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Lectures 20-21, Chapters 12-13 Regulations and risk assessment Neal Stewart Discussion questions 1. What are regulations supposed to achieve? 2. With GM crops being used so extensively, how are we assured of their health and environmental safety? 3. How is genetic engineering (biotechnology) regulated? 4. When is plant genetic engineering not regulated? 5. How do the risks posed by products of biotechnology compare to those posed by conventional technologies? 6. How do different countries regulate products of biotechnology? Plant genetic modification Any gene, any organism The new plant will pass the transgene to its progeny through seed. Recall… progression of transgenic plants • Input traits– commercialized fast from 1996 • Output traits—commercialized slowly from early 2000s • Third generation– pharma, oral vaccines, phytoremediation, phytosensors— emerging gradually. How might regulating these be more challenging. Bt maize Bt cotton Golden rice Engineered to deliver pro-vitamin A GFP canola Plants to detect landmines No TNT induction Using inducible promoter/GFP fusions +TNT Agriculture and Nature • • • • Are farms part of nature? Of the environment? Direct or indirectly? Impacts on nature and agriculture might be inter-related but the endpoints will be different Big picture—ecological impacts of agriculture • Major constraint is agriculture itself • Tillage and pesticide practices • Crop genetics (of any sort) is miniscule ag v wild tillage pesticides herbicides crop genetics Amount of genetic information less added to ecosystems Tran s gene Conventiona s l breeding Mutagenesi s Half genomes, e.g., wide crosses in hybrids Whole genomes, e.g., horticultural introductions or biological control Risk?? more Figure 12.1 Domestication of corn 9000 years ago? Teosinte Corn Domestication of carrot Daucus carota 300 to 1000 years ago? Queen Anne’s Lace 1700s orange carrots appear in Holland Brassica oleracea Wild cabbage Ornamental kale Late 1900s Kohlrabi Germany 100 AD Cauliflower 1400s Kale 500 BC Broccoli Italy 1500s Cabbage 100 AD Brussel sprouts Belgium 1700s Regulations What/why regulate • Biosafety– human and environmental welfare • Recombinant DNA (rDNA) triggers regulation in most countries • Transgenic plants and their products are pound for pound the most regulated organisms on earth • “Protect” organic agriculture • “Precautionary principle” US history of regulating biotechnology • Early 1970s recombinant organisms are possible (microbes)—plants in 1980s • Asilomar conference 1975 • NIH Guidelines 1976—regulating lab use • OSTP Coordinated Framework—1986 • Set up the USDA, EPA and FDA to regulate aspects of transgenic plants Regulatory agencies provide safeguards and requirements to assure safety— determination and mitigation of risks. Roles of agencies in US regulation of transgenic plants • USDA: Gene flow, agronomic effects • EPA: Gene flow, environmental/nontarget, toxicity when plants harbor transgenes for pest control • FDA: human toxicity/allergenicity Ecological Risk Assessment of Transgenic Plants Problem formulation—assessment and measurement endpoints exposure assessment hazard assessment Objectives At the end of this lecture students should… • Understand framework for assessing risks • Be able to define short-term and long-term risks for a transgenic plant application—i.e., define ecological endpoints • Understand exposure assessment and hazard assessments for today’s GM plants • Critically think about exposure and hazard assessments for upcoming GM plants Methods of risk analysis • Experimental approach (toxicology or ecology) – Controlled experiments with hypothesis testing – Cause and effect • Theoretical modeling • Epidemiological approach—association of effects with potential causes • Expert opinion Adapted from 2002 NRC report: Environmental Effects of Transgenic Plants Risk Likelihood of harm to be manifested under environmentally relevant conditions Joint probability of exposure and effect Qualitative is more reasonable than quantitative Risk analysis Johnson et al. 2007 Trends Plant Sci 12:1360 Ecological Risks Risk = exposure x hazard Risk = Pr(event) x Pr(harm|event) • The example gene flow • Exposure = probability hybridization • Hazard = consequences of ecological or agricultural change--severity of negative impact Ecological Risks Risk = exposure x hazard Risk = Pr(event) x Pr(harm|event) • Transgene persistence in the environment– gene flow – Increased weediness – Increased invasiveness • Non-target effects– killing the good insects by accident • Resistance management– insects and weeds • Virus recombination • Horizontal gene flow Public perception: Risk = visibility x hysteria Stated another way and with terms: Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency Impact Hazard Consequence