P Bustamante

H Budzinski

A Luna-Acosta

Persistent Organic Pollutants in a marine bivalve on the Marennes-Oléron Bay and the Gironde Estuary (French Atlantic coast) -Part 2: Potential biological effects

Keywords:

Contaminant effects on defence responses of ecologically and economically important organisms, such as the Pacific oyster Crassostrea gigas, are likely to influence their ability to resist infectious diseases, particularly at the young stages. The aim of this study was to explore the potential relationships between organic contaminants accumulated in the soft tissues of juvenile oysters, defence responses and physiological condition. Oysters were transplanted during summer and winter periods in different sites in the Marennes-Oléron Bay, the first area of oyster production in France, and in the Gironde Estuary, the biggest estuary in Occidental Europe. Among the battery of biochemical and physiological biomarkers applied in the present work (superoxide dismutase -SOD-, catalase, glutathione peroxidase -GPx-, malondyaldehyde -MDA-, catecholase, laccase and lysozyme in gills, digestive gland, mantle and haemolymph, glycogen, proteins and lipids in the digestive gland and the condition index at the whole organism level), MDA and lysozyme in the digestive gland and SOD, GPx and laccase in plasma contibuted to significantly discriminate sites in which oysters bioaccumulated different levels of heavy polycyclic aromatic hydrocarbons (HPAHs), polychlorobiphenyls (PCBs), polybromodiphenylethers (PBDEs), dichlorodiphenyltrichloroethanes (DDTs) and lindane. These results

strengthen the hypothesis that it is possible to differentiate sites depending on their contamination levels and biological effects by carrying out studies with transplanted juvenile oysters. In addition, correlations between antioxidant and immune defence responses and PAH and DDT body burdens in the first area of oyster production in France, the Marennes-Oléron Bay, and where massive oyster mortalities have been reported, suggest that the presence of organic chemical contaminants in the Marennes-Oléron Bay may influence defence responses in juveniles of C. gigas, and, therefore, could influence their ability to resist infectious diseases.

Introduction

The Pacific oyster, Crassostrea gigas (Thunberg 1753), is the leading aquaculture product at the worldwide level (FAO 2010), and is considered an important species for ecosystem functioning [START_REF] Gutierrez | Mollusks as ecosystem engineers: the role of shell production in aquatic habitats[END_REF]. Hence, the maintenance of healthy populations of this economically and ecologically important organism is of considerable relevance.

Like other bivalves, C. gigas has been extensively used in environmental risk assessment as pollutant concentrations in its tissues faithfully reflect the environmental concentrations (Ref). This sessile filter-feeder bivalve mollusc bioaccumulates contaminants in its tissues (e.g. [START_REF] Auffret | Monitoring of immunotoxic responses in oysters reared in areas contaminated by "Erika" oil spill[END_REF][START_REF] Livingstone | Biotechnology and pollution monitoring: Use of molecular biomarkers in the aquatic environment[END_REF], especially in areas with intense human activities, frequently contaminated with chemical compounds. Survey networks such as ROCCH (Réseau d'Observation de Contamination Chimique du Littoral, previously RNO, in France) have implemented monitoring programs known as Mussel Watch Programs (MWPs), using naturally settled populations of marine bivalves, such as C. gigas, as sentinel species for inorganic and organic contamination and have shown to contribute to the improvement of water quality in countries such as the USA (Melwani 2013). However, this type of monitoring possess no experimental control and cannot be used to evaluate short-term trends in contaminants distribution. Another type of survey, called active biomonitoring, consists on using caged bivalves transplanted to sites with different levels of contamination. The main advantages of active biomonitoring over traditional MWPs is 1) the experimental control, which is achieved using bivalves of similar genetic and environmental stocks at all test sites, pre-selecting test animal size or age group, and monitoring animals during the test, and 2) the possibility to examine both short-and long-term trends in contaminants distribution and related effects [START_REF] Besse | Relevance and applicability of active biomonitoring in continental waters under the Water Framework Directive[END_REF][START_REF] Galgani | Assessment of polycyclic aromatic hydrocarbon concentrations in mussels (Mytilus galloprovincialis) from the Western basin of the Mediterranean Sea[END_REF][START_REF] Gunther | Long-term bioaccumulation monitoring with transplanted bivalves in the San Francisco Estuary[END_REF].

Both types of monitoring analyze chemical body burdens in marine bivalves. In addition to chemical analyses, the use of biological markers or biomarkers, have been introduced in some monitoring programs since chemical analyses do not offer by themselves any type of indication of possible deleterious effects of contaminants on marine organisms [START_REF] Allan | A "toolbox" for biological and chemical monitoring requirements for the European Union's Water Framework Directive[END_REF][START_REF] Cajaraville | The use of biomarkers to assess the impact of pollution in coastal environments of the Iberian Peninsula: A practical approach[END_REF]. Biomarker responses (i.e. biochemical, cellular, physiological, behavioural or energetic responses of the organisms) on body fluids, cells, tissues, or on whole animals are considered as early-warning sensitive signals of deleterious effects of contaminants since they can be used to evaluate the exposure to and toxic effects of contaminants [START_REF] Bodin | Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplant into the northwest Mediterranean Sea[END_REF][START_REF] Livingstone | Biotechnology and pollution monitoring: Use of molecular biomarkers in the aquatic environment[END_REF][START_REF] Magni | Genotoxicity biomarkers and acetylcholinesterase activity in natural populations of Mytilus galloprovincialis along a pollution gradient in the Gulf of Oristano (Sardinia, wester Mediterranean)[END_REF][START_REF] Rodriguez-Ariza | Genetic diversity and metal tolerance of two marine species: A comparison between populations from contaminated and reference sites[END_REF], especially if several biomarkers are measured at the same time in the same animals [START_REF] Lagadic | The role of biomarkers in environmental assessment (5). Invertebrate populations and communities[END_REF]).

Among biomarker responses, defence mechanisms may affected by the presence of contaminants in the environment [START_REF] Lacoste | Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus[END_REF][START_REF] Perdue | Some relationships between gametogenic cycle and summer mortality phenomenon in the Pacific oyster (Crassostrea gigas) in Washington State[END_REF]). Enzymes such as superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT;EC 1.11.1.6) and glutathione peroxidase (GPx; EC 1.11.1.9) regulate the presence of reactive oxygen species (ROS) in the organism [START_REF] Neumann | Antimicrobial mechanisms of fish phagocytes and their role in host defense[END_REF], and when ROS overwhelm these antioxidant enzyme activities, this may lead to the lipid peroxidation of polyunsaturated fatty acids, resulting in the formation of reactive electrophile species, such as malondialdehyde (MDA), considered as a marker for oxidative stress [START_REF] Gerard-Monnier | Reactions of 1methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation[END_REF]. Humoral immune defences in bivalves, key enzymes such as phenoloxidase (PO, EC 1.14.18.1) and lysozyme (EC 3.2.1.17) participate in the inactivation of invading pathogens [START_REF] Hagger | Toxicity of tributyltin in the marine mollusc Mytilus edulis[END_REF], Söderhäll & Cerenius 1998), and therefore a depletion of these enzymes could influence the ability of the organism to resist infectious diseases. [START_REF] Lagadic | The role of biomarkers in environmental assessment (5). Invertebrate populations and communities[END_REF] suggests that energy reserves could also be considered as biomarkers reflecting sublethal changes from a stressful xenobiotic exposure. Among energy reserves in Ostreidae, the digestive gland is the main reserve organ of glycogen [START_REF] Gabbott | Seasonal changes in the specific activities of the pentose phosphate pathway enzymes, G6PDH and 6PGDH and NADP-dependent isocitrate dehydrogenase in the bivalves Mytilus edulis, Ostrea edulis and Crassostrea gigas[END_REF] and an important organ of reserve of lipids [START_REF] De | Seasonal variations on the biochemical composition and lipid classes of the gonadal and storage tissues of Crassostrea gigas (Thunberg, 1794) in relation to the gametogenic cycle[END_REF], especially when glycogen reserves are depleted [START_REF] Beninger | Seasonal variations in condition, reproductive activity, and gross biochemical composition of two species of adult clam reared in a common habitat: Tapes decussatus L. (Jeffreys) and Tapes philippinarum (Adams & Reeve)[END_REF]. Glycogen reserves in the digestive gland are generally used in the synthesis of lipids and/or proteins for gametogenesis [START_REF] De | Seasonal variations on the biochemical composition and lipid classes of the gonadal and storage tissues of Crassostrea gigas (Thunberg, 1794) in relation to the gametogenic cycle[END_REF][START_REF] Gabbott | Storage cycles in marine bivalve molluscs: a hypothesis concerning the relationship between glycogen metabolism and gametogenesis[END_REF]). Glycogen, protein and/or lipid reserves may be also depleted in the presence of contaminants [START_REF] Pellerin-Massicotte | Seasonal variability in biochemical composition of the polychaete Nereis virens (Sars) in two tidal flats with different geographic orientations[END_REF]. The condition index is often used as index endpoint, i.e. to evaluate bivalve condition and is considered as one of the best indicators of gross body state for environmental studies and thus, could also reflect sublethal changes from a stressful xenobiotic exposure [START_REF] Hyötylänen | Assessment of bioavailability and effects of chemicals due to remediation actions with caging mussels (Anodonta anatina) at a creosote-contaminated lake sediment site[END_REF].

