Dissolved azaspiracids are absorbed and metabolized by blue mussels (Mytilus edulis).

The relationship between azaspiracid shellfish poisoning and a small dinoflagellate, Azadinium spinosum, has been shown recently. The organism produces AZA1 and -2, while AZA3 and other analogues are metabolic products formed in shellfish. We evaluated whether mussels were capable of accumulating dissolved AZA1 and -2, and compared the toxin profiles of these mussels at 24 h with profiles of those exposed to live or lysed A. spinosum. We also assessed the possibility of preparative production of AZA metabolites by exposing mussels to semi-purified AZA1. We exposed mussels to similar concentration of AZAs: dissolved AZA1 + 2 (crude extract) at 7.5 and 0.75 µg L(-1), dissolved AZA1+2 (7.5 µg L(-1)) in combination with Isochrysis affinis galbana, and lysed and live A. spinosum cells at 1 × 10(5) and 1 × 10(4) cell mL(-1) (containing equivalent amounts of AZA1 + 2). Subsequently, we dissected and analysed digestive glands, gills and remaining flesh. Mussels (whole flesh) accumulated AZAs to levels above the regulatory limit, except at the lower levels of dissolved AZAs. The toxin profile of the mussels varied significantly with treatment. The gills contained 42-46% and the digestive glands 23-24% of the total toxin load using dissolved AZAs, compared to 3-12% and 75-90%, respectively, in mussels exposed to live A. spinosum. Exposure of mussels to semi-purified AZA1 produced the metabolites AZA17 (16.5%) and AZA3 (1.7%) after 4 days of exposure, but the conversion efficiency was too low to justify using this procedure for preparative isolation.

Effect of Azadinium spinosum on the feeding behaviour and azaspiracid accumulation of Mytilus edulis.

Azadinium spinosum, a small toxic dinoflagellate, was recently isolated and identified as a primary producer of azaspiracid toxins (AZAs). Previous experiments related to AZA accumulation in blue mussels upon direct feeding with A. spinosum revealed increased mussel mortality and had negative effects on the thickness of the digestive gland tubules. Therefore we conducted follow up experiments in order to study effects of A. spinosum on mussel feeding behaviour. Individual assessment of mussel feeding time activity (FTA), clearance rate (CR), filtration rate (TFR), absorption rate (AR), faeces and pseudofaeces production were carried out on mussel fed either toxic (A. spinosum) or non-toxic (Isochrisis aff. galbana (T-Iso)) diets. Furthermore, AZA accumulation and biotransformation in mussels were followed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A. spinosum had a significant effect on mussel feeding behaviour compared to T-Iso: CR was lower by a factor of 6, FTA by a factor of 5, TFR by a factor of 3 and AR even decreased to negative values for the last day of exposure. Even so, a rapid AZA accumulation was observed during the first hours of the trial; less than 6h of feeding were required to reach AZA concentration in mussel above regulatory level. In consistence with physiological observations, AZA concentration of about 200 µg kg(-1) did not increase further until the end of the study. AZA bioconversion was also found to be a fast process: after 3h of exposure AZA17, -19 and AZA7-10 were already found, with a proportion of AZA17 equal to AZA2. These results show a negative effect of A. spinosum on blue mussel feeding activity and indicate a possible regulation of AZA uptake by decreasing filtration and increasing pseudofaeces production.

Production and isolation of azaspiracid-1 and -2 from Azadinium spinosum culture in pilot scale photobioreactors.

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.

Azaspiracid accumulation, detoxification and biotransformation in blue mussels (Mytilus edulis) experimentally fed Azadinium spinosum.

Azadinium spinosum (Elbrächter and Tillmann), a small marine dinoflagellate, has been recently described as a de novo producer of azaspiracid-1 and -2 (AZA1 and -2) diarrhoeic toxins. A culture of A. spinosum was established in our laboratory and optimised for pilot-scale production of this organism, to evaluate and understand AZA1 and -2 accumulation and biotransformation in blue mussels (Mytilus edulis) fed with A. spinosum. Adult mussels were continuously exposed to A. spinosum over 1 week in 160 L cylindrical conical tanks. Three different diets were tested for contamination: 5000, 10 000 cells mL(-1) of A. spinosum and a mixture of 5000 cells mL(-1) of A. spinosum with 5000 cells mL(-1) of Isochrysis aff. galbana (T-Iso, CCAP 927/14). During the subsequent period of detoxification (2 weeks), contaminated mussels were continuously fed with 5000 cells mL(-1) of T-Iso. Kinetics of accumulation, detoxification and biotransformation were evaluated, as well as the toxin distribution and the effect of A. spinosum on mussel digestive gland tubules. M. edulis fed on A. spinosum in the three tested conditions; this finding confirmed our recent experiments feeding A. spinosum to mussels. The original algal toxins AZA1 and -2, as well as mussel metabolites AZA3 to 12, -17, -19, -21 and -23 were found during these trials. After as little as 6 h, azaspiracid contents in mussels reached the EU regulatory limit, and metabolites were observed in all conditions at approximately 25% of the total AZA content. This fraction exceeded 50% after 24 h, and continued to increase until the end of the study. AZA17 and -19 were found to be the main metabolites, with AZA17 concentrations estimated in the same order of magnitude as that of the main algal toxin, AZA1.

Influence of Crassostrea gigas (Thunberg) sexual maturation stage and ploidy on uptake of paralytic phycotoxins.

The purpose of this study was to assess paralytic phycotoxin uptake in diploid and triploid oysters at two stages of their sexual cycle corresponding to their status in early summer (June) and winter (November). Samples of diploid and triploid oysters were exposed to a toxic culture of Alexandrium minutum for 4 days in each season. No significant differences in filtration or clearance rates were observed during either November or June experiments. When diploid oysters were at resting stage (November), toxin uptake showed no significant difference between the ploidy classes. In contrast, when the diploid oysters were at the peak of their sexual maturation (June), the triploid oysters were seen to accumulate almost double the amount of paralytic toxins as the diploid ones.