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3 illustration ideas: 1. A x-ray picture of a fish tracking the endocrine system 2. a picture of a fish showing the phenotypic results of male sex reversal due to ethinylestradiol 3. a picture showing the route of ethinylestradiol into waterways

Environmental Issues
Ethinylestradiol is an emerging contaminant that enters rivers and larger waterways after passing through standard wastewater treatment. The semi synthetic hormone ethinylestradiol enters water systems through human consumption and excretion of pharmaceuticals, primarily oral contraceptives and hormone therapies. Research shows that ethinylestradiol is the most potent estrogen in sewage effluent. At certain levels present in municipal sewage effluents, ethinylestradiol induces endocrine disruptive effects in exposed fish. Endocrine disrupting chemicals are substances known to mimic or block natural hormones in exposed individuals. The United States Environmental Protection Agency (EPA) defines environmental endocrine disrupting chemicals as xenobiotics that interfere with natural hormones in the body. Studies have shown detrimental effects of estrogen contaminants, including ethinylestradiol, in a wide variety of wildlife species. Species that have been affected by endocrine disrupting compounds include frogs, fish, and alligators. Impact of Endocrine disrupting compounds on marine wildlife has begun to raise concerns of ethinylestradiol’s effects on human health. Ethinylestradiol has a high persistency and is prone to bioaccumulation in exposed organisms. Low levels of estrogens in the water can lead to many adverse effects on native fish including the feminization of males.

Effects on Fish
A balance of both genetic and environmental factors control sex determination and differentiation of fish. Alteration of environmental factors by Endocrine Disrupting Chemicals can impact sex assignment of exposed species. Laboratory Studies were done to assess the effects of varying quantities of ethinylestradiol in the water on wild roach (Rutilus rutilus). Sexually mature female and male roach were obtained from the Environment Agency’s National Coarse Fish Farm and were artificially induced to produce embryos 7-10 days post fertilization. Roach were exposed to varying levels of ethinylestradiol for up to two years and data was obtained on their sexual development. Ethinylestradiol exerted a wide range of effects including the disruption of normal sexual development, alteration of reproductive behaviors, reduction in reproductive success, and an overall reduction in fertilization success. At significant quantities of ethinylestradiol, complete sex reversal of male individuals was observed. Sex-reversed males had typical female ducts, with separate openings for the genital and urinary tracts and contained both female and male reproductive structures. The gonadosomatic index was used to measure the gonadol growth of each individual by comparing the gonad weight to the total body weight. All fish exposed to concentrations of 4 ng/L showed ovarian morphology making all individuals phenotypically female, indicating sex reversal of male species. Exposure to 4 ng/L also resulted in a large increase in plasma vitellogenin in male individuals. Vitellogenin is an estrogen dependent egg yolk precursor naturally found only in female fish. Intersex fish were characterized in several treatment groups. Intersex roach were characterized by the presence of primary oocytes scattered throughout their testicular tissue. The studies showed that females exposed to 4 ng/L concentrations contained smaller ovaries than the controls. The overall impact of a feminized population from long-term exposure of 4 ng/L can result in population failure in the wild. Assuming some intersex and sex reversed fish can produce functional gametes, due to a retarded maturation rate, gametes will be released at inappropriate times for successful reproduction.

The gamete quality of wild caught intersex roach has been analyzed. It was determined that wild-caught intersex roach that were moderately to severely feminized were less likely to produce sperm. The feminized individuals who were able to produce sperm produced up to 50% less sperm than normal males. The sperm quality of feminized males was also affected. Sperm motility decreased by 50%, reducing fertilization success by 75% compared to normal or less severely intersexed individuals. It has been estimated that environmentally relevant concentrations of ethinylestradiol in the water and its effects on native fish reduces the growth rate to zero.

