LONG TERM ADVERSE EFFECTS OF CARBAMAZEPINE AND GEMFIBROZIL ON MALE ZEBRAFISH (Danio rerio) REPRODUCTION

Pharmaceuticals are emerging surface water contaminants, and are manufactured, used, and released into environment in considerable amounts. Concerns have been raised due to the inherent potency and bioactivity of these molecules, which makes effects at low concentrations more likely. The ubiquitous presence and stability of pharmaceuticals brings up concerns about the frequency and length of exposures. However, the distribution and fate of these compounds in surface water bodies is not clear. There is limited information about the potential effects in non-target, especially aquatic, species vulnerable to cumulative or lifelong exposures. Carbamazepine (CBZ) and gemfibrozil (GEM) are two of the most frequently detected pharmaceuticals in surface water. This thesis examined sub-lethal adverse reproductive effects of chronic direct exposure of CBZ and GEM to F0 zebrafish and several generations of unexposed offspring; the effects of exposure on testicular steroidogenesis were also examined. Chronic exposure of zebrafish to CBZ and GEM reduced ex vivo production of 11KT in testes. In vivo, CBZ decreased reproductive output, 11-ketotestosterone (11KT), male courtship and aggression behaviours, and sperm morphology in F0 parents. The F1, F2 and F3 offspring of CBZ exposed males had lower reproductive output, altered courtship, aggression, sperm morphology and lower 11KT compared to fish from the unexposed lineage. The adverse effects persisted into the F3 generation which suggested transgenerational paternal effects. GEM decreased reproductive output in F0 parents and a reduction in 11KT, altered male courtship, aggression and sperm morphology. Unexposed F1 male offspring, but not other generations, had sub-lethal toxic effects from parental exposure. We therefore suggest that CBZ and GEM act as endocrine disruptors in fish and that chronic exposure may reduce male reproductive fitness.DissertationDoctor of Philosophy (PhD)Human pharmaceuticals reach aquatic environments through municipal wastewater. The bioactivity of pharmaceuticals at low concentrations has raised concerns about undesired effects in aquatic species like fish, which can experience chronic exposures. This thesis examined adverse reproductive effects of direct chronic exposure of carbamazepine and gemfibrozil to parental zebrafish and their un-exposed offspring for multiple generations. Exposure to both compounds reduced androgens and reproduction and altered behaviour, and sperm quality in males. Effects persisted in the unexposed offspring. Parental carbamazepine exposure impacted multiple generations. We suggest that carbamazepine and gemfibrozil may reduce male reproductive fitness by reducing male sex steroids

The Effects of Parental Carbamazepine and Gemfibrozil Exposure on Sexual Differentiation in Zebrafish (Danio rerio)

Endocrine-disrupting compounds (EDCs) interfere with the physiology of hormone systems. Traditionally, steroidogenic pharmaceuticals have been studied as EDCs however there has been growing evidence that non-steroidogenic pharmaceuticals can alter sex steroid levels and impair reproductive functions in fish. This is of concern as pharmaceuticals are detected in surface waters at the ng L-1 to µg L-1 range. Zebrafish (Danio rerio) were exposed to 10 µg L-1 of the pharmaceuticals carbamazepine and gemfibrozil for 6 weeks. Male-biased sex ratios were observed in the sexually mature offspring after paternal exposure, suggesting that sexual differentiation may be impacted in juveniles. Currently, the ability of pharmaceuticals to interfere with sexual differentiation of parentally exposed offspring is unknown. This thesis examined the gonad histology of juvenile zebrafish to understand how sexual differentiation was affected in the offspring of exposed parents. Paternal, but not maternal, exposure to carbamazepine resulted in a significantly faster sexual differentiation of the gonads and led to a male-biased sex ratio; these effects were not observed when both parents were exposed. Combined paternal and maternal exposure to gemfibrozil resulted in significantly faster sexual differentiation and paternal, but not maternal, exposure to gemfibrozil led to male-biased sex ratios. Interestingly, sex ratios observed in the juveniles did not always reflect those found in the same lineage at sexual maturity, suggesting a sex reversal, including a male to female transition, occurred past the juvenile sexual differentiation period in some fish. This thesis demonstrates that pharmaceuticals have the ability to disrupt sexual differentiation in the F1 offspring of exposed parents and that paternal exposure is most relevant for offspring effects.ThesisMaster of Science (MSc)Parental exposure to the environmentally-relevant pharmaceuticals carbamazepine or gemfibrozil led to male-biased sex ratios in adult offspring of zebrafish (Danio rerio), a common model organism. The development of the gonads in juveniles was investigated to determine how this process was impacted. Predominately, paternal exposure was found to result in a faster development of the testes and male-biased sex ratios. Interestingly, sex ratios in juveniles did not always reflect those in adults, suggesting a sex reversal may have occurred in adulthood. This study demonstrates the ability of pharmaceuticals to alter gonad development in offspring of exposed parents

