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This study investigated the potential application of biological processes for the removal of organic xenobiotics from the environment using open mixed cultures under aerobic conditions. The xenobiotics studied include phenol, bisphenol A (BPA), caffeine, diclofenac, ketoprofen, ibuprofen, and acetaminophen and, differently from most published studies, they were investigated as sole carbon and energy sources. The study was divided into two major phases namely batch and continuous studies. In batch experiments, phenol and acetaminophen were found biodegradable, while the others (BPA, caffeine, diclofenac, ketoprofen, and ibuprofen) were not biodegraded. For phenol, the lag time before the start of the biodegradation was observed to increase as the phenol concentration increased (in the range 0.2-0.5 g/L), with no biodegradation for phenol concentration of 1 g/L. However, phenol at concentration of 1 g/Lwas removed if the culture was previously acclimated with lower concentrations of phenol or with a mixture of peptone and yeast extract. In batch experiments, acetaminophen was only biodegraded after the addition of the peptone/yeast extract mixture. Phenol and acetaminophen degradation were also investigated using a continuous process, laboratory scale sequencing batch reactor (SBR). Data from the SBR shows that phenol and acetaminophen degradation increase as the SRT increases and the degradation is very low if the SRT is lower than a certain value (4 d) for phenol and (3 d) for acetaminophen. The ability of the biomass in the SBR to remove phenol increased as the number of cycles per day was increased, probably due to a reduction in the maximum phenol concentration the microorganisms were exposed to, which reduced the inhibiting effect of phenol. A maximum high chemical oxygen demand (COD) removal efficiency of 95% was recorded for phenol while 93% was observed for acetaminophen using SBR. A preliminary calibration of a kinetic model for phenol and acetaminophen biodegradation was carried out using oxygen profile data from selected SBR cycles.

The mercapturic acid pathway is an important detoxication mechanism in mammalian cells/tissues. However, work over the last decade has shown that several halogenated alkenes, once processed via this pathway, become substrates for the enzyme cysteine conjugate β-lyase. This latter enzyme cleaves these conjugates to ultimately yield potent nephrotoxins. The factors controlling the β-lyase enzyme's cellular distribution and substrate specificity have, however, yet to be elucidated. To this end, a cDNA for cysteine conjugate β-lyase has been isolated from a rat kidney cDNA library using immunological screening. The cDNA was sequenced and found to be previously unreported. Northern blot analysis of rat kidney and liver RNA showed the cDNA to be highly specific for a single mRNA, of 1.9kb in length, in the kidney which was in agreement with the expected size for a 48kD protein. This cDNA was used to investigate the tissue distribution of rat kidney β-lyase. Northern blot analysis of tissue RNA was in close agreement with the Western blot finding that the kidney β-lyase was of a structurally different form when compared to extrarenal forms of the enzyme. The cDNA was also used to examine the induction/reduction of rat kidney β-lyase activity by pretreatment of rats with the β-lyase substrate precursor NAc-PCBD. It was demonstrated that a non-toxic dose of 3mg/kg NAc-PCBD caused a significant increase in kidney cytosolic β-lyase as assessed by enzyme activity, enzyme protein levels and corresponding mRNA levels. In contrast, a toxic 10mg/kg dose caused a significant decrease in these latter parameters. Further cDNAs were isolated by immunological screening of a separate cDNA library but failed to be identified as corresponding to β-lyase. The possible reasons for this and the significance of the isolation and characterisation of the original cDNA are discussed at length.

