Guide to Risk Assessment and Biosafety in Biotechnology, GRABBGuide to Risk Assessment and Biosafety in Biotechnology, GRABBAn Initiative of the United Nations Environment Programme (UNEP)
| SECTION: | GENETICALLY MODIFIED MICROORGANISMS (GMMs) | ||||
| TITLE: | Ecological Issues Related to Release of Microorganisms | ||||
| BY: | Morris Levin | ||||
| LABEL: | ECO | ECO | UPDATED: | 31 Dec 1997 | |
II. Risk Assessment Protocols in Terrestrial Trials
III. Large scale issues
Containment, Dispersal and Confinement
Table 1 - General Comparison - EPA-OPPT Points to Consider/USDA-APHIS EPA-OPPT Points to Consider/USDA-APHIS Data Elements
Table 2: Examples of Co-Metabolism
By applying the tools of modern molecular biology it is possible to produce genetically engineered organisms (GEOs) tailored to particular uses. After laboratory development, field trials of transgenic products involve the potential for an adverse effect after release into the open environment (Witt 1990). The inherent danger to ecosystems must be assessed and managed through risk assessment protocols. Over the past decade, the use of agricultural products modified through biotechnology has moved beyond the laboratory stage for which specific protocols have been developed. Many crops grown from genetically altered seeds are widely marketed, and engineered microbes for agricultural use are widely available. Microbes are used for other environmental purposes, especially for bioremediation. Althoediation. Although engineered microbes for bioremediation are not widely available, general use of natural microbes, alone or as members of a consortia, is common.
Formal procedures for assessing risks on a case by case basis are well established. There are many issues that should be addressed by risk assessors during product development and testing; however, the present consensus is that any risks associated with these products will not be different from risks associated with conventional products of a similar nature. The fact that over 2000 of these field trials have gone forward in the US alone over the course of a decade with no known negative effects suggests that the risk assessment procedures in place have been successful in filtering out unsafe uses of GEOs (Medley 1996). In this chapter we examine these procedures in some detail to provide an understanding of the basis for the information required and applicability to large scale, commercial release.
The first field trial of a transgenic organism was of a bacterium designed to protect plants against frost. This test conducted in 1986, which successfully demonstrated the efficacy of the product and produced no adverse environmental effects (Levin and Strauss 1993), has been followed by over 2000 plant and microorganism field applications in the terrestrial environment (GeneExchange 1994a, 1994b; Beck and Ulrich 1993; PIP 1993; Chasseray and Duesing 1993; Levin 1996). However, these trials were, in fact, all limited scale trials. At issue is Whether the assessments conducted would assure safety for commercial application.
Although the basic questions that need to be asked remain the same, (see Chapter one, Points to consider), USEPA's Final Rule for commercial products (USEPA 1997) differs from earlier USEPA regulations by establishing alternate pralternate procedures requiring different levels of information from the applicant depending on the nature of the organism to be tested. The establishment of three levels of application, each with differing information requirements, reflects a desire to minimize impact on applicants while maximizing protection of public health and the environment. This change is possible because experience has demonstrated the lack of adverse effects of certain applications. A similar trend can be seen in the USDA regulations (see Chapter one). However, the basic requirement for an information set fulfilling the needs of a risk assessor based on a data base for the risk assessment is not changed.
To meet the requirements explicit in Points to Consider (Chapter 1), applicants and agencies must follow several steps before an open field trial or product registration is approved. First, the developer must present data describing the test organism and its potential ecological effects. In this chapter we will examine l examine the information requirements and their relation to large scale environmental issues underlying the need for risk assessment.
Notwithstanding the direct purpose of the assessment, or which agency is conducting the evaluation, all ecological risk assessment efforts require determination of two critical aspects of the proposed trial: the exposure anticipated and the hazard involved. Evaluating exposure involves determining what other organisms will be present at the test site and in what density. Evaluating hazard requires knowledge of how indigenous organisms, and other aspects of the local ecology, are likely to be affected by the test organisms.