Human Drug Pollution in the Aquatic System: The Biochemical Responses of Danio rerio Adults

To date, drug pollution in aquatic systems is an urgent issue, and Danio rerio is a model organism to study the toxicological effects of environmental pollutants. The scientific literature has analyzed the effect of human drug pollution on the biochemical responses in the tissues of D. rerio adults. However, the information is still scarce and conflicting, making it difficult to understand its real impact. The scientific studies are not consistent with each other and, until now, no one has grouped their results to create a baseline of knowledge of the possible impacts. In this review, the analysis of literature data highlights that the effects of drugs on adult zebrafishes depend on various factors, such as the tissue analyzed, the drug concentration and the sex of the individuals. Furthermore, the most influenced biochemical responses concern enzymes (e.g., antioxidants and hydrolase enzymes) and total protein and hormonal levels. Pinpointing the situation to date would improve the understanding of the chronic effects of human drug pollution, helping both to reduce it in the aquatic systems and then to draw up regulations to control this type of pollution

The toxicological evaluation of Sewage Effluents and Pharmaceuticals with the use of Zebrafish as a model organism

The presence of pharmaceuticals in the environment has been an issue of increasing concern and sewage treatment plants have been identified as the principal sources of pharmaceuticals and endocrine disrupting chemicals in the aquatic environment. In this study, zebrafish juveniles and adults were exposed to sewage effluents that had undergone treatment processes (A1-A7) at Hammarby Sjostad’s sewage treatment plant in order to evaluate the effectiveness of the treatment processes. This was conducted with the aid of the fish embryotoxicity test (FET), reproduction test and the fish sexual development test (FSDT). A reduction in the spawning ability and fecundity (number of eggs produced by the females) was observed in the reproduction test in sewage effluent A4 (Biofilter). The zebrafish exposed to sewage effluent A2 (after sedimentation treatment) had a higher number of successful spawnings than the controls, while the fish exposed to effluents A3(Outlet L1) and A5 (ozone) exhibited a decrease in spawning ability. The induction of vitellogenin was detected in male zebrafish exposed to A2 (after sedimentation treatment), A3 (Outlet L1) and A4 (Biofilter) treatment processes in the fish sexual development test(FSDT). In the FET conducted on the offsprings of the adult zebrafish exposed to the various sewage effluents, no effect was observed. In addition, eight pharmaceuticals from different therapeutic classes viz Clozapine (anti psychotic), Atenolol (β-blocker), Cimetidine (anti histamine), Fluoxetine (anti depressant), Loperamide (anti diarrhoeal), Verapamil (Calcium channel blocker), Bezafibrate (lipid lowering agent) and Cyclophosphamide (anti neoplastic) were evaluated for their potential toxicity to zebrafish embryos with the aid of the fish embryotoxicity test. The zebrafish embryos were exposed to five different concentrations of the pharmaceuticals in 96 well plates. The exposure concentrations were 1μg/L, 10μg/L, 100μg/L, 1mg/L and 10mg/L. Dimethyl sulphoxide(DMSO) and standardized fish water were used as controls. A concentration of 0.1% DMSO was maintained in the test solutions. A mixture of musk ketone and phenylthiourea was used as positive control. The endpoints monitored included hatching time, heart rate, tail extension, circulation, coagulation, spinal deformation, death and oedema. A decrease in the heart rate was observed in the zebrafish embryos exposed to the highest concentration of clozapine (10 mg/L) and abnormalities were observed in the embryos exposed to all the concentrations of clozapine. The results of this investigation indicate that the FET could be improved upon in order to render it more sensitive for the toxicity testing of substances. Also, ozonation appears to be an effective treatment technique in sewage treatment plants for the reduction of sewage effluents and pharmaceuticals

