Bioresource Technology 101 (2010) 8581–8586

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Bioresource Technology

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The effect and fate of antibiotics during the anaerobic digestion of pig manure

J.A. Álvarez *, L. Otero, J.M. Lema, F. OmilDepartment of Chemical Engineering, School of Engineering, University of Santiago de Compostela, Rúa Lope Gómez de Marzoa, 15782 Santiago de Compostela, Spain

a r t i c l e i n f o

Article history:Received 9 March 2010Received in revised form 10 June 2010Accepted 17 June 2010

Keywords:Oxytetracycline (OTC)Chlortetracycline (CTC)ManureAnaerobic methane production

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.06.075

* Corresponding author. Tel.: +34 981563100x1601E-mail address: juanantonio.alvarez@usc.es (J.A. Á

a b s t r a c t

This work studies the inhibitory effects and fate of the antibiotics oxytetracycline (OTC) and chlortetra-cycline (CTC) during the anaerobic digestion of pig manure. Both substances were added together inbatch assays at concentrations of 10, 50 and 100 mg L�1. Control assays only with antibiotics (abiotic)as well as without antibiotics (biotic) were also conducted. Methane production was reduced by 56%,60% and 62% at OTC and CTC concentrations of 10, 50 and 100 mg L�1, respectively. The IC50 level calcu-lated from these experiments was estimated to be around 9 mg L�1, a significant value considering thereported concentrations of these compounds in pig manure samples (up to 136 mg L�1). Strong adsorp-tion to solid matter was observed, which increased the stability of both substances. Antibiotic degrada-tion was thus much higher in control assays, without solids, than those determined from assays includinginoculum and manure substrate.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Antibiotics are widely used in pig farms to prevent infections,treat diseases as well as growth promoters (Kemper, 2008).According to the Animal Health Institute (AHI Animal HealthInstitute, 2008), antibiotics used for animal feeding in the UnitedStates (US) have increased from nearly 91 Mg in 1950 to9900 Mg (including 3000 Mg of tetracyclines) in 2004, 60–80% ofwhich were used for non-therapeutic purposes (AHI Animal HealthInstitute, 2008). In the European Union, 5000 Mg of antibioticswere used in 1999 for veterinary therapy (Sarmah et al., 2006).

Once ingested by an animal, these compounds can be metabo-lised following different pathways. They are eventually excreted,maintaining the same chemical structure or as metabolites thathave been transformed into epimers or isomers (Kemper, 2008).Between 17% and 76% of antibiotics administered to animals areexcreted via urine and faeces in an unaltered form or as metabo-lites of parent compounds (Jjemba, 2002). Previous studies havereported that bacterial activity may be inhibited by antibioticmetabolites produced in the gastrointestinal tract rather than bythe original molecule (Massé et al., 2000).

Oxytetracycline (OTC) is a common antibiotic with a broadrange of activity and low cost. OTC is administered to livestock ani-mals (including cattle, swine, poultry and fish) to promote growthand for prophylactic and therapeutic treatment. Chlortetracycline(CTC) is another antibiotic that is widely used in livestock produc-tion because it is active against a broad range of gram-positive andgram-negative bacteria. 4-epi-Oxytetracycline (EOTC) and 4-epi-

ll rights reserved.

6; fax: +34 981528050.lvarez).

Chlortetracycline (ECTC) are degradation products and epimers ofOTC and CTC, respectively. These compounds are strongly adsorbedin manure samples because they create complexes with metal ions,humic acids, proteins, particles and organic matter in the manurematrix (Loke et al., 2002, 2003).

The overuse of antibiotics in livestock feed leads to high concen-trations of antibiotics and their metabolites in manure, whichcould eventually be released into the environment. OTC and CTCconcentrations in manure samples have been reported to be inthe range of 0.006–136 and 0.003–46 mg L�1, respectively(Martínez-Carballo et al., 2007; Winckler et al., 2003; Choi,2007). The final release of antibiotics to the environment is of con-siderable concern because persistent antibiotic residues may leadto the development of antibiotic-resistant bacteria (Chee-Sanfordet al., 2001). In the last decade, a large group of pharmaceuticalsubstances have been detected in wastewater treatment plants(Carballa et al., 2008; Suarez et al., 2008), as well as the presenceof antibiotics or antibiotic metabolites in manure can inhibit thedigestion activity of anaerobic bacteria (Arikan et al., 2006; Arikan,2008).

