Aquatic receiving environments have long been used to dilute municipal wastewater effluents (MWWE) which are the largest discharge by volume into the aquatic environment in Canada. These treated effluents are a complex mixture of environmental contaminants that includes natural and synthetic hormones, pharmaceuticals, industrial chemicals, nutrients and ammonia. Discharge of MWWE may lead to serious problems in aquatic environments such as eutrophication, hypoxia as well as increased occurrence of disease and toxicity in resident aquatic biota. Reproductive impairment in fish has also been widely reported in association with exposure to wastewaters. Recently, concerns have been raised about the potential for municipal effluents to cause endocrine disruption in fish and other organisms. The effects of MWWE on fish and fish populations in Canada are currently poorly understood. The overall objective of this thesis is to contrast the impact of MWWE discharged into two Canadian rivers on sentinel fish species across levels of biological organization ranging from biochemical responses to changes at the fish community level. Results from these studies support the development of robust effects-based biological monitoring approaches to assess the effectiveness of regulations and remedial actions for minimizing the effects of MWWE. Understanding the temporal changes in physiological and reproductive parameters across the annual cycle of a sentinel species is necessary to optimize biomonitoring programs. The annual variability in terms of survival, reproduction and energy storage in the Greenside Darter (Etheostoma blennioides), a potential sentinel species for the Grand River, Ontario, was documented at a reference site across two years. Variation in energy storage and reproductive development indicated by somatic indices (i.e., relative organ size) and steroid production suggest that biomonitoring can be optimized for this species by sampling in late fall or early spring (pre-spawning). With this new knowledge, field studies conducted with small bodied species, including Rainbow Darter (E. caeruleum), Brook Stickleback (Culaea inconstans) and Fathead Minnow (Pimephales promelas) have demonstrated that, when sampled during the appropriate season (e.g., pre-spawning), they can be used as effective biomonitoring tools to detect fish responses associated with exposure to MWWEs. Two sentinel fish species, Rainbow Darter and Greenside Darter, were then used to examine the impact of two MWWE discharges on fish in the Grand River, Ontario, relative to reference sites in two seasons (fall and early spring). Fish responses, in terms of energy storage (condition factor, liver size), energy utilization (gonadosomatic indices) and reproduction (in vitro sex steroid production, cellular development and intersex) were assessed at each site. Both sentinel species were longer and heavier downstream of the wastewater outfalls. However, these larger fish did not demonstrate consistent increases in condition and liver somatic indices. MWWE-exposed male Rainbow and Greenside Darters had impaired capacity to produce androgens in vitro, lower gonadosomatic indices and altered sperm cell staging. Exposed female fish also had impaired capacity to produce estrogens in vitro, however, they did not demonstrate differences in oocyte development. Male Rainbow and Greenside Darters collected downstream of both MWWE discharges showed increased incidence of intersex (33 - 100%) in contrast to very low occurrences of this condition in upstream agricultural and urban reference sites. This increased incidence of intersex coincided with reductions in gonadosomatic indices and capacity to produce steroids, demonstrating the ability of MWWE to alter the reproductive systems of these fish. The fish communities downstream of the the MWWE outfalls demonstrated differences in abundance, diversity, and species composition when compared to reference sites. MWWE exposed sites had few of the darter species that dominate the fish community at reference sites. More mobile fish species such as suckers (Catostomidae spp.) and sunfish (Centrarchidae spp.) were more common downstream of the outfalls, with occurances becoming more pronounced downstream of the second sewage discharge. Wascana Creek, Saskatchewan, downstream of the wastewater treatment plant for the City of Regina can be up to 100% treated municipal wastewater. Brook Stickleback and Fathead Minnow exhibited delayed spawning and altered gonadal development downstream of the wastewater outfall. Exposed male Fathead Minnows were feminized, having lower expression of secondary sexual characteristics (i.e., loss of nuptial tubercles, dorsal pad, and dorsal fin dot) and induction of the female egg-yolk precursor protein, vitellogenin. Fathead Minnows also showed cellular damage to the gills and kidneys. These responses indicate exposure to a variety of environmental contaminants in the effluent such as ammonia as well as endocrine disruptors. The potential effect of MWWE discharges in these two Canadian watersheds on fish responses was demonstrated across various levels of biological organization including reduced sex steroid production, altered gonadal development, reduction in gonadosomatic indices, delayed spawning, and changes in fish assemblages. An effects-based monitoring approach using sentinel species can be successfully applied to detect changes associated with MWWE outfalls, as long as sampling of sentinel species is conducted during optimal time periods (i.e., when somatic indices are maximized and variability among individuals is minimized). MWWE can impair the reproductive potential of fish beyond a threshold where impacts are expressed at higher levels of organization such as populations or communities. It is essential to make mechanistic linkages between responses at different levels to determine the overall potential impact of effluents on fish. The collection of responses across multiple levels of biological organization can complement and support development of biomonitoring approaches that are focused at the population and community levels such as those being proposed for MWWE in Canada.