The Biological Effects of Pharmaceuticals in the Marine Environment

Environmental pharmaceuticals represent a threat of emerging concern for marine ecosystems. Widely distributed and bioaccumulated, these contaminants could provoke adverse effects on aquatic organisms through modes of action like those reported for target species. In contrast to pharmacological uses, organisms in field conditions are exposed to complex mixtures of compounds with similar, different, or even opposing therapeutic effects. This review summarizes current knowledge of the main cellular pathways modulated by the most common classes of environmental pharmaceuticals occurring in marine ecosystems and accumulated by nontarget species-including nonsteroidal anti-inflammatory drugs, psychiatric drugs, cardiovascular and lipid regulator agents, steroidal hormones, and antibiotics-and describes an intricate network of possible interactions with both synergistic and antagonistic effects on the same cellular targets and metabolic pathways. This complexity reveals the intrinsic limits of the single-chemical approach to predict the long-term consequences and future impact of pharmaceuticals at organismal, population, and community levels

A multibiomarker approach highlights effects induced by the human pharmaceutical gemfibrozil to gilthead seabream Sparus aurata

Lipid regulators are among the most prescribed human pharmaceuticals worldwide. Gemfibrozil, which belongs to this class of pharmaceuticals, is one of the most frequently encountered in the aquatic environment. However, there is limited information concerning the mechanisms involved in gemfibrozil effects to aquatic organisms, particularly to marine organisms. Based on this knowledge gap, the current study aimed to assess biochemical and behavioral effects following a sublethal exposure to gemfibrozil (1.5, 15, 150, 1500 and 15,000 μg L-1) in the estuarine/marine fish Sparus aurata. After the exposure to 1.5 μg L-1 of gemfibrozil, fish had reduced ability to swim against a water flow and increased lipid peroxidation in the liver. At concentrations between 15-15,000 μg L-1, the activities of some enzymes involved in antioxidant defense were induced, appearing to be sufficient to prevent oxidative damage. Depending on the organ, different responses to gemfibrozil were displayed, with enzymes like catalase being more stimulated in gills, whereas glutathione peroxidase was more activated in liver. Although there were no obvious concentration-response relationships, the integrated biomarker response version 2 (IBRv2) analysis revealed that the highest concentrations of gemfibrozil (between 150-15,000 μg L-1) caused more alterations. All the tested concentrations of gemfibrozil induced effects in S. aurata, in terms of behavior and/or oxidative stress responses. Oxidative damage was found at a concentration that is considered environmentally relevant, suggesting a potential of this pharmaceutical to impact fish populations.This research was supported through the COMPETE – Operational Competitiveness Program and National Funds through FCT – Foundation for Science and Technology, under the project “NANOAu – Effects of Gold Nanoparticles to Aquatic Organisms” (FCT PTDC/MAR-EST/3399/2012) (FCOMP-01-0124-FEDER-029435), through FCT/MCTES through national funds (PIDDAC), the cofounding by FEDER, within the PT2020 Partnership Agreement and Compete 2020 to CESAM (UID/AMB/50017 – POCI-01-0145-FEDER-007638) and UID/QUI/50006/2013. A. Barreto has a doctoral fellowship from FCT (SFRH/BD/97624/2013); L. G. Luis had a fellowship from FCT (BI/UI88/6881/2014). MO has financial support of the program Investigator FCT, co-funded by the Human Potential Operational Programme and European Social Fund (IF/00335(2015).info:eu-repo/semantics/publishedVersio

Reproductive performance of Zebra Fish (Danio rerio) exposed to palm oil mill eflluent in chronic toxicity

