Bioresource Technology 102 (2011) 5924–5931

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

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

a r t i c l e i n f o a b s t r a c t

Article history: The purposes of this study were to investigate the behavior of three tetracyclines including chlortetracy-
Received 29 October 2010 cline (CTC), oxytetracycline (OTC) and tetracycline (TC) and their degradation products in a pilot scale
Received in revised form 3 March 2011 swine manure composting, and also to study the degradation kinetics of CTC, OTC and TC. During the pilot
Accepted 3 March 2011
scale composting, CTC, OTC and TC were degraded by 74%, 92% and 70%, respectively. Several degradation
Available online 9 March 2011
products were found like 4-epitetracycline (ETC), 4-epioxytetracycline (EOTC), 4-epichlortetracycline
(ECTC), demeclocycline (DMCTC) and anhydrotetracycline (ATC). Both the simple and the adjusted
Keywords:
first-order kinetic models successfully fit the degradation process of CTC, OTC and TC during the com-
Swine Manure
Composting
posting, but the adjusted first-order kinetic model fit much better with the calculated half-lives of 8.2,
Degradation product 1.1 and 10.0 days, respectively.
Degradation kinetic Ó 2011 Elsevier Ltd. All rights reserved.
Tetracyclines

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 esti-
mated 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 antibiot-
ics (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 antibiot-
ics 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: yswei@rcees.ac.cn (Y. Wei).
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 environ-
ment 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 stabiliza-
tion of organic matter and reduction of pathogens and odors, com-
posting 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 Funam-
izu, 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 oxy-
tetracycline 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 com-
posting declined rapidly, with over 99% reduction occurred in less

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.03.007
 X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931 5925

than 10 days. Tylosin and monensin exhibited a gradual decline 2. Methods

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 tetracy-
clines’ 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 ioniz-
able functional groups (Chen and Huang, 2009) as shown in Fig. 1.
For instance, the 4-epimers such as 4-epitetracycline (ETC), 4-epi-
oxytetracycline (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 anhydro-
tetracyclines 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 tetracy-
clines 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 prod-
ucts 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 prod-
ucts 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 ani-
mal 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 tetracy-
cline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) as
well as their potential degradation products were investigated dur-
ing 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.
2.1. 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 tetracy-
clines 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 Materials
Swine Mushroom Mixture
manure residues
pH 7.68 7.66 8.46
Moisture (%) 66.3 25.1 56.9
EC (lS/cm) 1236 1580 1031
OM (%) 64.3 87.0 77.9
TKNa (g/kg) 30.36 24.82 29.97
TPa (g/kg) 12.01 4.69 11.93
C:N 9.01 16.77 11.02

EC: electrical conductivity; OM: organic matter; TKN: total kjeldahl nitrogen; TP:
total phosphorus.
Fig. 1. Chemical structure and property of tetracyclines (TCs) (Chen and Huang, a
Measured based on dry matter.
2009).
5926 X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931

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 revol-
ving evaporator under vacuum and then diluted to 100 mL with
the ultra pure water. For sample purification and pre-concentra-
tion, 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% meth-
anol aqueous solution. Finally, the cartridges were eluted with
9 mL methanol/ethyl acetate solution (1:9, v/v). The elute was col-
lected 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 deg-
radation products were determined by an ultra performance li-
quid 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 main-
tained 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 con-
stant 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 con-
ditions were optimized as follows: source temperature 120 °C,
desolvation temperature 350 °C, capillary voltage 3.0 kV, desolva-
tion gas flow 600 L/h, cone gas flow 50 L/h. Acquisition was done
in the multiple reaction monitoring mode (MRM) and the opti-
mized 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 de-
scribed 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-to-
run 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 ex-
pressed 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 6.69 427 410 410 31 16
(EATC) 154 34
Anhydrotetracycline 6.87 427 410 410 31 16
(ATC) 154 34
a-apo-oxytetracycline 5.55 443 426 426 31 16
(a-apo-OTC) 408 25
b-apo-oxytetracycline 6.82 443 426 426 31 16
(b-apo-OTC) 408 25
4-epitetracycline 4.32 445 410 410 28 19
(ETC) 427 15
Tetracycline 5.08 445 410 410 28 19
(TC) 427 15
4-epioxytetracycline 4.47 461 444 426 22 16
(EOTC) 426 19
Oxytetracycline 4.72 461 444 426 22 16
(OTC) 426 19
Demeclocycline 5.78 465 448 448 34 19
(DMCTC) 430 25
4-epichlortetracycline 5.93 479 444 444 34 22
(ECTC) 462 15
Chlortetracycline 6.34 479 444 444 34 22
(CTC) 462 15
 X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931 5927

Table 3
Recovery, LOD and LOQ, reproducibility and linearity of three tetracyclines and their degradation products.

