Xenobiotic Organic Compounds (XOCs) are present in wastewater and wastewater-impacted environmental systems. Pharmaceuticals and personal care products are a broad and varied category of chemicals that are included among these compounds. Although, these compounds have been detected at low levels in surface water, concerns that these compounds may have an impact on human health and aquatic life, have led to increased interest in how XOCs are removed during wastewater treatment. Recognizing specific mechanisms in recent literature and simulating those mechanisms responsible for the removal of XOCs is the main objective of this study. Conventional models, such as the popular activated sludge models (ASM1, ASM2, etc), do not sufficiently address the removal processes; therefore, a fate model is created to provide a means of predicting and simulating removal mechanisms along with experimental analyses. GPS-X is a multi-purpose modeling tool for the simulation of municipal and industrial wastewater treatment plants. This software package includes conventional models as built-in libraries, which can be used as bases on which new models can be created. In this thesis, the removal mechanisms of XOCs are recognized and investigated; a new library for GPS-X is also created to include XOCs. As a first step the uncalibrated fate model, which includes all mechanisms of interest with their process rates and state variables, is developed using in GPS-X software. A modified ASM1 (Mantis model) is used as a basis for developing the fate model. Since only a group of mechanisms is responsible for the removal of each compound the mechanisms are categorized in three different case studies as the next step. Thus, one submodel is associated with each case study. The model developer toolbar in GPS-X software is used to develop the model for these case studies. The first case study involves the removal of antibiotics, such as Sulfamethoxazole. The removal mechanisms used in this case are biodegradation, sorption, and parent compound formation, with co-metabolism and competitive inhibition effects being inserted into the structure of the model. Secondly, the removal of nonylphenol ethoxylates (NPEOs) occurs through abiotic oxidative cleavage, hydrolysis, and biodegradation. The third case study includes removal mechanisms of biodegradation and sorption for neutral and ionized compounds. In the calibration process, model parameters are tuned such that the model can best simulate the experimental data using optimization methods. A common error criterion is Sum of Squared Errors (SSE) between the simulated results and the measured data. By minimizing SSE, optimal values of parameters of interest can be estimated. In each case study different data sets were used for the validation process. To validate the calibrated model, simulated results are compared against experimental data in each case study. The experimental data set used in the validation process is different from that used for calibrating the model, which means the validation process data set was obtained from the different literature. By looking at the validation results, it is concluded that the proposed model successfully simulates removal of XOCs. Since the operating parameters of wastewater treatment plants, such as Solids Retention Time (SRT) and Hydraulic Retention Time (HRT) are crucial for the fate of XOC???s, a sensitivity analysis is carried out to investigate the effect of those parameters. Moreover, the pH effect is studied because it relates to the ionized XOCs. Sensitivity analysis results show that the fate model is more sensitive to model parameters i.e. biodegradation rate constant (kb) than the operational parameters, i.e. SRT and HRT. Furthermore, the responses showed sensitivity to pH, whereby acidic conditions provide a better environment for removing neutral forms and alkaline conditions were suitable for removing ionized forms, according to the ionized compound fate model.