Palm Oil Mill Effluent (POME) is potentially harmful to the aquatic environment. POME contains high organic material including COD (Chemical Oxygen Demand), BOD (Biochemical Oxygen Demand) TTS (Total Suspended Solid) and various type of heavy metals. of zebra fish (Danio rerio). Reproductin has an important role in producing new individuals which directly affect the population. Impaired reproductive performance potentially impairs juvenile production optimization. The present study investigated how sub-chronic toxicity of POME impact the reproductive performance used Completely randomized Design (CRD) in three treatments and four replicates based on value of LC50-96 hours (5.156 ml/l): Control (0 ml/L), treatment A 10% POME (0,5 ml/L), treatment B 20 % POME (1 ml/L). The fecundity, relative fecundity, GSI, and egg diameter were analyzed. Data was analyzed with Analysis of Variance (ANOVA) and followed with Least Significance Difference (LSD) test. Results showed that fecundity in treatment A (149 ± 38.70) and treatment B (85± 11.35) were significantly decreased compared to the control (219 ± 42.38) (P<0.05). While relative fecundity significantly decreased in treatment B (0.33 ± 0.13) rather than control (0,87 ± 0,14). Significantly decline is also observed on GSI in tretment A (4.79 ± 2.55%) and treatment B (2.55 ± 0.21%) compared to control (6.96 ± 1.70%). While the egg diameter only shows a significantly decline in treatment B (0.57 ± 0.18 mm) compared to control (0.71 ± 0.27 mm)

Pharmaceuticals And Personal Care Products: Emerging Contaminants In Aquatic Ecosystems

In recent years, the presence of pharmaceuticals and personal care products (PPCPs) in aquatic systems has led to research on their fate and effects. PPCPs have been found in mixture in wastewater effluents, surface, ground, and drinking water at low concentrations from areas of intense urbanization. Although adverse effects to human health from the current environmental concentrations are unlikely, the impacts to ecological receptors are not clear. We performed field and laboratory studies to quantify and evaluate effects of PPCPs on fish. First, a field study was conducted at the Baca National Wildlife Refuge, Colorado (2010-2012) because a portion of the Refuge receives discharges of treated water from the Aspen Wastewater Treatment Plant (WWTP). Water and fish samples were used to quantify the presence of PPCPs in surface and wastewater effluents, and to determine the potential impact of PPCPs in fish communities (using histology and gene expression analysis). We focused on fathead minnows ( Pimephales promelas ) since they have been the subject of relevant ecotoxicological research and are a good sentinel species. A total of 120 analytes were quantified using a combination of grab samples and polar organic chemical integrative samplers (POCIS). Although no PPCPs were detected from the grab samples, POCIS allowed for the detection of PPCPs in all our fish sites. High concentrations of N,N-Diethyl-meta-toluamide (DEET) and thirteen pharmaceuticals (triclocarban, triclosan, gemfibrozil, ibuprofen, progesterone, diphenhydramine, atenolol, caffeine, trimethoprim, levorphanol, cannabidiol, tetrahydrocannabinol (THC), and naproxen) were detected in all fish sites, including the reference site. Cellular changes in gonads and livers and significant changes in gene expression (steroidogenic acute regulatory protein, star and androgen receptor, ar ) were observed from female and male fathead minnows sampled from creeks contaminated with PPCPs. However, because we could not identify a clean reference site, we cannot affirm these changes are due to PPCP exposure. We conclude that POCIS is a sensitive method for the detection and quantification of PPCPs in small streams. Additional studies at the Refuge are needed to better understand the ecological impacts of PPCPs. Next, we conducted a laboratory study using the same PPCPs found in our field study and exposed adult fathead minnows for 48 hr to the highest environmental concentration of each chemical. Our goal was to evaluate molecular changes of a suite of genes known to respond after exposure to chemicals affecting lipid metabolism, and the endocrine and nervous systems. Fish were exposed to triclocarban (1.4 μg/L), DEET (0.6 μg/L) or to a PPCP mixture consisting of: atenolol (1.5 μg/L), caffeine (0.25 μg/L), diphenhydramine (0.1 μg/L), gemfibrozil (1.5 μg/L), ibuprofen (0.4 μg/L), naproxen (1.6 μg/L), triclosan (2.3 μg/L), progesterone (0.2 μg/L), triclocarban (1.4 μg/L), and DEET (0.6 μg/L). Vitellogenin ( vtg ) was up-regulated in livers of females and males exposed to triclocarban. Also, an up-regulation of hepatic lipoprotein (lpl ) and a down-regulation of ar and star were observed in testes. The group treated with DEET only showed a significant decrease in ar in females. In contrast, the PPCP mixture down-regulated vtg in females and males, and expression of estrogen receptor alpha (erα ), star, and thyroid hormone receptor alpha 1 (thra1 ) in testes. Our results show the molecular `estrogenic\u27 effects of triclocarban are eliminated (males) or reversed (females) when dosed in conjunction with several other PPCPs, once again showing that results from single exposures could be vastly different from those observed with mixtures

