Angelika Hilbeck

Degradation of the Cry1Ab protein within transgenic Bacillus thuringiensis corn tissue in the field.

Large quantities of Bacillus thuringiensis (Bt) corn plant residue are left in the field after harvest, which may have implications for the soil ecosystem. Potential impacts on soil organisms will also depend on the persistence of the Bt toxin in plant residues. Therefore, it is important to know how long the toxin persists in plant residues. In two field studies in the temperate corn‐growing region of Switzerland we investigated degradation of the Cry1Ab toxin in transgenic Bt corn leaves during autumn, winter and spring using an enzyme‐linked immunosorbent assay (ELISA). In the first field trial, representing a tillage system, no degradation of the Cry1Ab toxin was observed during the first month. During the second month, Cry1Ab toxin concentrations decreased to ≈ 20% of their initial values. During winter, there was no further degradation. When temperatures again increased in spring, the toxin continued to degrade slowly, but could still be detected in June. In the second field trial, representing a no‐tillage system, Cry1Ab toxin concentrations decreased without initial delay as for soil‐incorporated Bt plants, to 38% of the initial concentration during the first 40 days. They then continued to decrease until the end of the trial after 200 days in June, when 0.3% of the initial amount of Cry1Ab toxin was detected. Our results suggest that extended pre‐ and post‐commercial monitoring are necessary to assess the long‐term impact of Bt toxin in transgenic plant residues on soil organisms.

Effects of transgenic Bt corn litter on the earthworm Lumbricus terrestris

A 200‐day study was carried out to investigate the impact of transgenic Bacillus thuringiensis (Bt) corn on immature and adult Lumbricus terrestris in the field and in the laboratory. Another objective of this study was to develop test methods that could be used for standard testing of the impact of transgenic plants on different earthworm species in the field and in the laboratory. For this purpose two different experiments were involved, a laboratory experiment with adult L. terrestris and a field experiment with immature L. terrestris. No lethal effects of transgenic Bt corn on immature and adult earthworms were observed. Immature L. terrestris in the field had a very similar growth pattern when fed either (Bt+) or (Bt−) corn litter. No significant differences in relative weights of (Bt+) and (Bt−) corn‐fed adult L. terrestris were observed during the first 160 days of the laboratory trial, but after 200 days adult L. terrestris had a significant weight loss of 18% of their initial weight when fed (Bt+) corn litter compared to a weight gain of 4% of the initial weight of (Bt−) corn‐fed earthworms. Further studies are necessary to see whether or not this difference in relative weight was due to the Bt toxin or other factors discussed in the study. Degradation of Cry1Ab toxin in corn residues was significantly slower in the field than at 10 °C in the laboratory. Enzyme‐linked immunosorbent assay results indicated that earthworms in both experiments were exposed to the Bt toxin throughout the whole experimental time.

Science-Based Risk Assessment for Nontarget Effects of Transgenic Crops

Abstract Nontarget risk assessment for transgenic crops should be case specific, depending on the plant, the transgene, and the intended release environment. We propose an ecological risk-assessment model that preserves the strengths and avoids the deficiencies of two other commonly used models, the ecotoxicology and nonindigenous-species models. In this model, locally occurring nontarget species are classified into groups according to their ecological function. Within each group, ecological criteria are used to select the species that are most likely to be affected by the transgenic crop. Initial experimental assessments are conducted in the laboratory and consist of two kinds of test: toxicity tests using purified transgene product, and whole-plant tests using intact transgenic plants. For nontarget natural enemy species, it will also be important to evaluate both direct bitrophic impacts and indirect tritrophic impacts.

Effects of activated Bt transgene products (Cry1Ab, Cry3Bb) on immature stages of the ladybird Adalia bipunctata in laboratory ecotoxicity testing.

Insect-active Bacillus thuringiensis (Bt) proteins are expressed by several transgenic crop plants to control certain pests, but nontarget organisms such as ladybirds also can be exposed to these proteins in the field. We developed an improved ecotoxicity testing protocol and conducted feeding trials in a laboratory setting to test for possible adverse effects of different concentrations of microbially produced trypsin-activated Cry1Ab and Cry3Bb toxins on the coccinellid Adalia bipunctata. Larval/pupal mortality, development time, and overall body mass accumulation were recorded. Even at the lowest concentration (5 μg/ml), A. bipunctata larvae fed with the lepidopteran-active Cry1Ab toxin exhibited significantly higher mortality than the control group. In experiments with the coleopteran-active Cry3Bb, only a concentration of 25 μg/ml resulted in a marginally significantly higher mortality compared to the control. Both experiments revealed a slight decline in mortality at the highest concentration of 50 μg/ml, though this was statistically significant only in the Cry1Ab treatment. No differences were detected for development time and body mass of newly emerged adults. Dilutions of the expression vector pBD10—used as a control to exclude effects of the toxin production method—at concentrations between 10 and 100 μg/ml revealed no significant effects on either of the studied parameters. This suggests that the increased mortality of larvae in the toxin feeding trials was caused directly by the activated Bt toxins and raises questions regarding their commonly postulated specificity and their mode of action in A. bipunctata. Implications of the reported results for ladybird populations and their biological pest control functions in transgenic crop ecosystems are discussed.

