Anaerobic Decolorization of Reactive Black 5 by Clostridium sp. strain T4 and Associated Toxicity Increase Due to Aromatic Amine Formation

Reactive Black 5 (RB5) is a widely used azo dye with high environmental persistence and potential toxicity. This study evaluated RB5 decolorization and biotransformation by Clostridium sp. strain T4, isolated from a constructed wetland, and assessed associated toxicity changes. Decolorization was monitored by UV–Vis spectroscopy, and transformation products were analyzed by LC/MS, while acute toxicity was assessed using Daphnia magna . Strain T4 efficiently decolorized 50–200 mg L⁻¹ RB5 under anaerobic conditions. Spectral analysis confirmed reductive azo bond cleavage, and LC/MS tentatively identified aromatic amine metabolites, including 1,7-diamino-8-hydroxynaphthalene and 4-(ethylsulfonyl)aniline. Despite effective color removal, acute toxicity toward Daphnia magna increased during incubation, as indicated by declining EC₅₀ values. demonstrating increase in acute toxicity concurrent with decolorization. This inverse relationship demonstrates that anaerobic RB5 reduction promotes accumulation of toxic intermediates rather than detoxification. These results show that decolorization alone is not a reliable indicator of treatment safety and provide foundational ecotoxicity data for future hydraulic retention time in integrated anaerobic–aerobic azo dye wastewater treatment.