Murat Belivermiş

Michel Warnau

Marc Metian

François Oberhänsli

Jean-Louis Teyssié

Thomas Lacoue-Labarthe

Limited effects of increased CO 2 and temperature on metal and radionuclide bioaccumulation in a sessile invertebrate, the oyster Crassostreagigas

Keywords: Ocean acidification, bioaccumulation, Pacific oyster, metal, radiotracer, in vitro digestion

This study investigated the combined effects of reduced pH and increasedtemperature on the capacities of the Pacific cupped oyster Crassostrea gigasto bioconcentrateradionuclide and metals . Oysters were exposed to dissolved radiotracers ( 110m Ag, 241 Am, 109 Cd, 57 Co, 54 Mn, and 65 Zn)atthree pH (7.5, 7.8, 8.1) and two temperatures (21 °C and 24 °C)undercontrolled laboratory conditions. Although calcifying organisms are recognized as particularly vulnerable to ocean acidification, the oyster did not accumulate differently the studied metals when exposed under the different pH conditions.However temperature alone or in combination with pH somewhat altered the bioaccumulation of the studied elements. At pH 7.5, Cd was accumulated with an uptake rate constant two-fold higher at 24°C than 21°C.

Bioaccumulation of Mn was significantly affected by an interactive effect between seawater pH and temperature, with a decreased uptake rate at pH 7.5 when temperature increased(27 ± 1 vs. 17 ± 1 d -1 at 21°C and 24°C, respectively). Retention of Co and Mn tended also to decrease at the same pH with decreasing temperature. Neither pH nor temperature affectedstronglythe elements distribution between shell and soft tissues. Significant effects of pH were foundon the bio-accessibility of Mn, Zn and 241 Amduring experimental in vitrosimulation of human digestion.

INTRODUCTION

The increase of partial pressure of CO 2 (pCO 2 ) in the atmosphere due to anthropogenic activities is well recognized as a driver ofmajor global changes such as theelevation ofatmospheric temperature. The global mean surface air temperature is projected to increase by 1 to3.7°C by the end of the century (IPCC, 2013).Accordingly the temperature of ocean will increase by 0.6°C to 2.0°C for the top 100 metres (IPCC, 2013). Additionally, the ocean is a major sink for atmospheric CO 2 (it absorbs~25 % of the anthropogenic emissions) and thusthe increase inpCO 2 in the atmosphere implies an increase inpCO 2 in surface ocean waters, that causesthe "other" CO 2 problem [START_REF] Doney | Ocean acidification: the other CO 2 problem[END_REF], better known as"ocean acidification".

Marine organisms can be affectedby ocean acidificationthrough(1) limitation of available carbonates, mainly affecting calcifying organisms, (2) the increase in H + ions in the water resulting in decreasing pH -i.e. acidification of the surrounding environment-and (3) an increase in pCO 2 within their body (hypercapnia).In this context,attention should be paid tovulnerablespecies, which have key role(s) in the food chains, or are of high economic interest for aquaculture or fisheries.

The Pacific cupped oyster Crassostrea gigas is the most farmedoyster species in the world (FAO, 2014), with an annual production of 0.609 million tonnes for ca. US$ 1.3 billion(2012 data).Consequently, many researchers focused on this marine resource and recently investigated, among others, the responses of the Pacific cupped oyster to ocean acidification.Several studies assessed growth, calcification,development and survival of oystersunder realistic low pH and low carbonate concentrations conditions(for a review, see [START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF]. A particular attention has beenpaid tothe early life stages of the oyster, which are considered as the most vulnerable and therebyas a bottleneck for population dynamics [START_REF] Dupont | What meta-analysis can tell us about vulnerability of marine biodiversity to ocean acidification?[END_REF]. Overall, reduced shell size and thickness, developmental retardation and increased mortalityare the most common effectsof elevated pCO 2 on Crassostrea gigas(e.g. [START_REF] Parker | Comparing the effect of elevated pCO 2 and temperature on the fertilization and early development of two species of oysters[END_REF][START_REF] Gazeau | Effect of carbonate chemistry alteration on the early embryonic development of the pacific oyster (Crassostrea gigas)[END_REF][START_REF] Barton | The Pacific oyster, Crassostreagigas, shows negative correlation to naturally elevated carbon dioxide levels: Implications for near-term ocean acidification effects[END_REF].

Intraspecific variations have been highlighted,and it has been suggestedthat some populations could bepre-adapted to ocean acidification [START_REF] Parker | Comparing the effect of elevated pCO 2 and temperature on the fertilization and early development of two species of oysters[END_REF][START_REF] Parker | Populations of the Sydney rock oyster, Saccostreaglomerata, vary in response to ocean acidification[END_REF][START_REF] Timmins-Schiffman | Elevated pCO 2 causes developmental delay in early larval Pacific oysters, Crassostreagigas[END_REF].

The elevatedpCO 2 in seawater is expected to affect the metabolism though disturbances of acid-base regulation and respiration [START_REF] Pörtner | Biological impact of elevated ocean CO 2 concentrations: lessons from animal physiology and earth history[END_REF]. It is admitted that active organisms, such as fish, cephalopods and crustaceanshave naturally the required physiological machinery to deal with elevated extracellular pCO 2 [START_REF] Melzner | Physiological basis for high CO 2 tolerance in marine ectothermic animals: preadaptation through lifestyle and ontogeny?[END_REF]. In contrast, sessile organisms, such as oysters, are weak acid-base regulators and, in light of the current knowledge,tend to increase their energy demand to poorly compensate the body acidosis [START_REF] Parker | Predicting the response of molluscs to the impact of ocean acidification[END_REF]. This energy budget change is exacerbated when seawater temperature increases [START_REF] Lannig | Impact of ocean acidification on energy metabolism of oyster, Crassostreagigas -changes in metabolic pathways and thermal response[END_REF] andcould affect other performances such as somatic growth and survival [START_REF] Beniash | Elevated level of carbon dioxide affects metabolism and shell formation in oysters Crassostrea virginica[END_REF].

