【作者】 勉丽娜；

【导师】 康维钧；

【作者基本信息】 河北医科大学 ， 劳动卫生与环境卫生学， 2008， 硕士

【摘要】 苯酚是一种重要的苯系中间体,主要用于生产酚醛树脂、己内酰胺、双酚A、己二酸、苯胺、水杨酸等,此外还可用作溶剂和消毒剂。环境中的苯酚主要来源于工厂排出的废气和废水,进入大气,土壤和水体,从而对人体及其他生物产生危害。除此,还有一些日常生活使用的含苯酚产品也可以使人体暴露于苯酚,如润喉片,消毒剂等,在饮用水中、香烟的烟雾中也发现了苯酚。苯酚可以通过呼吸道,消化道及皮肤三种途径进入人体,国内关于苯酚的毒性研究基本上都是苯酚中毒的病例报告,人群流行病学调查及动物实验研究较少。由于苯酚广泛存在于环境中,因此研究苯酚的毒性即显得尤为重要。本文建立了活性炭富集-高效液相色谱法测定水中痕量苯酚,并通过研究苯酚与DNA的相互作用及其对斑马鱼胚胎发育的生态毒性诊断研究,以评价苯酚对生物体健康的影响。目的:1建立水体中苯酚含量测定的方法,并通过研究苯酚在水体中的暴露水平,以评价苯酚对环境的污染程度。2研究苯酚与DNA之间的相互作用模式。3观察苯酚对斑马鱼胚胎发育各阶段毒理学终点的影响,以评价苯酚及对硝基苯胺对生物体的单一毒性和联合毒性。方法:1用活性炭富集自来水及河水中的苯酚,富集后的活性炭加入0.5mol/L的NaOH,同时超声进行解吸,滤过后加H2SO4将pH值调至5左右,高效液相荧光检测器检测,激发波长为273nm,发射波长为300nm,流动相为乙腈:磷酸盐缓冲液(0.05mol/L,pH为6.87)= 45:55。2采用紫外分光光度法,荧光分光光度法观察了不同浓度DNA对苯酚的紫外光谱、荧光光谱的影响,同时研究了离子强度和温度对苯酚-DNA混合体系的影响,实验结果用Stern-Volmer方程进行数据处理。3不同浓度的苯酚、对硝基苯胺对受精后0小时的斑马鱼胚胎进行染毒,观察染毒后4、8、12、16、24、36、48、72小时不同的毒理学终点,并用LD50统计软件计算各个终点的EC50值。用联合效应相加指数法评价苯酚和对硝基苯胺联合对斑马鱼胚胎各发育阶段的联合效应。结果:1建立了检测水体中痕量苯酚的活性炭富集-高效液相色谱法,该方法标准曲线回归方程为:Y=1.1×10~3X + 19.37,r=0.9991,加标回收率为83.2%-88.2%,相对标准偏差为3.1%,方法的检出限为30ng/L。自来水中未检测到苯酚,河水中的苯酚含量为73.75μg/L。2在pH 7.0的磷酸盐缓冲溶液中,苯酚的荧光激发峰和发射峰分别位于273nm和299nm处。小牛胸腺DNA的加入对苯酚的荧光存在着猝灭作用,室温条件下测定ct-DNA对苯酚的动态猝灭常数为7.062×103 L/mol。3本实验各浓度组均未观察到苯酚、对硝基苯胺对斑马鱼胚胎的眼点、耳石发育及20秒内主动运动三个毒理学终点的影响。斑马鱼胚胎对苯酚最敏感的毒理学终点为48小时黑素细胞不发育,EC50 = 52.14 mg/L;斑马鱼胚胎对对硝基苯胺最敏感的毒理学终点为72小时发育畸形,EC50=26.11mg/L。苯酚和对硝基苯胺对斑马鱼胚胎的联合毒性因毒理学终点不同而表现不同,32小时无血液循环、48小时无心率,48小时心包水肿表现为弱的协同作用;32小时心律减慢表现为相加作用;24小时尾部延展,48小时卵凝结,72小时发育畸形和孵化率表现为弱的拮抗作用。结论1活性炭富集-高效液相色谱法测定水中苯酚的方法成本低,灵敏度高,测定样品结果满意。2 ct-DNA对苯酚的荧光猝灭作用属于动态猝灭,在体外实验条件下,苯酚与DNA之间未形成络合物。3苯酚的毒性小于对硝基苯胺,两者均可对斑马鱼胚胎的血液循环产生影响并致使斑马鱼胚胎发育畸形。两者的联合作用在不同的毒理学终点表现不同。更多还原

