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.

Combining chemical, bioanalytical and predictive tools to assess persistence, seasonality, and sporadic releases of organic micropollutants within the urban water cycle.

Using a novel liquid chromatography-tandem mass spectrometry method with large volume direct injection and quantitation via isotope dilution, we evaluated the presence of 55 organic micropollutants in wastewater effluents, and locations within the Bow River and Elbow River watersheds in and around the city of Calgary, Alberta, Canada. In addition to establishing baseline micropollutant data for water utility operations, our study aimed to enhance our understanding of micropollutant behavior in the urban water cycle, assess the contributions of three wastewater treatment plants (WWTPs) to downstream receiving waters, explain the potential causes of total estrogenicity measured using the yeast-estrogen screen assay (YES), and prioritize a subset of substances for continuous monitoring. With data spanning 48 months and 95 river km, our results indicate the extensive persistence of metformin (antidiabetic), seasonality of N,N­diethyl-m-toluamide (DEET, insect repellant), O-desmethylvenlafaxine (antidepressant metabolite), and sulfamethoxazole (antibiotic) in source waters, and sporadic detections of a well-known perfluoroalkyl substance (PFOA). The seasonality of pharmaceuticals at the sentinel downstream monitoring site appeared to coincide with river dilution while that of DEET was likely attributable to peak usage during the warmer months. Steroidal estrogens were rarely detected in wastewater effluents although total estrogenicity via YES was evident, suggesting the presence of less potent but more abundant non-steroidal estrogens (e.g., flame retardants, bisphenols, and phthalates). A conservative mass balance analysis suggests that the largest WWTP (serving a population of >1 million) consistently contributed the highest load of micropollutants, with the exception of metformin, which appeared to be influenced by a smaller WWTP (serving 115,000) that operates a different activated sludge process. We consider metformin, sucralose, diclofenac, and venlafaxine as more effective conservative tracers of wastewater pollution due to their notably higher concentrations and persistence in the Bow River compared to carbamazepine and caffeine, respectively. Finally, hierarchical clustering revealed a close association between E. coli and caffeine, supporting the use of caffeine as an indicator of short-term, untreated anthropogenic inputs. Overall, this study yields valuable insights on the presence, behavior, and sources of organic micropollutants in the urban water cycle and identifies indicators of anthropogenic impacts that are useful for prioritizing future monitoring campaigns in Calgary and elsewhere.

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.

Photodecomposition of pharmaceuticals and personal care products using P25 modified with Ag nanoparticles in the presence of natural organic matter.

The presence of pharmaceuticals and personal care products (PPCPs) in water remains a concern due to their potential threat to environmental and human health. Advanced oxidation processes (AOPs) have been receiving attention in water treatment studies to remove PPCPs. However, most studies have been focused on pure water containing a limited number of substances. In this study, the photocatalytic efficiency of commercially available titanium dioxide nanoparticles (P25) and P25 modified by silver nanoparticles (Ag-P25) were compared for their ability to degrade 23 target PPCPs (2 µg L-1) in realistic water matrices containing natural organic matter (Suwanee River NOM, 6.12 mg L-1). The experiments were completed under ultraviolet-light emitting diode (UV-LED) illumination at 365 and 405 nm wavelengths, with the latter representing visible light exposure. Under 365 nm UV-LED treatment, 99% of the PPCPs were removed using both P25 and Ag-P25 photocatalysts within 180 min of the treatment duration. The number of PPCPs removed dropped to 57% and 53% for P25 and Ag-P25 respectively under the 405 nm UV-LED irradiation. Dissolved organic carbon (DOC) and UV absorbance at 254 nm (UV254) measured at the end of the experiment indicated that the aromatic fraction of NOM was preferentially removed from the water matrix. Also, Ag-P25 was more effective in DOC removal than P25. The relationships of removal rate constants with physico-chemical properties of the substances were also determined. The molecular weight and charge were strongly associated with removal, with the former and the latter being positively and negatively correlated with the rate constants. The results of this work indicate that Ag-P25 is a promising photocatalyst to degrade persistent substances such as PPCPs and NOM even if they are present in a complex water matrix. The properties of individual substances can also be employed as an indication of their removal using this technology.

Improving Risk Assessment by Predicting the Survival of Field Gammarids Exposed to Dynamic Pesticide Mixtures.

