (28 %) PVC (32 %) PP (40 %)

activity at T3 for the exposure conditions because the control condition follows the same trend. The GST activity in control condition had no significant change at the three sampling times (ANOVA, p=0.9). The trend was similar to oysters exposed to 0.1 mg MP. L -1 , observing no significant difference between the three sampling times (ANOVA, p=0.5). In the 10 mg. L -1 MP condition, the GST activity increased at T3 (35.9 ± 5.4 U/mg of protein; ANOVA, p<0.05, Figure 4).

Malondialdehyde (MDA) assay: lipid peroxidation

In the control and 10 mg. L -1 MP conditions, the MDA activity in oysters had no significant difference at the three sampling times (respectively, ANOVA, p=0.2 and p=0.9). In the 0.1 mg. L -1 MP condition, the MDA activity increased between T2 (1.3 ± 0.2 µM/mg of protein) and T3 (1.9 ± 0.3 µM/mg of protein; ANOVA, p < 0.05) (Figure 4).

Laccase (LAC) assay: environmental stress

In control oysters, there were no significant differences of laccase activity between the three sampling times. The activity in oysters exposed to MPs (0.1 and 10 mg. L -1 MP) increased significantly between T2 (respectively 5.6 ± 0.6 and 4.1 ± 0.6 U/mg of protein) and T3 (respectively 7.3 ± 0.6 and 6.7 ± 0.6 U/mg of protein; ANOVA, p < 0.05, Figure 4).

Larval behaviour, development and growth

The recorded videos (ZEISS Axio Observer Z1) highlighted a swimming activity (maximum swimming speed) increasing with the larvae age. The swimming speed was faster in 72-hour than for 24-hour larvae (Figure 5). After being fixed with formalin (1 %), 72-h D-larvae were classified according to their developmental states (well-formed, malformed or in developmental arrest, Figure 6). D-larvae from unexposed adult oysters were well-formed (85.8 ± 1.4 %) and statistically different (p<0.05) from oysters exposed to 0.1 (p<0.01) (81.1 ± 1.3 %) and 10 mg. L -1 MP (p<0.01) (74.2 ± 1.7 %). Malformed D-larvae from the control and 0.1 mg. L -1 MP conditions were not statistically different, with respectively 8.5 ± 1.2 and 11.8 ± 1.0 % (p=0.08). On the contrary, malformed larvae in the 10 mg. L -1 MP condition were significantly higher than in the control condition (16.0 ± 1.8 % (p<0.01)). In addition, D-larvae with developmental arrest had no significant difference between the first two conditions (control and 0.1 mg.

L -1 MP, with respectively 5.7 ± 0.7 and 7.1 ± 0.9 %, p=0.6). In comparison with the control condition (p=0.03<0.05), the 10 mg. L -1 MP condition differed significantly, with 9.8 ± 1.2 %. Representative photographs of malformed and developmental arrest larvae are presented in Figure S5. Larval growth was slower in exposed than in control larvae (Figure 7). Significantly differences of size reductions of 10.6 % and 12.3 %, were observed in 72-hour larvae from parents exposed to 0.1 and 10 mg. L -1 MP. The total length of control D larvae was 63.0 ± 1.5 μm, and significantly greater than larvae of parents exposed to 0.1 mg. L -1 MP and up to 10 mg. L -1 MP measured 56.3 ± 1.0 μm and 55.3 ± 0.8 μm.

