Computational protocol: The treatment of wastewater containing pharmaceuticals in microcosm constructed wetlands: the occurrence of integrons (int1–2) and associated resistance genes (sul1–3, qacEΔ1)

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Protocol publication

[…] Standard wastewater parameters such as chemical oxygen demand (COD), ammonium nitrogen (N-NH4), ortophosphate phosporus (P-PO4), nitrate nitrogen (N-NO3), total organic carbon (TOC), and total nitrogen (TN) were analyzed according to the protocol of Nowrotek et al. ().High-performance liquid chromatography method was used to determine the SMX concentrations in the influent and effluents between days 1 and 86 of the experiment. The analytical protocol for this method was reported in Nowrotek et al. (). The limit of quantification (LOD) of SMX was equal to 0.2 mg/L.The effluent samples (taken on day 22) from the PhCs-P columns were analyzed by LC-MS/MS for the presence of TPs-SMX. The effluents from the corresponding control columns were taken as background samples during the analysis of the transformation products. The identification of the TPs-SMX was performed using ultra-performance liquid chromatography system (UltiMate 3000 RS; Dionex) coupled with a tandem mass spectrometer (AB SCIEX 4000 Q TRAP) equipped with an electrospray ionization source (ESI). In both cases, the chromatographic separation was performed using C18 Hypersil™ Gold column (250 mm × 4.6 mm; pore size 5 μm) (Thermo Scientific, Polygen, Poland) at an isocratic operation mode. The flow rate of the mobile phase through the chromatographic column was equal to 1.0 mL/min. The mobile phase was composed of a mixture of acetonitrile and phosphate buffer (pH = 5.7; 40/60; v/v) and 0.1% formic acid (40/60; v/v). The tandem mass spectrometry (with electrospray ionization) was performed in a positive ion mode. Crucial MS-MS detector parameters such as ionization voltage (IS), collision-assisted dissociation (CAD) gas, source temperature (TEM), sheath gas (GS1), desolvation gas (GS2), and curtain gas (CUR) were optimized by flow injection analysis (FIA) in order to obtain better ionization of the pattern compound. These parameters were as follows: TEM = 500 °C, IS = 4000 V, CAD = medium, CUR = 15 psi, GS1 = 35 psi, and GS2 = 35 psi. The identification of TPs-SMX was performed by means of LightSight® software (AB Sciex) using predicted multiple reaction monitoring (pMRM) mode. [...] Statistical testing was performed using the STATISTICA 12 software (Stat Soft Inc. ). The results concerning the removal of SMX, TOC, and N-NO3 have been analyzed according to the procedure discussed in Nowrotek et al. (). The chi-squared and Fisher’s exact tests were performed to determine the relationship between the presence of analyzed genes, concentration of SMX, and the presence of plants in columns. The hypothesis was rejected at a p value ≥0.05. […]

Pipeline specifications

Software tools LightSight, Statistica
Applications Miscellaneous, MS-based untargeted metabolomics
Organisms Phalaris arundinacea
Chemicals Diclofenac, Glutathione, Sulfamethoxazole