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Detoxification of Pacific oyster Crassostrea gigas fed on diets of Skeletonema costatum with and without silt, following PSP contamination by Alexandrium minutum ArchiMer
Gueguen, Marielle; Bardouil, Michele; Baron, Regis; Lassus, Patrick; Truquet, Philippe; Massardier, Julie; Amzil, Zouher.
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...
Tipo: Text Palavras-chave: Alexandrium minutum; Crassostrea gigas; Detoxification; Inorganic matter; Organic matter; PSP; Paralytic shellfish poisoning.
Ano: 2008 URL: http://archimer.ifremer.fr/doc/2008/publication-3896.pdf
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Direct evidence for toxin production by Pseudo-nitzschia plurisecta (Bacillariophyceae) and extension of its distribution area ArchiMer
Caruana, Amandine; Ayache, Nour; Raimbault, Virginie; Rétho, Michael; Hervé, Fabienne; Bilien, Gwenael; Amzil, Zouher; Chomérat, Nicolas.
We confirm an amnesic shellfish poisoning risk associated with domoic acid production by the species Pseudo-nitzschia plurisecta and determine the toxin profile, which includes three domoic acid isomers. Although the species was reported once in European waters, along the Spanish Atlantic coast, here we describe and characterize P. plurisecta collected during the first occurrence in French Atlantic waters. Three strains (IFR-PPL-15.1, 15.2 and 15.3) were isolated from French coastal water samples in June 2015. These strains were identified and characterized by microscopic observation, molecular (ITS region sequencing and phylogenetic analysis) and metabolite (toxins and pigments) analyses. The cell shape is lanceolate and the valve contains a central...
Tipo: Text Palavras-chave: Coastal monitoring; Domoic acid; Pigment composition; Pseudo-nitzschia plurisecta; Taxonomic identification; Toxic diatoms.
Ano: 2019 URL: https://archimer.ifremer.fr/doc/00505/61687/66033.pdf
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Effect of Dilution Rate on Azadinium spinosum and Azaspiracid (AZA) Production in Pilot Scale Photobioreactors for the Harvest of AZA1 and -2 ArchiMer
Jauffrais, Thierry; Sechet, Veronique; Truquet, Philippe; Amzil, Zouher; Herrenknecht, Christine; Hess, Philipp.
Azadinium spinosum, a small dinoflagellate has recently been discovered and identified as the primary producer of azaspiracid-1 (AZA) and -2. Since AZA poisoning has been reported following consumption of contaminated shellfish it is important to have these toxins available for toxicological studies, and a sustainable production of AZAs as calibrants in monitoring programs without having to rely on natural events. In order to address this concern, continuous pilot scale cultures were carried out to evaluate the feasibility of AZA production from A. spinosum. Algae were cultured using two 100 L chemostats in series (R1 and R2), with agitation and pH control. Four different dilution rates were tested (0.15, 0.2, 0.25 and 0.3 day−1) to evaluate chemostat...
Tipo: Text Palavras-chave: Solid phase adsorption; Chemostat; Photobioreactor; Microalgae; LC-MS/MS; Tangential flow filtration.
Ano: 2014 URL: http://archimer.ifremer.fr/doc/00152/26349/24437.pdf
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Effects of copper and butyltin compounds on the growth, photosynthetic activity and toxin production of two HAB dinoflagellates : the planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata ArchiMer
Couet, Douglas; Pringault, Olivier; Bancon-montigny, Chrystelle; Briant, Nicolas; Poulichet, Francoise Elbaz; Delpoux, Sophie; Yahia, Ons Kefi-daly; Hela, Bengharbia; Charaf, M'Rabet; Herve, Fabienne; Rovillon, Georges-augustin; Amzil, Zouher; Laabir, Mohamed.
