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Removal of pharmaceutical compounds in membrane bioreactors (MBR) applying submerged membranes
Desalination 261 (2010) 148–156
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Desalination j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l
Removal of pharmaceutical compounds in membrane bioreactors (MBR) applying submerged membranes José Luiz Tambosi a,b, Rênnio Felix de Sena a,b, Maxime Favier b, Wilhelm Gebhardt b, Humberto Jorge José a, Horst Friedrich Schröder b, Regina de Fátima Peralta Muniz Moreira a,⁎ a Laboratory of Energy and the Environment, Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis, SC, Brazil b Institute of Environmental Engineering, Environmental Analytical Laboratory, RWTH Aachen University, Krefelder Strasse 299, D-52056 Aachen, Germany
a r t i c l e
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Article history: Received 22 September 2009 Received in revised form 7 May 2010 Accepted 10 May 2010 Available online 17 June 2010 Keywords: Elimination Mass spectrometry (MS) Membrane bioreactor (MBR) Pharmaceuticals Wastewater
a b s t r a c t Pharmaceuticals such as non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics have been detected in sewage treatment plant (STP) effluents, surface and ground waters and even in drinking waters all over the world. These compounds of concern may induce toxic effects in aquatic organisms and disturb the ecological balance on their way from wastewater to drinking water treatment via surface water and bank filtration. Membrane bioreactors (MBRs) have gained significant popularity as an advanced wastewater treatment technology and might be effective in removing such pollutants. This paper evaluates the treatment of wastewater containing three NSAIDs (acetaminophen, ketoprofen and naproxen) and three antibiotics (roxithromycin, sulfamethoxazole and trimethoprim) in two MBRs with sludge retention times (SRTs) of 15 (MBR-15) and 30 (MBR-30) days over a period of 4 weeks. It was observed for both MBRs that the NSAIDs were removed with higher efficiency than the antibiotics, and the MBR-30 presented higher removal efficiencies than the MBR-15 for all compounds studied. Biological transformation products of acetaminophen, ketoprofen and naproxen produced by wastewater biocoenosis were identified in permeates from both MBRs. Further research in this field is required to assess the environmental risks associated with the presence of pharmaceuticals and their transformation products in the environment. © 2010 Elsevier B.V. All rights reserved.
1. Introduction With the rapid development of analytical techniques, it has been reported that many aquatic environments are polluted with pharmaceutically active compounds (PhACs) at low concentrations [1–4]. Different sources can be highlighted to explain the appearance of PhACs in the aquatic environment. It is currently widely accepted that the main sources of such pollution are sewage treatment plant (STP) effluents [5]. The occurrence of several PhACs has been reported in STP effluents as well as in surface and drinking water in Brazil [6,7], Canada [8,9], China [10,11], Germany [12,13], Italy [5,14], Spain [15,16], Switzerland [17,18] and the United States [19,20]. However, there is no regulation which sets limits for individual pharmaceutical compounds in drinking water.
⁎ Corresponding author. Tel.: + 55 48 37219448; fax: + 55 48 37219687. E-mail addresses: [email protected] (J.L. Tambosi), [email protected] (R.F. de Sena), [email protected] (M. Favier), [email protected] (W. Gebhardt), [email protected] (H.J. José), [email protected] (H.F. Schröder), [email protected] (R.F.P.M. Moreira). 0011-9164/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2010.05.014
PhACs such as non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics belong to classes of pharmaceuticals that are extensively used worldwide. In Spain, 55% of the Top 200 drugs consumed are ingested orally, and approximately 5% of them are NSAIDs [21]. In Germany the total consumption of antibiotics by men has been calculated as approx. 400 t per year. From the literature we know that about two thirds of these are excreted into the sewer system as the main route for emission [22]. Recent estimates indicate that in Europe, which holds about 26% of the international pharmaceutical market, more than 2000 different pharmaceutical products are used, and the annual consumption of antibiotic-type substances is similar in quantity to the application of some pesticides [23]. The frequent occurrence of PhACs in the aquatic environment as well as in treated drinking water has raised concern regarding their potential impact on environmental and public health. Some of the adverse effects caused by PhACs pollution include aquatic toxicity, increased resistance of pathogenic bacteria, genotoxicity, and endocrine disruption [24–26]. The presence of trace pharmaceutical and other xenobiotic compounds in treated drinking water is another public health concern, since little is known about potential chronic health effects associated with long-term ingestion of mixtures of these compounds contained in drinking water [27]. Thus, effective
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Table 1 Name, CAS no., sum and structural formula, pKa and log Kow and transformation products of pharmaceuticals under study [31–35]. Name
CAS no.
Sum formula
Acid/base/neutral
pKa
Log Kow
Acetaminophen
103-90-2
C8H9O2N
Structural formula
N
9.39
0.46
Ketoprofen
22071-15-4
C16H14O3
A
4.45
3.12
Naproxen
22204-53-1
C14H14O3
A
4.15
3.18
Roxithromycin
80214-83-1
C41H76N2O15
B
8.8–9.2
2.75
Sulfamethoxazole
723-46-6
C10H11N3O3S
A
1.8–5.7
0.89
Trimethoprim
738-70-5
C14H18N4O3
B
6.6–7.2
0.91
Acetaminophen-O-sulfate
–
C8H9O5NS
A
Acetaminophen–glutathione-conjugate
–
C18H24O8N4S
N
Acetaminophen transformation product
–
C12H10O2N
N
–
–
Ketoprofen transformation product [33]
–
C11H12O5
A
–
–
Naproxen transformation product [33]
–
C13H12O3
A
–
–
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Fig. 1. Scheme of the MBR pilot plant for the treatment of spiked pharmaceuticals.
removal of pharmaceutical compounds, along with other priority pollutants, from sources such as hospital and domestic wastewaters, prior to their discharge is an emerging issue in environmental science and engineering. The elimination of pharmaceuticals is affected by many factors, such as the dilution factor and temperature of the raw sewage, the
Fig. 2. Wastewater flow (a) and pressure drop (b) measured during the spiking period in MBR treatment with SRT of 15 (MBR-15) or 30 (MBR-30) days.
