The active synthetic constituent of oral contraceptives 17α- ethinylestradiol (EE2) and its natural steroidal estrogen analogues are being released into the aquatic environment mainly via wastewater treatment plants (WWTPs). Although steroidal estrogens have been frequently reported in very low concentrations in the environment, they have been placed at the top of the list of endocrine disrupting chemicals (EDCs) for their high estrogenic activity in non-target aquatic species like fish and frogs. Almost 30 years worth of intensive research has moved the problem of endocrine disruption in the aquatic environment from being a hypothesis to a well-known cause-and-effect story. However, the fate and effects of many pharmaceuticals, including EE2, remain poorly understood. Thus, the main objective of this thesis was to investigate EE2 bioaccumulation in fish by field and laboratory studies. An optimized sample preparation and analytical method protocol was achieved to detect EE2 in its biologically active form in whole fish tissue at trace levels (ng/g). Shorthead redhorse suckers (ShRHS) (Moxostoma macrolepidotum) collected in proximity to a WWTP in the St. Clair River have significantly accumulated EE2 with an average of 1.5 ng/g. EE2 bioaccumulation was limited to fish exhibiting intersex and with induced vitellogenin (VTG). EE2 was absent in fish from a reference site. Positive correlations between EE2 and lipid content as well as "15N supported the hypothesis of EE2 bioaccumulation in wild ShRHSs. A wider survey was carried out to explore patterns of EE2 bioaccumulation in the pelagic and benthic food-wed of the St. Clair IV River, Ontario. In the WWTP effluents and nearby surface waters, EE2 levels were extremely low and below our method detection limits. All of the seven sampled species from different trophic levels and the sediment samples collected from the impacted sites and the reference sites did not have any measurable EE2 concentrations. A laboratory controlled study where male goldfish (Carassius auratus) were exposed to EE2 via water versus food demonstrated that fish can rapidly accumulate EE2 by both routes of exposure. The uptake constant for water was 45 h-1 and the bioconcentration factor for EE2 in fish was 377. The fast uptake rate of EE2 via water coincided with a much slower elimination rate constant of 0.0786 h-1. The assimilation efficiency of 0.106 for EE2 by goldfish was determined by EE2 dietary exposure, which was used to predict EE2 accumulation under different exposure scenarios. The work presented here was the first demonstration of EE2 bioaccumulation in wild fish and the first to model EE2 bioaccumulation from water and dietary exposure in laboratory-exposed fish.

The active synthetic constituent of oral contraceptives 17α- ethinylestradiol (EE2) and its natural steroidal estrogen analogues are being released into the aquatic environment mainly via wastewater treatment plants (WWTPs). Although steroidal estrogens have been frequently reported in very low concentrations in the environment, they have been placed at the top of the list of endocrine disrupting chemicals (EDCs) for their high estrogenic activity in non-target aquatic species like fish and frogs. Almost 30 years worth of intensive research has moved the problem of endocrine disruption in the aquatic environment from being a hypothesis to a well-known cause-and-effect story. However, the fate and effects of many pharmaceuticals, including EE2, remain poorly understood. Thus, the main objective of this thesis was to investigate EE2 bioaccumulation in fish by field and laboratory studies. An optimized sample preparation and analytical method protocol was achieved to detect EE2 in its biologically active form in whole fish tissue at trace levels (ng/g). Shorthead redhorse suckers (ShRHS) (Moxostoma macrolepidotum) collected in proximity to a WWTP in the St. Clair River have significantly accumulated EE2 with an average of 1.5 ng/g. EE2 bioaccumulation was limited to fish exhibiting intersex and with induced vitellogenin (VTG). EE2 was absent in fish from a reference site. Positive correlations between EE2 and lipid content as well as "15N supported the hypothesis of EE2 bioaccumulation in wild ShRHSs. A wider survey was carried out to explore patterns of EE2 bioaccumulation in the pelagic and benthic food-wed of the St. Clair IV River, Ontario. In the WWTP effluents and nearby surface waters, EE2 levels were extremely low and below our method detection limits. All of the seven sampled species from different trophic levels and the sediment samples collected from the impacted sites and the reference sites did not have any measurable EE2 concentrations. A laboratory controlled study where male goldfish (Carassius auratus) were exposed to EE2 via water versus food demonstrated that fish can rapidly accumulate EE2 by both routes of exposure. The uptake constant for water was 45 h-1 and the bioconcentration factor for EE2 in fish was 377. The fast uptake rate of EE2 via water coincided with a much slower elimination rate constant of 0.0786 h-1. The assimilation efficiency of 0.106 for EE2 by goldfish was determined by EE2 dietary exposure, which was used to predict EE2 accumulation under different exposure scenarios. The work presented here was the first demonstration of EE2 bioaccumulation in wild fish and the first to model EE2 bioaccumulation from water and dietary exposure in laboratory-exposed fish.

