Vanessa Koehle-Divo

Benedicte Sohm

Laure Giamberini

Daniele Pauly

Justine Flayac

Simon Devin

Melanie Auffan

Catherine Mouneyrac

Sandrine

Koehle-Divo Vanessa

Sohm Bénédicte

Giamberini Laure

Pauly Danièle

Devin Simon

Auffan Mélanie

Devin Sandrine

A sub-individual multilevel approach for an integrative assessment of CuO nanoparticle effects on Corbicula fluminea

Keywords: Copper oxide nanoparticles (CuO NP), bivalve, gene expression, biochemical effects, filtration rate

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Introduction

Technological advances allow synthesis and incorporation of nanoparticles (NPs) in numerous daily commercial products. Their tiny size considerably enhances their specific surface area and confers new properties (surface reactivity, optic, catalytic, etc). Metal and metal oxide NPs are among the most manufactured NPs employed in a wide range of applications (Vance et al., 2015). Among them, copper oxide nanoparticles (CuO NPs) are used in various applications such as antimicrobial agents, agricultural biocides, catalysts or gas sensors [START_REF] Hou | Ecotoxicological effects and mechanism of CuO nanoparticles to individual organisms[END_REF][START_REF] Chibber | Can CuO nanoparticles lead to epigenetic regulation of antioxidant enzyme system[END_REF], Keller et al., 2017). Their increasing use leads inevitably to their release in the aquatic environment while their quantification and characterization in such complex matrices are not yet fully developed.

Although the predictive environmental concentration (PEC) values for CuO NPs are currently estimated to be less than few µg/L [START_REF] Keller | Predicted Releases of Engineered Nanomaterials: From Global to Regional to Local[END_REF], their production continues to increase from 580 tons produced in 2014 versus 1600 tons estimated for 2025 [START_REF] Hou | Ecotoxicological effects and mechanism of CuO nanoparticles to individual organisms[END_REF].

In the framework of nanotoxicology, the assessment of NP toxicity according to their physico-chemical properties remains a work in progress. Moreover, their biocide abilities raise the question of their (eco)toxicity towards non-target organisms [START_REF] Keller | Comparative environmental fate and toxicity of copper nanomaterials[END_REF]. In aquatic environments, Cu-based NPs would be accumulated in the sediments of freshwater and marine environments [START_REF] Keller | Comparative environmental fate and toxicity of copper nanomaterials[END_REF]. Benthic species should be of particular concern because of their close contact with contaminants and because their burrowing activities leads to a potential remobilization of contaminants (Roberts, 2012). The widespread endobenthic freshwater bivalve Corbicula fluminea is a good model for such assessments due to its ability to be both filter-and pelagic-feeder, but also due to its ability to strongly bioaccumulate a large amount of contaminants, such as metals [START_REF] Inza | Tissue distribution of inorganic mercury, methylmercury and cadmium in the Asiatic clam (Corbicula fluminea) in relation to the contamination levels of the water column and sediment[END_REF][START_REF] Shoults-Wilson | Comparison of metal concentrations in Corbicula fluminea and Elliptio hopetonensis in the Altamaha River system, Georgia, USA[END_REF][START_REF] Marescaux | Assessing filtration rates of exotic bivalves: dependence on algae concentration and seasonal factors[END_REF][START_REF] Hakenkamp | The impact of an introduced bivalve (Corbicula fluminea) on the benthos of a sandy stream[END_REF]. As estimated by [START_REF] Garner | Assessing the Risk of Engineered Nanomaterials in the Environment: Development and Application of the nanoFate Model[END_REF] for freshwater ecosystems, CuO NPs may accumulate in the aquatic environment over the long term in sufficient concentration to cause potential toxicity. To the best of our knowledge, there is only one publication assessing CuO NP (20-30 nm, uncoated) effects on a freshwater bivalve. This study was conducted on the endobenthic species Anodonta cygnea exposed to 0.25, 2.5 and 25 µg Cu/L over 12 days and showed histopathological alterations, significant bioaccumulation in the gills, and deleterious impacts in the filtration rate [START_REF] Moëzzi | Ecotoxicological impacts of exposure to copper oxide nanoparticles on the gill of the Swan mussel, Anodonta cygnea (Linnaeus, 1758)[END_REF]. However, in freshwater bivalves the assessment of CuO NP effects in the lowest levels of biological organization are still lacking, such as in molecular and biochemical responses.

The main toxic mechanism reported in nanotoxicology studies is the generation of reactive oxygen species (ROS) that can induce oxidative stress, resulting in further cyto-and genotoxicity [START_REF] Klaper | Molecular interactions of nanomaterials and organisms: defining biomarkers for toxicity and high-throughput screening using traditional and next-generation sequencing approaches[END_REF], Vale et al., 2016). A first line against reactive oxygen species (ROS) damages is the antioxidant defense pathway (Flora, 2009, Mustacich and[START_REF] Mustacich | Thioredoxin reductase[END_REF], that was affected by CuO NP in various aquatic organisms such as algae [START_REF] Melegari | Evaluation of toxicity and oxidative stress induced by copper oxide nanoparticles in the green alga Chlamydomonas reinhardtii[END_REF], fish (Villarreal et al., 2014), anemones [START_REF] Siddiqui | Comparative effects of dissolved copper and copper oxide nanoparticle exposure to the sea anemone, Exaiptasia pallida[END_REF] and marine bivalves [START_REF] Mouneyrac | Fate and effects of metal-based nanoparticles in two marine invertebrates, the bivalve mollusc Scrobicularia plana and the annelid polychaete Hediste diversicolor[END_REF]Kastsumiti et al., 2018). In bivalves, the antioxidant defense activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx)) were increased after CuO NP exposure in the marine species Mytilus galloprovinciallis and Scrobicularia plana exposed to 10 µg Cu/L for 3 to 21 days along with involvement of detoxification mechanisms (metallothionein (MT), glutathione-S-transferase (GST)) [START_REF] Buffet | Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles[END_REF][START_REF] Buffet | A Mesocosm Study of Fate and Effects of CuO Nanoparticles on Endobenthic Species (Scrobicularia plana, Hediste diversicolor)[END_REF][START_REF] Gomes | Effects of Copper Nanoparticles Exposure in the Mussel Mytilus galloprovincialis[END_REF][START_REF] Gomes | Accumulation and toxicity of copper oxide nanoparticles in the digestive gland of Mytilus galloprovincialis[END_REF][START_REF] Ruiz | Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis[END_REF]. These defenses were not sufficient to prevent damage since lipid peroxidation [START_REF] Gomes | Effects of Copper Nanoparticles Exposure in the Mussel Mytilus galloprovincialis[END_REF]2012), apoptosis [START_REF] Buffet | A Mesocosm Study of Fate and Effects of CuO Nanoparticles on Endobenthic Species (Scrobicularia plana, Hediste diversicolor)[END_REF] and genotoxicity [START_REF] Buffet | A Mesocosm Study of Fate and Effects of CuO Nanoparticles on Endobenthic Species (Scrobicularia plana, Hediste diversicolor)[END_REF][START_REF] Gomes | Genotoxicity of copper oxide and silver nanoparticles in the mussel Mytilus galloprovincialis[END_REF][START_REF] Ruiz | Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis[END_REF] were observed. As few dissolution was measured in each of these studies and as discussed by [START_REF] Ruiz | Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis[END_REF] regarding seawater, those effects might then result of CuO particles exposure rather than Cu 2+ .

