Assessment of the effects of atrazine on the cane toad Rhinella marina and the striped marsh frog Limnodynastes peronii

Atrazine is one of the most widely applied herbicides and is commonly detected in surface and groundwater samples around Australia. Reproductive and developmental abnormalities in amphibians have reportedly been linked to atrazine exposure. However, the use of Australian native frogs in understanding these effects is limited. This study investigated the effects of atrazine alone and also in combination with a common secondary stressor, elevated salinity, to an Australian non-native anuran species, the cane toad, Rhinella marina and a native species, the striped marsh frog, Limnodynastes peronii, at both acute and chronic level. This study also explored the longer-term effects of atrazine to the cane toad to determine the impacts on its development and reproduction. Variations in larval sensitivities to acute concentrations of atrazine were first determined at different larval developmental stages in both R. marina and L. peronii. The static acute test design involved six nominal concentrations of atrazine, including control, solvent control, 3, 6, 12, and 24 mg/L. Gosner stages (GS) 22–23 as hatchlings, GS 25–26, 28–29, 32–33 as pre-metamorphic, GS 36–37 as prometamorphic and GS 40–41 as metamorphic climax stages of cane toads and the first four sets of Gosner stages (GS 22–33) of striped marsh frogs were exposed to atrazine treatments for 96 hours. Results showed that late larval stages were more sensitive than early stages and significant differences (P ˂ 0.05) occurred in sensitivities across different pre-metamorphic larval stages in both test species. The striped marsh frog showed greater sensitivity to atrazine than the introduced cane toad. In both experimental species, GS 28–29 showed the best response regarding the iv concentration-dependent increase in sensitivities to atrazine compared to other larval stages. To determine the chronic effects of atrazine, GS 28–29 of both test species were exposed to five nominal concentrations of atrazine, including control, solvent control, 0.1, 1.0 and 10 µg/L for 21 days. Results revealed no significant effects from atrazine exposure to survivorship, snout vent length, body weight and development among treatments in both species. Only a small percentage of exposed animals exhibited kinky tail and gonadal anomalies in L. peronii and a low percentage of atrazineexposed cane toads showed both limb and tail anomalies. Interestingly, both species had a female biased sex ratio in all treatments. No significant effect of atrazine on gross and histological morphology of thyroid glands in R. marina was noted; however, no thyroid analysis was performed on L. peronii. In order to evaluate the longer-term effects of atrazine on cane toad reproduction, GS 29–31 of R. marina was exposed to four concentrations of atrazine, including solvent control, 0.1, 4 and 11 µg/L until completion of metamorphosis. The metamorphs obtained from atrazine exposure were reared until their sexual maturity without further atrazine exposure. A number of attempts at breeding, including natural breeding, injecting with Luteinizing Hormone Releasing Hormone (LHRH) and lucrin (synthetic LHRH) were performed; however, no offspring were obtained. Morphological abnormalities, survival, fecundity, erythrocytic nuclear abnormalities, and gonadal and liver histology were analysed to evaluate the potential effects of atrazine on subsequent generations and also to examine the possible reason for the unsuccessful breeding. Results indicated that a concentration of 0.1 µg/L caused significant mortality, and 4 µg/L produced the highest incidence of limb abnormalities and also v decreased the hepato-somatic index during post-exposure period. No significant erythrocytic nuclear abnormalities or liver malformations were detected; however, gonadal histology showed a relatively high percentage of animals containing gonadal anomalies in all atrazine-treated males, except at 0.1 µg/L. To explore the combined effects of atrazine and a common secondary stressor, both R. marina and L. peronii were exposed to mixtures of atrazine and increased salinity. At acute exposure, GS 25–26 of both test species were exposed to salinity alone (0 to 8% sea water) and also in combination with atrazine (0 to 24 mg/L) for 96 hours. Results revealed no acute effects of elevated salinity on either test species; however, cane toad tadpoles demonstrated greater sensitivity to the mixture of atrazine and salinity than atrazine alone, while L. peronii indicated no differences in sensitivity. For chronic exposure, GS 28–29 of both R. marina and L. peronii were firstly exposed to a range of salinity (0 to 8% sea water) alone for 21 days. Results showed that 2% to 8% sea water caused significant effects on growth and development in L. peronii, but not in R. marina. Secondly, five treatments including control, solvent control, 0.1, 1 and 10 µg/L of atrazine each mixed with 8% sea water were used to expose the GS 28–29 for 21 days. Significant effects were obtained on the growth and duration of metamorphosis in L. peronii from mixture treatments, but not in R. marina. Overall, it can be concluded that inter- and intra-species variations in sensitivities to atrazine may occur in anurans residing in Australia and native species may exhibit greater sensitivity to atrazine than the introduced cane toad. Interestingly, although low concentrations of atrazine may not cause any noteworthy adverse effects on the developing R. marina and L. peronii during the larval exposure period; however, the longer-term study with R. marina showed that deleterious impacts may occur during vi the post-exposure period, which may cause adverse effects on the fitness and reproduction of R. marina. This may ultimately affect the population of this species. Longer-term testing with native species is required to determine the potential effects of atrazine on these populations. From the mixture of atrazine and salinity studies, it can be suggested that elevated salinity may pose serious hazards to Australian native anurans. The similar responses obtained from chronic exposure to atrazine in both R. marina and L. peronii may raise the possibility of the cane toad as an indicator for native anurans; nevertheless, further studies are required to establish this.