Efeitos das exposições aguda e crónica a sinvastatina e ácido clofíbrico em Danio rerio

Lipid-regulating drugs are one of the most prescribed medications around the world, to control human cholesterol levels, to more than 20 million patients. Due to their wide usage, pharmaceuticals can be discarded, metabolized and excreted into the environment, potentially affecting aquatic organisms. Despite this increasing use of lipid-regulating drugs, particularly simvastatin and clofibric acid, are not fully characterized and understood in terms of their potential toxicological effects at the environmental level, therefore being necessary to investigate them. Therefore, it emerges a new concern on their effects related to the potential environmental impact, particularly in the aquatic environment. This work intended to characterize the toxicity due to an acute (120 hours post-fertilization) and chronical (60 days) exposure to antihyperlipidemic drugs, namely simvastatin (92.45, 184.9, 369.8, 739.6 and 1479.2 ng L-1) and clofibric acid (10.35, 20.7, 41.4, 82.8 and 165.6 μg L-1), in the freshwater species of zebrafish (Danio rerio). The concentrations hereby selected were implemented in both exposures. The analysis of effects focused on the histological observation of tissues in individuals, concerning sex determination and maturation stages of gonads, behavior (small and large distance, total distance and swimming time), biomarkers of oxidative stress (superoxide dismutase, catalase and glutathione peroxidase), biotransformation (glutathione S-transferases) and lipid peroxidation (thiobarbituric acid reactive substances). In terms of acute exposure, it was observed behavioral alterations in both compounds, simvastatin caused hyperactivity and clofibric acid provoked hypoactivity in all behavioral parameters. Moreover, it was observed a significant decrease in all biomarkers in individuals exposed to simvastatin from 184.9 to 1479.2 ng L-1, except for catalase, for which no significant differences were found. Glutathione peroxidase selenium-dependent activity also showed a significant increase at 92.45 ng L-1. On the other hand, in individuals exposed to clofibric acid, there was a significant increase in all biomarkers, typically from 41.4 to 165.6 μg L-1. However, in catalase and glutathione S-transferases, in the highest concentration, the activity was significantly decreased. This study suggests that the chronic exposure of Danio rerio to simvastatin and clofibric acid does not interfere with the sex ratio and maturation stages of individuals. As antioxidant defenses are important in terms of the capacity of the organism to overcome oxidative stress, along with effects in locomotion, it can affect the metabolism or even the survival of organisms. Therefore, further studies in terms of mode of action of these two compounds, including reproductive disruption effects in longer exposures, are required to observe and characterize the long-term effects of simvastatin and clofibric acid in the aquatic compartment and its organisms.Os antihiperlipidémicos são dos fármacos mais prescritos no mundo para o controlo dos níveis de colesterol, a mais de 20 milhões de pacientes. Devido ao seu amplo uso, os fármacos podem ser descartados, metabolizados e excretados no ambiente, potencialmente afetando organismos aquáticos. Apesar desta ampla utilização de antihiperlipidémicos, particularmente a sinvastatina e o ácido clofíbrico, os seus potenciais efeitos toxicológicos ao nível ambiental