Environ Sci Pollut Res (2016) 23:4422–4429 DOI 10.1007/s11356-015-5624-y

RESEARCH ARTICLE

Enrofloxacin degradation in broiler chicken manure under various laboratory conditions Marko Slana 1 & Marija Sollner-Dolenc 2

Received: 21 May 2015 / Accepted: 15 October 2015 / Published online: 27 October 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract The rate of degradation of enrofloxacin in broiler chicken manure has been characterized in the laboratory according to the CVMP guideline on determining the fate of veterinary medicinal products in manure. Degradation was followed in a flow-through system under aerobic and anaerobic conditions, in the dark and in the presence of light. The rate of degradation of enrofloxacin and the formation of its degradation products are dependent on laboratory conditions. A rapid degradation of enrofloxacin in the dark was noticed, where a shorter degradation half-life under aerobic (DT50 = 59.1 days), comparing to anaerobic conditions (DT50 = 88.9 days), was determined. The presence of light slowed down the enrofloxacin degradation half-life, which was significantly shorter under aerobic (DT50 =115.0 days), comparing to anaerobic conditions (DT50 =190.8 days). Desethyleneenrofoxacin was the only degradation product formed, its concentrations ranged from 2.5 to 14.9 %. The concentration of the degradation product was approximately 2.5-fold higher under aerobic conditions. Enrofloxacin degradation in sterile manure incubated under sterile conditions was marginal comparing to non-sterile conditions; after 120 days of incubation, approximately 80 % of enrofloxacin was still present in manure and only 1 % of desethylene-enrofloxacin was formed. The present work demonstrates that enrofloxacin degradation

Responsible editor: Philippe Garrigues * Marija Sollner-Dolenc [email protected] 1

Krka, d. d., Novo mesto, Šmarješka cesta 6, 8501 Novo mesto, Slovenia

2

Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, 1000 Ljubljana, Slovenia

in chicken manure is relatively fast when incubated in the dark under aerobic conditions which is the recommended incubation system for chicken manure according to CVMP guideline. Keywords Enrofloxacin degradation kinetics . Degradation product . Desethylene-enrofloxacin . Aerobic and aerobic incubation . Non-sterile and sterile conditions . Light/dark regime

Introduction The number of investigations of the fate in the environment of veterinary medicine products (VMPs) has been increasing in recent years. The amounts of active VMPs that are applied to animals in intensive rearing livestock facilities are relatively high, and transfer from the treated animals to the environment can occur. After treatment with a VMP, the active compound and/or its metabolites are excreted by the animal (Boxall et al. 2002 and 2004; De Liguoro et al. 2003; Gagliano and McNamara 1996; Slana and Sollner-Dolenc 2013; Schlüsener et al. 2006; Teeter and Meyerhoff 2003; Wang and Yates 2008; Wetzstein et al. 2002; Wu et al. 2011). The manure or slurry from such intensively reared animals is regularly used as a fertilizer. The amounts of the active compound and its metabolites thus released into the environment depend on their rate of degradation in the manure storage phase as well as after application to soil. According to Slana and Sollner-Dolenc (2013), the storage time (average over 3 months) and handling of the animal’s excreta depend on the species of animal (pasture or intensively reared), on the management of manure (anaerobic tanks, aerobic/anaerobic lagoons, anaerobic composts), and on the agricultural practice (slurry sprayed over the soil surface, incorporation of manure

