人参中代森锰锌和氟菌唑的残留降解动态研究
摘要
中国现已成为世界上主要的人参生产国和出口国,吉林省和黑龙江省是我国人参主要生产基地,其人参产量占全国总产量的九成以上,出口占全国的八成左右。但我国的人参产量和产值不成比例,产值很低。究其原因,一是我国人参产品质量问题,更重要的是我国人参产品农药残留量严重超标。导致农药残留超标的原因主要是农药的不合理的使用,特别是高毒、不宜降解的农药的大量使用。国外欧盟、美国、韩国、日本等国相继建立了人参上多种农药的检测方法和农药残留限量标准,而且标准也越来越高,而我国目前只建立了2种人参上使用的农药的残留检测方法,残留标准的制定也仅限于有机氯农药。所以,我国人参出口只能按进口国制定的农药限量标准来检测,被动地接受其残留检测结果,人参出口因农药残留超标屡遭退货现象时有发生。代森锰锌(Mancozeb,MZ)是乙撑双二硫代氨基甲酸盐(EBDCs)类杀菌剂,对人参黑斑病、灰霉病、疫病及苗期病害等均有较好的防治效果,是人参上使用量最大的农药品种。其在人参植株内的代谢物和环境中的降解产物为乙撑硫脲(ethylenethiurea,ETU),具有致畸、致癌和致突变作用。氟菌唑(Triflumizole)是高效、低毒、低残留的内吸性杀菌剂,兼具保护和治疗作用,广泛用于人参上防治灰霉病、黑斑病等病害,具有活性高、不易产生抗药性等特点。因此开展人参上代森锰锌、乙撑硫脲和氟菌唑的残留检测方法和残留降解研究,对规范人参上代森锰锌和氟菌唑的使用,建立其残留标准,提出农药最高限量,促进我国参与国际上人参农药使用标准和农药残留标准的制定,变被动检测为主动控制,突破国际贸易壁垒具有重要意义。本文研究内容与结果如下：
1.建立了代森锰锌的顶空气液色谱法(HS-GLC)口乙撑硫脲的高效液相色谱(HPLC)残留分析技术。代森锰锌的顶空气液色谱法(HS-GLC)残留分析技术：本研究通过向顶空反应瓶中加入SnCl,溶液16ml,EDTA四钠盐溶液3ml时,在0.2μg/mL～1.0μg/mL范围内浓度与峰面积呈良好的线性关系(R2=0.9991),代森锰锌的检出限为1.4×10-1mg/L。在0.5～2.0mg/kg的添加水平下添加回收率为88.8%～89.9%,相对标准偏差为0.46%～5.71%,人参根、茎、叶、果实和土壤的代森锰锌的平均回收率和相对标准偏差范围符合农药残留检测要求。乙撑硫脲的高效液相色谱(HPLC)残留分析技术：当溶液pH、淋洗液、氧化铝分别为11、175ml、5g时,前处理符合要求,空白样品图中没有杂质峰,且人参的根、茎、叶、土壤和果实回收率在90.64%～97.46%,变异系数在1.02%～4.54%之间。色谱条件为：流动相为甲醇：水=10：90(V/V),流速0.5ml/min,波长233nm时,液相色谱的最小检出质量为1.4×10-11g,最低检出浓度为0.07mg·L-1,故符合试验要求。
2.在建立了对人参制品中代森锰锌和乙撑硫脲残留分析方法的基础上,针对目前人参制品中农药残留较高的问题,测定了目前有代表性的人参制品中代森锰锌和乙撑硫脲残留量。
结果表明代森锰锌在活性参、红参、生晒参、保鲜参和人参茶中残留量在0.37mg/kg-1.67mg/kg之间,其中人参茶中残留量最高(平均值为0.81mg/kg),人参酒中没有检测到代森锰锌,可能是在人参酒中人参含量少原因。人参制品中代森锰锌的残留量均高于欧盟规定的限量标准(0.1mg/kg),但低于日本规定的限量标准(1mg/kg)。乙撑硫脲在活性参、红参、生晒参和保鲜参的残留实测值在0.22mg/kg～0.52mg/kg之间,而人参茶中平均为7.5mg/kg,人参茶需冲洗处理后使用。
3.采用顶空气液色谱法分析技术测定了代森锰锌在人参根、茎、叶、果实和土壤的残留降解动念和最终残留量。结果表明：施药剂量为推荐剂量的2倍(6250g a.i./hm2)时,施药1次,代森锰锌在根、茎、叶、土壤中原始沉积量分别为0.89mg/kg、1.34mg/kg、3.32mg/kg和1.21mg/kg；半衰期分别为12.9d、14.1d、12.3d和11.7d。施药1次,高剂量时(6250g a.i./hm2)测得根、茎、叶、土壤和果实中代森锰锌最终残留量为0.32mg/kg、0.21mg/kg、0.46mg/kg、0.18mg/kg和未检出(ND)mg/kg；施药剂量为推荐剂量(3125ga.i./hm2)时,施药1次,人参根、茎、叶、土壤和果实中代森锰锌最终残留量为0.18mg/kg、0.14mg/kg、0.31mg/kg、ND mg/kg和NDmg/kg。综合多方面因素,按照推荐剂量3125ga.i./hm2处理,根据国外人参上的残留标准和试验结果,建议我国代森锰锌在人参根、茎、叶和果实的最高残留限量(MRLs)可暂定为0.5mg/kg,施药次数为1次。
4.采用HPLC-UV分析技术测定了乙撑硫脲在人参根、茎、叶、土壤和果实上的残留降解动态。人参生长期施药量分别为3125g a.i./hm2、6250g a.i./hm2,施药1次,人参收获期根、茎、果实和土壤样本的乙撑硫脲残留量均低于0.3mg/kg,而叶残留量分别为0.31mg/kg和0.46mg/kg,残留量较高,不易直接药用和食用。当75%代森锰锌WP的施药剂量为推荐剂量的2倍(6250g/hm2)时,乙撑硫脲在根、茎、叶和土壤中原始沉积量分别为0.45mg/kg、0.78mg/kg、8.23mg/kg和0.98mg/kg,半衰期分别为12.93d、11.1d、12.3d和11.7.2d。根据欧盟规定的乙撑硫脲最高残留限量值(0.20mg/kg)和本研究试验结果,建议乙撑硫脲在人参根、茎上MRLs值为0.25mg/kg,而人参叶中的残留限量值无法确定。
5.为了减少代森锰锌在人参上的使用量和降低其在人参及其制品中的残留量,研究了代森锰锌的混用技术。采用菌丝生长速率法室内测定了代森锰锌、丙环畔、多菌灵及混剂对人参灰霉病菌和人参黑斑病菌菌丝生长的抑制作用,结果表明代森锰锌等药剂对人参灰霉病菌和人参黑斑病菌均有较好抑制作用,EC50值均较低。在此基础上进行了代森锰锌、丙环唑、多菌灵及其混剂对人参灰霉病和人参黑斑病的田间药效试验,结果表明代森锰锌对人参灰霉病菌和人参黑斑病菌均有较好防治效果,防治效果在72.1%-80.0%之间。其中代森锰锌：丙环唑(1：1)混剂无论是共毒系数还是田间防效均大于单剂和其他混剂,防治效果在84.3%-91.0%之间。为代森锰锌及其混剂在人参上的合理使用及标准的制定奠定了基础。
6.测定了代森锰锌：丙环唑(1：1)混剂中代森锰锌和乙撑硫脲在收获期的最终残留。在人参生长期施用代森锰锌：丙环唑(1：1)混剂1次,施药量分别为3125、6250g a.i./hm2,代森锰锌和乙撑硫脲在人参收获期采集根、茎、果实和土壤样本,残留量均低于0.3mg/kg,其残留量值明显低于单剂在人参中的残留。代森锰锌和丙环畔混用用于人参病害防治,在降低农药残留量同时提高了药效。
