The Diagnosis Of Eco-toxicity Of Paraquat To Zebrafish Embryo

For the past few years, herbicide has been widely used in agriculture and environmental greening, more people start to consider its effection to human health. Paraquat is one kind of important herbicide which is poisonous, and the way of detoxification is very difficult. As most of the herbicide would finally inflow into the water in the using process, it is inevitable that paraquat would take some negative effect to aquatic environment. At present, the method of extracting and testing about paraquat can not use widely, because the samples are hard to prepare. It is great important to find a test method which is sensitive, simple and economy used in analysis the toxicity of paraquat. The objective of this study is mainly to evaluate the effects of paraquat exposure to human health through investigating the interaction of paraquat with DNA and the toxicity of paraquat to zebrafish embryo.Objective:1 To establish a simple and sensitive method of HPLC for paraquat in plants, observe the metabolism condition of paraquat in spinach which in fridge and natural environment.2 To establish a new method of chemoluminescence for detecting paraquat. 3 To study the interaction of paraquat with DNA.4 To observe the effects of paraquat to zebrafish embryo and investigate the effects of paraquat to life in water.Methods:1 Put the sample into the mixed liquor of methanol and hydrochloric acid (pH4-5) one night and extract through ultrasonic sound, then extract the liquor with C18 tube and analyze the quantity of paraquat in plants with ion pair reversed-phase liquid chromatography. The mobile phase is phosphate buffer (0.18mol/L) with sodium hexanesulfonate (18mmol) and methyl Cyanides (9:1), then adjust pH=2 with diethylamine. The wavelength is 256nm. Drop paraquat solution (10.0μg/ml and 100.0μg/ml) in spinach and put the spinach into fridge and natural environment, observe the metabolism condition of paraquat in spinach respective after 24h, 48h and 72h.2 Chemiluminescence reaction would happen in the solution with Ag(III) and luminol, paraquat enhanced the signal of this system, and concentration of paraquat and the signal had a very nice linear relation. Taking samples 10.0g in a beaker, wash and douse for 1 hour with distilled water, and clean with ultrasonic for 15 minutes, taking washings for determination.3 The ultraviolet spectrophotometry and fluoro spectrophotometry were used for observing the effects of different DNA concentrations to the ultraviolet spectrum and fluorescence spectrum of paraquat-NR system, the effect of ionic strength to paraquat-DNA mixed system. The experimental data were plotted by Stern-Volmer equation.4 The Zebrafish embryos were exposed to a range of concentration of paraquat 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.Results:1 A method of high performance liquid chromatography for determination of paraquat in vegetable was developed.The linear equation of the method was Y=1.21×104X+46×102, r = 0.9997, the recoveries of standard addition was 82.6%, the relative standard deviation was 3.6%, the detection limit was 10ng/ml based on signal-to-noise ratio of 3:1.2 A method of flow-injection chemiluminescence for determination of paraquat in vegetable was developed.The linear equation of the method was△I=2.95×107C+3.76×102, r = 0.997, the recoveries of standard addition was in rang of 85.4%-109.2%, the relative standard deviation was 1.0%, the detection limit was 13.4×10-6mol/L based on signal-to-noise ratio of 3:1.3 At pH 7.3 Tris-HCL buffer solution, the ultraviolet absorption of paraquat was at 260nm, the ultraviolet spectrum and fluorescence spectrum of NR-ctDNA system changed regularly with paraquat.4 Effections were beginning 4 hours after development, no heartbeat, pericardial edema, no blood circulation and other endpoints were observed. The most sensitive endpoints to paraquat is hatching rate (72h), EC50=387.6mmol/L, malformations had no significant effects between two groups.Conclusions:1 The determination of paraquat with ion-pair HPLC is low cost, high sensitivity, and easy operation, the results of sample determination were satisfactory.2 The feature of flow-injection chemiluminescence for determination of paraquat in vegetable is convenience, high sensitivity and low cost.3 Both the experiments of ultraviolet spectrophotometry and fluorospectrophotometry indicate that there are electrostatic attraction and intercalation between paraquat and DNA.4 The most sensitive endpoints to paraquat is hatching rate (72h) in all of the endpoints, and the lower one is egg coagulation (4h), both of them are fatal index. The influence of paraquat to zebrafish embryo is lethal effect.