Previous studies in juveniles of C. gigas have shown that the responses of biomarkers such as SOD, CAT, GPx, MDA, catecholase, laccase and lysozyme can be modulated in experiments carried out in laboratory-controlled conditions with organic contaminants and that these responses are tissuedependent (Luna-Acosta et al. 2011a, 2012). However, although laboratory studies may help to identify biomarkers of interest, a lack of validation may occur when using these biomarkers in the field with organisms from contaminated and reference sites, e.g. body size and/or gross body state (which can be estimated through flesh dry weight, shell dry weight or condition index) may influence biomarker responses [START_REF] Packard | The use of percentages and size-specific indices to normalize physiological data for variation of body size: wasted time, wasted effort?[END_REF], and biomarker responses may vary depending on environmental factors, such as seawater temperature [START_REF] Oliver | Comparative form and function of oyster Crassostrea virginica hemocytes from Chesapeake Bay (Virginia) and Apalachicola Bay (Florida)[END_REF]. To date, no transplantation studies with juvenile Pacific oysters analysing biomarker responses have been carried out to our knowledge, despite the fact that massive summer mortalities, particularly of juveniles of C. gigas, have become a widespread concern in the world in recent decades [START_REF] Cheney | Summer mortality of Pacific oysters, Crassostrea gigas (Thunberg): initial findings on multiple environmental stressors in Puget Sound, Washington[END_REF][START_REF] Garnier | Evidence for the involvement of pathogenic bacteria in summer mortalities of the Pacific oyster Crassostrea gigas[END_REF][START_REF] Perdue | Some relationships between gametogenic cycle and summer mortality phenomenon in the Pacific oyster (Crassostrea gigas) in Washington State[END_REF], and that there is growing evidence that contaminants may be partly responsible for the observed increase in disease and mortality in C gigas, by adversely affecting their defence mechanisms [START_REF] Lacoste | Stress and stress-induced neuroendocrine changes increase the susceptibility of juvenile oysters (Crassostrea gigas) to Vibrio splendidus[END_REF][START_REF] Perdue | Some relationships between gametogenic cycle and summer mortality phenomenon in the Pacific oyster (Crassostrea gigas) in Washington State[END_REF].

Marennes-Oléron Bay is a suitable area to assess the effects that contaminants have on biomarker responses of the Pacific oyster C. gigas, since it is the first production site of C. gigas in France [START_REF] Soletchnik | Mortalité de l'huître Crassostrea gigas dans le bassin de Marennes-Oléron: étude sur la variabilité spatiale de son environnement et de sa biologie par le systeme d'informations geographiques (SIG)[END_REF], one of the most important production sites at the worldwide level (FAO 2010), and because this area accounts for high levels of a wide range of anthropogenic contaminants such as heavy metals, polycyclic aromatic hydrocarbons (PAHs) and persistent organic pollutants (POPs; [START_REF] Miramand | Sources et impacts potentiels des micropolluants chimiques sur un écosystème littoral exploité: L'exemple des côtes de Charente-Maritime[END_REF][START_REF] Munaron | Assessment of the quantities of herbicides and nutrients brought down by the river Charente to the coast and modelling of the dispersion of atrazine in the Marennes-Oléron bay[END_REF]). In the Marennes-Oléron Bay, contaminants are mainly transported by the Charente River and to a less extent by the Seudre River [START_REF] Miramand | Sources et impacts potentiels des micropolluants chimiques sur un écosystème littoral exploité: L'exemple des côtes de Charente-Maritime[END_REF][START_REF] Munaron | Assessment of the quantities of herbicides and nutrients brought down by the river Charente to the coast and modelling of the dispersion of atrazine in the Marennes-Oléron bay[END_REF], Luna-Acosta et al. 2010a). The bay is also influenced by inputs from the biggest estuary in occidental Europe, the Gironde Estuary [START_REF] Miramand | Sources et impacts potentiels des micropolluants chimiques sur un écosystème littoral exploité: L'exemple des côtes de Charente-Maritime[END_REF][START_REF] Munaron | Assessment of the quantities of herbicides and nutrients brought down by the river Charente to the coast and modelling of the dispersion of atrazine in the Marennes-Oléron bay[END_REF]). In addition, in previous studies, Bouin (B) has been considered as a reference site for chemical contamination [START_REF] Geffard | Kinetics of metal elimination in oysters from a contaminated estuary[END_REF].

Therefore, the main goals of this transplantation study were to evaluate: 1) PAH and POP body burdens in transplanted juvenile oysters after a three-month transplantation period in summer and winter, in different sites of the Marennes-Oléron Bay and the Gironde Estuary; 2) the potential effects of exposure to organic contaminants on antioxidant defence responses, immune defence responses, energy reserves and condition index in juveniles of C. gigas; 3) the potential effects of internal (i.e. physiological status) and/or external (i.e. environmental) factors on juvenile oyster biomarker responses; and 4) the potential use of transplanted juvenile oysters to differentiate sites depending on their contamination levels and biological effects.

Juvenile oysters bought in a hatchery were transferred in summer and winter to B and to different transplantation sites in the Marennes-Oléron Bay and the Gironde Estuary. After a three-month transplantation period, for each site and season, body burdens of PAHs and POPs (polychlorobiphenyls -PCBs-, polybromodimethylethers -PBDEs-and organochlorine pesticides -OCPs-), were analysed and biochemical biomarker responses (SOD, CAT GPx, MDA, catecholase, laccase and lysozyme) were measured in different tissues (gills, digestive gland, mantle and haemolymph). Additionally, energy reserves (glycogen, proteins and lipids) in the digestive gland and the condition index were analysed, and environmental data from French monitoring networks were collected.

Materials and methods

Study area and transplant design

Juvenile Pacific oysters C. gigas from same size, 3-4 cm in height (1 year-old), were purchased from the oyster hatchery France Naissain, located in Vendée (France) at the beginning of each transplantation period, in summer and winter. All animals were issued from the same breeding population in order to reduce genotype variability. In April 2008 and in October 2008, oysters were transplanted to Bouin (B; 46° 58' 28" N, 2° 00' 02" W), considered as a reference site (Fig. 1a), since it is located in a zone near the oyster hatchery where oyster were purchased and because it possesses different seawater physicochemical characteristics in comparison to the transplantation zone [START_REF] Geffard | Kinetics of metal elimination in oysters from a contaminated estuary[END_REF]. In April 2008 and in October 2008, oysters were also transplanted into four other sites, considered as the transplantation sites: Boyard (BOY; 45° 58' 0" N, 1° 15' 0" W), under the double influence of inputs from the Gironde and the Charente estuaries; Les Palles (LP; 45° 58' 0" N, 1° 08' 0" W), highly influenced by inputs from the Charente River; Mus du Loup (ML; 45° 46' 10" N, 1° 08' 30" W), mildly influenced by inputs from the Seudre River, and Cordouan (C; 45° 35' 11" N, 1° 10' 24" W), highly influenced by inputs from the Gironde Estuary (Fig. 1b). Transplantation was carried out for a period of three months (90 days), from April to June 2008 (summer season) and from October to December 2008 (winter season). For each site and season, a total of 100 individuals were placed in HDPE (high-density polyethylene) oyster bags, all with the same size (1 m x 0.5 m) and the same mesh size (2 cm). For each site, all individuals were transplanted in the same oyster bag and at the same location, and oyster bags were placed on oyster aquaculture tables. These tables were placed at a height of 40 cm from the substrate, and all located on the same hypsometric level (iso-altitude of about 1 m corresponding to low-tide water level, coefficient 90), in order to have an equivalent immersion time (<15% of the time over a tidal cycle) for all the individuals that were transplanted, regardless of the geographic location. Three months after each transplantation period (in June 2008 and in December 2008) and for each site (B, BOY, LP, ML and C), 5 oysters were randomly collected for condition index and chemical analyses, and 9 oysters were collected for biochemical biomarkers and energetic reserves analyses. Oysters were transported in ice bags and processed immediately after their arrival in the laboratory.

Sample procedure

Back in the laboratory, the 5 oysters collected for condition index and chemical analyses, were processed as follows: soft tissues were removed from the shells, homogenized using an Ultra Turrax (T25 basic, IKA-WERKE) and a Thomas-Potter homogenizer (IKA-Labortechnik RW 20.n, size 0.13-0.18 mm), freeze-dried and weighted (dry weight; dw) for flesh dw, shell dw and condition index analyses (n=5, per site and per season), and then pooled and frozen (-80ºC) for chemical analyses. For chemical analyses, three replicate analyses were carried out per site and per season (n=3, 1 sample replicate from 5 oysters analyzed in triplicate, per site and per season).

For the other biological analyses, the 9 oysters collected for biochemical biomarkers and energetic reserve analyses were processed as follows: pooled gills, digestive glands, mantles, and plasma of three oysters were used for each replicate sample, and three replicates were prepared per site and per season (n=9, 3 sample replicates from 3 oysters each, per site and per season). To collect these samples, after opening the oyster shells by cutting off the adductor muscle, a quantity of haemolymph (~0.3-0.5 ml for the three oysters) was withdrawn and centrifuged (260 g, 10 min, 4°C) to separate the cellular fraction from the plasma. The gills, digestive gland and mantle were removed from the soft tissues and homogenized at 4°C in 0.1 M Tris HCl buffer pH 7.0 (0.45 M NaCl, 26 mM MgCl2, 10 mM CaCl2; 0.5 ml of buffer.g -1 of fresh weight for the gills and the mantle, and 1 ml.g -1 of fresh weight for the digestive gland), using an Ultra Turrax (T25 basic, IKA-WERKE) and a Thomas-Potter homogenizer (IKA-Labortechnik RW 20.n, size 0.13-0.18 mm). The homogenate was centrifuged at 10 000 g for 10 min at 4°C. The resulting supernatant was collected for enzymatic analyses.

