Marc Metian
email: m.metian@iaea.org

Simon Pouil

Christine Dupuy

Jean-Louis Teyssié

Michel Warnau

Paco Bustamante

Influence of food (ciliate and phytoplankton) on the trophic transfer of inorganic and methyl-mercury in the Pacific cupped oyster Crassostrea gigas

Keywords: Bivalve, Bioaccumulation, Food pathway, Mercury, Radiotracers

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Introduction

Mercury (Hg) is a persistent global pollutant released in the coastal environment, mainly released by anthropogenic activities, including combustion of fossil fuels and gold mining (UNEP, 2013;[START_REF] Sundseth | Global sources and pathways of mercury in the context of human health[END_REF]. Estimated annual anthropogenic Hg emissions were about 2390 tonnes in 2015 [START_REF] Streets | Global and regional trends in mercury emissions and concentrations, 2010-2015[END_REF]). Mercury is a toxic, non-essential element that can be highly bioaccumulated by marine biota and biomagnified along the aquatic food webs, especially in its methylmercury form (MeHg; see e.g., [START_REF] Riisgård | Biomagnification of mercury in a marine grazing foodchain: algal cells Phaeodactylum tricornutum, mussels Mytilus edulis and flounders Platichthys flesus studied by means of a stepwise-reduction-CVAA method[END_REF][START_REF] Dijkstra | Experimental and natural warming elevates mercury concentrations in estuarine fish[END_REF][START_REF] Eagles-Smith | Modulators to mercury risk to wildlife and humans in the context of rapid global change[END_REF][START_REF] Harding | Bioaccumulation of methylmercury within the marine food web of the outer Bay of Fundy, Gulf of Maine[END_REF].

Mercury bioaccumulation processes have been well studied in laboratory conditions in low trophic level aquatic organisms, especially in bivalves, such as Mytilidae [START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF][START_REF] Amachree | Comparison of intermittent and continuous exposures to inorganic mercury in the mussel, Mytilus edulis: Accumulation and sublethal physiological effects[END_REF][START_REF] Raftopoulou | Comparative study of the accumulation and detoxification of Cu (essential metal) and Hg (nonessential metal) in the digestive gland and gills of mussels Mytilus galloprovincialis, using analytical and histochemical techniques[END_REF], Ostreidae [START_REF] Cunningham | Accumulation and depuration of mercury in the American oyster Crassostrea virginica[END_REF][START_REF] Denton | Influence of temperature and salinity on the uptake, distribution and depuration of mercury, cadmium and lead by the black-lip oyster Saccostrea echinata[END_REF][START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF], and Pectinidae (Metian et al., 2008;[START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF], acting as important vectors for "pumping" Hg to higher trophic levels [START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF].

Although bivalves are also exposed to Hg from the water and the sediment [START_REF] Gagnon | Bioavailability of sediment-bound methyl and inorganic mercury to a marine bivalve[END_REF][START_REF] Kidd | Bioaccumulation and Biomagnification of Mercury through Food Webs[END_REF], previous laboratory studies carried out in the 1970s highlighted that bivalves have substantial capacity to accumulate Hg from their diet especially in MeHg form (e.g., [START_REF] Cunningham | Accumulation, tissue distribution and elimination of 203 HgCl 2 and CH 3 203 HgCl in the tissues of the American oyster Crassostrea virginica[END_REF][START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF][START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF]. Assimilation Efficiency (AE) of inorganic Hg (iHg) in bivalves is variable and ranges from 5 to 60% [START_REF] Fowler | Factors affecting methyl and inorganic mercury dynamics in mussels and shrimp[END_REF][START_REF] Riisgård | Biomagnification of mercury in a marine grazing foodchain: algal cells Phaeodactylum tricornutum, mussels Mytilus edulis and flounders Platichthys flesus studied by means of a stepwise-reduction-CVAA method[END_REF][START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF]Metian et al., 2008b;[START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF]. Some studies have shown in different bivalves that MeHg is more readily bioavailable than iHg: AE of MeHg is higher than that of iHg, by a factor 1.5 to 4.0 (MeHg AE = 50-95%; e.g., [START_REF] Fowler | Factors affecting methyl and inorganic mercury dynamics in mussels and shrimp[END_REF][START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF][START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF]. These observations are valuable for interpreting the trophic transfer of iHg and MeHg from the base of food webs. Nevertheless, interpreting field data based on laboratory findings has some limitations. Indeed, the experimental characterization of the trophic transfer of Hg in bivalves has mainly been studied using phytoplankton species as diet, which does not fully reflect the diversity of the natural diet of bivalves in the wild.

