Previous European guidance for environmental risk assessment of genetically modified plants emphasized the concepts of statistical power but provided no explicit requirements for the provision of statistical power analyses. Similarly, whilst the need for good experimental designs was stressed, no minimum guidelines were set for replication or sample sizes. Furthermore, although substantial equivalence was stressed as central to risk assessment, no means of quantification of this concept was given. This paper suggests several ways in which existing guidance might be revised to address these problems. One approach explored is the `bioequivalence' test, which has the advantage that the error of most concern to the consumer may be set relatively easily. Also, since the burden of proof is placed on the experimenter, the test promotes high-quality, well-replicated experiments with sufficient statistical power. Other recommendations cover the specification of effect sizes, the choice of appropriate comparators, the use of positive controls, meta-analyses, multivariate analysis and diversity indices. Specific guidance is suggested for experimental designs of field trials and their statistical analyses. A checklist for experimental design is proposed to accompany all environmental risk assessments.

Genetically modified plants (GMPs) may soon be cultivated commercially in several member countries of the European Union (EU). According to EU Directive 2001/18/EC, post-market monitoring (PMM) for commercial GMP cultivation must be implemented, in order to detect and prevent adverse effects on human health and the environment. However, no general PMM strategies for GMP cultivation have been established so far. We present a conceptual framework for the design of environmental PMM for GMP cultivation based on current EU legislation and common risk analysis procedures. We have established a comprehensive structure of the GMP approval process, consisting of pre-market risk assessment (PMRA) as well as PMM. Both programs can be distinguished conceptually due to principles inherent to risk analysis procedures. The design of PMM programs should take into account the knowledge gained during approval for commercialization of a specific GMP and the decisions made in the environmental risk assessments (ERAs). PMM is composed of case-specific monitoring (CSM) and general surveillance. CSM focuses on anticipated effects of a specific GMP. Selection of case-specific indicators for detection of ecological exposure and effects, as well as definition of effect sizes, are important for CSM. General surveillance is designed to detect unanticipated effects on general safeguard subjects, such as natural resources, which must not be adversely affected by human activities like GMP cultivation. We have identified clear conceptual differences between CSM and general surveillance, and propose to adopt separate frameworks when developing either of the two programs. Common to both programs is the need to put a value on possible ecological effects of GMP cultivation. The structure of PMM presented here will be of assistance to industry, researchers, and regulators, when assessing GMPs during commercialization.

The prospect of the deliberate environmental release of genetically manipulated microorganisms has given rise to a great deal of polemic. Amongst the rational scientific concerns are those concerned with the fate of the released bacteria, the fate of the recombinant genes that they carry, the selective pressures acting upon them in different environmental situations and the long term effects on the environment and human health. All recombinant DNA is carried by vectors (plasmids, transposons or bacteriophage or remnants of these). Thus the way in which recombinant constructions are made may itself lead to potential biosafety concerns, irrespective of the host bacterium and the recombinant DNA fragment of primary interest. The purpose of the present review is to assess progress in improved vector design aimed at eliminating risks due to the way recombinant vectors are constructed. Improved vector constructions include the avoidance of the use, or removal, of antibiotic resistance genes, the use of defective transposons rather than plasmids in order to reduce horizontal transfer and the development of conditionally lethal suicide systems. More recently, new site-specific recombination systems have permitted transposon vectors to be manipulated following strain construction, but before environmental release, so that virtually all recombinant DNA not directly involved in the release experiment is eliminated. Such bacteria are thus pseudo-wild type in that they contain no heterologous DNA other than the genes of interest.

In the European Union, the definition of a GMO is technology-based. This means that a novel organism will be regulated under the GMO regulatory framework only if it has been developed with the use of defined techniques. This approach is now challenged with the emergence of new techniques. In this paper, we describe regulatory and safety issues associated with the use of oligonucleotide-mediated mutagenesis to develop novel organisms. We present scientific arguments for not having organisms developed through this technique fall within the scope of the EU regulation on GMOs. We conclude that any political decision on this issue should be taken on the basis of a broad reflection at EU level, while avoiding discrepancies at international level.