Controlled laboratory experiments were conducted to test the effects of copper (Cu2+) and butyltins (BuT) on the growth, photosynthetic activity and toxin content of two HABs (Harmful Algal Blooms) dinoflagellates, the planktonic Alexandrium catenella and the benthic Ostreopsis cf. ovata. Microalgae were exposed to increasing concentrations of Cu2+ (10−4 to 31 nM) or BuT (0.084 to 84 nM) for seven days. When considering the growth, EC50 values were 0.16 (±0.09) nM and 0.03 (±0.02) nM of Cu2+ for A. catenella and O. cf. ovata, respectively. Regarding BuT, EC50 was 14.2 (±6) nM for O. cf. ovata, while A. catenella growth inhibition appeared at BuT concentrations ≥27 nM. Photosynthetic activity of the studied dinoflagellates decreased with increasing Cu and...
Tipo: Text Palavras-chave: Alexandrium catenella; Ostreopsis cf. ovata; Cu; Butyltin; Photosynthesis; Toxins.
Ano: 2018 URL: https://archimer.ifremer.fr/doc/00418/52970/53896.pdf
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Effects of Organic and Inorganic Nitrogen on the Growth and Production of Domoic Acid by Pseudo-nitzschia multiseries and P. australis (Bacillariophyceae) in Culture ArchiMer
Martin-jezequel, Veronique; Calu, Guillaume; Candela, Leo; Amzil, Zouher; Jauffrais, Thierry; Sechet, Veronique; Weigel, Pierre.
Over the last century, human activities have altered the global nitrogen cycle, and anthropogenic inputs of both inorganic and organic nitrogen species have increased around the world, causing significant changes to the functioning of aquatic ecosystems. The increasing frequency of Pseudo-nitzschia spp. in estuarine and coastal waters reinforces the need to understand better the environmental control of its growth and domoic acid (DA) production. Here, we document Pseudo-nitzschia spp. growth and toxicity on a large set of inorganic and organic nitrogen (nitrate, ammonium, urea, glutamate, glutamine, arginine and taurine). Our study focused on two species isolated from European coastal waters: P. multiseries CCL70 and P. australis PNC1. The nitrogen...
Tipo: Text Palavras-chave: Pseudo-nitzschia; Toxic diatoms; Nitrogen; Amino acids; Domoic acid.
Ano: 2015 URL: http://archimer.ifremer.fr/doc/00308/41912/41188.pdf
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Effets de l'algue toxique Alexandrium catenella sur le système de défense de l'huître creuse Crassostrea gigas ArchiMer
Medhioub, Walid; Duchiron, Marie; Masseret, Estelle; Savar, Veronique; Amzil, Zouher; Laabir, Mohamed; Rolland, Jean-luc.
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Ano: 2013 URL: http://archimer.ifremer.fr/doc/00172/28332/26618.pdf
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Enhancement of domoic acid neurotoxicity on Diptera larvae bioassay by marine fungal metabolites ArchiMer
Ruiz, Nicolas; Petit, Karina; Vansteelandt, Marieke; Kerzaon, Isabelle; Baudet, Joseph; Amzil, Zouher; Biard, Jean-francois; Grovel, Olivier; Pouchus, Yves Francois.
Peptaibols are small linear fungal peptides which are produced in the marine environment. They exhibit neurotoxicity by forming pores in neuronal membranes. This work describes their combine effect with domoic acid, a neurotoxic phycotoxin, on Diptera larvae. The Acute toxicity bioassay on this biological model was tested with a panel of different toxins (microbial, algal or fungal). It allowed the discrimination of neurotoxins and non-neurotoxic toxins, and an evaluation of the toxicity level (MED and ED50) which were correlated with published LD50 in mice for neurotoxins tested. The highest activities on this test were found for Na+ channel blockers tetrodotoxin (ED50 = 0.026 mg/kg) and saxitoxin (ED50 = 0.18 mg/kg). Domoic acid was less active with an...
Tipo: Text Palavras-chave: Domoic acid; Peptaibol; Synergism; Neurotoxicity; Marine-derived fungal metabolite; Diptera larvae bioassay.
