Five Years Monitoring the Emergence of Unregulated Toxins in Shellfish in France (EMERGTOX 2018-2022).

Shellfish accumulate microalgal toxins, which can make them unsafe for human consumption. In France, in accordance with EU regulations, three groups of marine toxins are currently under official monitoring: lipophilic toxins, saxitoxins, and domoic acid. Other unregulated toxin groups are also present in European shellfish, including emerging lipophilic and hydrophilic marine toxins (e.g., pinnatoxins, brevetoxins) and the neurotoxin ß-N-methylamino-L-alanine (BMAA). To acquire data on emerging toxins in France, the monitoring program EMERGTOX was set up along the French coasts in 2018. Three new broad-spectrum LC-MS/MS methods were developed to quantify regulated and unregulated lipophilic and hydrophilic toxins and the BMAA group in shellfish (bivalve mollusks and gastropods). A single-laboratory validation of each of these methods was performed. Additionally, these specific, reliable, and sensitive operating procedures allowed the detection of groups of EU unregulated toxins in shellfish samples from French coasts: spirolides (SPX-13-DesMeC, SPX-DesMeD), pinnatoxins (PnTX-G, PnTX-A), gymnodimines (GYM-A), brevetoxins (BTX-2, BTX-3), microcystins (dmMC-RR, MC-RR), anatoxin, cylindrospermopsin and BMAA/DAB. Here, we present essentially the results of the unregulated toxins obtained from the French EMERGTOX monitoring plan during the past five years (2018-2022). Based on our findings, we outline future needs for monitoring to protect consumers from emerging unregulated toxins.

Physiological and Metabolic Responses of Freshwater and Brackish-Water Strains of Microcystis aeruginosa Acclimated to a Salinity Gradient: Insight into Salt Tolerance.

Proliferation of microcystin (MC)-producing Microcystis aeruginosa in brackish waters has been described in several locations and represents a new concern for public and environmental health. While the impact of a sudden salinity increase on M. aeruginosa physiology has been studied, less is known about the mechanisms involved in salt tolerance after acclimation. This study aims to compare the physiological responses of two strains of M. aeruginosa (PCC 7820 and PCC 7806), which were isolated from contrasted environments, to increasing salinities. After acclimation, growth and MC production rates were determined and metabolomic analyses were conducted. For both strains, salinity decreased the biovolume, growth, and MC production rates and induced the accumulation of polyunsaturated lipids identified as monogalactosyldiacylglycerol. The distinct salt tolerances (7.5 and 16.9) obtained between the freshwater (PCC 7820) and the brackish-water (PCC 7806) strains suggested different strategies to cope with the osmotic pressure, as revealed by targeted and untargeted metabolomic analyses. An accumulation of trehalose as the main compatible solute was obtained in the freshwater strain, while sucrose was mainly accumulated in the brackish one. Moreover, distinct levels of glycine betaine and proline accumulation were noted. Altogether, metabolomic analysis illustrated a strain-specific response to salt tolerance, involving compatible solute production.IMPORTANCE Blooms of Microcystis aeruginosa and the production of microcystins are major issues in eutrophic freshwater bodies. Recently, an increasing number of proliferations of M. aeruginosa in brackish water has been documented. The occurrence of both M. aeruginosa and microcystins in coastal areas represents a new threat for human and environmental health. In order to better describe the mechanisms involved in Microcystis sp. proliferation in brackish water, this study used two M. aeruginosa strains isolated from fresh and brackish waters. High salinity reduced the growth rate and microcystin production rate of M. aeruginosa In order to cope with higher salinities, the strains accumulated different cyanobacterial compatible solutes, as well as unsaturated lipids, explaining their distinct salt tolerance.

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.

Extraction and analysis by liquid chromatography - tandem mass spectrometry of intra- and extracellular microcystins and nodularin to study the fate of cyanobacteria and cyanotoxins across the freshwater-marine continuum.