In their natural environment, bivalves are generally opportunistic, omnivore, filter-feeders feeding on different prey: phytoplankton, resuspended benthic microalgae, detritus or other feed items available in their habitats. Thus, experimental studies highlighted the ability of mussels (Mytilus edulis and Perna viridis) to assimilate zooplanktonic rotifers in addition to phytoplankton and found that this food source can be an important part of the mussel's energy budget (Wong et al., 2003a(Wong et al., , 2003b)). Diet of oysters (Ostrea edulis and Crassostrea gigas) includes phytoplankton, heterotrophic protists, large bacteria, fungi and detritus [START_REF] Heral | Traditional oyster culture in France[END_REF][START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. Oysters retained efficiently particles > 4 µm (Barillé et al., 1993). Thereby, > 4 µm autotrophic protists (primary producers) are directly retained by benthic bivalves. When primary producers are lower than < 4 µm of size, heterotrophic protists, like ciliates, are a trophic link between these small primary producers (< 4 µm) and benthic bivalves [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. Thus, these authors experimentally demonstrated that 94% of ciliates and 86% of flagellates (size between 4 and 72 µm) were retained by the Pacific cupped oyster Crassostrea gigas.

Previous experimental works have shown the importance of diet in the trophic transfer of trace elements in bivalves (e.g., Wang and Fisher, 1996;[START_REF] Lee | Influence of microalgal biomass on absorption efficiency of Cd, Cr, and Zn by two bivalves from San Francisco Bay[END_REF]Metian et al., 2008a). Food quality was shown to have a major influence on trace element assimilation. For example, Wang and Fisher (1996) report that in mussels fed with seven different algal diets, the AEs of Ag varied by a factor 9 (3.8-33.8%) according to the diet. Similar findings were shown for Ag, 241 Am, Co, Cd, Cr, Mn, and Zn in several bivalve species including clams, mussels, oysters and scallops (e.g., [START_REF] Lee | Influence of microalgal biomass on absorption efficiency of Cd, Cr, and Zn by two bivalves from San Francisco Bay[END_REF][START_REF] Chong | Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum[END_REF]Metian et al., 2008a;[START_REF] Hédouin | Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: qualitative and quantitative aspects[END_REF]. Although such effects have not yet been demonstrated for assimilation of Hg, [START_REF] Pan | Uptake of Hg(II) and methylmercury by the green mussel Perna viridis under different organic carbon conditions[END_REF] suggested that different feed sources might lead to different bioavailability of Hg for bivalves.

The main objective of the present study was to investigate the influence of the diet composition on the trophic transfer of inorganic Hg (iHg) and methyl Hg (MeHg) in the Pacific cupped oyster, C. gigas. The depuration kinetics of dietary Hg were determined experimentally in oysters using radiotracer techniques. Thus, AE and efflux rate constant (k e ) of iHg and MeHg were compared in oysters fed on an autotrophic protist model (diatom Thalassiosira pseudonana, diameter: 2.5-15 µm) or on a heterotrophic protist model (ciliate protozoan, Uronema marina, 20 µm of length). Both protists are components of the oyster natural diet (Le [START_REF] Gall | Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: protozoa as a trophic link between picoplankton and benthic suspension-feeders[END_REF][START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF].

Materials and Methods

Acclimation of oysters

Pacific cupped oysters C. gigas a cosmopolitan bioindicator of trace element contamination (Rainbow and Philips, 1993;[START_REF] Rainbow | Biomonitoring of heavy metal availability in the marine environment[END_REF], were purchased from a shellfish farm in La Rochelle, France. Oysters were then transferred to the IAEA Environment Laboratories in the Principality of Monaco. Prior to the experimentation, specimens were acclimated to laboratory conditions (constantly aerated open-circuit aquarium; salinity: 36 ± 1; temperature: 19 ± 1°C; pH: 8; light/dark cycle: 12h/12h). During acclimation, bivalves were fed the phytoplanktonic Prymnesiophyceae Isochrysis galbana (10 5 cells mL -1 ). Recorded mortality was lower than 5% over the acclimation period (4 weeks).