Chemical analysis

A total of 31 polyaromatic hydrocarbon (PAH), 10 polychlorobiphenyl (PCB), 4 polybromodimethylether (PBDE), and 13 organochlorine pesticide (OCP) compounds were quantified and concentrations of organic contaminants were expressed in µg kg -1 dry weight (dw). Briefly, PAHs were measured using a gas chromatograph (HP 6890. Agilent technologies, Palo Alto, CA, USA) equipped with a splitless injector and coupled to a MSD 5975 mass spectrometer [START_REF] Baumard | Polycyclic aromatic hydrocarbons in recent sediments and mussels (Mytilus edulis) from the Western Baltic Sea: occurrence, bioavailability and seasonal variations[END_REF]. PCBs, PBDEs and OCPs were quantified using a gas chromatograph (HP 5890 Hewlett Packard, Palo Alto, Ca, USA) equipped with a splitless injector and coupled to an electron capture detector [START_REF] Thompson | Determination of polychlorinated biphenyls and chlorinated pesticides in environmental biological samples using focused microwave-assisted extraction[END_REF][START_REF] Tapie | Fast and efficient extraction methods for the analysis of polychlorinated biphenyls and polybrominated diphenyl ethers in biological matrices[END_REF]) and confirmed using a gas chromatograph (Agilent Technologies 7890A) coupled to MS/MS (Agilent technologies 7000). Among pesticides, lindane, 2,4'-and 4,4'dichlorodiphenyltrichloroethanes (DDT), 2,4'-and 4,4'-dichlorodiphenyldichloroethanes (DDE), and 2,4´-and 4,4´-dichlorodiphenyldichloroethylenes (DDD), hexachlorobenzene (HCB), heptachlor (HC) and trans-nonachlor (TNC) were quantified. In the text, we will refer to the sum of all DDTs and their metabolites (DDEs and DDDs) as 'DDTs' and to the sum of HCB, HC and TNC as 'HCB+HC+TNC'.

More details on the methodology and quality control are described in Luna-Acosta et al. (submitted for this volume).

Biological analysis

Superoxide dismutase (SOD) assay • SOD was determined by an indirect method [START_REF] Therond | Antioxidant enzymes in psoriatic fibroblasts and erythrocytes[END_REF], based on competition of SOD with iodonitrotetrazolium (INT) for dismutation of superoxide anion (O2 -).

In the presence of O2 -, INT is reduced into a red formazan dye that can be measured at 505 nm at 25°C (Ransod SD 125 Kit, Randox, France). One unit of SOD is defined as the amount of enzyme that promotes a 50% decrease in the rate of INT reduction.

Catalase (CAT) assay • Catalase activity was determined according to the method of [START_REF] Fossati | Use of 3,5-dichloro-2-hydroxibenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine[END_REF]. This assay method is based on the measurement of the hydrogen peroxide substrate remaining after the action of catalase. First, catalase converts hydrogen peroxide to water and oxygen and then this enzymatic reaction is stopped with sodium azide. An aliquot of the reaction mix is then assayed for the amount of hydrogen peroxide remaining by a colorimetric method (Catalase Kit CAT-100, Sigma).

Glutathione peroxidase (GPx) assay • GPx activity was determined according to the method of [START_REF] Paglia | Studies on the quantitative and qualitative characterisation of erythrocyte glutathione peroxidase[END_REF]. In the presence of glutathione reductase and substrates (i.e. reduced glutathione and cumene hydroperoxide), the decrease of absorbance at 340 nm is proportional to the reduction of the oxidised glutathione by NADPH, H+ (Ransel RS 504 Kit, Randox, France). One unit of GPx oxidizes 1 nmol of NADPH (ε = 6.22 mM -1 cm -1 ) per minute.

Phenoloxidase (PO, catecholase-and laccase-type) assay • In C. gigas, catecholase-and laccase-type PO activities have been detected in C. gigas (Luna-Acosta et al. 2010b). Spectrophotometric measurements of these PO activities were made as described in Luna-Acosta et al. (2010b). Assays were carried out in triplicate for each sample. Briefly, catecholase-type PO assay was carried out in the presence of 100 mM dopamine and 0.1 M Tris HCl buffer, pH 7.0 (0.45 M NaCl, 26 mM MgCl2, 10 mM CaCl2; Sigma-Aldrich, France). Catecholase activity was followed by monitoring the increase of absorbance at 490 nm for 4 h. Laccase assay was carried out in the presence of 50 mM of p-phenylenediamine (PPD), diluted in methanol, and 0.1 M Tris HCl buffer, pH 7.0. Laccase activity was followed by monitoring the increase of absorbance at 420 nm for 2 h. Non-enzymatic oxidation of the substrate was followed in wells without oyster sample and subtracted to oxidation of the substrate with oyster sample. One unit of catecholase specific activity and one unit of laccase specific activity corresponds to the amount of enzyme that catalyzes the production of 1 µmole of product (ε = 3 300 M -1 .cm -1 and 43 160 M -1 .cm -1 , respectively), per minute and per milligram of protein [START_REF] Eggert | The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterisation of the laccase[END_REF][START_REF] Espin | A continuous spectrophotometric method for determining the monophenolase and diphenolase activites of apple polyphenol oxidase[END_REF].

Lysozyme assay • Lysozyme assay was done in triplicate for each sample and compared against hen egg white lysozyme standards (0.4-40 µg.ml -1 ), in the presence of Micrococcus lysodeikticus (Sigma-Aldrich, France). One unit of lysozyme corresponds to the amount of enzyme that diminishes absorbance at 450 nm of 0.001 per minute at pH 7.0. at 25°C [START_REF] Soudant | Impact of season and rearing site on the physiological and immunological parameters of the Manila clam Venerupis (=Tapes, =Ruditapes) philippinarum[END_REF].

Malondialdehyde (MDA) assay • Lipid peroxides, derived from polyunsaturated fatty acids, are unstable and decompose to form a complex of series of compounds, of which the most abundant is MDA [START_REF] Gerard-Monnier | Reactions of 1methyl-2-phenylindole with malondialdehyde and 4-hydroxyalkenals. Analytical applications to a colorimetric assay of lipid peroxidation[END_REF]. The method to determine MDA levels in the gills and the digestive gland (MDA-586, Oxis Research, Portland, OR, USA) was based on the reaction of a chromogenic reagent, N-methyl-2-phenylindole (NMPI) with MDA at 45°C. One molecule of MDA reacts with two molecules of NMPI to yield a stable carbocyanine dye with maximum absorption at 586 nnm. The MDA in the sample was determined from the sample's absorbance at 586 nm and the standard curve was prepared using the MDA standard provided in the kit.

Glycogen assay • Digestive gland samples were freeze dried and weighed before determining glycogen, lipids and protein contents. Glycogen content (precipitated with absolute ethanol) was quantified using the phenol-sulphuric method as described by [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. Known glucose standards were processed in the same way as the samples and used to construct a calibration curve.

Lipid assay • Lipids were extracted according to the procedure of [START_REF] Folch | A simple method for the isolation and purification of total lipids from animal tissues[END_REF]. A double static extraction (2x12h) was carried out on the rehydrated samples with a chloroform:methanol mix (1:2 then 2:1, v/v) in polytetrafluoroethylene (PTFE)-capped tubes held at 4°C, under a nitrogen atmosphere.

Following the addition of a salt solution (NaCl 1%), the crude extract was separated into two phases. The lower chloroform phase containing lipids was washed gently with a new volume of the upper phase. After centrifugation for 10 min at 3000 g at 10°C and decantation, it was recovered and stored at -20°C until analysis. Total lipids were measured using a Iatroscan-Chromarod system (thin layer chromatographyflame ionization detection (TLC-FID)) connected to a Shimadzu CR3A integrator. Five successive volumes (1 µl) of a concentrated extract aliquot were spotted on a Chromarod SIII silica rod with a 2 µl Hamilton microsyringe. After drying in a dessicator, the rod was read directly by the Iatroscan without any development. A calibration curve based on a total lipid extract from cod liver oil, with similar lipid characteristics than oyster tissues, was used to calculate the lipid concentration of the sample extracts.

Protein assay • Protein concentration measured according to the Lowry method with slight modifications, by using bicinchoninic acid and copper sulphate 4% [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF]. Serum albumin was used as standard (Sigma-Aldrich, France). All enzyme activities were expressed in relation to protein concentration.

Condition index • Condition index in juvenile oysters was calculated by the method of [START_REF] Lobel | Relationship between body zinc concentration and allometric growth measurements in the mussel Mytilus edulis[END_REF], and expressed in mg flesh dry weight g -1 shell dry weight.

Statistical analysis

All values are reported as mean ± standard deviation (SD; n=5, per site and season, for flesh dry weight, shell dry weight and condition index analyses; n=3, per site and per season, for chemical analyses and n=9, per site and season, for biochemical biomarkers and energetic reserve analyses). Values were tested for normality (Shapiro test) and homogeneity of variances (Bartlett test). In some cases (HPAHs, PCBs, PBDEs, DDTs, lindane and HCB+HC+TNC body burdens, catecholase, laccase, lysozyme in gills, GPx, MDA, lysozyme, proteins and lipids in the digestive gland, SOD, GPx, catecholase, and lysozyme in mantle and SOD, CAT, laccase and lysozyme in plasma), logarithmic transformations (Log10) were used to meet the underlying assumptions of normality and homogeneity of variances. Since body size and/or gross body state may influence biomarker responses [START_REF] Packard | The use of percentages and size-specific indices to normalize physiological data for variation of body size: wasted time, wasted effort?[END_REF], two-way MANCOVAs (covariate: flesh dw, shell dw or condition index), were carried out to evaluate these possible effects, with site and season as fixed factors [START_REF] Zar | Biostatistical analysis[END_REF]. After verifying that the tested covariates had no significant effect on biomarker responses (p>0.05), a two-way MANOVA was used, with site and season as fixed factors. When the null hypothesis (H0: no difference between sites or within sites at different seasons) was rejected, significant differences were tested using Tukey's HSD test. Statistical significance was designed as being at the level of p < 0.05. In addition, a discriminant analysis (DA; using three different groups of sites depending on their degree of contamination, in order to test which biomarker responses significantly contributed to discriminate sites between these three groups) and the ordination technique of redundancy analysis (RDA; using forward selection of explanatory variables, in order to test which contaminants had a significant effect on the observed biomarker responses) were performed to evaluate the response of biomarkers to chemical analyses. Data were centered and standardized before analysis. The significance of the relationship between biomarker responses and contaminant body burdens were tested using a global Monte Carlo permutation test. Statistical analyses of data were performed using the software STATISTICA 7.0, with the exception of DA and RDA that were performed using the software XLSTAT 5.0.

