July 1, 1996
EP A/600/A-96/096
Assessing Risks from GMOs to Ecosystems and Human Health
R.J. Seidleri*, L.S. Watrudi, and S.E. Georges
1	U.S. Environmental Protection Agency,
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Western Ecology Division
200 S.W. 35th Street, Corvallis, OR. 97330 (USA)
2	U.S. Environmental Protection Agency,
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Environmental Carcinogenesis Division
Mail Drop 68
Research Triangle Park, N.C. 27711 (USA)
Corresponding author
(541) 754-4708, FAX (541) 754-4799, E-Mail seidler@mail.cor.epa.gov
Key Words: genetically engineered organism, risk assessment, health
effects, environmental effects
This document has »been subjected to the Agency's peer and administrative
review, and It has been approved for publication as an EPA document. Mention
of trade names or commercial products does not constitute endorsement or
recommendation for use.

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"Assessing Risks from GMOs to Ecosystems and Human
Health"
I. Introduction
Since the 1970s, scientists have been altering directly significantly the
genetic makeup of living creatures (Tzotzos, 1995). Techniques in
molecular biology have made it possible to incorporate genes from one
organism into virtually any other organism's genetic composition to
create a broad array of unique life forms. These unique organisms may
have traditional or totally new uses for applications ranging from
agriculture, food and beverage and pharmaceutical production, to
environmental clean up and energy production. Through this technology
industry anticipates promises of unique economic opportunities from
patented "new" life forms. These rapid technological developments in
molecular biotechnology are forcing society to think about new concepts
in biology and to contemplate the potential effects that recombinant DNA
technology can have on ecosystems and on human health (Levin, et al.,
1987; Levin and Strauss, 1990; Regal, 1990). Questions about proprietary
rights to novel germplasm, cloned genes, and the patenting of "new" life
forms have also been raised.
9
Genetically modified organisms (GMOs) have already been released
experimentally into the environment. Essentially these releases have been
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small scale tests, designed to primarily examine product efficacy, not
usually to evaluate potential environmental risks. Some negative
environmental perturbations have been noted in some studies where risk
assessment has been an experimental objective. However, since the
reported effects were limited in scale and communicated largely through
technical channels, few adverse public reactions to these results have
been noted (Leopold, 1995).
It has been over 20 years since the first recombinant microorganism was
constructed and probably half as many years since they were suggested
for use in the environment. There have already been more than 1,000
applications to allow the testing of genetically engineered organisms,
most of which have or will be released in the U. S. (Mellon and Rissler,
1993). Once released, interactions with biotic and abiotic factors can
disperse microbial agents over considerable distances(Seidler and Hern,
1988; Lighthart and Kim, 1989; Seidler, et al., 1994) and a desire for
properly conducted risk assessments is supported by most who are
involved with this technology .
The widespread environmental experimentation with GMOs raises
important questions. Can the risks of GMOs be assessed without actually
releasing the agents directly into the natural environment? Can the
impacts be anticipated and is knowledge of ecology sufficient to predict
with confidence th£ fate and survival of a microbial agent and how it
might interact with ecosystems and humans? Concern has been expressed
by numerous scientists regarding the release of GMOs before such risks
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are understood (Halvorsen, Pramer, and Rogul, 1985; Levin and Strauss,
1990; Sharpies, 1990). Clearly, the possible risks that GMOs may pose to
the natural environment must be estimated and assessed before decisions
are made as to their release.
In order to ensure public health and environmental safety, the United
States Government mandated that release of genetically engineered
microorganisms be regulated by Federal Register statutes (1986). The US
Environmental Protection Agency regulates microbial pesticides and
microbial "chemicals" through Subdivision M (Microbial Pesticides) of the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and the Toxic
Substances Control Act (TSCA). In the European community, release of
genetically engineered microorganisms and the microbial plant protection
products are regulated through Directive 90/220/EEC and Directive
91/414/EEC respectively. Under both of these Directives, risk
assessments of health and environmental effects of the products are
determined.
In response to the diversity of product opportunities as well as risk
assessment concerns raised through many organizations and individuals
around the world, a massive R&D effort is underway in Europe to address
risk assessment issues. Designated the "biotechnology programme", the
research is funded through the European Commission and designated as
part of the fourth European Community research and technological
«
development framework program for 1994-1998. Currently there are
about 528 laboratories from every Union country sharing a budget of ECU
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73 million. Further details of this program can be accessed through the
internet via the URL posting;
http://www.cec.Iu/en/comm/dg12/networks.html.
The bases for general risk assessment of GMOs are understandably
different from those associated with chemicals. GMOs are living
organisms and therefore, unlike chemicals that may become diluted, GMOs
have the potential to disperse to new habitats, colonize those sites, and
multiply. Their novel activities including the production of metabolic
products, enzymes, and toxins, will occur as long as the GMOs remain
metabolically active. Once established, living organisms cannot be
recalled. The last twelve years has seen a new era of environmental
research devoted in part to the risk assessment issues associated with
the environmental release of GMOs (Bourquin and Seidler, 1986; Levin, et
at., 1987). This research seemed justified considering the anticipated
widespread development and application of GMOs and their multitude of
capabilities.
An understanding of basic ecological and evolutionary concepts as to why
organisms may survive or flourish in natural ecosystems have changed
comp?red to only 15-20 years ago (Regal, 1990). Many of the risk
assessment decisions on safety issues will depend upon whether and how
one weighs new concepts within ecology and evolution relative to older
theories. For example, conventional wisdom has it that if one engineered
in a new trait, selection by the environment would return the organisms to
their original state. Exotic species introductions mostly fail to establish
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in nature because the new conditions are not conducive to their survival
(Williamson, 1994). However, the engineering of an already established
host begins with an ecological advantage. Genetic engineering may also
enhance that competitive advantage. When exotic species go out of
control it is not only because they have escaped natural constraints, but
because they sometimes find new ecological opportunities (Regal, 1990).
A thorough discussion of the ecological concepts involved in biological
introductions excluding GMOs along with reasons for their success or lack
of success in establishment is offered in this Handbook in the chapter by
Simberloff and Alexander. Most of the concepts discussed in that chapter
are probably directly applicable to GMOs as well.
The purposes of this chapter are to describe and characterize primarily
genetically engineered microorganisms and the relevant tools used for
their risk assessment, provide examples of observed and possible
environmental effects induced by GMOs, summarize methods available for
detecting effects (both human and environmental) , and provide
discussions on possible hazards associated with their repeated, large
scale environmental releases that is anticipated in the near future. Many
unknowns still exist about the environmental and human health
significance of r«sks from GMOs despite approximately 11 years of
research. However, much has also been learned about how to
experimentally detect possible risks and the nature of those risks have
now been documented; both subjects are topics to be discussed in this
chapter of the Handbook. The sections below will (a) illustrate specific
examples of genetically engineered microorganisms; (b) define the risk
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associated issues; (c) report known ecological and health effects of GMOs;
and (d) highlight continuing and future research needs for the use of GMOs.
II. Examples of GMO products
IIA. Bacteria
With the advent of modern molecular methods, the identification,
isolation and movement of genes from one organism to another, even
between species or taxa which may traditionally have breeding barriers,
has been greatly facilitated. Consequently, the variety of microbes that
can produce novel products or have novel and useful activities is
increasing dramatically. The scope of microbial activities being developed
ranges from crop protection and environmental restoration, to food,
beverage, pharmaceutical and specialty product production (Harlander
1992; Edgington 1994; Valigra 1994; Watrud & Seidler 1996). The
examples given below will illustrate a variety of specific applications for
genetically modified bacteria developed for either environmental or
contained applications.
IIA1. Crop protection (pest control for Insects, diseases)
9
Intentional selection and development of bacteria intended for large scale
environmental release have primarily been made for agricultural purposes,
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particularly for insect control. Between 1948 and 1995, twenty-five
bacterial products were registered for use in the United States as
microbial pesticides (Schneider personal communication). Eighteen of
those registrations occurred after 1992. In part, it has been this
increased interest in microbials for pest control, that has led to the
recent creation of a Division of Biological Pesticides within the United
States Environmental Protection Agency. The most widely used bacterial
species for biological control of insect pests is Bacillus thuririgiensis
subsp. kurstaki (B. t. kurstaki), which has a high degree of specificity for
lepidopteran insects. The active pesticidal ingredients in B. t. kurstaki
and in the related species B. t. subsp. tenebrionis, which has a high degree
of activity against certain coleopteran insect pests, are proteins
(McPherson et al. 1988; Hofte & Whiteley, 1989). Using both classical
breeding and recombinant methods, geneticists have tried to create
strains that express higher levels of one or both of the desired
insecticidal protein activities (Gawron-Burke & Baum 1991; Bosch et al.
1994). Several engineered strains of Bacillus thuringiensis have recently
been registered (Bill Schneider, personal communication). Additional
Bacillus species registered in the United States for the control of
coleopteran insect pests include B. popilliae and B. lentimorbus
(Schneider personal communication). In addition to efforts to improve
microbial formulations of Bacillus species, pesticidal genes isolated from
Bacillus species have been or are being cloned into a number of plant
species, including giajor food and fiber crops such as corn, cotton,
potatoes and woody species (reviewed in Watrud et al. 1996). A concern
resulting from the anticipated millions of acres to be planted in crops
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expressing B. t. proteins, is an increase in the rate of development of
resistance in insect pests (Tabashnik 1994). On the human health side, a
potential concern is whether allergenic reactions may develop in people
exposed to higher levels of the insecticidal proteins, due to increased
environmental and dietary exposures. Non-target effects of microbial
forms of B. t. kurstaki on insects and on microbial biota in soil have
recently been reviewed (Watrud & Seidler, 1996).
Bacillus species and Pseudomonas species have each been proposed and
evaluated for use as biological control agents for Rhizoctonia and
Pythium, fungi which cause damping off diseases of seedlings (Weller
1988; Whipps 1992; Lumsden et al. 1995). Bacterial treatments for
disease control have been evaluated as seed treatments, as root drenches
for transplants, and as dry or liquid soil treatments. Although short term
ecological and toxicological effects and other safety considerations of
microbial pesticides typically have been addressed (Levin 1995), studies
on long term non-target effects of bacterial pesticides have often been
limited (Watrud & Seidler, 1996).
Both fungi and bacteria pathogenic or phytotoxic to specific types of
plants have been proposed for weed control. Dacterial genera reported to
have herbicidal activity include isolates of Streptomyces, Bacillus and
Pseudomonas (Charudattan 1990; Hoagland1990; Stonard & Miller-
Wideman 1995).' ¥Ks with the fungal biocontrol agents which are
described below, a major concern of using biocontrol agents that are
themselves pathogens, is their actual degree of host specificity; i. e., will
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non-target crop or native species also be affected? Increases in the
competitiveness of existing plant pathogens due to the acquisition of
traits such as tolerance to pesticides, heavy metals or temperature
extremes, by genetic recombination, are also of potential concern.
IIA2. Plant growth promoting biofertilizers /rhizobacteria
Use of commercial inocula of nitrogen fixing strains of Rhizobium or
Bradyrhizobium for seed treatment of legumes has become a standard
agronomic practice. Attempts have also been made to boost the nitrogen
fixing capacity of the inocula by the use of recombinant methods (Paau
1991; Hall 1995). For graminaceous species such as barley and wheat,
non-symbiotic nitrogen fixers (Azospirillum lipoferum, A. brasiliensis, A.
chroococcum), have been tested in greenhouses and in the field for a
number of years, often yielding positive effects on plant growth and yield,
particularly in warmer climates (Dobereiner et al. 1988; Bhattari & Hess,
1993; Di Ciocco & Rodriquez-Caceres 1994; Zaady & Perevolotsky 1994).
Effects of environmental conditions on efficacy of inocula have also been
studied; however, ecological studies on non-target effects of
Azospirillum inocula have not been reported.
Several bacterial preparations (P. cepacia, P. flourescens, E$. subtilis),
have been examined as seed treatments which may permit or enhance plant
growth by inhibiting damping off fungi (Weller 1988; Whipps 1992;
Lumsden et at. 1995). The distinction between biological control agents
used for controlling soil-borne plant diseases and plant growth promoting
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rhizobacteria, may be largely a semantic one. To date, there are no
registrations in the United States under TSCA, for plant growth promoting
bacteria per se. However, approximately one fourth of the bacteria
registered as microbial pesticides in the U. S. have plant diseases as their
targeted pests.
IIA3. Biomass conversion agents
In addition to starch or sugar crops such as corn and sugarcane,
lignocellulosic wastes also can be used as substrates for the production
of ethanol by fermentation. Pre-treated mechanically and chemically, e.g.,
by milling and acid or alkaline hydrolysis, complex wastes then can be
subjected to fermentation. Traditional sources of lignocellulosic biomass
are the by-products of logging, wood and paper processing operations.
Short rotation woody or herbaceous species such as poplars and
switchgrass and agricultural residues including corn stover, seed hulls,
straws and peels are also potential commercial sources of lignocellulose.
Several species of engineered bacteria (Klebsiella planticola, Klebsiella
oxytoca, Zymomonas mobilis and Streptomyces lividans), have each been
considered for production of ethanol from agricultural residues or for
degradation of lignocellulosic residues from paper milling operations
(Trotter 1990; Helsot 1990; McCarthy & Williams, 1992; Wood & Ingram,
