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Effective techniques for assessing soil environmental pollution are required to develop protective policy. Chemical methods have been traditionally used to determine total concentration of pollutants and biologically linked measurements have been used to assess the bioavailable fraction of pollutants. Bioluminescence-based microbial bioassays have been shown to respond to the bioavailable fractions. Growth and bioluminescence of lux-marked E. coli HB101 and P. fluorescens 10586r were characterised and optimised for freeze-drying culture. Freeze-drying cultures have been used effectively because of their ease of use, rapid assay response and sensitivity to a wide range of pollutants. An assessment of Zn and Cd amended soil was investigated. Two different techniques (centrifugation and Rhizon sampler) were used to obtain the interstitial pore water of soils. The concentrations of Zn and Cd were significantly higher in the soil solution extracted using the centrifugation technique compared to the Rhizon sampler technique. The biosensors responded to the free metal concentrations in the soil solution. An assessment of the toxicity of 2,4-dichlorophenol, 2,4,6-trichlorophenol and pentachlorophenol, individually and in combinations, was tested in deionised water (pH 5.5), soil solutions, and soils using lux-marked E. coli HB101 and P. fluorescens 10586r. Toxicity interaction responses of the mixture chlorophenols were predicted using a model. Synergistic interactions were observed for the response of P. fluorescens 10586r pUCD60-7 to all combinations of chlorophenol tested, while the response of E. coli HB101 pUCD607 varied with the matrix solutions tested. Bioavailability of naphthalene was studied using cyclodextrin-based extractions caused to the luminescence response of Pseudomonas fluorescens KH44 pUTK21. Increasing the concentrations of beta-cyclodextrin (b-CD) and hydroxylpropyl-b-cyclodextrin (HPBC) in the extract solutions increased the apparent concentration of naphthalene in the soil solutions. The luminescence response of P. fluorescens HK44 was associated with bioavailable of naphthalene.

The objective of this research was to determine the potential of two anti-atrazine single-chain antibodies (scAbs) to detect very dilute atrazine concentration, specifically in the nanomolar and sub-nanomolar ranges. Analysis of environmental pesticide contamination is routinely done by high performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS). However, since the mid eighties there have been an increasing number of reports describing the use immunoassays to detect environmental pollutants. Although immunoanalysis using whole antibodies has been shown to be almost as effective as GC/MS (Thurman et al., 1990) the technology is still not widely used for pesticide analysis. This thesis will describe the increased sensitivity of single-chain antibody fragments, compared to whole antibodies, to detect low concentrations of atrazine and related triazines. The effect of monomeric and dimeric scAb conformations on the ability of enzyme linked immunosorbent assays (ELISAs) to detect atrazine and related triazines is investigated. The thermal and chemical stability of the scAbs, and a modified scAb containing an interchain disulphide bond, are compared with the parent monoclonal antibodies (MAbs) to determine their suitability in an environmental ELISA assay system. The expressions in Escherichia coli (E. coli) of two different anti-atrazine scAbs and related antibody fragments are studied, and an optimised expression protocol for these antibody fragments obtained. Three potentially toxic amino acid residues identified in one of the variable heavy (VH) domains (Knappik & Pluckthun, 1995) are mutated to less toxic amino acids, and their effects on antibody fragment expression observed.