Ostreopsis cf. ovata produces palytoxin analogues including ovatoxins (OVTXs) and a putative palytoxin (p-PLTX), which can accumulate in marine organisms and may possibly lead to food intoxication. However, purified ovatoxins are not widely available and their toxicities are still unknown. The aim of this study was to improve understanding of the ecophysiology of Ostreopsis cf. ovata and its toxin production as well as to optimize the purification process for ovatoxin. During Ostreopsis blooms in 2011 and 2012 in Villefranche-sur-Mer (France, NW Mediterranean Sea), microalgae epiphytic cells and marine organisms were collected and analyzed both by LC-MS/MS and hemolysis assay. Results obtained with these two methods were comparable, suggesting ovatoxins have hemolytic properties. An average of 223 μg·kg−1 of palytoxin equivalent of whole flesh was found, thus exceeding the threshold of 30 μg·kg−1 in shellfish recommended by the European Food Safety Authority (EFSA). Ostreopsis cells showed the same toxin profile both in situ and in laboratory culture, with ovatoxin-a (OVTX-a) being the most abundant analogue (~50%), followed by OVTX-b (~15%), p-PLTX (12%), OVTX-d (8%), OVTX-c (5%) and OVTX-e (4%). Ostreopsis cf. ovata produced up to 2 g of biomass per L of culture, with a maximum concentration of 300 pg PLTX equivalent cell−1. Thus, an approximate amount of 10 mg of PLTX-group toxins may be produced with 10 L of this strain. Toxin extracts obtained from collected biomass were purified using different techniques such as liquid-liquid partition or size exclusion. Among these methods, open-column chromatography with Sephadex LH20 phase yielded the best results with a cleanup efficiency of 93% and recovery of about 85%, representing an increase of toxin percentage by 13 fold. Hence, this purification step should be incorporated into future isolation exercises.

Following a review of official control data on shellfish in France, Ingril Lagoon had been identified as a site where positive mouse bioassays for lipophilic toxins had been repeatedly observed. These unexplained mouse bioassays, also called atypical toxicity, coincided with an absence of regulated toxins and rapid death times in mice observed in the assay. The present study describes pinnatoxin G as the main compound responsible for the toxicity observed using the mouse bioassay for lipophilic toxins. Using a well-characterised standard for pinnatoxin G, LC-MS/MS analysis of mussel samples collected from 2009 to 2012 revealed regular occurrences of pinnatoxin G at levels sufficient to account for the toxicity in the mouse bioassays. Baseline levels of pinnatoxin G from May to October usually exceeded 40 µg kg−1 in whole flesh, with a maximum in September 2010 of around 1200 µg kg−1. These concentrations were much greater than those at the other 10 sites selected for vigilance testing, where concentrations did not exceed 10 µg kg−1 in a 3-month survey from April to July 2010, and where rapid mouse deaths were not typically observed. Mussels were always more contaminated than clams, confirming that mussel is a good sentinel species for pinnatoxins. Profiles in mussels and clams were similar, with the concentration of pinnatoxin A less than 2% that of pinnatoxin G, and pteriatoxins were only present in non-quantifiable traces. Esters of pinnatoxin G could not be detected by analysis of extracts before and after alkaline hydrolysis. Analysis with a receptor-binding assay showed that natural pinnatoxin G was similarly active on the nicotinic acetylcholine receptor as chemically synthesized pinnatoxin G. Culture of Vulcanodinium rugosum, previously isolated from Ingril lagoon, confirmed that this alga is a pinnatoxin G producer (4.7 pg cell−1). Absence of this organism from the water column during prolonged periods of shellfish contamination and the dominance of non-motile life stages of V. rugosum both suggest that further studies will be required to fully describe the ecology of this organism and the accumulation of pinnatoxins in shellfish.

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The presence of Ostreopsis cf. ovata on the Mediterranean coast represents a serious concern to human health due to production of toxins - putative palytoxin and ovatoxins (ovatoxin-a, -b, -c, -d, -e, -f and -g). However, purified ovatoxins are not widely available and their toxicities are still unknown. In the present study, we report on HR LC-MS/MS analysis of a French O. cf. ovata strain (IFR-OST-0.3V) collected at Villefranche-sur-Mer (France) during a bloom in 2011. Investigation of this strain of O. cf. ovata cultivated in our laboratory by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS) confirmed the production of ovatoxins-a to -e and revealed the presence of a new ovatoxin analog, named ovatoxin-h. O. cf. ovata extracts were pre-purified by Sephadex LH-20 to obtain a concentrated fraction of ovatoxins (OVTXs). This method provided a recovery of about 85% of OVTXs and a cleanup efficiency of 93%. Different stationary phases were tested with this fraction of interest to elucidate the structure of the new OVTX congener and to obtain purified ovatoxins. Eight reversed phase sorbents were evaluated for their capacity to separate and purify ovatoxins. Among them Kinetex C18, Kinetex PFP and Uptisphere C18-TF allowed for best separations almost achieving baseline resolution. Kinetex C18 is able to sufficiently separate these toxins, allowing us to identify the toxins present in the extract purified by Sephadex LH-20, and to partly elucidate the structure of the new ovatoxin congener. This toxin possesses one oxygen atom less and two hydrogens more than ovatoxin-a. Investigations using liquid chromatography coupled to high resolution tandem mass spectrometry suggest that the part of the molecule where ovatoxin-h differs from ovatoxin-a is situated between C42 and C49. Uptisphere C18-TF was proposed as a first step preparative chromatography as it is able to separate a higher number of ovatoxins (especially ovatoxin-d and ovatoxin-e) and because it separates ovatoxins from unknown compounds, identified using full scan single quadrupole mass spectrometry. After pre-purification with Sephadex LH-20, purification and separation of individual ovatoxins was attempted using an Uptisphere C18-TF column. During recovery of purified toxins, problems of stability of OVTXs were observed, leading us to investigate experimental conditions responsible for this degradation.

Azaspiracid (AZA) poisoning has been reported following consumption of contaminated shellfish, and is of human health concern. Hence, it is important to have sustainable amounts of the causative toxins available for toxicological studies and for instrument calibration in monitoring programs, without having to rely on natural toxin events. Continuous pilot scale culturing was carried out to evaluate the feasibility of AZA production using Azadinium spinosum cultures. Algae were harvested using tangential flow filtration or continuous centrifugation. AZAs were extracted using solid phase extraction (SPE) procedures, and subsequently purified. When coupling two stirred photobioreactors in series, cell concentrations reached 190,000 and 210,000 cell·mL−1 at steady state in bioreactors 1 and 2, respectively. The AZA cell quota decreased as the dilution rate increased from 0.15 to 0.3 day−1, with optimum toxin production at 0.25 day−1. After optimization, SPE procedures allowed for the recovery of 79 ± 9% of AZAs. The preparative isolation procedure previously developed for shellfish was optimized for algal extracts, such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of more than 70% was achieved, and isolation from 1200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 of >95% purity. This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures.

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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.