Determination of bisphenols in beeswax based on sugaring out–assisted liquid–liquid extraction: Method development and application in survey, recycling and degradation studies

The methodology of sugaring out–assisted liquid–liquid extraction (SULLE) coupled with high-performance liquid chromatography–fluorescence detection was devised for quantifying bisphenol A (BPA) and bisphenol B (BPB) in beeswax. The effectiveness of SULLE was methodically explored and proved superior to the salting out–assisted liquid–liquid extraction approach for beeswax sample preparation. The analytical performance underwent comprehensive validation, revealing detection limits of 10 μg/kg for BPA and 20 μg/kg for BPB. The method developed was employed to analyse commercial beeswax (n = 15), beeswax foundation (n = 15) and wild-build comb wax (n = 26) samples. The analysis revealed BPA presence in four commercial beeswax samples and three beeswax foundation samples, with the highest detected residue content being 88 ± 7 μg/kg. For BPB, two beeswax foundation samples were positive, with concentrations below the limits of quantification and 85 ± 4 μg/kg, respectively. No bisphenols were detected in wild-build comb wax. Furthermore, the bisphenol removal efficacy of two recycling methods—boiling in water and methanol extraction—was assessed. The findings indicated that after four recycling cycles using water boiling, 9.6% of BPA and 29.2% of BPB remained in the beeswax. Whereas methanol extraction resulted in approximately 7% residual after one recycling process. A long-term study over 210 days revealed the slow degradation of bisphenols in comb beeswax. This degradation fitted well with a first-order model, indicating half-lives (DT50) of 139 days for BPA and 151 days for BPB, respectively. This research provides the first report on bisphenol contamination in beeswax. The low removal rate during the recycling process and the gradual degradation in beeswax underscore the significance of bisphenol contamination and migration in bee hives along with their potential risk to pollinators warranting concern. Furthermore, the developed SULLE method shows promise in preparing beeswax samples to analyse other analytes.