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into soil) of the specific country or area. Based on the different storage techniques and conditions, diversity in the degradation pattern of VMPs can be expected. According to the Guideline on Environmental Impact Assessment for Veterinary Medicinal Products that supports the VICH guidelines GL6 and GL 38 (Anon 5, 2009), the degradation pattern of the VMP can be studied in the field or in laboratory studies. The guideline’s preference is to demonstrate the VMP’s degradation in the laboratory where the laboratory conditions are well controlled. Recommended study designs and instructions for performing laboratory manure degradation studies, taking into account the species of animal involved, are given in the guideline for determining the fate of veterinary medicinal products in manure (Anon 8, 2011). It is recommended that such studies on cattle and pig manure are performed under anaerobic conditions, whereas the degradation study in chicken manure should be performed under aerobic conditions. Incubation under either condition should be performed in the absence of light. In the present study, the degradation of enrofloxacin (EF) in broiler chicken manure has been investigated under wellcontrolled laboratory conditions. The study was performed according to the guideline’s criteria (Anon 8, 2011). In addition to incubating the manure aerobically and in the dark, as advised by the guideline, anaerobic incubation in the dark, as well as aerobic and anaerobic incubations under laboratory lights, was performed. According to CVMP (Anon 8, 2011) and literature data (Anon 4 2007; Anon 5 2009; Chambers et al. 2001; Huijsmans et al. 2004; Slana and Sollner-Dolenc 2013), it was demonstrated that at intensively rearing facilities of chicken manure together with the bedding material (straw or wood chops) is stored outside approximately for 2– 4 months as manure heap under aerobic conditions before it is applied to soil as fertilizer. Contrary, the majority of pig and cattle manure is stored in liquid form (manure and urine) under anaerobic or semi-anaerobic conditions in manure tanks or lagoons before application to land occurs. EF (C19H22FN3O3) is a fluoroquinolone that is most commonly used to treat respiratory and digestive diseases of animals intended for human consumption. Its metabolism in target animals is poor and species dependent, 87–60 % of EF is excreted in unchanged form, the rest is excreted mainly in form of ciprofloxacin which has also antimicrobial activity (Gagliano and McNamara 1996; Unisol 2011; Zhou et al. 2008). In the open literature, 14C-Enrofloxacin aerobic degradation studies were performed with feces that were taken from non-treated cattle or cattle treated with EF. The manure test systems were maintained for 64 to 210 days. At the end of day 64, EF decreased from 78 to 54 % and trace amounts of 14CO2 were detected. The EF half-life (DT50) in non-treated/treated feces was 468/142 days in the study performed by Gagliano and McNamara (1996). In the study performed by Wetzstein

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et al. (2002), at the end of the incubation period (210 days), only 11–15 % of EF remained, resulting in a DT50 of 84 days. Approximately 18 % of applied EF was liberated as 14CO2 during the study, and three known metabolites (desethylene and 7-amino congeners of EF and CF) were identified at day 210. A study was published where degradation of EF and CF was investigated in chicken feces. EF was applied on a farm to 8900 chickens for 5 days at a dose of 10 mg/kg. After the animals departed, the manure was stored in two heaps for 63 days. After the heap was posted outside, the samples were taken at 30 and 80 cm above the ground. The initial concentrations of EF and CF in the heap were 22 and 1.8 mg/kg; after 63 days of storage, only 27.1 % of initial concentration was detected (Moraru et al., 2012). The fate of EF present in raw sewage was investigated. Sewage treatment for 8 months in the lagoon dramatically reduced the amount of EF to levels below the detection limit (LOD=0.6 μg/L). The results stress the importance of appropriate sludge management in limiting the impact of EF on the environment (Pierini et al. 2004). In the open literature, there is some evidence that EF may persists in manure. Half-lives of 84–468 days for its degradation in cattle manure have been determined (Gagliano and McNamara 1996; Wetzstein et al. 2002), but information about its degradation in chicken or pig manure is lacking. EF is often used to treat gastrointestinal and respiratory infections in chickens; therefore, the facilities for intensive rearing of chickens usually exhibit the greatest potential for contamination of the environment by EF in treated chicken manure. Thus, a study of EF degradation in chicken manure under laboratory conditions is needed to provide additional information about the release of EF into, and its persistence in, the environment.

Material and methods Chemicals Standards: enrofloxacin (EF) (Zhejiang Guobang Pharmaceutical Co., Ltd., China), ciprofloxacin HCl (CF) (analytical standard, Krka d.d., Novo mesto, Slovenia), desethylene-enrofloxacin (DES) (Pharmaffilates Analytics & Sinthetics Ltd., India), desethylene-ciprofloxacin (DES-CF) (USP Rockwille, USA), and pipemidic acid, internal standard (analytical standard, Krka d.d., Novo mesto, Slovenia). Other chemicals: acetonitrile (ACN) (HPLC grade, J.T. Baker, Netherlands), phosphoric (V) acid (H3PO4) (p.a., J.T. Baker, Netherlands), sodium dodecyl sulfate (SDS) (p.a., Merck, Germany), sodium hydroxide (NaOH) (p.a., Fluka, Switzerland), ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, USA), and potassium monohydrogen phosphate (K2HPO4) (Sigma-Aldrich, USA).

