The behavior of tetracyclines and their degradation products during swine manure composting

Bioresource Technology 102 (2011) 5924–5931

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The behavior of tetracyclines and their degradation products during swine manure composting Xiaofeng Wu a, Yuansong Wei a,⇑, Jiaxi Zheng a, Xin Zhao b, Weike Zhong b a b

Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, Beijing 100085, PR China Chinese Academy of Inspection and Quarantine, Beijing 100123, PR China

a r t i c l e

i n f o

Article history: Received 29 October 2010 Received in revised form 3 March 2011 Accepted 3 March 2011 Available online 9 March 2011 Keywords: Swine Manure Composting Degradation product Degradation kinetic Tetracyclines

a b s t r a c t The purposes of this study were to investigate the behavior of three tetracyclines including chlortetracycline (CTC), oxytetracycline (OTC) and tetracycline (TC) and their degradation products in a pilot scale swine manure composting, and also to study the degradation kinetics of CTC, OTC and TC. During the pilot scale composting, CTC, OTC and TC were degraded by 74%, 92% and 70%, respectively. Several degradation products were found like 4-epitetracycline (ETC), 4-epioxytetracycline (EOTC), 4-epichlortetracycline (ECTC), demeclocycline (DMCTC) and anhydrotetracycline (ATC). Both the simple and the adjusted first-order kinetic models successfully fit the degradation process of CTC, OTC and TC during the composting, but the adjusted first-order kinetic model fit much better with the calculated half-lives of 8.2, 1.1 and 10.0 days, respectively. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Veterinary antibiotics are extensively used at therapeutic levels in Concentrated Animal Feeding Operations (CAFOs) to treat and prevent diseases and at sub-therapeutic levels to promote animal growth and feed efficiency (Kemper, 2008; Phillips et al., 2004; Sarmah et al., 2006). For example, approximately 70% of the estimated 16 million kg of antibiotics consumed annually in the USA have been used for non-therapeutic purposes in 2000 (Sarmah et al., 2006). Since the administered veterinary antibiotics are poorly absorbed in animal tissues, as much as 40–90% of antibiotics (Halling-Sørensen et al., 1998; Kemper, 2008; Kumar et al., 2005; Phillips et al., 2004; Winckler and Grafe, 2001) are excreted in the form of their parent compounds or metabolites via urine and feces that end up in animal manure. Tetracyclines such as tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) are among the most common antibiotics used in animal husbandry (De Liguoro et al., 2003; Kumar et al., 2005; Sarmah et al., 2006). It is estimated that as high as 3000 tons of tetracyclines were produced in 2003 for both farm and companion animals in the United States (Arikan et al., 2007; Bao et al., 2009). High tetracyclines residues in animal

⇑ Corresponding author. Tel./fax: +86 010 62849109. E-mail address: [email protected] (Y. Wei). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.03.007

manure due to the wide usage and incomplete absorption can be detected in the level of mg/kg or even up to several hundreds of mg/kg (Bao et al., 2009; De Liguoro et al., 2003; Shen et al., 2009a; Zhao et al., 2010) and thus pose an increasing potential risk to human health and ecosystem safety with the application of manure as fertilizers in agricultural lands (Boxall et al., 2003; Kummerer, 2003). Therefore, it is necessary and important to treat and dispose animal manure before its land application to reduce the amount of veterinary antibiotics released into the environment and minimize the risk of the widespread development of resistant bacteria derived from residual antibiotics (Kemper, 2008; Sarmah et al., 2006). As an established and well-developed technology for stabilization of organic matter and reduction of pathogens and odors, composting is widely applied in animal manure treatment and reclamation, and the compost of animal manure is commonly used as soil conditioner and fertilizer in agriculture and forestry. Recently, it has been proved to be a feasible and effective approach to promote removal of antibiotics in animal manure (Arikan et al., 2007; Bao et al., 2009; Dolliver et al., 2008; Kakimoto and Funamizu, 2007; Kakimoto et al., 2007; Ramaswamy et al., 2010; Shen et al., 2009b). For instance, Arikan et al. (2007) presented that within the first six days of beef manure composting, levels of oxytetracycline in the compost mixture decreased dramatically and achieved a 95% reduction. In Dilliver’s research (Dolliver et al., 2008), the concentration of chlortetracycline during manure composting declined rapidly, with over 99% reduction occurred in less