The presence of microcystins (MCs) is increasingly being reported in coastal areas worldwide. To provide reliable data regarding this emerging concern, reproducible and accurate methods are required to quantify MCs in salt-containing samples. Herein, we characterized methods of extraction and analysis by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for nine MCs and one nodularin (NOD) variants in both cyanobacteria (intracellular) and dissolved forms (extracellular). Different approaches have been used to cope with salinity for the extraction of dissolved MCs but none assessed solid phase extraction (SPE) so far. It was found that salt had negligible effect on the SPE recovery of dissolved MCs using the C18 cartridge while an overestimation up to 67% was noted for some variants with a polymeric sorbent. The limits of detection (LOD) and quantification (LOQ) were 1.0-22 and 5.5-124 pg on column for the intracellular toxins, while 0.05-0.81 and 0.13-2.4 ng/mL were obtained for dissolved toxins. Extraction recoveries were excellent for intracellular (89-121%) and good to excellent for extracellular cyanotoxins (73-102%) while matrix effects were considered neglectable (<12% for 16/20 toxin-matrix combinations), except for the two MC-RR variants. The strategy based on the application of a corrective factor to compensate for losses proved useful as the accuracy was satisfactory (73-117% for intra- and 81-139% for extracellular cyanotoxins, bias <10% for 46/60 conditions, with a few exceptions), with acceptable precisions (intra- and inter-days variabilities <11%). We then applied this method on natural colonies of Microcystis spp. subjected to a salt shock, mimicking their estuarine transfer, in order to assess their survival and to quantify their toxins. The colonies of Microcystis spp. had both their growth and photosynthetic activity impaired at salinities from 10, while toxins remained mainly intracellular (>76%) even at salinity 20, suggesting a potential health risk and contamination of estuarine organisms.

Effects of salinity and nutrient stress on a toxic freshwater cyanobacterial community and its associated microbiome: An experimental study.

We aimed to evaluate the ability of naturally occurring colonies of Microcystis, embedded in a thick mucilage, to persist in estuarine waters. In two batch experiments, we examined the dynamics of microbial communities, including cyanobacteria and associated heterotrophic bacteria, sampled from the field during both a cyanobacterial bloom (non-limiting nutrient condition) and the post-bloom period (limiting nutrient condition), and subjected them to a salinity gradient representative of the freshwater-marine continuum. We demonstrated that both Microcystis aeruginosa and M. wesenbergii survived high salinities due to osmolyte accumulation. Specifically, prolonged exposure to high salinity led to betaine accumulation in the cyanobacterial biomass. The relative abundance of the mcyB gene remained around 30%, suggesting no selection for toxic genotypes with salinity or nutrient changes. Microcystins were predominantly intracellular, except at high salinity levels (>15), where more than 50% of the total microcystin concentration was extracellular. In both nutrient conditions, over 70% of the heterotrophic bacterial community belonged to the Gammaproteobacteria family, followed by the Bacteroidota. Bacterial community composition differed in both size fractions, as well as along the salinity gradient over time. Finally, genus-specific core microbiomes were identified and conserved even under highly stressful conditions, suggesting interactions that support community stability and resilience.

Spatio-temporal connectivity of a toxic cyanobacterial community and its associated microbiome along a freshwater-marine continuum.

Due to climate changes and eutrophication, blooms of predominantly toxic freshwater cyanobacteria are intensifying and are likely to colonize estuaries, thus impacting benthic organisms and shellfish farming representing a major ecological, health and economic risk. In the natural environment, Microcystis form large mucilaginous colonies that influence the development of both cyanobacterial and embedded bacterial communities. However, little is known about the fate of natural colonies of Microcystis by salinity increase. In this study, we monitored the fate of a Microcystis dominated bloom and its microbiome along a French freshwater-marine gradient at different phases of a bloom. We demonstrated changes in the cyanobacterial genotypic composition, in the production of specific metabolites (toxins and compatible solutes) and in the heterotrophic bacteria structure in response to the salinity increase. In particular M. aeruginosa and M. wesenbergii survived salinities up to 20. Based on microcystin gene abundance, the cyanobacteria became more toxic during their estuarine transfer but with no selection of specific microcystin variants. An increase in compatible solutes occurred along the continuum with extensive trehalose and betaine accumulations. Salinity structured most the heterotrophic bacteria community, with an increased in the richness and diversity along the continuum. A core microbiome in the mucilage-associated attached fraction was highly abundant suggesting a strong interaction between Microcystis and its microbiome and a likely protecting role of the mucilage against an osmotic shock. These results underline the need to better determine the interactions between the Microcystis colonies and their microbiome as a likely key to their widespread success and adaptation to various environmental conditions.

Sudden peak in tetrodotoxin in French oysters during the summer of 2021: Source investigation using microscopy, metabarcoding and droplet digital PCR.

