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Abstract
Amphibians have suffered serious global declines in recent decades. A probable cause of these declines is widespread exposure of pesticides for these non-target species. The objectives of this study were 1) identify pesticides in rainwater and surface waters (i.e. spray drift and runoff) following agricultural application in southern Georgia impacting amphibian habitats, 2) determine metabolic rate constants of select pesticides identified in these matrices in amphibian liver microsomes, 3) determine if the hydration status of an amphibian can affect the tissue concentration of pesticides, and 4) identify potential effects of exposure to pesticide mixtures on the metabolome of amphibians. To this end, surface water and rainwater samples including stemflow and throughfall were analyzed via GCxGC-ToF/MS for over 150 pesticides, overall, thirty-two different pesticides were detected in these matrices. To determine the metabolic rates for atrazine, triadimefon, and fipronil in amphibian microsomes the Michaelis-Menten equation was utilized to obtain KM and Vmax parameters. The Vmax ranged from 150834 pmol min-1 mg-1, while KM was measured from 5 to 147 M which resulted in calculated intrinsic clearance rates ranging from 0.5438.31 mL min-1 kg-1 for southern toads. The effect of hydration status on amphibians was determined by dehydrating amphibians for 0, 2, 4, 6, 8 or 10 hours prior to an 8 h exposure to pesticide-contaminated soil. Interestingly, increased time of dehydration resulted in lower tissue concentrations observed in amphibians for the five pesticides studied. Metabolomics was then utilized to identify biochemical fluxes and pathways impacted by pesticide exposure, both as singlets and in combinations, in terrestrial phase amphibians. Ultimately, this research can be utilized to create a dermal uptake model for pesticide exposure in amphibians, increasing the reliability of current regulatory efforts. Overall, this research details the consequence of pesticide exposures on amphibians and lays the foundation for identifying key biomarkers that can help identify pesticide exposure in future research efforts.