Elimination of Macrolides, Tiamulin, and Salinomycin ... - Springer Link

Arch. Environ. Contam. Toxicol. 51, 21–28 (2006) DOI: 10.1007/s00244-004-0240-8

Elimination of Macrolides, Tiamulin, and Salinomycin During Manure Storage M. P. Schlsener,1 M. A. von Arb,2 K. Bester1 1 2

Waste and Wastewater Management, University of Duisburg Essen, Campus Essen, Universitaetsstrasse 15, D-45141 Essen, Germany Civil, Architectural, and Environmental Engineering, University of Missouri, Rolla, 309 Butler-Carlton Hall, Rolla, Missouri 65409-0030, USA

Received: 1 December 2004 /Accepted: 22 August 2005

Abstract. The extensive use of veterinary drugs in livestock farming increases the risk that these compounds end up in the environment when manure is used as fertilizer. This study focuses on the fate of antibiotics in liquid manure tanks before the liquid manure is spread on fields. A 180-day degradation experiment of four commonly used antibiotics erythromycin, roxithromycin, salinomycin, and tiamulin in liquid manure was performed. The resulting half-lives during manure storage were calculated as follows: 41 days for erythromycin, 130 days for roxithromycin, and 6 days for salinomycin. A first-order degradation rate was calculated for these three antibiotics. The concentration of tiamulin remained unchanged during the entire experiment. No degradation of tiamulin was detected even after 180 days.

In recent years, the occurrence of pharmaceuticals in the agricultural environment has been reported (Halling-Sørensen et al. 1998; Kmmerer et al. 2001; Hirsch et al. 1999). Additionally, the occurrence of antibiotics in influents, effluents, sludge of sewage treatment plants (Giger et al. 2003; Gçbel et al. 2004) and surface waters (Christian et al. 2003; Kolpin et al. 2002) has been discussed. Furthermore, bacterial resistance to the majority of existing antibiotics were first reported by Neu (1992). Veterinary antibiotics such as tetracylines, sulfonamides, macrolides, ionophores, and pleuromutilins are commonly used to treat infections in livestock. Three different uses of antibiotics are currently considered significant in pig farming: (1) the treatment of infections with pleuromutilins and macrolides; (2) disease prevention, especially if pigs from different breeders are brought together for fattening; and (3) growth promotion; which is the continuous dosing of an antibiotic compound such as salinomycin to pigs to promote growth during the fattening phase. Sodium-monensin, sodium-salinomycin, flavophospholipol, and avilamycin are currently used for growth promotion in agriculture, but these antibiotics will

Correspondence to: K. Bester; email: [email protected]

probably be phased out in the European Union on January 1, 2006 (European Commission 2002). Sodium-salinomycin is still allowed as feed additive for pigs until October 2009 (European Commission 2004a) and for the prevention of coccidiosis until August 2014 (European Commission 2004b). A typical pig farm in Germany holds an average of 800 pigs (Federal Statistical Office Germany 2004). Depending on the actual infections in the livestock, approximately 1 to 5 kg each of the antibiotics are used during the fattening phase on such a farm. The pigs quickly absorb most of the compounds and excrete 50% to 90% (ß-lactam-antibiotics, tetracyclines, sulfonamides) of the initial amount after several days (Kroker 1983). Thus, large quantities of the pharmaceuticals are transferred to manure tanks along with the liquid manure. After the storage, the manure is dispersed on the fields, and the unmetabolized antibiotics contained in this manure may contaminate the soil and eventually the ground water. The occurrence and fate of veterinary drugs such as sulfonamides and tetracyclines in soil or manure has also been reported (Hamscher et al. 2002; Ingerslev et al. 2001; Thiele-Bruhn et al. 2004). Basic considerations about the fate of veterinary drugs have also been published by Tolls (2001). However, little is known about the fate of antibiotics in manure. In this medium, they may undergo diverse reactions resulting in complete or partial elimination of the parent compound (Kmmerer 2004). Three different degradation behaviours are possible: (1) complete elimination by mineralization of the antibiotics; (2) partial transformation; and (3) persistence of the compounds in the manure. These processes can be performed by biotic or abiotic means. Although the published literature has focused on sulfonamides and tetracyclines, this study focused on the fate of the macrolide antibiotics, tiamulin, and salinomycin during storage of liquid manure until the manure is spread on agricultural fields. Structural formulae of the analytes are shown in Figure 1. Manure can be stored approximately 180 days before being dispersed on fields (Agricultural Board NRW 2005, personal communication) and is most often stored in anaerobic lagoons or storage tanks. Manure storage in anaerobic lagoons, typically used in the United States and Canada, are large outdoor basins of liquid manure with air and sunlight on the surface but anaerobic in depth. Therefore, some oxygen may diffuse into the lagoon, and sunlight may cause phototransformations of antibiotics at the surface.

