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Residues of herbicides with the extensive applications may impact the soil ecosystem and ultimately threaten agricultural sustainability. However, the effects of long-term herbicide residues on soil multifunctionality and the soil microbial community remain poorly understood. Here, we evaluated relationships between soil multifunctionality and soil microbial communities with residual herbicide concentrations by surveying and analyzing 62 black soil samples collected from an agricultural area in northeastern China. Total residual herbicide concentrations varied from 35 to 568 µg/kg in the soil samples. The response of soil multifunctionality to increasing residual herbicide concentrations exhibited an inverted U-shaped relationship with a peak at approximately 310 µg/kg, with net mineralized organic nitrogen (Nm) and total nitrogen (TN) exhibiting the same trend. Microbial community richness was significantly lower in soil samples with high residual herbicide concentrations (> 310 µg/kg, HG) compared to low residual herbicide concentrations (< 310 µg/kg, LG). In addition, the relative abundances of specific keystone microbial genera differed significantly between LG and HG: norank_f_Acetobacteraceae, norank_f_Caldilineaceae, Candidatus_Alysiosphaera, and Gonytrichum. The relative abundances of these genera were also significantly correlated with soil multifunctionality. Structural equation models (SEMs) further showed that herbicide residues influenced soil multifunctionality by affecting these specific keystone genera. Our study demonstrates that long-term herbicide residues significantly impact the multifunctionality of agricultural black soil, where low concentrations stimulate while high concentrations inhibit, underscoring the need for reasonable application of herbicides to maintain soil ecosystem health.

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The use of glyphosate-based herbicides (GBHs) to control weeds has increased exponentially in recent decades, and their residues and degradation products have been found in soils across the globe. GBH residues in soil have been shown to affect plant physiology and specialised metabolite biosynthesis, which, in turn, may impact plant resistance to biotic stressors. In a greenhouse study, we investigated the interactive effects between soil GBH residues and herbivory on the performance, phytohormone concentrations, phenolic compound concentrations and volatile organic compound (VOC) emissions of two woodland strawberry (Fragaria vesca) genotypes, which were classified as herbivore resistant and herbivore susceptible. Plants were subjected to herbivory by strawberry leaf beetle (Galerucella tenella) larvae, and to GBH residues by growing in soil collected from a field site with GBH treatments twice a year over the past eight years. Soil GBH residues reduced the belowground biomass of the susceptible genotype and the aboveground biomass of both woodland strawberry genotypes. Herbivory increased the belowground biomass of the resistant genotype and the root-shoot ratio of both genotypes. At the metabolite level, herbivory induced the emission of several VOCs. Jasmonic acid, abscisic acid and auxin concentrations were induced by herbivory, in contrast to salicylic acid, which was only induced by herbivory in combination with soil GBH residues in the resistant genotype. The concentrations of phenolic compounds were higher in the resistant genotype compared to the susceptible genotype and were induced by soil GBH residues in the resistant genotype. Our results indicate that soil GBH residues can differentially affect plant performance, phytohormone concentrations and phenolic compound concentrations under herbivore attack, in a genotype-dependent manner. Soil GBH altered plant responses to herbivory, which may impact plant resistance traits and species interactions. With ongoing agrochemical pollution, we need to consider plant cultivars with better resistance to polluted soils while maintaining plant resilience under challenging environmental conditions.

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The widespread use of herbicides impacts non-target organisms, promotes weed resistance, posing a serious threat to the global goal of green production in agriculture. Although the herbicide residues have been widely reported in individual environmental medium, their presence across different media has received scant attention, particularly in Mollisols regions with intensive agricultural application of herbicides. A systematic investigation was conducted in this study to clarify the occurrence of herbicide residues in soil, surface water, sediments, and grains from a typical agricultural watershed in the Mollisols region of Northeast China. Concentrations of studied herbicides ranged from 0.30 to 463.49 µg/kg in soil, 0.31-29.73 µg/kg in sediments, 0.006-1.157 µg/L in water, and 0.32-2.83 µg/kg in grains. Among these, Clomazone was the most priority herbicide detected in soil, sediments, and water, and Pendimethalin in grains. Crop types significantly affected the residue levels of herbicides in grains. Clomazone posed high ecological risks in soil and water, with 86.4 % of water samples showing high risks from herbicide mixtures (RQ > 1). These findings aid in enhancing our comprehension of the pervasive occurrence and potential ecological risks of herbicides in different media within typical agricultural watersheds, providing detailed data to inform the development of targeted mitigation strategies.

