The substances that drive progress and improve our quality of life also pose unintended risks to both human health and ecosystems, necessitating careful assessment and management. Aquatic environments, among the most biodiverse and vital ecosystems on earth, provide essential ecological services and sustain countless species. Protecting these environments requires interdisciplinary collaboration across environmental sciences, such as toxicology and chemistry, to address the direct and indirect consequences of anthropogenic pollution. To enhance water quality, particularly while facing growing environmental challenges, it is crucial to bridge the gap between chemical analyses and effect-based methods (EBM). One category of emerging contaminants consists of endocrine-disrupting chemicals (EDC), which can exert harmful effects even at concentrations below 1 ng/L. They are pollutants that interfere with hormonal systems in organisms, potentially causing adverse health effects. To address present challenges, this thesis aims to apply effective methodologies for monitoring EDC in freshwater, with a focus on their occurrence, biological impact and the sustainability of their analysis. It builds on the
current state of the art and provides new insights into the assessment of endocrine disruptors in aquatic environments to improve monitoring strategies and support regulatory measures for protecting ecosystems and human health.

Sample preparation is crucial in these concentration ranges, with solid-phase extraction (SPE) being the most common method. Micropollutant concentrations in unenriched samples may be insufficient for detection, particularly in water samples with reduced contamination load. Although concentrations may fall below detection limits, biological activity may still be present, necessitating sample enrichment for reliable analysis. However, as enrichment can introduce masking effects due to high chemical loads, obscuring the endpoints of interest. Furthermore, standardized procedures for these processes are still lacking. EBM targeting EDCs primarily employs receptor-mediated
bioassays, which assess specific toxicity pathways. This study demonstrated the efficacy of applying various Arxula yeast-based bioassays in detecting endocrine activity (estrogen, androgen and progestogen) in aquatic samples. While surface water (SW) samples exhibited higher effect concentrations due to direct pollution sources like wastewater treatment plants, groundwater (GW) samples showed a dominance of inhibitory effects. Not all hormonal endpoints in Arxula yeast-based assays are suitable for GW and SW analysis. The Arxula Yeast Glucocorticoid Screen (A-YGS) exhibited reactivity to glucocorticoid receptor agonists but showed EC50 values in the μg/L range, limiting its applicability for detecting environmentally relevant concentrations. Antagonism in yeast-based assays remains challenging due to the lack of appropriate reference substances, particularly for the
progesterone and glucocorticoid receptors.

Estrogens were among the first group of chemicals to raise awareness of EDC exposure in the environment. Due to their known risks, the European Water Framework Directive (WFD) established strict environmental quality standards (EQS) for five different estrogens, including 17α-ethinylestradiol (EE2), which has the lowest EQS at 0.035 ng/L in surface waters. Chemical analyses such as gas chromatography-tandem mass spectrometry (GC-MS/MS) and liquid chromatography-tandem mass spectrometry (LC- MS/MS) have demonstrated the ability to detect all estrogens of interest. The validated chemical methods, now included in the new standardized ISO 13646:2025, exhibited strong
performance with acceptable repeatability and reproducibility. Therefore, a crucial clean-up step with for instance silica gel cartridges was required to accurately detect all target estrogens; without this step, compounds such as EE2 remained undetected. However, LC-MS/MS more frequently struggled with detection despite employing the purification step, whereas GC-MS/MS achieved a 0.4 pg/L detection limit for the critical EE2 (EQS: 35 pg/L) with a 10,000-fold enrichment. In surface water, wastewater influent and effluent, the limit of detection for targeted estrogens remained in the lower pg/L range.

In times of resource scarcity, environmental pollution and climate change evaluating analyses in terms of their sustainability provides insights into potential improvements. The application of software tools for green analytical chemistry (GAC) and white analytical chemistry (WAC) helps to assess sustainability aspects of different methods. While GAC focuses on environmental burden and safety, WAC incorporates additional factors such as performance and efficiency. The combined use of multiple metrics provides a more holistic assessment. Advancements in in-situ SPE and process miniaturization can significantly enhance the sustainability of EDC analysis. Automation, particularly in EBM, holds substantial potential to improve efficiency and reduce manual intervention in complex workflows. Additionally, effect-directed analysis (EDA) offers both green and analytical advantages by reducing the number of samples requiring extensive chemical characterization.