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Vulcanodinium rugosum is an emerging benthopelagic neuro-toxic dinoflagellate species responsible for seasonal Pinnatoxins and Portimines contaminations of shellfish and marine animals. This species is challenging to detect in the environment, as it is present in low abundance and difficult to be identified using light microscopy. In this work, we developed a method using artificial substrates coupled with qPCR (AS-qPCR) to detect V. rugosum in a marine environment. This sensitive, specific and easy-to-standardize alternative to current techniques does not require specialized expertise in taxonomy. After determining the limits and specificity of the qPCR, we searched for the presence of V. rugosum in four French Mediterranean lagoons using artificial substrates collected every two weeks for one year. The AS-qPCR method revealed its occurrences in summer 2021 in every studied lagoon and detected cells in more samples than light microscopy. As V. rugosum development induces shellfish contamination even at low microalga densities, the AS-qPCR method is accurate and relevant for monitoring V. rugosum in a marine environment.

oxic algae and their toxins represent an emerging public health issue, particularly due to global warming. The toxicological mechanisms of neurotoxic phycotoxins and their human health effects have been widely described (paralytic, neurological, amnesic, and ciguateric toxins). Food poisoning by bivalve shellfish (mussels, oysters...) can cause serious or even fatal neurological disorders, due to the accumulation in their flesh of natural toxins that develop in the water that surrounds them. However, retrospective study of human shellfish poisoning highlighted the difficulties in identifying cases related to neurotoxic phycotoxins by gathering clinical and biological diagnostic confirmations on the one hand, as well as environmental contaminations on the other. To improve the collection of shellfish poisoning data, the French Agency for Food, Environmental and Occupational Health and Safety (ANSES), French Poison Control Centers (PCCs), the French Research Institute for Exploitation of the Sea (Ifremer) and the French Defense Procurement Agency (DGA)have developed a prospective surveillance of neurotoxins from algal origin that combines monitoring of poisoning cases (clinical monitoring), of harmful algae and their toxins in shellfish production areas (environmental monitoring), as well as of toxins levels in seafood leftovers and in biological samples from poisoned subjects. This original clinical and environmental monitoring will allow us to have a more complete view of phycotoxin human exposures that are underreported, and to implement measures to protect consumers.

Présentation des projets de recherche, de l'équipe environnement, concernant le diagnostic de la contamination des eaux côtières de la Martinique, par différentes méthodes.

Le projet SECTOX porte sur l’étude de la contamination par les phycotoxines (PST et lipophiles dont DST) des huîtres creuses et des moules de la lagune de Thau ainsi que la dynamique temporelle et spatiale de cette contamination dans l’optique d’une gestion sectorisée de la lagune. Les campagnes de terrain ont été réalisées entre septembre 2019 et décembre 2020.La contamination des toxines lipophiles et des toxines hydrophiles (PST) a été suivie dans plusieurs matrices : coquillages et échantillonneurs passifs. Le suivi des espèces toxiques dans la colonne d’eau a été opéré selon plusieurs méthodes d’analyses et techniques d’échantillonnage. L’étude a mis en évidence que la contamination par les toxines lipophiles produites par une microalgue appartenant au genre toxique Dinophysis, impacte l’ensemble de la lagune de Thau sans qu’il soit possible de déterminer des dynamiques différentes entre les différents secteurs de la lagune. L’absence de prolifération du dinoflagellé Alexandrium pacificum (producteur de PST) sur la période suivie n’a pas permis de conclure sur l’existence d’une dynamique spatio-temporelle différente entre les secteurs de l’étang de Thau pour le risque de contamination par les PST. L’étude a aussi permis de tester de nouveaux outils (SPATT, échantillonneur passif pour les toxines) ainsi que la méthode de qPCR utilisant les outils de la biologie moléculaire pour l’identification et le dénombrement des espèces toxiques. Les résultats obtenus montrent que ces techniques sont prometteuses mais nécessitent une optimisation avant une utilisation en « routine » dans le volet sanitaire du réseau de surveillance REPHY.

