Hemolysis, fish mortality, and lc-esi-ms of cultured crude and fractionated golden alga (Prymnesium parvum)

Erythrocyte lysis and fish mortality assays, in combination with high performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) analysis, were investigated for bioassay-guided fractionation of cultured golden alga (Prymnesium parvum). Intracellular constituents from isolated cell pellets and extracellular supernatant growth medium were fractionated by a variety of common separation modes, including reversed phase and normal phase solid phase extraction step fractionation procedures. For reversed phase fractionation of extracellular growth medium, one fraction was obtained that displayed hemolytic activity and adversely affected fish survival. Effective dose concentrations for this sample were similar in both assays and the LC-ESI-MS analysis of the fraction showed a number of mass spectral signals which were distinct to this fraction. Fractions obtained from separation of an ethanol extract of the lyophilized cell pellet provided one sample that was highly hemolytic, but not toxic to fish. Discrepancies such as this, along with notable fish behavioral responses from other nonhemolytic cell pellet fractions, problems with the use of unbonded silica gel for fractionation, and misleading mass spectral signatures are interesting in the context of our current understanding of P. parvum toxicity and remain to be investigated further. This work provides an account of ongoing research aimed toward comprehensive elucidation of toxic constituents produced by golden alga for the purpose of providing a better understanding and means to potentially remediate the ecological impact of this harmful bloom organism.