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。
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 satisfactory
2 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.
引文
1 国家环境保护总局。水和废水监测分析方法 [ M ] 。北京:中国环境科学出版社,1989
2 中国药典二部,292
3 邵山,谢勤。紫外分光光度法测定苯酚滴耳液的含量。中国生化药物杂志,2002,23(2):90-93
4 倪翠芳,王建华。单扫描极谱法测定水中痕量苯酚。分析试验室,1999,18(6):75-78
5 冯立娟,辛长波。荧光法直接测定环境水中痕量苯酚。光谱实验室,2002,19(2):267-269
6 赵起越,岳志孝。气相色谱法分析固定废弃物中苯酚类物质。岩矿测试,2001,20 (4):279-283
7 吴宪龙,朱爱丽。高效液相色谱法测定环境水中痕量 4 ,4 -二氨基联苯、4-硝基酚和苯酚。色谱,1998,16 (6):536-538
8 王秀芳,张会平,肖新颜等。苯酚在竹炭上的吸附平衡和动力学研究。功能材料,2005,5(36):746-749
9 叶李艺,钟辉,张会平。对氯苯酚在活性炭上的吸附与脱附工艺。厦门大学学报(自然科学版),1998,37(4):542-546
10 Zhang Qisheng, Jiang Shuhai, Huang Helang.Research Adsorption Properties and Relative Factors of Bamboo Charcoal on 2,4-dichlorophenol. Editorial Committee. Mechanism and Science of Bamboo Charcoal and Bamboo Vinegar[M]. Beijing : China Forest ry Publishing House, 2001,168-173
11 Kei M , To shitatsu M , Yasuo H , et al . Removal of nitratenitrogen from drinking water using bamboo powder charcoal [J ]. Bioresource Technology , 2004 , (95) : 255 - 257
1 应萍君,唐赟,胡立军。一起苯酚污染水质事件的调查[J]。现代预防医学,2006,33(4):565
2 Fasman GD. Handbook of Biochemistry and Molecular Biology: Nucleic Acids[M]. Cleveland Ohio: CRC Press, 1975.73
3 Cao Y, He XW. Studies of Interaction between Safranine T and double helix DNA by spectral methods [J]. Spectrochim Acta PartA,1998,54(6)∶883-892
4 吴性良,朱万森,马林。分析化学原理[M]。北京:化学工业出版社,2004,364-365
5 Prativel G, Bermadou J, Meunier B. Carbon-hydrogen bonds of DNA sugar units as targets for chemical nucleases and drugs [J]. Angew Chem Int Ed Engl, 1995, 34 (7)∶746-769
1 Axel O. The use of a Refined Zebrafish Embryo Bioassay for the Assessment of Aquatic Toxicity. Research Note, 2000, 29(7): 32-40
2 张金荣,唐非,谷康定。自来水中有机污染物对细胞DNA的损伤作用。中国公共卫生[J],2004,20(7):785。
3 OECD. Fish, embryo toxicity test with the zebrafish. OECD Guideline for Testing of Chemicals, OECD TG212, 1998.
4 周宇,于红霞,丁关羽。氯代苯类有机污染物对斑马鱼胚胎联合毒性效应的研究。农业环境科学学报[J],2003,22(3):344-344
5 刘静玲,袁 星,郎佩珍。2,4—二硝基甲苯与共存硝基芳烃化合物对斜生栅列藻的联合毒性[J]。环境科学,1997,18(5):31-36
6 国家环境保护局.生物监测技术规范(水环境部分)[R]。北京:国家环境保护局,1986,95。
7 陆光华,袁星,赵元慧。苯酚苯胺及其衍生物对斜生栅列藻的急性毒性及QSAR研究。环境化学[J],2000,19(3):225-229
8 苏丽敏,孟庆俊,袁星。苯胺和硝基苯胺对大型蚤Daphnia magna的联合毒性。环境科学研究[J],2002,15(6):42-44。
9 Lange M. Comparison of testing acute toxicity on embryo of zebrafish, brachydanio rerio and RTG-2 eytotoxicity as possible alternatives to the acute fish test. Water Research[J], 1995, 30: 2087-2102
1 CARB. Air toxics emissions data collected in the Air Toxics Hot Spots Program CEIDARS Database as of January 29, 1999
2 Bruce RM, Santodonato J, and Neal MW. Summary review of the health effects associated with phenol. Toxicol. Ind. Health, 1987 3: 535-568
3 Griffiths GJ. Fatal acute poisoning by intradermal absorption of phenol. Med. Sci. Law, 1973 13:46-48
4 Dosemeci M, Blair A, Stewart PA,et al. Mortality among industrial workers exposed to phenol. Epidemiology, 1991 2(3):188-193
5 Deichmann WB, Keplinger ML. Phenols and phenolic compounds. In: Clayton GD, Clayton FE, eds. Patty's industrial hygiene and toxicology, 3rd ed. New York, NY: John Wiley and Sons, Inc, 1981 2567-2627
6 Stajduhar-Caric Z. Acute phenol poisoning.Singular findings in a lethal case. J Forensic Med, 1968 15:41-42
7 Boatto G, Nieddu M, Carta A, et al. Determination of phenol and o-cresol by GC/MS in a fatal poisoning case. Forensic Sci Int, 2004 139(2-3):191-194
8 Tanaka S, Choe N, Kita T, et al. Distribution of phenol in a fatal poisoning case determined by gas chromatography / mass spectrometry. J Forensic Sci, 1998 43(5):1086-1088
