Ecosystems At Risk When Estrogens Mix With Other Chemicals

New experiments reveal that the synthetic estrogen used by women for birth control causes wide ranging health effects in minnows, but that the effects were different from when the drug was tested alone compared with when it was mixed with wastewater effluent. The tests found that when the estrogen, called 17α-ethinylestradiol, showed up in the water along with municipal wastewater, it caused feminization of male fish, altered their DNA integrity, changed their immune cell numbers and the ability to breakdown pollutants.

New experiments reveal that the synthetic estrogen used by women for birth control causes wide ranging health effects in minnows, but that the effects were different from when the drug was tested alone compared with when it was mixed with wastewater effluent.

The tests found that when the estrogen, called 17α-ethinylestradiol, showed up in the water along with municipal wastewater, it caused feminization of male fish, altered their DNA integrity, changed their immune cell numbers and the ability to breakdown pollutants.

The study discussed here highlights the need for more research on the potential health effects of exposure to complex estrogenic mixtures such as wastewater effluent, as the effects may differ from effects of exposure to single compounds known to be in those mixtures.

Ecological overview:

Water flowing out of wastewater treatment plants carries estrogenic chemicals out into the environment. Over the past 15 years scientists have discovered natural and synthetic estrogenic chemicals in sewage water effluent, including 17α- ethinylestradiol (EE2) from birth control pills can affect reproduction and development of fish living in the waters downstream.


They cause male fish to start producing egg yolk (measured by detection of a yolk protein, vitellogenin), change blood concentrations of hormones, alter the expression of genes important for synthesizing hormones, decrease the size of testes and alter secondary sex characters, including coloration and behavior.

Lab studies have identified EE2 as one of the most powerful components in the wastewater. In one experimental study in a Canadian lake, EE2 caused the collapse of the an entire fish population.

This study is the first to examine the impact of EE2 on an array of health endpoints when it interacts with the complex mixture of chemicals present in wastewater effluent.

What did they do?

Filby et al. exposed adult fathead minnows for 21 days to different mixtures of EE2 alone, wastewater effluent and the two as a mixture and then looked at a wide array of health endpoints to detect effects. The effluent, called wastewater treatment plant effluent, or WWTPE, was collected from wastewater treatment plants in the United Kingdom.

They used two different WWTPE exposures, one that was strongly estrogenic and one that was only weakly estrogenic. Some minnows were exposed to the strongly estrogenic water. In a second experiment, another set of minnows were divided into two groups: Both were exposed to a weakly estrogenic WWTPE, but in addition, one of the groups also was exposed simultaneously to EE2. The final experimental group was exposed only to EE2.

The yeast estrogen screen (YES) assay was used to determine relative estrogenicity of the potent, weak and weak effluent plus EE2 treatments. The concentrations were 21.3 ± 9.12, 6.18 ± 0.96 and 11.1 ± 3.39 nanograms estradiol equivalents per liter (ng E2/L), respectively.

While the same amount of EE2 was used for the EE2 treatment only and the weak estrogen plus EE2, direct measurements of the actual concentrations revealed 11.9 ± 0.47 for the EE2 treatment and 4.46 ± 1.00 ng/L in the weak effluent plus EE2 treatment. The authors suggested that something in the weak effluent may have bound with the EE2, or that microbes could have digested it.

This water analysis showed that adding EE2 to the weak effluent increased the mixture's estrogenicity by 1.8-fold, to 11.1 nanograms estradiol equivalents.

Five health effects were measured in the exposed fish: 1) growth (tissue mass and insulin-like growth factor-1 expression), 2) genotoxicity (via DNA strand breakage in blood and/or gonad cells), 3) immunotoxicity (total white blood cell types (lymphocytes, granulocytes, monocytes and thrombocytes) and phagocyte activity), 4) metabolic responses (a measure of CYP1A enzyme activity and the expression of cyp1a, cyp3a and gst genes) and 5) endocrine effects (vitellogenin and estrogen and androgen receptor gene expression).

The liver enzyme genes, cyp1a, cyp3a, and gst are responsible for making certain pollutants easier to clear from the body. Gene expression was measured in the liver and/or gonads.

What did they find? In addition to the expected endocrine disruption (i.e., altered vitellogenin gene expression and protein induction and altered hormone receptor expression), effluent exposure was also associated with increased DNA strand breakage, a decreased number of lymphocytes, an increased number of granulocytes and thrombocytes, and altered expression of liver metabolizing enzyme genes. No effects on growth or phagocytosis were found in fish and so these endpoints were not measured during the second experiment.

For many of these endpoints, the impacts were greater in the strongly estrogenic effluent. Some were specific to either female or male fish.

An important finding from this study was that some of the responses to the single compound differed from the responses of fish exposed to the mixture.

Male fish exposed to all four treatments - the potent effluent, the weak effluent, the weak effluent plus EE2 and EE2 alone - had increased amounts of vitellogenin protein and gene expression compared to controls. In females, potent or weak WWTPE, had no effect on vitellogenin protein or gene expression, yet EE2 alone slightly increased vitellogenin protein. The authors suggest males were more sensitive to the effects of WWTPE estrogens than the females, which normally have higher blood estrogen levels.

