| Doxycycline,as a member of the tetracycline antibiotics group,is widely used in modern animal agriculture prevention and treatment of bacteria-borne diseases and improved growth rates.However,the widespread occurrence and accumulation of doxycycline in the environment has become an issue of global health concern because of its antibiotic resistance,toxic and mutagenic characteristics.Given this concern,the objectives of the present study were to:(1)establish a HPLC method of doxycycline and investigate the concentration and distribution of doxycycline residues in animal excretion,waste water and soil from farms and vegetable bases;(2)trace the behavior of adsorption,decompose and degradation of doxycycline in animal excretion;(3)evaluate the uptake of doxycycline by vegetable from hydroponic system and investigate the effect on MLTC-1 cell activity and toxicity.1.Occurrence of 13 veterinary drugs in livestock farm-amended soil The distribution of 13 veterinary drugs and long term investigation of tetracyclines residues were studied in soil fertilized with animal manures in Eastern China.The results showed that the detection frequencies of 13 veterinary drugs were beyond 80%in the soil samples among the depths of 0-20,20-40 and 40-60cm.The concentrations of the five class of drugs in soils showed the following trend:cyromazine>tetracyclines>sulfonamides>fluoroquinolones>florfenicol.Long term investigation in soil layers at 0-20 depth showed that tetracyclines were one of the predominant veterinary drugs in soils,which was related to the frequency use of this kind of veterinary drugs and the biological stability in soil.Overall,the use of animal manures as fertilizer is a main source of veterinary antimicrobial pollution in soil around the livestock farms.And it’s an effective way to reduce the antimicrobial’ pollution in soil by necessary management and treatment of the animal manures.2.Occurrences of doxycycline and other three tetracyclines in animal excretion and wastewater Concentrations and spatial distributions of doxycycline and other three tetracyclines(chlortetracycline,oxytetracycline,and tetracycline)were investigated in 28 animal manure samples and 28 wastewater samples from intensive animal farms(pig and chicken).The results showed that higher concentrations of tetracyclines were found in animal manure and wastewater samples and the detection frequencies were beyond 68%in manure samples and beyond 75%in wastewater samples except tetracycline.The concentrations of doxycycline and oxytetracycline in animal manure were at the range of 0.03-20.60mg/kg and 0.01-31.60mg/kg,respectively,within the level of PPM.The concentrations of them in wastewater were at the range of 0.59～220.96μg/L and 0.32～876.21μg/L,respectively,within the level of PPB.Generally,the concentrations of the four tetracyclines detected in animal manure and wastewater were at the similar contamination levels in both pig and chicken farms.However,the water pollution coming from pig farms is considered to be much more serious because of the higher production of wastewater compared with that from chicken farms.Thus,strict supervision and regulation should be strengthened in the process of pig farming.3.Analysis method of doxycycline in wastewater from farms by HPLC A HPLC method was developed for determination of doxycycline residues in domestic animal wastewater.Water sample was adjusted to pH 6 after centrifugation.The disposable solid phase extraction(SPE)column,Oasis HLB cartridge,was employed to clean up and concentrate in a gentle nitrogen stream in a water bath.SB-C18 was used for stationary phase,whereas the mobile phase comprised 65%oxalic acid,35%methanol/acetonitrile(1:1 v/v).Doxycycline residues in animal wastewater were detected by HPLC at wavelength of 350 nm.Linear calibration curves for doxycycline were found in a concentration range of 1-20.0μg/mL with a correlation coefficient of 0.9998.At the spiked levels of 10-50μg/mL,the average recoveries ranged from 91.55%to 95.14%.Relative standard deviations(RSDs)of the recoveries were from 1.66%to 6.45%within day and from 0.62%to 0.75%between days.The method detection limit(MDL)was 0.1 μg/mL and the method quantity limit(MQL)was 1μg/mL.The analysis method was accepted for doxycycline residues determination in animal wastewater.4.Analysis method of doxycycline in animal excretion and vegetable leaves by LC-MS/MS A LC-MS/MS method was developed for determination of doxycycline residues in animal excretion and vegetable leaves.The animal excretion sample was extracted with 0.1MNa2EDTA-McIlvainesolution(pH=4),while the vegetable leaves sample was extracted with methanoland0.1MNa2EDTA-McIlvainesolution(9:1,pH=6.0).Then the supernatant was moved to acleantube after centrifugation.SB-C18 