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2
When and Why to Consider
Bioconfinement
INTRODUCTION
Safety is an issue for all modern technology--from the design of auto-
mobiles to the delivery of information over the Internet--and confinement
of technology is frequently an important aspect of ensuring safety. Airbags
and radial tires promote drivers' safety; highly developed software pro-
motes computer users' privacy and security. The atomic energy industry
works to confine radiation; the chemical industry, to confine toxic chemicals;
the food industry, to prevent nonedible industrial rapeseed from contami-
nating canola oil for human consumption; and the horticultural industry, to
confine ornamental plants that might become invasive. The stewardship of
those industries bolsters public confidence in technology and its products at
the same time as it prevents the damage that can result from the movement
of products and byproducts into arenas for which they are not intended.
The success of any new technology depends on this attention to safety and
confinement and on building public trust by protecting human health, the
environment, and the security of intellectual and financial information.
Biotechnology should enjoy the same benefits from attention to safety
and confinement. As most traditionally improved organisms pose few safety
problems that require confinement, it is likely that most of the vast array of
proposed genetically engineered organisms (GEOs) will pose little threat to
public health or the environment, and they will require minimal confine-
ment, if any. However, as some traditionally improved organisms require
confinement, some GEOs will need some or substantial confinement. If
29
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30 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
field release of a GEO is proposed, it is important to consider whether
confinement is necessary, and, if so, how to attain it.
This chapter reviews the concept of risk and then reviews some of the
effects that could be expected from the release of GEOs. Avoiding or mini-
mizing damage generally requires that specific methods of confinement be
considered and that the consequences of failure (inadequate confinement
performance) are predicted. This chapter also includes a discussion of who
bears responsibility for deciding whether and how to confine GEOs and
their engineered genes.
WHAT IS RISK?
In many aspects of its deliberations, the Committee on the Biological
Confinement of Genetically Engineered Organisms found it necessary to
discuss "hazard," "harm," and "risk." The terminology used here is consis-
tent with past reports of the National Research Council (NRC, 1983, 1996,
2002b). To quote a recent report (NRC, 2002b), "as set forth by the NRC
(1983, 1996), a hazard is an act or phenomenon that has the potential to
produce harm, and risk is the likelihood of harm resulting from exposure to
the hazard ... risk is the product of two probabilities: the probability of
exposure and the conditional probability of harm, given that exposure has
occurred." Risk assessment typically involves asking questions: What can
go wrong? How likely is failure? What are the consequences of failure?
How likely are those consequences (assuming the triggering event occurs)?
How significant are those consequences? How certain are we about this
knowledge, whether it is qualitative or quantitative (NRC, 1996)?
Risk assessment involves (1) identifying potential harms, (2) identifying
potential hazards that might produce the harms, (3) defining exposure and
the likelihood of exposure, (4) quantifying the likelihood of harm given that
exposure has occurred, and estimating the severity of the harm (NRC,
2002b). The importance of social values to this determination is discussed
below.
Risk is sometimes described as a formula: exposure multiplied by
hazard. Although this can be useful shorthand, risk is not easily estimated.
Uncertainties can arise from random events (including human error) in the
physical world, lack of knowledge about the physical world, or lack of
knowledge about the applicability of risk-generating processes (NRC, 1996).
Ecological harm (consequences) is difficult to quantify, including damage to
an ecosystem or the extinction of a species, for example. Moreover, evaluating
harm requires consideration of social values that will define the significance
of predicted consequences. Clearly, exact quantification is impossible.
An evaluation of risk must consider cumulative risk--the combined
risks to human health or the environment posed by exposure to multiple
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 31
agents or stressors (US EPA, 2003a). Without cumulative risk analysis, it is
impossible to assess the hazard associated with bioconfinement failure. For
example, suppose failure would result in the release of a contaminant into
the environment. The hazard stemming from that failure could depend on
whether the environment is totally free of the contaminant, the contami-
nant already is present in the environment at a near-hazardous concentra-
tion, the environment already is stressed by other factors such that its
normal resilience is compromised, or there are some other substances in the
environment that would neutralize the contaminant or exacerbate its effects.
The risk of any new technology should be considered in the context of
preexisting relevant technologies. Doing so would likely involve an assess-
ment of relative risks and while this comparison is an important one for
consideration, it is also a challenging task. In the context of this discussion,
relative risk is, in theory, equal to the probability of harm utilizing the new
technology divided by the probability of harm utilizing the preexisting
technology. One challenge is that, as indicated above, risk assessment is not
readily susceptible to exact mathematical calculation. Another challenge is
identifying the appropriate preexisting technology to serve as a compara-
tor. Given the diversity of GEOs and the farming systems where they might
be used, there is a substantial amount of disagreement as to what pre-
existing agricultural technologies are appropriate to serve as comparators.
Furthermore, given the range and number of potential variables and risks
associated with the application of the technologies, identifying the relevant
risks for comparison would have to be done on a case-by-case basis. For the
bioconfinement technologies described in this report that have yet to be
fully developed or applied, discussion of relative risks is simply premature
at this time. Nonetheless, as developers of GEOs and the confinement
methods progress such comparisons will be attempted but will not be easy
to make.
Given the uncertainty involved in risk analysis and the fact that many
variables cannot be quantified, the committee determined that an alternate,
less formulaic, model for risk assessment would be valuable. Figure 2-1 is a
risk assessment matrix that assumes that a hazardous event has occurred..
The hazard of greatest concern is that with a high risk (probability of
occurrence) and high significance or severity of harm (black area). Social,
ecological, and economic considerations influence the significance of conse-
quences. Depending on the quality of information available, the axes could
consist of continuous values or more discrete categories (e.g., a 3×3 matrix
of highmediumlow rankings; Miller et al., in press).