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(1) Background: Aryloxyphenoxy-propionates and cyclohexanediones are herbicides most widely used in dicot crops worldwide. The main objective of the study was to determine the dynamics of herbicide residues in carrot, lettuce, cauliflower, and onion in order to suggest a low level of residues in harvested vegetables. (2) Methods: Small plot field trials were carried out in four vegetables in the Czech Republic. The samples of vegetables were collected continuously during the growing season. Multiresidue methods for the determination of herbicide residues by LC-MS/MS were used. Non-linear models of degradation of individual herbicides in vegetables were calculated using the exponential decay formula. Action GAP pre-harvest intervals for the 25% and 50% maximum residue limit (MRL) and 10 µg kg-1 limit (baby food) were established for all tested herbicides. (3) Results: The degradation dynamics of fluazifop in carrot, onion, and cauliflower was significantly slower compared to quizalofop and haloxyfop. The highest amount (2796 µg kg-1) of fluazifop residues was detected in cauliflower 11 days after application. No residue of propaquizafop and cycloxydim was detected in any vegetable samples. (4) Conclusions: Aryloxyphenoxy-propionate herbicide (except propaquizafop) could contaminate vegetables easily, especially vegetables with a short growing season. Vegetables treated with fluazifop are not suitable for baby food. Lettuce and cauliflower treated by quizalofop are not suitable for baby food, but in onion and carrot, quizalofop could be used. Propaquizafop and cycloxydim are prospective herbicides for non-residual (baby food) vegetable production.

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Recent genetically modified plants tend to include both insect resistance and herbicide tolerance traits. Some of these 'stacked' GM plants have multiple Cry-toxins expressed as well as tolerance to several herbicides. This means that non-target organisms in the environment (biodiversity) will be co-exposed to multiple stressors simultaneously. A similar co-exposure may happen to consumers through chemical residues in the food chain. EFSA, the responsible unit for minimizing risk of harm in European food chains, has expressed its scientific interest in combinatorial effects. However, when new data showed how two Cry-toxins acted in combination (added toxicity), and that the same Cry-toxins showed combinatorial effects when co-exposed with Roundup (Bøhn et al., 2016), EFSA dismissed these new peer-reviewed results. In effect, EFSA claimed that combinatorial effects are not relevant for itself. EFSA was justifying this by referring to a policy question, and by making invalid assumptions, which could have been checked directly with the lead-author. With such approach, EFSA may miss the opportunity to improve its environmental and health risk assessment of toxins and pesticides in the food chain. Failure to follow its own published requests for combinatorial effects research, may also risk jeopardizing EFSA's scientific and public reputation.

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The aim of this study was to examine the influence of glyphosate (GL) residues in feedstuffs on performance, energy balance and health-related characteristics of lactating dairy cows fed diets with different concentrate feed proportions. After an adaption period, 64 German Holstein cows (207 ± 49 d in milk; mean ± SD) were assigned to either groups receiving a GL contaminated total mixed ration (TMR) (GL groups) or an uncontaminated TMR (CON groups) during a 16 weeks trial. Contaminated feedstuffs used were legally GL-treated peas and wheat (straw and grain). GL and CON groups were subdivided into a "low concentrate" group (LC) fed on dry matter (DM) basis of 21% maize silage, 42% grass silage, 7% straw and 30% concentrate and a "high concentrate" group (HC) composed of 11% maize silage, 22% grass silage, 7% straw and 60% concentrate for ad libitum consumption. Body condition score, body weight, DM intake and milk performance parameters were recorded. In blood serum, ß-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA) and glucose were measured and energy balance was calculated. Milk was analysed for GL residues. At week 0, 7 and 15, general health status was evaluated by a modified clinical score. The average individual GL intake amounted for Groups CONLC, CONHC, GLLC and GLHC to 0.8, 0.8, 73.8 and 84.5 mg/d, respectively. No GL residues were detected in milk. GL contamination did not affect body condition score, body weight, DM intake, nutrient digestibility, net energy intake, net energy balance or BHB, glucose, NEFA and milk performance parameters; whereas concentrate feed proportion and time did affect most parameters. The clinical examination showed no adverse effect of GL-contaminated feedstuffs on cows' health condition. In the present study, GL-contaminated feedstuffs showed no influence on performance and energy balance of lactating dairy cows, irrespective of feed concentrate proportion.

