Does intersex matter? A case study of rainbow darter in the Grand River

Endocrine disrupting compounds (EDCs) are present in the environment and can have negative effects on the health of wildlife. Aquatic organisms residing near the outfalls of municipal wastewater effluent (MWWE) are chronically exposed to EDCs, including natural hormones, pharmaceuticals, and industrial chemicals. The vulnerability of aquatic organisms to these compounds is due to the evolutionary conservation of endocrine systems. Although numerous studies have indicated that compounds in MWWE including estrogenic and anti-androgenic contaminants, feminize male fish, it is still uncertain what the consequences of feminization of male fish are. Research on this topic since the early 1990’s has demonstrated that a multitude of compounds in MWWE are capable of binding to estrogen receptors in fish. Key biomarkers of estrogen exposure are the elevation of vitellogenin protein and gene expression levels, as well as the presence of female tissue in male gonads; a condition referred to as intersex. The feminization of male fish and intersex condition has been noted in populations of fish around the world including rainbow darter (Etheostoma caeruleum) in the Grand River, Ontario, Canada. Male rainbow darter collected from sites near MWWE outfalls have intersex condition as well as impaired androgen production and sperm development. The aim of the studies in this thesis was to assess the implications of feminized males using rainbow darter as a model. Several questions were addressed through field and laboratory studies. The first question addressed was whether exposure to MWWE, and intersex in particular, reduced the reproductive success of rainbow darter. To test this, fish were collected in the field during the spawning season, gametes were stripped, and manual fertilizations conducted at each of several sites. Fertilization success and embryo survival were lower at sites downstream of a MWWTP outfall when compared to a rural reference site. Additionally, when grouped into categories based on the severity of intersex (based on number and development of oocytes in testes), it was found that the most severely affected males had the lowest fertilization success. In contrast, no relationship was found between embryo survival and intersex severity, suggesting that egg quality may play a larger role in the survival of embryos. This study concluded that severe intersex condition, which is a marker of MWWE exposure, was an indicator of poor reproductive success in male rainbow darter. The second question addressed in this thesis was whether reproductive behaviour was altered in fish exposed to MWWE. To test this, rainbow darter were collected from sites in the Grand River along an urban gradient and two separate experiments conducted. The first experiment was a breeding competition experiment where three males, each from a different site, were placed into a spawning tank with a single female from either a reference or MWWE exposed site. Reproductive behaviour of both male and female rainbow darter collected near MWWTP outfalls were impaired. Males spent less time guarding the spawning area and females performed fewer nose digs. Both behaviours are important for reproductive success. The second experiment in this study assessed the response of males to a mirror-competitor. Males with severe intersex condition spent less time performing aggressive acts than males with no intersex condition. From these findings, this study concluded that MWWE exposure, as indicated by intersex severity in the second experiment, alters reproductive behaviour of rainbow darter and would likely lead to reduced reproductive fitness in the wild. The third study in this thesis tested whether observations of impaired reproductive success and behaviour are directly associated with MWWE as opposed to other urban stressors. In this study, male and female rainbow darter were collected from a reference site during the pre-spawning period and placed in breeding groups. Following a 10-day pre-exposure spawning period, rainbow darter breeding groups were exposed to 1 of 5 treatments (control, 17α-ethinylestradiol (EE2, a synthetic estrogen), 1%, 10%, or 20% MWWE) for 21 days. After the 21-day exposure, male behaviour was assessed in a mirror-competitor test. Fecundity (number of eggs) was lower in the highest (20%) dose of MWWE during the first week of the exposure but was comparable to other groups for the remainder of the experiment. Fertilization success was also lower during the exposure period compared to the pre-exposure period in MWWE exposed and EE2 exposed fish. Males exposed to 20% MWWE were less aggressive and more active than the control group. Although the responses of this experiment were less dynamic than those observed in fish exposed in the wild, they corroborated the findings that rainbow darter exposed to MWWE have reduced reproductive fitness. The final study in this thesis addressed the issue of repeatability of field studies of feminized males by comparing the variability of biological measures between fall and spring seasons among five years of collections as well as across levels of biological organization. Measures were most consistent in males collected during the fall. Additionally, measures at the tissue level, including sperm development and intersex incidence and severity, were the most consistent measures among years and seasons. This study concluded that the use of multiple biological measures from several levels of biological organization allowed for the same conclusion to be made about the reproductive health of rainbow darter in all years and both seasons of field collections. Throughout these studies, intersex was found to be one of the most reliable markers of exposure to endocrine disrupting compounds in municipal wastewater effluent. Additionally, from the sum of these studies, it can be concluded that highly prevalent and severe intersex in a population of fish indicates poor reproductive fitness. Whether this results in reduced population size or decreased genetic diversity is not known and is an important topic for future studies

