1. Lecture 13 Regulations and risk assessment Neal Stewart

2. Discussion questions 1. What are regulations supposed to achieve? 2. With GM crops being used so extensively, how can we be assured they are safe to eat and not harming the environment? 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 breeding technologies? 6. How do different countries regulate products of biotechnology?

3. Plant genetic modification The new plant will pass the transgene to its progeny through seed. Any gene, any organism

4. Recall… progression of transgenic plants Input traits– commercialized fast from 1996—also known as agronomic traits Output traits—commercialized slowly from early 2000s—AKA quality traits Third generation– pharma, oral vaccines, phytoremediation, phytosensors— emerging gradually. –How might regulating these be more challenging?

5. Bt maize

6. Bt cotton

7. Golden rice Engineered to deliver pro-vitamin A

8. GFP canola

9. Plants to detect landmines induction Using inducible promoter/GFP fusions No TNT +TNT

10. 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

11. Big picture—ecological impacts of agriculture Major constraint is agriculture itself Tillage and pesticide practices Crop genetics (of any sort) is miniscule

12. Trans genes Conventional breeding Mutagenesis Half genomes, e.g., wide crosses in hybrids Whole genomes, e.g., horticultural introductions or biological control Amount of genetic information added to ecosystems less more Risk??

13. Figure 13.1

19. Domestication of corn TeosinteCorn 9000 years ago?

20. Domestication of carrot Daucus carota 300 to 1000 years ago? Queen Anne’s Lace 1700s orange carrots appear in Holland

21. Brassica oleracea Wild cabbage Kohlrabi Germany 100 AD Kale 500 BC Cabbage 100 AD Cauliflower 1400s Broccoli Italy 1500s Brussel sprouts Belgium 1700s Ornamental kale Late 1900s

23. Regulations

24. 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”

25. 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 Coordinated framework empowered 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.

26. Roles of agencies in US regulation of transgenic plants USDA: Gene flow, agronomic effects such as weediness and pest properties EPA: Gene flow, environmental/non- target, toxicity when plants harbor transgenes for pest control FDA: human toxicity/allergenicity for plants used for food and feed

27. Ecological Risk Assessment of Transgenic Plants Problem formulation—assessment and measurement endpoints exposure assessment hazard assessment

28. 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 endpoints Understand exposure assessment and hazard assessments for today’s GE plants Critically think about exposure and hazard assessments for upcoming GE plants

29. 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

30. Risk vLikelihood of harm to be manifested under environmentally relevant conditions vJoint probability of exposure and effect vQualitative assessment is more reasonable than a quantitative assessment

31. Risk analysis Johnson et al. 2007 Trends Plant Sci 12:1360

32. Ecological Risks Risk = exposure x hazard Risk = Pr(event) x Pr(harm|event) The example of gene flow and its consequences Exposure = probability of hybridization Hazard = consequences of ecological or agricultural change--severity of negative impact

34. 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

35. Risk = Pr(GM spread) x Pr(harm|GM spread) Stated another way and with terms: ExposureImpact FrequencyHazard Consequence

36. 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 POTENTIAL PROBLEMS.

37. Example endpoints H, insect death: toxicology of insect resistance genes E, hybridization frequency: gene flow What are some ideal features of end points?

38. Gene flow model: Bt Cry1Ac + canola and wild relatives Diamondback moth larvae. http://www.inhs.uiuc.edu/inhsreports/jan-feb00/larvae.gif Brassica napus – canola contains Bt Brassica rapa – wild turnip wild relative

39. Brassica relationships Triangle of U

40. Bt Brassica gene flow risk assessment Is it needed? What kind of experiments? At what scale?

41. Tiered approach—mainly non- targets Wilkinson et al. 2003 Trends Plant Sci 8: 208

42. 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

43. Brassica napus, hybrid, BC1, BC2, B. rapa

44. Hybridization frequencies— Hand crosses– lab and greenhouse F 1 Hybrids BC 1 Hybrids CAQB1QB2TotalCAQB1QB2Total GT 169%81%38%62%34%25%41%33% GT 263%88%81%77%23%35%31%30% GT 381%50%63%65%24%10%30%20% GT 438%56% 50%7%30%36%26% GT 581%75%81%79%39%17%39%31% GT 650% 54%51%26%12%26%21% GT 731%75%63%56%30%19%31%26% GT 856%75%69%67%22% 21%22% GT 981%31% 48%27%28%23%26% GFP 150%88%75%71%18%33%32%27% GFP 269%88%100%86%26%20%57%34% GFP 319%38%19%25%10%22%11%15%

45. Gene flow model with insecticidal gene Wilkinson et al. 2003 Trends Plant Sci 8: 208

46. 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?