Compounds Recovery ± RSD (%, n = 3) LOD (lg/kg) LOQ (lg/kg) Reproducibility (%, n = 11) Linearity R2
Spiked level (mg/kg DW)
0.2 1.0 4.0
TC 71 ± 5.4 89 ± 9.2 88 ± 2.8 3.189 10.630 5.332 0.9960
ETC 51 ± 11.4 37 ± 15.7 36 ± 7.9 2.947 9.824 5.843 0.9990
ATC 21 ± 13.8 26 ± 6.6 21 ± 12.7 1.668 5.561 8.376 0.9970
EATC 3 ± 8.9 6 ± 1.2 6 ± 11.1 1.893 6.309 5.773 0.9997
OTC 66 ± 25.7 94 ± 17.1 73 ± 7.2 13.775 45.918 6.648 0.9984
EOTC 32 ± 28.8 35 ± 1.3 46 ± 2.2 4.444 14.814 13.054 0.9994
a-apo-OTC 4 ± 21.9 8 ± 13.5 3 ± 11.0 3.791 12.636 4.360 0.9979
b-apo-OTC 50 ± 6.7 43 ± 10.0 24 ± 14.5 12.346 41.154 13.326 0.9987
CTC 84 ± 1.9 83 ± 7.4 66 ± 4.1 6.707 22.357 6.789 0.9991
ECTC 124 ± 0.1 102 ± 4.8 48 ± 2.1 17.270 57.567 10.742 0.9989
DMCTC 53 ± 6.9 64 ± 6.1 52 ± 3.7 5.768 19.226 7.055 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):
dC=dt ¼ kkC
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
t 1=2 ¼  lnð1  a  ln 2=k Þ=a
00
3. Results and discussion
3.1. Method validation
Table 3 presented that there was pronounced difference be-
tween recovery of three parent tetracyclines and that of their deg-
radation 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-
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 pos-
sible 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.
3.2. Composting process
As shown in Fig. 2a, the ambient temperature increased contin-
uously 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 ex-
cept 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 temper-
ature observed during the composting.
In Fig. 2b and c, the pH value was kept about 8.5 with a little de-
cline 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, decomposi-
tion of organic matter could be inferred from its descending con-
tent 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
5928 X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931

70
Upper Central Lower Ambient OTC EOTC
2.0
60
1.5
50
Temperature ( C)

1.0
o

40
0.5
30
0.0

20

Concentration (mg/kg DW)

0.45 TC ETC ATC
10
0.30
0
0 10 20 30 40 50 60
0.15
Time (d)
(a) Temperature
0.00
9.5 2000
pH EC CTC ECTC DMCTC
3.0
9.0 1800
2.4

8.5 1600 1.8

EC(µS/cm)

1.2
pH

8.0 1400 0.6

0.0
7.5 1200
0 10 20 30 40 50 60
Time (d)
7.0 1000
Fig. 3. The behavior of tetracylines and their degradation products during the pilot
scale swine manure composting in the spring.
6.5 800
0 10 20 30 40 50
Time (d)
(b) pH and EC decreased but also several of their degradation products were de-
tected during the composting. Tetracyclines were considered to
70 90 be instable because of their unique chemical structure, and may
Moisture Organic matter undergo abiotic degradation conditions such as pH, temperature,
redox and light conditions and then generate degradation products
65 85
via epimerization, dehydration or other pathways (Halling-
Sørensen et al., 2003; Kühne et al., 2001). Although DMCTC was de-
Orgnic matter (%)

60 80 tected in this study, its initial concentration tended to be limited,

Moisture (%)

55
50
45
40
0 10 20 30 40
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 addi-
tives in livestock production. Degradation of these three tetracy-
clines can be observed because not only their concentration
less than 0.15 mg/kg DW. Except for DMCTC, the main degradation
75 products of tetracyclines in the pilot scale swine manure compost-
ing were their epimers as ECTC, EOTC together with ETC, and an-
70 other degradation product was dehydrated compound ATC,
which was also found rather limited. None of the 4-epianhydrotet-
racyclines of the three parent tetracyclines had been discovered
65
during the composting. The slight alkaline condition (Fig. 2b) in
the pile may be helpful to account for this because anhydrotetracy-
60 clines could only be formed when they are under strongly acidic
50
conditions. Both of ECTC and ICTC were found in beef manure com-
posting (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 anaero-
bic 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 de-
crease, 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
 X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931 5929

fed to the calves was recovered in the manure as CTC/ECTC and 3.5
ICTC, and the previous reported approximately 75% of CTC excre- CTC
tion rate for cow by Elmund et al. (1971). Both the parent CTC 3.0 Simple first-order model