M. P. Schlsener et al.

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Fig. 1. Structural formulae of (a) erythromycin, (b) roxithromycin, (c) salinomycin, and (d) tiamulin

Manure storage tanks are more commonly used in Europe. In a manure tank, manure is stored in the dark and has decreased air admittance. In these tanks, there are more anaerobic conditions than in lagoons.

roxithromycin, tiamulin, and salinomycin were below the limit of detection.

Chemicals Materials and Methods Materials 10 L liquid manure was collected in May 2002 directly from the manure tank of a local farmer. The manure in the tank was stirred for approximately 20 minutes before sampling. The temperature of the liquid manure was approximately 18°C. The pigs were not administered any of the four antibiotics for approximately 8 months before sampling. The blank values of erythromycin,

Acetonitrile (''HPLC-S gradient grade'') was purchased from Biosolv (Valkensward, Netherlands). Water (high-performance liquid chromatography [HPLC] grade) was obtained from Mallinckrodt Baker (Griesheim, Germany). Isooctane, methanol (SupraSolv grade), acetone, and ethyl acetate (analytic grade) were obtained from Merck (Darmstadt, Germany). Ammonium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, urea, and disodium ethylenediaminetetraacetic acid (EDTA) were of analytic grade and were purchased from Merck. Erythromycin and roxithromycin were provided by Sigma-Aldrich (Seelze, Germany). Salinomycin SV sodium salt 2.5-hydrate and tiamulin fumarate (Vetranal) were obtained from Riedel-de HaLn (Seelze, Germany). The synthesis of (E)-9-[O-

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Macrolides, Tiamulin, and Salinomycin During Manure Storage

(2-methyloxime)]-erythromycin has been described by Schlsener et al. (2003a), and the synthesis of anhydroerythromcin has been described by McArdell et al. (2003).

Degradation Experiment Thirty Erlenmeyer flasks were filled with 100 g fresh manure that was spiked with a mixture of antibiotics at a concentration of 2000 lg/kg to mimic excretion of treated animals. This concentration was based on the assumption that an administered dosage of 2 kg antibiotic is excreted completely by 800 pigs and that all of the liquid manure is stored in a 1000-m3 manure tank. This is the amount used by the local farmers (Agricultural Board NRW 2005, personal communication). The Erlenmeyer flasks were stored in the dark at 20°C (Memmert, Modell 800, Schabach, Germany) and closed with a fermenting tube to maintain anaerobic conditions. The loss of water during the experiment was compensated weekly by addition of HPLC-grade water after difference weighting. After a specified time period, the samples were homogenized, extracted, and analyzed. Three subsamples of 15 g homogenized manure each were analyzed.

Sample Pretreatment The manure was homogenized for 5 minutes at 25,000 rpm using an ultra turrax homogenizer (VF2/IKA, Staufen, Germany). Fifteen grams homogenized manure were transferred into 75-mL centrifuge glass tubes with a screw cap (Schott, Mainz, Germany), and 5 g urea was added. The samples were buffered to pH 8 by the addition of 6 mL phosphate buffer (3.4 g K2HPO4 and 0.1 g KH2PO4 dissolved in 100 mL HPLC-grade water).