Mining, fossil fuel combustion, industrial activities, and uncontrolled discharges are common sources of trace element and radionuclide releases in the environmentandare major contributorsto the contamination of coastal marine ecosystemsover the last century.Widespread in the environment, these contaminants are accumulated in organisms, and potentially bio-magnifiedalong the food chains [START_REF] Zhou | Biomonitoring: An appealing tool for assessment of metal pollution in the aquatic ecosystem[END_REF].Excessive accumulationof essential (e.g. Co, Mn or Zn) or non-essential (e.g.Ag, 241 Am, Cd) elements in organismscan induce toxic effects [START_REF] Rainbow | Trace metal concentrations in aquatic invertebrates: why and so what?[END_REF]. Since bioaccumulation of metals in biota depends on 1) the bioavailability of the element determined by physicochemical conditions of the environment, 2) the biological traits of organisms (e.g. metabolism, feeding strategy), and 3) the element properties with respect to biological tissues (e.g. ligand affinities, biological function), it appears pertinent to investigate the effect of ocean acidification on metal bioaccumulation capacities. Indeed pH and seawater chemistry changes caused by increased pCO 2 affect chemicalspeciation of metals and therefore their bioavailability to organisms [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF], especially for elements that form strong complexes with carbonates(e.g. Am -Choppin, 2006).Furthermore, increased temperature or hypercapnia (as discussed above) can influencethebioaccumulation of metals [START_REF] White | Regulation of zinc concentration by Palaemonelegans (Crustacea:Decapoda): zinc flux and effects of temperature, zinc concentration and moulting[END_REF][START_REF] Houlbrèque | Effects of increased pCO 2 on zinc uptake and calcification in the tropical coral Stylophora pistillata[END_REF],and can thus enhancetheirtoxicity(e.g. [START_REF] Pascal | The toxicological interaction between ocean acidity and metals in coastal meiobenthic copepods[END_REF][START_REF] Roberts | Ocean acidification increases the toxicity of contaminated sediments[END_REF].

This study aimsat assessing the bioaccumulationbehaviours of radionuclides/metals in a sessile species of economic importance, the Pacific cupped oyster,Crassostrea gigas,usually cultured in coastal areassubjected to metal contamination and high pCO 2 variations (e.g. [START_REF] Green | Death by dissolution: Sediment saturation state as a mortality factor for juvenile bivalves[END_REF].Radiotracer techniqueswereused to determine uptake and depuration parameters of 6 elements during experimental exposuresof oysters to dissolved radiotracers ( 110m Ag, 241 Am, 109 Cd, 57 Co, 54 Mn, and 65 Zn) at three pH (7.5, 7.8, 8.1) and two temperatures (21 °C and 24 °C) under controlled laboratory conditions [START_REF] Warnau | Radiotracer techniques: A unique tool in marine ecotoxicological studies[END_REF].In vitrohuman digestion simulations [START_REF] Versantvoort | Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food[END_REF] were also carried out todeterminethe bio-accessible fraction of metals and radionuclides in oysters that would be ingested by human (Metian et al., 2009a).

MATERIALS AND METHODS

Organisms, acclimationand experimentalconditions

One hundred and sixty-two adult oysters Crassostrea gigas (17.71 ± 3.36 h/12 h).Then, the pH and the temperature were progressively modified overone week until targeted values (see experimental procedure below) and acclimation to these targeted conditions was allowed for at least 15 days prior to the radiotracers exposure. During the whole acclimation period, oysters were fed daily with a mixt algal dietcomposed ofIsochrysis galbana and Skeletonema costatum.

The oysters were maintained under controlled temperature and pH in a crossed experimental design (2 temperatures x 3 pH levels). Due to technical constraints (mainly in term of radiotracer cost and waste water management) only one70-L tank could be dedicated per pCO 2temperature condition. In order to avoid other variation in other factors (e.g. light) the tanks were randomly interspersed on the bench.In addition seawater was very frequently renewed (every one to two days) and tanks were cleaned at each seawater renewal to prevent any "tank" effect due to the development of different biomasses or to the accumulation of detritus such as pseudo faeces, or bacterial proliferation, which might affect the metabolism of oysters or the bioavailability of the chemicals.The 3selected pH values were8.10 (~400 µatm), 7.80 (~800 µatm), 7.50 (~1800 µatm) andthe two temperatures were 21°C and 24°C in accordance to the projected values over the next twocenturies (Orr et al., 2005;Solomon et al., 2007; currentcondition: pH 8.1 -21°C; estimated conditions by 2100: pH 7.8 -24°C; estimated conditions by 2200: pH 7.5 -24°C).The pH was controlled in each aquarium to within ± 0.05 pH unit with a continuous pH-stat system (IKS, Aquastar©) that bubbled pure CO 2 into the tanksthat were continuously aerated with CO 2 -free air (see [START_REF] Lacoue-Labarthe | Temperature and pCO 2 effect on the bioaccumulation of radionuclides and trace elements in the eggs of the common cuttlefish Sepia officinalis[END_REF]. The pH values of the pH-stat system were calibratedevery week from measurements of pH on the total scale, using Tris/HCl buffer solution with a salinity of 38 p.s.u. and prepared according to [START_REF] Dickson | Determination of the pH of sea water using a glass / reference electrode cell[END_REF]. The pCO 2 was determined from pH and total alkalinity using the R package seacarb [START_REF] Gattuso | seacarb: Seawater Carbonate Chemistry[END_REF].

Radiotracer exposures.

