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.

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.

The presence of pharmaceuticals in the environment has raised an emerging interest due to the fact that they pose negative environmental impact and health hazards related to long-term toxicity effects. As conventional treatments are not able to totally remove these substances it is necessary to seek for alternative advanced technologies such as oxidation with chlorine dioxide (ClO2). The objective of this master thesis is thus to find the most optimal dose – reaction time of ClO2 for the oxidation and maximum removal of selected environmentally relevant pharmaceuticals. Factorial design and subsequent optimization with MODDE was selected as the best approach to find the optimal dose – time. Batch oxidation tests were conducted on 100mL aliquots treated with ClO2 using wastewater effluent from Henriksdal WWTP. Thereafter solid phase extraction and final determination of pharmaceuticals was carried out on a high performance liquid chromatography- triple quadrupole mass spectrometry (HPLC-MS/MS). Results showed that applying a dose of 5 mg ClO2/L and a reaction time of 10 minutes, it is possible to remove more than a half of the 17 analyzed substances. Besides most of the pharmaceuticals with high and moderate environmental risk, would pose a low risk for the environment after treatment with the optimal ClO2 dose – reaction time. Despite the fact that ClO2 could successfully degrade most environmentally relevant pharmaceuticals, deeper research concerning the formation of toxic by-products after oxidative treatment needs to be done before upscaling this technology to pilot or full scale as a suitable end of pipe technology for pharmaceuticals removal.

Wastewater reclamation and reuse have been increasingly practiced as sustainable strategies to meet water demands, particularly in regions threatened by water shortages. However, one of the biggest challenges for reusing wastewater effluents (WEs) as irrigation water is to remove emerging organic contaminants such as persistent and potentially bioaccumulated per- and polyfluoroalkyl substances (PFAS), whose presence may result in adverse impacts on crops, soils, aqueous ecosystems, and human health. Photocatalysis is an effective and promising technique to remediate PFAS in aqueous media. This dissertation aims to: i) Develop a novel, environmental-friendly, and low-cost treatment process for PFAS removal and degradation for water reuse; ii) Optimize the experimental conditions and investigate the removal mechanisms of PFAS with different structures in this novel process; iii) Scale up this treatment process and apply it to treatment of WEs in a point-of-use (POU) system. First, ultraviolet (UV) C /nanoscale zero-valent iron (nZVI, Fe0 nanoparticles (NPs)) system is used for the first time to induce PFAS photocatalytic removal from aqueous solution. Oxidative and/or reductive degradation of three representative PFAS - perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorooctane sulfonate (PFOS) was achieved using Fe0 NPs under UVC light both with and without presence of oxygen. However, no PFAS removal was observed either under visible light and in the dark, and much lower PFAS degradation was achieved under UVA light. Higher degradation and defluorination efficiencies were obtained for longer chain PFNA compared to PFOA, and higher degradation and defluorination of PFAS were achieved without presence of O2 compared to with O2. The degradation of PFOA and PFOS followed first order reaction kinetics with the highest efficiencies achieved of 97.6, >99.9, and 98.5% without presence of O2 for PFOA, PFNA, and PFOS, respectively. The degradation efficiencies increased with the increase of nZVI concentrations in the range of 1-100 mg/L. The degradation efficiency of PFOA using bare Fe0 NPs was higher than that using 1% PVP-coated Fe0 NPs in the initial 6 h. Second, the removal mechanism of PFAS in UVC/Fe0 NPs system was obtained by testing the concentrations of iron ions (Fe2+/Fe3+), intermediate products, and reactive oxygen species (ROS, e.g., ·O2- and ·OH) generated, and conducting ROS quenching experiments. The proposed degradation pathway of PFCAs (PFNA and PFOA) was initiated from PFOA/PFNA oxidation by transferring an electron of the carboxylate terminal group of PFOA/PFNA to the Fe(III)-carboxylate complex, then followed by decarboxylation−hydroxylation−elimination−hydrolysis (DHEH) pathway and the accompanying CO2 and F− release. The generated shorter chain PFCAs also underwent degradation with time in the system. This proposed degradation pathway was confirmed by the formation of shorter chain PFCAs, e.g. PFHpA, PFHxA, PFPeA, and PFBA, F- ions, and rapid consumption of Fe3+. For PFOS, besides H/F exchange pathway and chain-shortening (DHEH pathway) to form short chain PFAS during PFCA degradation, desulfonation to form PFOA followed by PFOA degradation also happened. These pathways were suggested by the formation of intermediates — trace amount of shorter chain PFCAs, 6:2 FTS, PFHpS, and F- ions. ·O2- and ·OH were not involved in PFOA degradation in the UVC/Fe0 NPs system with presence of O2, while they may be involved in PFOS degradation, e.g., desulfonation to form PFOA, which were suggested by the results of quenching experiments. And introducing H2O2 into the UVC/Fe0 NPs system resulted in lower PFOA degradation efficiency and defluorination efficiency, which also indicated that ·OH may not be involved in PFOA degradation. Hydrated electrons e-aq that can be involved in desulfonation, defluorination, and C-C bond scission processes were likely quenched by the presence of oxygen to reduce the degradation and defluorination efficiencies; plus, presence of Fe0 NPs may promote the generation of hydrated electrons. Last, UVC/Fe0 NPs system was used to degrade PFAS from WEs in both bench scale and in a scale up POU system. The degradation efficiencies of PFAS in WEs from both wastewater treatment plants (WWTP) were lower than that in deionized water, likely reflecting the complex compositions in the environmental media. Optimal degradation efficiencies of 90±1%, 88±1%, and 46±2% were obtained for PFNA, PFOS, and PFOA, respectively, each starting from 0.5 µg/L using bare Fe0 at pH 3.0 after 2 h. PFAS removal and bacterial inactivation were achieved simultaneously in the POU system using Fe0 NPs without and with rGO support under UVC irradiation in WEs, although the PFAS levels were still above the regulation levels for discard. These pilot tests provided more data and experiences for the real applications of UVC/Fe0 NP system to PFAS contaminated wastewater or other water matrix treatment. Overall, this research demonstrated a cost-effective and environment-friendly method — UVC/Fe0 NPs method for PFAS (i.e., PFOA, PFNA, and PFOS) degradation from WEs for water reuse both with and without presence of oxygen. The possible degradation mechanisms of PFAS with different structures were obtained by testing the concentrations of iron ions, intermediate products, and reactive oxygen species (ROS) involved in the reactions. The developed technology can be potentially applied to treat other environmental media (e.g., groundwater, landfill leachate) that are contaminated by PFAS from previous anthropogenic activities.