Based on a diverse consortia of research completed within the last 15 years, it has been found that Pharmaceutical Compounds (PCs) are present in detectable levels within a variety of environmental matrices, including tap water. This is largely attributed to anthropogenic activities as humans are the majority consumer of PCs. As a result, the primary method of disposal is via wastewater pathways resulting from human excretion of ingested PCs. Based on past research into PC fate via the wastewater treatment process, only limited biotic and abiotic transformations are achieved – most PC’s are detected in the effluents of WWTP’s. This suggests that improving the removal of PCs during the wastewater treatment process provides a promising strategy for limiting the conveyance of PCs to the environment. Historically, studies regarding PC fate in WWTPs have predominantly focused on the activated sludge process. However, fixed film (biofilm) wastewater treatment technologies continue to gain popularity at full scale wastewater treatment facilities. The limited studies which investigated fixed film wastewater treatment processes have reported that improved transformation efficiencies were observed relative to activated sludge systems. Based on these previous studies, it was postulated that the more diverse bacterial consortium present within the Integrated Fixed Film Activated Sludge (IFAS) process, a novel treatment process which has recently gained popularity in North America, may lead to improved transformation efficiencies (“removals”) of these very complex compounds. Only one previous study which investigated the transformation efficiencies of the IFAS process compared to a control was found. It was therefore considered that an additional investigation into the IFAS process warrants further investigation. Four IFAS Sequencing Batch Biofilm Reactors (SBBRs) and four control Sequencing Batch Reactors (SBRs) were operated with varied experimental conditions in a 22 factorial design to investigate whether an observable difference in the level of PC transformations would result via the IFAS process when compared to a control. Experimental conditions were characterized by varying the operating Solids Retention Time (SRT) and mixed liquor temperature. For all other operational parameters, best efforts were made to ensure both reactors were operated under equivalent conditions. This permitted a true assessment of the effects of the inclusion of IFAS media. Reactors were investigated through three phases of sampling, under which the performance of the reactors was investigated through the measurement of the following parameters: • Conventional parameters (tCOD, sCOD, TAN, NO3-N) within the initial and final samples; • Operational parameters (MLSS, MLVSS, ESS); and • The transformation efficiencies achieved for 5 PC (Carbamazepine, Sulfamethoxazole, Trimethoprim, Atenolol and Acetaminophen). During all three phases of PC sampling, the pilot reactors were found to have been performing as anticipated with respect to conventional contaminant removals. Organic removals were found to be statistically similar between the IFAS and control reactors across all four experimental conditions. Full nitrification was observed for all reactors with the exception of the control SBR operated under the low SRT, low temperature condition. The IFAS SBBRs were found to demonstrate improved nitrification kinetics when compared to their respective controls operated under the same experimental conditions. This was believed to be related to the more diverse bacterial consortia present as a result of the IFAS biofilms. All reactors were generally believed to be operating at steady state and were within an acceptable range of the target operating conditions. Due to complications associated with the analysis of samples, only CBZ, TRIM, ATEN and ACE could be successfully quantitated. CBZ was found to not have been transformed to any appreciable level across all conditions investigated through either the IFAS SBBRs or control SBRs. ACE was transformed at efficiencies greater than 99% under all conditions and in both IFAS and control reactors and therefore no comparison could be made. TRIM and ATEN demonstrated improved transformation efficiencies under all conditions within the IFAS reactors. The presence of IFAS media, SRT and temperature were all found to be statistically significant effects through ANOVA using a confidence limit of 95%.

Endocrine disrupting compounds (EDCs) in the environment have recently emerged as a major issue in Canada and around the globe. The primary objective of this thesis was to investigate the fate of EDCs in two wastewater treatment processes, membrane bioreactors (MBRs) and ultraviolet (UV) disinfection. Two submerged MBR systems using hollow fiber membranes from two membrane manufacturers were tested. The results from a bench-scale and a pilot scale MBR for the treatment of swine wastewater with high concentration of EDCs showed that over 94% of the estrogenic activity (EA) in the influent was reduced through the MBR process. Biological degradation was the dominant removal mechanism for the removal of EDCs in MBRs. Over 85% of the influent EA was reduced by biodegradation through the MBR process. The other MBR system was built to study the removal mechanisms of two estrogens in a hybrid MBR with the addition of powdered activated carbon (PAC). The effects of PAC dosing on MBR overall performance was studied as well. It was found that PAC dosing could increase the removal rates of 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) by 3.4% and 15.8%, respectively and result in a slower rate of trans-membrane pressure (TMP) increase during MBR operation, which could significantly reduce the operating cost for membrane cleaning and/or replacement. The operating cost for PAC dosing could be offset by the benefit achieved from reducing the cost for membrane maintenance. The slower rate of TMP increase in the PAC-MBR was associated with the lower concentrations of soluble extracellular polymeric substances and colloidal organic compounds in the PAC-MBR sludge. The degradation kinetics of three estrogens, estrone (E1), E2, and EE2 in de-ionized water by UV irradiation was studied. The experimental results showed both the apparent concentrations and overall EA of all three investigated estrogens in water decreased with direct UV irradiation. To further study the impact of UV on the overall EA of wastewater, the EA of pre-UV and post-UV samples from five wastewater treatment plants were measured in both liquid and solid phase by Yeast Estrogen Screen assay. It was found that the EA of wastewater decreased after UV disinfection in three of the investigated plants whereas it increased in the other two plants. This observation needs to be further studied because it might have significant impacts on the application of UV systems for wastewater disinfection.

