Chemodiversity of Brevetoxins and Other Potentially Toxic Metabolites Produced by Karenia spp. and Their Metabolic Products in Marine Organisms.

In recent decades, more than 130 potentially toxic metabolites originating from dinoflagellate species belonging to the genus Karenia or metabolized by marine organisms have been described. These metabolites include the well-known and large group of brevetoxins (BTXs), responsible for foodborne neurotoxic shellfish poisoning (NSP) and airborne respiratory symptoms in humans. Karenia spp. also produce brevenal, brevisamide and metabolites belonging to the hemi-brevetoxin, brevisin, tamulamide, gymnocin, gymnodimine, brevisulcenal and brevisulcatic acid groups. In this review, we summarize the available knowledge in the literature since 1977 on these various identified metabolites, whether they are produced directly by the producer organisms or biotransformed in marine organisms. Their structures and physicochemical properties are presented and discussed. Among future avenues of research, we highlight the need for more toxin occurrence data with analytical techniques, which can specifically determine the analogs present in samples. New metabolites have yet to be fully described, especially the groups of metabolites discovered in the last two decades (e.g tamulamides). Lastly, this work clarifies the different nomenclatures used in the literature and should help to harmonize practices in the future.

Guidance Level for Brevetoxins in French Shellfish.

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

Pinnatoxins' Deleterious Effects on Cholinergic Networks: From Experimental Models to Human Health.

Pinnatoxins (PnTXs) are emerging neurotoxins that were discovered about 30 years ago. They are solely produced by the marine dinoflagellate Vulcanodinium rugosum, and may be transferred into the food chain, as they have been found in various marine invertebrates, including bivalves. No human intoxication has been reported to date although acute toxicity was induced by PnTxs in rodents. LD50 values have been estimated for the different PnTXs through the oral route. At sublethal doses, all symptoms are reversible, and no neurological sequelae are visible. These symptoms are consistent with impairment of central and peripheral cholinergic network functions. In fact, PnTXs are high-affinity competitive antagonists of nicotinic acetylcholine receptors (nAChRs). Moreover, their lethal effects are consistent with the inhibition of muscle nAChRs, inducing respiratory distress and paralysis. Human intoxication by ingestion of PnTXs could result in various symptoms observed in episodes of poisoning with natural nAChR antagonists. This review updates the available data on PnTX toxicity with a focus on their mode of action on cholinergic networks and suggests the effects that could be extrapolated on human physiology.

Seasonal variations of phytoplankton community in relation to environmental factors in a protected meso-oligotrophic southern Mediterranean marine ecosystem (Mellah lagoon, Algeria) with an emphasis of HAB species.

The spatial and temporal variation of phytoplankton communities including HAB species in relation to the environmental characteristics was investigated in the protected meso-oligotrophic Mellah lagoon located in the South Western Mediterranean. During 2016, a biweekly monitoring of phytoplankton assemblages and the main abiotic factors were realized at three representative stations. Taxonomic composition, abundance, and diversity index were determined. In total, 227 phytoplankton species (160 diatoms and 53 dinoflagellates) were inventoried. There was a clear dominance of diatoms (62.9%) compared with dinoflagellates (36.8%). Diatoms dominated in spring and dinoflagellates developed in summer and early autumn in Mellah showing a marked seasonal trend. Data showed that the dynamic of the phytoplankton taxa evolving in the lagoon was mainly driven by temperature and salinity. For the first time, a number of potentially toxic species have been identified, including 2 diatoms (Pseudo-nitzschia group delicatissima, Pseudo-nitzschia group seriata) and 5 dinoflagellates (Alexandrium minutum, Alexandrium tamarense/catenella, Dinophysis acuminata, Dinophysis sacculus, Prorocentrum lima). These harmful species could threat the functioning of the Mellah lagoon and human health and require the establishment of a monitoring network. Finally, our study suggests that the observed decrease of the phytoplankton diversity between 2001 and 2016 could result from the reduction in water exchanges between the lagoon and the adjacent coast following the gradual clogging of the channel.

Variation in Health Technology Assessment and Reimbursement Processes in Europe.

