Deciphering the Estrogenic Activity of Aqueous Leachates from Elastomers by Effect-Directed Analysis

This study successfully used effect-directed analysis to identify a transformation product of N,N'-substituted p-phenylenediamines (PPD) rubber-antiozonants as the main driver of estrogenicity in aqueous leachates from elastomer membranes. First, two signals with estrogenic activity were detected via high-performance thin-layer chromatography (HPTLC) in a planar yeast estrogen screen (p-YES). After the active fractions were extracted from the HPTLC plate, they were analyzed by nontarget screening using liquid chromatography coupled to high-resolution mass-spectrometry. Via a prioritization process, the elucidation of the MS²-spectra, and a library research, 4-hydroxydiphenylamine (4HDPA) and N-phenyl-p-benzoquinone monoimine (QMI) were identified (m/z 186.0915 and 184.0757, respectively, in positive mode). 4HDPA is a hydrolysis product of PPD antiozonants, whereas QMI is formed by the oxidation of 4HDPA. By measuring the leachate within the YES medium, we found that QMI was transformed into 97 ± 1% of the estrogenic active compound 4HDPA. Therefore, YES results of redox couple compounds need to be carefully assessed due to potential transformations occurring in the YES medium itself. Finally, the estrogenic activity of 4HDPA was identified for the first time, and we confirmed that the overall estrogenicity observed in the elastomer leachates was predominantly caused by the elution and formation of 4HDPA by 120 ± 30%. Furthermore, 4HDPA was found in river and stream water at concentrations ranging between 7 and 20 ng/L.