Laboratory Study Design for Risk Assessment of

Non-Target Organisms非靶作用生物风险评估的实验室研究设计

Rick HellmichRick HellmichUSDA-ARS, Corn Insects & Plant Genetics Research Unit USDA-ARS, Corn Insects & Plant Genetics Research Unit 美国农业部－农业研究服务局，玉米昆虫和植物遗传研究所美国农业部－农业研究服务局，玉米昆虫和植物遗传研究所

Dept. Entomology, Iowa State UniversityDept. Entomology, Iowa State University昆虫系，依阿华州立大学昆虫系，依阿华州立大学

AgriculturalResearchService

Topics 主题 Preparing for risk assessment laboratory studies 风险评估的实验室研究准备 Experimental details 试验细节 Examples 例子

– Tier 1 study (Univ. NE)梯度 1 研究（内布拉斯加州大学）

– Tier 2 study (ARS & Univ. Guelph)梯度 1 研究（农业研究服务局和圭尔夫大学 ）

Research and collaboration possibilities 研究和合作的可能性

Preparing for RA Studies 风险评估研究的准备

Clear purpose and objectives 清晰的目的和目标– From problem formulation 从问题构成开始

Surrogate concept (case-by-case refinement) 替代概念（案例进化）

– Standard list of surrogate species 替代种属的标准清单– Supplemental species 补充种属

Species must be laboratory reared 种属须实验室培养– Use of artificial diet is preferable 最好使用人工饲料– Need to reliably deliver stage specific organisms 需要正确提供特定阶段的生物

Preparing for RA Studies 风险评估研究的准备

Most protocols are derived from insecticide tests 大部分试验方法都是来自于杀虫剂的试验 Modified to consider exposure pathway 考虑到暴露途径进行修改 Bt maize example Bt 玉米为例

– Toxin produced in leaf tissues 毒素表达在叶的组织– Less toxin produced in roots or pollen 在根和花粉毒素很少– Route of exposure is oral 暴露途径是口

Tiered or Iterative System 分级和迭代系统

Tier 1: maximum limit dose tests 梯度 1 ：最大限制剂量试验

– > 10X MHD (maximum hazard dose) or HEEE (high-end exposure estimation, 90th percentile)

> 10X MHD （最大损害剂量）和 HEEE （高端暴露量估计， 90％）

– Dose response 剂量反应 Tier 2: 1X plant material 梯度 2 ： 1X 植物材料

– Refined hazard characterization 精确的损害鉴定 Higher Tiers: 更高的梯度

– Semi-field and field tests 半田间和田间试验– Reproduction, life cycle 生殖，生命周期– Tri-trophic interactions 食物链交换作用

Triggers for moving to a higher tier should be well defined! 进一步做更高分级试验的阈值需要很好的界定

Experimental Details 试验细节 Consult relevant compliance standards and

guidelines 参考相关的标准和指导– Examples 例子

» USEPA 885 Microbial Pesticide Test Guidelines 美国环保局 885 微生物杀虫剂试验指导

885.4380 Honey bee testing, Tier I 885.4380 蜜蜂试验，梯度 I

» Candolfi, M.P., et al. 2000. Guidelines to Evaluate Side-Effects of Plant Protection Products to Non-Target Arthropods IOBC, BART, EPPO Joint Initiative. IOBC/WPRS (ISBN: 92-9067-129-7)

Candolfi, M.P., et al. 2000. 评价植保产品对非靶作用节肢动物的副作用。 (ISBN: 92-9067-129-7)

Most guidelines based on chemical or microbial insecticides 大部分指导都是基于化学和微生物杀虫剂的– Details for testing GE products often are lacking 测试转基因产品的试验细节还缺少

Bioassays 生物测定法 Organism – common and scientific names 生物－通俗和科学名 Stage tested 测试的阶段

– Egg, larvae (instar?), adult, multiple generations 卵、幼虫（中间形态？）、成虫、多代

Test system and conditions 测试系统和条件– Containment, e.g., testing chambers, arenas 容器，如测试皿、测试场– Temperature, relative humidity, light cycle 温度、相对湿度和光周期– Food and water delivery 食物和水的提供

Bioassays 生物测定 Length of test 试验时间长短

– Partial or whole stage 部分或全部阶段– Consider longer tests for GE products 对于转基因产品考虑更长时间