Cette expertise répond à la demande de la DDPP34 de réévaluation de la stratégie de suivi de la contamination en phycotoxines des coquillages des deux zones d’élevage de la lagune de Thau, dans un contexte de gestion non différenciée vis à vis du risque phycotoxinique de ces deux zones. Cette expertise s’appuie sur l’analyse des données spatialisées du projet SECTOX de dénombrements du genre Dinophysis dans l’eau (27 campagnes, 14 lieux) et de concentrations des huîtres creuses et des moules en toxines lipophiles (13 campagnes, 13 lieux), en 2019 et 2020. L'expertise conclut sur la pertinence du maintien des points de suivi historiques et sur le une proposition d’optimisation de la stratégie de surveillance du risque lipophile avec le rajout en REPHY sanitaire et REPHYTOX des lieux n°7 localisés à l’Ouest de la zone de Bouzigues-Loupian et n°12 localisés à l’Ouest de la zone de Marseillan pour une durée minimale de 3 ans.

L’Anses a été saisie le 3 décembre 2021 par la Direction générale de la santé (DGS) et la Direction générale de l’alimentation (DGAL) pour la réalisation de l’expertise suivante dont le titre initial était : « demande d'avis relatif aux risques liés aux efflorescences d'Ostreopsis spp. sur l’ensemble du littoral français»

Pinnatoxins (PnTX) and Portimines (Prtn), two toxins produced by the benthic dinoflagellate Vulcanodinium rugosum, are known to be lethal to mice after intraperitoneal or oral administration. They are also known to accumulate in shellfish such as mussels and clams, but their effect on fish and the upper food chain remains unknown. In this work, juveniles of the fish Liza ramada (Mullet) were exposed to a strain of V. rugosum producing PnTX G and Prtn A. The fishes’ viability and contamination were recorded at times interval. Results showed that L. ramada juveniles were able to feed on V. rugosum and that their tissues could be contaminated by PnTX G and Prtn A without impact on fish viability. Furthermore, the microalgae temporary cysts survived and germinated after fish gut passage. This study showed the potential of L. ramada to transfer PnTX and Prtn toxins to the upper food chain and to disseminate V. rugosum in environment.

French Mediterranean lagoons are frequently subject to shellfish contamination by Diarrheic Shellfish Toxins (DSTs) and Paralytic Shellfish Toxins (PSTs). To predict the effect of various environmental factors (temperature, salinity and turbidity) on the abundance of the major toxins producing genera, Dinophysis and Alexandrium, and the link with shellfish contamination, we analysed a 10-year dataset collected from 2010 to 2019 in two major shellfish farming lagoons, Thau and Leucate, using two methods: decision trees and Zero Inflated Negative Binomial (ZINB) linear regression models. Analysis of these decision trees revealed that the highest risk of Dinophysis bloom events occurred at temperature <16.3°C and salinity <27.8, and of Alexandrium at temperature ranging from 10.4 to 21.5°C and salinity >39.2. The highest risk of shellfish contaminations by DSTs and PSTs occurred during the set of conditions associated with high risk of bloom events. Linear regression prediction enables us to understand whether temperature and salinity influence the presence of Alexandrium and affect its abundance. However, Dinophysis linear regression could not be validated due to overdispersion issues. This work demonstrates the tools which could help sanitary management of shellfish rearing areas.

La consommation de coquillages bivalves (moules, huîtres...) peut être à l’origine de troubles neurologiques graves voire mortels, liés à l’accumulation dans la chair des mollusques de toxines naturelles dont les teneurs sont par ailleurs réglementées. En juin 2019, une intoxication spécifique liée à la consommation de moules contaminées par des toxines paralysantes a attiré l’attention de l’Anses et des Centres antipoison (CAP) sur une possible méconnaissance diagnostique des intoxications par des neurotoxines marines. Une étude rétrospective des cas d’intoxication par des coquillages enregistrés par les CAP de 2012 à 2019, et des données de surveillance réglementaire des zones de production conchylicoles, a permis d’identifier 15 cas probables d’intoxication par des neurotoxines. Une surveillance prospective des intoxications par des coquillages à l’origine de signes neurologiques a depuis été mise en place à partir des données des CAP.

