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Microsoft word - parent-karam-1-sigera.rtf

Effect of ultrasonic bath temperature on recovery rate of three veterinary antibiotics
added to swine manure

Parent, Elizabeth. ERSAM. Département des sols et de génie agroalimentaire, Pavillon Paul-
Comtois, Université Laval, Québec, Canada G1V 0A6.
Karam, Antoine. ERSAM. Département des sols et de génie agroalimentaire, Pavillon Paul-
Comtois, Université Laval, Québec, Canada G1V 0A6.
Parent, Léon Etienne. ERSAM. Département des sols et de génie agroalimentaire, Pavillon
Paul-Comtois, Université Laval, Québec, Canada G1V 0A6.
Land application of swine manures can increase antibiotic concentrations in agricultural soils.
For soil management and water quality purposes, it is important to determine the precise
conditions necessary to evaluate the concentration of antibiotics in manures and soils. The
objective of this study is to examine the effect of ultrasonic water bath temperature (T) on
recovery rate of three antibiotics added to fresh swine manure. The antibiotics tested were:
oxytetracycline (OTC), chlortetracycline (CTC) and tylosin (TYL). Antibiotics were applied to
the manure at a rate of 20 mg/L. Antibiotics were extracted from swine manure using a
mixture of Na2EDTA and McIlvaine buffer at pH 7 and ultrasonic agitation with temperature
ranging from 25 to 60 °C and SPE clean-up. Concentrations of the antibiotics in the manure extracts were analyzed using high performance liquid chromatography with ultraviolet detection. The use of Na2EDTA-pH 7 McIlvaine buffer for antibiotic ultrasonic dissolution from swine manure is enhanced with heating. The highest levels of extractable CTC and OTC
were obtained with ultrasonic bath temperature of 45 °C for CTC and OTC and 40 °C for
TYL. Compared with the control (T= 25 °C), the ultrasonic extraction temperature of 45 °C
increased the extraction recovery of CTC and CTO by a factor of 7.35 and 2.22, respectively.
The amount of extractable TYL was highest at T = 40 °C. Ultrasonic temperature between 40
and 45 °C during 10 min can be used to enhance extractability of CTC, OTC and TYL from
swine manures.

: antibiotic extraction, chlortetracycline, oxytetracycline, tylosin


Land application of manures from livestock which had received antibiotics in feed rations,
either as growth promoters or therapeutic agents, can increase antibiotic concentrations in
agricultural soils (Shi et al., 2010). If precipitation occurs before incorporating liquid manure
(slurry) into the soil for some time it is likely that any antibiotic residues in the slurry will be
transported towards surface waters in overland flow (Kay et al., 2005). For soil management
and water quality purposes, it is important to evaluate of the concentration of oxytetracycline
(OTC), chlortetracycline (CTC) and tylosin (TYL) in livestock manures (O’Connor et al.,
2007). However, determination of the concentration of manure-applied antibiotics is no easy
The conventional methods used to analyze tetracycline in water, soil and livestock manure
samples are high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection
(Kühne et al., 2000; Blackwell et al., 2004a) and LC-MS (Zhu et al., 2001; O’Connor et al.,
2007). Extraction buffer solutions have been employed in concert with ultrasonic energy in
order to dissolve target veterinary antibiotics for their subsequent measurement (Blackwell et
al., 2004a; Blackwell et al., 2005). However, little is known about the best temperature for
ultrasound extraction. The purpose of this paper is to report the effect of ultrasonic bath
temperature on levels of extractable OTC, CTC and TYL in antibiotic-treated swine manure.