9 Soares ER, Tift JP. Phenol poisoning: Three fatal cases. J Forensic Sci, 1982 27(3):729-731
10 Lo Dico C, Caplan YH, Levine B, et al. Phenol: Tissue distribution in a fatality. J Forensic Sci, 1989 34:1013-1015
11 Lister J. On a new method of treating compound fracture, abcess, etc., with observations on the condition of suppuration. Med Classics, 1867 2: 28-71
12 Duverneuil G , Ravier E. Toxicité suraigu? du phénol par voie transcutanée. Arch Mal Prof, 1962 23: 830-833
13 Evans SJ. Acute phenol poisoning. Br J Ind Med, 1952 9: 227-229
14 Cronin TD, Brauer RO. Death due to phenol contained in Foille. J Am Med Assoc, 1949 139: 777-779
15 Hinkel GK, Kintzel HW. Phenol poisoning of a newborn through skin resorption. Dtsch Gesundh, 1968 23: 2420 - 2422
16 Deichmann WB, Kitzmiller KV, Witherup BS. Phenol studies. VII. Chronic phenol poisoning, with special reference to the effects upon experimental animals of theinhalation of phenol vapor. Am. J. Clin. Pathol, 1944 14:273-277
17 Flickinger CW. The benzenediols: Catechol, resorcinol and hydroquinone. A review of the industrial toxicology and current industrial exposure limits. Am Ind Hyg Assoc J, 1976 37:596-606
18 von Oettingen WF, Sharpless NE. The toxicity and toxic manifestations of 2,2-bis (pchlorophenyl) -1,1,1- trichloroethane (DDT) as influenced by chemical changes in the molecule. J Pharmacol Exp Ther, 1946 88:400-413
19 Baj Z, Majewska E, Zeman K, et al. The effect of chronic exposure to formaldehyde, phenol and organic chlorohydrocarbons on peripheral blood cells and the immune system in humans. J Invest Allergol Clin Immunol, 1994 4:186-191
20 De Ceaurriz JC, Micillino JC, Bonnet P, et al. Sensory irritation caused by various industrial airborne chemicals. Toxicol Lett (Amst), 1981 9:137-144
21 Hoffman GM, Dunn BJ, Morris CR, et al. Two-week ( ten - day) inhalation toxicity and two-week recovery study of phenol vapor in the rat. Int J Toxicol, 2001 20:45-52
22 Langford CP, Bartlett R, Haddad LM. Phenol and related agents. In: Haddad LM, Shannon MW, Winchester JF, eds. Clinical management of poisoning and drug overdose. Philadelphia, PA: Saunders, 1998 956-960
23 Truppman ES , Ellenby JD. Major electrocardiograph changes during chemical face peeling. Plast Reconstr Surg, 1979 63: 44-48
24 Wilcosky TC, Tyroler HA. Mortality from heart disease among workers exposed to solvents. J Occup Med, 1983 25:879-885
25 Merliss RR. Phenol marasmus. Occup Med, 1972 14: 55-56
26 Kamijo Y, Soma K, Fukuda M, et al. Rabbit syndrome following phenol ingestion. Clin Toxicol, 1999 37:509-511
27 Shamy MY, El Gazzar RM El Sayed MA, et al. Study of some biochemical changes among workers occupationally exposed to phenol, alone or in combination with other organic solvents. Ind Health, 1994 32:207-214
28 Ciranni R, Barale R, Marrazzini A, et al. Benzene and the genotoxicity of its metabolites. I. Transplacental activity in mouse fetuses and in their dams. Mutat Res, 1988 208:61-67
29 Hsieh GC, Sharma RP, Parker RDR, et al. Immunological and neurobiochemical alterations induced by repeated oral exposure of phenol in mice. Eur J Pharmacol, 1992 228:107-114
30 Kim DH, Lee SK, Chun BY, et al. Illness associated with contamination of drinking water supplies with phenol. J Korean Med Sci, 1994 9:218-223
31 Foxall PJD, Bending MR, Gartland KI, et al. Acute renal failure following accidental cutaneous absorption of phenol:Application of NMR urinalysis to monitor the disease process. Human Toxicol, 1989 9 : 491-496
