Environmental Risk Assessment Process for Bt Crops Bt 农作物的环境风险评估过程 Tessa S. Milofsky, M.S. 泰莎. 米洛夫斯基, 硕士 U.S. Environmental Protection Agency Office of Pesticide Programs 美国环保局 杀虫剂项目办公室

Outline of presentation 报告纲要 Describe environmental risk assessment (ERA) process for insect-resistant Bt crops: 介绍抗虫Bt农作物环境风险评估(ERA)过程: Terms 术语 Conceptual framework 基本概念 Data requirements 必要数据 Tiering 阶段 Summarize ERA for MON810 corn MON810玉米环境风险评估概述

Terms 术语 Risk - the probability of adverse effects resulting from exposure to an environmental agent or mixture of agents 风险 – 暴露于环境因素或混合因素所导致的负面影响的可能性 Hazard - the inherent toxicity of the insecticidal protein 危害 –杀虫蛋白的内在毒性 Exposure - the concentration of insecticidal protein encountered by an organism in the environment 暴露 – 某种生物在环境中接触到的杀虫蛋白的浓度 High risk 高风险 Hazard 危害 Low ris 低风险 Exposure 暴露

Terms (cont.) 术语 (续) Risk Assessment - assessment of the probability that a harmful condition (hazard) will occur under a given set of conditions, using both hazard and exposure data 风险评估 – 利用危害性数据和暴露数据评测在一系列给定条件下, 有害条件(危害性)发生的可能性.

Risk assessment process 风险评估过程 Hazard Identification Measures of Effects Risk Characterization Exposure Assessment Problem Formulation 问题建立 Risk Conclusion 风险结论 Exposure Characterization 暴露描述 Effects Characterization 效果描述 危害性识别 效果测试 风险描述 暴露评估

Problem formulation 问题建立 Consider: 考虑 Available information 可利用的信息 Overall management goals 总体的管理目标 Scientific and public concerns 科学和公众的考虑 Identify: 识别 Assessment endpoints 评估终点 Test species 测试物种 Test endpoints 测试终点 Tiers required 必要的阶段

Risk assessment process 风险评估过程 Hazard Identification Measures of Effects Risk Characterization Exposure Assessment Problem Formulation 问题建立 Risk Conclusion 风险结论 Exposure Characterization 暴露描述 Effects Characterization 效果描述 危害性识别 效果测试 风险描述 暴露评估

Risk assessment at EPA EPA(美国环保局) 风险评估 Assessment based on: Data submitted to the Agency Advisory panel recommendations Consultations with scientific experts Public comments 评估基于: 递交于环保局的数据 顾问团的建议 与学术专家的咨询 公众的意见

Data requirements 必要的数据 Product characterization 产品特征 Human health 人类健康 Toxicity 毒性 Allergenicity 过敏性 Environmental hazard 环境危害 Insect resistance management (Bt crops only)昆虫抗药性管理(仅对Bt农作物)

Product characterization data requirements 产品特征中的必要数据 Molecular characterization Characterization of inserted genes (active and inert) Transformation system Inheritance and stability of characteristics Mode of action 分子特征 插入基因的特征 (有活性的和无活性的) 转录系统 遗传和稳定性特征 作用模式

Product characterization data requirements 产品特征中的必要数据 Test substance equivalence Protein expression levels Analytical methods Verify presence of intended protein in plant Check for cross reactivity 测试物质的等效性 蛋白表达水平 分析方法 确认目标蛋白存在于植物中 验证杂交

Environmental hazard data requirements 环境危害性中的必要数据 Toxicity to nontarget wildlife: Mammals Birds Fish Terrestrial and aquatic invertebrates Earthworms Plants 对非目标野生生物的毒性 哺乳动物 鸟类 鱼类 陆生水生无脊椎动物 蚯蚓 植物

环境危害性中的必要数据 Environmental hazard data requirements 在环境中的归趋: 对濒临灭绝物种的影响 Fate in environment: Soil fate Horizontal gene transfer Gene flow Effect on threatened and endangered species 在环境中的归趋: 在土壤中的归趋 横向基因转移 基因流动 对濒临灭绝物种的影响

