Asari (=Manila) clam, Ruditapes philippinarum, is the second bivalve mollusc in terms of production in the world and, in many coastal areas, can beget important socio-economic issues. In Europe, this species was introduced after 1973. In Arcachon Bay, after a decade of aquaculture attempt, Asari clam rapidly constituted neo-naturalized population which is now fished. However, recent studies emphasized the decline of population and individual performances. In the framework of a national project (REPAMEP), some elements of fitness, stressors and responses in Arcachon Bay were measured and compared to international data (41 publications, 9 countries). The condition index (CI = flesh weight/shell weight) was the lowest among all compared sites. Variation in average Chla concentration explained 30% of variation of CI among different areas. Among potential diseases, perkinsosis was particularly prevalent in Arcachon Bay, with high abundance, and Asari clams underwent Brown Muscle Disease, a pathology strictly restricted to this lagoon. Overall element contamination was relatively low, although arsenic, cobalt, nickel and chromium displayed higher values than in other ecosystems where Asari clam is exploited. Finally, total hemocyte count (THC) of Asari clam in Arcachon Bay, related to the immune system activity, exhibited values that were also under what is generally observed elsewhere. In conclusion, this study, with all reserves due to heterogeneity of available data, suggest that the particularly low fitness of Asari clam in Arcachon Bay is due to poor trophic condition, high prevalence and intensity of a disease (perkinsosis), moderate inorganic contamination, and poor efficiency of the immune system. (C) 2015 Elsevier Ltd. All rights reserved.

The originality of this project consists in the interdisciplinary approach gathering the impact of toxic phytoplankton blooms and metallic pollution on health (behaviour, stress, immunity, genetic) and disease development of the Manila clam, an economieally important species. The Manila clam Ruditapes philippinarum is the second exploited bivalve in the world. It was introduced in Arcachon bay during the 80's and is today exploited by professional fishermen (57 licences). A 50% decrease of the stock was observed during the last five years. Beyond fishing pressure, a PhD thesis identified several diseases as potential threats, like perkinsosis (=Dermo) and brown muscle disease (BMD). The project confirms the settlement of these pathologies with high prevalence and intensity, impacting clams growth and condition index. Perkinsosis and BMD rather develop in elevated mudflats with lower water turnover (north-western area). The hypothesis that the BMD agent would be a virus is still valid although it had not been yet demonstrated. Phytoplanctonic related toxins in Manila clams remain very low but the occurrence of spirolids justifies our project to experiment multi-contamination. Beyond the technological challenges (toxic culture, multitreatments and multi-responses protocol), this project highlighted effects of toxie algae and/or metal contamination on histologieal, immune, behavioural and molecular parameters. Our expertise was partly acquired with the assistance of the Fishing Committee and, in return, provided help this Committee for their own fishing strategies

Oysters (Crassostrea gigas) were exposed 4 days to cultures of the toxic dinoflagellate Alexandrium ostenfeldii (strain CCMP1773) that produces spirolides belonging to fast acting toxins (FAT) and let depurate for 7 days. During depuration, oysters were either fed the non-toxic algae Isochrysis galbana Tahitian clone (T. Iso) or starved. The objectives of this experiment were to evaluate (i) spirolide uptake and depuration by oysters (ii) spirolide effects on oysters and (iii) oyster recovery according to food supply during depuration.A. ostenfeldii cells were filtered and ingested by oysters while faeces contained numerous intact cells of the toxic diet. This suggested that ingested cells were not totally digested by oysters. Contents of spirolides (SPX) in digestive gland and remaining tissues during contamination and detoxification periods were measured by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Four different SPX analogues (13,19-didesMeC, 13-desMeC, 13-desMeD and trace of SPX-D) were detected. The 13,19-didesMeC-SPX dominated in both digestive gland and remaining tissues. After four days exposure, digestive gland (DG) contained 83% of the total initial spirolide concentration, whereas remaining tissues contained only 17%. During detoxification, spirolide content in DG was lower in fed than in unfed oysters but similar in remaining tissues.Exposure to A. ostenfeldii resulted in an inflammatory response consisting of hemocyte infiltration and diapedesis into the intestinal tract of the oysters. Percentage of active digestive tubules in oysters fed A. ostenfeldii was significantly lower than in control (prior exposition) oysters (36% and 61%, respectively). At the end of the detoxification period, there was a significant difference in the percentage of active digestive tubules (P<0.001) between fed and unfed oysters. When oysters were fed T-Iso following the A. ostenfeldii exposure, 80% of digestive tubules were active, thus revealing a rapid recovery after toxic algae exposure.Overall, both spirolide detoxification and recovery from their toxic effects are almost complete within 7 days after exposure to spirolide producers. Such informationmay help to resume faster oyster sales after toxic events involving FAT implying thus more frequent chemical analysis. (C) 2012 Elsevier B. V. All rights reserved.