Biological responses in fish exposed to municipal wastewater treatment plant effluent in situ

Effluents from municipal wastewater treatment plants (MWTPs) are complex mixtures of chemicals including endocrine-disrupting compounds (EDCs) and 17α-ethynylestradiol (EE2). The objective of this study was to evaluate selected responses of two fish species, in two different years, exposed in situ to MWTP effluent. Biological markers of exposure (plasma vitellogenin (VTG) and antioxidant enzymes) were measured in two species of male fish, rainbow trout (Oncorhynchus mykiss) and fathead minnow (Pimephales promelas), caged at sites associated with wastewater outfall. The estrogenicity of the final effluent in 2010 was determined to be 17.0 + 0.4 ng/L estrogen equivalents (EEQ) and reduced to 7.5 + 2.9 ng/L EEQ after infrastructure upgrades. Pharmaceuticals and personal care products in the effluent and surface water in both years confirmed the exposures at each downstream site. Despite the presence of estrogenic compounds in the MWTP effluent, no effluent-caged male fish demonstrated plasma VTG induction. Minnows and trout that received an intraperitoneal injection of 5 mg/g EE2 showed VTG induction at both field sites. In 2012, the liver somatic index (LSI) of both species increased with exposure, as did changes in antioxidant enzymes, and reactive oxygen species (ROS) activity. Multiple biological mechanisms are modified by effluent exposure, and multiple endpoints are needed to assess risk.Standards Development Branch|| Ontario Ministry of the Environment and Climate Change (OMECC)||the Canadian Water Network||Natural Sciences and Engineering Research Council|| Canada Research Chairsto MRS|| OMECC Laboratory Services Branch||Trent University||University of Ottawa ||Environment and Climate Change Canad

Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater

Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named “N200” focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.Ontario Ministry of the Environment, Conservation and Parks||Natural Sciences and Engineering Research Council of Canad

Near real-time determination of B.1.1.7 in proportion to total SARS-CoV-2 viral load in wastewater using an allele-specific primer extension PCR strategy

"The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome corona- virus 2 (SARS-CoV-2) has claimed millions of lives to date. Antigenic drift has resulted in viral variants with putatively greater transmissibility, virulence, or both. Early and near real-time detection of these variants of concern (VOC) and the ability to accurately follow their incidence and prevalence in communities is wanting. Wastewater-based epidemiology (WBE), which uses nucleic acid amplification tests to detect viral fragments, is a reliable proxy of COVID-19 incidence and prevalence, and thus offers the potential to monitor VOC viral load in a given population. Here, we describe and validate a primer extension PCR strategy targeting a signature mutation in the N gene of SARS-CoV-2. This allows quantification of B.1.1.7 versus non-B.1.1.7 allele frequency in wastewater without the need to employ quantitative RT-PCR standard curves. We show that the wastewater B.1.1.7 profile correlates with its clinical counterpart and benefits from a near real-time and facile data collection and reporting pipeline. This assay can be quickly implemented within a current SARS-CoV-2 WBE framework with minimal cost; allowing early and contemporaneous estimates of B.1.1.7 community transmission prior to, or in lieu of, clinical screening and identification. Our study demonstrates that this strategy can provide public health units with an additional and much needed tool to rapidly triangulate VOC incidence/prevalence with high sensitivity and lineage specificity"National Microbiology Laboratory||Water Services at the Cities of Ottawa and Barrie||Ottawa Public Health||Simcoe Muskoka District Health Unit|| Public Health Ontario||Ontario Wastewater Surveillance Initiativ

