Androgenic and Estrogenic Activity in Water Bodies Receiving Cattle Feedlot Effluent in Eastern Nebraska, USA

Importance of the Topic

The presence of hormonally active agents in surface waters poses a growing threat to aquatic ecosystems and wildlife health. Concentrated animal feeding operations (CAFOs) are recognized as point sources of natural sex steroids, synthetic anabolic agents, and their metabolites. Understanding the extent of androgenic and estrogenic contamination downstream of cattle feedlots is critical for assessing ecological risks, informing regulatory measures, and guiding water-quality monitoring strategies.

Objectives and Study Overview

This study investigated whether effluent from a large cattle feedlot in eastern Nebraska releases significant levels of hormonally active compounds into the Elkhorn River watershed. Specific goals were:

• To quantify total androgenic and estrogenic activity in water samples collected at multiple sites along the river and tributaries.
• To identify and measure concentrations of key anabolic steroids and natural hormones using analytical chemistry techniques.
• To compare bioassay-based activity estimates with instrumental measurements and assess their relative contributions.

Methodology and Instrumentation

Sampling was conducted over three years at six locations: a feedlot retention pond (site 1), a downstream canal (site 2), an intermediate exposure stream (site 3), three reference tributaries (sites 4–6), and two tap-water sources. Water volumes of 8–10 L were collected, preserved with sodium azide, and extracted by dichloromethane liquid–liquid partitioning. Extracts were split for parallel analyses:

• E-SCREEN and A-SCREEN bioassays using MCF7 cell lines to estimate total estrogenic (17β-estradiol equivalents) and androgenic (R1881 equivalents) activities.
• Gas chromatography–mass spectrometry (GC-MS and GC-HRMS) for targeted steroids, including estrone, 17β-estradiol, testosterone metabolites, trenbolone isomers, zeranol, and progestogens.
• Immunoaffinity cleanup and high-performance liquid chromatography followed by enzyme-immunoassay for select analytes (e.g., trenbolone, 19-nortestosterone).

Main Results and Discussion

Bioassay results revealed elevated hormonal activity at the feedlot site, declining progressively downstream:

• Site 1 exhibited the highest androgenic activity (~9.6 pM R1881 Eq) and estrogenic activity (~1.7 pM E2 Eq).
• Androgenic activity dropped by >50% at site 2 and approached reference levels (~2.5 pM) by site 6.
• Estrogenic activity was comparable at sites 1 and 2, then halved at downstream locations.
• No measurable activity was found in municipal tap water or blank controls.

Instrumental analysis identified estrone as the major estrogen in all feedlot-impacted samples (270–2,536 pg/L), accounting for up to 46% of total estrogenic activity. Natural androgen metabolites (trenbolone isomers, zeranol) were present at low picogram-per-liter levels, representing <1% of total androgenic bioactivity. The discrepancy between bioassay-derived activities and targeted analyte measurements indicates the presence of additional unidentified endocrine-active compounds and transformation products in feedlot effluent.

Benefits and Practical Applications of the Method

Combining in vitro cell-based bioassays with targeted analytical chemistry provides a comprehensive approach to water-quality assessment. Bioassays capture total hormonal effects of complex mixtures, while GC-MS and immunoassays allow for compound-specific identification and quantification. This integrated strategy can inform:

• Routine monitoring of endocrine activity in agricultural runoff.
• Risk assessments for aquatic wildlife and potential human exposure.
• Evaluation of best management practices to reduce hormone discharges from CAFOs.

Future Trends and Opportunities

Emerging needs and research directions include:

• Development of improved extraction and clean-up protocols for steroid conjugates and polar metabolites.
• High-throughput screening of broader classes of endocrine disruptors, including veterinary pharmaceuticals and transformation products.
• Expanded monitoring of sediments and biota to assess sorption and bioaccumulation.
• Integration of in vivo assays and endocrine biomarkers in sentinel species for ecological effect validation.
• Implementation of regulatory frameworks requiring routine endocrine activity testing of CAFO effluents.

Conclusion

Feedlot effluent in eastern Nebraska contributes measurable androgenic and estrogenic activity to receiving water bodies. Estrone is the dominant estrogenic compound, while natural androgen metabolites account for a small fraction of total androgenic effects. Bioassay–analytical coupling offers a powerful tool for detecting complex endocrine-active mixtures and guiding mitigation strategies to protect aquatic ecosystem health.

Reference

Soto AM, Calabro JM, Prechtl NV, Yau AY, Orlando EF, Daxenberger A, Kolok AS, Guillette LJ Jr, le Bizec B, Lange IG, Sonnenschein C. 2004. Androgenic and Estrogenic Activity in Water Bodies Receiving Cattle Feedlot Effluent in Eastern Nebraska, USA. Environmental Health Perspectives 112(3):346–352. doi:10.1289/ehp.6590