7.采用气质联用(GC-MS)技术研究了氟菌唑在人参上的残留降解动态和最终残留。按高剂量340g a.i./hm2施药,施药1次,施药后0、7、14、21、28d和收获期氟菌唑在人参根的原始沉积量分别为ND、ND、0.29、0.23、0.12和0.04mg/kg,半衰期为19.9d。土壤中的原始沉积量分别为0.25、0.16、0.11、0.12、0.10和0.03mg/kg,半衰期为25.4d。氟菌唑属于易降解农药。日本、加拿大、韩国等国家尚未制定氟菌唑在人参中最大残留限量标准,各国在蔬菜及药草中的最大残留限量值MRLs在0.5～5.0mg/kg之间。参照以上各国的残留限量规定及本研究田间试验残留动态数据,建议30%氟菌唑WP在人参生长期与其他杀菌剂交替施用,施药量不高于340g a.i./hm2,施药次数为1次：氟菌唑在人参和土壤上的最大残留量值MRL可暂定为0.25mg/kg,收获前21d不能施药。
综上所述,根据代森锰锌、乙撑硫脲在人参根、茎、叶、土壤和果实上的残留降解动态,人参制品中的残留量和田间试验结果,建议人参上代森锰锌使用量为3125-6250ga.i./hm2,每年施药1次,做获前28d不能施药。建议使用代森锰锌：丙环唑(1：1)的混剂,尽量不使用单剂。建议人参及制品中代森锰锌、乙撑硫脲和氟菌唑的最高残留限量值(MRLs)分别为0.5mg/kg、0.25mg/kg和0.25mg/kg。
China has become the main country for ginseng producing and exporting in the world. Jilin and Heilongjiang Province are the main production bases of ginseng in China, which ginseng production accounted for more than90%of the country's total output, export output outputs accounted for80%of the total country export output. However, China's ginseng production and value were out of proportion, the output value was very low. The first reason was the low quality of ginseng products in China, more importantly, pesticide residues in ginseng products in China was seriously overweight. The reasons leading to excessive pesticide residues in ginseng, were mainly due to the unreasonable use of pesticides, particularly use of pesticides being of highly toxic and uneasible degradation in a large-scale. Several countries including the European Union, the United States of America, South Korea, Japan and other countries have established detection methods of most of pesticides and pesticide maxium residue limits on ginseng, and the standards are getting higher and higher, but in China detection methods of residues on ginseng were established for only two kinds of pesticides, the standards of pesticide residue limits were also limited to organochlorine pesticides. Therefore, pesticide detect limits of exported ginseng were enacted by the importing countries and we had to accept passively the results of residue detection, the phenomenon were common that ginseng exports due to excessive pesticide residues had been repeatedly returned. Mancozeb(MZ) as the largest fungicide used on ginseng, belongs to ethylene-bis dithiocarbamate (EBDCs) fungicides, has better control effect to Alternaria black spot caused by Alternaria panax, gray mold disease caused by Botrytis cinerea, Phytophthora blight caused Phytophthora cactorum and seedling diseases of ginseng by Pythium sp.. However, ethylene thiourea (ETU), as the metabolite of Mancozeb in ginseng plants and environment could induce teratogenic, carcinogenic and mutagenic effect. Triflumizole as one of systemic fungicides with high efficiency, low toxicity, low-residue and both protective and therapeutic effect is widely used in prevention and treatment of gray mold, Alternaria black spot and other diseases on ginseng and difficult to produce resistance. Therefore, researchs on residue detection methods and residue degradation of Mancozeb, ETU and Triflumizole were carried out to regulate the application of Mancozeb and