The bucket and the searchlight are metaphors for opposing theories of the growth of scientific knowledge. The bucket theory proposes that knowledge is gained by observing the world without preconceptions, and that knowledge emerges from the accumulation of observations that support a hypothesis. There are many problems with this theory, the most serious of which is that it does not appear to offer a means to distinguish between the many hypotheses that could explain a particular set of observations. The searchlight theory proposes that preconceptions are unavoidable and that knowledge advances through the improvement of our preconceptions – our hypotheses – by continuous criticism and revision. A hypothesis is a searchlight that illuminates observations that test the hypothesis and reveal its flaws, and knowledge thereby increases through the elimination of false hypotheses. Research into the risks posed by the cultivation of transgenic crops often appears to apply the bucket theory; many data are produced, but knowledge of risk is not advanced. Application of the searchlight theory, whereby risk assessments test hypotheses that transgenic crops will not be harmful, seems to offer a better way to characterise risk. The effectiveness of an environmental risk assessment should not be measured by the size of the bucket of observations on a transgenic crop, but by the power of the risk hypothesis searchlights to clarify the risks that may arise from cultivation of that crop. These points are illustrated by examples of hypotheses that could be tested to assess the risks from transgenic crops and their hybrids becoming weeds or invading non-agricultural habitats.

Genes of Bacillus thuringiensis var. kurstaki (Berliner) that encode lepidopteran-specific toxins were engineered into maize for protection against the European Corn Borer, Ostrinia nubilalis (Hbn.). Recent data suggest that Lepidoptera may be negatively affected, if maize pollen contains high amounts of Bt toxin and is diposited on host plants near maize fields. Monitoring the environmental effects of commercial Bt maize fields requires effective use of limited financial and logistical resources. The aim of this study was to develop and apply tools for selecting relevant herbivore species for the field monitoring of environmental Bt toxin effects via pollen deposition. We first present a theoretical selection tree based on “risk index of Bt pollen for herbivores” (IBtp). Our index consists of five classes from zero (not relevant) to four (highly relevant) derived from data on potential temporal and spatial coincidence of pollen exposure (A), feeding mode (B), susceptibility to lepidopteran-specific Bt toxins (C) and hazard to rare and/or endangered species (“Red List”) (D). We then screened the Macrolepidoptera database LEPIDAT to identify relevant species in Germany. Finally, we also applied the index to species found in a local biocoenotic field study (Bonn, Western Rhineland, Germany). Approximately 7% of the German Macrolepidoptera species mainly occur in farmland areas and were selected as being potentially affected by Bt pollen exposure. Of these species, 14% (= 1% of total) were found to be potentially exposed on a regional scale. The combination of IBtp and database screening enables us to pre-select species for monitoring purposes.

We compared some fitness components of the wild squash species Cucurbita pepo spp. ovifera var. texana (C. texana) and three generations of hybrids (F1, BC1, and BC2) between C. texana and commercial transgenic squash CZW-3 over three consecutive years under field conditions of low (LDP) and high disease pressure (HDP) by Cucumber mosaic virus (CMV), Zucchini yellow mosaicvirus (ZYMV) and Watermelon mosaic virus (WMV). Transgenic squash CZW-3 expresses the coat protein (CP) genes of CMV, ZYMV, and WMV, and is resistant to these three aphid-borne viruses. Across all HDP trials, transgenic BC1 and BC2 hybrids expressing the three CP genes grew more vigorously, displayed resistance to CMV, ZYMV, and WMV, and produced a greater number of mature fruits and viable seeds than nontransgenic hybrid segregants and C. texana. Transgenic F1 hybrids behaved similarly to BC1 and BC2 hybrids but grew less vigorously than C. texana. In contrast, across all LDP trials, C. texana outperformed the transgenic and nontransgenic hybrid segregants. Further, only one back cross was necessary to recover individuals with most of the C. texana characteristics and yet maintain virus resistance. Our data suggest that C. texana acquiring CP transgenes upon hybridization and introgression could have a selective advantage if CMV, ZYMV, and WMV are severely limiting the growth and reproductibility of wild squash populations.