Discussion

The study aimed at evaluating the effects of environmentally-aged MPs (plastic materials from oyster farms) on C. gigas oysters. Over a two-month period, adult oysters were exposed to aged MPs obtained from a mixture of HDPE, PP and PVC. Their growth, survival, MP ingestion/excretion and the response of biomarkers were monitored. Adult oysters were fertilised in laboratory conditions to produce embryos. Then, the development and swimming activity of 72-hour oyster D-larvae (early stages of development) were observed. MP concentrations in the exposure medium were assessed through flow cytometry. Except for the 0.1 mg. L -1 MP condition at T3, concentrations measured were constant. In the latter condition at T3, the increase in MP concentration over time might be due to particles binding on the tank walls while water was renewed [START_REF] Summers | Agglomeration of nano-and microplastic particles in seawater by autochthonous and de novo-produced sources of exopolymeric substances[END_REF]. Our present study focused on ecotoxicological test concentrations, stronger than what is found in the marine environment [START_REF] Lenz | Microplastic exposure studies should be environmentally realistic[END_REF][START_REF] Paul-Pont | Constraints and priorities for conducting experimental exposures of marine organisms to microplastics[END_REF]. The previous study by [START_REF] Dubaish | Suspended microplastics and black carbon particles in the Jade system, southern North Sea[END_REF] determined concentrations in the North Sea of 1.7 MP.mL -1 (MP > 80 µm). In our work, the ranges of concentrations assayed reported means of 758 ± 60 and 3,322 ± 228 MP.mL -1 for the two theoretical mass concentrations (0.1 and 10 mg. L -1 MP). It is therefore estimated that our study would have used concentrations 445 and 1,954 higher than the natural environment. However, our concentrations are close to certain ecotoxicological studies that took into account doses of 1,000 MP.mL -1 [START_REF] Sussarellu | Oyster reproduction is affected by exposure to polystyrene microplastics[END_REF][START_REF] Paul-Pont | Exposure of marine mussels Mytilus spp. to polystyrene microplastics: toxicity and influence on fluoranthene bioaccumulation[END_REF]. According to literature, MP loads in seawater are within the μg.L -1 range, and the highest MP loads reported in the sea area round 0.08 to 0.3 mg.L -1 [START_REF] Lusher | Microplastic pollution in the Northeast Atlantic Ocean: validated and opportunistic sampling[END_REF][START_REF] Frias | Evidence of microplastics in samples of zooplankton from Portuguese coastal waters[END_REF]. Thereby, the theoretical 0.1 mg. L -1 MP concentration correspond to the contamination peak of MPs in the marine environment [START_REF] Beiras | Ingestion and contact with polyethylene microplastics does not cause acute toxicity on marine zooplankton[END_REF]. A recent study in the Bay of Biscay (South-West, Atlantic, France) near the study area where our MPs were collected indicated MP concentrations between 0.03 and 0.06 MPs.L -1 in water, with a size distribution between 4-2,000 µm, at the surface of the studied marine environment [START_REF] Lerebours | Étude spatio-temporelle de la contamination en microplastiques chez les moules et les huîtres en mer des Pertuis : hétérogénéité ? Oral communication on Polymers & Oceans congress[END_REF]. [START_REF] Wang | Quantitative and qualitative determination of microplastics in oyster, seawater and sediment from the coastal areas in Zhuhai, China[END_REF], reported higher concentrations of 15-20 MPs.L -1 in seawater from the coastal areas in Zhuhai (China), with a size range of 101-500 µm for 45 % of the detected MPs.

