Study on the enantioselective behaviors, activity, toxicity and mechanism of novel SDHI fungicide benzovindiflupyr to reduce the environmental risks.

Benzovindiflupyr (BEN) has emerged as one of the fastest-growing SDHI fungicides in recent years, but it is considered "very highly toxic" to aquatic fish, invertebrates and crustaceans (EC50 or LC50, 0.0035-0.056 mg/L, acute toxicity). The comprehensive study on bioactivity, toxicity, and degradation behaviors of BEN at the enantiomeric level would facilitate the development of a high-efficiency and low-risk application method. The bioactivities of 1S, 4R-(-)-BEN against five target pathogens (Alternaria alternata, Phoma multirostrata, Selerotium rolfsii, Magnaporthe oryzae, and Rhizoctonia solani) (EC50, 0.00562-0.329 mg/L, high-efficiency) were 6.7-1029 times higher than 1R, 4S-(+)-BEN, demonstrating significant enantioselectivity. For Danio rerio, 1S, 4R-(-)-BEN (LC50, 0.0360 mg/L, "very highly toxic") exhibited higher toxicity than 1 R, 4S-(+)-BEN, but the toxic interaction was concentration addition (TUrac, 0.94), indicating an enhanced toxicity in the presence of 1R, 4S-(+)-BEN. Molecular docking was employed to offer insights at the molecular level and elucidate the factors influencing enantioselectivity. The stronger binding affinity of 1S, 4R-(-)-BEN with SDH was in line with the quantitative experimental findings. The degradation of two BEN enantiomers in four different fruits followed the first-order degradation kinetics equation, and displayed enantioselectivity. The preferential degradation of 1R, 4S-(+)-BEN was found in pears and grapes, while varying enantioselectivity was found at different stages in tomatoes and watermelons. The residual concentrations of BEN in grapes were higher than the EU's MRL, which in the other three fruits were below the MRLs during the sampling. In conclusion, 1S, 4R-(-)-BEN proved to be the more effective monomer. Utilizing the pure monomer could not only reduce the dosage of racemate by about 44-59 %, but also mitigate the risk of introducing inefficient monomer into the environment (especially for fish).

Impacts of co-exposure to zearalenone and trifloxystrobin on the enzymatic activity and gene expression in zebrafish.

Although humans and animals are usually exposed to combinations of toxic substances, little is known about the interactive toxicity of mycotoxins and farm chemicals. Therefore, we can not precisely evaluate the health risks of combined exposure. In the present work, using different approaches, we examined the toxic impacts of zearalenone and trifloxystrobin on zebrafish (Danio rerio). Our findings showed that the lethal toxicity of zearalenone to embryonic fish with a 10-day LC50 of 0.59 mg L-1 was lower than trifloxystrobin (0.037 mg L-1). Besides, the mixture of zearalenone and trifloxystrobin triggered acute synergetic toxicity to embryonic fish. Moreover, the contents of CAT, CYP450, and VTG were distinctly altered in most single and combined exposures. Transcriptional levels of 23 genes involved in the oxidative response, apoptosis, immune, and endocrine systems were determined. Our results implied that eight genes (cas9, apaf-1, bcl-2, il-8, trb, vtg1, erß1, and tg) displayed greater changes when exposed to the mixture of zearalenone and trifloxystrobin compared with the corresponding individual chemicals. Our findings indicated that performing the risk assessment based on the combined impact rather than the individual dosage response of these chemicals was more accurate. Nevertheless, further investigations are still necessary to reveal the modes of action of mycotoxin and pesticide combinations and alleviate their effects on human health.

Toxicological Effect and Molecular Mechanism of the Chiral Neonicotinoid Dinotefuran in Honeybees.