47. Field level backcrossing Maternal Parent B. rapaTransgenic/germinatedHybridization rate per plant Location 134/56,8450.060% Location 244/50,1770.088% B. rapa total78/107,0220.073% Halfhill et al. 2004. Environmental Biosafety Research 3:73

48. Genetic Load  Negative effects of genetic load may hinder a hybrid’s ability to compete and survive  Said another way, genetic load could offset any fitness benefits conferred by a [potentially] fitness enhancing transgene GM CropWeed F 1 Hybrid BC X weed

49. Field level hybridization Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency

50. Genetic introgression Halfhill et al. 2003 Theor Appl Genet 107:1533 AFLPs

51. Generating transgenic “weeds” testing the consequences Brassica napus (AACC, 2n=38) Brassica rapa (AA, 2n=20) F 1 Generation (AAC, 2n=29) B. rapa BC 1 F 1 Generation (AAc, 2n=20 + 1 or 2) BC 2 F 1 Generation (AA, 2n=20) B. rapa BC 2 F 2 Generation (AA, 2n=20)     BC 2 F 1 Generation (AA, 2n=20)

52. Competition field design

53. Competition results NC GA Halfhill et al 2005 Mol Ecol 14:3177

54. Discussion question Which is more important: that a field test be performed for a transgenic crop for grain yield or environmental biosafety?

55. Transgenic pollen harms monarch larvae JOHN E. LOSEY, LINDA S. RAYOR & MAUREEN E. CARTER Although plants transformed with genetic material from the bacterium Bacillus thuringiensis (Bt ) are generally thought to have negligible impact on non-target organisms, Bt corn plants might represent a risk because most hybrids express the Bt toxin in pollen, and corn pollen is dispersed over at least 60 metres by wind. Corn pollen is deposited on other plants near corn fields and can be ingested by the non-target organisms that consume these plants. In a laboratory assay we found that larvae of the monarch butterfly, Danaus plexippus, reared on milkweed leaves dusted with pollen from Bt corn, ate less, grew more slowly and suffered higher mortality than larvae reared on leaves dusted with untransformed corn pollen or on leaves without pollen. 20 May 1999 Nature © Macmillan Publishers Ltd 1999 Registered No. 785998 England. Case of the Monarch Butterfly Slide courtesy of D. Bartsch

56. Monarch butterfly exposure to Bt cry1Ac

57. Slide courtesy of D. Bartsch Monarch Butterfly Larvae Photo: http://www.news.cornell.edu/releases/May99/Butterflies.bpf.html

58. Impact of Bt maize pollen (MON810) on lepidopteran larvae living on accompanying weeds ACHIM GATHMANN, LUDGER WIROOKS, LUDWIG A. HOTHORN, DETLEF BARTSCH, INGOLF SCHUPHAN* Molecular Ecology: Volume 15 Issue 9 Page 2677-2685, August 2006 In October 2001 PNAS– 6 papers delineated the risk for monarchs. Exposure assumptions tested made by Losey not close to realistic. Diamondback Moth Plutella xylostella www.agf.gov.bc.ca/.../images/diamondback3.jpg Cabbage Moth Pieris rapae www.butterfliesandmoths.org/pic/Pieris_rapae.jpg

59. Bt and Monarch Risk Model Sears et al. (2001) http://www.geo-pie.cornell.edu/issues/monarchs.html cls.casa.colostate.edu/.../images/larva.jpg www.smartcenter.org/ovpm/babymonarch-09.jpg