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than 10 days. Tylosin and monensin exhibited a gradual decline over time with removal rates ranging from 54% to 76%. Ramaswamy et al. (2010) found that more than 99.8% removal of salinomycin was achieved during the 38-day composting period. All the mentioned results above clearly showed that composting is effective to remove the residual antibiotics in animal manure. Factors of possibly affecting removal behavior of tetracyclines such as temperature, moisture and other abiotic conditions are of great attention (Bao et al., 2009; Dolliver et al., 2008; Wang and Yates, 2008). However, there is litter information on their fate concerning about the degradation products and degradation process during animal manure composting. It is known that generation of tetracyclines’ degradation products depends on different pH conditions because their molecular structure contains four connected benzene rings (lettered A through D from right to left) with multiple ionizable functional groups (Chen and Huang, 2009) as shown in Fig. 1. For instance, the 4-epimers such as 4-epitetracycline (ETC), 4-epioxytetracycline (EOTC) and 4-epichlortetracycline (ECTC) can be reversibly formed under mildly acidic conditions (pH 2–6). Strong acidic conditions (pH < 2) facilitate the formation of anhydrotetracyclines that could transform to their corresponding epimers in the same way. While the majority of anhydro-tetracyclines are stable, anhydro-oxytetracycline is quite unstable and quickly forms a-OTC and b-OTC. Although degradation products of tetracyclines once formed are not as active as their parent compounds, several products have potency at the same concentration level as their parents on environmentally relevant sludge and soil bacteria and some are even more toxic than their parents (Halling-Sorensen et al., 2002). Moreover, some metabolites can also be transformed back to the parent compounds (Kemper, 2008; Sarmah et al., 2006). However, there is lack of knowledge involving degradation products of tetracyclines as most researchers usually just focus on the degradation behavior of the parent compounds (Bao et al., 2009; Dolliver et al., 2008; Wang and Yates, 2008) and degradation products have not been regularly investigated. OTC or CTC and their degradation products in soil interstitial water have been reported (Halling-Sørensen et al., 2003; Søeborg et al., 2004), but during animal manure composting, only Arikan et al. (2009) contributed to the fate of CTC and concerned about the degradation products in a laboratory scale. Degradation processes of the other two common tetracyclines during animal manure composting have not been the subject of research recently. In this study, the behavior of three tetracyclines such as tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) as well as their potential degradation products were investigated during a pilot scale swine manure composting in the spring. Moreover, the degradation kinetics of the three parent tetracyclines were also studied and compared on the basis of both the simple and adjusted first-order kinetic model.

2. Methods 2.1. Chemicals and standards The three target parent tetracyclines analyzed in this study including tetracycline hydrochloride (TC, 97.5% purity, CAS No. 64-75-5), oxytetracycline hydrochloride (OTC, 98.5% purity, CAS No. 2058-46-0) and chlortetracycline hydrochloride (CTC, 99.0% purity, CAS No. 64-72-2) were purchased from Dr. Ehrestorfer Co. (Germany). Degradation products of these three parent tetracyclines with high purity (>97%) including 4-epitetracycline (ETC), anhydrotetracycline (ATC), 4-epianhydrotetracycline (EATC), 4-epioxytetracycline (EOTC), a-apo-oxytetracycline (a-apo-OTC), b-apo-oxytetracycline (b-apo-OTC), 4-epichlortetracycline (ECTC), demeclocycline (DMCTC) were obtained from Acros Organics (Geel, Belgium). All the stock solutions of these standards above were dissolved in methanol and stored at 20 °C in the darkness. Acetonitrile, methanol and ethyl acetate from J.T. Baker Co. (USA) were all of HPLC-grade. The HPLC-grade formic acid (88% purity, CAS No. 64-16-6) was purchased from Mallinckrodt Baker Inc. (USA). All of the following chemicals, including disodium hydrogen phosphate dodecahydrate, citric acid monohydrate and ethylenediamineteraacetic acid disodiumsalt (Na2EDTA), were of analytical pure grade. The ultra pure water was supplied by a Millipore Milli-Q system. 2.2. The pilot scale swine manure composting A pilot scale of swine manure composting was carried out in the spring to further investigate the fate behavior of tetracyclines and their degradation products. It was operated in the spring from March 16 to May 6, 2010. Raw materials for composting as shown in Table 1 were made up of swine manure and mushroom residues mixed at the ratio of 1:2 (v/v). The pilot scale composting operated in an open windrow system was mixed and turned manually every week. Meanwhile, water was irregularly added so as to maintain stable moisture content of the mixtures during the composting. The pile was about 5 m3 at the length of 5 m and the height of 1 m. Samples were taken on days 1, 4, 7, 14, 21, 28, 35 and 52, respectively, and collected by mixing the upper, central and lower portion of the three sites uniformly distributed in the pile to achieve high representativeness and homogeneity as possible, and then obtained by quartation. All the samples taken were stored at 20 °C for further analysis. 2.3. Analytical procedures Parameters of all composting materials including pH, electrical conductivity (EC), moisture content, organic matter, TKN, TP were Table 1 Characteristics of raw materials in the pilot scale swine manure composting. Characteristics

