## 2 Results and Discussion
### Concentrations of the Target Analytes  
The new method involves the use of filtration followed by sample clean-up and pre-concentration with the C18 SPE and subsequent analyses of extracts from SPE using the developed liquid chromatography-negative ion electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method which had an additive-free mobile phase. The application of this new method to the analyses of the target analytes in upstream, downstream, and raw and treated wastewater at five WWTPs in Saskatchewan was evaluated. As the matrix effects varied from soft to severe, the quantitative  
Figure 4: Percentage matrix effect for target analytes at Wastewater Treatment Plants in Saskatchewan. The average and median are represented by X and midline, respectively. Upper and lower box borders are the first and third quartile, respectively. The whiskers are minimum and maximum values. analysis was completed with standard addition calibration, and good linearity was obtained (R\({}^{2}>\) 0.94, see Supplementary Materials, Table S1; sample chromatogram shows BPA in wastewater compared to standard, Figure S2). The recoveries for SPE and pre-concentration steps were \(>\)85% as assessed from the deuterated surrogate (4 NP-d\({}_{4}\) and EQ) for all samples analyzed, which is a significant improvement as compared to other methods where recoveries could be \(<\)15% for surface water [1]. All the samples were obtained by WSA from the WWTPs, processed through the SPE sampling loading stage within 24 h, followed by completing the SPE, drying step, and LC-ESI-MS/MS within five days to ensure the stability of the target analytes. Duplicate samples were collected at approximately the same time for each location. The % relative standard deviation (%RSD) of the duplicates was \(<\)10% in all the samples except in a few cases of the more difficult target analytes in raw wastewater samples such as E3 (%RSD 3-22.5%) and PRDN (%RSD 4-19%). The frequency of detection of target analytes was low for surface water samples collected at upstream or downstream sites near the WWTPs in Saskatchewan as compared to wastewater collected from the WWTPs. Only selected target analytes were detected above the LOQ in surface water samples downstream of WWTPs, including BPA (11.8 \(\pm\) 0.8 ng/L at one location) and 4 NP (15.1 \(\pm\) 1.7 ng/L at one location). E2 was also detected in an upstream surface water sample at one location (60.2 \(\pm\) 2.6 ng/L). No other analytes were detected in surface water upstream to the WWTPs. The presence of E2 upstream could be due to a non-point agricultural source contributing to runoff to the upstream sample collection to this WWTPs as there was no detection of E2 downstream to the same location (LOQ for E2 is 5 ng/L). BPA was the most frequently detected xenoestrogen in raw wastewater (100% of samples) and treated wastewater (80% of samples), and the concentrations of BPA ranged from \(133.3\pm 12.4\) ng/L to \(403.0\pm 16.1\) ng/L in raw wastewater, and the concentration range was lower (\(<\)LOQ \(-175.5\pm 6.8\) ng/L) in the treated wastewater. The maximum concentration of BPA was low relative to influent at other Canadian WWTPs [22]. Concentrations of BPA in the effluent (treated wastewater) are generally significantly lower than raw wastewater at other WWTPs worldwide as was measured at the WWTPs in this study. The presence of BPA has been reported in wastewater when the WWTPs uses a UV lamp, which is a common summer practice for the WWTPs in Saskatchewan [11]. The highest concentration of 4 OP in raw wastewater was \(305.8\pm 17.1\) ng/L and at this WWTP 4 OP was not detected in treated wastewater, as well 4 NP was not detected in raw or treated wastewater. At one other WWTP, both 4 OP and 4 NP were detected in both raw and treated wastewater (\(132.9\pm 8.7\) ng/L and \(49.6\) ng/L \(\pm\) 7.4 ng/L in raw wastewater; \(110.6\pm 5.8\) ng/L and \(57.9\pm 6.9\) ng/L, respectively, in treated wastewater).