Terms (术语) Estimated environmental concentration (EEC) – estimated concentration within the ecosystem (usually used to describe concentrations in soil or water) 环境浓度估计值(EEC) – 在生态系统中浓度的估计值(通常用来描述在土壤和水中的浓度) High end exposure estimate (HEEE) - measured or estimated 90th percentile of possible exposure concentrations (usually used to describe levels of expression in the insect-resistant plant) 高端暴露估计值(HEEE) – 百分之九十可能暴露的测量或估计浓度(通常用来描述在抗虫作物中的表达水平) Maximum hazard dose (MHD) – the dose chosen to represent an extreme exposure scenario, calculated using the EEC or HEEE and incorporating an additional safety factor 最大危害剂量(MHD) – 用来表达极端暴露的剂量,由EEC或HEEE和另外一个合并的安全因素来计算.

Tiered progression 阶梯状过程 Tier I 第一级 Tier II 第二级 Tier III 第三级 Tier IV 第四级 Worst case scenario 最差案例设想 Field conditions 田间条件 Effects seen at screening level? 检测水平上的结果

Tiered progression Tier IV Tier III Tier II Tier I Semi-field test in green or screen house Long-term (full-life cycle) Plant material Actual field exposure (1x EEC/HEEE) Tier I Tier II Tier III Tier IV Screening level test Laboratory test Short-term (30-day) Microbe-derived protein with artificial diet Uses representative NTOs Exposure of 10x EEC/HEEE Full-field test Long-term Plant material Actual field exposure (1x EEC/HEEE) May have multiple locations Census or look at sentinel orgs. Laboratory test Plant material alone or mixed with artificial diet Exposure of 1-5x EEC/HEEE

阶梯状过程 第四级 第三级 第二级 第一级 温室中的半田间测试 长期 (完整生命周期) 植物材料 暴露于 (1x EEC/HEEE) 检测水平测试 实验室测试 短期 (30-天) 由人工食物培育的微生物起源的蛋白 使用有代表性的NTO 暴露于10x EEC/HEEE 第一级 第二级 第三级 第四级 完整田间测试 长期 植物材料 暴露于 (1x EEC/HEEE) 不同地点 普查或在一些机构查询 实验室测试 单独植物材料或人工混合食物 暴露于 1-5x EEC/HEEE

Tiering: an important message 阶梯过程: 重要的信息 Adverse effect at maximum hazard dose (MHD) 最大危害剂量(MHD)的副作用 ≠ Unreasonable effect 不合理作用 Adverse effect at estimated environmental concentration (EEC) or high end exposure estimate (HEEE) 环境浓度估计值(EEC)或高端暴露估计值(HEEE)表现的副作用 = Potential for unreasonable effect 潜在的不合理作用

An example: MON810 corn 例子: MON810 玉米 Pesticide: Cry1Ab protein cry1Ab gene derived from Bacillus thuringiensis kurstaki Activity against European corn borer, southwestern corn borer, and corn earworm Source of test substance: Plant material Bacteria-produced purified protein 杀虫剂: Cry1Ab 蛋白 cry1Ab 基因由Bacillus thuringiensis kurstaki 得来 可抗欧洲玉米螟, 西南玉米螟,和玉米田棉铃虫 测试物质来源 植物材料 细菌产生的纯蛋白

MON810 environmental toxicity data summary Indicator Species Common Name Tier Test Sub. Dose Result Hippodamia convergens Lady beetle I Bacteria-derived 20 ppm N O A E Chrysoperla carnea Green lacewing (larvae) 16.7 ppm Papilio polyxenes Black swallowtail (larvae) II Corn pollen 10,000 grains/cm2 Danaus plexippus Monarch (larvae) 2,000 grain pollen/cm2 Brachymeria intermedia Parasitic hymenoptera Folsomia candida and Xenylla grisea Collembola 200 ppm Corn leaf 50.6 ug/g dry tissue Apis mellifera Honey bee (larvae) Honey bee (adult) Daphnia magna Water flea 100 mg/L Eisenia fetida Earthworm