The weak WWTPE plus EE2 mix changed the immune response in fish when compared to the EE2 only exposure. Lymphocytes decreased, and thrombocytes (platelet precursors) and granulocytes increased in both females and males. For the EE2 alone, there was no effect on any of the white blood cell numbers.

The ability to metabolize natural and foreign chemicals circulating in the blood differed. Fish exposed to either potent or weak WWTPE had increased liver CYP1A enzyme (EROD) activity and cyp1a gene expression.

When male fish were exposed to the weak WWTPE and EE2 mixture or EE2 alone, the effects on both endpoints were reversed; that is, both EROD and cyp1a were inhibited relative to the weak WWTPE exposed fish. In females, there was a similar response in EROD activity. Although there was no significant difference in cyp1a gene expression from the weak WWTPE exposure, cyp1a expression from EE2 alone was inhibited.

The authors suggest that components of the WWTPE are by themselves enough to activate the metabolizing enzyme (EROD activity) and induce cyp1a gene expression, but that EE2 is inhibiting this biological effect. This is an important finding as the metabolizing of many pollutants, including polyaromatic hydrocarbons, PCBs, and dioxins, would potentially be affected by the presence of EE2.

What does it mean?

Sewage effluent contains a variety of estrogens combined with other pollutants in a complex chemical mix that can affect normal fish development, reproduction, metabolism, immune function, DNA integrity and endocrine function. These effects vary with the potency of, and the substances found in, the chemical concoctions. Importantly, the common synthetic estrogen, EE2, elicited different effects on the exposed fish by itself than it did when added to a weak effluent mix.

This study is important because the researchers thoroughly examined broad health effects from exposure to WWTPE. The findings are compelling in that they go beyond endocrine effects to identify changes to the immune, metabolic and genetic systems brought on by exposure to a complex mixture of chemicals.

More importantly, the mixtures altered how an individual estrogen affected the fish. Exposing fish to an estrogen alone or the same estrogen in a mix caused different health effects in the minnows. The results cast doubt on the accuracy of chemical safety testing regimes that rely solely on testing individual chemicals instead of mixtures, which is how exposure occurs in the environment.

The authors' conclude that "the effects of estrogens when administered alone cannot always predict their effects in complex environmental mixtures and these interactive effects have far-reaching implications for the use of some biomarkers (such as EROD) in environmental monitoring. A greater understanding of the mechanisms of interactive chemical effects is essential to fully understand the impacts of environmental mixtures like effluents for exposed organisms."

In Westernized countries, male fish held in cages or collected from the wild downstream of WWTPE discharge will have elevated vitellogenin protein, altered secondary sex characteristics and possibly be intersex from exposure to ubiquitous estrogens. In fresh and estuarine waterways, researchers also find diseased fish (e.g., increased parasite load and microorganism-associated lesions) or the more profound occasional die-offs. These very same waters receive WWTPE and other industrial and agricultural effluents. More than 10 years of research into the effects of WWTPE has proven a connection between exposure to these effluents and altered reproduction and development of fishes. Government regulatory agencies are beginning to notice and take appropriate steps toward protecting wildlife and human health.

The research by Filby et al. begins to link (now) obvious endocrine system effects from exposure to broader health consequences. The findings show that effluent mixtures can have different effects on health and reproduction than exposure to single pure compounds found in the mix. These results offer important insights as to how mixtures affect health and could guide discussions about proper regulation of WWTPE and perhaps other pollutant mixtures.


Esperanza M, MT Suidan, F Nishimura, Z-M Wang and GA Sorial. 2004. Determination of sex hormones and nonylphenol ethoxylates in the aqueous matrixes of two pilot-scale municipal wastewater treatment plants. Environmental Science &Technology 38(11):3028-3035.

Jobling S, M Nolan, CR Tyler, G Brighty and JP Sumpter. 1998. Widespread sexual disruption in wild fish. Environmental Science & Technology 32:2498-2506.

Khanal SK, B Xie, ML Thompson, SW Sung, SK Ong and J Van Leeuwen. 2006. Fate, transport and biodegradation of natural estrogens in the environment and engineered systems. Environmental Science & Technology 40(21):6537-6546.

Kidd KA, PJ Blanchfield, KH Mills, VP Palace, RE Evans, JM Lazorchak et al. 2007. Collapse of a fish population after exposure to a synthetic estrogen. Proceedings of the National Academy of Sciences 104(21):8897-8901.

Liney KE, S Jobling, JA Shears, P Simpson and CR Tyler. 2005. Assessing the sensitivity of different life stages for sexual disruption in roach (Rutilus rutilus) exposed to effluents of wastewater treatment works. Environmental Health Perspectives 113:1299-1307.

Martinovic D, WT Hogarth, RE Jones and PW Sorensen. 2007. Environmental estrogens suppress hormones, behavior and reproductive fitness in male fathead minnows. Environmental Toxicology and Chemistry 26(2):271-278.