Identification of Unknown Antiandrogenic Compounds in Surface Waters by Effect-Directed Analysis (EDA) Using a Parallel Fractionation Approach

Abstract

Among all the nuclear-receptor mediated endocrine disruptive effects, antiandrogenicity is frequently observed in aquatic environments and may pose a risk to aquatic organisms. Linking these effects to responsible chemicals is challenging and a great share of antiandrogenic activity detected in the environment has not been explained yet. To identify drivers of this effect at a hot spot of antiandrogenicity in the German river Holtemme, we applied effect-directed analysis (EDA) including a parallel fractionation approach, a downscaled luciferase reporter gene cell-based anti-AR-CALUX assay and LC-HRMS/MS nontarget screening. We identified and confirmed the highly potent antiandrogen 4-methyl-7-diethylaminocoumarin (C47) and two derivatives in the active fractions. The relative potency of C47 to the reference compound flutamide was over 5.2, whereas the derivatives were less potent. C47 was detected at a concentration of 13.7 μg/L, equal to 71.4 μg flutamide equivalents per liter (FEq/L) in the nonconcentrated water extract that was posing an antiandrogenic activity equal to 45.5 (±13.7 SD) FEq/L. Thus, C47 was quantitatively confirmed as the major cause of the measured effect in vitro. Finally, the antiandrogenic activity of C47 and one derivate was confirmed in vivo in spiggin-gfp Medaka. An endocrine disrupting effect of C47 was observed already at the concentration equal to the concentration in the nonconcentrated water extract, underlining the high risk posed by this compound to the aquatic ecosystem. This is of some concern since C47 is used in a number of consumer products indicating environmental as well as human exposure.

Effect‐Directed Analysis of Progestogens and Glucocorticoids at Trace Concentrations in River Water

“…For secondary fractionation, an aliquot of the primary fraction F18 corresponding to 3 L of water equivalent was concentrated into 2 mL of methanol:water (30:70), and aliquots of 400 µL were fractionated using an aminopropyl column (Unison NH2, 150 × 10 mm, 3-µm particle size, Imtakt) with methanol [B]:water [A] as mobile phases, both containing 0.1% formic acid. Aminopropyl has been identified in a previous study as a stationary phase highly orthogonal to C18 in RP chromatography (Muschket et al 2018). Gradient elution was carried out with a flow rate of 2.8 mL/min.…”

“…In a previous study, Weiss et al (2009) demonstrated that fractionation of complex mixtures may eliminate such masking effects. Combining effect-based monitoring and fractionation with chemical analysis of active fractions in effect-directed analysis (EDA) is a powerful tool to identify the drivers of endocrine disruption (Thomas et al 2004;Brack et al 2016;Hashmi et al 2018;Muschket et al 2018).…”

Prioritising site-specific micropollutants in surface water from LC-HRMS non-target screening data using a rarity score

“…Among these compounds were 7-diethylamino-4-methylcoumarin, 7-ethylamino-4-methylcoumarin and 7-amino-4-methylcoumarin, which were recently identified at this site as causative compounds for the observed anti-androgenicity [40]. The latter is used as optical brightener (or fluorescent whitening agent) for textiles and a constituent in cleaning detergents and washing powders [27] and has not been found anywhere else in surface water or wastewater [36].…”

Future water quality monitoring: improving the balance between exposure and toxicity assessments of real-world pollutant mixtures

“…Furthermore, it may involve mixture toxicity modeling, and finally hazard confirmation, which should account for effects at higher levels of biological organization, such as populations and communities under realistic exposure conditions [10]. The power of this approach has been demonstrated, e.g., for anti-androgenic effects detected in vitro in a small river in Germany impacted by treated wastewater [43]. Parallel fractionation with different stationary phases together with testing and chemical screening of the resulting fractions was able to reduce the number of candidate peaks to very few peaks, eventually identifying and confirming the fluorescent dye coumarin 47 as the driver of the measured effect in vitro and in vivo in Medaka embryos.…”