1992; Crawford et al. 1993; Sprenger 1993). Anaerobic digestion of
lignocellulosic and municipal wastes also has been evaluated as a means
of producing energy in the form of methane (Wyman & Goodman 1993). The
feasibility of producing lipids as a result of metabolism of sugars
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produced by photosynthetic activities of marine micro-algae such as
diatoms also has been proposed (De La Noue & De Pauw 1988; Wyman &
Goodman 1993). Specialty chemical production (e .g., for solvents such as
acetone), by engineered isolates of Clostridium acetobutylicum, also has
been studied (Mermelstein et al. 1993).
IIA4. Bioremediation and Mining Applications
A number of isolates of Pseudomonas species (P. putida, Burkholderia
cepacia) have been studied biochemically, genetically and to a lesser
degree ecologically, for their potential use and non-target effects in the
remediation of polluted soils, sediments and surface and ground waters
(Mulbry & Kearney 1991; Fan & Scow, 1993; Krumme et al. 1994;
Edgington 1994; Ramos et al. 1994). Laboratory selection and genetic
engineering for customized degradation of given aromatic compounds by
individual isolates has been actively pursued for a number of years
(Singleton 1994; Daubaras et al. 1995). However, intentional
environmental applications of engineered organisms for bioremediation
purposes have been few, limited in part by environmental and public
opinion concerns. Those issues, along with cost considerations, often
have led to management of both marine and terrestrial spills by physical
or chemical methods which enhance desired indigenous microbial
activities. These methods include the use of absorbents (straw or
*
sawdust or biopolymers), agitation to increase aeration and oxidation, pH
modification, and addition of fertilizers (organic and inorganic) and
organic substrates such as molasses or starch (King et al. 1992; Rouchard
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et al. 1993). Less frequently, non-engineered inocula have been added to
achieve the desired degradation and bio-transformations. In 1990, a
polyaromatic hydrocarbon degrading strain of Pseudomonas fluorescens,
HK44, containing an engineered bioluminescent reporter plasmid for
napthalene catabolism (King, et a!., 1990) was approved for field testing
in the U.S., under TSCA (Phil Sayre, personal communication). To date,
however, engineered inocula have been used primarily In contained
laboratory facilities or microcosms, or in bioreactors that have been
brought to contaminated field sites or to centralized treatment centers.
Recently, progress has been reported in the development of transformation
systems for Thiobacillus and Leptospirillum, genera which may prove to
be useful in management of leachates from mining operations (Rawlings &
Silver 1995). Other proposed applications for bacteria in mining include
the bioconcentration of valuable metals and for the detoxification of
waste streams by the adsorption or complexation of heavy metals by
microorganisms (Francis 1990; Wales & Sagar 1990; Sahoo et al. 1992;
Volesky & Holan 1995). Potential roles for using microbially based
processes to desulfurize coal with isolates of Thiobacillus,
Leptospirillum or Rhodococcus have also been proposed (Merretig et al.
1989; Kilband & Jackowski 1992; Mannivannan et al. 1994).
HAS. Food biotechnology /Specialty products
I
Historically, the longest term de facto or intentional applications of
bacterial activities by humans have been the use of fermented foods.
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Traditional and ethnic foods such as sausages and sauerkraut, soy sauce
and miso, salami and pepperoni, yogurt, cheese, and buttermilk, have a
long history of consumption world-wide. In addition to creating
characteristic flavors, aromas or textures, the microbial activities often
have enhanced the storage life of the meats, milk, fruit and grains which
have served as the substrates for the bacterial activities. Dairy products
produced as a result of controlled fermentations with known bacterial
inocula include yogurt, buttermilk, sour cream, and Swiss cheese
(Harlander 1992). As the sciences of microbiology, biochemistry and
genetics have advanced, the ability to select, improve and create strains
with desired activities has also increased. Specific examples of the
applications of biotechnology to the dairy industry include strain
improvement and development of DNA based diagnostics for both inocula
and contaminants in foods (Ramos and Harlander 1990; Mc Intyre &
Harlander 1993). Additional useful products resulting from bacterial
fermentations include organic acids such as citric acid which can be used
as a food preservative or flavoring agent, vitamins, and amino acids such
as lysine and methionine, which are used as food or feed supplements.
Organic acids and amino acids produced as a result of fermentations also
may serve as substrates for subsequent modifications, e. g., the chemical
combination of microbially produced phenylalanine with aspartic acid, to
produce the sweetener, aspartame (De Boer & Dijkuizen, 1994). Enzymes
used in food processing or detergent industries (i. e., proteases,
invertases, lipases fand amylases), also may be derived from bacterial
fermentations (Harlander 1992). In addition to the production of
fermented foods, bacterial activities have also been utilized to produce
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food ingredients ranging from thickeners, sugars, and vitamins, to
pigments, flavors, and amino acids (Harlander 1992; Gableman 1994).
Enzymes derived from bacteria (e. g., amylase, glucose isomerase,
pullulanase), are used in food and beverage processing for starch
liquefaction, high fructose corn syrup production and in beer production.
Biotechnological methods also are being used to select and modify strains
of Lactobacillus to improve the digestibility and preservation of silage
(Flores 1991; Wallace 1994).
Most examples given above have been taken from processes that generally
have been carried out indoors, in vats or fermentors. As the use of
recombinant strains increases, some concerns may arise as to the health
or environmental effects of those strains, or novel products produced by
them, in the event of spills or leaks. Potential adverse effects include
short term fish kills, resulting from lowered oxygen levels in
contaminated lakes or streams. Additional concerns include health risks
to susceptible individuals who may be immunocompromised or allergic to
recombinant proteins expressed in the released microbes.
IIA6. Pharmaceuticals
Many of the antibiotics in common use today are fermentation products or
synthetic derivatives of bacterial metabolites produced during closely
controlled fermentations. Prominent among the medically useful bacterial
species that have served as sources of numerous commercial antibiotics
are the genera Streptomyces, Actinomyces, and Bacillus. The well known
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antibiotics, streptomycin, actinomycin and bacitracin are produced
respectively by Streptomyces, Actinomyces and Bacillus (Glasby, 1992).
As techniques for transforming key antibiotic producing species of
microbes have become available, cloning of genes controlling metabolic
pathways involved in antibiotic synthesis have permitted the development
of strains which may serve as the source of new, novel and useful
antibiotics (Malpartida & Hopwood 1992; Hopwood 1993; McDaniei et al.
1993; Valigra 1994; Bedford et al. 1995; Kakinuma et al. 1995). The
availability of technologies to produce new types of antibiotic compounds
is becoming increasingly important as new antibiotic resistant strains of
human and veterinary pathogens continue to develop and spread in clinical
and outdoor environments. In addition to production of novel bacterial
compounds in bacterial hosts well suited for growth in fermentors,
recombinant technologies are being applied to develop microbially derived
vectors to optimize production of mammalian compounds in bacteria,
bacterial compounds in plant cell cultures, and monoclonal antibodies in
animal cell lines. Recombinant pharmaceuticals used for human and
veterinary growth enhancement, metabolic regulation or for therapeutic
purposes, include human and animal (bovine and porcine) growth hormones,
insulin, inteferons, blood clotting factors, tissue plasminogen activator,
and vaccines (Thayer 1992, 1994; Ladisch & Kohlman, 1992; Borman 1994;
Munn 1994). One concern of biotechnology applications for production of
pharmaceuticals is allergenicity of susceptible individuals to recombinant
proteins. Environmental concerns resulting from pharmaceutical
applications of biotechnology are limited primarily to events resulting
from spills or leaks, which might result in fish kills or other non-target
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effects.
IIB. Fungi
IIB1. Crop protection
Twelve of the forty-five microbial pest control agents registered in the
United States are fungi. The agents include Beauveria and Metarrhizium,
used for the control of insects, Colletotrichum, used to control the weed
joint vetch, in rice, Cronartium, used to control water hyacinths, and
Puccinia, used for control of nutsedge. Several fungi (Trichoderma and
Gliocladium), are registered for the control of control of the plant
pathogenic fungi Pythium and Rhizoctonia., which cause damping off
diseases of seedlings. In France and Italy, hypovirulent strains of the
causal organism of chestnut blight, En doth ia (Hyponectria) parasitica,
have been successfully used in the field, to treat the disease (MacDonald &
Fulbright, 1991; Nuss 1992). Some of the major ecological concerns
which arise with the increasing use of biological agents include non-
target effects of the released agents on beneficial organisms (James and
Lighthart 1994; Watrud & Seidler 1996), and the potential for increased
host range or competitiveness, resulting from genetic recombination
between released and indigenous strains (Cisaref al. 1994). Health
concerns center largely on potential allergenicity of the inocula to
sensitive individuals.
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IIB2. Mycorrhizae
Each of the major classes of mycorrhizai fungi can enhance inorganic
nutrient uptake in host plants and help alleviate the effects of stressors
ranging from drought, disease organisms, and heavy metals to agricultural
chemicals (Gildon & Tinker, 1983; Heggo & Angle, 1990; Hetrick et al.
1994). Among the three major classes of mycorrhizae, /. e., the
ectomycorrhizal fungi, the ericoid mycorrhizai fungi, and the vesicular-
arbuscular mycorrhizai (VAM) fungi, laboratory culture has become routine
primarily only for members of the ectomycorrhizal group. Accordingly, it
is the group of ectomycorrhizal fungi which includes the basidiomycete
genera Pisolithus and Laccaria, and the ascomycete genera Tuber and
Morchella, which has received the most commercial attention in terms of
inoculum production and genetic improvement.
Basidiomycete ectomycorrhizal inocula are used to inoculate conifer
species used in restoration of areas damaged by mining and smelting
operations. Among the ericoid mycorrhizai fungi that typically form
symbiotic associations with ericaceous plants, only two genera
(Hymenoscyphus and Oidiodendrori), can be routinely cultured in the
laboratory (Linderman, personal communication). The vesicular-
arbuscular mycorrhizai (VAM) fungi, which include the phycomycetous
genera Glomus and Gigaspora, can associate with most herbaceous
species, and at some life stages, with some woody species as well.
However, these fungi evade extended axenic culture in the laboratory.
Accordingly the VAM fungi routinely are maintained by being grown in
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association with the roots of host plants such as grasses, onions or
legumes. Most biotechnological work to date with mycorrhizal fungi has
focused on the development of molecular probes to identify effective
strains, particularly of ectomycorrhizal and VAM fungi (Simon et al. 1993;
Lanfranco et al. 1995; Paolocci et al. 1995; Tommerup et al. 1995). As
axenic culture methods become improved or available for the various
types of mycorrhizal fungi, the potential to transform mycorrhizal fungi
to select or create more competitive strains is expected to increase (Hall
1995). However, given the relative lack of specificity of ectomycorrhizal
fungi among conifers and of VAM fungi among herbaceous and some
hardwood species, the ability to mitigate the spread of (undesired)
strains, which may displace or outcompete desired strains, could be
environmentally or economically problematic.
IIB3. Biomass conversion
Yeasts, especially Saccharomyces spp., are the traditional organisms of
choice for production of ethanol from fruits and grains, potatoes and
sugarcane. Other yeast species, including Yarrowia isolates, also have
been evaluated for industrial purposes, including the biotransformation of
lipids (Helsot 1990). Isolates of the filamentous fungus Trichoderma,
noted above as a biological agent for plant disease control and producer of
enzymes, also have been proposed for use in the conversion of
lignocellulosic wastes for fuel production, and for the treatment of
cotton-based textiles. This industrial interest in Trichoderma
accordingly has resulted in research on the molecular biology and safety
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of the organism (Pentiila et al. 1991; Nevalainen et al. 1994). The oyster
mushroom Pleurotous ostreatus, which is edible, also has been evaluated
as a commercial candidate to degrade ligninocellulosic wastes (Kerem &
Hadar1995). In addition to their degradative activities, bacteria and fungi
may also serve in biomass conversions, biopulping or biobleaching, or as
pollution prevention agents by minimizing accumulation of toxic
metabolites. However, the possibility of toxic metabolite production by
the biomass conversion or remediating agents also needs to be considered
in risk evaluations.
IIB4. Bioremediation
Utilization of fungal activities in bioremediation may occur by
management of indigenous inocula, or by addition of exogenous inocula.
For example, composting may effect a remediation due to activities of
indigenous flora on straw or sawdust added to polluted soils. The white
rot fungus Phanaerochaete chrysosporium, has been reported to degrade a
number or compounds, including 2, 4, 5,-trichlorophenol (2,4,5-T), the
BTEX complex (benzene, toluene, ethylbenzene and xylene), and
pentachlorophenol (PCP) (Joshi & Gold, 1993; Yadav & Reddy, 1993).
Additional filamentous fungi evaluated as polyaromatic hydrocarbon
bioremediating agents include Acremonium and Curwinghamella (Pothuluri
et al. 1995). Filamentous fungi such as Rhizopus, the common bread mold,
and Absidia, each have been proposed for use in biosorption of heavy
metals for detoxification of waste streams (Volesky & Holan, 1995). As
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transformation methods and vectors for fungal systems become more
available, increased efforts in engineering industrial strains is
anticipated. One concern associated with the environmental release of
degradative fungal strains, many of which may be plant pathogens or
saprophytes in nature, is the development of more competitive pathogens
as the result of genetic recombination with indigenous pathogens or other
compatible strains. Another concern is the potential for adverse effects
on the quality and storage life of plant-based food and fiber crops.
Allergenic reactions in sensitive individuals either to the fungi or to
recombinant proteins produced by them, also are of concern.
IIB5. Food/Beverage Industries
Whether added intentionally as pure cultures, or inadvertently as
indigenous flora on fruits and grains, historically yeasts (especially
Saccharomyces species) have been used to produce alcoholic beers, ales
and wines. In more recent times, genetic selection and improvement of
yeasts have been explored to more consistently attain desired aromas and
flavors (Rank & Xiao, 1991; Colagrande et al. 1994). They are also being
used to produce light beers, with reduced starch, sugar and calorie
contents, and to help regulate the level of ethanol in final products. The
composition of high fructose com syrups used in non-alcoholic beverages
such as carbonated sodas and juices and in products such as jams, jellies,
and pancake syrups, also can be adjusted by the use of genetically
improved strains or enzymes such as invertases, amylases, glucose
isomerases, pullanases, derived from the selected strains (Harlander
