Fate and detection of microcystin-LR and its metabolites in the environment

Microcystins are hepatotoxins produced by selected strains of cyanobacteria. These toxins have been cited as human health hazards and have the potential for transfer through the foodweb. Current guidelines to protect human health from exposure to microcystins do not include microcystin metabolites. These metabolites can comprise a substantial portion of the total toxin pool of natural systems. Previous studies have shown that fish and zooplankton metabolize microcystins using the glutathione metabolic pathway. Metabolism is initiated by the attachment of a glutathione residue to microcystin-LR (MC-LR) followed by a series of enzymatic steps which result in three additional conjugated products: a cysteine-glycine intermediate, a cysteine intermediate, and a mercapturic acid conjugate. The work in this thesis addresses the occurrence of these metabolites in fish and Daphnia pulicaria. A tandem quadrupole mass spectrometry (LC-MS/MS) analytical technique was developed for the analysis of fish and zooplankton tissues. Both a quantitation ion and a confirmation ion were identified for MC-LR and each of the conjugate metabolic products. This LC-MS/MS method was coupled with an extraction and novel charcoal cleanup method to analyze MC-LR and its conjugates in fish reared in the laboratory and fish obtained from the field. Pomoxis nigromaculatus exposed to MC-LR from a natural system was highly variable in its MC-LR concentrations, with values ranging from non-detect up to 70 microg/kg. This variation was also observed in laboratory-reared catfish, suggesting that microcystin concentrations in tissues between individual fish were highly variable. The cysteine conjugate of MC-LR was detected in several catfish tissues, but no other GSH metabolites were identified. To evaluate the role of zooplankton in foodweb transfer of microcystins, Daphnia pulicaria was exposed to MC-LR through toxic algae and MC-LR was assimilated into their tissues. Rapid excretion of MC-LR was observed without metabolic alteration of the toxin. This suggests that the GSH metabolic pathway is not the dominant pathway in elimination of microcystins in Daphnia. Combined, the results here show that fish and zooplankton do not metabolize MC-LR using the same mechanism, which can potentially complicate analyses of these toxins and the implementation of human health guidelines.