Ano: 2010 URL: http://archimer.ifremer.fr/doc/00002/11279/7886.pdf
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Étude sur la prolifération de la micro algue Alexandrium minutum en rade de Brest ArchiMer
Chapelle, Annie; Le Bec, Claude; Amzil, Zouher; Dreanno, Catherine; Guillou, Laure; Klouch, Khadidja; Labry, Claire; Le Gac, Mickael; Pineau-guillou, Lucia; Siano, Raffaele; Abernot, Chantal; Andrieux, Francoise; Caradec, Florian; Destombe, Christophe; Dia, A.; Doner, Anne; Duval, Julia; Gouriou, Jeremie; Lazure, Pascal; Le Brun, Luc; Le Gal, Dominique; Malestroit, Pascale; Petton, Sebastien; Plus, Martin; Quere, Julien; Savar, Veronique; Schmitt, Sophie; Terre, A.; Youenou, Agnes.
Suite à l’événement de grande ampleur d’efflorescence d’Alexandrium minutum et des toxicités associées en Rade de Brest en 2012, un projet a été élaboré avec la Région Bretagne dans le cadre du développement du port de Brest. L’Ifremer a organisé un échantillonnage dans l’estuaire de la rivière de Daoulas, zone la plus touchée en 2012, au cours des printemps-été 2013 et 2014. Un suivi de la présence d’A. minutum dans les sédiments a également été réalisé en hiver 2014. De ces suivis, des mesures réalisées au laboratoire à ce jour, des simulations du modèle Prévimer ainsi que des résultats du réseau Rephy et Velyger, il ressort que: * La rade de Brest peut aujourd’hui être considérée comme une zone à risque pour Alexandrium minutum. En effet, depuis 2012...
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Ano: 2014 URL: http://archimer.ifremer.fr/doc/00254/36487/35029.pdf
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Etude sur la prolifération de la micro algue Alexandrium minutum en rade de Brest. Projet Daoulex. Rapport d’avancement n° 1 : Novembre 2013 ArchiMer
Chapelle, Annie; Le Bec, Claude; Le Gac, Mickael; Labry, Claire; Amzil, Zouher; Guillou, Laure; Dreanno, Catherine; Pineau-guillou, Lucia.
Suite à l’événement de grande ampleur d’efflorescence d’Alexandrium minutum et des toxicités associées en Rade de Brest en 2012, un projet a été monté avec la Région dans le cadre du développement du port de Brest. De juillet à août 2013, l’Ifremer a organisé un échantillonnage serré dans l’estuaire de Daoulas, zone la plus touchée en 2012. De ce suivi et des mesures réalisées au laboratoire à ce jour, ainsi que des résultats du réseau Rephy et Velyger, il ressort que : * Alexandrium minutum s’est développé à nouveau en rade de Brest en 2013 mais à des teneurs inférieures à 2012. * l’épisode de bloom et de toxicité supérieur aux seuils légaux s’est déroulé plus tard qu’en 2012, du 1er juillet jusqu’à 5 août. * les efflorescences et la toxicité ont touché...
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Ano: 2013 URL: https://archimer.ifremer.fr/doc/00191/30230/28644.pdf
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Evidence of the bioaccumulation of ciguatoxins in giant clams (Tridacna maxima) exposed to Gambierdiscus spp. cells ArchiMer
Roue, Melanie; Darius, Helene Taiana; Picot, Sandy; Ung, Andre; Viallon, Jerome; Gaertner-mazouni, Nabila; Sibat, Manoella; Amzil, Zouher; Chinain, Mireille.
Ciguatera Fish Poisoning (CFP) is a foodborne disease classically related to the consumption of tropical coral reef fishes contaminated with ciguatoxins (CTXs), neurotoxins produced by dinoflagellates of the Gambierdiscus genus. Severe atypical ciguatera-like incidents involving giant clams, a marine resource highly consumed in the South Pacific, are also frequently reported in many Pacific Islands Countries and Territories. The present study was designed to assess the ability of giant clams to accumulate CTXs in their tissues and highlight the potential health risks associated with their consumption. Since giant clams are likely to be exposed to both free-swimming Gambierdiscus cells and dissolved CTXs in natural environment, ex situ contamination...
Tipo: Text Palavras-chave: Giant clams; Ex situ contamination; Ciguatoxins; Gambierdiscus polynesiensis; LC-MS/MS; Neuroblastoma cell-based assay.