pH Moisture (%) EC (lS/cm) OM (%) TKNa (g/kg) TPa (g/kg) C:N

Fig. 1. Chemical structure and property of tetracyclines (TCs) (Chen and Huang, 2009).

Materials Swine manure

Mushroom residues

Mixture

7.68 66.3 1236 64.3 30.36 12.01 9.01

7.66 25.1 1580 87.0 24.82 4.69 16.77

8.46 56.9 1031 77.9 29.97 11.93 11.02

EC: electrical conductivity; OM: organic matter; TKN: total kjeldahl nitrogen; TP: total phosphorus. a Measured based on dry matter.

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analyzed according to the methods (Bao, 2000). The evolution of temperature that concerns about ambient temperature and temperature of the pile including upper, central and lower part was recorded every day. To determine tetracyclines and their degradation products, samples stored at 20 °C were firstly thawed, freeze-dried (ALPHA 1-2LD PLUS, Christ, Germany) and then sieved by nylon screen with mesh size of 100, and finally analyzed by using an improved method described by Shen (Shen et al., 2009a). Each sample at 1 g was extracted with 10 mL McIlvaine-Na2EDTA buffer prepared by mixing 0.1 mol/L citric acid monohydrate solution with 0.2 mol/L disodium hydrogen phosphate dodecahydrate solution, adding 0.1 mol/L ethylenediamineteraacetic acid disodiumsalt (Na2EDTA). After vortexed for 1 min, 5 mL acetonitrile was added to increase the extraction efficiency and then followed by intensively shaking for 30 min and centrifugation at 15,000 r/min of 4 °C for 5 min. The same extraction procedure was repeated twice, and then all the supernatant was combined and transferred into a new flask. The supernatant was evaporated to nearly 10 mL using the revolving evaporator under vacuum and then diluted to 100 mL with the ultra pure water. For sample purification and pre-concentration, a vacuum system (SUPELCO VISIPREP™ DL, USA) was applied. Firstly, the solid phase extraction (SPE) cartridges (Oasis HLB, 150 mg, 6 mL) were conditioned with 5 mL methanol followed by 5 mL McIlvaine-Na2EDTA buffer. After the conditioning step, the extract of samples was percolated through the cartridges at a flow rate of approximately 1.0 mL/min. Then the cartridges were sequentially rinsed with 5 mL ultra pure water and 5 mL 5% methanol aqueous solution. Finally, the cartridges were eluted with 9 mL methanol/ethyl acetate solution (1:9, v/v). The elute was collected and concentrated to dryness under a stream of nitrogen and then reconstituted with 1.0 mL acetonitrile containing 0.1% formic acid (1:9, v/v) followed by filtration with 0.22 lm nylon membrane before determined by UPLC–MS/MS. 2.4. Liquid chromatography and mass chromatography Concentrations of the three parent tetracyclines and their degradation products were determined by an ultra performance liquid chromatography with tandem mass spectrometry (Waters Corp., USA). Separation of tetracyclines and their degradation