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1992).
IIB6. Human and Veterinary Pharmaceuticals
Prominent among the strains of filamentous fungi which have served as
sources of antibiotics or as models for chemical synthesis are the genera
Perticillium and Cephalosporium, known for their synthesis of the broadly
used penicillins and cephalosporins (Weil et al. 1995). Strains of the
phycomycete Rhizopus, have served as gene sources, precursors or models
for the production of steroids such as the cortisones used as anti-
inflammatory agents and in the manufacture of steroid hormones used in
birth control pills (Breskvar et al. 1991; Vidyarthi & Nagar 1994). Genetic
engineering of yeasts as hosts for the production of heterologous proteins,
has also been explored (Buckholz & Gleeson 1991).
IIC. Viruses
Seven of the forty-five microbial pest control agents currently registered
with the United States Environmental Protection Agency are viruses used
to control lepidopteran insect pests of forest and agronomic species.
Efforts to select and modify viral insecticides, include using genetic
engineering to broaden the host range of viruses (Vlak 1993). Recent
approvals for field testing in the United States for engineered microbial
«
pesticides include recombinant insecticidal baculoviruses which express
insect specific toxin genes derived from scorpions (Bill Schneider,
personal communication). With the advent of genetic engineering of higher
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plants, viral elements including the genes encoding viral coat proteins of
plant pathogens, have been cloned into a number of food crops including
tomatoes, potatoes and cucurbit species. Other viral elements such as the
cauliflower mosaic virus (CaMV35S) promoter, also have been
instrumental in obtaining or optimizing expression of diverse genes in
engineered plants (Sanders et al. 1987). Engineered plants expressing
viral coat proteins have been demonstrated to confer protection to one or
more types of viruses, particularly the types which served as the source
of the viral coat proteins (reviewed in Watrud et al. 1996). In addition to
plant protection by expression of viral genes (coat proteins, ribozymes,
viral satellite RNAs), some plants may be protected from certain viruses
by treatment with either closely related viral strains or with avirulent
strains. The concept of cross protection using intact viruses has been
used to control tristeza, an aphid transmitted virus disease of citrus
crops (Fulton 1986). Aphids exposed to avirulent strains of the virus are
used to transmit the avirulent strains to citrus trees, thereby minimizing
the effects of virulent viral strains on the citrus trees. Other crops for
which the cross protection approach of using viral inocula to protect
crops against viral diseases is being evaluated include cacao (for swollen
shoot), papaya (for ringspot), avocado (for sun blotch), and stone and pome
fruits, such as peaches and apples (Whipps 1992). Hypovirulent strains of
the fungal causal agent of chestnut blight [Cryphonectria (Endothia)
parasitica)] reported to have been successfully used to control the
disease, have consistently shown a correlation with the presence of
virus-like double stranded RNAs (MacDonald & Fulbright 1991; Nuss 1992).
An engineered hypovirulent strain of Cryphonectria received approval for
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field testing in the U.S. in 1994 (Jane Rissler, personal communication).
Mammalian pathogens including poliomyelitis, influenza and sindbai
viruses, currently are being evaluated as expression vectors in animal cell
systems, for the production of heterologous proteins (Schlesinger 1995).
The use of transgenic plants as factories to produce viral components
such as coat proteins or other antibodies, which might be used in
production of vaccines for human or veterinary applications, also has been
proposed (Hiatt et al, 1989). Environmental concerns which emanate from
the use of virus-resistant transgenic plants include the selection for
novel, more competitive viruses, potentially with broader host range
arising as the result of genetic recombination. Another concern related to
the use of transgenic virus-resistant plants, is that they may serve as
reservoirs of inoculum for viral transmission via biological or mechanical
means, to non-protected plants. Allergenicity and pathogenicity to
sensitive individuals may cause potential health concerns following
exposure to viral expression systems and expression of foreign proteins.
III. Issues to Consider in Conducting Risk Assessments of GMOs.
A document written in 1984 (Bourquin and Seidler, 1986) developed and
guided the earliest strategies to provide methods for conducting risk
assessments of GMOs released to the environment. In that report,
research plans were developed that addressed the need for increased
research in microbfal ecology in order to provide knowledge on how to
conduct GMO environmental risk assessments. The focus of basic research
needed to conduct appropriate risk assessments is summarized as follows:
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•	Methods are needed to detect, identify, and enumerate released GMOs
•Methods are needed to determine fate and transport of the GMO
*	Genetic stability, i.e., propensity for gene transfer must be assessed
•Hazard Assessments (pathogenicity to non-target spp. including humans
and disruption of environmental processes) must be thoroughly
investigated
*A means to mitigate possible effects would be prudent.
Environmental risk of GMOs has been described as a function of the
exposure and the hazard posed by the GMO (Bourquin and Seidler, 1986;
Levin and Strauss 1990). This relationship has helped conceptually to
develop strategies for identifying relevant research needs to address the
ecological concerns of releasing GMOs to the environment. Thus, exposure
is a general function of organism survival and multiplication (cell
density), which in turn influences GMO transport in the environment.
Hazard is a function of ecological effects including competitiveness and
metabolic activities that perturb the habitat, as well as pathogenicity,
virulence, toxicity, and allergenicity. Therefore it is essential to
understand the fundamental ecology and physiology of the organism to be
released and the basic character of the release environment in order to
address questions concerning risk assessment of the GMO (U.S.
Environmental Protection Agency, 1992). These evaluations to date
largely have been conducted on a case-by-case basis because most
products are unique and previously have not been encountered by
regulators, ecologists, and the environment.
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In order to conduct a risk assessment of a GMO, information is needed on
at least five technical issues that comprise the exposure and hazard
subcomponents for a microorganism (Bourquin and Seidler, 1986; Levin, et
al., 1987; Hall, 1995). These include: 1) knowledge of how to detect and
enumerate the GMO; 2) estimates on organism survival, multiplication,
and potential competitiveness; 3) transport and subsequent colonization;
4) possible transfer of genetic traits; 5) the ability to cause adverse
environmental effects including a) disruption of environmental
processes, and b) pathogenicity, toxicity, infectivity. Useful technical
guidance applicable to further risk assessment issues can be found in the
July 1994 and November 1994 publications of the US EPA Risk Assessment
Forum and in Sayler and Sayre, 1995 . These technical issues are now
characterized in further detail and are presented as a central theme for
conducting risk assessments of GMOs.
IIIA. The need to detect, identify, and enumerate:
The most fundamental requirement for GMO risk assessments is the
necessity to have an absolutely specific means to detect and enumerate
the target GMO from environmental samples (Bourquin and Seidler, 1986;
Levin, et al., 1987). This makes it possible to detect and document any
hazard should one exist, and evaluate the components of the "exposure"
side of the risk assessment equation (survival, multiplication, transport
and competitiveness). The enumeration procedure must be so specific that
the GMO can be readily distinguished from indigenous microbes of the
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same species. This simple requirement may be complex in practice
because of the diversity of habitats that must be sampled and the lack of
any single methodology applicable to recovery of GMOs from all habitats
(Donegan, et al.,1991; Angle et al., 1994).
IIIB. Conditions Impacting Survival, Multiplication, and
Competitiveness In the environment:
By having a general understanding of the ecology of the GMO and knowledge
of the release habitat, certain generalities may be developed regarding
expectations for the survival and potential for regrowth of the test
organism. For example, if the GMO strain was isolated from the intestine
of an insect, it may be expected to better colonize that habitat when
reintroduced into nature than to colonize soil (Armstrong, Porteous, and
Wood, 1989). Such a microorganism obtained from an insect also might be
more readily transported off-site through colonization of other insects.
Knowledge of the maximum growth temperature of an organism will also
be useful in estimating hazards. For example, a recombinant organism
isolated from soil and having a maximum growth temperature of 32C,
would be most unlikely to colonize the gastrointestinal tract of a warm
blooded animal, let alone cause a disease in such a host. On the other
hand, Scanferlato et al. (1989) studied the survival of a genetically
engineered Erwinia carotovora in aquatic microcosms. This plant pathogen
did poorly and did not become established and did not survive beyond 32
days.
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Understanding the capabilities and conditions that may lead to growth in
the environment might be conveniently accomplished through pilot studies
in contained environments such as in microcosms or greenhouses
(Fredrickson and Seidler, 1989; Cripe, Pritchard, and Stern, 1992; Hood
and Seidler, 1995; Krimisky, et al., 1995). Gillett and Witt (1978) have
defined a microcosm as "... a controlled, reproducible laboratory system
which attempts to simulate the situation in a portion of the real world."
The intent is that the microcosm duplicate as faithfully as possible,
environmental conditions in order to simulate the proposed field site.
Perhaps the most relevant practical use of microcosms is that they can
provide results that can be used to recommend how often to sample, where
to sample, and what general concentrations the GMO may be anticipated in
the field environment. Furthermore, research has demonstrated that
studies in these contained environments may provide a first
approximation of anticipated fate/survival in the field (Bentjen at al.,
1988; Armstrong, 1989; Bolton et al, 1991; Angle et al., 1995; Wagner-
Dobler, et al., 1992). Other natural factors that influence survival may
otherwise be difficult to anticipate and may include an ability to persist
under starvation conditions; grow in association with other organisms
(intestines of animals, insects, rhizosphere of plants), or with decaying
tissue of plants or animals. All the latter situations also may be
simulated in microcosms.
A valid concept has been raised in risk assessment discussions: "what cell
densities are important in assessing risks and is there a threshold
response level below which any potential risks will diminish?" For
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example, in order to exhibit detectable metabolic activities,
approximately 106 to 10? cells/gm or per ml will be required to induce a
detectable event, whether it be production of transconjugants, change in a
substrate concentration, outcompeting indigenous microflora, etc. (Short,
Seidler, and Olsen, 1990; De-Leij, et al,, 1994; Doyle, et al., 1992; Olson,
et al., 1990; Ripp, Ogunseitan, and Miller, 1994; Walter, et al., 1991). If
the GMO has declined to less than 103 cells/gm it is very unlikely an
undesirable metabolic or ecological event effect would occur, let alone be
detected. It is conceivable however, that a critical environmental
substrate could allow the GMO to multiply and achieve significant levels
but there are few known examples where this has occurred except for
disease processes in animals and plants. For documented situations where
GMOs have multiplied significantly following their release into the
environment the reader is referred to: Armstrong, Porteous, and Wood,
1989; Raaijmakers, et al., 1995; De Leij, et al., 1995. Whatever the GMO is
designed for, it is likely that large cell densities (in excess of 107
cells/gm) will be necessary for it to carry out its intended function in the
environment. Periodic sampling in microcosm habitats over several weeks
to months will provide useful data for anticipating cell numbers that a
specific GMO might achieve.
IIIC. Fate (dispersal and transport) of GMOs:
With regard to transport and fate, it has been demonstrated with early
field experiments and on a smaller scale with microcosms that wind,
water and insects can transport GMOs from the original site of deposition
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(Armstrong, et al., 1989; 1990; Lighthart and Kim, 1989; Seidler and Hern,
1988; Seidler, et al., 1994). These studies revealed that once a GMO is
released into the environment, it can spread. This is not an unexpected
observation but few earlier studies failed to investigate the transport of
microbes with such specificity and detail. Transport with GMOs has been
illustrated not only through a simple physical change of location in soil,
but also a change in the habitat as well. Thus, it has been shown that
bacteria can travel from a leaves to insects, to soil (Armstrong, et al.,
1989, 1990). Therefore, when planning a field release locale
investigators, regulators, and the general public should realize that
dissemination may occur to a certain extent. Movement of the GMO will
not necessarily be a risk per se but mobility should be taken into
consideration as a component of the risk assessment relationship.
Mobility could lead to GMO establishment at new sites, perhaps in new
habitats, and therefore, may lead to new exposures.
There is obviously a need to understand fate and rate of transport to
evaluate exposures in the environment. Test procedures are available for
assessing fate of GMOs in microcosms. These model ecosystems can
provide very useful information that deal with GMO dispersal, transport,
survival, and potential effects but obviously they are scale limited (Angle,
et al., 1995).
It is presumed thai there are interactions of biotic and abiotic
components as well as fundamental ecological processes in microcosms
that reflect processes in the environment. Studies clearly have
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demonstrated that a key to the successful application of microcosms is
the necessity of controlling and monitoring environmental conditions
during the course of the experimental period (Armstrong, 1989; Wagner-
Dobler et al., 1992). An advantage of using microcosms is that they can be
constructed to contain fewer complexities compared to the natural
environment however, this simplification in itself can be a disadvantage
because the natural environment is not simple. Obviously microcosms are
closed systems while natural ecosystems are open.
HID. Gene flow and recombination
Gene transfer has been documented to occur within more than two dozen
bacterial genera and numerous additional species (Levin, et al., 1992). In
order for gene transfer to occur between microbes, certain physiological,
metabolic and cell density requirements must be met. Gene transfer under
artificial situations in the laboratory or in microcosms usually requires
millions of cells per unit volume of material in order to obtain detectable
transfers (Knudsen, et al., 1988; van Elsas and Smit, 1994). Because
these metabolic and cell density conditions are not thought to occur with
any regularity in the natural environment, some believe that gene transfer
among GMOs and the indigenous bacteria is an extremely infrequent event.
However, more recent data lead to other conclusions. The three well
characterized mechanisms by which bacteria may undergo genetic
exchange (transformation, transduction, and conjugation) are, in all
likelihood, operational in soil (van Elsas and Smit, 1994; Smit, et al.,
1991). Recently, studies from two laboratories have rigorously
