Lecture 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 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?
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—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?
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
Trans
genes
Conventional
breeding
Mutagenesis
Half genomes,
e.g., wide crosses
in hybrids
Risk??
Whole genomes, e.g.,
horticultural introductions or
biological control
more
Figure 13.1
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
• 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.
Roles of agencies in US regulation of
transgenic plants
• USDA: Gene flow, agronomic effects
such as weediness and pest
properties
• EPA: Gene flow, environmental/nontarget, toxicity when plants harbor
transgenes for pest control
• FDA: human toxicity/allergenicity for
plants used for food and feed
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 endpoints
• Understand exposure assessment and
hazard assessments for today’s GE plants
• Critically think about exposure and hazard
assessments for upcoming GE 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 assessment is more
reasonable than a quantitative
assessment
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 of gene flow and its
consequences
• Exposure = probability of 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
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 POTENTIAL PROBLEMS.
Example endpoints
• H, insect death: toxicology of insect
resistance genes
• E, hybridization frequency: gene flow
What are some ideal features of end points?
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
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%
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
 Said another way, genetic load could offset any
fitness benefits conferred by a [potentially] 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
Discussion question
•Which is more important: that a field test be
performed for a transgenic crop for grain yield
or environmental biosafety?
Case of the Monarch Butterfly
20 May 1999
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.
Nature © Macmillan Publishers Ltd 1999 Registered No. 785998 England.
Slide courtesy of D. Bartsch
Monarch butterfly exposure to
Bt cry1Ac
Monarch Butterfly Larvae Photo: http://www.news.cornell.edu/releases/May99/Butterflies.bpf.html
Slide courtesy of D. Bartsch
In October 2001 PNAS– 6 papers delineated the risk for monarchs.
Exposure assumptions tested made by Losey not close to realistic.
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
Diamondback Moth
Plutella xylostella
www.agf.gov.bc.ca/.../images/diamondback3.jpg
Cabbage Moth
Pieris rapae
www.butterfliesandmoths.org/pic/Pieris_rapae.jpg
Bt and Monarch Risk Model
cls.casa.colostate.edu/.../images/larva.jpg
Sears et al. (2001)
http://www.geo-pie.cornell.edu/issues/monarchs.html
www.smartcenter.org/ovpm/babymonarch-09.jpg
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
Field East
2 ha
Field West
4 ha
Farmer
Slide courtesy of D. Bartsch
Experimental Design: Field Study
Bt = Bt-maize Mon 810
INS = Isogenic variety with insecticide treatment
ISO = Isogenic variety, no insecticide treatment (Control)
Bt
INS
ISO
INS
Bt
5
4
3
2
1
ISO
Bt
INS
Bt
ISO
5
4
3
2
1
INS
ISO
Bt
ISO
INS
5
4
3
2
1
Bt
ISO
INS
6
6
6
ISO
INS
Bt
7
7
7
INS
Bt
ISO
8
8
8
Bearbeitunsrichtung
178 m
162 m
ca. 500 m
Bearbeitunsrichtung
182 m
141 m
162 m
186 m
237 m
248 m
Slide courtesy of D. Bartsch
Lepidopteron Larvae Exposure to
Bt cry1Ab
Insect collection
Species Identification
Slide courtesy of D. Bartsch
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
July
26. 27.
28
sample 1
August
29. 30. 31. 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 13. 14. 15.
sample 2
2001
flowering of maize
sample 1
sample 2
flowering of maize
2002
Slide courtesy of D. Bartsch
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
Abundant Species
Autographa gamma
Plutella
xylostella
Xanthorhoe flucata
Pieris rapae
Slide courtesy of D. Bartsch
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 not close.
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
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?