The free antibiotic swine manure was collected from an animal farm production (St. Joseph
Farm) located at Portneuf (Quebec, Canada). After sampling, manure sample was frozen
until sample preparation. Unless otherwise stated, all solvents were of HPLC grade.
Ultrapure water was obtained by running demineralized water through a Milli-Q water
purification system. All glass material was washed with Liqui-Nox soap and rinsed with
ultrapure water, methanol and ethylenediaminetetraacetic acid disodium salt (Na2EDTA).
McIlvaine buffer was prepared by mixing 0.2 M citric acid and 0.4 M Na2HPO4 in the right proportions to get pH 7. Antibiotic treatments were applied to the manures (2 g) as a solution of OTC, CTC or TYL at rates providing 0 and 20 mg antibiotic /L. The manure suspension was allowed to stand for 1 h at room temperature. The study compounds were simultaneously extracted from the manure suspension using 0.1 M Na2EDTA and pH 7 McIlvaine buffer (50:50) according to Blackwell et al. (2004a, b). Briefly, 2 ml of antibiotic-manure suspensions were accurately weighed into a 50-ml centrifuge tube immediately after homogenization and 8 ml of extraction buffer (0.1 M Na2EDTA: pH 7 McIlvaine buffer, 50:50) added. The tubes were vortex mixed for 30 s, sonicated in an ultrawave sonic bath (VWR Brand Ultrasonic Cleanersfor) 10 min at
25, 35, 40, 45, 50 and 60 ºC. The samples were then centrifuged at approximately 1160g for
15 min to separate the solid and liquid phases. Exactly 5 ml of the supernatant was
transferred into a 15-ml centrifuge tube and 50 μl of phosphoric acid and 50 μl of acetonitrile
were added to adjust the sample pH and to help in precipitating proteins in the extract. The
tubes were allowed to stand for 10 min and then centrifuged at approximately 1160g for
15 min. The three antibiotics were extracted from the solution by solid phase extraction
(SPE), using an Isolute SAX (strong anion exchange) cartridge to remove interfering humic
materials in tandem with an Oasis HLB (hydrophilic–lipophilic-balanced) cartridge to extract
the compounds. Concentrations of the antibiotics in the manure extracts were analyzed by
HPLC with UV detection. Briefly, a gradient elution over 25 min was carried out with a
tetrahydrofuran, acetonitrile and 0.05% trifluoroacetic acid in water mobile phase. A flow rate
of 1 ml/min was used for the extraction and the compounds were detected at 285 nm.

and Discussion
Increasing the temperature of ultrasonic water bath over 25 °C increased the levels of
extractable antibiotics. Total OTC and CTC extraction in McIlvaine buffer-Na2EDTA
increased markedly as the temperature of ultrasonic water bath increased from ambient laboratory temperature (25 °C) to 45 °C and then slightly decreased with increasing temperature (Fig. 1). Compared to the control treatment (25 °C), the temperatures of ultrasonic water bath, e.g., 35, 40, 45, 50 and 60 ºC produced respectively 1.05-, 2.25-, 7.35-, 2.41- and 1.97-fold increase in OTC. Changes in OTC concentrations in the extracts were similar to those of CTC. When compared to the control treatment (25 °C), the ultrasonic water bath temperatures, 35, 40, 45, 50 and 60 ºC produced respectively 1.02-, 1.68-, 2.22-, 1.66- and 1.46-fold increase in CTC. In contrast, the TYL concentration increased up to approximately 40°C and then remained relatively constant. When compared to the control treatment (25°C), the ultrasonic water bath temperatures, 35, 40, 45, 50 and 60 ºC produced respectively 1.04-, 1.50-, 1.36-, 1.50- and 1.48-fold increase in TYL. Recoveries of the study compounds in manure were 19.3–42.8% for OTC, 13.6 to 100% for CTC and 20.2-35.5% for TYL. The increase in recovery rate of CTC was significantly higher than in the case of OTC and TYL (Fig. 1). This result indicates that CTC, OTC and TYL extraction is temperature dependent. The results of studies by Stoob et al. (2006) showed that the temperature was the most important parameter for the extraction
efficiency of five sulfonamide antibiotics (sulfadiazine, sulfadimethoxine, sulfamethazine,
sulfamethoxazole, and sulfathiazole) in aged soil samples by a pressurized liquid extraction
This increase in extractable antibiotics concentrations can be attributed in part to a higher
solubility of OTC, CTC and TYL at temperature higher than 25 °C. According to O’Connor et
al., (2007) higher temperature generally increases the solubility of analytes in the extracting
solvent. An alternative explanation would be that higher temperature in combination with
agitation helped increase OTC, CTC and TYL desorption from manure sorption sites.
However, the use of ultrasonic temperature higher than 40-45 °C could have limitations (Fig.
1), e.g., co-extraction of unwanted soil-matrix components and potential thermal degradation
of antibiotics (O’Connor and Aga, 2007). In a laboratory incubation study of OTC degradation
kinetics in animal manure, Wang and Yates (2008) found that the increasing incubation
temperature from 15 to 25 °C accelerated the desorption process of OTC in manure, but the
degradation in manure at 35 and 45 °C was much faster than at 15 and 25 °C.


