Fate of tetracycline in enhanced biological nutrient removal process

Chemosphere 193 (2018) 998e1003

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Fate of tetracycline in enhanced biological nutrient removal process Hang Liu a, b, Yongkui Yang b, Huifang Sun c, Lin Zhao b, **, Yu Liu a, c, * a Advanced Environmental Biotechnology Centre, Nanyang Environment & Water Research Institute, Nanyang Technological University, 1 Cleantech Loop, Singapore, 637141, Singapore b School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China c School of Civil and Environmental Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore, 639798, Singapore

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Partial removal of tetracycline was achieved via biosorption and biodegradation.
A substantial amount of tetracycline was accumulated in sludge over a long period.
More than 40% of tetracycline was degraded biologically.
The more toxic biodegradation byproduct, phthalic anhydride was generated.
This study stresses the ecological risks of sludge disposal and effluent discharge.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 31 August 2017 Received in revised form 21 November 2017 Accepted 22 November 2017 Available online 23 November 2017

This study investigated the fate of tetracycline at four different concentrations of 20 mg L1, 50 mg L1, 2 and 5 mg L1 in the enhanced biological nutrient removal processes. At the tetracycline concentration below 50 mg L1, no obvious inhibition on the biological N&P removal was observed, while the inhibition appeared after the tetracycline concentration was increased to 2 and 5 mg L1. It was found that about 44%e87% of tetracycline was removed through biodegradation, while only 3%e6% of removal was due to biosorption. These results clearly suggested that a substantial amount of tetracycline eventually ended up in sludge with the tetracycline content of 23 mg to 4.5 g kg1 sludge depending on the tetracycline concentration. Obviously, this could pose an emerging challenge to the post sludge disposal and reuse. Furthermore, phthalic anhydride was detected as a biodegradation byproduct of tetracycline, which has been known to be more toxic than tetracycline to aquatic organisms. Consequently, this study offers indepth insights into the fate of tetracycline in the enhanced biological nutrient removal process, highlighting on the emerging ecological risks associated with sludge disposal and effluent discharge. © 2017 Elsevier Ltd. All rights reserved.

Handling Editor: Hyunook Kim Keywords: Tetracycline Enhanced biological nutrient removal Biosorption Biodegradation Toxicity

1. Introduction

* Corresponding author. School of Civil and Environmental Engineering, Nanyang Technological University, Singapore 639798, Singapore. ** Corresponding author. E-mail addresses: [email protected] (L. Zhao), [email protected] (Y. Liu). https://doi.org/10.1016/j.chemosphere.2017.11.136 0045-6535/© 2017 Elsevier Ltd. All rights reserved.

Antibiotics have been frequently detected in aquatic and soil environment due to the discharge of the effluent from municipal wastewater treatment plants (Zhang et al., 2013a; Xu et al., 2015; Mohapatra et al., 2016; Yang et al., 2016). So far, it is not yet totally clear about the fate of antibiotic in the municipal wastewater

