How Is Estrogenic Activity Measured in the Environment?

By Stefano Tommasone Ph.D.Reviewed by Lexie Corner

Certain compounds in the environment can mimic or interfere with the natural hormone estrogen, a property known as estrogenic activity.

These interactions are often linked to endocrine-disrupting chemicals (EDCs), which can alter hormonal systems and lead to effects such as reproductive disorders, developmental issues, and increased cancer risk.

Monitoring estrogenic activity provides a practical way to assess chemical contamination in water and sediment. It's also a key tool in evaluating ecological risk and the effectiveness of pollution control strategies, particularly in regions exposed to industrial, agricultural, or municipal waste.¹

Assays for Estrogen Detection

In vitro assays are used to detect estrogenic compounds. One example is the yeast estrogen screen (YES) assay, a simple and cost-effective method that utilizes genetically modified yeast cells that express human estrogen receptors and a reporter gene. When estrogenic compounds bind to the receptors, the reporter gene is activated, leading to a measurable response.

Another common assay is E-SCREEN, which uses MCF-7 human breast cancer cells that proliferate in response to estrogenic stimuli. In this case, the extent of cell proliferation is proportional to the estrogenic potency of the sample.

In the ER-CALUX assay (Estrogen Receptor–Chemically Activated LUciferase eXpression) human cells are engineered to produce luciferase in response to estrogen receptor activation. The luminescence generated provides a quantifiable measure of estrogenic activity, with detection limits in the low pg range.

As an alternative to bioassays, estrogen receptor binding assays can provide information on potential estrogenic activity by determining the affinity of compounds for estrogen receptors.

Several synthetic fluorescence compounds have been used recently to determine relative binding affinities of estrogenic compounds, but their high cost has limited large-scale use.²

Image Credit: Saiful52/Shutterstock.com

Tracking Estrogenic Pollution in the Environment

These assays are integral to environmental monitoring programs that assess estrogenic contamination across various sources, including water from rivers, lakes, and wastewater treatment plant effluents.

For example, E-SCREEN has been used to profile estrogenic activity in Maryland’s Coastal Bays (a eutrophic system of estuaries impacted by human activities), focusing on the determination of 17ß-estradiol, triclosan, and acetaminophen levels.³

Assays have also been used to evaluate the bioavailability and potential release of estrogenic compounds from sediments. In Guanabara Bay, Brazil, the YES assay identified possible harmful effects from micropollutants, highlighting the need for ongoing monitoring efforts.⁴

The data obtained from these assays inform regulatory decisions and ecological risk assessments. They guide the establishment of environmental quality standards and help evaluate the effectiveness of pollution control strategies, ultimately protecting both public health and the environment.

Are Current Testing Methods Enough? What Works and What Doesn’t

Measuring estrogenic activity using in vitro bioassays comes with several notable advantages, chief among them is their high sensitivity. These assays can detect extremely low concentrations of estrogenic compounds and provide valuable insight into their potential biological effects.

Cost-effectiveness and scalability are also key strengths. Compared to in vivo tests, in vitro assays require fewer resources and are better suited for screening large numbers of samples or compounds at relatively low cost, making them ideal for routine environmental monitoring.

However, there are limitations. The biological responses observed in vitro don't always align with outcomes in living organisms. As a result, in vivo studies are often needed to confirm whether a compound truly has endocrine-disrupting effects.

Other challenges include variability in assay performance. Differences in assay design, cell line sensitivity, sample preparation, and experimental conditions can all affect results. This variability can reduce consistency across studies and complicate broader analyses or comparisons, such as those needed for regulatory assessments.

Another key limitation is that in vitro bioassays typically don't identify the specific chemicals driving estrogenic activity. To fill that gap, analytical techniques are used to pinpoint and quantify individual compounds in environmental samples.

Among the most commonly used methods is liquid chromatography–mass spectrometry (LC-MS). For detecting very low concentrations, more advanced techniques like ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) offer greater precision, though they come with higher costs and require more specialized equipment.⁵

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What’s Next? New Approaches to Measuring Estrogenic Activity

One growing area of focus in measuring estrogenic activity is the use of integrated bioassays, methods that screen for multiple endocrine pathways at once, including androgenic, thyroid, and glucocorticoid activity. This approach offers a more complete picture of a compound’s endocrine-disrupting potential.