Triflumizole on ginseng, which has important significance to establish their residue limit standards and propose maximum residue limits (MRLs), and it is of great significance also for promoting China's participation in the formulation of ginseng pesticide standards and pesticide residue standards, changing from passive detection for active control, and breaking through lhe barriers in international trade. The contents and results of this dissertation are as follows:
1. Residual analysis techniques were established with headspace gas liquid chromatography (HS-GLC) for mancozeb (MZ) detection and high performance liquid chromatography (HPLC) for ethylene thiourea(ETU) detection in ginseng.
Residue analysis method with HS-GLC for MZ:the results showed that concentration and peak area showed a good linear relationship(R2=0.9991)by spiking16ml of SnCl2solution and3ml of EDTA sodium salt solution into the reaction flask of the headspace in the range of0.20μg/mL to1.00μg/mL. The limits of detection (LOD) for mancozeb were1.4×10-1mg/L, the recoveries of the method for mancozeb varied from88.8%to89.9%, and the relative standard deviations (RSDs)varied from0.46%to5.71%at spiking levels of0.5～2.0mg/kg. The average recoveries and the relative standard derivations of mancozeb could meet the requirements for the detection of residues in ginseng.
Residue analysis method with HPLC for ETU:the proposed solution pH, eluent, and alumina were11,175ml,5g, respectively, pre-treatment methods met the requirements of detection, there was no impurity peak in the blank sample chart, and recoveries of ETU in roots, stems, leaves. fruit of ginseng and soil varied from90.64%to97.46%, the relative standard deviations varied from1.02%to4.54%. The limits of detection of ETU were1.4x10-11g, the minimum detectable concentration of ETU was0.07mg-L-1by HPLC with chromatographic conditions: mobile phase of methanol:water=10:90(V/V), flow rate of0.5ml/min and wavelength of233nm. Therefore requirements were met for the detection.
2. In view of high residues of fungicides in current ginseng and its products, residues of mancozeb and ethylene thiourea in representative ginseng products were detected based on the establishment of methods for detection of pesticide residues. The measured residue value of MZ in active ginseng, red ginseng, suncured ginseng, fresh gingseng, ginseng tea, gingeng sugar ginseng wine varied from0.37mg/kg to1.67mg/kg. the measured residue value (0.81mg/kg) of MZ in ginseng tea was higher than others and no detection in ginseng wine that maybe there was little quantity of ginseng in ginseng wine. Mancozeb residues in ginseng and its products were higher than the EU limits (0.1mg/kg), but less than the limits (1mg/kg)imposed by Japanese. Residue measured value of Ethylene thiourea (ETU) in active ginseng, red ginseng, white ginseng and the preservation varied from0.22mg/kg to0.52mg/kg. there was an average7.5 mg/kg of ETU in ginseng tea, which need to be rinsed with hot water for drink.
