恩诺沙星在鸡粪中的残留及其生态毒理学研究

【作者】 吴银宝；

【导师】 汪植三；

【作者基本信息】 华南农业大学 ， 生态学， 2003， 博士

【摘要】 恩诺沙星是在畜禽养殖业中得到广泛应用的抗菌药物之一。恩诺沙星进入畜禽体后,不仅会在畜禽产品中残留,其活性代谢物还会随畜禽的排泄物进入环境,对生态环境造成影响。本文首先进行恩诺沙星在鸡体的排泄试验,确定健康鸡在灌服不同浓度的恩诺沙星后,恩诺沙星及其活性代谢物对环境的释放量以及鸡粪中恩诺沙星在自然光条件下的降解; 其次进行恩诺沙星部分化学及毒理学指标的测定,包括恩诺沙星的水溶解度、正辛醇/水分配系数、恩诺沙星在不同pH条件下的水解、恩诺沙星在自然光条件下的水解、恩诺沙星在不同类型土壤中的吸附和解吸规律、恩诺沙星对雨生红球藻(Haemafococcus pluvialis)和隆腺溞(Daphnia Carinata)的急性毒性等指标; 最后利用池塘微宇宙来研究恩诺沙星在水生生态系统中的降解,研究恩诺沙星对水体和淤泥中微生物的数量、各项水质指标等的影响,从而对恩诺沙星的环境安全性作出评价。选取40只80日龄左右的粤黄鸡,按体重大小随机分为四组,每组10只,公母各半,各组鸡体重相似,单笼饲养。给药前称重,根据鸡的体重确定给药量,给药剂量分别为2.5mg/kg体重、5mg/kg体重、7.5mg/kg体重,设空白对照组。给药后定时采集鸡粪样(粪尿混合),用高效液相色谱(HPLC)法测定粪样中的恩诺沙星及其主要代谢产物的含量。结果表明,健康鸡灌服恩诺沙星后,主要以恩诺沙星原形、其次以环丙沙星的形式随粪(尿)排出体外。鸡粪中环丙沙星含量为恩诺沙星的1/2~1/3。2.5mg/kg体重组和5mg/kg体重组在给药后6h恩诺沙星的排泄量达到最高峰,峰浓度分别为27.64μg/g鸡粪、34.28μg/g鸡粪; 环丙沙星达到峰浓度的时间为4h,峰浓度分别为7.69μg/g鸡粪、19.29μg/g鸡粪。7.5mg/kg体重组恩诺沙星排泄量达到最高峰的时间为9h,峰浓度为41.45μg/g鸡粪; 环丙沙星达到峰浓度的时间为6h,峰浓度为24.63μg/g鸡粪。至给药后第10d时鸡粪中检不出恩诺沙星和环丙沙星。取空白鲜鸡粪,加入恩诺沙星系列浓度标准液,使得各样品中制得的恩诺沙更多还原

