Sodium acetate

Aromatic amines and reference substances with a purity of >97 % were purchased from Sigma-Aldrich, Steinheim, Germany and Alfa-Aesar, Karlsruhe, Germany. The reagents used for hydrolysis and derivatization, namely concentrated hydrochloric acid (37 %), hydriodic acid (55 %, unstabilized, A.C.S.), sodium nitrite (≥97 %), sodium sulphite (≥98 %), alizarinsulfonic acid (98 %), and amidosulfonic acid (99, 3 %) were all products of Sigma-Aldrich. Sodium hydroxide (99 %) was from VWR, Darmstadt, Germany, and sodium acetate (≥99 %) from Applichem, Darmstadt, Germany. Analytical grade methanol was purchased from KMF Laborchemie, Lohmar, Germany, and the ultra-filtered water used was a product of PureLab Ultra (ELGA LabWater, Celle, Germany).

BPA was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). A solution of BPA (97%; CAS No. 80-05-7; MW: 228.29 g/mol) was prepared by dissolving 2 mg of the compound [28 (link)] in 2 mL of HPLC-grade methanol (Merck, Darmstadt, Germany) [25 (link),37 (link)] and added to the respirometer during the homogenization phase of the doping assay. Moreover, 2 mL of methanol was added to the respirometer during the control homogenization assay to avoid side effects generated by methanol addition in the doping test.
A total of 1 mg of carbamazepine was added during the doping phase to reach a concentration of 1000 µg/L in the respirometer, and 0.1 mg of ciprofloxacin was added in the same way to maintain a concentration of 100 µg/L [38 (link)].
Sodium acetate was obtained from AppliChem GmbH (Darmstadt, Germany). A stock solution of Sodium acetate (99%; CAS No. 127-09-3; MW: 82.03 g/mol) of 500 mg/L was prepared; then, three different dilutions of 35, 70, and 100% [39 (link)] were used in the assays.
Mixture assays were carried out by adding the three above compounds at concentrations identical to those in the single-doping test.

Feces samples were collected and stored at −20 °C until processing for DNA-based 16S rRNA gene sequencing. DNA was extracted using an established phenol–chloroform-based method^{65 (link)}. In short, 500 µl of extraction buffer (200 mM Tris (Roth), 20 mM EDTA (Roth), 200 mM NaCl (Roth), pH 8.0), 200 µl of 20% SDS (AppliChem), 500 µl of phenol:chloroform:isoamyl alcohol (PCI) (24:24:1) (Roth) and 100 µl of zirconia/silica beads (0.1 mm diameter) (Roth) were added to each feces sample. Samples were lysed and homogenized twice using a Mini-BeadBeater-96 (BioSpec) for 2 min. After centrifugation and additional extraction with PCI (24:24:1), DNA was precipitated using 500 µl isopropanol (J.T.Baker) and 0.1 volume of 3 M sodium acetate (AppliChem). Samples were incubated at −20 °C overnight and centrifuged at 4 °C at maximum speed for 20 min. The resulting DNA pellet was dried, resuspended in TE buffer (AppliChem) with 100 µg/ml RNase I (Sigma-Aldrich) and column purified (BioBasic Inc.) to remove PCR inhibitors.