The temperature of extracting solution affected the recovery of antibiotic from swine manure
samples. Temperature between 40 and 45 °C for 10 min can be used for the ultrasonic
dissolution of CTC, OTC and TYL from swine manures. Recoveries ranging from 30.4 (TYL)
to 100% (CTC) were obtained under the extraction temperature mentioned above. Further
investigations should focus on other ultrasonic bath parameters such as: volume of water;
extractant concentration; horizontal and vertical positions in the bath for sonicating samples
(central and bottom); sonication time.


Funding for this research was provided by the Natural Sciences and Engineering Research
Council of Canada. Project No RDCPJ385199-09.


Blackwell P. A., Boxall A. B. A., Kay P. and Noble H. (2005). Evaluation of a lower tier
exposure assessment model for veterinary medicines. Journal of Agricultural and Food
, 53 (6), 2192-2201.
Blackwell P.A., Holten Lützhøft H.-C., Ma H.-P., Halling-Sørensen B., Boxall A.B.A. and Kay
P. (2004a). Ultrasonic extraction of veterinary antibiotics from soils and swine slurry with SPE
clean-up and LC-UV and fluorescence detection. Talanta, 64 (4), 1058-1064.
Blackwell P. A., Holten Lützhøft H.-C., Ma H.-P., Halling- Sørensen B., Boxall A. B. A. and
Kay P. (2004b). Fast and robust simultaneous determination of three veterinary antibiotics in
ground water and surface water using a tandem solid-phase extraction with high-
performance liquid chromatography-UV detection. Journal of Chromatography A, 1045 (1-2),
Kay P., Blackwell P. A. and Boxall A. B. A. (2005). A lysimeter experiment to investigate the
leaching of veterinary antibiotics through a clay soil and comparison with field data.
Environmental Pollution, 134 (2), 333–341.
Kühne M., Ihnen D., Moller G. and Agthe O. (2000). Stability of tetracycline in water and
liquid manure. Journal of Veterinary Medicine A, 47 (6), 379-384.
O’Connor S. and Aga D. S. (2007). Analysis of tetracycline antibiotics in soil: Advances in
extraction, clean-up, and quantification. TrAC Trends in Analytical Chemistry, 26 (6), 456-
O’Connor S., Locke J. and Aga D. S. (2007). Addressing the challenges of tetracycline
analysis in soil: extraction, clean-up, and matrix effects in LC-MS. Journal of Environmental
, 9 (11), 1254-1262.
Stoob K., Singer H. P., Stettler S., Hartmann N., Mueller S. R. and Stamm C. H. (2006).
Exhaustive extraction of sulfonamide antibiotics from aged agricultural soils using
pressurized liquid extraction. Journal of Chromatography A, 1128 (1-2), 1-9.
Shi Y., Zhao M.-qiu, Wang J. and Song Y.-fang. (2010). Effect of antibiotics from organic
manure on protected vegetable soil-plant system: A review. Journal of Agro-Environment
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September 2010).
Wang Q and Yates S. R. (2008). A laboratory study of oxytetracycline degradation kinetics in
animal manure and soil. Journal of Agricultural and Food Chemistry, 56 (5), 1683-1688.
Zhu J., Snow D. D., Cassada D. A., Monson S. J. and Spalding R. F. (2001). Analysis of
oxytetracycline, tetracycline, and chlortetracycline in water using solid-phase extraction and
liquid chromatography-tandem mass spectrometry. Journal of Chromatography A, 928 (2),
Temperature, °C
Figure 1. Effect of ultrasonic bath temperature on recovery rate of oxytetracycline (OTC), chlortetracycline (CTC) and tylosin (TYL) in swine manure.



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