Exposure assessment of pesticides has substantially improved over time, with methods that now include a combination of advanced analytical techniques and fate/transport models to evaluate their spatiotemporal distribution. However, the current regulatory environmental risk assessment considers thresholds from laboratory studies completed under standardized conditions that do not reflect environmental dynamics. Using the General Unified Threshold model for Survival (GUTS) model framework, we predicted the impact of time-varying pesticide exposures on the survival of gammarids in a small agricultural stream. The LP50 values were used as an additional metric for assessing risks (defined in GUTS as a multiplication factor applied to the concentration time series to induce 50% mortality by the end of exposure). Although real-case exposures to individual pesticides were predicted to produce little to no impact on survival, the LP50 values indicate acute (LP50 ≤ 100) and/or chronic (LP50 ≤ 10) toxicities for azoxystrobin, chlorpyrifos, diazinon, and imidacloprid, while risk to propiconazole exposure was considered very low (LP50 ≫ 100). Finally, the model was extended to reflect mixture toxicity via concentration addition. It predicted risks under acute and chronic exposures to organophosphates and neonicotinoids. Given that gammarids are simultaneously exposed to multiple chemicals and other stressors throughout their lifetime, a decline in survival probabilities due to chemical stress can likely influence their overall fitness. We recognize that some assumptions require validation, but our work included a level of realism that can assist risk managers when evaluating the cumulative consequences of chemical exposure.

Coupling River Concentration Simulations with a Toxicokinetic Model Effectively Predicts the Internal Concentrations of Wastewater-Derived Micropollutants in Field Gammarids.

Although the exposure assessment of wastewater-derived micropollutants via chemical, bioanalytical, and modeling methods in environmental compartments is becoming more frequent, the whole-body burden (i.e., internal concentrations) in nontarget organisms is rarely assessed. An understanding of the internal concentration fluctuation is especially important when exploring the mechanistic linkage between exposure and effects. In this study, we coupled a simple river model with a first-order toxicokinetic (TK) model to predict the concentrations of wastewater-derived micropollutants in freshwater invertebrates (Gammarus spp.). We applied Monte Carlo simulations and conducted laboratory experiments to account for the uncertain input data and the lack of uptake/depuration rate constants required for the TK model. The internal concentrations in field gammarids were predicted well, and the estimates varied only by a factor of 0.1-1.9. Fast equilibrium may also be assumed such that bioconcentration factors (BCFs) are used together with the daily river dilution patterns to predict internal concentrations. While this assumption is suitable for compounds observed in our experiment to reach the steady state within 48 h in gammarids, the model overpredicted the concentrations of substances that reach this condition after longer periods. Nevertheless, this approach provides conservative estimates and simplifies the coupling of models as BCFs are slightly more accessible than the rate constants. However, if one is interested in a more detailed exposure information (e.g., peak concentration and the whole-body burden recovery after a spill), then the nonsteady-state formulation should be employed.

Modeling the exposure of wild fish to endocrine active chemicals: Potential linkages of total estrogenicity to field-observed intersex.

Decades of studies on endocrine disruption have suggested the need to manage the release of key estrogens from municipal wastewater treatment plants (WWTP). However, the proposed thresholds are below the detection limits of most routine chemical analysis, thereby restricting the ability of watershed managers to assess the environmental exposure appropriately. In this study, we demonstrated the utility of a mechanistic model to address the data gaps on estrogen exposure. Concentrations of the prominent estrogenic contaminants in wastewaters (estrone, estradiol, and ethinylestradiol) were simulated in the Grand River in southern Ontario (Canada) for nine years, including a period when major WWTP upgrades occurred. The predicted concentrations expressed as total estrogenicity (E2 equivalent concentrations) were contrasted to a key estrogenic response (i.e., intersex) in rainbow darter (Etheostoma caeruleum), a wild sentinel fish species. A predicted total estrogenicity in the river of ≥10 ng/L E2 equivalents was associated with high intersex incidence and severity, whereas concentrations <0.1 ng/L E2 equivalents were associated with minimal intersex expression. Exposure to a predicted river concentration of 0.4 ng/L E2 equivalents, the environmental quality standard (EQS) proposed by the European Union for estradiol, was associated with 34% (95% CI:30-38) intersex incidence and a very low severity score of 0.6 (95% CI:0.5-0.7). This exposure is not predicted to cause adverse effects in rainbow darter. The analyses completed in this study were only based on the predicted presence of three major estrogens (E1, E2, EE2), so caution must be exercised when interpreting the results. Nevertheless, this study illustrates the use of models for exposure assessment, especially when measured data are not available.

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.

Reduction of Intersex in a Wild Fish Population in Response to Major Municipal Wastewater Treatment Plant Upgrades.