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demonstrated that gene transfer from experimentally released bacteria
into indigenous bacteria has occurred in a watershed and an aquifer
(Fulthorpe and Wyndham, 1991; Zhous and Tiedje, 1995). Transduction and
conjugation also have been experimentally demonstrated to take place in
the phylloplane, rhizosphere, and epilithon of rocks in a stream (Farrand,
1992; Knudsen, et al., 1988; Kidambi, et a!.. 1994; Hill, et al., 1992).
Soil and its component parts have been demonstrated to be both a
hinderance (compartmentalization) and a stimulator (nutrients,
rhizosphere effect) of gene transfers. It also has been confirmed that
when the recombinant DNA is maintained in the chromosome as opposed to
on a transmissible plasmid, the incidence of gene transfer by conjugation
generally drops to undetectable levels as anticipated (Smit, et al., 1991;
Smit, van Elsas, and van Need, 1992). Thus, if one is prudent in the
molecular design of the recombinant organism and can avoid habitats that
promote cell contact, high cell densities, and is low in available
substrates for growth, the transfer of recombinant DNA into indigenous
bacteria is surely not a detectable event.
Recently, evidence also has accumulated that bacteriophage mediated
transduction plays a significant role in the transfer of both plasmid and
chromosomal DNA in aquatic ecosystems (Ripp and Miller, 1995). Using
lake water microcosms incubated in a freshwater lake, investigators
demonstrated that t particulate matter increased transduction up to 100-
fold. Furthermore, up to 40% of the Pseudomonas aeruginosa in natural
systems contain DNA sequences homologous to phage genomes indicating
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probable prior interaction with a transducing bacteriophage (Ogunseitan,
et al., 1992) .
It is well documented that gene transfer between bacteria requires
specific physiological, nutritional, and environmental conditions. These
conditions can be met in a variety of habitats where natural cell densities
are sufficient to promote necessary cell, bacteriophage, or naked DNA
interactions to allow genetic exchange to occur at detectable rates. DNA
transfers of recombinant genes have been facilitated by naturally
occurring mobilizing plasmids in river epilithon where bacterial numbers
are very high on rock surfaces (Hill, Weightman, and Fry, 1992).
IIIE. Characterizing Ecological Effects.
The experimental search for ecological effects from GMOs has taken a
tortuous and intellectually challenging route because of the potential
political, social, economic, and scientific consequences and challenges of
finding such an organism. Uncertainties over possible ecological effects
probably are derived from a combination of our uncertain and limited
knowledge of microbial ecology and the documented "horror" stories of
significant and adverse effects from introduct'ons of certain higher
species of plants and animals (Sharpies, 1990; Halvorson et al, 1985).
Scientists have tended to speculate that certain undesirable consequences
brought about by higher plant and animal introductions might be extended
to GMOs. This has perhaps sensationalized the anticipated discovery of
GMO-induced effects and made the documented effects seem less
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important.
Sharpies (1990) has compiled a list of characteristics that probably
assist in the establishment of new organisms in new niches. An
inspection of these traits reveals their applicability to microorganisms
as well as to higher organisms:
"the organisms with generalized requirements and broad tolerances seem
to make better invaders
exploiting a used or underused resource will facilitate establishment
unique physiological features, as in the ability to metabolize unusual
compounds may facilitate establishment
an organism may survive better if it is preadapted to survive when the
new habitat is like the one in which it evolved."
It may prove valuable to keep these concepts in mind as new GMOs are
constructed and examined for possible environmental perturbations.
Despite the verifications that GMOs may undesirable changes in a habitat
(Short, et al, 1991; Doyle, et al, 1991; Seidler, 1992; Doyle, et al., 1995),
questions remain as to how best to evaluate and detect these potential
detrimental effects with new GMOs. Another equally perplexing issue is a
criticism as to 'the, significance of an effect that is "transient" in nature
as opposed to one that is longer in duration. First, of course one must be
entirely certain that the recovery from a "transient" effect actually
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leaves the habitat in a "recovered" state that is unaltered from its
original structure and function. Most investigations have used only broad,-
general assays to monitor habitat changes induced by GMOs. However,
when detailed methodologies are used to track perturbations, evidence for
taxonomic changes in bacterial populations has been revealed, despite the
return in culturable bacterial numbers to control levels (Donegan, et al,
1995).
Many endpoints have been considered in evaluating possible effects from
GMOs (Table 1). Initially, it was not obvious which endpoints would be
the most logical to investigate. A "shotgun approach" was used in early
research with hopes of capturing GMO-induced alterations (Seidler, 1992;
Doyle, et al., 1995) . This approach resulted in the detection of effects
documented in changes of respiration, cell numbers, and certain soil
enzymes (Doyle et al., 1991; Short et al, 1991; Wang, et al., 1991).
Monitoring these population changes and other risk assessment
experiments can be conducted readily and, perhaps should be conducted, in
microcosms. Population trends, biochemical activities, mineralization or
leaching of soil enzymes and plant biomass trends also have been
investigated and documented to be useful when monitoring for GMO
perturbations (Bolten, et al., 1991).
The day may be approaching rapidly when experimental trials of GMOs will
be-routinely conducted in the open environment with little to no
preliminary microcosm investigations. This certainly has become the
trend in testing transgenic plants (Mellon and Rissler, 1993). Another
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approach to initial or preliminary assessments of GMOs may be to conduct
field trials with the unaltered parental strain. In this case there may be
doubt if the parental strain would accurately mimic key ecological traits
that pertain to risk assessment aspects of the GMO. Chances are that
differences in key metabolic characteristics of the GMO may significantly
alter its survival and this may influence its characteristics for exposure
or hazard assessments. Therefore, if experiments are to be conducted in
microcosms, this should be done using the (recombinant) GMO strain.
IMF. Health Effects
The deliberate environmental release of GMOs has initiated investigations
of potential health effects associated with exposure to these organisms.
Humans may come into contact with these microorganisms or their
products in the agricultural or industrial setting during production or
application (Grunnet and Hansen, 1978; Olenchock, 1988). Because soil,
water, or air may contain high concentrations of the microbial product,
the primary routes of exposure are through inhalation, ingestion, or skin
penetration (Levy, 1986).
The new Escherichia coli host-vector systems constructed during the
initial recombinant DNA studies in the 1970s raised several human health
issues (Gorbach, 1978a; Gorbach 1978b). Colonization of the intestinal
tract and transfer fo and expression of foreign genes by the intestinal
microbiota was of particular concern. Researchers studied the
colonization of the human Gl tract by E. coli strains K12 and B (and their
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derivatives) and found that the colonization of normal healthy adults did
not occur (Anderson, 1975; Levy et al, 1980; Smith, 1975; Smith, 1978),
However, E. coli strains were able to establish in the intestines of germ-
free and antibiotic-treated mice (Cohen et al, 1979; Laux et al., 1981;
Levy and Marshall, 1981; Levy et al., 1980). Gene transfer to the normal
human intestinal flora was plausible, but E. coli strains harboring
plasmids were demonstrated to have a selective disadvantage in their
colonizing ability (Anderson, 1978; Duval-lflah and Chappuis, 1984; Laux
et al., 1982).
Aside from the pesticide products that require health effects testing as
described in Subdivision M of FIFRA, little health effects research has
been done on biotechnology agents and their products or by-products. To
date, the general consensus has been that because the strains used for
biotechnology applications are of little risk to "normal" people, more
research emphasis has been placed on environmental effects. However, as
research into potential health effects is conducted, the health effects of
some biotechnology strains may be less certain than previously thought.
As summarized in Table 2, the majority of the published research has
dealt with microorganisms used for biodegradation or the by-products of
this process. Recently, Health Canada has completed an extensive study on
the pathogenicity of environmental and clinical P. aeruginosa (because of
its potential application for biodegradation) isolates looking at a variety
of endpoints which will be addressed in Section IIF(1) (Godfrey et al.,
1996).
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In general, there are two fundamental questions that should be answered
when considering health effects associated with biotechnology agents and
products: 1) Does the microorganism(s) involved impact health, and 2)
does the microbial product or by-product result in an increased health
risk? In order to address these questions, 3 general areas will be
explored and their relevance discussed. Included are 1) pathogenicity
(comprised of mortality, morbidity, and infectivity), 2) allergenicity of
both the product of interest and the microbial component, and 3) product
(or by-product) toxicity.
(1) Pathogenicity
The likelihood of an industrial release of a known pathogen is very low. In
fact, the Federal Register statutes (1986) regulating the release of GMOs
mandate that the organism used not be pathogenic. However, because of
the increased exposure potential and the possibility for the use of
identified or unknown opportunistic pathogenic agents, it is imperative to
determine if an organism has the probability to infect workers or the
general public. Therefore, several in vivo and in vitro systems have been
used to determine if biotechnology agents and their products are
potentially hazardous. Many of these use rodent models to determine
morbidity and establish LD5o values. For example, Roe et al., (1991) have
demonstrated that the solubilized parasporal crystalline protein of
Bacillus thuringientis subsp. israelensis has an LD50 of 1.3 mg/kg
following i.p. injection of the Swiss-Webster mouse . The LD50 in the CD
rat is 9 mg/kg. Following intranasal exposure, George et al. (1993) have
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established an LD5o of 1.05 x 107 CFU for P. aeruginosa strain AC869 in
the C3H/HeJ mouse.
In order to cause disease, an organism must evade or overwhelm the
defense barriers of its potential host and multiply. Therefore, survival
temperature range plays an important role in the infection process. As
stated earlier, if an organism cannot survive above 30°C, it is unlikely
that it will persist and multiply in a mammalian host. However, if the
microorganism can persist at the host temperature, it can attach to or
invade host cells. A list of temperature ranges for selected biotechnology
agents are presented in Table 3. Additionally, in order to achieve
colonization, an organism must avoid the host immune systems. Once
inside the cell, an organism may multiply and be disseminated throughout
the host. Pathogenicity factors, such as adhesins, extracellular enzymes,
and cytotoxins, may enhance disease progression. However, disease is an
inadvertent outcome of infection (Finlay and Falkow, 1989).
Production of pathogenicity factors by an invasive microorganism may
facilitate infection (Finlay and Falkow, 1989). Significant attention has
been given to pathogenicity factors attributed to P. aeruginosa and other
pseuriomonads (Table 4). Members of the genus Pseudomonas are used for
environmental applications because of their effectiveness in biocontrol
and versatile substrate utilization abilities. Even though most of these
microorganisms generally are considered harmless soil isolates, they may
cause opportunistic infections following exposure of susceptible
individuals to high concentrations. P. aeruginosa is recognized as an
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opportunistic human pathogen and is capable of causing disease,
especially in immunosuppressed hosts and patients with leukemia or
cystic fibrosis (Bodey et al., 1978; Schimpff,1980; Guiot et al., 1981).
Grimwood et al. (1993) have concluded that exoenzyme expression
correlates with lung deterioration in cystic fibrosis disease progression.
Some isolates of Burkholderia cepacia (formally Pseudomonas cepacia) are
involved in nosocomial infections (Martone et al., 1981) and can colonize
and cause pneumonia and septicemia in cystic fibrosis patients
(Rosenstein and Hall, 1980; Sajjan et al., 1992). P. putida, P. fluorescens,
and Xanthomonas maltophilia (formally Pseudomonas maltophilia) have
been shown to cause secondary infections in cancer patients and are
involved in other opportunistic infections (Gilardi, 1991; Anaissie et al.,
1986).
In order to establish regulations for biotechnology products, Health
Canada has investigated several endpoints for use in the risk analysis of
P. aeruginosa, a potential isolate of bioremediation products (Godfrey et
al., 1996). Clinical and environmental isolates have been screened for
exoenzyme production (Toxin A, protease, phospholipase C, exoenzyme S,
elastase), cytotoxicity, serotype, serum sensitivity, phagocytic killing,
opsonic phagocytosis, A and B band LPS probe reactivity, pilus phage
sensitivity, and restriction fragment length polymorphism type. Of all of
the endpoints measured, exoenzyme S is the only one to demonstrate a
good correlation to, mouse virulence (LD50 in the neutropenic mouse model)
with elastase production showing possible correlation to LD50. Because
the mechanism of pathogenicity by P. aeruginosa is multifactorial, the
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elimination of one pathogenicity factor, such as exoenzyme S, may not
eliminate pathogenicity (Godfrey et al., 1996). Others have suggested
that, with the exception of alginase production by P. aeruginosa cystic
fibrosis isolates, that there is no one factor that differentiates between
clinical and environmental isolates of P. aeruginosa and B. cepacia (Nicas
and Iglewski, 1986). However, Bevivino et al. (1994) have shown that
rhizosphere isolates do have several unique traits, not found in their
clinical counterparts, such as wider temperature range for growth,
nitrogen fixation ability, and indole acetic acid production. In this study,
clinical isolates produced protease and pyochelin, and adhered to human
cells.
(2) ailergenicity
A second category of deleterious outcomes following human exposure to
biotechnology agents and their products is an allergic reaction. This can
culminate in a skin reaction or a more serious respiratory response to the
allergen such as bronchitis and/or asthma. Because a variety of fungi can
cause severe respiratory reactions, it is especially important to consider
the potential ailergenicity of fungal pest control agents such as
Metarhizium anisophilae and Beauvaria bassiana. Additionally, bacterial
strains that secrete extracellular enzymes and/or polysaccharides may be
allergens.
An extensively studied enzyme product that elicits an allergic reaction is
alcalase (subtilisn). Following its introduction into the detergent
industry in the late 1960s, this alkaline protease, produced by Bacillus
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subtilis and supplied by Novo Industri A/S (Bagsvaerd, Denmark), was
identified as a Type I (IgE) respiratory allergen (Flindt et al., 1969; Pepys
et al., 1969). Franz et al (1971) reported respiratory symptoms in
detergent industry workers 3 to 8 hours following their work shift.
Another group at risk, the consumer, also has been shown to develop
respiratory symptoms from exposure to the enzyme component in the
detergent (Shapiro and Eisenberg, 1971).