Human health is adversely affected by exposure to xenobiotics at elevated levels, and the impact of minor exposure is often overlooked. Toxicological behavior of agrochemicals is commonly determined by their ability to bind to plasma protein, as they are significantly more toxic when unbound. Moreover, for pharmaceuticals, plasma protein binding determines their therapeutic effect since only the free form can diffuse to their target sites. In spite of separate investigations regarding pharmaceuticals and pesticides binding to plasma proteins, the binding behaviors of pharmaceuticals in the presence of pesticides have not been comprehensively explored. To determine the effect of xenobiotics binding to plasma protein, a targeted quantitative method coupling solid phase microextraction (SPME) with both liquid chromatography (LC)-mass spectrometry and gas chromatography (GC)-mass spectrometry was developed. The performance of a polydimethylsiloxane/divinylbenzene/ polydimethylsiloxane (PDMS/DVB/PDMS) biocompatible extraction phase was investigated for extraction of xenobiotics from plasma samples via direct immersion solid-phase microextraction (SPME) prior to gas chromatography-mass spectrometry. Under the optimum extraction settings, that included an attentive optimization of the fiber rinsing conditions, the microextraction device was able to endure 100 consecutive extractions from undiluted and diluted plasma with an overall reproducibility up to 28% for all the analytes except chlorpyrifos-methyl. Optimized conditions were used to validate a quantitative method using matrix-matched calibration. Accuracy and precision values for bovine plasma were within 96-132% and 0.05-5.82% respectively. LLOQs for all the analytes were at 1 µg L-1 and LDR ranged within 1-100 µg L-1. The applicability of this method to plasma from different species (human, rat, rabbit) was also investigated. This work represents the first step towards broader use of the biocompatible PDMS/DVB/PDMS extraction phases to analyze multiclass xenobiotics in plasma and other complex biofluids.Chapter 3 describes a quantitative method for the analysis of xenobiotics in plasma based on solid phase microextraction (SPME) coupled with liquid chromatography (LC)-mass spectrometry. Moreover, the developed method was applied to evaluate the matrix effects of plasma from four different species (human, bovine, rat, and rabbit). PAN/C18 SPME fiber, which has previously been demonstrated to have outstanding biocompatibility in plasma, was used for this analysis in order to address the knowledge gap found in the literature, as this fiber has not been evaluated for the analysis of pesticides in biofluids. A group of model analytes containing physically, and chemically different xenobiotics (drugs and pesticides) were selected to determine the matrix effects of plasma in terms of analyte-protein binding and the instrument associate matrix interferences. Absolute matrix effect values, which were calculated by the differences in responses obtained from standards spiked before and after extraction of the matrix, range between the acceptable level of 96-108% for analytes from different plasma sources except for methaqualone and chlorpyrifos-methyl in rat plasma. These results indicate that the developed SPME method can be successfully applied for the analysis of xenobiotics from complex matrices with minimal matrix effects. This study also evaluated the variance of matrix effects due to the differences of interspecies plasma protein binding of analytes by comparing plasma calibration curves from different species with the calibration curve from PBS. Bayesian Hierarchical Model (BHM) was used to correct the variance added to measurements from different plasma types. Correction factors were calculated for each analyte in each plasma type and PBS. The correction factors effectively mitigated measurement bias resulting from different calibration curve types used in human plasma quality control measurements. This work would help to develop cost-effective and efficient calibration strategies for biofluids by utilizing plasma from readily available sources, such as bovine plasma, for method optimization and validation prior to quantitative analysis of plasma available in limited quantities (e.g., rabbit and rat).The developed SPME methods were tested for two different applications. Two different applications were described in chapters 4 and 5. In the fourth project, a high-throughput method was developed for analysis of 79 pesticides, commonly used in agricultural practices, in human plasma, using biocompatible solid phase microextraction (SPME) coupled to liquid chromatography-tandem mass spectrometry. An SPME method was developed using a hydrophilic-lipophilic balanced/polyacrylonitrile (HLB/PAN) extraction phase and demonstrated negligible matrix effects. The performance of the developed SPME method was compared to a QuEChERS- quick, easy, cheap, effective, rugged, and safe method, the most common sample preparation and cleanup approach for pesticide analysis in complex matrices. Comparable accuracy and precision were achieved for both methods, with accuracy values within 70-120% and relative standard deviation

The decline in DNA sequencing costs and the increased efficiency of next generation sequencing platforms have led to a flood of raw sequence data that taxes the analytical capacities of the current bioinformatic and statistical tools. The number of microbiome studies and the sequencing depth of each is likely to continue to grow given their tremendous potential to uncover important roles in ecosystem and human health. Microbiome data is highly complex; it is compositional, multi-dimensional, overdispersed, and sparse. For these reasons, the analytical and statistical pipelines used to analyze these data should be carefully chosen to address both experimental design and the intrinsic features of the expected data. Here, I present three case studies to illustrate the use of deep 16S rRNA gene and metagenomic sequence analysis of environmental microbiomes. The first study assessed the impact of triclosan with and without biochar on soil bacterial communities. The second study used 16S rRNA amplicons to examine the effect of a glyphosate-based herbicide on the intestinal bacteria of mice. The last study used a shotgun-metagenomic approach to shed light on the genetic underpinnings of flurbiprofen degradation in a bacterial consortium enriched from active sludge.

