【作者】 施艳红；

【导师】 林华峰；

【作者基本信息】 安徽农业大学 ， 农业昆虫与害虫防治， 2018， 博士

【摘要】 膳食暴露评估是农药残留风险评估关键步骤之一。以膳食摄入总量为依据开展农产品中农药残留风险评估是其不确定性的主要来源。膳食摄入是生食蔬果中的农药残留危害人体健康最为主要的暴露途径,运用体外模拟方法测定不同蔬果中农药残留摄入人体后在模拟胃肠环境中的生物可给性,并以此评价农药残留的食用安全性已成为当前研究热点。本文以番茄、黄瓜、胡萝卜为生食蔬果代表,以噻虫嗪、吡虫啉、啶虫脒、噻虫啉等4种新烟碱类杀虫剂为对象,建立了模拟胃肠液中新烟碱类杀虫剂的残留分析方法以及基于SHIME模型的生物可给性体外测定方法,研究不同种类生食蔬果中4种新烟碱类杀虫剂的生物可给性及其影响因素,并以合肥市常见蔬果为例,运用生物可给性评估了蔬果中农药残留的膳食暴露风险。研究结果将为科学评价不同蔬果中农药残留的健康风险提供依据。主要研究结果如下:1、生食蔬果中4种新烟碱类杀虫剂在模拟胃肠液中残留分析方法的建立基于模拟人体胃肠消化的SHIME模型,建立了番茄、黄瓜和胡萝卜中4种新烟碱类杀虫剂在模拟胃肠液中的残留分析方法。添加回收结果表明,在0.5、1.0和5.0 mg/kg的添加水平下,番茄、黄瓜和胡萝卜中4种新烟碱类杀虫剂在模拟胃液中的添加回收率为73.90-117.15%,相对标准偏差为1.71-9.06%;在模拟肠液中的添加回收率为78.08-106.50%,相对标准偏差为0.35-9.62%。方法的线性关系良好(相关系数均大于0.999),4种新烟碱类杀虫剂的LOQ均为0.05 mg/L。该方法的准确性、精确性以及灵敏度均满足农药残留分析要求。2、基于SHIME模型的生物可给性体外测定方法影响因子的研究根据SHIME模型的模拟参数,分别研究了 pH值、消化时间和固液比(S/L)等影响因子对生物可给性体外测定方法的影响。结果表明,模拟胃肠液pH条件、消化时间及其膳食摄入后食物质量与胃肠液体积的比值(S/L)是影响生物可给性体外测定方法的重要因子。3种生食蔬果中4种新烟碱类杀虫剂在模拟胃液和肠液中的生物可给性总体上随着pH值的增高呈现先增后减的趋势,当模拟胃液、肠液的pH值分别为2.54、6.51时,4种新烟碱类杀虫剂的生物可给性相对较高;函数拟合明确了 S/L与4种新烟碱类杀虫剂的生物可给性之间存在良好的对数相关性,R2为0.8658-0.9843;而4种新烟碱类杀虫剂的生物可给性受消化时间影响较小,整体呈增加的趋势,并分别于2 h(胃阶段)、4 h(肠阶段)时达到释放平衡。基于人体正常生理条件,确定了测定番茄、黄瓜和胡萝卜中4种新烟碱类杀虫剂生物可给性的SHIME模型参数为:模拟胃液的pH 2.5,消化时间2 h;模拟肠液的pH 6.5,消化时间4 h;膳食摄入后的生食蔬果质量与模拟胃肠液体积的固液比值为1/100。3、生食蔬果中新烟碱类杀虫剂的生物可给性及膳食成分作用规律的研究在确定的SHIME模型下,测定了番茄、黄瓜、胡萝卜中4种新烟碱类杀虫剂在模拟胃肠液中的生物可给性。结果表明,噻虫嗪、吡虫啉、啶虫脒、噻虫啉在番茄、黄瓜、胡萝卜等3种生食蔬果中的生物可给性分别为39.44-75.28%、28.75-75.28%、55.27-90.92%和33.23%-78.33%。4种新烟碱类杀虫剂的生物可给性受其理化性质(水溶性、酸碱性等)及不同蔬果基质影响,在3种生食蔬果中的农药残留释放大小依次为:胡萝卜>黄瓜>番茄。参考《中国居民膳食营养素参考摄入量表(2016版)》中关于蛋白质、膳食纤维及植物油等主要膳食成分的百分含量,评价了不同膳食成分强化下生食蔬果中农药残留的生物可给性。结果表明,膳食纤维可使4种新烟碱类杀虫剂生物可给性降低18.38-67.91%,植物油可使4种新烟碱类杀虫剂生物可给性降低11.27-44.29%,但添加蛋白质会造成胡萝卜中4种新烟碱类杀虫剂生物可给性的增加。4、Caco-2细胞模型研究新烟碱类杀虫剂的转运吸收行为建立了 Caco-2细胞及其与肠道微生物共同培养的体外模型,研究4种新烟碱类杀虫剂在体外肠上皮模型中转运吸收行为。结果表明,4种新烟碱类杀虫剂在Caco-2细胞模型上的转运量与时间呈显著正相关性;6 h时,4种新烟碱类杀虫剂的转运量大小依次为:啶虫脒(883.80 ng)>噻虫嗪(649.61 ng)>吡虫啉(321.94 ng)>噻虫啉(282.39 ng)。ANOVA分析显示,4种新烟碱类杀虫剂在Caco-2细胞模型的双向转运吸收量差异显著(P<0.05),其在AP→BL测的转运吸收量较高,主要与Caco-2细胞模型双向膜的极性有关。相同时间内,新烟碱类杀虫剂的转运吸收量是苯醚甲环唑、己唑醇和螺螨酯的10000倍,表明水溶性较高的农药易被吸收,其对人体健康风险相对更大。研究肠道微生物对4种新烟碱类杀虫剂在体外肠上皮模型中转运的影响结果表明,肠道微生物对肠上皮细胞单层具有屏障作用,可减少4种新烟碱类杀虫剂的体外转运量,且影响大小具有时间效应。通过表观渗透系数(Papp)及渗透方向率(PDR)分析,发现4种新烟碱类杀虫剂的转运方式可能为主动运输。5、基于生物可给性评估生食蔬果中4种新烟碱类杀虫剂残留的膳食暴露风险建立了同时检测番茄、黄瓜、胡萝卜鲜果中4种新烟碱类杀虫剂的残留分析方法,并检测了合肥地区3种生食蔬果实际样品中4种新烟碱类杀虫剂的残留水平。结果表明,实际样品中4种新烟碱类杀虫剂的检出率为12-25%,噻虫嗪、吡虫啉、啶虫脒、噻虫啉在3种生食蔬果实际样品中的残留水平分别为ND-0.023 mg/kg、ND-0.031 mg/kg、ND-0.017 mg/kg、ND-0.016mg/kg,均未超出其最大残留限量值。基于检测的实际样品残留水平,采用残留平均值进行膳食暴露风险评估,并用生物可给性修正3种生食蔬果的膳食摄入残留量。结果表明,噻虫嗪、吡虫啉、啶虫脒、噻虫啉的风险商值(HQ)在5.27×10-5-9.85×10-3之间,均在可接受范围内。其中,黄瓜中4种新烟碱类杀虫剂的HQ相对较高,是番茄和胡萝HQ的5-100倍。基于生物可给性的农药膳食暴露风险数据显著低于以摄入农药总量为依据的风险评估方法,若不考虑生物可给性,生食蔬果中4种新烟碱类农药残留对人体健康风险将被高估14-65%。更多还原

【Abstract】 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%.更多还原