Intersex in fish downstream of municipal wastewater treatment plants (MWWTPs) is a global concern. Consistent high rates of intersex in male rainbow darter (Etheostoma caeruleum) have been reported for several years in the Grand River, in southern Ontario, Canada, in close proximity to two MWWTPs. The larger MWWTP (Kitchener) recently underwent upgrades that included the conversion from a carbonaceous activated sludge to nitrifying activated sludge treatment process. This created a unique opportunity to assess whether upgrades designed to improve effluent quality could also remediate the intersex previously observed in wild fish. Multiple years (2007-2012) of intersex data on male rainbow darter collected before the upgrades at sites associated with the MWWTP outfall were compared with intersex data collected in postupgrade years (2013-2015). These upgrades resulted in a reduction from 70 to 100% intersex incidence (preupgrade) to <10% in postupgrade years. Although the cause of intersex remains unknown, indicators of effluent quality including nutrients, pharmaceuticals, and estrogenicity improved in the effluent after the upgrades. This study demonstrated that investment in MWWTP upgrades improved effluent quality and was associated with an immediate change in biological responses in the receiving environment. This is an important finding considering the tremendous cost of wastewater infrastructure.

Photocatalytic decomposition of selected estrogens and their estrogenic activity by UV-LED irradiated TiO2 immobilized on porous titanium sheets via thermal-chemical oxidation.

The removal of endocrine disrupting compounds (EDCs) remains a big challenge in water treatment. Risks associated with these compounds are not clearly defined and it is important that the water industry has additional options to increase the resiliency of water treatment systems. Titanium dioxide (TiO2) has potential applications for the removal of EDCs from water. TiO2 has been immobilized on supports using a variety of synthesis methods to increase its feasibility for water treatment. In this study, we immobilized TiO2 through the thermal-chemical oxidation of porous titania sheets. The efficiency of the material to degrade target EDCs under UV-LED irradiation was examined under a wide range of pH conditions. A yeast-estrogen screen assay was used to complement chemical analysis in assessing removal efficiency. All compounds but 17ß-estradiol were degraded and followed a pseudo first-order kinetics at all pH conditions tested, with pH 4 and pH 11 showing the most and the least efficient treatments respectively. In addition, the total estrogenic activity was substantially reduced even with the inefficient degradation of 17ß-estradiol. Additional studies will be required to optimize different treatment conditions, UV-LED configurations, and membrane fouling mitigation measures to make this technology a more viable option for water treatment.

Photocatalytic decomposition of organic micropollutants using immobilized TiO2 having different isoelectric points.

Organic micropollutants found in the environment are a diverse group of compounds that includes pharmaceuticals, personal care products, and endocrine disruptors. Their presence in the aquatic environment continues to be a concern as the risk they pose towards both the environment and human health is still inconclusive. Removal of these compounds from water and wastewater is difficult to achieve and often incomplete, but UV-TiO2 is a promising treatment approach. In this study, the efficiency of titanium dioxide (TiO2) immobilized on porous supports were tested for treatment of target pharmaceuticals and their metabolites under UV-LED exposure, a potential low energy and cost effective alternative to conventional UV lamps. Immobilization was completed using two different methods: (1) dip coating of TiO2 onto quartz fiber filters (QFT) or (2) thermal-chemical oxidation of porous titanium sheets (PTT). Comparison against experimental controls (dark QFT, dark PTT, and photolysis using UV-LED only) showed that UV-LED/PTT and UV-LED/QFT treatments have the potential to reduce the concentrations of the target compounds. However, the treatments were found to be selective, such that individual pharmaceuticals were removed well using QFT and PTT but not both. The complementary treatment behavior is likely driven by electrostatic interactions of charged compounds with the membranes. QFT membranes are negatively charged at the experimental pH (4.5-5) while PTT membranes are positively charged. As a result, cationic compounds interact more with QFT while anionic compounds with PTT. Neutral compounds, however, were found to be recalcitrant under any treatment conditions suggesting that ionic interactions were important for reactions to occur. This behavior can be advantageous if specificity is required. The behavior of pharmaceutical metabolites is similar to the parent compounds. However, isomeric metabolites of atorvastatin with functional groups in para and ortho configurations behave differently, suggesting that the positioning of functional groups can have an impact in their interaction with the immobilized TiO2. It was also apparent that PTT can be reused after cleaning by heat treatment. Overall, these newly synthesized membrane materials have potential applications for treatment of trace organic contaminants in water.