With the increasing demand for pollinating services, the wellness of honeybees has received widespread attention. Recent evidence indicated honeybee health might be posed a potential threat by widely used neonicotinoids worldwide. However, little is known about the molecular mechanism of these insecticides in honeybees especially at an enantiomeric level. In this study, we selected two species of bees, Apis mellifera (A. mellifera) and Apis cerana (A. cerana), to assess the toxicity and molecular mechanism of neonicotinoid dinotefuran and its enantiomers. The results showed that S-dinotefuran was more toxic than rac-dinotefuran and R-dinotefuran to honeybees by oral and contact exposures as much as 114 times. A. cerana was more susceptible to highly toxic enantiomer S-dinotefuran. S-dinotefuran induced the immune system response in A. cerana after 48 h exposure and significant changes were observed in the neuronal signaling of A. mellifera under three forms of dinotefuran exposure. Moreover, molecular docking also revealed that S-dinotefuran formed more hydrogen bonds than R-dinotefuran with nicotinic acetylcholine receptor, indicating the higher toxicity of S-dinotefuran. Data provided here show that R-dinotefuran may be a safer alternative to control pests and protect pollinators than rac-dinotefuran.

Low-dose cadmium affects the enantioselective bioaccumulation and dissipation of chiral penflufen in zebrafish (Danio rerio).

Pesticides are currently extensively used in agriculture, forestry, animal husbandry, and environmental hygiene, and their residues have become a global environmental problem, which can easily form combined pollution with heavy metals. The present study examined the effects of chronic (28 days) aqueous exposure of chiral penflufen (rac-penflufen, R-(-)-penflufen and S-(+)-penflufen), a widely used fungicide, with/without cadmium (Cd), a highly toxic heavy metal in zebrafish (Danio rerio). After rac-penflufen individual or combined exposure with Cd, the bioaccumulation and residual levels of S-(+)-penflufen were significantly higher than R-(-)-penflufen, and the effects of Cd were insignificantly. But for penflufen enantiomer, the effects of Cd were more serious for R-(-)-penflufen, which could increase the bioaccumulation (up to1.73 times), inhibit the dissipation (up to 32.3%) and enhance the residue (up to 5.35 times) of R-(-)-penflufen in zebrafish, decreasing the enantioselectivity. However, significant increase of S-(+)-penflufen concentrations was only found in viscera under co-exposure of Cd. The tissue distribution of penflufen enantiomers were not affected by the presence of Cd, and no interconversion of the two enantiomers occurred regardless of the presence of Cd. These findings indicated that co-contamination with Cd could increase the persistence of R-(-)-penflufen in zebrafish, thus increasing the environmental risks. The significant differences of Cd effects on chiral pesticide enantiomer and racemate indicated that the combined pollution of heavy metal and chiral pesticide might have enantiomer-specific, which should raise concern, and the enantioselective mechanism deserve further study.

Enantioselective toxicity and mechanism of chiral fungicide penflufen based on experiments and computational chemistry.

Penflufen fungicide is widely used as a racemate, which has potential ecological risks to aquatic organisms, while its enantioselective toxicity data is limited. This study aimed to differentiate the enantioselective toxicity difference of penflufen enantiomers, and illuminate the enantioselective mechanism from the insight of enantiomer-protein specific binding. The semipreparative separation and absolute configuration of penflufen enantiomers were conducted. The acute toxicity of S-(+)-penflufen was 54 times higher than R-(-)-penflufen to Danio rerio, and the coexistence of R-(-)-penflufen could increase the exposure risk of S-(+)-penflufen. For chronic toxicity, after low-dose long-term exposure, rac-penflufen and S-(+)-penflufen inducted more serious oxidative stress than R-(-)-penflufen in D. rerio, and inhibited the succinate dehydrogenase (SDH) activity significantly. For target phytopathogen, the toxicity difference of S-(+)-penflufen and R-(-)-penflufen was up to 148 times for Rhizoctonia solani. Based on the toxic unit analysis, the toxic interactions of antagonistic effect and concentration addition were found between penflufen enantiomers, indicating the coexistence of R-(-)-penflufen could increase overuse and environmental risks. Computational chemistry was used to illuminate the enantioselectivity mechanism, and the lower binding energy between the active site of SDH and S-(+)-penflufen contributed to the higher toxicity. The higher target toxicity might be due to the hydrophobic pocket of CybL in R. solani was more benefited to S-(+)-penflufen binding SDH than Botrytis cinerea. These results could be helpful for further understanding the potential risk of chiral penflufen in the environment, demonstrating the importance of understanding the enantioselective difference of chiral pesticides, and providing a new insight for analyzing the enantioselective mechanism.