products was achieved with an Acquity UPLC™ C18 column (i.d. 2.1 mm  50 mm, 1.7 lm. Waters, USA). The column was maintained at 35 °C while the sample room was kept at 10 °C and the injection volume was 10 lL. The gradient of mobile phase consisted of acetonitrile (A) and 0.1% formic acid (B) with a constant flow rate at 0.30 mL/min was shown as follows: the initial 5% A was increased linearly to 17% in 5 min, and then increased to 30% over 1 min followed by an increase to 85% during the next 4 min. Finally the gradient was back to the initial 5% A and held for 1 min to maintain equilibration. The total run time was 11 min. The tandem mass spectrometry (MS) was performed using a Waters Micromass Quattro Premier XE triple quadrupole mass spectrometer equipped with an electrospray ionization source that operated in the positive ionization mode (ESI+). The operation conditions were optimized as follows: source temperature 120 °C, desolvation temperature 350 °C, capillary voltage 3.0 kV, desolvation gas flow 600 L/h, cone gas flow 50 L/h. Acquisition was done in the multiple reaction monitoring mode (MRM) and the optimized parameters used for identification and quantitation of three tetracyclines and their degradation products were summarized in Table 2. Peak integration and quantification was processed by MassLynx V 4.1 software. 2.5. Determination of recovery, limit of detection (LOD) and limit of quantification (LOQ), reproducibility and linearity To determine the extraction efficiency of all target compounds, samples were spiked with their standard solutions at three levels: 0.2 mg/kg dry weight (DW), 1.0 mg/kg DW and 4.0 mg/kg DW, respectively. Triplicate samples were extracted and analyzed as described above. The limit of detection (LOD) and limit of quantification (LOQ) of this method were evaluated by spiking samples with a mixture of all standards at a low concentration and calculated as the signal-to-noise (S/N) ratio was 3 and 10, respectively, based on the obtained peaks. The reproducibility was assessed by run-torun recoveries (eleven successive injections). Calibration curves were constructed from 0 to 300 lg/L with correlation coefficient R2 representing their linearity. The precision of method was expressed as the relative standard deviation (RSD) of triplicate measurements.

Table 2 The optimized MS/MS parameters used for identification and quantitation of three tetracyclines and their degradation products in MRM condition. Compounds

Retention time (min) Parent ions [M+H]+ Product ions (m/z) Quantitative ions (m/z) Cone voltage (V) Collision energy (eV)

4-epianhydrotetracycline (EATC) Anhydrotetracycline (ATC) a-apo-oxytetracycline (a-apo-OTC) b-apo-oxytetracycline (b-apo-OTC) 4-epitetracycline (ETC) Tetracycline (TC) 4-epioxytetracycline (EOTC) Oxytetracycline (OTC) Demeclocycline (DMCTC) 4-epichlortetracycline (ECTC) Chlortetracycline (CTC)

6.69

427

6.87

427

5.55

443

6.82

443

4.32

445

5.08

445

4.47

461

4.72

461

5.78

465

5.93

479

6.34

479

410 154 410 154 426 408 426 408 410 427 410 427 444 426 444 426 448 430 444 462 444 462

410

31

410

31

426

31

426

31

410

28

410

28

426

22

426

22

448

34

444

34

444

34

16 34 16 34 16 25 16 25 19 15 19 15 16 19 16 19 19 25 22 15 22 15

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X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931 Table 3 Recovery, LOD and LOQ, reproducibility and linearity of three tetracyclines and their degradation products. Compounds

TC ETC ATC EATC OTC EOTC a-apo-OTC b-apo-OTC CTC ECTC DMCTC

Recovery ± RSD (%, n = 3) Spiked level (mg/kg DW) 0.2 1.0

4.0

71 ± 5.4 51 ± 11.4 21 ± 13.8 3 ± 8.9 66 ± 25.7 32 ± 28.8 4 ± 21.9 50 ± 6.7 84 ± 1.9 124 ± 0.1 53 ± 6.9

88 ± 2.8 36 ± 7.9 21 ± 12.7 6 ± 11.1 73 ± 7.2 46 ± 2.2 3 ± 11.0 24 ± 14.5 66 ± 4.1 48 ± 2.1 52 ± 3.7

