Resistance to acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in Lolium multiflorum Lam. populations of Argentina.

BACKGROUND: Italian ryegrass (Lolium multiflorum Lam.) is one of the most troublesome grass weeds in Argentina. The extensive and repetitive use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides has induced resistance in this weed species. The objectives of this study were to quantify the resistance levels to ACCase-inhibiting herbicides in two resistant populations and to identify the target-site mutations associated with their resistance. RESULTS: Two resistant Italian ryegrass populations, Roldán and H2, were studied. Roldán was a suspected haloxyfop-resistant population, located in a wheat field from Santa Fe province with a history of ACCase-inhibiting herbicide use. The H2 population was obtained from the susceptible Hernandarias population (H0) after two cycles of selection with the herbicide quizalofop-ethyl. Whole-plant dose-response assays revealed that the resistant populations exhibited a high resistance to haloxyfop, with resistance factors (RF) exceeding 97-fold. Additionally, both populations showed a moderate resistance to pinoxaden (RF > 7), while maintaining susceptibility to clethodim. Partial chloroplastic ACCase sequences revealed isoleucine-to-asparagine substitution at position 2041 (Ile-2041-Asn) in both resistant populations. CONCLUSION: This work provides a better understanding of cross-resistance to ACCase-inhibiting herbicides in L. multiflorum populations and represents the first report of the target-site mutation Ile-2041-Asn conferring resistance in populations from Argentina. © 2024 Society of Chemical Industry.

High doses of clethodim-based herbicide GrassOut Max poses reproductive hazard by affecting male reproductive function and early embryogenesis in Swiss albino mice.

Clethodim is a widely used and approved class II herbicide, with little information about its impact on the reproductive system. Herein, we investigated the male reproductive toxicity of clethodim using a mouse model. GrassOut Max (26% clethodim-equivalent) or analytical grade clethodim (≥90%) were given orally to male mice for 10 d in varying doses. All parameters were assessed at 35 d post-treatment. Significant decrease in testicular weight, decreased germ cell population, elevated DNA damage in testicular cells and lower serum testosterone level was observed post clethodim based herbicide exposure. Epididymal spermatozoa were characterized with significant decrease in motility, elevated DNA damage, abnormal morphology, chromatin immaturity and, decreased acetylated-lysine of sperm proteins. In the testicular cells of clethodim-based herbicide treated mice, the expression of Erß and Gper was significantly higher. Proteomic analysis revealed lower metabolic activity, poor sperm-oocyte binding potential and defective mitochondrial electron transport in spermatozoa of clethodim-based herbicide treated mice. Further, fertilizing ability of spermatozoa was compromised and resulted in defective preimplantation embryo development. Together, our data suggest that clethodim exposure risks male reproductive function and early embryogenesis in Swiss albino mice via endocrine disrupting function.

Reduction in the Use of Some Herbicides Favors Nitrogen Fixation Efficiency in Phaseolus vulgaris and Medicago sativa.

In recent decades, the quality of agricultural soils has been seriously affected by the excessive application of pesticides, with herbicides being one of the most abundant. Continuous use of herbicides alters the soil microbial community and beneficial interactions between plants and bacteria such as legume-rhizobia spp. symbiosis, causing a decrease in the biological nitrogen fixation, which is essential for soil fertility. Therefore, the aim of this work was to study the effect of two commonly used herbicides (pendimethalin and clethodim) on the legume-rhizobia spp. symbiosis to improve the effectiveness of this process. Phaseolus vulgaris plants grown in pots with a mixture of soil:perlite (3:1 v/v), showed a 44% inhibition of nitrogen fixation rate with pendimethalin. However, clethodim, specifically used against monocots, did not induce significant differences. Additionally, we analyzed the effect of herbicides on root exudate composition, detecting alterations that might be interfering with the symbiosis establishment. In order to assess the effect of the herbicides at the early nodulation steps, nodulation kinetics in Medicago sativa plants inoculated with Sinorhizobium meliloti were performed. Clethodim caused a 30% reduction in nodulation while pendimethalin totally inhibited nodulation, producing a reduction in bacterial growth and motility as well. In conclusion, pendimethalin and clethodim application reduced the capacity of Phaseolus vulgaris and Medicago sativa to fix nitrogen by inhibiting root growth and modifying root exudate composition as well as bacterial fitness. Thus, a reduction in the use of these herbicides in these crops should be addressed to favor a state of natural fertilization of the soil through greater efficiency of leguminous crops.

