## 2 Materials and Methods
### Wastewater Samples Analysis by SPE-LC-MS/MS  
Of the collected sample volume, 500 mL was filtered using quantitative filter paper, 1.0 um glass microfiber, and 0.45 um nylon membrane. This volume was selected to ensure a representative water sample, but only 50 mL of the filtered sample was used. Therefore, only the concentrations of PhACs in the dissolved fraction of the sample are considered.  
For extraction, the SPE method was implemented based on previous work conducted in wastewater samples [23]. Briefly, cartridges were activated with 4 + 4 mL of methanol followed by 4 + 4 mL of reagent water. Wastewater and surface water samples were passed  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \begin{tabular}{c} **Reguengos de** \\ **Monsaraz** \\ **WWTP** \\ \end{tabular} & \begin{tabular}{c} **Borba** \\ **WWTP** \\ \end{tabular} & \begin{tabular}{c} **Redondo** \\ **WWTP** \\ \end{tabular} & \begin{tabular}{c} **Potel WWTP** \\ \end{tabular} \\ \hline Influents & 5 & 6 & 4 & 5 & 5 \\ Effluents & 6 & 5 & 4 & 4 & 5 \\ Upstream & 4 & 1 & 3 & — & 3 \\ Downstream & 4 & 4 & 3 & 3 & 3 \\ Receiving waters & Xarrama River & Monreal Stream & Borba Stream & \
\begin{tabular}{c} Agua Ruca \\ Stream \\ \end{tabular} &
\begin{tabular}{c} Pego Grande \\ Stream \\ \end{tabular} \\ \hline \hline \end{tabular} ASCP: activate sludge conventional process; LT-ASP: long-term activate sludge process; WSPs: wastewater stabilization ponds.  
\end{table}
Table 1: Cont.  
through cartridges at a flow rate of 5 mL/min. Afterward, the cartridges were rinsed with 4 mL of reagent water and dried under a nitrogen stream for 20 min. Cartridges were eluted with 4 + 4 mL of methanol at a flow rate of 2 mL/min. The organic extract was evaporated to dryness in a TurboVap system with a nitrogen stream (5 psi/35 \({}^{\circ}\)C). Samples were reconstituted with 1 mL of reagent water and filtered through a 0.45 \(\upmu\) polytetrafluoroethylene (PTFE) syringe filter.  
For quantification, standard addition calibration curves of PhACs were prepared by adding 100 \(\upmu\)L of sample and 100 \(\upmu\)L of standard solution with a minimum of six points. For the non-spiked calibration point, 100 \(\upmu\)L of reagent water was used instead of standard solution.  
For analysis, the LC-MS/MS method that was used had already been validated for PhACs determination in wastewaters, clams, and sludge samples [23; 24; 25]. To ensure maximum sensitivity, two chromatographic methods were used. One was an acidic method that uses two solvents, A (water + 0.01 mM ammonium acetate + 0.5% formic acid (\(v/v\))) and B (methanol) for the quantification of 14 PhACs. The other was a basic method that uses a solvent C (water + 0.05% ammonia (\(v/v\)) at 25%) and B (methanol), for the quantification of the remaining 12 PhACs. For both methods, the injection volume used in the chromatographic separation was 20 \(\upmu\)L. Regarding mass spectrometer conditions, two transitions (product ion) were determined for each compound, one for quantification (MRM1), and another for confirmation (MRM2), except for ibuprofen (IBUP), which only has one identifiable transition. Further details of the optimized chromatographic conditions and on the optimized mass spectrometer conditions are described in [26; 24].