## 5 Conclusions  
The purpose of these experiments was to investigate the response of darters (rainbow darter, greenside darter, and fantail darter) to Waterloo municipal wastewater treatment plant effluent in the Grand River after upgrading to secondary treatment with an increased solids retention time and nitrification. These upgrades were hypothesized to reduce the biological impacts on fishes downstream, as seen after upgrades to a second municipal wastewater treatment plant in the Waterloo region [12; 48]. Rainbow darters had a reduced intersex severity downstream of the Waterloo outfall after the upgrades to the treatment plant [77]. Improving municipal wastewater treatment plants to tertiary treatment to reduce the biological impacts on darters downstream is supported.  
This study aimed to characterize the metabolic and gill tissue responses of darters exposed to the improved Waterloo municipal wastewater treatment plant effluent and compare them to fish populations upstream of the outfall. However, rainbow and fantail darters showed an increased maximum metabolic rate, and therefore, an increased aerobic scope. Greenside darters were unaffected. Rainbow and fantail darters' sizes were unaltered when metabolic adjustments were seen, while the greenside darters were significantly larger and did not alter their aerobic scope, indicating that species-specific differences in metabolic adjustments may be occurring. Overall, there is no clear pattern in the impacts of the municipal wastewater treatment plant effluent or detrimental effects of effluent (i.e., contaminants) due to the variation in the darter's physiological responses at the various sites. The increased aerobic scope and body measurements indicate that fish living in the urbanized areas of the Grand River may have utilized the greater nutrient (i.e., food) and oxygen availability. Additionally, there were effects of effluent on the gill morphology of darters found in the Grand River. The results were not consistent across seasons, sites, or species, which should be considered in future studies. However, the evidence presented here suggests there were species-specific responses to changing water quality conditions. This study highlights potential effects and markers of contaminant exposure and the importance of looking at multiple species when assessing contaminant effects.  
Conceptualization, R.H., M.R.S., and P.M.C.; methodology, R.H., L.B., H.A.D., and P.M.C.; software, R.H. and L.B.; validation, R.H., L.B., H.A.D., and P.M.C.; formal analysis, R.H.; investigation, R.H., L.B., and H.A.D.; resources, L.B., H.A.D., M.R.S., and P.M.C.; data curation, R.H.; writing--original draft preparation, R.H.; writing--review and editing, R.H., M.R.S., and P.M.C.; visualization, R.H.; supervision, P.M.C.; project administration, M.R.S. and P.M.C.; funding acquisition, R.H., M.R.S., and P.M.C. All authors have read and agreed to the published version of the manuscript.  
This research was supported through the Natural Science and Engineering Research Council (#RGPIN-2015-05643) and the Canadian First Research Excellence Fund Global Water Futures initiative (PI: [PERSON]). R.H. was supported by a [PERSON] Elizabeth II Graduate Scholarship in Science and Technology.  
**Conflicts of Interest:** The authors declare no conflict of interest.  
Figure 17: (**a**) Sample of morphometric measurements of the gill. BET, basal epithelium thickness; ID, interlamellar diameter; SLL, secondary lamellar length; SLW, secondary lamellar width. (**b**,**c**). Examples of the gill pathologies that were scored. Five pathologies were scored out of a scale from 1–4 for severity and summed, where (**b**) shows edema and fusion of the secondary lamellae and (**c**) shows epithelial lifting, hyperplasia, and hypertrophy. See methods and Table 12 for descriptions of the pathologies.