Reference genes for the study of herbicide stress responses in Leptochloa chinensis (L.) Nees and estimation of ACCase expression in cyhalofop-butyl resistant populations.

Cyhalofop-butyl resistance in Leptochloa chinensis (L.) Nees is a threat to rice production. Qualitative changes to the acetyl-CoA carboxylase gene (ACCase) have been reported to induce cyhalofop-butyl resistance in some weed species, but the role of ACCase in cyhalofop-butyl resistance through quantitative changes remains uncertain. The accurate assessment of transcriptional changes in the functional genes associated with herbicide resistance in L. chinensis is challenging owing to the lack of available reference genes for expression normalization. Here, we selected nine candidate reference genes in L. chinensis and assessed their transcription stability in populations susceptible and resistant to cyhalofop-butyl. Transcription stability was compared under conditions of herbicide stress and control conditions using BestKeeper, NormFinder, and geNorm. Elongation factor 1 alpha, eukaryotic initiation factor 4A, and cap-binding protein CBP20 were the most stable reference genes under cyhalofop-butyl treatment. Transcription levels of ACCase were evaluated in seven resistant populations, one of which showed higher transcription than the susceptible population after 24 h cyhalofop-butyl treatment. However, the slight up-regulation of ACCase (approximately 2.0-fold) is unlikely to be responsible for the high resistance levels in these populations of L. chinensis.

Assessment of Carbendazim Residues and Safety in Celery Under Different Cultivation Conditions.

Although the carbendazim is widely used to manage spot blight in celery cultivation, information on residues identified is of interest. In this study, we examined the dissipation and residual amounts of carbendazim in celery and soil under different cultivation methods when using the suggested dose and ten times of that and the bioconcentration factor of carbendazim for celery. The results showed that when celery leaves were sprayed with the suggested dose, the half-lives in a celery field and greenhouse were 2.75 days and 3.29 days, respectively. When the soil matrix was sprayed with the recommended dose before cultivation, the half-lives of carbendazim residues were 16.86 days and 11.97 days. We also conducted a long-term dietary risk assessment using the corresponding criteria. The results showed that, in China, the use of carbendazim at a dose of 0.022 g/m2 is safer and more reasonable when the harvest interval is 28 days.

High-throughput multipesticides residue analysis in earthworms by the improvement of purification method: Development and application of magnetic Fe3 O4 -SiO2 nanoparticles based dispersive solid-phase extraction.

As a key representative organism, earthworms can directly illustrate the influence of pesticides on environmental organisms in soil ecosystems. The present work aimed to develop a high-throughput multipesticides residue analytical method for earthworms using solid-liquid extraction with acetonitrile as the solvent and magnetic material-based dispersive solid-phase extraction for purification. Magnetic Fe3 O4 nanoparticles were modified with a thin silica layer to form Fe3 O4 -SiO2 nanoparticles, which were fully characterized by field-emission scanning electron microscopy, transmission electron microscopy, Fourier-transform infrared spectroscopy, X-ray diffractometry, and vibrating sample magnetometry. The Fe3 O4 -SiO2 nanoparticles were used as the separation media in dispersive solid-phase extraction with primary secondary amine and ZrO2 as the cleanup adsorbents to eliminate matrix interferences. The amounts of nanoparticles and adsorbents were optimized for the simultaneous determination of 44 pesticides and six metabolites in earthworms by liquid chromatography with tandem mass spectrometry. The method performance was systematically validated with satisfactory results. The limits of quantification were 20 µg/kg for all analytes studied, while the recoveries of the target analytes ranged from 65.1 to 127% with relative standard deviation values lower than 15.0%. The developed method was subsequently utilized to explore the bioaccumulation of bitertanol in earthworms exposed to contaminated soil, verifying its feasibility for real sample analysis.

