anammox process

Accepted Manuscript Removal of PPCPs from the sludge supernatant in a one stage nitritation/anammox process T. Alvarino, S. Suarez, E. Katsou, J. Vazquez-Padin, J.M. Lema, F. Omil PII:

S0043-1354(14)00753-2

DOI:

10.1016/j.watres.2014.10.055

Reference:

WR 10974

To appear in:

Water Research

Received Date: 13 June 2014 Revised Date:

15 October 2014

Accepted Date: 25 October 2014

Please cite this article as: Alvarino, T., Suarez, S., Katsou, E., Vazquez-Padin, J., Lema, J.M., Omil, F., Removal of PPCPs from the sludge supernatant in a one stage nitritation/anammox process, Water Research (2014), doi: 10.1016/j.watres.2014.10.055. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Removal of PPCPs from the sludge supernatant in a one stage nitritation/anammox process

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T.Alvarino1, S. Suarez1,E. Katsou2,J.Vazquez-Padin3,J. M. Lema1 and F. Omil1*

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Compostela, E-15782 Santiago de Compostela, Spain

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Lane, Uxbridge Middlesex UB8 3PH, E-mail: [email protected]

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Department of Chemical Engineering, Institute of Technology, University of Santiago de

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Department of Mechanical, Aerospace and Civil Engineering, Brunel University, Kingston

Aqualia (FCC Group), Vigo WWTP, Avda. Ricardo Mella 180, E-36331 Vigo, Spain

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Abstract

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Pharmaceutical and personal care products (PPCPs) are extensively used and can therefore find

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their way into surface, groundwater and municipal and industrial effluents. In this work, the

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occurrence, fate and removal mechanisms of 19 selected PPCPs was investigated in an

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‘ELiminación Autótrofa de Nitrógeno’ (ELAN®) reactor of 200 L. In this configuration,

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ammonium oxidation to nitrite and the anoxic ammonium oxidation (anammox)processes occur

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simultaneously in a single-stage reactor under oxygen limited conditions. The ELAN® process

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achieved high removal (>80%) of the studied hormones, naproxen, ibuprofen, bisphenol A and

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celestolide, while it was not effective in the removal of carbamazepine (<7%), diazepam (<7%)

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Corresponding Author (phone: +34 881816805; fax: +34 881816702; e-mail: [email protected])

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and fluoxetine (<30%). Biodegradation was the dominant removal mechanism, while sorption

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was only observed for musk fragrances, fluoxetine and triclosan. The sorption was strongly

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dependent on the granule size, with smaller granules facilitating the sorption of the target

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compounds. Increased hydraulic retention time enhanced the intramolecular diffusion of the

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PPCPs into the granules, and thus increased the solid phase concentration. The increase of

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nitritation rate favored the removal of ibuprofen, bisphenol A and triclosan, while the removal of

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erythromycin was strongly correlated to the anammox reaction rate.

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Keywords: Pharmaceutical and personal care products (PPCPs); biodegradation; sorption;

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nitritation/anammox; granule size; biomass activity

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1. Introduction

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Pharmaceuticals and personal care products (PPCPs) comprise a wide number of compounds that

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are extensively used in households, veterinary medicine, agriculture and aquaculture. PPCPs in

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the aquatic environment (surface water and groundwater) and in wastewater effluents still

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constitute an emerging issue due to the lack of sufficient information concerning their

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occurrence, fate and ecotoxicological effects in sewage, surface, ground and drinking water

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(Ellis, 2006; Loos et al., 2010; Sun et al., 2014). The usual concentrations of PPCPs in sewage

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are in the range of ppb and ppt. However, significant variations in their concentrations are

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observed. The removal efficiency of PPCPs from municipal wastewater treatment plants

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(WWTPs) is wide ranging from negligible to almost complete removal, (Xia et al., 2005)

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depending on the compound, the operating conditions (i.e. hydraulic retention time, HRT), the

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biological treatment process (redox potential) and the technology that is applied (Carballa et al.,

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2004; Zorita et al., 2009). Results obtained from batch experiments have revealed that the

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nitrifying activated sludge is able to degrade several pharmaceuticals while sorption on activated

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sludge can be an important removal mechanism for substances that are characterized by high

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solids/water partition coefficients (Carballa et al., 2008; Shi et al., 2013). The PPCP compounds

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which are not degraded during the anaerobic digestion process are found in the anaerobic sludge

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reject water and particularly non lipophilic compounds present both in the primary and

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secondary sludge (Radjenovic et al., 2009).

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Several research works have investigated the fate and removal of selected pharmaceuticals in

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biological wastewater treatment processes, including membrane bioreactors (MBRs), (Reif et al.,

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2011; Sui et al., 2011) constructed wetlands (Matamoros and Bayona, 2006; Matamoros et al.,

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2007) and in advanced post-treatment processes (e.g. ozonation, advanced oxidation processes -

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AOPs, powder activated carbon adsorption, dense membrane technologies) (Margot et al., 2013).

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AOPs generally lead to the removal of the specific activity/toxicity of the pharmaceuticals.

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However, the degradation of a micropollutant can lead to the formation of toxic transformation

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products with a different mode of action than the parent compound (Postigo and Richardson,

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2013).

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In municipal WWTPs, the nitrogen load of the supernatant of the sewage sludge digesters can

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represent 10-30% of the total nitrogen input load when it is returned to the main inlet without

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previous treatment. Thus, in the last decades new technologies have emerged for the treatment of

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highly nitrogenous effluents (Yang et al., 2011; Frison et al., 2013;Du et al., 2014; Malamis et

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al., 2014). Similarly, the nitritation/anammox process has important advantages compared to the

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conventional nitrification-denitrification process: less energy demand and lower biomass

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production. Moreover, external carbon source is not needed and therefore, the organic matter can

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be used to increase the denitrification in the main wastewater line or to generate more methane in

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the anaerobic digesters (Carballa et al., 2007a; Vázquez-Padín et al., in press). In spite of some

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PPCPs were detected in the supernatant of the anaerobic digestion (Radjenovic et al., 2009),

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limited information is available on the fate and removal mechanisms of PPCPs (i.e. sorption,

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biodegradation, volatilization) by the nitritation/anammox process treating the anaerobic sludge

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reject water.

