The Government of Canada launched the Chemicals Management Plan (CMP) in 2006, which aims to reduce the risk posed by chemical substances to human health and the environment. In 2009, the CMP was expanded to include a wastewater monitoring program led by the Wastewater Science Unit of Environment and Climate Change Canada. The purpose of this program is to generate data on the concentrations of priority substances in wastewater and biosolids from representative municipal wastewater treatment plants (WWTPs) in Canada. Since 2009, the Wastewater Monitoring Program has included over 80 WWTPs. Wastewater and biosolids data from this monitoring program provide important information regarding a major pathway for chemicals entering the environment and the effectiveness of different wastewater treatment processes. However, data produced by federal departments and agencies have not historically been easily accessible to the public. In response, the Government of Canada launched the Open Government initiative as a commitment from the federal government to be transparent, accountable, and to engage with Canadians. This includes the sharing of data through an Open Government Portal. To date, the Wastewater Monitoring Program has published nine years of data (2010-2019) on seven priority substance groups to the Portal including: hexabromocyclododecane, bisphenol A, tetrabromobisphenol A, triclosan, nonylphenols and ethoxylates, halogenated flame retardants, and organophosphate flame retardants in wastewater and biosolids. Upcoming datasets include polybrominated diphenyl ether and other brominated flame retardants, and per- and polyfluorinated alkyl substances. This presentation will provide an overview of the monitoring program and a summary of the datasets that are and will be available through the Open Government Portal.

Groundwater containing a complex mixture of known and unknown contaminants was collected from an industrial site for characterization of toxic effects. The study involved a 60-day oral exposure to drinking water (control group), 10% v/v low impact water from a low impact well on-site compared to 0.01% v/v, 0.1% v/v, 1% v/v and 10% v/v of high impact groundwater concentration (n= 5 males and 5 females/group). The dose-response study showed a reduced plasma alpha-2 macroglobulin in groups exposed to low impact and high impact groundwater. The kidney histopathology in both low and high impact groups showed evidence of acute tubular necrosis and an increase in glomerular-specific nephrotoxic biomarker, plasma symmetric dimethylarginine, which was significantly elevated in the 1% and 10% v/v high impact groups. Cerebral blood flow directly correlated to the exposure concentrations of high impact groundwater. Conversely, diminishing rotarod performance test was associated with increased high impact groundwater concentration. In summary, the dose-response study showed evidence of immunotoxicity, nephrotoxicity and neurotoxicity. Based on the pattern of histopathology, hydrocarbons are suspected to be the class of contaminants driving toxicity in the groundwater samples and will be confirmed with further analyses.

Emerging contaminants such as pharmaceutical drugs have been detected in waters across the globe. Many pharmaceuticals are found at trace concentrations, but the continuous use and potential accumulation of some of these compounds can lead to potential effects on aquatic organisms. Wastewater treatment plants are not designed to remove pharmaceuticals, so these compounds are virtually unattained before the treated water is discharged. Consequently, WWTPs’ management have a key role in the removal and monitoring of pharmaceuticals. Pharmaceutical concentrations can be affected by temporal variations, the flow velocity of water receptors, the sorption capacity of sediments, and other abiotic dynamics in aquatic ecosystems. Currently, most experimental approaches have not considered these dynamics to evaluate the chemical activity, bioavailability, and toxicity of pharmaceuticals. The principal aim of this research is to enhance our understanding of the environmental risks associated with pharmaceuticals as a group of emerging contaminants. To this end, the presence of a suite of representative pharmaceuticals was measured upstream and downstream of two WWTPs located in the South Saskatchewan River basin and Wascana Creek, Saskatchewan, Canada, during three sampling campaigns (spring, summer, and fall of 2021), through both conventional and passive water sampling. According to the chemical analysis conducted, Amitriptyline with a concentration of 3353.8 ng/L was the most abundant compound in comparison to the seven pharmaceuticals evaluated in the water and sediment samples at the four sampling sites and across the three seasons. Generally, concentrations of analytes were higher downstream of WWTPs compared to upstream sites. The data collected indicates widespread contamination with psychoactive pharmaceuticals, which can have marked impacts on exposed organisms.

