Risk Assessment Of Genetically Modified Plants Research Articles

Risk assessment of genetically modified zoysiagrasses (Zoysia japonica) in a zebrafish model

Genetically modified (GM) plants have been mainly advanced for the mass production of agricultural plants. However, there are concerns about GM crops which cause side effects on ecosystems and human beings. Zebrafish is a better in vivo model and possess the advantages; small size, large clutches, transparency, low cost, and physiological similarity to mammals. The objective of this study was to assess the safety of non-GM and GM grasses (Zoysia japonica) including JG21, JG21-MS1, JG21-MS2 in zebrafish embryos by examining the survival rates, heart-beating rate, yolk sac edema size, cell death, and morphology. The survival rates, heart-beating rates, and yolk sac edema sizes in non-GM and GM grass-treated zebrafish were similar change with the control group. And, all the groups did not show a significant difference within less than 1.0-fold in cell death by fluorescence levels. These results indicate that the zebrafish model is a useful and simple tool and can be used for risk assessment of GM plants.

Allergy Risk Assessment for Newly Expressed Proteins (NEPs) in Genetically Modified (GM) Plants

Based on experience and scientific advancements over the past two decades, a revised approach for the assessment of the allergenic potential of newly expressed proteins (NEPs) in genetically modified (GM) plants is warranted. NEPs are most often not native to the crop genome, and thus regulatory reviews of the safety of GM plants include an assessment of the allergenic potential of NEPs. International standards for the assessment of allergenicity first developed in the mid-1990s required a series of characterization studies to be conducted that are, to some extent, still applicable today to the risk assessment of GM plants, with most modern versions represented in the Codex Alimentarius. This standardized guidance on allergenicity assessments, including the required characterization studies, presented two primary challenges. First, there was (and still is) no defined and accepted model (animal or in vitro) for directly testing allergy potential. Second, bioinformatic analyses were prescribed using thresholds for hazard identification that were neither universal for all allergens nor tested prior to the implementation of requirements into guidance documents. Herein, risk assessment principles are applied to structure the assessment of the allergenic potential of NEPs. This allergy risk assessment is built on a foundation of: 1) identifying hazard by assessing similarity to known allergens, and 2) assessing exposure when a hazard is identified. Supplementary studies such as IgE binding may need to be performed in special cases. These recommended revisions to current approaches to the assessment of allergy potential are designed to ensure a realistic, case-by-case approach, and consider updated molecular biology, genomics, and bioinformatic techniques that were unavailable when earlier allergy risk assessment approaches were established.
 doi: 10.21423/jrs-v09i1mcclain

A new ecotoxicological test method for genetically modified plants and other stressors in soil with the black fungus gnat Bradysia impatiens (Diptera): current status of test development and dietary effects of azadirachtin on larval development and emergence rate

BackgroundFew suitable and standardized test methods are currently available to test the effects of genetically modified plants (GMP) on non-target organisms. To fill this gap and improve ecotoxicological testing for GMP, we developed a new soil ecotoxicological test method using sciarid larvae as test organisms.ResultsBradysia impatiens was identified as a candidate species. Species of the genus Bradysia occur in high numbers in European agroecosystems and B. impatiens can be reared in the laboratory in continuous culture. A functional basic test design was successfully developed. Newly hatched larvae were used as the initial life stage to cover most of the life cycle of the species during the test. Azadirachtin was identified as a suitable reference substance. In several tests, the effects of this substance on development time and emergence rate varied for different temperatures and test substrates. The toxicity was higher at 25 °C compared to 20 °C and in tropical artificial soil compared to coconut fiber substrate.Conclusions and outlookResults suggest that the developed test system is suitable to enter a full standardization process, e.g., via the Organisation for Economic Co-operation and Development. Such a standardization would not only assist the risk assessment of GMP, but could include other stressors such as systemic pesticides or veterinary pharmaceuticals reaching the soil, e.g., via spreading manure. The use of sciarid flies as test organisms supports recommendations of EFSA, which stressed the ecological role of flies and encouraged including Diptera into test batteries.