BACKGROUND: It has been suggested that differences in health technology assessment (HTA) processes among countries, particularly within Europe, have led to inequity in patient access to new medicines. OBJECTIVES: To provide an up-to-date snapshot analysis of the present status of HTA and reimbursement systems in select European countries, and to investigate the implications of these processes, especially with regard to delays in market and patient access. METHODS: HTA and reimbursement processes were assessed through a review of published and gray literature, and through a series of interviews with HTA experts. To quantify the impact of differences among countries, we conducted case studies of 12 products introduced since 2009, including 10 cancer drugs. RESULTS: In addition to the differences in HTA and reimbursement processes among countries, the influence of particular sources of information differs among HTA bodies. The variation in the time from the authorization by the European Medicines Agency to the publication of HTA decisions was considerable, both within and among countries, with a general lack of transparency as to why some assessments take longer than others. In most countries, market access for oncology products can occur outside the HTA process, with sales often preceding HTA decisions. CONCLUSIONS: It is challenging even for those with considerable personal experience in European HTA processes to establish what is really happening in market access for new drugs. We recommend that efforts should be directed toward improving transparency in HTA, which should, in turn, lead to more effective processes.

Clinical trials of new drugs for the treatment of rheumatoid arthritis: focus on early disease.

The European Society for Clinical and Economic Aspects of Osteoporosis, Osteoarthritis and Musculoskeletal Diseases convened a task force of experts in rheumatoid arthritis (RA) and clinical trial methodology to comment on the new draft 'Guideline on clinical investigation of medicinal products for the treatment of RA' released by the European Medicines Agency (EMA). Special emphasis was placed by the group on the development of new drugs for the treatment of early RA. In the absence of a clear definition of early RA, it was suggested that clinical investigations in this condition were conducted in disease-modifying antirheumatic drugs naïve patients with no more than 1 year disease duration. The expert group recommended using an appropriate improvement in disease activity (American College of Rheumatology (ACR) or Simplified/Clinical Disease Activity Index (SDAI/CDAI) response criteria) or low disease activity (by any score) as primary endpoints, with ACR/European League Against Rheumatism remission as a secondary endpoint. Finally, as compelling evidence showed that the Disease Acrivity Score using 28-joint counts (DAS28) might not provide a reliable definition of remission, or sometimes even low disease activity, the group suggested replacing DAS28 as a measurement instrument to evaluate disease activity in RA clinical trials. Proposed alternatives included SDAI, CDAI and Boolean criteria.

Passive Sampling and High Resolution Mass Spectrometry for Chemical Profiling of French Coastal Areas with a Focus on Marine Biotoxins.

Passive samplers (solid phase adsorption toxin tracking: SPATT) are able to accumulate biotoxins produced by microalgae directly from seawater, thus providing useful information for monitoring of the marine environment. SPATTs containing 0.3, 3, and 10 g of resin were deployed at four different coastal areas in France and analyzed using liquid chromatography coupled to high resolution mass spectrometry. Quantitative targeted screening provided insights into toxin profiles and showed that toxin concentrations and profiles in SPATTs were dependent on the amount of resin used. Between the three amounts of resin tested, SPATTs containing 3 g of resin appeared to be the best compromise, which is consistent with the use of 3 g of resin in SPATTs by previous studies. MassHunter and Mass Profiler Professional softwares were used for data reprocessing and statistical analyses. A differential profiling approach was developed to investigate and compare the overall chemical diversity of dissolved substances in different coastal water bodies. Principal component analysis (PCA) allowed for spatial differentiation between areas. Similarly, SPATTs retrieved from the same location at early, medium, and late deployment periods were also differentiated by PCA, reflecting seasonal variations in chemical profiles and in the microalgal community. This study used an untargeted metabolomic approach for spatial and temporal differentiation of marine environmental chemical profiles using SPATTs, and we propose this approach as a step forward in the discovery of chemical markers of short- or long-term changes in the microbial community structure.

Effect of Nitrate, Ammonium and Urea on Growth and Pinnatoxin G Production of Vulcanodinium rugosum.