hydraulic and solids retention times and, predominantly, by the plant configuration. Many pharmaceuticals are very hydrophilic and, consequently, their adsorption onto sludge is limited, inhibiting the degradation of these compounds by bacteria during the treatment process. In addition, some of these compounds are difficult to degradable because of their structure which protects them against attack from the wastewater biocoenosis. These effects lead to a significant persistency [28]. Since conventional water and wastewater treatment processes are unable to act as a reliable barrier towards some recalcitrant pharmaceuticals, it is necessary to introduce and apply additional advanced treatment technologies to achieve sustainable protection of the environment. Membrane bioreactor (MBR) technology combines the biological degradation process using activated sludge with a direct solid–liquid separation by membrane filtration. The use of micro or ultrafiltration membrane technology (with pore size ranges from 0.05 to 0.4 µm) in MBR systems allows the complete physical retention of bacterial flocs and virtually all suspended solids within the bioreactor [29]. MBR treatment has gained significant popularity recently and could be applied to address the above-mentioned issues. Although many articles have reported the application of MBRs to the treatment of urban and industrial wastewaters, there are few papers which describe the behavior of emerging contaminants during MBR treatment. Kimura et al. [1] compared the removal of 7 different pharmaceuticals on treating municipal wastewater using an MBR in parallel with conventional activated sludge. Gebhardt and Schröder [3] studied the degradation of 4 different pharmaceuticals during municipal wastewater treatment applying biological treatment (conventional and membrane bioreactor) alone and in combination with advanced oxidation. Göbel et al. [4] studied the fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies including a conventional activated sludge treatment plant and an MBR pilot plant. Schröder [30] monitored the efficiency of MBR technology in the degradation and elimination of biodegradable polar compounds using mass spectrometry. In this study, the fate of six pharmaceuticals of high consumption worldwide, three NSAIDs (acetaminophen, ketoprofen and naproxen)
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and three antibiotics (roxithromycin, sulfamethoxazole and trimethoprim) was monitored during wastewater treatment in an MBR pilot plant. The qualitative and quantitative monitoring of the precursor compounds and the generation of biological transformation products during the MBR treatment was performed using liquid chromatography coupled to high resolution mass spectrometry (LC-(HR)MS). 2. Experimental 2.1. Chemicals The ultra-pure water used during the treatment or as the LC eluent component was obtained from a Milli-Q system (Millipore, Milford, MA, USA). All solvents used as mobile phases, for desorption of the pharmaceuticals and their potential degradation products extracted by solid phase extraction (SPE), were nanograde solvents purchased from LGC Promochem (Wesel, Germany). All other chemicals used were of “analytical reagent” or “residue analysis” purity grade. Gases applied were products of Linde, Germany, and were of 99.999% purity. 2.2. Pharmaceuticals The pharmaceutical compounds used in this study, three NSAIDs (acetaminophen, ketoprofen and naproxen) and three antibiotics (roxithromycin, sulfamethoxazole and trimethoprim), were purchased from Sigma-Aldrich. Relevant information on these pharmaceuticals is given in Table 1. Concentrated stock solutions were used for the analytical determination and for spiking purposes. To avoid degradation during the test period these methanolic solutions were kept at −18 °C. 2.3. Membrane bioreactor pilot plants Fig. 1 shows the scheme of the MBR pilot plants operated in this study. Wastewater used as feed for the MBR pilot treatment plant was taken continuously from the effluent of the pre-settling tank of the municipal STP of Aachen, Germany. MBR treatment was performed over a period of 4 weeks. In this period, the flow rate and pressure
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drop were nearly constant and no procedure for cleaning the membranes was applied (Fig. 2). The sludge retention time (SRT), sludge concentration (SC) and hydraulic retention time (HRT) of the pilot-plants were 15 days, 12 g/ L and 9 h for MBR-15, and 30 days, 12 g/L and 13 h for MBR-30, respectively. Both MBRs used in this study were equipped with 1.43 m2 of hollow-fiber ultrafiltration (UF) membranes (PURON, KMS Germany). The nominal pore size and material of the membrane were 0.04 µm and polyethersulfone (PES), respectively. The volumes of MBR-15 and MBR-30 were 260 L and 240 L, respectively. Aeration was continuously carried out in both MBRs. The plants were equipped with sensors for online measurement of temperature (mean 15.1 °C), pressure, filling level and wastewater flow. The pH (mean 7.2) and dissolved oxygen (2.8–3.2 mg/L) were determined manually. A steady-state was reached over 6 months before the tests started. The pharmaceutical compounds were spiked in each MBR to reach a concentration of 50 µg/Ld. During the week the spiking was performed with the addition of 2/3 (33.33 µg/L) of the total daily spiking quantity in the morning (10:00 a.m.) and 1/3 (16.67 µg/L) in the afternoon (4:00 p.m.). Spiking during the weekend was performed with the addition of the total amount of 50 µg/L at 4:00 in the afternoon. Every day, at 3:00 p.m., 500-mL samples of the permeates of each MBR were taken. In parallel, 16 L (MBR-15) or 8 L (MBR-30) of excess sludge was discharged in order to keep the sludge concentration (SC) constant in each MBR. 2.4. Sample preparation The pharmaceuticals present in the MBR permeate were concentrated using commercially available solid phase extraction (SPE) cartridges (1 mL) filled with 100 mg of Isolute ENV+ material from IST (Mid Glamorgan, Wales, UK). Prior to use they were handled as prescribed by the manufacturer. After the SPE procedure, the cartridges were rinsed with ultra-pure water to remove salts before they were dried in a gentle stream of nitrogen at 30 °C. The pharmaceuticals adsorbed were desorbed by addition of 6 × 1 mL of methanol. STP eluates were brought to dryness in a gentle stream of nitrogen at 60 °C. The dry residues containing the pharmaceuticals were reconstituted in 1 mL of methanol/water (1:1) and were used for injection during LC-MS analysis. 2.5. Analytical procedures Sodium chloride (NaCl) was applied as a tracer compound to ensure an ideal mixing in the MBRs and was monitored using a conductivity probe. Identification and quantification of spiked pharmaceuticals in MBR permeates were performed by means of a LTQ Orbitrap mass spectrometer (Thermo Electron) applying electrospray ionization (ESI), both in the positive mode (for roxithromycin, sulfamethoxazole, trimethroprim, acetaminophen and their metabolites) and in the negative mode (for ketoprofen, naproxen and their metabolites) as described by Gebhardt and Schröder [3]. To calculate the different efficiencies of the pharmaceutical removal from the spiked wastewater, the concentrations of the precursor drugs in the MBR and permeates were determined quantitatively using LC-MS after SPE. The PhAC concentrations in the samples were determined by peak area comparison of real samples to standard solutions. Transformation products were detected by extraction of selected mass traces from TICs recorded using high resolution MS (HRMS) and identified by LC-MSn. Their quantification was impossible because calibration standards were missing. 2.6. Liquid chromatographic (LC) and mass spectrometric (MS) data
Fig. 3. Mathematical model for NaCl dilution in MBRs with different SRTs: (a) MBR-15 with SRT of 15 days and (b) MBR-30 with SRT of 30 days.
LC separations were carried out with a Hypersil GOLD aQ column (RP5, 5 µm, spherical; 150 × 2.1 mm I.D.) equipped with a Hypersil
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GOLD aQ pre-column (10 × 2.1 mm I.D.), also filled with 5 µm spherical material (Thermo Electron, USA). Gradient elution was applied by means of (A) methanol/water 90:10 (v:v) in combination with (B) Milli-Q-purified water/methanol 90:10 (v:v), both containing 2 mM ammonium acetate. The gradient was programmed as follows: starting with 20% A/80% B the concentration was increased linearly to 90% A/10% B within 12 min. The composition was kept constant for 20 min. The overall flow rate was 0.2 mL/min. Instrument control, data acquisition and data processing were performed using Xcalibur 2.0 software (Thermo Electron).
biodegradation by microorganisms present in the wastewater. With the aim of estimating the contribution of the dilution factor in pharmaceutical compound removal, the tracer compound sodium chloride (NaCl) was used because it is well known that NaCl is neither adsorbed nor absorbed by sludge, it is not biodegraded by the microorganisms and it is not retained by ultrafiltration membranes. Therefore, a mathematical model based on a perfectly mixed reactor [36] was developed to simulate the concentration of NaCl in both MBR permeates, according to Eq. (1). C = C0 : exp
−Q :t V
ð1Þ
3. Results and discussion 3.1. Mathematical model for NaCl dilution in MBRs The main mechanisms responsible for the removal of pharmaceutical compounds in the MBR-system are sludge sorption and
where C is the NaCl concentration [g L−1]; C0 is the initial NaCl concentration [g L−1]; V is the tank volume [L]; Q is the wastewater flow [L min−1], and t is the time [min]. After the validation of the mathematical model (cf. Fig. 3), the total removal efficiency (sludge sorption + biodegradation + membrane
Fig. 4. (a) LC-ESI(+) total ion current (TIC) trace for a MBR permeate sample. Extracted ion mass trace of (b) acetaminophen (tR 3.92 min); (c) acetaminophen transformation product (tR 9.64 min); (d) acetaminophen transformation product (acetaminophen-O-sulphate) (tR 2.88 min); (e) roxithromycin (tR 14.25 min), (f) sulfamethoxazole (tR 6.43 min) and (g) trimethoprim (tR 6.72 min). (h) LC-ESI(−) TIC for MBR permeate sample as in (a), however, negatively ionized. Extracted ion mass traces of (i) ketoprofen (tR 11.80 min) and (j) naproxen (tR 12.38 min), (k) ketoprofen transformation product (tR 2.66 min) and (l) naproxen transformation product (tR 10.00 min), (m) positively ionized ESI MS2 product ion spectra of acetaminophen transformation products at m/z 200 and (n) m/z 232 (acetaminophen-O-sulphate).
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retention) of each pharmaceutical compound was determined for both MBR-15 and MBR-30 according to Eq. (2): Removalð%Þ =
C2 −C1 × 100 C2
where: C1
ð2Þ
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the experimental concentration determined for each pharmaceutical compound in each MBR permeate by (HP)LC(HR)MS analysis.
Fig. 5. Daily removal rates of pharmaceutical compounds and transformation products, generated during a 4-week examination period applying MBR treatment. Removal rates in [%]: SRT of 15 (MBR-15) □ or 30 (MBR-30) ■ days and concentrations of transformation products observed for SRT of 15 (MBR-15) ○ or 30 (MBR-30) ● days. Concentrations of transformation products expressed in peak area counts.
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C2
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the theoretical concentration of each pharmaceutical compound extrapolated by the mathematical model in each MBR permeate considering only the dilution factor.
To evaluate the removal efficiency of each spiked pharmaceutical compound for MBR-15 and MBR-30, the concentration of each compound (C1) was measured after treatment and related to the concentration predicted by Eq. (1) (C2), which considers only the effect of dilution.
biocoenosis immobilized on glass-foam beads [39]. The transformation products from the MBR treatment as well as the closed-loop confirmation experiments were determined without any extraction or concentration from the wastewater. Their recognition was facilitated by blank samples generated in parallel for comparison. Samples were directly injected and analyzed by LC-(HR)MS and identified from their product ion spectra obtained by LC-(HR)MSn under collision induced dissociation (CID) conditions as shown in Fig. 4m and n.