Results and discussion

PAH and POP body burdens in transplanted juvenile oyster

In the present study, over the different sampling stations (i.e. reference and transplantation sites) and seasons (i.e. summer and winter), body burdens ranged between 27±1 and 68±19 µg kg -1 dw for LPAHs, between 35±7 and 159±34 µg kg -1 dw for HPAHs, between 15±1 and 89±39 µg kg -1 dw for PCBs, between 0.4±0.0 and 2.9±0.7 µg kg -1 dw for PBDEs, between 2±0 and 15±0 µg kg -1 dw for DDTs, between 0.1±0.0 and 2.2±1.1 µg kg -1 dw for lindane, and between 0.7±0.0 and 3.5±1.0 µg kg -1 dw HCB+HC+TNC (Table 1). Transplanted oysters with the highest HPAH and DDT body burdens were observed in LP in summer and LP, BOY and ML in winter (Table 1). Surprisingly, transplanted oysters with the highest PCB, PBDE and lindane body burdens, were observed in B, considered as a reference site for chemical contamination in previous studies (e.g. [START_REF] Geffard | Kinetics of metal elimination in oysters from a contaminated estuary[END_REF]; Table 1). Results of the present study reveal that B should no longer be considered as a reference site and show that special care must be taken in future studies, since it should not be considered as a reference site for a certain type of contaminants, such as metals but no others, such as chemical organic contaminants. Previous authors have pointed out the difficulty to establish a reference site that is not polluted [START_REF] Davies | Integrated marine environmental monitoringof chemicals and their effects[END_REF], and some of authors have even argued that there is no such thing as a pristine environment [START_REF] Clark | The Long-Term Effect of Oil Pollution on Marine Populations, Communities and Ecosystems: Some Questions[END_REF]. Thus, B remains an interesting site in the present study, since it is located in a zone near the oyster hatchery where oyster were purchased and because it possesses different seawater physico-chemical characteristics in comparison to the transplantation zone [START_REF] Geffard | Kinetics of metal elimination in oysters from a contaminated estuary[END_REF]. Moreover, although it is difficult to determine the normal range of biomarker responses in the present study, the chemical compounds that are analyzed in the present study are not naturally present in animals so their presence unequivocally indicate the presence of environmental pollution and therefore findings of the present study are not compromised since relationships between biomarkers response and exposure can still be determined.

Another interesting result was that oysters from C, located in the Gironde Estuary, did not have the highest body burdens for the organic contaminants analysed in the present study (Table 1), suggesting that the Marennes-Oléron Bay is not subjected to chronic pollution by PAHs or POPs from the Gironde watershed. These results were not expected since the Marennes-Oléron Bay is known to be influenced by the historic polymetallic pollution of the Gironde Estuary, especially of Cadmium [START_REF] Pigeot | Cadmium pathways in an exploited intertidal ecosystem with chronic cadmium inputs (Marennes-Oléron, Atlantic coast, France)[END_REF][START_REF] Strady | Roles of regional hydrodynamic and trophic contamination in cadmium bioaccumulation by Pacific oysters in the Marennes-Oléron Bay (France)[END_REF], and therefore the Bay was expected to be influenced by other contaminants, such as PAHs and POPs, of the Gironde Estuary [START_REF] Laroche | Variation patthers in individual fish responses to chemical stress among estuaries, seasons and genders: the case of the European flounder (Patichthys flesus) in the Bay of Biscay[END_REF]. These results suggest also the presence of other contaminant sources in the Marennes-Oléron Bay, such as local inputs from agricultural or industrial activities, as more deeply discussed in Luna-Acosta et al. (submitted to this volume) and are in agreement with different studies (e.g. [START_REF] Greenfield | The usefulness of transplantation studies in monitoring of metals in the marine environment: South African experience[END_REF][START_REF] Hédouin | Validation of two tropical marine bivalves as bioindicators of mining contamination in the New Caledonia lagoon: field transplantation experiments[END_REF][START_REF] Orbea | Peroxisome proliferation and antioxidant enzymes in transplanted mussels of four basque estuaries with different levels of polycyclic aromatic hydrocarbon and polychlorinated biphenyl pollution[END_REF]) that show that transplant experiments with bivalves could be useful to assess the origin of chemical pollution. 2). SOD levels were generally significantly higher, especially in LP and BOY, except when they were measured in the gills (Table 2). CAT levels were also significantly higher, when measured in the digestive gland or in plasma (Table 2). GPx levels were significantly lower or higher, depending on the analysed tissue and season (Table 2). MDA levels were generally significantly higher in the gills and in the digestive gland, especially in winter (Table 2). Catecholase levels were higher when measured in the gills and the digestive gland and lower when measured in plasma, especially in summer (Table 2). Laccase levels were lower, independently of the season (Table 2), and lysozyme levels were lower, except when measured in winter in the digestive gland (Table 2). Thus, as a general trend, higher levels of SOD, CAT, GPx, MDA, catecholase, laccase and lysozyme were observed in winter.

Potential effects

Humoral immune defence responses of the present study are in agreement with previous studies that have reported lower immune defences in summer [START_REF] Samain | Genetically based resistance to summer mortality in the Pacific oyster (Crassostrea gigas) and its relationship with physiological, immunological characteristics and infection processes[END_REF]). However, the latter has generally been attributed to the reproductive cycle of bivalves in summer [START_REF] Samain | Genetically based resistance to summer mortality in the Pacific oyster (Crassostrea gigas) and its relationship with physiological, immunological characteristics and infection processes[END_REF], and in the present study oysters were juveniles. Results on MDA levels in the present study are in agreement with previous studies in bivalves that have reported higher oxidative stress in winter, but results on SOD, CAT and GPx are in disagreement with previous studies in bivalves that have reported that generally antioxidant defences in bivalves are lower in winter [START_REF] Manduzio | Seasonal variations in antioxidant defences in blue musselsMytilus edulis collected from a polluted area: major contributions in gills of an inducible isoform of Cu/Zn-superoxide dismutase and of glutathione S-transferase[END_REF]). Thus, factors other than the seasonality are likely to affect biomarker responses in the present study.

Concerning other biomarker responses analysed in the present study, glycogen, lipid and proteins in the digestive gland generally accounted for 5-15%, 60-70% and 10-20% of the total energy reserves, respectively (Fig. 2b,c), and varied also depending on the season and site (Fig. 2a). A significant decrease in lipid contents was observed between summer and winter, in oysters from B (Fig. 2a). When analysing results from other sites, low, not significant, levels in oysters from BOY, LP and C were concomitant with significant high levels in lipids and proteins. Low glycogen levels could be due to an exposure of the organism to high levels of contaminants [START_REF] Pellerin-Massicotte | Seasonal variability in biochemical composition of the polychaete Nereis virens (Sars) in two tidal flats with different geographic orientations[END_REF] or the use of glycogen reserves in oysters for the synthesis of lipids and/or proteins for gametogenesis [START_REF] De | Seasonal variations on the biochemical composition and lipid classes of the gonadal and storage tissues of Crassostrea gigas (Thunberg, 1794) in relation to the gametogenic cycle[END_REF][START_REF] Gabbott | Storage cycles in marine bivalve molluscs: a hypothesis concerning the relationship between glycogen metabolism and gametogenesis[END_REF]). High levels of glycogen in ML in summer, concomitant with low levels of lipids and proteins, could suggest a delay in the gametogenesis in this site [START_REF] Gauthier-Clerc | Delayed gametogenesis of Mya arenaria in the Saguenay fjord (Canada): a consequence of endocrine disruptors?[END_REF] or that organisms in this site were not exposed to high levels of contaminants. When comparing these results to the total flesh dry weight, the digestive gland weight in oysters from BOY, LP, ML and C was lower in summer than in winter (Fig. 2b), and this could be associated to gametogenesis [START_REF] De | Seasonal variations on the biochemical composition and lipid classes of the gonadal and storage tissues of Crassostrea gigas (Thunberg, 1794) in relation to the gametogenic cycle[END_REF]. However, further studies should be carried out to verify this hypothesis.

In the present study, higher condition index values were observed in summer in comparison to winter (Fig. 3c). These results are in agreement with previous studies in mussels and oysters [START_REF] Bodin | Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplant into the northwest Mediterranean Sea[END_REF][START_REF] Soletchnik | Seasonal changes in carbohydrate metabolism and its relationship with summer mortality of Pacific oyster Crassostrea gigas (Thunberg) in Marennes-Oléron bay (France)[END_REF]. Interestingly, significant differences between reference and transplantation sites were observed in summer but not in winter. In summer, a significant increase of the condition index was observed in a North-South gradient, with higher values observed in C: in comparison to the condition index of oysters from B, the condition index was ~1.3, ~1.3, ~1.7 and ~2-fold significantly higher in BOY, LP, ML and C, respectively. This increase in the condition index was not due to an important increase in shell dry weight (Fig. 3b), but to an important increase in flesh dry weight (Fig. 3a). However, no clear differences in contaminant body burdens between sites were observed in a North-South gradient.

To further evaluate potential relationships between biomarker responses and contaminant body burdens, a DA analysis was carried out dividing sites into three groups, based on contaminant body burdens (Table 1): group 1 corresponded to B in summer and winter (Bs and Bw, respectively), which showed to be the most contaminated site concerning PCB, PBDE and lindane contamination, group 2 corresponded to Les Palles in summer and winter, Boyard in winter and Mus du Loup in winter, which showed to be the most contaminated sites concerning HPAH and DDT contamination (Table 1), and group 3 corresponded to the remaining sites (Boyard and Mus du Loup in summer and Cordouan in summer and winter). Biomarker responses that significantly (p<0.001) contributed to discriminate sites into these three groups were: MDA in gills, SOD, GPx, MDA, lysozyme and proteins in the digestive gland, SOD in the mantle, SOD, GPx and laccase in plasma and the condition index. The RDA analysis was carried out with these biomarkers.