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Study design The degradation of EF was studied in broiler chicken manure gathered from healthy broiler chickens not treated with any kind of VMP during their life cycle. It was obtained from a local intensive rearing facility. The gathered chicken manure contained 61.2 % of dry material and had a pH of 8.5. Five days before the start of the study, it was acclimatized to the conditions present in the laboratory incubation room (25± 1 °C, moisture=50–65 %). A flow-through system was used, whereby the manure and sterile manure, incubated in flasks (duplicates at each sampling point), was aerated with air (aerobic incubation) or nitrogen (anaerobic incubation). A flowthrough system contained one 250-mL flask with distillated or sterilized water through which ventilated air or nitrogen was flowing into the next 500-mL flask where manure was incubated. The manure was exposed to an LED reflector (50 W, 5000 K) for incubation in the presence of light. The sterile manure was autoclaved for 30 min at 124 °C, after which sterilized distilled water was added to give the same dry content (61.2 %) as before sterilization. 0.2-μm sterile filters, sterilized laboratory equipment, and a flow-through system were used in the incubation of sterile manure. EF, dissolved in 1:1 (v/v) water/acetonitrile at 200 μg/mL, was applied to 50 g of manure to a final concentration of 4 mg/kg. The manure was incubated in duplicate flasks for 120 days. On days 0, 1, 3, 8, 16, 29, 41, 60, 90, and 120 for non-sterile incubation and days 0 and 120 for sterile incubation, samples of manure were extracted and analyzed with HPLC. Sample preparation At each sampling point, the manure samples were extracted using the method of Slana et al. (2014). Forty-four milliliters of extraction buffer was added to each portion of 25 g manure and the sample vortexed for 90 s. Each sample was then incubated in an ultrasonic bath for 15 min, then again shaken for 90 s before centrifugation at 3200 rpm. The liquid phase was decanted and the extraction procedure repeated three times. The liquid phase (combined extracts from all 4 extraction cycles) was filtered and analyzed for the presence of EF and its degradation products. HPLC method validation and analysis Manure extracts were analyzed at room temperature (23± 2 °C) by HPLC (Agilent 1100 with fluorimetric detection) as described by Slana et al. (2014). Mobile phase flow was 1.3 mL/min. The method was validated for EF, CF, DES, and DES-CF for the parameters presented in Table 1, in line with the European Commission, European Medicines Agency and OECD analytical guidance (Anon 1, 2000; Anon 2, 2002; Anon 6, 2010; Anon 7, 2011). Samples for method validation

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comprised manure extract that was extracted the same way as the incubated manure samples in the degradation study but spiked with (EF/CF/DES/DES-CF). A calibration curve was constructed using blank manure extracts spiked with concentrations 0.051, 0.102, 0.204, 0.476, 1.020, 2.040, 3.400, 6.800, and 13.600 mg/kg (R2 >0.99). Intra-day precision and accuracy were determined using blank manure extracts spiked at 0.425 (QC1), 1.7 (QC2), and 8.5 (QC3) mg/kg. The stabilities of the stock solution (EF/CF/DES/DES-CF dissolved in distilled water with the addition of 20 μL of 2 M NaOH) and of the working solution (EF/CF/DES/DES-CF dissolved in distilled water:ACN=1:1, v/v) were determined after 1 month at +5±3 °C. The stability of the manure extract was determined in spiked blank manure extract after 48 h (+23 ± 2 °C), after 10 days (+5±3 °C), and after 2 months (−18± 4 °C). Extraction recovery was established in residue-free manure spiked at 0.5, 1.0, and 2.0 mg/kg for EF, DES, CF, and DES-CF, with three replicates at each level. Theory/calculation The concentrations of EF, CF, DES, and DES-CF in manure extracts were determined by comparing values at each sampling point with the calibration curve obtained with spiked (EF/CF/ DES/DES-CF) blank manure extract. Calibration curves were obtained by plotting the peak/area ratios of EF/CF/DES/DESCF and internal standard (IS) versus the known soil concentrations of analyte by using weighted (1/concentration) linear regression analysis (Chemstation Plus, Agilent, 2004):
y ¼ k*X þ n R2

y—HPLC peak-height ratios of EF/CF/DES/DES-CF vs. IS x—EF/CF concentration (mg/kg) k—slope of calibration curve n—intercept of calibration curve R2—correlation coefficient From the averaged concentrations of duplicates, a degradation half-life (DT50) was calculated (in line with EMEA, FOCUS and OECD 307 guidelines (Anon 2, 2002; Anon 3, 2006; Anon 8, 2011) for each set of laboratory conditions to which the manure was exposed. For DT50 and DT90 (time when 90 % of the applied compound is degraded) calculations, the software R version 3.0.3 with the kinfit software package was used. For the statistical analysis, the best fitted of the four statistical models, SFO (simple first-order kinetics), FOMC (first-order multi compartment), DFOP (double-firstorder in parallel), or HS (first-order sequential bi-phasic) suggested by the software, was used.

