Determination of some physicochemical parameters of microcystins (cyanobacterial toxins) and trace level analysis in environmental samples using liquid chromatography.

Some physicochemical parameters of three microcystin standards, known to be potent hepatotoxins produced by cyanobacteria, were determined using well defined chromatographic measurements. The logarithm of their retention factor on octadecylsilica (ODS) with water as the eluent, which is an estimation of the hydrophobicity of a molecule, was assessed at pH 7 at 3.9, 4.2 and 4.4 for microcystins-YR, -LR and -RR, respectively. Though being rather hydrophobic, microcystins also possess polar functions, namely carboxylic acids, amino and amido groups. The ionization of carboxylic groups occurs at pH values of 3.3-3.4. In environmental waters, microcystins are neutral or anionic. They are readily soluble in water, the solubility of microcystin-LR being higher than 1 g/l. Owing to their hydrophobicity and their polar functions, microcystins remain in the aqueous phase rather than being adsorbed on sediments or on suspended particulate matter. In a river water spiked with microcystins at 5 micrograms/l, only 10% was adsorbed on particles and 7% on the sandy sediment after three days. A method using solid-phase extraction on ODS followed by high-performance--or micro--liquid chromatography was optimized to detect microcystins at trace level in water. A clean-up was introduced to eliminate part of the interfering compounds coextracted during the sample percolation. Good recoveries (75-80%) were obtained. The method was linear, reproducible (with relative standard deviations ranging from 5 to 8%) and enabled the determination of microcystins at levels as low as 30 ng/l in drinking water and 100-200 ng/l in surface waters. The production of toxins by two strains of cyanobacteria was evaluated. Variations in the microcystin-LR content in the cells and in the medium of Microcystis aeruginosa PCC7806 were recorded over a five-week period. Toxin production was not correlated to the biomass but depended on the growth stage and was maximal at the end of the exponential growth phase. The release of toxin in water occurred essentially in old cultures where microcystin-LR was determined at concentrations of 170 and 280 micrograms/l in the media of M. aeruginosa PCC7806 and M. aeruginosa PCC7813, respectively. Other microcystins are likely to be synthesized by these strains. But owing to the lack of standards, mass spectrometric detection is required for further identification. This study points out the need of having other standards for water quality monitoring.