Development of a bioanalytical phosphatase inhibition test for the monitoring of microcystins in environmental water samples

Abstract A phosphatase inhibition assay using commercially available components has been developed for the detection of cyanobacterial toxins in drinking and surface water samples. The assay is simple, economic and rapid. Results are obtained in 1 h on a 96-well microtitre plate. The test uses a type 2A protein phosphatase whose activity is inhibited proportionally to the toxin concentration and is measured by means of a colourimetric reaction. Two procedures of the tests have been optimised using the same phosphatase, but different working conditions. The first one allows a direct estimation of the concentration of microcystin-LR in the range 0.4–10 μg/l and is used to calculate the sample dilution required for the second procedure, which has been optimised in order to be much more accurate with a working range between 0.2 and 0.8 μg/l (concentration in the sample, which corresponds to 0.1–0.4 μg/l in the well). An enrichment using solid phase extraction on octadecyl silica has been described for samples containing less than 0.2 μg/l of microcystin. A repeatability of 8% at the IC 50 level has been obtained and the IC 50 value is 0.21 ± 0.02 μg/l (concentration in the well). Inhibition of enzyme activity by other toxins such as microcystin-YR and microcystin-RR has also been evaluated. Particular attention has been paid to the effect of the sample matrix on the assay. Several drinking and surface waters have thus been investigated. Validation of the assay has been performed by comparing the phosphatase inhibition assay results to the liquid chromatography results for the same samples. A good correlation ( R 2  = 0.96) has been observed for samples containing microcystin-LR.