Aquatic receiving environments have long been used to dilute municipal wastewater effluents (MWWE) which are the largest discharge by volume into the aquatic environment in Canada. These treated effluents are a complex mixture of environmental contaminants that includes natural and synthetic hormones, pharmaceuticals, industrial chemicals, nutrients and ammonia. Discharge of MWWE may lead to serious problems in aquatic environments such as eutrophication, hypoxia as well as increased occurrence of disease and toxicity in resident aquatic biota. Reproductive impairment in fish has also been widely reported in association with exposure to wastewaters. Recently, concerns have been raised about the potential for municipal effluents to cause endocrine disruption in fish and other organisms. The effects of MWWE on fish and fish populations in Canada are currently poorly understood. The overall objective of this thesis is to contrast the impact of MWWE discharged into two Canadian rivers on sentinel fish species across levels of biological organization ranging from biochemical responses to changes at the fish community level. Results from these studies support the development of robust effects-based biological monitoring approaches to assess the effectiveness of regulations and remedial actions for minimizing the effects of MWWE. Understanding the temporal changes in physiological and reproductive parameters across the annual cycle of a sentinel species is necessary to optimize biomonitoring programs. The annual variability in terms of survival, reproduction and energy storage in the Greenside Darter (Etheostoma blennioides), a potential sentinel species for the Grand River, Ontario, was documented at a reference site across two years. Variation in energy storage and reproductive development indicated by somatic indices (i.e., relative organ size) and steroid production suggest that biomonitoring can be optimized for this species by sampling in late fall or early spring (pre-spawning). With this new knowledge, field studies conducted with small bodied species, including Rainbow Darter (E. caeruleum), Brook Stickleback (Culaea inconstans) and Fathead Minnow (Pimephales promelas) have demonstrated that, when sampled during the appropriate season (e.g., pre-spawning), they can be used as effective biomonitoring tools to detect fish responses associated with exposure to MWWEs. Two sentinel fish species, Rainbow Darter and Greenside Darter, were then used to examine the impact of two MWWE discharges on fish in the Grand River, Ontario, relative to reference sites in two seasons (fall and early spring). Fish responses, in terms of energy storage (condition factor, liver size), energy utilization (gonadosomatic indices) and reproduction (in vitro sex steroid production, cellular development and intersex) were assessed at each site. Both sentinel species were longer and heavier downstream of the wastewater outfalls. However, these larger fish did not demonstrate consistent increases in condition and liver somatic indices. MWWE-exposed male Rainbow and Greenside Darters had impaired capacity to produce androgens in vitro, lower gonadosomatic indices and altered sperm cell staging. Exposed female fish also had impaired capacity to produce estrogens in vitro, however, they did not demonstrate differences in oocyte development. Male Rainbow and Greenside Darters collected downstream of both MWWE discharges showed increased incidence of intersex (33 - 100%) in contrast to very low occurrences of this condition in upstream agricultural and urban reference sites. This increased incidence of intersex coincided with reductions in gonadosomatic indices and capacity to produce steroids, demonstrating the ability of MWWE to alter the reproductive systems of these fish. The fish communities downstream of the the MWWE outfalls demonstrated differences in abundance, diversity, and species composition when compared to reference sites. MWWE exposed sites had few of the darter species that dominate the fish community at reference sites. More mobile fish species such as suckers (Catostomidae spp.) and sunfish (Centrarchidae spp.) were more common downstream of the outfalls, with occurances becoming more pronounced downstream of the second sewage discharge. Wascana Creek, Saskatchewan, downstream of the wastewater treatment plant for the City of Regina can be up to 100% treated municipal wastewater. Brook Stickleback and Fathead Minnow exhibited delayed spawning and altered gonadal development downstream of the wastewater outfall. Exposed male Fathead Minnows were feminized, having lower expression of secondary sexual characteristics (i.e., loss of nuptial tubercles, dorsal pad, and dorsal fin dot) and induction of the female egg-yolk precursor protein, vitellogenin. Fathead