Explanatory note on the determination of newly expressed protein levels in the context of genetically modified plant applications for EU market authorisation

Genetically modified organisms are subject to a risk assessment and regulatory approval before entering the European market. According to legislation (Directive 2001/18/EC, Regulation (EC) No 1829/2003 and Regulation (EU) No 503/2013) and the EFSA guidance documents on the risk assessment of food and feed from genetically modified (GM) plants and on the environmental risk assessment of GM plants, applicants need to perform a molecular characterisation of any DNA sequence inserted in the GM plant genome. When a GM plant is designed to produce one or more newly expressed proteins, an aspect of the risk assessment process is to characterise these proteins and to reliably determine their levels in the GM plant tissues. A number of methods to measure protein expression levels are used and several experimental factors within these methods are of critical importance in order to obtain reliable results. This explanatory note provides details on key methodological aspects for the determination of newly expressed protein levels that should be considered and reported by applicants in order to harmonise the information in GM plant applications submitted to EFSA.

Are Limits of Concern a useful concept to improve the environmental risk assessment of GM plants?

BackgroundThe European Food Safety Authority (EFSA) has introduced a concept for the environmental risk assessment of genetically modified (GM) plants which foresees the definition of ecological threshold values defining acceptable adverse effects of the GM plant on the environment (Limits of Concern, LoC).MethodsWe analysed the LoC concept by scrutinising its feasibility with regard to important aspects of the environmental risk assessment. We then considered its relationship with protection goals, the comparative safety assessment and the stepwise testing approach. We finally discussed its usefulness for assessing long-term effects, effects on non-target organisms and species of conservation concern.ResultsThe LoC concept is a possible approach to introduce ecological thresholds into environmental risk assessment in order to evaluate environmental harm. However, the concept leaves many important aspects open. Thresholds for environmental harm for protection goals need spatial and temporal differentiation from LoCs used for ERA indicators. Regionalisation of LoCs must be provided for as biodiversity levels and protection goals vary across the EU. Further guidance is needed with respect to the consequences, in case LoCs are exceeded and a link needs to be established between environmentally relevant results from the comparative safety assessment and the LoC concept. LoCs for long-term effects have to be evaluated by long-term monitoring. LoCs for non-target organisms need to be discriminated according to the species and parameters assessed.ConclusionsThe overall LoC concept is considered useful if LoCs are further specified and differentiated. Although LoCs will finally be determined by political decisions, they should be based on scientific grounds in order to increase confidence in the conclusions on the safety of GM plants.

Risk assessment of new sequencing information on GM maize event DAS‐59122‐7

The EFSA Panel on Genetically Modified Organisms (GMO Panel) has previously assessed genetically modified (GM) maize DAS-59122-7 as a single event as well as part of several stacked events. These maize events were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health, and the environment. On 26 July 2016, the European Commission received from Pioneer new nucleic acid sequencing data on maize event DAS-59122-7 and updated bioinformatic analyses using the new sequencing data. The European Commission tasked EFSA to analyse these data and to indicate whether the previous conclusions of the GMO Panel on the previously assessed GM maize events remain valid. The GMO Panel used the appropriate principles described in its guidelines for the risk assessment of GM plants to analyse the received data. The new sequencing data indicated three base pair (bp) differences compared to the sequencing data originally provided; two located in a non-coding region of the insert and one located in the 5′ genomic flanking region. These base pairs reported as differences in the new nucleic acid sequencing data on maize event DAS-59122-7 had already been present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, the GMO Panel concludes that the original risk assessment of event DAS-59122-7 as a single and as a part of stacked events remains valid.

Risk assessment of new sequencing data of GM maize event GA21

In 2007, 2010 and 2011, the European Food Safety Authority (EFSA) Panel on Genetically Modified Organisms (GMO Panel) concluded the assessment of genetically modified (GM) maize GA21, MIR604 x GA21 and MIR604 x GA21 x Bt11. These were xfound to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 July 2015, the European Commission (EC) received from Syngenta new nucleic acid sequencing data on maize event GA21 and updated bioinformatic analyses using the new sequencing data. The EC tasked EFSA to analyse these data and to indicate whether the previous conclusions of the EFSA GMO Panel on the above-listed GM maizes remain valid. The EFSA GMO Panel used the appropriate principles described in its guidelines for the risk assessment of GM plants to analyse the received data. Compared with the sequencing data originally provided, the new sequencing data indicated a one-base pair addition in a non-coding region of the insert, a three-base pair deletion in the 3’ flanking region of the insert, and a difference in the number of functional copies of the mepsps expression cassette. These differences were only recently identified, but it was confirmed that they had been present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment remain valid. The bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, the GMO Panel concludes that the original risk assessment of event GA21 as a single event, and as a part of stacked events, remains valid.