Earlier reports of the National Research Council contain lengthy dis-
cussions of risk and approaches to its analysis. There is extensive consider-
ation of risk in the report Understanding Risk (NRC, 1996), and Environ-
mental Effects of Transgenic Plants (NRC, 2002a) contains a detailed
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32 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
High
Harm
of
Severity
Low
Low High
Risk (probability of harm)
FIGURE 2-1 A risk assessment matrix. The horizontal axis denotes the risk, that
is, the probability of occurrence of a harm, and the vertical axis denotes the sever-
ity of the harmful consequence.
discussion of risk analysis, including analyses of two-part and whole-
organism models, and fault-tree and event-tree approaches. Discussions of
risk often distinguish among three related processes: risk assessment, risk
management, and risk characterization. The 1996 report sets forth an elabo-
rate description of risk characterization, which it defines as "a synthesis
and summary of information about a potentially hazardous situation that
addresses the needs and interests of decision makers and of interested and
affected parties and which is a prelude to decision making and depends on
an iterative, analyticdeliberative process" (NRC, 1996). The committee
recognizes those earlier discussions and suggests it would be useful to
describe a systematic approach to risk assessment and management, with-
out delving into the issue of risk characterization other than to note its
relevance and importance, particularly with respect to transparency and
public participation.
Table 2-1 presents a systematic approach to risk assessment and man-
agement. It considers exposure, hazard, risk reduction and prevention,
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 33
TABLE 2-1 Systematic Risk Assessment and Management
Step Key questions
Hazard What event posing harmful consequences could occur?
identification
Risk analysis How likely is the hazard?
What would be the harms from realization of the hazard, and how
severe are they, taking into account social values?
What is the risk assessment as shown on a matrix of risk (likelihood
of harm) plotted against severity of harm; see Figure 2-1, above)?
Each cell of the matrix should be accompanied by a qualitative
assessment of the response and a quantification of assurance
needed to reduce harm if the cell's conditions were to occur.
How well established is the knowledge used to identify the hazard,
estimate its risk, and predict harms?
Risk reduction What can be done (including bioconfinement and other
planning and confinement) to reduce risk, either by reducing the likelihood or
implementation mitigating the potential harms? Are there steps that can be taken
to prepare for remediation?
Risk tracking How effective are the implemented measures for risk reduction?
(monitoring)
Are they as good as, better than, or worse than planned?
What follow-up, corrective action, or intervention will be pursued
if findings are unacceptable?
Did the intervention adequately resolve the concern?
Remedial action What remedial action should be taken?
Transparency How transparent should the entire process be? How much and
and public what type of participation should there be in the steps above
participation (and in risk characterization) by the public at large, by experts,
and by interested and affected parties?
SOURCE: Adapted from Kapuscinski, 2001.
monitoring, remedial action, transparency, and public participation (adapted
from Kapuscinski, 2001).
Confinement should be undertaken in the context of an integrated
confinement system (ICS)--one that considers whether confinement might
be necessary from the beginning of GEO development and that has redun-
dancy of confinement as an operating principle (Chapter 6). Elements of
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34 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
this approach include a clear strategy that is adopted in advance of field
testing or release of the GEO. Adequate training of those who are respon-
sible for managing the confinement strategy is essential. Permanent employ-
ees should maintain accountability and consistency of standard operating
procedures. For pharmaceutical-producing GEOs, good management prac-
tices also will be necessary. An ICS should be subjected to periodic audit,
review, adjustment, and reporting. Each ICS should be supported by a
rigorous, comprehensive, and credible regulatory regime that includes
mechanisms for inspection and enforcement.
The risk assessment techniques discussed here would have been effec-
tive in predicting some notable events that have already occurred. Consider
the StarLink incident (Box 2-1). In this case a variety of corn was deregu-
BOX 2-1
Confinement Failure: StarLink Corn
The StarLink incident illustrates the failure of a regulatory-agencyindustry
attempt to prevent genetically engineered material from entering the human food
supply. The story offers lessons about the confinement of engineered genes and
their products.
Genes from Bt that produce various Cry proteins have been individually engi-
neered into plants to confer resistance to insect pests. These are commonly called
Bt crops. The most widespread use of genes for Cry proteins has been to confer
resistance to lepidopteran pests in maize.
The trade name StarLink was applied to maize varieties produced by Aventis
CropScience that were engineered to synthesize the Cry9 protein for resistance to
the European corn borer. The Cry9 protein was found to be more heat resistant
than other Cry proteins and potentially harder to digest based on in vitro studies,
suggesting possible allergenicity (US EPA, 1999). Because of the inconclusive
tests regarding possible allergic reactions in humans, the US EPA granted StarLink
varieties a "split registration" in 1998, designating it for animal feed and industrial
use, but not for human consumption (US EPA, 1998).
In September 2000, the Washington Post reported that traces from transgenes
of StarLink corn had been detected in the human food supply by an independent
laboratory for Genetically Engineered Food Alert, a coalition of environmental and
food safety organizations (Kaufman, 2000). Soon thereafter StarLink Bt gene was
found in a great variety of products intended for human consumption, in the United
States as well as in Japan and South Korea--major United States maize buyers.
The effect on the United States food industry was substantial, ranging from the
recall of food products to the temporary shutdown of grain mills (Taylor and Tick,
2003). Aventis voluntarily withdrew its registration, and to prevent further mixing of
StarLink material into the human food supply, it agreed to a buyback program with
USDA. The US EPA announced that it would no longer grant split registrations for
GEOs (Taylor and Tick, 2003).
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 35
lated with the proviso that it was not permitted to enter the human food
supply, and yet it did so. If the following factors were initially considered--
the possibility of pollen dispersal, retention of prior genotypes in the soil
seed bank, and the mixing of corn seeds in facilities that harvest, transport,
store, and process corn seedit would have been obvious that without very
stringent procedures confinement failure was inevitable.
