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.

Comparative Assessment of the Sensitivity of Fish Early-Life Stage, Daphnia, and Algae Tests to the Chronic Ecotoxicity of Xenobiotics: Perspectives for Alternatives to Animal Testing.

No-observed-effect concentrations (NOECs) are used in environmental hazard classification and labeling of chemicals and their environmental risk assessment. They are typically obtained using standard tests such as the fish early-life stage (FELS) toxicity test, the chronic Daphnia reproduction test, and the algae growth inhibition test. Given the demand to replace and reduce animal tests, we explored the impact of the FELS toxicity test on the determination of effect concentrations by comparing the FELS toxicity test and the Daphnia and algae acute or chronic toxicity tests. Lowest-observed-effect concentrations (LOECs) were used instead of NOECs for better comparison with median lethal or effect concentration data. A database of FELS toxicity data for 223 compounds was established. Corresponding Daphnia and algae toxicity tests were identified using established databases (US Environmental Protection Agency ECOTOX, Organisation for Economic Co-operation and Development QSAR Toolbox, eChemPortal, EnviroTox, and OpenFoodTox). Approximately 9.5% of the investigated compounds showed a 10-fold higher sensitivity with the FELS toxicity test in comparison with the lowest effect concentrations obtained with any of the other tests. Some of these compounds have been known or considered as endocrine disrupting, or are other non-narcotic chemicals, indicating that the higher sensitivity in the FELS toxicity test is related to a specific mechanism of action. Targeting these mechanisms by alternative test systems or endpoints, using fish embryos for instance, may allow reduction or replacement of the FELS toxicity test or may allow us to prioritize compounds for conduction of the FELS toxicity test. Environ Toxicol Chem 2019;39:30-41. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Automated Morphological Feature Assessment for Zebrafish Embryo Developmental Toxicity Screens.

Detection of developmental phenotypes in zebrafish embryos typically involves a visual assessment and scoring of morphological features by an individual researcher. Subjective scoring could impact results and be of particular concern when phenotypic effect patterns are also used as a diagnostic tool to classify compounds. Here we introduce a quantitative morphometric approach based on image analysis of zebrafish embryos. A software called FishInspector was developed to detect morphological features from images collected using an automated system to position zebrafish embryos. The analysis was verified and compared with visual assessments of 3 participating laboratories using 3 known developmental toxicants (methotrexate, dexamethasone, and topiramate) and 2 negative compounds (loratadine and glibenclamide). The quantitative approach exhibited higher sensitivity and made it possible to compare patterns of effects with the potential to establish a grouping and classification of developmental toxicants. Our approach improves the robustness of phenotype scoring and reliability of assay performance and, hence, is anticipated to improve the predictivity of developmental toxicity screening using the zebrafish embryo.