Science of the Total Environment 773 (2021) 145374 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Prospects on coupling UV/H2O2 with activated sludge or a fungal treatment for the removal of pharmaceutically active compounds in real hospital wastewater Josep Anton Mir-Tutusaus a, Adrián Jaén-Gil b,f, Damià Barceló b,c,f, Gianluigi Buttiglieri b,f, Rafael Gonzalez-Olmos d, Sara Rodriguez-Mozaz b,f, Glòria Caminal e, Montserrat Sarrà a,⁎ a Departament d'Enginyeria Química Biològica i Ambiental, Escola d'Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, H2O Building, Emili Grahit 101, 17003 Girona, Spain c Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA), Spanish Council for Scientific Research (CSIC), Jordi Girona 18-26, 08034, Barcelona, Spain d IQS School of Engineering, Universitat Ramon Llull, Via Augusta 390, 08017, Barcelona, Spain e Institut de Química Avançada de Catalunya (IQAC) CSIC, Jordi Girona 18-26, 08034, Barcelona, Spain f University of Girona, Girona, Spain H I G H L I G H T S G R A P H I C A L A B S T R A C T • Fungal treatment was the most efficient as first treatment. • Removal efficiency was higher than 90% for most of the treatment combinations. • Deconjugation was evidenced after both biological treatments. • Carbamazepine and its TPs highly re- moved (90%) by UV/H2O2 alone. • UV/H2O2 more convenient as a polishing than pre-treatment step. a r t i c l e i n f o a b s t r a c t Article history: Conventional active sludge (AS) process at municipal centralized wastewater treatment facilities may exhibit lit- Received 24 September 2020 tle pharmaceuticals (PhACs) removal efficiencies when treating hospital wastewater (HWW). Therefore, a ded- Received in revised form 19 January 2021 icated efficient wastewater treatment at the source point is recommended. In this sense, advanced oxidation Accepted 19 January 2021 processes (AOPs) and fungal treatment (FG) have evidenced promising results in degrading PhACs. The coupling Available online 2 February 2021 of the AOP based on UV/H2O2 treatment with biological treatment (AS or FG) treating a real non-sterile HWW, Editor: Dimitra A Lambropoulou was evaluated in this work. In addition, a coagulation-flocculation pretreatment was applied to improve the ef- ficiency of all approaches. Twenty-two PhACs were detected in raw HWW, which were effectively removed Keywords: (93–95%) with the combination of any of the biological treatment followed by UV/H2O2 treatment. Similar re- AOP moval results (94%) were obtained when placing UV/H2O2 treatment before FG, while a lower removal (83%) Fungal treatment was obtained in the combination of UV/H2O2 followed by AS. However, the latest was the only treatment combi- Hospital wastewater nation that achieved a decrease in the toxicity of water. Activated sludge Abbreviations: HWW, hospital wastewater; AOP, advanced oxidation process; CAS, conventional activated sludge; WRF, white-rot fungi; PhAC, pharmaceutically active compound; CBZ, carbamazepine; AS, activated sludge; TPs, transformation products; TU, toxicity unit; bld, below limit of detection; blq, below limit of quantification. ⁎ Corresponding author. E-mail address: Montserrat.Sarra@uab.cat (M. Sarrà). https://doi.org/10.1016/j.scitotenv.2021.145374 0048-9697/© 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 UV/H2O2 Moreover, deconjugation of conjugated PhACs has been suggested for ofloxacin and lorazepam after AS treat- Decentralized treatment ment, and for ketoprofen after fungal treatment. Monitoring of carbamazepine and its transformation products along the treatment allowed to identify the same carbamazepine degradation pathway in UV/H2O2 and AS treat- ments, unlike fungal treatment, which followed another degradation route. © 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction fungal treatment have also proved to remove PhACs from wastewater streams. Particularly white-rot fungi (WRF) have succeeded on Hospital wastewater (HWW) contains a complex mixture of hazard- degrading a wide range of pollutants thanks to its unspecific intracellu- ous chemicals and harmful microbes, which can pose a threat to the en- lar and extracellular enzymatic systems (Rodarte-Morales et al., 2011; vironment and public health. Although the contribution of hospital Nguyen et al., 2013; Cruz-Morató et al., 2013). Fungal operations per- facilities to the total volume uploaded in the municipal WWTP usually form reasonably well in terms of PhACs removal but some compounds range between 0.2 and 2% (Carraro et al., 2016), a specific directive or still remain in the effluents (Cruz-Morató et al., 2014). Advanced Oxida- guideline for the management of hospital wastewater effluents in tion Processes (AOPs) are being largely studied in regards to PhACs deg- Europe is missing, and national legal regulations quite rarely define radation with promising results, and an increasing number of articles how to manage and treat HWW before its disposal (Rodriguez-Mozaz has been published in recent years (Klavarioti et al., 2009). When the et al., 2017). Therefore, hospital effluents are usually discharged in the UV light is absorbed by H2O2, •OH radicals are formed by the photolysis municipal sewer system without any previous pretreatment. The com- of the –O–O– peroxidic bond. Hydroxyl radicals can react with organic mon practice of co-treating hospitals and urban wastewaters jointly at pollutants in different ways: by transferring electrons, by oxidising or- a municipal WWTP (centralized treatment) is considered as an inade- ganic compounds, by adding hydroxyl groups, by abstracting a hydro- quate solution for the removal of compounds such as some pharmaceu- gen atom or by