【Abstract】 Phenol is one of the important benzene series.The two major uses of phenol are as an intermediate in the production of phenolic resins and in the production of bisphenol A. It is also used in the production of caprolactam,hexanedioic acid, aniline, salicylic acid et al. Phenol is also used as a solvent, as a disinfectant. The mostly likely source of exposure to phenol is at manufacturing and hazardous waste sites; therefore, people living near landfills, or plants manufacturing phenol are the most likely populations to be exposed. Other possible direct exposure may occur through use of consumer products containing phenol. Such as throat lozenges and antiseptic lotions. Phenol has been found in drinking water, tobacco smoke。Phenol can enter body through three routes, respiratory tract, digestive tract and dermal. The toxicity studies about phenol in our country are mainly case reports of phenol poisoning, no human epidemiological surveys and animal experiments is available. Due to the extensive existance, therefore, it is great important to study the toxicity of phenol. The objective of this study is mainly to evaluate the effects of phenol exposure to human health through investigating the interaction of phenol with DNA, the toxicity of phenol to zebrafish embryo and the exposure levels of phenol in water.Objective:1 To establish a method for measuring the concentration of phenol in water, and evaluate the contaminated levels of phenol through measuring the concentration of phenol in water samples.2 To investigate the interaction between phenol and calf thymus DNA(ct-DNA).3 To observe the effects of phenol and p-nitroaniline to zebrafish embryo and evaluate the single and combined toxicity of them to organism.Methods:1 Trice amounts of Phenol in tap water and river water were enriched by activated carbon activated under 200℃, The enriched pheno1 was desorbed from activated carbon with 0.5mol/L NaOH solution as well as ultrasonic heating, sulphuric acid was added to the desorbed solution to adjust PH to 5-6. the solution was detected by fluorescence detector, the excitation wavelength is 273nm, the emission wavelength is 300nm, acetonitrile - 0.05mol/L ph=6.87 phosphate buffer solution (45: 55) as mobile phase.2 The ultraviolet spectrophotometry and fluorospectrophotometry were used for observing the effects of different DNA concentrations to the ultraviolet spectrum and fluorescence spectrum of phenol, the effects of ionic strength and temperature to phenol-DNA mixed system. The experimental data were plotted by Stern-Volmer equation.3 The Zebrafish embryos were exposed to a range of concentration of phenol and p-nitroaniline within 30 minutes after the eggs have been fertilized, and then different toxicological endpoints were observed at 4, 8, 12, 16, 24, 36, 48, 72 hours after exposure, EC50 values were calculated by LD50 software. The combined toxicity of phenol and p-nitroaniline were evaluated by additive index.Results:1 A method of Preconcentration with activated carbon and high performance liquid chromatography with fluorescence detection for determination of phenol in river water was developed.The linear equation of the method was Y = 1.1×103X + 19.37,r = 0.9991, the recoveries of standard addition was in range of 83.2%-88.2%, the relative standard deviation was 3.1%, the detection limit was 30ng/L based on signal-to-noise ratio of 3:1. phenol wasn’t determined in tap water and the concentration of phenol in river water is 73.75μg/L.2 At pH 7.0 phosphate buffer solution, the excitation and emission wavelength of phenol are 273 nm and 299 nm, respectively. The addition of ct-DNA to phenol solution resulted in fluorescence quenching and Under the room temperature circumstance, we got its quenching constant KSV=7.062×103 L/mol. 3 No significant effects were seen at several toxicological endpoints, such as the development of eye and ear, active movement within 20 seconds. the most sensitive endpoints to phenol and p-nitroaniline are melanocytes aplasia (48h), EC50=52.14 mg/L, developmental malformation (72h), EC50 = 26.11mg/L, respectively. Slight Synergistic effect of combined toxicity were observed at no blood circulation (32h), no heartbeat (48), pericardial edema (48h) . additive effect of combined toxicity were observed at significant slow cardiac rhythm. And slight antagonistic effect were observed at tail extension (24h), coagulation of eggs (48h), developmental abnormality (72h) and hatching rate (72h).Conclusions:1 The features of this method are low cost, high sensitivity, and easy operation, the results of sample determination were satisfactory2 The fluorescence quenching between phenol and ct-DNA belongs to dynamic quenching, not static quenching, which means they don’t form new adduct.3 Toxicity of phenol is less than that of p-nitroaniline, both can effect the blood circulation of zebrafish embryo, developmental abnormality and even death were also caused. the manifestation of the combined toxicity between phenol and p– nitroaniline were different at different endpoints.更多还原