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In this study, we investigated whether the implementation of the advanced biological process of

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the autotrophic nitrogen removal can lead to a higher removal of PPCPs compared to

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conventional biological processes. The investigation considered the effect of critical parameters,

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such as biomass activity, HRT, sludge particle size and operating time on the biodegradation and

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sorption of 19 selected PPCPs in an autotrophic nitrogen removal process treating the anaerobic

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supernatant generated from digested sewage sludge. The efficiency of the process was examined

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at pilot scale by operating the plant for 200 days. The combined effect of degradation, sorption

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and volatilization in the removal of the PPCPs was investigated. Furthermore, a deeper

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investigation of sorption kinetics and equilibrium was performed for those PPCPs in which

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sorption on anammox granules was important.

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2. Material and methods

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2.1. Chemicals

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The standard solution consisted of 19 PPCPs: three musk fragrances (galaxolide HHCB, tonalide

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AHTN and celestolide ADBI), four antibiotics (sulfamethoxazole SMX, trimethropim TMP,

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erythromycin ERY and roxithromycin ROX), three hormones (estradiol E2, estrone E1 and

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ethinylestradiol EE2), three antiphlogistics (ibuprofen IBP, naproxen NPX, diclofenac DCF),

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two antidepressants (fluoxetine FLX, citalopram CTL), an antiepileptic (carbamazepine CBZ), a

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tranquilizer (diazepam DZP), an antibacterial and antifungal agent (triclosan TCS) and a

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synthetic compound that is used for the production of plastics and epoxy resins (bisphenol A

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BSF). The substances were obtained from Sigma Aldrich (Steinheim, Germany). Stock solutions

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of individual compounds (1000 mg/L) were prepared in methanol and acetone and were stored at

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4oC. The organic solvents used were of HPLC grade.

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2.2. ELAN® pilot plant

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The removal efficiencies and mechanisms of the target substances were determined during the

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long term operation (200 days) of the ‘ELiminación Autótrofa de Nitrógeno’ ELAN® process

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(which stands for autotrophic nitrogen removal in Spanish), in a pilot scale (200 L) reactor. The

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technology was developed by the company Aqualia with the know-how of the University of

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Santiago de Compostela. In the ELAN® process, nitritation/anammox takes place

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simultaneously in a single reactor. ELAN® is a suitable process to treat wastewater characterized

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by low carbon to nitrogen (C/N) ratio, exhibiting lower oxygen requirements than the

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conventional process and without any external carbon source demand. A schematic diagram of

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the ELAN® pilot plant is given in Fig. 1.

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The removal of the selected PPCPs was studied in the ELAN® pilot plant treating the

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supernatant of an anaerobic sludge digester of the Lagares WWTP in Vigo (northwesternof

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Spain). The pilot plant was inoculated with 0.7 kgVSS/m3 of sludge from an airlift reactor that was

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operated in a continuous mode (Vázquez-Padín et al., 2011). The sequencing batch reactor

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(SBR) was operated in cycles of 3 hours with a volumetric exchange ratio of 10-30%. The

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dissolved oxygen (DO) concentration in the SBR was maintained at 0.6-1.2 mg/L during the

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feeding, in order to create a micro-aerobic environment. The operational cycle of the reactor was

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divided in micro-aerobic feeding (135 min), micro-aerobic reaction (30 min), settling (10 min)

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and discharge (5 min) stages. The temperature in the reactor was maintained at 29oC and the

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HRT ranged from 0.4-1.4d in order to study its influence in PPCPs removal. The biomass

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concentration was 7-9 gVSS/L. The reactor worked at a high SRT that ranged between 70-120 d.

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PPCPs were spiked at concentrations between 1 and 40 µg/L in order to maintain a constant inlet

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feeding due to the low concentration in the supernatant of certain PPCPs studied (below LOQ,

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Table S1 – supporting information).

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2.3. Analytical Methods

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2.3.1. Determination of conventional parameters

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Total and volatile suspended solids (TSS, VSS), pH, chemical oxygen demand (COD),

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ammonium, nitrite, nitrate, phosphorus were determined according to standard methods (APHA,

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AWWA, WEF, 1998). Ammonia and nitrite oxidation rates (AOR and NOR, respectively) and

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nitrogen removal rate by anammox bacteria (ANR) were estimated based on nitrogen mass

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balances and the stoichiometry of the process (Vázquez-Padín, 2009). The morphology and the

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size distribution of the granules were measured regularly using an Image Analysis procedure

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(Tijhuis et al., 1994) with a stereomicroscope (Stemi 2000-C, Zeiss). The biomass density was

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determined as gVSS/L of granules using dextran blue, following the methodology proposed by

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Beun et al. (1999). The sludge and the anaerobic supernatant characteristics are given in the

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supporting information (Tables S2 and S3).

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2.3.2 Sampling and determination of PPCPs

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Nine sampling campaigns (performed in triplicate) were carried out during six months to

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determine the dissolved and sorbed PPCPs concentrations. Additionally, the concentration of

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PPCPs in the supernatant of the sludge digester was measured before the addition of the

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substances. All samples were collected in amber glass bottles and stored at 4ºC. The PPCP

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content was determined both in the liquid and the solid phase. The influent and effluent samples

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were taken with a time delay of one HRT during the sampling campaigns. The samples were

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split into liquid and solid phase by settling (anammox granules settled in few seconds). Liquid

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phases were prefiltered (AP3004705, Millipore) and stored at 2 °C prior to analysis. In the case

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of the solid phase collected from the settled granules, the samples were frozen and lyophilized.

