Bisphenol a

The produced laccase of P. variabile was purified from fungal culture broth based on the method described by Forootanfar et al.
[28 (link)] and applied in pollutant removal experiments. Phenol, bisPhenol A (BPA), and 4-aminoantipyrine (4-AAP) were purchased from Merck (Darmstadt, Germany). 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) was obtained from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals were of analytical grade.

Bisphenol A (BPA) was purchased from Merck KGaA, Darmstadt, Germany, CAS Number: 80–05-7, Cat. No.: 803546. Nattokinase (NK) was purchased from Doctor’s Best (USA), Item Code: 286,737, UPC: 75,395,000,253.

Styrene (ST) (Sigma-Aldrich, St. Louis, MO, USA) has been purified through vacuum distillation. 2-Hydroxyethyl methacrylate (HEMA) (Sigma-Aldrich, St. Louis, MO, USA) and glycidyl methacrylate (GMA) (Merck, Darmstadt, Germany) have been purified by passing over short columns of activated basic alumina. Potassium persulfate (K₂S₂O₈) (KPS) (Merck, Darmstadt, Germany) has been recrystallized from an ethanol/water mixture and then vacuum-dried. β-cyclodextrin (BCD-OH) (≥95.0%, Wacker Chemie, Munich, Germany) was vacuum-dried before use for 24 h. p-Toluene sulfonyl chloride (Ts-Cl) (reagent grade, ≥98%, Merck, Darmstadt, Germany), sodium hydroxide (reagent grade, ≥98%, pellets (anhydrous), (Sigma-Aldrich, St. Louis, MO, USA), 1,4-Diaminobutane (99%, Sigma-Aldrich, St. Louis, MO, USA), acetone (for analysis, >99%, Chemical Company Iasi), dimethyl sulfoxide (DMSO) (Aldrich-anhydrous, Darmstadt, Germany), pyridine (Aldrich, Darmstadt, Germany), glucose (Aldrich anhydrous, Darmstadt, Germany), phenol (Merck, Darmstadt, Germany), sulfuric acid (Sigma-Aldrich, St. Louis, MO, USA), bisphenol A (Merck, Darmstadt, Germany), ethanol (Chimopar, Bucuresti, România) were used as received.

The reactions were performed on a Heidolph MR Hei-Tec, equipped with Radleys Heat-On heating mantle. The solvents (dichloromethane, methanol, 99.5% of purity) were obtained from commercial suppliers (TCI, Zwijndrecht, Belgium) and used without further purification unless otherwise stated. The P25 Aeroxide titanium(IV) oxide was purchased from Sigma-Aldrich, Taufkirchen, Germany. The other reagents (bisphenol A, methylene blue, and carbamazepine) were purchased from Merck, Darmstadt, Germany; TCI, Zwijndrecht, Belgium; and Fluorochem, Glossop, UK.

Cultures of D. magna were initiated from third brood Bham2 strain neonates and maintained at a density of 20 animals/L in borehole water (collected from the University of Birmingham). The cultures were held at 20 °C with a 16 h:8 h light:dark photocycle and fed daily with Chlorella vulgaris (100 µL/animal, 500 cells/µL) as previously reported [48 (link)]. Media changes were carried out three times per week during maintenance and daily during chemical exposures.
The single exposure concentration of bisphenol-A (>99%, Merck Life Science Ltd., Gillingham, Kent, UK) in this study was determined by conducting a range finding experiment using 0, 5, 8, 10, 12, and 20 mg/kg with each test vessel containing 500 µL/L of DMSO as a solvent carrier and a total of 8 biological replicates per concentration. Single daphnids in individual 80 mL vessels were exposed immediately after their 6th maternal brood (3–4 weeks old) with samples taken at 8, 24, 48, and 72 h.
For the sample set that was analysed in this work, three biological replicate samples were taken from the control animals and those that were exposed to a single BPA concentration of 5 mg/kg using the same protocol as described for the range finding.