Non-target organism risk assessment of MIR604 maize expressing mCry3A for control of corn rootworm

Abstract:  Event MIR604 maize expresses a modified Cry3A protein (mCry3A), for control of corn rootworm. As part of the environmental safety assessment of MIR604 maize, risks to non-target organisms of mCry3A were assessed. The potential exposure of non-target organisms to mCry3A following cultivation of MIR604 maize was determined, and the hypothesis that such exposure is not harmful was tested. The hypothesis was tested rigorously by making worst-case or highly conservative assumptions about exposure, along with laboratory testing for hazards using species taxonomically related to the target pest and species expected to have high exposure to mCry3A, or both. Further rigour was introduced by study designs incorporating long exposures and measurements of sensitive endpoints. No adverse effects were observed in any study, and in most cases exposure to mCry3A in the study was higher than the worst-case expected exposure. In all cases, exposure in the study was higher than realistic, but still conservative, estimates of exposure. These results indicate minimal risk of MIR604 maize to non-target organisms.     

Quality of laboratory studies assessing effects of <Emphasis Type="Italic">Bt</Emphasis>-proteins on non-target organisms: minimal criteria for acceptability

The potential risks that genetically modified plants may pose to non-target organisms and the ecosystem services they contribute to are assessed as part of pre-market risk assessments. This paper reviews the early tier studies testing the hypothesis whether exposure to plant-produced Cry34/35Ab1 proteins as a result of cultivation of maize 59122 is harmful to valued non-target organisms, in particular Arthropoda and Annelida. The available studies were assessed for their scientific quality by considering a set of criteria determining their relevance and reliability. As a case-study, this exercise revealed that when not all quality criteria are met, weighing the robustness of the study and its relevance for risk assessment is not obvious. Applying a worst-case expected environmental concentration of bioactive toxins equivalent to that present in the transgenic crop, confirming exposure of the test species to the test substance, and the use of a negative control were identified as minimum criteria to be met to guarantee sufficiently reliable data. This exercise stresses the importance of conducting studies meeting certain quality standards as this minimises the probability of erroneous or inconclusive results and increases confidence in the results and adds certainty to the conclusions drawn.     

Detection and drivers of exposure and effects of pharmaceuticals in higher vertebrates

Pharmaceuticals are highly bioactive compounds now known to be widespread environmental contaminants. However, research regarding exposure and possible effects in non-target higher vertebrate wildlife remains scarce. The fate and behaviour of most pharmaceuticals entering our environment via numerous pathways remain poorly characterized, and hence our conception and understanding of the risks posed to wild animals is equally constrained. The recent decimation of Asian vulture populations owing to a pharmaceutical (diclofenac) offers a notable example, because the exposure route (livestock carcasses) and the acute toxicity observed were completely unexpected. This case not only highlights the need for further research, but also the wider requirement for more considered and comprehensive ‘ecopharmacovigilance’. We discuss known and potential high risk sources and pathways in terrestrial and freshwater ecosystems where pharmaceutical exposure in higher vertebrate wildlife, principally birds and mammals, may occur. We examine whether approaches taken within existing surveillance schemes (that commonly target established classes of persistent or bioaccumulative contaminants) and the risk assessment approaches currently used for pesticides are relevant to pharmaceuticals, and we highlight where new approaches may be required to assess pharmaceutical-related risk.     

Assessing environmental risks of transgenic plants

By the end of the 1980s, a broad consensus had developed that there were potential environmental risks of transgenic plants requiring assessment and that this assessment must be done on a case-by-case basis, taking into account the transgene, recipient organism, intended environment of release, and the frequency and scale of the intended introduction. Since 1990, there have been gradual but substantial changes in the environmental risk assessment process. In this review, we focus on changes in the assessment of risks associated with non-target species and biodiversity, gene flow, and the evolution of resistance. Non-target risk assessment now focuses on risks of transgenic plants to the intended local environment of release. Measurements of gene flow indicate that it occurs at higher rates than believed in the early 1990s, mathematical theory is beginning to clarify expectations of risks associated with gene flow, and management methods are being developed to reduce gene flow and possibly mitigate its effects. Insect pest resistance risks are now managed using a high-dose/refuge or a refuge-only strategy, and the present research focuses on monitoring for resistance and encouraging compliance to requirements. We synthesize previous models for tiering risk assessment and propose a general model for tiering. Future transgenic crops are likely to pose greater challenges for risk assessment, and meeting these challenges will be crucial in developing a scientifically coherent risk assessment framework. Scientific understanding of the factors affecting environmental risk is still nascent, and environmental scientists need to help improve environmental risk assessment.     

