Abstract
Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis, were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles (n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57–66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.
Highlights
Amphibian population declines and morphological malformations recorded globally since the early 1990’s have been attributed to multiple stressors, whereby habitat loss associated with agricultural expansion is considered the predominant activity affecting lowland amphibian populations [1,2,3,4]
Analytical chemistry results obtained during exposure period 1 showed no significant difference between NEO concentrations in the culture media during the 24 h elapsed between daily solution changes (Supplementary Table S3)
With repeated application and runoff and due to unforeseen hydrologic and atmospheric processes, concentrations of NEO in waters under particular conditions could reach relatively high levels [16,67,74,75] such as those used in this study
Summary
Amphibian population declines and morphological malformations recorded globally since the early 1990’s have been attributed to multiple stressors, whereby habitat loss associated with agricultural expansion is considered the predominant activity affecting lowland amphibian populations [1,2,3,4]. Subsequent to habitat loss, pollutants such as anthropogenic chemicals are the major influence on amphibians [5]. Studies on the direct effects of pesticides on amphibian growth and survival are more routine than those conducted on indirect effects [6,7], yet even those studies are complicated with confounding aspects such as animal densities, proximity to the chemicals, forage, and predators. Results on the indirect biological or sublethal effects can show longer larval periods, developmental abnormalities, increased susceptibility to disease and predation, slowed swimming, as well as genotoxic and cytotoxic effects after concentration exposures well below those associated with mortality [8,9,10,11,12,13].
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