3.2.1. Chemicals

This study targeted 26 antimicrobials including: Azithromycin (AZM), cefadroxil (CFR), ciprofloxacin (CIP), clarithromycin (CLR), clindamycin (CLI), erythromycin (ERY), fleroxacin (FLRX), lincomycin (LCM), lomefloxacin (LOM), metronidazole (MTZ), nalidixic acid (NAL), norfloxacin (NOR), ofloxacin (OFX), oxytetracycline (OTC), pefloxacin (PEF), rifampicin (RIF), roxithromycin (ROX), sulfadiazine (SD), sulfadimethoxine (SDM), sulfamethazine (SDD), sulfamethoxazole (SXT), sulfathiazole (ST), tetracycline (TET), thiabendazole (TBZ), trimethoprim (TMP), and vancomycin (VAN). All pharmaceutical standards for target antimicrobials were of high purity grade (>90%). All compounds were purchased from Sigma–Aldrich (Darmstadt, Germany). Only trimethoprim was sourced from The National Drug Research Institute in Warsaw, Poland. Isotopically labeled compounds used as mixture of internal standards (1 μg/mL in methanol), i.e., azithromycin-C13, ciprofloxacin-D8, sulfamethoxazole-D4, clindamycin-D4, erythromycin-C13D3, ofloxacin-D8, tetracycline-D6, and trimethoprim-D9 (Toronto Research Chemicals, Toronto, Canada), were added to each sample before extraction (500 μL). Solvents, such as HPLC-grade methanol, acetonitrile (LiChrosolv), and formic acid (98%), were obtained from Merck (Darmstadt, Germany). Moreover, ultrapure water was obtained from a Millipore water purification system (Milli-Q water). All working solutions were prepared prior to analysis.

#### 3.2.2. Preparation of Water and Sewage Sludge Samples

Aqueous samples were filtered through glass fiber filters (GF/C, Whatman, Pittsburgh, PA, USA) and membrane filters (0.2 μm, Sartorius Goettingen, Germany). To 200 mL volumes of filtrate, 200 mg of ethylenediaminetetraacetic acid was added. Then, solid-phase extraction (SPE) (Oasis HLB cartridges, 3 mL, 400 mg, Waters Corp., Milford, MA, USA) using a Phenomenex vacuum system (Torrance, CA, USA) was performed. The elutions were made with pure methanol (3 × 2 mL). The eluents were

evaporated to dryness under a stream of nitrogen (99.999% purity, Multax, Poland) at 40 ◦C and reconstituted in a methanol-water mixture (10:90, *v*/*v*) (1 mL).

Sludge samples were centrifuged at 5000 rpm for 5 min and the supernatant was discarded. Five g of WWTP2-RS or 10 g of the other samples were placed into 50 mL polypropylene tubes, which were then mixed with 9 mL of 30 mMpotassium phosphate monobasic solution and 1 mL of methanol, and extracted for 20 min using 10 mL of acetonitrile with 1% formic acid and modified QUECHERS salts (4 g MgSO4, 1 g NaCl, 1 g Na3Citrate, and 0.5 g Na2Citrate•H2O). Next, the samples were centrifuged for 5 min at 5000 *g*. The samples were then cleaned by incubating 9 mL of extract with 500 mg octadecyl sorbent and 750 mg of MgSO4. They were then vortexed for 5 min at 1200 rpm and centrifuged for 5 min at 5000 rpm. Eight mL of the extract (organic layer) under a nitrogen stream at 40 ◦C and reconstituted in 0.5 mL of a mixture of methanol-water (10:90) was evaporated. The isotopically labeled compounds were used as an internal standards mixture to each sample before extraction (50 μL).

#### 3.2.3. Antimicrobial Detection by LC-MS/MS Analysis

Antimicrobial concentrations were analyzed by high-performance liquid chromatography coupled to mass spectrometry with a Hybrid Triple Quadrupole/Linear Ion trap mass spectrometer (QTRAP®4000, AB SCIEX, Framingham, MA, USA). LC analysis was performed using an Agilent 1260 Infinity (Agilent Technologies, Santa Clara, CA, USA) equipped with a degasser, thermostated autosampler, and binary pump, and connected in series to an AB Sciex 4000 QTRAP mass spectrometer equipped with a Turbo Ion Spray source that was operated in both positive mode and negative mode. The curtain gas, ion source gas 1, ion source gas 2, and collision gas (all high purity nitrogen) were set at 35 psi, 60 psi, 40 psi, and "medium" instrument units, respectively, and the ion spray voltage and source temperature were set at 5000 V and 600 ◦C, respectively. Chromatographic separation was achieved with a Kinetex RP-18 column (100 mm × 4.6 mm, 2.6 μm) supplied by Phenomenex (Torrance, CA, USA). The column was maintained at 40 ◦C and the flow rate was 0.5 mL/min. The mobile phase consisted of HPLC-grade water with 0.2% formic acid as eluent A and acetonitrile with 0.2% formic acid as eluent B. The gradient (%B) was as follows: 0 min. 10%, 1 min. 10%, 25 min. 90%, and 35 min. 90%. The injection volume was 10 μL. The target compounds were analyzed in multiple reaction monitoring (MRM) mode in positive ionization mode (ESI +), monitoring two transitions between the precursor ion and the most abundant fragment ions for each compound. Internal standards were attributed to analyzed compounds based on similarities between chemical structures of surrogate and analyzed compound (according to the Tanimoto similarity index). The LC-MS method was validated using three quality control levels (low, medium, and high) prepared on effluents. The interday precision higher than 15% was observed for cefadroxil (up to 22%), norfloxacin (up to 22%), lomefloxacin (up to 24%), and azithromycin (up to 35%) in case of wastewater and for cefadroxil (up to 23%), azithromycin (up to 26%), roxithromycin (up to 34%), and clarithromycin (up to 38%) in case of sediments. Significant matrix effect (lower than 85% or higher than 115%) was observed for 12 compounds in the case of wastewater and 21 in the case of sediments. Additionally to internal standard addition to control the matrix effect, each sample was tested without and after fortification with antimicrobials.

The following blanks were used: the HPLC blank (10% methanol) and the method blank (Milli-Q water, calcinated sand) to evaluate the contamination resulting from the complete preparation and analytical procedure. The positive control (water or sand fortified with pharmaceuticals) was also applied. Then, the obtained results were adjusted with recovery and matrix effect. The method detection limit (MDL) and method quantitation limit (MQL) for the entire method (including extraction) were determined as the amount of analyte in matrix spiked with signal-to-noise ratios (S/N) of 3:1 and 10:1, respectively. For the pharmaceuticals already present in samples, MDL and MQL were estimated by determining the S/N of the minimum measured concentrations and extrapolating to S/N values of 3 and 10, respectively.
