【作者】 倪新；

【导师】 薛明； Y.L.Ren；

【作者基本信息】 山东农业大学 ， 农业昆虫与害虫防治， 2008， 博士

【摘要】 溴甲烷替代研究是当前有害生物控制领域的热点问题之一,研究开发新的替代熏蒸剂,重新评估已有熏蒸剂,是溴甲烷替代研究工作的重点。挥发性硫化物(VSCs)氧硫化碳(COS)、二硫化碳(CS2)、二甲基二硫(DMDS)和甲酸乙酯(EtF)与广泛使用的溴甲烷都是重要的化学熏蒸剂,在防治有害生物方面发挥着重要作用。氧硫化碳(COS)已于上个世纪九十年代在澳大利亚登记使用,甲酸乙酯作为环境友好的粮食熏蒸剂,其制剂产品VapormatTM已在澳大利亚登记使用。同时,挥发性硫化物(VSCs)具有沸点低、嗅阈值低(PPb级)、化学活性强,稳定性差等特点,其采样和分析技术是国际公认的难题。为了解决挥发性硫化物(VSCs)和甲酸乙酯(EtF)检测的技术难题,明确蔬菜中挥发性硫化物(VSCs)和甲酸乙酯(EtF)的自然含量对熏蒸剂残留检测的影响,有效降低CO2增效作用对熏蒸剂毒力的影响,筛选出有潜力的溴甲烷替代熏蒸剂,解决甲酸乙酯应用推广的技术难题,明确植物体中挥发性硫化物(VSCs)的前体降解情况,揭示挥发性硫化物(VSCs)与前体之间的代谢关系,作者开展了蔬菜中挥发性硫化物(VSCs)和甲酸乙酯(EtF)检测技术、杀虫活性及前体降解研究。该研究可为进口食品挥发性硫化物(VSCs)及甲酸乙酯(EtF)熏蒸剂残留检测提供技术支持,还可应用其它科学领域中的挥发性硫化物(VSCs)和甲酸乙酯(EtF)检测。该研究将为溴甲烷替代熏蒸剂研究和有毒有害物质控制等方面提供技术支持,对溴甲烷替代熏蒸剂开发和应用具有十分重要意义。主要研究结果如下:1、甲酸乙酯(EtF)和二甲基二硫(DMDS)对仓储害虫具有良好的控制作用,应加快甲酸乙酯替代溴甲烷防治仓储害虫开发应用,同时,二甲基二硫(DMDS)是很有潜力的仓储害虫防治熏蒸剂,要深入进行二甲基二硫(DMDS)控制有害生物的技术研究,以期获得新的溴甲烷替代熏蒸剂。2、利用循环熏蒸系统(FTFS)测定了甲酸乙酯(EtF)、二甲基二硫(DMDS)和甲硫醚( DMS )对米象Sitophilus oryzae(L.)、赤拟谷盗Tribolium castaneum(Cherbst)和谷蠹Rhyzopertha dominica (F.)三种主要仓储害虫的成虫及混合虫态的熏蒸作用。循环熏蒸系统(FTFS)可以保持恒定的熏蒸剂浓度和较低的CO2浓度,有效解决了CO2增效作用对研究结果的影响,所取得甲酸乙酯(EtF)、二甲基二硫(DMDS)和甲硫醚(DMS)的毒力数据更真实准确。在25℃,相对湿度70%的条件下,甲酸乙酯(EtF)熏蒸处理6h对米象、赤拟谷盗和谷蠹成虫具有良好的控制作用,Ct50分别为107.8, 108.8和72.8mg h L-1。相同试验条件下甲酸乙酯(EtF) 43.34、80.72、99.08、117.74、146.49mg/L熏蒸处理6h,对赤拟谷盗和谷蠹混合虫态有很好的控制作用;80.72、99.08、117.74、146.49mg/L处理对米象的混合虫态杀虫效果较好,但对米象的蛹灭杀效果较差。在25℃,相对湿度50%的条件下,二甲基二硫(DMDS)15.69、17.02、17.80、18.02mg/L熏蒸处理6 h对三种仓储害虫的LC50值分别为16.86mg/L、16.95mg/L、15.31mg/L。在相同试验条件下,甲硫醚(DMS)51.81、53.02、53.51、54.50mg/L熏蒸处理6 h对赤拟谷盗成虫有较高控制作用,但对米象成虫无作用,谷蠹成虫对甲硫醚(DMS)也不敏感,LC50为51.11mg/L。3、采取程序升温、优化色谱条件、确定响应指数等方法,建立了硫化氢(H2S)、氧硫化碳(COS)、二硫化碳(CS2)、甲硫醚(DMS)、二甲基二硫(DMDS)五种挥发性硫化物(VSCs)良好分离的HS-GC-PFPD测定技术;利用程序升温等色谱分析技术,初步建立了蔬菜中甲酸乙酯(EtF)及其降解产物甲酸和乙醇的HS-GC-FID测定技术。4、利用上述技术测定了十字花科的花椰菜(Broccoli)、白菜花(Cauliflower)、卷心菜(Cabbage)、萝卜(Radish);菊科的生菜(Lettuce)和百合科的大蒜(Garlic)六种常见蔬菜中挥发性硫化物(VSCs)和甲酸乙酯(EtF)的自然含量及在酸性条件下的释放量。蔬菜中的挥发性硫化物(VSCs)和甲酸乙酯(EtF)在水和盐酸中是一个动态和缓慢的释放过程,其总量和种类相对比较丰富,蔬菜中甲酸乙酯(EtF)的自然含量和挥发性硫化物(VSCs)种类及含量差别较大,蔬菜不同部位的挥发性硫化物(VSCs)和甲酸乙酯(EtF)的自然含量不同。在水中,蔬菜释放的挥发性硫化物(VSCs)主要以氧硫化碳(COS)、甲硫醚(DMS)和二甲基二硫(DMDS)为主,蔬菜中硫化氢(H2S)、氧硫化碳(COS)、二硫化碳(CS2)、甲硫醚(DMS)、二甲基二硫(DMDS)和甲酸乙酯(EtF)含量分别在0.12～1.11mg/kg、0.05～4.98mg/kg、0.07～8.11mg/kg、0.08～3.53mg/kg、0.001～3.25mg/kg、0.04～0.71mg/kg之间,萝卜中的氧硫化碳(COS)和甲硫醚(DMS)含量较高,萝卜叶的硫化氢(H2S)含量、花椰菜中二硫化碳(CS2)含量、大蒜中的二甲基二硫(DMDS)含量和生菜中的甲酸乙酯(EtF)含量较高。在酸性条件下,蔬菜主要释放出甲硫醚(DMS)、氧硫化碳(COS)、二甲基二硫(DMDS)及部分二硫化碳(CS2)挥发性硫化物(VSCs)和甲酸乙酯(EtF),在5hrs内释放出的挥发性硫化物(VSCs)的量较小,萝卜中的氧硫化碳(COS)和二甲基二硫(DMDS)释放量较高,花椰菜中甲硫醚(DMS)释放量、生菜中的二硫化碳(CS2)和甲酸乙酯(EtF)释放量较高。在酸性条件下,蔬菜中甲硫醚(DMS)和甲酸乙酯(EtF)的释放量高于在水中的释放量,分别为0.08～12.18mg/kg和0.04～7.32mg/kg;蔬菜中氧硫化碳(COS)、二硫化碳(CS2)和二甲基二硫(DMDS)的释放量低于在水中的释放量。