Previous research indicates that pharmaceutically active compounds (PhACs) and endocrine-disrupting compounds (EDCs) are poorly removed during conventional drinking water treatment processes including coagulation; however, removal efficiency increases in the presence of natural organic matter (NOM). Therefore, this project investigates the link between various NOM types with EDC/PhAC removal. Bench-scale coagulation tests were conducted on three different source waters spiked with environmentally relevant levels (nominally 1000 ng/L) of EDCs/PhACs. Two different coagulants were used: polyaluminum chloride (PACl) and aluminum sulphate (alum). NOM was characterized using size exclusion liquid chromatography-organic carbon detection (LC-OCD). Results for Lake Ontario, Otonabee and Grand River water indicate that certain EDCs/PhACs are significantly removed during coagulation while others increase in concentration. Concurrently, particular NOM fractions (biopolymers and humic substances) are also being removed. Solvents used for EDC/PhAC spiking (acetone and acetonitrile) did not affect coagulation, but contributed to low molecular weight neutral and hydrophobic NOM fractions.

Endocrine disruption and high occurrences of intersex have been observed in wild fish associated with wastewater treatment plant (WWTP) effluents in the urbanized reaches of the Grand River watershed located in southern Ontario, Canada. WWTP effluent is a complex matrix with diverse aquatic environmental contaminants and stressors. This study aimed to: (1) characterize the spatio-temporal distribution and fate of antiandrogenic personal care products (triclosan, chlorophene, and dichlorophene), along with selected pharmaceuticals (carbamazepine, ibuprofen, naproxen, and venlafaxine) and the herbicide, atrazine in the Grand River watershed and (2) model the behaviour of these contaminants in the aquatic environment. Water sampling of 29 sites which covered six municipal WWTPs and ~100 km of river length was completed during summer low flows (July 2012). Monthly samples were also collected immediately upstream and downstream of a major WWTP (Kitchener) from August to November 2012. Many of the target pharmaceuticals and triclosan were detected in WWTP effluents in the Grand River watershed, especially those that did not nitrify (minimal treatment with high ammonia). Chlorophene was either undetected or was only found at trace levels in the effluents. Under low flow conditions, triclosan and several other pharmaceuticals exhibited a spatial pattern where concentrations increased directly downstream of the WWTPs, then decreased with distance downstream (dilution and/or degradation). Chlorophene, in contrast, was not found downstream of most of the WWTP outfalls but was first detected at a site 5 km upstream of a WWTP and then continued with relatively constant concentrations for approximately 29 km downstream. It was also only found during the summer sampling period. Atrazine was consistently found in all sampling locations which reflected the agricultural non-point source nature of this compound. The WASP 7.5 model (US Environmental Protection Agency) was adapted and calibrated to a reach of the Grand River associated with the Kitchener WWTP. The simulation of the fate and transport of the target compounds revealed that flow-driven transport processes (advection and dispersion) greatly influence their behaviour in the aquatic environment. However, fate mechanisms such as biodegradation and photolysis also potentially play an important role in the attenuation of most compounds. The exception was carbamazepine where it was shown to act as a conservative tracer compound for wastewater specific contaminants in the water phase. The fate model developed can be applied in the future to predict the fate of a wide variety of contaminants of emerging concern across the watershed to help define the exposure of these biologically active chemicals to sensitive ecosystems.