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Table 1 Validation parameters EF (RSD in %)

CF (RSD in %)

LLOQ (mg/kg)

0.051

0.102

0.051

0.051

LLOQ precision in %, N=6

112.9 (5.5) 0.051–13.60

103.1 (10.3) 0.102–13.60

102.5 (7.8) 0.051–13.60

115.0 (4.3) 0.051–13.60

96.8 (6.1)

87.3 (10.8)

90.8 (9.9)

91.6 (4.0)

96.1 (5.5) 96.8 (3.9)

91.1 (6.2) 101.5 (8.8)

98.7 (4.1) 95.6 (4.0)

94.9 (3.3) 90.1 (2.0)

102.0 (5.3) 97.0 (6.5)

91.5 (9.6) 91.7 (1.6)

92.1 (11.9) 99.0 (5.1)

97.6 (2.6) 96.6 (1.1)

95.1 (3.0) 95.7 (4.3)

100.0 (10.4) 98.2 (6.0)

93.6 (2.4) 99.4 (3.9)

90.9 (0.9) 100.1 (5.2)

95.4 (5.1)

100.8 (8.3)

98.8 (5.9)

99.8 (7.8)

110.0 (8.0) 103.8 (6.0)

110.1 (9.1) 102.7 (7.5)

97.5 (6.6) 101.1 (6.2)

100.6 (9.3) 100.2 (5.3)

92.2 (7.3) 97.6 (5.9)

86.3 (10.1) 96.0 (8.3)

96.2 (6.4) 97.6 (5.5)

91.5 (11.0) 95.5 (8.0)

98.1 (4.4) 110.0 (3.1)

74.3 (8.5) 105.7 (6.8)

94.9 (6.0) 111.7 (4.3)

102.2 (7.0) 112.2 (5.3)

0.5 mg/kg

84.7 (4.0)

78.6 (6.5)

91.6 (3.6)

66.1 (7.1)

1.0 mg/kg 2.0 mg/kg

87.6 (3.4) 89.9 (2.8)

82.7 (6.1) 79.3 (5.0)

93.0 (3.9) 94.9 (3.3)

70.2 (5.9) 68.3 (5.4)

Linearity (mg/kg)a Inter-day precision in %, N=3 QC1 QC2 QC3

DES (RSD in %)

DES-CF (RSD in %)

Intra-day precision in %, N=4 QC1 QC2 QC3 Stock solution stability at +5±3 °C in %, N=3b Working soln. stability at +5±3 °C in %, N=3b Manure extract stability at +23±2 °C in %b QC1 (48 h), N=3 QC3 (48 h), N=3 Manure extract stability at +5±3 °C in %b QC1 (10 days), N=3 QC3 (10 days), N=3 Manure extract stability at -18±4 °C in %b QC1 (2 months), N=3 QC3 (2 months), N=3 Extraction recovery in %, N=3

RSD standard deviation/mean, LLOQ lower limit of quantification, N number of replicates, QC1–3 quality control sample a

R2 >0.999 for all compounds

b

Stability=analyte concentration t=48 h or 10 days or 2 months/analyte concentration t=0

Results Incubation of chicken manure showed that EF degradation rate was dependent on laboratory conditions. The formation rate of the degradation product DES was also dependent on laboratory conditions. The EF and DES values (in %) obtained in manure, compared to those for the first incubation day (t= 0), are presented in Table 2. The rate of degradation of EF is fastest under dark, aerobic conditions (testing conditions recommended by the guideline), where approximately 38 % of EF was present in manure at the end of the incubation period (Table 2). The second fastest degradation of EF took place in the dark under anaerobic conditions, where approximately 45 % remained in the manure after incubation. The degradation was also relatively rapid under aerobic conditions in the presence of light, whereas significantly slower degradation occurred under anaerobic.

The formation of DES was significantly slower under anaerobic conditions comparing to aerobic conditions. Degradation of EF under sterile conditions was slower than under non-sterile conditions. At the end of incubation, approximately 80 % of the initially applied EF was still present in the manure. Additionally, around 1 % of DES was found in sterile manure incubated in the presence of light, whereas no DES was detected in the sterile manure incubated in the dark. The summary of the kinetic models used, goodness of fit, and the calculated DT50 and DT90 values for each incubation type are presented in Table 3 and Fig. 1.