initiating a radical (Kim et al., 2009a). ticals (PhACs), because highly polluted effluents can be detrimental for However, total mineralization of organic pollutants in both biologi- their removal by biological treatment (Verlicchi et al., 2015; Pauwels cal and AOP treatment of wastewater is highly unlikely (Cavalcante and Verstraete, 2006; Badia-Fabregat et al., 2015; Joss et al., 2006). et al., 2015) and transformation products (TPs) of these contaminants Therefore, the use of alternative wastewater treatments at the source are usually formed. These TPs are typically more biodegradable than point (decentralized treatment) has been highly recommended the corresponding parent compounds but, in some cases, they can be (Verlicchi et al., 2015; Pauwels and Verstraete, 2006; Joss et al., 2006; even more toxic. Cruz-Morató et al., 2014; Verlicchi et al., 2010) and extensive research Biological processes can be coupled to other treatment technologies, has been conducted in the development of appropriate decentralized like AOP: On the one hand, biological treatment such as CAS and fungal treatments for hospital effluents (Verlicchi et al., 2015). However, full- processes can enhance PhAC removal with AOP as pretreatment in scale dedicated treatment of hospitals effluents has only been imple- order to increase contaminant biodegradability (Changotra et al., 2019); mented in a limited number of places (Rodriguez-Mozaz et al., 2017). on the other hand, AOP as a post-treatment could not only improve over- In the case of psychiatric hospitals, on-site wastewater treatment can all pollutant removal efficiency but also reduce treatment economic cost be particularly recommended since the effluents contain remarkable and ecological footprint (Oller et al., 2011). Specifically in the case of loads of psychiatric drugs as well as their metabolites and transforma- wastewater with high amounts of PhACs, like HWW, the combination tion products (Herrmann et al., 2015; Yuan et al., 2013a). These type of bio-oxidation and AOP has been recently proven to be economically of pharmaceuticals are more recalcitrant than most of the PhACs in feasible (Changotra et al., 2020; Martínez et al., 2018). Some studies both conventional WWTP and in the natural environment (Baena- have discussed the coupling of selected AOPs with some biological pro- Nogueras et al., 2017; Mir-Tutusaus et al., 2017; Verlicchi et al., 2012; cesses, mainly CAS-based and using the AOP as a polishing step Calisto and Esteves, 2009). They have also been targeted as contami- (Giannakis et al., 2015; Sirtori et al., 2009; Vidal et al., 2004; Hörsch nants to be prioritized by several authors (Ashton et al., 2004) as well et al., 2003; Ahmed et al., 2017). The hypothesis in this work is that the as by the Global Water Research Coalition (2008). In addition, the use coupling of advanced oxidation with biologically based technologies of antidepressants has significantly increased in most OECD countries may improve PhAC removal, compared to individual treatments, espe- in the last years, as a reflection of the prevalence of mental illness, in- cially in a complex matrix like HWW. However, depending of the waste- crease in health coverage, new treatment opportunities and population water nature, AOP has reported both to increase the biodegradability of ageing (Organization for Economic Cooperation and Development, contaminants (Changotra et al., 2019) and to decrease it, and also to en- 2015). An increase in the worldwide consumption of this type of hance the effluent toxicity due to the formation of toxic by-products PhACs class can thus be foreseen in the next years. (Plahuta et al., 2014; Cuerda-Correa et al., 2020). Therefore, more thor- Wastewater treatment plants are among the main point sources of ough and wider-spectrum studies need to be performed to fully under- pharmaceutical release into the environment. Therefore, the improve- stand the interactions between the systems and to provide feasible ment of WWTP capabilities is of high importance (Verlicchi et al., combinations from which to choose. Therefore, the main objective of 2015; Collado et al., 2013; Fatta-Kassinos et al., 2011) and physical, this study was the evaluation of coupling an AOP process, in this work chemical and biological processes have been tested with varying de- the well-known UV/H2O2 treatment, with activated sludge or a fungal grees of success. Conventional activated sludge (CAS) process is the treatment treating real non-sterile HWW. The removal of a broad set of standard practice in conventional WWTPs, which usually does not PhACs was evaluated in each treatment separately, and in several combi- achieve high removal efficiencies of recalcitrant micropollutants nations of them. Moreover, a detailed evaluation of carbamazepine, con- (Verlicchi et al., 2015). Three main removal pathways are usually sidered as a model compound, and of some of its TPs was performed. accounted in activated sludge: microbial processes (biodegradation, ei- ther metabolic or co-metabolic), sorption onto sludge flocs and volatil- 2. Materials and methods ization (mainly during aeration). However, volatilization can be considered negligible for most PhACs (Joss et al., 2006). Some analgesics 2.1. Reagents and hospital wastewater and anti-inflammatories are well removed by CAS but other drug fami- lies such as psychiatric drugs and antibiotics are more resistant to bacte- All the PhACs and the corresponding isotopically labelled standards rial degradation (Yuan et al., 2013a). Other biological processes such as used in the analysis were of high purity grade (>90%) and they were 2 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 