MON810 环境毒性数据概述 无副作用 指示种 通用名称 阶段 测试物质 剂量 结果 Hippodamia convergens 瓢虫 I 细菌起源 20 ppm 无副作用 Chrysoperla carnea 绿草蛉(卵) 16.7 ppm Papilio polyxenes 黑凤蝶 (卵) II 玉米花粉 10,000 粒/cm2 Danaus plexippus 美国王斑蝶 (卵) 2,000 粒花粉/cm2 Brachymeria intermedia 寄生蜂 Folsomia candida and Xenylla grisea 跳虫 200 ppm 玉米叶子 50.6 ug/g 干组织 Apis mellifera 蜜蜂 (卵) 蜜蜂 (成体) Daphnia magna 水蚤 100 mg/L Eisenia fetida 蚯蚓

MON810 environmental toxicity data summary (cont.) Indicator Species Common Name Tier Test Sub. Dose Result Mus musculus Mouse I Bacteria-derived 4000 mg/kg N O A E Colinus virginianus Northern bobwhite quail II Corn grain 100,000 ppm Gallus domesticus Broiler chicken 50-60% corn grain in diet Ictalurus punctatus Catfish 50% corn grain in diet Estuarine and marine animal Waived: low potential for aquatic exposure to corn Nontarget plant Waived: Cry1Ab protein is nontoxic to plants Fate in soil DT50 8.3 d, DT90 33 d Whole plant DT50 1.6 d, DT90 15 d

MON810环境毒性数据概述(续) 无副作用 指示种 通用名称 阶段 测试物质 剂量 结果 Mus musculus 小鼠 I 细菌起源 4000 mg/kg 无副作用 Colinus virginianus 北方鹌鹑 II 玉米粒 100,000 ppm Gallus domesticus 布罗勒鸡 食物中含50-60%玉米 Ictalurus punctatus 鲶鱼 食物中含50%玉米 淡水动物和海洋动物 未测试: 由于在水中摄食玉米可能性低 非目标植物 未测试: 由于Cry1Ab 蛋白 对植物没有毒性 土壤中归趋 DT50 8.3 d, DT90 33 d 全植物 DT50 1.6 d, DT90 15 d

Other environmental toxicity issues 其他环境毒性问题 Conclusion 结 论 Horizontal gene transfer 横向基因转移(HGT Cry1Ab gene not expressed by a bacterial promoter in MON810 corn; if transferred to bacteria the gene would not be expressed Experiments conducted to assess the likelihood of HGT have not detected gene transfer under typical field conditions No significant risk of HGT with MON810 corn 在MON810 玉米中, Cry1Ab 基因不能被细菌的启动子启动表达; 如果转化到细菌中这个基因就不能表达. 测试HGT可能性的试验还没有检测到在典型田间条件下的基因转移 在MON810玉米上HGT没有显著的风险

Other environmental toxicity issues 其他环境毒性问题 Conclusion 结 论 Gene flow 基因流动 Gene capture and expression of Bt cry toxins by wild or weedy relatives of corn was unlikely to occur in the U.S., its possessions, or territories 在美国及周边地区, 野生玉米或与玉米有亲缘关系的野草中Bt cry 毒素的基因捕获和表达发生的可能性低 Endangered and threatened species considerations 对濒危物种的影响 No overlap with corn cultivation and endangered lepidopteran breeding habitats Nontarget data support conclusion that MON810 corn would have no adverse effect on endangered and/or threatened species 玉米的耕作地与濒临灭绝的鳞翅目昆虫的栖息地并没有重叠区 非目标数据支持以下结论:MON810玉米对濒危物种没有副作用

Risk assessment process 风险评估过程 Hazard Identification Measures of Effects Risk Characterization Exposure Assessment Problem Formulation 问题建立 Risk Conclusion 风险结论 Exposure Characterization 暴露描述 Effects Characterization 效果描述 危害性识别 效果测试 风险描述 暴露评估

Risk characterization 风险描述 Low probability of harm due to source of toxin and history of safe use No adverse effects at Tier I and Tier II test levels Low probability of risk to endangered species, due to pest biology, distribution, and exposure potential No observed adverse effects on population abundance in field census 低危害率归因于毒素的来源和安全使用的历史. 第一第二测试阶段没有副作用. 对濒危物种的低风险率归因于害虫的生物学特性,害虫分布和害虫的暴露潜在可能性. 在田间普查中没有观察到对种群的丰度有负面作用.

