| The objective of this study is to reveal the environmental behavior and ecotoxicologies of chlortetracycline (CTC) and its metabolites ECTC and ICTC in animal excretion when CTC was administered to animal as a feed drug additive. Firstly, a HPLC and LC-MS/MS analytical method were developed for determination of CTC and its metabolites, ECTC and ICTC, in animal manure and wastewater. And the occurrence of CTC, ECTC and ICTC were monitored in 82 animal manure samples and 55 wastewaters samples collected from intensitive animal farm and nearby. Then the residues and excretion of CTC, ECTC and ICTC in chicken and pig excretion were investigated when they were fed diet containing CTC. Afterwards, the environmental behavior including biogradation, sorportion and mobility of CTC and ECTC and ICTC in chicken and pig manure were studied according to OECD guidelines in laboratory. Meanwhile the ecotoxicologies of manure-derived CTC exposure on soil microorganism was studied.The studies included three sections:1) Development of analytical method for determination of CTC, ECTC and ICTC and investigation of three compounds in animal excretion and wastewater;2) Environmental behavior of CTC in animal excretion; and 3) Toxicology of manure-derived CTC exposure on soil microorganism. The six experiments were conducted as follows:1 Development of analytical method for determination of CTC and its metabolites in animal excreta and wastewater by HPLC and LC-MS/MSA HPLC and LC-MS/MS method were developed for determination of chlortetracycline (CTC) and its metabolites ECTC and ICTC residues in animal excrement and wastewater in this study. The first experiment was conducted for establishment analytical method to detect CTC in animal excretion by HPLC, the animal excrement samples were extracted with extraction solution composed of acetone-4mol.L -1hydrochloric acid-water (13:1:6, v/v/v), then filtrated and ready to detect. ZORBAX SB-C18 was used for stationary phase, whereas the mobile phase comprised 66.7% oxalic acid,13.3% methanol and 20% acetonitrile. CTC residues in animal excrement were detected by VWD at wavelength of 375nm. the calibration curves for CTC was linear in a concentration range of 0.5-10.Oug/ml (r2=0.9999).The recoveries at levels of 0.5,1.0 and 5.0mg.kg-1 fortified samples ranged from 79.6%-92.78%, with coefficient of variation of 3.88%-10.2%. The limits of detection were 0.5mg.kg-1.The second experiment was carried on for development of analysis of CTC ECTC and ICTC in animal excretion and wastewater by LC-MS/MS. Animal manure samples were extracted three times by 6mL 0.1 mol.L-1Na2EDTA-Mcllvaine buffer (pH=4) and clean up by Oasis HLB solid phase extraction column. Wastewater was filtrated by 0.65?m film then mixed with 5% Na2EDTA. Solution was clean up on Oasis HLB column followed by adjusting pH into 7. The analytes were separated by liquid chromatography and determined by liquid chromatography coupled to tandem mass spectrometry under electrospray ion and multiple reaction monitor (MRM) mode. The results indicated that the detection limits of CTC, ECTC and ICTC 20?g/kg in animal excretion and 10?g/L in wastewater respectively, and their average recoveries were 79.64%?97.23%, 75.34%?98.37% and 73.67%?95.08%,respectively. The inter-days relative standard deviations (RSDs) and intra-dayRSDs ranged from 1.78% to 6.18% and from 1.07% to 6.28%, respectively. The method has the advantage of simplicity, sensitivity and repeatability, and is suitable for simultaneous determination of CTC, ECTC and ICTC in animal excretion and wastewater.2 Investigation of CTC and metabolites ECTC and ICTC in animal manure and wastewater around the farm82 animal manure samples and 55 wastewater samples were collected in intensive animal farms and nearby during 2008 to 2009, the occurrence of chlortetracycline (CTC) and its metabolite ECTC and ICTC was investigated in manure and wastewater samples. In animal manure samples, the most frequently detected compounds were CTC (69.51%) with the maximum concentration of 72.8 mg.kg-1 DM, and followed by ECTC (51.22%) and ICTC (32.93%) with the maximum concentration of 26.37 mg.kg-1 DM and 13.67 mg.kg-1 DM, respectively. The highest concentration of CTC and ECTC were observed in pig manure, however, the highest level of ICTC was founded in dairy cow manure. Of 55 water samples investigated,28 was wastewater samples and 27 was surface water collected stream water around farms.CTC was dected in 57.1% wastewater samples and 51.9% surface water, respectively. ECTC was found in 39.3% wastewater samples and 40.7% surface water samples.ICTC has higher pollution rate (25%) in surface water than that in wastewater (10.7%). CTC, ECTC and ICTC were simultaneously detected in wastewater samples, the maximum pollution level of CTC, ECTC and ICTC were 29.12 ?g.L-1, 32.43 ?g.L-1 and 69.2?g.L-1, respectively. Concentration of ICTC was highest, which is different from the trend in manure samples. The investigation data showed that pollution load of CTC and its metabolites ECTC and ICTC in animal manure samples and wastewaters was serious, when CTC was fed to animals, which imply the environmental risk of persistence of antibiotics in feed.3 Residues and excretion of CTC and two metabolites in excrement when the chicken and pig were fed diet containing CTC36 21-day-old AA broiler chickens and 18 pigs (21.5±1.78Kg) were chosen as experimental animals to study the residues and excretion of CTC and two metabolites in animal excrement when CTC was fed to animal as feed drug additives in dietary. The classical metabolic total manure-collection method was used. The results showed that 62.88%,71.67% of CTC in took by chicken were excreted into chicken excrement in the form of CTC and two major metabolites ECTC and ICTC when chicken was fed with diet containing 40.45 mg.kg-1 and 197.23 mg.kg-1 CTC, of which CTC, ECTC and ICTC accounted for 35.69%,19.4%,7.79% and 