Concentration (mg/kg DW)

and its degradation product ECTC declined fast during the com- Adjusted first-order model
posting process and their removal rates were 74% and 82% sepa- 2.5
rately. By the end of composting, DMCTC went down to close to
2.0
its detection limit, achieving an 87% of reduction. The reduction
of OTC soon achieved 92% within one week, however, its degrada-
1.5
tion 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, 1.0
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 re- 0.5
moval rate of about 70%. Seventy-four percent of its degradation
product ETC was removed within the first week and another deg- 0.0
radation product ATC was maintained all the same at levels only 0 10 20 30 40 50 60
above its detection limit. Time (d)
The above results elucidated that all the three tetracyclines (a) CTC
could undergo degradation process and be removed during the
course of composting, and thus demonstrated that composting is 2.5
an effective alternative to remove residual antibiotics in animal OTC
manure as reported before (Arikan et al., 2007; Bao et al., 2009; Simple first-order model
2.0
Adjusted first-order model
Dolliver et al., 2008; Kakimoto and Funamizu, 2007; Kakimoto

Concentration (mg/kg DW)

et al., 2007; Ramaswamy et al., 2010).
1.5

3.4. Degradation kinetics of tetracyclines

1.0
The degradation data of three parent tetracyclines such as CTC,
OTC and TC during the pilot scale swine manure composting was 0.5
fitted by both the commonly used simple first-order kinetic and
adjusted first-order kinetic model (Fig. 4). Results as listed in
0.0
Table 4 indicated that both models can be used to describe the deg-
radation process of the three tetracyclines during the composting 0 10 20 30 40 50 60
as all of their correlation coefficients R2 were over 0.75. However, Time (d)
it was apparent that the adjusted first-order kinetic model could (b) OTC
fit much better to describe the degradation behavior of the three
parent tetracyclines than the simple first-order kinetic model. For 1.0
example, at the beginning of composting, the simple first-order
TC
kinetic model fitted the experimental data of OTC well, however, Simple first-order model
0.8
after one week the predicted concentrations of OTC were lower Adjusted first-order model
Concentration (mg/kg DW)

than the determined concentration of OTC which seemed to be

0.6
nearly constant, and thus could not describe the degradation pro-
cess accurately. Due to their molecular structure and physical–
0.4
chemical property, tetracyclines were apt to be adsorbed in the
environment (Thiele-Bruhn, 2003; Tolls, 2001). In this study, the
0.2
more satisfied fitting of the adjusted first-order kinetic model con-
firmed the existed behavior of sorption in the manure and also
proved that the availability of tetracyclines declined during the 0.0
composting. Interestingly, OTC appeared to obey the two models
most successfully among the three target tetracyclines with corre- -0.2
lation coefficients of 0.9286 and 0.9969, respectively. 0 10 20 30 40 50 60
The half-lives of CTC, OTC and TC calculated by the simple first- Time (d)
order kinetic model were 16.95, 2.66 and 22.36 days, respectively, (c) TC
while the obtained half-lives using the adjusted first-order kinetic
Fig. 4. Degradation kinetics of three tetracyclines (a) CTC, (b) OTC and (c) TC during
model were 8.25, 1.14 and 10.02 days, respectively, nearly half of
the pilot scale swine manure composting.
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 re-
search also testified that the degradation rates of residual tetracy- the composting, not only account for the increased temperature
clines in swine manure increased as temperature increasing and as mentioned before, but also a key factor to promote the degrada-
achieved the maximum at 55 °C (Shen et al., 2009b). Generally, pile tion of tetracyclines. As the decomposition temperatures of tetra-
temperature should maintain at 50–55 °C or above for at least cyclines are mostly above 170 °C, we proposed that it was
5–7 days according to the sanitary standard (GB7959-87) to inacti- synergy effect of biodegradation and thermal degradation that
vate the pathogens during composting, as shown in our study contributed to the degradation behavior happened during the com-
(Fig. 2). Therefore, such high pile temperature may be helpful to in- posting. Compared with longer half-lives of CTC and TC, the half-
crease the degradation of tetracyclines during the swine manure lives of OTC in both models were less than 3 days which means
composting. Microbial activity also played an important role in OTC was the rapidest degraded one among the three tetracyclines.
5930 X. Wu et al. / Bioresource Technology 102 (2011) 5924–5931
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 Simple first-order kinetic model
tetracyclines
C0 (mg/kg) Rate constant k (d1) R2 Half-life
t1/2(d)
CTC 2.5536 ± 0.3209 0.0409 ± 0.0123 0.7565 16.95
OTC 2.0158 ± 0.2695 0.2605 ± 0.0743 0.9286 2.66
TC 0.3473 ± 0.0326 0.0310 ± 0.0076 0.8075 22.36
It was observed that both the rate constant k00 and availability coef-
ficient a of OTC in the adjusted first-order kinetic model were high-
er 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 stock-
piled 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 degrada-
tion 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 ki-
netic models were less than 25 days, shorter than the period of
thermophilic stage, meaning that at least 50% of them had been de-
graded 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 degrada-
tion of CTC, OTC and TC occurred, with a removal rate of 74%,
92%, and 70% for CTC, OTC and TC, respectively, and their degrada-
tion behavior predominately took place in the thermophilic stage
of composting. Several degradation products were detected includ-
ing 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).
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