Study suggests increased biotoxicity due to anaerobic decolorization of diazo dye by wetland-derived Clostridium
Untreated synthetic dye effluents severely threaten aquatic ecosystems by blocking sunlight, depleting dissolved oxygen and releasing compounds with potential ecological toxicity. Among the various treatment strategies, biological treatment is considered a cost-effective and environmentally friendly alternative to chemical methods.
Reactive Black 5 (RB5), one of the world's most widely used diazo (azo) dyes in the textile industry, is known for its stability, water solubility and resistance to natural degradation. While certain bacterial genera have demonstrated the ability to decolorize RB5 via anaerobic metabolism, understanding of the formation and ecotoxicological effects of the resulting intermediate metabolites remains limited.
To address this gap, professor Satoshi Soda from the College of Science and Engineering at Ritsumeikan University in Japan, together with Dr. Kazuko Sawada, research assistant professor at Ritsumeikan University, and Abd. Aziz Amin, a lecturer at Universitas Brawijaya in Indonesia and a doctoral student at Ritsumeikan University, investigated RB5 decolorization, metabolite formation and the biotransformation pathway by a constructed wetland (CW)-derived Clostridium sp. strain T4.
Along with evaluating the wastewater treatment potential of the Clostridium sp. strain, the study also assessed the environmental implications of anaerobic RB5 decolorization. Their findings were published in the journal Environmental Technology & Innovation.
Soda explains the motivation behind the study, "We are involved in the Indonesia Linkage Program (Professional Human Resources Development Project Phase III), which enrolls Indonesian working professionals in our graduate program. Discussions with these students provided us with insights on environmental problems related to textile and batik wastewater treatment in Indonesia. Their insights prompted us to ask if dye decolorization actually leads to environmental detoxification."
The researchers isolated the Clostridium sp. strain T4 from the effluent of a laboratory-scale CW. At concentrations ranging from 50 to 200 mg/L, under anaerobic conditions, the bacterium achieved up to 97% decolorization within 3 days. As no dye removal was observed in uninoculated controls, it was confirmed that the process was biologically mediated.
Spectral analysis confirmed that the dye's azo chromophore was disrupted. The researchers suggest that flavin-dependent NADH-linked azoreductase activity is the most plausible mechanism underlying this process. Additionally, two aromatic amine metabolites, 4-(ethylsulfonyl)aniline and 1,7-diamino-8-hydroxynaphthalene, were identified.
However, acute toxicity increased as decolorization progressed. Using Daphnia magna for acute toxicity assessment, the researchers found that the 48-hour effective concentration (EC50) decreased from 64.4% before treatment to 20.9% after five days of anaerobic incubation. As lower EC50 values indicate higher toxicity, the findings suggest that "decolorization" does not equal "detoxification."
This inverse relationship between decolorization and detoxification highlights a key limitation of anaerobic azo dye treatment. Under anaerobic conditions, bacteria can effectively cleave azo bonds and eliminate visible color, but they lack the downstream oxidative capacity needed to further degrade aromatic amines, allowing these toxic intermediates to accumulate.
The study does not propose a new RB5 degradation pathway, since anaerobic azo bond cleavage and aromatic amine formation are well established. Instead, its novelty lies in linking efficient single-strain anaerobic decolorization with time-dependent metabolite accumulation and increasing ecotoxicity.
Soda said, "Anaerobic treatment should be regarded as a reductive first stage for decolorization rather than a complete remediation process. To ensure safer effluent quality, integration with subsequent aerobic or oxidative treatment is necessary to achieve further detoxification in azo dye-containing wastewater management systems."
Indonesia's batik industry, an internationally recognized cultural heritage, supports millions of livelihoods but poses severe environmental challenges. Untreated wastewater from dyeing and wax-removal processes, laden with synthetic dyes, heavy metals and silicates, frequently pollutes local waterways.
While wastewater management and dye removal are necessary, this study highlights the importance of minimizing and managing toxic intermediate byproducts.
Overall, the study findings demonstrate that wastewater treatment should be evaluated not only by color removal but also by ecotoxicological safety.
The research supports routine toxicity monitoring, improved wastewater treatment regulations and the development of safer treatment technologies to promote sustainable "green batik" production in Indonesia and other textile-producing countries while better protecting aquatic ecosystems and human health.
More information
Abd. Aziz Amin et al, Anaerobic decolorization of reactive black 5 by Clostridium sp. strain T4 and associated toxicity increase due to aromatic amine formation, Environmental Technology & Innovation (2026). DOI: 10.1016/j.eti.2026.105043
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Citation: Study suggests increased biotoxicity due to anaerobic decolorization of diazo dye by wetland-derived Clostridium (2026, July 13) retrieved 13 July 2026 from https://phys.org/news/2026-07-biotoxicity-due-anaerobic-decolorization-diazo.html
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