Molecular impairment-guided ecotoxicity assessment: Insights from copper and pH stress in zebrafish

Copper (Cu) toxicity is governed by the redox transition of Cu(II) to Cu(I), yet its temporal dynamics and modulation by water chemistry remain poorly understood. This study developed an omics-driven toxicokinetic framework evaluating Cu toxicity in zebrafish gills across varying pH levels. By interrogating the expression kinetics of key redox-switch genes (steap family/fdx1), we identified a 9-h molecular peak window serving as a highly sensitive signature for incipient toxicity, preceding macroscopic phenotypic shifts. A relatively high-dose of Cu (30 μg/L) advanced steap2/4 peaks to 6 h, indicating STEAP capacity-constrained reduction of Cu(II) to Cu(I). Fluorescent quantification of gill Cu(I) accumulation displayed no significant differences. Nonetheless, transcriptomic profiling established a condition-dependent hierarchical pathway impairment: necroptosis > ferroptosis > apoptosis > inflammatory > immune > stress response > oxidative stress response. Acidic regimes uniquely triggered necroptosis and profound metabolic dysfunction. Interaction with a Cu(I)-specific chelator abolished cell necrosis and reversed global transcriptional phenotypes, validating that intracellular Cu(I) trafficking drives systemic damage. This dual-analytical framework, coupling a "Cu toxic molecular switch" with a "gradation of injury pathways", to some extent, provides a temporally resolved predictive tool and offers a robust mechanistic foundation for risk assessment of metals in dynamic aquatic ecosystems.