In natural systems, contaminations consist in complex mixtures of compounds that are constantly subjected to processes of transformation and degradation. This can lead to difficulties in using controlled laboratory studies to estimate risk as the system is in flux. The objective of this study was to evaluate whether toxic effects determined for complex environmental mixtures using a controlled, laboratory-based short-term reproduction assay with fish would be predictive of those observed under a more environmentally relevant scenario (i.e., outdoor mesocosms). To do this, 21-day fish short-term reproduction assays were conducted by exposing adult fathead minnows (Pimephales promelas) to gradient of concentrations of groundwater samples collected from a legacy contaminated site under both laboratory and outdoor mesocosm scenarios. Under laboratory conditions fish showed a decrease in the cumulative number of eggs produced, a slight reduction in the number of nuptial tubercles of males and an increase of the presence of micronuclei in fish erythrocytes in a concentration dependent manner. In contrast, the mesocosm scenario revealed a greater cumulative number of eggs produced at the highest concentration of exposure. However, no significant differences were observed for the other endpoints assessed in the mesocosms. Several factors could have explained these differences in observations, including the aging of groundwater in mesocosms before fish addition (e.g., evaporation of toxic volatile compounds) and possible uptake of contaminants by plants and invertebrates. Overall, outcomes observed under laboratory conditions were protective of a more environmentally relevant scenario and they would be effective in ecological risk assessment.

Monitoring the presence and concentrations of pesticides in Ontario’s aquatic systems is necessary to understand the impacts on the aquatic environment. Ontario’s streams are susceptible to pesticide pollutants which are transported outside of the intended area of application from surrounding agricultural fields. Biofilms are a collective of microorganisms that grow on hard surfaces in aquatic ecosystems and have been shown to bioconcentrate pesticides in water. Biofilms are highly responsive to chemical and biological changes in the environment, and therefore have the potential to act as a cost-effective, integrated sampling tool to monitor pesticide exposures in aquatic ecosystems. The objective of this study is to determine whether biofilms can be used to provide an accurate representation of pesticide exposure in Ontario’s aquatic systems. Ten sites across Southern Ontario were sampled between May-September 2021. At each site, water, sediment and biofilm, colonizing both artificial and natural substrate, were collected and analyzed for the presence of ~500 pesticides. This data will be used to determine the distribution of pesticides in the three matrices (water, sediment, and biofilm) and will provide an assessment of how well each matrix characterizes the streams' exposure to pesticides. It is hypothesized that the partitioning of pesticides within water, sediment and biofilm will be related to the physicochemical properties of the detected pesticide. The development of a novel and cost-effective sampling tool would benefit Ontario’s current pesticide monitoring program and may greatly improve the reporting on pesticide pollutants and water quality in Southern Ontario’s aquatic environments.

Antidepressant drugs are present in freshwater ecosystems worldwide and the antidepressant venlafaxine has been detected at >2.0 μg/L. Laboratory studies have shown that although venlafaxine is not acutely lethal to aquatic organisms at environmentally relevant concentrations, it can cause sub-lethal and behavioural changes in fish. The chronic and indirect effects of persistent venlafaxine exposure to aquatic ecosystems have not been investigated, and hence this study was initiated. Limnocorrals (n=10, 2-m diameter, 1.5-m deep) were deployed in a lake at the IISD-Experimental Lakes Area and spiked with venlafaxine for a 10-week exposure to assess the potential risk chronic venlafaxine exposure poses to ecosystem health. Specifically, we assessed the responses of native phytoplankton, emergent insect, and macroinvertebrate communities during this period. For most endpoints, minimal differences were observed at environmentally relevant concentrations. Environmentally relevant concentrations were not directly lethal to aquatic biota but water quality impacts and shifts in community structure were noted in higher treatments. Indications of behavioural changes in fish could suggest reduced predation pressure and indirect effects on ecosystem structure. This study integrates the whole ecosystem response, providing a unique view into the long exposure, sublethal, effects of a commonly detected freshwater contaminant.

