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Bacillus thuringiensis var. israelensis (Bti) is presumed to be an environmental friendly agent for the use in either health-related mosquito control or the reduction of nuisance associated with mosquitoes from seasonal wetlands. Amphibians inhabiting these valuable wetlands may be exposed to Bti products several times during their breeding season. Up until now, information regarding effects on the non-targeted group of amphibians has to be considered rather inconsistent. On this account, we evaluated how three repeated exposures to frequently used Bti formulations (VectoBac®12AS, VectoBac®WG) in field-relevant rates affect European common frog (Rana temporaria) larvae. In a laboratory approach, we assessed potential effects with regard to enzymatic biomarkers (glutathione-S-transferase (GST), glutathione reductase (GR), acetylcholine esterase (AChE)), development, body condition and survival until the end of metamorphosis. Although survival and time to metamorphosis were not significantly affected, larval development tended to be shortened in the Bti treated water phase. Furthermore, exposure to Bti induced significant increases of GST (37–550%), GR (5–140%) and AChE (38–137%) irrespectively of the applied formulation, indicating detoxification, antioxidant responses as well as an alteration of neuronal activity. GST activity increased twice as much after two repeatedly executed Bti applications within a time period of 6 days. The examination of several biochemical markers is needed to fully evaluate the ecotoxicological risk of Bti for amphibian populations, especially in the context of worldwide amphibian declines. Nevertheless, following the precautionary principle, it may be advisable to implement certain thresholds for application numbers and intervals in order to ensure environmentally friendly mosquito control programs, especially in areas designated for nature conservation.VectoBac®12AS and ®WG causes no mortality in Rana temporaria tadpoles.Bti in field relevant rates increases the activity of GST, GR and AChE enzymes.Alterations in enzymes are independent of application rate and Bti formulation.Repeated Bti applications seem to increase the risk for biochemical alterations.