purchased from Sigma–Aldrich (Steinheim, Germany), US Pharmaco- 203 352 38 19 50 23 AS + UV/H2O2 9 1 2 6 2 peia USP (MD, USA), Europea Pharmacopeia EP (Strasbourg, France), Toronto Research Chemicals TRC (Ontario, Canada) and CDN isotopes ± ± ± ± ± ± ± ± ± ± ± (Quebec, Canada). Individual as well as isotopically labelled standard 1212 2773 7190 892 132 612 892 123 462 bld bld bld bld bld bld bld bld bld solutions were prepared according to Gros et al. (2012). Malt extract 47 46 93 was acquired from Scharlau (Barcelona, Spain) and glucose, ammonium 201 214 532 chloride and other chemicals were purchased from Sigma-Aldrich (Bar- 40 39 22 FG + UV/H2O2 5 1 0 5 5 4 celona, Spain). All other chemicals used were of analytical grade. In the ± ± ± ± ± ± ± ± ± ± ± ± UV/H2O2 experiments titanium (IV) oxysulfate reagent (1.9–2.1% from 1545 1800 5694 Sigma-Aldrich), H2O2 (30% w/v 100 vol. stabilized PRS from Panreac) 886 995 224 bld bld bld bld bld bld bld bld bld 60 79 97 95 and sodium thiosulfate (Panreac) were used. 6 2 The HWW was collected directly from the sewer manifold of Sant 175 363 130 893 28 55 11 48 71 0 3 8 9 Joan de Déu Hospital (Barcelona, Catalonia) in the NE of Spain. Fresh UV/H2O2 + AS samples were pretreated with a coagulation-flocculation process as de- ± ± ± ± ± ± ± ± ± ± ± ± ± scribed previously (Mir-Tutusaus et al., 2016). The pretreatment used 17,862 43 mg L−1 of coagulant Hyfloc AC50 and 4.8 mg L−1 of flocculant 1052 1247 5319 4718 1897 1214 1056 232 694 178 253 bld bld bld bld bld bld bld 83 Himoloc DR3000, both kindly provided by Derypol, S.A. (Barcelona, 3 Spain). Characterization of the wastewater samples in terms of PhAC 554 614 14 20 14 11 UV/H2O2 + FG 0 5 concentrations are summarized in Table 1. ± ± ± ± ± ± ± ± 2.2. HWW treatments 3778 6270 797 790 196 275 435 bld bld bld bld bld bld bld bld bld bld bld bld bld 94 Fig. 1 summarizes the experiments carried out on the coupling of 1386 AOP and the biological treatments. 390 499 100 35 95 40 27 20 75 53 13 30 19 0 1 0 7 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.2.1. Fungal treatment Trametes versicolor (ATCC#42530) was maintained on 2% malt agar 29,499 1045 2342 1713 3887 1053 1565 2439 6259 2952 4202 slants at 25 °C until use. Subcultures were routinely made. A mycelial 328 663 901 bld bld AS 11 41 96 73 3 suspension of T. versicolor was obtained as in Borràs et al. and pellets were obtained as previously described (Borràs et al., 2008; Blánquez 506 714 60 48 27 66 0 4 1 2 0 6 et al., 2006). An air-fluidized bed bioreactor was operated as a batch ± ± ± ± ± ± ± ± ± ± ± ± for 7 d. Fluidized conditions in the reactor was maintained by using 1 s air pulse every 4 s, resulting in an aeration rate of 0.8 Lmin−1. Nutri- Levels of selected PhACs (ng L−1) in the initial wastewater and after each treatment. Carbamazepine is discussed separately. 11,246 Final concentration (ng L−1) 3450 1578 1897 2504 ents for maintenance, namely, glucose and NH4Cl, were added with a 113 757 136 264 543 bld bld bld bld bld bld bld bld bld FG 90 3 molar C/N ratio of 7.5 at T. versicolor consumption rate (1200 mg glucosegDCW−1·d−1). Temperature was maintained at 25 °C and pH 1141 595 132 289 39 52 13 16 13 was controlled at 4.5 by HCl 1 M or NaOH 1 M addition. 0 4 1 ± ± ± ± ± ± ± ± ± ± ± ± 2.2.2. Activated sludge treatment UV/H2O2 22,009 All the biodegradation tests with activated sludge were performed 7128 2921 7655 2067 1295 581 154 bld bld bld bld bld bld bld bld 60 93 53 80 using 1 L lab-scale Applikon stirred tank reactor coupled with a 2 proportional-integral-derivative (PID) controller for pH, oxygen and 12,019 3506 1205 1051 temperature. Bioreactors were operated as batch for 24 h and each ex- Initial concentration 954 398 799 846 111 118 574 601 411 509 314 107 178 295 12 31 periment was conducted in duplicate. The biomass originated from Celrà WWTP (Catalonia, Spain, 20,000 equivalent inhabitants, 2100 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± m3d−1), with a hydraulic retention time of 48 h and a sludge retention ± (ng L−1) 107,987 27,569 26,939 13,955 time of 20–22 d (Collado et al., 2014). The experiments started at the 1448 3169 6883 6738 2052 1370 2188 1670 1970 4118 5766 414 898 538 225 latest 24 h after withdrawing the biomass from the full scale WWTP. 77 Biomass concentration during the experiments was 3 gTSS L−1 and aer- Hydrochlorothiazide obic conditions (>2.5 mg O2 L−1) were achieved with a continuous air Acetaminophen supply. pH was controlled at 7.5 and the temperature maintained at Carbamazepine Pharmaceutical Ciprofloxacin Atorvastatin 25 °C. All these parameters were selected based on the optimum condi- Venlafaxine Gemfibrozil Furosemide Ketoprofen Citalopram Lorazepam Trazodone Diclofenac Ranitidine Naproxen Ibuprofen Ofloxacin Valsartan tions needed for this treatment (Rubirola et al., 2014). Organic solution Atenolol (sodium acetate, propionate and yeast extract), phosphate buffer, trace and inorganic solution were added as described elsewhere (Collado et al., 2013). Analgesics and anti-inflammatories 2.2.3. UV/H2O2 treatment Photo-oxidation was carried out in a UV Laboratory Reactor System H1 and H2 antagonists from UV-Consulting Peschl® which consists of an immersion-type Antihypertensives photo-reactor with a working volume of approximately 550 mL. The Psychiatric drugs Lipid regulators UV lamp used was a 15 W Heraeus Noblelight TNN 15/32 low- Removal (%) pressure mercury vapor lamp emitting at 254 nm. The photo-reactor Antibiotics β-blockers Diuretics was covered with aluminum foil to minimize loss of UV light and Family Table 1 Total avoid any reflections, and magnetically stirred. Potassium ferrioxalate actionometry (Hatchard