Screening for suitable chemical acaricides against two-spotted spider mites, Tetranychus urticae, on greenhouse strawberries in China.

Effective and safe acaricides based on scientific data are needed for that no chemical acaricides has been registered for the control of two-spotted spider mite in strawberry crops in China. To identify suitable acaricides, the efficacy, persistence, and toxicity of eight acaricides (hexythiazox, fenpyroximate, chlorfenapyr, propargite, etoxazole, bifenazate, spirodiclofen, and pyridaben) on greenhouse strawberries were tested. The eight acaricides were ranked, from highest average efficacy at the recommended dosage to lowest, as follows: etoxazole > bifenazate > fenpyroximate > propargite > spirodiclofen > pyridaben > hexythiazox> chlorfenapyr. The average recoveries of the eight acaricides at the spiking levels of 0.05 and 0.5 mg/L ranged from 72.4% to 108.1% (relative standard deviation, 1.3-8.8%). The concentrations of hexythiazox, fenpyroximate, etoxazole, bifenazate, spirodiclofen, and pyridaben at 5 days after application were lower than the maximum residue limits (MRLs) specified by China, the European Union (EU), the Codex Alimentarius Commission, and Japan, but those of chlorfenapyr and propargite residues were 8.8 and 1.9 times higher than the MRLs in the EU. Only propargite posed a high chronic dietary risk to humans. Pyridaben and chlorfenapyr showed unacceptable ecotoxicology risks for honeybees (hazard quotient values of > 50). The recommended acaricides to control spider mites in greenhouse-grown strawberry crops are etoxazole, bifenazate, fenpyroximate, spirodiclofen, and hexythiazox based on the efficacy, persistence and toxicity.

Positive effects of an oil adjuvant on efficacy, dissipation and safety of pyrimethanil and boscalid on greenhouse strawberry.

Methylated vegetable oil adjuvants can enhance initial deposition and decrease the required dosages of pesticides sprayed on plants, so an oil adjuvant mixed with fungicides were used to prevent and control gray mold in greenhouse strawberry. As the persistence and dietary exposure risks from fungicides on strawberries after using adjuvants have not been assessed, the efficacy, dissipation and safety of pyrimethanil and boscalid in the presence and absence of a methylated vegetable oil adjuvant were evaluated. To better describe the actual use of fungicides in greenhouse strawberry, twice repeated application of fungicides were conducted follower by an optimized QuEChERS pre-treatment method. When applied at 60% of their recommended dosages with the adjuvant, the efficacy of pyrimethanil and boscalid for gray mold was similar to that shown by the treatment of 100% fungicides in absence of the adjuvant based on Duncan's Multiple-Range test, and their average residues increased to 89.0% and 89.3%, respectively. The adjuvant enhanced the accumulation effect of pyrimethanil residue by 31.7% after repeated applications, and the half-lives were similar (5.2 and 4.2 d). The adjuvant had comparable accumulation effects (1.75 and 1.83) and similar half-lives (5.4 and 5.5 d) for boscalid. In absence of adjuvant, the risk quotients (RQs) of pyrimethanil (0.41 and 0.33) and boscalid (0.49 and 0.63) after twice applications at pre-harvest interval were lower than 1. Adding the methylated vegetable oil adjuvant to fungicides would result in unprolonging half-life and acceptably low dietary exposure risk on strawberries, but lower dosage of fungicides were used.

Degradation of three fungicides following application on strawberry and a risk assessment of their toxicity under greenhouse conditions.

The health risk to humans of pesticide application on minor crops, such as strawberry, requires quantification. Here, the dissipation and residual levels of three fungicides (pyraclostrobin, myclobutanil, and difenoconazole) were studied for strawberry under greenhouse conditions using high-performance liquid chromatography (HPLC)-tandem mass spectrometry after Quick, Easy, Cheap, Effective, Rugged, and Safe extraction. This method was validated using blank samples, with all mean recoveries of these three fungicides exceeding 80%. The residues of all three fungicides dissipated following first-order kinetics. The half-lives of pyraclostrobin, myclobutanil, and difenoconazole were 1.69, 3.30, and 3.65 days following one time application and 1.73, 5.78, and 6.30 days following two times applications, respectively. Fungicide residue was determined by comparing the estimated daily intake of the three fungicides against the acceptable daily intake. The results indicate that the potential health risk of the three fungicides was not significant in strawberry when following good agricultural practices (GAP) under greenhouse conditions.