One study was performed through the direct nanoinjection of ethinylestradiol in medaka (Oryzias latipes). Embryos were directly injected with varying concentrations of ethinylestradiol in order to mimic the effects the contaminant has through maternal transfer. The exposed embryos were compared to control individuals after a single exposure of ethinylestradiol. Each egg was injected with a concentration of 0.005, 0.05, 0.5, or 2.5 ng/L. After growth, histological examination of adult gonads indicated complete sex reversal at injections of 0.5 ng/L in genetic males. Only two of the six males treated with 0.5 ng/L concentrations contained testes resembling control males with all stages of germ cells present. The remaining individuals had few if any sperm present. Individuals, both genetically male and female, exposed to the highest dosages contained very small ovaries with very few mature oocytes. Results of the study indicate the relationship between ethinylestradiol induced sex reversal in males with critical times of sexual differentiation. Maternal transfer of ethinylestradiol to embryos during critical times of sexual development and differentiation can produce irreversible effects on gonad development and reproductive abilities.

Since endocrine disrupting chemicals are classified as emerging contaminants, ethinylestradiol concentration in wastewater is currently unregulated. Several organizations are considering the problems of emerging contaminants on the environment and setting up direction and legal framework to protect freshwater resources. These organizations include the European Union, The North American Organization, and the International Program of Chemical Safety. However, it is important to realize that current wastewater treatment plant technologies are not efficiently designed to remove estrogenic compounds from sewage. While wastewater treatment plants reduce the concentration of estrogens in sewage before dumping effluents into rivers, some hormones can pass through treatment plans intact. In addition to the presence of ethinylestradiol in surface waters, studies have been conducted to test the vertical transmission of ethinylestradiol through sediments into groundwater. Estrogen compounds are moderately hydrophobic which allows their adsorption to sediment and possible transmission into groundwater. Research has shown this characteristic of ethinylestradiol allows the compound to move into the sediment under effluent receiving waters, downstream from wastewater treatment plants. This increases the life and transport of the substance. In addition, its hydrophobic properties allow it to bind to the solid sludge of activated sludge biological treatment plants. This solid sludge is then spread on fields as fertilizers, burned, or buried where it has the potential to affect other organisms or ultimately end up back in water systems. Activated sludge plant is the most inexpensive wastewater treatment technology to remove contaminants from sewage. It has been shown that estrogens are most efficiently removed from wastewater using the membrane bioreactor over the activated sludge plant. The membrane bioreactor leaves approximately 2% of estrogenic activity in effluents whereas the activated sludge plant leaves about 30% of estrogenic activity.

A study performed in Nebraska focused on determining if wastewater treatment plants were contributing to estrogen concentrations in surface waters of Nebraska and if these compounds were in sufficient enough quantities to cause the feminization of fish. Caged fish and Polar organic chemical integrative samplers (POCIS) were deployed at three sites downstream from wastewater treatment plants and two sites upstream of plants near Columbus, Grand Island, and Hastings. The results of the study showed that the greatest quantities of ethinylestradiol were found downstream from wastetwater treatment plants. Downstream from treatment plants in Columbus concentrations of ethinylestradiol were measured at 1.3 ng/L. Downstream from wastewater treatment plants in Grand Island and Hastings ethinylestradiol concentrations were much higher, ranging from 12.2-14.5 ng/L. The study also showed a positive correlation between feminized fish, particularly increases in vitellogenin, and their location relative to wastewater treatment plants. Studies show the greatest feminization occured downstream from treatment plants, where levels of estrogenic contaminants, measured by the POCIS were highest.

In the United Kingdom wastewater treatment effluents typically contain about 0.15 ng/L to 2.85 ng/L of ethinylestradiol. Although this is below the 4 ng/L required for complete feminization of the population, there are concerns about the feminizing effects of multiple endocrine disrupting chemical's additive effects. The presence of natural steroidal estrogens and other endocrine disrupting chemicals can be additive in their effects. Ethinylestradiol has been measured at concentrations of up to 15 ng/L in effluents and up to 5 ng/L in surface waters in Europe. Exposure of such large concentrations produces adverse and irreversible effects. Fish feminized from estrogenic compounds have been found in several waterways throughout the United States. Intersex fish have been found in the Washington D.C part of the Potomac River and the Los Angeles area of the Pacific Ocean. In Lake Mead in Las Vegas several male carp were turning into females. With further analysis, it was shown that the epidemic in Lake Mead was the result of endocrine disrupting chemicals.