The results of the RDA analysis are presented in the tri-plot ordination diagram of Fig. 4. Among contaminants that were analysed, HPAHs, PCBs, PBDEs, DDTs and lindane had a significant effect on the observed biomarker responses. The first two axes of the RDA analysis accounted for 77% of the overall variability of the data. Therefore, the other axes were neglected because they did not provide significant additional information. The first RDA axis (horizontal) accounted for 42% of the total, and HPAHs and DDTs strongly correlated with the first axis. In addition, MDA in the gills and the digestive gland, SOD in mantle and SOD and GPx in plasma correlated positively with the first axis and the condition index correlated negatively with the first axis (Fig. 4a).

The second RDA axis (vertical) accounted for 35% of the total variability of the data and the levels of PCBs, PBDEs and lindane strongly correlated with the second axis. In addition, GPx in the digestive gland and laccase in the plasma and mantle correlated positively with the second axis, and SOD in the digestive gland correlated negatively with the second axis (Fig. 4a).

Thus, results of the RDA analysis suggest that HPAH and/or DDT contamination increase SOD activities in the mantle, and SOD and GPx activities in plasma and that PCB, PBDE and/or lindane contamination increase GPx activities in the digestive gland (Fig. 4a). The observed results could be explained by the fact that PAHs can lead to the formation of ROS and enhance oxidative stress in aquatic organisms [START_REF] Giulio | Biochemical responses in aquatic animals: A review of determinants of oxidative stress[END_REF][START_REF] Winston | Oxidants and antioxidants in aquatic animals[END_REF]. Indeed, different studies have shown an increase in SOD activity in bivalves exposed to hydrocarbons [START_REF] Orbea | Antioxidant enzymes and peroxisome proliferation in relation to contaminant body burdens of PAHs and PCBs in bivalve molluscs, crabs and fish from the Urdaibai and Plentzia estuaries (Bay of Biscay)[END_REF][START_REF] Richardson | Antioxidant responses to polycyclic aromatic hydrocarbons and organochlorine pesticides in green-lipped mussels (Perna viridis): Do mussels "integrate" biomarker responses?[END_REF][START_REF] Sole | The use of biomarkers for assessing the effects of organic pollution in mussels[END_REF], suggesting that hydrocarbons induced oxidative stress by producing ROS such as O2 -. A significant correlation between PAH accumulation in the flesh and SOD and CAT activities in the gills has been found in mussels from the Mediterranean Sea [START_REF] Porte | Responses of mixed-function oxigenase and antioxidase enzyme system of Mytilus sp. to organic pollution[END_REF]. [START_REF] Sole | The use of biomarkers for assessing the effects of organic pollution in mussels[END_REF] also reported correlations between GPx activity in the digestive gland of molluscs collected on the Mediterranean coast and their organochlorine body burden and SOD activity in the digestive gland and PAH concentrations. [START_REF] Krishnakumar | Effects of chemical contaminants on the health of Mytilus edulis from Puget Sound, Washington. II. Cytochemical detection of subcellular changes in digestive cells[END_REF] reported higher CAT activity in the digestive gland of mussels from contaminated sites when compared to those sampled at a reference site in the North American Pacific coast. Consistently, increased antioxidant activities were found in oysters from contaminated sites by PCBs, PAHs and pesticides (Rodriguez-Ariza et al. 1993). In the present study, results suggest also that HPAH and/or DDT contamination increase MDA levels in the gills and digestive gland.

SOD activities in the digestive gland correlated negatively with the second RDA axis (Fig. 4a).

These results suggest that PCB, PBDE and/or lindane contamination decrease SOD activities in digestive gland. This could be due to the inhibition of the enzyme synthesis by these contaminants or to an enzyme inactivation caused by high tissue contaminant concentrations, as it has been suggested for other contaminants [START_REF] Borg | Cytotoxicity from coupled redox cycling of autoxidizing xenobiotics and metals[END_REF].

Laccase in plasma and mantle correlated positively with the second RDA axis (Fig. 4a). These results suggest that PCB, PBDE and/or lindane contamination increase laccase activities in plasma (Fig. 4a). Thus, laccase activity may enable a protection against bioaccumulated PCBs, PBDEs and lindane, and therefore, be a potential defence biomarker candidate in ecotoxicological studies, as suggested in previous studies (Luna-Acosta et al. 2011a, b). However, there is a lack of fundamental understanding of the activation and regulation of key factors of catecholase-type and laccase-type PO pathways in bivalves.

Thus, more studies on the potential roles of these enzymes in oysters are needed for a better understanding of the potential implications of their modulations in the presence of contaminants.

Results of the RDA analysis suggest also that PCB, PBDE and/or lindane contamination increase lysozyme activities in the digestive gland (Fig. 4a), suggesting that lysozyme play a role in the detoxification of PCB, PBDE and/or lindane in C. gigas.

Proteins in the digestive gland correlated negatively with the second RDA axis (Fig. 4a). This result suggests a negative correlation between protein contents in the digestive gland and PCB, PBDE and lindane levels and therefore are in agreement with the hypothesis that contaminants may exert an effect on oyster energy reserves, i.e. that the exposure to contaminants induces a lower accumulation of energy reserves or that energy reserves are deployed in the presence of these contaminants for the proper activation of defence mechanisms. However, no correlations were observed between lipid and glycogen levels in the digestive gland, and contaminant body burdens. Therefore, more studies are needed to better assess the relationship between contaminant contents and energy reserves in bivalves.

HPAHs and DDTs strongly correlated with the first RDA axis and the condition index correlated negatively with the first axis (Fig. 4a). This result suggests negative correlations between condition index and HPAH and DDT contents, suggesting that these contaminants may exert a negative effect in oyster condition index. However, the clear North-South gradient on condition index values in summer (Fig. 3c), suggests that other internal or external factors could have an effect on the condition index.

Potential effects of internal and external factors on juvenile oyster biomarker responses

Possible effects on biomarker responses could be attributed to internal factors, such as oyster body size or gross body state [START_REF] Packard | The use of percentages and size-specific indices to normalize physiological data for variation of body size: wasted time, wasted effort?[END_REF], estimated in the present study through flesh dw, shell dw and condition index. MANCOVA analyses revealed no significant effect of the tested covariates on biomarker responses, suggesting that oyster body size or gross body state had no effect on biomarker responses.

Possible effects on biomarker responses could also be attributed to external environmental factors.

Data collected from 2000 to 2006, and from 2008, from French monitoring networks in the same area of the present study (Table 3), suggests that differences in some environmental parameters between sites follow a North-South gradient: a slight increase of the temperature was observed in a North-South gradient in summer, higher levels of turbidity were observed in winter, especially in B, BOY and LP, and differences in phytoplankton abundance and species composition were observed between sites and seasons, with higher abundance of phytoplankton and species composition observed in summer, especially in C (Table 3). However, no clear modulations on biomarker responses were observed in a North-South gradient in the present study, with the exception of the condition index (Fig. 3c). It is important to mention that the condition index varies principally with the availability and/or the quality of the food [START_REF] Mourgaud | Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field[END_REF]. Therefore, results of the present study suggest that trophic quality is better in C than in the other sites, and also, that, in the present study, the condition index is more influenced by the seawater temperature or the presence and/or quality of food, than by contaminants. Among other environmental variables (salinity, presence of phycotoxins, faecal contamination), no particular differences were observed between sites and seasons (Table 3).

Overall, with the exception of the condition index, these results suggest that environmental factors, such as temperature, salinity, turbidity, phytoplankton abundance and species composition, phycotoxins and faecal contamination, were not the main causes of variations on biomarker responses between sites and seasons. However, further studies should be carried out to verify this hypothesis.

Use of transplanted juvenile oysters to differentiate sites depending on their contamination levels and biological effects

Site sample analysis (Fig. 4b) shows three well-defined groups, depending on levels of contaminants: 1) the first group, with Bs and Bw, is clearly defined by the levels of PCBs, PBDEs and lindane, 2) the second group, with Les Palles in summer (LPs) and winter (LPw), Boyard in winter (BOYw) and Mus du Loup in winter (MLw), is clearly defined by the levels of HPAHs and DDTs, and 3) the third group encompasses all the other sites, i.e. BOY in summer (BOYs), ML in summer (MLs) and Cordouan in summer (Cs) and winter (Cw). Similar correlations were observed between contaminant levels and MDA in gills and digestive gland and between contaminant levels and SOD in plasma and mantle (Fig. 4a), suggesting that, if only two tissues should be selected for in situ studies in C. gigas, measurement of MDA only in the digestive gland and measurement of SOD only in plasma, would be enough to discriminate sites with high levels of HPAH and DDT contamination. In addition, GPx in plasma could also contribute to discriminate sites with high levels of HPAH and DDT contamination. On the other hand, SOD and lysozyme in the digestive gland, and laccase in plasma could contribute to discriminate sites with high levels of PCB, PBDE and lindane contamination. Thus, in the present study, SOD, GPx, MDA, laccase, lysozyme and proteins in the digestive gland and SOD, GPx and laccase in plasma contributed to discriminate sites, with more evident bioaccumulation of organic pollutants, especially HPAHs, DDTs, PCBs, PBDEs and lindane.

In a transplantation study carried out in the Ebro Delta (NE Spain) with the freshwater clam (Corbicula fluminea) for a 3-month period, measured biomarker responses (antioxidant and esterase enzyme responses, and lipid peroxidation) showed marked differences across sites and months, depending on the levels of pesticides present in these sites [START_REF] Damasio | Identifying major pesticides affecting bivalve species exposed to agricultural pollution using multibiomarker and multivariate methods[END_REF]. In a transplantation study in the Cecine River (Italy), the freshwater painter´s mussel (Unio pictorum) was transplanted for 4 weeks (1 month) in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses [START_REF] Guidi | Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin[END_REF].