Excessive use and lack of appropriate disposal technology for industrial xenobiotics have resulted in the contamination of ecosystems globally impacting the self-regulating capacity of the biosphere. This often results in irreversible alterations of ecosystem’s structure and function, but the outcomes of these events on soil microbial communities (and their functional capabilities) are poorly understood. Assessing the impact of xenobiotics on soil microbial communities is of paramount importance as they play a vital role in ecosystem services and maintain soil health, which are key requirements for sustainable land use in terms of food security and environmental sustainability. Bacteria are the most abundant and diverse soil micro-flora and play a key role in the biogeochemical cycles of important elements including carbon (C), nitrogen (N), and phosphorus (P) and sulphur (S). The current work aimed to unravel the two-way interactions between xenobiotics and soil microbial communities; i.e., how soil microbial communities modulate xenobiotic persistence through biodegradation and what impacts xenobiotic have on soil microbial community’s structure and functions, with particular focus on widely used pesticides (chlorpyrifos and imidacloprid) and industrial solvents (trichloroethene). In Chapter 2, characterisation of active methanotrophs involved in trichloroethene (TCE) degradation under different methane (CH4) concentrations was evaluated. Methane (CH4) enriched methanotrophic consortia from three Australian soils (Sydney University, Victoria Park and Botany Industrial Park) were examined for their effectiveness in TCE (50μM) degradation at 1%, 10% and 33% CH4 concentration at 20oC. Only the methanotrophic consortium from Sydney University (SU) soil was able to co-metabolically degrade TCE. The (SU) methanotrophic growth and TCE degradation was accelerated under high CH4 concentration degrading up to 30% (within 2 days) and 20% (within 5 days) TCE under 33% and 10% CH4, respectively. No degradation of TCE was observed at 1% CH4 concentration or in the absence of CH4 suggesting the dependence on relatively high CH4 availability for TCE degradation. pmoA-based stable isotope probing (SIP), terminal restriction fragment length polymorphism (T-RFLP), clone library construction and sequencing of TCE degrading SU methanotroph consortium revealed the dominance of novel uncultivable Type I methanotrophs (distantly related to Methylovulum-88%) belonging to TRF-53 in TCE degradation. In Chapter 3, the effects of the pesticides chlorpyrifos (CP) and imidacloprid (IC) on soil microbial processes (e.g. biodegradation and respiration) and community structure were evaluated. Two soil treatments (from five sugarcane farms), one with no history of pesticide application (non-treated; 1H, 2H, 3H, 4H and 5H) and the other with ~20 years pesticide application (pesticide-treated; 1R, 2R, 3R, 4R and 5R), were used in this study. MicroRespTM, q-PCR and T-RFLP analyses were combined to explore the relationship between pesticide degradation and soil microbial communities in soils spiked (3 times) with 10 mg/kg of CP or IC, under lab conditions. The results showed that the half-lives of CP decreased with application frequency and were 23-47, 8-20 and 3-17 days following the first, second and third application, respectively (for soils from five sugarcane farms). In particular, the soils from 4R, 4H and 5R showed enhanced CP degradation even when not exposed to CP for last 13 years due to legacy effect of the pesticide. Parallel analyses of IC degradation (10 mg/kg) showed high persistence of this pesticide in soil where repeated application increased half-lives from 30-60 days for the first treatment to 45-65 days for second treatment. The application of both pesticides (CP and IC) reduced soil respiration (basal and substrate-induced) between 7-76% with the lowest respiration found in 5R and highest in 1R after the pesticides treatment, indicating that application of pesticides had an adverse impact on soil functional activity. The molecular analyses showed that both CP and IC significantly altered the soil bacterial community structure and reduced diversity, evenness and richness. In Chapter 4, sequential soil and liquid culture enrichments enabled the isolation of six bacterial CP degraders with sequence homologies to Xanthomonas sp. (3), Pseudomonas sp. (1), Rhizobium sp. (1) and Lysobacter sp. (1). The efficacy of the isolated strains: Xanthomonas sp. 4R3-M1, Pseudomonas sp. 4H1-M3 and Rhizobium sp. 4H1-M1 were further investigated for biodegradation of CP and its primary metabolic product, TCP (3,5,6-trichloro-2-pyridinol). The results indicated that all three bacterial strains utilised CP (10 mg/l) and TCP (as CP degradation product) in mineral salt media (MSM) as a sole source of C and N. Bacterial strains Xanthomonas sp. 4R3-M1 and Pseudomonas sp. 4H1-M3 could also degrade 10 mg/l TCP as a sole C- and N-source, when provided externally. Thus, these bacterial strains promise to be effective in practical application of bioremediation of both CP and TCP. In Chapter 5, using next-generation sequencing, the structure and potential functions of bacterial communities in pesticide-treated and non-treated reference sites was compared at finer levels. Across all soils, the functional beta diversity was correlated with taxonomic diversity indicating possible linkages between the structure and functioning of soil microbial communities. The pesticide-treated sites had higher relative abundance of Proteobacteria and Bacteroidetes, with Archaea exhibiting the opposite pattern. Metagenomic analysis revealed increases in the relative abundance of genes associated with key specialised functions (iron acquisition and metabolism, motility, cell signalling, stress response) at pesticide-treated sites. The results suggested impacts of long-term pesticide application on soil microbial community composition and potential functions. Despite, a CP legacy effect, no marked difference was observed in abundance of genes related to P-metabolism between pesticide-treated and non-treated sites. Overall, the results supported taxonomic and functional adaptations in the soil microbial communities following pesticide treatment. Overall, this study provides the novel insights into the interaction between xenobiotics and soil microbial communities both at structural (diversity, community structure) and functional (degradation) levels and should be considered in developing new bioremediation technologies and agronomic practices such as number and frequency of pesticide applications.

