In-vitro Bioaccessibility Of Neonicotinoids In Raw Vegetables And Fruits And Related Health Risk Assessment

Risk assessment of pesticide residues in food is a necessary approach to rule safety measures.However,the most widely used method for the evaluation of pesticide exposure is based on the ingestion of individual residue levels.This exposure assessment assumes that the maximum amount of a compound in the ingested food has been absorbed by the human gastrointestinal tract.This approach overlooks the extent to which compounds are solubilized from the food matrix after gastrointestinal digestion and become bioavailable,potentially resulting in overestimates of the resulting risks.In recent years,bioaccessibility studies have therefore received a great deal of attention,having been used to evaluate the contaminant.Pesticide residues in fruits and vegetables create human health risk through dietary intake which is a most important exposure pathway.The influence of bioaccessibility of pesticide residues in fruits and vegetables on dietary exposure estimate has already become a scientific problem which needs solving urgently in human health risk assessment of pesticide residues in food.In this study,In vitro simulated digestion model was used to thoroughly evaluate the bioaccessibility of four neonicotinoid residues in tomato,cucumber,and carrot and the affecting factors.Using the bioaccessibility to quantitatively analyze the health risk of pesticide residues in raw fruits and vegetables collected from selected markets in Hefei,Anhui province,based on the data of pesticide residues in selected samples of raw fruits and vegetables.The results are of great theoretical and practical value for the scientific assessment of the safety of edible fruits and vegetables.The main outcomes are as follow:1.The multi-residue analytical method for neonicotinoid residues in simulatedgastrointestinal juiceA method based on human SHIME simulation was developed and optimized for the analysis of four neonicotinoid residues applied on vegetables and fruits in simulated gastrointestinal juice.The recoveries of neonicotinoid residues in simulated gastric and intestinal juice were in the range of 73.90-117.15%and 78.08-106.50%,respectively,at spiking levels of 0.5,1,and 5 mg/kg.The RSD of three quintuplicates for the four neonicotinoid residues were 1.71-9.06%(gastric juice)and 0.80-9.62%(intestinal juice),respectively.The correlation coefficients(r2)of four neonicotinoid residues were greater than 0.999,indicating good linearity.The limit of quantification(LOQ)was determined as 0.05 mg/L.Results demonstrated the residue method can fulfil the requirements for pesticide residue analysis.2.Factors affecting the in vitro simulated digestion SHIME modelEffects of gastrointestinal pH,digestive times,and the solid-liquid(S/L)ratio on in vitro simulated digestion model was investigated.Results showed that the bioaccessibility of the four neonicotinoids reached their highest value at a pH of 2.54 and 6.51 in the simulated gastric and intestinal juice,respectively.A significant positive correlation was observed between the bioaccessibility and the S/L ratio in the gastrointestinal fluid.Curve fitting demonstrated a strong logarithmic relationship,and the R2 values ranged from 0.8658 to 0.9843.There is a concomitant decrease in the in vitro bioaccessibility of neonicotinoids with an increase in the content of digestive times,which reached balance at 2 and 4 h in simulated gastric and intestinal juice,respectively.Based on normal physiological conditions of human body,S/L ratio of 1:100 with an incubation time of 2 h at pH of 2.5 for gastric samples and 4 h at pH of 6.5 for intestinal samples,was suggested for in vitro human digestion assay by our investigation.3.Variability of neonicotinoids bioaccessibility in raw vegetables and fruitsNeonicotinoids bioaccessibility among differ gastrointestinal juice were investigated.Results showed that the bioaccessibility of thiamethoxam,imidacloprid,acetamiprid,and thiacloprid in vegetables and fruits was 39.44-75.28%,28.75-75.28%,55.27-90.92%,and 33.23%-78.33%,respectively.Significant differences were observed in the tomato,cucumber,and carrot for neonicotinoids bioaccessibility,indicating that the matrix is a major factor affecting bioaccessibility.The release of neonicotinoids bioaccessibility from the matrix was in the order of carrot>cucumber>tomato,which may be explained by the physicochemical property(solubility and acid-base property)of pesticides and matrix effects.The evaluation of the influence of dietary components was performed by protein,dietary fiber,or vegetable oil separately on samples.Dietary components were added and simulated according to Dietary Guidelines for Chinese Residents.Results showed that dietary components exhibited a marked influence on neonicotinoids bioaccessibility and the effects varied between the different matrices.The addition of dietary fiber and vegetable oil significantly reduced the 18.38-67.91%and 11.27-44.29%of bioaccessibility,respectively.However,the addition of protein could the bioaccessibility of neonicotinoids in carrot.Therefore,we recommend that consuming increased intake of dietary fiber could improve the safety of fruit and vegetables in daily life.4.In vitro analysis of pesticide transport using Caco-2 cellsIntestinal transport of neonicotinoids in human Caco-2 cells was investigated.significant time-response effects were observed for intestinal membrane permeability of neonicotinoids.After 6 h of incubation,translocation of the four pesticides was in the order of Acetamiprid(883.80 ng)>thiamethoxam(649.61 ng)>imidacloprid 321.94 ng)>thiacloprid(282.39 ng).ANOVA analysis showed that the AP?BL have a relatively higher transport efficiency,which may be related to polarity of two-way membrane.The translocation of neonicotinoids was almost 10000-fold that of difenoconazole,hexaconazole,and spirodiclofen,indicating that water solubility is one of the main factors affecting transport efficiency.Moreover,the gut microbiota significantly decreased the in vitro pesticide transport,and the decrease efficiency differ between AP?BL and BL?AP.Based on apparent permeability coefficient(Papp)and permeability directionrate(PDR),neonicotinoids were thought to active transporte by a membrane transporter.5.Risk assessment of neonicotinoids in raw vegetables and fruits based on the bioaccessibilityMarket monitoring showed that the frequently of neonicotinoids was 12-25%.Single residue was the main form of the four neonicotinoids in tomato,cucumber,and carrot in Hefei.The residue levels of thiamethoxam,imidacloprid,acetamiprid,and thiacloprid were ND-0.023 mg/kg,ND-0.031 mg/kg,ND-0.017 mg/kg,and ND-0.016 mg/kg,which did not exceed maximum residue limits(MRLs).Our risk assessment by means of the hazard quotient revealed that exposures to pesticide residues in the three tomato,cucumber,and carrot are far below levels that might pose a health risk.The HQ values of thiamethoxam,imidacloprid,acetamiprid,and thiacloprid were ranged from 5.27×10-5 to 9.85×10-3.The HQ values of cucumber were 5-100 times that of tomato and carrot,suggesting cucumber creates significantly higher risk to human health.Moreover,assessing the health risks by ingesting pesticides-contaminated food requires data on food consumption and pesticide concentration,and more importantly,the bioaccessibility of pesticides following gastrointestinal digestion.Without considering the oral bioavailabilities of neonicotinoids released from the vegetables and fruits,the risk assessment of pesticides can be overestimated by 14-65%.