Recommendations for the design of laboratory studies on non-target arthropods for risk assessment of genetically engineered plants

This paper provides recommendations on experimental design for early-tier laboratory studies used in risk assessments to evaluate potential adverse impacts of arthropod-resistant genetically engineered (GE) plants on non-target arthropods (NTAs). While we rely heavily on the currently used proteins from Bacillus thuringiensis (Bt) in this discussion, the concepts apply to other arthropod-active proteins. A risk may exist if the newly acquired trait of the GE plant has adverse effects on NTAs when they are exposed to the arthropod-active protein. Typically, the risk assessment follows a tiered approach that starts with laboratory studies under worst-case exposure conditions; such studies have a high ability to detect adverse effects on non-target species. Clear guidance on how such data are produced in laboratory studies assists the product developers and risk assessors. The studies should be reproducible and test clearly defined risk hypotheses. These properties contribute to the robustness of, and confidence in, environmental risk assessments for GE plants. Data from NTA studies, collected during the analysis phase of an environmental risk assessment, are critical to the outcome of the assessment and ultimately the decision taken by regulatory authorities on the release of a GE plant. Confidence in the results of early-tier laboratory studies is a precondition for the acceptance of data across regulatory jurisdictions and should encourage agencies to share useful information and thus avoid redundant testing.     

Risk analysis and management decisions for weed biological control agents: Ecological theory and modeling results

Biologically based control methods offer many advantages for the control of invasive plant species; however, these methods are not without risks to native species. Thus, there is a need for more effective and efficient methods of risk analysis for biological control agents. We show how the process of ecological risk assessment established by the United States’ Environmental Protection Agency may be adapted to improve assessment of the risks of proposed biological control agents. We discuss the risks posed by weed biological control agents, and present a simple individual-based model of herbivorous insect movement and oviposition on two species of host plant, a target invasive plant species and a non-target native species, in simulated landscapes. The model shows that risks of non-target impacts may be influenced by the details of the movement behavior of biological control agents in heterogeneous landscapes. The specific details of insect movement that appear to be relevant are readily measured in field trials and the general modeling approach is readily adapted to real landscapes. Current biological control risk assessments typically emphasize effects analysis at the expense of exposure analysis; the modeling approach presented here provides a simple and feasible way to incorporate exposure analyses. We conclude that models such as ours should be given serious consideration as part of a comprehensive strategy of risk assessment for proposed weed biological control agents.     

Risk analysis and management decisions for weed biological control agents: Ecological theory and modeling results

Biologically based control methods offer many advantages for the control of invasive plant species; however, these methods are not without risks to native species. Thus, there is a need for more effective and efficient methods of risk analysis for biological control agents. We show how the process of ecological risk assessment established by the United States’ Environmental Protection Agency may be adapted to improve assessment of the risks of proposed biological control agents. We discuss the risks posed by weed biological control agents, and present a simple individual-based model of herbivorous insect movement and oviposition on two species of host plant, a target invasive plant species and a non-target native species, in simulated landscapes. The model shows that risks of non-target impacts may be influenced by the details of the movement behavior of biological control agents in heterogeneous landscapes. The specific details of insect movement that appear to be relevant are readily measured in field trials and the general modeling approach is readily adapted to real landscapes. Current biological control risk assessments typically emphasize effects analysis at the expense of exposure analysis; the modeling approach presented here provides a simple and feasible way to incorporate exposure analyses. We conclude that models such as ours should be given serious consideration as part of a comprehensive strategy of risk assessment for proposed weed biological control agents.     