32 U.S. Air Force. Tolerance criteria for continuous inhalation exposure to toxic material. I. Effects on animals of 90-day exposure to phenol, CCl4 and a mixture of indole, skatole, H2S and methyl mercaptan. Wright-Patterson Air Force Base, OH: U.S. Air Force systems command, Aeronautical Systems Division, ASD technical report, 1961 61-519(I)
33 Conning DM, Hayes MJ. The dermal toxicity of phenol: An investigation of the most effective first-aid measures. Br J Ind Med, 1970 27:155-159
34 Berman E, Schlicht M, Moser V C, et al. A multidisciplinary approach to toxicological screening: I. Systemic toxicity. J Toxicol Environ Health, 1995 45:127-143
35 NCI. Bioassay of phenol for possible carcinogenicity. Bethesda, MD: U.S. Department of Health and Human Services. National Cancer Institute. NCI-CG-TR-203. 1980
36 Horch R, Spilker G, Stark GB. Phenol burns and intoxications. Burns, 1994 20:45-50
37 Brown VKH, Box VL, Simpson BJ. Decontamination procedures for skin exposed to phenolic substances. Arch Environ Health, 1975 30:1-6
38 Pullin TG, Pinkerton MN, Johnson RV, etal. Decontamination of the skin of swine following phenol exposure: A comparison of the relative efficacy of water versus polyethylene glycol/industrial methylated spirits.Toxicol Appl Pharmacol, 1978 43:199-206
39 Hunter DM, Timerding BL, Leonard RB, et al. Effects of isopropyl alcohol, ethanol, and polyethylene glycol/industrial methylated spirits in the treatment of acute phenol burns. Ann Emerg Med, 1992 21:1303-1307
40 Schmidt SH, Anniko M, Hellstr?m S. Electrophysiological effects of the clinically used local anesthetics lidocaine, lidocaine-prilocaine and phenol on the rat's inner ear. Eur Arch Otorhinolaryngol, 1990 248:87-94
41 Schmidt SH, Hellstr?m S. Phenol anesthesia of the tympanic membrane in purulent otitis media A structural analysis in the rat. Eur Arch Otorhinolaryngol, 1993 249:470-472
42 Spiller HA, Quandrani-Kushner DA, Cleveland P. A five year evaluation of acute exposures to phenol disinfectant (26%). J Toxicol Clin Toxicol, 1993 31:307-313
43 Liao TF, Oehme FW. Tissue distribution and plasma protein binding of [14C] phenol in rats. Toxicol Appl Pharmacol, 1981 57:220-225
44 Dalin NM, Kristoffersson R. Physiological effects of a sublethal concentration of inhaled phenol on the rat. Ann Zool Fenn, 1974 11:193-199
45 Cassidy MK, Houston JB. In vivo capacity of hepatic and extra hepatic enzymes to conjugate phenol. Drug Metab Dispos, 1984 12:619-624
46 Powell GM, Miller JJ, Olavesen AH, et al. Liver as major organ of phenol detoxification. Nature (London), 1974252:234-235
47 Quebbemann AJ, Anders MW. Renal tubular conjugation and excretion of phenol and pnitrophenol in the chicken: Differing mechanisms of renal transfer. J Pharmacol Exp Ther, 1973 184:695-708
48 Tremaine LM, Diamond GL, Quebbemann AJ. In vivo quantification of renal glucuronide and sulfate conjugation of 1-naphthol and p-nitrophenol in the rat. Biochem Pharmacol, 1984 33:419-427
49 Chapman DE, Namkung MJ, Juchau MR. Benzene and benzene metabolites as embryotoxic agents: Effects on cultured rat embryos. Toxicol Appl Pharmacol, 1994 128 (1) : 129 - 137
50 Moser VC, Cheek BM, MacPhail RC. A multidisciplinary approach to toxicological screening: III. Neurobehavioral toxicity. J Toxicol Environ Health, 1995 45:173-210
51 NTP. 1983b. Teratologic evaluation of phenol (CAS No. 108-95-2) in CD mice. Laboratory study: September 18, 1980 to January 12, 1981. National Toxicology Program. Research Triangle Park, NC: Research Triangle Institute
52 Zamponi GW, Ing D, French RJ. Arrhythmias during phenol therapies: A specific action on cardiac sodium channels? Circulation, 1994 89:914