Theoysters wereexposedfor 21 days to dissolved radiotracers: 110m Ag [as 110m AgNO 3 ; T ½ = 250 d], 241 Am [as 241 AmNO 3 ; T ½ = 432 y], 109 Cd [as 109 CdCl 2 ; T ½ = 464 d], 57 Co [as 57 CoCl 2 ; T ½ = 272 d], 54 Mn [as 54 MnCl 2 ; T ½ = 312 d] and 65 Zn [as 65 ZnCl 2 ; T ½ = 244 d].Uptake kinetics were followed during that period, after which seawater was no longer spikedin order to assess the metal retention capacities of the oysters.

During the exposure period, the seawater of each 70-L tankwas spiked with typically 5 μL ofradioactive stock solution ( 110m Ag, dissolved in 0.1M HCl, 241 Am in 1M HCl, 109 Cd in 0.5 M HCl, 57 Co 0.1 M in HCl, 54 Mn in 0.5 M HCl and 65 Zn in 0.5 M HCl). Seawater and spikes were renewed daily during the first week and then every second day to maintain good water quality and radiotracer concentrations as constant as possible [START_REF] Metian | Investigation of Ag in the king scallop Pectenmaximus using field and laboratory approaches[END_REF] as metal could be removed from seawater medium through absorption (in organisms) or adsorption on surface (e.g. aquarium glasses).For water renewal, each tank was emptied and then, immediately slowly refilled (30 min for ~ 60 L) with new seawater at the required temperature. The slow refill allowedfor immediate regulation of the targeted pCO 2 level by the IKS system. Before renewal of the seawater, the oysters were fed with the algal diet for 30 minutes, after which time the new seawater wasspiked with the required volumes of the radioactive stock solutions.

Activity of the radiotracers in seawater was checked before and after each spike renewal, yielding for the time-integrated activities in seawater for the 6 radiotracersshown in Table 1.

Five oysters from each aquarium were identified (tagged), weighted and whole-body radioanalyzed alive (same individuals each time)daily during the first week, and then every second day in order to follow the radiotracers uptake kinetics. During the counting sessions, oysters were held out of the seawater as briefly as possible(typically 10 to 20 minutes). At the end of the 21-d exposure period, 3 individuals (not belonging to the tag-identified batch) from each tank were collected and dissected.Shell andall soft tissues were separated and radioanalyzed in order to assess the distribution of radiotracers between these two compartments.

The remaining exposedoysters were placed in non-contaminated conditions (open-circuit 70-L tankwith same salinity, temperature, pH and light: dark conditions as previously indicated)for 38 days. Flowing seawater was adjusted in order to maintain pH values as constant as possible (seawater flux: 70 Lh -1 ). The five tag-identifiedoystersin each tank were radioanalyzed every day during the first week and then every second day in order to follow the depuration kinetics of the radiotracers.Three individuals from each tank were collected at the end of the depuration period and dissected for radio-analysis as described above.

In vitro digestion simulation.

At the end of the exposure and depuration periods, three oysters were collected in each tank in order to perform in vitro simulated digestion [START_REF] Versantvoort | Applicability of an in vitro digestion model in assessing the bioaccessibility of mycotoxins from food[END_REF]in orderto assess the bioaccessible fraction of elements for human consumers of oysters. The method consists of a three-step proceduresimulating quiet closely the human digestion processes. Homogenized oyster tissues were step by step exposed to artificial saliva, gastric juice and mixture of duodenal juice, bile and NaHCO 3 (chemicals and enzymes were purchased from Sigma®).

Following the in vitro digestion, the resulting chyme was centrifuged and the radiotracers activities in supernatant, considered as the bioaccessible element fraction, were counted. The detailed procedure has been previously described by Metian and colleague (Metian et al., 2009a).

Radioanalyses and data treatments

The radiotracers were counted using a high-resolution -spectrometry system consisting of five coaxial Germanium (N-or P-type) detectors (EGNC 33-195-R, Canberra ® and Eurysis ® ) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (Interwinner ® 6). The radioactivity of the samples (whole-body oysters, seawater, soft tissues, and supernatant and pellets) wasdetermined by comparison with standards of known activities and appropriate geometries and was corrected for background and physical decay of the radiotracers. The counting time was adjusted to obtain a propagated counting error of less than 5% (Rodriguez y [START_REF] Rodriguez Y Baena | Experimental evidence for 234 Th bioaccumulation in three Antarctic crustaceans: Potential implications for particle flux studies[END_REF].

The uptake of radiotracer was expressed as change in concentration factors (CF), which is the ratio between radiotracer activity in the whole-body oyster (Bq g -1 ) and the time-integrated radiotracer activity in seawater (Bq g -1 ) over time. Uptake kinetics were fitted using either a linear equation (Eq. 1) or a saturation exponential equation (Eq. 2):

𝐶𝐹 𝑡 = 𝑘 𝑢 𝑡 (Eq. 1) 𝐶𝐹 𝑡 = 𝐶𝐹 𝑠𝑠 (1 -𝑒 -𝑘 𝑒 𝑡 ) (Eq. 2)
where CF t and CF ss (CF ss = k u / k e ) are the concentration factors at time t (d) and at steadystate, respectively, and k e and k u are the biological depuration and uptake rate constants (d - 1 ),respectively [START_REF] Whicker | Radioecology : nuclear energy and the environment[END_REF].