H. Liu et al. / Chemosphere 193 (2018) 998e1003

treatment plants. For example, the concentrations of antibiotics in effluents were found to be as high as 3.1 mg L1 (Luo et al., 2014), €bel et al. (2007) reported that the elimination rates of while Go sulfonamides, macrolides, and trimethoprim were up to 70% in two activated sludge systems in Switzerland. The remaining antibiotics in effluents of WWTPs would be discharged into various water bodies, and eventually become the most pronounced source of antibiotics in river (Paiga et al., 2016), lake (Li et al., 2016) and ocean (Zhang et al., 2013b). In addition, sewage sludge also contained antibiotics (Subedi et al., 2014), e.g. 5.8 mg per gram of sludge was detected in an activated sludge process (Zhou et al., 2013). These indicate an ecological risk when such sludge is disposed of to landfill or used for agriculture. More importantly, antibiotics can trigger the proliferation and spread of antibiotic resistant bacteria (ARB) and the development of antibiotic resistance genes (ARG), which have been considered as the major microbial threat to the environment (Novo et al., 2013). Therefore, it is vital to better understand the fate of antibiotics in biological wastewater treatment process. Tetracycline (TET) has been widely used in human therapy and animal husbandry (Van Boeckel et al., 2014), with the global annual production of more than 20,000 tons (Zhang et al., 2013c). As such, it has been one of the most frequently detected antibiotics in the wastewater treatment plants. Li and Zhang (2013) found that sorption was the major removal mechanism of tetracycline in their activated sludge system, with negligible biodegradation. However, 63.3% and 67.4% of tetracycline were found to be removed through biodegradation in the activated sludge processes fed with 10 and 1 mg L1 of tetracycline, respectively (Song et al., 2016). In fact, it is still unclear about the fate of tetracycline in an enhanced biological nutrients removal process, especially relative contribution of biodegradation versus biosorption. In particular, little attention had been dedicated to examining the biodegradation byproduct of tetracycline and associated ecological toxicity in the enhanced biological nutrients removal process. Given such a situation, this study thus aimed to determine the transformation and fate of tetracycline in the enhanced biological nutrients removal processes which were operated at environmentally relevant concentrations of 20 and 50 mg L1 and significantly high concentrations of 2 and 5 mg L1 of tetracycline. The effects of tetracycline on the biological nitrogen and phosphorus removal were investigated, while extensive effort was devoted to generating a complete picture of the fate of tetracycline, possible biodegradation byproduct and associated ecological toxicity.

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Table 1 Experimental conditions. Reactor No.

Tetracycline concentration

Operation period

R0

0 mg L1 5 mg L1 20 mg L1 2 mg L1 50 mg L1 5 mg L1

1-150 d (Phase I) 151-230 d (Phase II) 1-150 d (Phase I) 151-230 d (Phase II) 1-150 d (Phase I) 151-230 d (Phase II)

R1 R2

Aldrich, St. Louis, MO, USA) at the concentrations of 20 mg L1, 50 mg L1, 2 mg L1 and 5 mg L1 were step-wise added into the three SBR over the operation time. The experimental conditions were summarized in Table 1. 2.2. Tetracycline analysis

2. Materials and methods

Tetracycline was extracted from sludge sample according the method by Wu et al. (2008). In this method, 50 mg of freeze-dried sludge pellet was suspended in 10 mL of the extraction buffer (i.e. 50 mM acetic acid þ10 mM EDTA, pH 3.6) by vortexing for 15 min. The suspension was centrifuged, and the harvested supernatant was then filtered through 0.2 mm nylon membrane. The filtrates were further used for analysis. To determine tetracycline concentration, solid phase extraction (SPE) with Oasis HLB cartridges (200 mg, Waters, Milford, MA, USA) was employed to concentrate the samples. The procedure of SPE was modified from the method by Miao et al. (2004). Briefly, the sample pH was adjusted to 3.0 with 3.0 M H2SO4, followed by addition of 0.5 g L1 Na2EDTA to prevent the potential chelation. The cartridges were sequentially preconditioned by 6 mL of acetonitrile, 6 mL of methanol and 6 mL of 50 mM Na2EDTA at pH 3.0. The samples were loaded to the preconditioned cartridges. Afterwards, the cartridges were washed with the same volume of MilliQ water and dried for 10 min under vacuum. Finally, tetracycline adsorbed onto the cartridges was eluted with methanol, and 96.6e102.1% of recovery of tetracycline were achieved in this study. Tetracycline in the above eluted samples was analyzed by an Agilent 1290 Infinity II liquid chromatography equipped with a ZORBAX Eclipse Plus C18 (2.1  50 mm, 1.8-mm) column and interfaced with Agilent 6460 Triple Quad mass spectrometer (Agilent Technologies, Palo Alto, CA, USA). Water with 0.1% formic acid and acetonitrile with 0.1% formic acid were used as mobile phases. The retention time of tetracycline was 3.04 min with precursor ion of m/z 445.2 and product ion of m/z 410.1.