There is currently a need to investigate chemical effects at different biological levels as mentioned by the Organization for Economic Co-operation and Development (OECD) for the development of an Adverse Outcome Pathway (AOP) approach (OECD, 2018). This approach aims to provide information of causally linked events at different biological levels of organization following the exposure to a chemical and leading to an adverse health effect, which should help for regulatory purposes. In the literature, studies relying on the impact of NP across different pathways and biological levels are underrepresented so far. In this context, the use of -omic tools such as the measurement of gene expressions, protein or metabolite activities should then be particularly informative by allowing the simultaneous monitoring of a high number of responses [START_REF] Snape | Ecotoxicogenomics: the challenge of integrating genomics into aquatic and terrestrial ecotoxicology[END_REF][START_REF] Mussali-Galante | Biomarkers of exposure for assessing environmental metal pollution: from molecules to ecosystems[END_REF].

In addition, the expected precocity of answers at such levels can provide an early warning signal of contamination effect that should have further repercussions on the organism physiology, and allow unraveling the pathways of toxicity mechanisms [START_REF] Bigot | Early defense responses in the freshwater bivalve Corbicula fluminea exposed to copper and cadmium: Transcriptional and histochemical studies[END_REF][START_REF] Chen | Effects of fluoxetine on behavior, antioxidant enzyme systems, and multixenobiotic resistance in the Asian clam Corbicula fluminea[END_REF].

The interactions of a contaminant with the different cellular components implicated from the cellular entry to organism response has to be taken into account. Indeed, the cellular uptake of metallic NP will occur via active transport (endocytosis) or passive diffusion through the cell membrane [START_REF] Beddoes | Understanding nanoparticle cellular entry: A physicochemical perspective[END_REF]. P-glycoprotein (PGP) and multidrug resistance-associated protein 1 (MRP1), as part of the multi-xenobiotic-resistance (MXR) system, can act as detoxifier due to their role of multidrug transporter [START_REF] Achard | Induction of a multixenobiotic resistance protein (MXR) in the Asiatic clam Corbicula fluminea after heavy metals exposure[END_REF]. If they manage to enter the cell, NPs can interact with cell targets, the first of them being proteins. In order to maintain the integrity of proteins, members of the Heat Shock Protein family may be solicited [START_REF] Fabbri | HSP expression in bivalves[END_REF]. As already mentioned, the induction of oxidative stress is expected in the presence of NP. Cells are armed with a battery of enzymes able to protect them against harmful effects of ROS. The most studied are superoxide dismutase (SOD) that allows the dismutation of O2 .-to H2O2, Catalase (Cat) that reduces H2O2 to water and oxygen, and glutathione peroxidase (GPx) that reduces H2O2 or other hydroperoxides [START_REF] Flora | Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid exposure[END_REF].

Glutathione-S-transferase (GST) is also studied because of its contributions to antioxidant, and more generally, antitoxic mechanisms [START_REF] Doyen | Molecular cloning and expression study of pi-class glutathione S-transferase (pi-GST) and selenium-dependent glutathione peroxidase (Se-GPx) transcripts in the freshwater bivalve Dreissena polymorpha[END_REF]. The contribution of Thioredoxin reductase (Trxr) which is mainly involved in antioxidant pathways, and its contribution in DNA synthesis, gene transcription, cell growth and apoptosis inhibition was also described [START_REF] Mustacich | Thioredoxin reductase[END_REF]. One major consequence of the ROS overproduction is the damage to membranes by peroxidation of their lipid components (Alves de [START_REF] Alves De Almeida | Oxidative stress in Perna perna and other bivalves as indicators of environmental stress in the Brazilian marine environment: Antioxidants, lipid peroxidation and DNA damage[END_REF]. In the presence of metallic toxicants, the cell may trigger the synthesis of metallothioneins (MT). Finally, implementing such a battery of defenses against toxic effects may present an energy cost for cells. The assessment of parameters involved in the metabolic management in the cells (e.g. triglyceride content, electron transport system (ETS) activity, lactate dehydrogenase (LDH) activity) can give insights in the physiological status of the organisms and inform about the favored metabolic pathways. Studying molecular and biochemical responses in the frame of the AOP approach implies the description of these first cellular events. Experiments with short duration of exposure (24h to 96h) may provide early response ensuing the first interactions of contaminants with cells [START_REF] Ankley | The Adverse Outcome Pathway: A Multifaceted Framework Supporting 21st Century Toxicology[END_REF][START_REF] Lee | Significance of adverse outcome pathways in biomarker-based environmental risk assessment in aquatic organisms[END_REF]. Following exposure, resident proteins trigger the response and molecular events are expected to be on first line before newly synthetized proteins can act. Therefore, working in the frame of short-term exposure duration may help in understanding the first steps of the cascade event leading to physiological impairments.