Minnows also showed cellular damage to the gills and kidneys. These responses indicate exposure to a variety of environmental contaminants in the effluent such as ammonia as well as endocrine disruptors. The potential effect of MWWE discharges in these two Canadian watersheds on fish responses was demonstrated across various levels of biological organization including reduced sex steroid production, altered gonadal development, reduction in gonadosomatic indices, delayed spawning, and changes in fish assemblages. An effects-based monitoring approach using sentinel species can be successfully applied to detect changes associated with MWWE outfalls, as long as sampling of sentinel species is conducted during optimal time periods (i.e., when somatic indices are maximized and variability among individuals is minimized). MWWE can impair the reproductive potential of fish beyond a threshold where impacts are expressed at higher levels of organization such as populations or communities. It is essential to make mechanistic linkages between responses at different levels to determine the overall potential impact of effluents on fish. The collection of responses across multiple levels of biological organization can complement and support development of biomonitoring approaches that are focused at the population and community levels such as those being proposed for MWWE in Canada.

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.

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.

Municipal wastewater effluent (MWWE) has the potential for aquatic degradation, as it is the largest, per volume, anthropogenic discharge in Canada and other areas in the world. With an increasing population in many areas, such as Southern Ontario, there is concern that infrastructure of wastewater treatment facilities will not be able to maintain adequate treatment and prevent further degradation of the environment. The Grand River watershed, in Southern Ontario, is predicted to have its population increase to 1.2 million people by 2031 (from 780,000 people in 2001). Although wastewater treatment has improved, concern remains for receiving environments due to inadequate treatment (i.e. Kitchener) and minimal dilution (i.e. Guelph). This research was conducted to understand current impacts of MWWE in the Grand River watershed on fish communities to support future management and protection. Study sites upstream and downstream were chosen for their proximity to the Guelph, Kitchener, and Waterloo MWWE outfalls, similarity in habitat, and wadeability. Habitat analysis indicated that there were no large physical differences among sites. Fish communities were collected in a standardized method with a backpack electroshocker at each site (six randomly selected 10 m by 10 m sub-sites for 5 min). Greenside Darter (Etheostoma blennioides) and Rainbow Darter (E. caeruleum), the most abundant species, were also analyzed for stable isotope signatures (δ13C and δ15N) at each site. Downstream of the Guelph outfall there were no changes in mean total catch per unit effort (CPUE) or mean total mass. Changes to diversity, resilience, and tolerance in the fish community were attributed to a decreased abundance of Greenside Darter and increased abundance of Rainbow Darter. Downstream of the Kitchener discharge, there was a trend towards decreasing mean total CPUE, especially for darter species, and an increase in mean total mass due to a community shift to larger species including Catostomids and Centrarchids. The changes in abundance of Rainbow Darter, Catostomids, and Centrarchids among reference and Kitchener MWWE exposed sites explained the pattern in resilience, tolerance, and diet classifications. Lower diversity downstream of all three MWWE outfalls can be attributed to the increase in Rainbow Darter abundance. Stable isotope signatures (δ13C and δ15N) of Greenside Darter did not change downstream of the Guelph and Waterloo discharges, but signatures of Rainbow Darter increased immediately below the two outfalls. This shift may be due to the Rainbow Darter being able to take advantage of a change in the environment (i.e. food availability), resulting in its increased abundance and changes in isotopic signature. Directly downstream of the Kitchener outfall both darter species had an increase in δ13C and a large decrease in δ15N, likely due to high nutrient inputs from the outfall. The Kitchener wastewater discharge is also associated with a decrease in abundance of fish and a shift in community structure. MWWEs are currently affecting the aquatic environment, including fish communities in the Grand River watershed. Future investments in infrastructure and watershed management should be made to mitigate degradation of water quality in this watershed.

