Development of a bacterial bioassay to assess xenobiotic toxicity in soils and groundwaters
Organic xenobiotic compounds in the environment are causing widescale concern. Knowledge of the toxicity of organic xenobiotic compounds to the soil microbial biomass is essential as soil health and sustainability are essential for ecosystem function. Traditionally, chemical analysis has been used to assess contamination impacts in soils, however, compound residue cannot provide information on toxicity in environmental matrices, the toxicity of complex chemical mixtures and bioavailability. Bioluminescence bioassays, utilising the naturally bioluminescent bacterium Vibrio fischeri have been widely used to investigate the toxicity of contaminants in soil, sediment and freshwater environments. To utilise the V. fischeri bioassay all samples must be adjusted to a near neutral pH and a salinity of 2 % NaCl. Application of this assay to assess toxicity in terrestrial environments means that samples are not assessed under natural pH and saline conditions. Bioluminescent terrestrial bacteria have been constructed by inserting the lux genes, encoding of bioluminescence in marine bacterium, into the plasmid or chromosomal genome of terrestrial bacteria. The plasmid lux marked strain studied in this thesis, Pseudomonas fluorescens 10586s pUCD607, was used to develop a bioassay which could assess the toxicity of substituted benzenes in aqueous solution. The EC50 values determined for benzene and 1,2-dichlorobenzene were comparable between lux marked P. fluorescens and V. fischeri. Bioluminescence responses to substituted benzenes were investigated with a view to understanding modes of toxic action. Observed stimulation of bioluminescence in response to a number of compounds was thought to be caused by the uncoupling of proton gradients by low organic xenobiotic concentrations. Viable cell counts confirmed that stimulation of bioluminescence was not as a result of an increase in the number of viable cells. At high substituted benzene concentrations an inhibition of bioluminescence was observed. Application of quantitative structure-activity relationships (QSARs) for chlorobenzenes, utilising P. fluorescens toxicity data and physiochemical characteristics, showed that the toxic responses of these non-polar compounds were a function of compound solubility and lipophicity. QSARs applied to assess chlorobenzene toxicity could not be used to predict the toxicity of polar compounds due to the differing modes of toxic action. Correlating P. fluorescens with QSARs developed for C. meneghiniana and Pimephales promelas showed good correlation between the freshwater organisms but a poor correlation between marine bacterium V. fischeri.