In bivalves, filtration has an important place in the regulation of all physiological processes because it governs the entry of water and food in organisms and thereby the entry of contaminants. It is also known that bivalves may reduce or stop the filtration process for a while when environmental conditions are stressful [START_REF] Castro | Feeding inhibition in Corbicula fluminea (O.F. Muller, 1774) as an effect criterion to pollutant exposure: Perspectives for ecotoxicity screening and refinement of chemical control[END_REF][START_REF] Farris | Freshwater bivalve ecotoxicology[END_REF]. The filtration rate is then a good candidate for an early physiological warning [START_REF] Hartmann | Establishing mussel behavior as a biomarker in ecotoxicology[END_REF].

The aim of this work was to provide a multiparameter and multiscale assessment (molecular, biochemical and physiological) of CuO NP effects on C. fluminea exposed to different concentrations (0, 20 and 100 µg CuO/L) in a short-term period (96 hours).

Considering the importance of NP size on their stability, solubility and surface reactivity [START_REF] Peng | Behavior and Potential Impacts of Metal-Based Engineered Nanoparticles in Aquatic Environments[END_REF][START_REF] Baker | Impacts of metal and metal oxide nanoparticles on marine organisms[END_REF], we expected to observe different effects between the two CuO NP. A battery of gene expressions and biochemical markers involved in the previously described pathways was monitored and a filtration test was performed at the end of the exposure period. Since our aim is to depict a global modification of biological parameters associated to NP exposure, these parameters will not be analyzed individually. Instead, we developed an integrated interpretation of obtained results thanks to multivariate statistical tools.

Materials and methods

All chemicals were analytical grade. Chemical and enzymes were purchased from Sigma (Lille, France) unless stated otherwise.

Tested Nanoparticles:

Two CuO NPs were selected in this study. The first one was provided as CuO NP powder with a particle size of 10-100 nm specified by the manufacturer (Intrinsiq Materials Limited).

These NPs were previously characterized by [START_REF] Buffet | Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles[END_REF]. Briefly, nanoparticles were uncapped and polyhedral with a size determined by transmission electron microscopy (TEM) of 29.5 nm in average while their hydrodynamic size in deionized water (DIW) determined by dynamic light scattering (DLS) ranged from 40 to 500 nm (194 nm average). Their mean specific surface area (SSA) was 25.3 m².g -1 . The mean zeta-potential was +26.3 mV, indicative of relative stability in DIW. The suspension appeared stable for approximatively 1 month. A stock suspension of 25 mg.L -1 was prepared in DIW according to [START_REF] Buffet | Fate of isotopically labeled zinc oxide nanoparticles in sediment and effects on two endobenthic species, the clam Scrobicularia plana and the ragworm Hediste diversicolor[END_REF] protocol, stored at room temperature and used during the whole experiment. The stock suspension was sonicated for 5 min (Sonorex super RK 510, 160 W) before each use and was called "30nm-NP" in this study.

The second tested CuO NPs, called "12nm-NP", were supplied by PlasmaChem GmbH (Germany) and suspended by the CEREGE laboratory in Milli-Q water at 518 mg CuO/L. They were previously characterized by [START_REF] Ortelli | Colloidal characterization of CuO nanoparticles in biological and environmental media[END_REF]. The particles were uncoated and semi-spherical with a size reported of 12 ± 8 nm measured by TEM. Their mean hydrodynamic size in DIW was 140.5 nm (DLS) and their SAA was 47 ± 1.7 m².g -1 . The zeta potential in ultrapure water was +28.1 ± 0.6 mV, indicating a relative stability in such media.

This stock suspension was homogenized by magnetic stirring for a few minutes before injections [START_REF] Ortelli | Colloidal characterization of CuO nanoparticles in biological and environmental media[END_REF].

Collection and acclimatization of organisms:

Freshwater Asian clams C. fluminea (3.1 ± 0.3 cm) were hand-collected in June 2016 in the Moselle River, La Maxe (49°09'42.9"N 6°11'55.0"E, France) at a water temperature of 21°C.

The physico-chemical characteristics of the site water were previously measured (NH4: 0.03 mg N/L; NO2: < 0.05 mg N/L; NO3: 1.69 mg N/L; PO4: 0.01 mg P/L; P tot: 0.10 mg P/L; Cl -: 214 mg/L; SO4: 52 mg/L; TAC: 2.3 meq; MEST: 15.4 mg/L; MVS: 37.2%; Ca 2+ : 117 mg/L; Mg 2+: 9.3 mg/L; Na + : 61.4 mg/L; K + : 4.2 mg/L; DCO: 52 mg/L; DBO5: 4 mg/L; Tot A: 9.54 meq/L; Tot C: 9.4 meq/L; Bal.: -0.7%; pH: 8.0; Cond: 1040 µS/cm; dissolve Cu: 1 µg/L; Cu tot.: 1.7 µg/L). After sampling, the clams were transported to the laboratory in their water of origin in plastic coolers. They were acclimated progressively to the experimental conditions in a temperate room (15 °C) for 3 days by gradually increasing the proportion of artificial water that will be used for the experiment. The artificial water was prepared using commercial salt (Tropic Marin ® , Tropicarium Buchshlag Dreieich, Germany) diluted in DIW at 1.5 psu. The elemental composition of this salt (major cations, major anions, nutrients and trace elements) was assessed by [START_REF] Atkinson | Elemental composition of commercial seasalts[END_REF]. The media was continuously aerated and a natural photoperiod was kept (16h light: 8h dark). One day before the experiment, two organisms per beaker were placed in 200 mL of water in order to acclimate them to the device.

