*3.2. Concentration and Removal of PhACs and Antibiotics in Assamra WWTP*

A total of 15 PhACs (excluding antibiotics) and one stimulant were detected in influent and effluent of Assamra WWTP. The combined concentration of PhACs (excluding antibiotics and caffeine; ∑PhACs) was 20,668–31,485 ng L−<sup>1</sup> in the influent and 4032–4394 ng L−<sup>1</sup> in the effluent, showing a significant reduction effect of treatment in Assamra WWTP (Figure 2, Table S4 in SI). The influent contained high levels of caffeine (27,737–53,223 ng L−1), but these were reduced effectively in Assamra WWTP, resulting in a concentration of 64–273 ng L−<sup>1</sup> in the effluent. Caffeine is highly biodegradable and can be used as an indicator of residual bioactivity [40].

The dominant PhACs in influent were paracetamol (anti-inflammatory; 14,891–24,309 ng L−1) which comprised 74% of ∑PhACs, carbamazepine (anti-epileptic; 2365–3020 ng L−1), which comprised 11% of ∑PhACs, atenolol (beta-blocker; 1723–1952 ng L<sup>−</sup>1), which comprised 7% of ∑PhACs, and metformin (anti-diabetic), which comprised 4% of ∑PhACs. In the effluent, carbamazepine was the dominant PhAC (3138–3352 ng L<sup>−</sup>1), comprising 78% of ∑PhACs, followed by metoprolol (beta-blocker; 10% of ∑PhACs). The removal rate of the dominant substances during WWTP treatment was: 99% for paracetamol, −22% for carbamazepine, 95% for atenolol, and 97% metformin (Figure 3). Poor removal has been reported previously for carbamazepine and hydrochlorothiazide in WWTPs in Spain [41]. Lajeunesse et al. also reported poor removal of carbamazepine in WWTPs in Canada [42]. Other studies investigating the removal of PhACs in middle-income countries (e.g., Jordan) report low removal efficiencies (<50%) for a number of PhACs, including carbamazepine [30]. Bisoprolol (beta-blocker) was present in higher effluent concentrations than influent (10-fold increase). Beta-blocker i.e bisoprolol is generally difficult to remove from wastewater [43]. The overall removal rate in Assamra WWTP was 81%–87% for ∑PhACs and 99% for caffeine. The concentrations of all measured substances are shown in Table S4.

In a previous study, Al-Mashaqbeh et al. [30] found that carbamazepine concentration was high in Assamra WWTP influent (1100 ng L−1) and effluent (850 ng L−1), resulting in low removal of the substance (23%). That study also reported a high occurrence of caffeine (156,000 ng L−1) and its metabolite (1,7-dimethylxanthine; 10,500 ng L−1) in influent, but high removal in Assamra WWTP plant (>99%), and very high concentrations (36,700 ng L−1) of paracetamol in influent, but efficient removal in the WWTP (99%). The results in the present study confirm these findings of Al-Mashaqbeh et al. [30].

**Figure 2.** Concentrations (ng L−1) of pharmaceutically active compounds (PhACs, excluding antibiotics) in Assamra wastewater treatment plant (WWTP) influent (*n* = 2) and effluent (*n* = 2), and in water from the Zarqa River at Sukhna station, (*n* = 2), Twahin Eledwan station (*n* = 2), Military station (*n* = 2) and Jerash stream (*n* = 2). S1, S2 = parallel samples.

**Figure 3.** Efficiency (%) of Assamra wastewater treatment plant in removing pharmaceutically active compounds (PhACs, excluding antibiotics) from the wastewater. S1, S2 = parallel samples.

Of the 18 antibiotics targeted in the analysis, only five substances were detected in the influent and six in the effluent of Assamra WWTP. These were clarithromycin, erythromycin, ofloxacin, metronidazole, and sulfamethoxazole in the influent, and these five plus ciprofloxacin in the effluent (Figure 4). Ofloxacin (fluoroquinolones class) showed the highest concentration in wastewater effluent, followed by erythromycin (macrolides class) and metronidazole (antiprotozoal class) (Figure 4, Table S5 in SI). None of the target substances in the penicillin class (amoxicillin, ampicillin, benzylpenicillin) was detected in the influent or effluent samples. This result is surprising since Almaaytah et al. [2] reported

that antibiotics in the classes fluoroquinolones, macrolides, penicillin, and antiprotozoal are the most commonly dispensed antibiotics in Jordan.

**Figure 4.** Concentrations of antibiotics (ng L−1) in Assamra wastewater treatment plant (WWTP) influent (*n* = 2) and effluent (*n* = 2), and in water from the Zarqa River at Sukhna station, (*n* = 2), Twahin Eledwan station (*n* = 2), Military station (*n* = 2), and Jerash stream (*n* = 2). S1, S2 = parallel samples.

The combined antibiotics (∑Antibiotics) concentration was 510–860 ng L−<sup>1</sup> in influent and 2300–2600 ng L−<sup>1</sup> in the effluent (Figure 4, Table S5). After passing through the treatment process, only the concentration of clarithromycin decreased (from 308 to 82 ng L−1; removal rate 73% on average). C.F. Couto et al. reviewed the WWTPs performances to remove the PhACs in various countries [44]. The reported studies of 12 kinds of antibiotics, including Ofloxacin and Erythromycin, all observed positive values as removal ratio. However, the present study showed 2- to 5-fold higher concentrations of all other antibiotics in effluent than the levels in influent. Ciprofloxacin, which was not detected in the influent, was found in the effluent samples. Hydrolyses of organic matter of wastewater of solid phase and hence the release of ciprofloxacin bound to solids into the liquid phase could have occurred, explaining the increase of the substance in the effluent. Moreover, antibiotics partition into water is generally based on the chemical and physical properties of the antibiotic itself [45]. For example, clarithromycin has high partition coefficients (log Kow = 3.16, Koc = 150, Table S2) and low solubility. These properties suggest that clarithromycin is likely to adsorb to the solids in wastewater, which explains this substance's reduction. In contrast, ofloxacin has low log Kow (−2) and high-water solubility (6.7 × 105 mg L−1) (Table S2), suggesting that it is water-soluble, explaining the high concentrations in the effluent.

In this study, we did not analyse the concentrations of antibiotics in the solid phase of raw wastewater or the WWTP sludge. It is likely that a fraction of the antibiotics initially partitioned to organic matter or accumulated in biomass in the wastewater was released after hydrolysis of the organic matter during biological treatment in the WWTP. This condition would partly explain the increase in antibiotic concentrations in effluent compared with influent, as also suggested in other studies [45,46]. Another explanation could be the presence of antibiotic conjugates or metabolites, which were cleaved back to their mother forms during the treatment process [46,47].
