A procedure is presented for developing environmental risk hypotheses associated with thedeployment of forest trees genetically modified to have altered wood properties and forselecting non-target species to test these hypotheses. Altered-lignin Pinusradiata trees intended for use in New Zealand are used as a hypothetical casestudy to illustrate our approach. Firstly, environmental management goals (such as woodproduction, flood control or preservation of biodiversity) were identified and linked tothe forest attributes they require. Necessary conditions for each attribute were listedand appropriate assessment endpoints for them developed. For example, biological controlof pests may be one condition necessary for a forest to have healthy trees, and thediversity and abundance of natural enemy species in the forest could be an appropriateassessment endpoint for measuring this condition. A conceptual model describing therelationships between an altered-lignin GM pine tree and potentially affectedinvertebrates and micro-organisms in a plantation forest was used to develop a set of riskhypotheses describing how the GM trees might affect each assessment endpoint. Becausepurified lignin does not represent the properties it imparts to wood, maximum hazard dosetests with non-target organisms, as are used to inform toxin risk assessment, cannot beconducted. Alternative experiments, based on current knowledge of the responses oforganisms to lignin, must be designed. A screening method was adapted and applied to adatabase of invertebrate species known to inhabit New Zealand pine forests to identify andprioritize non-target invertebrate species that could be used as experimental subjects forexamining these hypotheses. The screening model and its application are presented, alongwith a set of recommendations for pre-release tests with GM pines and potentially affectedinvertebrates and micro-organisms.

Several biological containment methods have been developed to reduce pollen dispersal; many of them only have a proof of concept in a model plant species. This review focuses on biological containment measures which were tested for their long-term efficiency at the greenhouse or field scale level, i.e. plastid transformation, transgene excission, cleistogamy and cytoplasmic male sterility (CMS). Pollen-mediated gene transfer in transplastomic tobacco could occur at very low frequencies if the predominant mode of inheritance is maternal. Transgene excision from tobacco pollen can be made highly efficient by coexpression of two recombinases. For cleistogamous oilseed rape it was shown that some flowers were partially open depending on genotypes, environment and recording dates. Reports on the stability of CMS in maize and sunflower indicated that there is a high variability for different genotypes under different environmental conditions and over successive years. But for both crop types some stable lines could be selected. These data demonstrate that the biological containment methods discussed are very promising for reducing gene flow but that no single containment strategy provides 100% reduction. However, the necessary efficiency of biological containment methods depends on the level of containment required. The containment level may need to be higher for safety purposes (e.g. production of special plant-made pharmaceuticals), while much lower containment levels may already be sufficient to reach coexistence goals. It is concluded that where pollen-mediated gene flow must be prevented altogether, combinations of complementary containment systems will be required.

Prunus species are important commercial fruit (plums, apricot, peach and cherries), nut (almond) and ornamental trees cultivated broadly worldwide. This review compiles information from available literature on Prunus species in regard to gene flow and hybridization within this complex of species. The review serves as a resource for environmental risk assessment related to pollen mediated gene flow and the release of transgenic Prunus. It reveals that Prunus species, especially plums and cherries show high potential for transgene flow. A range of characteristics including; genetic diversity, genetic bridging capacity, inter- and intra-specific genetic compatibility, self sterility (in most species), high frequency of open pollination, insect assisted pollination, perennial nature, complex phenotypic architecture (canopy height, heterogeneous crown, number of flowers produced in an individual plant), tendency to escape from cultivation, and the existence of ornamental and road side Prunus species suggest that there is a tremendous and complicated ability for pollen mediated gene movement among Prunus species. Ploidy differences among Prunus species do not necessarily provide genetic segregation. The characteristics of Prunu s species highlight the complexity of maintaining coexistence between GM and non-GM Prunus if there were commercial production of GM Prunus species. The results of this review suggest that the commercialization of one GM Prunus species can create coexistence issues for commercial non-GM Prunus production. Despite advances in molecular markers and genetic analysis in agroecology, there remains limited information on the ecological diversity, metapopulation nature, population dynamics, and direct measures of gene flow among different subgenera represented in the Prunus genus. Robust environmental impact, biosafety and coexistence assessments for GM Prunus species will require better understanding of the mechanisms of gene flow and hybridization among species within the Prunus species complex.