The purpose of this study was to assess paralytic phycotoxin uptake in diploid and triploid oysters at two stages of their sexual cycle corresponding to their status in early summer (June) and winter (November). Samples of diploid and triploid oysters were exposed to a toxic culture of Alexandrium minutum for 4 days in each season. No significant differences in filtration or clearance rates were observed during either November or June experiments. When diploid oysters were at resting stage (November), toxin uptake showed no significant difference between the ploidy classes. In contrast, when the diploid oysters were at the peak of their sexual maturation (June), the triploid oysters were seen to accumulate almost double the amount of paralytic toxins as the diploid ones.

Growth and spirolide production of the toxic dinoflagellate Alexandrium ostenfeldii (Danish strain CCMP1773) were studied in batch culture and a photobioreactor (continuous cultures). First, batch cultures were grown in 450 mL flasks without aeration and under varying conditions of temperature (16 and 22 degrees C) and culture medium (L1, f/2 and L1 with addition of soil extract). Second, cultures were grown at 16 degrees C in 8 L aerated flat-bottomed vessels using L1 with soil extract as culture medium. Finally, continuous cultures in a photobioreactor were conducted at 18 degrees C in L1 with soil extract; pH was maintained at 8.5 and continuous stirring was applied.This study showed that A. ostenfeldii growth was significantly affected by temperature. At the end of the exponential phase, maximum cell concentration and cell diameter were significantly higher at 16 degrees C than at 22 C. In batch culture, maximum spirolide quota per cell (approx. 5 pg SPX 13-desMeC eq cell(-1)) was detected during lag phase for all conditions used. Spirolide quota per cell was negatively and significantly correlated to cell concentration according to the following equation: y = 4013.9x(-0.858). Temperature and culture medium affected the spirolide profile which was characterized by the dominance of 13,19-didesMeC (29-46%), followed by SPX-D (21-28%), 13-desMeC (21-23%), and 13-desMeD (17-21%).Stable growth of A. ostenfeldii was maintained in a photobioreactor over two months, with maximum cell concentration of 7 x 10(4) cells mL(-1). As in batch culture, maximum spirolide cell quota was found in lag phase and then decreased significantly throughout the exponential phase. Spirolide cell quota was negatively and significantly correlated to cell concentration according to the equation: y = 12,858x(-0.8986). In photobioreactor, spirolide profile was characterized by higher proportion of 13,19-didesMeC (60-87%) and lower proportions of SPX-D (3-12%) and 13-desMeD (1.6-10%) as compared to batch culture. (C) 2011 Elsevier B.V. All rights reserved.

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. Contamination des mollusques bivalves par des phycotoxines : interactions entre niveaux de toxicité et réponses physiologiques Patrick Lassus, Philippe Soudant, Jean-Charles Massabuau, Michèle Bardouil, Régis Baron, Marielle Guéguen, Hansy Haberkorn, Christophe Lambert, Nelly Le Goïc, Claire Marcaillou, et al.

