The Ecotoxicology of Microbial Insecticides and Their Toxins in Genetically Modified Crops: An Overview.

The use of microbial insecticides and their toxins in biological control and transgenic plants has increased their presence in the environment. Although they are natural products, the main concerns are related to the potential impacts on the environment and human health. Several assays have been performed worldwide to investigate the toxicity or adverse effects of these microbial products or their individual toxins. This overview examines the published data concerning the knowledge obtained about the ecotoxicity and environmental risks of these natural pesticides. The data presented show that many results are difficult to compare due to the diversity of measurement units used in the different research data. Even so, the products and toxins tested present low toxicity and low risk when compared to the concentrations used for pesticide purposes. Complementary studies should be carried out to assess possible effects on human health.

Bioaccumulation and Phytotoxicity and Human Health Risk from Microcystin-LR under Various Treatments: A Pot Study.

Microcystin-LR (MC-LR) is prevalent in water and can be translocated into soil-crop ecosystem via irrigation, overflow (pollution accident), and cyanobacterial manure applications, threatening agricultural production and human health. However, the effects of various input pathways on the bioaccumulation and toxicity of MCs in terrestrial plants have been hardly reported so far. In the present study, pot experiments were performed to compare the bioaccumulation, toxicity, and health risk of MC-LR as well as its degradation in soils among various treatments with the same total amount of added MC-LR (150 µg/kg). The treatments included irrigation with polluted water (IPW), cultivation with polluted soil (CPS), and application of cyanobacterial manure (ACM). Three common leaf-vegetables in southern China were used in the pot experiments, including Ipomoea batatas L., Brassica juncea L., and Brassica alboglabra L. All leaf vegetables could bioaccumulate MC-LR under the three treatments, with much higher MC-LR bioaccumulation, especially root bioconcentration observed in ACM treatment than IPW and CPS treatments. An opposite trend in MC-LR degradation in soils of these treatments indicated that ACM could limit MC-LR degradation in soils and thus promote its bioaccumulation in the vegetables. MC-LR bioaccumulation could cause toxicity to the vegetables, with the highest toxic effects observed in ACM treatment. Similarly, bioaccumulation of MC-LR in the edible parts of the leaf-vegetables posed 1.1~4.8 fold higher human health risks in ACM treatment than in IPW and CPS treatments. The findings of this study highlighted a great concern on applications of cyanobacterial manure.

Gut remediation: a potential approach to reducing chromium accumulation using Lactobacillus plantarum TW1-1.

Some lactobacilli have protective effects against some heavy metals in mammals, but the underlying mechanism is not fully understood. To evaluate the remediation potency and the mechanism of Lactobacillus against chromium (Cr) in mice, Lactobacillus plantarum TW1-1 was orally administrated to Kunming mice for 7 weeks during exposure to 1 mM K2Cr2O7 in drinking water. Results showed that TW1-1 helped to decrease Cr accumulation in tissues and increase Cr excretion in feces, and may also attenuate alterations in oxidative stress and histopathological changes caused by Cr exposure. Moreover, the chromate reduction ability of fecal bacteria doubled after administration of TW1-1 upon Cr induction. MiSeq sequencing of fecal bacterial 16S rRNA genes revealed that the overall structures of gut microbiota was shifted by Cr exposure and partially restored by TW1-1. The abundances of 49 of the 79 operational taxonomic units altered by Cr were reversed by TW1-1. Based on these, we proposed a working model of TW1-1 against Cr: TW1-1 helps to remove Cr from the host and meanwhile acts as a regulator of gut microbiota, which aids in chromate reduction and provide protection against Cr. We call this process of remediation of heavy metal in the gut "gut remediation".

Procedure to select test organisms for environmental risk assessment of genetically modified crops in aquatic systems.

