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.

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.

Circulating plasma microRNAs (miRNAs) are well established as biomarkers of several diseases in humans and have recently been used as indicators of environmental exposures in fish. However, the role of plasma miRNAs in regulating acute stress responses in fish is largely unknown. Tissue and plasma miRNAs have recently been associated with excreted miRNAs; however, external miRNAs have never been measured in fish. The objective of this study was to identify the altered plasma miRNAs in response to acute stress in rainbow trout ( Oncorhynchus mykiss ), as well as altered miRNAs in fish epidermal mucus and the surrounding ambient water. Small RNA was extracted and sequenced from plasma, mucus, and water collected from rainbow trout pre- and 1 h-post a 3-min air stressor. Following small RNA-Seq and pathway analysis, we identified differentially expressed plasma miRNAs that targeted biosynthetic, degradation, and metabolic pathways. We successfully isolated miRNA from trout mucus and the surrounding water and detected differences in miRNA expression 1-h post air stress. The expressed miRNA profiles in mucus and water were different from the altered plasma miRNA profile, which indicated that the plasma miRNA response was not associated with or immediately reflected in external samples, which was further validated through qPCR. This research expands understanding of the role of plasma miRNA in the acute stress response of fish and is the first report of successful isolation and profiling of miRNA from fish mucus or samples of ambient water. Measurements of miRNA from plasma, mucus, or water can be further studied and have potential to be applied as non-lethal indicators of acute stress in fish.

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.