and Parker, 1956) was used to characterize 3 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 Fig. 1. Diagram of the coupled treatments. FG represents the fungal treatment; AOP, the UV/H2O2 treatment; AS, the activated sludge process. the intensity of the light, resulting in an irradiance of 0.049 W cm-2 were put in contact with the bacterium Vibrio fischeri. Bioluminescence (Benito et al., 2017). The experiments were carried out with 500 was recorded after 15 min exposure and compared with that of blank mL of wastewater, 15 mgL−1 of H2O2 and a reaction time of 10 min controls. Toxicity values are expressed in Toxicity Units (TUs). The pos- that corresponds to a UV dose of 29.4 J cm−2 . Sodium thiosulfate sible presence of trace sodium thiosulfate in the analysis was tested in a was added to interrupt the oxidation reaction (with stoichiometric previous work and had no toxic effect on luminescent bacteria at the excess of 20%). The presence of H2O2 was analyzed by a spectropho- added concentration (Jaén-Gil et al., 2019). tometric method using titanium (IV) oxysulfate (Shahbazi et al., 2014) to check that if it was completely quenched by the added so- 2.5. Statistical analysis dium thiosulfate. For removal calculations, values below limit of detection (bld) 2.3. Analysis of pharmaceutically active compounds and below limit of quantification (blq) were considered to have a concentration half of the limit of detection and half of the limit of Samples collected from the experiments were filtered through 0.45 quantification, respectively (EPA, 2000). Summary statistics were μm PVDF filters (Millipore, Barcelona, Spain) and kept in PET containers performed with R: A language and environment for statistical com- at −20 °C until PhAC analysis. Sample pretreatment was carried out puting (R Core Team, 2015). using the methodology described elsewhere (Gros et al., 2012). Briefly, samples were filtered through 1 μm glass fibber followed by 0.45 μm 3. Results and discussion PVDF membrane filters (Millipore; Billerica, MA, USA) and an appropri- ate volume of Na2EDTA was added to obtain a final concentration of General physicochemical parameters of the HWW studied were in 0.1% (w/w). Oasis HLB cartridges (60 mg, 3 mL) (Waters Corp.; the same range than previously sampled HWW from the same sewer Mildford, MA, USA) were conditioned with 5 mL of methanol followed manifold and from other hospitals (Mir-Tutusaus et al., 2017; Mir- by 5 mL of HPLC-grade water. After conditioning step, 25 mL and 50 Tutusaus et al., 2016). The use of a coagulation-flocculation pretreat- mL for raw and treated wastewater respectively were percolated ment can be highlighted as necessary, as several studies have reported through cartridges, rinsed with 6 mL of HPLC grade water and further low removal efficiencies while operating UV/H2O2 and fungal reactors dried with air for 5 min to remove remaining water. Elution was per- with raw wastewater, due to high suspended solids and microorgan- formed with 10 mL of pure methanol. 5 mL of the eluate was evaporated isms concentration, respectively (Verlicchi et al., 2015; Mir-Tutusaus under gentle nitrogen stream and reconstituted in 1 mL of methanol- et al., 2016). The coagulation-flocculation pretreatment reduced the ab- water (10: 90, v/v). Finally, 10 μL of 10 ngμL−1 internal standard mix sorbance at 650 nm to zero, the COD from 174 to 87 mg O2 L−1 and total was added in all samples. suspended solids from 108 to 16 mg L−1. Analysis was performed by using an Ultra-Performance Liquid Chro- On one hand, as presented in Fig. 1, the UV/H2O2 was studied as a matography system (Waters Corp. Mildford, MA, USA) coupled to a step for both removing micropollutants and incrementing the biode- quadrupole-linear hybrid ion trap mass spectrometer 5500 QTRAP (Ap- gradability of the effluent before the subsequent fungal or AS treat- plied Biosystems, Foster City, CA, USA) equipped with a Turbo V ion ments (Oller et al., 2011). On the other hand, fungal and AS spray source. Chromatographic separation was carried out by using an treatments were evaluated as a first step for reducing the pharma- Acquity HSS T3 column (50 mm × 2.1 mm i.d, 1.8 μm particle size; Wa- ceutical load prior to the UV/H2O2 stage, which would be considered ters Corp. Mildford, MA, USA) for positive ionization (PI) mode and an as a polishing step. Only 23 PhACs (out of the total 77 analyzed; Acquity BEH C18 column (50 mm × 2.1 mm i.d. 1.7 μm particle size; Wa- Table S1) were detected before the treatments, and only those de- ters Corp. Mildford, MA, USA) for negative ionization (NI) mode. All tected at concentrations at least 10 times their limit of quantification transitions were recorded by using Scheduled MRM™ algorithm moni- are discussed in the following sections. Table 1 shows the concentra- toring two SRM transitions for each compound; the first one for quanti- tions of the 23 compounds, before and after single and coupled treat- fication and the second one for confirmation of the compounds. ments. The analgesics and anti-inflammatories compounds were Concentrations were calculated by internal calibration and processed contributing the most to total PhACs concentration in raw wastewa- by using Analyst 1.5.1 software. ter, a common trend in urban and hospital wastewaters (Verlicchi et al., 2015; Verlicchi et al., 2010; Frédéric and Yves, 2014). However, 2.4. Toxicity analysis a high concentration of psychiatric drugs can be highlighted, most likely due to a large psychiatric ward located within the hospital pre- To determine the ecotoxicological effects on treated samples, decay mises. For example, the levels of carbamazepine and lorazepam percentages