Effects of nitrate and atrazine on larval development and sexual differentiation in the northern leopard frog Rana pipiens

Pollution from agrochemicals may be contributing to the global decline in amphibian populations. Environmentally relevant concentrations of nitrate and/or atrazine on anuran development and gonadal differentiation were tested. Four replicates of 20 tadpoles per tank (80/treatment) were exposed from Taylor-Kollros stage 2 to 3 to stage 23 to 34 to either 10 mg/L nitrate, 10 microg/L atrazine, a combined exposure of 10 mg/L nitrate plus 10 microg/L atrazine, or untreated controls. No treatment-dependent effects on weight, snout-vent or hind limb length, or time to forelimb emergence were observed. The proportions of females increased in all treatments compared to the controls, especially in the combined treatment (chi2 = 17.90, df = 6, p = 0.0065, combined = 66.4% female, control = 41% female). The frequency of intersex was low in all treatments. No treatment-related effects on the total number of spermatogenic cells were observed, but the ratio of cell types differed in that testes from animals in the treated groups exhibited more spermatogonia, fewer spermatocytes, and more spermatids than the control (significantly different, Kruskal-Wallis, p < 0.05). Ovaries from animals treated with nitrate or atrazine exhibited larger immature (previtellogenic) and mature (vitellogenic) follicles, but ovaries from the combined treatment had larger immature follicles only. Testicular oocytes were observed in the nitrate-only and atrazine-only treatments, and the control treatment, but not the combined treatment. Overall, this study has demonstrated changes in sex ratios that are more marked in response to combined nitrate/atrazine exposure than with these chemicals alone. Histological evidence suggests that premature maturation of gonad may occur as a result of nitrate and/or atrazine exposure during larval development.