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Increasing nitrogen (N) immobilization and weed interference in the early phase of implementation of conservation agriculture (CA) affects crop yields. Yet, higher fertilizer and herbicide use to improve productivity influences greenhouse gase emissions and herbicide residues. These tradeoffs precipitated a need for adaptive N and integrated weed management in CA-based maize (Zea mays L.)-wheat [Triticum aestivum (L.) emend Fiori & Paol] cropping system in the Indo-Gangetic Plains (IGP) to optimize N availability and reduce weed proliferation. Adaptive N fertilization was based on soil test value and normalized difference vegetation index measurement (NDVM) by GreenSeeker™ technology, while integrated weed management included brown manuring (Sesbania aculeata L. co-culture, killed at 25 days after sowing), herbicide mixture, and weedy check (control, i.e., without weed management). Results indicated that the 'best-adaptive N rate' (i.e., 50% basal + 25% broadcast at 25 days after sowing + supplementary N guided by NDVM) increased maize and wheat grain yields by 20 and 14% (averaged for 2 years), respectively, compared with whole recommended N applied at sowing. Weed management by brown manuring (during maize) and herbicide mixture (during wheat) resulted in 10 and 21% higher grain yields (averaged for 2 years), respectively, over the weedy check. The NDVM in-season N fertilization and brown manuring affected N2O and CO2 emissions, but resulted in improved carbon storage efficiency, while herbicide residuals in soil were significantly lower in the maize season than in wheat cropping. This study concludes that adaptive N and integrated weed management enhance synergy between agronomic productivity, fertilizer and herbicide efficiency, and greenhouse gas mitigation.

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BACKGROUND: MON89788 was the first genetically engineered soybean worldwide to express a Bt toxin. Under the brand name Intacta, Monsanto subsequently engineered a stacked trait soybean using MON89788 and MON87701-this stacked soybean expresses an insecticidal toxin and is, in addition, tolerant to glyphosate. After undergoing risk assessment by the European Food Safety Authority (EFSA), the stacked event was authorised for import into the EU in June 2012, including for use in food and feed. This review discusses the health risks associated with Bt toxins present in these genetically engineered plants and the residues left from spraying with the complementary herbicide. RESULTS: We have compared the opinion published by EFSA [1] with findings from other publications in the scientific literature. It is evident that there are several issues that EFSA did not consider in detail and which will need further assessment: (1) There are potential combinatorial effects between plant components and other impact factors that might enhance toxicity. (2) It is known that Bt toxins have immunogenic properties; since soybeans naturally contain many allergens, these immunogenic properties raise specific questions. (3) Fully evaluated and reliable protocols for measuring the Bt concentration in the plants are needed, in addition to a comprehensive set of data on gene expression under varying environmental conditions. (4) Specific attention should be paid to the herbicide residues and their interaction with Bt toxins. CONCLUSIONS: The case of the Intacta soybeans highlights several regulatory problems with Bt soybean plants in the EU. Moreover, many of the issues raised also concern other genetically engineered plants that express insecticidal proteins, or are engineered to be resistant to herbicides, or have those two types of traits combined in stacked events. It remains a matter of debate whether the standards currently applied by the risk assessor, EFSA, and the risk manager, the EU Commission, meet the standards for risk analysis defined in EU regulations such as 1829/2003 and Directive 2001/18. While this publication cannot provide a final conclusion, it allows the development of some robust hypotheses that should be investigated further before such plants can be considered to be safe for health and the environment. In general, the concept of comparative risk assessment needs some major revision. Priority should be given to developing more targeted approaches. As shown in the case of Intacta, these approaches should include: (i) systematic investigation of interactions between the plant genome and environmental stressors as well as their impact on gene expression and plant composition; (ii) detailed investigations of the toxicity of Bt toxins; (iii) assessment of combinatorial effects taking into account long-term effects and the residues from spraying with complementary herbicides; (iv) investigation into the impact on the immune and hormonal systems and (v) investigation of the impact on the intestinal microbiome after consumption. Further and in general, stacked events displaying a high degree of complexity due to possible interactions should not undergo a lower level of risk assessment than the parental plants.

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A rapid and sensitive method was developed for the determination of 50 herbicides in cereal grain by ultra-performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS). Using acetonitrile effectively extracted 22 kinds of triazine and other basic herbicides, and using 90:10 v/v acetonitrile-phosphate buffer (pH = 7.5) effectively extracted other 28 herbicides. Chromatographic separation was achieved using gradient elution with acetonitrile-water as a mobile phase for 22 triazine and phenylurea herbicides and with 5mM ammonium acetate aqueous solution containing 0.1% formic acid-acetonitrile as a mobile phase for other 28 herbicides. Using matrix-matched standard calibration curve effectively reduced the indirect matrix effects, ensured accurate quantification for these herbicides. The response was linear over two orders of magnitude with a correlation coefficients (r(2)) higher than 0.992. The limits of quantification for the herbicides varied from 0.2 to 25.6 µg kg(-1). The intra- and inter-day precisions (relative standard deviation, RSD) were 2.2-9.3% and 5.7-17.1%, respectively. The recovery varied from 61.6% to 110% with the RSD of 1.6-11.8%. Analyzing soybean, corn and wheat samples from 17 counties evaluated this method. The developed and validated method has high sensitivity, satisfactory recovery and precision, can ensure the multi-class multi-residue analysis at low µg kg(-1) level for the most herbicides in cereal grain.