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The dried sludge was extracted successively at room temperature in an ultrasonic bath three

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times with methanol and acetone and the solvent fractions were combined and evaporated in

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order to resuspend the samples in water. Solid-phase extraction (SPE) was used to pre-

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concentrate the samples of the liquid and the solid phase. The extracts were measured by liquid

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chromatography in the case of estrogens, neurodrugs, and antibiotics and by gas chromatography

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in the case of antiinflammatories, triclosan, bisphenol A and musk fragrances (Fernandez-

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Fontaina et al., 2014). GC/MS and LC/MSMS methods are described in supporting information.

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For this purpose, an Agilent liquid chromatograph API 400 GI312A connected with a triple

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quadruple mass spectrometer and a gas chromatograph Varian CP 3900 equipped with an

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autosampler CP-8400 and coupled to Varian mass ion trap spectrometer Saturn 2100were used.

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The limits of quantification (LOQ) and the recoveries in the liquid and solid phase are given in

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the supporting information (Table S1).

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2.4. Methodology for the PPCP mass balances

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A complete PPCP mass balance was developed under steady-state conditions (Equation 1) by

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considering the measured PPCP concentrations in both the liquid and the solid fractions

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(Carballa et al., 2007b). The dimensionless Henry’s law constant of PPCPs (H, supporting

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information; Table S1) was used to determine the volatilization fraction (Equation2), while

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sorption (Equation 3) is a function of the PPCP concentration in the solid phase in the effluent

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(qeff, µg/gTSS) and the level of total suspended solids in the effluent (TSSeff, mgvss/L). PPCPs

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biodegradation was considered taking into account a pseudo-first order kinetic model (Joss et al.,

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2006), where kbiol is the kinetic coefficient for each compound (Equation 4). =

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−

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=

=

∙

=

(eq. 1)

∙ ∙

(eq. 2)

∙

·

(eq. 3)

·

(eq. 4)

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∙

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+

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where Finf, Feff, Fstripped, Fsor and Fbiod are the mass flows (in µg/d) of the influent, the effluent

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(only dissolved), the gas emissions, sorbed onto solids and biodegraded respectively; f

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(Lair/Linfluent) is the air supply per unit of wastewater treated flow, Q (L/d) is the feed flow rate,

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VSS are the volatile suspended solids inside the reactor (gvss/L), Ceff(µg/L) and qeff (µg/g) are

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the effluent concentration of the compounds in the liquid and solid phase respectively, V is the

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volume of the reactor (L). 8

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In order to determine the influence of the primary substrate in the experiments at steady-state

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conditions, the PPCP removal rate (M, µg/gVSSd, Equation 5) was correlated with the biomass

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nitrifying activity rate (mgN/gVSSd) using the linear regression analysis.

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(eq. 5)

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2.5. Kinetic and equilibrium studies for fluoxetine sorption on anammox granules

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The amount of FLX sorbed onto sludge (qt, mg/g) at a given time (t, min) was studied as a

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function of the initial FLX concentration in the solution (C0,mg/L) and its remaining level in the

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liquid phase at time t (Ct, mg/L), where TSS is the concentration of the total suspended solids

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inside the reactor (gvss/L) (Equation 6).

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qt =

C0 − Ct TSS

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Sorption kinetic studies were performed in 500 mL flasks at constant temperature (25ºC) using

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sludge previously spiked with FLX. The experiments were conducted at pH 7 using a phosphate

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buffer, with initial FLX concentrations in the range of 2.5-10 mg/L. The concentration of the

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sludge granules was 1.2 gTSS/L. Continuous stirring was provided at 150 rpm. Samples of 2 mL

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were taken from a total volume of 200 mL at different time intervals for a period of 30 h and

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after filtration, the FLX concentration was measured by HPLC (Jasco X-LC with diode array).

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Each test was conducted in duplicate. The obtained kinetic data were modeled using the pseudo-

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first-order and pseudo-second-order equations, as well as, with the intraparticle diffusion model.

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Furthermore, Reichenberg’s diffusion equation was applied to determine the effective diffusion

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coefficient (Deff). The equations are summarized in Table S4 (supporting information).

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The same process and initial FLX concentrations were considered for the equilibrium studies.

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Samples were taken up to the point a constant FLX concentration was measured in the liquid.

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Τhe fit of the experimental data to the isotherm models (Langmuir and Freundlich), was done

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through linear regression analysis. The equations are summarized in Table S5 (supporting

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information).

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3. Results and discussion

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3.1. Nitritation and anammox activity

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The ELAN® process was operated for 200 days. During the first 10-20 days a high anammox

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activity rate was achieved (above 1000 mgN/Ld), while operating the system with an HRT of 0.4

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d(Fig. 2). After the first 60 d of operation the nitrogen removal efficiency decreased (from 70%

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to 45%), since both the anammox and nitritation activity rate decreased by ~50% (300 mgN/Ld)

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to recover the biomass activity, the HRT was increased up to 0.8 d. From day 120 onwards, both

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the anammox and nitritation rate recovered, reaching values higher than 800 and 500 mgN/Ld

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respectively. Consequently, the HRT was reduced to 0.5 d.

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3.2. Occurrence and fate of PPCPs

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Only IBP, NPX, DCF and BSF were detected in ppb concentrations in the anaerobic supernatant

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before the spiking of PPCPs (above 10 µg/L in the case of BSF and IBP) The presence of IBP

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can be explained by its high consumption leading to concentrations of several µg/L in raw

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sewage (Carballa et al., 2004) and hundreds of ng/g in the primary sludge (Radjenovic et al.,

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2009). The latter in combination with its moderate biodegradability during anaerobic sludge

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digestion (Carballa et al., 2007a) justifies the concentration that was detected in the anaerobic

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supernatant.

