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By Mindy Criser, Presented to Diane Henshel, Ph.D., School of Public and Environmental Affairs, November 15, 2001
I. Introduction
Estrogens are imperative for human growth and development. Both naturally produced estrogens and those that are
synthetically made and introduced into the body significantly affect human health. Since estrogens are produced and excreted by
humans they will inevitably end up in wastewater. The persistence of these hormones in water and the types of wastewater
treatment processes will affect their presence in our waterways. Once introduced into our waterways, these estrogens can affect
humans and wildlife by eliciting estrogenic responses. Thus, the introduction of estrogenic chemicals in the environment will not
only affect water quality, but will also affect the health of the wildlife and the humans in that environment.
II. Natural Estrogens
Estrogens are chemicals that are secreted by the endocrine glands, mainly in the ovaries. These chemicals are important for
reproductive processes and the normal development of females (ALtruis, 1999). Estrogens stimulate the development of the
reproductive structures and secondary sexual characteristics of the female, including breast, uterine, and vaginal development.
Estrogens are also necessary for skin and vascular system health, and bone homeostasis.
The female body naturally produces three estrogens: estradiol, estrone, and estriol. The principal estrogen secreted by the
ovaries is estradiol (E2) (Snyder, 1999). Estradiol is the most important estrogen for a woman’s development, and thus is also the
most potent naturally occurring estrogen (Highland, 1999; ALtruis, 1999). Its estrogenic potency is 12 times that of estrone and 80
times that of estriol. The main natural estradiol is 17-beta-estradiol (Snyder, 1999). Estrone (E1) is the dominant estrogen in
women once menopause occurs and the ovaries stop producing estradiol (Highland, 1999). Estriol (E3) is a minimal byproduct of
estrone metabolism in non-pregnant women; however, the placenta in pregnant women is a major source of estriol. In vitro, estriol
is more than 300 times more active than estradiol (Snyder, 1999).
III. Synthetic Estrogens
The main reasons for introducing synthetic estrogens into the body are for birth control (oral contraceptives) and relieving the
symptoms of menopause. The main estrogen in birth control pills is 17-alpha-ethynlestradiol (EE2) (Desbrow et al., 1998). Most
oral contraceptives contain between 30 and 50 micrograms of estrogen (Desbrow et al., 1998; Gardner, 1983). In America more
than 7 million women use oral contraceptives (Gardner, 1983). To treat menopause symptoms, women take Hormone
Replacement Therapy (HRT), which is a combination of estrogen and progesterone, and Estrogen Replacement Therapy (ERT),
estrogen only (NIA, 1999; ALtruis, 1999). Synthetic estrogens are important for not only controlling reproductive functions, but
also for maintaining women’s health once menopause occurs.
IV. Effects of Estrogens on Humans and Wildlife
Levels of vitellogenin (VTG), an egg protein precursor that is induced by estrogen, have been studied in male fish to determine
species exposure to estrogenic chemicals (Folmar et al., 1996; Routledge et al., 1998). The VTG gene is present in males but is
usually not expressed unless induced by estrogen-like chemicals. Exposure to exogenous estrogens at significant levels could
induce VTG synthesis in males. The VTG synthesis in fish exposed to treated sewage effluent confirms that estrogen
concentrations in treated effluent have an effect on wildlife. In rainbow trout, concentrations of E2 as low as 1 ng/L elicited a
vitellogenic response, as did concentrations as low as 25 ng/L of E1. (Routledge et al., 1998). EE2 has elicited responses in trout
at doses as low as 0.1 ng/L (Purdom et al., 1994). The 14-day no-observed-effect concentration (NOEC) determined by Thorpe et
al. (2000) for VTG induction in rainbow trout by 17-beta-estradiol was less than 5 ng/L. The lowest-observed-effect concentration
(LOEC) and the effective concentration (EC50) values were 9 and 15 ng/L, respectively.