Microplastics concentrations in experimental seawater

Microplastic in adult oysters tissues and faeces

MPs were not detected in tissues of control oysters. Like most animals, oysters lack enzymes and enzymatic pathways enabling to break down plastics [START_REF] Aladaileh | Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function[END_REF][START_REF] Wright | The physical impacts of microplastics on marine organisms: A review[END_REF]. After filtration-ingestion, MPs cannot be absorbed or used as energy, leading to their accumulation in the gills and digestive gland [START_REF] Teng | Toxic effects of exposure to microplastics with environmentally relevant shapes and concentrations: Accumulation, energy metabolism and tissue damage in oyster Crassostrea gigas[END_REF]. This work echoed previous results highlighting that bivalve accumulated MPs in both experimental and natural conditions [START_REF] Browne | Ingested microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis (L.)[END_REF][START_REF] Von Moos | Uptake and effects of microplastics on cells and tissue of the blue mussel Mytilus edulis L. after an experimental exposure[END_REF][START_REF] Magni | Evaluation of uptake and chronic toxicity of virgin polystyrene microbeads in freshwater zebra mussel Dreissena polymorpha (Mollusca: Bivalvia)[END_REF][START_REF] Teng | Microplastic in cultured oysters from different coastal areas of China[END_REF][START_REF] Birnstiel | Depuration reduces microplastic content in wild and farmed mussels[END_REF][START_REF] Ward | Selective ingestion and egestion of plastic particles by the blue mussel (Mytilus edulis) and eastern oyster (Crassostrea virginica): implications for using bivalves as bioindicators of microplastic pollution[END_REF]. The presence of MPs accumulated in the tissues at the end of exposure (T3) could potentially affect reproductive efforts and subsequently the quality of the gonads and embryo-larval development [START_REF] Sussarellu | Oyster reproduction is affected by exposure to polystyrene microplastics[END_REF]. One must recognize that the concentrations tested and MP in tissues were much higher than those in the environment [START_REF] Keisling | Low concentrations and low spatial variability of marine microplastics in oysters (Crassostrea virginica) in a rural Georgia estuary[END_REF]. Regarding the quantities of MPs contained in the biodeposits, these present results are comparable to the results published by [START_REF] Revel | Realistic environmental exposure to microplastics does not induce biological effects in the Pacific oyster Crassostrea gigas[END_REF]. Oysters tended to quickly excrete MPs [START_REF] Graham | Microplastics uptake and egestion dynamics in Pacific oysters, Magallana gigas (Thunberg, 1793), under controlled conditions[END_REF]. In a recent study [START_REF] Thomas | The world is your oyster: low-dose, long-term microplastic exposure of juvenile oysters[END_REF], MPs were detected in tissues and biodeposits. Oysters could excrete MPs in faeces through cleaning processes, preventing the gills from being clogged by particles (Jorgensen, 1981) and rejecting indigestible particles [START_REF] Wegner | Effects of nanopolystyrene on the feeding behavior of the blue mussel (Mytilus edulis L.)[END_REF]. Our results indicate low amounts of MPs in faeces. Although this suggests that oysters might excrete MPs through feces, a small fraction of these microparticles might persist in the tissues or be re-ingested by filtration, potentially leading to longer-term effects [START_REF] Farrell | Trophic level transfer of microplastic: Mytilus edulis (L.) to Carcinus maenas (L.)[END_REF][START_REF] Ribeiro | Microplastics effects in Scrobicularia plana[END_REF].

Oysters state of health

In the present study, MP exposure of adult oysters did not affect their growth, but their survival. The highest dose of exposure (10 mg. L -1 MP) led to a significant increase of mortality rate. Sussarellu et al.

(2016) obtained similar results, showing that oysters exposed to PS-MPs had disrupted energy flows, metabolism and homeostasis, increasing the mortality rate. [START_REF] Browne | Microplastic moves pollutants and additives to worms, reducing functions linked to health and biodiversity[END_REF] reported in Arenicola marina, accumulation of nonylphenol and triclosan from PVC that led to altered physiological stress, immune system responses and increasing mortality. Stabilisation of mortality were noted at 35 and 44 days for oysters exposed to respectively 0.1 and 10 mg. L -1 MP. The evolution in cumulative mortality of exposed individuals may suggest an adaptation to MP exposure [START_REF] Guo | Immune and stress responses in oysters with insights on adaptation[END_REF]. However, it remains complicated to conclude on the lethal effects of microplastics without setting up exclusion analyzes for other factors (i.e. bacteria, nutrition, toxic algae, etc.). In addition, an impact on fertilisation success was noted. The oysters exposed to MPs, having reached the stage of sexual maturation, had more males than females, unlike the control oysters. In addition, the quality of female gametes was poorer in adults exposed to MPs than in control. A previous study revealed that marine medaka (Oryzias melastigma) exposed to PS-MPs faced sex-specific endocrine disruption. [START_REF] Wang | Polystyrene microplastics cause tissue damages, sex-specific reproductive disruption and transgenerational effects in marine medaka (Oryzias melastigma)[END_REF] suggested that PS-MPs had sex-specific reproductive disruption and decreased sex hormone levels in female plasma by inhibiting their synthesis. [START_REF] Sussarellu | Oyster reproduction is affected by exposure to polystyrene microplastics[END_REF] indicated that the quality of gametes decreased following parental exposure to PS-MPs. This work reached similar conclusions on the quality of gametes from exposed parents.

