Fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction...

Global urban and industrial growth, with the associated environmental contamination, is promoting the development of rapid and inexpensive general toxicity methods. Current microbial methodologies for general toxicity determination rely on either bioluminescent bacteria and specific medium solution (i.e. Microtox(®)) or low sensitivity and diffusion limited protocols (i.e. amperometric microbial respirometry). In this work, fast and sensitive optical toxicity bioassay based on dual wavelength analysis of bacterial ferricyanide reduction kinetics is presented, using Escherichia coli as a bacterial model. Ferricyanide reduction kinetic analysis (variation of ferricyanide absorption with time), much more sensitive than single absorbance measurements, allowed for direct and fast toxicity determination without pre-incubation steps (assay time=10 min) and minimizing biomass interference. Dual wavelength analysis at 405 (ferricyanide and biomass) and 550 nm (biomass), allowed for ferricyanide monitoring without interference of biomass scattering. On the other hand, refractive index (RI) matching with saccharose reduced bacterial light scattering around 50%, expanding the analytical linear range in the determination of absorbent molecules. With this method, different toxicants such as metals and organic compounds were analyzed with good sensitivities. Half maximal effective concentrations (EC50) obtained after 10 min bioassay, 2.9, 1.0, 0.7 and 18.3 mg L(-1) for copper, zinc, acetic acid and 2-phenylethanol respectively, were in agreement with previously reported values for longer bioassays (around 60 min). This method represents a promising alternative for fast and sensitive water toxicity monitoring, opening the possibility of quick in situ analysis.