New federal regulations with regards to ammonia in wastewater effluent discharge will require over 1000 existing wastewater treatment facilities to be upgraded. Although biological treatment is the most common and economical means of wastewater ammonia removal, nitrification rates can be completely impeded at cold temperatures. Moving bed biofilm reactors (MBBR) have shown promise as an upgrade nitrifying unit at pilot-scale and full-scale applications with respect to low temperature nitrification. MBBR technologies offfer the advantages of less space requirement, utilizing the whole tank volume, no sludge recycling, and no backwashing, over other attached growth systems. Two laboratory MBBRs were used in this study to investigate MBBR nitrification rates at 20deg.C, after long-term exposure to 1deg.C, and at the kinetic threshold temperature of 5deg.C. Furthermore, the biologically produced solids from the MBBR system 20deg.C and after long-term exposure to 1deg.C, and the Arrhenius temperature correction models used to predict nitrification rates after long-term exposure to 1deg.C. The nitrification rates at 1deg.C over a four month exposure period as compared to the rate at 20deg.C were 18.7 + 5.5% and 15.7 + 4.7% for the two reactors. The nitrification rate at 5deg.C was 66.2 + 3.9% and 64.4 + 3.7% compared to the rate measured at 20deg.C for reactors 1 and 2, respectively, and as such was identified as the kinetic temperature threshold. The quantity of solids detached from the nitrifying MBBR biocarriers was low and did not vary significantly at 20deg.C and after long-term exposure to 1deg.C. Lastly, a temperature correction model based on exposure time to cold temperatures, developed by Delatolla et al. (2009) showed a strong correlation to the calculated ammonia removal rates relative to 20deg.C following a gradual acclimatization period to cold temperatures. Biofilm morphology along with biomass viability at various depths in the biofilm were investigated using variable pressure electron scanning microscope imaging (VPSEM) and confocal laser scanning microscope (CLSM) imaging in combination with viability live/dead staining. The biofilm thickness along with the number of viable cells showed significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant increases after long-term exposure to 1deg.C while the dead cell coverage did not show significant changes. Hence, this study observed higher cell activities at warm temperatures and a slightly greater quantity of biomass with lower activities at cold temperatures in nitrifying MBBR biofilms. Using DNA sequencing analysis, 'Nitrosomonas' and 'Nitrosospira' (ammonia oxidizers)as well as 'Ntrospira' (nitrite oxidizer) were identified in which no population shift was observed during 20deg.C and after long-term exposure to 1deg.C. Furthermore, a number of non-nitrifiers were identified int he biofilm during warm and cold temperatures presenting the possibility that their presence may have provided some form of protection to the nitrifiers during long-term temperature exposure.

