Development And Application Of Joint Toxicity Detection Method

ObjectiveWith the rapid development of industry,agriculture and medicine,a large number of heavy metal ions,organic toxic substances and medicine-related chemicals are discharged into the water environment,causing great harm to people’s health.Therefore,the detection of toxic substances and the risk assessment have become the focus of attention all over the world.The traditional toxicity detection methods are complicated,costly and time-consuming,so they are limited in practical application.It is imperative to develop a detection method with simple operation,low cost and more suitable for practical application.Considering of this,the present work developed a respiration substrate(RS)-less isolation method for toxicity detection.This method was based on the mechanism that the respiration intensity of microorganisms could be changed by the toxic substances,and the electrochemical method with artificial medium was employed for signal recording.RS was removed from the first step of previous isolation method,which was an effective strategy for improving acute toxicity assessment.Methods(1)RS-less isolation method:Escherichia coli(E.coli)or Saccharomyces cerevisiae(S.cerevisiae)were used as the test organism,K3[Fe(CN)6]was used as the electrochemical probe,electrochemical test was carried out by using a three-electrode system:microarray Pt electrode(27×25 pm)as the working electrode,Ag/AgCl(saturated KCI)as the reference electrode,Pt wire as auxiliary electrode.The effects of diluent methods,microbial concentrations and addition of respiratory substrates on the sensitivity of toxic detection were investigated.(2)The difference between test organisms:E.coli,S.cerevisiae and Misgurnus anguillicaudatus(a fish),were chosen as the test organism for typical prokaryotes,eukaryotes and vertebrates,respectively.The electrochemical method and the survival time were employed for recording the results obtained by the microorganisms and the fish.(3)The difference between methodologies:The biofilm sensor,morphology method and suspended microbes-based methods including one-pot method,RS-isolation method,RS-less isolation method,RS-less isolation method with added potassium ferricyanide were compared in this work.Results(1)RS-less isolation method:For E.coli,50% inhibiting concentration(IC50)values of Cu2+,Cd2+,Zn2+,Hg2+ and Ni2+ were 0.39 mg/L,5.99 mg/L,3.99 mg/L,0.23 mg/L and 5.74 mg/L,respectively.For 3,5-dichlorophenol(DCP),the adding of RS seems to amplify the final test results.Differently,for salicylic acid(SA)measurement,the sensitivity was reduced by adding RS-1/4 LB.Furthermore,E.coli in this method had a weak response to the antibiotics such as gentamicin,azithromycin and clarithromycin.The analysis of biodegradable organic matter in the actual water samples of local lakes showed that the RS-less method was more reasonable for practice.(2)The difference between test organisms:For S.cerevisiae,the IC50 values of Cu2+ and Zn2+ were 50 mg/L and no obvious toxic effect.For the combined Cu2+-Zn2+,E.coli showed significant synergistic effect(Vt<Vp).A synergistic effect was gradually replaced by antagonistic effect(Vt>Vp)depending on the increasing of Zn2+ concentration for S.cerevisiae.Differently,for fish as the test organism,both additive effect and slight antagonism effect were shown.(3)The difference between methodologies:In the enzyme-substrate assay,a 38% inhibition was obtained by 2 mg/L Zn2+.But for the Cu2+below 2 mg/L,the inhibition was negative.So the enzyme-substrate method was more sensitive than the respiratory toxicity assay for measuring of Zn2+ toxicity,and more sluggish for Cu2+.The combined effect of Cu2+-Zn2+ was dose-dependent and more complicated than that obtained by the respiratory toxicity assay.Conclusions(1)In this study,the RS-less isolation method was developed,which was sensitive for the detection of metal ions,organic toxic substances,but with poor sensitivity for antibiotic.(2)To consider these differences in the results obtained by this work,across different species of test organism and the different mechanisms of the method should be applied in comprehensively for evaluating the safety or toxicity of a chemical substance,rather than simply judging any possible threats.