Radiotracer and counting

Depuration kinetics of Hg in oysters were determined using high-specific activity inorganic 203 Hg purchased from Isotope Products Laboratories ( 203 Hg as HgCl 2 in 1M HCL, T 1/2 = 46.6 days, specific activity: 4 10 9 Bq g -1 at the beginning of the experiment). In order to assess the trophic transfer of methylmercury, MeHg was synthesized from 203 HgCl 2 according to the method described in [START_REF] Rouleau | Fast and high-yield synthesis of radioactive CH 3 203 Hg(II)[END_REF]. Briefly, this method is based on the methylation of inorganic 203 Hg by methylcobalamin and isolation of resulting CH 3 203 Hg using a hexane/benzene extraction procedure.

Exposed oysters were counted using a high-resolution γ-spectrometer system composed of three Germanium (N-or P-type) detectors Canberra® and Eurysis®) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (Interwinner® 4). The radioactivity was determined by comparison with standards of known activity and of appropriate geometry [START_REF] Cresswell | Aquatic live animal radiotracing studies for ecotoxicological applications: Addressing fundamental methodological deficiencies[END_REF], and corrected for counting efficiency and physical radioactive decay. The counting time was adjusted to obtain a propagated counting error less than 5% [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: Sea water and food exposures[END_REF][START_REF] Warnau | Cadmium bioconcentration in the echinoid Paracentrotus lividus: influence of the cadmium concentration in sea water[END_REF].

Trophic transfer experiment

Trophic transfer of iHg and MeHg in oysters was studied using protozoan and phytoplankton as diets. Ciliate (Uronema marina) and diatom (Thalassiosira pseudonana, clone 3H) cells were maintained in FAG and F/2 (without EDTA) medium respectively. Cultures were handled aseptically throughout the experimentation and exposed to 1 kBq 203 HgCl 2 L -1 or CH 3 203 Hg L -1 during their exponential growing phase (4-7 d). In terms of stable Hg concentration, this addition corresponded to 12.3 ng L -1 . After the radiolabeling period, the diatom culture was centrifuged (2500 g for 25 min). The protozoan culture was filtered (25µm mesh size; Osmonic® filters) and the filtrate centrifuged (1000 g for 15 min).

Forty oysters (weight: 68.6 ± 3.4 g wet wt, shell length: 89 ± 9 mm) had been randomly placed into four 13-L aquaria 14 days before the start of feeding experiment. Oysters were fed with radiolabeled feeds by resuspension of the centrifuged pellets (same concentration of 10 5 cells mL -1 for the two feeds) for 2 h in closed circuit. After the feeding period, all oysters were γ-counted and flow restored in the aquarium. Depuration was followed during 50 d in the same condition as for acclimation (see section 2.1). During the 50-d depuration period, oysters were fed daily with I. galbana (10 4 cells mL -1 ).

Ingestion of food was verified by the γ-counting of the organisms at the end of the 2-h period of feeding. Activity per cell was notably higher in protozoans than diatoms (mean activity of 1150 and 1200 µBq 203 Hg cell -1 for iHg and MeHg in ciliates, and 0.27 and 0.18 µBq 203 Hg cell -1 for iHg and MeHg in diatoms) whereas the quantity ingested was lower (mean quantity of 3.5 10 5 cells for the ciliate and 3.1 10 8 cells for the diatom, for both iHg and MeHg). Bioconcentration capacities of the prey are nevertheless not directly comparable.