3. Degradation dynamics and the final residues of mancozeb in root, stem, leaves and fruit of ginseng and soil were carried out by headspace gas-liquid chromatography analysis techniques.The results showed that spraying one time in one season at the highest dose of6250g a.i./hm2, original depositions of Mancozeb were0.89mg/kg in ginseng root,1.34mg/mg in ginseng stem,3.32mg/mg in leaves and1.21mg/kg in soil, respectively. Half-life were12.9d in ginseng root,14.1d in ginseng stem,12.3d in ginseng leaves and11.7d in soil, respectively; When spraying one time in one season at a dose of6250mg/kg, the final residues of MZ were0.32mg/kg in ginseng root,0.21mg/mg in ginseng stem,0.46mg/mg in leaves, ND mg/kg in fruit and0.18mg/kg in soil, respectivelly. When spraying one time in one season at a dose of3125mg/kg, the final residues of MZ were0.18mg/kg in ginseng root,0.14mg/mg in ginseng stem.0.31mg/mg in leaves. ND mg/kg in fruit and ND mg/kg in soil, respectivelly. Integrated a number of factors, in accordance with the recommended dose of3125g ai/hm2as well as according to foreign ginseng residue standards and test results by ourselves, maximum residue limits (MRLs) of mancozeb in ginseng roots, stems, leaves and fruit in China may be temporarily set at0.5mg/kg, spraying one time in one season.
4. The degradation dynamics of Ethylene thiourea residues in ginseng root, stem, leaves, soil and fruit were determined with HPLC-UV analytical techniques. Spraying MZ75%WP one time in one season at dosages of3125,6250g a. i./hm2, the results showed that residues of ETU at harvest time were lower than0.3mg/kg in root, stem, fruits and soil, but residue in leaf were0.31mg/kg and0.46mg/kg higher than0.3mg/kg which shouldn't be directly eaten and used for medicine. When spraying MZ75%WP at a dose of6250g a.i./hm2, the original depositions of ETU were0.45mg/kg in root,0.78mg/kg in stem,8.23mg/kg in leaf and0.98mg/kg in soil, respectively, the half-lives were12.93d in root,11.1d in stem,12.3d in leaf andl1.7d in soil, respectivelly. According to MRLs(0.20mg/kg) of the European union as well as test data by us, the MRLs of ETU may be temporarily set at0.25mg/kg in root and stem of ginseng, but it could not be determined in leaf.
5. In order to reduce the use of mancozeb on ginseng and reduce its residues in ginseng and its products, the techniques for mixure of MZ with other fungicides were studied. The toxicity of Mancozeb, Propiconazole, Carbendazim and mixture of Mancozeb with other fungicides to ginseng gray mold and Alternaria black spot were tested by mycelium growth rate methods in laboratory. The results showed that Mancozeb75%WP et al had stronger toxicity with lower EC50values to the pathogens. and their control effects in field trial were carried out and the results showed Mancozeb75%WP had good control efficiency of72.1%～80.0%to gray mold disease and Alternaria black spot. Among them, both the co-toxicity coefficient and control effect in mixture at a ratio of Mancozeb and Propiconazole1:1were better than single agent and other mixtured agents.
6. Final residue of Mancozeb and Propiconazole (1:1) mixture of mancozeb and ethylene thiourea at harvest time of ginseng were detected. Applying one time in ginseng growing season with mancozeb and propiconazole (1:1) mixture at dosages of3125,6250g ai/hm2, and collecting samples of roots, stems, fruits of ginseng and soil at harvest, all residues of MZ were lower than0.3mg/kg and lower than their reside value of MZ and ETU in single agents. It improved the efficacy and reduced residues of MZ in plants and soil that Mancozeb and propiconazole mixed with1:1used to control ginseng diseases.
7. Spraying triflumizole30%WP one time in one season.at highest dose of340g a.i./hm2, the results showed that original depositions of Triflumizole were ND. ND,0.29,0.23,0.12and0.04mg/kg in ginseng root collected at Od,7d,14d,21d,28d later after spraying and harvest time, half-life was19.9d in ginseng root, and original deposition of Triflumizole in soil were0.25,0.16,0.11,0.12,0.10and0.03mg/kg at Od,7d,14d,21d28d later after spraying and harvest time and half-life was25.4d, so Triflumizole is easy to degradate. the maximum residue limits of triflumizole in ginseng have not be established in Japan, Canada, Korea and other countries, and MRLs was from0.5and5mg/kg in vegetable and herbs. According to MRLs in vegetables, herbs and the dynamic residue data of Triflumizole on ginseng in field trial, Triflumizole30%WP could be applied with other fungicides alternatively at the dose less than340g a.i./hm2and spraying one time in one growing season, the MRLs of Triflumizole in ginseng and soil were temporarily set at0.25mg/kg and it was not pemitted to apply Triflumizole28d before harvest time.