Vulcanodinium rugosum, a recently described dinoflagellate species producing a potent neurotoxin (pinnatoxin G), has been identified in French Mediterranean lagoons and was responsible for recurrent episodes of shellfish toxicity detected by mouse bioassay. Until now, the biology and physiology of V. rugosum have not been fully investigated. We studied the growth characteristics and toxicity of a V. rugosum strain (IFR-VRU-01), isolated in the Ingril lagoon in June 2009 (North-Western French Mediterranean Sea). It was cultivated in Enriched Natural Sea Water (ENSW) with organic (urea) and inorganic (ammonium and nitrate) nitrogen, at a temperature of 25 °C and irradiance of 100 µmol/m²·s(-1). Results showed that ammonium was assimilated by cells more rapidly than nitrate and urea. V. rugosum is thus an osmotrophic species using urea. Consequently, this nitrogen form could contribute to the growth of this dinoflagellate species in the natural environment. There was no significant difference (Anova, p = 0.856) between the growth rate of V. rugosum cultivated with ammonium (0.28 ± 0.11 day(-1)), urea (0.26 ± 0.08 day(-1)) and nitrate (0.24 ± 0.01 day(-1)). However, the production of chlorophyll a and pinnatoxin G was significantly lower with urea as a nitrogen source (Anova, p < 0.027), suggesting that nutritional conditions prevailing at the moment of the bloom could determine the cellular toxicity of V. rugosum and therefore the toxicity measured in contaminated mollusks. The relatively low growth rate (≤0.28 day(-1)) and the capacity of this species to continuously produce temporary cysts could explain why cell densities of this species in the water column are typically low (≤20,000 cells/L).

Beta-N-methylamino-L-alanine: LC-MS/MS optimization, screening of cyanobacterial strains and occurrence in shellfish from Thau, a French Mediterranean lagoon.

ß-N-methylamino-L-alanine (BMAA) is a neurotoxic non-protein amino acid suggested to be involved in neurodegenerative diseases. It was reported to be produced by cyanobacteria, but also found in edible aquatic organisms, thus raising concern of a widespread human exposure. However, the chemical analysis of BMAA and its isomers are controversial, mainly due to the lack of selectivity of the analytical methods. Using factorial design, we have optimized the chromatographic separation of underivatized analogues by a hydrophilic interaction chromatography coupled to tandem mass spectrometry (HILIC-MS/MS) method. A combination of an effective solid phase extraction (SPE) clean-up, appropriate chromatographic resolution and the use of specific mass spectral transitions allowed for the development of a highly selective and sensitive analytical procedure to identify and quantify BMAA and its isomers (in both free and total form) in cyanobacteria and mollusk matrices (LOQ of 0.225 and 0.15 µg/g dry weight, respectively). Ten species of cyanobacteria (six are reported to be BMAA producers) were screened with this method, and neither free nor bound BMAA could be found, while both free and bound DAB were present in almost all samples. Mussels and oysters collected in 2009 in the Thau Lagoon, France, were also screened, and bound BMAA and its two isomers, DAB and AEG, were observed in all samples (from 0.6 to 14.4 µg/g DW), while only several samples contained quantifiable free BMAA.

A feedback mechanism to control apoptosis occurs in the digestive gland of the oyster crassostrea gigas exposed to the paralytic shellfish toxins producer Alexandrium catenella.

To better understand the effect of Paralytic Shellfish Toxins (PSTs) accumulation in the digestive gland of the Pacific oyster, Crassostrea gigas, we experimentally exposed individual oysters for 48 h to a PSTs producer, the dinoflagellate Alexandrium catenella. In comparison to the effect of the non-toxic Alexandrium tamarense, on the eight apoptotic related genes tested, Bax and BI.1 were significantly upregulated in oysters exposed 48 h to A. catenella. Among the five detoxification related genes tested, the expression of cytochrome P450 (CYP1A) was shown to be correlated with toxin concentration in the digestive gland of oysters exposed to the toxic dinoflagellate. Beside this, we observed a significant increase in ROS production, a decrease in caspase-3/7 activity and normal percentage of apoptotic cells in this tissue. Taken together, these results suggest a feedback mechanism, which may occur in the digestive gland where BI.1 could play a key role in preventing the induction of apoptosis by PSTs. Moreover, the expression of CYP1A, Bax and BI.1 were found to be significantly correlated to the occurrence of natural toxic events, suggesting that the expression of these genes together could be used as biomarker to assess the biological responses of oysters to stress caused by PSTs.