More than 10 years after registration of the first Bt maize cultivar in Europe, there still exists a remarkable lack of data on effects on Lepidoptera which would be necessary for a complete and comprehensive environmental risk assessment. So far only very few European butterfly species have been tested in this aspect. In our study the effect of transgenic Bacillus thuringiensis (Bt) maize pollen (event Bt-176) on the development and survival of neonate larvae of the Peacock butterfly, Inachis io (L.) was for the first time shown. The results of our study suggest that the Peacock butterfly may serve as a model organism for assessing potential side effects of new developed transgenic Bt crops on non-target butterflies in a GMO environmental risk assessment. The study was done under laboratory conditions by exposing larvae of the Peacock butterfly to various pollen doses of transgenic maize event Bt-176 (cv. PACTOL CB) or the conventional isogenic maize (cv. PACTOL) using a no-choice test. Larvae feeding for 48 h on nettle plants (Urtica dioica) that were contaminated with higher pollen concentrations from Bt-176 maize (205 and 388 applied pollen.cm-2) suffered a significantly higher mortality rate (68 and 85% respectively) compared to larvae feeding on leaves with no pollen (11%), or feeding on leaves with pollen from conventional maize (6 to 25%). At lower Bt maize pollen doses (23–104 applied pollen.cm-2), mortality ranged from 11–25% and there were no apparent differences among treatments. The corresponding LC50-and LC90-values for neonate larvae of the Peacock butterfly were 187 and 448 applied pollen grains.cm-2 of Bt-176, respectively. Weight of larvae surviving consumption of Bt-176 maize pollen declined between 10 and 81% with increased pollen doses (r =  −0.95). The highest weight reduction (81%) corresponded to the highest pollen concentration (388 pollen grains applied.cm-2). Ingestion of pollen from the conventional maize hybrid did not have negative effects on larval weight gain or survival rate.

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.

While significant progress has been made on the modification of crops for the benefit ofproducers, the same cannot be said in regards to eliciting the potential impact that thesecrops may have on the wider landscape and the diversity of life therein. Managementimpacts can create difficulties when making policy, regulation and licensing decisions inthose countries where agriculture has a significant social and ecological position in thelandscape. To begin to gauge the potential impacts of the management of a selection of GMcrops on an agricultural landscape, four key biodiversity stressors (Chemicals,Introgression, Nutrients and Management: CINMa) were identified and a grading systemdeveloped using published data. Upon application to five selected GM crops in a case studyarea, CINMa identifies areas in the wider landscape where biodiversity is likely to benegatively or positively impacted, as well as agricultural zones which may benefit fromthe land use change associated with the management of GM crops and their associated postmarket environmental monitoring.

Accumulation of information about natural hybridization between GM soybean (Glycine max) and wild soybean (Glycine soja) is required for risk assessment evaluation and to establish biosafety regulations in Japan. This is particularly important in areas where wild relatives of cultivated soybean are grown (i.e. East Asia including Japan). To collect information on temporal and spatial factors affecting variation in hybridization between wild and GM soybean, a two year hybridization experiment was established that included one wild soybean and five GM soybean cultivars with different maturity dates. Hybridization frequencies ranged from 0 to 0.097%. The maximum hybridization frequency (0.097%) was obtained from wild soybean crossed with GM soybean cv. AG6702RR, which were adjacently cultivated with wild soybean, with 25 hybrids out of 25 741 seedlings tested. Cultivar AG6702RR had the most synchronous flowering period with wild soybean. Ten hybrids out of 25 741 were produced by crossing with cv. AG5905RR, which had the second most synchronous flowering period with wild soybean. Most hybrids were found where GM and wild soybeans were adjacently cultivated, whereas only one hybrid was detected from wild soybean plants at 2 m, 4 m and 6 m from a pollen source (GM soybean). Differences in flowering phenology, isolation distance and presence of buffer plants accounted for half of the variation in hybridization frequency in this study. Temporal and spatial isolation will be effective strategies to minimize hybridization between GM and wild soybean.