The intrinsic toxicity of lepidopteran-active Bt proteins necessitates assessment of non-target risks associated with environmental release of transgenic crops expressing these proteins. Principles of ecological risk assessment provide a means for assessing non-target risks when information regarding exposure to the toxin and species-specific effects are lacking. This is shown for the case of Bt Cry1F maize release in Japan, where off-field pollen dissemination and effect on butterfly species is of concern. The specific ecological entity of concern for the assessment of the non-target impact of Cry1F maize pollen was Yamato-shijimi (pale grass blue butterfly), Pseudozizeeria maha (Kollar), a commonly occurring, susceptible species. Yamato-shijimi is widely adapted in Japan where it occurs in both rural and metropolitan settings, corresponding to the distribution and habitat of katabami (Oxalis corniculata (L.)), the larval host plant. The northern extent of Yamato-shijimi habitat lies to the south of major maize production regions in Japan, but exposure may occur elsewhere where maize and Yamato-shijimi co-occur. Screening level assessment of potential adverse effects to Yamato-shijimi in the field environment considered the probability for spatial-temporal co-occurrence of the life stages of concern (1st and 2nd instars) and the stressor (Cry1F protein expressed in maize pollen) at environmentally relevant concentrations. In the event of exposure to maize pollen, early instars of Yamato-shijimi feed exclusively on the underside of katabami leaves, which further limits the portion of the butterfly population that would be exposed. Projected levels of exposure to Cry1F pollen are below the toxicity level of concern and, thus, indicate negligible risk. Most sensitive species characterization (intergenera sensitivity) similarly shows negligible risk to other Japanese butterfly species of concern when distributed beyond the maize field or field margin.

Release of transgenic insect-resistant crops creates the potential not only for the insect pest to evolve resistance but for the escape of transgenes that may confer novel or enhanced fitness-related traits through hybridization with their wild relatives. The differential response of diamondback moth (Plutella xylostella) populations in eastern and western Canada to Bt-producing (GT) Brassica napus and the potential for enhanced fitness of GT B. napus and weedy GT Brassica rapa × B. napus hybrid populations (F1, BC1, BC2) were studied. Comparative bioassays using neonates and 4th instars showed that GT B. napus and GT B. rapa × B. napus hybrids are lethal to larvae from both populations. No measurable plant fitness advantage (reproductive dry weight) was observed for GT B. napus (crop) and GT B. rapa × B. napus hybrid populations at low insect pressure (1 larva per leaf). At high insect densities (>10 larvae per leaf), vegetative plant weight was not significantly different for GT B. napusand non-GT B. napus, whereas reproductive plant weight and proportion of reproductive material were significantly higher in GT B. napus. Establishment of the Bt trait in wild B. rapa populations may also increase its competitive advantage under high insect pressure.

Monitoring for escape of genetically modified (GM) oilseed rape (Brassica napus) during transport can be performed by means of roadside evaluations in areas where cultivation of this GM crop is not conducted, such as in Japan. We performed a survey of oilseed rape plants growing along a 20-km section of Japan's Route 51, one of the main land transportation routes in central Japan for imports of GM oilseed rape from the Port of Kashima into Keiyo District. Oilseed rape plants were found each year, but the number of plants varied substantially during the three years of our study: 2162 plants in 2005, 4066 in 2006, and only 278 in 2007. The low number in 2007 was probably caused by roadwork. Herbicide-resistant individuals were detected in the three consecutive years (26, 8, and 5 individuals with glyphosate resistance), but glufosinate-resistant plants (9 individuals) were detected only in 2005. The roadside plants occurred mainly along the inbound lane from Kashima to Narita. These plants are likely to have their origin in seeds spilled during transportation of cargo from the port, since there are no potential natural seed source plants for B. napus near Route 51. This is the first detailed report on the transition and distribution of herbicide-resistant oilseed rape plants following loss and spillage along Japanese roads.