Effect of temperature, salinity and nutrients on the growth and toxin content of the dinoflagellate Gymnodinium catenatum from the southwestern Mediterranean.

The dinoflagellate Gymnodinium catenatum is considered the primary cause of recurrent paralytic shellfish toxins (PSTs) in shellfish on the Moroccan Mediterranean coasts. The impacts of key environmental factors on the growth, cell yield, cell size and PST content of G. catenatum were determined. Results indicated that increasing salinity from 32 to 39 and nitrate concentrations from 441 µM to 1764 µM did not significantly (ANOVA, P-value >0.63) modify the growth rate of the studied species. Gymnodinium catenatum exhibited the highest growth rate at 24 °C. Cells arrested their division at 15 °C and at ammonium concentration above 441 µM, suggesting that this nitrogen form is toxic for G. catenatum. Furthermore, G. catenatum was unable to assimilate urea as a nitrogen source. In G. catenatum cells, eight analogues of saxitoxin were detected, belonging to the N-sulfocarbamoyl (C1-4, B1 and B2) and decarbamoyl (dc-GTX2/3) toxins. C-toxins contributed 92 % to 98 % of the molar composition of the PSTs. During the exponential growth, C2 tended to dominate, while C3 prevailed during the stationary phase. Toxin content per cell (ranging from 5.5 pg STXeq.cell-1 to 22.4 pg STXeq.cell-1) increased during the stationary growth phase. Cell toxin content increased with the concentrations of nitrate, ranging from 12.1 pg STXeq.cell-1 at 441 µM to 22.4 pg STXeq.cell-1 at 1764 µM during the stationary growth phase. The toxin content of G. catenatum showed the highest values measured at the highest tested temperatures, especially during the stationary phase, where toxicity reached 17.8 pg STXeq.cell-1 and 16.4 pg STXeq.cell-1 at 24 °C and 29 °C, respectively. The results can help understand the fluctuations in the growth and PST content of G. catenatum in its habitat in response to changing environmental variables in the Mediterranean Sea when exposed to increases in warming pressure and eutrophication.

Influence of environmental factors on the paralytic shellfish toxin content and profile of Alexandrium catenella (Dinophyceae) isolated from the Mediterranean Sea.

Laboratory experiments were designed to study the toxin content and profile of the Alexandrium catenella strain ACT03 (isolated from Thau Lagoon, French Mediterranean) in response to abiotic environmental factors under nutrient-replete conditions. This dinoflagellate can produce various paralytic shellfish toxins with concentrations ranging from 2.9 to 50.3 fmol/cell. The toxin profile was characterized by carbamate toxins (GTX3, GTX4 and GTX5) and N-sulfocarbamoyl toxins (C1, C2, C3 and C4). C2 dominated at 12-18 °C, but only for salinities ranging from 10 to 25 psu, whereas GTX5 became dominant at temperatures ranging from 21 to 30 °C at almost all salinities. There was no significant variation in the cellular toxin amount from 18 °C to 27 °C for salinities ranging between 30 and 40 psu. At salinities of 10 to 25 psu, the toxin concentrations always remained below 20 fmol/cell. Toxin content was stable for irradiance ranging from 10 to 70 µmol photons/m2/s then slightly increased. Overall, the toxin profile was more stable than the toxin content (fmol/cell), except for temperature and/or salinity values different from those recorded during Alexandrium blooms in Thau Lagoon.

Diabetes clinical trials: helped or hindered by the current shift in regulatory requirements?