Ano: 2016 URL: https://archimer.ifremer.fr/doc/00343/45415/45123.pdf
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Exposure to the Neurotoxic Dinoflagellate, Alexandrium catenella, Induces Apoptosis of the Hemocytes of the Oyster, Crassostrea gigas ArchiMer
Medhioub, Walid; Ramondenc, Simon; Vanhove, Audrey; Vergnes, Agnes; Masseret, Estelle; Savar, Veronique; Amzil, Zouher; Laabir, Mohamed; Rolland, Jean-luc.
This study assessed the apoptotic process occurring in the hemocytes of the Pacific oyster, Crassostrea gigas, exposed to Alexandrium catenella, a paralytic shellfish toxins (PSTs) producer. Oysters were experimentally exposed during 48 h to the toxic algae. PSTs accumulation, the expression of 12 key apoptotic-related genes, as well as the variation of the number of hemocytes in apoptosis was measured at time intervals during the experiment. Results show a significant increase of the number of hemocytes in apoptosis after 29 h of exposure. Two pro-apoptotic genes (Bax and Bax-like) implicated in the mitochondrial pathway were significantly upregulated at 21 h followed by the overexpression of two caspase executor genes (caspase-3 and caspase-7) at 29 h,...
Tipo: Text Palavras-chave: Shellfish; Toxins; Apoptosis; Gene expression.
Ano: 2013 URL: http://archimer.ifremer.fr/doc/00176/28744/27213.pdf
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Exposure to the Paralytic Shellfish Toxin Producer Alexandrium catenella Increases the Susceptibility of the Oyster Crassostrea gigas to Pathogenic Vibrios ArchiMer
Abi-khalil, Celina; Lopez-joven, Carmen; Abadie, Eric; Savar, Veronique; Amzil, Zouher; Laabir, Mohamed; Rolland, Jean-luc.
The multifactorial etiology of massive Crassostrea gigas summer mortalities results from complex interactions between oysters, opportunistic pathogens and environmental factors. In a field survey conducted in 2014 in the Mediterranean Thau Lagoon (France), we evidenced that the development of the toxic dinoflagellate Alexandrium catenella, which produces paralytic shellfish toxins (PSTs), was concomitant with the accumulation of PSTs in oyster flesh and the occurrence of C. gigas mortalities. In order to investigate the possible role of toxic algae in this complex disease, we experimentally infected C. gigas oyster juveniles with Vibrio tasmaniensis strain LGP32, a strain associated with oyster summer mortalities, after oysters were exposed to Alexandrium...
Tipo: Text Palavras-chave: Harmful algae; Environment; Interaction; Pathogens; Defense; Paralytic shellfish toxin.
Ano: 2016 URL: https://archimer.ifremer.fr/doc/00309/42016/41308.pdf
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Exposure to toxic Alexandrium minutum activates the detoxifying and antioxidant systems in gills of the oyster Crassostrea gigas ArchiMer
Fabioux, Caroline; Sulistiyani, Yeni; Haberkorn, Hansy; Hegaret, Helene; Amzil, Zouher; Soudant, Philippe.
Harmful algal blooms of Alexandrium spp. dinoflagellates regularly occur in French coastal waters contaminating shellfish. Studies have demonstrated that toxic Alexandrium spp. disrupt behavioural and physiological processes in marine filter-feeders, but molecular modifications triggered by phycotoxins are less well understood. This study analyzed the mRNA levels of 7 genes encoding antioxidant/detoxifying enzymes in gills of Pacific oysters (Crassostrea gigas) exposed to a cultured, toxic strain of A. minutum, a producer of paralytic shellfish toxins (PST) or fed Tisochrysis lutea (T. lutea, formerly Isochrysis sp., clone Tahitian (T. iso)), a non-toxic control diet, in four repeated experiments. Transcript levels of sigma-class glutathione S-transferase...
Tipo: Text Palavras-chave: Crassostrea gigas; Alexandrium minutum; Paralytic shellfish toxins; Antioxidant enzymes; Detoxification enzymes; MRNA.