Risk assessment of new sequencing data on GM maize event MIR604

In 2009 and 2010, the EFSA GMO Panel concluded the assessment of genetically modified (GM) maizes MIR604, MIR604 × GA21, MIR604 × Bt11 and MIR604 × GA21 × Bt11. These maizes were found to be as safe as their conventional counterparts and other appropriate comparators with respect to potential effects on human and animal health and the environment. On 23 July 2015, the European Commission (EC) received from Syngenta new nucleic acid sequencing data on maize event MIR604 and updated bioinformatic analyses using the new sequencing data. EC tasked EFSA to analyse these data and to indicate whether the previous conclusions of the EFSA GMO Panel on the above-listed GM maizes remain valid. The EFSA GMO Panel used the appropriate principles described in its guidelines for the risk assessment of GM plants to analyse the received data. The new sequencing data indicated a single base pair difference compared to the sequencing data originally provided, located in a non-coding region of the insert. which had already been present in the original plant material used for the risk assessment. Thus, with the exception of bioinformatics analyses, the studies performed for the risk assessment remain valid. The new sequencing data and the bioinformatic analyses performed on the new sequence did not give rise to safety issues. Therefore, the GMO Panel concludes that the original risk assessment of event MIR604 as a single and as a part of stacked events remains valid.

Development of Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae) on pollen from Bt-transgenic and conventional maize.

Maize (Zea mays) pollen is highly nutritious and can be used by predatory arthropods to supplement or replace a carnivorous diet. We demonstrate that maize pollen can be utilized by larvae of the green lacewing, Chrysoperla carnea (Neuroptera: Chrysopidae) under laboratory conditions. Complete development on maize pollen was not possible, but 25% of neonates reached the third instar. When only one instar was fed with pollen and the other two instars with eggs of Ephestia kuehniella (Lepidoptera: Pyralidae), 58–87% of the larvae reached the pupal stage. The experiments included pollen produced by nine cultivars: three genetically modified (GM) cultivars expressing the Bacillus thuringiensis proteins Cry1Ab or Cry3Bb1, their corresponding non-transformed near-isolines, and three conventional cultivars. Maize cultivars were grown in two batches in a glasshouse. Their pollen differed by up to 59% in total protein content, 25% in C:N ratio, and 14% in grain diameter, but the differences were inconsistent and depended on the batch. Lacewing performance was not affected by maize cultivar. For environmental risk assessment of GM plants, in planta studies must consider the variability among conventional cultivars, individual plants, batches, and environmental conditions when evaluating the ecological significance of differences observed between GM and near-isolines.

Potential use of an arthropod database to support the non-target risk assessment and monitoring of transgenic plants

Worldwide, plants obtained through genetic modification are subject to a risk analysis and regulatory approval before they can enter the market. An area of concern addressed in environmental risk assessments is the potential of genetically modified (GM) plants to adversely affect non-target arthropods and the valued ecosystem services they provide. Environmental risk assessments are conducted case-by-case for each GM plant taking into account the plant species, its trait(s), the receiving environments into which the GM plant is to be released and its intended uses, and the combination of these characteristics. To facilitate the non-target risk assessment of GM plants, information on arthropods found in relevant agro-ecosystems in Europe has been compiled in a publicly available database of bio-ecological information during a project commissioned by the European Food Safety Authority (EFSA). Using different hypothetical GM maize case studies, we demonstrate how the information contained in the database can assist in identifying valued species that may be at risk and in selecting suitable species for laboratory testing, higher-tier studies, as well as post-market environmental monitoring.