2.3.Experimental design

C. fluminea were exposed to 0 (controls), 20 and 100 µg CuO/L of each NP (30nm-NP and 12nm-NP) for 96 hours at 15°C in artificial water (1 C. fluminea/100 mL). The consideration of the SSA of each NP lead to 0.000506 m²/L and 0.00253 m²/L for 30nm-NP at 20 µg/L and 100 µg CuO/L, respectively, and 0.00094 m²/L and 0.0047 m²/L for 12nm-NP at 20 µg/L and 100 µg CuO/L, respectively. The water was renewed every day. No aeration and no feeding were performed in order to avoid any interference with NP. The natural photoperiod (16h light: 8h dark) was kept during the entire duration of the experiment.

The experiment was performed in beakers containing 200mL of medium and two clams.

Twelve beakers were used for each combination treatment x date (144 beakers in total, i.e. 288 clams). Twelve clams per treatment were sampled before water renewal at 24 and 96 hours for biochemical (6 individuals) and for molecular analyses (6 individuals). At these time steps, 5 individuals were transferred in clean water to depurate before the bioaccumulation measurements. The remaining clams in each treatment were in need of help in case of mortality, but were actually not used. After 96 hours, a filtration test was also conducted on individuals kept for the bioaccumulation measurement. The total copper concentrations were measured in the water column and in the organisms (digestive gland and remaining tissues).

2.4.Measurements of water and tissue copper concentrations

As the media was changed daily, the water was sampled for total Cu measurement immediately after NP introduction and after 24 hours (just before the next renewal) in 3 beakers per treatment and time selected randomly. This procedure was applied three times: at the start of the exposure and at the renewal at 24 and 72 hours of exposure. Five clams per treatment were dissected after 24 hours of depuration at each time step (0, 24, 96 hours) for Cu measurement. Then, the digestive gland and the remaining soft tissues were selected, freeze-dried, weighed and digested in 69% HNO3 for 24 hours at 60°C. Concentrations were determined by flame for concentrations above 10µg Cu/L (Perkin-ELMER Aanalyst 100) or by graphite furnace for concentrations below 10 µg/L (VARIAN Spectraa 800 with ZEEMAN correction) atomic absorption spectrophotometry for each individual clam. Certified water (SRM 3114) was used to check the accuracy of the quantification for the water samples (20.2 ± 1.7 µg Cu/L for 20 ± 1 µg Cu/L) and certified lobster hepatopancreas (TORT-2) to check the accuracy of the quantification for tissue samples (97.1 ± 2.6 µg Cu/g for 106 ± 10 µgCu/g). Metal concentration in clams are expressed as µg.g -1 dry weight (DW) tissue and µg.L -1 for water media.

Measurements of gene expression

Preparation of RNA extraction

C. fluminea digestive glands of 6 individuals per treatment were excised, rinsed in PBS 1%, and transferred into 1 mL of RNA later solution (Qiagen, Hilden, Germany) kept in 4°C.

Then, the tissues can be kept during a maximal duration of 1 month. After a manual removal of non-digestive tissue, digestive glands were stored in -80°C until subsequent analysis.

The total RNA was extracted from individual digestive gland using an RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturer protocol. Genomic DNA was digested after extraction with DNAse I and total RNA was purified with RNeasy mini kit.

RNA purity and quantity were assessed by optical density measurements (OD 260 nm and OD ratio 260/280 and 260/230). RNA integrity was assessed by capillary electrophoresis using Bioanalyseur 2100 (Agilent, CA, USA).

RT-qPCR analysis

RT-qPCR was then performed on 12 genes and the sequences used in this study are listed in Supporting data Table S1. Primers were designed using Primer3Plus and purchased from Eurofins Genomic (Ebersberg, Germany). The cDNA was synthetized in a final volume of 20 µL using 1 µg of RNA, 2.5 µM of random hexamer primers and SuperScript® III reverse transcriptase according to the manufacturer's instructions (Invitrogen, CA, USA). The qPCR reaction was performed as described in [START_REF] Koehlé-Divo | Corbicula fluminea gene expression modulated by CeO2 nanomaterials and salinity[END_REF]. Briefly, 45 ng of cDNA, water (no template control) or total RNA (45 ng/reaction; no RT control) were used as a template, from 200 to 300 nM of primers (see table S1) and Fast SYBR® green master mix (Applied Biosystem®, CA, USA ) in a reaction mixture with a final volume of 20 µL. The cycling conditions were 20 s at 95°C, followed by 40 cycles of 3 s at 95°C and 30 s at 60°C.

The melting curve was used to check the specificity of the amplicons and their sizes were verified on agarose gel. All PCR amplifications were performed on each biological replicate using the StepOnePlus RT-PCR system (Applied Biosystems®). Gene expression levels were analyzed using the relative quantification method (∆∆Ct) [START_REF] Livak | Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 -ΔΔCT Method[END_REF]. Two genes were selected as potential normalizing genes (S3 and β-actin), but only β-actin has revealed sufficient stability in our exposure conditions. β-actin was then used as a reference for normalization. The control condition (0 µg/L) was chosen as the reference condition. The final treated/control ratio and the pooled standard deviation were calculated based on the mean ∆Ct of the biological replicates. Statistical analyses were performed separately for each exposure duration (24 & 96 hours) as described below ( §2.8).

Biochemical biomarker analysis

After removal of the shell, the C. fluminea digestive gland was excised, kept in ice and stored at -80°C until analysis. Each digestive gland of six clams per treatment were separately treated as described in [START_REF] Sroda | Seasonal variability of antioxidant biomarkers and energy reserves in the freshwater gammarid Gammarus roeseli[END_REF] for cellular biomarker measurements.

The digestive glands were defrosted, weighed and crushed in a 50 mM phosphate buffer at pH 7.6 and at 4°C and supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM L-serine borate mixture as protease inhibitor at a 8/1 volume/weight ratio. The mixture was then centrifuged at 250xg at 4°C during 5 min and supernatant was divided in two parts: one for the assay of total protein, LPO, ACP (acid phosphatase) and ETS activities, while the other part was centrifuged during 20 min at 1,000xg at 4°C and the resulting supernatant was again centrifuged 50 min at 20,000xg. The final supernatant corresponded to the cytosolic fraction that was used for enzyme activity.