was used for stationary phase,whereas the mobile phase comprised methanol and 0.1%formic acid(60:40 v/v).The derivatized compounds were detected with MRM(ESI+).Linear calibration curves for doxycycline were found in a concentration range of 20-1000 ng/mL with a correlation coefficient of 0.9999.At the spiked levels of 50-500ng/g,the recoveries ranged from 66.42%to 81.93%in animal excretion with RSDs of 4.85%～7.29%within day and 2.23%～2.43%between day.The average recoveries in vegetable leaves samples ranged from 72.16%to 88.64%with RSDs of 3.51%-6.87%within day and 1.44%～1.82%between days.The method detection limit(MDL)was 5ng/g and the method quantity limit(MQL)was50.0 ng/g.The analysis method was accepted for doxycycline residues determination in animal excretion and soil.5.Degradation of doxycycline in poultry feces and its influencing factors The effects of concentration,light and bacteria on degradation kinetics of doxycycline in poultry manure were studied.The results showed that the decline of extractable residues of doxycycline in poultry manure was found to follow first-order kinetics and the degradation rate was negatively connected with the initial concentrations.The dissipation half-life of doxycycline in sterilization treatment was 140.28d and 69.27d in non-sterilization treatment under dark environment,while the dissipation half-life of doxycycline was 36.87d in non-sterilization treatment under light environment(P<0.01).Overall,both light and bacteria degradation are the main ways for doxycycline dissipation in poultry manure.But the degradation of doxycycline in chicken manure by light is stronger than that of microorganisms in this study.6.Adsorption dynamics of doxycycline in poultry manure The effects of pH,Ca2+,Na+,K+ions on adsorption and desorption dynamics of dynamics of doxycycline in poultry manure were studied.The results showed that doxycycline could be easily adsorbed on the surface of poultry manure with an adsorption rate of 90%and the adsorption dynamics followed the Freundlich function.The desorption rate increased as the pH decreased at the condition of pH<6(P<0.05),while it showed no variation when pH>6,indicating that it existed a big risk of doxycycline desorption when decomposing and manure in south-part of China.In low ionic strength medium,the desorption rate of Ca2+、was much higher,but not significant,than that of Na+ and K+,while it increased as the increasing of Ca2+ionic strength in high ionic strength medium(P<0.01).7.Uptake and accumulation of doxycycline by different kinds of vegetables Uptake and accumulation of doxycycline by four vegetables under doxycycline stress were studied to evaluate the food safety and phytoremediation in antibiotics environmental pollution.The results showed that the degradation rates of doxycycline in vegetable-planting treatment were beyond 95%,much higher than in the control.The dissipation half-lives of doxycycline in Green Lollo-Ross Lettuce,endive,Crassula tetragon and Veronica arvensis L.were 22.1d、1.4d、1.5d、2.3d and 2.8d,respectively.The difference was significant compared with the control group(P<0.01).The bioconcentration factors(BCFs)in GreenLollo-Ross Lettuce and endive were 7.20 and 4.95,respectively,much higher than in Crassula tetragon(0.76)and Veronicaarvensis L(0.49).The former belongs to the hyperaccumulate varieties,and is easy to produce food safety problem.They are suitable for phytoremediation of polluted environment by antibiotics.8.Effects of doxycycline exposure on the activity and toxicity of reproductive cells at low doses The effects of doxycycline on mouse leydig tumor cells(mLTC-1)were designed to study the influence and possible mechanism of veterinary antibiotics on progesterone synthetic and secretion of reproductive cells.The results showed that the doxycycline levels had a significant influence on mLTC-1 dynamics by using MTT and Annexin V-FITC/PI analysis.The concentrations of doxycycline above 20μM could significantly decrease the viability of mLTC-1 and increase the apoptosis(P<0.05).When the concentrations of doxycycline were 10μM,they had no significant effects on the viability and apoptosis of mLTC-1,but could inhibit progesterone synthetic(P<0.05).This inhibition was not significantly related to the expression of cAMP in MLTC-1 stimulated by hCG or forskolin.Further analysis by 2D gel showed that doxycycline stimulated the up regulation of HSP60,GRP75,IVD,and HCDH proteins in mLCT-1 cells,and these proteins were related to mitochondrial damage.It shows that doxycycline inhibits progesterone synthesis through the damage of mLCT-1 mitochondria and then inhibit stestosterone secretion,which is a potential risk on health of male reproductive function. |
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