Assessing Risks of Plant-Based Pharmaceuticals: I. Human Dietary Exposure

Protein-based drugs are the fastest growing class of drugs for the treatment of disease in humans and other animals. However, the current method of producing proteins for pharmaceutical application is predicted to fall short because of population growth and demographic trends. This study characterized human dietary risks using quantitative risk assessment techniques for three pharmaceutical proteins produced in field-grown maize. The three proteins were aprotinin, gastric lipase, and Escherichia coli heat-labile enterotoxin B subunit (LT-B). The human dietary risks from the three proteins inadvertently occurring in food were evaluated using three different exposure scenarios so that potential risks could be compared. The three exposure scenarios ranged in conservatism to evaluate the range of risk between the proteins and scenarios. Risk quotients (RQs) were calculated for all three scenarios to integrate exposure and effect (toxicity). The risk assessments revealed that the most conservative scenario produced higher RQs than the other two scenarios. The dietary risks from scenario 1 for aprotinin were three orders of magnitude greater than for scenario 2, and four orders of magnitude greater than for scenario 3. This risk assessment revealed that dietary risks will vary dramatically and depend on factors such as the specific pharmaceutical protein, protein expression, and exposure scenarios. The assessment also reinforced the need for case-by-case assessments.     

On risk and plant-based biopharmaceuticals

Research into plant-based expression of pharmaceutical proteins is proceeding at a blistering pace. Indeed, plants expressing pharmaceutical proteins are currently being grown in field environments throughout the USA. But how are these plants and proteins being assessed for environmental risk and how are they being regulated? Here, we examine the applicability of the risk assessment paradigm for assessing human and ecological risks from field-grown transgenic plants that express pharmaceutical proteins.     

Risk assessment system establishment for evaluating the potential impacts of imported Bacillus thuringiensis maize on a non-target insect,Tenebrio molitor

Large amounts of genetically modified grains producing Bacillus thuringiensis (Bt) toxins have been imported to Korea. Therefore, the establishment of a risk assessment system for evaluating the potential impacts of imported Bt maize on non-target insects is important. Before evaluating the environmental impacts of Bt grains of unknown origin, Cry protein types must first be identified in test Bt grains. Cry toxins of imported Bt maize grains were analyzed by ELISA. Because all tested Bt maize grains contained Cry1A, Tenebrio molitor, a non-lepidopteran species, was selected as the non-target insect species. A domestic maize strain that showed few differences in nutritional composition compared to the Bt maize grain was used as the alternative non-Bt control. Slightly increased survival rate and head capsule width of Bt maize-fed T. molitor were observed, indicating that Bt maize has no sub-chronic adverse effects on T. molitor. An ELISA test revealed that concentrations of Cry1A toxins slowly increased in the body of T. molitor when the insects were fed Bt maize. Such substantial amounts of Cry toxins remaining in the alimentary tract of larvae indicate that Cry toxins can be transferred to the higher trophic level of predatory insects. However, no Cry proteins were detected in the hemolymph of the Bt maize-fed larvae, suggesting that there is little possibility of Cry toxin exposure via T. molitor to the higher endoparasitoids. The risk assessment strategies and protocols established in this study may also be applicable to other imported Bt crops in Korea.     

Assessing the exposure risk and impacts of pharmaceuticals in the environment on individuals and ecosystems

The use of human and veterinary pharmaceuticals is increasing. Over the past decade, there has been a proliferation of research into potential environmental impacts of pharmaceuticals in the environment. A Royal Society-supported seminar brought together experts from diverse scientific fields to discuss the risks posed by pharmaceuticals to wildlife. Recent analytical advances have revealed that pharmaceuticals are entering habitats via water, sewage, manure and animal carcases, and dispersing through food chains. Pharmaceuticals are designed to alter physiology at low doses and so can be particularly potent contaminants. The near extinction of Asian vultures following exposure to diclofenac is the key example where exposure to a pharmaceutical caused a population-level impact on non-target wildlife. However, more subtle changes to behaviour and physiology are rarely studied and poorly understood. Grand challenges for the future include developing more realistic exposure assessments for wildlife, assessing the impacts of mixtures of pharmaceuticals in combination with other environmental stressors and estimating the risks from pharmaceutical manufacturing and usage in developing countries. We concluded that an integration of diverse approaches is required to predict ‘unexpected’ risks; specifically, ecologically relevant, often long-term and non-lethal, consequences of pharmaceuticals in the environment for wildlife and ecosystems.     