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Removal of PPCPs trough biodegradation and sorption was determined according to the

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equations 1-4. Accumulation on the biomass inside the reactor was negligible. As an example, in

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the case of AHTN the amount accumulated in the reactor due to sorption taking into account the

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whole operation period was of 0.033 µg/d, while the average AHTN flow in the influent was of

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4800 µg/d. Thus, the assumption of steady state was considered as valid and only the PPCPs in

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the solids leaving were considered in the mass balances.

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The estrogens under examination, NPX, IBP, BSF and ADBI were effectively (≥80%) removed

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in the ELAN® process when the anammox activity was higher than 600 mgN/Ld (Table 1). On

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the contrary, low removal was obtained for FLX, DCF, CBZ and DZP, which displayed a

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recalcitrant behavior. These PPCPs are characterized by low biodegradation constant (kbiol), in

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accordance to the low removal level obtained in the process (Table 1). The main PPCPs removal

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mechanism was biodegradation, while the contribution of sorption onto the sludge granules was

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negligible for the vast majority of the compounds (Fig. 3). Sorption was considerable in the case

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of musk fragrances, FLX and TCS, owing to their lipophilic character.

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PPCP removal efficiencies obtained in the ELAN® process were similar to the respective ones

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obtained under enriched nitrifying conditions (Fernandez-Fontaina et al., 2012a; Suarez et al.,

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2010). Redox conditions can be a key parameter that influences the biotransformationmetabolic

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route of pharmaceutical compounds. In the case of SMX, ahigher removal was obtained in the

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ELAN® process compared to the nitrifying reactors, since SMX is strongly dependent on the

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reduction potential (Banzhaf et al., 2012) and its removal is higher under anoxic than under

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aerobic conditions (Hai et al., 2011). In a previous work developed in an upflow anaerobic

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sludge blanket (UASB) –partial nitritation (PN) – anammox system, the last two autotrophic

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stages contributed by 93% to the removal of IBP, while the anaerobic step negatively affected its

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removal(de Graaff et al., 2011).To conclude, biodegradation was by far the most important

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removal mechanism of PPCPs (i.e. transformation) and was affected by the applied redox

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potential.

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IBP, estrogens (E1, E2, EE2) and BSF were highly biodegradable by the ELAN® biomass.

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Therefore, their kbiols were higher than 15 L/gVSSd, with the exception of EE2. In the case of

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musk fragrances and natural estrogens, similar results were obtained by Suarez et al. (2010) in a

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nitrification process. However, kbiols were higher (>75 L/gVSSd), since IBP is a readily

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biodegradable compound and the activity was measured in both studies, but with different

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biomass concentrations (1.6 and 9-10gVSS/L in the nitrifying reactor and in the ELAN® process

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respectively).

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3.3. Influence of nitritation and anammox activity

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High variability in the PPCP removal rate was observed due to the variation in the nitrogen

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removal. IBP, BSF and TCS removal was strongly dependent on nitritation, as revealed by the

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correlation between the nitritation rates and the specific micropollutant removal rates (Fig.4).

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Accordingly, Suarez et al. (2010) obtained high aerobic degradation rates for IBP, while anoxic

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degradation only occurred after long adaptation times (more than 340 d). The higher removals

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obtained when nitritation activities were high can be explained by the enhanced production of

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ammonium monooxygenase enzymes, which have been proved as a key factor for the

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cometabolic degradation of several PPCPs (Fernandez-Fontaina et al., 2012a). The enhanced

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nitrogen removal maintained in the ELAN® process also positively affected the biodegradation

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of E1, EE2 and ROX. However, it was not feasible to identify which microorganisms (anammox

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or nitritationbacteria) were mainly responsible, since significant correlation for both removal

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rates (anammox and nitritation) was observed.

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In the case of ERY, its removal rates were well correlated with the anammox activity(Fig.5);

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high anammox activities resulted in an enhanced removal of ERY (up to 73% during the early

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operation of the reactor); whereas very low ERY efficiencies (around20%) were observed when

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anammox activity was reduced by more than 70%. Finally, the recovery of anammox activity (by

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increasing HRT) resulted in a greater removal of ERY.

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3.4. Influence of hydraulic retention time

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The impact of HRT is significant in the removal of substances characterized with a medium kbiol

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and a high Kd. As shown in Fig.6, musk fragrances are retained by sorption and are significantly

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removed when the HRT is long enough, as previously reported by Suárez et al., (2008). In this

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work, the increase of HRT from 0.5 d to 1.4 d resulted in a significant increase in the removal of

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lipophilic compounds, particularly the granular structure of biomass seems to slow down the

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sorption process. On the contrary, in the study of Fernandez-Fontaina et al. (2012b) the removal

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of HHCB remained constant (> 80%) and was independent of the HRT, though a higher HRT

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was applied (1-4.6 d) in a nitrifying reactor under steady-state conditions. Compounds which

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were characterized by high kbiol constants, such as ibuprofen or estrogens, were readily

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transformed independent of the HRT. SRT was not an influential parameter forPPCPs

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biodegradation in the ELAN® process since, it was above 50d and thus, higher than the ‘critical’

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value of 10 d that has been reported as the minimum sludge age for nitrogen removal(Clara et al.,

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2005).

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3.5. Influence of particle size in sorption

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The release of non-biodegradable PPCPs into the environment can occur not only with the

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effluent of the treatment plant, but also with the disposal of the excess sludge. Thus, it is

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important to gather information concerning the presence of PPCPs both in the aqueous phase and

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onto biosolids. Musk fragrances and TCS are lipophilic compounds with high octanol-

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water partitioning coefficients that exhibit significant removal by sorption as shown in Fig. 3 (5-

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15%).