CONCERNS
The committee adopted the definition of "concern" used in the report
Animal Biotechnology: Science-based Concerns (NRC, 2002b). "Concern"
is used here to mean an "uneasy state of interest, uncertainty, and appre-
The potential sources of the widespread presence of StarLink material in the
human food supply are many. Opportunities for mixing genes, seeds, and maize
products start in the field and end at the store. Cleaning the facilities that harvest,
transport, and store maize seed does not necessarily remove every seed. Pro-
cessing also presents an opportunity to commingle products. If a farmer does not
rotate crops, ungerminated seed from an earlier planting or seed that dropped
from another year's crop could germinate and mix with the current year's variety.
StarLink genes also were found in varieties into which it had not been engineered,
strongly suggesting unintended cross-pollination from StarLink maize. It also is
clear that some farmers did not comply with the terms of US EPA registration:
Some admitted that they had sold their maize for human food, others stated that
they did not know how their maize would be used when they sold it (Pollack, 2001).
Although StarLink corn caused no proven adverse human health effects, other
effects have been substantial. Despite the fact that StarLink represented less than
1% of the U.S. maize crop, millions of dollars were spent to buy back grain, con-
sumer confidence in GEOs was shaken, and international trade relationships
became strained. Once a gene escapes into populations for which it is unintended,
the consequences can be enormous. On December 27, 2002, more than two years
after Aventis stopped selling StarLink seed, the Washington Post reported that
traces of StarLink material were detected in a shipment of U.S. maize on its way to
Tokyo for use in food products (Fabi, 2002).
More extensive information on the StarLink incident is presented in Post-Market
Oversight of Biotech Foods (Taylor and Tick, 2003), which includes a substantial
chronology of the incident. Each of the following publications concentrates on a
different aspect of the incident: The StarLinkTM Situation (Harl et al., 2001),
StarLink: Impacts on the United States Corn Market and World Trade (Lin et al.,
2001), and Channeling, Identity Preservation and the Value Chain: Lessons from
the Recent Problems with StarLink Corn (Ginder, 2001).
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36 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
hension" (NRC, 2002b). Ethical concerns--a subset--are discussed in
Chapter 1.
The committee attempted to base its analyses of risk and concern on
current natural and social scientific knowledge. As with its consideration of
risk, the criteria include the likelihood that a given result will occur; its
severity; and its significance, as would be determined at least in some
measure by public opinion or societal values.
Potential Effects
Genetically engineered organisms are controversial. Dozens of scholarly
reports have identified, reviewed, and evaluated the realized and potential
effects of their use, particularly transgenic crops and fish (e.g., Carpenter et
al., 2001; Colwell et al., 1985; Cook et al., 2000; Dale et al., 2002; Hails,
2000; Kapuscinski and Hallerman, 1991, 1994; Keeler and Turner, 1990;
Marvier, 2001; McHughen, 2000; NRC, 1989b, 2000, 2002a, 2002b; Pew
Initiative on Food and Biotechnology, 2003; Rissler and Mellon, 1996;
Scientists' Working Group on Biosafety, 1998; Snow and Moran-Palma,
1997; Tiedje et al., 1989; Traynor and Westwood, 1999; Winrock Inter-
national, 2000; Wolfenbarger and Phifer, 2000). As GEOs are developed
and released, their specific effects can be scrutinized.
Current and Future GEOs: A Brief Summary
The first commercially grown transgenic plant--the Flavr SavrTM tomato
(Kramer and Redenbaugh, 1994)--was released to the field a little more
than a decade ago. Since that time, millions of acres have been planted in
genetically engineered crops--mostly in the United States. Soybean, corn
(maize), canola (oilseed rape), and cotton are the major species used world-
wide, and most of those plants are restricted to three phenotypic classes:
herbicide resistance, insect resistance, and viral resistance (James, 2002).
Despite the narrow range of crops and phenotypes and despite the fact that
most of the acreage is restricted to a few countries, their rapid acceptance is
remarkable in the history of agriculture, outpacing even the acceptance of
corn hybrids (James, 2002). Not all field-grown plants that are used to
produce commercial products have been deregulated. In the United States,
some products have been produced from transgenic crops and viruses in
field tests that are regulated under the United States Department of Agricul-
ture (USDA) Animal and Plant Health Inspection Service (APHIS) notifica-
tion and permit processes (NRC, 2002a).
Transgenic animals are largely under development, and some have been
commercialized. In a few cases, transgenic mammals have been created to
secrete commercial biochemicals (http://www.nexiabiotech.ca/en/01_tech/
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 37
01.php). Those genetically engineered animals that have been commercial-
ized have not been released to the field. Some microorganisms have achieved
commercial success with applications in medicine and in food production.
As yet, they have been used in industrial situations, rather than in situations
calling for field release. For example, chymosin is the active enzyme in
rennet, which is used in cheese production. The gene for this enzyme has
been engineered into bacteria and yeast, and it now contributes substan-
tially to the commercial production of hard cheese in Europe and North
America (Mohanty et al., 1999). Prior to genetic engineering, chymosin was
obtained exclusively from rennet extracted from the stomachs of calves.
Benefits beyond the commercial success of transgenic crops also have
been noted. In the United States, insect resistance and herbicide tolerance
sometimes have permitted reduced pesticide use and have increased yields
(Benbrook, 2001; Fernandez-Cornejo and McBride, 2002). The gains in
yield and reductions in pesticide use have been modest in developed coun-
tries but more substantial in developing nations (e.g., Qaim and Zilberman,
2003). Herbicide resistance probably has facilitated the increasing practice
of "no-till" agriculture, which replaces mechanical weed control with chemi-
cal weed control, resulting in reduced soil erosion--but the details of how
and whether herbicide-resistant plants have improved soil quality require
more study (Wolfenbarger and Phifer, 2000).
