abstract

Microcystin and nodularin are cyanobacterial metabolites found worldwide in freshwater and marine environments. These toxins have been linked to various health effects in both animals and humans. These compounds accumulate in aquatic organisms probably as a result of irreversible binding to liver protein phosphatase. The aim of this study was to develop a sensitive assay to measure levels of toxin in plasma using LC/MS/MS technology and correlate it to the lesions observed in the liver tissues of test animals.

An LC/MS/MS method was developed using microcystin and nodularin standards on a Thermo LCQ Deca XP Plus. The HPLC consisted of a gradient run with 0.05% formic acid and acetonitrile with a Betasil C18 5-micron 2.1x150 mm column. The blank, spiked, and test plasma was treated with two volumes of acetonitrile to precipitate the protein. The protein was filtered and a 20 ul aliquot of the filtrate was injected into the LC/MS/MS. Catfish (Ictalurus punctatus) were dosed with a sublethal dose, 250 ug/kg, of either nodularin or microcystin intraperitoneally, and the control fish were dosed with the vehicle alone. The fish were sacrificed 24 hours after dosing and blood and tissues were collected.

The experimental fish treated with nodularin had plasma concentrations ranging from 85 ppb to 325 ppb; toxin was not detected in the control fish. Histopathological examination consistently revealed microscopic lesions (diffuse hepatocellular necrosis, dissociation, and vacuolation) in the livers of test fish; these lesions were not observed in the control fish. All test fish exposed intraperitoneally to microcystin had average plasma concentrations of 98 ppb. The histopathological examination revealed liver changes similar to those seen with nodularin. The control plasma, as expected, did not exhibit any peak at the retention time for microcystin.