Biomarkers activities

The activities of biomarkers (SOD, GST, MDA and LAC) that can be modulated by environmental pollutants were used as defense markers, providing information on the level of exposure and the In contrast to this study, [START_REF] Ribeiro | Microplastics effects in Scrobicularia plana[END_REF] revealed that the SOD activity in the digestive gland of Scrobicularia plana exposed to PS-MPs increased at the end of the exposure period.

GST is involved both in the biotransformation of xenobiotics (Dos [START_REF] Santos | Glutathione S-transferase activity in aquatic macrophytes and halophytes and biotransformation potential for biocides[END_REF] but also in the recycling of reduced glutathione and therefore in the fight against oxidants [START_REF] Fabioux | Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas[END_REF]. This phenomenon could be a reaction to an increased xenobiotic biotransformation, as underlined by [START_REF] Breitwieser | Short-term and long-term biological effects of chronic chemical contamination on natural populations of a marine bivalve[END_REF]. Excessive oxidative stress can lead to a significant increase in oxidative damage (Araújo et al., 2016), potentially due to the depletion of organisms due to the activation of defense mechanisms to fight against the polluted environment. Concerning the GST enzyme, also linked to oxidative stress, an increase in this enzymatic activity was noted in freshwater clams Corbicula fluminea exposed to PS-NP [START_REF] Li | Impacts of nanoplastics on bivalve: Fluorescence tracing of organ accumulation, oxidative stress and damage[END_REF]. GSTs are key players in the removal of reactive oxygen species (ROS), intervening indirectly by controlling the level of reduced glutathione.

If the balance is upset in favor of ROS, oxidative stress is generated due to environmental stress and xenobiotics could influence this balance by catalyzing the production of ROS, and altering the metabolic balance [START_REF] Donaghy | The known and unknown sources of reactive oxygen and nitrogen species in haemocytes of marine bivalve molluscs[END_REF][START_REF] Manduzio | The point about oxidative stress in molluscs[END_REF]. In addition, [START_REF] Wang | Polystyrene microplastics cause tissue damages, sex-specific reproductive disruption and transgenerational effects in marine medaka (Oryzias melastigma)[END_REF] noted an increased GST activity in the intestine and gills of the marine medaka (Oryzias melastigma) exposed to 20 and 200 µg PS-MP. L -1 , concentrations similar to this study.

The MDA content reflects the rate and intensity of lipid peroxidation (Qiao et al., 2019a, b). In the 0.1 mg. L -1 MP condition at T3, the MDA content increased. The increase in MDA content, decreased the antioxidant protection and immune modulation (Pan et al., 2009;[START_REF] Breitwieser | Short-term and long-term biological effects of chronic chemical contamination on natural populations of a marine bivalve[END_REF]. Indeed, constantly impacted by plastics [START_REF] Santana | Continuous exposure to microplastics does not cause physiological effects in the cultivated mussel Perna perna[END_REF]. The main limitations of biomarker measurements for assessing the quality of ecosystems are thus the difficulty in discriminating between "natural" adaptive responses and responses to chemical stress, in extrapolating responses from one scale of biological organization to another (cell-individual-population-community) and from one species to another [START_REF] Forbes | The use and misuse of biomarkers in ecotoxicology[END_REF]. In addition, the use of biomarkers such as SOD and GST lead to increased sensitivity in the results which could create bias in the observed effects.

