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.

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.

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

Municipal wastewater treatment plant (WWTP) effluent is one of several point sources of contaminants (nutrients, pharmaceuticals, estrogens, etc.) which can lead to adverse responses in aquatic life. Studies of WWTP effluent impacts on rainbow darter (Etheostoma caeruleum) collected downstream of WWTPs in the Grand River, Ontario have reported disruption at multiple levels of biological organization, including altered vitellogenin gene expression, lower levels of in vitro steroid production, and high frequency of intersex. However, major upgrades have occurred at treatment plants in the central Grand River over the last decade. Treatment upgrades to the Waterloo WWTP were initiated in 2009 but due to construction delays, the upgrades came fully on-line in 2017/2018. Responses in rainbow darter have been followed at sites associated with the outfall consistently over this entire time period. The treatment plant upgrade resulted in nitrification of effluent, and once complete there was a major reduction in effluent ammonia, selected pharmaceuticals, and estrogenicity. This study compared several key responses in rainbow darter associated with the Waterloo WWTP outfall prior to and post upgrades. Stable isotopes signatures in fish were used to track exposure to effluent and changed dramatically over time, corresponding to the effluent quality. Disruptions in in vitro steroid production and intersex in the darters that had been identified prior to the upgrades were no longer statistically different from the upstream reference sites after the upgrades. Although annual variations in water temperature and flow can potentially mask or exacerbate the effects of the WWTP effluent, major capital investments in wastewater treatment targeted at improving effluent quality have corresponded with the reduction of adverse responses in fish in the receiving environment.

In this study, the estrogenicity of two major wastewater treatment plant (WWTP) effluents located in the central reaches of the Grand River watershed in southern Ontario was estimated using population demographics, excretion rates, and treatment plant-specific removals. Due to the lack of data on estrogen concentrations from direct measurements at WWTPs, the treatment efficiencies through the plants were estimated using the information obtained from an effects-directed analysis. The results show that this approach could effectively estimate the estrogenicity of WWTP effluents, both before and after major infrastructure upgrades were made at the Kitchener WWTP. The model was then applied to several possible future scenarios including population growth and river low flow conditions. The scenario analyses showed that post-upgrade operation of the Kitchener WWTP will not release highly estrogenic effluent under the 2041 projected population increase (36%) or summer low flows. Similarly, the Waterloo WWTP treatment operation is also expected to improve once the upgrades have been fully implemented and is expected to effectively treat estrogens even under extreme scenarios of population growth and river flows. The developed model may be employed to support decision making on wastewater management strategies designed for environmental protection, especially on reducing the endocrine effects in fish exposed to WWTP effluents.

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.