Detergent industry workers were closely monitored once alcalase was
identified as the allergen. Several studies of workers have been reported
that correlate exposure to a positive skin prick test and total serum IgE
levels (How et al., 1978; Shapiro and Eisenberg, 1971; Weill et al., 1971).
Once exposure control was implemented in the detergent plant (e.g. dust
levels reduced, protective clothing worn by enzyme handlers), fewer
instances of respiratory allergenicity were reported. Additionally,
because acceptable exposure levels have been established for Alcalase, it
now is used as the benchmark for comparison of new enzyme products
(Sarlo et al., 1991).
A third area of potential allergen exposure arises from the consumption of
transgenic plants. The Food and Drug Administration has conceded that it
is possible for genetically modified plants to express foreign proteins
that could cause the food to be allergenic and now has a protocol to help
regulate potentially allergenic transgenic foods (Nestle, 1996).
Interestingly, Nordlee et al. (1996) identified a Brazil nut allergin in an
engineered soybean, which expressed brazil nut albumin. Therefore, it is
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important to determine if an engineered food has an allergenic potential
so that the consumer can be notified of potential risks.
(3) By-product toxicity
Use of bioremediation to reduce or eliminate hazardous waste is
economically important. The ultimate goal of waste management is to
eliminate toxicity of the waste and reduce risk of exposure to the
remaining hazardous components. There are many bacteria and fungi that
can metabolize, or co-metabolize, many complex chemicals either alone or
in consortia, both aerobically and anaerobically. However, the scenario at
the hazardous waste site or biodegradation facility may not be as
controlled as the laboratory incubator, so a biodegradation process may
not occur as readily or identically as that observed in the laboratory.
Therefore, because the reduction of one compound does not necessarily
indicate reduced toxicity of the resulting by-products, a variety of
methods can be used to determine if a chemical degradation process
reduces the overall toxicity (cytotoxicity, genotoxicity, teratogenicity,
neurotoxicity) of the target compound(s) or complex mixtures.
There are two general approaches to toxicity reduction: 1) if one or a few
chemicals are involved and if the metabolic pathway(s) is known, then the
toxicity of the metabolites can be established and the toxicity of the
process predicted, 'and 2) if the metabolic pathway is unknown or if the
target is a complex mixture of chemicals, then extracts from benchscale
biodegradation processes can be analyzed for a reduction in overall
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toxicity prior to large scale use. An example of the first scenario is that
of pentachlorophenol (PCP) degradation. The pathway for PCP degradation
from anaerobic sewage sludge studies is shown in Figure 1 (Mikesell and
Boyd, 1988). PCP induces hepatic carcinomas and adenomas in mice and
chromosomal aberrations in Chinese hamster ovary cells (DeMarini et al.,
1990; NTP, 1989). The metabolites 2,3,4,5-tetrachlorophenol and 3,4,5-
trichlorophenol are more genotoxic than the parental compound PCP in the
prophage induction bioassay in E coli (DeMarini et al., 1990).
Tetrachlorohydroquinone, a major PCP metabolite identified in rodent
studies, is genotoxic in V79 Chinese hamster cells and may be responsible
for oxidative DNA damage in vivo (Jansson and Jansson, 1991). A similar
scenario occurs following 2,4,5-trichlorophenoxyacetic acid (2,4,5-T)
metabolism by B. cepacia (George et al., 1992). One of the metabolites,
2,4,5-trichlorophenol is 100-fold more genotoxic than the parental
compound in the prophage induction bioassay. Therefore, if the parental
compounds PCP and 2,4,5-T are metabolized, the chlorinated
intermediates of these compounds may accumulate, and the resulting
toxicity may be considerably higher than that observed for the
unremediated waste. Because the metabolic pathway is known for PCP and
2,4,5-T, and the resulting intermediates are more toxic than the parental
compounds, strains can be constructed that do not accumulate the more
toxic intermediates or other microorganism with alternative degradative
pathways can be employed for the bioremediation process.
Toxicity tests have been used to monitor the toxicity of hazardous waste
sites and bioremediation (Table 5). Claxton et al. (1991a; 1991b) used the
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Salmonella assay to assess the genotoxicity during the bioremediation
efforts following the Alaskan oil spill (March 1989) in Prince William
Sound, Alaska. The efficacy of two fertilizers, one oil soluble and the
other water soluble, was studied. The reduction or generation of
toxicants during bioremediation was of particular interest. The results
concluded that the mutagenicity declined over time and that both the
naturally occurring processes and fertilizer supplement contributed to the
reduction in toxicity.
IV. Examples and Methods for Detecting Effects Caused by GMOs
1VA. Environmental Perturbations from GMOs
What kinds of environmental perturbations might be anticipated in a worst
case scenario involving a GMO? Are there any precedents in the literature
that might provide answers to this question? There are now many
examples of documented effects on beneficial organisms or ecosystem
processes induced following exposure to a GMO (Table 6). Overall, the
documented effects include the transfer of genetic material to indigenous
bacteria, production of toxic metabolites, changes in metabolic activity of
the soil community, competition resulting in the suppression of
indigenous microorganisms, and changes in the biodiversity of organisms
present in the habitat. These changes represent exactly the kinds of
perturbations that were speculated as possible occurrences early in the
risk assessment of GMOs (Halvorson, et al.,1985; Bourquin and Seidler,
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1986). GMOs that are effectors of ecosystem perturbations also represent
a large and diverse type of taxonomic and metabolic groups.
Perhaps more studies have been conducted with a plant/soil ecosystem to
explore possible GMO effects than with any other system. The first field
studies in the United States involved the release of Ice- Pseudomonas
syringae onto strawberries and potatoes to control frost damage (Seidler
and Hern, 1988;Seidler, et al., 1994). In those experiments it was learned
that despite reasonable precautions, the aerosol spray of the GMO resulted
in considerable drift off the main plot. Although elaborate monitoring
devices were employed, it was soon learned that large (150 mm dia) Petri
dishes could adequately serve as indicators of the extent of microbial
deposition on and off the plot. Only 0.001% of the sprayed P. syringae
drifted some 30 m off the central plot area. This still translated into a
considerable number of viable cells (est 107). Although this increased
transport occurred, no known undesirable consequences resulted. This
may not always be the case with other GMOs. Therefore, it is prudent to
anticipate dispersal of a GMO beyond the boundaries of a test, especially
in aerosol releases. Monitoring may easily be conducted as described
above to document drift for cases where legal or sensitive environmental
issues may be involved.
Two studies assessing similar potential GMO effects were carried out
independently in different parts of the world and the results merit special
mention. The studies conducted by Fredrickson et al, (1989) and by White,
et al., (1994) both evaluated the possible impacts caused by a different
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root colonizing GMO on the bacterial flora associated with wheat. In both
cases a Pseudomonas species was the GMO and in both cases the
introduced organism resulted in substantial reductions in indigenous
microbial populations found naturally on wheat seeds and root systems.
At least in one case the introduced organisms suppressed other
fluorescent pseudomonads, organisms that help to reduce the incidence of
take-all disease, a fungal disease of wheat.
In other studies, it was learned that GMOs placed on seeds or inoculated
directly into soil were capable of moving or being transported to the
aerial parts of plants. For example, insects could vector GMOs between
soil and plant parts and even on to new insects via contaminated leaves
(Armstrong, et al.,1989, 1990). In a recent study, plant growth promoting
fluorescent pseudomonads colonized the surface and interior tissues of
cotyledons in row crops following initial inoculation onto seeds
(Raaijmakers, et al., 1995). It is clear that at least with some
applications of GMOs, transport may be the rule rather than the exception
to their ecological fate.
Two documented cases have been described of GMO effects resulting from
the production of metabolites. These studies validate the potential
concern that an introduced taxon can significantly alter its surroundings.
Streptomyces lividans has been genetically modified to enhance its
production of extracellular lignin peroxidase and hydrogen peroxide to
facilitate the degradation of lignin (Wang, et al.,1989). Whenever the
recombinant strain was added to native soil supplemented with lignin,
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there was an increase in the amount of carbon dioxide respired over all
control treatments. Because GMO strains did not cause an increased
evolution in carbon dioxide in sterile soil, the researchers hypothesized
that the enzyme-induced lignin breakdown by the GMO provided fresh
substrate for indigenous soil microflora and this synergistic relationship
caused the enhanced mineralization of carbon. Thus, the lignin peroxidase
produced by the GMO had a significant short term impact on carbon
turnover rates that peaked out by day 6-9, presumably when the added
lignin had been biodegraded.
The synergism established between an added bacterium (GMO) and
components of the indigenous microbiota illustrate dramatically how a
GMO may interact with and influence the metabolic characteristics the
indigenous community. It would be of great interest to determine
whether the breakdown of natural or added lignin had any impact on other
soil parameters such as fertility, moisture holding capacity, pH, etc, and
what proportion of the resident lignin can be decomposed by this GMO. In
this case, microcosm studies could provide answers to both sets of
questions.
The second case of a deleterious GMO metabolite resultad from an
unanticipated combination of ecological and biochemical circumstances.
The use of GMOs as agents of bioremediation to facilitate environmental
cleanup is a most'worthy goal. However, the metabolic limitations and
capabilities of any altered metabolic traits should be well documented
prior to open field trials to evaluate efficacy. A case in point that
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illustrates this caution is the P. putida PPO301 (pRO103) organism that is
capable of degrading 2,4-dichlorophenoxyaeetic acid (2,4-D). When this
GMO was added to a soil microcosm containing added 2,4-D, the first
metabolic product of 2,4-D breakdown, 2,4-dichlorophenol(2,4-DCP),
accumulated to 70-90 ug/g of soil. The 2,4-DCP is more toxic than 2,4-D
and fungal propagules in the experimental treatment deceased from over 3
x 106 to undetectable levels by day 18 (Short, et al., 1991). Toxicity of
the 2,4-DCP also was demonstrated on Petri dish assays where relative
fungal counts were determined in the presence of increasing
concentrations of 2,4-DCP. Growth of all soil fungi was reduced by as
little as 10ug/ml of media of 2,4-DCP and by 25 ug/g of soil in other pure
culture assays. Interestingly, the 2,4-DCP did not reduce the numbers of
soil bacteria. The accumulation of 2,4-DCP was the result of two
factors. First, the bacterium could not degrade 2,4-D completely so the
2,4-DCP accumulated. Furthermore, the arid soil used in these studies did
not contain any indigenous microbes capable of degrading 2,4-D or 2,4-
DCP. Thus, in this soil with this GMO, 2,4-DCP accumulated. These
observations are also valuable because they validate the use of
microcosms for investigating ecological perturbations and the results
were easy to measure since changes in microbial populations were the
impacted endpoint.
IVB. Toxicological considerations
f
In this section, in vivo models and tier testing approaches will be
presented for use In determining the potential pathogenicity, infectivity,
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and allergenicity of- biotechnology agents and their products. In a previous
section [IIF (1)], endpoints that are characteristic for pathogenic strains
were described so pathogenicity and infectivity factors will not be
discussed further in this section.
(1) in vivo infectivity and pathogenicity models.
Several animal models have been used to detect potential health effects
of biotechnology agents and their products including conventional mice
(with and without fasting) and rats (Table 7). From these animal models,
LD5o values can be established. Aside from mortality, morbidity endpoints
including body and tissue weight loss, heart rate, hypothermia,
vasodilation, jejunal hemorrhaging, liver centrilobular congestion have
been detected (Roe et al., 1991; George et al., 1991; George et at., 1993).
Additionally, clearance (from the lungs, small and large intestine, cecum),
colonization (Gl tract), translocation (spleen, liver, mesenteric lymph
nodes), and pulmonary inflammatory response have been determined
following oral or intranasal exposure (George et al., 1989, 1991, 1993).
Other animal models that may be useful for determining the mortality and
morbidity of GMOs are: 1) pulmonary exposure of rats to agar or pgarose
encapsulated P. aeruginosa or other organisms (Cash et al., 1979; Starke
et al., 1987; Woods et al., 1980), 2) neutropenic mouse model to determine
LD5o (Godfrey et af., 1995), and 3) the Streptococcus
zooepidemicusfinfluenza virus pulmonary exposure mouse model
(Sherwood et al., 1988). The latter has been used to determine health
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effects of P. aeruginosa, B. cepacia, X. maltophilia, and B. thuringiensis
subsp. israelensis, B. thuringiensis subsp. kurstaki, and B. thuringiensis
subsp. aizawai (George et al., 1991; George et al., 1993; Kawanishi et al.,
1990).
(2) Allergenicity.
Sarlo and Clark (1992) have described a tier approach to determine the
respiratory allergenicity potential of low molecular weight chemicals.
This strategy has application for biotechnology chemical products and may
be suitable for protein products. The tier approach is shown in Figure 2.
If a chemical can modify a protein, then there is a potential to yield an
immunogenic chemical-carrier conjugate (Sarlo and Clark, 1992). The
ability of a biotechnology product to react with a protein demonstrates
its potential as an allergen. The Level 3 guinea pig injection model
involves subcutaneous injection of the potential allergen in an olive oil
carrier over a period of 6 weeks. At the end of the treatment regiment,
respiratory reactivity (following intratracheal challenge of the potential
allergen), active cutaneous anaphylaxis testing (intradermal injection
with the chemical protein conjugate using Evans blue dye intracardially
injected as the indicator), and allergen-specific IgE levels are measured
(EUSA).
Guinea pig inhalation tests constitute the final tier (level 4). Animals are
servsitized by exposure to aerosols for 5 days (3 h/day) and respiratory
rate and breath peak height continuously monitored. Two weeks following
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sensitization, animals are exposed to an aerosol and respiratory rate and
breath peak height determined over a 30 min period. Readers should refer
to the Sarlo and Clark (1992) reference for a more detailed description of
the Levels 1-4 procedures.
Skin testing of detergent factory workers has been used to identify atopic
individuals (How et al., 1978; Newhouse et al., 1970; Franz et al., 1971;
Juniper et al., 1977). Subjects were injected cutaneously with different
concentrations of alcalase protein in physiological saline and the skin
response observed (Sarlo et al., 1990; How et al., 1978).
(3) Tier testing approach for microbial and biochemical pest
control agents.
The United States Environmental Protection Agency regulates new
microbial and biochemical pest control products using the approach
