Predictive modelling of chemical mixture toxicity to amphibians in Discoglossus galganoi larvae and metamorphs

Abstract

In natural environments, amphibians are exposed to individual chemical substances, and regularly also to mixtures of chemicals. At the same time, prospective risk assessment methods that account for exposure to mixtures across substances, species and environmental conditions are not implemented, partly because the experimental assessment of mixtures is extremely resource-intensive. Due to their specific life cycle, as well as susceptibility to anthropogenic and biological stressors, amphibians are of particular relevance for the environmental risk assessment of chemicals. Therefore, we set out to develop a model that can account for toxic effects of chemical mixtures in amphibians, specifically anurans, that integrates established toxicokinetic-toxicodynamic modelling principles with an amphibian-specific dynamic energy budget model (AmphiDEB-TKTD). We calibrated the AmphiDEB-TKTD model to single-substance toxicity data for Flupyradifurone and 2,4-D in \textit{Discoglossus galganoi} larvae, and found that the model can reproduce observed effects on larval growth and metamorphosis traits. Also, we could use the data to identify the underlying physiological modes of action (PMoA), since growth and timing of metamorphosis are differentially affected through different PMoAs. A cross-validation with data on effects at Gosner stage 46 further clarified the physiological mode of action, and demonstrated that effects on Gosner stage 46 can be predicted from effects on larvae up to Gosner stage 42, if an appropriate PMoA has been identified. Concerning binary mixture toxicity, the model correctly predicted a dominance effect of 2,4-D in a mixture with Flupyradifurone. This can be explained through the different PMoAs of Flupyradifurone (decrease in growth efficiency) and 2,4-D (increase in maintenance costs), which qualitatively differ in the severity of their effects, regarding the propagation to different apical endpoints. We conclude that the AmphiDEB-TKTD model appears as a practical tool for the risk assessment of chemical mixtures for anurans, while also being useful for the investigation of mechanisms of toxicity. Since the model has only been validated on a substance combination of different PMoAs, further investigations should include the validation with substance combinations of identical PMoAs before more general claims about predictive capacity can be made. Furthermore, validation studies with additional species, especially ones exhibiting contrasting life histories, are desirable.