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.

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.

Marine biotoxins are algal metabolites that can accumulate in fish or shellfish and render these foodstuffs unfit for human consumption. These toxins, released into seawater during algal occurrences, can be monitored through passive sampling. Acetone, methanol and isopropanol were evaluated for their efficiency in extracting toxins from algal biomass. Isopropanol was chosen for further experiments thanks to a slightly higher recovery and no artifact formation. Comparison of Oasis HLB, Strata-X, BondElut C18 and HP-20 sorbent materials in SPE-mode led to the choice of Oasis HLB, HP-20 and Strata-X. These three sorbents were separately exposed as passive samplers for 24 h to seawater spiked with algal extracts containing known amounts of okadaic acid (OA), azaspiracids (AZAs), pinnatoxin-G (PnTX-G), 13-desmethyl spirolide-C (SPX1) and palytoxins (PlTXs). Low density polyethylene (LDPE) and silicone rubber (PDMS) strips were tested in parallel on similar mixtures of spiked natural seawater for 24 h. These strips gave significantly lower recoveries than the polymeric sorbents. Irrespective of the toxin group, the adsorption rate of toxins on HP-20 was slower than on Oasis HLB and Strata-X. However, HP-20 and Strata-X gave somewhat higher recoveries after 24 h exposure. Irrespective of the sorbent tested, recoveries were generally highest for cyclic imines and OA group toxins, slightly lower for AZAs, and the lowest for palytoxins. Trials in re-circulated closed tanks with mussels exposed to Vulcanodinium rugosum or Prorocentrum lima allowed for further evaluation of passive samplers. In these experiments with different sorbent materials competing for toxins in the same container, Strata-X accumulated toxins faster than Oasis HLB, and HP-20, and to higher levels. The deployment of these three sorbents at Ingril French Mediterranean lagoon to detect PnTX-G in the water column showed accumulation of higher levels on HP-20 and Oasis HLB compared to Strata-X. This study has significantly extended the range of sorbents for passive sampling of marine toxins. In particular, sorbents were included that had previously been evaluated for polyhalogenated contaminants, pharmaceuticals, phytochemicals or veterinary residues. Moreover, this study has for the first time demonstrated the usefulness of the polymeric Oasis HLB and Strata-X sorbents in laboratory and field studies for various microalgal toxins.

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