Tetrodotoxin (TTX) is a potent neurotoxin causing human intoxications from contaminated seafood worldwide and is of emerging concern in Europe. Shellfish have been shown to contain varying TTX concentrations globally, with concentrations typically higher in Pacific oysters Crassostrea gigas in Europe. Despite many decades of research, the source of TTX remains unknown, with bacterial or algal origins having been suggested. The aim of this study was to identify potential source organisms causing TTX contamination in Pacific oysters in French coastal waters, using three different techniques. Oysters were deployed in cages from April to September 2021 in an estuary where TTX was previously detected. Microscopic analyses of water samples were used to investigate potential microalgal blooms present prior or during the peak in TTX. Differences in the bacterial communities from oyster digestive glands (DG) and remaining flesh were explored using metabarcoding, and lastly, droplet digital PCR assays were developed to investigate the presence of Cephalothrix sp., one European TTX-bearing species in the DG of toxic C. gigas. Oysters analysed by liquid chromatography-tandem mass spectrometry contained quantifiable levels of TTX over a three-week period (24 June-15 July 2021), with concentrations decreasing in the DG from 424 µg/kg for the first detection to 101 µg/kg (equivalent to 74 to 17 µg/kg of total flesh), and trace levels being detected until August 13, 2021. These concentrations are the first report of the European TTX guidance levels being exceeded in French shellfish. Microscopy revealed that some microalgae bloomed during the TTX peak, (e.g., Chaetoceros spp., reaching 40,000 cells/L). Prokaryotic metabarcoding showed increases in abundance of Rubritaleaceae (genus Persicirhabdus) and Neolyngbya, before and during the TTX peak. Both phyla have previously been described as possible TTX-producers and should be investigated further. Droplet digital PCR analyses were negative for the targeted TTX-bearing genus Cephalothrix.

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.

Toxic dinoflagellate Centrodinium punctatum (Cleve) F.J.R. Taylor: An examination on the responses in growth and toxin contents to drastic changes of temperature and salinity.

To understand environmental effects affecting paralytic shellfish toxin production of Centrodinium punctatum, this study examined the growth responses, and toxin contents and profiles of a C. punctatum culture exposed to drastic changes of temperature (5-30 °C) and salinity (15-40). C. punctatum grew over a temperature range of 15-25 °C, with an optimum of 20 °C., and over a salinity range of 25-40, with optimum salinities of 30-35. This suggests that C. punctatum prefers relatively warm waters and an oceanic habitat for its growth and can adapt to significant changes of salinity levels. When C. punctatum was cultivated at different temperature and salinity levels, the PST profile included four major analogs (STX, neoSTX, GTX1 and GTX4, constituted >80 % of the profile), while low amounts of doSTX and traces of dc-STX and dc-GTX2 were also observed. Interestingly, though overall toxin contents did not change significantly with temperature, increases in the proportion of STX, and decreases in proportions in GTX1 and GTX4 were observed with higher temperatures. Salinity did not affect either toxin contents or profile from 25 to 35. However, the total toxin content dropped to approximately half at salinity 40, suggesting this salinity may induce metabolic changes in C. punctatum.

Exposure to the neurotoxic dinoflagellate, Alexandrium catenella, induces apoptosis of the hemocytes of the oyster, Crassostrea gigas.

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, suggesting that the intrinsic pathway was activated. No modulation of the expression of genes implicated in the cell signaling Fas-Associated protein with Death Domain (FADD) and initiation-phase (caspase-2) was observed, suggesting that only the extrinsic pathway was not activated. Moreover, the clear time-dependent upregulation of five (Bcl2, BI-1, IAP1, IAP7B and Hsp70) inhibitors of apoptosis-related genes associated with the return to the initial number of hemocytes in apoptosis at 48 h of exposure suggests the involvement of strong regulatory mechanisms of apoptosis occurring in the hemocytes of the Pacific oyster.

Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Paralytic toxins accumulation and tissue expression of α-amylase and lipase genes in the Pacific oyster Crassostrea gigas fed with the neurotoxic dinoflagellate Alexandrium catenella.