The list of potential benefits attributable to transgenic plants, animals,
and microorganisms is long and diverse, and it is difficult to name all of the
beneficial phenotypes that have been proposed for transgenic organisms.
However, some expected benefits have been discussed widely. One is the
dramatic growth enhancement in several lines of transgenic fish that are
approaching commercialization (NRC, 2002b). Also, some traits related to
coping with abiotic stresses have been targeted, such as drought and salinity
tolerance in crops (e.g., Garg et al., 2002) and cold tolerance in fish (e.g.,
Wang et al., 1995). Some phenotypes have been proposed to make food
healthier by increasing its nutritional value, by eliminating allergens and
antinutritional factors, and by extending the shelf life of fruits and veg-
etables. Other phenotypes pertain to nonfood products processed from
plants, such as the alteration of the chemical composition of wood fibers
from trees to reduce the cost of paper production, and the production of
expensive or hard-to-synthesize specialty chemicals, such as pharmaceuticals
in transgenic crop plants (Pew Initiative on Food and Biotechnology, 2002),
aquatic plants and moss (Spencer, 2003; Wagner, 2003), algae (Mayfield,
2003), and fish (Aquagene, 2003). Viruses and microorganisms that are
natural pathogens of insects and other pests could be engineered into more
effective agents of biological control. Transgenic plants, microorganisms,
and algae have been proposed for use in environmental remediation to
detoxify polluted soils and waters (Cai et al., 1999).
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38 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
Some of the proposed uses are novel; others are natural extensions of
current use. Nontransgenic bacteria already are used to degrade environ-
mental pollutants. Table 2-2 lists some GEOs and phenotypes that have
been proposed, are under development, or are in use. Only time will reveal
which predicted benefits of those organisms are realized, and a thorough
discussion of those benefits is beyond the scope of this report.
Gene Dispersal and Persistence
Before considering specific categories of GEOs that may be candidates
for bioconfinement, it is useful to examine how genes and organisms move,
and whether transgenes are likely to be favored by natural selection. The
ecological spread of transgenes is accomplished by horizontal transfer, dis-
persal of whole organisms, or dispersal of gametes that may move transgenes
past the point of intentional release and into new environments and organ-
isms. The persistence of transgenes depends largely on how they affect the
organisms' evolutionary fitness, as discussed below.
How Transgenes Disperse
Horizontal transfer occurs when genetic material from one organism
becomes incorporated into an unrelated organism (NRC, 2002a); it is natu-
rally occurring genetic engineering. Gene transfer among unrelated bacteria
is relatively common (Syvanen, 2002); transfer among unrelated eukaryotes
happens less frequently. The rate of horizontal transfer that is detected as
evolutionary change is extremely low--it is far less the mutation rate--
although it is surprisingly fast over evolutionary time. And some plants, for
example, appear to have acquired genes from other types of organisms,
even from other kingdoms (Adams et al., 1998). And some flowering plants
apparently have acquired mitochondrial genes from fungi hundreds of times
over the past 100 million years (Palmer et al., 2000).
Horizontal transfer is largely viewed as a source of unanticipated con-
sequences. Transgenic plant DNA can transform bacteria in sterile soil
microcosms (Nielsen et al., 2000). There is no data yet, however, to suggest
that the rate of horizontal transfer would be any faster for transgenic
organisms (Syvanen, 2002). But relevant data remain few (Nielsen et al.,
1998); further research to identify the mechanisms and ecological implica-
tions of horizontal gene transfer (Traavik, 2002) would be helpful.
Dispersal of whole organisms includes the movement of juvenile and
adult animals, fertilized eggs and seeds, spores, and vegetative propagules
such as offshoots and fragments of plants and algae. Dispersal can occur
passively--in the same ways pollen and seeds are transported, for example--
or by anthropogenic means: during international trade, in the ballast water
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 39
TABLE 2-2 Genetically Engineered Organisms
Species Engineered Trait Application Development
Finfish
Mud loach Increased growth rate, Aquaculture Research
improved feed conversion, and for human
likely sterility after insertion of food
mud loach growth hormone
driven by mud loach -actin
regulatory region (Nam et al.,
2001a,b)
Channel catfish Enhanced bacterial resistance Aquaculture Research
after insertion of moth peptide for human
antibiotic, cecropin B gene food
(Dunham et al., 2002)
Medaka Facilitation of better detection Industrial Research;
of mutations (presumably caused uses; method has
by environmental pollution) after environmental been patented
insertion of a bacteriophage uses
vector (serves as a mutational
target). After exposure to
mutagenic agent, vector DNA
is removed and inserted into
indicator bacteria where mutant
genes can be easily measured
(Winn, 2001a, b; Winn et al.,
1995, 2000, 2001)
Atlantic salmon Increased growth rate and food Aquaculture Method has
conversion efficiency after for human been patented;
insertion of Chinook salmon food seeking Food
growth hormone gene that and Drug
operates year-round, thereby Administration
fostering steady growth through approval
the year rather than summer
growth (Cook et al., 2000;
Hew and Fletcher, 1996)
Red sea bream Increased growth rates after Aquaculture Research
insertion of an "all fish" growth for human
hormone consisting of ocean food
pout antifreeze protein gene
promoter and Chinook salmon
growth hormone (Zhang et al.,
1998)
continued
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54 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
Precisely how to identify what combination of confinement measures to
undertake--if any--is addressed above.
HOW MUCH CONFINEMENT IS ENOUGH?
If some type of confinement is deemed necessary, careful consideration
should be given to how much will be sufficient. In some cases, appropriate
confinement might be obtained by conventional, non-biological, methods.
For example, sufficient isolation might be achieved by growing a minor
crop far from stands of the same variety and away from populations of wild
relatives. That might reduce viable pollen flow to acceptable levels. Other
steps would ensure that seed and vegetative propagules did not find an
environment where they could become established.