2.1. Experimental setups

2.3. Tetracycline biodegradation byproduct and toxicity analysis

Three sequencing batch reactors (SBR), each with a working volume of 5.5 L, were operated at 6-h cycle consisting of 2 min feeding, 100 min anaerobic reaction, 200 min aerobic reaction, followed by settling and decanting. The reactors were covered by aluminium foil to avoid the possible photodegradation of tetracycline and algal growth. The aeration rate in the aerobic phase was controlled at 0.8 L min1, while nitrogen gas was introduced in the anaerobic phase. The solid retention time (SRT) and hydraulic retention time (HRT) was maintained at 18 d and 16.5 h, respectively. The synthetic wastewater with the following composition was used: CH3COONa 528.73 mg L1, NH4Cl 315.32 mg L1, KH2PO4 20.56 mg L1, K2HPO4$3H2O 34.47 mg L1, NaHCO3 412.5 mg L1, FeSO4$7H2O 20.6 mg L1, MgSO4$7H2O 20.6 mg L1, CaCl2$2H2O 20.6 mg L1 and 1 mL of trace element solution (Li et al., 2013a). All the chemicals were purchased from VWR International (Radnor, PA, USA) and Sigma-Aldrich (St. Louis, MO, USA). Tetracycline (Sigma-

An Agilent Q-TOF mass spectrometer (6550 iFunnel Q-TOF LCMS, Agilent Technologies, Santa Clara, CA, USA) was used to identify the byproduct produced from biodegradation of tetracycline. The elemental composition was deduced from the exact m/z values obtained by the TOF-MS, while the structure was identified according to the fragmentation pattern. The detailed chromatographic and MS conditions were provided in the Supporting Information (Text S1). Quantitative structure-activity relationship (QSAR) analysis calculated by the Ecological Structure-Activity Relationship model (ECOSAR) program was used to estimate the toxicity of tetracycline and the degradation byproduct (Zhang et al., 2016a). The prediction of no effect concentration (PNEC) and risk quotient (RQ) were calculated according to the method by Carlsson et al. (2006). PNEC was derived from EC50 (Half Effect Concentration) of green algae divided by the assessment factor 1000. The risk characterization

1000

H. Liu et al. / Chemosphere 193 (2018) 998e1003

described as RQ (RQ ¼ Concentration/PNEC) reflected the estimated ecotoxicity of the compound.

exposure to 3 mg L1 of antibiotics, while phosphorus removal was more susceptible to antibiotic than nitrogen removal (Meng et al., 2015).

2.4. Other analytical methods 3NHþ 4 -N, PO4 -P and mixed liquid suspended solids (MLSS) were determined according to Standard Method (APHA, 2005). Extracellular polymeric substances (EPS) of the activated sludge were extracted according to the method by Cai and Liu (2016). Dissolved organic carbon (DOC) was measured by a total organic carbon (TOC) analyzer (Shimadzu Co., Kyoto, Japan). All the tests were performed in triplicate.

3. Results and discussion 3.1. Effect of tetracycline on biological nutrient removal 3Fig. 1 showed the removal of DOC, NHþ 4 -N and PO4 -P at different tetracycline concentrations. Compared to the control free of tetracycline (R0), 93.1 ± 1.2% of DOC, 99.6 ± 0.3% of NHþ 4 -N and 93.1 ± 5.9% of PO34 -P were removed on average at the tetracycline concentrations of 20 and 50 mg L1. These suggested that inhibition by tetracycline at the environmental concentrations was insignificant on microorganisms involved in the biological N&P removal process. However, when the tetracycline concentrations in the reactors were increased to 2 and 5 mg L1, severe inhibition appeared, which was evidenced by reduced DOC and NHþ 4 -N removal. It was also found that the inhibition of tetracycline was more profound on the phosphorus-accumulating organisms (PAOs), e.g. the P-removal was reduced from 93.1 ± 6.2% free of tetracycline to 63.4 ± 8.8% when the tetracycline concentration was increased to 5 mg L1. Amorim et al. (2014) also reported that the activity of PAOs in a granular sludge SBR was significantly affected after the prolonged

3.2. Biodegradation of tetracycline The tetracycline in the reactors could be removed through biosorption and biodegradation. Eq. (1) showed a mass balance on tetracycline in the reactors:

TETin ¼ TETsor þ TETdeg þ TETout

(1)

where TETin is influent tetracycline, TETsor is the amount of tetracycline biosorbed onto sludge, TETdeg is the amount of tetracycline biodegraded and TETout is the amount of tetracycline in effluent. Hence, the amount of tetracycline biodegraded can be calculated as follows:

TETdeg ¼ TETin  TETout  TETsor

(2)

and the total amount of tetracycline biosorbed onto the sludge can be determined by Eq. (3):

TETsor ¼

X

TETsorc

  TETsorc ¼ Cs;end  Cs;beg  X þ Cs;end  DXinc

(3) (4)

where TETsor-c is the amount of tetracycline removed by biosorption per cycle, Cs,beg and Cs,end is the amount of tetracycline biosorbed by per unit biomass at the beginning and the end of each cycle, X is the amount of biomass at the end of each cycle and DXinc is the amount of biomass increased during oneecycle operation.

3Fig. 1. Removal of DOC (a), NHþ 4 -N (b) and PO4 -P (c) at different tetracycline concentrations. Left: The scatter of the removal efficiency; Right: The statistics analysis with the 25th percentile, the median and the 75th percentile of the removal efficiency.

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Fig. 2. Amount of tetracycline removed by biodegradation. a: Amount of tetracycline biodegraded in Phase I; b: Amount of tetracycline biodegraded in Phase II.

Fig. 2 showed the amount of tetracycline removed by biodegradation at different concentrations. It was found that the biodegradation of tetracycline was significantly enhanced with the prolonged operation, especially at the lower concentrations of 20 and 50 mg L1. When the tetracycline concentrations were increased to 2 and 5 mg L1, an initial lag phase of biodegradation was observed. In addition, R2 exhibited much stronger ability to degrade tetracycline than R0. This could be attributed to the longterm exposure of R2 to trace-level tetracycline in Phase I. It had also been reported that the biodegradation of erythromycin in an anaerobic SBR was enhanced with the step-wise increase in erythromycin concentration (Aydin et al., 2016). These was probably due to the prior acclimation of microorganisms in the lowconcentration antibiotic environment. 3.3. Biosorption of tetracycline Fig. 3 showed the tetracycline content in sludge. During the first 120-day operation (Phase I), the tetracycline content in sludge tended to gradually decrease from 30.8 to 23.4 mg g1 MLSS in R1 and from 67.9 to 47.2 mg g1 MLSS in R2. When the influent tetracycline concentrations were increased to 2 and 5 mg L1, the tetracycline content in sludge was found to increase sharply at the early stage. Antibiotics could be biosorbed by microorganisms and

1001

Fig. 3. Tetracycline content in sludge. a: Phase I sludge; b: Phase II sludge.

EPS in activated sludge. The sorption of sulfamethazine by EPS accounted for 62% of the total sulfamethazine sorption at the initial biosorption stage and decreased to 35% subsequently (Xu et al., 2013). In this study, the EPS content increased by more than 20 mg TOC g1 MLSS in the first 15 days of Phase II (Fig. S2). Therefore, the increased biosorption of tetracycline should be related to the increased EPS content. Li et al. (2013b) reported that the maximum biosorption capacity of tetracycline in an upflow anaerobic sludge bioreactor was 3.0 mg g1. However, the sludge in Phase II was able to remove 4.5 mg g1 MLSS via biosorption. It should be noted that the tetracycline contents in sludge were as high as 23 mg kg1 to 4.5 g kg1 in the long-term operation. It had been reported that nearly half of the antibiotics were reported to be persistent during the sludge treatment processes, such as anaerobic digestion and aerobic composting (Martín et al., 2012). Moreover, antibiotics were found to be more persistent in biosolids-amended soil than aquatic environment (Verlicchi and Zambello, 2015). Consequently, this study raises serious concern about the current sludge disposal practice.

3.4. Contributions of biodegradation and biosorption to tetracycline removal Overall, 47%e91% of tetracycline at the tested concentrations could be remove through biodegradation and biosorption (Fig. 4).

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H. Liu et al. / Chemosphere 193 (2018) 998e1003 Table 2 The calculated risk quotient of tetracycline (TET) and phthalic anhydride (TP 149) at the beginning and end of one cycle (based on acute toxicity to green algae).

