Identifying transcriptomic indicators of tertiary treated municipal effluent in longnose dace (Rhinichthys cataractae) caged under semi-controlled conditions in experimental raceways.

To evaluate effects of tertiary treated wastewater treatment plant effluent (MWWE) on transcriptomic responses in longnose dace (Rhinichthys cataractae; LND) we conducted a semi-controlled study in experimental raceways (Advancing Canadian Water Assets facility) imbedded in the Pine Creek treatment plant (Calgary, AB). LND collected from a reference site in the Bow River (REF) were caged in raceways containing either 5 % Pine Creek effluent (PC) or Bow River water (BR; control) over 28 d. Liver transcriptomes were analyzed in males and females sampled on days 7, 14 and 28 from BR and PC, and compared to REF fish on day 0. Concurrent with the caging, selected environmental substances of concern were analyzed in the BR and PC. Significantly different unigenes (SDUs) in females (vs males) within both BR and PC raceways increased over time and compared to REF fish. Moreover, SDUs in females and males within the same treatment (i.e., BR, PC) showed a temporal increase as well as compared to REF fish. Time was the dominant factor affecting SDUs, whereas sex and treatment had less of an impact on the transcriptome profiling. Gene Set Enrichment Analysis of BR vs PC over time revealed effects on genes involved in growth, metabolism of carbohydrates and lipids, and immune system on day 7; however, by day 28, 80-100 % of the transcripts localized to enriched biomarkers were associated with tissue immune responses in both sexes. Exposure to 5 % effluent had significant effects on female liver somatic index but no effects were observed on other phenotypic health indices in either sex. BR was used as the source of reference water, but analyses showed trace amounts of ESOCs. Analyses did not point towards definitive response patterns that could be used in field-based ecotoxicogenomic studies on the impacts of well-treated MWWE but suggested compromised adaptive immune responses.

Intersex manifestation in the rainbow darter (Etheostoma caeruleum): Are adult male fish susceptible to developing and recovering from intersex after exposure to endocrine active compounds?

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.

Wild fish responses to wastewater treatment plant upgrades in the Grand River, Ontario.

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.

Multi-year prediction of estrogenicity in municipal wastewater effluents.

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.

Assessing recovery of in vitro steroid production in male rainbow darter (Etheostoma caeruleum) in response to municipal wastewater treatment plant infrastructure changes.

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.

How Does Reference Site Selection Influence Interpretation of Omics Data?: Evaluating Liver Transcriptome Responses in Male Rainbow Darter (Etheostoma caeruleum) across an Urban Environment.

Studies quantifying the influence of reference site selection on transcriptomic profiles in aquatic organisms exposed to complex mixtures are lacking in the literature, despite the significant implications of such research for the interpretation of omics data sets. We measured hepatic transcriptomic responses in fish across an urban environment in the central Grand River watershed (Ontario, Canada). Adult male rainbow darter (RBD) (Etheostoma caeruleum) were collected from nine sites at varying distances from two major municipal wastewater treatment plants (MWWTPs) (Waterloo, Kitchener), including three upstream reference sites. The transcriptomic response in RBD was independently compared with that of fish from each of the three reference sites. Data collected in fish downstream of the Waterloo MWWTP (poorest effluent quality) suggested that ∼15.5% of the transcriptome response was influenced by reference site selection. In contrast, at sites where the impact of MWWTPs was less-pronounced and fish showed less of a transcriptome response, reference site selection had a greater influence (i.e., ∼56.9% of transcripts were different depending on the site used). This study highlights the importance of conducting transcriptomics studies that leverage more than one reference site, and it broadens our understanding of the molecular responses in fish in dynamic natural environments.

Returning to normal? Assessing transcriptome recovery over time in male rainbow darter (Etheostoma caeruleum) liver in response to wastewater-treatment plant upgrades.

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.

Getting More Ecologically Relevant Information from Laboratory Tests: Recovery of Lemna minor After Exposure to Herbicides and Their Mixtures.

Recovery after exposure to herbicides-atrazine, isoproturon, and trifluralin-their binary and ternary mixtures, was studied under laboratory conditions using a slightly adapted standard protocol for Lemna minor. The objectives of the present study were (1) to compare empirical to predicted toxicity of selected herbicide mixtures; (2) to assess L. minor recovery potential after exposure to selected individual herbicides and their mixtures; and (3) to suggest an appropriate recovery potential assessment approach and endpoint in a modified laboratory growth inhibition test. The deviation of empirical from predicted toxicity was highest in binary mixtures of dissimilarly acting herbicides. The concentration addition model slightly underestimated mixture effects, indicating potential synergistic interactions between photosynthetic inhibitors (atrazine and isoproturon) and a cell mitosis inhibitor (trifluralin). Recovery after exposure to the binary mixture of atrazine and isoproturon was fast and concentration-independent: no significant differences between relative growth rates (RGRs) in any of the mixtures (IC10Mix, 25Mix, and 50Mix) versus control level were recorded in the last interval of the recovery phase. The recovery of the plants exposed to binary and ternary mixtures of dissimilarly acting herbicides was strictly concentration-dependent. Only plants exposed to IC10Mix, regardless of the herbicides, recovered RGRs close to control level in the last interval of the recovery phase. The inhibition of the RGRs in the last interval of the recovery phase compared with the control level is a proposed endpoint that could inform on reversibility of the effects and indicate possible mixture effects on plant population recovery potential.