的试验 Specific measurement endpoints 特定测量指标

– Life-table parameters, e.g., mortality, fecundity 生命表法参数，如死亡率和生殖力– Other endpoints are possible, e.g., weight, behavior, but risk

assessors should agree beforehand how to interpret 其它可能指标，如体重、行为，但是风险评价因子需要事先对怎样解

释有共识 Test material (origin and form) 试验材料（来源和型）

– Derived from bacteria or plant 来源于细菌或植物– Should be equivalent to form expressed in plant 应该于植物中表达

的一样 Dose 剂量

– Consider > 10X MHD or HEEE 考虑 > 10X MHD 或 HEEE Treatment application 处理的采用

– Protein in artificial diet, plant material, etc. 蛋白在人工饲料、植物材料中，等。

Quality Control 质量控制 Assures function of test system 保证试验系统的运行

– Low negative control mortality 低隐性对照死亡率– Homogeneity of test material 试验材料的同质度 – Stability of insecticidal compound 杀虫药物的稳定性– Negative control 阴性对照

» Carrier/buffer minus test material 载体／缓冲液减去试验材料– Positive control (determine test material is ingested) 阳性对照（确定试验材料被吞噬）

» Orally active insecticide 口服活性杀虫剂» Nature of compound (e.g., stomach poison, growth regulator) 试验物的性质（如，胃毒性、生长调控）

– Statistics 统计» Sufficient statistical power (maximize discriminatory ability) 足够统计幂（区分能力最大化）» Power tests, required replicates 幂试验，需要重复» T test, ANOVA, etc. T检验、方差分析， 等

Good laboratory practices (GLP) recommended 推荐良好实验室质量管理体系 （ GLP) Quality control assures repeatability and interpretability; facilitates

transportability of data 质量控制确保重复性和可解释性；有助于数据交流

Higher Tier Testing 更高梯度试验 Usually involves semi-field or field tests

sometimes involves life-cycle or tri-trophic evaluations

通常包括半田间或田间试验，有时包括生命周期或食物链交互作用评价

Problematic because of complexity and high intrinsic uncertainty; problems of low statistical power

因为很复杂和高内在不确定性，所以会有问题。低的统计学能力也会导致问题。

Conduct only when tests can reduce uncertainty in risk assessment and only when justified by detection of unacceptable risks at lower tiers of testing

只有当试验能减少风险评价的不确定性和当在低分级试验证实不可接受的风险时才进行更高分级试验

acute mammal 急性哺乳动物试验– rodent 啮齿类

acute avian 急性禽类试验– game bird & waterfowl 猎禽和水鸟

acute fish 急性鱼试验– cold & warm water spp 冷和温水种属

acute invertebrate 急性非脊椎动物试验– daphnia 水蚤

non-target insects 非靶作用昆虫试验– honey bee larvae 蜜蜂幼虫– parasitic hymenoptera 寄生性膜翅目– green lacewing 草蛉虫– lady beetle 瓢虫– Collembola 弹尾目

Tier 1 Test Examples 梯度 1 试验例子

other tests 其它试验– Degradation rates of proteins in

soil

蛋白质在土壤中的降解速度– Plant tissue expression data and

degradation rates

植物组织表达数据和降解速度

Dose-Response of Dose-Response of Danaus plexippusDanaus plexippus Larvae to Purified Bt ToxinsLarvae to Purified Bt Toxins

Danaus plexippusDanaus plexippus 幼虫对纯幼虫对纯 BtBt 毒素的毒素的剂量反应关系剂量反应关系

Blair D. Siegfried, and Terrence SpencerBlair D. Siegfried, and Terrence Spencer

Department of EntomologyDepartment of Entomology昆虫系昆虫系

UNIVERSITY OF NEBRASKA-LINCOLNUNIVERSITY OF NEBRASKA-LINCOLN内布拉斯加州大学内布拉斯加州大学

NebraskaNebraskaInstitute of Agriculture and Natural Institute of Agriculture and Natural Resources Resources 农业和自然资源研究所农业和自然资源研究所

Objectives Objectives 目标目标 Provide comparative Provide comparative

assessment of neonate assessment of neonate monarch larval susceptibility monarch larval susceptibility to purified Bt toxinsto purified Bt toxins