5、初步探明了蔬菜中挥发性硫化物(VSCs)前体降解动态。利用“黑箱方法”将含硫氨基酸(SCAA)和硫甲基蛋氨酸(SMM)代谢和降解的复杂过程简化,选择蛋氨酸(Met)、胱氨酸(Cystine)、半胱氨酸(Cys)三种含硫氨基酸(SCAA)和硫甲基蛋氨酸(SMM)作为挥发性硫化物(VSCs)的可能前体,采用HS-GC-PFPD技术,研究蔬菜中挥发性硫化物(VSCs)前体的降解。结果表明:含硫氨基酸(SCAA)和硫甲基蛋氨酸(SMM)是蔬菜中挥发性硫化物(VSCs)的重要前体物质。在不同条件下,含硫氨基酸(SCAA)产生的挥发性硫化物(VSCs)种类和释放量有明显差异,在不同时间挥发性硫化物(VSCs)释放量也存在明显差异。硫甲基蛋氨酸(SMM)是蔬菜中甲硫醚(DMS)主要的前体物质,胱氨酸(Cystine)是蔬菜中甲硫醚(DMS)的前体物质之一。在酶存活和酶失活的情况下,半胱氨酸(Cys)和胱氨酸(Cystine)均可显著减少蔬菜中的氧硫化碳(COS)释放量。在酶存活的情况下,氧硫化碳(COS)、甲硫醚(DMS)和二甲基二硫(DMDS)释放量范围分别在1.06mg/L～26.79mg/L、0.58mg/L～9.16mg/L和0.68mg/L～11.23mg/L之间。在酶失活的情况下,氧硫化碳(COS)、甲硫醚(DMS)和二甲基二硫(DMDS)释放量范围分别在0.61mg/L～5.71mg/L、0.27mg/L～3.55mg/L和0.08mg/L～2.49mg/L之间。更多还原

【Abstract】 Research on methyl bromide alternatives is one of the hot issues in the area of current pest control. To study and develop new alternative fumigant, re-evaluate current fumigants are the focus of methyl bromide alternatives research. The widely used methyl bromide, ethyl formate (EtF) and volatile sulfide compounds (VSCs) such as carbonyl sulfide (COS), carbon disulfide (CS2), dimethyl disulfide (DMDS) are all important fumigants for pest control. Carbonyl sulfide has been registered in Australia in the 1990s to control pests in food products. As an environment-friendly fumigant, VapormateTM, a formulation of ethyl formate with CO2 has been registered in Australia. Meanwhile volatile sulfide compounds (VSCs), having a low boiling point, low-threshold olfactory (PPb level), being chemically active and with poor stability characteristics, makes sampling and analytical techniques publicly known international problems. In order to solve the technical problems of detection of volatile sulfide compounds (VSCs) and ethyl formate, to clarify the impact of fumigant residual detection on the natural level of Volatile Sulfide Compounds (VSCs) and ethyl formate in vegetables, to keep the toxicity of fumigants from being enhanced by carbon dioxide (CO2) and to find out methyl bromide alternative fumigants, to solve the difficult technical problems of wide usage and application of ethyl formate, to clarify the degradation process of VSCs’precursors and reveal the metabolism relationship between VSCs and its precursors, the author done research on determination of natural level in vegetables and toxicity of VSCs and ethyl formate and degradation of VSCs’precursor. This study could provide technical support for the residue detection of VSCs and ethyl formate in imported foodstuffs. This study also could provide technical support for methyl bromide