Model-driven high-throughput zebrafish embryo assay for evaluating whole effluent toxicity variation across 100...

The zebrafish embryo is a valuable model for evaluating whole effluent toxicity (WET). However, the widely recognized acute toxicity indicator, based on International Organization of Standardization (ISO) methods, requires large numbers of embryos and is often time-consuming due to its complex experimental procedures. In this study, we propose an alternative to the conventional reliance on ISO standards by developing a model-driven high-throughput assay that utilizes actual wastewater, enabling rapid LC₁₀ (the lethal concentration at which 10 % of the test organisms are affected) prediction through machine learning techniques and multidimensional indicators derived from streamlined experimental procedures. We compared three streamlined toxicity assays-developmental toxicity, behavioral toxicity, and vascular toxicity-along with five different models. Among these, the Lasso model based on behavioral toxicity emerged as the most effective, achieving an R² value of 0.893 while reducing experimental time by 5- to 8-fold. Furthermore, fivefold cross-validation confirmed its robust predictive accuracy. The application of this model-driven high-throughput assay across 100 wastewater treatment plants in China highlights the crucial role of biological treatment, particularly aerobic processes and secondary sedimentation, in reducing toxicity, thereby providing valuable insights into their functions. This high-throughput assay not only surpasses the ISO standard method in efficiency but also substantially decreases embryo usage, facilitating rapid WET assessments of actual wastewater with larger sample sizes.