No scientific consensus on GMO safety

A broad community of independent scientific researchers and scholars challenges recent claims of a consensus over the safety of genetically modified organisms (GMOs). In the following joint statement, the claimed consensus is shown to be an artificial construct that has been falsely perpetuated through diverse fora. Irrespective of contradictory evidence in the refereed literature, as documented below, the claim that there is now a consensus on the safety of GMOs continues to be widely and often uncritically aired. For decades, the safety of GMOs has been a hotly controversial topic that has been much debated around the world. Published results are contradictory, in part due to the range of different research methods employed, an inadequacy of available procedures, and differences in the analysis and interpretation of data. Such a lack of consensus on safety is also evidenced by the agreement of policymakers from over 160 countries - in the UN’s Cartagena Biosafety Protocol and the Guidelines of the Codex Alimentarius - to authorize careful case-by-case assessment of each GMO by national authorities to determine whether the particular construct satisfies the national criteria for ‘safe’. Rigorous assessment of GMO safety has been hampered by the lack of funding independent of proprietary interests. Research for the public good has been further constrained by property rights issues, and by denial of access to research material for researchers unwilling to sign contractual agreements with the developers, which confer unacceptable control over publication to the proprietary interests.The joint statement developed and signed by over 300 independent researchers, and reproduced and published below, does not assert that GMOs are unsafe or safe. Rather, the statement concludes that the scarcity and contradictory nature of the scientific evidence published to date prevents conclusive claims of safety, or of lack of safety, of GMOs. Claims of consensus on the safety of GMOs are not supported by an objective analysis of the refereed literature.

Identifying indicator species for post-release monitoring of genetically modified, herbicide resistant crops

In Europe, regulations for release and placing-on-the-market of genetically modified (GM) crops require post-release monitoring of their impact on the environment. Monitoring potential adverse effects of GM crops includes direct effects as well as indirect effects, e.g. GM crop specific changes in land and pest management. Currently, there is a gap in the pre-release risk assessments conducted for regulatory approval of GM herbicide resistant (HR) crops. Since the relevant non-selective herbicides have been registered many years ago, in current dossiers requesting regulatory approval of GM HR crops, the environmental impacts of the corresponding non-selective herbicides are either entirely omitted or the applicant simply refers to the eco-toxicological safety assessments conducted for its original pesticide approval that do not address environmental issues arising in conjunction with the cultivation of GM HR crops. Since the ‘Farm-scale Evaluations’, it is clear that consequences for farmland biodiversity can be expected. The objective of this project was to identify relevant indicator species for the long-term impact of GM HR maize cultivation and the application of their corresponding non-selective herbicides, glyphosate and glufosinate. In this article, we describe the outcome of a modified Event Tree Analysis, essentially a funnel-like procedure allowing to reduce the large number of potentially affected non-target species to those with greatest ecological relevance and highest risk to be adversely affected based on a number of ecological criteria. This procedure allowed us to identify a total of 21 weed-Lepidoptera associations that we proposed for post release monitoring of GM HR maize in Germany.

Transgene Expression and Bt Protein Content in Transgenic Bt Maize (MON810) under Optimal and Stressful Environmental Conditions

Bt protein content in transgenic insect resistant (Bt) maize may vary between tissues within plants and between plants growing under different environmental conditions. However, it is unknown whether and how Bt protein content correlates with transgene expression, and whether this relationship is influenced by stressful environmental conditions. Two Bt maize varieties containing the same transgene cassette (MON 810) were grown under optimal and stressful conditions. Before and during stress exposure, the upper leaves were analysed for transgene expression using quantitative RT-PCR and for Bt content using ELISA. Under optimal conditions there was no significant difference in the transgene expression between the two investigated Bt maize varieties whereas Bt protein content differed significantly. Transgene expression was correlated with Bt protein content in only one of the varieties. Under stressful environmental conditions we found similar transgene expressions as under optimal conditions but Bt content responded differently. These results suggest that Bt content is not only controlled by the transgene expression but is also dependent on the genetic background of the maize variety. Under stressful conditions the concentration of Bt protein is even more difficult to predict.

Inter-laboratory comparison of Cry1Ab toxin quantification in MON 810 maize by enzyme-immunoassay

Abstract A laboratory ring trial was performed in four laboratories for determination of Cry1Ab toxin in leaf material of MON 810 maize using a standardised enzyme-linked immunoassay protocol. Statistical analysis was carried out by the ISO 5725-2 guidelines, sample standard deviation and standard error, within-laboratory and inter-laboratory SD and SE were calculated. Measured inter-laboratory average values were 12.5±4.0, 15.3±4.6 and 72.6±17.8 µg/g for three lyophilised samples, and 27.8±4.3 µg/g for a frozen sample, yet, Cry1Ab concentrations ranged 66.5–160.1% of the corresponding IA. Determined concentrations by in-house protocols were statistically not different in one laboratory and different in two laboratories from the corresponding values by the joint protocol. Results emphasise the importance of a standardised protocol among laboratories for comparable quantitative Cry1Ab toxin determination. However, even when using a standardised protocol, significant differences still occur among toxin concentrations detected in different laboratories, although with a smaller range of variation.