Enantioselective fate and risk assessment of chiral fungicide pydiflumetofen in rice-fish and wheat farming systems.

Fungicides have been widely used for reducing the losses caused by plant diseases. Rice and wheat are the most basic food crops, and the potential risks after applying fungicides are worthy of attention. Especially rice-fish farming system is an ecological symbiosis system that is beneficial to both environmental and ecological protection. However, the application of pesticides will stress the ecosystem, and the pesticide residues in rice and fish would be transmitted along the food chain, which is harmful to human health. Here, the enantioselective behaviors of chiral pydiflumetofen in rice-fish and wheat farming systems were clarified. In the rice-fish farming system, pydiflumetofen enantiomers were preferentially attached to the plants, entering the paddy water and settling into the paddy soil, and then accumulating and dissipating in the fish. With the growth of rice, it was transported to rice fruits. The wheat farming system was similar. Enantioselective dissipation occurred in carp (Cyprinus carpio), brown rice and wheat soil, and S-(+)-pydiflumetofen was preferentially dissipated. In other words, R-(-)-pydiflumetofen showed higher concentrations, especially in carp, which meant R-(-)-pydiflumetofen was more easily accumulated in the environment, and posed a greater potential risk to the farming system. The pydiflumetofen residues in brown rice and wheat were lower than MRLs from the EFSA (0.02 mg/kg) and eCFR (0.3 mg/kg), respectively. What deserves attention is that the MRL of pydiflumetofen in fish is not clear. Meanwhile, pydiflumetofen in paddy soil and wheat soil had a persistent residual effect, and the risks could not be ignored. Combined with the previous research, developing S-(+)-pydiflumetofen products will help to reduce the dosage and reduce the risks to environment and people. This study evaluated the environmental fate and risk of chiral pydiflumetofen from the perspective of farming system, and would provide data support for its rational use and risk assessment.

Mixture toxicities of tetrachlorantraniliprole and tebuconazole to honey bees (Apis mellifera L.) and the potential mechanism.

The extensive use of pesticides has negative effects on the health of insect pollinators. Although pollinators in the field are seldom exposed to individual pesticides, few reports have assessed the toxic impacts of pesticide combinations on them. In this work, we purposed to reveal the combined impacts of tetrachlorantraniliprole (TET) and tebuconazole (TEB) on honey bees (Apis mellifera L.). Our data exhibited that TET had greater toxicity to A. mellifera (96-h LC50 value of 298.2 mg a.i. L-1) than TEB (96-h LC50 value of 1,841 mg a.i. L-1). The mixture of TET and TEB displayed acute synergistic toxicity to the pollinators. Meanwhile, the activities of CarE, CYP450, trypsin, and sucrase, as well as the expressions of five genes (ppo, abaecin, cat, CYP4G11, and CYP6AS14) associated with immune response, oxidative stress, and detoxification metabolism, were conspicuously altered when exposed to the mixture relative to the individual exposures. These results provided an overall comprehension of honey bees upon the challenge of sublethal toxicity between neonicotinoid insecticides and triazole fungicides and could be used to assess the intricate toxic mechanisms in honey bees when exposed to pesticide mixtures. Additionally, these results might guide pesticide regulation strategies to enhance the honey bee populations.

Study on the enantioselective bioaccumulation and dissipation of uniconazole enantiomers in earthworm-soil microcosm through supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS).