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According to Figure 7, the concentration of musk fragrances in the solid phase was influenced by

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the contact time and the diameter size of the sludge granules. Fig.7a shows that the relative

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amount of musk fragrances sorbed onto sludge increased continuously with the operating time of

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the reactor. It was observed that while the solid phase concentration was more or less constant, a

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high variability in the liquid phase concentrations occurred. Concretely, it seems that once

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PPCPs are sorbed onto sludge, desorption was too slow to reach equilibrium, most probably

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relatedto the granular structure of the biomass. Therefore, the solid-liquid equilibrium was not

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achieved and these compounds cannot be associated with a unique Kd for each one as commonly

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used for flocculent biomass. Musk fragrances must penetrate the granule in order to be sorbed

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inside; thus a fast sorption onto the surface is followed by an intramolecular diffusion

308

mechanism (Shi et al., 2011). The mechanism of the process was studied via the examination of

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the sorption kinetics and the equilibrium (section 3.6).

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After two months of system operation, an increase of the granular sludge size (from 2.9 to 4.5

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mm) resulted in a decrease in the musk fragrances concentration in the sludge (Fig.7) due to the

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reduction of the specific surface of the granules. On the contrary, four months later the opposite

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effect was observed; musk fragrances concentration in the solid phase increased as a

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consequence of the particle size reduction (from 4.5 to 2.3 mm), as well as, the higher contact

315

time, which enhanced the intramolecular diffusion. The effect of the particle size on sorption was

316

previously examined by Reif et al. (2011) who demonstrated higher AHTN concentration on the

317

activated sludge in the MBR than in the conventional activated sludge process due to the lower

318

sludge particle size in the first case.

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3.6. Sorption mechanisms onto anammox granules

321

FLX is a hydrophobic substance (logKow 4.2) with medium sorption efficiency in the ELAN

322

operation. Previous works indicated that even with flocculent biomass (in which solid-liquid

323

phase equilibrium would be more easily expected rather than in granular systems), a high

324

variability in FLX sorption coefficients (Kd) have been reported in the range of 1000-10000 L/kg

325

TSS (Fernandez-Fontaina et al., 2012b; Hörsing et al., 2011). This work has shown that sorption 15

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is a significant removal mechanism for FLX. The kinetics and equilibrium of this compound are

327

studied in detail in this section.

328

The kinetic parameters and the isotherm constants for FLX sorption onto anammox granules are

329

summarized in Table 2. The kinetic parameters were derived by fitting the linear equations to

330

experimental data using linear regression analysis. The experimental data fitted well to both the

331

pseudo-first and pseudo-second-order models with high coefficient of determination (R2).The

332

solid phase concentration at equilibrium predict from both models (qe,mod) was close to that

333

obtained experimentally (qe,exp). The rate constants of the pseudo-second order model (k2)

334

decreased with increasing the initial FLX concentration; a similar trend was obtained for the

335

sorption of EE2 onto activated sludge by Feng et al. (2010). In fact, a fast sorption followed by

336

anintraparticle diffusion in nitrifying granules was observed by Shi et al. (2011) in the case of

337

tetracycline (TC).

338

Moreover, the application of the intraparticle diffusion model revealed two distinct diffusion

339

stages for all the examined concentrations, indicating that more than one diffusion step took

340

place for the diffusion of FLX into the sludge granules. The latter is clearly seen in Fig.S1 that is

341

given as supporting information. The slope of each linear part provides an indication of the FLX

342

sorption rate. Both stages are attributed to intraparticle diffusion, probably for diffusion into the

343

macropores and mesopores respectively. FLX diffusion rate was significantly higher in the first

344

stage of the process compared to that of the second stage. This is reflected by the values of the

345

intraparticle diffusion constants given in Table 2, where ki1> ki2 for all the examined cases. The

346

application of Reichenberg’s model enabled the determination of the effective diffusion

347

coefficient (Deff) of FLX onto the granules. The mean radius of the anammox granules was 4.5

348

mm. The Deff was in the range of 1.8-3.1·10-14 m2/s.

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Comparing the two-parameter isotherm equations with the experimental data, it can be

350

concluded that the Freundlich model exhibited the highest coefficient of determination (R2) and

351

the lowest chi-square value. Anammox granules are a highly heterogeneous mixture consisting

352

of several functional groups. Thus, the use of Freundlich model provided a better description of

353

FLX sorption process, as it considered the heterogeneity of the sorbent. The latter is in

354

accordance to the results obtained by Shi et al. (2011) for TC sorption onto nitrifying granules.

355

Furthermore, in line with our findings, the results of previous research studies have shown that

356

sorption of estrogens follow the Freundlich isotherm equation (Ren et al., 2007).

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4. Conclusions

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In this work the fate and removal mechanisms of 19 PPCPs was investigated in the completely

360

autotrophic nitrogen removal process. The process includes the biological processes of nitritation

361

and anammox.The fate of the substances was determined during the long term operation of the

362

ELAN® process. The following were concluded: -

The removal efficiencies of the ELAN® reactor were similar to the respective ones

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obtained in nitrifying reactors, although the combination of anoxic-aerobic processes

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enhanced the removal of SMX and TMP.In any case further investigation on the behavior

366 367 368

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of the novel process is needed in larger scale (full scale). Several parameters should be taken into consideration for the selection of the most appropriate integrated treatment scheme, such as optimization of the cost / benefit ratio, elimination of background

369

contaminationand by-products formation, adaptability in existing WWTPs the scale of

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the process and the broad spectrum of action.

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-

correlation was found between the anammox activity and the removal of ERY.

372 373

-

The increase of the HRT positively influenced the removal of lipophilic compounds, such asmusk fragrances.