提供可比较的对新生帝王蝶幼虫对提供可比较的对新生帝王蝶幼虫对纯纯 BtBt 毒素敏感性的评价毒素敏感性的评价

Determine comparative Determine comparative

susceptibility of later instarssusceptibility of later instars 测定晚龄的可比较的敏感性测定晚龄的可比较的敏感性

Bioassays Bioassays 生物测定生物测定 Eggs from field-collected adult females or Eggs from field-collected adult females or

commercial provider commercial provider

卵来源于采集田间雌性成虫或供应商卵来源于采集田间雌性成虫或供应商 Neonate (<24 hr)Neonate (<24 hr)

新生（小于新生（小于 2424 小时）小时） Late 2Late 2ndnd (4-day) and Early 4 (4-day) and Early 4thth (7-day) (7-day)

晚第晚第 22 （第（第 44 天）和早第天）和早第 44 （第（第 77 天）天） Bioassay diet: Multispecies Lepidopteran Bioassay diet: Multispecies Lepidopteran

diet with 2% powdered leaves from diet with 2% powdered leaves from common milkweed common milkweed

生物测定饲料：多种属鳞翅目饲料，另加生物测定饲料：多种属鳞翅目饲料，另加 22 ％％普通乳草叶粉末普通乳草叶粉末

Development on Artificial DietDevelopment on Artificial Diet在人工饲料中生长在人工饲料中生长

Bioassays (Cont.) Bioassays (Cont.) 生物测定（续生物测定（续）） Purified Cry1Ab, Cry1Ac, Cry1F, Cry9C incorporated into Purified Cry1Ab, Cry1Ac, Cry1F, Cry9C incorporated into

dietdiet 纯纯 Cry1Ab, Cry1Ac, Cry1F, Cry9C Cry1Ab, Cry1Ac, Cry1F, Cry9C 加入饲料加入饲料 2 replicates for each toxin; 5 Bt concentrations/rep2 replicates for each toxin; 5 Bt concentrations/rep 每个毒素有每个毒素有 22 个重复；个重复； 55个个 BtBt浓度剂量／重复浓度剂量／重复 Larval weight & mortality recorded at 7 daysLarval weight & mortality recorded at 7 days 77 天内记录幼虫体重和死亡率天内记录幼虫体重和死亡率 Weight determined before and after exposure for later Weight determined before and after exposure for later

instars (instars (Cry1Ab onlyCry1Ab only)) 晚龄（只有晚龄（只有 Cry1AbCry1Ab ）在暴露前后测量体重）在暴露前后测量体重 MortalityMortality data: probit analysis; data: probit analysis; 死亡率数据：概率单位分析死亡率数据：概率单位分析 Growth InhibitionGrowth Inhibition data: non-linear regression data: non-linear regression

生长抑制数据：非线性回归分析生长抑制数据：非线性回归分析

Bioassay Bioassay MethodologyMethodology生物测定方法生物测定方法

Results at 7 daysResults at 7 days77 天结果天结果

Control 对照100 ng/ml Cry 9C

Sublethal EffectsSublethal Effects亚致死作用亚致死作用

00

2525

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7575

100100P

erc

en

tag

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0.10.1 11 1010ng Cry1Ab/mLng Cry1Ab/mL

Mortality Mortality 死亡死亡

Growth Inhibition Growth Inhibition 生长抑制生长抑制

Cry1Ab Dose ResponseCry1Ab Dose Response

Cry1Ab Cry1Ab 剂量反应剂量反应

Comparative Toxicity (1999-2000)可比较的毒性（ 1999－ 2000）ToxinToxin LC LC5050

** EC EC50 50 **

毒素 毒素 1999 2000 1999 20001999 2000 1999 2000

Cry1AbCry1Ab 1.37 1.37 4.764.76 0.130.13 1.281.28

Cry1AcCry1Ac 18.5418.54 -- -- 9.489.48 ---- Cry9CCry9C 313.9313.9 345.7345.7 25.625.6 111.2111.2

Cry1FCry1F >10,000>10,000 >30,000>30,000 >10,000>10,000 5,2005,200

**ng toxin/ml of diet ng toxin/ml of diet ng ng 毒素／毒素／mlml 饲料饲料

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Mort

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11 1010 100100

Cry1Ab (ng/ml)Cry1Ab (ng/ml)

