Does atrazine influence larval development and sexual differentiation in Xenopus laevis?

Abstract

Debate and controversy exists concerning the potential for the herbicide atrazine to cause gonadal malformations in developing Xenopus laevis. Following review of the existing literature the U.S. Environmental Protection Agency required a rigorous investigation conducted under standardized procedures. X. laevis tadpoles were exposed to atrazine at concentrations of 0.01, 0.1, 1, 25, or 100 microg/l from day 8 postfertilization (dpf) until completion of metamorphosis or dpf 83, whichever came first. Nearly identical experiments were performed in two independent laboratories: experiment 1 at Wildlife International, Ltd. and experiment 2 at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (IGB). Both experiments employed optimized animal husbandry procedures and environmental conditions in validated flow-through exposure systems. The two experiments demonstrated consistent survival, growth, and development of X. laevis tadpoles, and all measured parameters were within the expected ranges and were comparable in negative control and atrazine-treated groups. Atrazine, at concentrations up to 100 microg/l, had no effect in either experiment on the percentage of males or the incidence of mixed sex as determined by histological evaluation. In contrast, exposure of larval X. laevis to 0.2 microg 17beta-estradiol/l as the positive control resulted in gonadal feminization. Instead of an even distribution of male and female phenotypes, percentages of males:females:mixed sex were 19:75:6 and 22:60:18 in experiments 1 and 2, respectively. These studies demonstrate that long-term exposure of larval X. laevis to atrazine at concentrations ranging from 0.01 to 100 microg/l does not affect growth, larval development, or sexual differentiation.

FIG. 1.

(a) Snout-to-vent length (mean ± SE) in experiment 1 (WLI) (left panels) and 2 (IGB) (right panels). Statistical significance is marked by asterisk (p < 0.05). (b) Weight (mean ± SE) in experiment 1 (WLI) (left panels) and 2 (IGB) (right panels). Statistical significance is marked by asterisk (p < 0.05). (c) Time to metamorphosis (mean ± SE) in experiment 1 (WLI) (left panels) and 2 (IGB) (right panels). Statistical significance is marked by asterisk (p < 0.05).

FIG. 2.

(a) Cumulative percentage of males completing metamorphosis on each study day in both experiments at IGB. (b) Cumulative percentage of females completing metamorphosis on each study day in both experiments at IGB. (c) Cumulative percentage of males completing metamorphosis on each study day in both experiments at WLI. (d) Cumulative percentage of females completing metamorphosis on each study day in both experiments at WLI.

FIG. 3.

Proportion of frogs (%) that were histologically determined to be male, female, or mixed sex. Statistical difference are marked by asterisk *p < 0.05.

FIG. 4.

Gonads of Xenopus laevis after completion of metamorphosis demonstrating clearly differentiated male and female phenotypes by testes (a) and ovaries (b) of negative control animals. The mixed sex gonad (c) exhibiting testicular as well as ovarian tissue was obtained from the positive control of 0.2 μg E2/l treatment.