alternatives research and toxic and harmful substances control. It will be of significance for new fumigants of methyl bromide alternatives to be developed and applied. The main results are as follows:1. Ethyl formate and DMDS have good control over storage pests, and should be strongly promoted for treatment of storage pests as methyl bromide alternative. At the same time, DMDS is of great potential value as a fumigant for storage pest control. It is necessary to carry out a thorough study on pest control technology with DMDS, with an expectation of obtaining a new fumigant of methyl bromide alternative.2. Investigation of the toxicity of fumigation on adult and mixed stages of the three most tolerant insect species -- Sitophilus oryzae (L.), Rhyzopertha dominica (F.) and Tribolium castaneum (Herbst) -- under a range of EtF、DMS and DMDS doses in a Flow-through Fumigation System (FTFS) shows that the FTFS procedure offers a constant concentration of fumigant with low CO2 concentrations and ensures that the fumigant toxicity is effectively not compromised by CO2. In comparison with chamber fumigation method, the toxicity data of EtF、DMS and DMDS obtained from this experiment is more accurate by using FTFS.Ethyl formate could provide good disinfestations of all three adults’species at 25°C and 70% r.h. for the 6 h exposure. The Ct products of ethyl formate for adult Sitophilus oryzae, Tribolium castaneum and Rhyzopertha dominica at 25°C and 70% RH for the 6 h exposure were respectively: Ct50 107.8, 108.8 and 72.8 mg h L-1.It was found that under same experimental conditions high control over mixed stage cultures of T. castaneum and R. dominica can be obtained by ethyl formate at 25°C and 70% RH for the 6 h exposure to 43.34、80.72、99.08、117.74 and 146.49mg/L. The dose rates of EtF of 80.72、99.08、117.74 and 146.49mg/L gave good control of mixed stage cultures of S. oryzae in 6 h exposures at 25°C and 70% RH, but poor control of S. oryzae pupae.The LC50 of DMDS for adult S. oryzae, T. castaneum and R. dominica can be obtained by DMS at 25°C and 50% RH for the 6 h exposure to15.69、17.02、17.80 and 18.02 mg/L were respectively: 16.86 mg/L、16.95 mg/L、15.31mg/L. It was found that high control of adult T. castaneum 25°C and 50% RH for the 6 h exposure to 51.81、53.02、53.51 and 54.50 mg/L. While adult S. oryzae had no response and adult R. dominica remained more tolerant to DMS. In this condition, LC50 of adult R. dominica was 51.11 mg/L.3. To establish technicality of HS-GC-PFPD with good separation of five species of VSCs’chromatographic peak and HS-GC-FID