The most stringent confinement is necessary when field releases of
GEOs or their transgenes have sufficient potential to create substantial
problems. If stringent confinement is to be applied to released organisms,
the standard methods of spatial or temporal isolation will not suffice. For
example, the standard contamination tolerated by breeders of high-quality
"foundation" seed generally is 10 (NRC, 2000). That purity is attained by
3
spatial and temporal isolation, often in conjunction with the use of border
or barrier crops that will interfere with pollen flow (Kelly and George,
1998). Generally a 660-ft buffer is considered sufficient to reduce back-
ground contamination of maize fields to 0.10%--often considered accept-
able. However, almost one-third of some 300 maize fields in the Corn Belt
exhibited background contamination that ranged from 1.5% to 15.6% at
that distance (Burris, 2001).
Redundancy of methods is usually necessary to achieve stringent con-
finement levels. The 2002 APHIS requirements for growing transgenic maize
for pharmaceutical compounds in the field call for substantial spatial and
temporal isolation (USDA, 2002). Those requirements, however, are only
for preventing dispersal of genes by pollen. APHIS requires additional
methods to prevent gene movement by seed or gene persistence in a soil
seed bank.
Maximum confinement will not be necessary for most organisms. GEOs
that pose no hazard or whose risk level is so low as to be tolerable would
not require containment. The need for confinement might vary with the
fitness impact of the allele in question as well as with whether immigration
is anticipated to be recurrent or a single event. What if the allele will have
no impact on fitness? If the allele is expected to have a neutral impact on
fitness but immigration is expected to be recurrent, then the allele is expected
to increase, generation by generation. If such an allele is predicted to create
a significant hazard in locations for which it was not intended and that
recurrent immigration is going to occur, then the most stringent confine-
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 55
ment conditions are advised. If immigration would occur once or a few
times, then less stringent conditions might be permissible, depending on the
acceptable frequency for that allele.
What if the allele would prove detrimental in populations or environ-
ments for which it was not intended? If only one or a few immigration
events are anticipated, the allele will not persist in a population, and if
immigration is recurrent, the allele will be maintained at a frequency between
zero and one. In this case, the acceptable allele frequency is important. The
fact that the allele confers a fitness disadvantage will result in a decline in
the average fitness of the recipient population. The fitness change also
could be so severe as to drive the affected population to the point of
extinction (e.g., Huxel, 1999; Wolf et al., 2001).
Extinction by hybridization can result from more complicated situa-
tions. If an immigrant allele reduces viability but dramatically increases
mating success, the antagonistic effects on different fitness components can
lead to eventual extinction after a single immigration event (Muir and
Howard 1999, 2001, 2002). Whether such local extinction would be a
problem or a benefit would depend on the population. For example, an
increased extinction risk would be a problem for an endangered species, but
a benefit for one considered a noxious invasive.
Clearly, to reduce chances of escape, there is a need for as many cost-
effective tools as possible. The appropriate amount of confinement might
be possible now, but the method could be so expensive as to preclude its
use. Bioconfinement methods offer an opportunity to expand the number
and diversity of tools available.
Finally, there could be organisms for which the possible hazard is so
great that it would be best never to release the product. A recent document
(USDA, 2002) states that some species are "inappropriate for the produc-
tion of pharmaceuticals" when grown under field conditions. The cited
example is the oilseed rape species Brassica rapa. Its traits include multiple-
year seed dormancy, bee pollination, and sexual compatibility with weed
species that could be found in adjacent fields.
NEED FOR BIOCONFINEMENT
What sorts of GEOs might require confinement? Some GEOs may raise
environmental concerns, as described throughout this report. When great
harm to the environment is probable, confinement is warranted. Food safety
and food purity issues also could motivate confinement. Other reasons
could be social, ethical, political, or economic. For example, the security of
intellectual property has long been recognized as a motivation for prevent-
ing the unintended escape or theft of living biological material of value
(e.g., Ellstrand, 1989). Transgenic crops that are commercialized in the
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56 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
United States might be prohibited elsewhere, leading to the need to segregate
genetically engineered products for export. In addition, APHIS currently
requires strict confinement for GEOs that are field-released under "Notifi-
cation" and "Permit" as part of its performance standards (USDA, 1997).
New, more stringent requirements for organisms that produce products
intended for use in pharmaceutical and other industrial compounds were
released by APHIS in early 2003 (USDA, 2003).
The StarLink incident of 2000 illustrates the need for effective confine-
ment in maize (see Box 2-1). Although StarLink corn was released for
animal consumption only, it rapidly entered the general maize supply and
within a year its presence was detected "in nearly one-tenth of 110,000
grain tests performed by United States federal inspectors" (Haslberger,
2001). Generally, it is thought that mixing of seed was responsible for most
of the unintended movement. However, the Cry9C protein also was detected
in other, supposedly genetically pure, non-StarLink varieties, suggesting
that cross-pollination was partly responsible for its spread (Taylor and
Tick, 2003). Presence may or may not indicate a high level of contamina-
tion. Current detection techniques are quite sensitive, capable of detecting a
single contaminating grain out of thousands in a bulk sample. It is highly
unlikely that the Cry9C protein produced by this variety poses any kind of
risk, but the fact that the gene moved so rapidly demonstrates how quickly
unintentional movement can occur. In any case, regulatory agencies have
seen that granting approval to genetically engineered plants that are intended
solely for animal consumption is inadvisable if other varieties are used in
human food production.
PREDICTING THE CONSEQUENCES OF FAILURE
Failure of bioconfinement presents several challenges. The most obvious
apply to the unintentional escape of transgenes. The specific problem will
be the risk that is intended to be averted by the bioconfinement method.