There is much discussion of the probability of transgene flow from transgenic crop varieties to landraces and wild relatives in centers of origin or diversity, and its genetic, ecological, and social consequences. Without costly research on the variables determining gene flow, research on transgene frequencies in landrace (or wild relative) populations can be valuable for understanding transgene flow and its effects. Minimal research requirements include (1) understanding how farmer practices and seed systems affect landrace populations, (2) sampling to optimize Ne/n (effective /census population size), (3) minimizing variance at all levels sampled, and (4) using Ne to calculate binomial probabilities for transgene frequencies. A key case is maize in Mexico. Two peer-reviewed papers, based on landrace samples from the Sierra Juárez region of Oaxaca, Mexico, reached seemingly conflicting conclusions: transgenes are present (Quist and Chapela, 2001, Nature414: 541–543; 2002, Nature416: 602) or “detectable transgenes” are absent (Ortiz-García et al., 2005, Proc. Natl. Acad. Sci. USA102: 12338–12343 and 18242). We analyzed these papers using information on Oaxacan maize seed systems and estimates of Ne. We conclude that if Quist and Chapela’s results showing presence are accepted, Ortiz-García et al.’s conclusions of no evidence of transgenes at detectable levels or for their introgression into maize landraces in the Sierra de Juárez of Oaxaca are not scientifically justified. This is because their samples are not representative, and their statistical analysis is inconclusive due to using n instead of Ne. Using estimates of Ne based on Ortiz-García et al.’s n, we estimate that transgenes could be present in maize landraces in the Sierra Juárez region at frequencies of ~1–4%, and are more likely to be present in the 90% of Oaxacan landrace area that is not mountainous. Thus, we have no scientific evidence of maize transgene presence or absence in recent years in Mexico, Oaxaca State, or the Sierra Juárez region.

This review addresses the possible ecological effects of transgenic plants on micro-organisms in the field, hence, in the phytosphere and in the soil matrix. The important steps involved in the interaction between plant DNA and bacteria and the factors that influence the horizontal gene transfer (HGT) process will be discussed. HGT is a process in which two partners are involved, even if indirectly. In the first section, aspects concerning bacteria, such as their physico-chemical, biological and genetic characteristics, are described. Parameters affecting transgenic DNA fate in the environment are described in the second section. Subsequently, terrestrial habitats are evaluated in terms of their capacity to favor horizontal gene transfer. Finally, we focused on several studies in order to evaluate possible perturbations of soil bacterial community composition due to cultivation of transgenic plants in the field.

Over the last 20 years, there has been much research aimed at improving environmental risk assessment of transgenic crops. Despite large amounts of data, decisions to allow or prohibit the release of transgenic crops remain confused and controversial. We argue that part of the reason for confusion is the lack of clear definitions of components of the environment that should be protected, and, as a consequence, there is no way to judge the relevance of data collected under the auspices of ‘environmental risk assessment’. Although this criticism applies to most aspects of environmental risk assessment of transgenic crops, it is most pertinent to effects that might result from an increase in plant fitness, often referred to as increased weediness. Environmental risk assessment of weediness is regarded as complicated: an increase in the fitness of a transgenic plant compared with non-transgenic counterparts will be the result of an interaction between the altered plant phenotype and an enormous number of environmental variables. This has led to the idea that risk assessment of weediness needs to “understand” these interactions, with the implication that exhaustive data are required. Here we argue that environmental risk assessment of the weediness of transgenic plants need not be complicated. Analysis of the conditions that must be met for increased weediness to occur suggests a series of studies that starts with simple tests in the laboratory under “worst case” assumptions, and becomes increasingly complex and realistic should the simpler studies not indicate negligible risk with sufficient certainty. We illustrate how the approach might work for assessing the risks of increased weediness using the example of possible introgression of a gene for Turnip mosaic virus (TuMV) resistance from oilseed rape to certain wild Brassica species.