Thus evaluating levels of hazard and exposure requires risk assessors to consider potential adverse effects. The level of scrutiny will depend upon how well known the GEO is and the extent to which it has been genetically manipulated. The National Academy of Sciences (AS 1989) and an independent group of scientists (Tiedje et al.edje et al. 1989) have characterized this criterion in terms of the organism's "familiarity." Many other workers (Levin and Strauss), national and international groups (National Academy of Science (NAS,a,b,c,) have described the process of risk assessment in general and as related to GEMs in particular. The National Academy of Science, in two separate reports (AS b,c) stressed the importance of familiarity - the amount of background information available about the parent organisms and the host (ie the GEM). In their view, there is a risk continuum. On one end of this continuum are easily contained, well understood host organisms such as cows with introduced bovine growth hormone. At the high risk end are less well-known, less easily controllable organisms such as microbes engineered for bioremediation.
It is clear that the more information available about the parent and GEM microorganisms, the greater confidence one can have when predicting the behavior and overall effects of the GEM. Tiedge et Tiedge et al dissect this general statement into a series of attributes to consider and discussed the need for more scrutiny when less information is available about any particular attribute. Thus, if the phenotypic characteristics of the host microorganisms are well known and the inserted DNA is well characterized and/or from a closely related species, less concern is generated and a lesser degree of scrutiny is required. The specific information requirements (eg how detailed the description of the organism or method of DNA insertion should be), however, need be described only in very general terms.
Determining the risk of using a particular microorganism -naturally occurring or genetically engineered- in any environmental setting requires a synthesis of potential adverse effects associated with the specific agent (ie. HAZARD) and the level of EXPOSURE of populations sensitive to the identified Hazard. The probability and magnitude of the effect and the options available to the regulatory bodyatory body determine the risk management procedures employed. It is important to keep in mind that the potential risk associated with the use of any microorganism in environmental situations is related to the phenotype and genotype of the microorganism. Alteration of the genotype by one or a few genes may or may not affect the potential for environmental effect, and that effect may be positive, negative or neutral.
With a product such as a well studied bacterium engineered to contain a marker gene, this could mean simply asking if there are other closely related organisms for which it could be mistaken. For example, endowing a bacterium with the ability to utilize lactose to permit detection on a specific medium could result in misidentification of that organism by persons using standard taxonomic tests based on this property, or, alternatively, the ability to multiply in presence of lactose and thus fill a new niche. Concern about gene transfer can easily be understood. A gene new to a speciesa species can spread within the species or to it's relatives. The possibility of mutation before or after transfer must also be considered. Gene transfer to related species is less of a problem in the US where there are relatively few related species to most organisms of commercial interest but more of a problem where diversity is much greater. In some areas - Costa Rica in particular - this is a local issue of major importance. Costa Rica has more species within it's boundaries than the entire US. Gene stability and expression issues are, of course, closely related to gene transfer questions.
Critical issues in determining risk may include taxonomic position and life history. For example the more closely the test GEO is related to indigenous organisms the greater the likelihood of gene exchange in the case of bacteria or of cross breeding in plants.
This means that introduced transgenic plants must be evaluated for their ability to cross-pollinate with native species- IE are the IE are there related plants in the area and will the pollen reach and fertilize them. Examples of gene transfer between plants resulting in adverse effects are well known (eg pearl millet genes to wild millet, yielding shibra, a weed, and sorghum to Johnson Grass, resulting in a more aggressive type of Johnson grass).
Examples of special conditions that may be imposed include confining the trial to a limited area, requiring protective physical constraints, or establishing strict monitoring and mitigation procedures. In the case of microbes, unless specific techniques resulting in lack of ability to exchange genetic material naturally are employed, gene exchange must be assumed, and the consequences considered. In the terrestrial environment, physical containment may include berms to catch run-off, fences to restrict entry, and bags over flowers to trap pollen. To ensure worker protection the use of such items as hoods and respirators may be required. These practices, obviously, are limited to imited to small scale releases.
The safety record is excellent for terrestrial applications of biotechnology products. There have been over 4000 releases world-wide over more than a decade, and none has resulted in observed adverse effects. This rate of success has been achieved without legislation specifically targeting GEOs and with each agency using its own protocols and organizational structure. This positive outcome can best be attributed to three factors: a bias toward conservative decision making on the part of risk assessors and managers in all agencies; the comparatively high degree of information available about terrestrial products and ecology; and the relative safety of biotechnology products compared, for example, to toxic substances and other environmental contaminants.