Endocrine disruption and high occurrences of intersex have been observed in wild fish associated with wastewater treatment plant (WWTP) effluents in the urbanized reaches of the Grand River watershed located in southern Ontario, Canada. WWTP effluent is a complex matrix with diverse aquatic environmental contaminants and stressors. This study aimed to: (1) characterize the spatio-temporal distribution and fate of antiandrogenic personal care products (triclosan, chlorophene, and dichlorophene), along with selected pharmaceuticals (carbamazepine, ibuprofen, naproxen, and venlafaxine) and the herbicide, atrazine in the Grand River watershed and (2) model the behaviour of these contaminants in the aquatic environment. Water sampling of 29 sites which covered six municipal WWTPs and ~100 km of river length was completed during summer low flows (July 2012). Monthly samples were also collected immediately upstream and downstream of a major WWTP (Kitchener) from August to November 2012. Many of the target pharmaceuticals and triclosan were detected in WWTP effluents in the Grand River watershed, especially those that did not nitrify (minimal treatment with high ammonia). Chlorophene was either undetected or was only found at trace levels in the effluents. Under low flow conditions, triclosan and several other pharmaceuticals exhibited a spatial pattern where concentrations increased directly downstream of the WWTPs, then decreased with distance downstream (dilution and/or degradation). Chlorophene, in contrast, was not found downstream of most of the WWTP outfalls but was first detected at a site 5 km upstream of a WWTP and then continued with relatively constant concentrations for approximately 29 km downstream. It was also only found during the summer sampling period. Atrazine was consistently found in all sampling locations which reflected the agricultural non-point source nature of this compound. The WASP 7.5 model (US Environmental Protection Agency) was adapted and calibrated to a reach of the Grand River associated with the Kitchener WWTP. The simulation of the fate and transport of the target compounds revealed that flow-driven transport processes (advection and dispersion) greatly influence their behaviour in the aquatic environment. However, fate mechanisms such as biodegradation and photolysis also potentially play an important role in the attenuation of most compounds. The exception was carbamazepine where it was shown to act as a conservative tracer compound for wastewater specific contaminants in the water phase. The fate model developed can be applied in the future to predict the fate of a wide variety of contaminants of emerging concern across the watershed to help define the exposure of these biologically active chemicals to sensitive ecosystems.

Biosolids are the nutrient-rich organic residual materials resulting from the treatment of domestic sewage at a wastewater treatment facility, and are increasingly land-applied for agricultural and land-reclamation purposes as part of the wastewater management process. While the presence and fate of estrogenic endocrine-disruptors (eEDCs) in wastewater has been extensively studied, much less focus has been given to examining the presence and fate of eEDCs during biosolids treatment. In particular, little work has been done to measure the net estrogenic potency of biosolids using in vitro bioassays, such as the Yeast Estrogen Screen (YES) assay. This is despite the fact that widespread land-application of biosolids provides for the direct introduction of eEDCs into terrestrial and aquatic environments. The relative scarcity of bioassay-based net estrogenicity data for sludges and biosolids is in large part due to the analytical challenges involved in working with such a complex sample matrix. Comprehensive sampling at wastewater treatment plants in Guelph and London, ON, demonstrated that the estrogenicity of anaerobically-treated biosolids is considerably lower (12.0-19.7 ng/g estradiol-equivalents) than that reported in earlier published studies. The results of the present study were made possible due to the development of a sample preparation methodology that overcame the toxic effects that sludge and biosolid samples typically exert on yeast cells in the YES assay. An anti-estrogenicity assay was also applied for the first time to sludges/biosolids to measure the extent to which antagonistic compounds ‘block’ the response of the YES assay. The results of these tests suggest that although the net estrogenicity of anaerobically treated solids is indeed low, up to twice the amount of estrogenicity measured by the YES assay may be masked in biosolids by the presence of antagonistic compounds. While aerobic treatment conditions reduced net estrogenicity to at-or-below detectable levels, net estrogenicity remained relatively constant throughout the unit processes of the anaerobic treatment train. Biosolid ageing during storage led to an overall decrease in net estrogenicity of both conventionally-treated “restricted use” and advanced-treated “unrestricted use” anaerobic biosolids. However, levels of net estrogenicity were observed to spike during the early stages of storage, particularly under freeze/thaw conditions. / Natural Science and Engineering Research Council of Canada (NSERC) PGS-D3 scholarship, Water Environment Association of Ontario, Canadian Water Network