Liquid–Liquid Extraction The buffered manure was extracted with 40 mL ethyl acetate by shaking for 20 minutes on a horizontal shaker (Kottermann, type 4020, Haenigsen, Germany) at 150 minutes–1. After shaking and before phase separation, 25 lL internal standard solution (10 mg (E)-9[-O-(2-methyloxime)]- erythromycin dissolved in 100 mL acetonitrile) was added to the mixture, and the centrifuge glass was shaken by hand for 1 minute. The phases were separated by centrifugation at 800 g (1350 rpm) for 20 minutes (BeckmannCoulter, Avanti J25, Unterschleissheim, Germany). The organic phase was removed and stored. The aqueous phase was mixed with 6 mL EDTA solution (3.7 mg disodium EDTA dissolved in 100 mL HPLC-grade water), and the mixture was extracted again with 40 mL ethyl acetate with shaking (20 minutes) and centrifugation (800 g for 20 minutes). The organic phases from the first and the second extraction were combined, and the sample volume was decreased to 5 mL by means of a rotary evaporator at 60°C and 320 hPa. The residue was dissolved in 20 mL isooctane, and the volume was decreased again to 10 mL at 60°C and 170 hPa.

SPE Clean-Up Diol solid-phase extraction cartridges from UCT (2000 mg, Bristol, PA) were conditioned once with 10 mL isooctane at a flow rate of 5 mL/min. A solid-phase extraction manifold (IST, Grenzach-Wyhlen, Germany) with polytetrafluoroethylene stopcock and outlet was used. The manure extract (10 mL) was passed through the cartridge

at a speed of 5 mL/min (vacuum). The cartridge was washed once with 10 mL isooctane to remove lipids and dried for 20 minutes by sucking air through the column followed by a wash step with 10 mL water to remove salt. The analytes were eluted twice from the cartridge with 4 mL (3/2, v/v) mixture of acetonitrile: 0.1 M aqueous ammonium acetate solution at a flow rate of 5 mL/minutes. An aliquot of 0.8 mL eluate was transferred to a 1.5-mL autosampler vial for HPLC–mass spectrometry (MS)–mass spectrometry (MS) analysis.

HPLC The HPLC separations were performed using a Phenosphere-Next RP18 column (2 mm i.d., length 150 mm, particle size 3 lm) and a SecurityGuard (Phenomenex, Torrance, CA) at 25°C € 1°C. The flow rate was 0.2 mL/min. The HPLC gradient was established by mixing two mobile phases: phase A was 10 mM aqueous ammonium acetate solution, and phase B was pure acetonitrile. Chromatographic separation was achieved with the following gradient: 0 to 1 minute 10% B, 1 to 14 minutes 10% to 100% B, 14 to 29 minutes 100% B, 29 minutes to 30 minutes 100% to 10% B, and 30 to 35 minutes 10% B. Ten lL of each sample were injected. The HPLC system consisted of a GINA 50 autosampler, a P 580A HPG HPLC pump, a degasser unit DEGASYS DG-1210, and a column oven STG 585 (all from Dionex, Idstein, Germany). The dead time of the HPLC system was 1.8 minutes.

Mass Spectrometry The triple-quadrupole mass spectrometer (TSQ 7000, Finnigan-MAT, Bremen, Germany) was equipped with an atmospheric pressure chemical ionization (APCI 2) source and operated under the following conditions: capillary temperature 180°C; sheath gas 40 psi; corona current 5 lA; vaporizer temperature 450°C; auxiliary gas off; q0 offset )4.4 V; collision cell pressure 2.0 mTorr; collision gas argon; and multiplier 1900 V. The potential difference between the capillary and the tube lens was held at 70 V. The cycle time was 1.0 seconds during the chromatographic determination of antibiotics. The data obtained were processed using Xcalibur 1.2 software. The silica capillary of the APCI 2 source was replaced by a steel capillary to decrease tailing of antibiotics adsorbing on the silica surface (Pfeifer et al. 2001). APCI was preferred because this ionization is less vulnerable to matrix effects than electrospray ionization (ESI) (Bester et al. 2001). A post-column Valco divert valve was used to direct most of the nonsignificant HPLC flow of a sample to waste. Diverting the flow minimized contamination of the MS source: 0 to 8 minutes divert to waste, 8 to 28 minutes flow to mass spectrometer, and 28 to 35 minutes divert to waste. An additional flow of 50 lL/min water acetonitrile (3:7 v/v) pumped by a LC-10 AT high-pressure liquid chromatographer (Shimadzu, Duisburg, Germany) compensated the missing flow from the high-pressure liquid chromatographer during waste positing operation. Automatic data acquisition was triggered using a short contact closure signal of the autosampler. To gain higher selectivity, selected reaction monitoring (SRM) was chosen. Key parameter settings for SRM are listed in Table 1.

