Introduction 1 2 3 1 4 12 13 −1 2 11 14 16 11 17 Appendix Experimental Materials and standards Chemical standards of carbamazepine, lansoprazole, loratidine, famotidine, trimethoprim, ofloxacin, atenolol, metoprolol, azithromycin dihydrate, erythromycin hydrate, fluoxetine hydrochloride, ranitidine hydrochloride, sulfamethoxazole, propranolol hydrochloride, indomethacin, acetaminophen, mefenamic acid, clofibric acid, bezafibrate, mevastatin, and sotalol hydrochloride were purchased from Sigma–Aldrich (Steinheim, Germany), propyphenazone, pravastatin, and paroxetine hydrochloride from LGC Promochem (London, UK), ketoprofen, diclofenac, gemfibrozil, ibuprofen, and naproxen from Jescuder (Rubí, Spain), glibenclamide from SIFA Chemicals (Liestal, Switzerland), and hydrochlorothiazide from Pliva (Zagreb, Croatia). All pharmaceutical standards were of high-purity grade (>90%). 13 3 3 7 10 4 −1 −1 v v Membrane bioreactor (MBR) 2 17 The MBR was operated in parallel with the CAS process (aeration tank and secondary settling tank). The biocenosis of the MBR was grown from inoculated sludge from the municipal WWTP (aeration basin) and cultivated over a period of approximately 1 month to reach steady-state conditions. The hydraulic retention time was set to 14 h by regulating the effluent flow and the SRT was infinite, because no sludge was discharged from the reactor. −1 Wastewater-treatment plant (WWTP) 3 −1 Sampling and sample preparation primary sedimentation tank effluent, as the inflow to the conventional treatment plant and membrane bioreactor, CAS effluent, and MBR effluent. 18 −1 v v Chemical analysis 18 18 1 Table 1 MRM transitions of the compounds analyzed Compound MRM 1 MRM 2 MRM 3 Ibuprofen 205→161   Ketoprofen 253→209 253→197  Naproxen 229→170 229→185  Diclofenac 294→250 294→214  Indomethacin 356→297 356→255  Acetaminophen 152→110 152→93  Mefenamic acid 240→196 240→180  Propyphenazone 231→201 231→189  Clofibric acid 213→127 213→85  Gemfibrozil 249→121   Bezafibrate 360→274 360→154  Pravastatin 447→327   Mevastatin 391→185 391→159  Carbamazepine 237→194 237→192  Fluoxetine 310→44 310→148  Paroxetine 330→192 330→123  Lansoprazole 370→252 370→205  Famotidine 338→189 338→259  Ranitidine 315→176 315→130  Loratidine 383→337 383→267 383→259 Erythromycin 734.5→158 734.5→576.4  Azithromycin 749.5→591.4 749.5→158  Sulfamethoxazole 254→92 254→156  Trimethoprim 291→230 291→261  Ofloxacin 362→316   Atenolol 267→190 267→145  Sotalol 273→255 273→213  Metoprolol 268→133 268→159  Propranolol 260→166 260→183  Hydrochlorothiazide 296→269 296→205  Glibenclamide 494→369   18 −1 2 S N Table 2 −1 Compound Recovery (%) −1 Influent MBR effluent CAS effluent Influent MBR and CAS effluent Ibuprofen a 68.8 (11.0) 90.4 (11.0) 98.0 20.0 Ketoprofen 62.8 (2.94) 71.3 (3.11) 59.1 (0.897) 190 74.0 Naproxen 49.2 (20.0) 59.4 (1.28) 53.4 (2.31) 79.0 20.0 Diclofenac 83.3 (1.17) 94.9 (10.0) 95.0 (12.6) 160 40.0 Indomethacin 113 (2.95) 120 (5.63) 110 (3.78) 150 31.0 Acetaminophen 123 (17.0) 108 (10.5) 56.0 (7.61) 20.9 5.35 Mefenamic acid 93.3 (1.95) 92.1 (1.02) 91.5 (5.29) 5.70 1.85 Propyphenazone 60.0 (8.00) 71.0 (4.00) 71.0 (4.00) 4.80 1.45 Clofibric acid 86.0 (10.8) 104 (6.87) 74.5 (1.40) 16.3 3.75 Gemfibrozil 91.0 (8.47) 87.5 (1.36) 108 (17.2) 8.70 2.20 Bezafibrate 106 (3.43) 94.4 (9.30) 89.4 (4.62) 18.5 4.35 Pravastatin 85.6 (2.56) 78.0 (12.2) 96.0 (19.5) 120 30.9 Mevastatin 103 (8.61) 134 (15.6) 123 (9.86) 9.30 1.30 Carbamazepine 84.0 (7.84) 89.5 (5.20) 88.0 (9.24) 2.20 0.600 Fluoxetine 46.7 (2.34) 93.7 (17.6) 59.0 (1.60) 19.8 1.70 Paroxetine 62.2 (2.15) 109 (5.73) 71.4 (1.49) 3.50 0.650 Lansoprazole 70.0 (10.0) 87.0 (5.00) 86.0 (4.00) 10.9 4.20 Famotidine 58.2 (7.76) 55.4 (6.30) 66.6 (5.39) 3.10 0.40 Ranitidine 