Auchenorrhyncha (planthoppers and leafhoppers) are herbivorous organisms that can ingest Cry proteins from genetically engineered Bt-crops depending on their feeding behaviour. Consequently, they might be directly affected by non-target Bt-protein action and more importantly serve as a source of Cry protein exposure to beneficial predatory arthropods. During a three year field study, we surveyed the community of Auchenorrhyncha in Diabrotica-resistant Bt-maize DKc5143-Bt (event MON88017), its near-isogenic line and two conventional hybrids using sweep netting and custom made sticky traps. Zyginidia scutellaris (Herrich-Schäffer) (Hemiptera: Cicadellidae) represented more than 60% of all captured individuals, indicating that it is the dominant leafhopper within the maize community. The statistical analysis of Z. scutellaris data using confidence intervals for the ratios of mean abundance showed no consistent differences between the Bt-maize and the near-isogenic cultivar, indicating no negative impact of event MON88017. The two conventional hybrids Benicia and DK315 exhibited differences in terms of Z. scutellaris densities, which were greater than those observed between MON88017 and the near-isogenic line, but also not consistent over the years. Six more species accounted for an additional 39% of all captured specimens, while ten more species were found only as single individuals and can be considered vagrants from neighbouring habitats. These results inform future field work on the non-target impact of Bt-maize on this group of arthropods and monitoring approaches to assess biological control function by surveying herbivore communities.

We assessed the effects of cultivating two genetically modified (GM) glyphosate-tolerant soybean varieties (Glycine max (L.) Merr.) derived from Event 40-3-2 and a Japanese conventional variety on arthropods under field conditions, with weed control using glyphosate and conventional weed control for two years. Plant height and dry weight of the conventional variety were significantly larger than those of the GM varieties, but the GM varieties bore more pods than the conventional variety. We found arthropods of nine taxonomic orders (Araneae, Acari, Thysanoptera, Homoptera, Heteroptera, Coleoptera, Diptera, Lepidoptera, and Hymenoptera) on the plants. The arthropod incidence (number per plant unit weight pooled for each taxonomic order) on the soybean stems and leaves generally did not differ significantly between the GM and conventional varieties. However, the incidence of Thysanoptera and total incidence (all orders combined) were greater on the GM variety in the second year. The weed control regimes had no significant influence on the arthropod incidence on the soybean stems and leaves. The number of flower-inhabiting Thysanoptera (the dominant arthropod in the flowers) was not significantly different between the GM and conventional varieties. Asphondylia yushimai (Diptera, Cecidomyiidae) was more numerous on the pods of the GM variety in both years. Neither the soybean variety nor the weed control regime significantly affected the density of soil macro-organisms. However, the glyphosate weed control affected arthropods between the rows of plants by decreasing the abundances of Homoptera, Heteroptera, Coleoptera and Lepidoptera, and diversity of arthropods.