<p>The study and application of materials possessing size dimensions in the nano scale range and, as a result, unique properties have led to the birth of a new field; nanotechnology. Scientists and engineers have discovered and are exploiting the novel physicochemical characteristics of nanoparticles (NPs) to enhance consumer products and technologies in ways superior to their bulk counterparts. Escalating production and use of NPs will unavoidably lead to release and exposure to environmental systems. This introduction of emerging potential contaminant NPs will provide new and interesting challenges for exposure and risk forecasting as well as environmental endurance.</p><p>The ultimate goal of this research is to develop a framework that incorporates experimental and computational efforts to assess and better understand the exposure of metal and metal-oxide NPs released to wastewater treatment plants (WWTPs) and further implications on land application units (LAUs) where biosolids can be applied. The foundation of the computational effort is comprised of Monte Carlo mass balance models that account for the unique processes affecting NP fate and transport through the different technical compartments of a WWTP and LAU. Functional assay and bioreactor experiments in environmental media were used to determine parameters capable of describing the critical processes that impact the fate of NPs in wastewater. </p><p>The results of this research indicate that a simplified, but still environmentally relevant nano-specific exposure assessment is possible through experimentation to parameterize adapted models. Black box modeling efforts, which have been shown in previous studies, show no disadvantage relative to discretization of technical compartments as long as all key transport and fate mechanisms are considered. The distribution coefficient (_), an experimentally determined, time-dependent parameter, can be used to predict the distribution of NPs between the liquid and solid phase in WWTPs. In addition, this parameter can be utilized a step further for the estimation of the more fundamental, time independent attachment efficiency between the NPs and the solids in wastewater. The NP core, size, and surface coating will influence the value of these parameters in addition to the background particle characteristics as the parameters are specific to the environmental system of study. For the metal and metal-oxide NPs studied, preferential overall association of approximately 90% or greater with the solid phase of wastewater was observed and predicted. </p><p>Furthermore, NP transformations including dissolution, redox reactions, and adsorption can potentially impact exposure. For example, experimental results showed that nano-CeO2 is reduced from Ce(IV) to Ce(III) when in contact with wastewater bacteria where Ce2S3 will likely govern the Ce(III) phase in biosolids. From the literature, similar transformations have been observed with Ag and ZnO NPs to Ag2S and ZnS. With respect to TiO2 NPs, studies indicated that due to high insolubility, these NPs would not undergo transformation in WWTPs. The distribution and transformation rate coefficients can then be used in fate models to predict the NP species exposed to aquatic and terrestrial systems and environmentally relevant concentrations released from WWTPs. </p><p>Upon completion of the WWTP model, the predicted concentrations of NPs and NP transformation byproducts released in effluent and biosolids were attainable. A simple mass balance model for NP fate in LAUs was then developed to use this output. Results indicate that NP loading on LAUs would be very low but that build up over time to steady state could result in mass concentrations on the order of the typical level for the background metal in soil. Transport processes of plant uptake and leaching were expected to greatly impact the solid phase concentration of the NPs remaining in the LAU, while rainfall did not impart a significant influence upon variation between low and high annual amounts. The significance of this research is the introduction of a method for NP exposure assessment in WWTPs and subsequently in LAUs. This work describes and quantifies the key processes that will impact Ag, TiO2, CeO2 and ZnO NP fate and transport, which can inform future studies, the modeling community and regulatory agencies.</p> / Dissertation