Radiotracer depuration kinetics wereexpressed in terms of change in percentage of remaining activity (i.e., radioactivity at time t divided by the initial radioactivity measured in the individual at the beginning of the depuration period * 100) with time.The depuration kinetics werefitted bya simple exponentialmodel (Eq. 3): A t = A 0 e -ke t (Eq. 3) where A t and A 0 are the remaining activities at time t (d) and 0, respectively, k e is the biological depuration rate constant (d -1 ) [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotuslividus: sea water and food exposures[END_REF]. The biological half-life (d) ofthe radiotracer can then be calculated according to the relation (Eq. 4): T b1/2 = ln2 / k e (Eq. 4)

All statistics and graphics were performed using R freeware [START_REF] Core | R: A language and environment for statistical computing[END_REF]. Model constants and their statistics were estimated by iterative adjustment of the modelsusing linear and non-linear mixed effect models (lme and nlme functions from the package "nlme"; [START_REF] Pinheiro | {nlme}: Linear and Nonlinear Mixed Effects Models[END_REF], in whichindividual oyster identity hadbeen considered a random factor. Marginal R 2 representing the variance explained by the fixed factor (i.e. time) has been applied to linear models using the r.squaredGLMM function (package "MuMIn"; Bartoń, 2014).Comparison of uptake and eliminationconstant ratesamongthe different pH/temperature conditions was performed using two-way ANOVA on k u and k e calculated for each individual oyster (the best fitting model obtained for the entire set of oysters was applied to individuals).A Chi-square test was used to compare bioaccessible fractions of metals among the different oyster groups. The level of significance for statistical analyses was always set at α =0.05.

RESULTS

During the whole experimental period (i.e.three and five weeks of uptake and depuration phase, respectively) where the oysters were exposed to six different conditions (combinations of 2 temperatures and 3 pH; see Materials and Methods),limited growth of individual (< 1%) was measured and mortality was relatively high (18.5-29.6% according to tank) although no connexion between mortality and specific exposure conditioncould be detected. Specific mortality of oysters with condition of exposure werethe following: 18.5% for 7.5 (pH) -21°C (temperature), 26% for 7.8 -21°C, 26% for 8.1 -21°C, 29.6% for 7.5 -24°C, 18.5% for 7.8 -24°C,and 26% for 8.1 -24°C.

The whole-bodybioaccumulation kinetics of 110m Ag, 241 Am, 109 Cd, 57 Co, 54 Mn, and 65 Zn in oysters and their respective model parameters are shownin Figure 1 and Table 2. Uptake of all elements except 241 Am were best fitted by linear models.The actinide displayed an exponential uptake pattern, tending to reach a steady-state value rangingbetween 360 and470 according to pHtemperature conditions (Table 2) after 3 weeks of exposure. At the end of the exposure period and whatever the pH/temperature conditions,the estimated uptake rate constant (k u ) allowed ranking radiotracers taken up linearlyaccording to their bioaccumulation capacities:

110m Ag > 65 Zn > 54 Mn > 109 Cd ~57 Co.

At the end ofthe exposure period, oysters were placed in non-contaminating conditions and depuration of radiotracers wasfollowed for 37 days (Figure S1). The whole-body depuration kinetics of all elements were best fitted by a one-compartment exponential model (Table 3).

However it was not possible to determine accurately the depuration kinetic parameters for 109 Cd as the activity rapidly dropped below the detection limits of gamma spectrometers. All radiotracers were efficiently retained by Crassostrea gigas(T b1/2 between 50 and> 600 days).

Statistical analyses carried out on individual uptake rate constants (k u ; two-way ANOVA; Table 4) revealed that neither pH nor temperature significantly affected the bioaccumulation of metals in oysters(uptake and depuration biokinetics)except 109 Cd and 54 Mn. The highest temperature affectedCd accumulation(Figure 1,Table 4), which displayed an uptake rate constant at pH 7.5 two-fold higherat 24°C than at 20°C (23 ± 1 vs. 11 ± 1 d -1 , respectively; Table 4, interactive effectpHx temperature: p< 0.1).In the case of Mn, k u was significantly affected by an interactive effect between seawater pH and temperature, with a decreased uptake rate constant at pH 7.5 when temperature increased(27 ± 1 vs. 17 ± 1 d -1 at 21°C and 24°C, respectively; see Figure 1 andTable 4).Regarding the depuration rate constant of 241 Am, a significant interactive effectwas observed at pH 7.8 where T b1/2 was two-fold lower at 24°C than at 21°C (Tukey test; p = 0.049;Table 4). Retention of Co and Mntendedalso to decrease at the same pH when comparing warmerwith colder temperatures (Table 4; pHx temperature; p < 0.1).

When looking atthe distribution of the radiotracers betweenshells and soft tissues (Table 5), 241 Am, 57 Co and 54 Mn were mainlyassociatedwiththe shells (> 94%), whereas 109 Cd wasequally distributed between the two compartments and 110m Ag and 65 Zn were mainly found in soft tissues (76-98% and 67-92%, respectively). It is also noteworthy that 65 Zn fraction in soft tissues increased between the end of uptake period and the end of depurationperiod, at all pH and temperature conditions, suggesting a fasterdepuration of Zn associated with shellsthanthat withsoft tissues when non-contaminatingconditions were restored.Neither pH nor temperature affectedstrongly distribution ofmetals and radionuclide betweenshells and soft tissues, except that the fraction of 110m Ag associated with soft tissues at the end of the uptake phasewere lower in oysters maintained at 24°C when compared to those keptat 21°C (p < 0.05, Mann Whitney U test; Table 5).Finally, the radiotracer CFs reached in the oyster shells at the end of the uptake phase were not affected significantly by pCO 2 conditions (Table S1).

The in vitro digestion simulations revealed that the bioaccessible fraction of metals and radionuclide varied from 30% ( 241 Am in oyster maintained at 24°C and pH 7.8) to more than 90% ( 57 Co in all oysters; Figure 2). Significant effects of pH were highlighted for 241 Am, 109 Cd, 54 Mn and 65 Zn (Table 6). For these elements, the bioaccessible fraction at the end of the uptake phase tended to increase with decreasing pH except at pH 8.1 -24°C. At the end of the depuration phase, a similar trend was observed in oysters maintained at 24°C whereas a decrease in bioacessibilitywith decreasing pH was noted for the21°C-maintained oysters.