60. Experimental Goals Does growing of Bt-maize harm non-target Lepidoptera under field conditions? Compare growing of Bt-maize with conventional insecticide treatment Is the presented experimental design a useful approach for monitoring non-target Lepidoptera? * Note: this study did not specifically look at how Bt pollen effect monarch larvae. Examined other lepidopteron larvae native to Germany which are commonly found within corn fields Slide courtesy of D. Bartsch

61. Farmer Field East Field West 500m 4 ha 2 ha Slide courtesy of D. Bartsch

62. Experimental Design: Field Study Bt 6 ISO 7 ISO 8 INS 8 INS 6 Bt 8 INS 7 ISO 6 Bt 7 Bearbeitunsrichtung 178 m 162 m 141 m 186 m Bt 5 ISO 5 ISO 3 INS 5 INS 3 Bt 4 ISO 2 Bt 1 Bt 2 INS 1 ISO 1 INS 2 INS 4 ISO 4 Bt 3 Bearbeitunsrichtung 237 m 248 m 162 m 182 m ca. 500 m Bt = Bt-maize Mon 810 INS = Isogenic variety with insecticide treatment ISO = Isogenic variety, no insecticide treatment (Control) Slide courtesy of D. Bartsch

63. Lepidopteron Larvae Exposure to Bt cry1Ab Slide courtesy of D. Bartsch Insect collectionSpecies Identification

64. Field Test Results Lepidopteron larvae were not affected by the pollen of Mon 810 under field conditions Sometimes pollen shed and development of lepidopteron larvae barely overlapped 2001 2002 Slide courtesy of D. Bartsch

65. Field Test Results Choice of a lepidopteron monitoring species will be difficult because –species must be abundant –theoretical prediction of the presence of abundant species is not easy –occurrence and abundance of species depends on alot of variables ( e.g. climatic conditions, landscape structure around the fields, management options) Slide courtesy of D. Bartsch

66. Abundant Species Autographa gamma Plutella xylostella Pieris rapae Xanthorhoe flucata Slide courtesy of D. Bartsch

67. Monarch butterfly In October 2001 PNAS– 6 papers delineated the risk for monarchs. Exposure assumptions made by Losey were not close. What’s riskier? Broad spectrum pesticides or non-target effects?

68. Tiered approach—mainly non- targets Wilkinson et al. 2003 Trends Plant Sci 8: 208

69. Tier 1: Lab Based Experiments 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) Examples of insect bioassays

70. Tier 3: Field Studies Tier 2: Semi-Field/Greenhouse Greenhouse Study: Transgenic Tobacco Field Trials: Transgenic Canola Photo courtesy of C. Rose Photo courtesy of R. Millwood

71. 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)

72. 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 I Tier II Tier III Tier IV Lab Microbial protein High dose Lab PIP diet Expected dose Long-term Lab Semi-field Field Assessment Testing Jeff Wolt

73. Non-target insect model Wilkinson et al. 2003 Trends Plant Sci 8: 208

74. Examples…identifying Endpoints for Risks, Exposure, Hazards Plant system (crop, weeds, communities, etc) Phenotype Biotic interactions Abiotic interactions

75. 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

76. 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.

77. Risk assessment links research to risk management Problem Formulation Exposure & effects characterization Risk Characterization Risk Management Risk Assessment Data Acquisition, Verification, & Monitoring Jeff Wolt

78. An example of agricultural risk that is not regulated The evolution of weed resistance to herbicides

79. 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 Conyza canadensis

82. Spread of glyphosate resistance in Conyza

83. Copyright ©2004 by the National Academy of Sciences Baucom, Regina S. and Mauricio, Rodney (2004) Proc. Natl. Acad. Sci. USA 101, 13386-13390 Fig. 1. The proportion of soybean acreage sprayed with glyphosate from 1991 to 2002 relative to other herbicides

85. Resistant biotype 1 Susceptible biotype 14 DAT rate in lbs ae/Ac C.L. Main UTC1.121.52.25380.380.75

86. RR weed risk assessment research Is it needed? What kind of experiments? At what scale? Other weeds?

87. Environmental benefits of transgenic plants

89. 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.

90. When transgenic plants are not regulated The case of the ancient regulations

91. 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.

92. Transgenic plants would be regulated by the USDA if they contain some of these vectors

93. 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

94. What factors should trigger regulation?