For a long time, the environmental risk assessment (ERA) of genetically modified (GM) crops focused mainly on terrestrial ecosystems. This changed when it was scientifically established that aquatic ecosystems are exposed to GM crop residues that may negatively affect aquatic species. To assist the risk assessment process, we present a tool to identify ecologically relevant species usable in tiered testing prior to authorization or for biological monitoring in the field. The tool is derived from a selection procedure for terrestrial ecosystems with substantial but necessary changes to adequately consider the differences in the type of ecosystems. By using available information from the Water Framework Directive (2000/60/EC), the procedure can draw upon existing biological data on aquatic systems. The proposed procedure for aquatic ecosystems was tested for the first time during an expert workshop in 2013, using the cultivation of Bacillus thuringiensis (Bt) maize as the GM crop and 1 stream type as the receiving environment in the model system. During this workshop, species executing important ecological functions in aquatic environments were identified in a stepwise procedure according to predefined ecological criteria. By doing so, we demonstrated that the procedure is practicable with regard to its goal: From the initial long list of 141 potentially exposed aquatic species, 7 species and 1 genus were identified as the most suitable candidates for nontarget testing programs. Integr Environ Assess Manag 2017;13:974-979. © 2017 SETAC.

Metal-contaminated potato crops and potential human health risk in Bolivian mining highlands.

This study assessed metals in irrigation water, soil and potato crops impacted by mining discharges, as well as potential human health risk in the high desert near the historic mining center of Potosí, Bolivia. Metal concentrations were compared with international concentration limit guidelines. In addition, an ingested average daily dose and minimum risk level were used to determine the hazard quotient from potato consumption for adults and children. Irrigation water maximum concentrations of Cd, Pb and Zn in mining-impacted sites were elevated 20- to 1100-fold above international concentration limit guidelines. Agricultural soils contained total metal concentrations of As, Cd, Pb and Zn that exceeded concentration limits in agricultural soil guidelines by 22-, 9-, 3- and 12-fold, respectively. Potato tubers in mining-impacted sites had maximum concentrations of As, Cd, Pb and Zn that exceeded concentration limits in commercially sold vegetables by 9-, 10-, 16- and fourfold, respectively. Using conservative assumptions, hazard quotients (HQ) for potatoes alone were elevated for As, Cd and Pb among children (range 1.1-71.8), in nearly all of the mining-impacted areas; and for As and Cd among adults (range 1.2-34.2) in nearly all of the mining-impacted areas. Only one mining-impacted area had a Pb adult HQ for potatoes above 1 for adults. Toxic trace elements in a major regional dietary staple may be a greater concern than previously appreciated. Considering the multitude of other metal exposure routes in this region, it is likely that total HQ values for these metals may be substantially higher than our estimates.

Ecological risk assessment for DvSnf7 RNA: A plant-incorporated protectant with targeted activity against western corn rootworm.

MON 87411 maize, which expresses DvSnf7 RNA, was developed to provide an additional mode of action to confer protection against corn rootworm (Diabrotica spp.). A critical step in the registration of a genetically engineered crop with an insecticidal trait is performing an ecological risk assessment to evaluate the potential for adverse ecological effects. For MON 87411, an assessment plan was developed that met specific protection goals by characterizing the routes and levels of exposure, and testing representative functional taxa that would be directly or indirectly exposed in the environment. The potential for toxicity of DvSnf7 RNA was evaluated with a harmonized battery of non-target organisms (NTOs) that included invertebrate predators, parasitoids, pollinators, soil biota as well as aquatic and terrestrial vertebrate species. Laboratory tests evaluated ecologically relevant endpoints such as survival, growth, development, and reproduction and were of sufficient duration to assess the potential for adverse effects. No adverse effects were observed with any species tested at, or above, the maximum expected environmental concentration (MEEC). All margins of exposure for NTOs were >10-fold the MEEC. Therefore, it is reasonable to conclude that exposure to DvSnf7 RNA, both directly and indirectly, is safe for NTOs at the expected field exposure levels.

Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

Corn rootworm-active RNA DvSnf7: Repeat dose oral toxicology assessment in support of human and mammalian safety.