Developmental abnormalities of reproductive organs and abnormal sex hormone concentrations have been attributed to
exposure to exogenous estrogens. For example, female alligators on Lake Apopka in Florida that had plasma 17-beta-estradiol
concentrations of almost double that of normal females also exhibited abnormal ovarian features (Guillette Jr. et al., 1994). Male
juvenile alligators at Lake Apopka had depressed plasma testosterone concentrations, poorly organized testes, and abnormally
small phalli. Exposure to exogenous estrogens at critical growth and development periods has been shown to cause feminization
of males. When exposed to estrogenic compounds during specific developmental periods, developing embryos of alligators and
several turtle species exhibited sex reversal (male to female) (Bull et al., 1988). High concentrations of 17-beta-estradiol have also
been shown to inhibit growth in juvenile rainbow trout (Thorpe et al., 2000). These reproductive and developmental effects caused
by exposure to estrogens and other endocrine disrupting chemicals pose serious threats to species viability and also cause concern
for human health.
Sufficient research has not been done to determine the effects of exogenous estrogens on humans; however, many current
health problems could very likely be attributed to exposure to estrogenic chemicals. The increases in testicular cancer and the
decreases in sperm counts are two current health issues that have been linked to endocrine disrupting chemicals (Joffe, 2001;
Toppari et al., 1996).
V. Presence of Estrogens in Wastewater
Since estrogens are not only produced in the body but also introduced synthetically, they will inevitably end up in one form or
another in wastewater. Studies have indicated that women can excrete in urine around 7 µg of E1, 2.4 µg of 17-beta-estradiol, and
4.6 µg of E3 per day (Adlercreutz et al., 1986). In feces, women can excrete around 0.5 µg of E1, 0.4 µg of 17-beta, and 1.25 µg
of E3 per day (Adlercreutz et al., 1994). Since estrogen production and excretion varies greatly depending on stage of the
menstrual cycle and on age, a normal female is reported to excrete between 10 and 100 µg of E2 per day (Routledge, 1999).
Pregnant women can excrete 259 µg per day of E2 during the later stages of pregnancy (Fotsis & Adlercreutz, 1987). Males also
produce and excrete estrogens, though in much smaller concentrations. One study showed that males can excrete 1.6 µg of E2 per
day. Thus, estrogens are being released constantly into wastewater.
Studies show that detectable levels of estrogens are found in the effluent from wastewater treatment plants (WWTP). The
stability of the estrogens in water will contribute to their continued presence in wastewater. Synthetic hormones are generally
more stable in water because they are less soluble (Snyder, 1999). For example, EE2 solubility in pure water and sewage-
treatment water was reported to be 3 times less soluble than natural steroidal estrogens (Desbrow et al., 1998; Tabak et al., 1981).
EE2’s low solubility contributes to its increased resistance to biodegradation as compared with natural estrogens and will cause it
to remain in wastewater for longer periods of time.
Even after wastewater treatment processes have occurred, estrogens show up in wastewater. A study of 14 sewage-treatment
plants surrounding Cincinnati, Ohio by Tabak et al. (1981) indicated that primary treatment processes remove 5 to 25 % of
synthetic hormones and 35 to 55% of natural hormones, while systems using both primary and secondary treatment could remove
20 to 40% of synthetic hormones and 50 to 70% of natural hormones. The presence of estrogens in treated effluent result from
“their partial or complete resistance to biodegradation during the treatment process” (Desbrow, et al., 1998). Laboratory
biodegradation studies have shown that EE2 is highly stable and persists in activated sludge with no detectable degradation
occurring after 120 hours of treatment (Aherne & Briggs, 1989).