Organic and inorganic compounds are present as contaminants in varying concentrations throughout our water cycle. Examples of these contaminants include the endocrine disrupting compounds (EDCs) bisphenol-A (BPA) and 17β-estradiol (E2) from plastics and pharmaceutical use. It can be necessary to obtain the concentration of these compounds within the water cycle for analysis by interested parties such as research groups, regulatory agencies, and private organizations. These concentrations, however, can be too dilute within the initial sample for analysis. Therefore it is necessary to concentrate the compound of interest (analyte) prior to analysis. One such way to do this is by way of Solid Phase Extraction (SPE). SPE uses a small cartridge which contains chromatographic packing material to chemically extract analytes from a water sample onto a solid phase. To increase concentration, these analytes are then transferred (eluted) to a substantially smaller volume of organic solvent for eventual analyses. These commercially available cartridges are relatively inexpensive, approximately $5 each. However, these cartridges are labeled as single use. In large-scale analyses, this can quickly add up to a sizable percentage of the analysis budget. Additionally, sizable waste volumes can be generated from these analyses in the form of non-degradable polypropylene plastic. If these cartridges can be re-used, material costs as well as waste volumes can be substantially reduced. However, little is known regarding how the quality of analysis degrades with cartridge re-use. The objective of this project is to evaluate the number of times SPE cartridges can be reused without compromising the results of the subsequent analyses. Based on a review of prior literature, I identified and developed protocols for extracting analytes (BPA and E2) from water via SPE, then analyzing them with gas chromatography and mass spectrometry (GC-MS). These protocols have been developed to mimic those employed by research labs, industry, and other entities for which the results of this study would be most applicable. The only deviation is the re-use of the cartridge rather than disposal and replacement. One type of commercially available SPE cartridge (Oasis HLB, Waters Inc., Milford, MA) was used and two water types were tested. The water was spiked with fixed concentrations of BPA and E2, and then analyzed by way of SPE/GC-MS. For both water types, I performed multiple SPE runs on 10 cartridges each. I tracked the history of GC-MS peak areas, which indicate apparent analyte concentration. Peak area data were analyzed as a function of the number of analyses performed (run number), and evaluated for statistically significant changes as well as overall trends. Statistically significant change and/or trends would indicate that the cartridge had exceeded the maximum allowable number of re-uses and would thereby identify the number of times the “single-use” cartridge can reliably be re-used. Peak area history for 20 SPE runs per cartridge for pure water samples and 10 SPE runs for wastewater effluent showed no statistically significant changes or trends on peak area. This indicates that cartridges can be re-used at least 10 times without compromising the integrity of water sample analysis for the EDCs considered in this study.

In this thesis, we have analysed the effects of EE2 on non-reproductive behaviours and fertility. We have showed that two doses of EE2 in male adult short-term exposures evokes opposite behaviours in the novel tank test. A lower dose induced increased bottom-dwelling, a sign of increased anxiety and a higher dose increased surface-dwelling, which would likely expose themselves to predation in a natural environment. Increased shoaling was observed in both exposures, possibly affecting feeding and reproduction opportunities. Fertility analysis of these fish demonstrated a complete inhibition of spawning in the highest dose group. To investigate mechanisms behind the spawning failure, we examined expression levels of genes involved in zebrafish sex differentiation and maintenance of gonadal function. We found downregulated transcription levels of male-predominant genes, suggesting a demasculinization of the testes contributing to functional sterility in these fish. We have demonstrated that non-reproductive behaviour in zebrafish is highly sensitive to EE2 exposure during development. After exposing male and female zebrafish to low doses of EE2 followed by remediation in clean water until adulthood, the fish displayed increased anxiety and shoaling behaviour, demonstrating persistent effects of EE2. Furthermore, behavioural effects were transferred to their progeny. Decreased fertilisation success of the developmentally exposed fish was observed in both sexes when mated to untreated animals of the opposite sex. These fertility effects persisted although the fish had a long remediation period, implying likely reduced fitness of fish populations in aquatic environments. Based on our findings on non-reproductive behaviours and fertility, we performed RNAsequencing analysis of the brain and testes in order to investigate possible biological mechanisms behind the persistent effects. There is a need for biomarkers allowing detection of both reversible and irreversible effects in animals exposed to estrogenic substances, hopefully contributing to better risk assessments for EDCs. Results from RNA-sequencing would serve as a basis for continued studies in pursuit of potential biomarkers.