Calibration The calibration was performed as an internal standard calibration in the presence of manure matrix to account for matrix effects (Bester


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Table 1. Retention time and MS conditions for the analyses of macrolides, salinomycin, and tiamulin Antibiotic

Retention time (min)

Precursor ion

Product ion

Collison energy (eV)

Erythromycin Roxithromycin Salinomycin Tiamulin

11.4 12.9 24.1 13.6

734.5 837.5 768.7 496.6

576.5 679.5 733.6 192.3

)22 )25 )22 )27

eV eV eV eV

Table 2. RSD, LOD and LOQ of macrolides, salinomycin, and tiamulin in manuresa Antibiotic

RSD (%)

LOD (lg/kg)


36 15 22 15

1.0 0.8 3.2 0.4

LOQ (lg/kg) 3.4 2.7 10.7 1.4

a

LOD: S/N = 3:1 and LOQ: S/N = 10:1. More detailed information of the validated method is given in Schlsener et al. (2003a). LOD = Limit of detection. LOQ = Limit of quantification. RSD = Relative standard deviation.

Fig. 2. Concentration–time plots of the 4 antibiotics during an incubation of 16 single experiments in liquid manure: (a) erythromycin, (b) roxithromycin, (c) salinomycin, and (d) tiamulin. Each point is the average of 3 extractions of one 100-g batch et al. 2001; Pfeifer et al. 2002). The details of the procedure are described in Schlsener et al. (2003a). Quality-assurance data such as relative standard deviation, limit of quantification (LOQ), and limit of detection are listed in Table 2.

Results The concentrations (c) of erythromycin, roxithromycin, salinomycin, and tiamulin during the degradation experiment are displayed in Figure 2. To obtain detailed insight, the data are shown on a natural log scale in Figure 3. Each point is the

average of three replicate extractions of a single incubation. From these data, kinetic data such as half-lives are calculated and listed in Table 3. Erythromycin (Fig. 2a) shows a typical first-order degradation curve according to equation (1).

c ¼ c0 ekt

ð1Þ

The natural logarithm of the concentration divided by the starting concentration (c0) (ln c/c0) versus time (t) plot (Fig. 3a) shows a straight line with a good regression

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Fig. 3. Plots of the natural logarithm of the concentration/starting concentration (c/c0) versus time of the 4 antibiotics during an incubation of 16 single experiments in liquid manure: (a) erythromycin, (b) roxithromycin, (c) salinomycin, and (d) tiamulin. Each point is the average of 3 extractions of 1 100-g batch Table 3. Slope (k) of the linear regression, regression coefficient (R2), and half-life of macrolides, salinomycin, and tiamulin during manure storage Antibiotic

Slope

R2


)0.017 )0.005 )0.135 —

0.98 0.95 0.97 —

(Table 3). Thus, the degradation of erythromycin follows a first-order degradation. From Equation 2, a half-life of 41 days was calculated for erythromycin in manure:

t12 ¼

ln2 k

ð2Þ

The kinetics of roxithromycin (Fig. 2b) was similar to those of erythromycin but had a slower elimination. From the natural logarithm of the c/c0 versus time plot (Fig. 3b), a half-life of 132 days can be calculated by a kinetic expression. The degradation kinetics of salinomycin (Fig. 2c) are different than those of the macrolides. The concentration of salinomycin remained constant for approximately 4 days before significant elimination started. If the elimination had been caused by biodegradation, the microorganisms responsible for degradation would have needed a lag-phase to adapt to salinomycin before they were able to metabolize this compound. After 40 days, the concentration of salinomycin was below the LOQ of the analytic method. A first-order

Half-life (d) 41 € 1 130 € 10 5.1 € 0.3 >200

degradation and a half-life of 5 days could be calculated from the linear regression of the natural logarithm/time plot starting from day 4. The same experiment with tiamulin (Figs. 2d and 3d) produced completely different results. The concentration of tiamulin remained constant during the course of the entire experiment. No degradation was detectable even after 180 days. Tests for higher-order kinetics were applied to all four degradation plots, but no linear correlation could be fitted. The SD of the three replicates was in the same range or better as the SD determined during the validation procedure for this analytic method. The calculated half-lives and figures are based on the SD of the validated method.

