Ecotoxicity of neutral red (dye) and its environmental applications

• Ecotoxicity of neutral red was tested to support enhanced methanogenesis from coal.
• EC50: 0.11mM (acute toxicity), 1-35.5mM (microbial viability & root elongation).
• Significant hormesis observed in seedling root & shoot growth between 0.25-10mM NR.
• Mean NR concentration deployed for enhanced methanogenesis was below LOAEL.

Neutral red (NR) is a synthetic phenazine with promising prospect in environmental biotechnology as an electron shuttle. Recently, NR injections into coal seam associated groundwater in Australia (final dissolved NR concentration: 8 µM±0.2) were shown to increase methanogenesis up to ten-fold. However, information about NR toxicity to ecological receptors is sorely lacking. The main aim of this study was to investigate the concentration dependent toxicity of NR in microorganisms and plants. Acute toxicity of NR was determined by the modified Microtox™ assay. Microbial viability was determined using Escherichia coli and Bacillus subtilis. Germination and early growth of plants was studied using Lactuca sativa, Daucus carota, Allium cepa and an Australian native Themeda triandra. Lastly, mutagenicity of the coal seam associated groundwater was assessed using the Ames test. The EC50 of acute NR toxicity was determined to be 0.11 mM. The EC50 of microbial viability was between 1 and 7.1 mM NR. Among the concentrations tested, only 0.01, 0.10 and 100 mM of NR significantly affected (p<0.001) germination of L. sativa. The EC50 for root elongation in seeds was between 1.2 and 35.5 mM NR. Interestingly, root elongation in seeds was significantly stimulated (p<0.001) between 0.25 and 10 mM NR, showing a hormetic effect. A significant increase in mutagenicity was only observed in one of the three wells tested. The results suggest that the average dissolved NR concentration (8 µM±0.2) deployed in the field trial at Lithgow State Coal Mine, Australia, appears not to negatively impact the ecological receptors tested in this study.