3.3. Removal of pharmaceutical compounds 3.2. Determination and identification of transformation products The pharmaceutical compounds acetaminophen, ketoprofen, naproxen, roxithromycin, sulfamethoxazole and trimethoprim were selected as target compounds for the experiments to monitor their elimination from wastewater during MBR treatment while in parallel we tried to determine the already known [33] as well as not yet known biological transformation products. The concentrations of the various pharmaceutical compounds and their transformation products during the spiking period were determined by LC-MS applying electrospray ionization (ESI) under high resolution MS conditions. With the results for a selected sample shown in Fig. 4 the compound identification and quantification procedures were performed. It can be clearly observed that in the positively generated total ion current (TIC) trace of the selected sample (cf. Fig. 4a) there are a larger number of peaks than in the negatively generated TIC (cf. Fig. 4h). For roxithromycin in the TIC an intensive signal, easily recognizable compared to other compounds, is shown in Fig. 4a at tR = 14.25 min and also in the extracted mass trace in Fig. 4e. The drug compounds acetaminophen, sulfamethoxazole and trimethoprim, however, which are not easily recognizable in the positive TIC, can be identified in their extracted ion mass traces (cf. Fig. 4b,f,g). Two acetaminophen transformation products can also be detected, but recognition was only possible in their extracted ion mass traces with prior knowledge of their exact masses (cf. Fig. 4c,d). Biochemical degradation is the most important elimination mechanism for the target compounds in wastewater, however, this process often results in transformation products which are more stable than their precursor drugs. In our experiments, some of the transformation products observed by Quintana et al. [33] for ketoprofen and naproxen were also identified, as shown in Fig. 4k and l. Thus, with the extraction of the mass traces of potential transformation products from the TICs recorded in HRMS mode the presence of the ketoprofen transformation product 3-(hydroxycarboxymethyl)hydratropic acid as an intermediate compound and the naproxen transformation product O-desmetyl-naproxen could easily be confirmed. The generation of the keto hydratropic acid by oxidation of the transformation product hydroxy hydratropic acid, however, was not observed because of the low SRT applied in our experiments. In the human body acetaminophen will be detoxified and excreted as acetaminophen-O-sulphate, -glucoronide and -glutathione conjugates [37,38]. Two of these metabolites have also been observed as transformation products in wastewater treatment process (cf. Table 1). Acetaminophen-O-sulphate at tR = 2.88 min (cf. Fig. 4d) and acetaminophen–glutathione at tR = 4.06 min, as well as a new second-order transformation product of acetaminophen–glutathione at tR = 9.64 min (cf. Fig. 4c), were observed in the positively ionized TICs of the MBR treated wastewater extract. The latter could also be recognized in its selected mass trace of m/z 200 at tR = 9.64 min. A glucoronic acid transformation product, however, generated as the predominant metabolite during metabolic degradation and excretion in the human body, was not found. For confirmation of these results the same compounds were generated in parallel by biodegradation in a closedloop system operated under aerobic conditions using MBR wastewater
Fig. 5 shows the removal rates of pharmaceutical compounds spiked into two MBRs with different SRTs over a period of 4 weeks. Each compound showed a distinct behavior during the spiking period which will be discussed below. As can be seen in Figs. 5 and 6, complete removal of acetaminophen in both MBRs took place during the spiking period. Acetaminophen showed the highest removal efficiency (around 100%) since its structure allows the unrestricted access of bacteria and enzymes to the sterically unprotected molecule, which is consequently modified. This disappearance of acetaminophen, however, could be attributed to biological conversion, as also observed in human metabolism, resulting in quite stable transformation products, that is, conjugates of acetaminophen, first reported herein. In addition, we verified a new second-order transformation product, resulting from the degradation of the acetaminophen–glutathione-conjugate, as shown in Table 1. These results are in agreement with those reported by Kim et al. [40] who obtained 99% acetaminophen removal during the treatment of municipal sewage in an MBR pilot plant, but who did not recognize that the ‘removal’ simply reflected a change in the structure. Concerning the ketoprofen behavior, an almost complete removal of the precursor drug was also observed for this compound in both MBRs (cf. Figs. 5 and 6). The compound ketoprofen has a hydrophobic nature (log Kow N 3) and acid character. According to Quintana et al. [33], for polar compounds like acidic pharmaceuticals, microbial degradation is the most important removal process in activated sludge wastewater treatment, while the retention by hydrophobic adsorption onto the membrane will not reduce the concentration. Quintana et al. [33] reported that during microbial degradation of ketoprofen, two new transformation products of high relative intensity could be detected and identified by means of LC-MS. A large variability in ketoprofen removal efficiencies in STPs has been reported in the literature: 98% by Thomas and Foster [41], 0–80% by Nakada et al. [42], 48–69% by Stumpf et al. [6], 8–53% by TauxeWuersch et al. [17] and Lindqvist et al. [43] observed a removal of
Fig. 6. Mean removal rates of pharmaceutical compounds during a 4-week monitoring period applying MBR treatment with SRT of 15 (MBR-15) or 30 (MBR-30) days.
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51–100%. Kimura et al. [1] compared the elimination of ketoprofen in parallel in municipal wastewater treatment using either an MBR pilot plant or a conventional activated sludge treatment (CAST) plant. Wastewater treatment in the CAST plant led to a concentration of 300 ng/L of ketoprofen in the effluent while in the permeate after MBR treatment only around 10 ng/L was present. The treatment of naproxen, with physicochemical properties comparable to those of ketoprofen, however, led to lower elimination rates of 86 and 89% in MBR-15 and MBR-30, respectively (cf. Figs. 5 and 6). This behavior of naproxen can be partially explained by its more stable chemical structure (naphthalene ring system). Naproxen removal in STPs has been studied by several authors and the following efficiencies are reported in the literature: 66% by Ternes [12], 0–80% by Nakada et al. [42], 40–100% by Metcalfe et al. [44], 100% by Thomas and Foster [41], 15–78% by Stumpf et al. [6], 93% by Andreozzi et al. [5], 40–55% by Carballa et al. [45] and 55–98% by Lindqvist et al. [43]. According to a study by Quintana et al. [33] on the microbial degradation of pharmaceuticals, the result for naproxen was low with approximately 60% of transformation in 28 days and only one transformation product could be detected. Kimura et al. [1] compared the removal of naproxen in municipal sewage effluents using either an MBR pilot plant or CAST and found that CAST led to a concentration of 50 ng/L of naproxen, while a concentration of approximately 20 ng/ L was reached after MBR treatment. Kim et al. [40] reported a 41% removal of naproxen from domestic sewage after treatment in a pilot scale MBR. For both compounds, ketoprofen and naproxen, we also observed the generation of metabolites rather than the complete mineralization as reported by Quintana et al. [33]. Roxithromycin has a moderately hydrophobic nature (log Kow =2.75) and basic character. This compound has the most complex chemical structure of the target compounds and acts as an antibacterial agent. These properties can partially explain the lower removal efficiencies of 57 and 81% compared to the NSAID compounds which were eliminated or metabolized with efficiencies of around 100% (acetaminophen and ketoprofen) and 89% (naproxen) (cf. Figs. 5 and 6). Similar results for roxithromycin removal in an MBR have been reported by Göbel et al. [4], where the elimination varied between 39% for an SRT of 16 days and 60% for higher SRTs (33 and 60 days). Sulfamethoxazole, also possessing antibacterial properties, was eliminated by only 55% and 64% in the MBR-15 and MBR-30 treatment processes (cf. Fig. 6), respectively. This compound has a hydrophilic nature (log Kow b 1) with two ionizable amine groups. As a result, in an aqueous solution, sulfamethoxazole can be present in positive, neutral, and negative forms. At pH values between the pKa values of the compound (pH 1.4 and 5.8), sulfamethoxazole is present predominantly as a neutral species, while above the second pKa value of the compound (pH 5.8) it becomes a negatively charged species. These physicochemical properties give an indication that in the MBR system studied (pH 7.2) the sludge adsorption mechanism played a negligible role, due to electrostatic repulsion between the negatively charged groups of the compound and the negatively charged surfaces of the sludge. Therefore, biodegradation can be considered as the main mechanism responsible for the removal, however, this processes will be hindered by the antibacterial properties of this compound. Göbel et al. [4], who studied the elimination of sulfamethoxazole, reported an elimination efficiency of around 80%, independently of the SRT. Considering the antibiotic compounds studied, the highest removal efficiencies were observed for trimethoprim (86 or 94%; cf. Figs. 5 and 6). This can be partially explained by its basic character and reduced antibacterial potency compared to sulfamethoxazole and roxithromycin. As observed for the other drugs, the removal efficiency for MBR-30 was higher than for MBR-15. Göbel et al. [4] studied the elimination of trimethoprim in an MBR and reported comparable elimination rates for SRTs of 16 and 33 days (30%), while 87% of removal was obtained for SRTs in the range of 60–80 days.
