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Abstract: The present study examined in vitro 11‐ketotestosterone and testosterone production by the testes of rainbow darter (Etheostoma caeruleum) collected from selected reference sites and downstream of 2 municipal wastewater treatment plants (MWWTPs; Waterloo and Kitchener) on the central Grand River (Ontario, Canada), over a 6‐yr period (2011–2016). The main objective was to investigate if infrastructure upgrades at the Kitchener MWWTP in 2012 resulted in a recovery of this response in the post‐upgrade period (2013–2016). Two supporting studies showed that the fall season is appropriate for measuring in vitro sex steroid production because it provides stable detection of steroid patterns, and that the sample handling practiced in the present study did not introduce a bias. Infrastructure upgrades of the Kitchener MWWTP resulted in significant reductions in ammonia and estrogenicity. After the upgrades, 11‐ketotestosterone production by MWWTP‐exposed fish increased in 2013 and it continued to recover throughout the study period of 2014 through 2016, returning to levels measured in reference fish. Testosterone production was less sensitive and it lacked consistency. The Waterloo MWWTP underwent some minor upgrades but the level of ammonia and estrogenicity remained variable over time. The production of 11‐ketotestosterone and testosterone in rainbow darter below the Waterloo MWWTP was variable and without a clear recovery pattern over the course of the present study. The results of the present study demonstrated that measuring production of sex steroids (especially 11‐ketotestosterone) over multiple years can be relevant for assessing responses in fish to environmental changes such as those resulting from major infrastructure upgrades. Environ Toxicol Chem 2018;37:501–514. © 2017 SETAC [ABSTRACT FROM AUTHOR]

• Intersex was induced in a seasonally spawning native fish species. • Exposure to estrogen while testes are fully developed resulted in intersex fish. • Adult male fish did not recover from intersex after onset of the condition. • Exposure to estrogen is consistent with impacts in wild fish exposed to wastewater. For over a decade, intersex has been observed in rainbow darter (RD) (Etheostoma caeruleum) populations living downstream wastewater treatment plants (WWTPs) in the Grand River, Ontario, Canada. To further our understanding of intersex development in adult male fish, the current study addressed three objectives: i) can intersex be induced in adult male fish, ii) is there a specific window of exposure when adult male fish are more susceptible to developing intersex, and iii) can pre-exposed adult male fish recover from intersex? To assess intersex induction in adult male fish, wild male RD were exposed in the laboratory for 22 weeks (during periods of spawning, gonadal regression, and gonadal recrudescence) to environmentally relevant concentrations of 17α-ethinylestradiol (EE2) including nominal 0, 1, and 10 ng/L. Intersex rates and severity at 10 ng/L EE2 were similar to those observed historically in adult male populations living downstream WWTPs in the Grand River and confirmed previous predictions that 1–10 ng/L EE2 would cause these adverse effects. To assess a window of sensitivity in developing intersex, male RD were exposed to nominal 0, 1 or 10 ng/L EE2 for 4 weeks during three different periods of gonadal development, including (i) spawning, (ii) early recrudescence and (iii) late recrudescence. These short-term exposures revealed that intersex incidence and severity were greater when RD were exposed while gonads were fully developed (during spawning) compared to periods of recrudescence. To assess if RD recover from intersex, wild fish were collected downstream WWTPs in the Grand River and assessed for intersex both before and after a 22-week recovery period in clean water that included gonadal regression and recrudescence. Results showed that fish did not recover from intersex, with intersex rates and severity similar to those both before and after the transition to clean water. This study further advances our knowledge on intersex manifestation in adult male fish including their sensitivity to endocrine active compounds during different periods of their annual reproductive cycle and their limited ability to recover from intersex after onset of the condition. [ABSTRACT FROM AUTHOR]

The present study measured hepatic transcriptome responses in male rainbow darter ( Etheostoma caeruleum) exposed to 2 municipal wastewater-treatment plants (MWWTPs; Kitchener and Waterloo) over 4 fall seasons (2011-2014) in the Grand River (Ontario, Canada). The overall goal was to determine if upgrades at the Kitchener MWWTP (in 2012) resulted in transcriptome responses indicative of improved effluent quality. The number of differentially expressed probes in fish downstream of the Kitchener outfall (904-1223) remained comparable to that downstream of Waterloo (767-3867). Noteworthy was that year and the interaction of year and site explained variability in more than twice the number of transcripts than site alone, suggesting that year and the interaction of year and site had a greater effect on the transcriptome than site alone. Gene set enrichment analysis revealed a gradual reduction in the number of gene ontologies over time at exposure sites, which corresponded with lower contaminant load. Subnetwork enrichment analysis revealed that there were noticeable shifts in the cell pathways differently expressed in the liver preupgrade and postupgrade. The dominant pathways altered preupgrade were related to genetic modifications and cell division, whereas postupgrade they were associated with the immune system, reproduction, and biochemical responses. Molecular pathways were dynamic over time, and following the upgrades, there was little evidence that gene expression profiles in fish collected from high-impact sites postupgrade were more similar to those in fish collected from reference site. Environ Toxicol Chem 2017;36:2108-2122. © 2017 SETAC [ABSTRACT FROM AUTHOR]

Stable isotopes (δ 15 N and δ 13 C) were used to assess the changes in exposure and assimilation of sewage-derived nutrients in an aquatic food web following changes in effluent quality over an 8 year period at two municipal wastewater treatment plants (WWTPs) that discharge to the Grand River, in southern Ontario. Upgrades at the Kitchener WWTP started in late 2012 to enhance nitrification, while the Waterloo WWTP had a series of construction issues at the plant that resulted in a deterioration of its effluent quality over the study period (2007–2014). Fish (rainbow darter, Etheostoma caeruleum ) and primary consumers (benthic invertebrates) were sampled in the receiving waters associated with each outfall. Upgrades at the Kitchener WWTP resulted in improved effluent quality with total annual ammonia output dropping by nearly sixfold (583–100 t), while the Waterloo WWTP increased its total annual ammonia output by nearly fourfold (135–500 t) over the duration of the study. Downstream of the Kitchener WWTP, the reduction in total ammonia output negatively correlated with changes in δ 15 N of rainbow darter from being depleted (prior to the upgrade) to reflecting signatures similar to those at the upstream reference site. The biota downstream of the Waterloo WWTP showed the opposite trend, going from slightly enriched, to being depleted relative to the upstream reference sites. δ 13 C was consistently higher downstream of both WWTPs regardless of changing effluent quality, and annual variability in δ 13 C was associated with annual river discharge. In a laboratory based dietary switch study conducted with rainbow darter, the isotope half-life in muscle (29 days for δ 15 N and 33 days for δ 13 C) were determined and these rapid changes were consistent with responses in muscle of wild fish. This is a unique study that was able to contrast two WWTPs in the same watershed as they underwent major changes in treatment processes. Stable isotopes were very effective as a tool to trace the changes in aquatic biota due to changes in wastewater effluent quality, both improvements and deterioration over time. [ABSTRACT FROM AUTHOR]