Discussion and conclusions The study was conducted in line with the guideline on determining the fate of veterinary medicinal products in manure.

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Environ Sci Pollut Res (2016) 23:4422–4429 Measured /initial concentrations of EF and DES under sterile and non-sterile conditions (values in %) Incubation conditions Anaerobic, dark

Aerobic, light

Incubation day

EF (RSD)

Aerobic, dark DES (RSD)

EF (RSD)

DES (RSD)

EF (RSD)

DES (RSD)

EF (RSD)

DES (RSD)

0 1

100 (0.2) 92.1 (1.5)

n.d. n.d.

100 (0.2) 92.7 (1.0)

n.d. n.d.

100 (1.3) 90.7 (3.2)

n.d. n.d.

100 (1.3) 93.9 (3.3)

n.d. n.d.

3 8

87.8 (3.0) 89.1 (1.5)

n.d. n.d.

79.0 (0.7) 83.9 (2.8)

n.d. 0.8 (0.1)

94.4 (4.4) 87.8 (2.8)

n.d. 1.8 (0.3)

79.5 (6.0) 77.2 (2.5)

n.d. 2.7 (0.4)

16

83.4 (4.6)

1.5 (0.2)

70.9 (0.6)

2.0 (0.5)

80.6 (5.5)

3.2 (0.2)

71.8 (2.0)

3.5 (0.2)

29 41

53.6 (3.5) 42.5 (2.2)

7.0 (0.9) 7.2 (0.4)

73.6 (2.2) 68.2 (3.1)

0.8 (0.2) n.d.

66.2 (6.0) 77.2 (3.7)

5.2 (0.9) 2.0 (0.3)

67.6 (8.0) 65.2 (4.6)

2.2 (0.2) 2.2 (0.5)

60 90

41.4 (1.9) 39.3 (5.1)

9.5 (0.7) 11.0 (1.0)

54.2 (4.0) 48.7 (1.9)

0.9 (0.2) 2.1 (0.3)

72.6 (3.9) 50.1 (1.9)

2.8 (0.1) 10.3 (0.5)

62.3 (3.8) 65.8 (7.3)

1.8 (0.3) 2.2 (0.6)

120

38.1 (4.3)

12.8 (0.8)

44.9 (3.6)

2.5 (0.3)

45.7 (4.1)

14.9 (0.6)

61.9 (5.2)

5.2 (0.7)

Sterile incub Incubation day

Aerobic, dark EF (RSD) DES (RSD)

Anaerobic, dark EF (RSD) DES (RSD)

Aerobic, light EF (RSD) DES (RSD)

Anaerobic, light EF (RSD) DES (RSD)

0 120

100 (1.3) 81.9 (3.3)

100 (1.3) 79.3 (2.8)

100 (2.2) 81.5 (4.9)

100 (2.2) 71.1 (2.5)

n.d. n.d.

n.d. n.d.

Anaerobic, light

n.d. 0.9 (0.3)

n.d. 0.7 (0.1)

n.d not detected

Manure degradation was studied under four sets of conditions and showed that EF degradation rate and DES formation are dependent on laboratory conditions. At the end of the incubation period (120 days), 38–62 % of the initially applied EF was still present in the manure. Further, 2.5–15 % of DES was formed. EF was degraded much more slowly in sterile manure, with approximately 80 % of the initially applied EF present at termination of the study and negligible formation of DES (1 % only in the presence of light). EF degradation under laboratory conditions can therefore be considered mainly as a biotic process. EF is degraded most rapidly in the dark under the aerobic conditions, which are also the testing conditions recommended by the manure degradation guideline (Anon 8, 2011). Overall, degradation of EF has been shown to be faster when manure is incubated in the dark. Further, aerobic conditions appear to speed up EF degradation and also contribute to greater formation of the degradation product DES. The results demonstrate that presence of light during incubation did not significantly accelerate the degradation process of EF or influenced the diversity or speed of degradation product Table 3 DT50 and DT90 values obtained with the best fitted statistical model Statistical model R2 Chi (χ) DT50 (days) DT90 (days)