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3.4. Mechanisms influencing the removal of pharmaceutical compounds The elimination of pharmaceutical compounds can occur through various mechanisms in MBRs. Sorption onto sludge is one of the mechanisms that takes into account the absorption and adsorption factors. According to Carballa et al. [15], absorption refers to the hydrophobic interactions of the aliphatic and aromatic groups of a compound with fats present in the sludge or with the lipophilic cell membrane of the microorganisms (depending on their Kow value), while adsorption refers to the electrostatic interactions of positively charged groups of dissolved chemicals with the negatively charged surfaces of the microorganisms (characterized by the dissociation constant pKa). Göbel et al. [4] studied the elimination of pharmaceuticals by MBRs and CAST and concluded that the contribution of activated sludge adsorption in the case of pharmaceutical compounds was less than 6%, i.e., negligible, because this is within the analytical variance of the method. Another mechanism responsible for the removal of pharmaceutical compounds in MBRs is the physical retention by the membranes. However, it seems that this mechanism would not have led to retention of the pharmaceutical targets studied herein because the molecular weight cut off (MWCO) of ultrafiltration MBR membranes is around 100–200 kDa. Sorption onto the membranes is also limited because of the available membrane surface area. Pharmaceutical compounds which are non-polar will sorb onto the biomass and will therefore be removed indirectly during the retention of the solids by the membranes. Polar pharmaceuticals, with a low tendency to adsorb to the lipophilic sludge surface will neither be eliminated by adsorption nor by biodegradation because the interaction with the wastewater biocoenosis essential for the biodegradation process will be too short. Fig. 6 shows the mean removal rates for our target pharmaceuticals applying MBR treatment over 4 weeks. It can be observed that the NSAIDs were removed with higher efficiency than the antibiotics, which can be partially explained by their less complex chemical structure, as reported by Kimura et al. [1]. These authors compared the removal of 7 pharmaceutical compounds using MBRs and CAST and concluded that the former offer a much better removal for the compounds ketoprofen and naproxen. On the other hand, the most important property in terms of reducing the removal efficiency, i.e., the biodegradation, was the antibacterial potency of the antibiotic compounds. In addition, the observed “elimination”, which led to a reduction in the concentrations of the precursor compounds in parallel, could be due to a biodegradation process which resulted in transformation products which are more stable than the precursor drugs, as in the case of ketoprofen, naproxen and acetaminophen. 4. Conclusions The performance of two MBR pilot plants with submerged membranes, operated with different SRTs, for the removal of pharmaceutical compounds, was investigated in this study. The results obtained showed that the MBR-30 presented higher removal efficiencies than the MBR-15 for all compounds. The compounds acetaminophen and ketoprofen had the highest removal efficiencies, while roxithromycin and sulfamethoxazole exhibited persistence to microbial attack and were removed to a lesser extent in both MBRs. Concerning the potential mechanisms responsible for the removal of the target pharmaceuticals in the MBRs (sludge sorption + biodegradation + membrane retention), it was not possible to determine exactly the extent to which each mechanism contributed to the removal efficiency. However, in general terms, membrane retention using micro- or ultrafiltration membranes can be neglected, whereas biodegradation plays an important role, since higher removal efficiency was obtained for higher SRTs. Nevertheless, the elimination
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by MBR treatment using ultrafiltration was only partially successful and therefore, persistent pharmaceuticals in small concentrations and their transformation products were discharged with the wastewater into the environment. This discharge could be reduced with the application of additional treatment steps using advanced treatment techniques, e.g., activated carbon adsorption, ozone oxidation, advanced oxidation processes (AOP), nanofiltration (NF) or reverse osmosis (RO). In conclusion, the results indicated the importance of investigating the environmental fate of pharmaceuticals and their largely unknown polar transformation products. Additional research is thus urgently required firstly to recognize the parent drugs and their transformation products and secondly to assess the effect of these micropollutants on the environment. Acknowledgments The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Deutscher Akademischer Austausch Dienst (DAAD, German Academic Exchange Service) for the financial support. Analytical support from the staff of the Environmental Analytical Laboratory of the Institute of Environmental Engineering of RWTH Aachen University is also greatly appreciated. References [1] K. Kimura, H. Hara, Y. Watanabe, Removal of pharmaceutical compounds by submerged membrane bioreactors (MBRs), Desalination 178 (2005) 135–140. [2] A.M. Comerton, R.C. Andrews, D.M. Bagley, P. Yang, Membrane adsorption of endocrine disrupting compounds and pharmaceutically active compounds, J. Membr. Sci. 303 (2007) 267–277. [3] W. Gebhardt, H.Fr. Schröder, Liquid chromatography-(tandem) mass spectrometry for the follow-up of the elimination of persistent pharmaceuticals during wastewater treatment applying biological wastewater treatment and advanced oxidation, J. Chromatogr. A 1160 (2007) 34–43. [4] A. Göbel, C.S. McArdell, A. Joss, H. Siegrist, W. Giger, Fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies, Sci. Total Environ. 372 (2007) 361–371. [5] R. Andreozzi, R. Marotta, N. Paxéus, Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment, Chemosphere 50 (2003) 1319–1330. [6] M. Stumpf, T.A. Ternes, R.D. Wilken, S.V. Rodrigues, W. Baumann, Polar drug residues in sewage and natural waters in the state of Rio de Janeiro, Brazil, Sci. Total Environ. 225 (1999) 135–141. [7] M. Favier, R.F. Sena, H.J. José, U. Bieling, H. Fr. Schröder, Liquid chromatographytandem mass spectrometry for the screening of pharmaceuticals and metabolites in various water bodies in Florianópolis, Santa Catarina, Brazil. Proceedings Ecosan-Fortaleza, Conference; 26.–28.11.2007 in Fortaleza, Ceará, Brazil. [8] T.A. Ternes, M. Stumpf, J. Mueller, K. Haberer, R.D. Wilken, M. Servos, Behavior and occurrence of estrogens in municipal sewage treatment plants—I. Investigations in Germany, Canada and Brazil, Sci. Total Environ. 225 (1999) 81–90. [9] X.S. Miao, F. Bishay, M. Chen, C.D. Metcalfe, Occurrence of antimicrobials in the final effluents of wastewater treatment plants in Canada, Environ. Sci. Technol. 38 (2004) 3533–3541. [10] A. Gulkowska, H.W. Leung, M.K. So, S. Taniyasu, N. Yamashita, L.W.Y. Yeung, B.J. Richardson, A.P. Lei, J.P. Giesy, P.K.S. Lam, Removal of antibiotics from wastewater by sewage treatment facilities in Hong Kong and Shenzhen, China, Water Res. 42 (2008) 395–403. [11] W. Xu, G. Zhang, X. Li, S. Zou, P. Li, Z. Hu, J. Li, Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta (PRD), South China, Water Res. 41 (2007) 4526–4534. [12] T.A. Ternes, Occurrence of drugs in German sewage treatment plants and rivers, Water Res. 32 (1998) 3245–3260. [13] K. Kümmerer, Drugs in environment: emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources — a review, Chemosphere 45 (2001) 957–969. [14] S. Castiglioni, R. Fanelli, D. Calamari, R. Bagnati, E. Zuccato, Methodological approaches for studying pharmaceuticals in the environment by comparing predicted and measured concentrations in River Po, Italy, Regul. Toxicol. Pharmacol. 39 (2004) 25–32. [15] M. Carballa, F. Omil, J.M. Lema, Removal of cosmetic ingredients and pharmaceuticals in sewage primary treatment, Water Res. 39 (2005) 4790–4796. [16] J.L. Santos, I. Apacicio, E. Alonso, Occurrence and risk assessment of pharmaceutically active compounds in wastewater treatment plants. A case study: Seville city, Environ. Int. 33 (2007) 596–601 (Spain).