Considering the mild contamination gradient in the investigated area, the overall results suggested that some oxidative biomarkers, as well as those evaluating chromosomal and cell damages, were highly sensitive and could be profitably applied to caged painter's mussels for environmental quality assessment in freshwater. In a transplantation study in the South coast of Portugal, transplanted mussels Mytilus galloprovincialis had significant alteration in some biomarkers that reflected the type of contaminants present in each site (metals or PAHs), and the biomarkers that better differentiate the impact of different contaminants at each site were metallothioneins, cytochrome P450, SOD and CAT, measured in the digestive gland. Thus, results of the present study in are in agreement with previous in situ studies in which biomarker responses have shown marked differences across sites depending on the level of contamination.

Conclusion

In conclusion, among the battery of biomarkers applied in the present work (including antioxidant defence mechanisms, humoral immune defence mechanisms, and physiological biomarkers), MDA and lysozyme in the digestive gland and SOD, GPx and laccase in plasma allowed to significantly discriminate sites in which oysters bioaccumulated different levels of HPAHs, DDTs, PCBs, PBDEs and lindane. In combination with other recent studies, these results strengthen the hypothesis that it is possible to differentiate sites depending on their contamination levels and biological effects by carrying out studies with transplanted juvenile oysters.

Interestingly, SOD, GPx and MDA levels were positively correlated to HPAH and DDT body burdens and were associated to different sites in the Marennes-Oléron Bay (LP, BOY and ML). These results suggest a potential oxidative stress, leading to lipid peroxidation in juvenile oysters, and seems to be related not to external inputs from the Gironde Estuary, but to local inputs of HPAHs and DDTs, probably from the Charente and the Seudre rivers. An increase in oxidative stress and lipid peroxidation could affect defence mechanisms in juvenile oysters, which may contribute to the increase in disease and mortality of C. gigas, observed in the Marennes-Oléron Bay. Therefore, more studies should be done to further examine the effects of these organic contaminants on C. gigas.

  of exposure to organic contaminants on antioxidant defence responses, immune defence responses, energy reserves and condition index in juveniles of C. gigas General trends of biomarker responses in oysters from transplantation sites in the Marennes-Oléron Bay and the Gironde Estuary are presented in this paragraph in comparison to biomarker responses in oysters from B (Table

Figure 1 .

 1 Figure 1. Estuarine sampling areas in French Atlantic coast. Location of reference (B, Bouin) and transplantation sites: Boyard (BOY), Les Palles (LP), Mus du Loup (ML) in the Marennes-Oléron Bay and Cordouan (C) in the Gironde Estuary.

Figure 2 .

 2 Figure 2. Flesh dry weight (a), shell dry weight (b) and condition index (c) in C. gigas from reference site (Bouinwhite bars, B), and transplantation sites (Boyard -light grey bars, BOY-, Les Palles -medium grey bars, LP-, Mus du Loup -dark grey bars, ML-and Cordouan -black bars, C-), in summer (S) and in winter (W). Data are expressed as mean ± SD. For clarity of results, only significant differences between reference and transplantation sites are shown; * p<0.05, ** <p0.01, *** p<0.001. Superscript letters indicate significant (p<0.05) differences between B samples from summer and winter.

Figure 3 .

 3 Figure 3. Energy reserves (glycogen, lipids, proteins) in the digestive gland of C. gigas. a. Energy reserves from reference site (Bouin, white bars), and transplantation sites (Boyard -light grey bars-, Les Palles -medium grey bars-, Mus du Loup -dark grey bars-and Cordouan -black bars-), in summer (S) and in winter (W). Data are expressed as mean ± SD. For clarity of results, only significant differences between reference and transplant sites are shown; * p<0.05, ** <p0.01, *** p<0.001. Superscript letters indicate significant (p<0.05) differences between B samples from summer and winter. b. Levels of energy reserves (glycogen in white, lipids in black, proteins in grey) are expressed in percentage (%) relative to the total flesh dry weight. c. Levels of energy reserves (glycogen in white, lipids in black, proteins in grey) are presented in percentage (%) relative to the total fresh dry weight, with the total fresh dry weight standardized for all sites and seasons to 100%. B: Bouin, BOY: Boyard, LP: Les Palles, ML: Mus du Loup, C: Cordouan, S: Summer, W: Winter.

Figure 4 .

 4 Figure 4. Redundancy analysis (RDA) ordination diagram with (a) contaminant contents (straight arrows), and biochemical parameters (dotted arrows) and (b) sampling sites. First axis is horizontal, second axis is vertical. The first axis displays 42% of the remaingroupng variation in contaminants in oysters, the second axis another 35%. Only biomarkers that significantly (p<0.05) contributed to discriminate sites depending on their degree of contamination were used to carry out the RDA analysis. HPAHs: heavy polycyclic aromatic hydrocarbons; PCBs: polychlorobiphenyls; PBDEs: polybromodimethylethers ; DDTs: dichlorodiphenyltrichloroethanes, dichlorodiphenyldichloroethanes and dichlorodiphenyldichloroethylenes; SOD: superoxide dismutase; GPx: glutathione peroxidase; MDA : malondialdehyde ; G : gills ; DG : digestive gland ; M : mantle ; P : plasma; B: Bouin; BOY: Boyard; LP: Les Palles; ML: Mus du Loup; C: Cordouan; s: summer; w: winter; 1: replicate 1; 2: replicate 2; 3: replicate 3.

Table 1 .

 1 Contaminant contents in oyster's flesh at two seasons after 3 months of transplantation. Mean ± SD (µg kg -1 dw), n =3 (i.e. 3 replicates from 1 pool of 5 oysters). B: Bouin; BOY: Boyard; LP: Les Palles; ML: Mus du Loup; C: Cordouan; dw: dry weight; nd: not detected. PAHs: polycyclic aromatic hydrocarbons; LPAHs: low molecular weight PAHs; HPAHs: high molecular weight PAHs; PCBs: polychlorobiphenyls; PBDEs: polybromodimethylethers; DDTs (DDT and metabolites: dichlorodiphenyltrichloroethanes (2,4´-DDT and 4,4´-DDT) + dichlorodiphenyldichloroethylenes (2,4´-DDE and 4,4´-DDE) + dichlorodiphenyldichloroethanes (2,4´-DDD and 4,4´-DDD)); HCB: hexachlorobenzene; HC: heptachlor; TNC: trans-nonachlor.

		Concentration (µg kg -1 dw)
	Contaminants	Summer					Winter
		B	BOY	LP	ML	C	B	BOY	LP	ML	C
	LPAHs	68±6	58±9	47±14	27±1	45±25	69±16	68±19	60±12	41±5	44±5
	HPAHs	60±1	81±10	141±11	54±10	35±7	87±10	167±25	145±3	159±34	76±12
	PCBs	81±9	30±3	23±4	15±1	39±12	89±39	47±3	30±3	17±6	44±4
	PBDEs	0.9±0.0	0.4±0.4	0.4±0.3	0.4±0.0	0.9±0.2	2.9±0.7	0.6±0.3	1.0±0.7	0.6±0.1	0.5±0.1
	DDTs	6±1	8±1	6±1	2±0	3±0	6±3	15±0	10±1	4±0	8±2
	Lindane	2.1±0.4	0.6±0.1	0.4±0.0	0.1±0.1	0.1±0.0	2.2±1.1	0.5±0.2	1.3±0.3	0.6±0.1	0.7±0.4
	HCB+HC+TNC	2.2±0.4	2.4±0.9	2.0±0.3	0.8±0.0	0.8±0.0	1.8±0.8	3.5±1.0	4.1±1.9	0.7±0.0	1.9±0.1

Acknowledgments

This study was supported by a PhD grant from the Conseil Général de the Charente-Maritime for A. Luna-Acosta. Authors wish to thank M. Baudrimont and D. Fichet for supporting this study through the 'Défi Cadmium' project, financed by Région Poitou-Charentes, Région Aquitaine, Conseil Général de la Charente-Maritime, Conseil Général de la Gironde, and Agence de l'Eau Adour-Garonne. CPER A2E is also acknowledged for financial support. Authors acknowledge also R. Galois and M. Metian for their scientific advice for lipid and glycogen content and condition index analyses, M. Caille, J. Laheux and M.

Pelmoine for their assistance during the study, and the Ifremer networks ROCCH and RAZLEC, the 'Site Ifremer consacré à l'environnement littoral' and especially O. Le Moine, P. Soletchnik, S. Guesdon, P-G. Sauriau, P. Miramand and T. Guyot, for data and references from Marennes-Oléron Bay. Authors acknowledge also C. Trichet and D. Vilday for their technical assistance during the study. Authors wish to warmly thank also T. Caquet and P. Legendre for their scientific advice and help in statistical analyses.