In conclusion, it was suggested that MZ were applied one time in one growing season at dosage from3125g a.i./hm2to6250g a.i./hm2and could not be used28days before harvest time according to the residue dynamics of MZ, ETU in root, stem, leaf, fruit of ginseng and soil. residue in ginseng and its products, and results of MZ for control effect in ginseng field trial. The MRLs of MZ, ETU and Triflumizole in ginseng and its products were suggested to be0.5mg/kg,0.25mg/kg and0.25mg/kg, respectively.
1.建立了代森锰锌的顶空气液色谱法(HS-GLC)口乙撑硫脲的高效液相色谱(HPLC)残留分析技术。代森锰锌的顶空气液色谱法(HS-GLC)残留分析技术：本研究通过向顶空反应瓶中加入SnCl,溶液16ml,EDTA四钠盐溶液3ml时,在0.2μg/mL～1.0μg/mL范围内浓度与峰面积呈良好的线性关系(R2=0.9991),代森锰锌的检出限为1.4×10-1mg/L。在0.5～2.0mg/kg的添加水平下添加回收率为88.8%～89.9%,相对标准偏差为0.46%～5.71%,人参根、茎、叶、果实和土壤的代森锰锌的平均回收率和相对标准偏差范围符合农药残留检测要求。乙撑硫脲的高效液相色谱(HPLC)残留分析技术：当溶液pH、淋洗液、氧化铝分别为11、175ml、5g时,前处理符合要求,空白样品图中没有杂质峰,且人参的根、茎、叶、土壤和果实回收率在90.64%～97.46%,变异系数在1.02%～4.54%之间。色谱条件为：流动相为甲醇：水=10：90(V/V),流速0.5ml/min,波长233nm时,液相色谱的最小检出质量为1.4×10-11g,最低检出浓度为0.07mg·L-1,故符合试验要求。
2.在建立了对人参制品中代森锰锌和乙撑硫脲残留分析方法的基础上,针对目前人参制品中农药残留较高的问题,测定了目前有代表性的人参制品中代森锰锌和乙撑硫脲残留量。
结果表明代森锰锌在活性参、红参、生晒参、保鲜参和人参茶中残留量在0.37mg/kg-1.67mg/kg之间,其中人参茶中残留量最高(平均值为0.81mg/kg),人参酒中没有检测到代森锰锌,可能是在人参酒中人参含量少原因。人参制品中代森锰锌的残留量均高于欧盟规定的限量标准(0.1mg/kg),但低于日本规定的限量标准(1mg/kg)。乙撑硫脲在活性参、红参、生晒参和保鲜参的残留实测值在0.22mg/kg～0.52mg/kg之间,而人参茶中平均为7.5mg/kg,人参茶需冲洗处理后使用。
3.采用顶空气液色谱法分析技术测定了代森锰锌在人参根、茎、叶、果实和土壤的残留降解动念和最终残留量。结果表明：施药剂量为推荐剂量的2倍(6250g a.i./hm2)时,施药1次,代森锰锌在根、茎、叶、土壤中原始沉积量分别为0.89mg/kg、1.34mg/kg、3.32mg/kg和1.21mg/kg；半衰期分别为12.9d、14.1d、12.3d和11.7d。施药1次,高剂量时(6250g a.i./hm2)测得根、茎、叶、土壤和果实中代森锰锌最终残留量为0.32mg/kg、0.21mg/kg、0.46mg/kg、0.18mg/kg和未检出(ND)mg/kg；施药剂量为推荐剂量(3125ga.i./hm2)时,施药1次,人参根、茎、叶、土壤和果实中代森锰锌最终残留量为0.18mg/kg、0.14mg/kg、0.31mg/kg、ND mg/kg和NDmg/kg。综合多方面因素,按照推荐剂量3125ga.i./hm2处理,根据国外人参上的残留标准和试验结果,建议我国代森锰锌在人参根、茎、叶和果实的最高残留限量(MRLs)可暂定为0.5mg/kg,施药次数为1次。
4.采用HPLC-UV分析技术测定了乙撑硫脲在人参根、茎、叶、土壤和果实上的残留降解动态。人参生长期施药量分别为3125g a.i./hm2、6250g a.i./hm2,施药1次,人参收获期根、茎、果实和土壤样本的乙撑硫脲残留量均低于0.3mg/kg,而叶残留量分别为0.31mg/kg和0.46mg/kg,残留量较高,不易直接药用和食用。当75%代森锰锌WP的施药剂量为推荐剂量的2倍(6250g/hm2)时,乙撑硫脲在根、茎、叶和土壤中原始沉积量分别为0.45mg/kg、0.78mg/kg、8.23mg/kg和0.98mg/kg,半衰期分别为12.93d、11.1d、12.3d和11.7.2d。根据欧盟规定的乙撑硫脲最高残留限量值(0.20mg/kg)和本研究试验结果,建议乙撑硫脲在人参根、茎上MRLs值为0.25mg/kg,而人参叶中的残留限量值无法确定。
5.为了减少代森锰锌在人参上的使用量和降低其在人参及其制品中的残留量,研究了代森锰锌的混用技术。采用菌丝生长速率法室内测定了代森锰锌、丙环畔、多菌灵及混剂对人参灰霉病菌和人参黑斑病菌菌丝生长的抑制作用,结果表明代森锰锌等药剂对人参灰霉病菌和人参黑斑病菌均有较好抑制作用,EC50值均较低。在此基础上进行了代森锰锌、丙环唑、多菌灵及其混剂对人参灰霉病和人参黑斑病的田间药效试验,结果表明代森锰锌对人参灰霉病菌和人参黑斑病菌均有较好防治效果,防治效果在72.1%-80.0%之间。其中代森锰锌：丙环唑(1：1)混剂无论是共毒系数还是田间防效均大于单剂和其他混剂,防治效果在84.3%-91.0%之间。为代森锰锌及其混剂在人参上的合理使用及标准的制定奠定了基础。
6.测定了代森锰锌：丙环唑(1：1)混剂中代森锰锌和乙撑硫脲在收获期的最终残留。在人参生长期施用代森锰锌：丙环唑(1：1)混剂1次,施药量分别为3125、6250g a.i./hm2,代森锰锌和乙撑硫脲在人参收获期采集根、茎、果实和土壤样本,残留量均低于0.3mg/kg,其残留量值明显低于单剂在人参中的残留。代森锰锌和丙环畔混用用于人参病害防治,在降低农药残留量同时提高了药效。
7.采用气质联用(GC-MS)技术研究了氟菌唑在人参上的残留降解动态和最终残留。按高剂量340g a.i./hm2施药,施药1次,施药后0、7、14、21、28d和收获期氟菌唑在人参根的原始沉积量分别为ND、ND、0.29、0.23、0.12和0.04mg/kg,半衰期为19.9d。土壤中的原始沉积量分别为0.25、0.16、0.11、0.12、0.10和0.03mg/kg,半衰期为25.4d。氟菌唑属于易降解农药。日本、加拿大、韩国等国家尚未制定氟菌唑在人参中最大残留限量标准,各国在蔬菜及药草中的最大残留限量值MRLs在0.5～5.0mg/kg之间。参照以上各国的残留限量规定及本研究田间试验残留动态数据,建议30%氟菌唑WP在人参生长期与其他杀菌剂交替施用,施药量不高于340g a.i./hm2,施药次数为1次：氟菌唑在人参和土壤上的最大残留量值MRL可暂定为0.25mg/kg,收获前21d不能施药。