Bt水稻田重要非靶标节肢动物暴露于Cry2Aa蛋白的程度分析

根据风险=危险×暴露的原理,在实验室条件下评价转基因作物对非靶标节肢动物影响时,所选择的代表性非靶标生物通常是在农田系统中较高地暴露于转基因外源杀虫蛋白的节肢动物种.为了弄清Bt稻田主要节肢动物暴露于Cry蛋白的程度,选择合适的非靶标节肢动物,用于转基因抗虫水稻的风险评价,本文采用酶联免疫技术检测了水稻不同生长期从转cry2Aa基因水稻田采集的不同节肢动物体内Cry2Aa蛋白的含量.结果表明: 不同节肢动物种体内的Cry蛋白含量差异显著.一些节肢动物体内不含Cry蛋白,而一些节肢动物体内含有较高的Cry蛋白;相对于花期后采集的节肢动物,在Bt水稻花期采集的节肢动物,特别是捕食性节肢动物体内的Cry蛋白含量较高;寄生性节肢动物体内未检测到Cry蛋白.这为在实验室条件下评价转基因水稻对农田非靶标节肢动物的影响奠定了基础.     

Selecting non-target species for risk assessment of entomophagous biological control agents: Evaluation of the PRONTI decision-support tool

The release of entomophagous biological control agents can pose risks to non-target invertebrate species in the release area and beyond. Pre-release risk assessment of these agents often involves tests with non-target species; however, selecting appropriate test species can be difficult when there is a large number to choose from. The PRONTI (priority ranking of non-target invertebrates) tool has been developed to aid this selection process. This automated tool prioritises species for testing using five criteria: (1) direct and indirect hazards posed by the agent, (2) likelihood of exposure to the hazards, (3) ecological impacts that may result from that exposure, (4) species’ anthropocentric value and (5) testability. Criteria (1) and (2) produce a risk estimate that drives the ranking process. In a test of PRONTI’s ability to identify non-target species at most risk from a proposed biological control agent, we used a generalist predator already present in New Zealand, the Asian paper wasp Polistes chinensis, as if it were the agent in a hypothetical biocontrol programme aimed at lepidopteran pests in New Zealand kiwifruit orchards. A ranked list of 340 invertebrate taxa known to occur in kiwifruit orchards was produced. To validate the risk estimates for a direct attack by P. chinensis on each taxon, wasps were introduced to kiwifruit orchards and prey taxa identified. Risk estimates were accurate except where identified prey taxa had not previously been recorded from kiwifruit orchards.     

Evaluating the necessity of additional aquatic plant testing by comparing the sensitivities of different species

At present, at least three and up to five plant species are required to assess the potential risks of herbicides to non-target aquatic plants. Several regulatory authorities are considering whether there should be further requirements based on concerns about the possible selectivity of herbicides (e.g., specific modes of action against dicotyledonous plants). The relative sensitivity of a range of aquatic plants is assessed in our work in order to evaluate the implications of differences in species sensitivity for aquatic risk assessment of herbicides. We therefore present results from ecotoxicological tests performed at Syngenta Crop Protection AG on various aquatic plants and compare them to available studies and results in literature. The criterion used for sensitivity ranking is the EC50 (median effect concentration) value, which allows a better comparison of values from different testing methods and conditions. The overall results obtained in the present work show that the aquatic risk assessment procedure for herbicides based on Lemna sp. and algae is sufficiently protective while identifying potential toxicity to non-target plants. Only few exceptions concerning herbicides with selective modes of action (e.g., auxin simulators) may require additional species testing for proper risk assessment.     

Environmental Fate of Neonicotinoids and Classification of Their Potential Risks to Hypogean,Epygean, and Surface Water Ecosystems in Brazil

Due to their reported high toxicity to honey bees, ecotoxicological studies into the side-effects of neonicotinoid insecticides have focused almost exclusively on these organisms. The fate of neonicotinoids and potential toxic side-effects on other (especially non-standard) organisms have received considerably less attention. In the present study, the environmental distribution and leaching potential of neonicotinoids registered for agricultural use in Brazil (acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam) were studied by applying several environmental fate models and indices. Potential risks to various environmental compartments were evaluated by applying ranking indices to the maximum application rates recommended in Brazil. Although bees were indeed found to be the most sensitive organism, the neonicotinoids also indicated potential environmental risks to other organism groups. Due to the greater maximum application rates recommended in Brazil as compared to other parts of the world, environmental risk and resistance potential for at least imidacloprid appears especially high in Brazil. Attention should thus also be allocated to organisms other than bees and to resistance potential when performing an environmental risk assessment of neonicotinoids if they are used at relatively high application rates.     