RAa PNECb (mg L1) RQc Total RQ

Beginning of one cycle

End of one cycle

TET

TP 149

TET

TP 149

1.00 0.24 4.17 4.17

0 0.05 0

0.37 0.24 1.56 10.16

0.43 0.05 8.60

a RA represent relative abundance, which was normalized by the initial signal intensity of TET. b PNEC represent predicted no effect concentration, PNEC values were derived from the acute EC50 to green algae value divided by the assessment factor 1000. c RQ represent risk quotient, RQ ¼ RA/PNEC.

Fig. 4. Contributions of biosorption and biodegradation towards tetracycline removal at different concentrations.

However, it should be noted that substantial amount of tetracycline still remained in the effluent, which was much higher than the reported prediction of no effect concentration of tetracycline (e.g. 90 ng L1 for M. aeruginosa) (Jiang et al., 2014). In addition, no significant differences in the proliferation of tetracycline resistance genes were observed at 5 and 50 mg L1 of tetracycline against 10 mg L1 of tetracycline (Zhang et al., 2016b). These in turn suggested that antibiotics discharged through the effluent may trigger the spread of antibiotic resistant genes among natural bacterial populations, and this has been recognized as an emerging ecological issue. As can be seen in Fig. 4, most of tetracycline was removed through biodegradation, while the contribution of biosorption towards the observed removal of tetracycline appeared insignificant. Although tetracycline is not readily biodegradable (Kim et al., 2005; Prado et al., 2009), more than 40% of tetracycline was still removed biologically in this study (Fig. 4). The enhanced tetracycline biodegradation could be attributed to the relatively long SRT of 18 d and the gradual acclimation strategy adopted in this study. Obviously, long SRT facilitated the enrichment of various enzymes that serve as the catalysts of biological reactions (Stadler and Love, 2016). On the other hand, long SRT also promoted the growth of slow-growing bacteria essential for antibiotics biodegradation (Vuono et al., 2016). 3.5. Biodegradation byproduct of tetracycline and its ecological toxicity In this study, the Q-TOF LC-MS was employed to identify the

biodegradation byproduct of tetracycline (Fig. 5), while chemical structure of the degradation byproduct was determined according to the accurate mass (Table S1) and the fragments information from MS2 spectrum (Fig. S3). Subsequently, TP 149 in Fig. 5 was identified as phthalic anhydride, which hydrolysate had been reported to have potential effect on biologically active substances, such as tetracycline-type antibiotics (Bang et al., 2011). Fig. 5 showed the relative abundances of tetracycline and phthalic anhydride determined from the signal intensity. It was found that 43.1% of tetracycline was converted to phthalic anhydride. The QSAR analysis was employed to predict the acute and chronic toxicity of tetracycline and phthalic anhydride (Table S2). Except for the 48-h LC50 (Half Lethal Concentration) to daphnid, the LC50, EC50 and ChV (Chronic Value) of phthalic anhydride to fish and green algae were much lower than those of tetracycline, indicating that phthalic anhydride was more toxic to these standard aquatic organisms than tetracycline. These were also supported by the calculated RQ representing the potential environmental risks (Table 2). Therefore, it appears necessary to include biodegradation byproduct and related toxic effect in the assessment of biological treatment processes of wastewater and subsequent ecological risks.

4. Conclusions This study investigated the fate of tetracycline in the enhanced biological nutrient removal process over a long-term operation. Tetracycline was found to be removed mainly through biodegradation, while the contribution of biosorption towards removed tetracycline was insignificant, e.g. generally less than 6%. The performance of the biological nutrient removal (e.g. nitrogen and

Fig. 5. MS spectrums, molecular structures and relative abundances of tetracycline (TET) and phthalic anhydride (TP 149). a: MS spectrum and molecular structure of tetracycline; b: MS spectrum and molecular structure of phthalic anhydride; c: Relative abundances of tetracycline and phthalic anhydride.