Atrazine is one of the most widely used herbicides in the world. It is primarily used in the production of corn in the United States. Although it may marginally increase crop yields, atrazine is also an endocrine disruptor in non-target organisms. Its moderate solubility in water allows for atrazine to contaminate surface and ground waters far removed from the point of application to soil. Although atrazine can be degraded abiotically, its primary mode of attenuation in natural environments is through bacterial degradation. Full mineralization of atrazine to CO2, H2O, Cl- and NH4+ has been demonstrated in Pseudomonas ADP, which contains the complete suite of atz atrazine catabolic genes.The overall hypothesis of this study is that the microorganisms and catabolic pathways reported in the literature do not universally account for the atrazine biodegradation observed in different natural environments. Furthermore, it is hypothesized that in situ pre-enrichment methods yield atrazine degraders uncultivable by classical laboratory enrichment, including anaerobic bacteria. The discovery of atrazine catabolic genes other than those in the atz pathway and the demonstrated involvement of consortia of bacteria in atrazine biodegradation suggest that the full diversity of environmental atrazine biodegradation has yet to be elucidated. In order to further elucidate the bacteria and genes responsible for atrazine biodegradation in different environments, locations with different exposures to atrazine were chosen for study. Among them were a farm location that was the site of multiple pesticide spill events throughout its history and a constructed wetland that receives river water containing agricultural runoff. Samples from these sites were assessed for atrazine biomineralization via biometer studies. In addition to traditional environmental sampling, these sites were also sampled with Bio-Sep beads to allow for in situ pre-enrichment for atrazine-degrading microbes. Soil and sediment samples were enriched for atrazine-degraders in liquid media, which included atrazine as the sole source of energy, C and N. Atrazine-degrading mixed cultures were screened via 16S-rDNA-PCR-DGGE, and mixed cultures displaying unique banding patterns were chosen for 16S rDNA clone library formation and sequencing. These isolates were also screened for eight atrazine catabolic genes via PCR.Atrazine mineralization was demonstrated in the sampling sites, but never exceeded 30% total mineralization of the added [U-ring-14C]-atrazine. Atrazine degrading pure and mixed cultures were successfully retrieved, with Acidovorax, Hydrogenophaga, Ralstonia, Variovorax, and Xanthobacter most prevalent among the mixed culture clone libraries and Arthrobacter spp. dominating the isolate collection.

In mammals, sulfation as catalyzed by the cytosolic sulfotransfereases (SULTs) is known to be involved in the metabolism and homeostasis of key endogenous compounds such as thyroid/steroid hormones, as well as in the detoxification of xenobioticsincluding drugs. The present study constitutes part of an overall effort in establishing the zebrafish as a model for studying drug sulfation. By searching GenBank database, we have identified sequences encoding three new zebrafish SULTs. These three novel zebrafish SULTs, designated SULT3 ST4, SULT3 ST5 and SULT1 ST9, were cloned, expressed, purified, and characterized. SULT3 ST4 showed strong activity toward endogenous compound such as dehydroepiandrosterine (DHEA), pregnenolone, and 17β-estradiol. SULT3 ST5 showed weaker, but significant, activities toward endogenous compounds such as DHEA and corticosterone, and xenobiotics including mestranol, β-naphthylamine, β-naphthol, and butylated hydroxyl anisole (BHA). SULT1 ST9, on the other hand, appeared to be mostly involved in the metabolism and detoxification of xenobiotics such as β-naphthol, β-naphthylamine, caffeic acid and gallic acid. pH-dependency and kinetic studies were performed using these three enzymes with DHEA, β-naphthol, β-naphthylamine, and 17β-estradiol as substrates. RT-PCR was carried out to investigate the expression of these three novel zebrafish SULTs during various developmental stages from embryogenesis to maturity.

The earthworms, Lubricus terrestris and Eisenia foetida, were used as non mammalian surrogate models to assess the immunotoxicpotential of xenobiotic to mammals. Assays were developed and optimized for detecting spreading activity and phagocytosis of rabbit red blood cell (RRBC), bacteria, and yeasts by macrophage-like coelomocytes of L. terrestris.

This research is part of an overall program to develop and use a suite of acute and sublethal toxicity biomarkers, and testing protocols for use in assaying potential effects of complex mixtures of xenobiotics such as found in soils containing agricultural biocides and petrochemical wastes dredged sediments, and hazardous waste sites (HWS). The purpose of this study was to evaluate four biomarkers of sublethal pathology that could be used in an integrative model of multiple toxicity endpoints with the earthworm Lumbricus terrestris.