Tests with zebrafish embryos have gained wide acceptance as an alternative test model for drug development and toxicity testing. In particular, the behavioral response of the zebrafish embryo is currently seen as a useful endpoint to diagnose neuroactive substances. Consequently, several behavioral test methods have been developed addressing various behavioral endpoints such as spontaneous tail coiling (STC), photomotor response (PMR), locomotor response (LMR) and alternating light/dark-induced locomotor response (LMR-L/D). Although these methods are distinct in their application, most of their protocols differ quite strongly in the use of experimental parameters and this is usually driven by different research questions. However, if a single mode of action is to be diagnosed, then varying experimental parameters may cause incoherent behavioral responses (hypo-or hyperactivity) of zebrafish during toxicity assessment. This could lead to inconclusiveness of behavioral test results for use within a prospective and diagnostic risk assessment framework. To investigate the influence of these parameters, we conducted a review of existing behavioral assays to address the following two questions: (1) To what extent do varying experimental parameters influence observed effects in published behavioral test methods? (2) Is the observed behavior change (hypo-or hyperactivity) of zebrafish embryos consistent with the expected mode of action of a chemical? We compiled a set of 18 substances which are anticipated to be neuroactive. We found that behavioral changes are not only affected by chemicals but also variation in the use of experimental parameters across studies seems to have a high impact on the outcome and thus comparability between studies. Four parameters, i.e., exposure concentration, exposure duration, endpoint parameter and developmental stage were the most influential parameters. Varying combinations of these parameters caused a non-reproducible outcome for the hyperactivity expected for the organophosphates; chlorpyrifos and diazinon. We highlighted that the STC test shows a higher capacity to predict the hyperactivity of organophosphates, while PMR and LMR-L/D were more suitable to predict the hypoactivity expected for anticonvulsants. We provide a list of recommendations which, when implemented, may help to exclude the risk of bias due to experimental parameters if similar goals are desired.

Soil properties like organic matter (OM) content show great variation, making it hard to predict the fate and effects of a chemical in different soils. We therefore addressed the question: can we remove the complexity of the soil matrix and yet accurately predict soil toxicity from porewater exposures? Folsomia candida was exposed to imidacloprid in natural (LUFA 2.2 [4.02% OM], Grassland [12.6% OM]) and artificial soils (OECD 5 [6.61% OM], OECD 10 [10.8% OM]), in pore water extracted from spiked LUFA 2.2 soil and in water. Toxicity decreased with increasing OM content except for Grassland soil, which had the highest OM content but the lowest clay content, suggesting a role of clay minerals in the binding of imidacloprid. Distribution coefficients for imidacloprid based on toxicity (Toxicity-Kd) were derived by comparing effect concentrations in LUFA 2.2 soil and in water. Using these Toxicity-Kds to recalculate soil LC50s/EC50s to porewater concentrations, the differences in LC50/EC50s almost disappeared. The recalculated porewater LC50s did not differ by more than a factor of 0.55–1.43 from the LC50 obtained upon water exposure. This similarity suggests that the toxicity in the soil is dependent on porewater concentrations and can be obtained from water exposure. The porewater test and the corresponding “pore-water extrapolation concept” developed in this study may be used to predict the toxicity of chemicals in the soil and extrapolate among different soils.