The active synthetic constituent of oral contraceptives 17α- ethinylestradiol (EE2) and its natural steroidal estrogen analogues are being released into the aquatic environment mainly via wastewater treatment plants (WWTPs). Although steroidal estrogens have been frequently reported in very low concentrations in the environment, they have been placed at the top of the list of endocrine disrupting chemicals (EDCs) for their high estrogenic activity in non-target aquatic species like fish and frogs. Almost 30 years worth of intensive research has moved the problem of endocrine disruption in the aquatic environment from being a hypothesis to a well-known cause-and-effect story. However, the fate and effects of many pharmaceuticals, including EE2, remain poorly understood. Thus, the main objective of this thesis was to investigate EE2 bioaccumulation in fish by field and laboratory studies. An optimized sample preparation and analytical method protocol was achieved to detect EE2 in its biologically active form in whole fish tissue at trace levels (ng/g). Shorthead redhorse suckers (ShRHS) (Moxostoma macrolepidotum) collected in proximity to a WWTP in the St. Clair River have significantly accumulated EE2 with an average of 1.5 ng/g. EE2 bioaccumulation was limited to fish exhibiting intersex and with induced vitellogenin (VTG). EE2 was absent in fish from a reference site. Positive correlations between EE2 and lipid content as well as "15N supported the hypothesis of EE2 bioaccumulation in wild ShRHSs. A wider survey was carried out to explore patterns of EE2 bioaccumulation in the pelagic and benthic food-wed of the St. Clair IV River, Ontario. In the WWTP effluents and nearby surface waters, EE2 levels were extremely low and below our method detection limits. All of the seven sampled species from different trophic levels and the sediment samples collected from the impacted sites and the reference sites did not have any measurable EE2 concentrations. A laboratory controlled study where male goldfish (Carassius auratus) were exposed to EE2 via water versus food demonstrated that fish can rapidly accumulate EE2 by both routes of exposure. The uptake constant for water was 45 h-1 and the bioconcentration factor for EE2 in fish was 377. The fast uptake rate of EE2 via water coincided with a much slower elimination rate constant of 0.0786 h-1. The assimilation efficiency of 0.106 for EE2 by goldfish was determined by EE2 dietary exposure, which was used to predict EE2 accumulation under different exposure scenarios. The work presented here was the first demonstration of EE2 bioaccumulation in wild fish and the first to model EE2 bioaccumulation from water and dietary exposure in laboratory-exposed fish.

The active synthetic constituent of oral contraceptives 17α- ethinylestradiol (EE2) and its natural steroidal estrogen analogues are being released into the aquatic environment mainly via wastewater treatment plants (WWTPs). Although steroidal estrogens have been frequently reported in very low concentrations in the environment, they have been placed at the top of the list of endocrine disrupting chemicals (EDCs) for their high estrogenic activity in non-target aquatic species like fish and frogs. Almost 30 years worth of intensive research has moved the problem of endocrine disruption in the aquatic environment from being a hypothesis to a well-known cause-and-effect story. However, the fate and effects of many pharmaceuticals, including EE2, remain poorly understood. Thus, the main objective of this thesis was to investigate EE2 bioaccumulation in fish by field and laboratory studies. An optimized sample preparation and analytical method protocol was achieved to detect EE2 in its biologically active form in whole fish tissue at trace levels (ng/g). Shorthead redhorse suckers (ShRHS) (Moxostoma macrolepidotum) collected in proximity to a WWTP in the St. Clair River have significantly accumulated EE2 with an average of 1.5 ng/g. EE2 bioaccumulation was limited to fish exhibiting intersex and with induced vitellogenin (VTG). EE2 was absent in fish from a reference site. Positive correlations between EE2 and lipid content as well as "15N supported the hypothesis of EE2 bioaccumulation in wild ShRHSs. A wider survey was carried out to explore patterns of EE2 bioaccumulation in the pelagic and benthic food-wed of the St. Clair IV River, Ontario. In the WWTP effluents and nearby surface waters, EE2 levels were extremely low and below our method detection limits. All of the seven sampled species from different trophic levels and the sediment samples collected from the impacted sites and the reference sites did not have any measurable EE2 concentrations. A laboratory controlled study where male goldfish (Carassius auratus) were exposed to EE2 via water versus food demonstrated that fish can rapidly accumulate EE2 by both routes of exposure. The uptake constant for water was 45 h-1 and the bioconcentration factor for EE2 in fish was 377. The fast uptake rate of EE2 via water coincided with a much slower elimination rate constant of 0.0786 h-1. The assimilation efficiency of 0.106 for EE2 by goldfish was determined by EE2 dietary exposure, which was used to predict EE2 accumulation under different exposure scenarios. The work presented here was the first demonstration of EE2 bioaccumulation in wild fish and the first to model EE2 bioaccumulation from water and dietary exposure in laboratory-exposed fish.

