## 4 Discussion  
The Waterloo MWWTP process upgrades included an increased solids retention time, added aeration/nirtification process, and UV disinfection, which improves effluent quality and odor and improved process efficiency. This upgrade followed guidelines set by the Fisheries Act Canada and the Ministry of Justice in 2012, requiring all MWWTPs to be upgraded to at least secondary treatment. The upgrades took place from 2009 until 2018; these measures were implemented to reduce BOD, TSS, and chlorine and ammonia levels. Although upgrades to the Waterloo MWWTP occurred prior to this study, there were measurable amounts of pharmaceuticals and personal care products detectable throughout the Grand River, where the levels increased downstream from the MWWTP (Figure 2). Of the highest contaminant concentrations detected, carbamazepine (antiepileptic), diclofenac (anti-inflammatory), and venlafaxine (antidepressant) were persistent pharmaceuticals that are not easily broken down and remain at high concentrations after release into the river, even after upgrades to the MWWTP [12, 44, 45]. Concentration limits for effects of CECs are difficult to set due to the potential synergistic, antagonistic, or additive effects of contaminant combinations [46, 47]. Although concentrations have significantly decreased compared to pre-upgrade concentrations, they may still have the potential to cause sub-lethal effects on fish populations [12, 48]. In the summer, there is a decrease in the flow in the river and this may be exposing the fishes downstream to a higher concentration of contaminants than in other seasons due to less dilution [49]. Tables 1 and 2 demonstrate that seasonal variance caused changes throughout the river. Responses documented at US2 and DS2 may indicate that fish were responding to other stressors upstream, such as non-point sources of agricultural and urban inputs. These inputs should be considered for future studies, as effects can be hard to differentiate from the MWWTP inputs [1, 48]. There were no significant changes in the FMR in any species, site, or season. RBDs could modulate their metabolic rate in response to living in effluent but perhaps the improvement in treatment has reduced their need for detoxification (lower ammonia and contaminant levels) [4]. The metabolic rates measured in this study were in the range of those of other fishes that are closely related [50]. Round Goby showed no increase in routine metabolic rate when exposed to effluent and is thought to be quite tolerant of MWWTP effluent [51]. In contrast, bluegill sunfish had an increased FMR when exposed to effluent [52]. Therefore, because the hypothesized response to effluent (i.e., contaminant) exposure was not seen, the effects of the MWWTP may have been reduced due to the upgrades. Figure 5: (**a**) The average SLW of the GSDs in summer 2019 (\(p<0.05\), \(n=11\), \(8\), \(24\), \(11\)). (**b**) The average pathology scores of the GSDs in summer 2019 (\(p<0.05\), \(n=11\), \(8\), \(24\), \(11\)). An asterisk represents a significant difference compared to US1 using a one-way ANOVA and Dunnett’s multiple comparisons test. GSD, greenside darter; US1, upstream 1; US2, upstream 2; DS1, downstream 1; DS2, downstream 2. Recently, [PERSON] et al. demonstrated that field-realistic exposure to an antidepressant (fluoxetine) disrupted foraging and aggression in mosquitofish [53]. While there may be no change in routine metabolism, a change in behavior could free up enough energy for detoxification and maintenance functions [54; 55]; however, this remains to be studied for darker species found in the Grand River. The aerobic scope was increased in RBDs and FTDs at DS1 and DS2 in summer 2019; additionally, there is some evidence of increased body measurements downstream from the MWWTP. Increased energy availability in fish exposed to effluent could help to explain the increase in AS [23; 56].