Determination of Metabolites The extract from day 192, the extract of blank manure, and a standard solution were measured in a full scan HPLC-MS run in ESI positive mode (API 2000, Applied Biosystems, Darmstadt) with the same HPLC conditions described previously. For the sake of improved sensitivity, these experiments


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Fig. 4. Full scan HPLC-MS run of the extract from day 192. Metabolites of salinomycin could be detected at 19.7 and 20.1 minutes and a metabolite of erythromycin at 14.8 minutes. Residues of tiamulin (15.9 minutes) and roxithromycin (15.7 minutes) and an unknown at 20.7 minutes were noted. HPLC-MS: high-pressure liquid chromatography–mass spectrometry

Table 4. Measured mass of the three peaks at 19.7, 20.1, and 20.7 minutes of a high-resolution full-scan HPLC–ESI–TOF–MS run Time (min)

Measured mass (amu)

Suggested elemental composition

Monoisotopic mass (amu)

Difference to theoretical value (ppm)

19.7

509.3466 526.3726 531.3286 509.3482 526.3729 531.3293 524.3586 529.3149

[C29H48O7+H]+ [C29H48O7+NH4]+ [C29H48O7+Na]+ [C29H48O7+H]+ [C29H48O7+NH4]+ [C29H48O7+Na]+ [C29H46O7+NH4]+ [C29H46O7+Na]+

509.3473 526.3744 531.3292 509.3473 526.3744 531.3292 524.3488 529.3142

1.4 3.4 1.1 1.8 2.8 0.2 1.9 1.3

20.1

20.7

Suggested elemental composition, monoisotopic molecular weight of the suggested composition, and difference of the mass of the suggested elemental composition and measured mass in ppm. HPLC–ESI–TOF–MS = High-pressure liquid chromatography–electrospray ionization–time of flight mass spectrometer.

were performed on this instrument and not on the somewhat older TSQ 7000. All chromatograms were compared with each other. Four new peaks were determined in the extract of day 192 (Fig. 4) in comparison with the blank and standard solution. The same HPLC experiment with a high-resolution mass spectrometer with ESI (Bio TOF III, Bruker, Bremen, Germany) resulted in high-resolution mass spectra (HR-MS) of the chromatographic peaks at 19.7, 20.1, and 20.7 minutes. By means of ISOFORM Version 1.02 (National Institute for Standards and Technology) and the HR-MS data, the empirical formula of all three peaks could be determined (Table 4).

Applied Biosystems, Darmstadt, Germany) of the ammonia adduct of C29H48O7 (Fig. 5a) showed a similar spectrum as the product ion scan of the ammonia adduct of salinomycin (Fig. 5b). It seems that the newly formed metabolite resulted by a cleavage of salinomycin. VTrtesy et al. (1987) described in their work a microbial decomposition product of salinomycin with the same empirical formula. The peak at 20.7 minutes could be identified by HR-MS as C29H46O7 (Table 4). The concentration of this metabolite was too small for further mass spectrometric experiments.

Metabolites of Erythromycin Metabolites of Salinomycin Peaks at 19.7 minutes and 20.1 minutes were identified as two isomers of C29H48O7. The product ion scan (API 2000,

By means of two SRM transitions — 716 > 158 @ 45 eV and 716 > 558 @ 25eV — and the same retention time of a standard solution, the peak at 14.8 minutes was identified as


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Fig. 5. Product ion scan of the ammonia adduct of the metabolite of salinomycin, 526 (a), and the ammonia adduct of salinomycin, 768 (b)

anhydroerythromycin, a well-known metabolite of erythromycin (Goodman Gillman et al. 1990).