The synthetic estrogen 17α-ethinylestradiol (EE2) is an endocrine disrupting chemical (EDC) of concern due to its persistent nature and widespread presence in the aquatic environment. In mammals, effects of developmental EDC exposure on reproduction and behavior not only persist to adulthood after discontinued exposure, but are also inherited by several consecutive unexposed generations. The results presented in this thesis demonstrate that non-reproductive behavior in fish is highly sensitive to the influence of EE2 during development and the effects do not appear to be restored after a long recovery period in clean water. We have shown that exposure to low doses of EE2 during development results in increased anxiety in two fish species (zebrafish and guppy) and their offspring. We have also demonstrated that the effects of EE2 on anxiety are apparent in both sexes and are transgenerationally transmitted to two consecutive generations of unexposed offspring in the guppy. In order to investigate the possible biological mechanisms of the observed persistent effects on non-reproductive behavior, we also performed an RNA sequencing analysis of the whole-brain transcriptome in developmentally exposed zebrafish after remediation in clean water until adulthood. Differential expression of 33 genes in males and 62 genes in females were observed as a result of EE2 exposure, with only one gene affected in both sexes. Functional analysis revealed cholesterol biosynthesis and circadian rhythm to be the top two affected pathways in males and females, respectively. Both pathways have previously been implicated in anxiety behavior and represent possible candidates connecting the transcriptome alterations to the observed behavioral phenotype. The study represents an initial survey of the fish brain transcriptome by means of RNA sequencing after long-term recovery from developmental exposure to an estrogenic compound.

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.

17α-ethinylestradiol (EE2) is a synthetic hormone, derivative of the natural hormone estradiol. EE2 is one of the the most prescribed drugs in the world. It belongs to the estrogenic endocrine disrupter chemicals. These compounds are able to alter functions of the endocrine system and cause adverse effects in the organism, offspring and (sub)population. In this thesis, there are observed effects of 17α-ethinylestradiol on enzyme activities of main enzymes involved in phase I of xenobiotic biotransformation, i.e. cytochromes P450 (CYP), in vitro. Isoforms of CYP subfamilies 1A, 2B, 2C, 2E and 3A were studied in rats and humans. Each CYP isoform was incubated with EE2 at two concentrations, 10μM EE2 and the concentration corresponding to the substrate concentration in the specific marker reactions of individual CYP isoforms. The results indicate, that in rat liver microsomes the activity of all studied isoforms except CYP1A2 was decreased in the presence of EE2. When EE2 was added to the incubation mixture at the concentration of the reaction substrate, the greatest decrease in enzyme activity was observed for CYP2C6, with the remaining activity only 36%. In human liver microsomes, the activity of CYP2B6, CYP2C9, CYP2E1 and CYP3A4 was also effected by EE2. As in the case of rat model, CYP2C subfamily...

Many exogenous and endogenous compounds are referred to as endocrine disruptors (EDCs), as they interfere with natural synthesis, signaling and metabolism of endogenous hormones. Common exogenous endocrine disruptors are benzo(a)pyrene (BaP) and 17α-ethinylestradiol (EE2). Endogenous endocrine disruptor 17β-estradiol (E2) is frequently present in the environment as well. In this thesis, the effect of the mentioned EDCs and their combinations on gene and protein expression of CYP1B1, 3A1 and 3A2 in rat liver, kidney and lung was determined. Protein expression was studied using Western blot method and specific antibodies; gene expression was assessed by quantitative PCR. Moreover, the effect of tested EDCs and their combinations on BaP metabolism and CYP3A specific activity (measured as testosterone 6β-hydroxylation) were studied in liver microsomal samples. It was confirmed, that BaP significantly increases CYP1B1 expression in rat liver and lung both alone and together with EE2 or E2. Pretreatment of rat with E2 and BaP increases the ability of BaP to induce CYP1B1 expression. On the contrary, EE2, E2 and their combination decrease the CYP1B1gene expression. The rate of BaP metabolites formed in liver microsomal samples increases in rats pretreated with BaP and its combinations. In liver, there was...