Table 2. Biomarker responses in the gills, digestive gland, mantle and plasma in C. gigas from reference, i.e. Bouin, and transplantation sites, i.e. Boyard, Les Palles, Mus du Loup and Cordouan, in summer and winter. Data are expressed as mean ± SD (n=3 pools from 3 individuals). For clarity of results, only significant differences between reference and transplantation sites are shown; * p<0.05, ** <p0.01, *** p<0.001. 10 5-6 10 5-6 10 5-6 10 5-6 10 5-6 ----10 3-4 Paralia sulcata (Diatomophyceae) -10 5-6 10 5-6 10 5-6 10 3-4 -10 3-4 10 3-4 10 3-4 10 5-6 Skeletonema costatum (Diatomophyceae)

10 5-6 ---10 5-6 10 3-4 <10 3 <10 3 <10 3 -Thalassiosira sp. (Diatomophyceae) ----10 5-6 10 3-4 ----Gymonodiniaceae (Dinophyceae) ----10 5-6 -----Phytoplancton producing phycotoxins (cells l -1 ) a -Alexandrium sp. (Diatomophyceae)


domain: chim chim.cata sde

text:
Flora Chambon

Franck Rataboul

Catherine Pinel

Amandine Cabiac

Emmanuelle Guillon

Nadine Essayem
email: nadine.essayem@ircelyon.univ-lyon1.fr

Conversion of cellulose to 2,5-hexanedione using tungstated zirconia in hydrogen atmosphere

Keywords: cellulose hydrolysis, solid acid catalysts, Lewis acidity, tungstated zirconia, hydride transfer, 2, 5-hexanedione

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction:

For environmental and sustainable development concerns, researchers are doing a spectacular efforts in using renewable resources for the production of biofuels and platform chemicals, especially the lignocellulosic one due to its large availability and non-edibility. Cellulose, the main component of the lignocellulose is a biopolymer composed of glucose units linked by β-1,4-glycosidic bonds which constitutes a material of choice since it is seen as a glucose reserve. Intermolecular and intramolecular hydrogen bonds existing in the crystalline network of the cellulose confers to it a high resistance to chemical conversion in conventional solvents [START_REF]Biomass recalcitrance. Deconstructing the Plant Cell Wall for Bioenergy[END_REF]. However, it is known that hydrolysis of cellulose into sugars can be catalysed by strong mineral acids such as H 2 SO 4 , HCl and HF. Because mineral acids are inexpensive, this method remains the most often used in research pilot development or in the few processes at industrial scale [START_REF]Biorefineries-Industrial processes and products[END_REF]. This method using liquid acids produces glucose but liquid acids cause corrosion and waste elimination remains problematic. Moreover, if acid hydrolysis conditions are severe (high temperature and/or concentration), a large fraction of sugars are degraded to undesired oligomers and tars. Later, enzymatic catalysis has been introduced with very selective cellulases for glucose production. However, these enzymes remain expensive and have still a rather low activity [START_REF]Biorefineries-Industrial processes and products[END_REF]. Recently, to try to overcome these drawbacks, other methods using heterogeneous catalysis in hydrothermal conditions appeared as an important breakthrough. From the pioneering works of Fukuoka in 2007, we can distinguish three main routes of cellulose transformation based on the nature of the bioproducts derived from glucose: hydrogenation, hydrogenolysis, or dehydration [START_REF] Cabiac | [END_REF]. The first one is based on noble metals such as Ru/C [4] or Pt/Al 2 O 3 [Erreur ! Signet non défini.] and leads to the formation of sorbitol directly from cellulose. The second routes produces shorter polyols in hydrogenolysis conditions, typically using transition metal catalysts and higher temperatures, i.e. ethylene glycol formation over Ni/WC at 245°C [5]. The third one concerns the use of BrØnsted acid catalysts such as sulfonated carbon [6,7,8] under milder conditions of temperature to give glucose and its derivatives formed via BrØnsted acid catalized mechanism: 5-HMF and levulinic acid.

Beside these three main ways of cellulose transformation, a fourth one can be considered, using Lewis acids as catalysts to produce lactic acid in aqueous media [9] or lactate in alcoholic media [10]. In a previous study, we have shown that solid Lewis acids, such as tungstated alumina and tungstated zirconia, promote selectively the cellulose depolymerisation in lactic acid in water. We proposed that the solid Lewis acid sites intervene in cooperation with the pool of hydroxonium ions provided by the water selfprotolysis at 190°C to depolymerise selectively a crystalline cellulose in Lactic acid. The Lewis sites are proposed to promote C-C and C-O scissions via coordination of the hydroxyl groups of the soluble polysaccharides with the Lewis centers.

The present paper reports the influence of H 2 /He atmosphere on cellulose conversion using tungstated zirconia as catalyst in hot water (190°C). Indeed, since noble metal free ZrW were previously reported to interact positively with molecular hydrogen in an acidic demanding reaction, alkane isomerisations in gas phase [11], we wanted to check if such interaction might occur in the aqueous medium and influence the cellulose conversion.

Experimental section:

Tunsgtated zirconia synthesis

Tungstated zirconia was prepared by anionic exchange between a tungstic acid (Aldrich) solution in hydrogen peroxide (Aldrich, H 2 O 2 30%) and zirconium hydroxide according to a patented method [12]. Zirconium hydroxide was prepared by precipitation at pH 9 with a zirconyl chloride solution (ZrOCl 2 .8H 2 O, Aldrich) and ammonia solution 1.8 M. After agitation of the suspension for 20 minutes, the mixture was centrifuged and the precipitate washed several times with deionised water to remove chloride ions. The solid was then dried at 110°C for 24 h. Then, 10 g of zirconium hydroxide were exchanged with the tungstic acid solution (0.25 M) in hydrogen peroxide (30%, 150 mL) for 15 minutes. The exchanged solid was filtered and dried at 80°C for 24 h. Finally, the solid was calcined at 700°C under air flow for 3 h [13]. The XRD pattern of ZrW shows the exclusive formation of the tetragonal ZrO 2 phase as expected from the optimized method of preparation via anionic exchange [14]. The W loading and the BET surface area equal 11.8 wt% and 219 m 2 .g -1 respectively.

In situ FTIR studies FTIR spectra of self-supported pellets of ZrW were recorded with a Brucker Vector 22 spectrometer in the absorption mode with a resolution of 2 cm -1 . The samples were placed in an IR cell equipped with CaF 2 windows and treated in situ. The wafer of ZrW samples was first pretreated at 400°C under flowing oxygen for 2 hours. Pyridine vapor pressure was introduced in the cell for 15 min at ambient temperature and the ZrW pellet was desorbed at 150°C for 1 hour in order to remove the physisorbed pyridine species. Then 100 torrs of hydrogen were introduced in the cell at ambient temperature, and the ZrW pellet, previously saturated with pyridine, was treated at increasing temperature in H 2 atmosphere. Finally, hydrogen was vacuum treated at ambient temperature, in order to compare the pyridine spectra before and after hydrogen treatment.

Classical Batch experiments

Untreated microcrystalline cellulose was purchased from Sigma-Aldrich and used as received (degree of polymerisation = 250, crystalline index 70%, particles size 20 nm).

Reactions were performed in a 100 ml Parr Hastelloy autoclave equipped with a Rushton turbide. The autoclave was filled with cellulose (1.6g), catalyst (0.68g) and de-ionized water (65 ml). The autoclave was flushed three times with He at room temperature, and then heated up at 190°C (5°C/min). The pressure was adjusted to 5 MPa with He or H 2 for 24 hours. The reaction was stopped by cooling the autoclave with an ice bath. The pressurized gas was evacuated, the reaction mixture was filtered and the liquid phase and the residual solid were collected.

Kinetic experiments

The Parr Hastelloy autoclave was connected to a 20 ml stainless steel sampling vessel, pressurized with He or H 2 at 5 MPa. Typically, the autoclave was filled with catalyst (0.68g) and de-ionized water (48 ml). A suspension of cellulose (0.8g) in de-ionized water (18 g) was introduced into the pressurized sampling vessel connected to the autoclave. The autoclave is flushed three times with He and then heated up to 190°C. When the temperature is reached, the cellulose suspension in the sampling vessel is introduced into the autoclave under 5 MPa of He or H 2 .This is the time zero of the reaction.

Analytical methods

The liquid phase was analyzed by a HPLC system with an ICE COREGEL 107H column (300mm x 7.8mm, 80°C) and a Refractive Index detector using acidified water as eluent (0.5 mL/min, 0.01% mol H 2 SO 4 ). The liquid phase was also analyzed on a Shimadzu TOC-V SCH Total Organic Carbon (TOC) analyzer (720°C, Pt/Al 2 O 3 catalyst, IR detector).

The solubilisation percentage, also defined as the conversion percentage, was calculated as the ratio of the total mass of carbon in the liquid phase obtained from TOC analysis and the initial mass of carbon in the charged cellulose: Conv (%) = Solubilisation (%) = 100*(mgC liquid phase /mgC initial cellulose )

The carbon yields of the products detected by HPLC were calculated as the molar ratio of the product i and the initial glucosyl units present in the initial cellulose, corrected by the number of carbon atoms: Yield i (%)= 100* (nC i /6)*(n i /n glucosyl units ) nC i : number of carbon atoms in the product i n i : number of moles of the product i determined by the HPLC analysis n glucosyl units : initial number of moles of glucosyl units in the cellulose sample = m cellulose /162

The yield of soluble oligosaccharides and polymers (SOP) was deduced from the difference between the conversion and sum of the yields of products analysed by HPLC:

Yield SOP (%) = conv(%)-Σyield i (%)

Results and discussion

Influence of hydrogen or helium atmosphere on the cellulose conversion over ZrW

The results of cellulose conversion at 190°C for 24 hours under hydrogen or helium atmosphere in the presence or absence of ZrW was summarized in Table 1. Blank experiments, done without solid catalysts and under H 2 or He, are very close and lead to cellulose conversions of 31-32% without any selective formation of chemicals. When the reaction was performed using ZrW, under He atmosphere, a lactic acid yield of 18.5% was obtained with a cellulose conversion of 35%. Beside, under hydrogen, in the presence of ZrW, the cellulose conversion was raised to 65%, together with yields of 14% in lactic acid and of 24% in 2,5-hexanedione, a product rarely reported to our knowledge. 2,5-hexanedione was identified by GC-MS (S1: supplementary information). A comparative experiment, performed with W free ZrO 2, lead to limited yields in lactic acid and 2,5-hexanedione, 4% of each, but the cellulose conversion was enhanced by 10% compared to the blank experiments.

These results highlight that hydrogen atmosphere have a remarkable promoting effect on the rate and on the selectivity of cellulose reaction in the presence of tungstated zirconia. Thus, these data strongly suggest that the Lewis acid centres provided by W atoms, which were previously identified as the active centres responsible for lactic acid formation [9] could be also involved in the formation of the 2,5 hexanedione in the presence of molecular hydrogen.

FTIR studies of hydrogen-ZrW Lewis acid sites interactions.

