β-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.

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.

Background: An ultrahigh-performance LC (UHPLC)–tandem MS (MS/MS) method for determination of paralytic shellfish poisoning toxins and tetrodotoxin (TTX) in bivalve molluscs was developed. To be used for regulatory testing, it needed to be validated through collaborative study. Objective: The aim was to conduct a collaborative study with 21 laboratories, using results to assess method performance. Methods: Study materials incorporated shellfish species mussels, oysters, cockles, scallops, and clams and were assessed to demonstrate stability and homogeneity. Mean concentrations determined by participants for blind duplicate samples were used to assess reproducibility, repeatability, and trueness. Results: Method performance characteristics were excellent following statistical assessment of participant data, with method trueness showing excellent method accuracy against expected values. No significant difference was found in the trueness results determined by different chromatographic column types. Acceptability of the between-laboratory reproducibility for individual analytes was evidenced by >99% of valid Horwitz ratio values being less than the 2.0 limit of acceptability. With excellent linearity and sensitivity fit-for-purpose over a range of mass spectrometer instruments, the UHPLC-MS/MS method compared well against other detection methods. It includes additional paralytic shellfish toxin (PST) analogues as well as TTX, which, to date, have not been incorporated into any other hydrophilic marine toxin official method of analysis. Conclusions: The results from this study demonstrate that the method is suitable for the analysis of PST analogues and TTX in shellfish tissues and is recommended as an official alternative method of analysis for regulatory control. Highlights: A new mass spectrometric method for PST and TTX has been validated successfully through collaborative study.

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.

Azadinium spinosum, a small dinoflagellate isolated from the North Sea, is a producer of azaspiracids (AZAs), a group of biotoxins associated with human illness following ingestion of contaminated shellfish. Using batch and continuous cultures of A. spinosum, the present study investigated the effects of different environmental and nutritional factors (salinity, temperature, photon flux density, aeration, culture media, nitrogen sources, phosphate source, and N/P ratios) on growth, maximum cell concentration, and AZA cell quota. Azadinium spinosum grew in a wide range of conditions fro 1 C to 6 C and salinities from 30 to 40, under irradiances ranging from 50 μmol m − s −1 to 250 μmol m − s −1 , with or without aeration. Growth and maximum cell concentration were highest at a salinit of 35, at te peratures between 1 C and C, and with aeration Concerning cell quota, the ost significant effect was observed at low te perature the cell quota was ore than ti es higher at 1 C (220 fg cell −1 than at te peratures between 1 C and 6 C. A. spinosum grew on all media tested with only slight differences in growth rate and AZA cell quota. In continuous culture, lowering the concentration of nutrients (0.5 strength of a modified K-medium) in the inflow improved AZA cell quota whereas higher concentration (doubling the normal strength of Kmedium) improved maximal cell concentration. A. spinosum grew on different sources of nitrogen tested (nitrate, urea, ammonium) with almost no effect on toxin cell quota and growth, except that adding ammonium caused a decrease in growth. These first experiments on Azadinium spinosum increased our knowledge on factors affecting its growth and toxin production; furthermore, these results allowed and improved particularly A. spinosum production in pilot scale photobioreactors for AZA isolation.