The Role of Wastewater Testing for SARS-CoV-2 Surveillance

Wastewater testing for SARS-CoV-2 is relatively new; however, it builds on existing public health surveillance infrastructure. There is a limited but growing evidence base for its use, despite notable interpretation challenges. Wastewater testing results have helped to inform public health policy and interventions during the COVID-19 pandemic in Ontario and other jurisdictions. Wastewater testing for SARS-CoV-2 is useful for early detection of outbreaks and surges as well as population-wide surveillance of COVID-19 that is complementary to clinical testing. Further, it offers an efficient means of SARS-CoV-2 surveillance for specific settings such as correctional facilities, shelters, and university residences. Wastewater testing can also be used for the detection and monitoring of variants of concern (VOCs)

Wastewater to clinical case (WC) ratio of COVID-19 identifies insufficient clinical testing, onset of new variants of concern and population immunity in urban communities

Clinical testing has been the cornerstone of public health monitoring and infection control efforts in communities throughout the COVID-19 pandemic. With the extant and anticipated reduction of clinical testing as the disease moves into an endemic state, SARS-CoV-2 wastewater surveillance (WWS) is likely to have greater value as an important diagnostic tool to inform public health. As the widespread adoption of WWS is relatively new at the scale employed for COVID-19, interpretation of data, including the relationship to clinical cases, has yet to be standardized. An in-depth analysis of the metrics derived from WWS is required for public health units/agencies to interpret and utilize WWS-acquired data effectively and efficiently. In this study, the SARS-CoV-2 wastewater signal to clinical cases (WC) ratio was investigated across seven different cities in Canada over periods ranging from 8 to 21 months. Significant increases in the WC ratio occurred when clinical testing eligibility was modified to appointment-only testing, identifying a period of insufficient clinical testing in these communities. The WC ratio decreased significantly during the emergence of the Alpha variant of concern (VOC) in a relatively non-immunized community’s wastewater (40-60% allelic proportion), while a more muted decrease in the WC ratio signaled the emergence of the Delta VOC in a relatively well-immunized community’s wastewater (40-60% allelic proportion). Finally, a rapid and significant decrease in the WC ratio signaled the emergence of the Omicron VOC, likely because of the variant’s greater effectiveness at evading immunity, leading to a significant number of new reported clinical cases, even when vaccine-induced community immunity was high. The WC ratio, used as an additional monitoring metric, complements clinical case counts and wastewater signals as individual metrics in its ability to identify important epidemiological occurrences, adding value to WWS as a diagnostic technology during the COVID-19 pandemic and likely for future pandemics.Ontario's Ministry of Environment, Conservation and Parks||Alberta Healt

Community Surveillance of Omicron in Ontario: Wastewater-based Epidemiology Comes of Age

Wastewater-based surveillance of SARS-CoV-2 RNA has been implemented at building, neighbourhood, and city levels throughout the world. Implementation strategies and analysis methods differ, but they all aim to provide rapid and reliable information about community COVID-19 health states. A viable and sustainable SARS-CoV-2 surveillance network must not only provide reliable and timely information about COVID-19 trends, but also provide for scalability as well as accurate detection of known or unknown emerging variants. Emergence of the SARS-CoV-2 variant of concern Omicron in late Fall 2021 presented an excellent opportunity to benchmark individual and aggregated data outputs of the Ontario Wastewater Surveillance Initiative in Canada; this public health-integrated surveillance network monitors wastewaters from over 10 million people across major population centres of the province. We demonstrate that this coordinated approach provides excellent situational awareness, comparing favourably with traditional clinical surveillance measures. Thus, aggregated datasets compiled from multiple wastewater-based surveillance nodes can provide sufficient sensitivity (i.e., early indication of increasing and decreasing incidence of SARS-CoV-2) and specificity (i.e., allele frequency estimation of emerging variants) with which to make informed public health decisions at regional- and state-levels.Ontario Ministry of the Environment, Conservation and Parks|| Genome Canada and Ontario Genomics (OGI-209)||NSERC (ALLRP 555041-20 to C.O.)||Ontario Clean Water Agenc

Emergence and Spread of the SARS-CoV-2 Omicron Variant in Alberta Communities Revealed by Wastewater Monitoring

