Residues,Bioaccumulation And Risk Assessments For Difenoconazole And Propiconazole In A Procambarus Clarkii-Rice Co-Culture Model

Difenoconazole and propiconazole are triazole fungicides.It is widely used for the control of rice sheath blight,bakanae,and bacterial streaks.Difenoconazole and propiconazole are mainly sprayed on the leaves.Difenoconazole and propiconazole that are not sprayed on the stems and leaves of rice will enter the paddy field water and then transfer to the sediment.This will cause environmental pollution in the paddy field.With the promotion of the rice-fish integrated cultivation,the rice-shrimp integrated cultivation is the most mature and widely used one;and 83.54%of the Procambarus clarkii in the market are from the rice-shrimp integrated cultivation.However,the toxicity and residue of difenoconazole and propiconazole on Procambarus clarkii are not reported.To prevent the transfer of difenoconazole and propiconazole from the tissue of Procambarus clarkii to the human body via the food chain,which will affect human health.In this study,the main animal used in the integrated cultivation of rice and shrimp in China,Procambarus clarkii,was used as the research object in the study.On the one hand,the enrichment effect of Procambarus clarkii on difenoconazole and propiconazole was studied under indoor conditions.On the other hand,study on the residue of difenoconazole and propiconazole in rice and shrimp integrated cultivation.The main results of this paper are as follows:（1）For the determination of the difenoconazole and propiconazole residues in water samples,paddy field sediment,waterweed,shrimp feed,muscle,hepatopancreas and gill tissue of Procambarus clarkii,a HPLC-MS/MS method was established.The treatment method for water sample was to use a hydrophile-lipophilic equilibrium extraction column（HLB）to enrich and purify difenoconazole and propiconazole.Other substrates such as sediment,water grass,crayfish muscle,hepatopancreas and gill tissue were extracted with acetonitrile/water（4:1）,NaCl and anhydrous MgSO₄ as salting-out agents.And the purification method was the combination of neutral alumina column,PSA and GCB.Difenoconazole and propiconazole had a good linear relationship in the range of 0.1～50μg/L,the correlation coefficient was 0.9950～0.9999.The detection limit of this newly established method was 0.5μg/kg.The recovery rate of spiked matrix was80.47%～110.87%.And the relative standard deviation range was 1.13%～13.96%.The residue detection of difenoconazole and propiconazole can be done with this method,which had good precision,sensitivity and accuracy.（2）During the acute toxicity test,it was found that when the concentration of difenoconazole was 1000 mg/L,Procambarus clarkii did not die,and the toxicity to Procambarus clarkii was low.And the 96h half lethal concentration of propiconazole to Procambarus clarkii was 0.52 mg/L,which was classified as high toxicity according to the toxicity grade.Results of the enrichment test showed that Procambarus clarkii had a low enrichment to difenoconazole and propiconazole.The enrichment amount and enrichment coefficient of difenoconazole and propiconazole in different tissues showed a gill>hepatopancreas>muscle effect.The concentration of enrichment in tissues increased as exposure increased.In contrast,the enrichment coefficient in the same tissue decreased with the increase exposure concentration of difenoconazole and propiconazole.（3）The field experiment was conducted to study the residues of difenoconazole and propiconazole in a rice and shrimp integrated cultivation field.The residual fate of difenoconazole and propiconazole in paddy field water,paddy field sediment,waterweed and Procambarus clarkii’gill,hepatopancreas muscle was investigate under this mode.The results of the field simulation experiment showed that in the experiment of the recommended dose of difenoconazole and propiconazole and twice the recommended dose of difenoconazole and propiconazole,the rate at which difenoconazole and propiconazole were broken down in paddy field water,paddy sediment,and waterweed was faster,but it was hard to get rid of the residue.In paddy field water,difenoconazole and propiconazole behave according to the first-order kinetic model.The correlation coefficient was0.8975～0.9655,and the half-life was 0.82～15.75d.There was a gradual increase and decrease in the concentrations of difenoconazole and propiconazole in paddy sediment,waterweed,and Procambarus clarkii’gill,hepatopancreas,muscle.At the recommended dose,the maximum residue of difenoconazole in sediment and waterweed was 201.72μg/kg and 440.00μg/kg,and that of propiconazole is 155.56μg/kg and 4340.04μg/kg.The distribution characteristics of the fungicides difenoconazole and propiconazole in Procambarus clarkii were gill>hepatopancreas>muscle.There was a positive correlation between residual quantity and exposure concentration.The residues of difenoconazole were significantly different among the three kinds of tissue in Procambarus clarkii（P<0.01）;and the propiconazole residues in the muscle and hepatopancreas of Procambarus clarkii were significantly lower than those in the gill tissue（P<0.05）.After 28 days of application,difenoconazole was not detected in the edible part of Procambarus clarkii;and the residual of propiconazole in the edible part of Procambarus clarkii,hepatopancreas and muscle tissue was 20.25μg/kg and 33.39μg/kg.（4）Depending on the residual amount in the field test of Procambarus clarkii,the dietary health risk was evaluated.According to the dosage,application times,and application time in field experiments,difenoconazole and propiconazole,two agricultural fungicides,were marketed 21d and 28d after application time,respectively.There were no health risks to humans caused by the residual difenoconazole and propiconazole content in Procambarus clarkii.