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 sam ples dominated by the cyanobacterium Anabaena circinalis Rabenhorst, c ollected from across Australia. The ability to produce PSPs has been m aintained in several non-axenic strains of A, circinalis kept in cultu re, whereas strains that were non-toxin-producing when isolated have r emained as such. PSPs were detected and quantified by highperformance liquid chromatography (HPLC), and the structures were confirmed by ele ctrospray mass spectroscopy. The concentration of toxins in PSP-positi ve samples ranged from 50 to 3400 mu g . g(-1) dry weight. Toxin profi les were always dominated by the N-sulfocarbamoyl-11-hydroxysulfate C toxins, C1 and C2 (44-85 mol%), with the remainder consisting of gonya utoxins-2, -3, and -5, decarbamoylgonyautoxins-2 and -3, saxitoxin, an d decarbamoylsaxitoxin. N-1-hydroxy PSPs, commonly found in marine din oflagellates, were absent, suggesting that A. circinalis lacks the enz yme responsible for N-1-hydroxylation. On a dry weight basis, the amou nt of toxin in cultured Anabaena circinalis (strain ACMB06) rose signi ficantly (P < 0.05) over time from 570 to 3400 mu g . g(-1) cells in l ate stationary phase. However, there was no significant trend in cellu lar toxin quota (toxin per cell) over the life of the culture; this ma y be explained by variation in cell mass. On average, batch cultures o f Anabaena circinalis contained 19% extracellular toxin, which increas ed slightly over the growth cycle and had a composition similar to tha t of the intracellular toxins. As cultures aged the formation of decar bamoyl toxins and increases in the alpha-/beta-epimer ratios of C toxi ns and gonyautoxins were observed. The variation in these components d uring stationary phase in culture was sufficient to explain the variat ion 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 toxici ty of the sample or bloom over time. The transformations of PSPs, whic h occur during aging and sample storage, render the comparison of PSPs by HPLC unreliable for phenotyping Anabaena circinalis, unless strain s are cultured, harvested, and analyzed under standard conditions.