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Bioaccumulation of 17α-Ethinylestradiol in FishAl-Ansari, Ahmed 04 April 2012 (has links)
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.
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Bioaccumulation of 17α-Ethinylestradiol in FishAl-Ansari, Ahmed 04 April 2012 (has links)
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.
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Bioaccumulation of 17α-Ethinylestradiol in FishAl-Ansari, Ahmed 04 April 2012 (has links)
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.
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Bioaccumulation of 17α-Ethinylestradiol in FishAl-Ansari, Ahmed January 2012 (has links)
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.
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Thermodynamics of steroid estrogen sorption to activated sludgeXu, Kai, Harper, Willie F., January 2008 (has links) (PDF)
Thesis (M.S.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references (p. 54-60).
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Estudo da degradação eletroquímica, fotolítica, fotocatalítica e eletrofotocatalítica do hormônio etinilestradiol / Study of electrochemical degradation, photochemical, and photocatalytic eletrofotocatalitica the hormone ethinylestradiolEduardo, Emerson Jeronymo 18 September 2009 (has links)
Nos últimos anos, a pesquisa ambiental tem se defrontado com sérios problemas em relação a algumas substâncias presentes no meio aquático em pequenas concentrações. Os desruptores endócrinos tais como produtos farmacêuticos, hormônios naturais e sintéticos, pesticidas, substancias tensosativas, polímeros de baixa massa molar e diversos outros contaminantes orgânicos presentes em efluentes municipais e industriais, são exemplos dessas substancias que contribuem para alterar o funcionamento do sistema endócrino. Varias técnicas já foram empregadas na degradação dessas substancias contida no meio aquático, mas não se demonstraram eficazes.Este trabalho tem por objetivo estudar a degradação do Hormônio etinilestradiol que é um agente terapêutico e um método contraceptivo muito usado. É preocupante o crescente uso desta substância, pois é encontrada no meio aquático de várias partes do mundo.Para a degradação do hormônio etinilestadiol testou!se quatro técnicas de degradação, (a) eletrólise, (b) fotólise, (c) fotocatálise e (d) eletrofotocatálise.O eletrodo de trabalho utilizado foi de carbono vítreo em meio de KCl para a eletrólise. Como catalisador utilizou!se o Ti/TiO2 na fotocatálise e foteletrocatálise. Os processos degradativos foram acompanhados por espectroscopia UV-vis e cromatografia HPLC.Foram feitos testes de identificação da formação de clorato e hipoclorito nas amostras finais, pois estas espécies podem auxiliar na degradação de substâncias orgânica nos processos eletroquímicos e fotoquímicos. / In recent years, the environmental research has been facing serious problems in relation to some substances in the aquatic environment in small concentrations, such as the endocrine desruptores pharmaceuticals, natural and synthetic hormones, pesticides, tensosativas substances, polymers of low molar mass and different other organic contaminants present in municipal and industrial effluents, are examples of substances that contribute to change and the functioning of the endocrine system. Several techniques have been used for the degradation of these substances in the aquatic environment, but none demonstrated effectiveness.This work aimed to study the degradation of the hormone ethinyl estradiol which is a therapeutic agent and is widely used in contraceptive method. It is worrying the increasing use of this substance because it is found in various parts of the world.It was tested four techniques of degradation of the hormone, (a) electrolysis, (b) photolysis, (c) photocatalysis and (d) photoelectrocatalysis. The working electrode of glassy carbon was used in KCl medium for electrolysis. As catalyst, was used Ti/TiO2 in the photocatalysis and photoelectrocatalysis. The degradation process was followed by UV-vis spectroscopy and HPLC chromatography.It was carried out tests to identify the formation of chlorate and hypochlorite in the samples once these species can help the degradation in electrochemical and photochemical process.
