Comparison of sensitivity to methyl methanesulphonate among tadpole developmental stages using the alkaline single-cell gel electrophoresis (comet) assay

Abstract

In a previous study, we demonstrated that tadpoles are suitable organisms for monitoring small bodies of water (e.g., creeks, ponds, and drainage ditches) for genotoxicity using the alkaline single-cell gel DNA electrophoresis (SCG) or "comet" assay [Ralph and Petras, 1997]. 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. In this initial study, most of the tadpoles collected were in the early stages of larval development, but this is not always possible. The present study evaluated the sensitivity of tadpoles, at different stages of larval development, to a range of concentrations of the genotoxicant methyl methane-sulphonate (MMS). Four specific phases of Rana clamitans (green frog) larval development were examined: first-year limbless tadpoles (Stage I as defined by Taylor and Kollros [1946]), second-year limbless tadpoles (Stages II-III), second-year tadpoles with only hindlimbs (Stages X-XVIII), and second-year tadpoles with all four limbs evident and a tail undergoing resorption (Stages XXII-XXIII). Twenty-four hour exposures to MMS of tadpoles in the three earliest phases produced a significant (P < 0.01) added variance component among tadpoles for DNA damage and there were significant increases (P < 0.05) in the length:width ratios of the DNA patterns at concentrations as low as 1.56 mg/I. However, tadpoles in the last phase studied (both pairs of limbs present) showed no significant (P > 0.05) added variance component and no significant increases (P> 0.05) in DNA damage upon exposure to any of the MMS doses tested. A nested ANOVA indicated that, for each of the tested concentrations of MMS, but not the dechlorinated water control, there was significant heterogeneity (P < 0.05) in DNA damage when tadpoles of all four phases studied were compared. However, when tadpoles of the lost phase of development were removed from the comparison, there was no significant heterogeneity (P > 0.05) among tadpoles of the remaining three phases. Possible reasons for this insensitivity to MMS as animals enter the metamorphic climax were considered. The results indicate that pooling of the early tadpole phases of R. clamitans for SCG environmental genotoxicity biomonitoring is acceptable.