*2.2. Sample Collection*

In a single sampling event, wastewater and river water samples were collected in the ZR catchment area (Figure 1). The samples of influent and effluent were collected at Assamra WWTP (*n* = 4). River water samples were collected manually from the top 30 cm water layer of the river by filling high-density polyethylene (HDPE) plastic bottles from the center of the river at four locations: (i) Sukhna station (5.45 km from the main ZR), in a tributary unaffected by Assamra WWTP (*n* = 2); (ii) Twahin Eledwan station (28.74 km from Assamra WWTP) (*n* = 2) and (iii) Military station (47.73 km from Assamra WWTP) (*n* = 2), both downstream of Assamra WWTP; and (iv) Jerash stream, a groundwater stream feeding into ZR (*n* = 2) (Figure 1, Table S1 in Supporting Information (SI)). Two samples were collected from each site, with an interval of 1 h between the samples. All samples were kept frozen at −20 ◦C and transported to Sweden for analysis of PhACs.

**Figure 1.** (**A**) Location of the Zarqa River (ZR) basin in Jordan. (**B**) Sampling sites for Assamra wastewater treatment plant (WWTP) influent and effluent (*n* = 4) and for river water at Sukhna station (*n* = 2), Twahin Eledwan station (*n* = 2), Military station (*n* = 2), and Jerash stream (*n* = 2). Part A of the diagram is modified from Shigei et al. (2020).

#### *2.3. PhACs Target Analyses*

The term PhACs is used hereafter to refer to all substances which have therapeutic effects other than antibiotics. Antibiotics were considered separately because of potential health and environmental impacts. A total of 15 PhACs were included in the analysis (atenolol, amlodipine, bisoprolol, carbamazepine, citalopram, diazinon, fluoxetine, ketoconazole, metformin, metoprolol, oxazepam, paracetamol, propranolol, risperidone, and sertraline). In addition, the concentrations of 18 antibiotics (ciprofloxacin, clarithromycin, clindamycin, doxycycline, erythromycin, ofloxacin, linezolid, metronidazole, moxifloxacin, norfloxacin, tetracycline, trimethoprim, amoxicillin, ampicillin, benzylpenicillin, fusidic acid, rifampicin, and sulfamethoxazole) and one stimulant (caffeine) were analysed. The chemical properties of each compound are shown in Table S1. Isotopically labelled internal standards (IS) used in the analyses were diclofenac 13C6, hydrochlorothiazide 13C6, carbamazepine 13C15N, and ibuprofen-d3.

#### *2.4. Extraction and Analyses of PhACs and Other Parameters*

All influent, effluent, and river water samples were extracted using solid-phase extraction with Oasis HLB cartridges (200 mg, 6 cc; Waters Corporation, Manchester, UK) according to the extractions method previously described in Dalahmeh et al. [36], Dalahmeh et al. [37], nd Gros et al. [38]. Before extraction, the samples were spiked with 100 μL of IS mixture containing 1 ng μL−<sup>1</sup> diclofenac 13C6, hydrochlorothiazide 13C6, carbamazepine 13C15N, and ibuprofen-d3.

The mass of the target PhACs, antibiotics, and caffeine compounds was determined using high-performance liquid chromatography coupled with mass spectrophotometry (HPLC-MS/MS). All analyses were carried out at the Swedish Environmental Institute (IVL) laboratories using a binary Shimadzu AD20 UFLC HPLC system with automatic sample changer and column furnace coupled to an ABSciex API-4000 mass spectrometer. Samples were analysed under positive and negative electrospray ionization (ESI) mode using a Waters XBridge BEH C18 column (100 mm × 2.1 mm with 3 μm opening size). The eluents used in the mobile phase were A: 10 mM acetic acid in deionized water and B: methanol. The gradient used was a linear gradient from 0–90% methanol for 17 min, with a final plateau at 90% methanol for 4 min before a rapid return to 100% A and a final recovery and equalization of 2 min. The concentration of each analyte was quantified using an eight-point calibration curve (0, 5, 10, 20, 50, 100, 200, and 500 ng).

Besides the target PhACs, antibiotics, and caffeine, the river water, influent and effluent samples were analysed for pH, chemical oxygen demand (COD), electrical conductivity (EC), total suspended solids (TSS), and total solids (TS). All analyses of liquid samples were performed according to Standard Methods for Examination of Water and Wastewater APHA [39], using the following protocols: pH (4500-H and B), biochemical oxygen demand (BOD5; 5210-B), TSS, and TS (2540-B-D). The pH was measured by an electrode that measures the concentration of H ion by millivolts. The Chemical Oxygen Demand was measured by oxidizing the water sample by oxidizing agent (potassium dichromate) followed by open reflux digestion at 1500 ◦C for 2 h, then back titration for the remaining dichromate using sodium thiosulfate. The electrical conductivity of water was measured using a conductivity cell immersed in a 50 mL sample. Total solid was measured by gravimetric method, through drying the sample in an oven at 105 ◦C overnight until the crucible has a constant weight. Then, the difference in weights showed the total solids that exist in a sample (summation of total dissolved solids and total suspended solids). Total suspended solid was also measured by gravimetric method, by weighing the washed dried filter paper, then filtering the sample and drying it in an oven at 105 ◦C, the difference showing the concentration of suspended solids.

#### *2.5. Quality Control*

Method blank was prepared using 500 mL of pure MilliQ®® water spiked with 100 μL IS. The blank sample was extracted following the same procedure as used for the other liquid samples. The blank sample did not show detectable levels of any of the measured substances. All detectable concentrations lower than 1 ng L−<sup>1</sup> were recorded as <1 ng L<sup>−</sup>1.