Methomyl, imbraclaobrid and clethodim induced cytomixis and syncytes behaviors in PMCs of Pisum sativum L: Causes and outcomes.

Cytomixis is a common phenomenon observed in meiotic cells such as anther which is influenced by various factors. Use of pesticides is a common practice in agriculture. However, it is not known whether pesticides can induce cytomixis in plant cells and induce genetic variation. To understand this, the present study was planned to assess the cytomixis and syncytes behaviors in PMCs of Pisum sativum L. Seeds of P. sativum (Family: Fabaceae) were treated with different concentrations of commonly used pesticides methomyl (ME), imbraclaobrid (IM) and clethodim (CL). Seeds were treated with various concentrations (0.1, 0.2, 0.3, 0.4 and 0.5% of ME, IM and CL prepared in water) for 1 and 3 h. Effect of pesticides on pollen fertility, frequency of cytomixis, and kind of cytomixis cells was assessed. In the cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), and various stages of meiosis (PI, MI, AI and TI) with cytomixis cells were observed. In addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) was assessed. During the microsporogenesis in P. sativum, the occurrence of cytomixis and syncytes at various stages of meiosis I were seen. The formation of cytoplasmic channels and direct fusing of pollen mother cells (PMCs) were both seen to cause cytomixis, with the former being more common than the latter. The percentage of PMCs with cytomixis and syncytes cells increased with increase in the concentration of pesticides. The result of the present investigation indicates that commonly used pesticides ME, IM, and CL have a significant effect on pollen fertility, frequency of cytomixis, and kind of cytomixis cells, the cytomictic channel (CC) and direct fusion (DF), in addition, frequency of syncytes cell and their various stages of meiosis I (PI, MI, AI and TI) in pollen mother cells (PMCs) on P. sativum.

ARTP Mutagenesis of Schizochytrium sp. PKU#Mn4 and Clethodim-Based Mutant Screening for Enhanced Docosahexaenoic Acid Accumulation.

Schizochytrium species are one of the best oleaginous thraustochytrids for high-yield production of docosahexaenoic acid (DHA, 22:6). However, the DHA yields from most wild-type (WT) strains of Schizochytrium are unsatisfactory for large-scale production. In this study, we applied the atmospheric and room-temperature plasma (ARTP) tool to obtain the mutant library of a previously isolated strain of Schizochytrium (i.e., PKU#Mn4). Two rounds of ARTP mutagenesis coupled with the acetyl-CoA carboxylase (ACCase) inhibitor (clethodim)-based screening yielded the mutant A78 that not only displayed better growth, glucose uptake and ACCase activity, but also increased (54.1%) DHA content than that of the WT strain. Subsequent optimization of medium components and supplementation improved the DHA content by 75.5 and 37.2%, respectively, compared with that of mutant A78 cultivated in the unoptimized medium. Interestingly, the ACCase activity of mutant A78 in a medium supplemented with biotin, citric acid or sodium citrate was significantly greater than that in a medium without supplementation. This study provides an effective bioengineering approach for improving the DHA accumulation in oleaginous microbes.

Rapid On-Farm Testing of Resistance in Lolium rigidum to Key Pre- and Post-Emergence Herbicides.

Overreliance on herbicides for weed control is conducive to the evolution of herbicide resistance. Lolium rigidum (annual ryegrass) is a species that is prone to evolve resistance to a wide range of herbicide modes of action. Rapid detection of herbicide-resistant weed populations in the field can aid farmers to optimize the use of effective herbicides for their control. The feasibility and utility of a rapid 7-d agar-based assay to reliably detect L. rigidum resistant to key pre- and post-emergence herbicides including clethodim, glyphosate, pyroxasulfone and trifluralin were investigated in three phases: correlation with traditional pot-based dose-response assays, effect of seed dormancy, and stability of herbicides in agar. Easy-to-interpret results were obtained using non-dormant seeds from susceptible and resistant populations, and resistance was detected similarly as pot-based assays. However, the test is not suitable for trifluralin because of instability in agar as measured over a 10-d period, as well as freshly-harvested seeds due to primary dormancy. This study demonstrates the utility of a portable and rapid assay that allows for on-farm testing of clethodim, glyphosate, and pyroxasulfone resistance in L. rigidum, thereby aiding the identification and implementation of effective herbicide control options.