Nevertheless, these results must be considered with caution due to the low number of oysters analysed per condition (n = 3).

DISCUSSION

Contamination of coastal waters is a worldwide concern. Ocean acidification and thus modified chemistry of seawater maychangethe way contaminants will interact with marine biota.For example, it affects the chemical speciation of cations, especially those that form complexes with carbonates and potentially increases their bioavailability for biota (e.g. [START_REF] Byrne | The influence of temperature and pH on trace metal speciation in seawater[END_REF]) by increasing the free-ion form in acidified conditions. Among theelements considered in the present study, only 241 Am is known as being able to bind to carbonates but with limited change inspeciation in the tested pH range, largely dominated by hydroxyl-Am form [START_REF] Choppin | Actinide speciation in aquatic systems[END_REF].According to previous work and metal speciation model as a function of seawater pH, metals that are mainly bound to chloride (such as Ag andCd)or those that are predominantly found in the free ion form (e.g. Co, Mn, Zn) arenot strongly affected by decreasing pH. Indeed, the free metal ion concentrations of these elements increasedfrom only few percent when pH decreased from 8.1 to 7.5 [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF].We thus assume that, forthe seawater pH range tested in the context of ocean acidification, bioaccumulation wouldbe less affected by change in metal speciationitself than byresulting change in the biological mechanisms of metal uptake and/or elimination.

In the current study, we examined the bioaccumulation patterns of sixmetals and oneactinidein the Pacific cupped oyster Crassostrea gigasunder elevated conditions of temperature and pCO 2 , using the levels projected for the end of the current century. Like other sessile invertebrates living in estuarine, intertidal and subtidal zones, C. gigasusesto be exposed to broad ranges of pH and temperatures in their habitats, potentially leading to an adaptation to a wide range of abiotic conditions [START_REF] Lannig | Impact of ocean acidification on energy metabolism of oyster, Crassostreagigas -changes in metabolic pathways and thermal response[END_REF]. For example, metabolic depression is an adaptive mechanism used by shelled intertidal molluscs to preserve energy during low tides. This physiological plasticity is time-limited and might not be adapted to longterm,continuous exposure to hypercapnic conditions. In the present study, the higher mortality observed at the lowest pH and highest temperature may suggest that energetic balance were pushed to the edge when both stressorswere combined [START_REF] Lannig | Impact of ocean acidification on energy metabolism of oyster, Crassostreagigas -changes in metabolic pathways and thermal response[END_REF].

The distribution of radiotracers between soft tissues andshells was in accordance with previous studies: 241 Am, Co, Mn were mainly adsorbed on the calcareous structures whereas Cd, Zn and Ag fraction associated with soft tissueswas gradually increasing during the depuration period (Metian et al., 2009b;[START_REF] Hédouin | Delineation of heavy metal contamination pathways (seawater, food and sediment) in tropical oysters from New Caledonia using radiotracer techniques[END_REF][START_REF] Metian | Characterization of 241 Am and 134 Cs bioaccumulation in the king scallop Pectenmaximus: investigation via three exposure pathways[END_REF]. CFs of 241 Am, Co and Mnin shellsat the end of the uptake phase were not significantly affected by seawater pCO 2 conditions, implying that the adsorptioncapacity of shellsis not strongly affected by decreasing pH for these metals. This might be explained by the fact that 1) increasing protons concentration resulting from decreasing pHdoes not significantly compete with metallic cations for shell bindingsites and/or 2) the binding sites are not affected by hypercapnia in the conditions tested (shellsof C. gigas -mainly made of calcite-are considered less vulnerable tocalcareous dissolution than aragonitic shells; [START_REF] Gazeau | Impact of elevated CO 2 on shellfish calcification[END_REF].

The main result of our study is that no effect of increasing pCO 2 andtemperature has been detected on Ag,Cd and Znbioaccumulation capacities in C.gigas soft tissues. Only few studies have investigated so far the impact of warming and hypercapnia on metal accumulation and/ortheir effects on marine organisms [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Pascal | The toxicological interaction between ocean acidity and metals in coastal meiobenthic copepods[END_REF][START_REF] Lacoue-Labarthe | Effects of ocean acidification on trace element accumulation in the early-life stages of squid Loligo vulgaris[END_REF][START_REF] Lacoue-Labarthe | Temperature and pCO 2 effect on the bioaccumulation of radionuclides and trace elements in the eggs of the common cuttlefish Sepia officinalis[END_REF][START_REF] Ivanina | Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenariamercenaria[END_REF][START_REF] Roberts | Ocean acidification increases the toxicity of contaminated sediments[END_REF][START_REF] Ivanina | Immunomodulation by the interactive effects of cadmium and hypercapnia in marine bivalves Crassostreavirginica and Mercenariamercenaria[END_REF] and contrasting responses (metal-or species-dependent)were observed. For instance, Cd accumulationwas lowered in cuttlefish embryosand Cd toxicity decreased in benthic copepod with decreasing pCO 2 [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Pascal | The toxicological interaction between ocean acidity and metals in coastal meiobenthic copepods[END_REF]presumably due to increasing competition between metallic cations and protons for biological binding sites.Similarly, a lower Cd uptake was observed in mantle cells of clams Mercenaria mercenaria, supposedly due to a decrease intransmembranechannel activities with metabolic depression of clams in short-term hypercapnic conditions [START_REF] Ivanina | Short-term acute hypercapnia affects cellular responses to trace metals in the hard clams Mercenariamercenaria[END_REF].In contrast, we noted in this study a higher Cd uptake inC. gigas when oysters were exposed to pH 7.5 (~1700 µatm) and 24°C for three weeks. Similar observation of enhanced Cd bioaccumulation in C. virginica maintained at pH 7.8 (~ 800 µatm) during 4-5 weeks were reported by Götze and co-authors [START_REF] Götze | Interactive effects of CO 2 and trace metals on the proteasome activity and cellular stress response of marine bivalves Crassostrea virginica and Mercenaria mercenaria[END_REF]. The mechanistic explanation of this Cd bioaccumulation modulation remains unclear. However it is noteworthy that, in contrast to others bivalves that maintain a comparatively constant internal pH by decreasing their metabolic rates and/or dissolving their shell [START_REF] Berge | Effects of increased sea water concentrations of CO 2 on growth of the bivalve Mytilusedulis L[END_REF], C. gigaswas shown to have elevated standard metabolic rate under hypercapnia when exposed to temperature stress [START_REF] Lannig | Impact of ocean acidification on energy metabolism of oyster, Crassostreagigas -changes in metabolic pathways and thermal response[END_REF]. This is turn couldincrease Cd ion uptake in oyster tissues.