presented in Subdivision M of the Pesticide Testing Guidelines (Anderson
et al., 1989). The Tier I toxicology guidelines, which address acute oral,
dermal, pulmonary, and intravenous toxicity and pathogenicity as well as
eye irritation and infection, hypersensitivity, cell culture tests (viral
agents) are suitable for GMOs. Tier II testing describes methods for acute
and subchronic toxicity/pathogenicity studies, and Tier III focuses on 1)
reproductive and fertility effects, 2) oncogenicity studies, 3)
I
immunodeficiency studies, and 4) a primate infectivity and pathogenicity
study. Even though the guidelines are established for regulation of pest
control agents, they have some applicability for determining health
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effects of GMOs.
IVC. Reliability of endpoint measurements
In order to assure workers and the population at large, the safety of
environmentally released GMOs must be established. If a potential
environmental release is not from a pathogenic genus, health or
environmental effects testing on this particular microbial strain may not
have been done. Even though the probability is low, there is a potential for
GMOs to harbor genes harmful to human health or the environment. By
developing, validating, and using methods that examine the potential
effects of the newly generated strains, such as those described in the
previous sections, researchers and industry can reassure the workers and
the general public that the isolates are safer.
In order to reliably evaluate measurements conducted on environmental
aspects of GMO risk assessments, properly designed ecologically relevant
experiments are essential. It is crucial that any prerelease studies be
conducted with environmental materials that will ultimately receive the
GMO. Thus, local water, soil, plants, etc should be employed. This
commonsense approach to evaluating survival, transport, and effects will
help to ensure that relevant and hopefully reliable information on the
ecology of the organism will be obtained. As indicated in Table 1,
r
numerous experimental endpoints have been tested or proposed to measure
risks associated with GMOs. A recent publication discusses many of these
endpoints (Stotzky, et a!., 1993) and the reader is referred to that report
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for a more detailed analysis.
One of the keys to taking reliable endpoint measurements is the ability to
control the environment in which microcosm analyses may be evaluated.
Without proper environmental controls to measure and control light,
temperature, soil moisture etc., there is little hope of either simulating
natural conditions or to obtaining repeatable data on basic ecological
parameters such as GMO survival. How and when to sample the
environment to measure endpoints can best be obtained from published
data on other GMOs and from trial and error experiences. Basic
microbiological techniques that ensure sample integrity is maintained
free from extraneous contaminants, a reliable procedure to detect and
enumerate the exact strain under test, and a reasonable culturing milieu
are all necessities. As stated earlier, we also have found when conducting
field trials that a quality assurance plan must be in place to insure the
quality and repeatability of the data collected (Seidler and Hern, 1988).
The determination of potential health effects of GMOs is not a precise
science. The selection of an animal model to determine morbidity and
mortality of all relevant GMOs is difficult. It is important to consider
clearance rate of the GMO from the animal tissues because human
pathogens do not necessarily cause disease in animal models. Exoenzyme
and toxin production, in combination with the toxicology data, are helpful
f
in determining if there is a potential for adverse health effects. However,
the pathogenicity factors described previously are from the Pseudomonas
species because of the widespread use of the pseudomonads in
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bioremediation and other environmental applications (predominantly P.
aeruginosa has been studied). Additionally, Godfrey et al. (1995) have
shown that only the pathogenicity factor exoenzyme S correlates with
LD50 in P. aeruginosa isolates tested even though Grimwood et al. (1993)
have demonstrated a correlation between several exoenzymes and cystic
fibrosis lung deterioration. Therefore, the possession of pathogenicity
factors by a biotechnology agent may impact the potential risk associated
with exposure.
However, if the GMO of interest does cause mortality, morbidity, or it
colonizes a selected animal and if it has some of the recognized
pathogenicity factors (a pest control agent for example), then the
potential to cause adverse human health effects may increase. This
information, along with the exposure data, should be considered prior to
release of the GMO.
Generally speaking, healthy adults should not experience any adverse
reactions following exposure to GMOs. However, because some of the
GMOs proposed for environmental release may cause opportunistic
infections, there are several groups of individuals that may be at a
greater risk following exposure due to their immune status. These groups
include 1) young children and infants, 2) the elderly, 3) chemotherapy
patients, 4) individuals with an active illness, 5) convalescents using
antibiotics or other'medications, and "6) atopic persons . For example,
isolates of B. cepacia, an organism found in the soil, cause disease in
cystic fibrosis victims (Rosenstein and Hall, 1980). Clostridium difficile
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can colonize the healthy, human adult intestinal tract in low numbers but
causes pseudomembranous colitis in patients on antibiotic therapy
(Bartlett, 1979). Green et ai. (1990) have examined the public health
implications of a B. thuringiensis var. kurstaki spray in Oregon and have
found that B. thuringiensis var. kurstaki is culturable from clinical
specimens from exposed individuals. Three patients with prior medical
problems harbored B. thuringiensis var. kurstaki which could not be ruled
out as a causative disease agent. However, detrimental effects from
controlled human exposure (oral and inhalation) to B. thuringiensis have
produced no adverse effects (Fisher and Rosner, 1959).
It is important to identify potential allergens so that atopic individuals
can be identified and their exposure reduced. The detergent industry-
alcalase experience is a case in point (Franz et al., 1971). Once
researchers recognized alcalase as an allergen and identified atopic
individuals, industrial exposures were reduced in the following ways: 1)
workers who handled the raw materials were outfitted with protective
clothing, 2) dust levels were reduced in the plants so that employees'
exposures were lessened, and 3) atopic individuals were placed in jobs
where exposure was reduced or eliminated. The industry continues to
closely monitor its workers even though the allergenicity-related
complaints have declined.
Otherwise healthy individuals may have some risk following exposure to
GMOs or their products. One point to consider is the transfer of antibiotic
resistance genes from the environmentally released GMOs to other
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microorganisms and mammals, including humans. The spread of the
antibiotic resistance genes has the potential to reduce the effectiveness
of the presently-used antibiotics to fight disease. One case in point is
multidrug-resistant Mycobacterium tuberculosis (Pearson et al., 1992).
Because the organism is resistant to a host of currently used antibiotics,
it is more difficult to eradicate the disease. Several groups have
demonstrated the flow of antibiotic resistance genes from
microorganisms to humans. Marshall et al. (1990) described the spread of
an E. coli strain that harbored antibiotic resistance markers on a plasmid
from the microflora of animals to humans. A study by Linton (1986)
reported a similar finding suggesting that E. coli resistance traits can
transfer from animal microflora to human microflora, ultimately
rendering potential pathogens resistant to drugs and therefore much more
difficult to treat. The probability of other (non-antibiotic) introduced
genes to transfer from the GMOs to humans may result in some unexpected
transfer of the engineered traits to humans. The long term effects of this
transfer are unknown; however, there is the potential for expression of
these traits.
Finally, the toxicity of both the by-products and end products should be
considered. This is especially important in terms of bioremediation
where hazardous waste reduction and elimination may result in production
and accumulation. Therefore, it is important to have an understanding of
the. remediation pnocess and the ultimate biological activity (toxicity) of
the finished product, not just the chemical analysis that measures
elimination of the parental compound(s).
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V. Future risk assessment concerns and research needs
The use of microorganisms and management of microbial activities long
predates the use of pure cultures as inocula. However, it has not been
until the last 10-20 years, with the advent and growth of recombinant
techniques that allow the development of novel genotypes and activities,
that public concerns have been raised about the safety of using microbes,
particularly for intentional environmental releases. To date, three of the
four engineered microbes approved for commercial use in the United
States, under the Federal Fungicide, Insecticide and Rodenticide Act, are
non-viable formulations of engineered microbes (P. fluorescens containing
toxin genes from various Bacillus thuringiensis subspecies). Engineered
microbes approved for small scale experimental environmental release
under the Toxic Substances Control Act (TSCA), include P. fluorescens (P.
aureofaciens) containing the lac zy gene from £ coll, P. fluorescens
strain HK44 for PAH degradation, and R. meliloti containing antibiotic
resistance and nitrogen fixation (nif) genes. A request for commercial
registration approval under TSCA, of a R. meliloti strain containing an
enhanced nitrogen fixation (nif) capability, is currently under review by
the United States Environmental Protection Agency. To date, with the
exception of widely used agronomic inocula such as nitrogen fixing
bacteria (Rhizobium spp.and Bradyrhizobium spp.), silage inocula such as
Lactobacillus spp end the microbial insecticide B. t kurstaki, relatively
few inocula have been intentionally released to the environment on a large
scale basis. Most of the non-agronomic applications noted above have
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either been experimental ones, performed under semi-contained conditions
of laboratory microcosms or greenhouses, or they are being used under the
contained conditions of fermentation tanks or other types of bioreactors.
Where field data are available, numerous examples have been reported for
presumably transient effects of both biologicals and chemicals, on
populations and activities of soil biota, nutrient cycling and diversity
(Watrud & Seidler 1996). As the size of field tests and commercial uses
increase, questions about the effects of multiple applications and long
term use of biotechnology products also arise. These include questions of
both fate of the biologicals (organisms, nucleic acids, proteins), and
ecological effects on non-target organisms and communities, nutrient
cycling processes and on below ground and above ground biological
diversity. However, in the absence of long term monitoring studies, and
lack of consensus on suitable biological indicators or methodology, long
term impacts remain unknown.
Scientific issues which repeatedly have been raised regarding the release
of recombinant microbes include their fate and their potential adverse
effects on non-target species. In traditional chemical risk or
ecotoxicological assessments, single species have typically been tested
for short periods of time, under laboratory conditions. Pending results
with the first "tier" of indicator organisms, additional organisms may be
tested or longer term tests may be carried out. With the current
increased interest 1n the use of biologicals, traditional ways of measuring
persistence, transport and effects (for chemicals), need to be re-
evaluated for their suitability in assessing the effects and risks of
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biologicals. Unlike chemicals, biologicals can reproduce; nucleic acids,
enzymes or toxins associated with biological agents may retain biological
activity in natural environments for extended periods of time. Genetic
exchange between introduced and indigenous flora can result in transport
and persistence of the biological activity beyond the site and time of
introduction. Experience and data from large-scale, repeated or long term
uses of engineered microbes, is limited. A need for research therefore
continues, to answer questions regarding fate and effects of the
introduced microbes and microbial products, not only at the points of
release, but also in areas adjacent, downwind or downstream from the
points of application or release. Methods need to be developed not only to
look at single species or single site effects, but to examine effects on
communities of organisms in different ecosystems in diverse geographic
areas. For example, if a product is designed for agronomic use, such as
crop protection, non-target effects perhaps should be examined not only
within the agronomic system or on the target pest, but also on adjacent
agronomic, native, weedy and horticultural species in the surrounding
area. Studies on the effects of engineered microbes on soil foodweb
components (plant symbionts, nematodes, litter arthropods, protozoans
and microbes), on nutrient cycling, plant biomass and nutrient status and
on plant community diversity, may be useful to identify early indicators
of adverse effects to the diversity, functioning and sustainability of
ecosystems. To facilitate the capability to handle large sample numbers,
automated methods? of sample preparation, and analysis may be needed.
Given a suitable data base, development of predictive models to permit
estimation of risks from novel biologicals may also be useful. A need for
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longer term monitoring of effects on local and geographic bases should be
considered, since as we have learned from persistent chemicals such as
dichloro diphenyl tricholoroethane (DDT) and the para chloro biphenyls
(PCBs), ecological effects may not be apparent until decades after their
initial introduction.
In summary, we have presented an overview of the complex interactions of
genetically engineered microorganisms with the human and natural
environments. It has become clear from this information that risk
assessments of these interactions is complex and at present at a very
preliminary stage of development. Additional model systems are needed
involving both humans and the natural environment in order to provide
reassurances that properly focused risk assessments can indeed provide
reliable predictions about the potential risks of these novel life forms.
Currently available data from short term experiments point towards
minimal risks associated with GMOs. However, there is neither sufficient
commercial production nor consumer utilization, of these diverse new
products to definitively draw any conclusions about the human safety and
ecological effects resulting from the long term use and repeated
applications of recombinant organisms or products derived from them.
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Woods, D.E., Sokol, P.A., Bryan, L.E. et al. (1991) In vivo regulation of
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rearrangement. Journal of Infectious Diseases, 163, 143-149.
Wyman, C. E.; Goodman, B. J. (1993) Biotechnology for production of fuels,
chemicals, and materials from biomass. Applied Biochemistry &
Biotechnology, 39,41-59.
Yadav, J. S.; Reddy, C. A. (1993) Degradation of benzene, toluene,
ethylbenzene, and xylenes (BTEX) by the lignin-degrading basidiomycete
Phanerochaete chrysosporium. Applied and Environmental Microbiology,
59,756-762.
Zaady, E.; Okon, Y.; Perevolotsky, A. (1994) Growth response of
Mediterranean herbaceous swards to inoculation with Azospirillum
brasilense. Journal of Range Management, 47,12-15,
Zhou, J.Z., Tiedje, J.M. (1995) Gene transfer from a bacterium injected into
an aquifer to an indigenous bacterium. Molecular Ecology, 4,613-618.
105