This study assessed the impact of Pinus radiata (D. Don) genetically modified (GM) by biolistic insertion of the LEAFY and nptII genes on rhizosphere microbial communities of field grown trees. Rhizosphere soil was sampled quarterly for two consecutive years. A culture-independent approach was used to characterise the microbial communities based on PCR and denaturing gradient gel electrophoresis (DGGE) of 16S/18S rDNA gene fragments, and internal transcribed spacer (ITS) fragments amplified from total rhizosphere DNA. Trees from two independent transformation events were sampled, together with non-modified control trees of the same parental genotype. DGGE profiles of rhizosphere general Bacteria did not differ between GM and control trees with one exception (summer 2006 sample). For Alphaproteo- and Actinobacteria, significant differences between treatments were detected in one out of eight samplings. Small seasonal shifts could be detected in all bacterial communities. General fungal and ectomycorrhizal communities did not differ significantly between GM and control trees with the exception of summer 2006, when ectomycorrhizal communities associated with GM trees from one transformation event differed from those associated with control trees. Small seasonal shifts of general fungal and ectomycorrhizal communities were seen over the two-year sampling period. More detailed analysis of microbial communities at one sampling date (using amplified rDNA restriction analysis (ARDRA) and 16S/18S rDNA sequencing) revealed significant differences in four ARDRA groups between one GM treatment and the control (bacteria), and significant differences in one ARDRA group between the two GM treatments (fungi). When data from all sampling dates are considered together, the low incidence of statistical differences in the microbial communities associated with the genetically modified and control trees suggests that there was no significant impact of this genetic modification on rhizosphere microbial communities.

The cultivation of genetically modified (GM) Bt maize (Zea mays L.) events MON810 and Bt11 is permitted in Uruguay. Local regulations specify that 10% of the crop should be a non-GM cultivar as refuge area for biodiversity, and the distance from other non-GM maize crops should be more than 250 m in order to avoid cross-pollination. However, the degree of cross-fertilization between maize crops in Uruguay is unknown. The level of adventitious presence of GM material in non-GM crops is a relevant issue for organic farming, in situ conservation of genetic resources and seed production. In the research reported here, the occurrence and frequency of cross-fertilization between commercial GM and non-GM maize crops in Uruguay was assessed. The methodology comprised field sampling and detection using DAS-ELISA and PCR. Five field-pair cases where GM maize crops were grown near non-GM maize crops were identified. These cases had the potential to cross-fertilize considering the distance between crops and the similarity of the sowing dates. Adventitious presence of GM material in the offspring of non-GM crops was found in three of the five cases. Adventitious presence of event MON810 or Bt11 in non-GM maize, which were distinguished using specific primers, matched the events in the putative sources of transgenic pollen. Percentages of transgenic seedlings in the offspring of the non-GM crops were estimated as 0.56%, 0.83% and 0.13% for three sampling sites with distances of respectively 40, 100 and 330 m from the GM crops. This is a first indication that adventitious presence of transgenes in non-GM maize crops will occur in Uruguay if isolation by distance and/or time is not provided. These findings contribute to the evaluation of the applicability of the “regulated coexistence policy” in Uruguay.

Transgenic fish in development for aquaculture could escape from farms and interbreed with wild relatives in the nearby environment. Predicting whether escapes would result in transgene introgression is a major challenge in assessing environmental risks of transgenic fish. Previous studies have simulated gene flow from transgenic fish using mathematical modeling of fitness traits to predict the relative selective value of transgenic genotypes. Here, we present the first study of gene flow over the full life cycle in openly-breeding populations of transgenic animals, along with measurement of fitness traits. We conducted two invasion experiments in which we released two lines of growth-enhanced transgenic fish (T67 and T400), Japanese medaka (Oryzias latipes), into populations of wild-type (W) medaka in structured mesocosms. After several generations, the frequency of transgenic fish varied across replicates in the first invasion experiment (6 months), but the frequency of transgenic fish decreased in the second experiment (19 months). We also measured selected fitness traits in transgenic and wild-type medaka because these traits could be used to predict the relative selective value of a genotype. We found that: T400 males were more fertile than W males; offspring of W females lived longer than those with transgenic mothers; and W and T67 females reached sexual maturity sooner than T400 females. In contrast with other research that reported larger transgenic males had a mating advantage, we found that W males obtained more matings with females than T males; genetic background effects may account for our differing results as we compared W and T fish derived from different strains. The decreasing frequency of transgenic fish in the second invasion experiment suggests that transgenic fish had a selective disadvantage in the experimental environment. Our finding of transgenic advantage of some fitness traits and wild-type advantage in others is consistent with our invasion experiment results.