Interestingly, it has been previously shownthat the non-essential element Ag is more efficiently accumulated, up to 2.5 fold, in embryos and larvae of cuttlefish and squidwhen exposed to elevated pCO 2 ranged from 600 to 1500 µatm [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Lacoue-Labarthe | Effects of ocean acidification on trace element accumulation in the early-life stages of squid Loligo vulgaris[END_REF]. This higher Ag uptake might be due to the Na mimetic properties of this element and caused by the enhanced acid-base regulation combined to the ionic regulation machinery. Indeedthe Na + /H + -exchanger,an ionic channel, isexpected involved in Ag uptake [START_REF] Webb | Bioaccumulation and distribution of silver in four marine teleosts and two marine elasmobranchs: influence of exposure duration, concentration, and salinity[END_REF][START_REF] Grosell | Branchial versus intestinal silver toxicity and uptake in the marine teleost Parophrys vetulus[END_REF] in mobileorganisms when they are exposed to environmental hypercapnia [START_REF] Melzner | Physiological basis for high CO 2 tolerance in marine ectothermic animals: preadaptation through lifestyle and ontogeny?[END_REF][START_REF] Hu | Elevated seawater PCO 2 differentially affects branchial acid-base transporters over the course of development in the cephalopod Sepia officinalis[END_REF][START_REF] Hu | Branchial NH 4 + -dependent acid-base transport mechanisms and energy metabolism of squid (Sepioteuthis lessoniana) affected by seawater acidification[END_REF]. In oysters, no significant change of Ag uptake was observed in soft tissuesas a consequence of change in seawater pCO 2 . This is congruent with the less-developed ionexchange machinery in sessile invertebrates, and their subsequentlimited ion regulation capacities [START_REF] Lannig | Impact of ocean acidification on energy metabolism of oyster, Crassostreagigas -changes in metabolic pathways and thermal response[END_REF][START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF]. This limited physiological capacity to compensate environmental acidosis might also explain the absence of modulation of the Zn bioaccumulation in soft tissues according topCO 2 conditions. Indeed, Zn is a co-factor of the carbonic anhydrase (CA) that catalyses the conversion of CO 2 to bicarbonate and vice versa, and that is involved in the acid-base regulation and calcification in shelled mollusc [START_REF] Gazeau | Impacts of ocean acidification on marine shelled molluscs[END_REF]. Beniash and co-authors (2010) have reported an increase inCA expression levels in the mantle of C. virginica exposed to 3500 µatm, but the latter partial pressure was much higher than those tested in the present study.

Oysters area very important marine resource and constitute a major source of animal protein for humans. They are also rich in essential elements such as Fe and Zn [START_REF] Tacon | Fish Matters: Importance of Aquatic Foods in Human Nutrition and Global Food Supply[END_REF]. Preliminary experiments werecarried outto determine whetherlower pH and higher temperatures may affect metal and radionuclide bioaccessibilty to human consumers. Results indicated that 241 Am, Mn, and Zn bioaccessibility tended to be enhanced when oysters were exposed to high temperature and lower pH. Although these results have to be considered with caution, they raise the question ofthe possible impact of environmental hypercapnia on metal detoxification mechanisms in oysters and subsequent subcellular storage of elements. Oysters are well known to accumulate efficiently trace elements such as Zn in soft tissues in the form of metabolically inert, metal-rich granules (e.g. [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM)[END_REF].These Mg/Ca carbonate granules are known to have the physiological role of buffering the extracellular pH [START_REF] Viarengo | Mechanisms of heavy metal cation homeostasis in marine invertebrates[END_REF] and might thus be easily dissolved when oysters are experiencing hypercapnia. In this context, the increased seawater pCO 2 is susceptible to affect the metal detoxificationstrategiesand thus the Trophically Available Fraction (TAM; [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM)[END_REF]that drives the bioaccessibility and transfer of metals to higher trophic levels.

Finally, the present study brought new data on the potential impact of ocean acidification on metal accumulation in a marine resource of high economic importance and raised the question of the seafood safety that could be worsen by future ocean conditions. Despite the limited number of oysters, the experimental design offered enough statistical power (with power = 90%, n=5, standard deviation = sd max in Table 2) to detect a putative 2-fold Ag uptake increase and a 3-fold Cd uptake decrease and corroborate previous observations made in active (mobile) organisms [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF][START_REF] Lacoue-Labarthe | Effects of ocean acidification on trace element accumulation in the early-life stages of squid Loligo vulgaris[END_REF]. We therefore assume that our results bring a new insight on the potential effect of environmental factors such as combined pCO 2 and temperature on the transfer and integration of metals in coastal organisms. However, our experimental approach(oysters were acclimated totemperature and pCO 2 conditions for two weeks before the experiments) implies that the responses observed with respect to metal accumulation efficiency reflected oyster physiological acclimation performances. They are therefore relevant to a coastal or estuarine context where high and short-term variations of these factors do occur [START_REF] Melzner | Future ocean acidification will be amplified by hypoxia in coastal habitats[END_REF]. Hence our results do not allow figuring out what could be the impact of ocean acidification and warming over longer timescale, during which adaptation processes will set up. Further studies should be carried out to highlight finer modulations of metal uptake at tissue and subcellular levels, taking into account the high variability of responses between oyster populations [START_REF] Parker | Populations of the Sydney rock oyster, Saccostreaglomerata, vary in response to ocean acidification[END_REF], and the adaptive capacities of organisms facing long-term major changes.