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Table 1. Experimental endpoints relevant to measuring possible ecological
risks associated with GMOs in the environment (adpated from Seidler,
1992).
Impact of GMO on numbers or kinds of viable bacteria
metabolic activity (respiration, kinetics of CO2 evolution, substrate
induced respiration)
numbers of Gram-negative bacteria present
bacterial populations in the rhizosphere and rhizoplane
species diverstiy
biomass of colonized plants
microbial biomass
biomass of plant shoots
nitrogen content of wheat shoots
total viable fungi
numbers of cellulose utilizers
number of chitin utilizers
numbers of denitrifiers
numbers of nitrifiers
numbers of protozoa
nutritional groups of bacteria present
activity of soil enzymes
kinetics of nitrogen transformations
numbers of nonsymbiotic dinitrogen fixers
competitive ability of GMOs
effects of MPCAs on mycorrhizal colonization of roots
effects of MPCAs on mycorrhizal colonization and plant growth
numbers of soil nematodes, mites, Collembola
trophic groups of nematodes
transfer of recombinant DNA to indigenous microorganisms
106

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Table 2. Examples of biotechnology agents and their products where
health effects studies have been reported in the scientific literature.
Application
Microbial Strain or Product
Pest Control
Bacillus thuringiensis subs p.

israelensis

Bacillus thuringiensis subsp.

kurstaki

Pseudomonas syringae

Autograph a californica
Biodegradation
Pseudomonas sp.

P. aeruginosa

P. putida

Burkholderia (Pseudomonas) cepacia

Xanthomonas (Pseudomonas)

maltophilia

Mycobacterium sp.

Flavobacterium sp.