41.5 (9.85) 75.8 (14.8) 125 (11.7) 1.40 0.300 Loratidine 72.6 (1.81) 78.0 (6.97) 64.5 (4.98) 8.00 2.75 Erythromycin 67.7 (3.15) 50.0 (13.0) 66.6 (12.0) 12.4 2.00 Azithromycin 30.0 (7.00) 73.0 (2.00) 63.0 (3.00) 1.00 0.300 Sulfamethoxazole 33.7 (2.76) 95.5 (9.24) 78.3 (1.08) 16.1 3.10 Trimethoprim 58.8 (3.29) 128 (6.58) 60.8 (3.87) 1.30 0.350 Ofloxacin 142 (19.0) 135 (5.45) 138 (4.47) 29.3 7.85 Atenolol 83.5 (33.8) 60.8 (10.8) 131 (15.5) 1.70 0.750 Sotalol 47.1 (2.91) 31.9 (3.05) 52.0 (3.63) 4.80 0.700 Metoprolol 36.7 (1.44) 120 (2.64) 76.7 (1.43) 6.30 1.60 Propranolol 60.2 (0.506) 90.8 (4.02) 70.5 (5.27) 2.60 0.300 Hydrochlorothiazide 39.8 (7.43) 58.9 (1.62) 73.4 (22.9) 4.50 0.900 Glibenclamide 100 (11.7) 107 (10.3) 98.5 (11.7) 19.2 2.30 a n Results and discussion −1 −1 3 3 −1 −1 −1 Table 3 Average daily output loads of the investigated pharmaceuticals for Rubí WWTP Pharmaceutical −1 Mean Range Analgesics and anti-inflammatory drugs Naproxen 37.0 10.8–76.9 Ketoprofen 17.1 11.4–36.3 Ibuprofen 56.3 7.39–137.9 Diclofenac 27.3 17.3–43.8 Indomethacin 1.93 nd–2.73 Acetaminophen 4.55 1.06–9.2 Mefenamic acid 0.44 0.27–0.85 Propyphenazone 0.68 0.43–0.96 Anti-ulcer agent Ranitidine 2.77 0.55-5.30 Psychiatric drug Paroxetine 0.08 a Antiepileptic drug Carbamazepine 5.21 1.44-6.71 Antibiotics Ofloxacin 6.93 2.40–11.2 Sulfamethoxazole 3.06 1.42–5.81 Erythromycin 2.29 0.95–4.51 β-blockers Atenolol 21.0 7.70–33.2 Metoprolol 3.32 1.14–5.43 Diuretic Hydrochlorothiazide 33.7 21.2–46.0 Hypoglycaemic agent Glibenclamide 0.74 nd–0.98 Lipid regulator and cholesterol lowering statin drugs Gemfibrozil 54.3 30.1–73.9 Bezafibrate 21.6 10.9–50.8 Clofibric acid 1.75 0.40–3.43 Pravastatin nd nd a 4 −1 −1 19 20 Table 4 Summary of the performance of the MBR system Property Influent MBR effluent CAS effluent −1 a 1.600 (1.770) 26.72 (15.69) total −1 508.2 (124.3) 48.58 (22.47) 111.6 (53.35) −1 67.67 (24.29) 10.89 (3.470) 27.33 (13.75) 4 −1 49.13 (15.79) 1.010 (0.4200) 48.41 (12.87) pH 7.52 (0.300) 7.08 (0.270) 7.63 (0.160) a n 1 2 3 Fig. 1 a b c d e f g h  Fig. 2 a b c d e f g h  Fig. 3 a b c d e f  −1 −1 5 biotransformation/biodegradation, adsorption by the sludge (excess sludge removal), and stripping by aeration (volatilization). Table 5 Mean removal of selected pharmaceuticals by the MBR and CAS processes Compound Elimination (%) in: a b Analgesics and anti-inflammatory drugs Naproxen 99.3 (1.52) 85.1 (11.4) Ketoprofen 91.9 (6.55) 51.5 (22.9) Ibuprofen 99.8 (0.386) 82.5 (15.8) Diclofenac 87.4 (14.1) 50.1 (20.1) Indomethacin 46.6 (23.2) 23.4 (22.3) Acetaminophen 99.6 (0.299) 98.4 (1.72) Mefenamic acid 74.8 (20.1) 29.4 (32.3) Propyphenazone 64.6 (13.3) 42.7 (19.0) Anti-ulcer agents Ranitidine 95.0 (3.74) 42.2 (47.0) Psychiatric drugs Paroxetine 89.7 (6.69) 90.6 (4.74) Antiepileptic drugs Carbamazepine c No elimination Antibiotics Ofloxacin 94.0 (6.51) 23.8 (23.5) Sulfamethoxazole 60.5 (33.9) 55.6 (35.4) Erythromycin 67.3 (16.1) 23.8 (29.2) B-blockers Atenolol 65.5 (36.2) No elimination Metoprolol 58.7 (72.8) No elimination Diuretics Hydrochlorothiazide 66.3 (7.79) 76.3 (6.85) Hypoglycaemic agents Glibenclamide 47.3 (20.1) 44.5 (19.1) Lipid regulator and cholesterol lowering statin drugs Gemfibrozil 89.6 (23.3) 38.8 (16.9) Bezafibrate 95.8 (8.66) 48.4 (33.8) Clofibric acid 71.8 (30.9) 27.7 (46.9) Pravastatin 90.8 (13.2) 61.8 (23.6) a,b n a n b c K H 21 16 2 3 9 11 For ketoprofen, diclofenac, bezafibrate, and gemfibrozil removal by the MBR system was very high and uniform (>90%), with the exception of two sampling programme. It is assumed this variation could have been a result of reduced microbial activity or altered sorption and flocculation conditions. No plausible explanation can be given for the drastically