Simultaneous determination and dietary risk assessment of clethodim and its metabolites in different fruits and vegetables.

Clethodim is one of the most widely used herbicides in agriculture and, after field application, is metabolised to several metabolites. The potential toxicological negative effects of these compounds are poorly understood. Thus, recently, within the risk assessment framework, the European Food Safety Authority (EFSA) proposed to include the minor metabolites in the definition of clethodim residue. In this work, an easy to use and reliable UHPLC method coupled with a triple quadrupole MS/MS was developed and validated for the detection and quantification of the herbicide clethodim and related metabolites clethodim sulphone, clethodim sulphoxide, metabolites M17R and M18R in apple, grape, olive and rice. The five analytes were extracted by using a modified QuEChERS procedure, while the active ingredients were determined in multiple reaction monitoring (MRM) ion-switching mode. The proposed method showed calibration curve linearity with r2 ≥ 0.990 for all active ingredients (a.is.) both in solvent and matrix extracts. Limits of quantitation (LOQ) of the five compounds ranged from 9.44 µg/kg for M17R in olive extract to 11.01 µg/kg for clethodim in apple extract. Recoveries values ranged from 86% to 119% at two concentration levels (LOQ and 10xLOQ), while the intraday and interday precisions of the method were both below 10% in all cases. The method was successfully used for the quantification of the five a.is. in different food matrices. Furthermore, chronic dietary risk was investigated using a hazard quotients (HQ) method based on European dietary habits. The chronic dietary exposure risk quotients ranged from 1.0 × 10-5 (lower bound scenario) to 2.7 × 10-4 (upper bound scenario) which were significantly lower than 1. Data obtained indicate that the dietary exposure risks were acceptable for clethodim and its major and minor metabolites applied in apple, table grape, rice and table olive.

Comparison of Efficacy and Detection of Clethodim and Glyphosate Applied with Dicamba and 2,4-D through Tank Mixture and Sequential Applications.

Greenhouse studies were planted at the R.R. Foil Plant Science Research Center in Starkville, MS. In the efficacy trial, pots were seeded with barnyardgrass (Echinochloa crus-galli), broadleaf signalgrass (Urochloa platyphylla), and giant foxtail (Setaria faberi). In the lab detection trial, only barnyardgrass was seeded. Both studies consisted of 16 treatments with four replications per treatment. The treatments consisted of clethodim, glyphosate, dicamba, and 2,4-D applied singularly and in combination with each other. Each herbicide combination was applied with three application methods: tank mixture, sequential applications where the synthetic auxin was applied first (auxin applied first), and sequential applications where glyphosate or clethodim was applied first (auxin applied second). The auxin applied second method had higher visual estimations of control ratings and lower biomass weights compared to the other two methods. The auxin applied second method had more glyphosate and clethodim detected with the use of liquid chromatography tandem mass spectrometry.

The combined effect of clethodim (herbicide) and nitrogen variation on allelopathic interactions between Microcystis aeruginosa and Raphidiopsis raciborskii.

The large-scale use of herbicides deteriorates water quality and threatens aquatic biodiversity. Unfortunately, there are few studies on the ecological effects of herbicides on toxin-producing strains of cyanobacteria under changing nutrient conditions. The objective of the present study was to investigate the effects of the herbicide clethodim and nitrogen variation on the allelopathic interactions and toxin production of Microcystis aeruginosa BCCUSP232 and Raphidiopsis raciborskii (formerly known as Cylindrospermopsis raciborskii) ITEPA1. M. aeruginosa had increased cell density when exposed to the clethodim (H +) (23.55 mg/L), whereas the highest cell density of R. raciborskii was observed in the treatment with clethodim plus limited nitrogen. Also, the cell-free exudate of R. raciborskii significantly stimulated the growth of M. aeruginosa on day 3 of the experiment. The concentration of chlorophyll-a in M. aeruginosa cultures generally increased in all the treatments, while in R. raciborskii cultures, the opposite occurred. Total microcystins (MCs) content of M. aeruginosa in the mixed cultures was 68% higher in nitrogen-enriched conditions than the control. A similar increase in MC content occurred in M. aeruginosa unialgal culture treated with R. raciborskii exudate. Total saxitoxin concentration was 81% higher in mixed cultures of R. raciborskii simultaneously exposed to high nitrogen and clethodim. Similarly, unialgal cultures of R. raciborskii exposed to either high nitrogen or clethodim had higher saxitoxins concentrations than the control. The intracellular H2O2 content of M. aeruginosa cultures decreased, whereas, in R. raciborskii cultures, it increased during exposure to high nitrogen and clethodim. Only R. raciborskii had a significant variation in peroxidase activity. The activities of glutathione S-transferase of both strains were higher in the presence of clethodim. These results revealed that nitrogen enrichment and the presence of clethodim might lead to the excessive proliferation of M. aeruginosa and R. raciborskii and increased production of cyanotoxins in aquatic environments.

