As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

Fish live in continuous contact with various stressors and antigenic material present within their environments. The impact of stressors associated with wastewater-exposed environments on fish has become of particular interest in toxicology studies. The objectives of this study were to examine potential effects of wastewater treatment plant (WWTP) effluent-associated stressors on innate cytokine expression within the gills of darter species (Etheostoma spp.), using both field and laboratory approaches. Male and female darters (rainbow, greenside, fantail, and johnny darters) were collected upstream and downstream of the Waterloo WWTP in the Grand River, Ontario. Gill samples were collected from fish in the field and from a second subset of fish brought back to the laboratory. Laboratory fish were acutely exposed (96-h) to an environmentally relevant concentration of venlafaxine (1.0 μg/L), a commonly prescribed antidepressant. To assess the impacts of these stressors on the innate immunity of darters, the expression of key innate cytokines was examined. Minor significant effects on innate cytokine expression were observed between upstream and downstream fish. Moderate effects on cytokine expression were observed in venlafaxine-exposed fish compared to their control counterparts however, changes were not indicative of a biologically significant immune response occurring due to the exposure. Although the results of this study did not display extensive impacts of effluent and pharmaceutical exposure on innate cytokine expression within the gills, they provide a novel avenue of study, illustrating the importance of examining potential impacts that effluent-associated stressors can have on fundamental immune responses of native fish species.

Introduction Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021–2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined. Methods Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021. Results Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener ( r = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1–N2) concentration in wastewater ( n = 99–191) was strongly correlated to the CBED in the communities ( r = 0.620–0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization ( n = 99–191) at Humber AMF and Kitchener and crAssphage normalization ( n = 29–57) correlations at all three sites were not significantly different from raw N1–N2 correlations with CBED. Discussion Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites.

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020-November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021-June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems.

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

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.

The Grand River (GR) extends throughout the majority of Southern Ontario with its final outlet at Lake Erie and accommodates thirty wastewater treatment plants (WWTP) with varied filtration processes. Many WWTPs are unable to effectively eliminate several contaminants of concern (CECs) from final released effluent, leading to measurable concentrations in surface waters and ultimately chronically exposing aquatic species to mixed CECs. Exposures to CECs have reported impacts on oxidative stress, measurable through reactive oxygen species (ROS) and the antioxidant defense response. This research focuses on the effects of WWTP effluent on four Etheostoma (Darter) species endemic to the GR. Objectives of this study examined if any oxidative stress markers are present in darter brains downstream from the effluent release point compared to an upstream reference site relative to the Waterloo, ON WWTP across two separate years (Fall 2020 and 2021). This was assessed using transcriptional and enzyme analysis of antioxidant enzymes (SOD, GPX, CAT) and an enzyme involved in serotonin synthesis. In fall 2020, significant differences in transcript expression of markers were found between sites and sexes in greenside darters (GSD) with SOD and CAT showing increased expression downstream. Changes in transcript expression aligned with antioxidative enzyme activity where interactive effects with sex-related differences were observed in fish collected the Fall of 2020. In contrast, transcription markers measured in Fall 2021 were increased upstream compared to downstream species. Continued investigation on the impacts of pharmaceutical exposures in non-target organisms is crucial to further the knowledge of WWTP effluent impacts.

The Grand River watershed is the largest in southern Ontario and assimilates thirty wastewater treatment plants (WWTP) with varied degrees of treatment. Many WWTPs are unable to effectively eliminate several contaminants of emerging concern (CECs) from final effluent, leading to measurable concentrations in surface waters. Exposures to CECs have reported impacts on oxidative stress measured through antioxidative enzymes (SOD, CAT, GPX). This study focuses on the effects of WWTP effluent on four Etheostoma (Darter) species endemic to the Grand River, by investigating if increased antioxidative response markers are present in darter brains downstream from the effluent outfall compared to an upstream reference site relative to the Waterloo, ON WWTP across two separate years (Oct 2020 and Oct 2021). This was assessed using transcriptional and enzyme analysis of antioxidant enzymes and an enzyme involved in serotonin synthesis, tryptophan hydroxylase (tph). In fall 2020, significant differences in transcript markers were found between sites and sexes in GSD with SOD and CAT showing increased expression downstream, in JD with both sexes showing increased SOD downstream, and an interactive effect for tph in RBD. Changes in transcripts aligned with enzyme activity where interactive effects with sex-related differences were observed in fish collected fall 2020. In contrast, transcripts measured in fall 2021 were increased upstream compared to downstream species in RBD and GSD. This study additionally displayed yearly, species and sex differences in antioxidant responses. Continued investigation on the impacts of CECs in effluent in non-target species is required to better understand WWTP effluent impacts.