The pacific oyster Crassostrea gigas was experimentally exposed to the neurotoxic Alexandrium catenella and a non-producer of PSTs, Alexandrium tamarense (control algae), at concentrations corresponding to those observed during the blooming period. At fixed time intervals, from 0 to 48 h, we determined the clearance rate, the total filtered cells, the composition of the fecal ribbons, the profile of the PSP toxins and the variation of the expression of two α-amylase and triacylglecerol lipase precursor (TLP) genes through semi-quantitative RT-PCR. The results showed a significant decrease of the clearance rate of C. gigas fed with both Alexandrium species. However, from 29 to 48 h, the clearance rate and cell filtration activity increased only in oysters fed with A. tamarense. The toxin concentrations in the digestive gland rose above the sanitary threshold in less than 48 h of exposure and GTX6, a compound absent in A. catenella cells, accumulated. The α-amylase B gene expression level increased significantly in the time interval from 6 to 48 h in the digestive gland of oysters fed with A. tamarense, whereas the TLP gene transcript was significantly up-regulated in the digestive gland of oysters fed with the neurotoxic A. catenella. All together, these results suggest that the digestion capacity could be affected by PSP toxins.

New insights into the dynamics of causative dinoflagellates and the related contamination of molluscs by paralytic toxins in the southwestern Mediterranean coastal waters of Morocco.

The distribution of the two potentially toxic dinoflagellates Gymnodinium catenatum and Alexandrium spp. was investigated in the Mediterranean Moroccan Sea from March 2018 to March 2019. The cockle Acanthocardia tuberculata and the smooth clam Callista chione were collected at four stations, and their toxin levels were assessed using the mouse bioassay. The toxin profile was analysed by LC-MS/MS in G. catenatum and in the bivalves harvested in M'diq and Djawn. The species G. catenatum was present throughout the year, whereas Alexandrium spp. was less abundant. The paralytic shellfish toxin (PST) level in cockles was, on average, six times above the sanitary threshold; GTX5 was the major contributor to the total PST level, followed by dc-STX and STX. The toxin level of the smooth clam was considerably lower than that of the cockle; GTX5 and C-toxins were the dominating analogues. Our results suggest the responsibility of G. catenatum for the recurrent PST contamination in the Moroccan Mediterranean Sea, with a west-east gradient.

In situ use of bivalves and passive samplers to reveal water contamination by microcystins along a freshwater-marine continuum in France.

Cyanobacteria are a potential threat to aquatic ecosystems and human health because of their ability to produce cyanotoxins, such as microcystins (MCs). MCs are regularly monitored in fresh waters, but rarely in estuarine and marine waters despite the possibility of their downstream export. Over a period of two years, we monthly analyzed intracellular (in phytoplankton) and extracellular (dissolved in water) MCs at five stations along a river continuum from a freshwater reservoir with ongoing cyanobacterial blooms to the coast of Brittany, France. MCs were quantified using two integrative samplers placed at each site: solid phase adsorption toxin tracking (SPATT) samplers for collecting extracellular MCs and caged mussels (Anodonta anatina and Mytilus edulis) filter-feeding on MC-producing cyanobacteria. The MC transfer was demonstrated each year during five months at estuarine sites and sporadically at the marine outlet. SPATT samplers integrated extracellular MCs, notably at low environmental concentrations (0.2 µg/L) and with the same variant profile as in water. The mussel A. anatina highlighted the presence of MCs including at intracellular concentrations below 1 µg/L. M. edulis more efficiently revealed the MC transfer at estuarine sites than water samplings. Bivalves showed the same MC variant profile as phytoplankton samples, but with differential accumulation capacities between the variants and the two species. Using SPATT or bivalves can give a more accurate assessment of the contamination level of a freshwater-marine continuum, in which the MC transfer can be episodic. MC content in M. edulis represents a potent threat to human health if considering updated French guideline values, and particularly the total (free and protein-bound) MC content, highlighting the necessity to include cyanotoxins in the monitoring of seafood originating from estuarine areas.

Tetrodotoxins in French Bivalve Mollusks-Analytical Methodology, Environmental Dynamics and Screening of Bacterial Strain Collections.

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.

Demonstrated transfer of cyanobacteria and cyanotoxins along a freshwater-marine continuum in France.