Although some researchers have studied the fate of antibioticsin specific environmental compartments, such as soil interstitialwater or anaerobic lagoons (Kemper, 2008), and in different biolog-ical processes (Arikan et al., 2007), there is a little informationregarding the effect and removal of antibiotics during the anaero-bic digestion of manure (Arikan et al., 2006; Arikan, 2008).

Thus, the objective of this work was to study the inhibitory ef-fect of these compounds on methane production during anaerobicdigestion of pig manure and the removal kinetics and fate of OTCand CTC under anaerobic conditions using discontinuous batchassays.

Table 1Experimental design of the batch assays carried out in this work.

Assay Inoculum(g VSS L�1)

Manure(g COD L�1)

CTC(mg L�1)

OTC(mg L�1)

Inoculum + manure control 2 2 0 0Antibiotic control 0 0 50 50Inoculum + manure + Antibiotic 2 2 10 10Inoculum + manure + Antibiotic 2 2 50 50Inoculum + manure + Antibiotic 2 2 100 100

8582 J.A. Álvarez et al. / Bioresource Technology 101 (2010) 8581–8586

2. Methods

2.1. Chemicals

OTC (CAS no. 2058-46-0, C22H24N2O9, MW: 460.4), 4-EOTC (CASno. 14206-58-7, C22H24N2O9, MW: 460.4), CTC (CAS no. 64-72-2,C22H23ClN2O8, MW: 478.9) and 4-epi-chlortetracycline (CAS no.101342-45-4, C22H23ClN2O8, MW: 478.9) were purchased (97–100% purity) from Acros Organics N.V. (Fair Lawn, NJ). All other re-agents used in this study were of analytical grade. The water usedin the experiments was of Milli-Q quality. McIlvaine buffer wasprepared by mixing aqueous solutions of 0.1 mol L�1 citric acidand 0.2 mol L�1 disodium hydrogen phosphate (62:38, v/v). Meth-anolic oxalic acid (0.01 mol L�1) was prepared by dissolving oxalicacid in methanol.

2.2. Batch assay methodology

Pig manure was collected from a sewer of a 150-pig fattenerand sow farm, where both faeces and urine had accumulated. Man-ure VSS (volatile suspended solids) and COD (chemical oxygen de-mand) concentrations were 11.7 and 28.9 g L�1, respectively. Batchassays were carried out in 500-mL glass flasks with coiled butylrubber stoppers. All tests were performed in duplicate assays un-der the following operating conditions: 35 �C, 120 rpm, 2 g VSS L�1

of inoculum (granular biomass from an anaerobic internal circula-tion digester (IC) treating brewery wastewater) and 2 gCOD L�1 ofmanure as assay substrate. OTC and CTC were not initially presentin pig manure samples and were both added to assays at concen-trations of 10, 50 and 100 mg L�1. Control assays without antibiot-ics (only with inoculum and substrate) and with only antibiotics(without inoculum and substrate) were also performed to evaluatethe biomass methanogenic activity without antibiotics and todetermine the stability of those compounds under abiotic anaero-bic conditions, respectively.

Anaerobic conditions were maintained by using an anaerobicbasal medium composed of cysteine (0.5 g L�1) and NaHCO3

(5 g L�1) at a pH of 7.0–7.2. Before flushing the liquid and head-space with N2, 1.2 mL of Na2S (20 g L�1) was added to each assayas a reducing agent (Molina et al., 2008). An initial volume of385 mL was used in all assays. A pressure transducer was used tomeasure increase in pressure. The biogas was sampled regularlyat days 0, 7, 14, 21 to determine methane production. Biogas com-position (N2, CH4, CO2 and H2S percentage) was analysed by gaschromatography (HP, 5890 Series II) equipped with a thermal con-ductivity detector (Molina et al., 2008). The detailed characteristicsof the experimental design are shown in Table 1.