Interaction of acetyl-CoA carboxylase enzyme inhibiting herbicides with auxin herbicides on ryegrass / Interação de herbicidas inibidores da enzima acetil-CoA carboxilase com herbicidas auxínicos sobre azevém

ABSTRACT: This study aimed to evaluate the antagonistic effect of the mixture ofacetyl coenzyme-A carboxylase (ACCase) enzyme inhibiting herbicides and auxin herbicides in Lolium multiflorum and to determine mechanisms to mitigate this possible effect. The first experiments were conducted by associating the herbicide clethodim (108 g a.i. ha−1), quizalofop-p-ethyl (54 g a.i. ha−1), and clethodim + quizalofop-p-ethyl (108+54 g a.i. ha−1) with 2,4-D (1005 g a.e. ha−1) or triclopyr (720 g a.e. ha−1), in addition to the sole application of the respective graminicides. Another experiment included clethodim (54; 81; 108; 162; 216 g a.i. ha−1), quizalofop-p-ethyl (27; 40.5; 54; 81; 108 g a.i. ha−1), and clethodim + quizalofop-p-ethyl (54+27; 81+40.5; 108+54; 162+81; 216+108 g a.i. ha−1) mixed with 2,4-D (1005 g a.e. ha−1), or triclopyr (720 g a.e. ha−1), in addition to the control treatments without herbicide application. In the second experiment, herbicides clethodim (108 g a.i. ha−1), quizalofop-p-ethyl (54 g a.i. ha−1), and clethodim + quizalofop-p-ethyl (108+54 g a.i. ha−1) in combination with the herbicides 2,4-D (1005 g a.e. ha−1) or triclopyr (720 g a.e. ha−1)had malathion (1000 g a.i. ha−1) or glyphosate (720 g a.e. ha−1) mixed, in addition to the sole applications of the graminicides. The herbicide clethodim + quizalofop-p-ethyl did not present an antagonistic interaction with the auxin herbicides, and obtained 85% weed control. To obtain control similar to the sole application of this graminicide, the dose of the herbicide clethodim needs to be increased by 20%. However, the mixture of the herbicide quizalofop-p-ethyl with 2,4-D and triclopyr affects the ryegrass control. The use of strategies that increase the absorption of ACCase herbicides or the inhibition of P450 enzymes are ways to mitigate the antagonistic effect caused by the association of the two auxin herbicides.

RESUMO: O objetivo do trabalho foi avaliar o efeito antagônico da mistura de herbicidas inibidores da enzima ACCase e herbicidas auxínicos em Lolium multiflorum e determinar mecanismos de mitigação deste possível efeito. No primeiro momento foram conduzidos experimentos associando o herbicida clethodim (108 g i.a. ha-1), quizalofop-p-ethyl (54 g i.a. ha-1) e clethodim + quizalofop-p-ethyl (108 + 54 g i.a. ha-1) com 2,4-D (1005 g e.a. ha-1) ou triclopyr (720 g e.a. ha-1), além da aplicação isolada dos respectivos graminicidas. Outro experimento contou com clethodim (54; 81; 108; 162; 216 g i.a ha-1), quizalofop-p-ethyl (27; 40,50; 54; 81; 108 g i.a ha-1) e clethodim + quizalofop-p-ethyl (54 + 27; 81 + 40,50; 108 +54; 162 +81; 216 + 108 g i.a ha-1) em mistura com 2,4-D (1005 g e.a ha-1) ou triclopyr (720 g e.a. ha-1), além das testemunhas sem aplicação herbicida. No segundo momento os herbicidas clethodim (108 g i.a. ha-1), quizalofop-p-ethyl (54 g i.a. ha-1) e clethodim + quizalofop-p-ethyl (108 + 54 g i.a. ha-1) em associação com os herbicidas 2,4-D (1005 g e.a ha-1) ou triclopyr (720 g e.a. ha-1), contaram com adição de malathion (1000 g i.a. ha-1) ou glyphosate (720 g e.a. ha-1), além das aplicações isoladas dos graminicidas. O herbicida clethodim + quizalofop-p-ethyl não apresentou interação antagônica com os herbicidas auxínicos, obtendo controle de 85%. Já para o herbicida clethodim é necessário o aumento de dose em 20 % é capaz de obter controle similar a aplicação isolada deste graminicida. Porém para o herbicida quizalofop-p-ethyl a mistura com os herbicidas 2,4-D e triclopyr repercute na diminuição do controle do azevém. O uso de estratégias que aumente a absorção do herbicida inibidor de ACCase ou a inibição das enzimas P450 são formas de mitigar o efeito antagônico causado pela associação destes dois tipos de herbicidas.