374 375

An increase in the nitritation rate favored the removal of IBP, BSF and TCS, while a

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-

The decrease of the particle size of the granular sludge applied in the ELAN® process enhanced the sorption of FLX onto the granules due to the higher specific surface.A

377

deeper examination of the process kinetics and equilibrium showedthat pseudo-first order

378

and pseudo-second order equations are in good agreement with the experimental data,

379

while the Freundlich isotherm model described better the FLX sorption process. Finally,

380

the intraparticle diffusion model revealed a two-stage diffusion process.

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Acknowledgments

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This research was supported by the Spanish Ministry of Education and Science through

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NOVEDAR_Consolider (CSD2007-00055) project, by the Spanish Ministry of Science and

386

Innovation through INNOTRAZA (CTQ2010-20240) project, by the Xunta de Galicia through

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MicroDAN (EM 2012/087) and GRC (2013-032) project and a student grant awarded by the

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Fundación Segundo Gil Dávila.

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Table 1. PPCPs removal efficiencies in ELAN® process and PPCPs biological constants

Effluent

Removalb

kbiol

(µg/L)

(µg/L)

(%)

(L/gVSSd)

ADBI

15.1±6

2.25±1

82±2

1.3±0.6

HHCB

7.9±4

1.7±0.7

76±5

0.8±0.3

AHTN

19.2±4

4.2±1.5

70±11

0.5±0.3

CBZ

17.3±5

16.3±6.8

7±1

ND

DZP

19±4

18.3±6.6

6±1

IBP

18.9±4

0.4±0.3

98±1

NPX

8.8±3

DCF

16.8±4

FLX

3.1±2

ROX

6.1±2

SMX

0.3±0.2

TMP

7.1±2

SC ND

38.0±7

80±1

16.8±0.2

10.3±3

36±1

0.9±0.1

2.2±1

29±1

0.1±0.05

3±1

50±1

0.3±0.1

0.2±0.1

57±2

0.3±0.1

4.1±2

45±1

0.2±0.1

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1.5±0.3

3.3±2

0.8±0.7

75±1

0.5±0.3

E2

0.5±0.2

0±0

98±1

27.0±12

E1

1±0.2

0±0

100±1

53.0±14

EE2

0.8±0.2

0.1±0.05

87±1

2.0±1

BSF

14.6±5

0.7±1

96±1

33.0±30

TCS

7.1±3

2±0.4

75±13

0.7±0.3

CTL

0.1±0.1

0±0

67±1

ND

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ERY

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Influenta

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(kbiol, L/gVSSd)

After PPCP spike, bAverage value (operation days 26,75, 150, 173 and 190)

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Table 2. Parameters for the sorption of FLX onto sludge in the ELAN® process derived from i) kinetic and ii) equilibrium studies

Pseudo-first order

qe,exp (mg/g)

R2

0.74 2.12 3.13 3.43 Experimental data qe,exp (mg/g) 0.74 2.12 3.13 3.43

0.9993 0.9979 0.9979 0.9997

k1 (min-1)

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R2 0.5838

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KL (L/mg) 0.8371

k2 (g/mgmin)

0.0026 0.0020 0.0030 0.0018 0.0039 0.0018 0.0033 0.0011 Reichenberg Deff (m2/s) Ri12 -14 1.81 × 10 0.958 3.04 × 10-14 0.995 2.47 × 10-14 0.999 3.05× 10-14 0.983 Equilibrium studies

Langmuir qm (mg/g) 14.20

qe,mod (mg/g) 0.76 2.05 3.26 3.09

Pseudo-second order qe,mod (mg/g)

0.97 2.40 3.73 3.58 Intraparticle Diffusion Ri22 ki1 0.9999 0.0250 0.9999 0.0750 0.9999 0.1340 0.9999 0.1109

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2.5 5 7.5 10 PPCP (mg/L) 2.5 5 7.5 10

Experimental data

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Kinetic studies

n 1.27

Freundlich KF (mg1-1/n l1/n g-1) 7.16

R2 0.9841 0.9999 0.9996 0.9984 ki2 0.0101 0.0235 0.0241 0.0305

R2 0.9874

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(a)

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Fig 1.a) Integration of ELAN® process in the conventional WWTP; b) ELAN® pilot scale plant

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

1200 1000 800 600 400 200 0 0

40

80

120

160

Operation time (d) Anammox activity

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Fig 2. Biomass activity and HRT in ELAN® process

HRT (d)

SC

Nitritation

HRT

1400

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Biomass activity (mgN/Ld)

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200

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100

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60 40 20 0

PPCP Volatilization

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Fig 3.PPCPs removal mechanisms in ELAN® process

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Sorption

SC

Removal (%)

80

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5

4

BSF

TCS

3 2 1

TCS

4

0

20 40 60 80 Nitritation rate (mgN/gVSSd)

100

BSF

3 2 1 0

0

IBP

0

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IBP

PPCP removal rate (µg/gVSSd)

PPCP removal rate (mgI/gVSSd)

5

30 60 90 120 150 Specific anammox activity (mgN/gVSSd)

Fig. 4. Influence of the specific nitritation rate and anammox activity in the removal of IBP,

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BSF and TCS

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2 1.5 1

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ERY removal rate (µ µgERY/gVSSd)

2.5

0.5 0

50 100 150 Specific anammox activity (mgN/gVSSd)

200

SC

0

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Fig 5. Influence of anammox activity in the removal of ERY

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ADBI

HHCB

AHTN

20

10 5 0 0

0.2

0.4

0.6 HRT (d)

\

0.8

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Fig 6. Influence of HRT in musk fragrances removal

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Musk fragrances removal (µ µg/Ld)

25

1

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ADBI

HHCB

AHTN

TCS

10

8000 6000 4000 2000 0 0

100 Time (d)

(a)