The monitoring of genetically modified organisms (GMOs) after deliberate release is important in order to assess and evaluate possible environmental effects. Concerns have been raised that the transgenic crop, Bt maize, may affect butterflies occurring in field margins. Therefore, a monitoring of butterflies was suggested accompanying the commercial cultivation of Bt maize. In this study, baseline data on the butterfly species and their abundance in maize field margins is presented together with implications for butterfly monitoring. The study was conducted in Bavaria, South Germany, between 2000–2002. A total of 33 butterfly species was recorded in field margins.A small number of species dominated the community, and butterflies observed were mostly common species. Observation duration was the most important factor influencing the monitoring results. Field margin size affected the butterfly abundance, and habitat diversity had a tendency to influence species richness. Sample size and statistical power analyses indicated that a sample size in the range of 75 to 150 field margins for treatment (transgenic maize) and control (conventional maize) would detect (power of 80%) effects larger than 15% in species richness and the butterfly abundance pooled across species. However, a much higher number of field margins must be sampled in order to achieve a higher statistical power, to detect smaller effects, and to monitor single butterfly species.

Hybridization and introgression between crops and wild relatives may have important evolutionary and ecological consequences such as gene swamping or increased invasiveness. In the present study, we investigated hybridization under field conditions between crop lettuce (Lactuca sativa) and its wild relative prickly lettuce (L. serriola), two cross-compatible, predominantly autogamous and insect pollinated species. In 2003 and 2004, we estimated the rates of hybridization between L. sativa and L. serriola in close-to-reality field experiments carried out in two locations of Northern Switzerland. Seeds set by the experimental wild plants were collected and sown (44 352 in 2003 and 252 345 in 2004). Progeny was screened morphologically for detecting natural hybrids. Prior to the experiment, specific RAPD markers were used to confirm that morphological characters were reliable for hybrid identification. Hybridization occurred up to the maximal distance tested (40 m), and hybridization rates varied between 0 to 26%, decreasing with distance. More than 80% of the wild plants produced at least one hybrid (incidence of hybridization, IH) at 0 m and 1 m. It equaled 4 to 5% at 40 m. In sympatric crop-wild populations, cross-pollination between cultivated lettuce and its wild relative has to be seen as the rule rather than the exception for short distances.

Recapitulating how genetic modification technology and its agro-food products aroused strong societal opposition in the European Union, this paper demonstrates how this opposition contributed to shape the European regulatory frame on GM crops. More specifically, it describes how this opposition contributed to a de facto moratorium on the commercialization of new GM crop events in the end of the nineties. From this period onwards, the regulatory frame has been continuously revised in order to slow down further erosion of public and market confidence. Various scientific and technical reforms were made to meet societal concerns relating to the safety of GM crops. In this context, the precautionary principle, environmental post-market monitoring and traceability were adopted as ways to cope with scientific uncertainties. Labeling, traceability, co-existence and public information were installed in an attempt to meet the general public request for more information about GM agro-food products, and the specific demand to respect the consumers' and farmers' freedom of choice. Despite these efforts, today, the explicit role of public participation and/or ethical consultation during authorization procedures is at best minimal. Moreover, no legal room was created to progress to an integral sustainability evaluation during market procedures. It remains to be seen whether the recent policy shift towards greater transparency about value judgments, plural viewpoints and scientific uncertainties will be one step forward in integrating ethical concerns more explicitly in risk analysis. As such, the regulatory frame stands open for further interpretation, reflecting in various degrees a continued interplay with societal concerns relating to GM agro-food products. In this regard, both societal concerns and diversely interpreted regulatory criteria can be inferred as signaling a request – and even a quest – to render more explicit the broader-than-scientific dimension of the actual risk analysis.

