*3.2. SMX Detection*

First, 1000 mL of effluent was collected at days 30, 60, 120, 240, and 360, to measure the SMX concentrations. Then, 200 g wetland media was collected from the bottom, middle, and surface layers on day 360. Both water samples and wetland media were taken in triplicate. Water samples were filtered

through 0.45 μm fiber filters [16]. Wetland media were extracted by solid-phase extraction (Waters, Millford, MA, USA) [47]. Liquid chromatography-mass spectrometry (LC-MS/MS, Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap; Thermo Fisher Scientific, Waltham, MA, USA) was used to analyze the concentrations of SMX in water and wetland media. The mobile phase was composed of pure 30% acetonitrile and 70% water solution (*v*/*v*) [39]. Hypersil GOLD C18 column (Acquity UPLC BEH C18; 100 mm × 2.1 mm, 3 μm) was used in this study. The capillary voltage was 3.8 (±) kV, the collision energy was 8 eV, and the capillary temperature was set to 350 ◦C. SMX products were detected by the LC-MS/MS in positive mode [16].

#### *3.3. DNA Extraction and ARG Analysis*

Genomic DNA was extracted from e ffluents (200 mL) and wetland media (5 g) using a DNA extraction kit (MoBio, Carlsbad, CA, USA) at each sampling point in Section 2.2. The DNA concentrations were tested using ND-1000 NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The 16S rRNA gene and two *sul* genes (*sulI* and *sulII*) were quantified by a Real-Time PCR System (CFX96, Bio-Rad). The reaction was 25 μL on 96-well plates (Bio-Rad, Shanghai) containing 12.5 μL SYBR Green qPCR mix (Bio-Rad, Shanghai), 0.5 μL of each forward and reverse primers (Bio-Rad, Shanghai), 1 μL DNA templates, and 10.5 μL ddH2O [48]. PCR protocol and primer sequences of *sul* genes were based on previous studies [49,50].