In this work, the enantioselective bioaccumulation and dissipation of uniconazole enantiomers in earthworm-soil microcosm were studied. A fast enantioseparation method of uniconazole through supercritical fluid chromatography-tandem mass spectrometry (SFC-MS/MS) was established. The CHIRALCEL OZ-3 column and a mixture of CO2 and methanol (80:20, v/v) were used within 1.0 min to separate uniconazole enantiomers. The recoveries of uniconazole enantiomers in earthworm and soil samples ranged from 83.3 to 113%, and the intra-day and inter-day relative standard deviation values were lower than 11%. In earthworms, the bioaccumulation concentrations of uniconazole enantiomers increased with time and reached the maximum on the 7th day and then decreased. The elimination of uniconazole enantiomers in earthworms followed the first-order kinetics equation, and the elimination half-lives were approximately 7 days. In artificial soil, the dissipation of uniconazole enantiomers was slow, and the dissipation half-lives were both 25.7 days. No enantioselectivity occurred in the earthworm-soil microcosm. These results may reduce the uncertainty of environmental risk assessment for uniconazole.

Stereoselective, Diastereoselective Dissipation and Risk Assessment of Chiral Metconazole in Soybean, Peanut, Cabbage, Celery, Tomato, and Soil.

The stereoselective behaviors and dietary risks of metconazole (MZE) in soil and five vegetables were investigated. The results showed that there was species-specific stereoselective and diastereoselective dissipation, and the half-lives ranged from 0.69 to 8.17 days. cis-(+)-1S,5R-MZE was preferentially dissipated in soybean pods, cabbages, celeries, and tomatoes, which was contrary to soybean plants and soil. trans-(+)-1R,5R-MZE was preferentially dissipated in peanut plants, peanut shells, celeries, and tomatoes, while trans-(-)-1S,5S-MZE was preferentially dissipated in soybean plants. cis-MZE was preferentially dissipated in the test vegetables and soil, except celery. The stereoisomeric excess changes were higher than 10%, indicating that the stereoselectivity and diastereoselectivity should be considered in the risk assessment of MZE in soybean plants, pods, and peanut plants. The acute and chronic dietary intake risks of rac-MZE for different groups of people were acceptable. The preferentially dissipated and high activity cis-(+)-1S,5R-MZE with lower toxicity might be suitable for application as monocase.

Comparative acute toxicity of twenty-four insecticides to earthworm, Eisenia fetida.

In this study, we used two different types of bioassay, a contact filter paper toxicity bioassay and a soil toxicity bioassay, to compare the acute toxicity of twenty-four insecticides belonging to six chemical categories on earthworm species, Eisenia fetida. Results of the contact filter paper toxicity bioassay indicated that neonicotinoids were super toxic to E. fetida (48 h-LC(50) value ranged from 0.0088 to 0.45 µg cm(-2)), pyrethroids were very toxic (48 h-LC(50) values ranged from 10.55 to 25.7 µg cm(-2)) and insect growth regulators (IGRs) were moderately toxic (48 h-LC(50) values ranged from 117.6 to 564.6 µg cm(-2)) to the worms. However, antibiotics, carbamates and organophosphates induced variable toxicity responses in E. fetida, and were very to extremely toxic (48 h-LC(50) values ranged from 3.64 to 75.75 µg cm(-2)). Results of the soil toxicity bioassays showed a different pattern of toxicity except that neonicotinoids were the most toxic even under the soil toxicity bioassay system. The acute toxicity of neonicotinoids was higher than those of antibiotics, carbamates, IGRs and organophosphates. In contrast, pyrethroids were the least toxic to the worms under the soil toxicity bioassay system. It was concluded that irrespective of bioassay systems, earthworms were more susceptible to neonicotinoids than other modern synthetic insecticides.

Assessment of toxicity risk of insecticides used in rice ecosystem on Trichogramma japonicum, an egg parasitoid of rice lepidopterans.