Data analysis

Depuration of radiotracers was expressed as the percentage of remaining radioactivity (radioactivity at time t divided by the initial radioactivity measured in the organism at the beginning of the depuration period). The depuration kinetics of the radiotracers were best fitted using either a single-component (Eq. ( 1)) or a double-component (Eq. ( 2)) exponential model (decision based on F test and ANOVA tables for two fitted model objects):

(1)

(2)

where A t and A 0 are the remaining activities (%) at time t (d) and 0, respectively; k e is the depuration rate constant (d -1 ); 's' and 'l' are the subscripts for the 'short-lived' and 'longlived' components, respectively. The short-lived component represents the loss kinetics of the radiotracer fraction that is weakly associated to the organisms and rapidly eliminated, whereas the long-lived component describes the loss kinetics of the radiotracer fraction that is tightly bound to the organism [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: Sea water and food exposures[END_REF]. The long-lived component allows estimating the assimilation efficiency (AE) of the radiotracer ingested with food (AE = A 0l ).

For each exponential component (s and l), a biological half-life can be calculated (T b1/2s and T b1/2l ) from the corresponding depuration rate constant (k es and k el , respectively) according to the relation T b1/2 = ln2/k e . Kinetic parameters were determined using the R freeware 3.5.2 (R Development Core Team, 2018). They are considered significantly different when 95% their confidence intervals do not overlap [START_REF] Payton | Overlapping confidence intervals or standard error intervals: What do they mean in terms of statistical significance[END_REF].

Results

To evaluate the influence of diet on Hg assimilation in C. gigas, depuration kinetics of the iHg and MeHg were followed after a pulse-chase feeding, using radiolabelled food items (diatoms and ciliates). Whole-body depuration kinetics of MeHg in oyster fed with diatoms were described by a two-component exponential model (Fig. 1 1) regardless which prey had been ingested.

The assimilation efficiency (AE) of Hg depended both on the food and Hg speciation.

Estimated 203 Hg AE varied significantly (p < 0.05, Fig. 2) according to the prey type. Indeed, AE of 203 Hg by oysters when fed with ciliates was 30.7 ± 2.4%, whereas its assimilation was very high when fed with diatoms (AE = 85.7 ± 4.0%). MeHg was always better assimilated than iHg (AE > 60%). Its AE was significantly lower (p < 0.05, Fig. 2) when oysters were fed with diatoms (60.7 ± 4.1%) than when fed with ciliates (96.2 ± 1.4% Fig. 2, Table 1).

Depuration rate constants (k el ) values obtained for MeHg when oysters were fed with diatoms (k el = 0.012 ± 0.003 d -1 , Table 1) were significantly higher (p<0.05, Fig. 2) than when fed with ciliates (k el = 0.001 ± 0.001 d -1 , Table 1), indicating that their retention was shorter when fed with diatoms (T b1/2l = 56 ± 13 d) than when fed with ciliates (614 ± 302 d). For iHg, no significant difference between k el was observed (average values ranging from 0.014 to 0.024 d -1 , i.e., T b1/2 from 28 to 42 days, p > 0.05, Fig. 2, Table 1).

Discussion

Dietary pathway is now recognized as a major source of Hg bioaccumulation in marine invertebrates (e.g., [START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF][START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North Eastern Atlantic waters: Influence of geographical origin and feeding ecology[END_REF][START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF]. The assimilation efficiency (AE) and efflux rate constant (k e ) or retention time (T b1⁄2 ) are critical parameters in assessing the dietary uptake of trace elements [START_REF] Reinfelder | Assimilation efficiencies and turnover rates of trace elements in marine bivalves: a comparison of oysters, clams, and mussels[END_REF] and numerous studies have been dedicated to estimate these parameters in different marine organisms (e.g., [START_REF] Warnau | Biokinetics of selected heavy metals and radionuclides in the common Mediterranean echinoid Paracentrotus lividus: Sea water and food exposures[END_REF]Wang and Wong, 2003a;Metian et al., 2008b;[START_REF] Lacoue-Labarthe | Bioaccumulation of inorganic Hg by the juvenile cuttlefish Sepia officinalis exposed to 203 Hg radiolabelled seawater and food[END_REF]. However, our understanding of the factors influencing the trophic transfer of Hg in bivalves is still limited.