Gene flow via seed or pollen is a basic biological process in plant evolution. The ecological and genetic consequences of gene flow depend on the amount and direction of gene flow as well as on the fitness of hybrids. The assessment of potential risks of transgenic plants should take into account the fact that conventional crops can often cross with wild plants. The precautionary approach in risk management of genetically modified plants (GMPs) may make it necessary to monitor significant wild and weed populations that might be affected by transgene escape. Gene flow is hard to control in wind-pollinated plants like beet (Beta vulgaris). In addition, wild beet populations potentially can undergo evolutionary changes which might expand their geographical distribution. Unintended products of cultivated beets pollinated by wild beets are weed beets that bolt and flower during their first year of planting. Weed beets cause yield losses and can delay harvest. Wild beets are important plant genetic resources and the preservation of wild beet diversity in Europe has been considered in biosafety research. We present here the methodology and research approaches that can be used for monitoring the geographical distribution and diversity of Beta populations. It has recently been shown that a century of gene flow from Beta vulgaris ssp. vulgaris has not altered the genetic diversity of wild Beta vulgaris L. ssp. maritima (L.) Arcang. in the Italian sugar beet seed production area. Future research should focus on the potential evolution of transgenic wild beet populations in comparison to these baseline data. Two monitoring models are presented describing how endpoints can be measured: (1) “Pre-post” crop commercialization against today's baseline and (2) “Parallel” to crop commercialization against GMP free reference areas/populations. Model 2 has the advantage of taking ongoing changes in genetic diversity and population dynamics into account. Model 1 is more applicable if gene flow is so strong that most areas/populations contain GMPs. Important traits that may change the ecology of populations are genes that confer tolerance to biotic and abiotic stress. An assessment of environmental effects can realistically only be based on endpoints and consequences of gene introgression, which may include economic values of biodiversity in littoral and other ecosystems containing wild beet. In general, there is still a great need to harmonize worldwide monitoring systems by the development of appropriate methods to evaluate the environmental impact of introgressed transgenes.

We have developed a screening method that can be used during the problem formulation phase of risk assessment to identify and prioritize non-target invertebrates for risk analysis with any transgenic plant. In previously published protocols for this task, five criteria predominated. These criteria have been combined by our method in a simple model which assesses: (1) the possible level of risk presented by the plant to each invertebrate species (through measurements of potential hazard and exposure, the two principal criteria); (2) the hypothetical environmental impact of this risk (determined by the currently known status of the species' population in the ecosystem and its potential resilience to environmental perturbations); (3) the estimated economic, social and cultural value of each species; and (4) the assessed ability to conduct tests with the species. The screening method uses information on each of these criteria entered into a specially designed database that was developed using Microsoft $^{\circledR}$ Access 2003. The database holds biological and ecological information for each non-target species, as well as information about the transgenic plant that is the subject of the risk assessment procedure. Each piece of information is then ranked on the basis of the value of the information to each criterion being measured. This ranking system is flexible, allowing the method to be easily adapted for use in any agro-ecosystem and with any plant modification. A model is then used to produce a Priority Ranking of Non-Target Invertebrates (PRONTI) score for each species, which in turn allows the species to be prioritized for risk assessment. As an example, the method was used to prioritize non-target invertebrates for risk assessment of a hypothetical introduction of Bacillus thuringiensis (Bt) Cry1Ac-expressing Pinus radiata trees into New Zealand.

Monitoring is a statutory requirement for the cultivation of genetically modified (GM) crops in the European Community. Questionnaires for farmers to report on observations of effects linked with the cultivation of GM crops can form a useful part of a monitoring regime. A questionnaire for GM maize (Zea mays L.) was designed, with questions focusing on potential effects related to the GM maize grown, as well as on background information about cultivation methods and on individual field situations. In this paper we present the methodological approach of the monitoring regime, the structuring of the data, and the contents and structure of the questionnaire. The statistical requirements and background for an appropriate evaluation and interpretation of the data are described. Results of interviews made from 2001 to 2005 are also presented. It is envisaged that this approach will be developed for monitoring other cultivated GM plants and traits, and may be applicable in monitoring certain non-farmed environments.