The host microbiome (mainly bacteria) is essential for host immune function, metabolism, and digestion. Alterations in these microbes, known as dysbiosis, generally results in adverse effects to the host, including diseases. Dysbiosis can be induced from exposures to various anthropogenic contaminants including constituents of municipal wastewater treatment effluents (MWWEs), namely, pharmaceuticals, antibiotics, and excess nutrients. Despite MWWEs being one of the largest dischargers to aquatic ecosystems, impacts of these contaminants on exposed organism microbiomes, especially in aquatic insects, is unclear. In addition, some aquatic contaminants may transfer to riparian habitats through predation on emergent insects that were exposed to contaminants as larvae, and subsequently alter microbiomes of terrestrial predators. Our study evaluated whether MWWEs altered microbiomes of freshwater larval and adult insects and their riparian spider predators using effluent-associated bacteria and stable nitrogen isotopes (δ15N) to confirm effluent exposure. We analyzed microbiome compositions through sequencing of the V3-V4 hypervariable region of the 16S rRNA gene and analyzed food web dynamics with stable carbon (δ13C) and nitrogen (δ15N) isotopes. We found that insects and spiders were enriched in δ15N at one site downstream of wastewater outfalls, indicating exposure to effluents and transfer of nutrients to terrestrial ecosystems. Microbiomes of most larval and adult insects were altered downstream of wastewater outfalls and had lower relative abundances of endosymbiont bacteria, shifts in bacterial diversities, increases in abundances of effluent-associated bacteria, and downregulation of some biosynthesis pathways than those collected at upstream sites. However, spider microbiomes had little evidence of dysbiosis, and were distinct from those of adult insects, despite a close association in their isotopic signatures. Overall, this study provides evidence of biological impacts from MWWEs to exposed insects and suggests that changes in microbial communities of invertebrates may be used as an effective indicator of effluent exposure as part of monitoring frameworks. / Thesis / Master of Science (MSc) / Municipal wastewater treatment facilities release contaminants into aquatic ecosystems that may affect the health of exposed organisms, including their microbiome, which contains bacteria essential for host digestion and immune function. Alterations in microbiomes of contaminant-exposed aquatic insects are poorly characterized and such effects may be transferred to terrestrial ecosystems through emergent insects. This study evaluated microbiomes and food web dynamics of freshwater insects and riparian spiders upstream and downstream of wastewater treatment facilities in the Bow River, AB. Results indicate that microbiomes of some downstream aquatic insects had lower relative abundances of endosymbiont bacteria, shifts in bacterial diversities, and increases in abundances of effluent-associated bacteria than those collected at upstream sites, but no such changes were observed in the spider predators. This study improves our understanding of how freshwater insect microbiomes are altered by municipal wastewater effluents and suggests that directly effluent-exposed organisms are more at risk of dysbiosis.

The feasibility of using solid phase micro-extraction (SPME) as an in vivo sampling tool for analysis of trace environmental contaminants in fish exposed to municipal wastewater effluents (MWWEs) was validated using controlled laboratory and field experiments. SPME was compared with traditional extraction techniques， including solid phase extraction (SPE) in water and solid-liquid extraction (SLE) in fish tissues to assess relative efficiencies. All three techniques were used to quantify the presence of eight compounds of interest in fish exposed to MWWEs in the laboratory, as well as in wild and field caged fish upstream and downstream of three wastewater treatment plants in the Grand River watershed. Atrazine, carbamazepine, naproxen, diclofenac, gemfibrozil, bisphenol A, fluoxetine and ibuprofen were selected as target compounds due to their diverse chemical characteristics and frequent detection in surface waters and sediments around the world. The distribution coefficients between various sample matrices (water, fish) and extraction phases (SPME fibers) were compared, as were extraction profiles and bioconcentration factors of target analytes in muscle of fish exposed to MWWEs under laboratory conditions, during field caging studies, or collected (wild) from the Grand River. Poly(dimethylsiloxane) (PDMS) medical grade tubing was utilized as the SPME extraction phase, which when kinetically calibrated, were effective at extracting and quantifying the target analytes from both water and fish tissue relative to traditional techniques. Caged and in wild fish exposed to MWWEs from all three municipal treatment plants bio-accumulated detectable levels of several of the target chemicals. All target analytes (except for fluoxetine) were identified in the MWWEs and exposed fish by SPME at low concentrations (ng/L). The presence and concentration of the targeted analytes in both water and wild fish living in the Grand River watershed varied with season and proximity to the wastewater outfalls. Results demonstrate that properly applied SPME can detect and quantify selected contaminants in fish tissues, surface water, and wastewater effluents. In vivo SPME allows for non-lethal sampling of fish, which creates the opportunity for monitoring contaminant exposure in receiving environments influenced by MWWEs or non-point-source runoff while minimizing the impact on the organisms.

The feasibility of using solid phase micro-extraction (SPME) as an in vivo sampling tool for analysis of trace environmental contaminants in fish exposed to municipal wastewater effluents (MWWEs) was validated using controlled laboratory and field experiments. SPME was compared with traditional extraction techniques， including solid phase extraction (SPE) in water and solid-liquid extraction (SLE) in fish tissues to assess relative efficiencies. All three techniques were used to quantify the presence of eight compounds of interest in fish exposed to MWWEs in the laboratory, as well as in wild and field caged fish upstream and downstream of three wastewater treatment plants in the Grand River watershed. Atrazine, carbamazepine, naproxen, diclofenac, gemfibrozil, bisphenol A, fluoxetine and ibuprofen were selected as target compounds due to their diverse chemical characteristics and frequent detection in surface waters and sediments around the world. The distribution coefficients between various sample matrices (water, fish) and extraction phases (SPME fibers) were compared, as were extraction profiles and bioconcentration factors of target analytes in muscle of fish exposed to MWWEs under laboratory conditions, during field caging studies, or collected (wild) from the Grand River. Poly(dimethylsiloxane) (PDMS) medical grade tubing was utilized as the SPME extraction phase, which when kinetically calibrated, were effective at extracting and quantifying the target analytes from both water and fish tissue relative to traditional techniques. Caged and in wild fish exposed to MWWEs from all three municipal treatment plants bio-accumulated detectable levels of several of the target chemicals. All target analytes (except for fluoxetine) were identified in the MWWEs and exposed fish by SPME at low concentrations (ng/L). The presence and concentration of the targeted analytes in both water and wild fish living in the Grand River watershed varied with season and proximity to the wastewater outfalls. Results demonstrate that properly applied SPME can detect and quantify selected contaminants in fish tissues, surface water, and wastewater effluents. In vivo SPME allows for non-lethal sampling of fish, which creates the opportunity for monitoring contaminant exposure in receiving environments influenced by MWWEs or non-point-source runoff while minimizing the impact on the organisms.

The development of an analytical method for the determination of total organic-bound chlorine (TOCl) produced during the chlorination of municipal wastewater effluents is presented. Sewage effluent from the Denton, Texas municipal treatment plant was chlorinated at high chlorine doses (1000 - 4000 ppm), as well as typical treatment levels. Chlororganics present in the wastewater, before and after chlorination, were concentrated by adsorption on Amberlite XAD-2 macroreticular resin, followed by elution with diethyl ether. After concentration, the extracts were analyzed for TOC1 by microcoulometry. Analysis of wastewater extracts revealed the production of substantial amounts of new chlorinated organics when effluents were treated with chlorine. The method shows good precision and estimated accuracy is favorable.