## 1 Introduction  
In the Grand River, located in Southwestern Ontario, contaminant inputs from effluent discharged by municipal wastewater treatment plants (MWWTPs) enter the water and have effects on the fish populations living downstream from the outfall. The effluent post-treatment contains contaminants of emerging concern (CECs), such as pharmaceuticals and personal care products (PPCPs), endocrine-disrupting compounds (EDCs), and other substances that can cause sub-lethal effects [1; 2]. In past studies, effects on reproduction, growth, sex ratios, metabolism, and molecular signatures have all been identified as maladaptive phenotypes in downstream locations from MWWTPs in this area, which were attributed to effluent exposure [3; 4; 5]. Rainbow darters downstream from the MWWTPs in the Grand River have experienced changes in gene expression, skewed sex ratios, altered gonad size, and intersex or feminized fish populations are found, largely due to the presence of EDCs [6; 7]. Additionally, run-off from urban developments and intense agricultural areas, along with stormwater drainages, are other sources of contamination and water quality degradation [8; 9]. Due to increasing human populations, urbanization, and agriculture processes, in combination with climate change, water quality downstream from MWWTPs is at risk and may lead to impacts on the ecology of the river. MWWTPs receive waste that contains mixed solids, inorganic, and organic contaminants, and treatment is meant to minimize the concentration of contaminants that are released back into the water. Initially, the waste is put through a primary treatment step to settle solids that will move to digestion and form biosolids. Liquids are moved to a secondary treatment, which decreases the biological oxygen demand (BOD) and nitrogenous compounds. The final step, the tertiary treatment, is more filtration or oxidation and is the most valuable step to limit contaminants and nutrients that will be released as a final effluent back into the river system. The tertiary treatment step is not a required process but many facilities are upgrading to incorporate tertiary treatment when upgrading to the required secondary treatment levels and comply with federal and provincial legislation [10; 11]. Recently, the Waterloo MWWTP has undergone upgrades to improve the water quality, i.e., reduce ammonia, biological oxygen demand, total suspended solids, and chloride, by increasing the solids retention time and nitrification/aeration [6; 12]. Water quality parameters, such as temperature, total suspended solids (TSS), BOD, chlorine, un-ionized ammonia and nitrogen compounds, conductivity, total phosphates, and pH extremes are all found to be highest or the most variable immediately downstream from MWWTPs and become diluted further downstream [8; 13; 14]. Increased TSS, BOD, and turbidity from effluent increase the water temperature and hypoxia, due to increased sunlight absorption and metabolism of microorganisms causing eutrophication [13; 15]. Un-ionized ammonia and nitrogen compounds can impact the ability of organisms to survive and reproduce in their environment due to direct toxicity. Damage to gill tissue, pH imbalances, osmo- and oxy-regulatory transport disruption, and immunocompromised are all possible effects of exposure to increased levels of un-ionized ammonia and nitrogen compounds, with the effects being amplified when exposed to multiple stressors [16; 17]. The upgrades greatly reduced the concentrations of many chemicals, [PERSON] et al. [12] reported that concentrations of numerous CEC's remain detectable in the treated effluent, including EDCs (synthetic hormones, personal care products), SSRIs (selective serotonin reuptake inhibitors, i.e., antidepressants), analgesics and anti-inflammatory drugs (NSAIDs), beta-blockers, antiepileptics) [12]. The remaining mixture of contaminants may have the potential to continue to alter ecosystem health and the extent of these effects are largely unknown because of the complexity of the effluent mixtures [18].