Determining the effects of genetically modified (GM) crops on non-target organisms is essential as many non-target species provide important ecological functions. However, it is simply not possible to collect field data on more than a few potential non-target species present in the receiving environment of a GM crop. While risk assessment must be rigorous, new approaches are necessary to improve the efficiency of the process. Utilisation of published information and existing data on the phenology and population dynamics of test species in the field can be combined with limited amounts of experimental biosafety data to predict possible outcomes on species persistence. This paper presents an example of an approach where data from laboratory experiments and field studies on phenology are combined using predictive modelling. Using the New Zealand native weevil species Nicaeana cervina as a case study, we could predict that oviposition rates of the weevil feeding on a GM ryegrass could be reduced by up to 30% without threat to populations of the weevil in pastoral ecosystems. In addition, an experimentally established correlation between feeding level and oviposition led to the prediction that a consistent reduction in feeding of 50% or higher indicated a significant risk to the species and could potentially lead to local extinctions. This approach to biosafety risk assessment, maximising the use of pre-existing field and laboratory data on non-target species, can make an important contribution to informed decision-making by regulatory authorities and developers of new technologies.

Regulators are acutely aware of the need for meaningful risk assessments to supportdecisions on the safety of GM crops to non-target invertebrates in determining theirsuitability for field release. We describe a process for developing appropriate, testablerisk hypotheses for invertebrates in agroecosystems that might be exposed to plantsdeveloped by GM and future novel technologies. An existing model (PRONTI) generates aranked list of invertebrate species for biosafety testing by accessing a database ofbiological, ecological and food web information about species which occur in croppingenvironments and their potential interactions with a particular stressor (EcoInvertebase). Our objective in this contribution is to explore and further utilise theseresources to assist in the process of problem formulation by identifying potentiallysignificant effects of the stressor on the invertebrate community and the ecosystemservices they provide. We propose that for high ranking species, a conceptual food webusing information in Eco Invertebase is constructed, and using an accepted regulatory riskanalysis framework, the likelihood of risk, and magnitude of impact for each link in thefood web is evaluated. Using as filters only those risks evaluated as likely to extremelylikely, and the magnitude of an effect being considered as moderate to massive, the mostsignificant potential effects can be identified. A stepwise approach is suggested todevelop a sequence of appropriate tests. The GM ryegrass plant used as the “stressor” inthis study has been modified to increase triacylglyceride levels in foliage by 100% toincrease the metabolisable energy content of forage for grazing animals. The high-ranking“test” species chosen to illustrate the concept are New Zealand native speciesWiseana cervinata (Walker) (Lepidoptera: Hepialidae),Persectania aversa (Walker) (Lepidoptera: Noctuidae), and theself-introduced grey field slug, Deroceras reticulatum (Müller).

In recent times, it has become imperative for countries to define and implement policy in biosafety due to the widespread adoption of genetically modified crops. As such, countries wishing to utilise transgenic technologies in the development of advanced agricultural products must have regulations in place coupled with trained personnel in national competent authorities able to contribute effectively to the decision-making process. Capacity building initiatives play an important role in supporting such individuals, institutions and governmental authorities by providing training and/or physical structures/equipment and technical assistance. There are many types of capacity building activities; however not all have the same relevance in different regions of the world. For capacity building to be effective, a strategic approach incorporating a variety of forms and disciplines is desired. This commentary discusses the importance of factors such as: the targeting of support to relevant beneficiary(ies); the identification of specific needs and the incorporation of socio-economic conditions when elaborating effective strategies designed to help building capacity. Moreover, the importance of interaction and collaboration amongst the various capacity builders is also discussed such that unnecessary duplication of efforts and best use of available human and economic resources results.