The aim of this study was to evaluate the importance of biotransformation of paralytic shellfish toxins during the detoxification process in contaminated oysters. Mathematical models based upon the detoxification patterns of digestive gland and other tissues were developed. It was demonstrated that biotransformations do not seem to play an important role in digestive gland or other tissue detoxification kinetics with our data set. Moreover, different toxin transfers from digestive gland toward other tissues were investigated. No significant transfer was highlighted in our data set. These first conclusions were drawn after comparing the results obtained from 13 biotransformations and identifiable transfer scenarios. Finally, to determine a more robust model, all 12 states corresponding to toxic compounds and tissues were aggregated into a single state model. The best adjustment was obtained with a simple one-compartment model based on total flesh toxicity with elimination rate expressed by a function depending on initial concentrations of GTX3 and GTX2 (i.e. the two major toxic compounds found in contaminated oysters). (C) 2011 Elsevier B.V. All rights reserved.

RÉSUMÉ Michèle BARDOUIL·, Madeleine BOHEC·, Serge BOUGRIER b, Patrick LASSUS· and Philippe TRUQUET a a Institut Français de Recherche pour l'Exploitation de la Mer (Ifremer). Centre de Nantes, B.P. 1105, 44311 Nantes Cedex 03, France. b CREMA, B.P. 5, 17137 L'Houmeau, France. Received 18/04195. in revised form 18110/95, accepted 20110/95. An experimental flow-through system allowing determination of the feeding behaviour of individual molluscs was used to study oysters exposed to mixed diets composed of varying proportions of the diatom Thalassiosira weissflogii and two strains (toxic and nontoxic) of the dinoflagellate Alexandrium larnarense. Our results show that, when compared to a T. weissflogii unialgal diet, even a diatomltoxic dinoflagellate ratio as low in biomass as 90/10 reduced clearance rates and biodeposit production by oysters. Consumption was slightly but significantly decreased for a 50/50 diatomltoxic dinoflagellate mixture. For the toxic dinoflagellate unialgal diet, ingestion, absorption and consumption were completely inhibited. Thus, the inclusion of low amounts of toxic A. lamarense in a diet composed of T. weissflogii significantly altered pseudofaeces production and probably oyster filtering capacity , whereas no significant effect was observed with the non-taxie dinoflagellate. Réponse écophysiologique de C. gigas (Thunberg) à l'introduction de proportions variables de dinoflagellés toxiques dans le régime alimentaire. A raide d'un dispositif expérimental en circuit ouvert pennettant de mesurer le comportement alimentaire individuel de bivalves, des huîtres (Crassoslrea gigasl ont été exposées à des régimes alimentaires mixtes comprenant des proportions variables de la diatomée Thalassiosira weissflogii et de deux souches, l'une toxique, l'autre non toxique, du dinoflagellé Alexandrium ramarense. Les résultats montrent que même pour un rapport aussi faible que 90110 en biomasse du régime diatomée / dinoflagellé toxique, le taux de filtration et la production de biodépôts sont réduits par rapport au témoin (diatomé: seule). La consommation n'est diminuée de façon significative que pour un melan~e 50/50 du même mélange tandis que l'ingestion, l'absorption et la consommauon SOn! complètement inhibés pour un régime composé uniquement du ~.nofla~ellé toxique. Il apparaît donc que l'introduction, même faible, d'Alexar;dnum tO<lq.ue dans un régime constitué de T. weissflogii altère la capacité des hultr~ à prodUire . 1 ue l'introduction dans le des pseudofèces et probablement aussI à filtrer, a ors q f"'fi'f . oduit aucun e .et SlgDJ IcaU . même récrime d'un Alexandrium non toxlque ne pr o OceanologicaAcla, 1996, 19, 2,177-182. -.~--------------------------------177