Organic contaminants from industrial and/or domestic effluents may be harmful to humans directly or indirectly by degrading the quality of the aquatic environment. Consequently these contaminants must be reduced to levels that are not harmful to humans and the environment before disposal. Chemical, physical and biological methods exist for the removal of these pollutants from effluents. Among the available chemical methods, heterogeneous photocatalytic oxidation has been found particularly effective in removing a large number of persistent organics in water. In this study, photocatalytic degradation was explored for the removal of reactive azo-dye (textile dye), triclocarban (disinfectant), clopyralid (herbicide) and three endocrine disrupting compounds (EDCs) (estrone, 17ß-estradiol and 17α-ethinylestradiol) from synthetic effluents. The major factors affecting the photocatalytic processes including the initial concentration of the target compounds, the amount of catalyst, the light intensity, the type of catalyst, the electron acceptor, the irradiation time and the pH were studied. Other oxidation techniques including (O3, H2O2, UV) were also studied. Generally UV light is used in combination with titanium dioxide, as photocatalyst, to generate photoinduced charge separation leading to the creation of electron-hole pairs. The holes act as electron acceptors hence the oxidation of organics occur at these sites. These holes can also lead to the formation of hydroxyl radicals which are also effective oxidants capable of degrading the organics. The results obtained in this study indicated that photolysis (i.e. UV only) was found to have no effect on the degradation of reactive azo-dye (RO16). However, complete photocatalytic degradation of 20 mg/L (3.24×10-2 mM) RO16 was achieved in 20 minutes in the presence of 1g/L TiO2 Degussa P25 at pH 5.5. Comparison between various types of catalysts (i.e. Degussa P25, VP Aeroperl, Hombifine N) gave varied results but Degussa P25 was the most effective photocatalyst hence it was selected for this study. For RO16 the optimum catalyst concentration was 0.5 g/L TiO2 with initial concentration of 20 mg/L RO16. It was found that the disappearance of RO16 satisfactorily followed the pseudo first-order kinetics according to Langmuir-Hinshelwood (L-H) model. The rate constant was k= 0.0928 mol/min. Photodegradation of TCC was studied in 70%v acetonitrile: 30%v water solutions. UV light degraded TCC effectively and the reaction rates increased with decreasing initial concentration of TCC. UV/TiO2 gave unsatisfactory degradation of triclocarban (TCC) since only 36% were removed in 60 minutes with initial concentration of TCC 20 mg/L. The degradation of clopyralid and the EDCs was studied using three oxidation systems UV/TiO2, UV/H2O2 and O3. Complete degradation of clopyralid (3,6-DCP) was achieved with UV/TiO2 in about 90 minutes at an optimum catalyst concentration of 1g/L. Zero-order kinetics was found to describe the first stage of the photocatalytic reaction in the concentration range 0.078-0.521 mM. At pH 5 the rate constant was 2.09×10-6-4.32×10-7 M.s-1.Complete degradation of all the three EDCs was achieved with UV/H2O2 in 60 minutes at catalyst concentration of (2.94×10-2 M). On the other hand complete degradation of the EDCs was achieved in just 2 minutes with ozonation. For high concentration EDCs, TiO2/UV gave low efficiency of degradation as compared with ozone and H2O2/UV. First-order kinetics was found to describe the photocatalytic reaction of the EDCs. / Education Service Department of the Libyan Government

This work is aimed at the study of the effect of 17α-ethinylestradiol (EE2) on the production and characteristics of ligninolytic enzymes (laccase, Mn-dependent peroxidase and lignin peroxidase) in I. lacteus, T. versicolor, P. chrysosporium and P. ostreatus cultures grown on two types of liquid media. Enzyme activity production in fungal cultures was affected by the composition of culture medium. In the case of P. chrysosporium, the addition of EE2 to the complex- medium cultures led to a MnP activity stimulation and simultaneously LiP production was partially repressed in these cultures. In the mineral MM medium, no effect of EE2 on enzyme production by P. chrysosporium was observed. In EE2 treated MM cultures of P. ostreatus lower MnP activities were found when compared to biotic controls. In the case of T. versicolor cultures, the addition of EE2 to the complex medium caused laccase and LiP stimulation in the cultures. In the MM medium, however, only laccase production was affected by EE2. I. lacteus MnP production was partially repressed by EE2 in MM medium. In contrast to that, significantly higher MnP activities were detected in complex- medium I. lacteus cultures after the treatment with EE2. Further EE2 degradation by the fungal cultures was studied. The highest degradation effeciency was...