Table 1. Carbonate system parameters during the uptake and depuration phase of the experiment on the bioaccumulation of metal and radionuclide in oyster exposed The indicative average partial pressure of CO 2 (pCO 2 ) were calculated for a seawater salinity of 38 and a total alkalinity of 2540 µmol.kg -1 average.

Experiment phase Temperature

(°C) pH T pCO 2 (µatm)

110m Ag (Bq.ml -1 ) 241 Am (Bq.ml -1 ) 109 Cd (Bq.ml -1 ) 57 Co (Bq.ml -1 ) 54 Mn (Bq.ml -1 ) 65 Zn (Bq.ml -1 ) Uptake 20.9 ± 0.9 7.43 ± 0.03 2161 ± 177 0.06 ± 0.05 0.15 ± 0.08 0.28 ± 0.05 0.44 ± 0.09 0.25 ± 0.08 0.38 ± 0.12 20.8 ± 0.9 7.79 ± 0.03 854 ± 54 0.06 ± 0.05 0.14 ± 0.07 0.25 ± 0.02 0.43 ± 0.09 0.21 ± 0.10 0.37 ± 0.13 20.8 ± 0.8 8.03 ± 0.02 451 ± 27 0.06 ± 0.04 0.13 ± 0.07 0.26 ± 0.03 0.44 ± 0.09 0.19 ± 0.11 0.36 ± 0.12 23.7 ± 0.2 7.52 ± 0.03 1732 ± 125 0.07 ± 0.03 0.14 ± 0.07 0.27 ± 0.03 0.44 ± 0.08 0.23 ± 0.09 0.39 ± 0.11 24.0 ± 0. along the uptake phase. Mean ± SD of CF are plotted for the day 24 at the end of the uptake. Parameters of models and statistics are reported in Table 2 and4

  g wet wt) were purchased at the "Satmar" farm in Oleron Island, Charentes-Maritimes, France, in January 2013. At the IAEA-EL premises, the oysters were randomlydistributed among six 70-L glass aquaria and acclimated for 2 weeks in0.45-µm filtered,UV-sterilized natural seawater (aerated open circuit 70-L aquarium; seawater flux: 70 L h -1 ; salinity: 38 p.s.u.; light/dark cycle:12

  53 error; T b½ : biological half-life (d), k e : depuration rate constant; R 2 : regression coefficient.Significant differences 54 of k e are indicated by *(p<0.05), **(p<0.01), or ***(p<0.005)

Figure 2 :Figure S1 .

 2S1 Figure 2: Radiotracer bioaccessibility (%, mean ± SD, n=3) from oyster soft tissues to human consumers

Table 2 .

 2 Parameters of the uptake kinetics of 110m Ag, 241 Am, 109 Cd, 57 Co,54 Mn, 65 Zn in oysters exposed for 21 days to dissolved radiotracers under three pHs and two

		1	7.88 ± 0.08	699 ± 157	0.07 ± 0.04	0.14 ± 0.08	0.25 ± 0.03 0.42 ± 0.11 0.18 ± 0.12 0.38 ± 0.11
		24.0 ± 0.1	8.08 ± 0.05	396 ± 63	0.07 ± 0.04	0.15 ± 0.08	0.28 ± 0.05 0.45 ± 0.09 0.19 ± 0.12 0.38 ± 0.12
		21.6 ± 0.1	7.45 ± 0.04	2023 ± 180	-	-	-	-	-	-
		21.6 ± 0.1	7.85 ± 0.03	742 ± 61	-	-	-	-	-	-
	Loss	21.2 ± 0.2	8.03 ± 0.01	444 ± 16	-	-	-	-	-	-
		24.0 ± 0.2	7.54 ± 0.11	1689 ± 355	-	-	-	-	-	-
		23.8 ± 0.1	7.79 ± 0.07	867 ± 149	-	-	-	-	-	-
		24.0 ± 0.1	8.06 ± 0.04	413 ± 50	-	-	-	-	-	-

Table 3 .

 3 Parameters of the depuration kinetics of 110m Ag, 241 Am, 57 Co, 54 Mn, and 65 Zn in oysters previously 50 exposed for 21 days to dissolved radiotracers under 3 pHs and 2 temperatures conditions and maintained for 37