on bacterial bioluminescence were measured by using a (4118 and 538 ng L−1, respectively) were considerably higher than Microtox™ Model 500 Toxicity Analyzer (Strategic Diagnostics Inc. the levels found previously in urban wastewater (1200 ng L−1 and Newark, DE, US). For this purpose, samples were adjusted at pH 7 and n.d., respectively) (Verlicchi et al., 2012). The same could be ob- centrifuged in glass vials to remove any suspended solids. Then, samples served with citalopram and venlafaxine, measured at a much higher 4 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 concentration (898 and 5766 ng L−1, respectively), than the concen- their effectiveness in PhAC degradation is lower when treating matrices trations observed in urban wastewater (5 and 287–371 ng L −1, with high COD or TSS content (Kim et al., 2009b). Overall, placing the respectively) (Yuan et al., 2013a; Mackuľak et al., 2015). UV/H2O2 after the biological treatment could effectively increase the total PhACs removal, compared to the single treatment, up to 95%. 3.1. Performance of individual treatments 3.2.2. UV/H2O2 coupled with biological treatments Fungal and AS operations were carried out in parallel, with the same Overall removal values of UV/H2O2 coupled with the biological treat- initial wastewater, and their removal efficiencies were compared. The sin- ments, as post treatments, can be found in Table 1. gle fungal step removed 90% of total PhACs concentration, 66% excluding Fig. 3 presents the cumulative removal efficiencies of UV/H2O2 analgesics and anti-inflammatories, which are commonly degraded in coupled with the biological treatments. The UV/H2O2 step alone WWTPs (and accounted for roughly half of influent wastewater PhACs completely removed the analgesics and anti-inflammatories acetamin- load). The fungus completely removed acetaminophen, diclofenac, ibu- ophen, diclofenac and naproxen, the antibiotic ofloxacin, furosemide, profen, ciprofloxacin, furosemide, hydrochlorothiazide, ranitidine, atorva- hydrochlorothiazide, ranitidine, atorvastatin, and the psychiatric drug statin, gemfibrozil, 2-hydroxycarbamazepine (2-hydroxyCBZ) and trazodone. It also removed by more than 80% valsartan, atenolol, trazodone (Table 1). High removal was achieved for analgesics and anti- 10,11-epoxyCBZ (by far the major contributor to the psychiatric drugs inflammatories (94%) as well as for antibiotics (91%) and around 42% family), 2-hydroxyCBZ and citalopram. Carbamazepine and gemfibrozil for psychiatric drugs, in accordance with previous studies (Cruz-Morató were poorly degraded by UV/H2O2 in accordance with previous studies et al., 2014; Mir-Tutusaus et al., 2017; Cruz-Morató et al., 2013; Lucas (Wols et al., 2013). It is noteworthy to remember that HWW was et al., 2016). pretreated with coagulation-flocculation to lower its COD and TSS con- In the case of the AS treatment, 73% of the initial pharmaceutical tent, which could have allowed the good observed removals with UV/ load was eliminated, 92% disregarding the analgesics and anti- H2O2. inflammatories. AS completely removed the analgesics and anti- Placing the UV/H2O2 step before a biological treatment generally inflammatories acetaminophen, ibuprofen and naproxen (Table 1), aims at increasing the biodegradability of biorecalcitrant compounds in line with the literature regarding these compounds and CAS (Oller et al., 2011). This was confirmed in this work with both AOP + bi- (Verlicchi et al., 2012). AS also removed nearly 75% of the antibiotic ologic treatment couplings. Regarding the first coupling (UV/H2O2 + FG ciprofloxacin, probably by adsorption to the biomass as it occurs in treatment, Fig. 3A), although each single treatment was already able to full-scale WWTPs (Verlicchi et al., 2012). Gemfibrozil is usually re- remove more than 70% of the initial pharmaceutical load, this value ported as recalcitrant in CAS (Verlicchi et al., 2010). In contrast it raised up to 95% when the two treatments were coupled. In the case was partly removed in the present CAS experiment. In general, the of the other coupling (UV/H2O2 + AS, Fig. 3B) a similar trend was ob- AS treatment exhibited lower removal efficiencies than fungal treat- served: i.e. UV/H2O2 eliminated 74% of ibuprofen and the following bio- ment for most of the compounds analyzed, and in agreement with logical treatment increased its removal up to 100%. Likewise, in the case removal efficiencies usually reported in WWTPs (Verlicchi et al., of gemfibrozil an increase of the removal efficiency from 45% to 60% was 2012; Wang et al., 2014). Fungal operation was thus more efficient observed, while the removal of any other compound was considerably than reference AS operation for HWW treatment and it could be affected. In fact, coupling the two treatments only increased the overall regarded, in some cases, as a standalone treatment of such PhAC- removal from 80% (UV/H2O2) to 83% (UV/H2O2 + AS). However, AS polluted streams. might obtain benefits from a previous pretreatment with UV/H2O2: for UV/H2O2 removed 80% of the total PhACs load while the fungal treat- example to degrade part of the antibiotics, with some of them known ment and the AS removed 90% and 73% respectively. The removal of to be toxic to the bacterial community in AS (Collado et al., 2013). psychiatric drugs in the UV/H2O2 treatment was 69% while in the FG Though promising, some reports have expressed concern about the and AS treatments was 55 and 24% respectively. Contrarily, the removal application of AOPs for micropollutants removal because the TPs gener- of antibiotics with UV/H2O2 was 67%, while with the biological treat- ated in these oxidation processes may have higher