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Desalination j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d e s a l
Removal of pharmaceutical compounds in membrane bioreactors (MBR) applying submerged membranes José Luiz Tambosi a,b, Rênnio Felix de Sena a,b, Maxime Favier b, Wilhelm Gebhardt b, Humberto Jorge José a, Horst Friedrich Schröder b, Regina de Fátima Peralta Muniz Moreira a,⁎ a Laboratory of Energy and the Environment, Department of Chemical Engineering and Food Engineering, Federal University of Santa Catarina, Campus Universitário, Trindade, 88040-900, Florianópolis, SC, Brazil b Institute of Environmental Engineering, Environmental Analytical Laboratory, RWTH Aachen University, Krefelder Strasse 299, D-52056 Aachen, Germany
a r t i c l e
i n f o
Article history: Received 22 September 2009 Received in revised form 7 May 2010 Accepted 10 May 2010 Available online 17 June 2010 Keywords: Elimination Mass spectrometry (MS) Membrane bioreactor (MBR) Pharmaceuticals Wastewater
a b s t r a c t Pharmaceuticals such as non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics have been detected in sewage treatment plant (STP) effluents, surface and ground waters and even in drinking waters all over the world. These compounds of concern may induce toxic effects in aquatic organisms and disturb the ecological balance on their way from wastewater to drinking water treatment via surface water and bank filtration. Membrane bioreactors (MBRs) have gained significant popularity as an advanced wastewater treatment technology and might be effective in removing such pollutants. This paper evaluates the treatment of wastewater containing three NSAIDs (acetaminophen, ketoprofen and naproxen) and three antibiotics (roxithromycin, sulfamethoxazole and trimethoprim) in two MBRs with sludge retention times (SRTs) of 15 (MBR-15) and 30 (MBR-30) days over a period of 4 weeks. It was observed for both MBRs that the NSAIDs were removed with higher efficiency than the antibiotics, and the MBR-30 presented higher removal efficiencies than the MBR-15 for all compounds studied. Biological transformation products of acetaminophen, ketoprofen and naproxen produced by wastewater biocoenosis were identified in permeates from both MBRs. Further research in this field is required to assess the environmental risks associated with the presence of pharmaceuticals and their transformation products in the environment. © 2010 Elsevier B.V. All rights reserved.
1. Introduction With the rapid development of analytical techniques, it has been reported that many aquatic environments are polluted with pharmaceutically active compounds (PhACs) at low concentrations [1–4]. Different sources can be highlighted to explain the appearance of PhACs in the aquatic environment. It is currently widely accepted that the main sources of such pollution are sewage treatment plant (STP) effluents [5]. The occurrence of several PhACs has been reported in STP effluents as well as in surface and drinking water in Brazil [6,7], Canada [8,9], China [10,11], Germany [12,13], Italy [5,14], Spain [15,16], Switzerland [17,18] and the United States [19,20]. However, there is no regulation which sets limits for individual pharmaceutical compounds in drinking water.
⁎ Corresponding author. Tel.: + 55 48 37219448; fax: + 55 48 37219687. E-mail addresses: [email protected] (J.L. Tambosi), [email protected] (R.F. de Sena), [email protected] (M. Favier), [email protected] (W. Gebhardt), [email protected] (H.J. José), [email protected] (H.F. Schröder), [email protected] (R.F.P.M. Moreira). 0011-9164/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.desal.2010.05.014
PhACs such as non-steroidal anti-inflammatory drugs (NSAIDs) and antibiotics belong to classes of pharmaceuticals that are extensively used worldwide. In Spain, 55% of the Top 200 drugs consumed are ingested orally, and approximately 5% of them are NSAIDs [21]. In Germany the total consumption of antibiotics by men has been calculated as approx. 400 t per year. From the literature we know that about two thirds of these are excreted into the sewer system as the main route for emission [22]. Recent estimates indicate that in Europe, which holds about 26% of the international pharmaceutical market, more than 2000 different pharmaceutical products are used, and the annual consumption of antibiotic-type substances is similar in quantity to the application of some pesticides [23]. The frequent occurrence of PhACs in the aquatic environment as well as in treated drinking water has raised concern regarding their potential impact on environmental and public health. Some of the adverse effects caused by PhACs pollution include aquatic toxicity, increased resistance of pathogenic bacteria, genotoxicity, and endocrine disruption [24–26]. The presence of trace pharmaceutical and other xenobiotic compounds in treated drinking water is another public health concern, since little is known about potential chronic health effects associated with long-term ingestion of mixtures of these compounds contained in drinking water [27]. Thus, effective
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Table 1 Name, CAS no., sum and structural formula, pKa and log Kow and transformation products of pharmaceuticals under study [31–35]. Name
CAS no.
Sum formula
Acid/base/neutral
pKa
Log Kow
Acetaminophen
103-90-2
C8H9O2N
Structural formula
N
9.39
0.46
Ketoprofen
22071-15-4
C16H14O3
A
4.45
3.12
Naproxen
22204-53-1
C14H14O3
A
4.15
3.18
Roxithromycin
80214-83-1
C41H76N2O15
B
8.8–9.2
2.75
Sulfamethoxazole
723-46-6
C10H11N3O3S
A
1.8–5.7
0.89
Trimethoprim
738-70-5
C14H18N4O3
B
6.6–7.2
0.91
Acetaminophen-O-sulfate
–
C8H9O5NS
A
Acetaminophen–glutathione-conjugate
–
C18H24O8N4S
N
Acetaminophen transformation product
–
C12H10O2N
N
–
–
Ketoprofen transformation product [33]
–
C11H12O5
A
–
–
Naproxen transformation product [33]
–
C13H12O3
A
–
–
150
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Fig. 1. Scheme of the MBR pilot plant for the treatment of spiked pharmaceuticals.
removal of pharmaceutical compounds, along with other priority pollutants, from sources such as hospital and domestic wastewaters, prior to their discharge is an emerging issue in environmental science and engineering. The elimination of pharmaceuticals is affected by many factors, such as the dilution factor and temperature of the raw sewage, the
Fig. 2. Wastewater flow (a) and pressure drop (b) measured during the spiking period in MBR treatment with SRT of 15 (MBR-15) or 30 (MBR-30) days.
hydraulic and solids retention times and, predominantly, by the plant configuration. Many pharmaceuticals are very hydrophilic and, consequently, their adsorption onto sludge is limited, inhibiting the degradation of these compounds by bacteria during the treatment process. In addition, some of these compounds are difficult to degradable because of their structure which protects them against attack from the wastewater biocoenosis. These effects lead to a significant persistency [28]. Since conventional water and wastewater treatment processes are unable to act as a reliable barrier towards some recalcitrant pharmaceuticals, it is necessary to introduce and apply additional advanced treatment technologies to achieve sustainable protection of the environment. Membrane bioreactor (MBR) technology combines the biological degradation process using activated sludge with a direct solid–liquid separation by membrane filtration. The use of micro or ultrafiltration membrane technology (with pore size ranges from 0.05 to 0.4 µm) in MBR systems allows the complete physical retention of bacterial flocs and virtually all suspended solids within the bioreactor [29]. MBR treatment has gained significant popularity recently and could be applied to address the above-mentioned issues. Although many articles have reported the application of MBRs to the treatment of urban and industrial wastewaters, there are few papers which describe the behavior of emerging contaminants during MBR treatment. Kimura et al. [1] compared the removal of 7 different pharmaceuticals on treating municipal wastewater using an MBR in parallel with conventional activated sludge. Gebhardt and Schröder [3] studied the degradation of 4 different pharmaceuticals during municipal wastewater treatment applying biological treatment (conventional and membrane bioreactor) alone and in combination with advanced oxidation. Göbel et al. [4] studied the fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies including a conventional activated sludge treatment plant and an MBR pilot plant. Schröder [30] monitored the efficiency of MBR technology in the degradation and elimination of biodegradable polar compounds using mass spectrometry. In this study, the fate of six pharmaceuticals of high consumption worldwide, three NSAIDs (acetaminophen, ketoprofen and naproxen)
