Abstract: Environmental risk assessments (ERA) support regulatory decisions for the commercial cultivation of genetically modified (GM) crops. The ERA for terrestrial agroecosystems is well-developed, whereas guidance for ERA of GM crops in aquatic ecosystems is not as well-defined. The purpose of this document is to demonstrate how comprehensive problem formulation can be used to develop a conceptual model and to identify potential exposure pathways, using Bacillus thuringiensis (Bt) maize as a case study. Within problem formulation, the insecticidal trait, the crop, the receiving environment, and protection goals were characterized, and a conceptual model was developed to identify routes through which aquatic organisms may be exposed to insecticidal proteins in maize tissue. Following a tiered approach for exposure assessment, worst-case exposures were estimated using standardized models, and factors mitigating exposure were described. Based on exposure estimates, shredders were identified as the functional group most likely to be exposed to insecticidal proteins. However, even using worst-case assumptions, the exposure of shredders to Bt maize was low and studies supporting the current risk assessments were deemed adequate. Determining if early tier toxicity studies are necessary to inform the risk assessment for a specific GM crop should be done on a case by case basis, and should be guided by thorough problem formulation and exposure assessment. The processes used to develop the Bt maize case study are intended to serve as a model for performing risk assessments on future traits and crops.
Abstract: 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.
Abstract: Beetles (Coleoptera) are a diverse and ecologically important group of insects in agricultural systems. The Environmental Risk Assessment (ERA) of genetically modified Bt-crop varieties with insect resistances thus needs to consider and assess the potential negative impacts on non-target organisms belonging to this group. We analysed data gathered during 6 years of field-release experiments on the impact of two genetically modified Bt-maize varieties (Ostrinia-resistant MON810 and Diabrotica-resistant MON88017) on the occurrence and field densities of Coleoptera, especially the two families Coccinellidae and Chrysomelidae. Based on a statistical analysis aimed at establishing whether Bt-maize varieties are equivalent to their near-isogenic counterparts, we discuss the limitations of using field experiments to assess the effects of Bt-maize on these two beetle families. The densities of most of the beetle families recorded in the herb layer were very low in all growing seasons. Coccinellidae and Chrysomelidae were comparatively abundant and diverse, but still low in numbers. Based on their role as biological control agents, Coccinellidae should be a focus in the ERA of Bt-plants, but given the large natural variability in ladybird densities in the field, most questions need to be addressed in low-tier laboratory tests. Chrysomelidae should play a negligible role in the ERA of Bt-plants, since they occur on-crop as secondary pests only. Species occurring off-crop, however, can be addressed in a similar fashion as non-target Lepidoptera in Cry1Ab expressing Bt-maize.
Abstract: In Europe, Bt-corn resistant against the European Corn Borer has until now been the only genetically modified plant to be grown commercially. With the advent of the Western Corn Rootworm Bt-corn varieties with resistance against Coleoptera will become important. The cultivation of Bt-plants may have negative impacts on non-target organisms, i.e. all species not explicitly targeted by a given Bt-crop. One prominent non-target group in corn are the herbivorous plant bugs (Heteroptera: Miridae). They are common, abundant and exposed to the Cry-protein. We therefore assessed the potential impact of the cultivation of the Cry3Bb1-expressing Bt-corn variety MON88017 and three conventional varieties on this group. Trigonotylus caelestialium (Kirkaldy) was the most abundant plant bug at the experimental field. There was no evidence for a negative impact of MON88017 on this species, despite its considerable exposure to Cry3Bb1 demonstrated with ELISA. The conventional corn varieties, however, had a consistent and significant influence on the field densities of this species over all three growing seasons.
Abstract: Fatty acid (FA) profiles of the Bt-maize line MON88017 expressing the Cry3Bb1 protein and its near-isogenic line DKC5143 were examined. Plant compartments under study included leaves taken from different internodes and roots. Sample preparation involved pressurized liquid extraction (PLE) of the biomass, transmethylation of the extracted lipids to give fatty acid methyl esters (FAMEs), and finally GC-MS analysis. The essential quality parameters for the FA profiles included total FA and sum of saturated FA, as well as double-bond index (DBI). FA profiles of the roots--characterized by high concentrations of homomorphic FA including palmitic and stearic acid, along with low concentrations of polyunsaturated surrogates--revealed high similarity between the genetically modified and the near-isogenic line. In contrast, FA profiles of the leaves showed significant differences: higher total FA concentrations and higher DBI were observed for the near-isogenic line. This was overwhelmingly associated with lower concentrations of alpha-linolenic acid (18:3omega3,6,9ccc) in the genetically modified leaf samples. These differences were particularly pronounced for leaves taken from the fourth elongated, above-ground internode. Given the large reported variability in the population of maize lines, MON88017 and its near-isogenic line can be regarded as equivalent with regard to their fatty acid profiles, despite the differences observed for the leaves. Further experiments are needed to assess whether the genetic modification of Bt-maize plants might induce unintended effects with regard to FA profiles.