2.3. Extraction of OTC, EOTC, CTC and ECTC

Concentrations of water-soluble and total (soluble and solid-extractable) OTC, EOTC, CTC and ECTC in batch assay samples weredetermined in duplicate. Solid-extractable concentrations werecalculated as the difference between total and water-soluble con-

centrations. To determine water-soluble concentrations of OTC,EOTC, CTC and ECTC, 5 mL samples were centrifuged (5000 rpm,20 min), then 0.5 mL aliquots of the supernatant were diluted(1:8) in a separate tube and vortexed for 30 s. The mixture wastransferred to a 2-mL amber autosampler vial and analysed byLC–MS.

To measure total concentrations of OTC, EOTC, CTC and ECTC, amethod was adapted from Capone et al. (1996). Briefly, 2 mLsamples were extracted three times with 3 mL of 0.1 mol L�1

Na2EDTA-McIlvaine buffer (pH 4) by vortexing for 30 s followedby sonication for 5 min in a 100 W sonication bath (Bronson Ultra-sonics, Danbury, CT). After each extraction, the extracts were sub-jected to centrifugation (5000 rpm, 5 min), the supernatants werepassed through pre-washed Waters 60-mg HLB (hydrophilic–lipo-philic balance) Oasis� cartridges (Waters Corp., Milford, MA) toconcentrate antibiotics and remove the extraction buffer. The car-tridges were pre-washed with 5 mL of methanol followed by 10 mLof 0.1 Mol L�1 Na2EDTA-McIlvaine buffer. After the extracts wereloaded, the cartridges were flushed with 20 mL distilled water with8 mL of 0.01 M methanolic oxalic acid. The eluents were diluted(1:10) and vortexed for 30 s. The resulting mixtures were trans-ferred to 2 mL amber autosampler vials and analysed by LC–MS/MS.

2.4. LC–MS/MS analysis

Concentrations of OTC, EOTC, CTC and ECTC were quantifiedusing an 1100 Agilent LC–MS/MS with a ZORBAX Eclipse XDB-C18 column (2.1 mm � 150 mm). The injection volume was 2 lL.A mobile-phase gradient was necessary to separate the com-pounds. The respective compositions of solvents A and B were0.1% aqueous formic acid and methanol, respectively. The solventsgradient increased from 0% to 20% B from 0–23 min, then rampedback to 0% B at 33 min. The flow rate was 0.20 mL/min. Six-pointcalibration curves, determined in water for water-soluble samplesand in methanol for total concentrations, were generated by inject-ing 2 lL of standard solutions ranging from 0.2 to 1.5 mg L�1.

The method detection limits (MDL) were estimated based onthe intensity of standard solutions that represented a signal-to-noise (S/N) level of 3/1. The minimum level (ML) of quantificationwas calculated as 3.18 times the MDL. The ML for OTC, EOTC, CTCand ECTC was 0.04 mg L�1. Atmospheric pressure ionisation-tan-dem mass spectrometry was performed on a benchtop triple quad-rupole mass spectrometer (model API4000 from AppliedBiosystems) operated in electrospray ionisation mode. The sourceparameters were as follows: capillary voltage was set at 5.0 kVand source temperature was 600 �C. Nitrogen was used as colli-sion-induced decomposition gas to fragment the parent ions. Theparent and daughter ions used for compound identification andquantitation were 461.1/426.1 for OTC and EOTC and 479.1/444.1for CTC and ECTC. Optimisation was performed by direct infusionof the standards from a syringe pump (10 lL min�1) mixed withthe LC effluent (100% A; 200 lL min�1). The detector was a photo-multiplier set at 1900 V. Analyte concentrations were calculated bythe external standard method.

2.5. Determination of recoveries and kinetic calculations

To determine extraction efficiencies, duplicate assays of 2 and5 g VSS L�1 of inoculum and 2 and 5 g COD L�1 of manure werespiked with 10 mg L�1 of OTC and CTC. The assays were incubatedfor 10 min and OTC and CTC were measured as described above.OTC recovery results were 80.8% and 87.4% for samples of 2 and5 g L�1, respectively. In the same way, CTC recovery results were83.8% and 81.0% for samples of 2 and 5 g L�1, respectively. In gen-eral, OTC recoveries were higher than CTC recoveries, as conse-

Table 2Summary of methane reduction in manure degradation assays in the presence of OTCand CTC.