reduced efficiency of removal of clofibric and mefenamic acid by MBR in two sampling programmes; otherwise these were eliminated with efficiencies between 65 and 90%. High and steady removal (>80%) in the MBR was also observed for ranitidine and ofloxacin. In conventional treatment all these pharmaceuticals were eliminated with a wide range of efficiencies, always lower than those obtained by the MBR. Better removal of readily biodegradable micropollutants by the MBR could be because of the smaller flock size of the sludge, which enhances mass transfer by diffusion and therefore increases elimination. Taking into consideration the composition of sludge originating from a membrane bioreactor (specialized microorganisms, large amount of active biomass in suspended solids) improved removal is to be expected; this was confirmed by our experiments. 14 22 2 7 8 9 11 23 24 8 25 N 4 26 Efficiency of removal of atenolol, metoprolol, pravastatin, erythromycin, and indomethacin varied in both MBR and CAS treatment. This could not be explained. Fluctuation of elimination efficiency was also observed for propyphenazone (44.8–82.9% for MBR and 6.82–62.6% for CAS) and glibenclamide (14.8–73.7% for MBR and 11.9–79.7% for CAS). Effluent concentrations greater than those recorded for the influent could be explained by the presence of input conjugate compounds that are transformed into the original compounds during treatment. Because these conjugates were not included in the analysis, no firm conclusion can be made about their biotransformation, especially because sampling inaccuracy can also lead to errors. Conclusion Several pharmaceutical products (e.g. ibuprofen, naproxen, acetaminophen, ketoprofen, diclofenac, bezafibrate, gemfibrozil, ranitidine, ofloxacin, hydrochlorothiazide, and paroxetine) with high rates of attenuation can be expected to be completely removed from wastewater by adsorption or degradation, or a combination of both, during membrane treatment. For most of the compounds investigated MBR effluent concentrations were significantly lower than in the effluent from conventional treatment. Elimination of hydrochlorothiazide and paroxetine was slightly better in CAS treatment. Some substances (e.g. carbamazepine) were not removed by either MBR or CAS treatment. No relationship was found between the structures of target compounds and their removal during wastewater treatment, however. The range of variation of the efficiency of removal by the MBR system was small for most of the compounds; in conventional treatment greater fluctuations were observed and removal efficiency was found to be much more sensitive to changes in operating conditions (temperature, flow rate, etc). Although membrane technology seems a promising means of removal of pharmaceutical compounds, the MBR process investigated would not completely halt discharge of micropollutants. Membrane treatment processes should be optimized by modification of the membranes (variation of the materials and reduction of molecular mass cut-off limits) and/or by modification of the treatment process (inoculation of special microorganisms). The efficiencies of diverse microbial populations in elimination of selected pharmaceuticals, and optimization of design and operating conditions of a laboratory-scale MBR will be the main objectives of our future investigations. That would provide guidelines for scale-up of a biological pilot plant and its evaluation by integration into an industrial process water-recycling system. Because of the current lack of information on the behaviour of pharmaceuticals in surface and wastewaters, however, further studies are required on the occurrence, fate, and effects of these substances in the environment.