200

(b)

2.3

2.9

4.5 mm

8 6 4 2 0 ADBI

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10000

Musk fragrances concentration solid phase (µgPPCP/gTSS)

Musk fragrance solid phase/liquid phase concentration (L/KgTSS)

12000

HHCB AHTN Musk fragrances

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Fig 7. Influence of (a) contact time and (b) granule diameter (D) in musk fragrances, fluoxetine and triclosan sorption

FLX

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Highlights

- Biodegradation is the dominant removal mechanism for PPCPs

- HRT affects the removal of lipophilic compounds

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- Sorption is dependent on the biomass particle size and contact time

- Increase of nitritation rate favors the removal of ibuprofen, bisphenol A, triclosan

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- Anammox activity is strongly correlated to the removal of erythromycin

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Removal of PPCPs from the sludge line in a one stage nitritation/anammox process

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Supporting Information

T.Alvarino1, S. Suarez1,E. Katsou2,J.Vazquez-Padin3,J. M. Lema1 and F. Omil1

Department of Chemical Engineering, Institute of Technology, University of Santiago de

Compostela, E-15782 Santiago de Compostela, Spain

Department of Mechanical, Aerospace and Civil Engineering, Brunel University, Kingston

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Lane, Uxbridge Middlesex UB8 3PH, E-mail: [email protected]

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Aqualia (FCC Group), Vigo WWTP, Avda Ricardo Mella 180, E-36331 Vigo, Spain

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PPCPs determination and quantification: - GC-MS Analytes were determined by GC-MS using a Varian (Walnut Creek , CA) SP 3900 gas chromatograph connected to an ion-trap mass spectrometer (Varian Saturn 2100T). Separations were carried out in a HP5-MS capillary column (30 m x 0.25 mm i.d., d.f. 0.25µm) purchased from Agilent (Wilmintong, DE). Helium (99.999 %) was used as carrier gas at constant flow of 1 ml min-1. The GC oven was programmed as follows: 70ºC (held for 2 min), at 25ºC min-1 to 150ºC, 3ºC min-1 to 180ºC and finally 8ºC min-1 to 280ºC (held for 15 min). The GC-MS interface and the ion trap temperatures were set at 280ºC and 220ºC, respectively. Injections (1 µL) were made in the splitless mode (split 1:50 at 2 min), with the injector at 280ºC. The mass spectrometer was operated in the electron impact ionization mode (70eV). Mass spectra were recorded in the range from 44-550 m/z units. Concentrations of the analytes in samples were determined using internal standard calibration method using Dihydrocarbamazepine. Meclofenamic acid and Bisphenol F as surrogates and PCB-30 like internal standard.

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- LC-MS/MS The liquid chromatography system consisted in an Agilent Technologies (San Jose, CA, USA) 1100 Series HPLC system with a binary pump, vacuum degasser, HTC-Pal (CTC Analytics, Zwingen, SW) autosampler. The LC was interfaced to an API-4000 triple quadrupole mass spectrometer equipped with a turbo ion spray interface (Applied Biosystems, Foster City, CA, USA). Nitrogen was employed as ESI and collision gas. Curtain pressure, ion source gas and gas pressure in collision cell were 45 psi, 40 psi and 6 psi, respectively. The spray voltage was 4.0 kV and operates in positive mode. Source temperature was set at 450ºC. Quantification of all analytes was made by recording the most intense transitions in multiple-reaction-monitoring (MRM). LC separation was carried out on a 250 mm x 4.6 mm (4 µm) Synergie Max-RP columns with C12 guard cartridge from Phenomenex (Torrance, CA, USA) at room temperature. A dual eluent system of 0.1% formic acid (A) and methanol (B) was used. The flow rate was 0.7 mL/min and the gradient was as follows: 0 min (85%A), 3.5 min (85%A), 10 min (20%A), 13.10 min (10%A) and 25 min (10%A). Injection volume was set to 5 µL.

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Recoveries in liquid phase: we added the compounds in known amounts (diluted in acetone and methanol) in the liquid phase. Solid-phase extraction (SPE) was used to pre-concentrate the samples of the liquid phase and the extracts were measured by liquid chromatography in the case of estrogens, neurodrugs, and antibiotics and by gas chromatography in the case of antiinflammatories, triclosan, bisphenol A and musk fragrances. The experiment was done in triplicate. Recoveries in solid phase: we took the granules, broke in small particles, frozen and lyophilized. After that, we added the compounds in known amounts diluted in acetone and we waited until the total evaporation of the acetone. Then, the same process was followed in all the solid samples: The dried sludge was extracted successively at room temperature in an ultrasonic bath three times with methanol and acetone and the solvent fractions were combined and evaporated in order to resuspend the samples in water. Solid-phase extraction (SPE) was used to preconcentrate the samples and the extracts were measured by liquid chromatography in the case of estrogens, neurodrugs, and antibiotics and by gas chromatography in the case of

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antiinflammatories, triclosan, bisphenol A and musk fragrances. The experiment was done in triplicate and the real concentration before the addition was measured in the sludge.

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Table S1. PPCP limits of quantification, Henry’s law constant, octanol-water coefficient, acid dissociation constant, solubility and recoveries.