Compound Concentration(mg L�1)

CH4 productiondecrease (%)

Reference

OTC 125 No inhibition Lallai et al.

J.A. Álvarez et al. / Bioresource Technology 101 (2010) 8581–8586 8583

quence that CTC has a higher transformation rate (Loftin et al.,2008).

First-order kinetic model was used to describe OTC and CTC re-moval under the different conditions tested. Regression coeffi-cients and degradation rate constants were calculated for bothcompounds.

(2002)250OTC 3.1 27 Arikan et al.

(2006)CTC 5 20 Sanz et al.

(1996)40 50152 80

OTC 1 2 Loftin et al.(2005)5 5

25 7CTC 1 32

5 3325 44

OTC andCTC

10 45.2 This work50 56.5

100 64.1

3. Results and discussion

3.1. Effect of OTC and CTC concentrations on methane production

Batch assays were incubated at 35 �C for 31 days. Methane pro-duction in each assay is shown in Fig. 1. The addition of 10 mg L�1

of OTC plus 10 mg L�1 of CTC resulted in a significant reduction ofmethane production during anaerobic digestion of pig manure. Inthis sense, the increase of the concentration of both substances,up to 50 and 100 mg L�1 caused a further increase but not so pro-nounced as observed with the lowest concentration. Methane pro-duction reduction according to manure control assay was 45.2%,56.5% and 64.1% when 10, 50 and 100 mg L�1 of both antibioticswere added, respectively, which corresponds to a reduction of itsspecific methanogenic activity of 56.2%, 59.8% and 62.3%, respec-tively. As expected, the maximum decrease of methane productionand methanogenic activity (64.1% and 62.3%, respectively) oc-curred in the assay containing the highest concentration of bothantibiotics (100 mg L�1).

Previous studies have reported reduction in methane produc-tion during anaerobic digestion of pig manure under the presenceof these compounds (Sanz et al., 1996; Arikan et al., 2006). In thiswork both antibiotics were tested together, so that their combinedeffect was studied. Table 2 summarises these results together withothers from previous reports. There is some disagreement aboutthe inhibitory effect exerted by these compounds. With concentra-tions such as 3.1 mg OTC L�1, Arikan et al. (2006) showed a 27%reduction in CH4 production. However, some studies have reportedan absence of inhibition even at OTC concentrations up to 125–250 mg L�1 (Lallai et al., 2002). Most reports indicated a slightlyhigher reduction of methane production caused by CTC than thatobserved by OTC. Sanz et al. (1996) and Loftin et al. (2005) reportedthat the methane production was reduced from 20% to 80% whenthe concentrations of CTC increased from 2 to 150 mg L�1. Theseinconsistencies might be the result of the different histories ofthe sludges used as well as the operational conditions maintainedin each work, including inoculum and manure source, inoculum/manure ratio, antibiotic concentration, reactor size, batch or con-tinuous operation, etc. For instance, the absence of inhibition re-ported by Lallai et al. (2002) might be the result of theacclimation experienced by their inoculum, since it was collected

0.0

0.10.2

0.30.4

0.50.6

0 5 10 15 20 25 30 35Time (d)

CH

4-C

OD

(g)

Manure control 10 OTC + 10 CTC50 OTC + 50 CTC 100 OTC + 100 CTC

Fig. 1. Methane production in the executed assays (g CH4-COD). OTC and CTC wereadded to the assays at 10, 50 and 100 mg L�1. Values are the means from duplicateassays and SD are shown as error bars.

from the biomass of an anaerobic digester that was treating pigmanure containing antibiotics.

In this study, the combined addition of CTC and OTC resulted inslightly higher reductions of methane production than those re-ported in bibliography when CTC and OTC were spiked separately.

The IC50 value for these compounds was estimated from thecorrelation between the methanogenic activity and their initialconcentration, using lineal interpolation method. An initial con-centration of around 8.9 mg L�1 of both antibiotics reduced meth-anogenic activity by 50%. Taking into account that OTC and CTChave been reported to be in pig manure at concentrations up to136 and 46 mg L�1, respectively (Martínez-Carballo et al., 2007;Winckler et al., 2003); which are several order of magnitude higherthan the IC50 values determined in this work, potential inhibitionproblems during the anaerobic treatment of pig manure shouldbe seriously taken into account.