Biomarker analysis was then performed on the automated spectrophotometer analyser (Konelab 20 XTi, Thermo Scientific) by using protocols described in [START_REF] Bertrand | The influence of salinity on the fate and behavior of silver standardized nanomaterial and toxicity effects in the estuarine bivalve Scrobicularia plana: Salinity influences Ag NM-300K behavior and toxicity in clam[END_REF].

Briefly, total protein content ([prot]), triglyceride content (TRI), lipid peroxidation (LPO)), acid phosphatase (ACP) and electron transport system (ETS) mitochondrial activities were measured using the whole homogenate whereas total protein content ([prot]), total antioxidant capacity (TAC), glutathione-S-transferase (GST), lactate deshydrogenase (LDH) and superoxide dismutase (SOD) activities were measured in cytosolic fractions. The SOD activity was assessed using the WST-1 method [START_REF] Ukeda | Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3′-${$1-[(phenylamino)-carbonyl]-3, 4tetrazolium$}$-bis (4-methoxy-6-nitro) benzenesulfonic acid hydrate reduction by xanthinexanthine oxidase[END_REF] adapted to C. fluminea.

Statistical analyses were performed separately for each exposure duration (24 & 96 hours) as described below ( §2.8).

Filtration rate measurements

Five clams per treatment were placed separately in 50 mL of neutral red solution of 5 mg.L -1 in the dark at 15 °C at the end of the exposure. Clams were placed in the middle of each beaker and were allowed to filter for 2 hours. Then, clams were picked up and placed in clean artificial water to let them depurate for 24 hours. After depuration, the clams were used for Cu measurements as described in §2.4.The beakers containing the neutral red solution were acidified to pH 5.0 with HCl 37% immediately after removal of the organisms and agitated for homogenization. Then, 1 mL was sampled twice in each beaker and the absorbance was read at 530 nm. The neutral red concentration in each beaker was then calculated using the standard neutral red curve. Filtration rate (f) was calculated according to [START_REF] Coughlan | The estimation of filtering rate from the clearance of suspensions[END_REF]: f = (V/(n x t)) x log (C0/Ct) with V corresponding to the total volume of neutral red solution (100 mL), n the number of individuals, t the time (h), C0 and Ct the initial and final neutral red concentrations in the beakers, respectively.

Statistical analysis

Statistical analyses were conducted using R (R Core Team, 2014). For the filtration rate and the bioaccumulation results, the homoscedasticity and the normality were checked by Levene and Shapiro tests, respectively. As the conditions of use were confirmed, these data were analyzed using a one-way anova test in order to compare both NPs separately from their respective controls for the filtration rate results. Significant results were defined with a threshold of p < 0.05. A two-way anova was performed on the bioaccumulation dataset in order to compare each condition over time.

The gene expression dataset as well as the biochemical markers were analyzed using multivariate analyses. First, a manova (multivariate ANOVA) was performed to assess if significant differences could be depicted according to the tested factors. These results helped us to design each Partial Least Square Discriminant Analyses (PLS-DA) highlighting the actors of those differences. Only the variables (i.e. gene expressions or biochemical biomarkers) with a Variable Importance in Projection (VIP) score above 0.8 were considered.

Finally, Hotelling T² tests were performed on PLS-DA results to more thoroughly explore the differences between the exposure conditions.

Results

Water and tissue Cu concentrations

The control beakers contained 0.56 ± 0.38 µg Cu/L throughout the entire duration time.

Measurements of total Cu in the contaminated waters are reported in fig. 1 and are expressed as percentage of recovery compared to the nominal introduced concentrations. Cu concentrations in water immediately after the introduction of NPs were close to the intended concentrations for both 20 µg CuO/L treatments with 98 ± 10 % and 92 ± 8 % recovery for 30nm-NP and 12nm-NP, respectively. High recoveries were also measured for both 100 µg CuO/L treatments with 30nm-NP and 12nm-NP presenting 82 ± 4 % and 84 ± 8 % recovery, respectively. Cu concentrations were also measured before each renewal (after 24 hours) and showed a decrease of 74 ± 1 % in all NP treatments (20 & 100 µg/L) after 24 hours in the test media.

Except for some slight variations, measurements of total Cu concentrations in the C. fluminea digestive gland (fig. 2) showed no significant accumulation of copper during the whole experiment duration. Additional measurements of Cu were performed on the remaining soft tissues (see fig. S1) but no significant accumulation was measured either.

Gene expression

The Manova analysis performed on the 24 hour gene expression dataset indicated a significant concentration effect (F= 3.54-60 df, p < 0.05) and a significant interaction of NP type with NP concentration (F= 5.44-60 df, p < 0.001) while no effect of NP type alone (F= 1.02-29 df, p= 0.37) was recorded. The pairwise comparisons (fig. 3, Table, left side) indicated significant differences between control organisms and the ones exposed to the highest concentration (100 µg CuO/L) for 30nm-NP while the 20 µg CuO/L treatment for 12nm-NP was different from both control and 100 µg/L treatments. The PLS-DA presented in fig. 3 (left side) highlighted these differences. The horizontal axis separated 30nm-NP treatments by lowered expression of genes involved in antioxidant defenses (CAT, GST-pi, Trxr, CuZnSOD, MnSOD & Se-GPx) when exposure concentration increased. Both control and 100 µg/L treatments for 12nm-NP were relatively close while 20 µg/L treatment was different, mainly along the vertical axis, presenting more PGP and MRP1 transcripts than the two other groups.

The Manova analysis conducted on the 96 hour gene expression dataset indicated a significant impact of NP type alone (F= 172-29 df, p < 0.001) and of its interaction with NP concentration (F= 4.14-60 df, p < 0.01) while no significant difference was shown for the concentration effect alone (F= 1.14-60 df, p = 0.37). The pairwise comparisons (fig. 3, table right side) only showed a clear discrimination between both NP types (30nm-NP & 12nm-NP) at the highest exposure concentration (100 µg/L). The associated PLS-DA is presented in fig. 3 (right side). Both NP types at 100 µg/L were separated by the vertical axis that showed a higher mRNA content of MT and GST-pi in 30nm-NP exposed organisms compared to those exposed to 12nm-NP.