Teosinte in Europe – Searching for the Origin of a Novel Weed

A novel weed has recently emerged, causing serious agronomic damage in one of the most important maize-growing regions of Western Europe, the Northern Provinces of Spain. The weed has morphological similarities to a wild relative of maize and has generally been referred to as teosinte. However, the identity, origin or genetic composition of ‘Spanish teosinte’ was unknown. Here, we present a genome-wide analysis of single-nucleotide polymorphism (SNP) data for Spanish teosinte, sympatric populations of cultivated maize and samples of reference teosinte taxa. Our data are complemented with previously published SNP datasets of cultivated maize and two Mexican teosinte subspecies. Our analyses reveal that Spanish teosinte does not group with any of the currently recognized teosinte taxa. Based on Bayesian clustering analysis and hybridization simulations, we infer that Spanish teosinte is of admixed origin, most likely involving Zea mays ssp. mexicana as one parental taxon, and an unidentified cultivated maize variety as the other. Analyses of plants grown from seeds collected in Spanish maize fields and experimental crosses under controlled conditions reveal that hybridization does occur between Spanish teosinte and cultivated maize in Spain, and that current hybridization is asymmetric, favouring the introgression of Spanish teosinte into cultivated maize, rather than vice versa.

Science-based risk assessment requires careful evaluation of all studies

1077 according to the table they provide in their Supplementary Notes, only one study of the 55 reviewed was actually able to be confirmed as meeting all of the quality criteria they assessed. Additionally, in the Supplementary Notes, the authors state that the three quality criteria of test substance purity, homogeneity and equivalence between the purified and plant-expressed protein “are not addressed by the majority of the published studies and are thus not presented in detail.” Since few (if any) of the 55 published studies can therefore be said to meet the nine quality criteria Romeis et al. identify as important, all studies could, thus, arguably be presented as “nonconclusive.” In drawing the conclusion that only studies finding negative effects are “nonconclusive,” Romeis et al.1 also selectively report information by failing to mention the debates surrounding these socalled “outlier” studies that they deem to be generating “false-positive effects,” according to their standards. These debates have taken place in peer-reviewed journals4–6 and have involved the scientists responsible for those studies responding to the claims and accusations that their results are invalid due to faulty study designs. For Romeis et al.1 to hide such scientific debate from readers and simply assert that the research is unreliable and nonconclusive following their selective evaluation is not only misrepresentative, it falsely claims a scientific consensus when the issues in fact remain open. In these published debates, it is particularly interesting to note that whereas the scientists finding possible harm recognize and explicitly acknowledge the inherent openness of science and thus the need for continual collective exchange and further research, Romeis et al.1 and other critics of the work seem to present their own science as conclusive and final, with no need for further investigation required (of either body of work). Surely a diversity of approaches, methods and possibly findings is not only normal, but quintessentially what constitutes good science, and also crucial for its public trustworthiness. An equally problematic form of selective reporting lies in the way Romeis et al.1 present the results from the meta-analyses. They state that numerous experimental studies and field observations have provided conclusive evidence that Cry1Ab protein expressed in Bt maize does not cause adverse effects on arthropods, and that supporting data for this have been analyzed in reviews and meta-analyses. What they fail to report, however, is that in at least one of the metaanalyses they cite7, the results did in fact 8. Klein, R.J. BMC Genet. 8, 58 (2007). 9. Spencer, C.C.A., Su, Z., Donnelly, P. & Marchini, J. PLoS Genet 5, e1000477 (2009). 10. http://www.genomicsengland.co.uk/100k-genomeproject/ 11. http://www.fastcompany.com/3018598/for-99-thisceo-can-tell-you-what-might-kill-you-inside-23andmefounder-anne-wojcickis-dna-r 12. http://gds.nih.gov/pdf/PTC_for_IRBs_and_Institutions_ revised5-31-11.pdf 13. Dorfman, R. Nat. Biotechnol. 31, 785–786 (2013). 1. http://www.councilforresponsiblegenetics.org/ ViewPage.aspx?pageId=88 2. Wojcicki, A. Nat. Biotechnol. 31, 1075–1076 (2013). 3. http://www.washingtonpost.com/wp-dyn/content/ article/2010/05/12/AR2010051205156.html 4. Vayena, E. & Prainsack, B. Nat. Biotechnol. 31, 16–17 (2013). 5. Wray, N.R. Nat. Rev. Genet. 14, 507–515 (2013). 6. Gibson, G. Nat. Rev. Genet. 13, 135–145 (February 2012). 7. Do, C.B., Hinds, D.A., Francke, U. & Eriksson, N. PLoS Genet. 8, e1002973 (2012).