44th Instar th Instar 第四龄第四龄

22nd Instar nd Instar 第二龄第二龄

11st Instar st Instar 第一龄第一龄

Comparative Susceptibility of Larval Instars to Cry1Ab幼虫对 Cry1Ab 的可比较敏感性

Tier 2 Monarch Test梯度 2 帝王蝶试验Objective: Compare monarch neonates susceptibility to pollen from Bt maize Cry1Ab events MON810, BT11 and 176 deposited on milkweed leaves

目标：比较新生帝王蝶对花粉的敏感性，花粉来自于存积在乳草叶上的有 Cry1Ab 的 MON810, BT11 和 176 Bt 玉米

Methods 方法 Pollen from field-collected plants 田间采集植物的花粉– Cry1Ab (Events MON810, BT11 and 176) Cry1Ab （来自于 MON810, BT11 和 176 ）– Pollen sieved with 90 micron screen 用 90

微米筛子过滤花粉 Milkweed leaves harvested morning of

experiment 乳草叶在试验的早上收获 Pollen sprayed onto leaf discs 花粉喷洒在叶子上 Leaf discs put into arena with 2.5%

agar 叶子和 2.5 ％胶一起放入试验皿

Methods (cont.) 方法（续） 1 neonate (< 24 hrs) put into each

arena 1只新生幼虫（小于 24小时）放入一个试验皿中

4 or 5 pollen densities and 0 control tested; 6 replicates per test

4 或 5 花粉密度和 0 花粉对照；每测试有 6个重复

Larval weight & mortality recorded at 4 days

在 4天内记录幼虫体重和死亡率

170 Mesh Sieve (90 Micron) 170网筛（ 90 微米）

Siftings 筛屑

Sieved pollen过筛的花粉

Punch Leaf Disks 打孔叶子

Applying Pollen To Leaf Disks 往叶子上撒花粉

Arena 试验皿

0

10

20

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Cry1Ab Event Bt11

Cry1Ab Event Mon810

Mean weight (mg)平均重量（ mg ）

None Bt non-Bt

100- 300

301- 600

601- 1000

1001- 1600

> 1600

Pollen grains per cm2

花粉颗粒每平方厘米

Lab Bioassays - Cry1Ab Event 176实验室生物测定－ Event 176 的 Cry1Ab 96 Hour Larval Weight 96小时幼虫体重

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Pollen per cm2

花粉每平方厘米

Hybrids - NK4394 & Max 21 (Bt)杂交－ NK4394 & Max 21 (Bt)

0 100 200 400

Cry1Ab < Cry9C < Cry1F Older instars less susceptible to Cry1Ab

晚龄幼虫对 Cry1Ab 更不敏感 Only event 176 pollen consistently

affected larvae

只有 event 176 花粉一直影响幼虫

Monarch larvae sensitivity to Bacillus thuringiensis- purified proteins and pollen帝王碟幼虫对苏云金芽孢杆菌纯蛋白和花粉的敏感性Richard L. Hellmich, Blair D. Siegfried, Mark K. Sears, Diane E. Stanley-Horn, Michael J. Daniels, Heather R. Mattila, Keith G. Bidne, and Leslie C. Lewis

PNAS 2001

Research & Collaboration Possibilities科研和合作可能性 There is a need to harmonize lab-based tests

for GE products 对转基因产品有必要协调实验室方法

– Tests for standard list of surrogate species标准清单替代种属的试验

Identify and develop tests for supplemental species

区别和发展补充种属的试验– Regionally or ecologically relevant arthropods 地区或生态上来说重要的节肢动物

Summary 摘要 Surrogate concept and tiered system are important tools

for risk assessment 替代概念和分级系统是风险评估很重要的工具 Higher tier tests should only be conducted 更高分级试验只有当以下情况才进行

– when justified by detection of unacceptable risks at lower tiers of testing

当低分级试验测到不可接受的风险时– when they can reduce uncertainty in risk assessment 当在风险评估可以减少不确定性时

Laboratory tests 实验室试验 – Well defined 很好地定义– Quality control facilitates repeatability of tests and

transferability of data 质量控制有助于试验的可重复性和数据交流

Many opportunities for research and collaboration 科研和合作的很多机会