	51
	52	days in non-contaminated conditions.
		Element	Temp (°C)	pH	A 0l  SE (%)	k el	T b½  SE(d)
		54 Mn	21	7.5	98.2 ± 1.4	0.007 ± 0.001***	97 ± 13
				7.8	101.0 ± 1.4	0.006 ± 0.001***	119 ± 19
				8.1	99.3 ± 1.2	0.006 ± 0.001***	118 ± 16
			24	7.5	96.3 ± 2.5	0.003 ± 0.002	217 ± 121
				7.8	99.9 ± 0.9	0.013 ± 0.001***	56 ± 3
				8.1	97.8 ± 1.3	0.005 ± 0.001***	110 ± 18
		57 Co	21	7.5	99.7 ± 1.7	0.006 ± 0.001***	120 ± 24
				7.8	99.0 ± 1.6	0.005 ± 0.001***	132 ± 26
				8.1	99.0 ± 1.7	0.007 ± 0.001***	95 ± 15
			24	7.5	98.9 ± 2.6	0.003 ± 0.002	236 ± 143
				7.8	100.4 ± 1.3	0.013 ± 0.001***	55 ± 4
				8.1	97.3 ± 1.6	0.006 ± 0.001***	115 ± 21
		65 Zn	21	7.5	97.2 ± 1.5	0.007 ± 0.001***	100 ± 16
				7.8	99.6 ± 2.5	0.008 ± 0.002***	92 ± 21
				8.1	99.6 ± 1.2	0.005 ± 0.001***	153 ± 26
			24	7.5	97.4 ± 3.7	0.006 ± 0.003*	125 ± 59
				7.8	95.8 ± 1.5	0.005 ± 0.001***	149 ± 34
				8.1	98.8 ± 1.3	0.005 ± 0.001***	134 ± 22
		110m Ag	21	7.5	99.9 ± 1.7	0.005 ± 0.001***	137 ± 31
				7.8	102.1 ± 2.0	0.006 ± 0.001***	110 ± 22
				8.1	101.8 ± 1.2	0.003 ± 0.001***	260 ± 72
			24	7.5	92.3 ± 4.6	0.001 ± 0.003	613 ± 1173
				7.8	98.9 ± 1.5	0.003 ± 0.001	273 ± 102
				8.1	100.9 ± 1.1	0.004 ± 0.001***	185 ± 33
		241 Am	21	7.5	95.1 ± 1.5	0.012 ± 0.001***	56 ± 5
				7.8	97.8 ± 1.9	0.009 ± 0.001***	77 ± 11
				8.1	96.7 ± 1.6	0.011 ± 0.001***	61 ± 6
			24	7.5	98.0 ± 2.5	0.006 ± 0.001 ***	107 ± 30
				7.8	97.1 ± 2.0	0.016 ± 0.002***	42 ± 4
				8.1	92.8 ± 3.3	0.007 ± 0.002***	88 ± 27
		All kinetics were best fitted by a one-compartment exponential model: A 0 : assimilation efficiency; SE: standard

Table 4 .

 4 Two-way ANOVA parameters testing the effects of three pH(7.5, 7.8 and 8.1) and two temperatures (21 and 24°C) on the uptake kinetic parameters, 56 i.e. k u (see Figures 1 and Table 1), and on the loss kinetics parameters, i.e. k e (see Figures 2 and Table 2) for all elements 57

		Parameter		pH			Temperature			pH X Temperature
			df	MS	F	df	MS	F	df	MS	F
		110m Ag -k u	2	3042	0.816	1	2018	0.541	2	8735	1.172
		241 Am -k u	2	235	2.144	1	303	2.772	2	199	1.817
		109 Cd -k u	2	64	2.713 	1	143	6.061*	2	65	2.735 
		57 Co -k u	2	22	1.682	1	0.05	0.004	2	38	2.860 
		54 Mn -k u	2	32	1.192	1	26	0.969	2	132	4.835*
		65 Zn -k u	2	380	1.847	1	522	2.537	2	207	1.006
		110m Ag -k el	2	1.6 10 -3	1.787	1	1.1 10 -3	1.282	2	1.4 10 -3	1.551
		241 Am -k el	2	2.8 10 -5	2.070	1	1.1e-6	0.081	2	9.9 10 -5	7.367**
		57 Co -k el	2	1.4 10 -5	0.785	1	8.3 10 -5	4.620*	2	4.8 10 -5	2.714 
		54 Mn -k el	2	4.2 10 -5	2.215	1	6.7 10 -5	3.529	2	3.3 10 -5	1.762 
		65 Zn -k el	2	1.1 10 -4	1.848	1	2.0 10 -5	0.344	2	5.7 10 -5	0.972
	58	df = degree of freedom; MS = mean squares. Probability levels for significant effects: p < 0.01 (**), p < 0.05 (*), p < 0.1 (  ).
	59
	60
	61

Table 5 .

 5 54 Mn,57 Co,65 Zn, 110m Ag, 109 Cd,and 241 Am distribution (%; mean ± SD) among soft tissue and shell of oysters exposed for 21 days of the uptake (n=3) and 37 days of 62 depuration phase (n=5) to dissolved radiotracers at three different pH and two temperatures

	63

Table 6 .

 6 Chi-square test on the effects of three pH levels on bioaccessible fraction of metals in oyster tissue 65 following in vitro digestion. Crassostrea gigas. Uptake kinetics of 110m Ag, 241 Am, 109 Cd, 57 Co, 54 Mn, 65 Zn (CF) in the oysters exposed to dissolved radiotracers at three different 81 pHs -pH 8.1, pH 7.8, and pH 7.5, and two temperatures (21°C in blue and 24°C in red). Points are omitted for clarity and only kinetics models are delineated 82

	66
	Digestion time	Temperature	54 Mn	57 Co	65 Zn	110m Ag	109 Cd	67 68 Am 241
	End of the uptake	21 °C		8.71*	2.35	6.55*	0.78	3.96	69 70 1.69
			24 °C		19.44***	0.10	17.13***	0.30	12.13**	22.77***
									71
	End of the loss	21 °C		19.15***	1.87	14.57***	1.78	-	72 3.12
			24 °C		7.72*	0.74	16.21***	0.28	-	27.51***
									73
	74	df (degree of freedom): 2
	75	* p <0.05 ** p <0.01	*** p <0.001
	76
	77

Acknowledgements

The IAEA is grateful to the Government of the Principality of Monaco for the support provided to its Environment Laboratories.Murat Belivermiş was supported by a post-doctoral scholarshipprovided byCouncil of Higher Education of Turkish Republic. Michel Warnau is an Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium).Authors are grateful to Florian Rivello, Kevin Calabro, andMarie-Yasmine Dechraouifor theirtechnical assistance in the laboratory.