Unveiling Whole Water Neurotoxicity through Sensitive Electrophysiological Functional Profiling: Beyond Conventional Morphological Metrics

Abstract Neuroactive contaminants typically coexist as complex mixtures in aquatic ecosystems, exerting deleterious effects through combined exposures. However, current assessment approaches based on morphological endpoints (e.g., neurite outgrowth) lack sufficient sensitivity to capture functional neurotoxicity of these mixtures. To address this gap, this study establishes in vitro functional neurotoxicity testing (iFNT), an ultrasensitive bioassay leveraging high-density microelectrode arrays to profile cortical network electrophysiology. Validated using reference toxicants, their mixture, and whole water samples, iFNT achieves 5- to 500-fold higher sensitivity than the neurite outgrowth assay. This heightened sensitivity enables clearer discrimination of functional phenotypes for both individual compounds at environmentally relevant concentrations (10–8 M) and whole water samples without preconcentration, providing a powerful tool for evaluating real-world neurotoxic risks. Alarmingly, conventional monitoring indicators exhibited a pronounced mismatch with iFNT profiles across wastewater treatment stages, demonstrating that seemingly acceptable routine readings masked the persistence of latent neurotoxicity. This study challenges morphology-based paradigms, revealing that accurate whole water risk assessment necessitates integrating functional biological endpoints into future monitoring frameworks.