Wastewater monitoring of SARS-CoV-2 allows for early detection and monitoring of COVID-19 burden in communities and can track specific variants of concern. Targeted assays enabled relative proportions of SARS-CoV-2 Omicron and Delta variants to be determined across 30 municipalities covering >75% of the province of Alberta (pop. 4.5M) in Canada, from November 2021 to January 2022. Larger cities like Calgary and Edmonton exhibited a more rapid emergence of Omicron relative to smaller and more remote municipalities. Notable exceptions were Banff, a small international resort town, and Fort McMurray, a more remote northern city with a large fly-in worker population. The integrated wastewater signal revealed that the Omicron variant represented close to 100% of SARS-CoV-2 burden prior to the observed increase in newly diagnosed clinical cases throughout Alberta, which peaked two weeks later. These findings demonstrate that wastewater monitoring offers early and reliable population-level results for establishing the extent and spread of emerging pathogens including SARS-CoV-2 variants.Alberta Healt

Data from - An assessment of the spatial and temporal variability of biological endpoint responses of rainbow darter collected along an urban gradient.

Municipal wastewater effluent (MWWE) and its constituents, such as chemicals of emerging concern, pose a potential threat to the sustainability of fish populations by disrupting key endocrine functions in aquatic organisms. While studies have demonstrated changes in biological markers of exposure of aquatic organisms to groups of chemicals of emerging concern, the variability of these markers over time has not been sufficiently described in many wild fish species. The aim of this study was to assess the spatial and temporal variability of biological markers in response to MWWE exposures and to test the consistency of these same responses between seasons and among years. Rainbow darter (<i>Etheostoma caeruleum</i>) were collected in spring and fall seasons over a five-year period in the Grand River, ON, Canada. In addition to surface water chemistry (nutrients and selected pharmaceuticals), measures were taken across levels of biological organization in rainbow darter. The measurement of hormone production, gonad development, and intersex severity were temporally consistent and suggested impaired reproduction in male fish collected downstream of MWWE outfalls.  In contrast, ovarian development and hormone production in females appeared to be influenced more by urbanization than MWWE.  Measures of gene expression and somatic indices were highly variable between sites and years respectively, and were inconclusive when determining the impacts of MWWE overall. Robust biomonitoring programs must consider these factors in both the design and interpretation of results, especially when spatial and temporal sampling of biological endpoints is limited.  Assessing the effects of contaminants and other stressors on fish in watersheds would be greatly enhanced by an approach that considers natural variability in the endpoints being measured

An Assessment of the Spatial and Temporal Variability of Biological Responses to Municipal Wastewater Effluent in Rainbow Darter (<i>Etheostoma caeruleum</i>) Collected along an Urban Gradient

<div><p>Municipal wastewater effluent (MWWE) and its constituents, such as chemicals of emerging concern, pose a potential threat to the sustainability of fish populations by disrupting key endocrine functions in aquatic organisms. While studies have demonstrated changes in biological markers of exposure of aquatic organisms to groups of chemicals of emerging concern, the variability of these markers over time has not been sufficiently described in wild fish species. The aim of this study was to assess the spatial and temporal variability of biological markers in response to MWWE exposure and to test the consistency of these responses between seasons and among years. Rainbow darter (<i>Etheostoma caeruleum</i>) were collected in spring and fall seasons over a 5-year period in the Grand River, Ontario, Canada. In addition to surface water chemistry (nutrients and selected pharmaceuticals), measures were taken across levels of biological organization in rainbow darter. The measurements of hormone production, gonad development, and intersex severity were temporally consistent and suggested impaired reproduction in male fish collected downstream of MWWE outfalls. In contrast, ovarian development and hormone production in females appeared to be influenced more by urbanization than MWWE. Measures of gene expression and somatic indices were highly variable between sites and years, respectively, and were inconclusive in terms of the impacts of MWWE overall. Robust biomonitoring programs must consider these factors in both the design and interpretation of results, especially when spatial and temporal sampling of biological endpoints is limited. Assessing the effects of contaminants and other stressors on fish in watersheds would be greatly enhanced by an approach that considers natural variability in the endpoints being measured.</p></div