Ano: 2015 URL: https://archimer.ifremer.fr/doc/00275/38601/38623.pdf
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Extended evaluation of polymeric and lipophilic sorbents for passive sampling of marine toxins ArchiMer
Zendong, Zita; Herrenknecht, Christine; Abadie, Eric; Brissard, Charline; Tixier, Celine; Mondeguer, Florence; Sechet, Veronique; Amzil, Zouher; Hess, Philipp.
Marine biotoxins are algal metabolites that can accumulate in fish or shellfish and render these foodstuffs unfit for human consumption. These toxins, released into seawater during algal occurrences, can be monitored through passive sampling. Acetone, methanol and isopropanol were evaluated for their efficiency in extracting toxins from algal biomass. Isopropanol was chosen for further experiments thanks to a slightly higher recovery and no artifact formation. Comparison of Oasis HLB, Strata-X, BondElut C18 and HP-20 sorbent materials in SPE-mode led to the choice of Oasis HLB, HP-20 and Strata-X. These three sorbents were separately exposed as passive samplers for 24 h to seawater spiked with algal extracts containing known amounts of okadaic acid (OA),...
Tipo: Text Palavras-chave: Passive sampling; Oasis HLB; Strata-X; LOPE; PDMS; SPATT.
Ano: 2014 URL: https://archimer.ifremer.fr/doc/00186/29756/28187.pdf
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First Evidence of Palytoxin and 42-Hydroxy-palytoxin in the Marine Cyanobacterium Trichodesmium ArchiMer
Kerbrat, Anne Sophie; Amzil, Zouher; Pawlowiez, Ralph; Golubic, Stjepko; Sibat, Manoella; Darius, Helene Taiana; Chinain, Mireille; Laurent, Dominique.
Marine pelagic diazotrophic cyanobacteria of the genus Trichodesmium (Oscillatoriales) are widespread throughout the tropics and subtropics, and are particularly common in the waters of New Caledonia. Blooms of Trichodesmium are suspected to be a potential source of toxins in the ciguatera food chain and were previously reported to contain several types of paralyzing toxins. The toxicity of water-soluble extracts of Trichodesmium spp. were analyzed by mouse bioassay and Neuroblastoma assay and their toxic compounds characterized using liquid chromatography coupled with tandem mass spectrometry techniques. Here, we report the first identification of palytoxin and one of its derivatives, 42-hydroxy-palytoxin, in field samples of Trichodesmium collected in...
Tipo: Text Palavras-chave: Cyanobacteria; Trichodesmium; Palytoxin; 42-hydroxy-palytoxin; Clupeotoxism.
Ano: 2011 URL: https://archimer.ifremer.fr/doc/00040/15143/12576.pdf
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First report on amnesic and diarrhetic toxins detection in French scallops during 2004-05 monitoring surveys ArchiMer
Amzil, Zouher; Royer, Florence; Sibat, Manoella; Fiant, Liliane; Gelin, Muriel; Le Gal, Dominique; Francoise, Sylvaine.
In the context of the French phytoplankton and phycotoxins monitoring network (REPHY), shellfish scallops were harvested systematically during the authorised fishing season for the purpose of investigating paralytic toxins (PSP-mouse test), amnesic toxins (ASPchemical analysis), and lipophilic toxins (DSP-mouse test). For all shellfish samples that tested positive in the DSP mouse bioassay used for lipophilic toxins detection, liquid chromatography coupled with mass spectrometry (LC-MS/MS) was used to search for the following lipophilic toxins: okadaic acid, dinophysistoxins, pectenotoxins, azaspiracids, yessotoxins, spirolides, and gymnodimine. In order to investigate the presence of okadaic acid esters (DTX3), alkaline hydrolysis was performed on all...
Tipo: Text Palavras-chave: Scallops; Domoic acid; Okadaic acid and derivatives; Dinophysis.
Ano: 2009 URL: http://archimer.ifremer.fr/doc/00015/12577/9447.pdf
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First report on azaspiracid and yessotoxin groups detection in French shellfish ArchiMer
Amzil, Zouher; Sibat, Manoella; Royer, Florence; Savar, Veronique.