Intergenerational effects on D-larvae

The present study also focused on the offspring obtained from adult oysters exposed to MPs over a twomonth period during gametogenesis [START_REF] Enríquez-Díaz | Gametogenesis, reproductive investment, and spawning behavior of the Pacific giant oyster Crassostrea gigas: evidence of an environment-dependent strategy[END_REF]. The larval growth was slower in the MPs-exposed groups in comparison to the control group. This result is consistent with those obtained by [START_REF] Sussarellu | Oyster reproduction is affected by exposure to polystyrene microplastics[END_REF] and [START_REF] Bringer | Experimental ingestion of fluorescent microplastics by pacific oysters, Crassostrea gigas, and their effects on the behaviour and development at early stages[END_REF], suggesting that MPs exposure can delay C. gigas larval growth. In this work, the exposure conditions revealed a significant increase in developmental abnormalities, including malformations and developmental arrest. Thus, when parents were exposed to high MP concentrations, developmental abnormalities were more frequent in their offspring. A lot studies highlighted that micro-pollutants such as heavy metals and pesticides, caused developmental defects in oysters [START_REF] Mai | Embryotoxic and genotoxic effects of heavy metals and pesticides on early life stages of Pacific oyster (Crassostrea gigas)[END_REF][START_REF] Gamain | Combined effects of pollutants and salinity on embryo-larval development of the Pacific oyster, Crassostrea gigas[END_REF][START_REF] Gamain | Combined effects of temperature and copper and S-metolachlor on embryo-larval development of the Pacific oyster, Crassostrea gigas[END_REF][START_REF] Bringer | Coastal ecosystem inventory with characterization and identification of plastic contamination and additives from aquaculture materials[END_REF]. [START_REF] Tallec | Nanoplastics impaired oyster free living stages, gametes and embryos[END_REF] reported greater toxicity of smaller plastic particles. In comparison with PS-MPs (2 µm), PS-NPs (500 and 50 nm) had greater impacts on the fertilisation success and embryo-larval development of oyster C. gigas. Our results revealed a shift in the swimming behaviour of larvae from parents exposed to MPs.

Indeed, circular trajectories increased, in comparison to rectilinear trajectory. [START_REF] Gamain | Assessment of swimming behavior of the Pacific oyster D-larvae (Crassostrea gigas) following exposure to model pollutants[END_REF] demonstrated that, under optimal developmental conditions, C. gigas D-larvae tended to have a rectilinear swimming behaviour (2-D analysis). In a previous study, Bringer et al. (2020a) noted that circular trajectories were significantly increased in larvae exposed to HDPE-MPs (4-13 µm). For both MP concentrations, a decrease in maximum speed recorded was noted in 24-and 72-hour D-larvae.

These results are consistent with previous studies (Bringer et al., 2020a & b) in which D-larvae exposed to MPs (HDPE and polymer cocktail) showed a decrease in maximum swimming speed. Yin et al.

(2019) indicated that black rockfish Sebastes schlegelii exposed to MPs (0.19 mg. L -1 PS-MP of 15 µm) had a restricted swimming speed and range of motion, affecting their hunting behaviour and exploration skill.

Altogether our data reinforced the hypothesis that MPs impacted both the physiology and behaviours of oysters C. gigas. This study relied on the use of secondary MPs -aged in the marine environment -as well as on plastic materials from oyster farming.

Conclusions and perspectives

To conclude, this experiment showed that the cocktail of MPs obtained from oyster material and aged in the marine environment can be toxic for adult oysters and the larval generation. The results indicated that exposure to MPs could affect survival, responses to defense biomarkers, and fertilization success, in adults (C. gigas) exposed for 2-months. D-larvae obtained from exposed oysters, having reached a mature stage, exhibited alterations in swimming behavior, development and growth. Bad weather and degradation of plastics from mariculture could therefore represent a possible threat to oyster farming and surrounding marine life. In the future, it would be useful to study the life cycle of aquaculture plastics and the potential risk of additives present on plastics on the Crassostrea gigas study model and the health of marine ecosystems in general.

Figure 1 .

 1 Figure 1. Experimental design of adult oysters exposed to MPs cocktail obtained from different aged-plastic materials used in French oyster farms.

Figure 2 .

 2 Figure 2. Experimental design of adult oysters exposed to environmentally-aged MPs cocktail for two months.

1

  MP, the mortality rate reached 23.3 % (Figure 3.a). Over the exposure period, the cumulative mortality rate differed significantly between the conditions tested (Figure 3.b).

Figure 3 .

 3 Figure 3. (a) Survival monitoring of adult oysters exposed to MPs for two months: control (without pollutants); 0.1 mg. L -1 MP and 10 mg. L -1 MP. (b) Histogram of cumulative mortalities for each exposure condition. Expressed in mean ± SEM. Different letters indicate significant differences between concentrations (p<0.05, Kaplan-Meir Test, n=90/condition).