The horizontal axis described by MRP1, PGP and Trxr expressions did not allow a clear separation between groups, but 12nm-NP exposed organisms tended to display an expression slightly more pronounced for these genes than 30nm-NP exposed ones did. This is in agreement with the results observed at 24 hours when MRP1 and PGP displayed more transcripts only in organisms exposed to the lower concentration.

Biochemical markers

The PLS-DA analyses using biochemical biomarkers measured in C. fluminea digestive gland after exposure to the two CuO NPs (30nm-NP & 12nm-NP) are presented in fig. 4. The Manova analysis performed on the 24 hour-and 96 hour-biochemical marker datasets did not indicate any significant effect of NP concentration (24h: F = 0.452-29 df, p = 0.65; 96h: F = 0.2.92-29 df, p = 0.07), NP type (24h: F= 1.954-60 df, p= 0.11; 96h: F= 1.994-60 df, p= 0.11) or interaction between them (24h: F = 0.1.674-60 df, p = 0.17; 96h: F = 0.864-60 df, p = 0.49). The associated Hotelling T² tests were in accordance with the Manova's results, showing no significant difference between all exposure groups at each time step.

Filtration rate

The filtration rates measured at the end of the exposure (96 hours) are shown in fig. 5. No significant difference was obtained between both controls (t=2.883.8 df, p = 0.48). The organisms exposed to 30nm-NP presented a significant decrease of filtration rate when exposed to the highest concentration (100 µg CuO/L) and the ones exposed to 12nm-NP presented a significant decrease of filtration activity for both exposure concentrations (20 & 100 µg CuO/L).

Discussion

The aim of this study was to define if different sizes of CuO NP may have different effects on C. fluminea exposed in a short-term period to concentrations of copper potentially found in the environment. In freshwater ecosystems, the Cu concentrations can range from 0.04 to 294 µg/L and can even reach 20 mg/L in extremely polluted areas (Tran et al., 2003 and references therein). These concentrations generally relate to dissolved and micrometric Cu, but the nanoparticulate forms could be a part of this contamination. The selected concentrations in this study were included in the Cu concentration range measured in the environment, but also in those estimated by [START_REF] Chio | Assessing the potential risks to zebrafish posed by environmentally relevant copper and silver nanoparticles[END_REF] for Cu NP in Taiwanese rivers with a confidence interval of 95% (10 -920 µg/L), but they were more than 200 times higher than the PEC values reported more recently in water by other authors [START_REF] Liu | Multimedia Environmental Distribution of Engineered Nanomaterials[END_REF][START_REF] Garner | Assessing the Risk of Engineered Nanomaterials in the Environment: Development and Application of the nanoFate Model[END_REF]. Although the tested concentrations (20 and 100 µg/L) seem then relatively high from an environmental point of view, they have allowed the recovery of the contaminant in the exposure media. While most studies focused on behavioral or physiological traits, molecular parameter assessment is still scarce in aquatic ecotoxicology despite its sensitivity [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]. This is due to the limited genomic information for sentinel and non-model organisms [START_REF] Calzolai | Transcriptomics and proteomics[END_REF][START_REF] Mussali-Galante | Biomarkers of exposure for assessing environmental metal pollution: from molecules to ecosystems[END_REF], such as bivalve species. Among bivalves, most of the studied organisms are marine species and mostly represented by Mytilus galloprovincialis [START_REF] Rocha | Ecotoxicological impact of engineered nanomaterials in bivalve molluscs: An overview[END_REF][START_REF] Revel | Omics tools: New challenges in aquatic nanotoxicology?[END_REF]. The measurement of expression of known genes for C. fluminea is relevant since these genes might be engaged in key responses against contamination and will give additional information regarding NP toxicity in freshwater species. In addition, the sensitivity and the precocity of answer at such a biological level will allow the detection of early warning signals during a short-term period, as the one applied in this work. This monitoring may also help to provide knowledge about mechanistic events that can be integrated afterward in AOP approaches for risk characterization [START_REF] Revel | Omics tools: New challenges in aquatic nanotoxicology?[END_REF].

As many parameters influenced NP fate in the environment, the measurement of the effective exposure concentration appeared as a first crucial indication. Our results clearly showed a drop of the concentration of Cu in the water after 24 hours. As usually observed, the loss might be explained by the process of NP homoaggregation and/or heteroaggregation, which is enhanced when the concentration increases [START_REF] Bundschuh | Nanoparticles in the environment: where do we come from, where do we go to?[END_REF][START_REF] Baker | Impacts of metal and metal oxide nanoparticles on marine organisms[END_REF]Melegeri et al., 2013;[START_REF] Baek | Microbial toxicity of metal oxide nanoparticles (CuO, NiO, ZnO, and Sb2O3) to Escherichia coli, Bacillus subtilis, and Streptococcus aureus[END_REF][START_REF] Lead | Nanomaterials in the environment: Behavior, fate, bioavailability, and effects-An updated review[END_REF]. The fast aggregation kinetic of NP between the injection and the sampling would be responsible for the lower values measured than the intended ones (much lower for 100 µg/L treatments). The resulting sedimentation is then responsible for the reduction of NP concentration in the water. The adsorption of NP on different abiotic and biotic surfaces can also contribute to this reduction.

Even if the concentrations measured after each renewal were slightly lower than intended values, they remained close to them. Thus, the daily media renewal ensures that the pressure of NP exposure was maintained during the whole experiment in a range of concentrations from 26 to 98% of the intended values depending on the treatment.