H. Liu et al. / Chemosphere 193 (2018) 998e1003

phosphorus) was only adversely affected at the higher tetracycline concentration of 2 and 5 mg L1. It should be realized that even at the environmentally relevant concentration of 20 mg L1 tetracycline, the tetracycline content in the sludge was as high as 23 mg kg1 sludge. In China, about 6.25 million tons dry solids of waste activated sludge was produced in 2013 (Yang et al., 2015), this implies that tremendous amount of antibiotics may enter the environment through the sludge disposal. It was also revealed in this study that phthalic anhydride as the byproduct generated through the biodegradation of tetracycline was much more toxic, implying that the environmental impact assessment of antibiotics should also include biodegradation byproduct and its toxicity, especially when water recycle and reuse are concerned. Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.chemosphere.2017.11.136. References Amorim, C.L., Maia, A.S., Mesquita, R.B.R., Rangel, A.O.S.S., van Loosdrecht, M.C.M., Tiritan, M.E., Castro, P.M.L., 2014. Performance of aerobic granular sludge in a sequencing batch bioreactor exposed to ofloxacin, norfloxacin and ciprofloxacin. Water Res. 50, 101e113. APHA, 2005. Standard Methods for the Examination of Water and Wastewater. American Public Health Association, Washington, DC. Aydin, S., Ince, B., Ince, O., 2016. Assessment of anaerobic bacterial diversity and its effects on anaerobic system stability and the occurrence of antibiotic resistance genes. Bioresour. Technol. 207, 332e338. Bang du, Y., Lee, I.-K., Lee, B.-M., 2011. Toxicological characterization of phthalic acid. Toxicol. Res. 27 (4), 191. Cai, W., Liu, Y., 2016. Enhanced membrane biofouling potential by on-line chemical cleaning in membrane bioreactor. J. Membr. Sci. 511, 84e91. Carlsson, C., Johansson, A.-K., Alvan, G., Bergman, K., Kühler, T., 2006. Are pharmaceuticals potent environmental pollutants?: Part I: environmental risk assessments of selected active pharmaceutical ingredients. Sci. Total Environ. 364 (1), 67e87. € bel, A., McArdell, C.S., Joss, A., Siegrist, H., Giger, W., 2007. Fate of sulfonamides, Go macrolides, and trimethoprim in different wastewater treatment technologies. Sci. Total Environ. 372 (2e3), 361e371. Jiang, Y., Li, M., Guo, C., An, D., Xu, J., Zhang, Y., Xi, B., 2014. Distribution and ecological risk of antibiotics in a typical effluentereceiving river (Wangyang River) in north China. Chemosphere 112, 267e274. Kim, S., Eichhorn, P., Jensen, J.N., Weber, A.S., Aga, D.S., 2005. Removal of antibiotics in wastewater: effect of hydraulic and solid retention times on the fate of tetracycline in the activated sludge process. Environ. Sci. Technol. 39 (15), 5816e5823. Li, B., Zhang, T., 2013. Removal mechanisms and kinetics of trace tetracycline by two types of activated sludge treating freshwater sewage and saline sewage. Environ. Sci. Pollut. R. 20 (5), 3024e3033. Li, C., Liang, S., Zhang, J., Ngo, H.H., Guo, W., Zheng, N., Zou, Y., 2013a. N2O reduction during municipal wastewater treatment using a two-sludge SBR system acclimatized with propionate. Chem. Eng. J. 222, 353e360. Li, J., Cheng, W., Xu, L., Jiao, Y., Baig, S.A., Chen, H., 2016. Occurrence and removal of antibiotics and the corresponding resistance genes in wastewater treatment plants: effluents' influence to downstream water environment. Environ. Sci. Pollut. R. 23 (7), 6826e6835. Li, K., Ji, F., Liu, Y., Tong, Z., Zhan, X., Hu, Z., 2013b. Adsorption removal of tetracycline from aqueous solution by anaerobic granular sludge: equilibrium and kinetic studies. Water Sci. Technol. 67 (7), 1490e1496. Luo, Y.L., Guo, W.S., Ngo, H.H., Nghiem, L.D., Hai, F.I., Zhang, J., Liang, S., Wang, X.C.C., 2014. A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment. Sci. Total Environ. 473, 619e641. ~ oz, M.D., Santos, J.L., Aparicio, I., Alonso, E., 2012. DistriMartín, J., Camacho-Mun bution and temporal evolution of pharmaceutically active compounds alongside sewage sludge treatment. Risk assessment of sludge application onto soils.

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