综上所述,根据代森锰锌、乙撑硫脲在人参根、茎、叶、土壤和果实上的残留降解动态,人参制品中的残留量和田间试验结果,建议人参上代森锰锌使用量为3125-6250ga.i./hm2,每年施药1次,做获前28d不能施药。建议使用代森锰锌：丙环唑(1：1)的混剂,尽量不使用单剂。建议人参及制品中代森锰锌、乙撑硫脲和氟菌唑的最高残留限量值(MRLs)分别为0.5mg/kg、0.25mg/kg和0.25mg/kg。
China has become the main country for ginseng producing and exporting in the world. Jilin and Heilongjiang Province are the main production bases of ginseng in China, which ginseng production accounted for more than90%of the country's total output, export output outputs accounted for80%of the total country export output. However, China's ginseng production and value were out of proportion, the output value was very low. The first reason was the low quality of ginseng products in China, more importantly, pesticide residues in ginseng products in China was seriously overweight. The reasons leading to excessive pesticide residues in ginseng, were mainly due to the unreasonable use of pesticides, particularly use of pesticides being of highly toxic and uneasible degradation in a large-scale. Several countries including the European Union, the United States of America, South Korea, Japan and other countries have established detection methods of most of pesticides and pesticide maxium residue limits on ginseng, and the standards are getting higher and higher, but in China detection methods of residues on ginseng were established for only two kinds of pesticides, the standards of pesticide residue limits were also limited to organochlorine pesticides. Therefore, pesticide detect limits of exported ginseng were enacted by the importing countries and we had to accept passively the results of residue detection, the phenomenon were common that ginseng exports due to excessive pesticide residues had been repeatedly returned. Mancozeb(MZ) as the largest fungicide used on ginseng, belongs to ethylene-bis dithiocarbamate (EBDCs) fungicides, has better control effect to Alternaria black spot caused by Alternaria panax, gray mold disease caused by Botrytis cinerea, Phytophthora blight caused Phytophthora cactorum and seedling diseases of ginseng by Pythium sp.. However, ethylene thiourea (ETU), as the metabolite of Mancozeb in ginseng plants and environment could induce teratogenic, carcinogenic and mutagenic effect. Triflumizole as one of systemic fungicides with high efficiency, low toxicity, low-residue and both protective and therapeutic effect is widely used in prevention and treatment of gray mold, Alternaria black spot and other diseases on ginseng and difficult to produce resistance. Therefore, researchs on residue detection methods and residue degradation of Mancozeb, ETU and Triflumizole were carried out to regulate the application of Mancozeb and Triflumizole on ginseng, which has important significance to establish their residue limit standards and propose maximum residue limits (MRLs), and it is of great significance also for promoting China's participation in the formulation of ginseng pesticide standards and pesticide residue standards, changing from passive detection for active control, and breaking through lhe barriers in international trade. The contents and results of this dissertation are as follows:
1. Residual analysis techniques were established with headspace gas liquid chromatography (HS-GLC) for mancozeb (MZ) detection and high performance liquid chromatography (HPLC) for ethylene thiourea(ETU) detection in ginseng.
Residue analysis method with HS-GLC for MZ:the results showed that concentration and peak area showed a good linear relationship(R2=0.9991)by spiking16ml of SnCl2solution and3ml of EDTA sodium salt solution into the reaction flask of the headspace in the range of0.20μg/mL to1.00μg/mL. The limits of detection (LOD) for mancozeb were1.4×10-1mg/L, the recoveries of the method for mancozeb varied from88.8%to89.9%, and the relative standard deviations (RSDs)varied from0.46%to5.71%at spiking levels of0.5～2.0mg/kg. The average recoveries and the relative standard derivations of mancozeb could meet the requirements for the detection of residues in ginseng.
Residue analysis method with HPLC for ETU:the proposed solution pH, eluent, and alumina were11,175ml,5g, respectively, pre-treatment methods met the requirements of detection, there was no impurity peak in the blank sample chart, and recoveries of ETU in roots, stems, leaves. fruit of ginseng and soil varied from90.64%to97.46%, the relative standard deviations varied from1.02%to4.54%. The limits of detection of ETU were1.4x10-11g, the minimum detectable concentration of ETU was0.07mg-L-1by HPLC with chromatographic conditions: mobile phase of methanol:water=10:90(V/V), flow rate of0.5ml/min and wavelength of233nm. Therefore requirements were met for the detection.
2. In view of high residues of fungicides in current ginseng and its products, residues of mancozeb and ethylene thiourea in representative ginseng products were detected based on the establishment of methods for detection of pesticide residues. The measured residue value of MZ in active ginseng, red ginseng, suncured ginseng, fresh gingseng, ginseng tea, gingeng sugar ginseng wine varied from0.37mg/kg to1.67mg/kg. the measured residue value (0.81mg/kg) of MZ in ginseng tea was higher than others and no detection in ginseng wine that maybe there was little quantity of ginseng in ginseng wine. Mancozeb residues in ginseng and its products were higher than the EU limits (0.1mg/kg), but less than the limits (1mg/kg)imposed by Japanese. Residue measured value of Ethylene thiourea (ETU) in active ginseng, red ginseng, white ginseng and the preservation varied from0.22mg/kg to0.52mg/kg. there was an average7.5 mg/kg of ETU in ginseng tea, which need to be rinsed with hot water for drink.