Notre étude a porté sur le diagnostic de la présence des perturbateurs endocriniens dans le golfe de Gabès (Tunisie), leurs effets sur la reproduction d’une espèce bioindicatrice de pollution, la palourde R. decussatus ainsi que leurs mécanismes d’action. Pour atteindre ces objectifs, nous avons fait appel à plusieurs outils méthodologiques, des expositions in vivo (effluents urbains, effluents industriels, 17β-œstradiol) et des prélèvements in situ afin d’évaluer le risque encouru par l’écosystème du golfe de Gabès tout entier en utilisant une approche multiparamétrique basée sur différents niveaux de l’organisation biologique (allant du niveau biochimique jusqu’au niveau populationnel). Nous avons mis en évidence la présence des composés reprotoxiques tout au long de la côte Sud-Est de la Tunisie. Les résultats obtenus ont révélé une corrélation entre la contamination chimique (cadmium, activités œstrogéniques et androgéniques au niveau du sédiment) et certains paramètres biologiques liés à la reproduction (diamètre ovocytaire, vitellogénine, glycogène) et un site très divergent de tous les autres sites (El hofra). Cette station d’El hofra semble être touchée par une perturbation endocrinienne liée probablement à une synergie entre la contamination par le Cd et la présence des composés à activité œstrogénique et androgénique dans le milieu. En parallèle à cette étude in situ, trois expérimentations au laboratoire ont été réalisées. Les résultats ont montré que les effluents de la station d’épuration de Sfax (cocktail de contaminants) sont capables d’affecter la reproduction de la palourde en induisant la synthèse de la vitellogénine et stimulant la reproduction chez les deux sexes. L’exposition des palourdes à un contaminant pur, le 17β-œstradiol, a révélé qu’il peut contribuer à expliquer les perturbations du cycle reproducteur observées dans l’expérience précédente. Cette hormone naturelle a un effet et sans doute un rôle dans la physiologie de la palourde. Dans le but d’étudier son mécanisme d’action via le récepteur aux œstrogènes (RE), nous avons cloné une partie de l’ADNc codant pour les RE. Cependant, l’analyse de la réponse transcriptionnelle n’a pas montré de changement significatif dans l'expression du gène aussi bien chez les mâles que chez les femelles suite à une exposition aux œstrogènes (17β-œstradiol, effluents urbains et industriels). / Our study aimed to assess the presence of endocrine disrupting compounds in the Gulf of Gabes area (Tunisia), their effects on the reproductive potential of the marine bivalve, Ruditapes decussatus as well as elucidating their mechanisms of action. To achieve these goals, several methodologies have been deployed, such as laboratory in vivo exposures (to urban or industrial effluents, estradiol), and in situ sampling to assess the risk of such substances on this ecosystem by using a multiparametric approach based on different levels of biological organization in clams (from biochemical level to the population level). We have demonstrated the presence of reprotoxic compounds along the south-eastern coast of Tunisia. The results revealed, a strong correlation between chemical contamination (cadmium, estrogenic and androgenic activities in sediment) and some reproductive parameters (oocyte diameter, vitellogénine and glycogen levels) and, a particular site (El hofra) that differs from all the other studied sites in this polluted context. Clams from this station seem to suffer from endocrine disruption probably resulting from a synergistic effect between both cadmium contamination and the presence of chemicals acting as estrogens and androgens in the environment. In parallel to this in situ study, three laboratory experiments were performed. The results showed that exposure to a mixture of chemical compounds released by the Sfax wastewater treatment plant had an adverse impact on the reproductive potential of R. decussatus, by inducing vitellogenin synthesis and stimulating reproduction in both sexes. Results of the exposure of organisms to 17β-estradiol alone, revealed that the estrogenic compound could contribute to the observed effects on the reproductive cycle of the test animals in the previous experiment. This natural hormone has induced deleterious effects and may play a role in the physiology of the clam. To study its mechanism of action via estrogen receptors, a partial cDNA of the gene was isolated and characterized from the clam R. decussatus. However, analysis of the transcriptional response did not show a significant change in the gene expression in males as well as in females following exposure to estrogens (17β-estradiol, urban and industrial effluents).