Two characterization studies were undertaken to assess the concentrations and environmental loadings of 125 pharmaceuticals, personal care products, illicit drugs and their metabolites (PPCPs) in screened municipal wastewaters being discharged into Juan de Fuca Strait from two marine outfalls in the Capital Regional District, British Columbia, Canada. Two up-stream pump stations were also sampled. The PPCP concentration profiles were generally similar between the four sampling locations. Qualitative seasonal patterns in PPCP concentrations were also observed, primarily due to rainfall events that diluted wastewater contaminants during the winter. Increases in wastewater flow volumes following a rain event appeared to result in consistent shifts in PPCP concentration profiles for at least three of the four sites. Results indicated that the concentrations of PPCPs were similar to those observed in influents from other jurisdictions. Predicted environmental concentrations were predominantly well below literature concentration thresholds known to induce acute or chronic effects in organisms in the environment. However, there was slight potential for adverse chronic effects as a result of the predicted environmental concentrations of ibuprofen around the outfalls based on comparison to literature environmental effects thresholds. In general, sub-lethal and chronic effects endpoints were relatively limited in availability in the literature, as were literature thresholds derived from exposures to PPCP mixtures. Additional adverse chronic effects of these substances may be discovered in the future. Comparisons were made to regional prescription rates and population demographics to determine whether these factors could be good predictors of PPCP concentrations or loadings. Although wastewater concentrations and loadings were proportional to both prescription rates and population size, the regression relationships were statistically weak or insignificant. As such, prescription rates and population size could not be used to accurately predict pharmaceutical wastewater concentrations and loadings on their own. No qualitative relationships were observed between wastewater PPCP concentrations and either population age or gender breakdown. Overall, wastewater flow volumes, derived population equivalents and analytical method variability were also important factors to consider. Minor proportional deviations were observed following a preliminary loading comparison based on the relative population equivalent sizes of each of the four wastewater system catchment areas. These deviations could have been a result of disproportional hospital loading inputs and/or wastewater system inflow and infiltration. Comparisons were also made between the concentrations of PPCPs and the concentrations of conventional wastewater parameters typically used to characterize bulk wastewater loadings (i.e., carbonaceous biochemical oxygen demand, biological oxygen demand, total suspended solids, volatile suspended solids). Only 18 of the 125 PPCPs were positively correlated with all four conventional parameters. This suggests that designing and optimizing treatment plants to efficiently reduce conventional parameter loadings may not lead to as efficient or consistent reductions in the concentrations of all of the assessed PPCPs. However, the PPCP results were based on analyses of the filtered aqueous fraction of the wastewater samples, whereas the conventional parameter results were based on whole unfiltered effluent samples. As such, there was no direct link between the two sets of results. / Graduate / 0306

Current municipal wastewater treatment plants (MWWTP) technologies are insufficiently removing emerging contaminants of concern. These emerging contaminants are an issue as many are known endocrine disrupting compounds (EDCs). EDCs are contaminants that can have severe and irreversible impacts on highly conserved endocrine systems that are critical during developmental periods in vertebrates as well as during adult life. Many EDCs have non-monotonic dose-response curves yet they are not often tested at low, environmentally relevant concentrations. EDC research to date has focused heavily on xenoestrogenic compounds whereas thyroid hormone (TH) disruption has been largely overlooked. TH is conserved in all vertebrates and plays crucial roles in neural development, basal metabolism, and thermoregulation. TH is comprised of thyroxine (T4), often known as the transport form of TH, and triiodothyronine (T3), the more bioactive form of TH. A TH spike occurs in the perinatal period of humans, and when disrupted, this spike can cause severe developmental defects. An analogous, but perhaps more overt, TH spike occurs in amphibians. TH is the sole hormone that drives amphibian metamorphosis, thus providing an excellent model for TH action. Our lab has previously developed the cultured tailfin (C-fin) assay, which uses biopsies from premetamorphic Rana catesbeiana tadpole tailfins cultured in the presence of an exogenous chemical of concern to assess perturbations to TH- and stress-responsive gene transcript levels by QPCR. This thesis uses the C-fin assay to assess the efficacy of removal of biological TH- and stress-altering activity in conventional municipal wastewater treatment systems. We first assess the successive levels of a full-scale conventional activated sludge (CAS) MWWTP in its ability to reduce perturbations of mRNA transcript levels of the critical TH receptors alpha (thra) and beta (thrb), and stress responsive gene transcripts superoxide dismutase (sod), catalase (cat) and heat shock protein 30 (hsp30). Secondary treatment of wastewater effluents removes cellular stress perturbations when compared to influents, but thr disruptions remain after conventional secondary wastewater treatment. We then assess three pilot-sized conventional secondary MWWTP configurations run at two operational conditions. The C-fin assay results suggest that the current understanding of operational conditions and the efficiency of complex MWWTP configurations is not clear-cut when assessed by biological endpoints such as the transcript abundance perturbations in the C-fin assay. Finally, the C-fin assay is used to investigate transcript profiles of genes of interest when the tissues are treated with the endogenous hormones T3, T4, and estradiol (E2). Our results indicate that T4 acts as more than solely a T3-prohormone and that gene expression levels in response to the two different forms of TH can be T3 or T4 specific. E2 effects, although implicated in altering TH-mediated responses in other contexts, do not affect TH-responsive gene transcripts in the C-fin. The data presented use the novel C-fin assay to challenge and advance the currently accepted views of TH-action, as well as develop necessary yet practical biological knowledge for management of emerging contaminant release from MWWTPs. / Graduate / 0383 / 0768 / 0307 / polaw@uvic.ca

Municipal wastewater effluent (MWWE) is an important source of pollution in the aquatic environment impacting fish. MWWE is a complex mixture of chemicals including pharmaceuticals, personal care products, industrial chemicals and pesticides. A link between reproductive endocrine disruption and MWWE exposure has been established in fish, but less is known about the effects of MWWE on non-reproductive endocrine disruption. The overall objective of this thesis was to examine the impacts of MWWE exposure on the stress response and intermediary metabolism in rainbow trout (Oncorhynchus mykiss). In fish, the primary adaptive organismal stress response involves the activation of hypothalamic-sympathetic-chromaffin axis to produce catecholamines, predominantly epinephrine, and the hypothalamic-pituitary-interrenal (HPI) axis to produce cortisol. Both of these hormones play a key role in elevating plasma glucose levels that is essential to fuel the increased energy demand associated with stress. Along with the organismal stress response, the cellular stress response, involving the synthesis of a suite of heat shock proteins (hsps), also plays an important role in protecting cellular protein homeostasis in response to stressors, including toxicants. The impact of MWWE on stress-related pathways were identified using a low-density trout cDNA microarray enriched with genes encoding for proteins involved in endocrine-, stress- and metabolism-related processes. This was further confirmed by assessing plasma hormone and metabolite levels and stress-related targeted genes and proteins expression and enzyme activities in select tissues in rainbow trout. Studies were carried out in controlled field (caging) and laboratory experiments to examine the impacts of MWWE on stress and tissue-specific metabolic responses in rainbow trout. Further in vitro studies using rainbow trout hepatocytes in primary cultures were carried out to investigate the mechanism of action of two pharmaceuticals, atenolol and venlafaxine, found in relatively high concentrations in MWWE in impacting the stress-mediated glucose response. In caged fish, MWWE exposure significantly elevated plasma cortisol and glucose concentrations, and altered the mRNA abundance of a number of stress-related genes, hormone receptors, glucose transporter 2 and genes related to immune function. When fish were exposed to an acute handling stress following a 14 d exposure to MWWE, the cortisol response was abolished and the glucose response was attenuated. The effects on cortisol did not correlate with changes in the expression of genes involved in cortisol biosynthesis, but were associated with an increase in hepatic glucocorticoid receptor (GR) protein expression. Upon further investigation in controlled laboratory studies, MWWE exposure elevated constitutive hsp 70 and hsp90 expression after 8 d exposure, which correlated with a decrease in glycogen levels in the liver in fish exposed to a high concentration of MWWE compared to control fish, pointing to a MWWE-induced increase in liver energy demand. By 14 d, glycogen stores were replenished, and this was commensurate with increases in liver gluconeogenic capacity, including increases in the activities of phosphoenolpyruvate carboxykinase (PEPCK) and alanine aminotransferase (AlaAT), along with a decrease in liver GR expression. In the heart, GR protein expression increased in treated fish, and the activity of pyruvate kinase increased, indicating