Recovery

LOQ

H (mg·m-3air/

(ng/L)

-3

PPCP

s (mg/L)ab

pKad

water)ª

60

7.3·10-1

6.1

-

HHCB

60

5.4·10-3

4.6-6.4

-

AHTN

60

5.1·10-3

CBZ

480

4.4·10-9

DZP

480

1.5·10-7

IBP

25

6.1·10-6

NPX

32

1.4·10-8

DCF

12

1.9·10-10

ERY

1.2

FLX

solid

phase

phasec

1.8

75-110

76-94

1.2

60-84

72-84 87-107

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liquid

SC

mg·m

Log Kowª

Recovery

-

0.22

70-110

2.6

13.9

17.7

89-99

2.7

3.35

50

25-35

3.5-5.2

5.3

21

100-110

3.2

4.2

16

110-120

4.6

4.2

2.4

110-120

2.2·10-27

2.7

8.9

1.4

90-100

1.2

3.6·10-6

4.2

10.5

60

65-80

ROX

1.2

2.0·10-29

2.8

9.2

0.02

35-60

SMX

6

2.6·10-11

0.7

5.8

610

65-80

TMP

6

9.8·10-13

1.2

6.9

400

95-105

E2

12

1.5·10-9

3.9

10.4

3.6

80-95

E1

12

1.6·10-8

3.2

10.4

30

70-85

EE2

12

3.3·10-10

2.8-4.2

10.6

11.3

60-90

BSF

32

2.1·10-10

3.3

9.6-11.3

1000

85-90

94-100

TCS

60

1.5·10-7

4.76

7.9

2-12

95-100

70-90

CTL

1.2

-

1.39

9.78

4

85-90

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5.4-6.6

38-42

ª Syracuse Research Corporation; bsolubility at 25 ºC; c: recoveries of the lipophilic compounds; d: Joss et al., 2005

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treatment plant (WWTP) in Vigo (northwesternof Spain)

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Table S2. Characteristics of anaerobic supernatant and treated effluent of the Lagareswastewater

Influent

Effluent

pH

6.5-7.6

6.5-7.6

Conductivity (mS/cm)

4.8-6.9

2.2-4.6

Alkalinity (mgIC/L)

416-615

N-NH4 (mg/L)

440-650

COD (mg/L)

220-320

P-PO4 (mg/L)

46-63

49-62

TSS (mg/L)

0.03-0.22

0.02-0.37

0.02-0.10

0.02-0.35

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25-160

45-180

156-230

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VSS (mg/L)

SC

Parameter

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Table S3. Sludge characteristics MLSS (gvss/L)

Diameter size

Granules

Sedimentation rate

(mm)

density(gvss/L)

(m/h) 39.5

9.9

2.9

32.7

80

9.1

4.5

-

140

8.9

2.3

31

72

36

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SC

20

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Time (d)

Fig. S1. Plot of FLX sorbed onto sludge versus the square root of time for various initial

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concentrations (intraparticle diffusion model)

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Table S4. Kinetic and diffusion models used for FLX sorption Equation

Parameters

Pseudo-first order, linear

ln(qe − qt) = lnqe− k1t

qe, k1

(Ho, 2004)

Pseudo-second order linear

t 1 1 = + t 2 qt q k 2q e e

qe, k2

(Ho and McKay, 1998)

Intraparticle diffusion

q t = k id t 0.5

kid

(Allen et al., 1989)

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F > 0.85

(Reichenberg, 1953; Lagoa

Deff

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Reichenberg

π 2F  πF  − 2π 1 −  3 3   Bt = -0.4977 - ln(1 - F)

F ≤ 0.85 Bt = 2π −

Reference

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Kinetic / Difussion model

and Rodrigues, 2009)

where B is a time constant, Ceis theliquid phase equilibrium FLX concentration (mg/L), F is the fractional attainment of equilibrium at time t (=qt/qe) ,k1is the rate constant of the pseudo-first-

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order model (min−1), k2 is the rate constant of the pseudo-second-order model (g/mgmin) kid is the constant of intraparticle diffusion (mg/gmin1/2), qt is the solid phase concentration of FLX (or any other compound) which has been sorbed on the granule at time t and qe is the solid phase

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concentration of FLX sorbed on the granule at equilibrium conditions.

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Table S5. Isotherm equations used for FLX sorption Param Equation

Langmuir,linear

Ce 1 1 = + Ce qe qmKL qm

Freundlich,linear

lnq e = lnK

+

1 lnC e n

KL, qm (Langmuir, 1916)

KF, n

(Freundlich, 1906)

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F

Reference eters

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Isotherm

where, qe is the solid phase equilibrium FLX concentration (mg/g), qm is the maximum sorption

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capacity (mg/g), KL is the Langmuir constant (L/mgor L/mol), KF is the Freundlich constant (mg1-1/n L1/n g-1), n is the Freundlich affinity constant,Ce is the liquid phase concentration of the compound in the solution at equilibrium conditions and Ct is the liquid concentration of the

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compound at time t.

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References Allen, S.J., McKay, G. and Khader, K.Y.H., 1989. Intraparticle diffusion of a basic dye during adsorption onto sphagnum peat. Environ. Pollut. 56, 39-50.

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Freundlich, H.M.F., 1906. Uber die adsorption in losungen. J. Phys. Chem. 57, 385-470.

Ho, Y.-S., 2004. Citation review of Lagergren kinetic rate equation on adsorption reaction. Scientometrics 59, 171-177.

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Ho, Y.S. and McKay, G., 1998. Kinetic models for the sorption of dye from aqueous solution by wood. J. Environ. Sci. Health B: Process Saf. Environ. Protect. 76, 183-191.

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Joss, A., Keller, E., Alder, A.C., Göbel, A., McArdell, C.S., Ternes, T., Siegrist, H., 2005. Removal of pharmaceuticals and fragrances in biological wastewater treatment. Water Res. 39, 3139-3152

Lagoa, R. and Rodrigues, J.R., 2009. Kinetic analysis of metal uptake by dry and gel alginate

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particles. Biochem. Eng. J. 46, 320-326.

Langmuir, I., 1916. The constitution and fundamental properties of solids and liquids. J. Am. Chem. Soc. 38, 2221-2295.

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Reichenberg, D., 1953. Properties of ion-exchange resin in relation to their structure, III.

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Kinetics of exchange. J. Am. Chem. Soc. 75, 589-597.