89 ± 9.2 37 ± 15.7 26 ± 6.6 6 ± 1.2 94 ± 17.1 35 ± 1.3 8 ± 13.5 43 ± 10.0 83 ± 7.4 102 ± 4.8 64 ± 6.1

LOD (lg/kg)

LOQ (lg/kg)

Reproducibility (%, n = 11)

Linearity R2

3.189 2.947 1.668 1.893 13.775 4.444 3.791 12.346 6.707 17.270 5.768

10.630 9.824 5.561 6.309 45.918 14.814 12.636 41.154 22.357 57.567 19.226

5.332 5.843 8.376 5.773 6.648 13.054 4.360 13.326 6.789 10.742 7.055

0.9960 0.9990 0.9970 0.9997 0.9984 0.9994 0.9979 0.9987 0.9991 0.9989 0.9996

DW: dry weight.

2.6. Kinetic model It is well known that in most cases degradation of organic pollutants in the environment follows the simple first-order kinetic model, which is described as follow:

dC=dt ¼ kC where C represents the concentration of target compound at the time t and k is the rate constant. Then the half-live can be expressed as

t 1=2 ¼ ln 2=k However, when the target compound is inclined to be adsorbed in the soil, manure or other matrices, the degradation process may deviate from the first-order kinetic model as the available target organic compound that can be degraded decreases because of sorption and fixation (Thiele-Bruhn, 2003). Hence, the simple first-order kinetic model needed to be improved and the adjusted first-order kinetic model was developed based on the decreasing availability of target organic compound to give a more accurate and clearer information on the degradation process of organic compounds, which is shown as follow (Wang and Yates, 2008):

tetracyclines were not so pleasant. This phenomenon may be due to strong matrix effect in the complicated composting samples. Difference of physiochemical characteristics could be another possible reason, especially for those anhydro-tetracyclines, because they are formed under strong acidic situations while the whole pretreatment process was maintained in weak acidic conditions to achieve simultaneous determination the parent tetracyclines and their degradation products. Although the determination of tetracyclines in animal manure was a hot topic (KarcI and BalcIoglu, 2009; Martinez-Carballo et al., 2007; Zhao et al., 2010), there was limited reports concerning about the recoveries of their degradation products. It was reported by Arikan et al. (2006) that the recoveries of a-apo-OTC and b-apo-OTC in beef manure were 40% and 25%, respectively. The same low recovery had been previously reported by Fedeniuk et al. (1996). Loke et al. (2003) found that the recovery of a-apo-OTC in the manure-containing matrix was 18.3%, slightly higher than our findings. The results indicated good reproducibility and great linearity of the calibration curve with all R2 above 0.9960. LOD and LOQ for the target compounds were in the range of 1.668–17.270 lg/kg and 5.561–45.918 lg/kg (Table 3), suggesting a high sensitivity of the method.

dC=dt ¼ kkC 3.2. Composting process where k is defined as the concentration ratio of the available portion to the total target compound and is assumed as a function of t expressed as k = k0eat where k0 is the value obtained when t = 0 and a is called availability coefficient. The called degradation rate constant k00 = kk0 is defined, and then we acquire the equation 00

dC=dt ¼ k Ceat Consequently, the half-life is derived as 00

t 1=2 ¼  lnð1  a  ln 2=k Þ=a

3. Results and discussion 3.1. Method validation Table 3 presented that there was pronounced difference between recovery of three parent tetracyclines and that of their degradation products. Better recoveries of parent tetracyclines could be observed as in the range of 71–89%, 66–94% and 66–84% for TC, OTC and CTC, respectively. The recovery of DMCTC was in the range of 52–64%. Except for some variances in ECTC recovery at three levels, the other two epimers’ recoveries like ETC and ECTC were in the range of 32–51%. However, recoveries of anhydro-