Thus, the problem could be environmental; for example, the creation of a
new or more difficult-to-control pest that is developed because of the intro-
duction of a gene that makes an organism more invasive. There also could
be effects on human or animal health. A gene introduced into a crop for the
production of industrial chemicals might inadvertently move into crops
intended for human consumption. The problem could be economic. A pat-
ented transgene, considered intellectual property, can be stolen. The prob-
lem could be the serious decline, displacement, or extinction of a species
with social or cultural significance. One could quantify specific risk as the
likelihood of failure and the magnitude of escape. Other problems are subtler.
The bioconfinement phenotype itself could cause havoc in organisms
for which it is not intended, as in the following example. Because of the
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automatic and substantial fitness advantage of asexual genotypes relative to
sexual genotypes due to the evolutionary "cost of sex" (e.g., Charlesworth,
1989; Williams, 1975), the application of seed apomixis--an often-touted
method of bioconfinement--could result in the rapid spread of an asexual
genotype through wild, sexually reproducing populations (see Chapter 3
for a detailed discussion). The consequence could be a drastic reduction in
genetic diversity and in the potential for evolutionary response (van Dijk
and van Damme, 2000).
The failure itself could result in altered public perception of biosafety,
decreasing the credibility of the biotechnology industry or government regu-
lators, or both. The StarLink incident led to the realization that current
systems do not provide for the segregation of genetic material. A flurry of
attention from the popular media about a bioconfinement method that fails
could result in similar public mistrust for methods that are designed to keep
transgenes in their place, leading to a loss of public confidence in the food
supply and damage to the viability of the biotechnology industry as a
whole.
Clearly, the failure of GEO bioconfinement can, in some circumstances,
result in substantial consequences. What factors must be considered to
predict some of the risks that could result from the failure of bioconfinement?
The consequences of bioconfinement failure must be assessed on a case-
by-case basis. A science-based risk analysis of the consequences of bio-
confinement failure should consider at least the following factors: the organ-
ism involved; the trait or traits that have been introduced into the organism;
the genomic, physical, and biotic environments in which the failure could
occur and that could experience the effects of a failure; the possible effects
on human health; the bioconfinement techniques involved; and the social
and behavioral factors that could affect consequences.
The most important factors in determining environmental consequences
could be the ecological phenotype and ecological novelty of the GEO. For
example, mud loach (Misgurnus mizolepis) (Chinese weatherfish) is an
aquacultural species native to China and Korea. It has been genetically
engineered such that no extraneous DNA was used (by inserting a gene and
a promoter that originated from the same species--the mud loach itself;
Nam et al., 2001a, 2001b). Hatchlings showed dramatically accelerated
growth--at a maximum, 35-fold faster than non-genetically-engineered sib-
lings. The largest hatchling weighed 413 g, and, with a length of 41.5 cm,
exceeded the size of 12-year-old normal broodstock (89 g, 28 cm). The time
required to attain marketable size (10 g) was 3050 days after fertilization;
nonengineered fish require at least 6 months. There also was significantly
improved feed-conversion efficiency, up to 1.9-fold. There appeared to be
no gross abnormalities other than the size increase, but most individuals
died after their body weight exceeded 400 g. Thus, despite the fact that the
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58 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
transgenic organism has no exogenous genes, it has a novel phenotype that
is obviously (ecologically) quite different from that of the original (Nam et
al., 2001a, 2001b). The degree of transgenic novelty (the number of geneti-
cally engineered changes) and the taxonomic or phylogenetic distance between
the host organism and the novel genes are not likely to determine conse-
quences (NRC, 2002b). This is also consistent with a previous NRC report
where it was noted that both small and large genetic changes can have
significant environmental consequences and that the consequences of biotic
novelty are strongly influenced by the "genomic environment, physical
environment, and biotic environment" (NRC, 2002a).
The significance of undefined consequences depends in part on social
values and the context in which the failure occurs. Potential loss of cultur-
ally symbolic varieties or species is often a focus for social action because
they represent social or spiritual values. For example, a failure leading to
the decline of a species will have greater significance if that species has high
symbolic importance in that location (sugar maples in New Hampshire,
blue crabs in the Chesapeake Bay, corn to the Hopi Tribe, wild rice (Zizania)
to the Ojibwa and Menomonii, or salmon to sport and commercial fishing
and to Native Americans in the Pacific Northwest) than if it has economic
or ecological importance alone. In addition, the decline of a symbolic species
is a likely indicator of environmental harm.
WHO DECIDES?
Decisions about bioconfinement involve asking whether bioconfinement
measures should be applied in a given case and, if so, which measures
should be adopted and whether they should be applied alone or in conjunc-
tion with other types of confinement. The decision makers come from the
genetic engineering industry, including the GEO developers (including aca-
demic and government scientists), related industries (such as the wholesale
and retail food industries) that could be affected by an escape or by any
resulting loss of public confidence; insurance companies; government regu-
lators; and private citizens who might sue to enforce environmental laws.
Decisions about private legal action arising from damage caused by GEOs
might indirectly affect whether bioconfinement or other confinement mea-
sures are undertaken.
Industry
Bioconfinement should be considered early in the development of a
GEO. Whether public or private, for-profit or nonprofit, the enterprise that
develops a GEO is in the best position to determine the advisability and
viability of bioconfinement because that person or group uniquely possesses
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 59
the information necessary to conduct such analyses. The enterprise has an
interest in achieving regulatory approval for field testing and marketing so
that it can avoid negative publicity and to protect itself from liability. The
industry has an interest in bioconfinement because the escape of one or a
few GEOs could jeopardize the viability of the entire industry. The com-
mittee is not aware of any industrywide standards (binding or nonbinding)
for bioconfinement.
