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

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.