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Estudo da degradação eletroquímica, fotolítica, fotocatalítica e eletrofotocatalítica do hormônio etinilestradiol / Study of electrochemical degradation, photochemical, and photocatalytic eletrofotocatalitica the hormone ethinylestradiolEmerson Jeronymo Eduardo 18 September 2009 (has links)
Nos últimos anos, a pesquisa ambiental tem se defrontado com sérios problemas em relação a algumas substâncias presentes no meio aquático em pequenas concentrações. Os desruptores endócrinos tais como produtos farmacêuticos, hormônios naturais e sintéticos, pesticidas, substancias tensosativas, polímeros de baixa massa molar e diversos outros contaminantes orgânicos presentes em efluentes municipais e industriais, são exemplos dessas substancias que contribuem para alterar o funcionamento do sistema endócrino. Varias técnicas já foram empregadas na degradação dessas substancias contida no meio aquático, mas não se demonstraram eficazes.Este trabalho tem por objetivo estudar a degradação do Hormônio etinilestradiol que é um agente terapêutico e um método contraceptivo muito usado. É preocupante o crescente uso desta substância, pois é encontrada no meio aquático de várias partes do mundo.Para a degradação do hormônio etinilestadiol testou!se quatro técnicas de degradação, (a) eletrólise, (b) fotólise, (c) fotocatálise e (d) eletrofotocatálise.O eletrodo de trabalho utilizado foi de carbono vítreo em meio de KCl para a eletrólise. Como catalisador utilizou!se o Ti/TiO2 na fotocatálise e foteletrocatálise. Os processos degradativos foram acompanhados por espectroscopia UV-vis e cromatografia HPLC.Foram feitos testes de identificação da formação de clorato e hipoclorito nas amostras finais, pois estas espécies podem auxiliar na degradação de substâncias orgânica nos processos eletroquímicos e fotoquímicos. / In recent years, the environmental research has been facing serious problems in relation to some substances in the aquatic environment in small concentrations, such as the endocrine desruptores pharmaceuticals, natural and synthetic hormones, pesticides, tensosativas substances, polymers of low molar mass and different other organic contaminants present in municipal and industrial effluents, are examples of substances that contribute to change and the functioning of the endocrine system. Several techniques have been used for the degradation of these substances in the aquatic environment, but none demonstrated effectiveness.This work aimed to study the degradation of the hormone ethinyl estradiol which is a therapeutic agent and is widely used in contraceptive method. It is worrying the increasing use of this substance because it is found in various parts of the world.It was tested four techniques of degradation of the hormone, (a) electrolysis, (b) photolysis, (c) photocatalysis and (d) photoelectrocatalysis. The working electrode of glassy carbon was used in KCl medium for electrolysis. As catalyst, was used Ti/TiO2 in the photocatalysis and photoelectrocatalysis. The degradation process was followed by UV-vis spectroscopy and HPLC chromatography.It was carried out tests to identify the formation of chlorate and hypochlorite in the samples once these species can help the degradation in electrochemical and photochemical process.
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Fishy behavior : persistent effects of early-life exposure to 17α-ethinylestradiolVolkova, Kristina January 2015 (has links)
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.
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Élimination des oestrogènes par Ozonation et Photocatalyse : cas de l’éthinylestradiol / Elimination of oestrogenes by ozonation and photocatalyse : case of ethinylestradiolKoumeir, Diala 16 December 2009 (has links)
Actuellement, les systèmes d’épuration des eaux n’ont pas été conçus pour éliminer les substances médicamenteuses notamment les hormones (estrogènes). Ces molécules sont ainsi déversées dans le milieu naturel et contaminent les rivières. Le 17 α-éthinylestradiol a été choisie comme molécule modèle pour illustrer la présence d’un perturbateur endocrinien dans une eau résiduaire. Cette molécule présente un réel risque pour la faune et la flore (Féminisation des poissons mâles, diminution de la reproduction…). L’objectif de ce travail est d’évaluer l’efficacité de la photocatalyse et de l’ozonation pour éliminer l’Ethinylestradiol dans les eaux. Le procédé d’oxydation utilisant l’ozone a été appliqué dans un premier temps sur des solutions concentrées d’éthinylestradiol (EE2 = 10 mg/L) préparées dans une eau ultrapure tamponnée afin de mieux comprendre les mécanismes d’élimination de ce composé et d’évaluer l’efficacité en terme de taux de conversion de EE2 et de la minéralisation du substrat. Les expériences réalisées ont permis de montrer que l’ozone est un oxydant très efficace pour oxyder l’éthinylestradiol. L’oxydation se fait essentiellement par réaction directe de l’ozone sur la molécule d’EE2 alors que sa minéralisation nécessite la conversion de l’ozone en radicaux hydroxyles qui vont ensuite réagir sur la structure saturée de la molécule organique. L’étude de l’ozonation de solutions EE2 à différentes concentrations a permis d’établir une relation entre la concentration initiale en polluant dans l’eau et la dose d’ozone à introduire pour éliminer ce composé à un pH proche de la neutralité. Cette relation a été utilisée pour estimer la dose d’ozone à appliquer pour une eau contaminée par EE2 à une concentration proche de celle rencontrée dans les eaux résiduaires et comparée à celle obtenue sur une eau provenant de la station d’épuration du Bourget du Lac. L’efficacité du procédé photocatalytique a été évaluée en travaillant sur les vitesses de disparition et de minéralisation de EE2 en présence de TiO2 «Degussa P25» et TiO2 PC500. Les cinétiques d’adsorption et de dégradation photocatalytiques ont été évaluées dans une gamme de concentration allant de 1 à 10 mg/L en travaillant à différentes masses de photocatalyseur. Les constantes d’adsorption et de vitesse, modélisés avec les modèles de Langmuir et Langmuir-Hinshelwood respectivement révèlent, (1) des propriétés d’adsorption et de photocatalyse plus importante pour le photocatalyseur TiO2 «Degussa P25», (2) des constantes d’adsorption différentes en présence ou en absence d’irradiation et aucune proportionnalité entre quantité de EE2 adsorbée et concentration en photocatalyseur. Cette absence de corrélation est expliquée en considérant la taille des particules. La formation d’agrégats de plus en plus important est observée en augmentant la masse de TiO2. La minéralisation de EE2 est totale après moins d’une heure d’irradiation en travaillant avec une puissance lumineuse de 5,5 mW/cm2 et 10 mg/L de EE2 indiquant l’efficacité de ce procédé. / Currently, systems for water treatment have not been designed to eliminate drug substances such as hormones (estrogen). These molecules are thus discharged into the environment and contaminate rivers. The 17 α-ethinylestradiol has been chosen as model molecule to illustrate the presence of an endocrine disrupter in water waste. This molecule presents a real risk to wildlife (feminization of male fish, reduced reproduction ...). The objective of this work is to evaluate the effectiveness of photocatalysis and ozonation to remove Ethinylestradiol waters. The oxidation process using ozone was applied initially in concentrated solutions of ethinylestradiol (EE2 = 10 mg / L) prepared in ultrapure water buffered to better understand the mechanisms of elimination of this compound and evaluate the effectiveness in terms of conversion rate of EE2 and mineralization of the substrate. Experiments have shown that ozone is a very effective oxidant for oxidation of ethinylestradiol. Oxidation occurs mainly by direct reaction of ozone on the molecule while EE2 mineralization involves the conversion of ozone into hydroxyl radicals which will then react on the structure of the saturated organic molecule. The study of ozonation of EE2 solutions at different concentrations has established a relationship between the initial concentration of pollutant in the water and the dose of ozone introduced to eliminate this compound at a pH near neutrality. This relationship was used to estimate the dose of ozone applied to water contaminated with EE2 at a concentration close to that found in wastewater and compared to that obtained water from the treatment plant Bourget du Lac. In the second part of our work, we studied the advanced oxidation process "Photocatalysis". The adsorption and photodegradation of EE2 were performed on the surface of TiO2 Degussa P25 "and under UV radiation (λ = 365 nm). The results were modeled using the model of Langmuir and Langmuir Hinshelwood respectively. These models allowed us to determine for different masses of TiO2 quantities of EE2 adsorbed on the surface of TiO2 and the constants of adsorption in the dark under UV light, and the rate constants. These values were compared to those obtained under the same conditions but on a different type of catalyst TiO2 PC500. The measurement of particle size of TiO2 shows the formation of agglomerates increasingly important function of the mass of catalyst TiO2 PC500 and it allowed us to explain the values of the constants of adsorption and kinetic results
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Bioavailability of organic contaminants in riversOnogbosele, Cyril Oziegbe January 2015 (has links)
In rivers, association of organic contaminants with dissolved organic carbon may limit freely dissolved or bioavailable fractions and toxicity of organic contaminants. Consequently, assessment of toxicity of organic contaminants on the basis of their total chemical concentrations may lead to overestimation of risks to organic contaminants. Therefore, to achieve reliable and accurate risks assessment for organic contaminants, determination of bioavailability is important. The influence of humic acid on the bioavailability of organic contaminants in rivers was studied, using three chemicals with different properties as model contaminants, which at the start of the study were detected in wastewater effluents. It was hypothesized that in the presence of dissolved organic carbon, a fraction of the total concentration of an organic contaminant would not be bioavailable in river water. Therefore, the aim of the study was to determine bioavailability and its impact on toxicity. Bioavailability in the presence of humic acid was determined chemically and using a yeast estrogen screen assay. The chemical method comprised solid-phase extraction and liquid chromatography-mass spectrometry to determine freely dissolved and the fraction of the chemicals associated with dissolved organic carbon. The results indicated increased binding to dissolved organic carbon with the hydrophobicity of the test compounds except for perfluorooctane sulfonate. The dissolved organic carbon-water partition coefficient for ethinylestradiol was determined to be Log KDOC 2.36. Log KDOC values of 4.15 and 4.41 at 10 and 100 mg/L humic acid, respectively, were derived for hexabromocyclododecane indicating greater binding than ethinylestradiol due to the more hydrophobic character. The yeast estrogen screen was used as a biological method to measure the effect of humic acid on the bioavailability of ethinylestradiol and a more hydrophobic compound, dichlorodiphenyltrichloroethane. Results of the yeast estrogen screen indicated that the presence of humic acid had no effect on bioavailability of either of the chemicals.
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