toxicity than the cor- ments the removal efficiencies were 91% in FG and 36% in the AS. The re- responding parent compounds (Yang et al., 2017). However, in some moval of analgesics and anti-inflammatories with UV/H2O2 was similar cases a reduction in ecotoxicity to larval zebrafish could be observed than with the biological treatments. in the effluent of several advanced wastewater treatment systems, sug- gesting a lack of negative short-term biological effects (Angeles et al., 3.2. Performance of the coupled treatments 2020). The toxicity values evaluated in raw wastewater and after the treatments performed in this study are presented in Fig. 4. As it can be 3.2.1. Biological treatments coupled with UV/H2O2 observed, UV/H2O2 diminished the toxicity of flocculated HWW by al- The combinations FG + UV/H2O2 and AS+UV/H2O2 are shown in most one TU. In contrast, the UV/H2O2 did increase the effluent toxicity Fig. 2A and B, respectively. Placing the UV/H2O2 after the biotreatment when placed after any biological treatment. This behavior could be at- aimed at degrading the remaining PhACs and the corresponding TPs tributed to the complexity of the matrix: the degradation of some com- produced by the first-placed biological processes. Psychiatric drugs pounds can lead to less toxic effluents (Cavalcante et al., 2015; Bilińska were the most recalcitrant family in this combination of treatments, al- et al., 2016; Díaz-Garduño et al., 2016) while degradation of others though carbamazepine, citalopram and venlafaxine were > 80% re- might not affect the toxicity (Dantas et al., 2008; González et al., moved in the best-case scenario. Further discussion on CBZ and its TPs 2007) and other compounds might create more toxic intermediates can be found in Section 3.4. (Calza et al., 2006; Méndez-Arriaga et al., 2008; Le et al., 2017). There- In the FG + UV/H2O2 combination (Fig. 2A), the UV/H2O2 removed fore, toxicity increase/decrease after UV/H2O2 might be regarded as only partially the residual PhACs content after the fungal treatment. Ex- matrix-specific, as a function of the pharmaceutical load and category. ceptions were CBZ TPs, produced by the biotransformation and Despite it has not been measured in this work, we cannot discard that deconjugation of carbamazepine, and ofloxacin, whose removal was the overall mineralization could have a direct impact on the toxicity improved by the UV/H2O2 treatment. Placing the UV/H2O2 after the AS (Perisic et al., 2016; Sági et al., 2018). The FG treatment consistently in- (Fig. 2B), instead, improved significantly the overall PhACs removal, creased the toxicity of the effluent, although previous studies with anal- even though the removal efficiency of AS was lower than the one ob- ogous wastewater showed opposite results (Cruz-Morató et al., 2014; tained by FG. AS can indeed decrease the COD, improving the effective- Mir-Tutusaus et al., 2017; Cruz-Morató et al., 2013). It was likely that ness of the subsequent UV/H2O2: since OH radicals produced in UV/ some fungal products interfered with Vibrio fischeri survival. Contrarily, H2O2 treatment have non-selective reactivity to organic materials, AS was the only treatment that constantly reduced the acute toxicity of 5 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 Fig. 2. Cumulative removal percentages of PhACs with FG + UV/H2O2 (A) and AS + UV/H2O2 (B) couplings. Dashed bars represent the removal of the fungal (A) and activated sludge (B) treatments; grey bars the overall removal of the corresponding biological step followed by UV/H2O2 process. the effluent, whether it was placed before or after the UV/H2O2. In all mechanism used by several organisms to detoxify xenobiotics as part cases, it is important to highlight that there are many non-analyzed of the Phase II metabolism and it involves the covalent addition of a compounds that can be contributing to sample toxicity. molecule to a compound (Badia-Fabregat et al., 2015; Sanchez and Kauffman, 2010). In the human liver, for example, the conjugation 3.3. Lin/deconjugation of compounds leads to the formation of water-soluble compounds that can be excreted through urine, being glucuronidation the most common conjugation The increase in the concentration of some PhACs during biological pathway in the biotransformation of xenobiotics (Sanchez and wastewater treatment has been reported in the literature and it is usu- Kauffman, 2010). Therefore, while both PhACs TPs and conjugated ally attributed to desorption from solids and also to conjugation/ forms are commonly present in wastewater, the analytical methods deconjugation phenomena of PhACs metabolites Conjugation is a are mostly targeting only parent compounds and non-conjugated 6 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 Fig. 3. Cumulative removal percentages of PhACs with UV/H2O2 + FG (A) and UV/H2O2 + AS (B) couplings. Dashed bars represent the removal of the UV/H2O2 treatment; grey bars, the removal of the coupled fungal (A) and activated sludge (B) treatments. molecules. Deconjugation in activated sludge and fungal treatments has 10,11-epoxyCBZ, acridone and lorazepam (Jelic et al., 2015) can be ex- been described elsewhere, but in general it can occur in biological sys- creted to some extent as conjugates. tems where specific enzymes exist that break the covalent bond in Without taking into account CBZ and its TPs (discussed in detail in conjugated compounds (Badia-Fabregat et al., 2015; Jelic et al., 2015). Section 3.4), the compounds detected at higher-than-initial concentra- It probably occurred in the present study as it can be inferred by the tion were treatment-specific: concentration of ofloxacin and lorazepam increase