Toxicity assessment of chlorophenols using a mediated microbial toxicity assay

Abstract

While direct toxicity assessment (DTA) is now widely recognised as a useful tool for environmental risk assessment, many existing tests fail to meet end-user needs. This article describes the significant progress made to the MICREDOX® DTA assay, developed at Lincoln Ventures Ltd, brought about by miniaturising this assay to a multi-well plate format combined with limiting current microelectrode transduction. The benefits have been reduced: preparation time, reduced assay time, lower material costs and a higher level of replication achieved. To validate the precision of the miniaturised format, the concentrations required to cause a 50% decrease in signal (EC₅₀) by an archetypal group of toxicants, the chlorophenols, were determined using two terrestrial bacterial strains, Escherichia coli K12 and Klebsiella oxytoca 13183. The assay time was then reduced by stepwise adjustment of the incubation time, from 60 down to 5 min, and the EC₅₀s reported by E. coli to each of the toxicants after 45, 30, 15 and 5 min incubations were determined. The results obtained match closely with those reported by the Activated Sludge Respiration Inhibition Test and confirm the miniaturised multi-well plate MICREDOX® DTA assay reliably reports representative EC₅₀ values for these toxicants. The previously described trends of increasing toxicity with increasing chlorine substitution and the observation that meta-substituted chlorophenols are more toxic than their ortho-substituted counterparts are also confirmed. The ability to monitor toxicity using terrestrial organisms, in volumes amenable to multi-well microtitre plates and incubations requiring only a few minutes, facilitates the rapid generation of highly reproducible, easy to operate and inexpensive DTA measurements.

Keywords:

• direct toxicity assessment
• microelectrode amperometry
• chlorophenols
• Escherichia coli
• Klebsiella oxytoca

Acknowledgements

The authors gratefully acknowledge funding from the New Zealand Foundation for Research, Science and Technology and the New Zealand Ministry of Education for awarding an Enterprise Scholarship to Amy Scott. We thank Dr Joanne Hay (Lincoln Technology) for editorial input and Dr Richard John (Griffith University) for providing the microelectrodes used in this study. The method for measuring the amount of substrate in a microbially catalysed reaction is covered by a patent (PCT/NZ97/00158), awarded in 1998.