Sourgrass control in response to the association of 2, 4-d to ACCase inhibitor herbicides / Controle de grama azeda em resposta à associação de 2, 4-d a herbicidas inibidores de ACCase

ACCase inhibitor herbicides are widely used to control poaceae such as sourgrass (Digitaria insularis). However, these herbicides are selective for eudicots, which requires the use of broadleaf herbicides in areas with broadleaf and narrow leaf weeds. The objective of this study was to evaluate the effect of the association ACCase inhibitors and 2,4-D for the control of sourgrass. Two experiments were carried out under greenhouse conditions. The first experiment evaluated the effect of associating 2,4-D and graminicides to control of sourgrass, using dose-response curves for the herbicides clethodim, quizalofop-P-tefuril and clethodim + quizalofop-P-tefuril. In the second experiment, due to the results of the first one, the necessary period between the application of 2,4-D and the graminicides was evaluated in order to avoid antagonism in sourgrass. The results show that the herbicide clethodim, at the recommended dose, has its efficiency reduced in about 8% when applied in association with 2,4-D. As for the herbicide quizalofop-P-tefuril, this effect was higher, causing a reduction of 39% in control. However, the association of 2,4-D to the mixture of clethodim + quizalofop-P-tefuril did not result in antagonism. It takes nine and 12 days between the application of 2,4-D and the graminicides clethodim and quizalofop-P-tefuril, respectively, for there to be no antagonism.

Os herbicidas inibidores da ACCase são amplamente utilizados no controle de poáceas, tal como o capim-amargoso. No entanto, esses herbicidas são seletivos para plantas eudicotiledôneas, o que demanda a utilização de herbicidas latifolicidas, tais como as auxinas sintéticas, em áreas que existam plantas daninhas de folhas largas e folhas estreitas. O objetivo deste trabalho foi avaliar o efeito da associação de inibidores da ACCase e 2,4-D sobre o controle de capim-amargoso. Foram realizados dois experimentos em casa de vegetação. No primeiro experimento foi avaliado o efeito da associação de 2,4-D aos graminicidas no controle de capim-amargoso, utilizando-se curvas de dose-resposta para os herbicidas clethodim, quizalofop-P-tefuril e clethodim + quizalofop-P-tefuril. No segundo experimento, em decorrência dos resultados do primeiro, foi avaliado o período necessário entre a aplicação de 2,4-D e os graminicidas para que não ocorra antagonismo em capim-amargoso. Os resultados demonstram que para o herbicida clethodim, na dose recomendada, a redução de controle foi de cerca de 8% quando o 2,4-D foi aplicado em associação. Já para o herbicida quizalofop-P-tefuril, esse efeito foi superior, causando redução de 39% no controle. Porém, a associação do 2,4-D à mistura de clethodim + quizalofop-P-tefuril não resultou em antagonismo. São necessários nove e 12 dias entre a aplicação de 2,4-D e os graminicidas clethodim e quizalofop-P-tefuril, respectivamente, para que não ocorra antagonismo.

Simultaneous Analysis and Dietary Exposure Risk Assessment of Fomesafen, Clomazone, Clethodim and Its Two Metabolites in Soybean Ecosystem.