Genetically modified (GM) crops have been developed and commercialized that utilize double stranded RNAs (dsRNA) to suppress a target gene(s), producing virus resistance, nutritional and quality traits. MON 87411 is a GM maize variety that leverages dsRNAs to selectively control corn rootworm through production of a 240 base pair (bp) dsRNA fragment targeting for suppression the western corn rootworm (Diabrotica virgifera virgifera) Snf7 gene (DvSnf7). A bioinformatics assessment found that endogenous corn small RNAs matched ∼450 to 2300 unique RNA transcripts that likely code for proteins in rat, mouse, and human, demonstrating safe dsRNA consumption by mammals. Mice were administered DvSnf7 RNA (968 nucleotides, including the 240 bp DvSnf7 dsRNA) at 1, 10, or 100 mg/kg by oral gavage in a 28-day repeat dose toxicity study. No treatment-related effects were observed in body weights, food consumption, clinical observations, clinical chemistry, hematology, gross pathology, or histopathology endpoints. Therefore, the No Observed Adverse Effect Level (NOAEL) for DvSnf7 RNA was 100 mg/kg, the highest dose tested. These results demonstrate that dsRNA for insect control does not produce adverse health effects in mammals at oral doses millions to billions of times higher than anticipated human exposures and therefore poses negligible risk to mammals.

Adaptation of the ToxRTool to Assess the Reliability of Toxicology Studies Conducted with Genetically Modified Crops and Implications for Future Safety Testing.

To determine the reliability of food safety studies carried out in rodents with genetically modified (GM) crops, a Food Safety Study Reliability Tool (FSSRTool) was adapted from the European Centre for the Validation of Alternative Methods' (ECVAM) ToxRTool. Reliability was defined as the inherent quality of the study with regard to use of standardized testing methodology, full documentation of experimental procedures and results, and the plausibility of the findings. Codex guidelines for GM crop safety evaluations indicate toxicology studies are not needed when comparability of the GM crop to its conventional counterpart has been demonstrated. This guidance notwithstanding, animal feeding studies have routinely been conducted with GM crops, but their conclusions on safety are not always consistent. To accurately evaluate potential risks from GM crops, risk assessors need clearly interpretable results from reliable studies. The development of the FSSRTool, which provides the user with a means of assessing the reliability of a toxicology study to inform risk assessment, is discussed. Its application to the body of literature on GM crop food safety studies demonstrates that reliable studies report no toxicologically relevant differences between rodents fed GM crops or their non-GM comparators.

GM crops and the rat digestive tract: a critical review.

The aim of this review is to examine the relationship between genetically modified (GM) crops and health, based on histopathological investigations of the digestive tract in rats. We reviewed published long-term feeding studies of crops containing one or more of three specific traits: herbicide tolerance via the EPSPS gene and insect resistance via cry1Ab or cry3Bb1 genes. These genes are commonly found in commercialised GM crops. Our search found 21 studies for nine (19%) out of the 47 crops approved for human and/or animal consumption. We could find no studies on the other 38 (81%) approved crops. Fourteen out of the 21 studies (67%) were general health assessments of the GM crop on rat health. Most of these studies (76%) were performed after the crop had been approved for human and/or animal consumption, with half of these being published at least nine years after approval. Our review also discovered an inconsistency in methodology and a lack of defined criteria for outcomes that would be considered toxicologically or pathologically significant. In addition, there was a lack of transparency in the methods and results, which made comparisons between the studies difficult. The evidence reviewed here demonstrates an incomplete picture regarding the toxicity (and safety) of GM products consumed by humans and animals. Therefore, each GM product should be assessed on merit, with appropriate studies performed to indicate the level of safety associated with them. Detailed guidelines should be developed which will allow for the generation of comparable and reproducible studies. This will establish a foundation for evidence-based guidelines, to better determine if GM food is safe for human and animal consumption.

Characterising microbial protein test substances and establishing their equivalence with plant-produced proteins for use in risk assessments of transgenic crops.

