Ecotoxicity of bauxite residue leachates during amendment-assisted revegetation: Multi-organ oxidative and genotoxic effects and gut microbiota shifts in zebrafish

Vegetation restoration is increasingly applied to mitigate environmental risks from bauxite residue disposal areas; however, how amendment-assisted revegetation alters leachate chemistry and affects aquatic organisms remains unclear. In this study, separate groups of adult zebrafish (Danio rerio) were exposed for 96 h to leachates collected after 30, 90, and 150 days of restoration. Organ-specific oxidative stress and genotoxicity, together with gut microbiota responses, were evaluated. Amendment-assisted revegetation significantly reduced leachate pH, electrical conductivity, and the concentrations of several PTEs. However, biological responses showed temporal and non-monotonic patterns, with persistent oxidative and genotoxic responses observed in some tissues. Multivariate analyses revealed that organismal responses were associated with bulk chemical parameters, selected PTEs (e.g., Ni), and major cations (e.g., Na⁺ and Ca²⁺), while gut microbiota showed temporal shifts characterized by changes in alpha diversity and taxonomic composition, including enrichment of Proteobacteria and depletion of Fusobacteriota. These findings demonstrate that improvements in bulk chemical indicators do not necessarily translate into immediate or uniform biological recovery, highlighting a potential temporal decoupling between chemical improvement and biological recovery during revegetation. This study provides new insights into the ecological implications of amendment-assisted restoration and supports more risk-informed management of bauxite residue leachates.