Risk conclusion 风险结论 This assessment finds no hazard to the environment from cultivation of MON810 corn for a time-limited registration period Supplemental long term effects studies would provide added weight to support the Agency’s conclusions 这一评估在一定的注册时间期限内并没有发现MON810玉米的耕作对环境有危害作用. 补充的长期效果研究将对环保局的结论提供更加有力的支持.

Post-registration data requirements for MON810 Long-term soil fate study (Tier II) Used to evaluate potential adverse effects on soil organisms Long-term field study (Tier IV) Used to evaluate potential adverse effects on nontarget invertebrates in a corn agroecosystem Study Type Tier Test Sub. Dose Result Fate in soil II Corn plant Field exposure NOAE Field study IV

注册期限后MON810的必需数据 长期土壤归趋研究 (阶段 II) 长期农田实验 (阶段 IV) 用来评价对土壤中生物的潜在毒副作用 用来评价对玉米田农业生态系统中的非目标生物的潜在毒副作用 研究类型 阶段 测试物质 剂量 结果 土壤归趋 II 玉米植株 田间暴露 无副作用 田间实验 IV

Why have we required field studies for Bt crops? 为什么需要Bt农作物的田间研究? Advisory panels have stated the following: Field evaluations should be conducted to determine the impact of insect-resistant crops on nontarget organisms Field studies (are) the most direct way to assess nontarget impact Community-level interactions cannot be easily measured in laboratory experiment Laboratory tests may not fully represent the level or form of harm that may occur in the field 顾问团有如下声明: 田间评价应可确定抗虫作物对非目标生物的影响. 田间研究是评价这种影响的最直接的方法. 实验室中的实验很难测出群落水平的相互影响. 实验室测试不能完全代表农田中发生的危害水平或危害形式.

Limitations of Tier IV full-field studies 第四阶段田间实验的局限性 Full-field studies (census) may result in significant within-plot variation 田间研究(普查)会导致显著的土地内变化 Difficult to: 很难 identify useful hypothesis 鉴别有用的假设 prove hypotheses 证明假设 compare/contrast treatments with meaningful controls in field 对比处理组和对照组

Tier IV full-field study: sampling canopy-dwelling invertebrates 第四阶段-田间实验: 林间生活的无脊椎动物取样 Flying insect trap 飞虫捕捉器 Whole plant survey 全植物调查

Tier IV full-field study: sampling surface-dwelling invertebrates 第四阶段-田间实验: 地表生活的无脊椎动物取样 Pitfall trap for ground-dwelling invertebrates 土地上生活的无脊椎动物陷阱 Carabid beetle 步行虫 Rove beetle 隐翅虫

What are the alternatives? 其他选择 Follow the tier progression: I→II→ III→IV Require higher-tier (Tiers III & IV) testing only when lower-tier tests indicate potential risk If higher-tier testing is required, investigate Tier III semi-field level alternatives 按着阶梯状过程: I→II→ III→IV 仅当低阶段测试表明有潜在的风险时,才需进行高阶段(第三第四阶段)测试. 如果高阶段测试是必须的,那么第三阶段半田间调查是可选择的

Why might Tier III semi-field tests be a better option than Tier IV full-field tests? 为什么第三阶段半田间测试相对于第四阶段田间测试可能是更好的选择? Guaranteed exposure Higher likelihood of measuring effects, because test system is confined Can readily relate results to those obtained in laboratory Can introduce laboratory-reared, or field-collected individuals Can regulate number of pests 暴露是有保证的 由于测试系统是有限的,所以得到结果的可能性更高 可以更好的和实验室得到的结果联系起来 可以引进实验室缺少的或只能在田间收集的个体 可以更好的控制害虫的数目

Why might Tier III semi-field tests be a better option than Tier IV full-field tests? 为什么第三阶段半田间测试相对于第四阶段田间测试可能是更好的选择? Greater number of replicates possible Can be compared with other treatments Con of Tier III tests Difficult to design 繁殖的可能性更高 可以和其他实验组比较 第三阶段的缺陷 很难设计

Tier III semi-field study: litter bag method 第三阶段半田间实验: 小袋法

A take-home message 重点总结 Risk assessment is always a dynamic process 风险评估往往是个动态的过程.