No abstract

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.

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.

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.

Several experiments using a self-regulated system were conducted to define the factors likely to influence the uptake of paralytic shellfish poison (PSP) by oysters in the Penzé estuary (France, Brittany). Each 4-day experiment was carried out in a recirculated sea water system using 15 Pacific oysters (Crassostrea gigas) separated from each other and supplied with unfiltered natural seawater containing alternatively toxic (Alexandrium minutum) or non-toxic (Skeletonema costatum) algal diets. The food supply and exposure times to toxic diets were determined according to field studies of the upstream and downstream movement of patches containing A. minutum. The experimental parameters corresponded roughly to the hydrological conditions generally observed in June when tidal coefficients are lowest and blooms occur: (i) A. minutum concentrations in sea water of 200, 5000 and 10 000 cell ml −1 ; (ii) inorganic matter consisting of 5 and 15 mg L −1 of calcinated muddy sediments; and (iii) low and high tide salinities of 25 and 35% , respectively. Significant experimental contamination (greater than the 80 µg STX equiv. 100 g −1 sanitary threshold) occurred after 4 days of exposure for the monospecific A. minutum diet (20−200 cell ml −1) and alternated A. minutum and S. costatum diets (5000 and 20 000 cell ml −1 , respectively). Contamination levels were less than the sanitary threshold for alternated A. minutum/S. costatum diets of 200 and 20 000 cell ml −1 , respectively, and for a monospecific A. minutum diet (1000−10 000 cell ml −1). In the last case, the accumulation rate was quite low, possibly because of inhibition of the filtration rate related to a lower biodeposit production rate and decreased feeding time activity. The addition of inorganic matter appeared to play a significant role in the observed increase of toxin uptake, whereas salinity was not a determining factor for toxin accumulation rates. These last observations were corroborated by statistical analysis and stepwise multiple linear regressions integrating all or some of the experimental parameters.