The influence of the hydrogen atmosphere was evaluated by FTIR spectroscopy in gas phase assuming that if a peculiar interaction exist in the gas phase this might hold true in the liquid phase. Thus, we studied the interaction of the Lewis acid centres with molecular hydrogen after saturation of dehydrated ZrW sample with pyridine and subsequent evacuation at 150°C for 1h to remove the physisorbed pyridine species. Thereafter, hydrogen was introduced in the IR cell and the temperature was increased stepwise to 250°C.

Pyridine adsorption on ZrW resulted in the appearance of bands (Fig. 1-a) due to pyridine adsorbed on Lewis sites (1445 cm -1 ) and BrØnsted acid sites (1538 and 1643 cm -1 ). Upon hydrogen introduction and further temperature increase, the band due to pyridine coordinated to the Lewis sites at 1445 cm -1 was shifted to 1440 cm -1 , wavenumbers value rather ascribed to physisorbed pyridine, in good agreement with the formation of new bands in the range 1550-1650 cm -1 . Evacuation of ZrW at 150°C after hydrogen introduction (Fig. 1.e) led to the disappearance of the previous bands, and to a decrease of the intensity of the Lewis band at 1445 cm -1 and a parallel increase of the intensity of the BrØnsted band at 1643 cm -1 although the most characteristic vibration of the pyridinium species at 1538 cm -1 remained almost unchanged. Nevertheless, these IR results have shown that molecular hydrogen have modified the Pyridine IR spectra. This might indicate that molecular hydrogen interacts directly with the Lewis acid sites of ZrW but this could also results from a slight reduction of the W species. Note that in situ formation of BrØnsted acid sites ascribed to slight reduction of WOx domains was already suggested by some authors [15] but this was also denied by others [16].

This issue remains controversial.

The observed increase of the cellulose conversion under hydrogen atmosphere compared to helium could be correlated, in a simple way, to in situ formation of new BrØnsted sites from hydrogen heterolytic splitting promoted by ZrW. However, if such a phenomenon might intervene and explain the increase of the cellulose conversion, it does not explain the product distribution changes observed on ZrW when helium was replaced by hydrogen.

Kinetic studies: mechanism pathways proposition

The products of cellulose conversion obtained in the previous batch experiments, after 24h of reaction, are the results of successive transformations. Over ZrW catalysts, in neutral atmosphere, lactic acid formation is favoured, while under hydrogen, the selectivity shifts towards 2,5-hexanedione. This peculiar behaviour in term of selectivity as a function of the reaction atmosphere (He or H 2 ) is not straightforward.

To address this issue several kinetic experiments were carried out in the presence of ZrW under hydrogen or helium atmosphere.

Figures 2 and3 presents the kinetic results showing the evolution with time of cellulose conversion and of the main products yields: lactic acid, 2,5-hexanedione, acetol, 5-HMH, carboxylic acids. In a general rule, the higher selectivity of the main products, lactic acid, acetol or 2,5-hexanedione, is observed initially whatever the conditions employed. This could indicate either they are primary products or that the disappearance rate of eventual intermediary primary products is high.

First of all, we can observe that the cellulose conversion is largely improved in the presence of the solid Lewis acid ZrW in the presence of hydrogen as well initially than after 30 hours of reaction ( Fig. 2a ,S2). ZrW used in hydrothermal conditions and under hydrogen leads to an initial rate of cellulose conversion increased by one fourth compared to the blank experiment (Table 2) and to a cellulose conversion of 72% after 30 hours of reaction. To explained this initial enhancement of the initial rate we can propose, based on the previous FTIR studies, that hydrogen contacting ZrW might provide supplementary protonic acidity which might participate, in cooperation with the self protolysis of water, to the cellulose hydrolysis in hot water [17]. In the blank experiment, after 8 hours of reaction, we can observe that the cellulose conversion achieve 40% (S2) and looking to the product yields (S3), no product is formed with a selectivity higher than 6% after 30h of reaction. One can also remark that the sum of the yields of products detected by HPLC represents roughly 50% of the cellulose conversion (S2), indicating than half of the product are soluble oligomers (humins), not detected by HPLC.

The presence of ZrW under helium atmosphere does not increased the cellulose conversion but has improved the selectivity of the transformation since the sum of the yields of products detected by HPLC are equivalent to the cellulose conversion determined by TOC analysis (Fig. 2a). This indicates that humins formations are suppressed as regards to the blank experiment (S2). Fig. 2b clearly shows that ZrW catalyst has a strong impact on the reaction selectivity: the high selectivity towards lactic acid is observed all along the transformation (Fig. 2b), the lactic acid yield continuously increases with the reaction time and achieves 20% after 30 h of reaction. Note, that initially the yield in 5-HMF equals that in lactic acid but with the course of the reaction, lactic acid becomes the dominant products, the yields in other products remain below 5% (levulinic, formic,acetic acids, acetol, 5-HMF and pyruvaldehyde).

As previously proposed [9], it seems that the presence of a solid Lewis acid in the reaction medium limits further transformations of the first oligosaccharides issued from the cellulose depolymerisation in humins, process catalyzed by the pool of hydroxonium ions provided by water self-protolysis. Accordingly, the first soluble species are proposed to be coordinate to the Lewis acid sites at the surface of the catalyst, and could undergo successive transformation steps leading to the selective formation of lactic acid.

It is proposed that solid Lewis acid sites could coordinate hydroxyl function groups, and provide carbocations at the catalyst surface by deshydroxylation. We propose that this is the driving force to convert selectively cellulose in lactic acid in hydrothermal conditions as previously suggested [9].

According to the above results, the following mechanisms are suggested (Scheme1):

We can propose that a Lewis acid site could coordinate the primary hydroxyl function of a glucose unit at the reductive end of a soluble oligosaccharide chain (Scheme 1, (3)). The protolysis of the glucosidic bond provides a glucose unit coordinated to a superficial Lewis center (4). Then, this could lead to a pull of OH -species, giving a primary carbocation on the C6 carbohydrate (5) and a superficial specie OH -L. This intermediate ( 5) could be stabilized by intramolecular hydride transfer to give specie (6) (primary to tertiary carbocation). Then a β-scission could produce the 3 carbons intermediates (7a and 7b).

The specie 7a could lead to acetol (8) formation via keto-enolic equilibrium. Attack of the superficial specie OH -L could also produce lactic acid (9). Furthermore, the specie 7b could undergo a successive chain mechanism giving formation of pyruvaldéhyde (10) or via βscission and attack of superficial specie OH -L, the formation of formic acid (12) and acetic acid could be explained.

The most efficient catalytic system, not only on the initial rate of cellulose conversion but also on the final cellulose conversion, is ZrW used under hydrogen atmosphere. Indeed, in these conditions, the initial rate of cellulose conversion is increased by one fourth as regards to the blank experiment (Table 2) and the final cellulose conversion reached 65% in the batch experiment while, under He, the cellulose conversion is roughly two times lower. As proposed above, this can be explained by the additional formation of Brφnsted sites due to the interaction of hydrogen with the Lewis acid centers but we can not exclude that the coordination of the reductive end of the first soluble oligosaccharide by the Lewis center might shift the cellulose solubilisation equilibrium.

Concerning the evolution of the products with time (Fig. 2b), we can observe high initial selectivities in acetol and in a lesser extent in lactic acid and 2,5-hexanedione, formic acid and pyruvaldehyde. After 30h of reaction, 2,5-hexadione and acetol became the main products.

To rationalize the kinetic data observed for cellulose conversion over ZrW in the presence hydrogen, we propose that fast hydride transfer steps induced by heterolytic cleavage of hydrogen interacting with the Lewis center of ZrW could account for the selectivity changes.

Therefore Scheme 2 proposes a reaction mechanism including hydride transfer steps to explain the formation of acetol and 2,5-hexanedione in the presence of ZrW/H 2 system. As said previously, the first soluble oligosaccharides (1) are generated from cellulose hydrolysis catalyzed by the hydroxonium ions issued from water self-protolysis at 190°C. The glucose unit at the reductive end of the first soluble oligosaccharide (2) could be coordinated via the primary hydroxyl group to the Lewis acid site of ZrW and undergo a chain mechanism as proposed in Scheme 2. The remaining oligosaccharide chain (3b) may undergo a fast hydride transfer step in the presence of ZrW/H 2 system (3b) and then produces a partially dehydroxylated polysaccharide end (4). This specie could undergo further classic protolysis steps and produce partially dehydroxylated C6 species (5).

Lewis acid sites of the solid catalyst could further coordinate specie (5) and remove the primary hydroxyl group to produce a primary carbocation (6) which rapidly give a more stable tertiary carbocation (7) after an intramolecular hydride transfer step. A β-scission of specie 7 could form 2,5-hexanedione (8) after several elementary steps. Further β-scission and hydride transfer steps on specie 7 could give acetol (9).

Conclusions

It is well known that the major barrier of cellulose conversion is its initial depolymerisation, i.e its resistance to hydrolysis in mild conditions. We have shown here that the system H 2 /ZrW increases by two fold the cellulose depolymerisation in hot water without loss of carbons in humins formation.

The action of solid Lewis acid catalyst on the cellulose transformation is proposed to be centered on the first soluble species generated by cellulose glycosidic bonds cleavage by the hydroxonium ions of aqueous media. BrØnsted solid acid sites was proposed to boost the initial depolymerisation of cellulose while the presence of solid Lewis acid sites might shift the equilibrium via coordination of the first soluble species, which might prevent their evolution towards humins via a classical protonic chain mechanism. The interaction between solid Lewis acid sites and the soluble species led to the selective formation of lactic acid in absence of hydrogen: hydroxyl groups abstraction is proposed to be the driving force.

The addition of hydrogen in the medium has several interesting effects. It permits not only to raise the cellulose conversion but also to change the selectivity of the reaction toward 2,5hexanedione formation. ZrW/H 2 system is proposed to promote fast hydride transfer steps.

Novel mechanism pathways have been proposed in this study, to highlight the conjugated effect of the aqueous media and the heterogeneous Lewis acid catalysis on the cellulose conversion proposed to catalyze two main elementary steps: hydride transfers and hydroxyls group abstraction.