Experimental endpoints • • • • • Hypothesis testing Tiered experiments– lab, greenhouse, field Critical P value Relevancy Comparisons– ideal vs pragmatic world HYPOTHESES MUST BE MADE— WE CANNOT SIMPLY TAKE DATA AND LOOK FOR PROBLEMS! Example endpoints • H, insect death: toxicology of insect resistance genes • E, hybridization frequency: gene flow What are some ideal features of end points? Risk analysis Johnson et al. 2007 Trends Plant Sci 12:1360 Balancing exposure and hazard • R = E x H: an example from the world of gene flow • R= E x H: an example from the world on non-targets Johnson et al. 2007 Trends Plant Sci 12:1360 Gene flow model: Bt Cry1Ac + canola and wild relatives Brassica napus – canola contains Bt Diamondback moth larvae. http://www.inhs.uiuc.edu/inhsreports/jan-feb00/larvae.gif Brassica rapa – wild turnip wild relative Brassica relationships Triangle of U Bt Brassica gene flow risk assessment • Is it needed? • What kind of experiments? • At what scale? Tiered approach—mainly nontargets Wilkinson et al. 2003 Trends Plant Sci 8: 208 Ecological concerns • Damage to non-target organisms • Acquired resistance to insecticidal protein • Intraspecific hybridization • Crop volunteers • Interspecific hybridization • Increased hybrid fitness and competitiveness • Hybrid invasiveness www.epa.gov/eerd/BioTech.htm Brassica napus, hybrid, BC1, BC2, B. rapa Hybridization frequencies— Hand crosses– lab and greenhouse F1 Hybrids BC1 Hybrids CA QB1 QB2 Total CA QB1 QB2 Total GT 1 69% 81% 38% 62% 34% 25% 41% 33% GT 2 63% 88% 81% 77% 23% 35% 31% 30% GT 3 81% 50% 63% 65% 24% 10% 30% 20% GT 4 38% 56% 56% 50% 7% 30% 36% 26% GT 5 81% 75% 81% 79% 39% 17% 39% 31% GT 6 50% 50% 54% 51% 26% 12% 26% 21% GT 7 31% 75% 63% 56% 30% 19% 31% 26% GT 8 56% 75% 69% 67% 22% 22% 21% 22% GT 9 81% 31% 31% 48% 27% 28% 23% 26% GFP 1 50% 88% 75% 71% 18% 33% 32% 27% GFP 2 69% 88% 100% 86% 26% 20% 57% 34% GFP 3 19% 38% 19% 25% 10% 22% 11% 15% Gene flow model with insecticidal gene Wilkinson et al. 2003 Trends Plant Sci 8: 208 In the UK, Wilkinson and colleagues predict each year… •32,000 B. napus x B. rapa waterside populations hybrids are produced •16,000 B. napus x B. rapa dry populations hybrids are produced But where are the backcrossed hybrids? Field level backcrossing Maternal Parent F1 hybrid Transgenic/germinated Hybridization rate per plant Location 1 983/1950 50.4% Location 2 939/2095 44.8% F1 total 1922/4045 47.5% B. rapa Transgenic/germinated Hybridization rate per plant Location 1 34/56,845 0.060% Location 2 44/50,177 0.088% B. rapa total 78/107,022 0.073% Maternal Parent Halfhill et al. 2004. Environmental Biosafety Research 3:73 Genetic Load Negative effects of genetic load may hinder a hybrid’s ability to compete and survive Negative epistatic effects of genetic load could trump any fitness benefits conferred by a fitness enhancing transgene GM Crop Weed F1 Hybrid Weed BCX weed Field level hybridization Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency percentage of B. napus-specific markers Genetic introgression Bn F1 100 BC1F1 BC2F1 BC2F2 Bulk 75 50 25 0 CA x GT1 2974 x GT1 2974 x GT8 Halfhill et al. 2003 Theor Appl Genet 107:1533 AFLPs Generating transgenic “weeds” testing the consequences ´ Brassica rapa (AA, 2n=20) B. rapa B. rapa ´ ´ Brassica napus (AACC, 2n=38) F1 Generation (AAC, 2n=29) BC1F1 Generation (AAc, 2n=20 + 1 or 2) BC2F1 Generation (AA, 2n=20) ´ BC2F1 Generation (AA, 2n=20) BC2F2 Generation (AA, 2n=20) Competition field design Competition results b a a 150 NC a 750 600 120 b Wheat seed mass per m2 (g) 90 c c c c 450 c c 60 B. rapa BC2F2 Bt BC2F2 GT1 c Wheat Only a 180 B. rapa BC2F2 Bt BC2F2 GT1 d Wheat Only 500 a ab GA ab 400 150 bc c 120 B. rapa BC2F2 Bt BC2F2 bc c bc c GT1 Wheat Only B. rapa BC2F2 Bt BC2F2 GT1 Halfhill et al 2005 Mol Ecol 14:3177 300 Wheat Only Wheat vegetative dry weight per m2 (g) b BMC Biotechnol 2009 9:83 Figure 1. Genetic Load Study: Productivity. Average vegetative dry weight and seed yield (2e +4 = 20,000 seeds, 1e + 5 = 100,000 seeds, etc.) of non-transgenic Brassica napus (BN), Brassica rapa (BR) and transgenic BC1/F2 hybrid lines (GT1, GT5 and GT9) grown under non-competitive (A and C) and competitive field conditions (B and D). Columns with the same letter do not differ statistically (P < 0.0001). Error bars represent ± standard error of the means. Note that different Y-axis scales are used among figure panels. Discussion question •Which is more important: that a field test be performed for grain yield or environmental biosafety? Monarch butterfly exposure to Bt cry1Ac Monarch butterfly What’s riskier? Broad spectrum pesticides or non-target effects? In October 2001 PNAS– 6 papers delineated the risk for monarchs. Exposure assumptions made by Losey were far off. Tiered approach—mainly nontargets Wilkinson et al. 2003 Trends Plant Sci 8: 208 Tier 