Elevated Winter Stream Temperatures below Wastewater Treatment Plants Shift Reproductive Development of Female Johnny Darter Etheostoma nigrum: A Field and Histologic Approach

Abstract

River water temperatures are increasing globally, particularly in urban systems. In winter, wastewater treatment plant (WWTP) effluent inputs are of particular concern because they increase water temperatures from near freezing to ~7-15 °C. Recent laboratory studies suggest that warm overwinter temperatures impact the reproductive timing of some fishes. To evaluate winter water temperature's influence in the wild, we sampled Johnny Darter Etheostoma nigrum from three urban South Platte River tributaries in Colorado upstream and downstream of WWTP effluent discharge sites. Fish were collected weekly during the spring spawning season of 2021 and reproductive development was determined from histological analysis of the gonads. Winter water temperatures were approximately 5-10 °C greater ~300 m downstream of the WWTP effluent compared to upstream sites, and approximately 3°C warmer at sampling sites ~5000 m downstream of the effluent discharge. Females collected downstream of WWTP effluent experienced accelerated reproductive development compared to upstream by 1-2 weeks. Water quality, including total estrogenicity, and spring water temperatures did not appear to explain varying reproductive development. It appears that small increases in winter water temperature influence the reproductive timing in E. nigrum. Further investigations into how shifts in reproductive timing influence other population dynamics are warranted.

Keywords: Johnny Darter; histology; spawning; urban rivers; water temperature; winter.

Figure 1.

Sampling site for Johnny Darter Etheostoma nigrum in 2021 on the Big Thompson River (BT), St. Vrain Creek (SV), and the Cache la Poudre River (CP) on the Front Range in Northern Colorado. Gray areas indicate the extent of maps below it, or river reaches sampled. Stars indicate sampling locations and triangles indicate wastewater treatment plant (WWTP) effluent input. FU refers to far upstream, U to upstream, D to downstream, and FD to far downstream sampling sites in relation to the WWTP effluent input.

Figure 2.

Examples of (a) previtellogenic, (b) early vitellogenic, and (c) late vitellogenic stages of ovaries. (d) spawning gonad with hydrated oocytes, post-ovulatory follicles and the next generation of oocytes at early vitellogenesis. The scale bars represent 500 μm.

Figure 3.

Mean weekly average temperatures in the winter season (December–February) at the Cache la Poudre (CP) and the Big Thompson and St. Vrain far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites in winter 2020–2021 and 2021–2022. The FU site on the Big Thompson was not established for temperature monitoring until spring 2021. The weekly average temperature was similar within sites each winter. Error bars represent 95% confidence intervals.

Figure 4.

Weekly average temperatures for each week sampling occurred in spring of 2021 at the Cache la Poudre (CP) site, and the Big Thompson and St. Vrain far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites. The solid horizontal line represents previously documented spawning temperatures for Johnny Darter Etheostoma nigrum in Colorado (17 °C) [9] and the dashed line represents the coolest spawning temperature recorded elsewhere in their range (11.7 °C) [29]. Spring 2021 temperatures were not recorded at FU site on the Big Thompson due to its recent site establishment. No water temperature data at the CP site were recorded for weeks 12 and 13 due to logger error. Weeks 12–18 of 2021 occur during the last two weeks in March through the first week in May. Error bars represent 95% confidence intervals.

Figure 5.

Principal component analysis of pesticides and personal care products (PPCPs) and nutrients (NNPs) detected at each site during the (a) pre-spawn (weeks 12–13), (b) spawn (week 16), and (c) post-spawn (weeks 32 and 41) sampling periods. (d) PCA on the average concentration of detected chemicals at each site during 2021. “N” refers to the number of PPCPs and NNPs detected across all sites during that sampling occasion.

Figure 6.

17β-estradiol equivalents (E2-EQs, ng/L) at the Cache la Poudre (CP) and the Big Thompson and St. Vrain far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites displayed as mean at each site during each sampling event. Water samples were collected prior to sampling weeks 12/13, 16, 32, and 41. The water sample from the U site on the St. Vrain on week 12/13 was taken independently of fish sampling because the site was not established for fish collection until week 14. High flows prevented the sampling of the Big Thompson on week 32, which was delayed until week 41. Mean derived using n = 3–6 technical replicates per sample. Error bars represent means + SD.

Figure 7.

Mean relative abundance of Vtg3 mRNA transcripts (relative number of copies, normalized) in the liver of male Johnny Darters Etheostoma nigrum collected at the Cache la Poudre (CP) and the Big Thompson and St. Vrain far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites on weeks 12, 13, 16, 32, and 41. The centerline of each box represents the median, and whiskers represent minimum and maximums. The dashed line indicates the relative abundance of Vtg3 mRNA transcripts detected in pooled female E. nigrum liver RNA samples (analyzed in duplicate).

Figure 8.

(a) Proportions of females sampled from the Big Thompson in March (week 12), April (weeks 14–17), and May (week 18) far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites that were previtellogenic (Pre), early vitellogenic (Early) or late vitellogenic (Late). (b) The proportion of total females with evidence of recent or incipient spawning activity (mature or post-ovulatory follicles present). All females fall into either the Pre, Early, or Late category but may also have evidence of recent or incipient spawning activity. For example, a female could be classified as early vitellogenic, but may also have post-ovulatory follicles present (evidence of recent spawning activity) because Johnny Darter Etheostoma nigrum are fractional spawners. Red highlighted columns indicate sites subject to warmer overwinter water temperatures (downstream of the WWTP) and blue highlighted columns indicate sites subject to cooler overwinter water temperatures (upstream of the WWTP). The Big Thompson was not sampled on week 13.

Figure 9.

(a) Proportions of females sampled from the St. Vrain in March (week 13) and April (weeks 14–17) far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites that were previtellogenic (Pre), early vitellogenic (Early) or late vitellogenic (Late). (b) The proportion of total females with evidence of recent or incipient spawning activity (mature or post-ovulatory follicles present). All females fall into either the Pre, Early, or Late category, but may also have evidence of recent or incipient spawning activity. For example, a female could be classified as early vitellogenic, but may also have post-ovulatory follicles present (evidence of recent or incipient spawning activity) because Johnny Darter Etheostoma nigrum are fractional spawners. Red highlighted columns indicate sites subject to warmer overwinter water temperatures (downstream of the WWTP) and blue highlighted columns indicate sites subject to cooler overwinter water temperatures (upstream of the WWTP). The St. Vrain was not sampled on week 12 or 18, and the U site was not established until week 14.

Figure 10.

Proportions of females afflicted with mild and severe follicle atresia and males with testicular oocytes at the CP site, and the BT and SV far upstream (FU), upstream (U), downstream (D), and far downstream (FD) sites. Error bars represent 95% confidence intervals. Severe atresia refers to females where follicle atresia affected >75% of vitellogenic follicles. Horizontal lines at 0 represent that no sampled fish had follicular atresia. For example, female Johnny Darter Etheostoma nigrum were sampled at CP, but none had severe atresia.