As shown in Fig. 2a, the ambient temperature increased continuously during the pilot scale swine manure composting in the spring, and the pile temperature in the central portion of the pile was obviously much higher than that of the other two portions. Central temperature in the pile soon went up to about 55 °C within 5 days and nearly maintained above 55 °C for the whole period except for some occasional down, meeting with the requirement of sanitary standard for the non-hazardous treatment of night soil in China (GB 7959-87). High level of microbial activity accounted for the rapid increase of pile temperature and the high pile temperature observed during the composting. In Fig. 2b and c, the pH value was kept about 8.5 with a little decline in the maturity stage while EC had a general ascending trend. Moisture content was observed in the range of 50–65% suitable for composting due to irregularly water addition. Besides, decomposition of organic matter could be inferred from its descending content during the whole composting process. 3.3. Behavior of tetracyclines and their degradation products in the pilot scale swine manure composting Fig. 3 illustrated the behavior of tetracyclines during the pilot scale swine manure composting process. The initial concentrations

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70 Upper

Central

Lower

Ambient

OTC

2.0

EOTC

60 1.5 1.0

o

Temperature ( C)

50 40

0.5

30

0.0

Concentration (mg/kg DW)

20 10 0

0

10

20

30

40

50

60

Time (d)

(a) Temperature

ETC

ATC

0.30 0.15 0.00

2000

9.5

pH

EC

CTC

3.0

9.0

1800

8.5

1600

8.0

1400

7.5

1200

ECTC

DMCTC

2.4 1.8

EC(µS/cm)

pH

TC

0.45

1.2 0.6 0.0 0

10

20

30

40

50

60

Time (d) 1000

7.0

Fig. 3. The behavior of tetracylines and their degradation products during the pilot scale swine manure composting in the spring.

800

6.5 0

10

20

30

40

50

Time (d)

(b) pH and EC 90

70

Moisture (%)

65

85

60

80

55

75

50

70

45

65

40

0

10

20

30

40

50

Orgnic matter (%)

Organic matter

Moisture

60

Time (d)

(c) Moisture content and organic matter Fig. 2. Evolution of (a) temperature, (b) pH and EC, (c) moisture content and organic matter during the pilot scale swine manure composting in the spring.

of three parent tetracyclines CTC, OTC and TC in the swine manure were 2.9, 1.6, and 0.4 mg/kg DW, respectively, the same range as reported (Dolliver et al., 2008; Kumar et al., 2005; Tylova et al., 2010), but different from those reported in previous researches (Arikan et al., 2009; Bao et al., 2009; Ramaswamy et al., 2010). This may be attributed to different application amounts of feed additives in livestock production. Degradation of these three tetracyclines can be observed because not only their concentration

decreased but also several of their degradation products were detected during the composting. Tetracyclines were considered to be instable because of their unique chemical structure, and may undergo abiotic degradation conditions such as pH, temperature, redox and light conditions and then generate degradation products via epimerization, dehydration or other pathways (HallingSørensen et al., 2003; Kühne et al., 2001). Although DMCTC was detected in this study, its initial concentration tended to be limited, less than 0.15 mg/kg DW. Except for DMCTC, the main degradation products of tetracyclines in the pilot scale swine manure composting were their epimers as ECTC, EOTC together with ETC, and another degradation product was dehydrated compound ATC, which was also found rather limited. None of the 4-epianhydrotetracyclines of the three parent tetracyclines had been discovered during the composting. The slight alkaline condition (Fig. 2b) in the pile may be helpful to account for this because anhydrotetracyclines could only be formed when they are under strongly acidic conditions. Both of ECTC and ICTC were found in beef manure composting (Arikan et al., 2009), however, the standard of ICTC was not commercially available so it cannot be determined in our study. In addition, a-apo-OTC and b-apo-OTC had been observed in anaerobic tests (Loke et al., 2003), but they could not be detected in this study either. As shown in Fig. 3, not only did the parent tetracyclines decrease, but also the degradation products went down during the swine manure composting. It was interesting to find that ECTC was following the similar trend as its parent CTC. At the very beginning, the concentration of ECTC was nearly as much as that of CTC. This phenomenon was possibly related with the inadequate absorption of veterinary antibiotics as pig feed resulting in a large proportion of them excreted as metabolites in the swine manure, as reported by Arikan et al. (2009) that roughly 65% of the CTC

