A simple and highly repeatable colorimetric toxicity assay method using 2,6-dichlorophenolindophenol as the redox color indicator and whole eukaryote cells

Abstract

A simple and highly reproducible toxicity assay method was studied by employing 2,6-dichlorophenolindophenol (DCIP) as a redox color indicator, baker’s yeast Saccharomyces cerevisiae, and a thermostable three-consecutive-stir unit. The absorbance of DCIP was decreased by increasing the metabolism activity of S. cerevisiae to intake glucose as an organic substance. By optimizing the measurement conditions, we obtained highly sensitive responses to glucose between 0.75 and 30 mg/L (eight points, n = 3) with an incubation time of the reaction mixture of 10 min at 30 °C. An excellent value of 1.15% was obtained as the average of the repeatability from eight points. Next, for the characterization of this method, we investigated the influence on the colorimetric response of dissolved substances, such as inorganic ions and surfactants, in natural water. Furthermore, the colorimetric responses to several toxicants were examined using Cu²⁺, Mn²⁺, Zn²⁺, Cr³⁺, and Fe³⁺ as heavy-metal ions and simazine as an agricultural chemical. As a result, notable colorimetric responses were obtained for Cu²⁺ and Mn²⁺ at several concentrations, and the results were compared with those obtained using river water as a real sample. In the stability test, responses to 30 mg/L glucose were obtained for 28 days when the yeast cell suspension was stored at 4 °C (response reduction, 43.9%; average of the relative standard deviation for nine testing days, 22.7%; average of repeatability, 1.01%).