The frequency of cyanobacterial proliferations in fresh waters is increasing worldwide and the presence of associated cyanotoxins represent a threat for ecosystems and human health. While the occurrence of microcystin (MC), the most widespread cyanotoxin, is well documented in freshwaters, only few studies have examined its occurrence in estuarine waters. In this study we evaluated the transfer of cyanobacteria and cyanotoxins along a river continuum from a freshwater reservoir through an interconnecting estuary to the coastal area in Brittany, France. We sampled regularly over 2 years at 5 stations along the river continuum and analysed for phytoplankton and cyanotoxins, together with physico-chemical parameters. Results show that cyanobacteria dominated the phytoplanktonic community with high densities (up to 2 × 106 cells mL-1) at the freshwater sites during the summer and autumn periods of both years, with a cell transfer to estuarine (up to 105 cells mL-1) and marine (2 × 103 cells mL-1) sites. While the temporal variation in cyanobacterial densities was mainly associated with temperature, spatial variation was due to salinity while nutrients were non-limiting for cyanobacterial growth. Cyanobacterial biomass was dominated by several species of Microcystis that survived intermediate salinities. Intracellular MCs were detected in all the freshwater samples with concentrations up to 60 µg L-1, and more intermittently with concentrations up to 1.15 µg L-1, at the most upstream estuarine site. Intracellular MC was only sporadically detected and in low concentration at the most downstream estuarine site and at the marine outlet (respectively <0.14 µg L-1 and <0.03 µg L-1). Different MC variants were detected with dominance of MC-LR, RR and YR and that dominance was conserved along the salinity gradient. Extracellular MC contribution to total MC was higher at the downstream sites in accordance with the lysing of the cells at elevated salinities. No nodularin (NOD) was detected in the particulate samples or in the filtrates.

First report on azaspiracid and yessotoxin groups detection in French shellfish.

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 scallops (Aequipecten opercularis) from Northern Brittany; (2) during the summer of 2007, YTX and its major metabolites (45-hydroxy-YTX, homo-YTX, carboxy-YTX) in shellfish from the Mediterranean coast. Regarding YTX-group, the toxin profiles evolution in mussels during summer showed that: (i) the carboxy-YTX depuration rate was much slower than the YTX and 45-hydroxy-YTX ones; (ii) the homo-YTX concentration, which was initially very weak, increased significantly during the last depuration phase, which seems to reveal a YTX-group high metabolisation level in mussels. This paper reports for the first time on AZA and YTX-groups detection in French shellfish.

Exposure to the Paralytic Shellfish Toxin Producer Alexandrium catenella Increases the Susceptibility of the Oyster Crassostrea gigas to Pathogenic Vibrios.

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 catenella. Exposure of oysters to A. catenella significantly increased the susceptibility of oysters to V. tasmaniensis LGP32. On the contrary, exposure to the non-toxic dinoflagellate Alexandrium tamarense or to the haptophyte Tisochrysis lutea used as a foraging alga did not increase susceptibility to V. tasmaniensis LGP32. This study shows for the first time that A. catenella increases the susceptibility of Crassostrea gigas to pathogenic vibrios. Therefore, in addition to complex environmental factors explaining the mass mortalities of bivalve mollusks, feeding on neurotoxic dinoflagellates should now be considered as an environmental factor that potentially increases the severity of oyster mortality events.

Relationship between valve activity, microalgae concentration in the water and toxin accumulation in the digestive gland of the Pacific oyster Crassostrea gigas exposed to Alexandrium minutum.

The complexity of the relationships between Alexandrium minutum (A.m.) concentration in the water ([A.m.](w)), Paralytic Shellfish Poisoning contamination in the digestive gland ([PSP](dg)) and valve behavior was explored in oysters Crassostrea gigas. Two experiments were conducted, during which oysters' valve behaviour were analyzed. Oysters, first acclimated for 10-days with the non harmful microalgae Heterocapsa triquetra (H.t.), were exposed to four microalgae mixtures at constant total concentrations of 10×10(3)cells ml(-1) (experiment-1) and 5×10(3)cells ml(-1) (experiment-2): 100% A.m.; 50% A.m.-50% H.t.; 25% A.m.-75% H.t.; 100% H.t. At the end of experiment-2, [PSP](dg) were measured. At 10×10(3)cells ml(-1), the microalgal ingestion decreased (p<0.05) with increasing [A.m.](w) but not at 5×10(3)cells ml(-1) (p>0.05). The frequency of microclosures specifically increased with [A.m.](w) (p<0.05) and the opening duration with [PSP](dg) (p<0.0001). Oysters exhibiting the maximum increase in opening duration also exhibited the highest [PSP](dg). The results are discussed in terms of oyster physiology and origin of the behavioral response.