não estão totalmente caracterizados e compreendidos, sendo assim necessário investigá-los. Portanto, surge uma nova preocupação quanto ao seu potencial impacto ambiental, particularmente no ambiente aquático. Este trabalho visa caracterizar a toxicidade decorrente da exposição aguda (120 horas pós-fertilização) e crónica (60 dias) a drogas antihiperlipidémicas, nomeadamente sinvastatina (92.45, 184.9, 369.8, 739.6 e 1479.2 ng L-1) e ácido clofíbrico (10.35, 20.7, 41.4, 82.8 e 165.6 μg L-1), na espécie de peixe de água doce peixe-zebra (Danio rerio). As concentrações selecionadas foram implementadas em ambas as exposições. A análise dos efeitos incidiu sobre a observação histológica de tecidos dos indivíduos, no que diz respeito à determinação do sexo e dos estádios de maturação das gónadas, comportamento (movimentos curtos e longos, tempo de natação e distância total de natação), e biomarcadores de stress oxidativo (superóxido dismutase, catalase e glutationa peroxidase), biotransformação (glutationa S-transferases) e peroxidação lipídica (substâncias reativas ao ácido tiobarbitúrico). No caso da exposição aguda, foram observadas alterações de comportamento em animais expostos a ambos os compostos, sendo que a sinvastatina originou hiperatividade e o ácido clofíbrico provocou hipoatividade em todos os parâmetros comportamentais. Para além disso, foi observada a inibição significativa em todos os biomarcadores em indivíduos expostos a sinvastatina em concentrações de 184.9 a 1479.2 ng L-1, exceto na catalase, parâmetro para o qual não foram reportadas diferenças significativas. A atividade da glutationa peroxidase selénio dependente também aumentou significativamente para níveis de 92.45 ng L-1. Por outro lado, em indivíduos expostos a ácido clofíbrico, houve um aumento significativo em todos os biomarcadores, geralmente em peixes expostos a concentrações de 41.4 a 165.6 μg L-1. No entanto, no caso da catalase e das glutationa S-transferases, na concentração mais elevada, a atividade diminuiu significativamente. Este estudo sugere que as exposições crónicas de Danio rerio a sinvastatina e ácido clofíbrico não interferem na proporção de sexo e nos estádios de maturação dos indivíduos. Como as defesas antioxidantes são importantes relativamente à capacidade do organismo no combate ao stress oxidativo, juntamente com efeitos a nível da locomoção, podem afetar o metabolismo ou até mesmo a sobrevivência dos organismos. Assim, terão de ser investigados o modo de ação destes dois compostos, incluindo os efeitos ao nível da disrupção reprodutiva em exposições mais prolongadas, de forma a observar e caracterizar os efeitos a longo-prazo da sinvastatina e do ácido clofíbrico no compartimento aquático e nos seus organismos.This work was partially supported by Strategic Funding UID/Multi/04423/2019 through national funds provided by FCT and European Regional Development Fund (ERDF), in the framework of the programme PT2020. Thanks are due to ECO-R-pharmplast - Ecotoxicity of realistic combinations of pharmaceutical drugs and microplastics in marine ecosystems, Fundação para a Ciência e a Tecnologia, FCT (reference POCI-01-0145-FEDER-029203). Thanks are also due for the financial support to CESAM (UID/AMB/50017/2019), to FCT/MEC through national funds, and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020.Mestrado em Biologia Marinha Aplicad

Characterization of endocrine disrupting potentials of municipal effluents from six wastewater treatment plants across Canada

