Effect of culture and bloom development and of sample storage on paralytic shellfish poisons in the cyanobacterium Anabaena circinalis

Paralytic shellfish poisons (PSPs) were detected in 24 of 31 bloom samples dominated by the cyanobacterium Anabaena circinalis Rabenhorst, collected from across Australia. The ability to produce PSPs has been maintained in several non-axenic strains of A, circinalis kept in culture, whereas strains that were non-toxin-producing when isolated have remained as such. PSPs were detected and quantified by highperformance liquid chromatography (HPLC), and the structures were confirmed by electrospray mass spectroscopy. The concentration of toxins in PSP-positive samples ranged from 50 to 3400 mu g . g(-1) dry weight. Toxin profiles were always dominated by the N-sulfocarbamoyl-11-hydroxysulfate C toxins, C1 and C2 (44-85 mol%), with the remainder consisting of gonyautoxins-2, -3, and -5, decarbamoylgonyautoxins-2 and -3, saxitoxin, and decarbamoylsaxitoxin. N-1-hydroxy PSPs, commonly found in marine dinoflagellates, were absent, suggesting that A. circinalis lacks the enzyme responsible for N-1-hydroxylation. On a dry weight basis, the amount of toxin in cultured Anabaena circinalis (strain ACMB06) rose significantly (P < 0.05) over time from 570 to 3400 mu g . g(-1) cells in late stationary phase. However, there was no significant trend in cellular toxin quota (toxin per cell) over the life of the culture; this may be explained by variation in cell mass. On average, batch cultures of Anabaena circinalis contained 19% extracellular toxin, which increased slightly over the growth cycle and had a composition similar to that of the intracellular toxins. As cultures aged the formation of decarbamoyl toxins and increases in the alpha-/beta-epimer ratios of C toxins and gonyautoxins were observed. The variation in these components during stationary phase in culture was sufficient to explain the variation in relative PSP composition observed among natural bloom samples. Because decarbamoylgonyautoxins are much more toxic than C toxins on a molar basis, these transformations also lead to an increase in toxicity of the sample or bloom over time. The transformations of PSPs, which occur during aging and sample storage, render the comparison of PSPs by HPLC unreliable for phenotyping Anabaena circinalis, unless strains are cultured, harvested, and analyzed under standard conditions.