Norcardia sp.
Product
r
AJcalase/subtilisin
107

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By-product
2,4,5-Trichlorophenoxyacetate

metabolites

Pentachlorophenol metabolites

Crude oil degradation metabolites
9
108

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References for Table 2.
Pest Control
Bacillus thuringiensis subsp. israelensis: Mayes, 1989; Held, 1986, Kawanishi, 1990
Bacillus thuringiensis subsp. kurstaki: Sherwood, 9191a, 1991b
Pseudomonas syringae: Olenchock, 1988; Goodnow, 1990
Autographa californica: Hartig, 1989; Hartig, 1992
Biodegradation
Pseudomonas sp.: Kaiser, 1981
P. aeruginosa: George, 1989,1990, 1993
P. putida: Kaiser, 1981
Burkholderia (Pseudomonas) cepacia: George, 1991, 1990
Xanthomonas (Pseudomonas) maltophilia: George 1989, 1990, 1993
Mycobacterium sp.: Kaiser, 1981
Flavobacterium sp.: Kaiser, 1981
Norcardia sp.: Kaiser, 1981
Product
Alcalase/subtilisin: Sario, 1991; Sarlo, 1990
By-product
2,4,5-Trichlorophenoxyacetate metabolites; George, 1992
Pentachlorophenol metabolites: DeMarini, 1990
Crude oil degradation metabolites: Claxtori, 1991a; 1991b
109

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Table 3. Temperature range for survival of some bacterial species of
biotechnology relevance.
Biotechnology Agent
Pseudomonas aeruginosa
P. putida
P. fluorescens
P. aureofaciens
P. syringae
Temperature Optimum (°C)a
37
25-30
25-30
-30
-25-30
Burkholderia cepacia
-30-35
Xanthomonas maltophilia
35
Bacillus thuringiensis
B. subtilis
B. popliae
B. lentimorbis
30-40 (10-40)b
30-40 (10-50)
-30
-30
Thiobacillus ferroxidans
30-35 (10-37)
Sulfolobus sp.
>50-87
Rhizobium meliloti
25-30 (4-42.5)b
Bradyrhizobium japonicum
25-30 (25-42)b
a Bergeys's Manual of Systematic Bacteriology, volume 1 (Krieg et al., 1984;, volume 2
(Sneath et al., 1986), and volume 3 (Staley et al., 1989)
b Numbers in parentheses indicate temperature range.
110

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Table 4. Pathogenicity Factors identified in Pseudomonas and
Burkholderia spp.
Trait
Pseudomonas
aeruginosa
Burkholderia
cepacia
Pseudomonas
fluorescens
Pseudomonas
putida
Protease
X
X
X

Exotoxin A
X



Elastase
X
X
X

Hemolysin
X

X

Phospholipase C
(Lecithinase)
X

(X)


X



Pyochelin
X
X


Lipase
X
X
X
X
Enterotoxin
X



Adhesins:




Alginate/Mucoid
polysaccharide
X
X


Exoenzyme S
X



Pili/Fimbriae
X
X


111

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Table 4 references:
Trait
Pseudomonas
aeruginosa
Burkholderia
cepacia
Pseudomonas
fluorescens
Pseudomonas
putida
Protease
Grimwood et
al, 1989
Woods &
Sokoi 1986
Liu
Bevivino et
al., 1994
Tan/Miller
1992
Wilson &
Miles 1975

Exotoxin A
Woods et al
1982
Woods et al
1991
Grimwood et
al 1989
Woods &
Sokol 1986



Elastase
Woods et al
1982
Liu
McKevitt
Oakley et al,
1963

Hemolysin
Woods/Sokol
1986
Liu

Wilson &
Miles 1975

Phospholipase C
(Lecithinase)
Grimwood
1989
Woods et al.,
1991

Wilson &
Miles 1975
Liu
Esselman
and Liu 1961

Collagenase
Liu



Pyochelin
Woods etal.,
1991
Bevivino et al
1994


Lipase
Jorgensen
1991
Wolfarth
1992
Jorgensen
1991
Tan/Miller
1992
Wolfarth
1992
Wolfarth
1992
Enterotoxin
Liu



112

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Adhesins:




Alginate/Mucoid
Marcus1989
Straus 1989


polysaccharide
Liu




Pedersen




1992



Exoenzyme S





Woods/Que




1987




Woods et al




1991




Grimwood et




al 1989




Woods Sokol




1986



Pili/Fimbriae

Kuehn 1992



Ramphal
Goldstein



1984
1995



Farinha 1994




Tang 1995



t
113

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Table 5. Use of bioassays to determine the toxicity of bioremediation processes and
hazardous waste.
Chemical
Matrix
Bioassay
Reference
PCB metabolites
—
Sister chromatid exchange
Salmonella reversion
Sayler
1982
Chlorophenols
—
Prophage induction (£. coll)
DeMarini
1990
2,4,5-T
—
Salmonella reversion
Prophage induction (£ coll)
George
1992
Pesticide waste
soil
Salmonella reversion
Somich
1990
Benzofajpyrene
soil
sludge
Salmonella reversion
Miller
1988
Wood preserving
waste
soil
water
MicrotoxTM
Salmonella reversion
Aspergillus nidulans
Bacillus subtilis DNA-repair
Aprill 1990
Donnelly
1987 a,b
Petroleum waste
soil
Microtox™
Salmonella reversion
Seed germination
Plant
Aprill 1990
Wang
1990
Hazardous waste
complex mixture
soil
water
Salmonella reverion
SOS (E. coli umu)
Prophage induction (E coli)
Donnelly
1988
McDaniels
1993
Houk
DeMarini
1988
114

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Table 6. Genetically engineered microorganisms documented to have
caused an ecological effect and/or transmitted DNA to another organism
(adapted from Seidler, 1992, Doyle, et al., 1995 ).
Alcaligenes
Alcaligenes eutrophusA
E0106(pRO101)
Degraded chlorinated phenols
and transferred plasmid to indigenous
bacteria; natural selection
favors recipients
tranisently altered the bacterial
diversity of aquatic microcosm
compared to uninoculated lake water
Fulthorpe and
Wyndham
(1991)
Nakatsu, et
al., (1995)
Leser
(1 995)
Enterobacter cloacae
multiples, gene transfer inside insect gut
Armstrong,
et al.
(1 990)
Pseudomonas putida
Pseudomonas sp. RC1
Degraded 3-chlorobenzoate and transferred
degradative capacity to indigenous Pseudomonas
sp.
Reduced colonization of wheat rhizoplane by
indigenous fluorescent pseudomonads, which
help in suppressing take-all disease of wheat
by the fungus Gaumannomyces graminis
var. tritici
Pertsova et al.
(1984)
Fredrickson et
al. (1989)
Pseudomonas sp. B13
gene transfer of 3-chlorobenzoate activity
into Alcaligenes in a natural aquifer
Zhou and
Tiedje,
(1995)
'This strain is not genetically engineered
Pseudomonas putida
PPO301 (pRROI 03)
accumulates 2,4-dichlorophenol from 2,4-D
degradation which reduces CO2 evolution from	Doyle et al.
soil and decreased number of fungal	(1991);
propagules in soil to undetectable	Short et al.
levels	(1991)
115

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Pseudomonas cepacia
AC100	Degraded 2,4,5-T; caused changes in taxonomic
diversity of indigenous microbiota, possibly as Bej et al.
a result of metabolic activity by the GMO	(1891)
Pseudomonas fluorescens
P.fluorescens
lux-modified
denitrifier
Pseudomonas aureofaciens
Kmr, xylE, lacZY
chromosomal modification
plant growth promoting
fluorescent pseudomonads
plasmid DNA transferred to several
genera of indigenous bacteria
caused short term reduction in
overall microbial diversity
in very small pore size soil
caused large perturbation (up to
100-fold) changes in microbial
populations on seeds and root
systems of spring wheat. Changes
were transient but could still be detected
in colony morphology differences
when cultured from maturing plant roots
unexpected dispersal to plant shoots
following seed inoculation
Smit, et al.
(1991)
White, et al.
(1994)
De-Leij, et al.
(1 994)
Raaijmakers,
et al.
(1995)
Streptomyces lividans
(TK23-3651 (pSE5)
Increased transiently the CO2 from soil
microcosms when lignocellulose was
added (pSE5 codes for enhanced
production of extracellular lignin
peroxidase and H2O2;
Wang, et al.
(1991)
Lactobacillus plantarum
Recombinant strains containing a
Clostridium thermocelium derived
cellulose gene inoculated into grass
minisilos sucessfully proliferated and
competed with epiphytic lactic acid
bacteria in silage
Sharp, et al
(1992)
116

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Agent/Product
Species
Strain
Route
Reference
28 K polypeptide of
B. thuringiensis subsp.
israelensis
mouse
rat
rat
Japanese
Quail
Swiss-Webster
Sprague-Dawley
CD
i.p.
oral
i.p.
i.p.
s.c.
i.v.
oral
i.p.
s.c
i.v.
i.n.
Roe
Mayes
1989
Mayes
1989
Roe 1991
Roe
Roe
Roe
Roe
Roe
Roe
Roe
B. thuringiensis subsp.
israelensis
mouse
CD-1
i.n.
i.t.
Kawanishi
1990
B. thuringiensis subsp.
kurstaki
mouse
CD-1
i.n.
i.t.
Kawanishi
1990
B. thuringiensis subsp.
aizawai
mouse
CD-1
i.n.
i.t.
Kawanishi
1990
B. thuringiensis subsp.
wuhanensis
mouse
CD-1
i.n.
Sherwood
1991
P. syringae
rat
Sprague-Dawley
inh.
Goodnow
1990
P. putida
rat
Sprague-Dawley
oral
i.e.
i.p.
inh.
Kaiser
1981
P. aeruginosa
1
mouse
CD-1
C3H/HeJ
oral
i.n.
i.n.
George
1989
George
1991
George
1993
117

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B. cepacia
mouse
CD-1
oral
i.n.
George
1990
George
1991
X. maltophilia
mouse
CD-1
oral
George


C3H/HeJ
i.n.
1989




George




1993
i.p,, intraperitoneal^; i.e., intracutaneous; s.c., subcutaneous; i.n., intranasal; i.t, intratracheal;
i.v., intravenous.
118

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Figure 1. Degradation pathway for pentachlorophenol.
9
119

-------
CI
CI
xx
CI CI
CI CI
C02+CH4
CI
Pentachlorophenol
CI
2,3,4,5"
Tetrachlorophenol
CI
3,4,5-
Trichlorophenol
3,5-	3-
Dichlorophenol Chlorophenol

-------
LEVEL 1: STRUCTURE-ACTIVITY RELATIONSHIP
•	Literature Search
•	Discussions with Chemists/Biochemists
~ \
(+)	H
LEVEL 2: IN-VITRO REACTIVITY
•	Reactivity with Protein-Conjugate Formation
~ \
(+)	H
i
LEVEL 3: IN-VIVO REACTIVITY-IMMUNOGENICITY
•	Guinea Pig Injection Model
~ \
(+)	H
LEVEL 4: IN-VIVO REACTIVITY-ALLERGENICITY
9
• Guinea Pig Inhalation Model
Figure 2. Proposed multilevel approach for assessment of new chemicals as respiratory
allergens. Positive results at one level should lead to further testing at the next level with
the final test using the inhalation model. If a negative results is obtained at one level, then
no further testing should be done with the chemical in question.

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NHEERL-COR-2024A
TECHNICAL REPORT DATA
(Please read instructions on the reverse before completing)
1 REP0RTNE°P A/600/A-96/096
2.
3. RECIPIEf
4. TITLE AND SUBTITLE
Assessing Risks from GMOs to Ecosystems and Human Health
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
'R. J. Seidler; 'L. S. Watrud; 2S. E. George
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
'U.S. EPA, NHEERL, Corvallis, OR
2U.S. EPA, NHEERL, Research Triangle Park, NC
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
US EPA ENVIRONMENTAL RESEARCH LABORATORY
200 SW 35th Street
Corvallis, OR 97333
13. TYPE OF REPORT AND PERIOD COVERED
Symposium Paper
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
Invited review article to appear in The Handbook of Environmental Risk Assessment
16. ABSTRACT
Scientists have been altering the genetic makeup of living creatures since the 1970s. Techniques in molecular biology
have made it possible to incorporate genes from one organism into virtually any other organism's genetic composition
to create a broad array of new life forms. This manuscript is a literature review of the status of the field of assessing
the health and ecosystem safety of genetically modified microorganisms (GMOs).
17. KEY WORDS AND DOCUMENT ANALYSIS
a. DESCRIPTORS
b. IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
genetically engineering organism, risk
assessment, health effects,
environmental effects


18. DISTRIBUTION STATEMENT
19. SECURITY CLASS (This Report)
21. NO. OF PAGES
121
20. SECURITY CLASS (This page)
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION IS OBSOLETE

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