Related industries that use or depend on GEOs, such as the wholesale
and retail food industries, also have an interest in maintaining the safety of
their products and the public confidence in that safety. Thus, they have an
interest in confinement and, by extension, in bioconfinement. The U.S. food
industry, for example, supports strong regulations that would ensure the
segregation of pharmaceutical crops from those that enter the U.S. food
supply. After the Prodigene incident, the Grocery Manufacturers of America
met with senior USDA officials and congressional staff members to call for
stricter regulation of pharmaceutical crops (Fox, 2003).
Insurance Companies
To the extent that GEO developers can obtain insurance against the
possibility of escape or failure of bioconfinement, the risk of loss shifts to
the insurer, who then would have an interest in adequate confinement,
including bioconfinement. Apparently, insurance is available for genetic
engineering under liability insurance policies, and only a few markets con-
tain specific coverage or exclusions of genetic engineering applications
(Epprecht, 1998). There is scant experience with losses involving GEOs. At
the same time, societal views about the acceptability and value of GEOs--
and even of particular bioconfinement methods (such as the use of termina-
tor genes)--are in flux and can influence the insurance risk (e.g., with
respect to product liability). Furthermore, the risk associated with some
escapes could be extraordinarily high. It thus would seem that the insurance
industry operates in a sea of uncertainty. The committee is unaware of any
confinement or bioconfinement requirements imposed by insurance compa-
nies individually or collectively.
Government
In the United States, GEOs and associated bioconfinement measures
are regulated by a mosaic of laws and agencies. The Coordinated Frame-
work for the Regulation of Biotechnology Products was adopted by federal
agencies in 1986 (51 Fed. Reg. 23302 [June 26, 1986]) in response to
concerns about how best to provide federal oversight for products of bio-
technology. Where those laws apply, they take precedence over the private
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60 BIOCONFINEMENT OF GENETICALLY ENGINEERED ORGANISMS
decisions described above, with the exception of decisions concerning pri-
vate lawsuits for damages.
The Coordinated Framework is based on two premises. The first, which
is consistent with the judgment of several reports (NRC, 1989a,b, 2000,
2002a), is that the potential risks associated with GEOs fall into the same
general categories as those established for traditionally bred organisms (but
see NRC, 2002b, for another judgment). The second is that the statutes
written for non-GEOs should provide an adequate basis for regulating
GEOs.
The coordinated framework is intended to provide a harmonized regu-
latory approach and to ensure the safety of biotechnology research and
products by using existing statutory authority and building on agency expe-
rience with agricultural, pharmaceutical, and other products developed
through traditional techniques of genetic modification. The development of
the framework anticipated that agencies might need to develop specific
regulations or guidelines under existing statutory authority. It also antici-
pated institutional evolution in accord with experience, including modifica-
tions made through administrative or legislative action. Finally, the coordi-
nated framework specifies that interagency coordination mechanisms are
necessary to address all manner of policy and scientific questions (Council
on Environmental Quality and the Office of Science and Technology Policy,
2001).
The regulatory approach articulated by the coordinated framework
invokes many statutes, as well as their implementing regulations and guide-
lines that could apply to GEOs. Some apply to specific products or activities
and are administered by a single agency; others apply generally and are thus
of interest to virtually all agencies. The committee finds that the complexity
of federal oversight could hamper the effectiveness of bioconfinement imple-
mentation, as discussed below.
Determining which law applies to a GEO under the coordinated frame-
work can entail a complicated analysis, involving such factors as the stage
of development (Is the GEO contained in the laboratory? Is it being field
tested? Is it ready for commercial use?), its uses (Is it intended for bio-
remediation of pollution or for biocontrol of another organism? Is it intended
to be a human food or drug or an animal biologic? Might it eventually be
used as food even though that is not its current use?), the type of possible
hazards (Could it harm plants? Could its genetic material cause a plant to
become a noxious weed? Could it release pollutants into the atmosphere or
water?), the type of organism (Is it an animal, plant, or microorganism?),
and whether regulatory agencies have reached consensus on how the GEO
should be regulated.
Depending on that analysis, many laws and several agencies could be
involved in regulating a particular GEO or bioconfinement method, and
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WHEN AND WHY TO CONSIDER BIOCONFINEMENT 61
several other statutes that are not currently used to regulate GEOs could be
brought to bear. Some of them are now applied to invasive species, and the
experience with and laws that regulate nonindigenous species might be
helpful in regulating GEOs (Council on Environmental Quality and the
Office of Science and Technology Policy, 2001).
Federal statutes and guidelines embody different approaches and con-
tain different authorities, standards, and enforcement provisions. Other
federal agencies, including the Office of Management and Budget, for
example, affect the way regulatory agencies interpret and apply the statutes
for which they are responsible. Congress also weighs in by controlling
funds. Congress prohibited US EPA from expending any money to enforce
a regulation issued under the Clean Air Act. Thus, each agency exercises
regulatory authority differently. To the extent that laws regulate confine-
ment and bioconfinement, they trump the decisions of the private parties
discussed above.
Several agencies impose specific confinement requirements. APHIS, for
example, requires physical confinement where crops genetically engineered
to produce industrial chemicals or pharmaceuticals are field tested. The
confinement requirements include a 50-ft perimeter fallow zone around the
field test site; restriction on the production of food and feed crops in the
field test site and in the fallow zone the next season if volunteer plants could
be inadvertently harvested with that season's crop; the use of dedication of
mechanical planters and harvesters for the duration of the test, and cleaning
of that equipment in accordance with protocols for tractors and tillage
attachments; the use of dedicated facilities for storing equipment and the
regulated GEO; and, for field tests of open-pollinated pharmaceutical corn,
a prohibition against growing any other corn one mile of the field test site
for the duration of the field test (Federal Register, 2003).
