Algal organelle integrity-based high-throughput alternative to conventional bioassay for whole wastewater toxicity...

Reliable assessment of whole wastewater toxicity (WWT) is essential for the rational design of wastewater treatment processes and safeguarding effluent quality. However, conventional bioassays (e.g., algal growth inhibition tests) remain inherently time- and labor-intensive, especially limiting the production of rapid, large-scale, and unbiased data. Here, we developed an algal organelle integrity‑based high-throughput assay for rapid WWT assessment based on integrating multiplexed fluorescence quantification of subcellular damage with machine learning prediction. Algal organelle-specific responses to 25 target chemicals, their mixture, and real wastewater samples were effectively captured within an optimized exposure duration of 12 h. The proposed bioassay achieves logarithmic scaling between experimental time and sample size through batch-based parallel processing, reducing the total testing time by 2.57-fold compared to using standard method of ISO 8692:2012. Furthermore, beyond improved throughput, the performance of the proposed assay was validated using 585 real wastewater samples collected from 100 wastewater treatment plants across China. It revealed that influent toxicity (R² = 0.7991, p < 0.0001) rather than process complexity governed effluent toxicity reduction (p = 0.6581), emphasizing the need for a paradigm shift toward biotoxicity-oriented wastewater treatment. Overall, the high-throughput assay provides a cost-effective and broadly applicable tool for streamlining large-scale wastewater risk assessment, with strong potential for adoption in practical WWT monitoring device.