Bivalves are filter-feeders and are known to bioaccumulate various contaminants including nanoparticles [START_REF] Hanna | Accumulation and Toxicity of Copper Oxide Engineered Nanoparticles in a Marine Mussel[END_REF][START_REF] Rocha | Ecotoxicological impact of engineered nanomaterials in bivalve molluscs: An overview[END_REF][START_REF] Buffet | Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles[END_REF], but they were also reported to pack NP in pseudofeces [START_REF] Montes | Uptake, accumulation, and biotransformation of metal oxide nanoparticles by a marine suspension-feeder[END_REF][START_REF] Conway | Effects and Implications of Trophic Transfer and Accumulation of CeO2 Nanoparticles in a Marine Mussel[END_REF] and in feces [START_REF] Hull | Filter-Feeding Bivalves Store and Biodeposit Colloidally Stable Gold Nanoparticles[END_REF]Ward and Kach, 2009), then reducing the bioaccumulated content.

In our study, neither the digestive gland nor the rest of the soft tissues showed any significant bioaccumulation. Bioaccumulation of CuO NP was reported in marine bivalves to concentrations as low as 10 µg/L in Scrobicularia plana [START_REF] Buffet | Behavioural and biochemical responses of two marine invertebrates Scrobicularia plana and Hediste diversicolor to copper oxide nanoparticles[END_REF]2013) and even after 3 days of exposure in Mytilus galloprovincialis [START_REF] Gomes | Effects of Copper Nanoparticles Exposure in the Mussel Mytilus galloprovincialis[END_REF]2012;2013[START_REF] Core | R: A language and environment for statistical computing[END_REF].

The absence of bioaccumulation recorded in C. fluminea remains difficult to explain, but the short duration of the exposure period might be a first point. Secondly, a short depuration period was applied. Other authors do not systematically do so and may have overestimated the actual accumulated concentrations including for example the Cu passing through the lumen of the digestive tracts. Finally, copper is an essential element that can also lead to toxic effects [START_REF] Gomes | Effects of Copper Nanoparticles Exposure in the Mussel Mytilus galloprovincialis[END_REF]. Bivalves are able to act for its elimination as well as to adopt a protective behavior such as valve closure or reducing filtration. C. fluminea was already reported to quickly respond to Cu contamination by closing their valves, even at low doses < 5 µg/L [START_REF] Tran | Copper detection in the Asiatic clam Corbicula fluminea: optimum valve closure response[END_REF][START_REF] Castro | Feeding inhibition in Corbicula fluminea (O.F. Muller, 1774) as an effect criterion to pollutant exposure: Perspectives for ecotoxicity screening and refinement of chemical control[END_REF], which reduced the organism exposure to the contaminated media. The filtration test we performed clearly showed that clams pre-exposed to both NP types reduced their filtration activity when further placed in clean water. Besides a protective response, the CuO NP could have altered the gills [START_REF] Moëzzi | Ecotoxicological impacts of exposure to copper oxide nanoparticles on the gill of the Swan mussel, Anodonta cygnea (Linnaeus, 1758)[END_REF], what may lead to a reduction of the filtration efficiency. Our experimental design does not enable us to monitor filtration activity directly during the NP exposure, but we assume that if filtration was reduced during the exposure (as it was during the test), it might partly explain why significant accumulation has not occurred. The absence of significant bioaccumulation observed does not necessarily mean an absence of contact or uptake, as already observed in other studies, using relatively low exposure concentrations, in which the absence of bioaccumulation of CeO2 NP [START_REF] Koehlé-Divo | Genotoxicity and physiological effects of CeO2 NPs on a freshwater bivalve (Corbicula fluminea)[END_REF][START_REF] Garaud | Multibiomarker assessment of cerium dioxide nanoparticle (nCeO2) sublethal effects on two freshwater invertebrates, Dreissena polymorpha and Gammarus roeseli[END_REF] was measured in bivalve species while strong biochemical and/or genotoxic effects were highlighted. In order to be sure that there was effectively no bioaccumulation, the use of labelled NP as tracer should be particularly relevant since labelling make them easily detectable, even in small quantities [START_REF] Dybowska | Synthesis of isotopically modified ZnO nanoparticles and their potential as nanotoxicity tracers[END_REF].

At the molecular level, the measurement of gene expression allowed us to evidence significant effects linked to the applied exposure. After 24 and 96 hours, significant effects were associated to the interaction of both the type and the concentration of the tested NP.

After 24 hours of exposure, the results suggest that 30nm-NP may be less oxidant than 12nm-NP, because the level of transcripts of genes involved in antioxidant defense was reduced. At 96 hours, an increase of transcripts of detoxification genes was noticed (MT and GSTpi), but genes of antioxidant defense were not significantly affected. As mentioned in [START_REF] Klaper | Molecular interactions of nanomaterials and organisms: defining biomarkers for toxicity and high-throughput screening using traditional and next-generation sequencing approaches[END_REF], the oxidative stress response can dissipate quickly and other molecular pathways such as drug resistance or detoxification genes can be induced at low exposure concentrations. Indeed, in the case of exposure to 12nm-NP, it seems that the mechanisms of cellular excretion (MRP1 and PGP) were elicited, especially after 24 hours at 20 µg/L. Our work demonstrated for the first time in the clam C. fluminea significant but not striking effects of CuO NP exposure at the molecular level. Coherent pathways were involved in the response we detected, that was based mainly upon detoxification (30nm-NP) and cellular elimination (12nm-NP). Our final results showed that the markers investigated at the biochemical level did not respond to NP contamination regardless of NP type or concentration for both times monitored. The absence of NP effect at this biological level was already reported in M. galloprovincialis exposed to 10 µg Cu/L of CuO NP for 24 hours [START_REF] Ruiz | Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis[END_REF] and for 3 days [START_REF] Gomes | Accumulation and toxicity of copper oxide nanoparticles in the digestive gland of Mytilus galloprovincialis[END_REF] but it must be duly noted that detoxification mechanisms (MT, SOD, CAT, GPx and/or GST) and/or marker of damages (i.e. LPO) were enhanced at a longer timescale ranging from 7 to 21 days [START_REF] Gomes | Accumulation and toxicity of copper oxide nanoparticles in the digestive gland of Mytilus galloprovincialis[END_REF][START_REF] Ruiz | Short-term effects on antioxidant enzymes and long-term genotoxic and carcinogenic potential of CuO nanoparticles compared to bulk CuO and ionic copper in mussels Mytilus galloprovincialis[END_REF]. A same trend was observed in C. fluminea exposed to 10 and 100 µg/L of CeO2 NP where none of the monitored biochemical markers have been modified after 2 days of exposure but significant damages (i.e. apoptosis) were observed after 6 days of exposure [START_REF] Koehlé-Divo | Genotoxicity and physiological effects of CeO2 NPs on a freshwater bivalve (Corbicula fluminea)[END_REF].