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The Potomac River Basin is significant for both public and ecological health as it flows directly into the ecologically-sensitive Chesapeake Bay. It is a drinking water source for about 5 million people living in Maryland, Virginia, and Washington D.C. The discovery of intersex fish, an indicator of poor ecological health, in the Chesapeake Bay occurred in the 2000s, and has led to a series of studies in the watershed to determine the sources and magnitude of endocrine disruption. Endocrine disrupting compounds (EDCs) are exogenous chemicals that interfere with the endocrine system and can cause detrimental health effects at low concentrations. This study aims to understand a best management practice referred to as planned indirect potable reuse (IPR) and its impacts on EDCs. The Occoquan Watershed is a planned IPR subwatershed of the Potomac River Basin. Water samples were collected at the water reclamation plant discharge (Upper Occoquan Service Authority), up- and downstream of that location along Bull Run, and at the water treatment plant intake (Frederick P. Griffith WTP) in the Occoquan Watershed to assess planned IPR. Samples were also collected at a water treatment plant (James J. Corbalis WTP) along the Potomac River for comparison as an unplanned IPR location. These two groups of samples were analyzed for EDCs (categorized into two groups: estrogen hormones and other synthetic organic compounds (SOCs)), nutrients, and other water quality parameters. The infrequency of estrogen hormones and SOC patterns indicate planned and unplanned IPR are both viable approaches to provide safe drinking water / Master of Science / Our river systems are important to maintain both for human and environmental health. The Potomac River Basin is the area of land drained by the Potomac River and its tributaries. The Potomac River Basin is significant for both public and ecological health as it flows directly into the ecologically-sensitive Chesapeake Bay. It is a drinking water source for about 5 million people living in Maryland, Virginia, and Washington D.C. The discovery of intersex fish, or when a single fish has both male and female characteristics, occurred in the Chesapeake Bay in the 2000s. Fish health is often an indicator of poor environmental health, and in this case endocrine disruption. This discovery led to a series of studies in the watershed to determine the sources and magnitude of endocrine disruption. Endocrine disrupting compounds (EDCs) are external chemicals that interfere with the endocrine system once they enter the body of a human or another organism, and can cause detrimental health effects even at low concentrations. This study aims to understand a best management practice, or a type of water pollution control, referred to as planned indirect potable reuse (IPR) and its impacts on EDCs. IPR occurs when wastewater from a community is discharged into to a river or a reservoir, and then downstream it is withdrawn from that same source for drinking water purposes. This can be either planned or unplanned. Planned IPR is becoming more common as population, especially in urban areas, increases. The Occoquan Watershed is a planned IPR subwatershed of the Potomac River Basin. Water samples were collected at the water reclamation plant discharge (Upper Occoquan Service Authority), up- and downstream of that location along Bull Run, and at the water treatment plant intake (Frederick P. Griffith WTP) in the Occoquan Watershed to assess planned IPR. Samples were also collected at a water treatment plant (James J. Corbalis WTP) along the Potomac River for comparison as an unplanned IPR location. These two groups of samples were analyzed for EDCs (categorized into two groups: estrogen hormones and other synthetic organic compounds (SOCs)), nutrients, and other water quality parameters. The infrequency of estrogen hormones and SOC patterns indicate planned and unplanned IPR are both viable approaches to provide safe drinking water.