Figure 4 .

 4 Figure 4. Effects of environmental MPs on oxidative stress (Superoxide Dismutase (SOD) and Glutathione Stransferase (GST)), lipid peroxidation (Malondialdehyde (MDA) and environmental stress Laccase (LAC) in C. gigas adult digestive gland. The different sampling times correspond to: T1 (D17), T2 (D35) and T3 (D56). Three MP conditions were tested: 0 (control), 0.1 and 10 mg. L -1 MP. The histogram data are expressed in mean ± SEM.Within series, the letters (a/b, A/B and α/β) above each bar indicate significant differences (p < 0.05, ANOVA and Tukey test, n=5/condition) between different sampling times for each condition.

  Compared to control larvae (153.3 ± 12.2 µm. s -1 ), 24-hour D-larvae (Figure5.a) from exposed adult oysters swam significantly slower (respectively 97.6 ± 9.0 and 93.1 ± 5.0 µm. s -1 at 0.1 and 10 mg. L -1 MP). 48-hour larvae (Figure5.b) from parents exposed to 0.1 mg. L -1 MP had a lower swimming activity. 72-hour larvae (Figure5.c) from parents exposed to the highest MP concentration (10 mg. L -1 MP, 155.6 ± 26.4 μm. s -1 ) swam slower than oyster larvae from other conditions (respectively 194.5 ± 12.2 and 158.5 ± 16.0 μm. s -1 in the control and 0.1 mg. L -1 MP conditions). In addition, over a two-month period, the swimming trajectories of larvae from exposed parents were characterised (Figure5). 24-hour D-larvae from unexposed parents (control condition) displayed rectilinear swimming trajectories (60.6 ± 6.4 %, Figure5.a). 48-and 72-hour larvae had predominant rectilinear swimming paths (57.5 ± 12.4 % and 54.3 ± 5.8 % respectively, Figure5.b & c). 24-hour D-larvae from parents exposed to 0.1 and 10 mg. L -1 MP mainly swam in circular trajectories, for 47.4 ± 8.1 and 65.0 ± 8.9 % of the larvae, respectively (Figure5.a). Forty-eight hour D-larvae from the 0.1 mg. L -1 MP condition showed predominant circular trajectories (61.8 ± 7.4 %), in higher proportion than the control condition (32.5 ± 10.4 %). D-larvae from the 10 mg. L -1 MP condition had motionless trajectories (31.1 ± 6.0%), in higher proportion than the control condition (10.0 ± 3.8%, Figure5.b). 72-hour larvae from parents exposed to 0.1 and 10 mg. L -1 MP displayed fewer rectilinear trajectories (respectively, 28.6 ± 9.4 and 23.8 ± 6.5 %), in comparison with larvae from the control condition (54.3 ± 5.8 %, Figure5.c).

Figure 5 .

 5 Figure 5. Maximum speed and trajectories (rectilinear, circular or motionless) of 24-(a), 48-(b) and 72-hour (c)

Figure 6 .

 6 Figure 6. Percentages of well-formed, abnormal and developmental arrest in 72-hour C. gigas larvae. Over a twomonth period, genitor oysters were exposed to three conditions: 0 (control), 0.1 and 10 mg. L -1 MP. For the three replicates, values are expressed in mean ± SEM. Different letters indicate significant differences between concentrations (p < 0.05, ANOVA and Tukey test, n=2400 larvae/condition).

Figure 7 .

 7 Figure 7. Larva total length (μm) of C. gigas after 24-, 48-and 72-h of development from parents exposed for 2 months to different concentrations of MPs cocktail : 0 (control), 0.1 and 10 mg. L -1 MP. Values are means ± SEM. Different letters indicated significant differences between different concentrations and development time (p < 0.05, ANOVA and Tukey test, n=60/condition).

  response capacities of oysters exposed to cocktail of MPs. Oxidative stress biomarkers were chosen for their early stress responses in bivalves(Valavanidis et al. 2006). Milinkovitch et al. (2015) confirmed that SOD was an early indicator of oxidative stress. In their study,[START_REF] Revel | Tissue-specific biomarker responses in the blue mussel Mytilus spp. exposed to a mixture of microplastics at environmentally relevant concentrations[END_REF] indicated that the SOD activity in the digestive gland of blue mussels (Mytilus edulis) exposed for 10 days to a mixture of MPs (PE, PP) was no affected. Our results show a decrease in SOD over time and with increasing gonadal maturity in all conditions tested, including condition control. Similar effects have been demonstrated in C. gigas with a decrease in SOD due to seasonal variations(Luna-Acosta et al., 2010b).