【Abstract】 Enrofloxacin was one of the most extensive antibiotics applied in animal husbandry. After application, it would not only remain in the product of animals, but also enter the environment through the excrement with its active metabolite. Firstly, the excretion experiment was carried out in healthy chickens to determine the concentration of enrofloxacin and its active metabolite in chicken feces. These chickens were administered by gavage at different dosage. Then the degradability of enroflixacin in chicken dung was tested under natural illumination outdoors. Secondly, some of chemistry and toxicity indices were measured such as the water solubility, n-octanol-water partition coefficients, hydrolytic characteristic in a series of buffers ranging from pH 1 to 10 at 50℃ and in natural aquatic environment, absorption and desorption in different soil, EC50 to Haemafococcus pluvialis and Daphnia Carinata. Finally, aquatic microcosm was constructed to study the degradation of enrofloxacin, and its effects on the microorganism and aquatic quality. Finally, the environmental safety of enrofloxacin was evaluated. Forty 80-days-old broiler chickens of Ling-Nang-Huang were divided into four groups randomly, 10 chickens each group, including 5 male and 5 female. Each chicken was fed in single coop. The first group (control) wasn’t treated by enrofloxacin. The others were administered with enrofloxacin by gavage at different dosage of 2.5mg/kg, 5mg/kg and 7.5mg/kg body weight, respectively. Chicken feces (the excrements mixed with urine) was collected at appointed time after oral administration to determine the concentration of enrofloxacin and its active metabolite by high-performance liquid chromatography (HPLC) with fluorescence detection. Following oral administration, enrofloxacin was excreted in healthy chicken with the parent form of enrofloxacin (main form) and metabolite ciprofloxacin. The concentration of ciprofloxacin in chicken feces was about half to third of the concentration of enriofloxacin. The concentration of enrofloxacin in chicken feces reached the peak at the 6th hour after oral administration in two groups of 2.5mg/kg and 5.0mg/kg body weight, and the peak concentration was 27.64μg/g chicken feces and 34.28μg/g chicken feces respectively; The concentration of ciprofloxacin reached the peak at the 4th hour and the peak concentration was 7.69μg/g chicken feces and 19.29μg/g chicken feces respectively. In the groups of 7.5mg/kg body weight the concentration of enrofloxacin reached the peak at the 9th hour after oral administration and the peak concentration was 41.45μg/g chicken feces; The concentration of ciprofloxacin reached the peak at the 6th hour an the peak concentration was 24.63μg/g chicken feces. Enrofloxacin and ciprofloxacin were not detected in chicken dung at day 10 after oral administration. Chicken feces samples were obtained from poultry that had not been exposed to any fluroquinolones (FQs) within the previous 30d. Chicken feces mixed with different concentration of enrofloxacin standard solution made the enrofloxacin concentration in chicken feces to be 0.4, 2, 10mg/g. The samples were deposited outdoors and collected at different time to determine the concentration of enrofloxacin and its metabolite by high-performance liquid chromatography (HPLC). Under daylight illumination, the degradation of enroflixacin in chicken feces slowed downgradually duing the process of experiment. The degradation of enrofloxacin in chicken feces could be described by the first order kinetic equation of Ct= C0e-Kt. The half-life calculated by this equation was 2.23d. Solubility of enrofloxacin in water at 25℃ was determined by three methods as the method prescribed in《Pharmacopoeia of People’s Republic of China》, the flask-shaking method and the shaking method. The solubility of enrofloxacin in water was 161.33mg/L, 243.28mg/L and 202.56mg/L, respectively. According to the standard, enrofloxacin was a chemical difficult to dissolve. Of three methods, the method prescribed in pharmacopoeia need the shorter time, but the result was lower; the other two methods need more time, but the results were more accurate. The n-octanol-water partition coefficient of enrofloxacin at 25℃ determined by flask-shaking method was 4.45. This indicated that enrofloxacin was a lipophilic chemical and was easily enriched by organism. The hydrolyzation of enrofloxacin at 50℃ in a series of buffers ranging from pH 1 to 10 was measured. After 5d, the rate of dissociation was lower than 10%. According to extrapolation, the half-life of enrofloxacin hydrolyzation in water at normal temperature and aphotic condition was longer than one year. It indicated that enrofloxacin was very stable in water at the above condition. The variety of pH didn’t affect the dissociation of enrofloxacin. The hydrolyzation of enrofloxacin in natural water was determined under the circumstance of having or not having germ. The result indicated that the dissociation of enrofloxacin in water was abiotic hydrolyzation and photolysis was the main form of hydrolyzation. Absorption and desorption of enrofloxacin in three soil picked from different places were studied. The balanceable time of absorption and desorption was 34h and 44h respectively. Enrofloxacin could be absorbed by soil strongly and the quantity of absorption was 99% of total enrofloxacin in aquatic phase. The absorption regulation data were fitted to the Freundlich equation CS= kfCe1/ n. The desorption of soil toenrofloxacin depended on the concentration and the quantity of desorption was only 1‰ of the absorption. The EC50 (Effective concentration for 50% reduction in growth) of enrfloxacin to Haemafococcus pluvialis was carried out. At 25 ℃ and 24h, the maximum concentration was 50μg/ml that could not have inhibition in growth to alga; the minimum concentration was 180μg/ml that made alga die all. The 24h and 96h EC50 of enrofloxacin was 119.67μg/ ml and 152.29μg/ ml, respectively. Along with the time extension, the tolerance of alga to enrofloxacin increased and the growth of alga recovered gradually. At 72h and 96h, the group under the concentration of 160μg/ ml growed better than the control group. The acute toxicity of enrofloxacin to Daphnia Carinata at 25℃ was studied. At 24h, the minimum concentration was 1μg/ml that could not make Daphnia die and the minimum concentration was 150μg/ml that made Daphnia die all. The 24h-EC50 and 48h-EC50 of enroflocxacin to Daphnia Carinata were 54.03μg/ml and 34.26μg/ml, respectively. The 48h-LC50 (Lethal concentration for 50%) was 48.27μg/ ml. Aquatic microcosmos was set up to study the degradation of enrofloxacin and its effect on the aquatic ecosystem. Three experiments were done. The outdoors experiments were done in the shed and the indoors experiment done near the window. Every experiment included 5 series concentration of enrofloxacin and 1 blank group. The results were as follows: The degradation of enrofloxacin in aquatic microcosmos was very fast, 50% of primary concentration or below of enrofloxacin in water could be reached in 5h after administration. With the proceeding of experiment, the speed of degradation slowed down. The degradation in low concentration would be kept for a long time; Within the scope of concentration, enrofloxacin had no significant effect on the sum of aerobic bacteria, fungus, actinomycetes, nitrite bacteria and nitrate-reducer bacteria in water and sediment; The DO, PO3-P and COD of water in microcosmos had not be affected significantly by enrofloxacin; Ammonia-N content increased at theprophase ( in 6d), but had not significantly difference compared to the blank group; TN content of water increased significantly; TP content was decreased significantly. The degradation of enrofloxacin was very fast, it didn’t affect the function of aquatic microcosmos. But enrofloxacin couldn’t be degraded thoroughly in short time at low concentration, and it was very stable in water under aphotic condition, was affected less by pH value of environment, had lipophilic characteristic and was easily enriched by organism, so the potential ecological risk could not be overlooked after enrofloxacin entered into aquatic ecosystem, especially in the deep floor of aquatic ecosystem or the pond that had small diaphaneity and feeble illumination According to enrofloxacin characteristics of the strong absorption, feeble desorption and weak movement in soil, it could be speculated that the risk of enrofloxacin in terricolous ecosystem was bigger than in aquatic ecosystem.更多还原