The different responses monitored in this study for both tested NP may be explained by their differing sizes and specific surface areas. Because of their smaller size, 12nm-NPs display a higher specific surface area. Consequently, for a given mass, the surface of 12nm-NP available to interact with the organisms will be larger. Per mass unit, the exposure concentrations are similar, but once normalized by the specific surface area, the exposure concentrations are twice as high for 12nm-NP. In addition, the difference of particle size may lead to a higher amount of 12nm-NP particles per mass concentration than 30nm-NP [START_REF] Quik | Heteroaggregation and sedimentation rates for nanomaterials in natural waters[END_REF][START_REF] Singh | NanoGenotoxicology: The DNA damaging potential of engineered nanomaterials[END_REF]. Moreover, the kinetics of transformation as dissolution are expected to be faster for 12nm-NP because of their higher specific surface area [START_REF] Auffan | Chemical stability of metallic nanoparticles: A parameter controlling their potential cellular toxicity in vitro[END_REF][START_REF] Singh | NanoGenotoxicology: The DNA damaging potential of engineered nanomaterials[END_REF]. Although their uncoated surfaces may have led to their fast aggregation in the exposure media, and by this, to the reduction of their surface reactivity, their interactions with the biological surfaces should allow their disaggregation through mechanical actions (gill cilia, labial palps) and/or their dissolution due to the composition change (proteins, pH) [START_REF] Rocha | Ecotoxicological impact of engineered nanomaterials in bivalve molluscs: An overview[END_REF]Ward and Kach, 2009;[START_REF] Canesi | Bivalve molluscs as a unique target group for nanoparticle toxicity[END_REF][START_REF] Griffitt | Effects of particle composition and species on toxicity of metallic nanomaterials in aquatic organisms[END_REF]. The high sensitivity of C. fluminea to copper could perhaps explain their physiological responses and the entry of CuO NPs and ions in the cells could have also contributed to the observed responses by interacting with cellular components. Finally, our results are in agreement with those obtained in the literature which have shown different effects of NP according to their size on ecotoxicological endpoints, the smaller being usually more toxic than the bigger ones [START_REF] Hu | Toxicity of copper oxide nanoparticles in the blue mussel, Mytilus edulis: A redox proteomic investigation[END_REF][START_REF] Peng | Behavior and Potential Impacts of Metal-Based Engineered Nanoparticles in Aquatic Environments[END_REF].

Conclusion

In this study, the short-term exposure of the Asian clam C. fluminea to relatively low concentrations of NPs (20 & 100 µg CuO/L) compared to the available literature highlighted significant modifications at different scales of biological organization from physiological to molecular levels. Integrated gene expression analysis revealed a different mechanism of action depending on NP treatment, with one enhancing antitoxic related mRNAs while the other presented gene expression modification linked to cellular elimination pathway. This analysis was performed on a restricted number of genes due to the lack of gene sequences in the clam C. fluminea. The filtration rate appeared also useful for the assessment of NP toxicity and induced an avoidance response of C. fluminea by valve closure. On a longer time scale, the reduction of the filtration rate and the gene expression modification could be increased and also lead to further side-effects. In the future, with the next generation sequencing advances, the omic tool will allow a screening of a larger set of genes involved in several pathways, like cell proliferation, cell signaling, or DNA repair. Although C. fluminea is considered as relatively tolerant to contaminants compared to other bivalves [START_REF] Castro | Feeding inhibition in Corbicula fluminea (O.F. Muller, 1774) as an effect criterion to pollutant exposure: Perspectives for ecotoxicity screening and refinement of chemical control[END_REF], these NPs were shown here as reactive in a short-term exposure and using a waterborne contamination. In regard to our results, the assessment of these NP impacts on C. fluminea should be addressed in more complex as well as in longer timescale experiments which can be achieved by the setting up of mesocosm experiments. The multi-scale approach is particularly relevant for a better evaluation and understanding of a potential toxicity on individuals and contribute to increased information regarding the first steps of an AOP approach. Such an assessment should be further developed at higher biological levels, including population and communities.

Figure 1: Percentages of Cu recovery compared to nominal Cu concentrations injected in the water column for each CuO NP treatment. These measurements are the mean ± SD (n=3) of the initial concentrations (initial) and concentrations after 24 hours (before renewal) for each NP treatment, obtained from samples collected at the start and after 24 and 72 hours of experiment (See §2.4).

.

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: Total Cu concentrations (mean ±SD, n=5) in the digestive gland of C. fluminea exposed to two different CuO NPs during 0, 24 and 96 hours. No significant differences between groups were pointed out by statistical tests.

Figure 5 :

 5 Figure 5: Filtration rate of C. fluminea exposed for 96 hours to two different CuO NPs (30nm-NP & 12nm-NP) at three concentrations (0, 20 and 100 µg/L). The presented results are mean ± SD). Groups with different letters (lowercase for 30nm-NP, capital for 12nm-NP) are significantly different.

Acknowledgements

Financial supports were provided by the French National Agency (ANR-3-CESA-0014/NANOSALT project) and CPER Lorraine-ZAM (Contrat Projet Etat Région Lorraine, Zone Atelier Moselle). This work is also a contribution to the Labex Ressources 21 (ANR-10-LABX-21-01, Strategic metal resources of the 21st century). The authors gratefully acknowledge CNRS for funding the iCEINT International Consortium for the Environmental Implications of NanoTechnology. KOEHLE-DIVO Vanessa received financial support for salary from French Research ministry. The authors gratefully acknowledge Sharon Kruger for her English corrections.