A commercial formulation, 37% dispersible oil suspension (DOS) (fomesafen, clomazone, and clethodim), is being registered in China to control annual or perennial weeds in soybean fields. In this paper, a liquid chromatography tandem mass spectrometry method with QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation was developed for the simultaneous determination of fomesafen, clomazone, clethodim, and its two metabolites (CSO and CSO2) in soybean, green soybean, and soybean straw samples. The mean recoveries of our developed method for the five analytes in three matrices were ranged from 71% to 116% with relative standard deviations (RSDs) less than 12.6%. The limits of quantification (LOQs) were 0.01 mg/kg in soybean, 0.01 mg/kg in green soybean, and 0.02 mg/kg in soybean straw while the limits of detection (LODs) ranged from 0.018 to 0.125 µg/kg for these five analytes. The highest final residual amount of CSO2 in green soybean samples (0.015 mg/kg) appeared in Anhui, and the highest in soybean straw samples was 0.029 mg/kg in Guangxi, whilst the terminal residues of fomesafen, clomazone, clethodim and CSO were lower than LOQs (0.01 mg/kg) in all samples. Furthermore, these terminal residues were all lower than the maximum residue limits (MRLs) set by China (0.1 mg/kg for fomesafen and clethodim, 0.05 mg/kg for clomazone) at harvest. Additional chronic dietary risk was evaluated using a risk quotients (RQs) method based on Chinese dietary habits. The chronic dietary exposure risk quotients were 4.3 for fomesafen, 0.12 for clomazone, and 19.3 for clethodim, respectively, which were significantly lower than 100. These results demonstrated that the dietary exposure risk of fomesafen, clomazone, and clethodim used in soybean according to good agricultural practices (GAP) was acceptable and would not pose an unacceptable health risk to Chinese consumers. These results not only offer insight with respect to the analytes, but also contribute to environmental protection and food safety.

Resistance to clethodim in Italian ryegrass (Lolium perenne ssp. multiflorum) from Mississippi and North Carolina.

BACKGROUND: Clethodim, an acetyl-CoA carboxylase (ACCase)-inhibiting herbicide, is one of the few postemergence chemical control options available to growers of Mississippi to manage glyphosate and/or other herbicide resistant Italian ryegrass populations. Recently, clethodim failed to adequately control Italian ryegrass populations across Mississippi. A sethoxydim, also an ACCase inhibitor, -resistant Italian ryegrass population from North Carolina was cross-resistant to clethodim. This research characterized the magnitude and mechanisms of clethodim resistance in the Mississippi and North Carolina Italian ryegrass populations via whole-plant herbicide dose response, cross resistance, and metabolism studies, and molecular analysis. RESULTS: Two clethodim-resistant biotypes from Mississippi, MS24 and MS37, were 10- and 4-fold resistant, respectively, relative to a susceptible (SUS1) biotype. A North Carolina biotype, NC21, was 40-fold resistant to clethodim compared to SUS1. Two additional biotypes from North Carolina, NC22 and NC 23, recorded shoot dry weight reduction of only 17-30% of nontreated at the highest clethodim dose of 2.17 kg ha-1 , (8×). The NC22 biotype was cross-resistant to sethoxydim, fluazifop, quizalofop, and pinoxaden. Metabolic inhibitors such as piperonyl butoxide and 4-chloro-7-nitrobenzofurazan did not affect resistance of MS37, MS51, and NC22 biotypes to fenoxaprop, clethodim, or pinoxaden. The MS37 biotype had three target site mutations, I2041N, C2088R, and G2096A. Another clethodim-resistant biotype from Mississippi, MS51, had only the C2088R substitution. The NC22 and NC23 biotypes had I1781L, I2041N, and D2078G replacements. CONCLUSION: This study shows that the mechanism of resistance to clethodim in Italian ryegrass from Mississippi and North Carolina is due to target site modifications in the ACCase gene leading to broad cross-resistance to other ACCase-inhibiting herbicides. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Clethodim exposure induced development toxicity and behaviour alteration in early stages of zebrafish life.