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.     

Bait consumption and residual concentrations of diphacinone in the Wellington tree weta (

To investigate the potential for mortality or sublethal effects in the tree weta (Hemideina crassidens) as the result of exposure to baits used for rodent control, and the potential secondary hazard to non-target species, captive weta were offered Ditrac® wax block bait containing the anticoagulant diphacinone. Bait consumption was recorded daily for the first week and then weekly. Weta were sampled in groups of four following 1, 4, 8, 6, 3, and 64 days of exposure to bait and analysed to determine the concentration of diphacinone residues in their bodies. Any changes in feeding behaviour, survival, and bodyweight were recorded. Weta found Ditrac wax block baits palatable even in the presence of natural plant food, showing steady consumption of bait over time. No mortality or weight loss was attributable to the intake of Ditrac bait. All weta that ate bait had detectable diphacinone in their bodies, but did not accumulate diphacinone, i.e. whole-body concentrations did not increase with the amount of diphacinone bait eaten over time. Field use of diphacinone bait is likely to present a low risk of mortality to weta, but the risk posed by secondary diphacinone exposure to non-target species that eat weta requires further investigation.     

Chlorpyrifos: Ecotoxicological Risk Assessment for Birds and Mammals in Corn Agroecosystems

A tiered risk assessment was conducted for the use of granular and liquid formulations of chlorpyrifos in corn agroecosystems in the U.S. The initial screening Tier I assessment suggested that under high-exposure scenarios the granular and some spray formulations present potential hazards to birds. Higher tiered probabilistic risk assessments were conducted separately for the granular and liquid formulations. The probabilistic assessment indicated that risk to birds from exposure to granular formulation is small and that this route of exposure would not be a significant source of mortality. Similarly, the assessment of potential exposure of birds to food items contaminated with chlorpyrifos showed that the risk from exposure via food was small, even if it was assumed that birds feed only on the treated fields. Although they have potentially greater sensitivity to chlorpyrifos, effects in nestling birds consuming food items from fields treated with granular chlorpyrifos were negligible. However, risks to young birds may be greater where the major source of food is from fields treated with liquid formulations of chlorpyrifos. A review of field studies showed that wildlife mortality incidents associated with use of either granular or liquid formulations of chlorpyrifos are not widely apparent in agroecosystems. Based on the multiple lines of evidence, we conclude that the presumption that chlorpyrifos use in corn agroecosystems will result in extensive mortality of terrestrial wildlife, particularly birds and mammals, is not supported by the scientific evidence.     

Leveraging existing data for prioritization of the ecological risks of human and veterinary pharmaceuticals to aquatic organisms

Medicinal innovation has led to the discovery and use of thousands of human and veterinary drugs. With this comes the potential for unintended effects on non-target organisms exposed to pharmaceuticals inevitably entering the environment. The impracticality of generating whole-organism chronic toxicity data representative of all species in the environment has necessitated prioritization of drugs for focused empirical testing as well as field monitoring. Current prioritization strategies typically emphasize likelihood for exposure (i.e. predicted/measured environmental concentrations), while incorporating only rather limited consideration of potential effects of the drug to non-target organisms. However, substantial mammalian pharmacokinetic and mechanism/mode of action (MOA) data are produced during drug development to understand drug target specificity and efficacy for intended consumers. An integrated prioritization strategy for assessing risks of human and veterinary drugs would leverage available pharmacokinetic and toxicokinetic data for evaluation of the potential for adverse effects to non-target organisms. In this reiview, we demonstrate the utility of read-across approaches to leverage mammalian absorption, distribution, metabolism and elimination data; analyse cross-species molecular target conservation and translate therapeutic MOA to an adverse outcome pathway(s) relevant to aquatic organisms as a means to inform prioritization of drugs for focused toxicity testing and environmental monitoring.     

Genetically modified crops and aquatic ecosystems: considerations for environmental risk assessment and non-target organism testing

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.