Lectures 20-21, Chapters 12-13Regulations 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

The new plant will pass the transgeneto its progeny through seed.

Any gene, any organism

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

induction

Using induciblepromoter/GFP fusions

No TNT +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

Transgenes

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 les

s

more

Risk??

Figure 12.1

Domestication of corn

Teosinte Corn

9000 years ago?

Domestication of carrotDaucus carota

300 to 1000 years ago?

Queen Anne’s Lace

1700s orange carrots appear in Holland

Brassica oleracea

Wild cabbage

KohlrabiGermany 100 AD

Kale 500 BC

Cabbage 100 AD

Cauliflower 1400s

Broccoli Italy 1500s

Brussel sproutsBelgium 1700s

Ornamental kaleLate 1900s

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/non-target, 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

ObjectivesAt 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

RiskRisk

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 RisksRisk = 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 RisksRisk = 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

Risk = Pr(GM spread) x Pr(harm|GM spread)

Stated another way and with terms:

Exposure ImpactFrequency 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

Diamondback moth larvae. http://www.inhs.uiuc.edu/inhsreports/jan-feb00/larvae.gif

Brassica napus – canolacontains Bt

Brassica rapa – wild turnipwild 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 non-targets

Wilkinson et al. 2003 Trends Plant Sci 8: 208

Ecological concernsEcological concerns

• Damage to non-target organisms• Acquired resistance to insecticidal

protein • Intraspecific hybridization

• Crop volunteers

• Interspecific hybridization• Increased hybrid fitness and

competitiveness

• Hybrid invasivenesswww.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 backcrossingMaternal 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%

Maternal ParentB. 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%

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

Weed

F1 Hybrid

BCX

weed

Field level hybridizationThird-tier Risk = Pr(GM spread) x Pr(harm|GM spread)

Exposure Frequency

CA x GT1 2974 x GT1 2974 x GT8

pe

rce

nta

ge o

f B

. n

ap

us-

spe

cific

ma

rke

rs

0

25

50

75

100

Bn F1

BC1F1

BC2F1

BC2F2 Bulk

Genetic introgression

Halfhill et al. 2003 Theor Appl Genet 107:1533

AFLPs

Generating transgenic “weeds”testing the consequences

Brassica napus

(AACC, 2n=38)

Brassica rapa(AA, 2n=20)

F1 Generation(AAC, 2n=29)

B. rapa

BC1F1 Generation (AAc, 2n=20 + 1 or

2)

BC2F1 Generation (AA, 2n=20)

B. rapa

BC2F2 Generation (AA, 2n=20)

BC2F1 Generation (AA, 2n=20)

Competition field design

Competition results

Whe

at s

eed

mas

s pe

r m

2 (g)

60

90

120

150

Whe

at v

eget

ativ

e dr

y w

eigh

t per

m2 (g

)

450

600

750

c

c c

b

aa

b

c c

c

B. rapa BC2F2 BtBC2F2

GT1 WheatOnly

a b

120

150

180

300

400

500

bc

ab

c c

a

bc

ab

bcc

ac d

B. rapa BC2F2 BtBC2F2

GT1 WheatOnly

B. rapa BC2F2 BtBC2F2

GT1 WheatOnly

B. rapa BC2F2 BtBC2F2

GT1 WheatOnly

NC

GA

Halfhill et al 2005 Mol Ecol 14:3177

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.

BMCBiotechnol20099:83

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

In October 2001 PNAS– 6 papers delineated the risk for monarchs.

Exposure assumptions made by Losey were far off.

What’s riskier?

Broad spectrum pesticides

or

non-target effects?

Tiered approach—mainly non-targets

Wilkinson et al. 2003 Trends Plant Sci 8: 208

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

Tier 3: Field StudiesTier 2: Semi-Field/Greenhouse

Greenhouse Study: Transgenic Tobacco

Field Trials: Transgenic Canola

Photo courtesy of C. Rose

Photo courtesy of C. Rose

Photo courtesy of R. Millwood

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 assessment &tiers of testingtiers 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

LabMicrobial protein

High dose

LabPIP diet

Expected dose

Long-term Lab Semi-field

Field

Assessment

Testing

Jeff Wolt

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 Risk assessment links research to risk managementresearch to risk management

ProblemFormulation

Exposure & effectscharacterization

RiskCharacterization

Risk Management

Risk Assessment

Data Acquisition, Verification, & Monitoring

Jeff Wolt

An example of agricultural risk that is not regulated

The evolution of weed resistance to herbicides

• 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

Spread of glyphosate resistance in Conyza

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

Resistantbiotype 1

Susceptiblebiotype

14 DATrate inlbs ae/Ac

C.L. Main

UTC 1.12 1.5 2.25 3 80.38 0.75

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 BRS7 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?