Discussion Before manure is spread on fields, the manure is stored up to 180 days in tanks in the region of North-Rhine-Westphalia, Germany. Erythromycin is used mostly as a one-time application in the beginning of the fattening phase of swine. A onetime application means one application during 10 to 20 days in the lifetime of a pig for the treatment of an infection. With a half-life of 41 days, the excreted erythromycin is up to 95% degraded during a 180-day storage in manure tanks. If the application of erythromycin is stopped a considerable time before the manure is applied to the fields, the contamination of the soil with high concentrations of this antibiotic can be prevented. Because the metabolite of erythromycin, anhydroerythromycin, has no antibiotic activity (Goodman Gillman et al. 1990) a risk for the environment for this metabolite is probably lower than that for the parent compound. Roxithromycin is not currently used as a veterinary antibiotic; however, it is still used in human medicine. This leads to significant concentrations in wastewater (Gçbel et al. 2004). This degradation experiment with liquid manure may give some basic insight into the behavior of roxithromycin in sewage treatment, especially in the anaerobic processes in these plants, i.e., the digester. As a result of an incomplete elimination of roxithromycin in the digester, roxithromycin may enter the environment if digested sludge is used as fertilizer on agricultural fields. Salinomycin shows a rapid degradation with a half-life of 5 days; therefore, the application of salinomycin may be less problematic. However, salinomycin is used as a feed additive

for the prevention of the coccidiosis and for growth promotion. Growth promoting is the continuous application of an antibiotic to promote weight gain in the fattening phase of pigs. In both cases, a continuous flow of freshly excreted salinomycin comes from the stables to the manure tank. A 99% degradation of the respective antibiotic in the manure tanks requires approximately 38 days. Thus, if there is not enough time for the complete degradation of salinomycin, the soil may be contaminated with salinomycin if this manure is dispersed on the fields. The newly formed metabolite of salinomycin has, according to VTrtesy et al. (1987), no antibiotic activity and is no longer capable to complex sodium or potassium. However, there has been no full-risk evaluation of this compound yet. In contrast, tiamulin showed no degradation. When manure containing tiamulin is spread on the fields, the soil will be contaminated. These conclusions are coherent with other studies (Schlsener et al. 2003a, 2003b) in which tiamulin and salinomycin were determined in liquid manure with concentrations of 11 lg/kg and 43 lg/kg, respectively, 2 months after application. Also, tiamulin was detected in soil that was fertilized with manure that contained tiamulin a considerable time before sampling (Schlsener et al. 2003b). Haller et al. (2002) and Tolls (2001) demonstrated that the antibiotics tylosin, sulfonamides, and tetracyclines are persistent in soil and manure as well. Gavalchin and Katz (1994) reported the half-life of chlortetracycline to be >30 days, whereas that of tylosin was 5 days in a manure–soil matrix under aerobic conditions at 20°C. Also, Hamscher et al. (2002) and Pfeifer et al. (2002) found high concentrations of tetracycline and sulfonamides in liquid manure several months after the antibiotics had been administered to the pigs. From the data presented here, it seems that the use of some macrolides and polyether antibiotics would be preferable to sulf-

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onamides and tetracyclines, and tiamulin should be avoided, if environmental aspects are taken into account.

Conclusion In this study, it was demonstrated that tiamulin, which is a pleuromutilin compound, is persistent in manure. Its use should be avoided if environmental issues are taken in consideration. Additionally, it was shown that macrolides, as well as the polyether antibiotic salinomycin, are degraded under the conditions prevalent in manure tanks. Metabolites of salinomycin and erythromycin were detected in this experiment, and a new metabolite was found. However, some questions about the persistence of these metabolites remain, and more basic discussions on the use of antibiotics in industrial agriculture should take place. Further degradation experiments of other antibiotics, such as sulfonamides and tetracyclines, in liquid manure under anaerobic conditions are necessary to create guidelines for farmers that will give sufficient time between termination of antibiotic use in their livestock and manure spreading.

Acknowledgments. The authors are indebted to the farmer in NorthRhine-Westphalia who cooperated in the manure sampling and provided data on the application of antibiotics during conventional farm management. The HR-MS measurement of W. Karow, MS facility of the University Duisburg-Essen, is also acknowledged. This study was supported by the Ministry for Education, Science and Research of NRW, Germany.

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