## 1 Introduction
There exist provincial guidelines on the limits of concentrations of known contaminants (Provincial Water Quality Objectives) in the surface waters, which were set, in part, to minimize the disruption to aquatic life. The upgrades help to reach or exceed these guidelines by causing a reduction in the concentration of chemicals and contaminants in the effluents, but there are still many CECs that remain in the discharged effluent that can affect aquatic life [12; 18]. The guideline list includes organics, pesticides, and heavy metals, but does not include CECs that are hard to degrade or settle into biomass, with the possibility of being taken up into the body. Fish, as keystone species, can serve as bioindicators to help assess the health of ecosystems [19], but the effects of wastewater on the physiology of different fish species in rivers, such as the Grand River is currently limited [19; 20]. Although extensive studies have been conducted on rainbow darter in the Grand River in response to treatment plant upgrades, few studies have examined the effects of treated effluent on the physiology of a group of closely related species with varying degrees of tolerance [6]. Some species can maintain a homeostatic balance in stressful environments better than others, but not without consequence. When a fish encounters a stressor (e.g., contaminant), a general adaptation response occurs, where alarm, resistance, and exhaustion phases may progress [21]. A fish will undergo a stress response, which includes altering its metabolism via energy substrate mobilization due to cortisol release, for example, due to increased energy demand of the gills for detoxification [22]. However, fish can be attracted to and live downstream of WWTPs, likely because there is an increase in nutrients, which allows for increased energy toward growth and reproduction, as demonstrated by the increased body condition downstream from MWWTPs, despite the presence of harmful contaminants [5; 23; 24]. A valuable endpoint to measure the effects of chronic exposure to contaminants is the metabolic rate, partly due to this late role in the stress response and its indicator of overall animal health [25]. In fish, aerobic scope (AS) is the difference between the maximum metabolic rate (MMR) and the routine metabolic rate (RMR); it is a measure of external environmental effects on fish and internal compensation and adjustment [26]. RMR is the minimum amount of energy required for physiological life and maintenance; major movement (swimming, food gathering, predator avoidance), growth, and reproduction require additional energy up to the maximum amount of oxygen uptake, i.e., the MMR. In the field, the field metabolic rate (FMR) is measured, which is typically higher than the RMR due to a shorter acclimation period and unknown feeding status [27]. Measuring differences in AS to understand the impacts of an environmental stressor, such as MWWTP effluent, can provide insights into how species are compensating for exposure to these stressors in wastewaters. While [PERSON] et al. demonstrated a negative impact on the metabolic rate in rainbow darters, there has not been a cross-species comparison on the impacts of municipal effluent on metabolic rate [4]. In addition to metabolism, the gills are an ideal organ to study effluent effects, as they control several homeostatic functions at the whole animal level (e.g., respiration and ionoregulation) and are at the interface between the internal and external environment of the organism. There is a significant metabolic cost to respiration (10-70%), and less so for osmoregulation (2-4%), which can increase when a fish needs to divert energy toward the essential processes [28]. The gill surface can be changed either by being restructured when undergoing an osmorespiratory compromise or in poor water quality conditions to avoid becoming damaged [29]. Damage to the gills is important because it can have effects on the key functions of the organ, such as respiration, osmoregulation, acid-base regulation, and nitrogen excretion.