There's also increasing interest in developing environmental sensors and field-deployable assays for rapid, on-site testing. These tools are especially valuable for early-warning systems or for monitoring remote locations where laboratory access may be limited.⁶

High-throughput screening (HTS) is making it easier and faster to identify estrogenic compounds. One example is the development of assays like E-Morph, which detect subtle structural changes in cells, specifically at adherens junctions, which are connections that help cells stick together. These changes occur when estrogen receptor signaling is blocked by estrogenic compounds, offering a novel way to assess their activity.⁷

As bioassay datasets grow in complexity and volume, artificial intelligence (AI) and machine learning are becoming essential tools. These technologies can analyze large datasets, detect patterns, and predict estrogenic activity, all of which enhance the speed and precision of environmental assessments.

HTS is often combined with in silico methods, such as similarity searches or conformal prediction models, to efficiently screen massive chemical libraries. This helps prioritize compounds with potential estrogenic effects for further testing.

Meanwhile, omics techniques like transcriptomics and metabolomics are also being used in endocrine disruption research. By linking gene expression or metabolic profiles with bioassay results, researchers can better understand how estrogenic compounds influence biological systems and predict how different organisms might respond to exposure.⁸

Together, these emerging tools are building on the foundation of current assays to support more comprehensive, efficient, and context-sensitive assessments of endocrine-disrupting activity.

Interested in related challenges and innovations in environmental and chemical monitoring? Check out these articles:

• Chemical Warfare Between Algae Threatens Kelp Forest Recovery
• Commercial Solutions for Reliable Food Contaminant Analysis
• Bacteria Can Degrade PFAS and Its Toxic Byproducts
• The Science of Antidotes: How Toxins Are Neutralized

References and Further Reading

1. Lerdsuwanrut, N., Zamani, R. Akrami, M. (2025). Environmental and Human Health Risks of Estrogenic Compounds: A Critical Review of Sustainable Management Practices. Sustainability, 17, 491. Available: https://www.mdpi.com/2071-1050/17/2/491
2. Wang, C., Li, C., Zhou, H. Huang, J. (2014). High-Throughput Screening Assays for Estrogen Receptor by Using Coumestrol, a Natural Fluorescence Compound. SLAS Discovery, 19, 253-258.https://doi.org/10.1177/1087057113502673. Available: https://www.sciencedirect.com/science/article/pii/S2472555222073245
3. Elfadul, R., Jesien, R., Elnabawi, A., Chigbu, P. Ishaque, A. (2021). Analysis of Estrogenic Activity in Maryland Coastal Bays Using the MCF-7 Cell Proliferation Assay. International Journal of Environmental Research and Public Health, 18, 6254. Available: https://www.mdpi.com/1660-4601/18/12/6254
4. Santos, A. D. D. O., Nascimento, M. T. L. D., Argolo, A. D. S., Felix, L. C., Santos, R. F. D., Freitas, A. D. S. D., Hauser-Davis, R. A., Fonseca, E. M. D., Bila, D. M. Baptista Neto, J. A. (2024). Estrogenic activity and acute toxicity assessments of sediments from a chronically polluted estuarine area in southeastern Brazil. Environmental Quality Management, 33, 183-194.https://doi.org/10.1002/tqem.22104. Available: https://onlinelibrary.wiley.com/doi/abs/10.1002/tqem.22104
5. Huang, F., Karu, K. Campos, L. C. (2021). Simultaneous measurement of free and conjugated estrogens in surface water using capillary liquid chromatography tandem mass spectrometry. Analyst, 146, 2689-2704.10.1039/D0AN02335C. Available: http://dx.doi.org/10.1039/D0AN02335C
6. Guo, W., Van Langenhove, K., Vandermarken, T., Denison, M. S., Elskens, M., Baeyens, W. Gao, Y. (2019). In situ measurement of estrogenic activity in various aquatic systems using organic diffusive gradients in thin-film coupled with ERE-CALUX bioassay. Environment International, 127, 13-20.https://doi.org/10.1016/j.envint.2019.03.027. Available: https://www.sciencedirect.com/science/article/pii/S0160412019304234
7. Klutzny, S., Kornhuber, M., Morger, A., Schönfelder, G., Volkamer, A., Oelgeschläger, M. Dunst, S. (2022). Quantitative high-throughput phenotypic screening for environmental estrogens using the E-Morph Screening Assay in combination with in silico predictions. Environment International, 158, 106947.https://doi.org/10.1016/j.envint.2021.106947. Available: https://www.sciencedirect.com/science/article/pii/S0160412021005729
8. Messerlian, C., Martinez, R. M., Hauser, R. Baccarelli, A. A. (2017). 'Omics' and endocrine-disrupting chemicals - new paths forward. Nat Rev Endocrinol, 13, 740-748.10.1038/nrendo.2017.81.