1: Lab Based Experiments Examples of insect bioassays www.ces.ncsu.edu/.../resistance%20bioassay2.jpg Bioassays to determine the resistance of the two-spotted spider mite to various chemicals www.ars.usda.gov/.../photos/nov00/k9122-1i.jpg A healthy armyworm (right) next to two that were killed and overgrown by B. bassiana strain Mycotech BB-1200. (K9122-1) Tier 2: Semi-Field/Greenhouse Tier 3: Field Studies Photo courtesy of C. Rose Photo courtesy of C. Rose Greenhouse Study: Transgenic Tobacco Photo courtesy of R. Millwood Field Trials: Transgenic Canola Goals of Field Research 1. Hypothesis testing 2. Assess potential ecological and biosafety risks (must be environmentally benign) 3. Determine performance under real agronomic conditions (economic benefits) Tiers of assessment & tiers of testing level of concern degree of uncertainty … arising from a lower tier of assessment drives the need to move toward a higher tier of data generation and assessment Tier IV Tier III Tier II Tier I Jeff Wolt Lab Microbial protein High dose Lab PIP diet Expected dose Long-term Lab Semi-field Field Assessment Testing Non-target insect model Wilkinson et al. 2003 Trends Plant Sci 8: 208 Examples…identifying Endpoints for Risks, Exposure, Hazards • Plant system (crop, weeds, communities, etc) • Phenotype • Biotic interactions • Abiotic interactions Class to give examples—discussion—setting up experiments Expert knowledge is important • Biotechnology – Transformation methods – Transgene – Regulation of expression • Ecology – – – – – – Plant Insect Microbial Populations Communities Ecosystems • Agriculture – Agronomy – Entomology • Regulator acceptance – Developed world – Developing world • Public acceptance – Finland and EU – Where GM crops are widely grown – New markets Features of good risk assessment experiments • Gene and gene expression (dose) – Relevant genes – Relevant exposure • • • • • Whole plants Proper controls for plants Choose species Environmental effects Experimental design and replicates Andow and Hilbeck 2004 BioScience 54:637. Risk assessment links research to risk management Data Acquisition, Verification, & Monitoring Risk Assessment Problem Formulation Jeff Wolt Exposure & effects characterization Risk Management Risk Characterization An example of agricultural risk that is not regulated The evolution of weed resistance to herbicides Conyza canadensis • Marestail or horseweed—found widely throughout North America and the world • Compositae • First eudicot to evolve glyphosate resistance • Resistant biotypes appeared in 2000, Delaware—resistant Conyza in 20+ US states and four continents, e.g. in countries such as Brazil, China, and Poland • 2N = 18; true diploid; selfer Spread of glyphosate resistance in Conyza Fig. 1. The proportion of soybean acreage sprayed with glyphosate from 1991 to 2002 relative to other herbicides Baucom, Regina S. and Mauricio, Rodney (2004) Proc. Natl. Acad. Sci. USA 101, 13386-13390 Copyright ©2004 by the National Academy of Sciences Resistant biotype 1 14 DAT rate in lbs ae/Ac Susceptible biotype C.L. Main UTC 0.38 0.75 1.12 1.5 2.25 3 8 RR weed risk assessment research • • • • Is it needed? What kind of experiments? At what scale? Other weeds? Environmental benefits of transgenic plants Big environmental benefits Herbicide tolerant crops have increased and encouraged no-till agriculture– less soil erosion. Over 1 million gallons of unsprayed insecticide per year. When transgenic plants are not regulated The case of the ancient regulations USDA APHIS BRS 7 CFR Part 340.0 Restrictions on the Introduction of Regulated Articles (a) No person shall introduce any regulated article unless the Administrator is: (1) Notified of the introduction in accordance with 340.3, or such introduction is authorized by permit in accordance with 340.4, or such introduction is conditionally exempt from permit requirements under 340.2(b); and (2) Such introduction is in conformity with all other applicable restrictions in this part. 1 1 Part 340 regulates, among other things, the introduction of organisms and products altered or produced through genetic engineering which are plant pests or which there is reason to believe are plant pests. The introduction into the United States of such articles may be subject to other regulations promulgated under the Federal Plant Pest Act (7 U.S.C. 150aa et seq.), the Plant Quarantine Act (7 U.S.C. 151 et seq.) and the Federal Noxious Weed Act (7 U.S.C. 2801 et seq.) and found in 7 CFR parts 319, 321, 330, and 360. Transgenic plants would be regulated by the USDA if they contain some of these vectors Not regulated by USDA http://www.aphis.usda.gov/biotechnology/downloads/reg_loi/Ceres_switchgrass_TRG108E_loi.pdf http://www.aphis.usda.gov/biotechnology/downloads/reg_loi/Ceres_switchgrass_responses.pdf What factors should trigger regulation?