The AE of Hg ingested with marine phytoplankton has been quantified in a few bivalve species, including clams (Ruditapes philippinarum), oysters (Saccostrea cucullata), mussels (Mytilus galloprovincialis, Perna viridis, Septifer virgatus) and scallops (Chlamys nobilis, C.

varia, Pecten maximus) [START_REF] Fowler | Factors affecting methyl and inorganic mercury dynamics in mussels and shrimp[END_REF][START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF]Metian et al., 2008b[START_REF] Metian | Delineation of heavy metal uptake pathways (seawater and food) in the variegated scallop Chlamys varia, using radiotracer techniques[END_REF][START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF]. In the present study, the AEs of MeHg and iHg in C.

gigas (61-96% for MeHg and 31-86% for iHg) were quantified for two diets, the diatom Thalassiosira pseudonana and the ciliate Uronema sp. The efficient assimilation of MeHg is presumably due to rapid desorption of the ingested MeHg within the digestive tract, the desorbed MeHg being then rapidly assimilated due to its high lipophilicity [START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF]. The AEs observed in our study for MeHg are corroborating those observed in other bivalves (50-95%) fed with phytoplankton [START_REF] Fowler | Factors affecting methyl and inorganic mercury dynamics in mussels and shrimp[END_REF][START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF][START_REF] Pan | Uptake of Hg(II) and methylmercury by the green mussel Perna viridis under different organic carbon conditions[END_REF]Wang, 2011). Regarding iHg, we found AE of 86 ± 4.0% in oysters fed with T. pseudonana, i.e. a value higher than the one found by [START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF] (AE:

31 ± 2%) in the rock oyster Saccostrea cucullata fed on the same phytoplankton species. In contrast, the latter value is similar to the AE we observed when C. gigas was fed with the ciliate Uronema sp (AE: 31 ± 2%). Such contrasting findings suggest that trophic transfer of iHg is both species-and feed-dependent.

Another mechanism partially accounting for difference in AEs between iHg and MeHg in oysters fed on diatoms is how Hg is stored in the phytoplankton cells as shown for other trace elements (e.g., [START_REF] Reinfelder | The assimilation of elements ingested by marine copepods[END_REF]Wang et al., 1996;Metian et al., 2008a). Indeed, trace elements in algal cytoplasm are assumed to be more bioavailable than the fraction associated with organelles (Wang and Fisher, 1996;[START_REF] Lee | Influence of microalgal biomass on absorption efficiency of Cd, Cr, and Zn by two bivalves from San Francisco Bay[END_REF]. Using subcellular fractioning technique, previous studies have shown that ~40-60% of iHg was found in the bioavailable fraction in the diatoms Thalassiosira sp., whereas the proportion of MeHg in the bioavailable fraction was much higher (~70-95%; Wu andWang, 2011, 2013). Nevertheless, [START_REF] Lee | Methylmercury uptake by diverse marine phytoplankton[END_REF] also highlighted that the proportion of MeHg in the cytoplasm of algal cells was dependent on environmental (culture) conditions. Indeed, the percentage of total cellular MeHg in the cytoplasmic fraction varied by a factor of 1.2-2.4 across six phytoplankton species maintained at two different temperatures (4 and 18°C) in the dark or under a 14:10 light-dark cycle. To the best of our knowledge, the effects of culture conditions on the storage of trace elements have never been investigated in ciliates. Further investigations are needed to assess how the subcellular partitioning (see [START_REF] Wallace | Subcellular compartmentalization of Cd and Zn in two bivalves. II. Significance of trophically available metal (TAM)[END_REF] of Hg in diatom and ciliate may affect the trophic transfer of iHg and MeHg in the oysters.

Our study demonstrated that there was a marked difference in the assimilation of Hg (MeHg and iHg) in the oyster C. gigas in response to the different diets tested. A number of studies have examined the assimilation of other trace elements from different feeds (i.e., phytoplankton species) in oysters (e.g., [START_REF] Ettajani | Determination of cadmium partitioning in microalgae and oysters: contribution to the assessment of trophic transfer[END_REF][START_REF] Ke | Bioaccumulation of Cd, Se, and Zn in an estuarine oyster (Crassostrea rivularis) and a coastal oyster (Saccostrea glomerata)[END_REF][START_REF] Hédouin | Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: qualitative and quantitative aspects[END_REF][START_REF] Pan | Mercury accumulation in marine bivalves: Influences of biodynamics and feeding niche[END_REF] and highlighted considerable variability in trace element assimilation according to the diets. As an example, [START_REF] Hédouin | Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: qualitative and quantitative aspects[END_REF] found that Co, Mn and Zn were better assimilated when tree oysters Isognomon isognomon was fed with