In all cases, however, after exposure and hazard have been characterized by risk assessors, the results are passed on to a risk manager for final review and disposition. These findings provide a basiside a basis for the manager to reach a decision. The manager may: (1) deny the application; (2) request additional data; (3) require a change in the product; (4) approve the application with special conditions attached; or (5) approve the application as presented.
The EPA Points to Consider and the elements covered in the USDA Application Form are fundamentally the same and were developed by groups of scientists to assure that all pertinent risk assessment information is available to the reviewer (Table 1). The differences between the two (e.g. Taxonomic position, toxicity, production process) reflect emphasis on microbes for EPA and plants for USDA and emphasis on industry by EPA. A comparison of general biotechnology risk assessment protocols promulgated by other nations and international organizations with those set out in the Points to Consider also finds substantial agreement. The scope of information an applicant for a permit must present at present as a basis for review is remarkably similar. A recent survey of nations in the Organization for Economic Cooperation and Development (OECD 1995) demonstrated a 90% agreement among their equivalents of the Points to Consider. One major difference is that Canada and Australia place more emphasis on requirements regarding the degree of familiarity with the host organism than do other nations and that Canada requires review of naturally occurring microorganisms.
An important consideration is that risk assessment protocols are generally designed for small-scale experimental trials. Requirements for assessing the risks involved in large-scale commercial uses of transgenic organisms are less well defined. It cannot be assumed that data requirements for field tests will suffice for commercial releases, or that results from small-scale trials can be extrapolated to aid in understanding the risks involved in large-scale activities (Burke et al. 1994; Snow 1997). For example, if a food crop gene crop genetically engineered to express production of a bacterial toxin that serves as a pesticide is planted on a commercial scale, there is a much greater risk that insects will develop resistance than would be present in a small-scale trial, since the time scale and the size of the population exposed would be much larger.
A recent international symposium considered issues associated with large-scale uses of transgenic plants (Burke et al. 1994). Participants discussed the data that should be required and the risk assessment methodologies that would be most appropriate to insure credible evaluation of commercial releases. Minimizing potential impact on biodiversity, biogeochemical cycles, community structure, and other ecological factors was a primary focus. Although it was generally felt that engineered plants, because of their physical characteristics (nonmotile, controtile, controllable), need not be considered major hazards, the necessity of developing specific tests to identify such potential adverse effects as invasiveness and persistence was noted.
Crawley et al. (1993) have described an experimental approach that can be used to demonstrate the lack of invasiveness of a particular plant relative to the parent plant. This method of collecting and analyzing data allows risk assessment decisions to be based on actual data on a case-by-case basis. Generalization may be possible across closely related species and similar locations.
Appraising the effects of past dispersals and invasions of exotic organisms into new environments, however, can provide useful risk assessment information. For example, there have been many deliberate introductions of nonindigenous species for various purposes (Howarth 1991). Data generated by these introductions can be analyzed to glean insights into the influence of new species on existing populations. Howarth (199Howarth (1991) describes case studies of the introduction of insects (eg. Levuana iridescens), snails (eg. Gonaxis kibweziensis), fish (Gambusia spp), and plants (eg. Tibulus terrestris). Among the critical characteristics he identifies that limit or enhance environmental effects are persistence, habitat range, phenotypic plasticity, host availability in the case of parasites, and behavior. Although these factors are drawn from analysis of non-transgenic introductions, Howarth (1991) notes that they are almost identical to those essential for assessment of the risks associated with the release of genetically engineered microbes, plants, and fish.
Other examples of introductions, whether intentional or unintentional, and whether the organism persisted or suffered local extinction, indicate the potential effects of introductions into the marine environment. Global sea-going commercial activities often carry species outside their native habitats. Generally, organly, organisms are adapted to their environment and will not persist in another setting. Occasionally a species transported to a foreign site will find an ecological niche in which it can establish itself (Iwama et al. 1992). In most terrestrial cases no effects have been noted; in a few cases beneficial effects occur; in a very few cases, adverse effects are observed (Simberloff 1985; Sharples 1991). In his survey of biological invasions, Holdgate (1986) notes that data support the pattern that 10% of exotic species introductions may succeed, and of these only 10% may produce adverse effects. However, invasive exotic species that do establish themselves, though rare, can cause dramatic ecological disturbance. Well known examples include kudzu in the southeastern United States and the brown snake in Guam (Howarth 1991). In contrast, the introduction of the Vedalia beetle in California is an example of a successful, beneficial introduction of an exotic species. In this case, crop damage caused by indigenous hernous herbivorous pests was significantly reduced.