3.2. Antibiotic fate during anaerobic digestion

The evolution of the concentrations of OTC and CTC and theirmetabolites during anaerobic assays are shown in Fig. 2. Significanttransformations of the parent compound appeared to occur alreadyduring an approximate hour that elapsed between the addition ofthe tetracyclines to the vials and the extraction of the first sample.This is especially significant in the case of CTC, so that the amountof the intermediate in the first sample was almost 50% of the con-centration of the parent compound.

Fig. 2 shows the distributions of the different compounds in thewater and solid phases, as well as their total concentrations. TotalOTC concentrations in the different assays decreased from 13.5,56.9 and 95.0 mg L�1 at day 0 to 5.7, 26.6 and 30.7 at day 21,respectively (Fig. 2A). Different trends in OTC concentration wereobserved for the water and solid phase. In the case of water-solubleOTC, concentrations decreased during the assay period, whereassolid-extractable OTC concentrations remained almost constantthroughout the 21 days of the assays (Fig. 2A).

Furthermore, the metabolite EOTC was produced in all assaysduring the first 7 days, reaching maximum concentrations of 0.9,3 and 5 mg L�1 in the 10, 50 and 100 mg OTC L�1 assays, respec-tively. The degree of epimerisation of the added OTC thus was 5–10%, since no OTC was present in the initial pig manure. After7 days, EOTC was removed from the water-soluble fraction, butthe concentration remained constant in the solid phase during

OTC (10 mg/L)

0

5

10

15

20

mg

/L

OTC (50 mg/L)

0

20

40

60

80

mg

/L

OTC (100 mg/L)

020406080

100120

0 7 14 21Time (d)

mg

/L

Total OTC water-soluble OTC solid-extractable OTC

4-epi OTC (10 mg/L)

0.0

0.5

1.0

1.5

4-epi OTC (50 mg/L)

0

1

2

3

4

4-epi OTC (100 mg/L)

0

2

4

6

8

0 7 14 21Time (d)

Total EOTC water-soluble EOTC solid-extractable EOTC

CTC (10 mg/L)

0

5

10

15

mg

/L

CTC (50 mg/L)

0

20

40

60

mg

/L

CTC (100 mg/L)

020406080

100

0 7 14 21Time (d)

mg

/L

Total CTC water-soluble CTC solid extractable CTC

4-epi CTC (10 mg/L)

0

2

4

6

8

4-epi CTC (50 mg/L)

0

10

20

30

4-epi CTC (100 mg/L)

0102030405060

0 7 14 21Time (d)

Total ECTC Water-soluble ECTC solid-extractable ECTC

OTC and CTC control assay

01020304050

0 7 14 21Time (d)

mg

/L

OTC EOTC CTC ECTC

A

B

C

Fig. 2. Solid-extractable, water soluble and total concentrations of measured antibiotics during anaerobic discontinuous assays. A: OTC and EOTC; B: CTC and ECTC; C:antibiotic concentration during the 50 mg L�1 OTC and CTC spike control assay at pH 7 and 35 �C. Values are the means from duplicate assays and SD are shown as error bars.

Table 3First-order kinetic degradation rate constant (k) and adjustment regression coefficient(r2) of OTC and CTC degradation.

Compound Assay antibiotic concentration (mg L�1) k (d�1) r2

OTC 10 0.052 0.90250 0.045 0.856

100 0.058 0.966CTC 10 0.184 0.986

50 0.216 0.983100 0.169 0.974

8584 J.A. Álvarez et al. / Bioresource Technology 101 (2010) 8581–8586

the 21 days of incubation (Fig. 2A), similar to the behaviour of theparent compound.

Total CTC concentration decreased from 9.8, 46.1 and74.0 mg L�1 at day 0 to 0.9, 4.0 and 7.5 mg L�1 at day 21, respec-tively (Fig. 2B). The rate of transformation appeared to be morerapid than that observed for OTC. This difference is more pro-nounced in the water phase, where CTC concentrations werereduced down to 0.3, 2.0, 5.8 mg L�1 at day 7 for the 10, 50, and100 mg L�1 assays, respectively (Fig. 2B). CTC concentrations alsodecreased in the solid fraction, although at a slower rate than that

J.A. Álvarez et al. / Bioresource Technology 101 (2010) 8581–8586 8585

observed in the aqueous phase. The epimerisation of CTC occurredfaster and more thoroughly than that observed for OTC (Fig. 2).Finally, ECTC disappeared at high rate, being almost completelyremoved at the end of the operation (Fig. 2B).