Another example is that the US EPA sometimes requires refuges or
buffer zones for certain genetically engineered plants (Bt corn and cotton)
(US EPA, 2002; Elias, 2002). In 2002, the US EPA filed complaints against
two companies for noncompliance with agency requirements not to grow
experimental GE corn too close to other crops and to use trees and other
corn to form a windblock next to the GE crops. The complaints were
settled through legal agreements between EPA and both companies, with
each company agreeing to pay a fine (Elias, 2002). Additionally, EPA fined
one of those companies a second time, for failing to notify the agency of test
results indicating the presence of the experimental gene in seeds grown near
the experimental plants, and for failing to submit maps identifying the
location of such seeds (EPA, 2003b).
The Food and Drug Administration (FDA) may require confinement
(including bioconfinement) of transgenic animals under the terms of the
Federal Food, Drug, and Cosmetics Act (FFDCA), 21 U.S.C. 321-397. The
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U.S. Fish and Wildlife Service and the National Marine Fisheries Service are
permitted by the Endangered Species Act (16 U.S.C. 1531-1544) to require
bioconfinement for protecting a listed species.
Laws concerning confidentiality of information also affect how GEOs
and their confinement are regulated by the federal government. Trade secrets
and confidential commercial informationoften called confidential busi-
ness information (CBI)are protected under the statutes that are used to
regulate GEOs. The CBI provisions in those statutes differ considerably, as
do the regulations issued thereunder and the procedures used by the agencies
involved. For example, FFDCA prohibits FDA from sharing CBI with any
other federal agency, even if that agency is engaged in evaluating or regulat-
ing the same GEO. In contrast, other statutes used to regulate GEOs do not
contain any such prohibition. Each agency administers its own program for
protecting CBI on a statute-by-statute basis. Thus, US EPA has two separate
CBI programs, and authorization for access to CBI under one does not
allow access under the other. The result is that a scientist involved in
regulating a GEO under one statute cannot share CBI, including confine-
ment-related CBI, with a scientist involved in regulating the same GEO
under another statute unless each is qualified to know CBI under both
statutes. Similarly, each agency applies different amounts of scrutiny to
assertions by business entities about what constitutes CBI. CBI is not
available to the public (for examples of the use of CBI, see Chapter 3,
Tables 3-2 and 3-3).
The regulatory system described above is obviously complex, leading to
differing legal authorities and responsibilities, potentially overlapping juris-
dictions, and differing statutory standards for regulating GEOs and deter-
mining confinement (including bioconfinement). This could lead to a host
of problems in regulating confinement. For example, US EPA has no inde-
pendent authority to require information about where a GEO granted
nonregulated status by USDA is grown, which impedes US EPA's ability to
monitor confinement requirements for that GEO. US EPA does not have
the regulatory authority to enforce the confinement requirements built into
the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) in any
event (Bratspies, 2002). US EPA may set regulatory thresholds for GEOs,
but FDA has no such authority (Taylor and Tick, 2003).
Coordination is needed, but there are no central mechanisms to ensure
consistency in confinement, including bioconfinement, or to ensure coherence
in setting priorities (Bratspies, 2002). The splintered approach to protecting
CBI further interferes with agencies' ability to take a coordinated approach
to GEO confinement.
Indeed, a mismatch exists between the application of existing laws and
the actual practice of regulating GEOs. Because the statutes initially were
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enacted for radically different purposes, there is no unifying vision of or
approach to the questions and challenges posed by GEOs and their confine-
ment and there is no concordance with the coordinated framework's evident
goal of smoothing the path for genetic engineering technology (Bratspies,
2002). The approaches normally used in carrying out those statutes do not
always fit GEO confinement. For example, US EPA typically applies split
registration for traditional chemical pesticides by requiring labeled con-
tainers with a clear indication of registered use. This practice cannot be
extended to Bt crops, for example, because they do not stay in neatly
labeled bottles, and the postharvest distribution, storage, and processing of
the corn do not parallel the use of a typical pesticide.
Citizen Suits to Enforce Environmental Laws
Private citizens have a role in enforcing relevant legal authority: Some
statutes allow them to bring action to enforce environmental laws, either to
direct the agency involved to enforce a law or to seek a civil penalty (which,
if recovered, would go to the government). Citizens can sue to enforce the
National Environmental Policy Act and the Endangered Species Act as they
apply to the bioconfinement of GEOs. Citizen suits seeking to compel
government agencies to enforce the law are permitted by other environ-
mental statutes including the Clean Water Act (sections 304 & 305, 42
U.S.C. sec. 7604, 33 U.S.C. 1365), and cases frequently are brought under
such provisions. Of the statutes used to regulate GEOs, only the Toxic
Substances Control Act (TSCA) contains such a provision (U.S.C. sec.
2619). In a related type of action, however, it is possible to petition USDA,
FDA, and US EPA, respectively, under the terms of the Plant Protection Act,
FFDCA, and the FIFRA regarding some GEO-related activities and to chal-
lenge the agencies' decisions regarding those petitions in court under the
judicial review provisions of those statutes.
Private Action for Damage
An indirect decision maker about bioconfinement is found in private
causes of action for compensation for damage that results from escape,
which typically would be subject to state law. One example with respect to
nonbiological confinement is a class action lawsuit brought by farmers who
claimed they were harmed when StarLink corn was discovered in the human
food supply in 2000. The biotechnology companies that created and dis-
tributed StarLink agreed to pay $110 million to settle the case (Pesticide
and Toxic Chemical News, 2003). Private legal action also can be used to
enforce or invalidate patent rights, including in the context of a failure of
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confinement (Monsanto Canada Inc. & Monsanto Co. v. Percy Schmeiser
& Schmeiser Enterprises Ltd., 2001 FCT 256, confirmed in Percy Schmeiser
& Schmeiser Enterprises Ltd. v. Monsanto Canada Inc. & Monsanto Co.,
2002 FCA 309, on appeal to the Canadian Supreme Court as of May
2003). Such law is federal, thus implicating lawmakers at the federal level.
Representative terms from entire chapter:
engineered organisms