Glycaemic control is an inadequate surrogate marker of cardiovascular event reduction in patients with type 2 diabetes. Clinical trials to date have been unsuccessful in identifying a therapeutic approach that addresses the underlying problem in diabetes (glycaemic control) and reduces cardiovascular risk. The potential for some agents to increase the risk of cardiovascular events has led to substantial changes in regulatory requirements for new anti-diabetic therapies. These requirements, while key to ensuring the cardiovascular safety of new agents, fail to emphasize the need to show clinical benefits, such as less visual impairment, less need for dialysis, or fewer cardiovascular events and deaths. Changes in test results such as glycaemic control, serum creatinine, micro-albuminuria, or retinopathy are inadequate surrogates. Regulators should consider the potential advantages of offering extended patent protection in order to encourage companies to conduct long-term trials in diabetes and many other chronic medical conditions. Cooperative efforts among physicians, clinical trialists, regulators, and sponsors are needed to address unresolved issues including re-defining therapeutic targets that are meaningful to patients with diabetes, determining the appropriate length of follow-up for future trials, and considering the ethical and operational challenges of non-inferiority designs.

Artificial Substrates Coupled with qPCR (AS-qPCR) Assay for the Detection of the Toxic Benthopelagic Dinoflagellate Vulcanodinium rugosum.

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.

Modelling spatiotemporal distributions of Vulcanodinium rugosum and pinnatoxin G in French Mediterranean lagoons: Application to human health risk characterisation.

Consumption of seafood contaminated by phycotoxins produced by harmful algae is a major issue in human public health. Harmful algal blooms are driven by a multitude of environmental variables; therefore predicting human dietary exposure to phycotoxins based on these variables is a promising approach in health risk management. In this study, we attempted to predict the human health risks associated with Vulcanodinium rugosum and its neurotoxins, pinnatoxins (PnTXs), which have been regularly found in Mediterranean lagoons since their identification in 2011. Based on environmental variables collected over 1 year in four Mediterranean lagoons, we developed linear mixed models to predict the presence of V. rugosum and PnTX G contamination of mussels. We found that the occurrence of V. rugosum was significantly associated with seawater temperature. PnTX G contamination of mussels was highest in summer but persisted throughout the year. This contamination was significantly associated with seawater temperature and the presence of V. rugosum with a time lag, but not with dissolved PnTX G in seawater. By using the contamination model predictions and their potential variability/uncertainty, we calculated the human acute dietary exposures throughout the year and predicted that 25% of people who consume mussels could exceed the provisional acute benchmark value during the warmest periods. We suggest specific recommendations to monitor V. rugosum and PnTX G.

The European Medicines Agency review of cabazitaxel (Jevtana®) for the treatment of hormone-refractory metastatic prostate cancer: summary of the scientific assessment of the committee for medicinal products for human use.

On March 17, 2011 the European Commission issued a marketing authorization valid throughout the European Union for Jevtana® (Sanofi-Aventis, Paris, France) for the treatment of patients with hormone-refractory metastatic prostate cancer previously treated with a docetaxel-containing regimen. The active substance of Jevtana® is cabazitaxel acetone solvate, an antineoplastic agent that acts by disrupting the microtubular network in cells. The recommended dose of cabazitaxel is 25 mg/m2 administered as a 1-hour i.v. infusion every 3 weeks in combination with oral prednisone or prednisolone, 10 mg, administered daily throughout treatment. In the main study submitted for this application, a 2.4-month longer median overall survival time and a 30% lower risk for death were observed for cabazitaxel, compared with mitoxantrone. The most common side effects with cabazitaxel were anemia, leukopenia, neutropenia, thrombocytopenia, and diarrhea. This paper summarizes the scientific review of the application leading to approval in the European Union. The detailed scientific assessment report and product information, including the summary of product characteristics, are available on the European Medicines Agency Web site (http://www.ema.europa.eu).

Liza ramada Juveniles after Exposure to the Toxic Dinoflagellate Vulcanodinium rugosum: Effects on Fish Viability, Tissue Contamination and Microalgae Survival after Gut Passage.

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.

Prediction of Alexandrium and Dinophysis algal blooms and shellfish contamination in French Mediterranean Lagoons using decision trees and linear regression: a result of 10 years of sanitary monitoring.

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.

Human poisonings by neurotoxic phycotoxins related to the consumption of shellfish: study of cases registered by the French Poison Control Centres from 2012 to 2019.