These results are consistent with those obtained by Arikan et al.(2006) and Arikan (2008), who reported a significant removal ofthe parent compounds during the first 10 days of incubation. Inthe following days, OTC degradation occurred over a very long time(60–70 days) whereas CTC removal continued at much slower rate.Those authors also found a similar trend of biotransformation be-tween the parent and the intermediate compounds (EOTC andECTC), as well as the removal of these intermediates. However,those authors do not guarantee the absence of intermediates inthe initial manure used for their assays.

Furthermore, Fig. 2 shows that at higher initial concentrationsof OTC and CTC in the assays, it was observed higher concentra-tions of the compounds into the water-soluble fraction. On theother hand, the fraction that was adsorbed onto the sludge and so-lid matter, around 18–20 mg OTC L�1 and 25–27 mg CTC L�1, wasthe same in the 50 and 100 mg L�1 assays and remained constantthroughout the incubations. As the inoculum and manure concen-tration used for all assays were 2 g VSS L�1 and 5 g COD L�1,respectively, the adsorption of these substances seems to be lim-ited by the available superficial area of the inoculum and pig man-ure in the assays.

The persistence of the fraction of both compounds associatedwith solids, especially significant in the case of OTC, might be ex-plained by their ability to form strong complexes with divalent cat-ions, which are abundant in pig manure, as well as their capacity tobe adsorbed onto proteins, particles and organic matter (Loke et al.,2003). Fig. 2B clearly shows that CTC removal first occurred in theliquid phase. Once completed, the concentration in the solid phasebegan to decrease while the liquid phase concentration was main-tained at a near zero concentration. These data suggest that CTCdesorption from the solid phase was the rate-limiting step.

3.3. Antibiotic fate in abiotic control assays

The results obtained from abiotic control assays indicated thatboth antibiotics are extremely unstable. Fig. 2C shows that at35 �C and pH 7, OTC and CTC were almost totally removed fromthe medium in a very short period of time (40% and 60% for OTCand CTC removal in the first hour, respectively). Both EOTC andECTC were detected and quickly degraded. After 7 days, only 6%of the initial amount of OTC remained in the assay, having been de-graded or transformed the whole amount of CTC (Fig. 2C). Thisunstable behaviour was also observed by Loftin et al. (2008),who conducted discontinuous assays with OTC and CTC separatelyand reported half-life times lower than those obtained in this workat pH 7 and 35 �C.

3.4. First-order kinetic model

Table 3 shows the degradation rate constants and regressioncoefficients of first-order kinetic equations used to describe thedegradation of both compounds in the different assays.

The degradation constants determined in this work differ fromprevious estimates of Arikan et al. (2006) and Arikan (2008), whoreported lower first-order degradation constants (0012 and0039 d�1 for OTC and CTC, respectively). This inconsistency mightbe caused by the higher organic matter content in the assays(50 g L�1 of cattle manure), which could have increased the stabil-ity of both compounds due to their strong adsorption onto the solidfraction.

4. Conclusions

The antibiotics CTC and OTC cause a significant inhibition on theanaerobic digestion of pig manure, being the IC50 in the range of9 mg L�1, much lower than the highest concentrations reportedin this kind of wastes (136 mg L�1). Both compounds exert a highaffinity for suspended solids, which cause that the fraction avail-able in the liquid phase is low during anaerobic treatment and,consequently, the time needed for biodegradation increases, beingthe rate-limiting step the degradation of the fractions present inthe solid phase. Their degradation has been properly describedby a first-order kinetic model.

Acknowledgements

This work was supported by project CTQ2007-66265/PPQ/MICROFARM and PSE-120000-2007-16/ PROBIOGAS of the Minis-try of Education and Science of Spain, as well as by a postdoctoralcontract (Juan de La Cierva) to Dr Juan A. Álvarez, funded by Min-istry of Education and Science of Spain.

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