Both chemical and biological methods are essential for control of insects, for example, lepidopterans, on rice. Thus, it is important to know the effect of chemicals on the biological control agents. In this study, we assessed the toxicity of commonly used insecticides on a biological control agent, Trichogramma japonicum Ahmead (an egg parasitoid of rice lepidopterans) by using a dry film residue method. Results showed that thirty insecticides from seven chemical classes exhibited various degree of toxicity to this parasitoid. Among the seven classes of chemicals tested, organophosphates (chlorpyrifos, fenitrothion, phoxim, profenofos, and triazophos) and carbamates (carbaryl, carbsulfan, isoprocarb, metolcarb, and promecarb) exhibited the highest intrinsic toxicity to T. japponicum, with an LC50 of 0.035 (0.029-0.044) to 0.49 (0.34-0.87) mg active ingredient (a.i.) L(-1), followed by antibiotics (abamectin, emamectin benzoate, and ivermectin), phenylpyrazoles (butane-fipronil, ethiprole, and fipronil), pyrethroids (cyhalthrin, cypermethrin, fenpropathrin, and lambda-cyhaothrin), and neonicotinoids (acetamiprid, imidacloprid, imidaclothiz, nitenpyram, thiacloprid, and thiamethoxam). Moreover, the insect growth regulator insecticides (chlorfluazuron, fufenozide, hexaflumuron and tebufenozide) exhibited the lowest toxicity to the wasps with an LC50 of 3,383 (2406-5499) to 30206 (23107-41008) mg ai. L(-1). Risk quotient analysis showed that phenylpyrazoles, pyrethroids, insect growth regulators, neonicotinoids (with the exception of thiamethoxam), and antibiotics (with the exception of abamectin) are classified as safe agents to the parasitoid, while organophosphates and carbamates are classified as slightly, moderately, or highly toxic agents to the parasitoid. The data presented in this paper provided useful information on the selection of compatible insecticides with T. japonicum.

Diverse mechanisms associated with cyhalofop-butyl resistance in Chinese sprangletop (Leptochloa chinensis (L.) Nees): Characterization of target-site mutations and metabolic resistance-related genes in two resistant populations.

Resistance of Chinese sprangletop (Leptochloa chinensis (L.) Nees) to the herbicide cyhalofop-butyl has recently become a severe problem in rice cultivation. However, the molecular mechanisms of target-site resistance (TSR) in cyhalofop-butyl-resistant L. chinensis as well as the underlying non-target-site resistance (NTSR) have not yet been well-characterized. This study aimed to investigate cyhalofop-butyl resistance mechanisms using one susceptible population (LC-S) and two resistant populations (LC-1701 and LC-1704) of L. chinensis. We analyzed two gene copies encoding the entire carboxyltransferase (CT) domain of chloroplastic acetyl-CoA carboxylase (ACCase) from each population. Two non-synonymous substitutions were detected in the resistant L. chinensis populations (Trp2027-Cys in the ACCase1 of LC-1701 and Leu1818-Phe in the ACCase2 of LC-1704), which were absent in LC-S. As Trp2027-Cys confers resistance to ACCase-inhibiting herbicides, the potential relationship between the novel Leu1818-Phe mutation and cyhalofop-butyl resistance in LC-1704 was further explored by single-nucleotide polymorphism (SNP) detection. Metabolic inhibition assays indicated that cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) contributed to cyhalofop-butyl resistance in specific resistant populations. RNA sequencing showed that the P450 genes CYP71Z18, CYP71C4, CYP71C1, CYP81Q32, and CYP76B6 and the GST genes GSTF11, GSTF1, and GSTU6 were upregulated in at least one resistant population, which indicated their putative roles in cyhalofop-butyl resistance of L. chinensis. Correlation analyses revealed that the constitutive or inducible expression patterns of CYP71C4, CYP71C1, GSTF1, and GSTU6 in L. chinensis were strongly associated with the resistant phenotype. For this reason, attention should be directed towards these genes to elucidate metabolic resistance to cyhalofop-butyl in L. chinensis. The findings of this study improve the understanding of mechanisms responsible for resistance to ACCase-inhibiting herbicides in grass-weed species at the molecular level, thus aiding in the development of weed management strategies that delay the emergence of resistance to this class of pest control products.

Enantioselectivity of indoxacarb during the growing, processing, and brewing of tea: Degradation, metabolites, and toxicities.

