Genotoxicity monitoring of small bodies of water using two species of tadpoles and the alkaline single cell gel (comet) assay.

To monitor genotoxicity in small bodies of water (e.g., creeks, ponds, and drainage ditches) we examined tadpole erythrocytes of two species: Rana clamitans and Rana pipiens,using the alkaline single cell gel DNA electrophoresis (SCG) or "comet" assay. This approach involves detection, under alkaline conditions, of cell DNA fragments which on electrophoresis migrate from the nuclear core, resulting in a "comet with tail" formation. Fifty-six samples, a total of 606 tadpoles, from 18 sites in southern Ontario, collected between 1993 and 1995, were examined. Samples of R. clamitans tadpoles collected in 1994 and 1995, from regions with heavy agricultural activity, gave significantly higher (P < 0.001) DNA length to width ratios than samples of R. clamitans tadpoles collected from sites in the Bruce Peninsula and near the French River, which have little or no agriculture. Samples of R. pipiens tadpoles collected in 1994 from sites on the outskirts of Windsor, Ontario, sites which receive genotoxic inputs from nearby industries, gave significantly higher (P < 0.001) DNA ratios than samples from agricultural areas and the Bruce Peninsula. R. clamitans tadpoles showed significant annual variation in DNA damage which was greater in samples of tadpoles collected from agricultural areas than from the Bruce Peninsula. The higher levels of DNA damage in tadpoles collected from agricultural areas may be due to the pesticides used, and the increased variation in DNA damage in the same areas is likely due to the impact of crop rotation, including leaving fields fallow, the timing of rainfall, and/or the application of pesticides. R. clamitans tadpoles, especially those collected from agricultural areas, also showed significant seasonal variation in DNA damage. There was no significant (P > 0.05) seasonal or annual variation in the levels of DNA damage in R. pipiens tadpoles collected from the Tallgrass Prairie. This study indicates that both species are suitable for use in the alkaline SCG assay and as in situ sentinel organisms for environmental biomonitoring.