Abstract

Anatoxin, 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 and liver using LC/MS/MS technology and correlate it to the lesions observed in the livers of experimental animals.

Catfish (Ictalurus punctatus) were dosed with anatoxin, microcystin, or nodularin by intraperitoneal injection (IP) or orally. All fish were sacrificed 24 hours later and blood and tissues were collected for histopathological examination and analysis by LC/MS/MS. The toxicologic analysis of samples was done using a LC/MS/MS method specifically developed for anatoxin, microcystin, and nodularin using pure standard reference materials.

The plasma samples collected from both the IP and oral control groups did not exhibit any peaks at the retention times for anatoxin, microcystin or nodularin. All the fish in the IP treatment groups had correlating positive findings in plasma: anatoxin concentrations ranged from 65 to 150 parts per billion (ppb), microcystin from 83 to 230 ppb, and nodularin was found to be between 85 and 325 ppb. The fish treated orally did not show the presence of any detectable toxin except for one fish from the nodularin treatment group that had a plasma nodularin level of 548 ppb.

No tissue residues were found in the orally-treated anatoxin fish or the IP and oral control groups. The fish treated with anatoxin intraperitoneally had residues present in both liver (range 40-230 ppb) and the target organ, brain (65-150 ppb). The fish treated intraperitoneally with microcystin had liver concentrations varying from 260 to 1650 ppb; as expected, the IP control group was negative. Out of seven fish, five in the oral microcystin group had liver residues varying from 270 to 1230 ppb; none of the fish livers in the oral control group showed any detectable nodularin. The amount of nodularin detected in the livers of IP-dosed fish varied from 100 to 300 ppb. The orally-dosed fish contained less than 30 ppb of nodularin except for two fish with levels of 185 and 400 ppb.

Histopathological examination consistently revealed microscopic lesions (diffuse hepatocellular necrosis, dissociation, and vacuolation) in the livers of all test fish; these lesions were not observed in the control fish.