Do genetic modifications in crops affect soil fungi? a review

The use of genetically modified (GM) plants in agriculture has been a topic in public debate for over a decade. Despite their potential to increase yields, there may be unintended negative side-effects of GM plants on soil micro-organisms that are essential for functioning of agro-ecosystems. Fungi are important soil organisms and can have beneficial or harmful effects on plants. Their benefits to agro-ecosystems come from their activities as free-living saprobes breaking down soil organic matter thereby releasing nutrients to the crops, as well as from mutualistic interactions. On the other hand, soil-borne plant pathogenic fungi can cause severe damage in crops. Understanding of the impact of GM plants on the dynamics and functioning of soil fungi is essential to evaluate the possible risks of introduction of GM plants for ecosystem functioning. In recent years, over 50 studies have addressed the effects of various GM traits in crops on soil fungal community structure and function. These studies showed that GM crops can have positive, negative, or neutral effects on both free-living and plant-associated soil fungi. The observed discrepancy in results of these studies is discussed. This is done by highlighting a number of case studies. New methods developed in recent years have enabled microbial ecologists to get a better picture on the functioning and assembly of soil fungal communities. This review presents and discusses two of the most promising methods which are also readily usable in risk assessment of GM plants on soil fungi and that could help answer remaining key questions in the field.

Applying a weed risk assessment approach to GM crops

Current approaches to environmental risk assessment of genetically modified (GM) plants are modelled on chemical risk assessment methods, which have a strong focus on toxicity. There are additional types of harms posed by plants that have been extensively studied by weed scientists and incorporated into weed risk assessment methods. Weed risk assessment uses robust, validated methods that are widely applied to regulatory decision-making about potentially problematic plants. They are designed to encompass a broad variety of plant forms and traits in different environments, and can provide reliable conclusions even with limited data. The knowledge and experience that underpin weed risk assessment can be harnessed for environmental risk assessment of GM plants. A case study illustrates the application of the Australian post-border weed risk assessment approach to a representative GM plant. This approach is a valuable tool to identify potential risks from GM plants.

EFSA’s scientific activities and achievements on the risk assessment of genetically modified organisms (GMOs) during its first decade of existence: looking back and ahead

Genetically modified organisms (GMOs) and derived food and feed products are subject to a risk analysis and regulatory approval before they can enter the market in the European Union (EU). In this risk analysis process, the role of the European Food Safety Authority (EFSA), which was created in 2002 in response to multiple food crises, is to independently assess and provide scientific advice to risk managers on any possible risks that the use of GMOs may pose to human and animal health and the environment. EFSA's scientific advice is elaborated by its GMO Panel with the scientific support of several working groups and EFSA's GMO Unit. This review presents EFSA's scientific activities and highlights its achievements on the risk assessment of GMOs for the first 10years of its existence. Since 2002, EFSA has issued 69 scientific opinions on genetically modified (GM) plant market registration applications, of which 62 for import and processing for food and feed uses, six for cultivation and one for the use of pollen (as or in food), and 19 scientific opinions on applications for marketing products made with GM microorganisms. Several guidelines for the risk assessment of GM plants, GM microorganisms and GM animals, as well as on specific issues such as post-market environmental monitoring (PMEM) were elaborated. EFSA also provided scientific advice upon request of the European Commission on safeguard clause and emergency measures invoked by EU Member States, annual PMEM reports, the potential risks of new biotechnology-based plant breeding techniques, evaluations of previously assessed GMOs in the light of new scientific publications, and the use of antibiotic resistance marker genes in GM plants. Future challenges relevant to the risk assessment of GMOs are discussed. EFSA's risk assessments of GMO applications ensure that data are analysed and presented in a way that facilitates scientifically sound decisions that protect human and animal health and the environment.

Foraging activity of bumblebees (Bombus terrestris L.) on Bt-expressing eggplants

A greenhouse experiment was setup to study foraging behavior of the bumblebee Bombus terrestris L. on Cry3Bb-expressing genetically modified (GM) eggplants and their near-isogenic control. Commonly, more bumblebees visited GM eggplants compared to near-isogenic control, but this difference was only marginally significant. The mean length of feeding bouts was similar on the two treatments. Neither the number of flowers produced nor their size could explain bumblebees’ tendency to prefer GM eggplants. Volatile compounds were extracted from five plants per genotype and separated using gas chromatography. Thirteen compounds were identified and five of them appeared significantly more abundant in GM eggplants. Six of the identified compounds [(+)-limonene, Z-jasmone, p-cymene, α-pinene, methyl-salicilate, and (−)-limonene] were tested in electrophysiological bioassays with antennas detached from young bumblebees, and a response was recorded in all six cases. Experimental results indicate that pollination activity of bumblebees is compatible with this GM eggplant event as a food source and that chemical cues may have an important role in plant identification. The implications for environmental risk assessment of GM plants are discussed.