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and three antibiotics (roxithromycin, sulfamethoxazole and trimethoprim) was monitored during wastewater treatment in an MBR pilot plant. The qualitative and quantitative monitoring of the precursor compounds and the generation of biological transformation products during the MBR treatment was performed using liquid chromatography coupled to high resolution mass spectrometry (LC-(HR)MS). 2. Experimental 2.1. Chemicals The ultra-pure water used during the treatment or as the LC eluent component was obtained from a Milli-Q system (Millipore, Milford, MA, USA). All solvents used as mobile phases, for desorption of the pharmaceuticals and their potential degradation products extracted by solid phase extraction (SPE), were nanograde solvents purchased from LGC Promochem (Wesel, Germany). All other chemicals used were of “analytical reagent” or “residue analysis” purity grade. Gases applied were products of Linde, Germany, and were of 99.999% purity. 2.2. Pharmaceuticals The pharmaceutical compounds used in this study, three NSAIDs (acetaminophen, ketoprofen and naproxen) and three antibiotics (roxithromycin, sulfamethoxazole and trimethoprim), were purchased from Sigma-Aldrich. Relevant information on these pharmaceuticals is given in Table 1. Concentrated stock solutions were used for the analytical determination and for spiking purposes. To avoid degradation during the test period these methanolic solutions were kept at −18 °C. 2.3. Membrane bioreactor pilot plants Fig. 1 shows the scheme of the MBR pilot plants operated in this study. Wastewater used as feed for the MBR pilot treatment plant was taken continuously from the effluent of the pre-settling tank of the municipal STP of Aachen, Germany. MBR treatment was performed over a period of 4 weeks. In this period, the flow rate and pressure
151
drop were nearly constant and no procedure for cleaning the membranes was applied (Fig. 2). The sludge retention time (SRT), sludge concentration (SC) and hydraulic retention time (HRT) of the pilot-plants were 15 days, 12 g/ L and 9 h for MBR-15, and 30 days, 12 g/L and 13 h for MBR-30, respectively. Both MBRs used in this study were equipped with 1.43 m2 of hollow-fiber ultrafiltration (UF) membranes (PURON, KMS Germany). The nominal pore size and material of the membrane were 0.04 µm and polyethersulfone (PES), respectively. The volumes of MBR-15 and MBR-30 were 260 L and 240 L, respectively. Aeration was continuously carried out in both MBRs. The plants were equipped with sensors for online measurement of temperature (mean 15.1 °C), pressure, filling level and wastewater flow. The pH (mean 7.2) and dissolved oxygen (2.8–3.2 mg/L) were determined manually. A steady-state was reached over 6 months before the tests started. The pharmaceutical compounds were spiked in each MBR to reach a concentration of 50 µg/Ld. During the week the spiking was performed with the addition of 2/3 (33.33 µg/L) of the total daily spiking quantity in the morning (10:00 a.m.) and 1/3 (16.67 µg/L) in the afternoon (4:00 p.m.). Spiking during the weekend was performed with the addition of the total amount of 50 µg/L at 4:00 in the afternoon. Every day, at 3:00 p.m., 500-mL samples of the permeates of each MBR were taken. In parallel, 16 L (MBR-15) or 8 L (MBR-30) of excess sludge was discharged in order to keep the sludge concentration (SC) constant in each MBR. 2.4. Sample preparation The pharmaceuticals present in the MBR permeate were concentrated using commercially available solid phase extraction (SPE) cartridges (1 mL) filled with 100 mg of Isolute ENV+ material from IST (Mid Glamorgan, Wales, UK). Prior to use they were handled as prescribed by the manufacturer. After the SPE procedure, the cartridges were rinsed with ultra-pure water to remove salts before they were dried in a gentle stream of nitrogen at 30 °C. The pharmaceuticals adsorbed were desorbed by addition of 6 × 1 mL of methanol. STP eluates were brought to dryness in a gentle stream of nitrogen at 60 °C. The dry residues containing the pharmaceuticals were reconstituted in 1 mL of methanol/water (1:1) and were used for injection during LC-MS analysis. 2.5. Analytical procedures Sodium chloride (NaCl) was applied as a tracer compound to ensure an ideal mixing in the MBRs and was monitored using a conductivity probe. Identification and quantification of spiked pharmaceuticals in MBR permeates were performed by means of a LTQ Orbitrap mass spectrometer (Thermo Electron) applying electrospray ionization (ESI), both in the positive mode (for roxithromycin, sulfamethoxazole, trimethroprim, acetaminophen and their metabolites) and in the negative mode (for ketoprofen, naproxen and their metabolites) as described by Gebhardt and Schröder [3]. To calculate the different efficiencies of the pharmaceutical removal from the spiked wastewater, the concentrations of the precursor drugs in the MBR and permeates were determined quantitatively using LC-MS after SPE. The PhAC concentrations in the samples were determined by peak area comparison of real samples to standard solutions. Transformation products were detected by extraction of selected mass traces from TICs recorded using high resolution MS (HRMS) and identified by LC-MSn. Their quantification was impossible because calibration standards were missing. 2.6. Liquid chromatographic (LC) and mass spectrometric (MS) data
Fig. 3. Mathematical model for NaCl dilution in MBRs with different SRTs: (a) MBR-15 with SRT of 15 days and (b) MBR-30 with SRT of 30 days.
LC separations were carried out with a Hypersil GOLD aQ column (RP5, 5 µm, spherical; 150 × 2.1 mm I.D.) equipped with a Hypersil
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GOLD aQ pre-column (10 × 2.1 mm I.D.), also filled with 5 µm spherical material (Thermo Electron, USA). Gradient elution was applied by means of (A) methanol/water 90:10 (v:v) in combination with (B) Milli-Q-purified water/methanol 90:10 (v:v), both containing 2 mM ammonium acetate. The gradient was programmed as follows: starting with 20% A/80% B the concentration was increased linearly to 90% A/10% B within 12 min. The composition was kept constant for 20 min. The overall flow rate was 0.2 mL/min. Instrument control, data acquisition and data processing were performed using Xcalibur 2.0 software (Thermo Electron).
biodegradation by microorganisms present in the wastewater. With the aim of estimating the contribution of the dilution factor in pharmaceutical compound removal, the tracer compound sodium chloride (NaCl) was used because it is well known that NaCl is neither adsorbed nor absorbed by sludge, it is not biodegraded by the microorganisms and it is not retained by ultrafiltration membranes. Therefore, a mathematical model based on a perfectly mixed reactor [36] was developed to simulate the concentration of NaCl in both MBR permeates, according to Eq. (1). C = C0 : exp
−Q :t V
ð1Þ
3. Results and discussion 3.1. Mathematical model for NaCl dilution in MBRs The main mechanisms responsible for the removal of pharmaceutical compounds in the MBR-system are sludge sorption and
where C is the NaCl concentration [g L−1]; C0 is the initial NaCl concentration [g L−1]; V is the tank volume [L]; Q is the wastewater flow [L min−1], and t is the time [min]. After the validation of the mathematical model (cf. Fig. 3), the total removal efficiency (sludge sorption + biodegradation + membrane
Fig. 4. (a) LC-ESI(+) total ion current (TIC) trace for a MBR permeate sample. Extracted ion mass trace of (b) acetaminophen (tR 3.92 min); (c) acetaminophen transformation product (tR 9.64 min); (d) acetaminophen transformation product (acetaminophen-O-sulphate) (tR 2.88 min); (e) roxithromycin (tR 14.25 min), (f) sulfamethoxazole (tR 6.43 min) and (g) trimethoprim (tR 6.72 min). (h) LC-ESI(−) TIC for MBR permeate sample as in (a), however, negatively ionized. Extracted ion mass traces of (i) ketoprofen (tR 11.80 min) and (j) naproxen (tR 12.38 min), (k) ketoprofen transformation product (tR 2.66 min) and (l) naproxen transformation product (tR 10.00 min), (m) positively ionized ESI MS2 product ion spectra of acetaminophen transformation products at m/z 200 and (n) m/z 232 (acetaminophen-O-sulphate).
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retention) of each pharmaceutical compound was determined for both MBR-15 and MBR-30 according to Eq. (2): Removalð%Þ =
C2 −C1 × 100 C2
where: C1
ð2Þ
153
the experimental concentration determined for each pharmaceutical compound in each MBR permeate by (HP)LC(HR)MS analysis.
Fig. 5. Daily removal rates of pharmaceutical compounds and transformation products, generated during a 4-week examination period applying MBR treatment. Removal rates in [%]: SRT of 15 (MBR-15) □ or 30 (MBR-30) ■ days and concentrations of transformation products observed for SRT of 15 (MBR-15) ○ or 30 (MBR-30) ● days. Concentrations of transformation products expressed in peak area counts.
154
C2
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the theoretical concentration of each pharmaceutical compound extrapolated by the mathematical model in each MBR permeate considering only the dilution factor.
To evaluate the removal efficiency of each spiked pharmaceutical compound for MBR-15 and MBR-30, the concentration of each compound (C1) was measured after treatment and related to the concentration predicted by Eq. (1) (C2), which considers only the effect of dilution.
biocoenosis immobilized on glass-foam beads [39]. The transformation products from the MBR treatment as well as the closed-loop confirmation experiments were determined without any extraction or concentration from the wastewater. Their recognition was facilitated by blank samples generated in parallel for comparison. Samples were directly injected and analyzed by LC-(HR)MS and identified from their product ion spectra obtained by LC-(HR)MSn under collision induced dissociation (CID) conditions as shown in Fig. 4m and n.