Microplastics (<5mm diameter) are an emerging concern to the health of various ecosystems. To date, the overwhelming majority of microplastic research has focused on their impacts on aquatic ecosystems, leaving a knowledge gap surrounding their effects on terrestrial ecosystems. One source that could potentially contribute a significant amount of microplastic contamination to soil ecosystems are biosolids. Biosolids are a nutrient-rich field amendment that are derived from wastewater treatment processing. In December 2021, crude biosolid samples were collected from six wastewater treatment plants in hopes of characterizing the magnitude and type of microplastic within biosolids that are used for land application. Additionally, in April and May 2022, soil samples were taken from thirteen Southern Ontario farm fields that had been amended with biosolids within the last four years. To ensure proper random sampling, a statistical model that incorporated the slope, slope length and steepness factor (LS-factor), and topographical wetness index of each field was used to generate 20 sampling points per field. Microplastic quantification was performed via density separation with olive oil and FTIR spectroscopy. This data will be used to determine a quantitative assessment of the exposure levels of microplastics in biosolid-amended fields. These results will then be coupled with data from microplastic toxicity tests with Eisenia andrei to perform an environmental risk assessment. Toxicity tests will be performed with varying types (polystyrene, polyester, polypropylene) and sizes of polymer, ranging from 6 um to 1000 um.

Environmental DNA (eDNA) may be captured through collection of bulk environmental samples and analyzed using DNA barcoding approaches to detect biological species without organism capture or sighting. Whole community biodiversity assessment is possible through eDNA metabarcoding, using universal primers and next-generation sequencing technology. However, limitations imposed by the taxonomic coverage of existing universal primer sets and their corresponding databases remains a significant challenge for community detection. Accurate, reliable inferences of community composition using eDNA require optimal primer selection through in vitro validation for the community of interest. In this study, we aimed to evaluate and compare taxonomic coverage using two existing universal primer sets for eDNA metabarcoding of fishes in the Grand River watershed. Water was sampled from the Grand and Eramosa Rivers on three properties with permission from the rare Charitable Research Reserve. eDNA was extracted and amplified by PCR using two primer sets, each targeting a short region of a mitochondrial gene: 12S rRNA and cytochrome oxidase I (COI). A second-round PCR affixed multiplexing indices and Illumina adaptors prior to sequencing on the Illumina MiSeq platform. Demultiplexed sequence reads were processed using MetaWorks(10.1.0), including pairing, trimming, merging, denoising, and deduplicating reads before taxonomic assignment of ESVs using classifiers trained on the MitoFish and NCBI GenBank databases, for 12S and COI, respectively. Using a locally-curated mock community as a positive control, primer sets will be evaluated based on the alpha diversity of the detected fish communities and compared to identify potential gaps in taxonomic coverage. The conclusions will highlight limitations surrounding taxonomic coverage and inform primer selection in future eDNA studies of local fish communities towards the optimization of this innovative community assessment approach in Canadian freshwater environments.

The Grand River, Waterloo is an outlet for many wastewater treatment plants (WWTP), which release contaminants into the environment. Darters (Etheostoma spp.) living downstream of the Waterloo WWTP have been shown to be under metabolic stress, however, the mechanisms controlling this stress response are not fully understood. This study hypothesized that microRNA, small post-transcriptional regulators, play a role in regulating mitochondrial gene expression in these fish. In mammalian cell culture, microRNA have been shown to be transported into mitochondria, where they are called mitomiRs and influence expression of mitochondrial genes involved in oxidative phosphorylation. Despite these findings in mammals, no study in fish has demonstrated the presence of mitomiRs, nor examined how they influence mitochondrial gene expression. To test the hypothesis, rainbow, fantail and Johnny darters were collected from upstream and downstream of the Waterloo WWTP, and the mitochondrial fractions of their livers were isolated. Bioinformatic analysis identified candidate mitomiRs and their target mitochondrial transcripts, which were quantified using qPCR. In addition, cytochrome c oxidase (COX) activity was measured for each species, and COX2 protein abundance was quantified in rainbow darters. MitomiRs miR-1, let-7a, miR-20 and miR-122 were detected in darter mitochondria, and as predicted, miR-1 was upregulated downstream in rainbow darters, with a corresponding decrease in COX activity. In addition, one sex-specific mitomiR, miR-20 was also identified. This novel research demonstrated that mitomiRs are present within fish and predicted their mode of action within darters to be mainly suppressing transcript translation. Future work could define how mitomiRs regulate mitochondrial gene expression in fish.