Isochrysis galbana than when fed with Emiliana huxleyi or Heterocapsa triquetra. Similar findings were reported for the oyster C. gigas with significantly higher AE of Cd in oysters fed with prasinophyte Tetraselmis suecica (AE = 20%) than when fed with the diatom Skeletonema costatum (AE = 9%; [START_REF] Ettajani | Determination of cadmium partitioning in microalgae and oysters: contribution to the assessment of trophic transfer[END_REF]. Observed differences were mainly explained by the subcellular fractioning of the trace elements within the phytoplankton species.

To the best of our knowledge, ciliates were never considered as feed to assess the trophic transfer of trace elements in bivalves. [START_REF] Twining | Trophic transfer of trace metals from protozoa to mesozooplankton[END_REF] compared the AE of iHg in 3 species of calanoid copepods fed with ciliate (Uronema sp.) and phytoplankton (dinoflagellate Oxyrrhis marina). They found higher iHg AE (44-53%) in copepods fed with ciliates than in those fed with dinoflagellates (13-14%). For other trace elements (Ag, Cd, Fe, and Zn), AEs in copepods fed with ciliates were higher than when fed with dinoflagellates or diatoms [START_REF] Fisher | Assimilation of metals in marine copepods and its biogeochemical implications[END_REF][START_REF] Mason | Uptake, toxicity, and trophic transfer of mercury in a coastal diatom[END_REF], which was correlated to the higher percentage of trace elements within cytoplasm of the ciliates. In the present study, we found that MeHg was more assimilated in oysters fed with ciliates than when fed with diatoms whereas the opposite trend was observed for iHg. In addition to interspecific differences in AE of an element from a same diet, the observed differences for the iHg assimilation in the oyster C. gigas and the copepods Acartia tonsa, A. hudsonica and Temora longicornis, may be related to the iHg exposure pathways of the ciliates and therefore to the form of storage of iHg. Indeed, [START_REF] Twining | Trophic transfer of trace metals from protozoa to mesozooplankton[END_REF] exposed ciliates through their diet (i.e., radiolabeled bacteria) while in the present study, ciliates were directly exposed to Hg (iHg and MeHg) from the dissolved pathway. While bioaccumulation of waterborne trace elements is based on transport mechanisms across the cell membrane, ingested matrix enters within digestive vacuoles (where pH 3 was measured; [START_REF] Fok | The correlation of digestive vacuole pH and size with the digestive cycle in Paramecium caudatum[END_REF], concentrating the trace elements inside the ciliate cells [START_REF] Twining | Trophic transfer of trace metals from protozoa to mesozooplankton[END_REF] and thus, potentially enhancing their bioavailability for consumers such as bivalves.

Although subcellular partitioning in prey is an important parameter influencing assimilation of trace elements, there is another important process to take into account: the rate at which a trace element passes through the digestive tract (Wang and Wong, 2003b). A long gut passage time (GPT) allows a more efficient assimilation by increasing the absorption time of the ingested trace elements through the intestinal epithelium. In earlier experimental studies, GPT has been shown to be dependent of the ingested diets. In the oyster C. rivularis fed with 4 different phytoplankton species and sediment [START_REF] Ke | Bioaccumulation of Cd, Se, and Zn in an estuarine oyster (Crassostrea rivularis) and a coastal oyster (Saccostrea glomerata)[END_REF] found that GPT of Cd, Se and Zn varied by a factor of 10 to 20 with the highest values obtained when oysters fed on T. pseudonana. In the present study, the differences in AE of Hg (iHg and MeHg) in the Pacific cupped oyster C. gigas are therefore likely to result from (1) a difference in Hg bioavailability in the ciliate (Uronema sp.) and the diatom (T. pseudonana) and (2) a GPT dependent of the diets. In this study, our results indicated that, for oysters, differences in the bioavailability of Hg or in GPT depending on diets is not the only explanation on the variability of Hg assimilation. Indeed, the comparison of efflux rate constants (k el ) reveals that the retention of ingested Hg is also diet-dependent (especially for MeHg) indicating that excretion processes are also affected by the diet. Overall, the lower k el observed for MeHg indicates a strong retention of this compound in oysters (T b1/2l ranging from 56 to 614 d).