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The degradation data of three parent tetracyclines such as CTC, OTC and TC during the pilot scale swine manure composting was fitted by both the commonly used simple first-order kinetic and adjusted first-order kinetic model (Fig. 4). Results as listed in Table 4 indicated that both models can be used to describe the degradation process of the three tetracyclines during the composting as all of their correlation coefficients R2 were over 0.75. However, it was apparent that the adjusted first-order kinetic model could fit much better to describe the degradation behavior of the three parent tetracyclines than the simple first-order kinetic model. For example, at the beginning of composting, the simple first-order kinetic model fitted the experimental data of OTC well, however, after one week the predicted concentrations of OTC were lower than the determined concentration of OTC which seemed to be nearly constant, and thus could not describe the degradation process accurately. Due to their molecular structure and physical– chemical property, tetracyclines were apt to be adsorbed in the environment (Thiele-Bruhn, 2003; Tolls, 2001). In this study, the more satisfied fitting of the adjusted first-order kinetic model confirmed the existed behavior of sorption in the manure and also proved that the availability of tetracyclines declined during the composting. Interestingly, OTC appeared to obey the two models most successfully among the three target tetracyclines with correlation coefficients of 0.9286 and 0.9969, respectively. The half-lives of CTC, OTC and TC calculated by the simple firstorder kinetic model were 16.95, 2.66 and 22.36 days, respectively, while the obtained half-lives using the adjusted first-order kinetic model were 8.25, 1.14 and 10.02 days, respectively, nearly half of the former results. Thermal degradation might contribute to the degradation of tetracyclines during the animal manure composting as suggested by Arikan et al. (2007). In addition, our previous research also testified that the degradation rates of residual tetracyclines in swine manure increased as temperature increasing and achieved the maximum at 55 °C (Shen et al., 2009b). Generally, pile temperature should maintain at 50–55 °C or above for at least 5–7 days according to the sanitary standard (GB7959-87) to inactivate the pathogens during composting, as shown in our study (Fig. 2). Therefore, such high pile temperature may be helpful to increase the degradation of tetracyclines during the swine manure composting. Microbial activity also played an important role in

CTC Simple first-order model Adjusted first-order model

Concentration (mg/kg DW)

3.0 2.5 2.0 1.5 1.0 0.5 0.0

0

10

20

30 Time (d)

40

50

60

(a) CTC 2.5 OTC Simple first-order model Adjusted first-order model

2.0 Concentration (mg/kg DW)

3.4. Degradation kinetics of tetracyclines

3.5

1.5

1.0

0.5

0.0

0

10

20

30 Time (d)

40

50

60

(b) OTC 1.0 TC Simple first-order model Adjusted first-order model

0.8

Concentration (mg/kg DW)

fed to the calves was recovered in the manure as CTC/ECTC and ICTC, and the previous reported approximately 75% of CTC excretion rate for cow by Elmund et al. (1971). Both the parent CTC and its degradation product ECTC declined fast during the composting process and their removal rates were 74% and 82% separately. By the end of composting, DMCTC went down to close to its detection limit, achieving an 87% of reduction. The reduction of OTC soon achieved 92% within one week, however, its degradation product EOTC seemed to be constant after it increased to about 0.25 mg/kg DW in the first week. As far as TC was concerned, its initial concentration was less than 0.5 mg/kg DW and decreased to about 0.1 mg/kg DW at the end of the composting, with a removal rate of about 70%. Seventy-four percent of its degradation product ETC was removed within the first week and another degradation product ATC was maintained all the same at levels only above its detection limit. The above results elucidated that all the three tetracyclines could undergo degradation process and be removed during the course of composting, and thus demonstrated that composting is an effective alternative to remove residual antibiotics in animal manure as reported before (Arikan et al., 2007; Bao et al., 2009; Dolliver et al., 2008; Kakimoto and Funamizu, 2007; Kakimoto et al., 2007; Ramaswamy et al., 2010).