The French Phytoplankton and Phycotoxins monitoring network (REPHY) recently found positive or dubious negative shellfish samples using lipophilic toxins mouse bioassay. These samples were analyzed by liquid chromatography (LC) in combination with mass spectrometry (MS) to detect the following toxins: okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs), azaspiracids (AZAs), yessotoxins (YTXs), spirolides (SPXs) and gymnodimines (GYMs). Over the 2006-2007 period, chemical analyses revealed various lipophilic toxin profiles according to shellfish sampling locations. In addition to OA and/or PTX-2 and their derivatives, several other compounds were found for the first time in France: (1) during the summer of 2006, AZA-1 and AZA-2 in Queen...
Tipo: Text Palavras-chave: LC MS/MS analyses; Shellfish; Yessotoxin group; Azaspiracid group; Lipophilic toxins.
Ano: 2008 URL: http://archimer.ifremer.fr/doc/2008/publication-4560.pdf
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From the sxtA4 Gene to Saxitoxin Production: What Controls the Variability Among Alexandrium minutum and Alexandrium pacificum Strains? ArchiMer
Geffroy, Solene; Lechat, Marc-marie; Le Gac, Mickael; Rovillon, Georges-augustin; Marie, Dominique; Bigeard, Estelle; Malo, Florent; Amzil, Zouher; Guillou, Laure; Caruana, Amandine.
Paralytic shellfish poisoning (PSP) is a human foodborne syndrome caused by the consumption of shellfish that accumulate paralytic shellfish toxins (PSTs, saxitoxin group). In PST-producing dinoflagellates such as Alexandrium spp., toxin synthesis is encoded in the nuclear genome via a gene cluster (sxt). Toxin production is supposedly associated with the presence of a 4th domain in the sxtA gene (sxtA4), one of the core genes of the PST gene cluster. It is postulated that gene expression in dinoflagellates is partially constitutive, with both transcriptional and post-transcriptional processes potentially co-occurring. Therefore, gene structure and expression mode are two important features to explore in order to fully understand toxin production processes...
Tipo: Text Palavras-chave: Alexandrium; Saxitoxins; SxtA4; Copy number variation; SxtA; Expression; Isoform.
Ano: 2021 URL: https://archimer.ifremer.fr/doc/00682/79402/81944.pdf
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Growth and toxin production of Azadinium spinosum in batch and continuous culture ArchiMer
Jauffrais, Thierry; Sechet, Veronique; Herrenknecht, Christine; Tillmann, Urban; Krock, Bernd; Amzil, Zouher; Hess, Philipp.
Azaspiracids are lipophilic marine biotoxins causing gastrointestinal symptoms similar to DSP toxins. Since 1995, azaspiracids have been encountered in Europe, Africa and more recently in North and South America and Japan. The biological primary producer remained undiscovered during many years and has now been identified as Azadinium spinosum. The organism was grown using K modified medium, at 18°C with a PFD of 200 μmol.m-2.s-1 and a photoperiod of 16L/8D. Batch cultures were carried out using 75mL and 10L flasks, while continuous cultures were produced in 100L chemostats. Cells were recovered using centrifugation or filtration. Different extraction solvents and procedures as well as evaporation modes were evaluated for yield. Quantitation was carried out...
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Ano: 2010 URL: http://archimer.ifremer.fr/doc/00152/26348/24431.pdf
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Gut passage times in two bivalve molluscs fed toxic microalgae: Alexandrium minutum, A-catenella and Pseudo-nitzschia calliantha ArchiMer
Gueguen, Marielle; Lassus, Patrick; Laabir, Mohamed; Bardouil, Michele; Baron, Regis; Sechet, Veronique; Truquet, Philippe; Amzil, Zouher; Barille, Laurent.
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 (...
Tipo: Text Palavras-chave: Risk assessment; Toxic phytoplankton; Uptake; Gut passage; Mytilus edulis; Crassostrea gigas.
Ano: 2008 URL: http://archimer.ifremer.fr/doc/2008/publication-3897.pdf
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