Study On The Evaluation Of Combined Toxicity And Mechanism Of Three Typical Disinfectants On Vibrio-qinghaiensis Sp.-Q67

Disinfectants play an important role in controlling infectious diseases and viruses.However,a series of environmental pollution problems caused by them began to be exposed with the use of disinfectants.At present,it has been reported that disinfectants have toxicity to non-target organisms in the environment.Therefore,three commonly used disinfectants,quaternary ammonium salts,guanidine disinfectants and chlorine-containing disinfectants:benzalkonium bromide（BEN）,didodecyl dimethyl ammonium bromide（DID）,domiphen bromide（DOM）and tetradecyl dimethyl benzyl ammonium chloride（TET）,hlorhexidine diacetate（CHD）,chlorhexidine（CHL）,polyhexamethylene biguanidine（POL）,calcium hypochlorite（CAL）,were selected as research objectives.Vibrio-qinghaiensis sp.-Q67（Q67）was selected as test organism,and the toxicity characteristics of eight disinfectants and their mixtures in different exposure time（0.25,2,4,8 and 12 h）were systematically analyzed by time-dependent toxicity microplate analysis method.Representative binary and multi-component mixtures of disinfectants were designed by direct equipartition ray design（Equ Ray）method and uniform design ray（UD-Ray）method.Furthermore,the toxicity interactions of each mixture system were evaluated by concentration addition（CA）model.The interaction intensities were evaluated by deviation from CA（d CA）model.Effects of eight disinfectants and their mixtures on cell structure and morphology of Q67 were observed by scanning electron microscope（SEM）.Protein and nucleic acid leakage in Q67 cells were determined by coomassie brilliant blue and diphenylamine colorimetry method.Finally,the effects of eight disinfectants and their mixtures on the structure of nucleic acids and proteins in Q67 cells at a certain concentration(EC₅₀-12h)were determined by UV spectroscopy.Based on this,the toxicity mechanisms of eight disinfectants and their mixtures were preliminarily determined.The main results obtained in this study are as follows:（1）The toxicities of eight disinfectants to Q67 show a good dose-effect relationship,and change with the prolongation of exposure time.All of them have strong long-term toxicity.The main toxic action sites of four quaternary ammonium disinfectants and three guanidine disinfectants are located in the cell membrane,and four quaternary ammonium disinfectants and three guanidine disinfectants can significantly destroy the Q67 cell structure and lead to the outflow of DNA and soluble proteins.Under the action of four quaternary ammonium salts disinfectants and three guanidine disinfectants,Q67 cells began to deform,the DNA and soluble protein contents in the culture medium increased,and the DNA in Q67 cells denatured.CAL can obviously destroy the cell structure of Q67,lead to the outflow of DNA and soluble proteins,and can also cause the destruction of DNA and protein in cells.（2）Toxicity of binary,ternary and quaternary mixtures of four quaternary ammonium salts depend on single component,and each mixture ray show obvious toxicity to Q67(p EC₅₀>4.6).The toxicity interactions between binary,ternary and quaternary mixtures of four quaternary ammonium salts are mainly additive and synergism.For the mixture with synergism,the synergism between its components is time-dependent and concentration ratio dependent,i.e.the intensity of synergism varies with the exposure time and concentration.The maximum synergism of each mixture ray mainly appears in the initial exposure and intermediate concentration region,and their effects increase 20%～40%.（3）Toxicity of ternary mixtures of three guanidine disinfectants depend on individual components,and the mixture rays show obvious toxicity to Q67(p EC₅₀>4.7).The toxicity interactions between ternary mixtures of three guanidine disinfectants are mainly antagonism and their effects decrease 40%～60%.（4）The toxicity of binary mixture of CAL and quaternary ammonium salts depend on single component,and the mixture rays show obvious toxicity to Q67(p EC₅₀>3).Binary mixtures of CAL and quaternary ammonium salts disinfectants’toxicity change with prolonged exposure time,but not significantly.The toxicity of each binary mixture ray has obvious concentration ratio dependence.Binary mixtures of CAL and quaternary ammonium salts disinfectants have obvious antagonism,and some rays show weak synergism.The toxicity interaction of each mixture ray is concentrated in the middle and high concentration region,and effect increased or decreased 20%and 30%～40%,respectively.The five-component mixtures of CAL and quaternary ammonium salt disinfectants show obvious toxicity to Q67(p EC₅₀>3.9)with obvious time-dependence and concentration ratio dependence.At different concentration ratios and different exposure time,the toxicity interactions of five-component mixtures system change,but the interactions are mainly addition,and some rays produce weak synergism and weak antagonism at a certain exposure time.（5）Binary mixtures of CAL and guanidine disinfectants change in toxicity with prolonged exposure time,but not significantly.The toxicity of each binary mixture ray has concentration ratio dependence.The antagonism between CAL and guanidine disinfectant is obvious,and their effects decrease 70%～80%.The quaternary mixture of CAL and guanidine disinfectant show obvious toxicity to Q67(p EC₅₀>3).The antagonism between quaternary mixture of CAL and guanidine disinfectant is also obvious,and effects decrease70%～80%.（6）Eight-component mixtures of CAL and two kinds of disinfectant show obvious toxicity to Q67(p EC₅₀>3.6),and also has concentration ratio dependence and time-dependence toxicity.The toxicity interactions of eight-component mixtures are mainly antagonism,and effect decreases 70%.（7）According to the mechanism experiment,four quaternary ammonium salt disinfectant mixtures,three guanidine disinfectant mixtures and the mixtures of CAL and two kinds disinfectants all can destroy the cell structure and lead to the leakage of macromolecular substances such as nucleic acid and protein.Meanwhile,four quaternary ammonium salt disinfectant mixtures,three guanidine disinfectant mixtures and the mixtures of CAL and two kinds of disinfectants can cause DNA of Q67 denaturation after entering bacteria.Mixtures containing CAL can also cause protein of Q67 denaturation.