Effect-directed identification and fate analysis of potent antiandrogenic coumarin derivatives in a central European river

Surface waters are adversely affected by the discharge of wastewater that is containing numerous anthropogenic micropollutants. In consequence, endocrine disrupting effects like androgenicity and antiandrogenicity are frequently observed in the aquatic environment. Even if a large list of androgen axis disruptors is available, linking of adverse effects to responsible chemicals is hampered by a great share of this compound group, which is remaining unknown. Structure elucidation of these “unknowns” in environmental mixtures of typically thousands of compounds is extremely challenging even despite powerful tools like effect-directed analysis (EDA) are available. In this thesis, a novel, multidimensional fractionation approach was developed to support EDA of surface waters with endocrine disrupting activity. In chapter 2, four reversed phases were selected with a focus on the separation of androgens and antiandrogens. To this end, a representative mixture of 39 endocrine disrupting compounds (EDCs) was separated on a set of 17 columns with widely differing bond chemistries. After exclusion of columns with poor peak shapes and chromatographic resolution those stationary phases displaying the highest degree of orthogonal separation selectivity for EDCs were depicted using principal component analysis and Spearman rank correlation: aminopropyl-, octadecyl-, and pyrenyl ethyl silica phase. Additionally, a pentafluorophenyl silica phase was chosen due to promising results reported in the literature. A surface water sample with antiandrogenic activity was fractionated in parallel on the four selected columns. The resulting coverage of the virtual two-dimensional separation space with non-target peaks confirmed the high degree of orthogonality of the selected stationary phases. Besides, the separation of co-eluting isobaric compounds led to a 4.8fold increase of the number of detected peaks. Consequently, the fractionation system is facilitating the chemical identification of organic micropollutants by chemical target, suspect and non-target screening using high resolution mass spectrometry. In chapter 3, the established fractionation approach was applied to minimize the complexity of a surface water sample collected close to the effluent of a waste water treatment plant in the river Holtemme in Germany. In concert with a miniaturized luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS non-target screening the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47), as well as the two less active derivatives 4-methyl-7-ethylaminocoumarin (C47T1) and 4-methyl-7-aminocoumarin (C47T2) were identified. The measured in vitro effect was quantitatively confirming C47 as the major cause of antiandrogenicity. Furthermore the antiandrogenic activity of C47 was also observed in vivo in spiggin-gfp Medaka already at the concentration equal to the concentration in the non-concentrated water extract. The longitudinal and temporal distribution of C47 and its derivatives along the whole river stretch was investigated in chapter 4. A constant exposure of the aquatic ecosystem by the three antiandrogens was observed at all sites downstream of the wastewater treatment plant of Silstedt, which was identified as the continuous source that is releasing C47, C47T1 and C47T2 in the gram per day range. Moreover, all compounds were detected in sediment and biota represented by the ubiquitous species Gammarus pulex. In this context, an experimental evaluation did not confirm typically proposed hydrophobic interaction to organic carbon as the driving force of partitioning into sediment but suggested cation binding of these aromatic amines to sediment. Finally, the coumarin derivatives were assessed as persistent as C47 is solely partially degraded to the less antiandrogenic potent C47T1 and C47T2 in the WWTP while a further attenuation of the compounds within the river was solely attributed to dilution by groundwater inflow