CONTEXT: In June 2019, a paralytic shellfish poisoning (PSP) case related to the consumption of mussels contaminated by saxitoxins at a concentration below the regulatory threshold came to the attention of the French Agency for Food, Environmental and Occupational Health and Safety (ANSES). This pointed to probable undetected human cases of poisoning by neurotoxic phycotoxins. METHODS: We conducted a retrospective study of poisoning cases by bivalve shellfish (oysters, mussels and scallops) recorded by the French Poison Control Centres (PCC) from 2012 to 2019. All medical records were reviewed by a toxicologist.Cases that could be related to neurotoxic phycotoxins were selected and described. Diagnosis was based on symptoms compatible with ingestion of contaminated shellfish and on contamination data for the shellfish production area (analysed by the French Research Institute for Exploitation of the Sea, Ifremer), or notifications to the European Rapid Alert System for Food and Feed when the origin of the shellfish was known. RESULTS: Among the 619 shellfish poisoning cases recorded by the PCCs from 2012 to 2019, 22% (n = 134) had reported at least one neurological symptom (headache, dizziness or paraesthesia). Review of medical records for the 134 patients led to suspicion of 14 cases of PSP and one case of amnesic shellfish poisoning. Five patients experienced persistent neurological symptoms. Marine toxins were not tested for in the blood or urine of these patients. CONCLUSION: This retrospective identification of cases strongly suspected of being related to neurotoxic phycotoxins led ANSES, PCCs and Ifremer to develop a specific questionnaire and to recommend actions to take when neurological symptoms related to shellfish consumption are reported to a PCC. Daily monitoring of shellfish poisoning cases registered in the national PCCs database was also implemented in order to rapidly detect any suspicious cases, alert the competent authorities, and warn the general population.

Development of harmful algal blooms species responsible for lipophilic and amnesic shellfish poisoning intoxications in southwestern Mediterranean coastal waters.

Mediterranean waters have undergone environmental changes during the last decades leading to various modifications of the structure of phytoplankton populations, especially Harmful Algal Blooms (HABs) species. Monitoring of the potentially toxic phytoplankton species was carried out biweekly in the western Mediterranean coast of Morocco from March 2018 to March 2019. Lipophilic Shellfish Toxins (LSTs) using LC-MS/MS and Domoic Acid (DA) using HPLC-UV were measured in the exploited mollusks, the cockle Acanthocardia tuberculata and the smooth clam Callista chione. We also determined the prevailing environmental factors in four surveyed sites (M'diq bay, Martil, Kaa Asras, and Djawn) selected to cover a variety of coastal ecosystems. Results showed that Pseudo-nitzschia spp. a DA producer species, was abundant with a pick of 50 × 103 cells l-1 on October 2018 in Djawn. Dinophysis caudata was the dominate Dinophysis species and showed a maximum density of 2200 cells l-1 on July in Djawn. Prorocentrum lima, an epibenthic dinoflagellate, appeared rarely in the water column with densities <80 cells l-1. Gonyaulax spinifera and Protoceratium reticulatum were found occasionally with a maximum density of 160 cells l-1. Karenia selliformis was detected only five times (<80 cells l-1) throughout the survey period. LC-MS/MS analyses revealed the presence of OA/DTX3, PTX-2, PTX-2 sa, and PTX-2 sa epi in the cockle at concentrations of up to 44.81 (OA/DTX-3+PTXs) ng g-1 meat. GYM-A was detected in the clam at concentrations of up to 4.22 ng g-1 meat. For the first time, AZAs and YTXs were detected in the southwestern Mediterranean with maximum values of 2.49 and 10.93 ng g-1 meat of cockle, respectively. DA was detected in moderate concentrations not exceeding 5.65 µg g-1 in both mollusks. Results showed that the observed toxic algae in the water column were responsible from the analysed toxins in the mollusks. It is likely that the southwestern Mediterranean waters could see the development of emergent species producing potent toxins (YTXs, AZAs, GYM-A). These dinoflagellates have to be isolated, ribotyped, and their toxin profiles determined.