formation. This is contrary to data published by Gagliano and McNamara (1996), Li et al. (2011), Lin et al. (2010), Martens et al. (1996), Parshikov et al. (2000), SchmittKopplin et al. (1999) and Wetzstein et al. (1999) who demonstrated that EF is susceptible to light and is degraded into various degradation products in pond sediments and in water solutions at different pH and in the presence or absence of humic acid. The accessibility of light to EF was probably lower in manure comparing to other matrixes which reflected in lower degradation rate. The DT50 values of 59.1 to 190.8 days obtained in the present study are comparable to those of 84 days determined in cattle manure incubated under aerobic conditions in the dark (Wetzstein et al. 2002). In the latter case, the degradation products 7-amino enrofloxacin and CF were found, as well as DES which was also detected in chicken manure in the present study. The DT50values of 142 to 468 days determined in cattle manure by Gagliano and McNamara (1996) are much longer than those reported for the cattle manure degradation performed by Wetzstein et al. (2002). The reason for the

Aerobic, dark

Anaerobic, dark

Aerobic, light

Anaerobic, light

SFO 0.865 10.8 % 59.1 196.3

DFOP 0.962 4.2 % 88.9 378.4

HS 0.914 5.9 % 115.0 427.6

SFO 0.69 8.8 % 190.8 633.9

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a

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c EF (% of applied)

EF (% of applied)

Time (days)

b

Time (days)

d

EF (% of applied) EF (% of applied)

Time (days)

Time (days)

Fig. 1 Kinetics of EF degradation under various laboratory conditions degradation rate is plotted against days of incubation. a Aerobic conditions in the dark, b anaerobic conditions in the dark, c aerobic conditions in the presence of light, and d anaerobic conditions in the presence of light

significant difference in the EF degradation rates in the reported papers is not clear, because of the poor study description by Gagliano and McNamara (1996) a comparison of both studies is not possible. However, according to Wetzstein et al. (2002), a higher variety of degradation products in cattle manure seems to occur, comparing to chicken manure under laboratory conditions. This is probably due to the fact that ruminants possess a sophisticated metabolic system which is also rich in microorganisms and fungi that probably contribute to EF degradation significantly. According to Randhawa et al. (Randhawa and Kullar 2011) strains of the fungi Rhizopus and Mucor can among others be found in cow rument. For both families, intensive degradation (up to 78 %) of fluoroquinolones with many formed degradation products was demonstrated (Martens et al. 1996; Parshikov et al. 2000; Wetzstein et al. 1999). Contrary to published EF laboratory degradation data are EF and CF degraded fast under the environmental conditions. A field study was performed by Boxall et al. (2004) where turkey litter from intensively rearing facility, after the rearing cycle has finished, was stored outside for 1 month. Turkeys were treated with EF at 10 mg/kg/day for 14 consecutive days.

EF and CF were detected in the manure heap at 2.92 and 0.28 μg/kg, respectively, whereas none was detected 21 and 90 days after the manure was applied to soil. The field study demonstrated that the degradation of EF and CF in manure amended soil under environmental conditions was faster than comparing the EF degradation in manure under controlled laboratory conditions. If faster EF dissipation in the environment occurred because of faster degradation of EF in manure or due to the synergistic effect of manure and soil is not clear and should be further investigated in the field. In conclusion, it has been shown that EF is degraded relatively rapidly in broiler chicken manure under the laboratory conditions recommended by guideline on determining the fate of veterinary medicinal products in manure. The EF degradation was faster under aerobic conditions in the dark. The formation of DES was the highest under aerobic conditions, where similar production rate in the presence or absence of light occurred. For other VMPs, the following DT50 in manure have been determined: tiamulin>180 days (Schlüsener et al. 2006), oxytetracycline≤30 days (De Liguoro et al. 2003; Wang and Yates 2008; Wu et al. 2011), florefnicol=10 days (Aquaflor 2004), flubendazole >102 days (Kreuzig et al.

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2007a), sulfadiazine>102 days (Kreuzig et al. 2007b), and doxycycline=52.5 days (Szatmari et al. 2011). Based on the comparison of the DT50 between different VMPs, a classification of EF as a semi-rapid degradable compound, which has low probability of accumulating and to produce secondary poisoning in the environment, may be appropriate. However, the question if EF degradation in the field is similar to that in the laboratory still remains, which can therefore be a prospect for future work. Published field studies indicate (Moraru et al., 2012; Boxall et al., 2004) that EF degradation in nature may be faster than in laboratory, but a well-designed and reported study to demonstrate the degradation pattern of EF under environmental/field conditions and under conditions occurring on the intensively rearing farms may be a challenge for the future.

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