an increase in glycolytic capacity. Subjecting the MWWE exposed fish to a secondary handling disturbance (acute stress) led to an attenuated plasma cortisol and glucose response compared to the control group. This corresponded with a reduced liver gluconeogenic capacity and a lower liver and heart glycolytic capacities, reflecting a disturbance in the energy substrate repartitioning that is essential to cope with stress. While it is difficult to establish causative agents from a complex mixture such as MWWE, the two pharmaceutical that were tested impacted glucose production. Specifically, atenolol and venlafaxine disrupted the epinephrine-induced glucose production, but did not modify cortisol-mediated glucose production in trout hepatocytes. The suppression of epinephrine-mediated glucose production by atenolol and venlafaxine was abolished by cAMP analogue (8-bromo cAMP) or glucagon (a metabolic hormone that increases glucose production). This suggests that both drugs disrupt β-adrenoceptor signaling, while it remains to be determined if the response is receptor isoform-specific. Altogether MWWE exposure disrupts the organismal and cellular stress responses in trout. Key targets for MWWE impact leading to the impaired cortisol and metabolic responses to stress include liver and heart GR expression, liver gluconeogenic capacity, and liver, heart and gill glycolytic capacities. Most significantly, MWWE impairs the ability to metabolically adjust to a secondary acute stressor, which is an important adaptive process that is integral to successful stress performance. From an environmental stand-point, long-term exposure to MWWE will lead to reduced fitness and will compromise the capacity of fish to cope with additional stressor, including escape from predators.

Fast paced population growth in urban areas of southern Ontario is putting increased pressure on the surrounding aquatic environment. The City of Guelph uses the Speed River to assimilate its municipal wastewater effluent. With a projected 57% population increase in the watershed by 2031, the assimilative capacity of the river may be challenged in the coming years. The Guelph Wastewater Treatment Plant uses tertiary treatment methods greatly reducing ammonia, suspended solids and phosphate concentrations in the effluent. However there are still impacts detectable related to excessive nutrients released into this relatively small river (6th order) which promotes algae and aquatic macrophyte growth. There is also concern about a variety of emerging contaminants that may enter the river and impact the health of the ecosystem. The research in this thesis examined the seasonal and spatial variability and extent of the impacts of the wastewater effluent on the riffle fish communities in the Speed River. Stable isotope signatures (δ13C and δ15N) were used to understand the changes in the dominant benthic fish species, Rainbow Darters (Etheostoma caeruleum) and Greenside Darters (E. blennioides), relative to changes in invertebrate signatures and their abundance. Rainbow Darters were extremely abundant relative to Greenside Darters at the site immediately downstream of the effluent outfall, particularly in August. The benthic invertebrate community was distinctly different downstream of the effluent outfall, especially in the summer, with a reduced abundance of Elmidae beetle larvae and increased abundance of isopods (Caecidotea intermedius) compared to upstream. δ13C and δ15N of the two darters species were similar at all sites in May and July, but in August and October Rainbow Darter signatures were more enriched in the two heavier isotopes at sites downstream of the effluent outfall. The vast majority of invertebrate taxa sampled were also enriched at the downstream sites. An analysis of Rainbow and Greenside Darter stomach contents revealed that Rainbow Darters incorporated more isopods and other invertebrates in their diet, especially at the immediate downstream sites suggesting that they are more adaptable to the altered downstream environment. The feeding habits of Greenside Darters appear to change between July and August in response to changes in habitat and food availability. They are potentially consuming food organisms with less enriched isotopic signatures, which results in their isotopic signatures not rising during these months like most of the invertebrates and other fish. Alternatively, the Greenside Darters may move across the stream to feed on invertebrates that remain unexposed to the wastewater effluent. These impacts, although subtle, may be a reflection of the Speed River ecosystem being compromised by nutrient inputs from the wastewater effluent. With the impending increase in demand on the treatment plant (e.g., population growth), ongoing treatment and infrastructure improvements may be needed in the future to maintain the current ecosystem structure.

The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.