Automated measurement of the spontaneous tail coiling of zebrafish embryos as a sensitive behavior endpoint using a workflow in KNIME.

Neuroactive substances are the largest group of chemicals detected in European surface waters. Mixtures of neuroactive substances occurring at low concentrations can induce adverse neurological effects in humans and organisms in the environment. Therefore, there is a need to develop new screening tools to detect these chemicals. Measurement of behavior or motor effects in rodents and fish are usually performed to assess potential neurotoxicity for risk assessment. However, due to pain and stress inflicted on these animals, the scientific community is advocating for new alternative methods based on the 3R principle (reduce, replace and refine). As a result, the behavior measurement of early stages of zebrafish embryos such as locomotor response, photomotor response and spontaneous tail coiling are considered as a valid alternative to adult animal testing. In this study, we developed a workflow to investigate the spontaneous tail coiling (STC) of zebrafish embryos and to accurately measure the STC effect in the KNIME software. We validated the STC protocol with 3 substances (abamectin, chlorpyrifos-oxon and pyracostrobin) which have different mechanisms of action. The KNIME workflow combined with easy and cost-effective method of video acquisition makes this STC protocol a valuable method for neurotoxicity testing.•Video acquisition duration of 60 s at 25 ± 1 hpf was used•20 embryos exposed per dish and acclimatized for 30 min before video acquisition•Capability to inspect and correct errors for high accuracy.

Assessing Combined Effects for Mixtures of Similar and Dissimilar Acting Neuroactive Substances on Zebrafish Embryo Movement.

Risk assessment of chemicals is usually conducted for individual chemicals whereas mixtures of chemicals occur in the environment. Considering that neuroactive chemicals are a group of contaminants that dominate the environment, it is then imperative to understand the combined effects of mixtures. The commonly used models to predict mixture effects, namely concentration addition (CA) and independent action (IA), are thought to be suitable for mixtures of similarly or dissimilarly acting components, respectively. For mixture toxicity prediction, one important challenge is to clarify whether to group neuroactive substances based on similar mechanisms of action, e.g., same molecular target or rather similar toxicological response, e.g., hyper- or hypoactivity (effect direction). We addressed this by using the spontaneous tail coiling (STC) of zebrafish embryos, which represents the earliest observable motor activity in the developing neural network, as a model to elucidate the link between the mechanism of action and toxicological response. Our objective was to answer the following two questions: (1) Can the mixture models CA or IA be used to predict combined effects for neuroactive chemical mixtures when the components share a similar mode of action (i.e., hyper- or hypoactivity) but show different mechanism of action? (2) Will a mixture of chemicals where the components show opposing effect directions result in an antagonistic combined effect? Results indicate that mixture toxicity of chemicals such as propafenone and abamectin as well as chlorpyrifos and hexaconazole that are known to show different mechanisms of action but similar effect directions were predictable using CA and IA models. This could be interpreted with the convergence of effects on the neural level leading to either a collective activation or inhibition of synapses. We also found antagonistic effects for mixtures containing substances with opposing effect direction. Finally, we discuss how the STC may be used to amend risk assessment.

Optimization of the spontaneous tail coiling test for fast assessment of neurotoxic effects in the zebrafish embryo using an automated workflow in KNIME®.

Neuroactive chemicals are frequently detected in the environment. At sufficiently high concentrations or within mixtures, they could provoke neurotoxic effects and neurological diseases to organisms and humans. Fast identification of such neuroactive compounds in the environment could help in hazard assessment and risk mitigation. Behavior change is considered as an important endpoint and might be directly or indirectly connected to a neuroactive mode of action. For a fast evaluation of environmental samples and pure substances, we optimized the measurement of a behavioral endpoint in zebrafish embryos - the spontaneous tail coiling (STC). Evaluation of results is automated via the use of a workflow established with the KNIME® software. Analysis duration and developmental stage were optimized to 1 min and 25 ± 1 hpf respectively during measurement. Exposing the embryos in a group of 10 or 20 and acclimatizing for 30 min at room temperature proved to be reliable. The optimized method was used to investigate neurotoxic effects of 18 substances with different modes of action (MoA). The STC test accurately detected the effect of 8 out of 11 neuroactive substances (chlorpyrifos, chlorpyrifos-oxon, diazinon, paraoxon-methyl, abamectin, carbamazepine, propafenone and diazepam). Aldicarb and nicotine showed subtle effects which were considered to be conditional and imidacloprid showed no effect. For substances with unknown neuroactive MoA, 3 substances did not provoke any effect on the STC (pyraclostrobin, diuron and daunorubicin-hydrochloride) while 4 other substances provoked an increased STC (hexaconazole, aniline, dimethyl-sulfoxide and 3,4-dichloroaniline). Such unexpected effects indicate possible neuroactive side effects or unknown mechanisms of action that impact on the STC. In conclusion, the optimized STC parameters and the automated analysis in KNIME® indicate opportunities for the harmonization of the STC test and further development for prospective and diagnostic testing.

Extrapolation of imidacloprid toxicity between soils by exposing Folsomia candida in soil pore water.

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.