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A time-saving and organic solvent efficient method to simultaneously determine six kinds of herbicide residues in tobacco using solid-phase extraction for sample clean-up and preconcentration and the highly sensitive ultraperformance convergence chromatography method was developed. Parameters for ultraperformance convergence chromatography, including the choice of stationary phase and modifiers, autobackpressure regulator pressure, column temperature, and the flow rate of mobile solvents, were optimized. The herbicide residues of napropamide, alachlor, quizalofop-ethyl, diphenamid, metolachlor, and clomazone in tobacco samples were successfully separated and detected at levels as low as 0.0043-0.0086 mg/kg within 5 min using a nonpolar high strength silica C18 selectivity for bases column and methanol as the cosolvent of the mobile phase of carbon dioxide (75-99.9%, v/v). Analysis of tobacco samples had recoveries of 69.8-95.0%, limit of quantitation of 0.0127-0.0245 mg/kg, limit of detection of 0.0043-0.0086 mg/kg, and correlation coefficient of >0.9990. Results support this method as an efficient alternative to current methodologies for the determination of herbicide residues in tobacco.

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The phytoextraction capacity of Italian ryegrass (Lolium multiflorum L.) to remove terbuthylazine (TBA) from aqueous solution has been assessed using a plant-based biotest (RHIZOtest). Three TBA concentrations (0.5, 1.0 and 2.0 mg L(-1)) were chosen to evaluate the tolerance capacity of the ryegrass. Even though the treatments negatively affected plants, they were able to remove up to 30-40% of TBA. In addition, some enzymatic activities involved in the response to TBA-induced stress were determined. Glutathione S-transferase (GST) has been activated with a TBA-dose dependent trend; ascorbate peroxidase (APX) activities have been induced within the first hours after the treatments, followed by decreases or disappearance in plants exposed to two higher dosages. In conclusion, this case-study highlights that the combination of ryegrass and RHIZOtest resulted to be effective in the remediation of aqueous solutions polluted by TBA.

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This article describes the nutrient and elemental composition, including residues of herbicides and pesticides, of 31 soybean batches from Iowa, USA. The soy samples were grouped into three different categories: (i) genetically modified, glyphosate-tolerant soy (GM-soy); (ii) unmodified soy cultivated using a conventional "chemical" cultivation regime; and (iii) unmodified soy cultivated using an organic cultivation regime. Organic soybeans showed the healthiest nutritional profile with more sugars, such as glucose, fructose, sucrose and maltose, significantly more total protein, zinc and less fibre than both conventional and GM-soy. Organic soybeans also contained less total saturated fat and total omega-6 fatty acids than both conventional and GM-soy. GM-soy contained high residues of glyphosate and AMPA (mean 3.3 and 5.7 mg/kg, respectively). Conventional and organic soybean batches contained none of these agrochemicals. Using 35 different nutritional and elemental variables to characterise each soy sample, we were able to discriminate GM, conventional and organic soybeans without exception, demonstrating "substantial non-equivalence" in compositional characteristics for 'ready-to-market' soybeans.

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A new method was developed for the determination of eight triazine herbicide residues in cereal and vegetable samples by on-line sweeping technique in micellar electrokinetic capillary chromatography (MEKC). Some factors affecting analyte enrichment and separation efficiency were examined. The optimum buffer was composed of 25 mM borate, 15 mM phosphate, 40 mM sodium dodecylsulfate (SDS) and 3% (v/v) of 1-propanol at pH 6.5. The separation voltage was 20 kV and the sample was injected at 0.5 psi for 240 s. The detection wavelength was set at 220 nm with the capillary temperature being at 25 °C. Under the optimized conditions, the enrichment factors were achieved from 479 to 610. The limits of detection (LODs, S/N = 3) ranged from 0.02 to 0.04 ng/g and the limits of quantification (LOQs) of eight triazine herbicides were all 0.1 ng/g. The average recoveries of spiked samples were 82.8-96.8%. This method has been successfully applied to the determination of the triazine herbicide residues in cereal and vegetable samples.

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