Karenia selliformis strain GM94GAB was isolated in 1994 from the north of Sfax, Gabes gulf, Tunisia. This species, which produces gymnodimine (GYM) a cyclic imine, has since been responsible for chronic contamination of Tunisian clams. A study was made by culturing the microalgae oil enriched Gaillard f/2 medium. The influence of growing conditions oil toxin content was studied, examining the effects of (i) different culture Volumes (0.25 to 40 litre flasks), (ii) two temperature ranges (17-15 degrees C et 20-21 degrees C) and (iii) two salinities (36 and 44). Chemical analyses were made by mass spectrometry coupled with liquid chromatography (LC-MS/MS). Results showed that (i) the highest growth rate (0.34 +/- 0.14 div d(-1)) was obtained at 20 degrees C and a salinity of 36, (ii) GYM content expressed as pg eq GYM cell(-1) increased with Culture time. The neurotoxicity of K. selliformis extracts was confirmed by mouse bioassay. This study allowed us to calculate the minimal lethal dose (MLD) of gymnodimine (GYM) that kills a mouse, as a function of the number of K. selliformis cells extracted.

The effects of an artificial bloom of the toxin-producing dinoflagellate, Alexandrium minutum, upon physiological parameters of the Pacific oyster, Crassostrea gigas, were assessed. Diploid and triploid oysters were exposed to cultured A. minutum and compared to control diploid and triploid oysters fed T. Isochrysis. Experiments were repeated twice, in April and mid-May 2007, to investigate effects of maturation stage on oyster responses to A. minutum exposure. Oyster maturation stage, Paralytic Shellfish Toxin (PST) accumulation, as well as several digestive gland and hematological variables, were assessed at the ends of the exposures. In both experiments, triploid oysters accumulated more PSTs (approximately twice) than diploid oysters. Significant differences, in terms of phenoloxidase activity (PO) and reactive oxygen species (ROS) production of hemocytes, were observed between A. minutum-exposed and non-exposed oysters. PO in hemocytes was lower in oysters exposed to A. minutum than in control oysters in an early maturation stage (diploids and triploids in April experiment and triploids in May experiment), but this contrast was reversed in ripe oysters (diploids in May experiment). In the April experiment, granulocytes of oysters exposed to A. minutum produced more ROS than those of control oysters; however, in the May experiment, ROS production of granulocytes was lower in A. minutum-exposed oysters. Moreover, significant decreases in free fatty acid, monoacylglycerol, and diacylglycerol contents in digestive glands of oysters exposed to A. minutum were observed. Concurrently, the ratio of reserve lipids (triacylglycerol, ether glycerides and sterol esters) to structural lipids (sterols) decreased upon A. minutum exposure in both experiments. Also, several physiological responses to A. minutum exposure appeared to be modulated by maturation stage as well as ploidy of the oysters.

In the Gulf of Gabes (Tunisia, Eastern Mediterranean sea), the grooved carpet shell Ruditapes decussatus has been seen to contain persistent levels of gymnodimine (GYM) for several years. The present experimental work represents the first attempt to assess detoxification kinetics of fast-acting toxins (FAT) in marine molluscs fed specific diets of non-toxic algae (Isochrysis galbana).To find an optimal detoxification method, two experiments were performed in which clams were first fed the toxic dinoflagellate Karenia selliformis to artificially contaminate them with GYM, thus simulating the effect of natural toxic episodes. As a second step, the same clams were fed a non-toxic algae, I. galbana. to speed up the detoxification process.Changes in toxin content over the whole experiment were assessed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis.The first results revealed (i) faster detoxification rates in digestive gland (DG) when clams were fed on L galbana compared with a starved control (no food) and (ii) a typical detoxification pattern, i.e. a rapid drop in toxin content within the first days followed by a secondary slower decrease. GYM levels could be reduced approximately to less than 5% within 7-8 days in clams fed I. galbana, according to the initial toxin levels of 1400 and 9400 mu g GYM/kg of DG, respectively. At the end of the second experiment, DSP mouse bioassay was negative when GYM was less than 100 mu g/kg DG. (C) 2009 Elsevier B.V. All rights reserved.