Guidance on selection of comparators for the risk assessment of genetically modified plants and derived food and feed

This opinion provides guidance in the area of comparators taking into account the requirements for the molecular characterisation, the food and feed and the environmental risk assessments. A key step in the risk assessment of genetically modified (GM) plants and derived food and feed is the identification of intended and unintended differences and equivalences between the GM plant and its comparator(s), taking into account the range of natural variation. In line with Regulation (EC) No 1829/2003 and Directive 2001/18/EC, the EFSA GMO Panel has, to date, required the use of non-GM lines with comparable genetic background as comparators. In the case of vegetatively propagated crops, these are the isogenic lines. In the case of sexually propagated crops these are non-GM lines as close as possible genetically to the GM plant under assessment. The identification and production of such comparators is becoming increasingly challenging due to the increasing complexity of GM plants, e.g. those developed by combining (stacking) events through conventional crosses, or those in which extensive compositional changes are targeted. Consequently, the EFSA GMO Panel has developed this guidance on the selection of comparators for the risk assessment of GM plants and derived food and feed. Whilst considering the requirements of Directive 2001/18/EC and Regulation (EC) No 1829/2003, the EFSA GMO Panel provides options which introduce flexibility in the selection of comparators based on sound scientific principles. This document addresses the selection of comparators for GM plants containing single or multiple events stacked by either conventional breeding, or by other approaches such as re-transformation, co-transformation and the use of multiple gene cassettes. The EFSA GMO Panel also considers situations where additional comparators may be required on a case-by-case basis and scenarios where appropriate comparators are not available (e.g. where extensive compositional changes are targeted). The EFSA GMO Panel recognises the different requirements for comparators for the molecular characterisation, food and feed and environmental components of the risk assessment.

Guidance for risk assessment of food and feed from genetically modified plants

This document provides updated guidance for the risk assessment of food and feed containing, consisting or produced from genetically modified (GM) plants, submitted within the framework of Regulation (EC) No 1829/2003 on GM food and feed. The risk assessment strategy for GM plants and derived food and feed proposed seeks to deploy appropriate approaches to compare GM plants and derived food and feed with their respective comparators. The underlying assumption of this comparative approach is that traditionally cultivated crops have gained a history of safe use for consumers and/or domesticated animals. The document provides guidance on how to perform the comparative analysis of the relevant characteristics of the GM plant. The document addresses the details of the different components of the risk assessment: the molecular characterisation, which provides information on the structure and expression of the insert(s) and on the stability of the intended trait(s); the toxicological assessment, which addresses the impact of biologically relevant change(s) in the GM plant and/or derived food and feed resulting from the genetic modification; the assessment of potential allergenicity, of the novel protein(s) as well as of the whole food derived from the GM plant; the nutritional assessment to evaluate whether food and feed derived from a GM plant is not nutritionally disadvantageous to humans and/or animals. In addition every section of the document addresses specifically the requirements for GM plants containing a combination of transformation events, providing guidance on how to establish that the combination is stable and that no interactions occurs between the events that may raise safety concerns. The document does not cover the environmental risk assessment of GM plants which is addressed in a stand-alone environmental risk assessment (ERA) guidance document developed by the EFSA GMO Panel.

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1), Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line, a leaf folder-resistant single trait GM line, and a rice leaf blast-resistant and leaf folder-resistant stacked GM hybrid line, respectively. The FISH signals were clearly detected on the arms of one homologous chromosome pair for LS28, and on the arms of another chromosome pair for Cry1Ac1 when using the transformation vector pSBM AtCK containing the rice leaf blast-resistant gene (LS28) and pMJ-RTB containing the leaf folder-resistant gene (mCry1Ac1) as a probe, respectively. As expected, we detected two pairs of FISH signals, each on the arms of different chromosome pairs in the stacked GM rice line LS28×Cry1Ac1 when using both pSBM AtCK and pMJ-RTB as probes. These results indicate that the transgenes are located at specific homologous loci and show position stability among generations in both single trait and stacked GM rice lines. The usefulness and the necessity of FISH to detect inserted genes in transformed plants will be discussed for the purpose of future studies to develop breeding programs and conduct risk assessment of GM plants.