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from ‘not detected’ to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex

This work aims to study the spatio-temporal evolution of the genus Nitzschia longissima, one of the most important genera of marine plankton diatoms, from 3 sampling stations in the Nador lagoon and during 2 seasons (spring and summer 2018), Using Nitzschia longissima, as a study system, one of the most diverse and abundant genera among marine planktonic diatoms. This species counts, in addition to the form Nitzschia longissima forma parva Grunow, three varieties namely Nitzschia longissima var. closterium (W. Smith) Van Heurck, Nitzschia longissima var. longissima (Breb.) Ralfs and Nitzschia longissima var. reversa Grunow. Nitzschia Longissima genus density was high during the warm season (Summer 2018) with a value of 8000 cells/liter, and low during the cold seasons (Spring 2018), which may be caused by water temperature and zooplankton community structure; and underwater light intensity was an important factor influencing the spatial distribution of Nitzschia density.

Ce rapport a été réalisé à la demande de la Direction Départementale de la Protection des Populations de l’Hérault (DDPP34). Les résultats en toxines lipophiles réglementées (Acide Okadaïque, Dinophysis toxines et Pectenotoxines uniquement) dans les moules et les huîtres prélevées au point « Parc Leucate 2 » dans le cadre du REPHYTOX, ainsi que les dénombrements du phytoplancton toxique du genre Dinophysis réalisés dans le cadre du REPHY dans la lagune de Leucate, sont présentés sur des graphiques permettant une visualisation temporelle des épisodes de contamination entre janvier 2014 et décembre 2020.

Alexandrium minutum and Alexandrium pacificum are representatives of the dinoflagellate genus that regularly proliferate on the French coasts and other global coastlines. These harmful species may threaten shellfish harvest and human health due to their ability to synthesize neurotoxic alkaloids of the saxitoxin group. However, some dinoflagellates such as A. minutum, and as reported here A. pacificum as well, may also have a beneficial impact on the environment by producing dimethylsulfoniopropionate-DMSP, the precursor of dimethylsulfur-DMS and sulfate aerosols involved in climate balance. However, environmental conditions might influence Alexandrium physiology towards the production of harmful or environmentally friendly compounds. After assessing the influence of two salinity regimes (33 and 38) relative to each species origin (Atlantic French coast and Mediterranean Lagoon respectively), it appears that DMSP and toxin content was variable between the three experimented strains and that higher salinity disadvantages toxin production and tends to favor the production of the osmolytes DMSP and glycine betaine. Hence, this key metabolite production is strain and species-dependent and is influenced by environmental conditions of salinity which in turn, can diversely affect the environment. Widespread coastal blooms of A. minutum and A. pacificum, although being a risk for seafood contamination with toxins, are also a DMSP and DMS source that potentially contribute to the ecosystem structuration and climate. Regarding recent advances in DMSP biosynthesis pathway, 3 dsyB homologs were found in A. minutum but no homolog of the diatom sequence TpMMT.

Mediterranean costal lagoons have been exposed to anthropic eutrophication for decades. Thau lagoon is one of the largest among them and provides many ecosystem services including shellfish farming. Considerable efforts were made between the 1970s and the late 2000s to improve the wastewater treatment systems in the lagoon watershed. A decrease in nutrient inputs to Thau lagoon was subsequently observed, evidence for the start of a recovery process in Thau lagoon, following oligotrophication. This decrease has significant consequences for ecosystem communities, above all primary producers. In our study, we characterised and quantified long-term changes by analysing long monitoring time-series (1998-2016) of phytoplankton biomass, abundance and composition of the taxa, nutrients and climate conditions, using univariate and multivariate statistics. Our results revealed that two decades of mitigation actions in the Thau lagoon watershed have led to a significant progressive reduction in nutrient concentrations and in phytoplankton biomass (-60%), associated with a decrease in diatom abundance (-66%), particularly affecting Skeletonema spp. The changes were associated with a community shift characterised by a shift in phytoplankton taxonomic dominance from Skeletonema-Chaetoceros to Chaetoceros-Pseudo-nitzschia. The median proportion of dinoflagellates relative to diatoms increased, although the total dinoflagellate abundance did not change significantly. We hypothesise that the shift in phytoplankton communities is the result of the mixed effects of the reduction in nutrient inputs and of climatic-related variables. Finally, we present a conceptual scheme of the main drivers of phytoplankton community structure to clarify the ecosystem functioning of a Mediterranean lagoon used for shellfish farming, under oligotrophication and climate change.