0.6 0.4 0.2 0.0 -0.2 0

10

20

30

40

50

60

Time (d)

(c) TC Fig. 4. Degradation kinetics of three tetracyclines (a) CTC, (b) OTC and (c) TC during the pilot scale swine manure composting.

the composting, not only account for the increased temperature as mentioned before, but also a key factor to promote the degradation of tetracyclines. As the decomposition temperatures of tetracyclines are mostly above 170 °C, we proposed that it was synergy effect of biodegradation and thermal degradation that contributed to the degradation behavior happened during the composting. Compared with longer half-lives of CTC and TC, the halflives of OTC in both models were less than 3 days which means OTC was the rapidest degraded one among the three tetracyclines.

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Table 4 Fitting results for degradation kinetics of three parent tetracyclines during the pilot scale swine manure composting using both the simple first-order kinetic model and adjusted first-order kinetic model. Parent tetracyclines

Simple first-order kinetic model C0 (mg/kg)

Rate constant k (d1)

R2

Half-life t1/2(d)

C0 (mg/kg)

Adjusted first-order kinetic model Rate constant k00 (d1)

Availability coefficient a

R2

Half-life t1/2(d)

CTC OTC TC

2.5536 ± 0.3209 2.0158 ± 0.2695 0.3473 ± 0.0326

0.0409 ± 0.0123 0.2605 ± 0.0743 0.0310 ± 0.0076

0.7565 0.9286 0.8075

16.95 2.66 22.36

3.1159 ± 0.4330 2.8935 ± 0.3414 0.4275 ± 0.0319

0.1130 ± 0.0514 0.6939 ± 0.1435 0.1006 ± 0.0271

0.0757 ± 0.0428 0.2371 ± 0.0446 0.0801 ± 0.0252

0.8567 0.9969 0.9467

8.25 1.14 10.02

It was observed that both the rate constant k00 and availability coefficient a of OTC in the adjusted first-order kinetic model were higher than those of CTC and TC. The higher the value of a was, the more the OTC was available. The higher rate constant k00 of OTC was, the faster degradation process and shorter half-life of OTC were. The fate of tetracyclines during the composting had been the subject of numerous studies (Arikan et al., 2007; Bao et al., 2009; De Liguoro et al., 2003; Dolliver et al., 2008) and the results of half-life differed to some degree. Arikan et al. (2007) reported a calculated half-life of OTC at approximately 3.2 days during beef manure composting which was comparable with our result, but De Liguoro et al. (2003) found that the half-life of OTC in the stockpiled manure-bedding mixture was 30 days and the compound was still detectable after 5 months. Assuming the first-order decay, the half-life of CTC in the managed manure composting was only 1 day reported by Dolliver et al. (2008), much shorter than our study. Similarly, according to the first-order kinetic, Bao et al. (2009) presented that the CTC half-lives were 11.0 days in broiler manure and 12.2 days in layer-hen manure, which were consistent with our finding. Furthermore, it could be inferred from Fig. 4 that the degradation behavior of three parent tetracyclines CTC, OTC and TC during the swine manure composting predominately took place in the thermophilic stage as their concentration almost reached the plateau in 3 weeks except that the concentration of OTC achieved constant even within 10 days. The calculated half-lives of CTC, OTC and TC based on both the simple and adjusted first-order kinetic models were less than 25 days, shorter than the period of thermophilic stage, meaning that at least 50% of them had been degraded by this time. Besides, pile temperature of the central part in this period was kept above 50 °C (Fig. 2), which was advantageous to their degradation. 4. Conclusions During the pilot scale swine manure composting, the degradation of CTC, OTC and TC occurred, with a removal rate of 74%, 92%, and 70% for CTC, OTC and TC, respectively, and their degradation behavior predominately took place in the thermophilic stage of composting. Several degradation products were detected including ECTC, EOTC, ETC, DMCTC and ATC. Both the simple and the adjusted first-order kinetic models fit their degradation process, but the adjusted first-order kinetic model was much better. Acknowledgements This work is financially supported by the National 863 Program of China (No. 2007AA06Z344) and National Natural Science Fund of China (No. 50578156 and No. 21077122). References Arikan, O.A., Sikora, L.J., Mulbry, W., Khan, S.U., Rice, C., Foster, G.D., 2006. The fate and effect of oxytetracycline during the anaerobic digestion of manure from therapeutically treated calves. Process Biochem. 41 (7), 1637–1643.

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