| Objective: Environmental pollution caused by OPs is increasingly serious,so urgent need one method called on-line monitoring and can reflect pollution situation continuously, timely, accurately. Compared with traditional instrument analysis,the AChE analysis has more advantages including sensitive reaction,?operability, low cost and portability, so it is suitable for on-line monitoring.?But because the price of AChE is expensive, and the sensitivity of AChE purified from different sources to OPs is diverse, to some extent the advantage of this method is inhibited; moreover, AChE is now widely used for detecting OPs'single toxity, and the research about joint toxity is lack,so the analysis result is not comprehensive.?The purpose of this study is to get AChE with high activity and sensitivity to OPs, then the enzyme is used to analyse the single toxicity and joint toxicity of OPs, and the testing results will be compared with luminescent bacteria'results,it will improve the goodness of AChE analysis in toxicity on-line monitoring, in addition it can provide foundations for the formulating of evaluation criterion about the OPs'toxity and the development of on-line monitoring.Methods: Selected carp that is more sensitive to water environment and is easily accessed as the source of enzyme, then extracted AChE from fish head.The target enzyme was obtained through a series of steps including optimization of extraction method, the single factor experiment and orthogonal experiment, salting-out, dialysis, gel chromatography and so on. Then the sensitivity of AChE to OPs was detected based on the inhibition to the activity of AChE, and the joint toxicity of binary mixtures of OPs were detected, the joint action of mixture was evaluated with the co-toxicity coefficient method.At last, the single toxicity and joint toxicity were detected by T3 luminescent bacteria through calculatiing the value of EC50,and the joint action was evaluated by toxic unit method, additive index method and hybrid toxic index evaluation method. Finally, the results of two methods were compared.Results: The best reaction conditions between the enzyme and the substrates are as follows: 200mmol/L substrates, 20μL enzyme fluid, pH 8.0, bathing for 30min at 30℃,after a series of purification the enzyme with high activity getting up 5171.76μmol/min/mg was obtained.?The results show that the sensitivity of AChE to different kind of OPs are diverse:dichlorvos>trichlotfon>acephate>omethoate>triazophos,and the?detection limit of five kinds of OPs are 1.69μg/ml,3.78μg/ml,4.03μg/ml,7.32μg/ml and 8.03μg/ml. The relative luminous efficiency of luminous bacteria and the concentration of OPs is negatively correlated though the study of single toxicity, and the correlation coefficient is between 0.93 and 0.98. The joint effect of OPs were analyzed by using luminescent bacteria and AChE self-made, the results show the mode of action of binary mixtures are combined synergies and some additive effect, and the results of the two methods are consistent.Conclusion:The activity of AChE from carp brain is high,and it's sensitive to OPs, so the goodness of AChE is improved on the application of on-line monitoring of toxicity.The joint toxicity of OPs was detected using AChE and luminescent bacteria,we find the mode of joint action of two methods are consistent,and the detection results of AChE about joint toxity are credible,and we can infer that the joint toxicity of mixture in is not simple addition, and the joint action should be take into account in formulating evaluation criterion.
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