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There are significant worries about the impact of heavy metal pollutants in soils during urbanisation and industrialisation in the developing world. Routine chemical analysis of soils is used to characterise the concentration of metals translocated by point source and diffuse actions. This fails to put in context the bioavailability or potency of these analytes. Biosensors offer a novel and direct method for hazard assessment of soils impacted with anthropogenic contamination. However, few significant examples of thorough applications of the biosensor in environment have been confirmed. This thesis makes use of comprehensively characterised biosensors for the applications to different environmental matrices. The objective was to apply and evaluate the performance of biosensors in determining the bioavailable fraction of heavy metals in the environment in conjunction with chemical analysis data. Both constitutively marked and metal induced biosensors were optimised and applied, but only the constitutively marked biosensor was responsive to a range of soils and elements of concern. As the matrix that the biosensors were exposed to became increasingly complex, the biosensors' performance became more difficult to interpret. For the metal induced biosensor, the response to standard aqueous samples was predictable as the relationship between chemical analysis data and biosensor predicted data was strong. By contrast, the performance of the metal induced biosensor was poor in soil samples which were historically contaminated (Chapter 9). This could become impacted by the soil characterisation, such as the presence of eo-contaminants, soil associated carbon or variations in soil pH. The constitutively-marked biosensors offered greater predictability of response and this is the reason why these are widely adopted in soil applications. Bacterial biosensors provide a useful tool for assessing the bioavailable fraction of analytes in soils and for complementing chemical analysis. If matrix matched control samples can be collected, then this technology can be applied to a wide range of contrasting soils with a suite of contaminants to aid in the development of generic soil hazard evaluation.

The luxCDABE genes (i.e. full cassette) were inserted into the bacterial strain used in this study, E. coli HB101, using a multi copy plasmid, (pUCD607). A number of experiments were carried out in this thesis to study the potential of using the biosensor, E. coli HB101 (pUCD607), for ecotoxicity testing. Growth and bioluminescence of E. coli HB101 (pUCD607) were characterised and optimised, as well as the stability of this biosensor to a range of environmental parameters. The biosensors were found to be sensitive to a range of pollutants and provided a highly consistent bioluminescence response under conditions likely to be encountered in environmental toxicity testing. Assessment of the potential of the lux-based bioassay for revealing the combination of toxicities of metals was carried out. This is necessary because samples in ecotoxicity testing may contain a number of types of pollutants. The biosensors showed high sensitivity of response to mixed metals and identified the combined toxicity of the mixture. Biosensor bioluminescence could also be used to diagnose the distinction between toxicity after 15 and 30 minutes exposure. Results suggested that exposure time is an important factor affecting on the toxicity of metals in mixtures. The results from challenging lux-based biosensors with heavy metals with various concentration of Cl- demonstrated the effect of Cl- ion complex formation for metals on the bioluminescence of E. coli HB101 (pUCD607). The bioluminescence response of the biosensor also related to the results from computer modelling (GEOCHEM) and was used to diagnose the effect of Cl- on metal toxicity. The results showed that E. coli HB101 (pUCD607) is sensitive to complexes of metals such as are formed by Cl- ion. Immobilised cells of E. coli HB101 (pUCD607) were used to investigate the toxicity of metals, both singly and in mixture. This work aimed to assess the potential of immobilised cells, as an alternative form of biosensor, for use in ecotoxicity testing. Bioluminescence response of immobilised cells varied with the test solution pH and showed that it has great potential for use in low pH environments. Immobilised cell biosensors demonstrated sufficient sensitivity to identify the toxicity of individual and mixed metals. High levels of bioluminescence of immobilised cells lasted up to 5 hours after bead production, suggesting that biosensor immobilisation increases the flexibility of the toxicity assay.

Atrazine, a member of the s-triazine herbicides, appears on the EC Red List of chemicals that cause environmental concern, with residues occurring frequently in sub-soils and aquifers world-wide. Microbial metabolism is considered to be the major mechanism of complete removal of atrazine from the soil environment. Laboratory-based studies were carried out to investigate the microbial mineralisation of atrazine in soil, and to develop novel methods for potential acceleration of breakdown. In initial investigations, simple microcosm experiments were carried out to identify the importance of different fractions (water-soluble and water-insoluble) of plant residues to the dynamics, and extent of atrazine mineralisation. The amendment of soil with different plant residue fractions initially inhibited (for the water-soluble fraction) or enhanced (for the water-insoluble fraction) dynamics of atrazine mineralisation. However, by the end of the incubation, there was no overall effect on atrazine mineralisation. This suggested that increasing the soil organic matter content may render the herbicide less bioavailable to the degrader population. Investigations carried out to determine which component of the microbial biomass was more important in atrazine mineralisation showed that substantial removal of the fungal and Gram-negative bacterial population inhibited atrazine mineralisation in the soil. Experiments to selectively enrich the soil for fungal or bacterial atrazine degraders isolated a mixed bacterial culture able to accelerate atrazine mineralisation when inoculated into soil. The effect of the presence of the earthworm Lumbricus terrestris on the indigenous atrazine degrading microflora was investigated. Results indicated that atrazine mineralisation could be accelerated, but this was not primarily due to increases in microbial biomass, but possibly due to physio-chemical changes brought about by the earthworm and subsequent alterations in atrazine bioavailability.

Different sample preparation methodologies were evaluated for the determination of pollutants in different matrices. The methods investigated were chosen on the merits of decreased sample preparation time and low toxic solvent consumption with the aim to provide viable alternatives to more laborious methods, such as, Soxhlet extraction. Techniques were developed to extract and quantify organic pollutants from contaminated soils and water. The results from shake flask extraction of aged phenolic contaminated soils were used in attempt to relate sorption to both the soil, and pollutant properties. This was to help gain an understanding of the transport and fate of phenols in different environmental situations. The partitioning (sorption/desorption) of radiolabelled phenols between aqueous solution and soil was investigated using a modified shake flask technique. This provides additional information which can be related to soil characteristics, hence pertaining to the fate mechanisms involved for phenols in the environment. The results from these investigations suggest that more than one factor contributes to the sorption of phenols in soils.

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