Chiral pesticides are unique hazardous materials. Here, we systematically studied the potentially harmful products of enantioselective indoxacarb degradation throughout tea growth, processing, and brewing and tested their toxicity to tea geometrid larvae and honeybees. The half-lives of S-indoxacarb and R-indoxacarb during tea growth were 2.6 d and 3.3 d, respectively. There was a trend toward the production of S-indoxacarb from R-indoxacarb. The degradation products IN-JT333, IN-MK638, IN-MF014, and IN-KG433 were also characterized in tea growth and processing and detected. IN-JT333, previously known as a direct insecticidal compound produced by the enzymatic transformation of indoxacarb in insects, was first found in plant samples. The fixation and rolling of green tea and the rolling of black tea were the most important steps that affected indoxacarb and its degradation products. The leaching rates of R-indoxacarb and S-indoxacarb were slightly higher in green tea than in black tea. The maximum leaching rates of IN-MK638 and IN-MF014 during the brewing process reached 89.9% and 94.1%, respectively. Contact toxicity tests with honeybees and tea geometrid larvae in the lab showed that the relative toxicities of the compounds could be ranked as follows: S-indoxacarb > indoxacarb (3S + 1R) ≫ R-indoxacarb. TEST toxicity predictions showed that relative toxicities were ranked IN-KG433 > indoxacarb > IN-JT333 > IN-MK638 > IN-MF014. The toxicity of the degradation product IN-KG433 is higher than that of indoxacarb itself, and its maximum leaching rate is as high as 88.2%. It therefore transfers readily from processed tea to the tea infusion during the brewing process. These findings indicate the need to pay attention to the risk of metabolites and enantiomeric differences and provide new, comprehensive insight into the risk factors for indoxacarb in tea and are relevant to the study of other chiral pesticides.

Stereoselective bioaccumulation and dissipation of pyrisoxazole in earthworm-soil microcosm.

Pyrisoxazole is a chiral fungicide with high sterilizing activity to the plant pathogenic bacteria. It has two chiral C atoms, which bring four stereoisomers. The present work was the first time to explore the stereoselective bioaccumulation behavior of pyrisoxazole in earthworms by chiral liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS). The absolute configurations of pyrisoxazole stereoisomers were confirmed by circular dichroism (CD) coupled with calculated electronic circular dichroism (ECD) method, and the elution order in Lux Cellulose-3 column was as follows: (-)-3S, 5R-pyrisoxazole, (+)-3R, 5S-pyrisoxazole, (+)-3S, 5S-pyrisoxazole and (-)-3R, 5R-pyrisoxazole. The recoveries of pyrisoxazole stereoisomers in earthworm and soil samples ranged from 80.8 % to 101 % with the RSD lower than 6.3 %. In bioaccumulation progress, (+)-3R, 5S-pyrisoxazole was accumulated preferentially in earthworms, and the bioaccumulation concentrations of high-activity (-)-3S, 5R-pyrisoxazole were the lowest. There were no stereoselective bioaccumulation between (+)-3S, 5S-pyrisoxazole and (-)-3R, 5R-pyrisoxazole, while there was diastereoselectivity between Z-pyrisoxazole and E-pyrisoxazole with higher E-pyrisoxazole concentrations. In the whole bioaccumulation process, the BAF values of (+)-3R, 5S-pyrisoxazole were significantly higher than (-)-3S, 5R-pyrisoxazole, and the BAF values of (-)-3S, 5R-pyrisoxazole were the lowest. The dissipation of pyrisoxazole stereoisomers in the artificial soil was very slow and had no stereoselectivity, and the existence of earthworms had little effects on the dissipation of pyrisoxazole stereoisomers, which indicated that the stereoselective behaviors of pyrisoxazole in earthworms were caused by the stereoselective enrichment and dissipation of earthworms themselves. Taken together, (-)-3S, 5R-pyrisoxazole was recommend as a commercial product. This study played a positive role in guiding the development of environmentally friendly pesticides and provided database for the environmental and biological risk assessment of pyrisoxazole.