The variability of processes involved in transgene dispersal—case studies from Brassica and related genera

We strive to predict consequences of genetically modified plants (GMPs) being cultivated openly in the environment, as human and animal health, biodiversity, agricultural practise and farmers' economy could be affected. Therefore, it is unfortunate that the risk assessment of GMPs is burdened by uncertainty. One of the reasons for the uncertainty is that the GMPs are interacting with the ecosystems at the release site thereby creating variability. This variability, e.g. in gene flow, makes consequence analysis difficult. The review illustrates the great uncertainty of results from gene-flow analysis. Many independent experiments were performed on the individual processes in gene flow. The results comprise information both from laboratory, growth chambers and field trials, and they were generated using molecular or phenotypic markers and analysis of fitness parameters. Monitoring of the extent of spontaneous introgression in natural populations was also performed. Modelling was used as an additional tool to identify key parameters in gene flow. The GM plant may affect the environment directly or indirectly by dispersal of the transgene. Magnitude of the transgene dispersal will depend on the GM crop, the agricultural practise and the environment of the release site. From case-to-case these three factors provide a variability that is reflected in widely different likelihoods of transgene dispersal and fitness of introgressed plants. In the present review, this is illustrated through a bunch of examples mostly from our own research on oilseed rape, Brassica napus. In the Brassica cases, the variability affected all five main steps in the process of gene dispersal. The modelling performed suggests that in Brassica, differences in fitness among plant genome classes could be a dominant factor in the establishment and survival of introgressed populations. Up to now, experimental analyses have mainly focused on studying the many individual processes of gene flow. This can be criticised, as these experiments are normally carried out in widely different environments and with different genotypes, and thus providing bits and pieces difficult to assemble. Only few gene-flow studies have been performed in natural populations and over several plant generations, though this could give a more coherent and holistic view. The variability inherent in the processes of gene flow in Brassica is apparent and remedies are wished for. One possibility is to expose the study species to additional experiments and monitoring, but this is costly and will likely not cover all possible scenarios. Another remedy is modelling gene flow. Modelling is a valuable tool in identifying key factors in the gene-flow process for which more knowledge is needed, and identifying parameters and processes which are relatively insensitive to change and therefore require less attention in future collections of data. But the interdependence between models and experimental data is extensive, as models depend on experimental data for their development or testing. More and more transgenic varieties are being grown worldwide harbouring genes that might potentially affect the environment (e.g. drought tolerance, salt tolerance, disease tolerance, pharmaceutical genes). This calls for a thorough risk assessment. However, in Brassica, the limited and uncertain knowledge on gene flow is an obstacle to this. Modelling of gene flow should be optimised, and modelling outputs verified in targeted field studies and at the landscape level. Last but not least, it is important to remember that transgene flow in itself is not necessarily a thread, but it is the consequences of gene flow that may jeopardise the ecosystems and the agricultural production. This emphasises the importance of consequence analysis of genetically modified plants.

Risk assessment of conventional crop plants in analogy to transgenic plants

The risk assessment of genetically-modified plants pursuant to Annex II B of EU Directive 94/15/EC assumes that it is possible to infer the environmental impacts of a crop plant from its characteristics, so most of Annex II should also be applicable to conventional plants. To test this, we surveyed reports on the ecological impacts of the cultivation of non-transgenic crop plants with novel or improved traits and, in three cases, investigated whether Annex II B would have been adequate to indicate the effects. Such an assessment appears to be feasible only if the time frame on which it is based is short, so that long-term effects cannot be assessed. Secondly, the plant must be genetically homogenous which is not always granted, e.g. with forest-trees. Thirdly, the cultivation area must be defined. Differences in the behaviour of foreign plants between their original and cultivation habitats may be ecologically relevant and should be assessed. In the (few) cases where direct inference of the observed effects was possible from inherent traits, these effects often correlated with poor adaptation to local environmental conditions. The ecological impacts of traits that had been introduced in order to overcome poor adaptation may differ widely according to the way in which the traits are exploited. In practice, the effects of agricultural measures are more important than the effects of gene transfer and invasiveness, although the latter currently play a major role in risk assessment. In the light of these deliberations, a modification of Annex II B of EU Directive 94/15/EC is suggested.