3.3. Removal of pharmaceutical compounds 3.2. Determination and identification of transformation products The pharmaceutical compounds acetaminophen, ketoprofen, naproxen, roxithromycin, sulfamethoxazole and trimethoprim were selected as target compounds for the experiments to monitor their elimination from wastewater during MBR treatment while in parallel we tried to determine the already known [33] as well as not yet known biological transformation products. The concentrations of the various pharmaceutical compounds and their transformation products during the spiking period were determined by LC-MS applying electrospray ionization (ESI) under high resolution MS conditions. With the results for a selected sample shown in Fig. 4 the compound identification and quantification procedures were performed. It can be clearly observed that in the positively generated total ion current (TIC) trace of the selected sample (cf. Fig. 4a) there are a larger number of peaks than in the negatively generated TIC (cf. Fig. 4h). For roxithromycin in the TIC an intensive signal, easily recognizable compared to other compounds, is shown in Fig. 4a at tR = 14.25 min and also in the extracted mass trace in Fig. 4e. The drug compounds acetaminophen, sulfamethoxazole and trimethoprim, however, which are not easily recognizable in the positive TIC, can be identified in their extracted ion mass traces (cf. Fig. 4b,f,g). Two acetaminophen transformation products can also be detected, but recognition was only possible in their extracted ion mass traces with prior knowledge of their exact masses (cf. Fig. 4c,d). Biochemical degradation is the most important elimination mechanism for the target compounds in wastewater, however, this process often results in transformation products which are more stable than their precursor drugs. In our experiments, some of the transformation products observed by Quintana et al. [33] for ketoprofen and naproxen were also identified, as shown in Fig. 4k and l. Thus, with the extraction of the mass traces of potential transformation products from the TICs recorded in HRMS mode the presence of the ketoprofen transformation product 3-(hydroxycarboxymethyl)hydratropic acid as an intermediate compound and the naproxen transformation product O-desmetyl-naproxen could easily be confirmed. The generation of the keto hydratropic acid by oxidation of the transformation product hydroxy hydratropic acid, however, was not observed because of the low SRT applied in our experiments. In the human body acetaminophen will be detoxified and excreted as acetaminophen-O-sulphate, -glucoronide and -glutathione conjugates [37,38]. Two of these metabolites have also been observed as transformation products in wastewater treatment process (cf. Table 1). Acetaminophen-O-sulphate at tR = 2.88 min (cf. Fig. 4d) and acetaminophen–glutathione at tR = 4.06 min, as well as a new second-order transformation product of acetaminophen–glutathione at tR = 9.64 min (cf. Fig. 4c), were observed in the positively ionized TICs of the MBR treated wastewater extract. The latter could also be recognized in its selected mass trace of m/z 200 at tR = 9.64 min. A glucoronic acid transformation product, however, generated as the predominant metabolite during metabolic degradation and excretion in the human body, was not found. For confirmation of these results the same compounds were generated in parallel by biodegradation in a closedloop system operated under aerobic conditions using MBR wastewater
Fig. 5 shows the removal rates of pharmaceutical compounds spiked into two MBRs with different SRTs over a period of 4 weeks. Each compound showed a distinct behavior during the spiking period which will be discussed below. As can be seen in Figs. 5 and 6, complete removal of acetaminophen in both MBRs took place during the spiking period. Acetaminophen showed the highest removal efficiency (around 100%) since its structure allows the unrestricted access of bacteria and enzymes to the sterically unprotected molecule, which is consequently modified. This disappearance of acetaminophen, however, could be attributed to biological conversion, as also observed in human metabolism, resulting in quite stable transformation products, that is, conjugates of acetaminophen, first reported herein. In addition, we verified a new second-order transformation product, resulting from the degradation of the acetaminophen–glutathione-conjugate, as shown in Table 1. These results are in agreement with those reported by Kim et al. [40] who obtained 99% acetaminophen removal during the treatment of municipal sewage in an MBR pilot plant, but who did not recognize that the ‘removal’ simply reflected a change in the structure. Concerning the ketoprofen behavior, an almost complete removal of the precursor drug was also observed for this compound in both MBRs (cf. Figs. 5 and 6). The compound ketoprofen has a hydrophobic nature (log Kow N 3) and acid character. According to Quintana et al. [33], for polar compounds like acidic pharmaceuticals, microbial degradation is the most important removal process in activated sludge wastewater treatment, while the retention by hydrophobic adsorption onto the membrane will not reduce the concentration. Quintana et al. [33] reported that during microbial degradation of ketoprofen, two new transformation products of high relative intensity could be detected and identified by means of LC-MS. A large variability in ketoprofen removal efficiencies in STPs has been reported in the literature: 98% by Thomas and Foster [41], 0–80% by Nakada et al. [42], 48–69% by Stumpf et al. [6], 8–53% by TauxeWuersch et al. [17] and Lindqvist et al. [43] observed a removal of
Fig. 6. Mean removal rates of pharmaceutical compounds during a 4-week monitoring period applying MBR treatment with SRT of 15 (MBR-15) or 30 (MBR-30) days.
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51–100%. Kimura et al. [1] compared the elimination of ketoprofen in parallel in municipal wastewater treatment using either an MBR pilot plant or a conventional activated sludge treatment (CAST) plant. Wastewater treatment in the CAST plant led to a concentration of 300 ng/L of ketoprofen in the effluent while in the permeate after MBR treatment only around 10 ng/L was present. The treatment of naproxen, with physicochemical properties comparable to those of ketoprofen, however, led to lower elimination rates of 86 and 89% in MBR-15 and MBR-30, respectively (cf. Figs. 5 and 6). This behavior of naproxen can be partially explained by its more stable chemical structure (naphthalene ring system). Naproxen removal in STPs has been studied by several authors and the following efficiencies are reported in the literature: 66% by Ternes [12], 0–80% by Nakada et al. [42], 40–100% by Metcalfe et al. [44], 100% by Thomas and Foster [41], 15–78% by Stumpf et al. [6], 93% by Andreozzi et al. [5], 40–55% by Carballa et al. [45] and 55–98% by Lindqvist et al. [43]. According to a study by Quintana et al. [33] on the microbial degradation of pharmaceuticals, the result for naproxen was low with approximately 60% of transformation in 28 days and only one transformation product could be detected. Kimura et al. [1] compared the removal of naproxen in municipal sewage effluents using either an MBR pilot plant or CAST and found that CAST led to a concentration of 50 ng/L of naproxen, while a concentration of approximately 20 ng/ L was reached after MBR treatment. Kim et al. [40] reported a 41% removal of naproxen from domestic sewage after treatment in a pilot scale MBR. For both compounds, ketoprofen and naproxen, we also observed the generation of metabolites rather than the complete mineralization as reported by Quintana et al. [33]. Roxithromycin has a moderately hydrophobic nature (log Kow =2.75) and basic character. This compound has the most complex chemical structure of the target compounds and acts as an antibacterial agent. These properties can partially explain the lower removal efficiencies of 57 and 81% compared to the NSAID compounds which were eliminated or metabolized with efficiencies of around 100% (acetaminophen and ketoprofen) and 89% (naproxen) (cf. Figs. 5 and 6). Similar results for roxithromycin removal in an MBR have been reported by Göbel et al. [4], where the elimination varied between 39% for an SRT of 16 days and 60% for higher SRTs (33 and 60 days). Sulfamethoxazole, also possessing antibacterial properties, was eliminated by only 55% and 64% in the MBR-15 and MBR-30 treatment processes (cf. Fig. 6), respectively. This compound has a hydrophilic nature (log Kow b 1) with two ionizable amine groups. As a result, in an aqueous solution, sulfamethoxazole can be present in positive, neutral, and negative forms. At pH values between the pKa values of the compound (pH 1.4 and 5.8), sulfamethoxazole is present predominantly as a neutral species, while above the second pKa value of the compound (pH 5.8) it becomes a negatively charged species. These physicochemical properties give an indication that in the MBR system studied (pH 7.2) the sludge adsorption mechanism played a negligible role, due to electrostatic repulsion between the negatively charged groups of the compound and the negatively charged surfaces of the sludge. Therefore, biodegradation can be considered as the main mechanism responsible for the removal, however, this processes will be hindered by the antibacterial properties of this compound. Göbel et al. [4], who studied the elimination of sulfamethoxazole, reported an elimination efficiency of around 80%, independently of the SRT. Considering the antibiotic compounds studied, the highest removal efficiencies were observed for trimethoprim (86 or 94%; cf. Figs. 5 and 6). This can be partially explained by its basic character and reduced antibacterial potency compared to sulfamethoxazole and roxithromycin. As observed for the other drugs, the removal efficiency for MBR-30 was higher than for MBR-15. Göbel et al. [4] studied the elimination of trimethoprim in an MBR and reported comparable elimination rates for SRTs of 16 and 33 days (30%), while 87% of removal was obtained for SRTs in the range of 60–80 days.