Abstract: Bt-maize MON88017, its near-isogenic line DKC5143, and the two conventional varieties DK315 and Benicia were subjected to tetramethylammonium hydroxide (TMAH)-induced thermochemolysis to reveal molecular level lignin patterns. MON88017 is genetically modified to express the Cry3Bb1 protein aimed at the Western corn rootworm Diabrotica virgifera virgifera, a serious threat for European maize production. The results indicated that roots of the Bt-maize were characterized by a slightly enhanced total lignin content (by approximately 7%) compared to the near-isogenic line, whereas the molecular-based patterns, expressed by the relative fractions of p-hydroxyphenyl, guaiacyl, and syringyl breakdown products (P-, G-, and S-units, respectively) were virtually identical for both lines. No effects regarding either total lignin or molecular-based lignin patterns could be observed for leaves, indicating that biogenesis of lignin was not pleiotropically affected by the genetic modification. Significant differences for both total lignin and different lignin proxies existed between the conventional maize lines. Molecular level lignin analysis by means of TMAH-induced thermochemolysis is able to distinguish conventional maize varieties. Further work is necessary to evaluate lignin-related pleiotropic effects in genetically modified maize plants. The validation and application of a commonly accepted method for lignin analysis, capable of characterizing lignin at the molecular level, is a prerequisite.
Abstract: BACKGROUND: Insect-resistant transgenic corn is meeting a growing adoption in Europe. Corn is also the major substrate for biogas production, which has vastly increased in recent years. In areas with high insect pest pressure Bt-corn silage will consequently be fed into biogas production facilities. To assess the potential risks of transgenic plants as energy crops we investigated the fate of the coleopteran-specific protein Cry3Bb1 from MON88017 corn in the utilization chain of a farm-scale biogas production facility using ELISA.
RESULTS: The mean Cry3Bb1 protein content in chaffed MON88017 plant material was 15 µg g-1. In silage the content decreased rapidly, with less than 20 ng g-1 detectable after 8 months. From the start of fermentation in the biogas reactor, only trace amounts of Cry3Bb1 could be detected. Conventional corn harvested alongside MON88017 with the same equipment showed a presence of transgenic plant material of around 0.5%. Analyses performed at two laboratories showed consistent and systematic differences in the concentrations of Cry3Bb1 measured with ELISA due to methodical differences.
CONCLUSIONS: An extensive degradation of Cry3Bb1 was observed in silage despite differences in ELISA measurements. The potential risk associated with the use of MON88017 silage is discussed on this basis.
Abstract: 1 Auchenorrhyncha (Planthoppers and Leafhoppers) are not only pests of many crops, but they are also non-target organisms with respect to Bt-protein expressing genetically modified plants. As herbivorous arthropods, planthoppers and leafhoppers ingest Cry proteins depending on their feeding behaviour. Consequently, they are directly exposed to these entomotoxic proteins and can also serve as a source of Cry protein exposure to predatory arthropods. Auchenorrhyncha, therefore, can be reasonably used in the risk assessment of genetically modified crops.
2 During a 2 year field study, we evaluated four different methods in terms of their feasibility to assess the impacts of plant-incorporated protectants from Bt-maize and of insecticide use on this group of arthropods. Visual assessment of plants, sweep netting, yellow traps and custom made sticky traps were utilised in field plots of Bt-maize MON810, untreated near-isogenic maize and insecticide treated near-isogenic maize and compared in their capability to reflect the diversity and abundance of Auchenorrhyncha species.
3 Zyginidia scutellaris (Herrich-Schäffer) (Cicadomorpha: Cicadellidae) represented more than 94% of all captured individuals in both years. The analysis of Z. scutellaris data showed no consistent differences between Bt-maize MON810 and the untreated near isogenic hybrid, showing no negative impact of MON810 on this species. The insecticide treatment, on the other hand, was not equivalent to the isogenic maize in terms of Z. scutellaris densities. Based on the collected data and on practical considerations we recommend the combined use of transect-wise sweep netting and sticky traps for the sampling of Auchenorrhyncha in maize.
Abstract: Biogas plants fuelled with renewable sources of energy are a sustainable means for power generation. In areas with high infestation levels with the European corn borer, Ostrinia nubilalis (Hbn.), it is likely that transgenic Bt-maize will be fed into agricultural biogas plants. The fate of the entomotoxic protein Cry1Ab from MON810 maize was therefore investigated in silage and biogas production-related materials in the utilization chains of two farm-scale biogas plants. The Cry1Ab content in silage exhibited no clear-cut pattern of decrease over the experimental time of 4 months. Mean content for silage was 1878 +/- 713 ng Cry1Ab g(-1). After fermentation in the biogas plants, the Cry1Ab content declined to trace amounts of around 3.5 ng g(-1) in the effluents. The limit of detection of the employed ELISA test corresponded to 0.75 ng Cry1Ab g(-1) sample material. Assays with larvae of O. nubilalis showed no bioactivity of the reactor effluents. The utilization of this residual material as fertilizer in agriculture is therefore deemed to be ecotoxicologically harmless.
