As a nondestructive means of environmental monitoring, bird feathers have been used to analyze levels of per- and polyfluoroalkyl substances (PFASs) in specific environments. In this study, feather samples from 10 waterbird species around Poyang Lake were collected, and a pretreatment method for PFASs in feathers was optimized. The results showed that a combined cleaning method using ultrapure water and n-hexane effectively removed external PFASs. Twenty-three legacy and emerging PFASs were identified in the feathers of waterbirds, of which hexafluoropropylene oxides (HFPOs), chlorinated polyfluoroalkyl ether sulfonates (Cl-PFESAs), and sodium p-perfluorinated noneoxybenzene sulfonate (OBS) were reported for the first time, with their concentrations ranging from 0.060–2.4 ng·g−1 dw, 0.046–30 ng·g−1 dw, and lower than the method detection limit to 30 ng·g−1 dw, respectively. Compound- and species-specific bioaccumulation of PFASs was observed in the feathers of different waterbird species, suggesting that different PFAS types can be monitored through the selection of different species. Moreover, the concentrations of most PFCAs (except perfluorobutyric acid), perfluorooctane sulfonate (PFOS), and perfluorooctane sulfonamide (FOSA) were significantly positively correlated with δ15N (p < 0.05), while the concentrations of HFPOs, Cl-PFESAs, and OBS had significant positive correlations with δ13C. This indicates that the bioaccumulation of legacy and emerging PFASs in waterbird feathers is affected by their trophic level, feeding habits, and foraging area.
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