Applications of redox indicators for evaluating redox conditions in environmental samples

Redox sensors based on immobilized redox indicators have been developed and characterized as to their applicability for evaluating environmental redox conditions. Unique methodology was developed to immobilize indicators to both agarose films and cellulose filter papers, allowing contact of the indicator with both dissolved and adsorbed redox-active species and microbes in environmental samples. The agarose films are placed in a novel flow cell allowing the absorbance of the indicator to be monitored continually while an environmental sample (e.g., groundwater or wastewater slurry) is pumped through the cell with minimal filtering. The absorbance of a given indicator decreases when conditions become sufficiently reducing and can be used to define a "redox potential". The immobilized redox indicators are reversibly reduced and oxidized by reductants and oxidants in the sample.;Three primary redox indicators with quite different formal reduction potentials at pH 7 (E70'), thionine, cresyl violet, and phenosafranine, establish a redox scale and were shown to couple to reductants produced under distinct microbial redox conditions. Thionine (E7 0' = +52 mV on agarose) is reduced by Fe(II) (at Fe(II) levels > 100 muM and pH 7 or greater) and is useful for identifying Fe(III)-reducing conditions, and cresyl violet (E70' = -81 mV on agarose) couples to sulfide ([S(-II)] of 1 - 100 muM at pH 6-8) and is useful for identifying sulfate-reducing conditions. Phenosafranine (E70' = -267 mV on agarose) is reduced under methanogenic conditions. Indicators can be used in pairs to delineate different types of reduction conditions (e.g., Fe(III)- from sulfate-reducing). Moreover, the degree of reduction of thionine or cresyl violet by a given level of Fe(II) or sulfide in simple solutions corresponds reasonably well with that predicted by equilibrium models in most cases. It was demonstrated that reduction of redox indicators can be used to predict the onset of redox transformations of As(V) to As(III) and trichloroethylene to cis-dichoroethylene.;The effect of different redox species on the Pt electrode potential (E Pt) and determination of hydrogen with redox indicators were also investigated. In soil slurries under anaerobic conditions, no evidence was found that E Pt is controlled by inorganic redox couples. The feasibility of determining H2 by its reduction of a redox indicator in the presence of palladium catalyst was demonstrated. H2 levels as low as 0.0002 atm (160 nM in solution) were detected with thionine. Although adsorption of thionine on Pd prevented achieving detection limits suitable for environmental samples, criteria were established for attaining this goal in future studies.