3. Degradation dynamics and the final residues of mancozeb in root, stem, leaves and fruit of ginseng and soil were carried out by headspace gas-liquid chromatography analysis techniques.The results showed that spraying one time in one season at the highest dose of6250g a.i./hm2, original depositions of Mancozeb were0.89mg/kg in ginseng root,1.34mg/mg in ginseng stem,3.32mg/mg in leaves and1.21mg/kg in soil, respectively. Half-life were12.9d in ginseng root,14.1d in ginseng stem,12.3d in ginseng leaves and11.7d in soil, respectively; When spraying one time in one season at a dose of6250mg/kg, the final residues of MZ were0.32mg/kg in ginseng root,0.21mg/mg in ginseng stem,0.46mg/mg in leaves, ND mg/kg in fruit and0.18mg/kg in soil, respectivelly. When spraying one time in one season at a dose of3125mg/kg, the final residues of MZ were0.18mg/kg in ginseng root,0.14mg/mg in ginseng stem.0.31mg/mg in leaves. ND mg/kg in fruit and ND mg/kg in soil, respectivelly. Integrated a number of factors, in accordance with the recommended dose of3125g ai/hm2as well as according to foreign ginseng residue standards and test results by ourselves, maximum residue limits (MRLs) of mancozeb in ginseng roots, stems, leaves and fruit in China may be temporarily set at0.5mg/kg, spraying one time in one season.
4. The degradation dynamics of Ethylene thiourea residues in ginseng root, stem, leaves, soil and fruit were determined with HPLC-UV analytical techniques. Spraying MZ75%WP one time in one season at dosages of3125,6250g a. i./hm2, the results showed that residues of ETU at harvest time were lower than0.3mg/kg in root, stem, fruits and soil, but residue in leaf were0.31mg/kg and0.46mg/kg higher than0.3mg/kg which shouldn't be directly eaten and used for medicine. When spraying MZ75%WP at a dose of6250g a.i./hm2, the original depositions of ETU were0.45mg/kg in root,0.78mg/kg in stem,8.23mg/kg in leaf and0.98mg/kg in soil, respectively, the half-lives were12.93d in root,11.1d in stem,12.3d in leaf andl1.7d in soil, respectivelly. According to MRLs(0.20mg/kg) of the European union as well as test data by us, the MRLs of ETU may be temporarily set at0.25mg/kg in root and stem of ginseng, but it could not be determined in leaf.
5. In order to reduce the use of mancozeb on ginseng and reduce its residues in ginseng and its products, the techniques for mixure of MZ with other fungicides were studied. The toxicity of Mancozeb, Propiconazole, Carbendazim and mixture of Mancozeb with other fungicides to ginseng gray mold and Alternaria black spot were tested by mycelium growth rate methods in laboratory. The results showed that Mancozeb75%WP et al had stronger toxicity with lower EC50values to the pathogens. and their control effects in field trial were carried out and the results showed Mancozeb75%WP had good control efficiency of72.1%～80.0%to gray mold disease and Alternaria black spot. Among them, both the co-toxicity coefficient and control effect in mixture at a ratio of Mancozeb and Propiconazole1:1were better than single agent and other mixtured agents.
6. Final residue of Mancozeb and Propiconazole (1:1) mixture of mancozeb and ethylene thiourea at harvest time of ginseng were detected. Applying one time in ginseng growing season with mancozeb and propiconazole (1:1) mixture at dosages of3125,6250g ai/hm2, and collecting samples of roots, stems, fruits of ginseng and soil at harvest, all residues of MZ were lower than0.3mg/kg and lower than their reside value of MZ and ETU in single agents. It improved the efficacy and reduced residues of MZ in plants and soil that Mancozeb and propiconazole mixed with1:1used to control ginseng diseases.
7. Spraying triflumizole30%WP one time in one season.at highest dose of340g a.i./hm2, the results showed that original depositions of Triflumizole were ND. ND,0.29,0.23,0.12and0.04mg/kg in ginseng root collected at Od,7d,14d,21d,28d later after spraying and harvest time, half-life was19.9d in ginseng root, and original deposition of Triflumizole in soil were0.25,0.16,0.11,0.12,0.10and0.03mg/kg at Od,7d,14d,21d28d later after spraying and harvest time and half-life was25.4d, so Triflumizole is easy to degradate. the maximum residue limits of triflumizole in ginseng have not be established in Japan, Canada, Korea and other countries, and MRLs was from0.5and5mg/kg in vegetable and herbs. According to MRLs in vegetables, herbs and the dynamic residue data of Triflumizole on ginseng in field trial, Triflumizole30%WP could be applied with other fungicides alternatively at the dose less than340g a.i./hm2and spraying one time in one growing season, the MRLs of Triflumizole in ginseng and soil were temporarily set at0.25mg/kg and it was not pemitted to apply Triflumizole28d before harvest time.
In conclusion, it was suggested that MZ were applied one time in one growing season at dosage from3125g a.i./hm2to6250g a.i./hm2and could not be used28days before harvest time according to the residue dynamics of MZ, ETU in root, stem, leaf, fruit of ginseng and soil. residue in ginseng and its products, and results of MZ for control effect in ginseng field trial. The MRLs of MZ, ETU and Triflumizole in ginseng and its products were suggested to be0.5mg/kg,0.25mg/kg and0.25mg/kg, respectively.
引文
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