in good chromatographic peak separation of EtF, formic acid and ethanol by methods adopted for temperature programming, optimizing chromatography conditions and determining response’s index and so on.4. To determine natural level of VSCs and EtF in broccoli, cabbage, cauliflower, radish, lettuce and garlic and emission of VSCs and EtF in acidity by using the above technicality of HS-GC-PFPD and HS-GC-FID.Natural level of EtF and VSCs varies in different vegetables and parts. The different vegetables also contain various VSCs’species. The major releases of VSCs’species in vegetables were COS, DMS and DMDS. High natural level of COS and DMS in radish and high natural level of H2S, CS2, DMDS and EtF in different vegetables are respectively: radish leaves, broccoli, garlic and lettuce. Natural level of H2S, COS, DMS, DMDS and EtF in vegetables ranged between 0.12～1.11mg/kg、0.05～4.98mg/kg、0.07～8.11 mg/kg、0.08～3.53mg/kg、0.001～3.25mg/kg and 0.04～0.71mg/kg respectively. The major releases of vegetables in acidity were EtF, DMS, COS, DMDS and part of CS2. The emission of VSCs in acidity was smaller within five hours and different vegetables released different species and various emission of VSCs. High COS and DMDS emission were found in radish, high CS2 and EtF in lettuce and high DMS in broccoli in acidity. Compared to natural level of VSCs and EtF in vegetables, DMS and EtF emission of vegetables in acidity were much higher than in water, being respectively, 0.08 ~ 12.18 mg / kg and 0.04 ~ 7.32mg/kg.5. Initial detection for the VSCs’precursor degradation trends in vegetables has been found. To simplify the complex process of metabolism and degradation of SCAA and SMM with“Black Box method”, choosing Met, Cystine, Cys of three SCAA and SMM as possible precursor of VSCs, to research VSCs’precursor degradation in vegetables. The results indicate that SCAA and SMM are important precursor substances of VSCs in vegetables. There were obvious differences in the variety and emission of VSCs under different conditions, and in the release of VSCs at different times. SMM is the main precursor substance of DMS, and Cystine is one of the precursor substances of DMS in vegetables. Cys and Cystine can notably reduce the release of COS in cell-free or enzyme-free extraction. In cell-free extraction, the emissions of COS, DMS, DMDS ranged between 1.06mg/L～26.79mg/L、0.58mg/L～9.16mg/L, 0.68mg/L～11.23mg/L respectively. In enzyme-free extraction, the emissions of COS, DMS, DMDS ranged between 0.61mg/L～5.71mg/L、0.27mg/L～3.55mg/L, 0.08mg/L～2.49mg/L respectively.更多还原