Clethodim is one of the most widely used herbicides in agriculture, however, its potential toxic effects on organisms and the underlying toxicity mechanism are still poorly understood. In this study, zebrafish embryos at 6 h post-fertilization (hpf) were exposed to 10 mg/L, 20 mg/L, and 30 mg/L clethodim for up to 24 hpf, and zebrafish larvae at 6 days post-fertilization (dpf) were exposed to the same density gradient for 24 h. Our results showed that clethodim could cause head and cardiovascular malformations in embryos: blurred brain ventricles, unapparent brain regions, condensation of nucleus and cytoplasm in brain cells, increased intercellular space, developmental malformations of eyes and ears, reduced neonatal neurons, disorder migration of neural ridge cells; morphological aberrations of the vascular ICM, slowing of heart beat and blood flow, reduction of circulating red blood cells, and delayed development of head and tail blood vessels. These defects could be a result of clethodim-induced oxidative stress and decreased acetylcholinesterase (AChE) activity, which in turn affected the expression of neurodevelopmental genes, decreased ATPase activity, and ultimately led to developmental malformations. The swimming behaviour of zebrafish larvae was observed to decrease with increasing concentration of clethodim exposure, but the angular velocity and mobility increased. These could be due to reduced AChE activity and disturbed gene expression of GABA, dopamine and glutamatergic neurotransmitter systems, which thus altered the locomotor behaviour. In summary, we found that clethodim induces developmental toxicity and neurotoxicity in zebrafish embryos and larvae.

Review of the existing maximum residue levels for clethodim according to Article 12 of Regulation (EC) No 396/2005.

According to Article 12 of Regulation (EC) No 396/2005, EFSA has reviewed the maximum residue levels (MRLs) currently established at European level for the pesticide active substance clethodim. To assess the occurrence of clethodim residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Directive 91/414/EEC, the MRLs established by the Codex Alimentarius Commission and European authorisations reported by Member States (including the supporting residues data). Based on the assessment of the available data, MRL could not be proposed because an overall consumer risk assessment could not be finalised. Risk managers should be made aware that the genotoxic potential of the clethodim metabolite 3-chloroallyl alcohol, the aglycon of 3-chlorolallyl alcohol glucoside (M14A/M15A) could not be concluded and no toxicological reference values could be derived for this metabolite. Until a conclusion on the toxicological properties of the metabolite is reached, a decision on the residue definition for risk assessment cannot be made which is a prerequisite to perform a reliable dietary risk assessment.

Clethodim exposure induces developmental immunotoxicity and neurobehavioral dysfunction in zebrafish embryos.

Clethodim is one of the most widely used herbicides in agriculture, but its potential negative effects on aquatic organisms are still poorly understood. This study examined the effects of clethodim on zebrafish at aspects of early stage embryonic development, immune toxicity, cell apoptosis and locomotor behavior. Firstly, clethodim exposure markedly decreased the survival rate, body length, and heart rate and resulted in a series of morphological abnormalities, primarily spinal deformities (SD) and yolk sac edema, in zebrafish larvae. Secondly, the number of immune cells was substantially reduced but the levels of apoptosis and oxidative stress were significantly increased in a dose-dependent manner upon clethodim exposure. Thirdly, we evaluated the expression of some key genes in TLR signaling including TLR4, MyD88, and NF-κB p65 and they were all up-regulated by exposure to 300 µg/L clethodim. Meanwhile, some proinflammatory cytokines such as TNF-α, IL-1ß, IL8, and IFN-γ were also activated in both the mock and the TLR4-KD conditions. Moreover, the locomotor behaviors and the enzymatic activities of AChE were obviously inhibited but the levels of acetylated histone H3 were greatly increased by clethodim exposure. In addition, incubation of zebrafish larvae with acetylcholine receptor (AChR) agonist carbachol can partially rescue the clethodim-modulated locomotor behavior. Taken together, our results suggest that clethodim has the potential to induce developmental immunotoxicity and cause behavioral alterations in zebrafish larvae. The information presented in this study will help to elucidate the molecular mechanisms underlying clethodim exposure in aquatic ecosystems.

Simultaneous determination and method validation of clethodim and its metabolites clethodim sulfoxide and clethodim sulfone in tobacco by LC-MS/MS.

A simple method was developed and validated for the simultaneous determination of clethodim, clethodim sulfoxide, and clethodim sulfone in soil and tobacco by liquid chromatography with tandem mass spectrometry. The three target compounds were extracted from tobacco and soil with acetonitrile, and the extracts were purified using octadecyl silane. The proposed method showed satisfactory linearity (R2 ≥ 0.9973) for the target compounds. The limits of detection and quantitation of the three analytes in all matrices were 0.024-0.06 and 0.08-0.2 mg/kg, respectively. The recovery was tested in blank soil and tobacco leaf samples and calculated to be 74.8-104.4% with relative standard deviations of 1.9-12.1%. The developed method was successfully applied to the analysis of residues of clethodim, clethodim sulfoxide and clethodim sulfone in real soil and tobacco samples. The results indicated that the developed method can meet the requirements for the analysis of trace amounts of all three analytes in soil and tobacco.