Bats are potentially exposed to pesticides via foraging in croplands. Common pesticides like organophosphates are neurotoxic for vertebrates and even low doses can impair essential processes such as locomotion and cognition. These sublethal effects are usually studied using molecular biomarkers with limited ecological relevance. Behavioral studies, in contrast, represent a more informative yet sensitive approach. Spatial navigation, for example, is an ecologically relevant behavior that is modulated by cellular pathways potentially targeted by neurotoxicants. We evaluated whether bats’ ability to memorize and navigate novel spaces was negatively affected by environmental relevant doses of chlorpyrifos, a common organophosphate insecticide. We also tested how the behavioral response correlated with molecular biomarkers. We orally dosed captive big brown bats (Eptesicus fuscus) with chlorpyrifos and studied exploratory behavior in two testing arenas. We evaluated similarity of stereotype flight trajectories in a flight tent, and associative memory in a Y-maze. We quantified molecular biomarkers including cholinesterase (ChE) activity and employed non-targeted proteomics in bat brain tissue. Bats exposed to chlorpyrifos were less explorative and made more incorrect choices in the Y-maze, but the consistency of their flight trajectories was unaffected. Exposed bats had 30% lower ChE activity, showed down-regulation of proteins involved in memory (VP37D), learning and sound perception (NOX3). Other important nervous system processes such as synaptic function, plasticity, oxidative stress, and apoptosis were enriched in chlorpyrifos-exposed bats. These results support the sensitivity of behavior as a biomarker of toxicity and the importance of considering other levels of organization to help explain the mechanisms underlying altered behavior due to human activities.

Few studies have examined the effects of oil spills on the early life stages of amphibians and this is seen as a significant knowledge gap in freshwater protection. To address this, parallel oil spill studies were conducted at the International Institute for Sustainable Development-Experimental Lakes Area in northwestern Ontario. The Freshwater Oil Spill Remediation Study (FOReSt) used in-lake enclosures to determine the effectiveness of non- invasive oil spill remediation techniques in different shoreline habitats and the Floating Wetland Treatments to Enhance Remediation (FloWTER) study examined the efficacy of plant-microbe relationships for in situ degradation of oil-derived hydrocarbons. In 2021, uptake and effects of in situ chronic exposure to conventional heavy crude oil (CHV) (FOReSt) or a CHV water-accommodated fraction (FLoWTER) on the growth and development of wood frog (Lithobates sylvaticus) tadpoles were characterized. Within the FLoWTER mesocosms, there were no apparent treatment effects when examining total length, snout-vent length and weight. Bioaccumulation of total polycyclic aromatic hydrocarbons (TPAH) in tadpole whole bodies were also not statistically different among treatments. Qualitative histology analysis also supports these results, as there were no apparent differences when examining cellular changes within the liver. Similar results were observed in the FOReSt study regarding apical endpoints and histology analysis; however, bioaccumulation of TPAH in the in-lake enclosures found higher concentrations of TPAHs in oiled compared to non-oiled treatments. This research provides much needed data on the toxicological effects of CHV on amphibians to inform risk assessment related to the over-land transportation of crude oil and potential impacts of freshwater oil spills.

Widespread use of plastic materials has led to the ubiquitous distribution of microplastics (plastic particles <5 mm) in the environment. Within freshwaters, large quantities of microplastics are discharged through wastewater effluents, but their fate and bioaccumulation remain understudied. A persisting issue is the lack of ideal bioindicators to monitor microplastics in these ecosystems. Fingernail clams (Sphaeriidae) are common bivalves with potential to be novel indicator organisms for microplastics pollution. This study investigates microplastics in fingernail clams near municipal wastewater treatment plants (WWTPs) in the Grand River, Ontario. Fingernail clams were collected upstream and downstream of two WWTPs and at two reference sites. Clams were measured and weighed, and microplastics extracted from the tissues using a 10% KOH solution. Suspected microplastics were visually identified and characterized. The concentration of these microparticles varied significantly among sites, with a mean of 1.39 ± 0.82 and a maximum of 7 microparticles per clam. These microparticles were elevated in clams immediately downstream of the Hespeler wastewater outfall compared to the upstream site, but this trend was not seen at the Caledonia WWTP. Differences could be due to the populations served by the plants or level of wastewater treatment employed. Suspected plastics were mainly fibers (92%) and consisted of clear (54%) or blue (21%) colours. Overall, microparticles are accumulating in fingernail clams in the Grand River, highlighting their ability to be used as a bioindicator for microplastics. Future work will determine the chemical composition of the suspected plastic particles and compare the microplastics found in fingernail clams to data from mussels, water, and sediment from the same sites.