Most commercial transgenic crops are genetically engineered to produce new proteins. Studies to assess the risks to human and animal health, and to the environment, from the use of these crops require grams of the transgenic proteins. It is often extremely difficult to produce sufficient purified transgenic protein from the crop. Nevertheless, ample protein of acceptable purity may be produced by over-expressing the protein in microbes such as Escherichia coli. When using microbial proteins in a study for risk assessment, it is essential that their suitability as surrogates for the plant-produced transgenic proteins is established; that is, the proteins are equivalent for the purposes of the study. Equivalence does not imply that the plant and microbial proteins are identical, but that the microbial protein is sufficiently similar biochemically and functionally to the plant protein such that studies using the microbial protein provide reliable information for risk assessment of the transgenic crop. Equivalence is a judgement based on a weight of evidence from comparisons of relevant properties of the microbial and plant proteins, including activity, molecular weight, amino acid sequence, glycosylation and immuno-reactivity. We describe a typical set of methods used to compare proteins in regulatory risk assessments for transgenic crops, and discuss how risk assessors may use comparisons of proteins to judge equivalence.

Spontaneous renal tumors in two rats from a thirteen week rodent feeding study with grain from molecular stacked trait lepidopteran and coleopteran resistant (DP-ØØ4114-3) maize.

A thirteen week feeding study was conducted by feeding young adult male and female Sprague Dawley [Crl:CD®(SD)] rats diets containing grain from genetically modified (GM) DP-ØØ4114-3 maize that was either untreated (4114) or treated in the field with glufosinate ammonium (4114GLU). Control rats were fed diets containing the same concentration of near isogenic, non-GM maize grain (091) or one of three types of commercially available non-GM maize grain. At the end of the in-life phase, renal tubule tumors were reported in two male rats consuming diets containing 4114 maize grain. An expert panel of pathologists was convened as a Pathology Working Group (PWG) to review coded kidney histology sections from control (091) and treated (4114 and 4114GLU) male rats. The objectives were for the panel to characterize the histopathologic findings and to interpret their relationship to consumption of the indicated diet. The PWG concluded unanimously that the kidney tumors were characteristic of amphophilic-vacuolar (AV) tumors and AV atypical tubular hyperplasia which represent a distinctive phenotype that has been reported to occur sporadically in young Sprague Dawley Rats. The PWG determined that the neoplasms and atypical tubular hyperplasias were multicentric and bilateral which typifies tumors of familial origin. Degenerative/regenerative or cytotoxic changes consistent with nephrotoxicity leading to tumor induction were not observed in these rats and thus supports the conclusion that tumors were unrelated to consumption of the test diet. It was the unanimous opinion of the PWG that the proliferative renal tubule cell lesions were spontaneous and not related to consumption of diets containing 4114 maize grain.

Thirteen week rodent feeding study with grain from molecular stacked trait lepidopteran and coleopteran protected (DP-ØØ4114-3) maize.

The results from a subchronic feeding study conducted in Sprague­Dawley rats fed with diets containing grain from 4114 (OECD unique identifier: DP-ØØ4114-3) maize that was untreated (4114) or sprayed in field with glufosinate ammonium (4114GLU) in a design similar to previous studies are reported. The test material, 4114 maize, is a hybrid maize produced by transformation with a DNA construct encoding 4 different transgenic proteins for resistance to lepidopteran pests, coleopteran pests, and tolerance to the herbicidal active ingredient glufosinate ammonium. There were a total of 144 rats divided into 12 groups of 12 rats/sex/group. All experimental diets were formulated by Purina Mills, LLC (St. Louis, MO) in accordance with the standards of Purina Mills Labdiet® Certified Rodent LabDiet® 5002. The incorporation rate of maize grain in all diets was 32% (wt/wt). No biologically significant, treatment related differences in body weight, food consumption, clinical pathology parameters (hematology, blood chemistry, urinalysis, or organ weight) were observed in rats consuming the diets containing 4114 maize grain compared with rats fed conventional maize diets. A number of histologic observations were noted in this study but were background lesions and representative of what would be expected for rats of this age and strain. An independent panel of experts determined certain observations to be spontaneous and not related to the test diet. Accordingly, these results support the conclusion that 4114 maize grain is as safe and nutritious as conventional maize grain.