Photolysis of clethodim herbicide and a formulation in aquatic environments: Fate and ecotoxicity assessment of photoproducts by QSAR models.

The photochemical fate of the herbicide clethodim in natural waters was investigated under simulated and natural sunlight radiation. This herbicide exhibited a rapid degradation rate in simulated aquatic environment with half-lives ranged from 27.9min to 4.6h. The commercial formulation of clethodim showed a faster degradation with half-lives from 19.3min to 1.4h. It has also been demonstrated that the photolytic behavior of clethodim was affected by the water composition and the radiation intensity. Nine major photoproducts were identified and their distribution was dependent on the experimental conditions. Photodegraded solutions of clethodim were shown to be more toxic to the bacteria Vibrio fischeri than the herbicide itself, reaching the maximum toxicity when the herbicide is completely degraded. QSAR analysis of the fate, ecotoxicological and physicochemical endpoints of the degradation products provided positive alerts for several identified by-products. Environmental fate and transport estimates showed that all photoproducts, unlike the active substance, are potential leachers. Moreover, predicted vapor pressures suggested that dermal contact and ingestion are the most probable exposure routes for workers and general population to both clethodim and its photoproducts. These results highlight the importance of the degradation products in attaining a complete knowledge of the fate and behavior of an herbicide in the environment. To our knowledge, this is the first study to report a detailed QSAR study on clethodim photoproducts under environmental conditions. These results provide a very valuable information that will guide further experimental studies leading to a better pesticide risk assessment.

Acetyl-CoA carboxylase overexpression in herbicide-resistant large crabgrass (Digitaria sanguinalis).

BACKGROUND: The occurrence of herbicide-resistant weed biotypes is increasing and this report of an acetyl-CoA carboxylase (ACCase) inhibitor-resistant Digitaria sanguinalis L. Scop. from southwestern Ontario is another example. The identified weed escaped control in an onion and carrot rotation in which graminicides were used for several consecutive years. Our goal was to characterize the level and mechanism of resistance of the biotype. RESULTS: The biotype was resistant to all five ACCase inhibitor herbicides tested. Gene-expression profiling was performed because none of the mutations known to confer resistance in the ACCase gene were detected. RNASeq and quantitative reverse-transcriptase PCR (qRT-PCR) results indicated that transcription of ACCase was 3.4-9.3 times higher in the resistant biotype than the susceptible biotype. ACCase gene copy number was determined by qPCR to be five to seven times higher in the resistant compared with the susceptible biotype. ACCase gene overexpression was directly related to the increase of the ACCase gene copy number. CONCLUSION: Our results are consistent with the hypothesis that overexpression of the herbicide target gene ACCase confers resistance to the herbicide. This is the first reported case of target gene duplication conferring resistance to a herbicide other than glyphosate. © 2017 Society of Chemical Industry See related Article.

Inheritance of evolved clethodim resistance in Lolium rigidum populations from Australia.

BACKGROUND: In Australia, the extensive use of clethodim for the control of Lolium rigidum has resulted in the evolution of many clethodim-resistant L. rigidum populations. Five clethodim-resistant populations of L. rigidum were analysed for the inheritance of clethodim resistance. RESULTS: Reciprocal crosses were made between resistant (R) and susceptible (S) populations. Within crosses, dose-responses of reciprocal F1 families of all populations except A61 were similar to each other, indicating that clethodim resistance in these populations is encoded on the nuclear genome. The level of dominance observed in the dose-response experiments ranged from partial to complete within the herbicide rate used. In the A61 population, within each cross, the response of F1 from the maternal and paternal parent was different, indicating that resistance is inherited through the female parent. All backcross populations segregated in a different manner. Only one population, FP, fitted a single-gene model (1:1). Two populations fitted two-gene models: a 3:1 inheritance model for F4 and a 1:3 inheritance model for A91. For population E2, no clear pattern of inheritance was determined, suggesting more complex inheritance. CONCLUSION: The results of this study indicate that different patterns of clethodim resistance in L. rigidum exist. © 2016 Society of Chemical Industry.