## 2 Materials and Methods  
In July 2019 (summer 2019) and October 2019 (fall 2019), samples of the three darter species were collected from sites surrounding the Waterloo MWWTP (US1, US2, DS1, and DS2) to capture seasonal differences in darter responses (Figure 1). All animal use and collection was approved by the University of Waterloo Animal Care Committee and followed the guidelines of animal use from the Canadian Council of Animal Care (Animal Use Protocols: #40315; #40318; #40989). Additionally, archived gill samples that were available from fish collected in October 2013 and 2014 (prior to MWWTP upgrades) were used for comparison between pre- and post-upgrades. US1 and US2 were upstream from the Waterloo MWWTP. US1 (43 deg35'07.54\" N, 80 deg28'54.08\" W) was located upstream from urban development and was an agriculturally dominated area, US2 (43 deg30'17.41\"N, 80 deg28'28.61\"W) was in an urban area. DS1 (43 deg28'24.69\" N, 80 deg28'23.99\" W) was immediately downstream from the MWWTP. DS2 (43 deg26'40.2\" N, 80 deg24'02.7\" W) was approximately 10.5 km downstream from DS1 and was also in an urban area. At least 14 random samples of each fish species (RBD, GSD, FTD) were collected in the morning (starting at 9 a.m.) using a backpack electro-fisher and dip nets; they were then placed into buckets that were aerated and kept at river temperature. In fall 2019, no female GSDs were collected. Animal collections were approved by the Animal Care Committee at the University of Waterloo under AUPP#40318. Fish smaller than 4.0 cm were not included, as they were previously found to be immature. Fish were used for respiratory trials (detailed below) only in the summer and fall of 2019. Following the respiratory, the fish were measured for total length (\(\pm\)0.1 cm) and weight (\(\pm\)0.01 g) and sacrificed via spinal severance. Body condition (\(k\)) was calculated using \(k=m/(l^{3})\times 100\), where \(m\) is the mass of fish in grams and \(l\) is the length in cm. Fish were then dissected immediately on-site for gill samples in all seasons. The second and third gill arches were extracted directly into [PERSON]'s solution (0.0065 mM formalin, 0.0066 mM 95% ethanol, 0.0011 mM glycerol, 0.0017 mM glacial acetic acid) for histological processing. At each site, water quality measurements (temperature, dissolved oxygen, conductivity, total dissolved solids, pH, and turbidity) were taken using a YSI Proplus multimeter (YSI Incorporated, Yellow Springs, OH, USA). In summer 2019, grab samples of river water were collected in 125 mL  
Figure 1: Map of the sampling locations used for collecting the darters used for this study. Orange markers indicate the general locations of capture, green indicates the general location of the effluent outfall. US1, upstream 1; US2, upstream 2; MWWTP, municipal wastewater treatment plant; DS1, downstream 1; DS2, downstream 2 (Google MyMaps; Map data ©2020, CNES/Airbus, First Base Solutions, Landsat/Copernicus, Maxar Technologies). -amber glass bottles for the analysis of nutrients and pharmaceuticals and general contaminants using LC-MS techniques, as previously described by [PERSON] et al. [5]. Samples of river water (250 mL) were preserved using 1 mL 49% sulfuric acid and measured for concentrations of total ammonia, nitrite, and nitrate by Maxcam Analytics (Mississauga, ON, Canada). Intermittent flow resiprometry was used to quantify the FMR and MMR in the field following capture. Eight 65 mL chambers, one reserved for background respiration, were used to measure fish respiration. Water was pumped directly from the river to the reservoir and an open-loop pump moved water through the chambers and out of the reservoir; this was a modified setup like that found in [PERSON] et al. (see Appendix A Figure A1) [38]. When the open-loop pump was turned off, each chamber maintained its flow using a closed-loop pump to mix water and measure oxygen via a fiber optic oxygen probe for continuous measurements (PyroScience, Aachen, Denmark).