Over the past three decades, concerns have been raised regarding the potential adverse effects of certain natural and synthetic chemicals that can disrupt the endocrine systems of humans and wildlife. These endocrine disrupting chemicals (EDCs) have been reported to cause developmental and reproductive effects at low concentrations (ng/L) in many vertebrate species, particularly in aquatic organisms such as fish. One of the most prevalent sources of EDCs in aquatic environments is municipal wastewater effluents (MWWEs). This is because conventional wastewater treatment systems are inefficient at removing many of the diverse contaminants present in raw sewage, including EDCs. Although multiple initiatives have been initiated to establish standardized testing and monitoring criteria for EDCs in the environment worldwide, our understanding of the contribution of MWWEs to endocrine disruption in Canadian surface waters is incomplete. Therefore, the main aims of this project were to 1) further our understanding of the contribution of MWWEs to the contamination of freshwater bodies in Canada with EDCs, and to 2) characterize the removal efficiency of EDCs by six wastewater treatment plants (WWTPs) across Canada. Specifically, this study explored the presence of EDCs and their potencies in influents and effluents as a function of wastewater treatment level/system, climate/seasonality and population size served by the WWTP using a combination of three in vitro bioassays and targeted chemical analysis. The MVLN, MDAkb2, and H295R Steroidogenesis assays were applied to assess (anti-)estrogenic, (anti-)androgenic and steroidogenesis disrupting potentials, respectively, of extracts of influents and effluents collected throughout the year from the WWTPs of the cities of Saskatoon (SK), Regina (SK), Guelph (ON), Kitchener (ON), Quebec City (QB) and Montreal (QB). In parallel, targeted chemical analysis was performed to determine the presence of selected chemicals with proven or suspected endocrine activities, and results were correlated with bioactivities determined in vitro. Overall, influents showed great androgenic activities regardless of treatment plant while significant estrogenic potentials were only observed in a few cases such as Regina effluent and Montreal influent. With the exception of Montreal, high to moderate treatment efficiencies of WWTPs occurred for the removal of androgens, while low or no removal of substances with estrogenic properties was observed. Significant anti-estrogenic and anti-androgenic potentials were detected in most of the influent and effluent samples, regardless of the treatment level. In general, WWTPs representing less advanced treatment technologies were less efficient at removing certain endocrine active substances. In particular, effluents from the two lagoon-based facilities, Regina and Montreal, had significant remaining estrogenic and androgenic activities, respectively. Furthermore, population size seemed to play an important role regarding EDC removal, with WWTPs serving greater than 500,000 habitants showing decreased removal of compounds with endocrine activities in general. However, given the limited sample size (only two of the cities investigated had populations greater than 500,000 inhabitants) it cannot be determined with certainty whether this decreased removal efficiency was a result of population size or simply insufficient capacities of the WWTPs. Thus, additional studies including more treatment facilities with different treatment levels and larger population sizes should be conducted to determine whether population does significantly affect the removal of EDCs. Furthermore, our original hypothesis that extremely cold temperatures would result in decreased efficiency of EDC removal due to reduced biological activity and light exposure was not always supported by the results. Samples collected during the spring season had the highest endocrine activities overall, which could potentially be a result of colder months. However, neither early nor late winter samples showed a comparable effect on removal efficiency. The observation that spring samples had the greatest endocrine activities may be of significant ecological concern as this season coincides with the spawning season of many fishes. This concern was further corroborated by two parallel studies that investigated the impacts of MWWEs collected from the Regina and Saskatoon WWTPs on fish. These studies observed general inhibition of reproductive functions such as delayed maturation, degeneration of gonadal tissues, reduction in the expression of secondary sex characteristics, and significant reduction of fecundity in fathead minnows exposed to both diluted effluents or that were collected downstream of the WWTP outflow of Regina. The observation that antagonistic effects at both the ER and AR represented the most prevalent endocrine potentials was also supported by chemical analysis that revealed greater concentrations of compounds with the ability to act as ER and AR antagonists, while there were low concentrations or no presence of chemicals previously shown to agonistically interact with these receptors. The results obtained by the combination of in vitro and the two parallel in vivo and chemical analysis demonstrated that in vitro assays can be used as a cost-effective tool for prioritizing potential endocrine disrupting impacts of MWWEs in aquatic environments. The significant endocrine activity, in particular, antagonism of sex steroid receptors, warrants further investigations to characterize the actual risks they may pose to aquatic wildlife. This is particularly true in cases where WWTPs utilize primary and/or outdated lagoon-based treatment technologies, such as Regina and Montreal. Furthermore, in cases where effluent flow is proportionally higher than that of the receiving water body, which can be encountered in many urban municipalities in semi-arid regions such as Regina in southern Saskatchewan, or in situations where the population is greater than WWTPs’ treatment capacity, bypassing untreated sewage, downstream ecosystems may be of particular risk