The effluent from municipal wastewater treatment plants is a major point source of contamination in Canadian waterways. The improvement of effluent quality to reduce contaminants, such as pharmaceuticals and personal care products, before being released into the environment is necessary to reduce the impacts on organisms that live in the river downstream. Here, we aimed to characterize the metabolic and gill physiological responses of rainbow (Etheostoma caeruleum), fantail (Etheostoma flabellare), and greenside (Etheostoma blennioides) darters to the effluent in the Grand River from the recently upgraded Waterloo municipal wastewater treatment plant. The routine metabolism of darters was not affected by effluent exposure, but some species had increased maximum metabolic rates, leading to an increased aerobic scope. The rainbow darter aerobic scope increased by 2.2 times and the fantail darter aerobic scope increased by 2.7 times compared to the reference site. Gill samples from effluent-exposed rainbow darters and greenside darters showed evidence of more pathologies and variations in morphology. These results suggest that darters can metabolically adjust to effluent-contaminated water and may also be adapting to the urban and agricultural inputs. The modification and damage to the gills provide a useful water quality indicator but does not necessarily reflect how well acclimated the species is to the environment due to a lack of evidence of poor fish health.

Aquatic organisms are continuously exposed to multiple environmental stressors working cumulatively to alter ecosystems. Wastewater-dominated environments are often riddled by a myriad of stressors, such as chemical and thermal stressors. The objective of this study was to examine the effects of an environmentally relevant concentration of a commonly prescribed antidepressant, venlafaxine (VFX) [1.0 μg/L], in addition to a 5°C increase in water temperature on zebrafish metabolism. Fish were chronically exposed (21 days) to one of four conditions: (i) 0 μg/L VFX at 27°C; (ii) 1.0 μg/L VFX at 27°C; (iii) 0 μg/L VFX at 32°C; (iv) 1.0 μg/L VFX at 32°C. Following exposure, whole-body metabolism was assessed by routine metabolic rate (RMR) measurements, whereas tissue-specific metabolism was assessed by measuring the activities of major metabolic enzymes in addition to glucose levels in muscle. RMR was significantly higher in the multi-stressed group relative to Control. The combination of both stressors resulted in elevated pyruvate kinase activity and glucose levels, while lipid metabolism was depressed, as measured by 3-hydroxyacyl CoA dehydrogenase activity. Citrate synthase activity increased with the onset of temperature, but only in the group treatment without VFX. Catalase activity was also elevated with the onset of the temperature stressor, however, that was not the case for the multi-stressed group, potentially indicating a deleterious effect of VFX on the anti-oxidant defense mechanism. The results of this study highlight the importance of multiple-stressor research, as it able to further bridge the gap between field and laboratory studies, as well as have the potential of yielding surprising results that may have not been predicted using a conventional single-stressor approach.

The objective of this study was to assess the effects of municipal wastewater treatment plant effluent on the energetics and stress response of rainbow darter (Etheostoma caeruleum). Male and female rainbow darter were collected upstream and downstream of the Waterloo WWTP in the Grand River watershed, ON, Canada. To assess the effects of wastewater treatment plant effluent on whole-body and tissue specific metabolic capacity, closed-chamber respirometry and muscle-enzyme activity analyses were performed. Plasma cortisol was also collected from fish before and after an acute air-exposure stressor to evaluate the cortisol stress response in fish exposed to additional stressors. Male and female rainbow darter collected downstream of the effluent had higher oxygen consumption rates, while differences in enzyme activities were primarily associated with sex rather than collection site. No impairment in the cortisol stress response between downstream and upstream fish was observed, however baseline cortisol levels in female fish from the downstream site were significantly higher compared to other baseline groups. Stress-induced cortisol levels were also higher in female fish from both sites when compared to their male counterparts. Overall, this study demonstrates that chronic exposure to WWTP effluent impacts whole-body metabolic performance. This study was also able to demonstrate that sex-differences are a key determinant of various metabolic changes in response to physiological stress, thereby, providing a novel avenue to be considered and further explored.

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.

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.