Toxicological parameters determined via FETAX: The effects of diesel fuel exposure on three developmental stages of Xenopus laevis

Despite the ubiquity of diesel fuel and its potential for polluting the environment, the United States Environmental Protection Agency (USEPA) reports that no information is available for six of eight basic toxicological tests on this complex hydrocarbon mixture. In the present study, three toxicological parameters, Lethal Concentration 50 (LC50), Ecological Effective Concentration 50 (EEC50), and No-Observed Effect Concentration (NOEC), were determined for diesel fuel No. 2 for three developmental stages of Xenopus laevis (African clawed frog). EEC50 is similar to an EC50 concentration, but the number of embryos that died after 96hrs of exposure to the toxicant was included in its calculation. Although the number of embryos that died in these non-lethal end-point experiments was relatively small, similar to those of the negative control, their inclusion is justified by the fact that halted as well as abnormal development of embryos would affect the population exposed to a toxicant. The experimental protocol utilized was based on the procedures outlined in the Standard Guide for Conducting the Frog Embryo Teratogenesis Assay-Xenopus (FETAX). FETAX is an approved method by both the USEPA and American Society for Testing and Materials (ASTM) for conducting 96 hour exposure experiments for single or mixtures of chemicals. The three developmental stages of embryos used to start exposure experiments included Nieuwkoop and Faber (N+F) Stage 8 (mid blastula), N+F Stage 14--16 (neurula), and N+F Stage 24--26 (tail bud formation).; Lethal Concentration 50%--96 hours experiments showed that N+F Stage 24--26 embryos were the most sensitive [LC50--96hrs: 8.922% (v/v)], N+F Stage 14--16 embryos were the most resistant [LC50--96hrs: 26.140% (v/v)], and N+F Stage 8 embryos were intermediate in sensitivity [LC50--96hrs: 16.203% (v/v)]. There was no statistically significant difference between developmental stages in the EEC50s [N+F Stage 8: 0.081% (v/v); N+F Stage 14--16: 0.078% (v/v); N+F Stage 24--26: 0.108% (v/v)] or the NOECs [N+F Stage 8: 0.017% (v/v); N+F Stage 14--16: 0.017% (v/v); N+F Stage 24--26: 0.015% (v/v)].; This project illustrates important factors to be considered when attempting to determine toxicological parameters for complex hydrocarbon mixtures. Each of above LC50s were determined by combining and treating multiple exposure experiments as one data set, and the relative sensitivities among developmental stages were made by comparing the LC50--96hrs concentrations only. However, it was found that genetic variation of the embryos as contributed by using different breeding pairs and the age of a given breeding pair were significant sources of variation. These sources of variation invalidated the use of the prescribed FETAX protocol, "range-finding" followed by confirmation exposure experiments, in this study for the determination of the LC50--96hrs concentrations. NOECs determined via the traditional method of using statistical analyses to determine the exposure concentration where it was not significantly different from the negative control proved to be overestimates. That is, the percentages of embryos with abnormalities at the NOECs determined by statistical methods were much higher than those of the negative controls. Therefore, the recommended method of determination of NOECs is to use probit analysis and extrapolate the 10% effect level which was more congruent to actual experimental data for all three developmental stages.