Sussarellu

  et al. (2016) andPalais et al. (2012) suggested that oysters exposed to particles can modify their energy flow and induced antioxidant and detoxification processes.Laccase (LAC) is a marker of environmental stress(Luna-Acosta et al., 2010a). In the two exposure conditions (0.1 and 10 mg. L -1 MP) at T3, the LAC activity increased over time. The environmental study conducted by[START_REF] Breitwieser | Biomonitoring of Mimachlamys varia transplanted to areas impacted by human activities (La Rochelle Marina, France)[END_REF] correlated an increase of the LAC activity in Mimachlamys varia over time in presence of metals (Cu, Cr, Mn, Sn, and As). Bivalves are able to detect pollutants in their environment and respond to stress by increasing their LAC activity[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]. At T3the last sampling time -LAC and MDA biomarkers were significantly increased in comparison to control group. Exposure to MPs should be investigated on a long-term basis to understand adaptive responses or toxic effects on bivalves. Understanding mechanisms triggered by organisms to fight against chronic exposure to MPs is key to conduct risk assessments. It is crucial as coastal areas are

Table 1 .

 1 Theoretical and measured MP concentrations in FSW at different sampling times.

	Conditions	Sampling	Theoretical	Measured	Measured
		time	(mg. L -1 MP)	(MP. µL -1 )	(MP. mL -1 )
		T1	0.1	0.68 ± 0.067	677 ± 67.6
	MPs cocktail	T2	0.1	0.66 ± 0.05	655 ± 53.0
		T3	0.1	0.94 ± 0.06	943 ± 60.7
		T1	10	3.30 ± 0.32	3,297 ± 319.0
	MPs cocktail	T2	10	3.47 ± 0.14	3,466 ± 142.7
		T3	10	3.21 ± 0.22	3,205 ± 221.3

Table 2 .

 2 Concentrations of environmental MP cocktail (PE, PP and PVC) in oyster tissues (MPs.g -1 ) and biodeposits (cumulate faeces and pseudo-faeces, MPs.mg -1 ) at three sampling times (T1, T2 and T3). Data are expressed in mean ± SEM. Different letters indicate significant differences between concentrations (p < 0.05, ANOVA and Tukey post-hoc test).

	Sampling time		T1		T2		T3
	Conditions	Tissues	Biodeposits	Tissues	Biodeposits	Tissues	Biodeposits
	Control	0	0	0	0	0	0
	MP 0.1 mg. L -1	2.1 ± 0.8 a 0.084 ± 0.02 a 6.3 ± 2.8 a	0.78 ± 0.2 a	5.4 ± 2.5 a	0.93 ± 0.2 a
	MP 10 mg. L -1	11.3 ± 3.6 b	6.4 ± 2.2 b	23.6 ± 9.2 b	7.6 ± 2.7 b	21.4 ± 7.3 b	7.3 ± 3.1 b

Acknowledgements

Arno Bringer received a PhD grant (Comité Régional de la Conchyliculture de la Charente-Maritime CRC17) as well as financial support from the Région Nouvelle Aquitaine and Comité Départemental de la Charente Maritime (CD17) to develop this research. The AQUAECOs project (Amélioration de la QUAlité Environnementale sur les zones COnchylicoles des Pertuis Charentais), funded by a partnership with CRC17 and OFB (French Biodiversity Office), contributed to this study. The funding was partly supported by the University of La Rochelle, the University of Bordeaux, the Centre National de la Recherche Scientifique (France). The authors thank the France Naissain hatchery for the oyster specimens as well as Oriane Titeca for her proofreading services.