Despite the analogy that is often drawn between introductions of exotic and genetically engineered organisms, differences exist that must be taken into account. The nature and extent of the impact will depend largely upon the phenotypic expression of the inserted genes. Co-Metabolism (Table 2) provides a case in point. Microbes capable of increased mineralization or production of diverse metabolites will multiply if provided with specific Co-Substrates. If this ability is introduced in a related species, ability to establish in new environments with subsequent production of byproducts will occur.
Evidence from ecological literature suggests that generalized nutritional requirements and broader tolerances to environmental conditions coupled with ability to exploit additional resources will result in successful establishment of introduced organisms.
When considering implications og implications of large scale release vis a vie biogeochemical issues, the enormous magnitude of global elemental cycles must also be considered. The Sulfur cycle, which circulates 600 tons of Sulfur annually between sediments and the atmosphere varying the chemical between elemental Sulfur, H2S, SO2, and S04 and the Carbon cycle which has over 18,000,000 billion tons of carbon in its 7 reservoirs (or pools) are two examples. While 99.75% of Carbon is stored in limestone, shale and other sedimentary deposits, over 45 billion tons exists in forms which could be affected by microbial action. Thus, while only 0.018% of carbon is present as dead vegetative matter, this represents approximately 3,400 billion tons. A small change in this reservoir could have significant environmental implications.
The establishment of monitoring procedures for persistence and spread of a GEO is required as part of the information package f package for approval of the release. In general the procedures involve development or application of already existing techniques for identifying the organism and enumerating in environmental samples. These procedures have been developed and are, in most cases, well accepted. A problem exists with bacteria which may convert to a Viable, non Cultural (VAC.) phase in nature. VAC. microbes have lost the ability to multiply in conventional laboratory situations. Use of standard laboratory media will indicate no microorganisms present, while extremely time consuming, logistically difficult tests demonstrate that some viable microorganisms are present. It has been shown that the VAC. organisms are capable of reversion to normal state when exposed to the appropriate conditions.
For example, Linder and Oliver demonstrated that conversion of Vibrio vulnificus to VAC. occurred after 24 days in a microcosm. They studied the occurrence of VAC. in V. vulnificus and, for comparison, that of Eat of Escherichia coli in artificial-seawater microcosms at 5 C. They reported that while total counts remained constant, comparison to plate counts suggested nonculturability by day 24. In contrast, direct viable counts indicated that the cells remained viable throughout 32 days of incubation. As an indication of the metabolic changes that occurred as cells entered the state of nonrecoverability, membrane fatty acid analyses were performed. At the point of nonculturability of V. vulnificus , the major fatty acid species (C16) had decreased 57% from the Time 0 level, concomitant with the appearance of several short-chain acids. Although the bacteria were still recoverable, a similar trend was observed with E. coli. Mouse infectivity studies with the vibrio suggested loss of virulence. Total cell counts were monitored by acridine orange epifluorescence, metabolic activity by direct viable counts, and culturability by plate counts on selective and nonselective media.
Rice Rice and Oliver, noted that the marine barophile CNPT-3 after stress by starvation exhibited a significant reduction in cell size and biovolume. The starved cells demonstrated a greater tendency to attach at in situ pressure (400 atm) and temperature (5 C) than at 1 atm, and the extent of attachment increased with increasing duration of starvation. However, Magarinos et al, looking at a variety of biochemical, physiological and serological properties, including LD50, of Pasteurella piscida observed no effect.
Similar data is available for many gram negative bacteria. The procedures for monitoring VAC. microbes are tedious. Thus, additional research into monitoring procedures is required to assure that the released organisms are not spreading and/or are declining in number.
Microorganisms are transported by a variety of routes such as: wind, water, mechanical, means, and biological vectors.
ectors.Effectiveness of aerial dispersal (aerosols) is primarily influenced by the way the microbe is introduced to the atmosphere and its ability to survive environmental stress (e.g. desiccation, UV light). Soil particles are raised by wind or when the ground is heated. Microorganisms adhere to insects or mites which can then be dispersed by wind currents.