Inactivation of allergens and toxins.

Plants are replete with thousands of proteins and small molecules, many of which are species-specific, poisonous or dangerous. Over time humans have learned to avoid dangerous plants or inactivate many toxic components in food plants, but there is still room for ameliorating food crops (and plants in general) in terms of their allergens and toxins content, especially in their edible parts. Inactivation at the genetic rather than physical or chemical level has many advantages and classical genetic approaches have resulted in significant reduction of toxin content. The capacity, offered by genetic engineering, of turning off (inactivating) specific genes has opened up the possibility of altering the plant content in a far more precise manner than previously available. Different levels of intervention (genes coding for toxins/allergens or for enzymes, transporters or regulators involved in their metabolism) are possible and there are several tools for inactivating genes, both direct (using chemical and physical mutagens, insertion of transposons and other genetic elements) and indirect (antisense RNA, RNA interference, microRNA, eventually leading to gene silencing). Each level/strategy has specific advantages and disadvantages (speed, costs, selectivity, stability, reversibility, frequency of desired genotype and regulatory regime). Paradigmatic examples from classical and transgenic approaches are discussed to emphasize the need to revise the present regulatory process. Reducing the content of natural toxins is a trade-off process: the lesser the content of natural toxins, the higher the susceptibility of a plant to pests and therefore the stronger the need to protect plants. As a consequence, more specific pesticides like Bt are needed to substitute for general pesticides.

Exposure to farm crops, livestock, and farm tasks and risk of glioma: the Upper Midwest Health Study.

Some studies of brain cancer have found an excess risk for farmers. The National Institute for Occupational Safety and Health previously found no increased glioma risk for ever (vs. never) being exposed to pesticides on a farm among 798 cases and 1,175 population-based controls (adult (ages 18-80 years) nonmetropolitan residents of Iowa, Michigan, Minnesota, and Wisconsin). For this analysis (1995-1998), 288 cases and 474 controls (or their proxies) who had lived on farms at age 18 years or after were asked about exposure to crops, livestock, and farm tasks. Logistic regression was used to calculate odds ratios adjusted for age, age group, sex, state, and education. Never immediately washing up (adjusted odds ratio (OR) = 3.08, 95% confidence interval (CI): 1.78, 5.34) or changing clothes (OR = 2.84, 95% CI: 1.04, 7.78) after applying pesticides was associated with increased glioma risk. Living on a farm on which corn, oats, soybeans, or hogs were raised was associated with decreased risk (corn-OR = 0.37, 95% CI: 0.20, 0.69; oats-OR = 0.63, 95% CI: 0.40, 1.00; soybeans-OR = 0.69, 95% CI: 0.48, 0.98; hogs-OR = 0.63, 95% CI: 0.43, 0.93). Negative associations may be due to chance or a "healthy farmer" effect. Farmers' increased risk of glioma may be due to work practices, other activities, or an inverse association with allergies (reported by other investigators).

Pesticide residues in conventionally grown and organic New Zealand produce.

A study was carried out to determine the prevalence and concentrations of pesticide residues in conventionally grown and organic produce (bananas, broccoli, grapes, lettuce, potatoes, tomatoes, wine). Sampling was stratified, with organic produce being over-sampled, when available. All food samples were analysed, as received, by a multi-residue pesticide residue screen. Residues were found in 130 of 307 conventionally grown food samples (42%) and in nine of 41 organic food samples (22%), including six of eleven (55%) organic tomato samples. Only four organic samples (9.8%) contained multiple residues, while 24% of conventionally grown food samples contained multiple residues. Nine conventionally grown food samples (2.6%) contained pesticide residues that exceeded the maximum residue limit (MRL). Where direct comparisons were possible between conventionally grown and organic produce, the mean concentration of residues was usually lower in the organic produce, but was generally higher than would be expected from spray drift or other adventitious sources. While the presence of these residues does not represent a significant risk to human health, their presence is inconsistent with consumer expectations for organic produce.