Even if degradation during treatment occurs, the results are not necessarily beneficial. When estrogens are excreted in urine,
they are in a water-soluble conjugate form (Snyder et al., 1999; Desbrow, et al., 1998). These conjugates can be broken down
during wastewater treatment and in the environment to form the more potent free estrogen. For example, certain microorganisms
that are present in sewage sludge, such as E. coli, have been shown to effectively convert glucuronated estradiol into an estrogenic
form (Routledge et al., 1999). The free estrogen form, which is also the form excreted in feces, is what circulates through the body
eliciting estrogenic responses. When these free estrogen forms enter the environment, they can create estrogenic responses in the
wildlife and humans that they come in contact with. Concentrations as high as 3.7 ng/L of the natural 17-beta-estradiol and 0.8
ng/L of the synthetic EE2 were found by a study of effluent from four municipal WWTP’s in south central Michigan (Snyder et
al., 1999). These concentrations are greater than those found to elicit VTG responses in rainbow trout.
VI. Current Regulations
There are currently no federal or state regulations concerning the presence of estrogens in wastewater. The Environmental
Protection Agency was mandated in 1996 by the Food Quality Protection Act and the amended Safe Drinking Water Act to
implement an Endocrine Disruptor Screening Program (EDSP) (EPA, 2001). EDSP is working to provide methods and procedures
for detecting and characterizing endocrine activity of chemicals to allow for the evaluation of potential risks and the development
of good policies.
VII. Wastewater in the City of Indianapolis
There are two advanced wastewater treatment (AWT) facilities within the City of Indianapolis, the Belmont and Southport
plants (City of Indianapolis, 2000). AWT facilities include tertiary treatment, which removes ammonia and a high percentage of
BOD and suspended solids, and involves disinfection. After treatment, effluents are discharged to the West Fork of the White
River. During wet weather events, wastewater flowing into the Belmont plant receives primary treatment (screens and
sedimentation tanks) but exceeds the secondary treatment (bacterial degradation of waste) capacity. Thus, this wastewater receives
only primary treatment and is then discharged without disinfection to the White River. There are 134 combined sewer outfall
points, primarily in the older sections of the City of Indianapolis. The estimated annual overflow volume within the Indianapolis
sub-sewer system is 4,045 to 5,473 million gallons per year.
VIII. Policy Recommendations
Due to the natural production of estrogens and their need for reproductive control and women’s health, any policies and
regulations will have to address estrogens once they reach wastewater rather than restricting production and use. The large
amounts of untreated wastewater being discharged into the White River are a cause of great concern. The presence of estrogens in
this water poses a serious health risk to wildlife along the White River and to humans that are exposed to the untreated sewage.
Since untreated wastewater poses many additional problems than introducing estrogens into the environment, such as the discharge
of feces and other pollutants, increasing the amount of wastewater that is treated by AWT’s should be the first step in addressing
this problem. The current facilities either need to be expanded to handle larger flow volumes or another facility should be built. In
addition to increasing volume capacity, the processes within the AWT facilities should be improved to ensure greater removal of
estrogens. A plant with good reduction in biological oxygen demand (BOD) will also have a good reduction in the biodegradable
hormones (Tabak et al., 1981). Furthermore, enrichment of the activated sludge process with microorganisms with higher estrogen
removing capabilities can shorten the persistence of the hormones in wastewater, as can increasing the contact time of the
wastewater with the sludge.
The concentration of estrogens being discharged from the AWT facilities and in CSO effluent should be measured to determine
the current flow of estrogens into the White River to assist in the development of appropriate polices for their removal. Also,
measurements prior to the implementation of any procedures for decreasing estrogen concentrations will also determine the
effectiveness of the procedures. Studies done determine the risk to Indianapolis’s wildlife and human population would also be
useful for creating appropriate policy and treatment procedures. Factors that also need to be considered when designing
appropriate treatment plans include the size of the receiving waterbody, effluent quality, and weather conditions (especially rainfall
rates which will dilute the effluent) (Sumpter, 1995). Rivers of higher water quality showed less estrogenic activity downstream of
effluent discharges than rivers of lower water quality.


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