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3.4. Mechanisms influencing the removal of pharmaceutical compounds The elimination of pharmaceutical compounds can occur through various mechanisms in MBRs. Sorption onto sludge is one of the mechanisms that takes into account the absorption and adsorption factors. According to Carballa et al. [15], absorption refers to the hydrophobic interactions of the aliphatic and aromatic groups of a compound with fats present in the sludge or with the lipophilic cell membrane of the microorganisms (depending on their Kow value), while adsorption refers to the electrostatic interactions of positively charged groups of dissolved chemicals with the negatively charged surfaces of the microorganisms (characterized by the dissociation constant pKa). Göbel et al. [4] studied the elimination of pharmaceuticals by MBRs and CAST and concluded that the contribution of activated sludge adsorption in the case of pharmaceutical compounds was less than 6%, i.e., negligible, because this is within the analytical variance of the method. Another mechanism responsible for the removal of pharmaceutical compounds in MBRs is the physical retention by the membranes. However, it seems that this mechanism would not have led to retention of the pharmaceutical targets studied herein because the molecular weight cut off (MWCO) of ultrafiltration MBR membranes is around 100–200 kDa. Sorption onto the membranes is also limited because of the available membrane surface area. Pharmaceutical compounds which are non-polar will sorb onto the biomass and will therefore be removed indirectly during the retention of the solids by the membranes. Polar pharmaceuticals, with a low tendency to adsorb to the lipophilic sludge surface will neither be eliminated by adsorption nor by biodegradation because the interaction with the wastewater biocoenosis essential for the biodegradation process will be too short. Fig. 6 shows the mean removal rates for our target pharmaceuticals applying MBR treatment over 4 weeks. It can be observed that the NSAIDs were removed with higher efficiency than the antibiotics, which can be partially explained by their less complex chemical structure, as reported by Kimura et al. [1]. These authors compared the removal of 7 pharmaceutical compounds using MBRs and CAST and concluded that the former offer a much better removal for the compounds ketoprofen and naproxen. On the other hand, the most important property in terms of reducing the removal efficiency, i.e., the biodegradation, was the antibacterial potency of the antibiotic compounds. In addition, the observed “elimination”, which led to a reduction in the concentrations of the precursor compounds in parallel, could be due to a biodegradation process which resulted in transformation products which are more stable than the precursor drugs, as in the case of ketoprofen, naproxen and acetaminophen. 4. Conclusions The performance of two MBR pilot plants with submerged membranes, operated with different SRTs, for the removal of pharmaceutical compounds, was investigated in this study. The results obtained showed that the MBR-30 presented higher removal efficiencies than the MBR-15 for all compounds. The compounds acetaminophen and ketoprofen had the highest removal efficiencies, while roxithromycin and sulfamethoxazole exhibited persistence to microbial attack and were removed to a lesser extent in both MBRs. Concerning the potential mechanisms responsible for the removal of the target pharmaceuticals in the MBRs (sludge sorption + biodegradation + membrane retention), it was not possible to determine exactly the extent to which each mechanism contributed to the removal efficiency. However, in general terms, membrane retention using micro- or ultrafiltration membranes can be neglected, whereas biodegradation plays an important role, since higher removal efficiency was obtained for higher SRTs. Nevertheless, the elimination
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by MBR treatment using ultrafiltration was only partially successful and therefore, persistent pharmaceuticals in small concentrations and their transformation products were discharged with the wastewater into the environment. This discharge could be reduced with the application of additional treatment steps using advanced treatment techniques, e.g., activated carbon adsorption, ozone oxidation, advanced oxidation processes (AOP), nanofiltration (NF) or reverse osmosis (RO). In conclusion, the results indicated the importance of investigating the environmental fate of pharmaceuticals and their largely unknown polar transformation products. Additional research is thus urgently required firstly to recognize the parent drugs and their transformation products and secondly to assess the effect of these micropollutants on the environment. Acknowledgments The authors would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Deutscher Akademischer Austausch Dienst (DAAD, German Academic Exchange Service) for the financial support. Analytical support from the staff of the Environmental Analytical Laboratory of the Institute of Environmental Engineering of RWTH Aachen University is also greatly appreciated. References [1] K. Kimura, H. Hara, Y. Watanabe, Removal of pharmaceutical compounds by submerged membrane bioreactors (MBRs), Desalination 178 (2005) 135–140. [2] A.M. Comerton, R.C. Andrews, D.M. Bagley, P. Yang, Membrane adsorption of endocrine disrupting compounds and pharmaceutically active compounds, J. Membr. Sci. 303 (2007) 267–277. [3] W. Gebhardt, H.Fr. Schröder, Liquid chromatography-(tandem) mass spectrometry for the follow-up of the elimination of persistent pharmaceuticals during wastewater treatment applying biological wastewater treatment and advanced oxidation, J. Chromatogr. A 1160 (2007) 34–43. [4] A. Göbel, C.S. McArdell, A. Joss, H. Siegrist, W. Giger, Fate of sulfonamides, macrolides, and trimethoprim in different wastewater treatment technologies, Sci. Total Environ. 372 (2007) 361–371. [5] R. Andreozzi, R. Marotta, N. Paxéus, Pharmaceuticals in STP effluents and their solar photodegradation in aquatic environment, Chemosphere 50 (2003) 1319–1330. [6] M. Stumpf, T.A. Ternes, R.D. Wilken, S.V. Rodrigues, W. Baumann, Polar drug residues in sewage and natural waters in the state of Rio de Janeiro, Brazil, Sci. Total Environ. 225 (1999) 135–141. [7] M. Favier, R.F. Sena, H.J. José, U. Bieling, H. Fr. Schröder, Liquid chromatographytandem mass spectrometry for the screening of pharmaceuticals and metabolites in various water bodies in Florianópolis, Santa Catarina, Brazil. Proceedings Ecosan-Fortaleza, Conference; 26.–28.11.2007 in Fortaleza, Ceará, Brazil. [8] T.A. Ternes, M. Stumpf, J. Mueller, K. Haberer, R.D. Wilken, M. Servos, Behavior and occurrence of estrogens in municipal sewage treatment plants—I. Investigations in Germany, Canada and Brazil, Sci. Total Environ. 225 (1999) 81–90. [9] X.S. Miao, F. Bishay, M. Chen, C.D. Metcalfe, Occurrence of antimicrobials in the final effluents of wastewater treatment plants in Canada, Environ. Sci. Technol. 38 (2004) 3533–3541. [10] A. Gulkowska, H.W. Leung, M.K. So, S. Taniyasu, N. Yamashita, L.W.Y. Yeung, B.J. Richardson, A.P. Lei, J.P. Giesy, P.K.S. Lam, Removal of antibiotics from wastewater by sewage treatment facilities in Hong Kong and Shenzhen, China, Water Res. 42 (2008) 395–403. [11] W. Xu, G. Zhang, X. Li, S. Zou, P. Li, Z. Hu, J. Li, Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta (PRD), South China, Water Res. 41 (2007) 4526–4534. [12] T.A. Ternes, Occurrence of drugs in German sewage treatment plants and rivers, Water Res. 32 (1998) 3245–3260. [13] K. Kümmerer, Drugs in environment: emission of drugs, diagnostic aids and disinfectants into wastewater by hospitals in relation to other sources — a review, Chemosphere 45 (2001) 957–969. [14] S. Castiglioni, R. Fanelli, D. Calamari, R. Bagnati, E. Zuccato, Methodological approaches for studying pharmaceuticals in the environment by comparing predicted and measured concentrations in River Po, Italy, Regul. Toxicol. Pharmacol. 39 (2004) 25–32. [15] M. Carballa, F. Omil, J.M. Lema, Removal of cosmetic ingredients and pharmaceuticals in sewage primary treatment, Water Res. 39 (2005) 4790–4796. [16] J.L. Santos, I. Apacicio, E. Alonso, Occurrence and risk assessment of pharmaceutically active compounds in wastewater treatment plants. A case study: Seville city, Environ. Int. 33 (2007) 596–601 (Spain).
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