Municipal wastewater effluent (MWWE) has the potential for aquatic degradation, as it is the largest, per volume, anthropogenic discharge in Canada and other areas in the world. With an increasing population in many areas, such as Southern Ontario, there is concern that infrastructure of wastewater treatment facilities will not be able to maintain adequate treatment and prevent further degradation of the environment. The Grand River watershed, in Southern Ontario, is predicted to have its population increase to 1.2 million people by 2031 (from 780,000 people in 2001). Although wastewater treatment has improved, concern remains for receiving environments due to inadequate treatment (i.e. Kitchener) and minimal dilution (i.e. Guelph). This research was conducted to understand current impacts of MWWE in the Grand River watershed on fish communities to support future management and protection. Study sites upstream and downstream were chosen for their proximity to the Guelph, Kitchener, and Waterloo MWWE outfalls, similarity in habitat, and wadeability. Habitat analysis indicated that there were no large physical differences among sites. Fish communities were collected in a standardized method with a backpack electroshocker at each site (six randomly selected 10 m by 10 m sub-sites for 5 min). Greenside Darter (Etheostoma blennioides) and Rainbow Darter (E. caeruleum), the most abundant species, were also analyzed for stable isotope signatures (δ13C and δ15N) at each site. Downstream of the Guelph outfall there were no changes in mean total catch per unit effort (CPUE) or mean total mass. Changes to diversity, resilience, and tolerance in the fish community were attributed to a decreased abundance of Greenside Darter and increased abundance of Rainbow Darter. Downstream of the Kitchener discharge, there was a trend towards decreasing mean total CPUE, especially for darter species, and an increase in mean total mass due to a community shift to larger species including Catostomids and Centrarchids. The changes in abundance of Rainbow Darter, Catostomids, and Centrarchids among reference and Kitchener MWWE exposed sites explained the pattern in resilience, tolerance, and diet classifications. Lower diversity downstream of all three MWWE outfalls can be attributed to the increase in Rainbow Darter abundance. Stable isotope signatures (δ13C and δ15N) of Greenside Darter did not change downstream of the Guelph and Waterloo discharges, but signatures of Rainbow Darter increased immediately below the two outfalls. This shift may be due to the Rainbow Darter being able to take advantage of a change in the environment (i.e. food availability), resulting in its increased abundance and changes in isotopic signature. Directly downstream of the Kitchener outfall both darter species had an increase in δ13C and a large decrease in δ15N, likely due to high nutrient inputs from the outfall. The Kitchener wastewater discharge is also associated with a decrease in abundance of fish and a shift in community structure. MWWEs are currently affecting the aquatic environment, including fish communities in the Grand River watershed. Future investments in infrastructure and watershed management should be made to mitigate degradation of water quality in this watershed.