of the concentration of some compounds (discussed after increased only after the AS treatment, while ketoprofen only after the Table 1). Specifically, atenolol (Yuan et al., 2013b), citalopram fungal treatment. This fact could mean that the two treatments had dis- (Dalgaard and Larsen, 1999), ketoprofen, naproxen, ibuprofen, tinct deconjugation capacities. In an attempt to shed light into the sub- diclofenac, gemfibrozil, ofloxacin, carbamazepine, 2-hydroxyCBZ, ject, ketoprofen can be considered as an example: it is well removed by 7 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 Fig. 4. Toxicity values of the raw HWW (white bars), after the first treatment (grey bars) and at the end of the coupled treatments (black bars). fungal treatments in spiked defined matrices (Mir-Tutusaus et al., 2016) (CBZD). A minor pathway is the transformation of CBZ to 2,3- but an increase in its concentration has been observed with real matri- epoxyCBZ to produce 2-hydroxyCBZ and 3-hydroxyCBZ. Human me- ces (Badia-Fabregat et al., 2015; Mir-Tutusaus et al., 2017). Similarly, tabolites include also several glucuronides of CBZ, CBZE, CBZD, 2- ketoprofen concentration rose in the fungal treatments both before hydroxyCBZ and 3-hydroxyCBZ, which were not analyzed in this and after the UV/H2O2, proving that conjugated forms of ketoprofen study (Lynn et al., 1978). were present in HWW and that UV/H2O2 did not deconjugate –nor The concentration of some of these TPs as well as CBZ in wastewa- remove– them. Nevertheless, deconjugation of PhACs could not be con- ter before and after each treatment can be found in Table 2. The ini- firmed in this study as no conjugated compounds were quantified. Thus, tial load of CBZE in this study was 100-fold higher than 2- the concentration of the whole set of PhACs, their TPs, metabolites and hydroxyCBZ (Table 2), because CBZE is the metabolite from the conjugates was potentially undervalued to an unknown degree. main route for CBZ transformation in humans and it is produced and excreted at much higher concentration than CBZ itself (Thorn 3.4. Fate of carbamazepine and transformation products et al., 2011). In fact, CBZE was the compound with the highest con- centration in the raw HWW analyzed. In general, UV/H 2O2 and AS Monitoring of parent compounds is as important as monitoring of seem to follow the same degradation pathway, namely, CBZE, their TPs, both already present in the raw water and newly generated, acridine-9-carbaldehyde, acridine and acridone. White-rot fungal in order to properly evaluate the real efficiency of a treatment. In this pathway resembles the human CBZE pathway (Jelic et al., 2012); study CBZ has been taken as model compound and some of its TPs and therefore, 2-hydroxyCBZ and 3-hydroxyCBZ could also be gener- have been quantified. CBZ, in fact, is frequently found in the environ- ated by WRF, as both humans and fungus have similar cytochrome ment worldwide (Zhang and Geißen, 2010) and it is usually refractory P450 systems. In fact, fungi C. elegans and U. ramanniana were to biodegradation with conventional wastewater treatments. For this reported to produce such compounds when metabolizing CBZ reason it remains stable through the aquatic compartments (Zhang (Kang et al., 2008). and Geißen, 2012), and several approaches have been tested to remove The UV/H2O2 treatment removed 90% of the overall initial concen- it. Different CBZ TPs may be formed based on the process/treatment. A tration of CBZ and TPs. It removed around 50% of CBZ, although UV/ summary of CBZ transformation pathways in humans, UV/H2O2, H2O2 has been reported to degrade up to a 70% of CBZ in some wastewa- white-rot fungus and AS can be found in Fig. 5 and it embodies a com- ters (Giannakis et al., 2015). In contrast, 98% removal of the main pendium of different studies (Golan-Rozen et al., 2015; Mathieu et al., metabolite, CBZE, was achieved whereas acridone concentration in- 2011; Pearce et al., 2009; Lekkerkerker-Teunissen et al., 2012; Kaiser creased. Acridone is a byproduct of CBZE degradation although its in- et al., 2014; Kosjek et al., 2009; Jarrott, 1999; Lynn et al., 1978; Thorn crease does not account for the complete degradation of CBZE; AOPs et al., 2011; Jelic et al., 2012). Several TPs are depicted but only 2- are able to degrade the acridine-9-carbaldehyde, acridine and acridone hydroxyCBZ, 10,11-epoxyCBZ and acridone could be included in the (Kosjek et al., 2009), so acridone must have been degraded too. present study. The raw HWW already contained human metabolites The fungal treatment removed 54% of CBZ, being the single of the CBZ: the parent compound, CBZ, is primarily metabolized in the treatment with the highest CBZ reduction. This is in accordance with human liver generating 10,11-epoxyCBZ (CBZE) as main metabolite previous works, with achieved removal values of around 50% (Mir- (Thorn et al., 2011). It is then further transformed to form acridine, Tutusaus et al., 2017). CBZE concentration decreased by 56% and acridone and the non-pharmaceutically active 10,11-dihydroxyCBZ acridone increased substantially, as expected when taking into account 8 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 Fig. 5. Transformation pathways of CBZ: in humans (solid lines), white-rot fungi (dotted lines), AS (short-dashed lines) and UV/H2O2 (long-dashed lines). Analyzed compounds are presented in bold. Only TPs relevant to this study have been represented (Golan-Rozen et al., 2015; Mathieu et al., 2011; Pearce et al., 2009; Lekkerkerker-Teunissen et al., 2012; Kaiser et al., 2014; Kosjek et al., 2009; Jarrott, 1999; Lynn et al., 1978). the fungal transformation pathway (Fig. 5) and in agreement with