As early as 1999, the EUROHAB initiative reported an urgent need for research on phycotoxin accumulation, detoxification and biotransformation rates in exploited shellfish species. It also strongly recommended a second objective of developing commercial systems for the cleansing of shellfish once contaminated with toxin. As a result, EU Framework Programs and regional programs funded a series of R & D projects aiming to consolidate our understanding of contamination/detoxification pathways, together with the development of industrial scale detoxification processes and mechanisms. These studies attempted to accelerate the depuration process, for DSP in blue mussels, PSP in Pacific oysters and Manila clams, and ASP in King Scallops. Bulk culture protocols were undertaken for either toxic (Alexandrium, Pseudo-nitzschia) or non-toxic (Skeletonema, Isochrysis) algae to balance the often random nature of toxic episodes, and to fulfil industry requirements for detoxification. Technical improvements were derived from these studies. They included a practical protocol for washing ASP from the edible parts of contaminated scallops, and the manufacture of an algal paste that could be used as detoxification material instead of live cells. Difficulties were experienced with a decrease in Alexandrium or Pseudo-nitzschia toxicity when grown in bulk cultures, and the wide individual variations of shellfish toxin content compromised some experiments. However, supplying non-toxin containing food to these bivalves was confirmed as one of the most efficient means of speeding up detoxification, although the time needed to reach the regulatory level varied according to toxin type and shellfish species.

The occurrence of new phytoplankton species in a coastal area may be explained by the import of shellfish containing whole live algal cells in their digestive tracts. Indeed, shellfish containing toxic algal cells can induce both primary contaminations in safe areas ( initially free from toxic microalgae), and secondary contaminations of other shellfish growing in the same area via the faeces of the imported animals. To mitigate this problem, shellfish need to be placed in a separate holding tank and their intestinal content purged. For a deeper understanding of the risks associated with transferring contaminated shellfish, oysters ( Crassostrea gigas) and mussels ( Mytilus edulis) were purposely fed either Alexandrium minutum or A. catenella ( Dinophyceae) or Pseudo- nitzschia calliantha ( Bacillariophyceae) toxic algae for 2 h. They were then transferred into individual tanks where they were continuously fed with a non- toxic alga, Tetraselmis suecica. Biodeposit production, faeces composition, and filtration rates were monitored for the shellfish over a 6- h period. The effect of temperature differences and different initial toxic algae concentrations were compared. This study revealed a relationship between temperature and cell lysis in the oyster digestive tract. It also indicated that toxic algae concentration did not seem to influence gut passage time in oysters, while a significant effect was observed in mussels, and confirmed the existence of a difference between oyster and mussel feeding patterns.

Contamination of shellfish by paralytic shellfish poisoning ( PSP) toxins poses an economic threat to shellfish farmers. As contaminated shellfish cannot be harvested for long periods of time, it would be very useful to develop processes to optimise and shorten their detoxification. In this study, Pacific oysters Crassostrea gigas were first experimentally contaminated over a period of 13 days with a continuous flow of toxic Alexandrium minutum cultures at concentrations ranging from 150 to 200 cell ml(-1) ( toxin content after 13 days of contamination 438 mu g STX equiv. 100 g(-1) wet weight). Then, two different detoxification treatments were tested and showed detoxification rates greater than those observed in coastal environments. The first treatment consisted of feeding oysters on Skeletonema costatum, at a concentration of 2000 cell ml(-1) to speed up detoxification rates. The second detoxification method used the same Skeletonema costatum diet, supplemented with silt particles at a concentration of 20 mg L-1. A control was also set up by placing contaminated oysters in seawater with no additional algal food. The detoxification experiment lasted 8 days. Toxin contents were analysed by liquid chromatography with fluorescence detection ( LC- FD). The S. costatum diet significantly reduced the time needed for oysters to reach the sanitary threshold ( 80 mu g STX equiv. 100 g(-1) wet weight), but no effect of the silt supplement could be demonstrated conclusively. These different detoxification methods did not influence toxin biotransformations as observed in oyster tissues, i. e. epimerisation and decarbamoylation of gonyautoxins 2 and 3.