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.

This study aims to detect how many microplastics and what kind are released from the wastewater treatment plant (WWTP) Skebäck, in Örebro. The study was limited to the analysis of three filters with 50 μm mesh size and one filter with 300 μm mesh size. The samples were taken at different times, two in the fall of 2019 and one in the spring of 2020. Visual characterization was used for the quantification of microplastics, and a lower and upper bound was used. The lower bound represents particles that were deemed identifiable as plastic with high certainty, while the upper bound also includes particles that may have been microplastic. An additional ATR-FTIR analysis was performed on selected microplastics >300 μm.  The presence of microplastics in the effluent from Skebäcks WWTP could be confirmed. The quantity of microplastics per m​3​ (MP/m​3​) trapped on the 50 μm filters were quantified in a range between 0 MP/m​3​ to 291 MP/m​3​ for the lower bound, and 72 MP/m​3​ to 435 MP/m​3​ for the upper bound. The 300 μm filter had considerably less microplastics than the 50 μm filter with 1.8 MP/m​3​. The quantification of fibers on the 50 μm filter and 300 μm filters was not possible due to high blank contaminations. According to the concentration of 63 MP/m​3​ of the lower bound count on the 50 μm filters and the amount of water flowing through Skebäck in 2019, 17 818 935 m​3​, 1.1 billion microplastic particles were released into Svartån that year. In comparison the highest value of the upper bound count, 435 MP/m​3​, gave a release of 7.7 billion microplastic particles. Using the concentration of the 300 μm filter 1.8 MP/m​3​, 32 million microplastics/year were released from Skebäck in 2019. The amount of spheres 50-300 μm released in the effluent from Skebäck was estimated to be 3.7 kg in 2019.

Vitellogenin in Pimephales promelas was used to assess estrogenicity of a local municipal effluent. Vitellogenin induction in male P. promelas increased in frequency and magnitude with increased exposure duration and was greater ("=0.05) than controls after 2 and 3 weeks of exposure. The level of vitellogenesis induced by effluent exposure was high compared to similar studies. A spring season evaluation followed. Biomarkers in P. promelas were used to assess the efficacy of a treatment wetland to remove toxicity and estrogenicity in final treated wastewater effluent. Comparisons were made with an effluent dominated stream and laboratory controls. Vitellogenin, GSIs (gonado-somatic indices), HSIs (hepato-somatic indices) and secondary sexual characteristics were biomarkers used in P. promelas models to assess aqueous estrogenicity. Biological indicators used to assess general fish health included hematocrit and condition factors. The estrogenic nature of the effluent was screened, concurrent with fish exposure, with GC/MS analysis for target estrogenic compounds including: 17-b estradiol, estrone, ethynylestradiol, Bisphenol A, nonylphenolic compounds, phthalates, and DDT. Plasma vitellogenin measured in P. promelas was significantly elevated (p < 0.0001) at the inflow site of the wetland and stream sites. GSIs for these exposures were less (a=0.001) at the wetland inflow site. At wetland sites closest to the inflow, secondary sexual charateristics, tubercle numbers and fat pad thickness, were less (a=0.0001). Hematocrit and condition factors were less (a=0.001) at sites closer to the wetland inflow. Seasonal variation was examined by repeating the effluent characterization in summer. Additionally, summer testing included exposure to an effluent dilution series. Fish condition heavily influenced interpretation of the results. Pre-acclimation exposure to spawning stresses may have altered many of the biological markers measured. Results are discussed relative to fish health and pre-exposure environment. Toxicity assessed with P. promelas biomarkers was compared with Ceriodaphnia dubia and Vibrio fischeri toxicty tests on this effluent. Biomarkers of fish health were somewhat less sensitive than C. dubia test endpoints, but more sensitive than V. fisheri.