39.57%,23.36%,8.74%,respectively. To compare with chicken, there were only 59.27% and 67.83% of CTC excreted by pig into faces and urine in the form of CTC, ECTC and ICTC when pig was fed 40.78 mg.kg-1 and 216.31mg.kg-1 CTC. Of which, CTC accounted for 31.45% and 36.43%, ECTC were 19.33% and 21.65%, ICTC were only 8.49% and 9.75%, respectively in mixture of pig faces and urine. The results showed part of CTC was digested and metabolized into ECTC and ICTC by animal when it was fed into animal, and there are 59.27%-71.67% of CTC excreted via the excrement into the environment in the form of parent compound CTC and two major metabolites ECTC and ICTC; of which CTC and ECTC accounted for a large proportion. For two kinds of animals, CTC and its metabolite residues in chicken excrement were higher than that in pig excrement, which might be related to the short digest tract of chicken.4 Degradation kinetics of CTC, ECTC and ICTC in chicken manure and pig slurriesTwo experiments were conducted to under better the degradation kinetics in different conditions.Experiment I:The objective of this study was to determine the degradation kinetics of CTC, ECTC and ICTC during the incubation of manure from medicated pig and chickens. The pig and chicken manure were collected in the Experiment 3 "Excretion and residue of CTC". The all manure samples were loaded into triplicate and incubated at 22.5?. The CTC,ECTC and ICTC concentration in chicken manure decreased approximately 53.88%(from 23.32mg.kg"1 to10.23mg.kg-1),50.7%(from 14.62 to 7.21 mg.kg-1 and 58.9%(from 3.77 tol.55 mg.kg-1) during the 48 days, yielding a half-life of about 44.15,51.34 and 33.81 days, respectively. And in pig slurries samples, the concentration of CTC and ECTC declined roughly 56.54%(from 22.18 mg.kg-1 to 9.64mg.kg-1) and72.5%(from 12.62mg.kg-1 to3.47mg.kg-1) during incubation period, with the half-life of 37.46d and 26.35d, shorter then that in chicken manure. However, the concentration of the ICTC slightly reduce by 18.35%(from 4.96 mg.kg-1 to 4.05 mg.kg-1) during the early 7days, then increased by 72.1%(from 4.05 mg.kg-1 to 6.972 mg.kg-1) during incubation period. The degradation kinetics of CTC, ECTC and ICTC were met with the first order kinetics model, except of ICTC in pig slurries.Experiment II:This experiment was to investigate the degradation mechanism with CTC and Chicken manures were divided into five groups at three initial concentrations and sterilized and dark treatment. Results showed that sterilization treatment affected significantly the degradation rate of CTC, and dark treatment also slowed down the degradation of CTC, which imply that the microorganism was the major factor of CTC biogradation, and the light also contribute to the reduction of CTC. Meanwhile, initial concentration of CTC was also an important factor, low concentration of CTC seems to be degradated rapidly.5 Sorption and mobility of CTC in pig and chicken manure during batch experimentThe sorption and mobility of CTC, a feed drug additive, in pig manure and chicken manure were investigated in laboratory according to the OECD guideline 106. The results showed that CTC was strongly and rapidly adsorbed to both manures and Freundlich model could describe preferably the CTC nonlinear sorption isotherms. The adsorbed CTC was difficult to be desorbed by rain with adsorption H of 1.522 for pig manure and 1.329 for chicken manure, respectively. Only 18.3%?20.4%?18.7%?19.4% and 55.7%?57.6% adsorbed CTC were desorbed by methanol,3mol/LNaCl and 3mol/LMgCl2. The sorption of CTC on manure were affected by solution pH and ionic strength, the amount of adsorbed CTC on manure decreased with an increase of pH value and ionic strength, and greater sorption effect in the Ca systems than the Na systems was observed. These results suggest that organic matter and cation exchange mainly contribute to the sorption of CTC on manure.6 Ecotoxicity effect of manure-derived CTC exposure on soil microorganismA field test was carried out to investigate the effect of manure-derived CTC exposure on soil microorganism when manure land application. Four 2m X 2m square agricultural soil fields were chosed and divided into four treatments:Control group (without any fertilizer application), fertilized with CTC-free chicken manure, and fertilized with CTC chicken manure. The population of soil microorganisms (bacteria, fungi and actinomycetes), soil microbial respiration, and soil bacteria resistance level to CTC and bacteria diversity were monitored during different period. The results show that:The soil microbial population was significantly inhibited during the first 15d in the order of bacteria> actinomycetes?fungi after the application of 40.0 and 100mg.kg-1 CTC, then the inhibition effect was decreased gradually. Soil respiration was inhibited during the first 15 days, and the inhibition effect was gradutely weaken afterwards. In addition, CTC exposure in manure-soil system increased CTC-resistant bacteria amount and proportion accounting for the total of cultured bacteria in soil, indicating manure-derived CTC increased risk of bacteria resistance to CTC. The 16S-rDNA DGGE electrophoresis pattern showed that the soil microbial diversity was significantly affected on the 7d when soil bacteria was exposed to 40 and 100mg.kg-1 CTC, then bacteria, exposed to 40 mg.kg-1 CTC, was found to return back on the 14d.To sum up, the results obtained in all the experiments showed that CTC, as feed additive, was administrated to animal,59.27%-71.67% of CTC, ECTC and ICTC was excreted into animal manure, then enter environment. CTC can be absorbed strongly on animal manure and was found to be difficult to degrade. When CTC-containing animal manure was applied into fish pond or soil, the fish and soil microorganism were affected during the first exposure time. It shows the risk of CTC. |
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