pre- in turn undervalued total removal capabilities of the treatment vious studies (Mir-Tutusaus et al., 2017). CBZE and 2-hydroxyCBZ might (Badia-Fabregat et al., 2015). have been present as glucuronides in raw HWW and biological pro- The AS process removed CBZ and CBZE less than the fungal treat- cesses might have deconjugated such glucuronides, which would have ment, but it did not accumulate acridone. CBZ is known to be very Table 2 Levels of CBZ and CBZ TPs (ng L−1) in the initial HWW and after each treatment. Pharmaceutical Initial concentration (ng L−1) Final concentration (ng L−1) UV/H2O2 FG AS UV/H2O2 + FG UV/H2O2 + AS FG + UV/H2O2 AS + UV/H2O2 Carbamazepine 4118 2067 1897 2952 790 1897 1545 892 10,11-epoxyCBZ 28,505 622 12,675 15,655 1195 1254 2442 bld 2-hydroxyCBZ 335 35 bld 5465 bld 1045 bld 605 Acridone 493 542 5710 257 1402 1544 153 251 Total 33,451 3266 20,282 24,329 3387 5740 4141 1747 Removal (%) 90 39 27 90 83 88 95 9 J.A. Mir-Tutusaus, A. Jaén-Gil, D. Barceló et al. Science of the Total Environment 773 (2021) 145374 recalcitrant and poorly degraded in CAS, while acridone has been re- CRediT authorship contribution statement ported to be removed up to a 40% (Kosjek et al., 2009). Other unmea- sured intermediates could have been accumulated during the Josep Anton Mir-Tutusaus: Investigation, fungal treatment Writing treatment before reaching acridone. As it was commented for the fungal original draft. Adrian Jaen-Gil: Investigation and PhACs analysis. Damià process, in fact, some compounds could have been present as glucuro- Barcelo: Supervision and reviewing. Gianluigi Buttiglieri: Investiga- nides that the AS could deconjugate. This pathway was confirmed by tion, activated sludge treatments Rafael Gonzalez-Olmos: Investigation the observed increase in 2-hydroxyCBZ (which is not a byproduct of and H2O2/UV treatments Sara Rodríguez-Mozaz: Conceptualization, CBZ in AS), supporting the hypothesis of deconjugation of already pres- supervision analysis and Reviewing. Gloria Caminal: Conceptualization, ent 2-hydroxyCBZ glucuronides. Supervision and Reviewing. Montserrat Sarrà: Supervision and In terms of coupled treatments, an increase of 2-hydroxyCBZ was Reviewing. observed with both possibilities (UV/H2O2 + AS and AS + UV/H2O2). Contrarily, the increase in acridone produced by UV/H2O2 can be lim- Declaration of competing interest ited with AS + UV/H2O2, that resulted the most convincing strategy to remove CBZ and its TPs. This combination removed, in fact, around The authors declare that they have no known competing financial 80% of the initial CBZ, as if the removal percentages of UV/H2O2 and interests or personal relationships that could have appeared to influ- AS separately were additive. Removal of CBZ and acridone were ence the work reported in this paper. lower than in other combinations, and 2-hydroxyCBZ increased. Nonetheless, considering that 10,11-epoxyCBZ was the most present Acknowledgments compound and that it was completely removed, 94% overall removal of CBZ and its TPs could be achieved. In general, both combinations of This work has been funded by the Spanish Ministry of Economy and the tested biotreatments with UV/H2O2 could eliminate most of ini- Competitiveness (project CTM 2013-48545-C2-1-R) and partly sup- tial CBZ. This is in accordance with other studies that used electro- ported by the Generalitat de Catalunya (Consolidated Research Groups oxidation or UV/H2O2 combined with a membrane bioreactor, even 2017-SGR-0014, 2017 SGR 1124 and 2017-SGR1404). J. A. Mir- though of course, a part of the biodegradation of the activated sludge Tutusaus acknowledges the predoctoral grant from UAB. S. Rodriguez- the effect of the membrane filtration and related phenomena should Mozaz and R. Gonzalez-Olmos acknowledge the “Redes de Excelencia be accounted for in that case (Monteoliva-García et al., 2020; Ouarda 2015” program (CTM2015-71054-REDT). S. Rodriguez-Mozaz and G. et al., 2018). In fact, the UV/H2O2 already removed 90% of the CBZ and Buttiglieri acknowledge the Ramon y Cajal research fellowships (RYC- its TPs even in real, flocculated HWW. The best coupling approach 2014- 16707 and RYC-2014-16754) as well as CLEaN-TOUR improved this removal only by an additional 5% (up to 95%, with (CTM2017-85385-C2-1-R), from the Spanish Ministry of Economy and AS+UV/H2O2). Nevertheless, if we take into account also the conju- Competitiveness. Adrián Jaén-Gil acknowledges the predoctoral grant gates and/or other TPs, then placing UV/H2O2 after a biological treat- 2019FI_B2_00202 from AGAUR and co-financed by the European Social ment, as a polishing step, would be the optimal treatment train Fund. ICRA researchers thank funding from CERCA program. We would configuration: the biological treatment would deconjugate and re- like to thank Sant Joan de Déu Hospital staff for their collaboration dur- move part of the compounds and UV/H 2O 2 would degrade the re- ing the sampling campaign and Aleix Benito for his support in the labo- maining pollutants and byproducts. ratory with the UV/H2O2 experiments. 4. Conclusions References Ahmed, M.B., Zhou, J.L., Ngo, H.H., Guo, W., Thomaidis, N.S., Xu, J., 2017. Progress in the In terms of overall PhACs removal, three out of the four considered biological and chemical treatment technologies for emerging contaminant removal treatment trains (namely UV/H2O2 + AS, UV/H2O2 + FG, FG + UV/ from wastewater: a critical review. J. Hazard. Mater. 323, 274–298. H2O2 and AS+UV/H2O2), showed high removal values (93–95%), Angeles, L.F., Mullen, R.A., Huang, I.J., Wilson, C., Khunjar, W., Sirotkin, H.I., McElroy, A.E., Aga, D.S., 2020. 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