Microplastics are present in municipal wastewater treatment plant (WWTP) effluents; however, it is unclear whether these contaminants are ingested by biota living downstream of these outfalls. This study examined whether microplastic levels in caged biota, resident fish, and environmental samples were elevated near the Waterloo and Kitchener WWTP outfalls along the Grand River in the fall of 2019. Amphipods (Hyalella azteca), fluted-shell mussels (Lasmigona costata), and rainbow trout (Oncorhynchus mykiss) were caged at one upstream reference site and two impacted sites downstream of the Kitchener WWTP for 14 (amphipods and trout) or 28 (mussels) days. Rainbow darter (Etheostoma caeruleum) were collected using a backpack electrofisher from 10 sites up and downstream of both the Kitchener and Waterloo WWTPs, along with surface water and sediment samples. Whole body Hyalella, fish digestive tracts, and fluted-shell mussel tissues (hemolymph, digestive glands, and gills) were digested in 20% potassium hydroxide. Environmental samples were processed using filtration and density separation, then visual identification of microplastics was done. Elevated particle counts were found in rainbow trout digestive tracts at the Kitchener outfall site, compared to the upstream reference and downstream farfield sites. Additionally, particle concentrations in sediment were significantly higher at the Waterloo outfall, compared to all other sites (except for one upstream location). However, whole Hyalella, fluted-shell mussel tissues (hemolymph, digestive glands, and gills), digestive tracts of rainbow darter, and surface waters did not show elevated counts downstream of these discharges. Across all samples, fibers were the most common morphology, and blue and clear particles were prevalent in samples collected near WWTPs. Overall, these findings suggest that the Kitchener and Waterloo WWTPs could be important sources of particles to the Grand River, adding to our understanding of the fate of this contaminant in freshwater ecosystems. / Thesis / Master of Science (MSc)

MSc Thesis - The effect of wastewater effluent on the gut microbiome of rainbow darter / The microbiome plays an important role in host physiology and can be influenced by species, diet, and environment. Municipal wastewater effluent contains a mixture of chemicals including antibiotics and antimicrobials that may affect the gut microbiome of fish living downstream of these discharges. Thus, this study examines the effect of wastewater treatment plant (WWTP) effluent on the gut microbiome of wild rainbow darter (Etheostoma cearuleum), and examines how the gut microbiome of wild fish changes in the lab. Fish were collected from sites upstream and downstream of 2 major WWTPs along the central Grand River and gut contents were aseptically sampled. After extracting gDNA, nested PCR of the V3-V4 region of the 16S rRNA gene, and Illumina sequencing were performed. The gut microbiome of exposed fish had increased bacterial diversity and was dominated by Proteobacteria, which has been linked to altered health outcomes in mammals. Next, rainbow darters were collected from a reference site on the Grand River. Fish were sampled in the field, after a 14 day lab acclimation, and after a 28 day exposure to environmental stressors (WWTP effluent or triclosan, an antimicrobial found in WWTP effluent). Surprisingly, there were no changes in the microbiome after exposure to environmental stressors. Major changes were observed between the field and laboratory fish suggesting that environment and diet are important factors influencing the gut microbiome. Changes in the gut microbiome continued up to 42 days in the lab, indicating longer acclimation periods may be needed. This study showed that effluents altered the gut microbiome of fish in the field, but not in the laboratory for unknown reasons. Laboratory studies indicated that transitioning to a new environment may require greater than 14 days before achieving a stable microbiome. / Thesis / Master of Science (MSc) / Wastewater is the largest source of pollution affecting Canada’s aquatic ecosystems; effluents contain antibiotics and antimicrobials that can affect fish and other aquatic life. The gut microbiome of fish is influenced by host species, its diet, and the environment, and thus contaminants released via wastewater effluents may alter the gut microbiome of fishes in receiving waters. This study found that the gut microbiota of rainbow darter fish exposed to wastewater effluents in the central Grand River (Waterloo/Kitchener, Ontario) were dominated by Proteobacteria and had increased diversity. Wild fish transitioned to the lab were dominated by Firmicutes and had decreased bacterial diversity in the gut compared to those in the wild. Altogether, these results suggest that wild fish exposed to wastewater effluents had altered gut microbiomes; transitions to new environments and laboratory acclimation periods are important considerations when studying the fish gut microbiome.