Unexpected Discovery of the Food Additive Nonivamide as a Main Estrogenic Contributor in the Three Gorges Reservoir

Thousands of natural and synthetic substances in the environment have been reported to exhibit estrogenic effects. However, targeted analysis of a limited number of typical estrogens or estrogenic candidates is insufficient for key estrogenic driver identification since many estrogenic substances are poorly characterized or even unknown. In this study, chemometric methods were applied to link <i>in vitro</i> bioassays and nontarget analysis to identify key drivers of estrogenic effects in complex environmental mixtures. The Three Gorges Reservoir (TGR), which is located in the middle and upper Yangtze River and provides drinking water to over 7 million people, was selected as the study area. Sediment samples were collected during flood and nonflood period to investigate toxic effects and chemical distribution. Bioassays, including cytotoxicity tests and yeast two-hybrid tests, revealed that samples collected during the flood period induced significantly higher estrogenic effects than those from the nonflood period. Nontarget screening by high resolution mass spectrometry (HRMS) detected 47,321 unique features, with 527 compounds being identified successfully (level 2a). Chemometric analysis by correlation and weighted contribution analysis indicated that <i>N</i>-[(4-hydroxy-3-methoxyphenyl)methyl]nonanamide (nonivamide) was significantly positively correlated with estrogenic activity (<i>p</i> < 0.05). Target analysis and the yeast two-hybrid assay confirmed that nonivamide induced up to 25.2% of the estrogenic effect of sediments at environmental concentrations. This study is the first report on nonivamide occurrence in environments and its unexpected estrogenic effects, highlighting its potential ecological and health implications.