The hydrology of soil water and groundwater flow and the proximity of open bodies of water such as lakes, rivers, streams, and water supplies for irrigation are among the primary physical determinants of water-borne dispersal from a terrestrial experimental plot. Rain or irrigation water also serve as a means of transport. Microorganism are dispersed by rain or irrigation water that washes the surfaces of plants. Rain splashes can throw microbial laden droplets from plant surfaces into the air.
Microorganisms can be dispersed over short and long distances through the successive handling of plants, through the use of contaminated tools and other equipment, through the transport of contaminated soil, plants, seeds and nursery stock. Likewise, any activity that generates aerosols represents a potential route of dispersal for microorganisms contained in the aerosol droplet. Mechanical disturbances such as tilling introduces microorganisms into the air (along with the soil).
In nature, animals can serve as vectors for microorganisms. Bacteria may be transported by browsing and burrowing mammals, soil arthropods, earthworms, and insects. Insects may carry micro-organisms on the surfaces of their bodies and deposit them on plant surfaces or in the wounds that insects make on the plants during feeding. .
A risk assessment is not complete withoutomplete without a discussion of contingency plans to contain the released organisms and/or to mitigate any observed untoward effects. This topic has been discussed in detail for terrestrial applications (Cask, 1992, Vidaver and Stotzky, 1992). It has been pointed out that containment in the absolute sense is not possible, but that confinement in the sense of restricting dispersal and growth is possible. Techniques to accomplish confinement include altering the site (berms, ditches), use of chemicals to lower population densities in the case of microorganisms, or, in the case of plants, destruction of organisms or reproductive portions of the organisms.
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| Organism | Co substrate | Growth Substrate | Product | Reference |
| Soil organism | Diazinona | root exudates | Increased mineralization | Baker & Woods, 1977 |
| Soil organism | Parathionb | root exudates | Increased mineralization | Hsu & Bartha, 1979 |
| < | ||||
| Mycobacterium vaccae | cyclohexane | propane | Cyclohexanone | Beam & Perry, 1974; Perry, 1979 |
| Nocardia corallina | toluene | hexadecane | 2,3 dihydroxbenzoate;alpha methylmuconate | Jamison et al., 1969; 1971 |
| Pseudomonas putida | 2 chloronaththalene | napthalene | Chloro-2-hydroxyl- oxohexadieneoate | Morris & Barnsley, 1982 |
| Pseudomonas sp. B13 | Isomeric cresols | phenol | Methyl catechols | Knackmuss & Hellwig, 1978 |
| Pseudomonas sp. B13 | Various chlorophenols | phenol | Various chlorocatechols | Knackmuss & Hellwig, 1978 |
a. Diazon = 0,0-diethyl-1-0-[2-isopropyl-4-methyl-6-pyrimidyl]phosphorothioate
b. Parathion = 0,0-diethyl-1-0-p-nitropheion = 0,0-diethyl-1-0-p-nitrophenyl-1-phosphorothioate
Baker, P.B. and D.R. Woods. Co-metabolism of the ixodicide Amitraz. J. Appl. Bacteriol., 42:187-196, 1977.
Beam, H.W. and J.J. Perry. Microbial degradation of cycloparaffinic hydrocarbons via co-metabolism and commensalism. J. Gen. Microbiol., 82:263-169, 2974.
Hsu, T.-S. and R. Bartha. Accelerated mineralization of two organophosphate insecticides in the rhizosphere. Appl. Environ. Microbiol., 37:36-41, 1979.
Jamison, V.W., R.L. Raymond and J.O. Hudson. Microbial hydrocarbon co-oxidation. III. isolation and characterization of an ,-'-dimethyl-cis, cis-muconic acid - producing strain of Nocardia corallina. Appl. Microbiol., 17:853-856, 1969.
Jamison, V.W., R.L. Raymond and J.O. Hudson. Hydrocarbon co-oxidation by Nocardia corallina V-49. Developments in Industrial Microbiol., 12:99-105, 1971.
Knackmuss, H.-J. and M. HellwH.-J. and M. Hellwig. Utilization and cooxidation of chlorinated phenols by Pseudomonas sp. B13. Arch. Microbiol., 117:1-7, 1978.
Perry, J.J. Microbial cooxidations involving hydrocarbson. Microbiol. Rev., 43:59-72, 1979.