A multi-pumping flow system for acute toxicity bioassay using the Vibrio fischeri bacteria

Abstract

A multi-pumping flow system was developed for automation of the acute toxicity bioassay using the Vibrio fischeri bacteria. Solenoid micro-pumps were exploited to improve mixing conditions, and to accomplish the required in-line dilutions of the tested compounds by modifying the sample volume and exploiting the partial overlap between the sample zone and the bacterial suspension. A spiral flow cell, placed at the emission window of a spectrofluorimeter, was used for signal measurements and a lab-made water bath based on the Peltier effect was used for temperature control of the bioassay. A 120 μL of the bacterial suspension was selected in order to minimize its consumption and, consequently, the costs of the assay. The contact time between the bacterial suspension and sample was reduced to 5 min to increase the sampling throughput and to avoid luminescence fading due to the short bacteria lifetime. The coefficients of variation were estimated at 2.4 and 2.0% (n = 10), in the absence and in the presence of 0.60 mg L⁻¹ Zn(II), respectively. The EC₅₀ values for emerging contaminants (parabens, caffeine, acetaminophen, diclofenac, and salicylic acid) agreed at the 95% confidence level with those obtained with the commercial BioTox™ kit performed in a microplate. The proposed flow system is then a simple, fast, robust, and accurate alternative for acute toxicity determination, using low sample and bacterial suspension volumes. Furthermore, it presented other advantages in relation to batch and previously proposed flow-based bioassays, such as in-line osmotic adjustment and sample dilutions, and the evaluation of toxicity kinetics for every assayed sample.