| Vibrio qinghaiensis sp.Q67 cells contain lux gene cluster,which can encode the synthesis of luciferase so as to send optical signal.When V.qinghaiensis sp.Q67 were stressed by toxic and harmful substances,the optical signal would be weakened.Therefore,the toxicity of chemical substance can be reflected by the change of luminescence intensity.At present,excessive pesticide residues are still a major problem affecting food safety.Excessive pesticide residues in food will not only damage human health,but also affect the sustainable development of agricultural production.However,the conventional detection methods for pesticide residues usually take a long time,which is limited for food with a short shelf life and fast circulation.In this study,V.qinghaiensis sp.Q67 were used as the toxicity test strain,the acute and joint toxicity of five pesticides（pirimicarb（PIR）,carbaryl（CAR）,glyphosate（GLY）,dimethoate（DIM）,acephate（ACE））were studied,the cell damage mechanism on V.qinghaiensis sp.Q67 were explored and the results were compared with toxic effects.In addition,V.qinghaiensis sp.Q67 detection method was applied to the detection of pesticide residues in two kinds of juice,and the specific results are as follows:（1）The individual acute toxicity effects of five pesticides on V.qinghaiensis sp.Q67were studied.Mass concentrations of five pesticides were positively correlated with inhibition Rate（IR%）of V.qinghaiensis sp.Q67,with correlation coefficients fitted to Hill equation ranging from 0.981 to 0.994.The acute toxicity of PIR/CAR/GLY/DIM/ACE to V.qinghaiensis sp.Q67 was different.Concentration for 50%of maximal effect(EC50)and its95%confidence intervals were 33.17（29.90-36.60）,43.89（40.40-47.28）,40.72（35.60-45.80）,265.67（247.69-281.65）and 562.68（519.31-609.60）mg/L,respectively.The order of acute toxicity was PIR>GLY>CAR>DIM>ACE.V.qinghaiensis sp.Q67 also had different degrees of luminescence response when pesticides were detected by national standard(IRPIR:8.80%;IRCAR:4.5%;IRGLY:-5.46%;IRDIM:-3.04%;IRACE:-5.95%).Compared with the detection results of GDYN-1012SC,a commercially available rapid detection instrument for pesticide residues,V.qinghaiensis sp.Q67 detection method can not only achieve qualitative objectives,but also respond to extremely low concentrations of five pesticides.Moreover,there is a certain mathematical relationship between IR%and pesticide concentration,so it has higher detection accuracy.（2）The joint toxicity of ten groups binary pesticides on V.qinghaiensis sp.Q67 were determined by equal toxicity ratio method.The results showed that the correlation coefficient of the fitting curve was 0.980～0.998,which could be used to predict the joint toxicity of binary pesticides to V.qinghaiensis sp.Q67.The acute toxicity of the ten binary compounds was PIR+DIM>CAR+DIM>CAR+GLY>PIR+GLY>CAR+ACE>PIR+ACE>GLY+ACE>GLY+DIM>PIR+CAR>DIM+ACE>DIM+CAR>DIM+ACE.Toxic Unit method was used to evaluate the joint toxicity results.PIR+DIM and CAR+DIM showed synergistic effect,while CAR+GLY,PIR+GLY,CAR+ACE and PIR+ACE showed additive effect.The joint toxic effect of GLY+ACE,GLY+DIM,PIR+CAR and DIM+ACE was antagonistic.The results of binary joint toxicity also evaluated by Isobole Plots method.The study suggested that the binding sites and destruction modes of the same type of pesticides were highly consistent,so there was competition between the same type of pesticides when mixing,which led to the antagonistic effect of the joint toxicity.（3）The joint toxicity of seven groups of five-component pesticide mixtures on V.qinghaiensis sp.Q67 were designed by uniform design ray method.The results showed that R1,R3,R5 and R6 were synergistic at low concentration and antagonistic at high concentration in the first 45 min,the toxicity was antagonistic at all concentrations within45-60 min,so there was a certain time dependence.The joint toxicity of R2,R4 and R7rays was always antagonistic within 60 min,which did not change with concentration and time,and there was no time-dependent toxicity.The IR%of the five-component mixture was the highest in R3 and the lowest in R6.In addition,the correlation between the joint toxicity of the mixture and the concentration ratio of each component in the system was explored.It was found that the concentration ratio of CAR was always the highest correlation with the toxicity of the mixture（R2=0.947）.The fitting equation after exposure for 15 min was as follows:p EC50=-5.13p CAR+2.61.（4）Based on the toxicity results above,the damage mechanism of five pesticides and the five-component mixture of R3 on V.qinghaiensis sp.Q67 cells was further studied at their respective EC50concentrations.The results showed that all treatments inhibited the growth of V.qinghaiensis sp.Q67,significantly damaged the cell membrane and cell wall structure,affected the cell metabolism,and led to the morphological change.On the whole,PIR,CAR and GLY had more serious damage to cells,while DIM and ACE had less damage to cell structure,which was consistent with the aforementioned results of toxicity of five pesticides,and to some extent confirmed the feasibility of judging the toxicity of pesticides by the changes in the luminescence intensity of V.qinghaiensis sp.Q67.（5）The edible safety evaluation system of V.qinghaiensis sp.Q67 in orange juice and apple juice was preliminarily established.There was a positive correlation between IR%and pesticide concentration in this two systems.In the orange juice,The EC50values of PIR,CAR,GLY,DIM,ACE and their 95%confidence intervals were 17.89（45.08-54.92）,10.92（43.89-56.11）,18.22（46.47-53.53）,261.91（48.95-51.05）and 470.24（46.07-53.93）mg/L,respectively.In the apple juice,The EC50values and their 95%confidence intervals were31.95（46.54-53.46）,20.91（46.28-53.72）,33.31（48.46-51.54）,233.61（45.31-54.69）,and308.89（48.13-51.87）mg/L,respectively.The order of acute toxicity of five pesticides in orange and apple juice both was CAR>PIR>GLY>DIM>ACE.According to the results of luminescence inhibition rate changes,15 min was selected as the incubation time to evaluate the edible safety of the two juices,and the evaluation criteria were preliminarily established.Orange juice:when IR%<10,samples are safe;when IR%∈（10,15）,the safety of samples are suspicious and further quantitative measurement is needed;when IR%>15,the samples are unsafe and need to be removed from the shelves immediately.Apple juice:Samples are safe when IR%<20;sample safety are suspect when IR%∈（20,25）;sample are unsafe when IR%>25.In the two kinds of juice,at the 0.1,0.5,1,2,5,10,20 times of maximum residue limits required by national standard,the luminescence intensity of V.qinghaiensis sp.Q67 had different degrees of response,indicating that this method met the detection requirements. |