Dietary pathway is an important contributor to the global bioaccumulation of Hg in bivalves (e.g., [START_REF] Blackmore | The transfer of cadmium, mercury, methylmercury, and zinc in an intertidal rocky shore food chain[END_REF]Wang, 2004, Metian et al., 2009). The present study has shown that the trophic transfer of Hg (iHg and MeHg) in the oyster C. gigas is influenced by its diet. As suggested by [START_REF] Hédouin | Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: qualitative and quantitative aspects[END_REF], such experimental data need to be considered to better interpret bioaccumulation data obtained in the framework of biomonitoring programs.

Conclusion

Taking given in Table 1. Symbols denote significant difference.

  into account the major importance of the dietary pathway to the Hg bioaccumulation in bivalves, a special attention should be paid to the factors influencing the trophic transfer of Hg in this group. The present study provides new insights regarding the influence of very different feeds, a heterotrophic protist (the ciliate Uronema sp.) and an autotrophic protist (the diatom T. pseudonana) on the AE of iHg and MeHg in the Pacific cupped oyster (C. gigas). Such information can help refining both Hg bioaccumulation models and interpretation of data from field surveys and biomonitoring programs.

Figure 1 .

 1 Figure 1. Influence of type of food (ciliate Uronema sp. and diatom Thalassiosira

Figure 2 .

 2 Figure 2. 95% confidence intervals of the main kinetic parameters: (A) Assimilation

Table 1 ;

 1 R 2 : 0.71) while depuration kinetics from oysters fed with ciliates were described by a two-component exponential model (Fig.1 and Table 1; R 2 : 0.92). A non-negligible fraction (30-96%) of the two radiotracers was strongly to very strongly retained in oysters (average T b½l ranging from 28 to 614 d, Table

and Table

1

; R 2 : 0.56) while depuration kinetics from oysters fed with ciliates were described by a one-component exponential model (Fig.

1

and Table

1

; R 2 : 0.20). Conversely, depuration kinetics of iHg from oyster fed with diatoms were described by a one-component exponential model (Fig.

1 and

Table 1 .

 1 Parameters (mean ± SE, n = 5) of the whole-body depuration kinetics of iHg and MeHg in the Pacific cupped oyster (C. gigas) fed on ciliate (Uronema sp.) and diatom (Thalassiosira pseudonana). Depuration parameters: A 0s and A 0l (=AE): activity (%) lost according to the short-and the long-lived exponential component, respectively; T b½ : biological half-life (d) [T b½ = ln2/k e ]; O and T: one-component and two-component exponential model, respectively. R 2 : determination coefficient. Probability of the model adjustment: NS p > 0.05, * p < 0.05, *** p < 0.001.

	Feed	Element Model A 0s ± SE	k es ± SE	T b½s ± SE	A 0l (=AE) ± SE	k el ± SE	T b½l ± SE	R 2
		MeHg	O	-		-	96.16 ± 1.39***	0.001 ± 0.0006*	614 ± 302***	0.20
	Ciliate
		iHg	T	69.27 ± 4.28***	1.86 ± 0.41***	0.4 ± 0.1*** 30.67 ± 2.40***	0.014 ± 0.003*** 48 ± 11***	0.92
		MeHg	T	39.29 ± 7.26***	1.78 ± 1.06 NS	0.4 ± 0.2 NS	60.71 ± 4.11***	0.012 ± 0.003*** 56 ± 13***	0.56
	Diatom
		iHg	O	-	-	-	85.72 ± 4.04***	0.024 ± 0.003*** 28 ± 3***	0.71

Acknowledgments

The IAEA is grateful for the support provided to its Environment Laboratories by the Government of the Principality of Monaco. The IUF (Institut Universitaire de France) is acknowledged for its support to PB as a Senior Member. MW is an Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium). The authors would like to express their gratitude to Dr Claude Rouleau for sharing his expertise on methylation of 203 Hg.