Co-existing antibiotics alter the enantioselective dissipation characteristics of zoxamide and drive combined impact on soil microenvironment.

Co-existence of antibiotics (ABX) in soil may expand the environmental harm of pesticide pollution. Our study investigated the combined effects of five antibiotics chlortetracycline (CTC), oxytetracycline (OTC), tetracycline (TC), sulfamethoxazole (SMX), enrofloxacin (ENR) on enantioselective fate of zoxamide (ZXM) and soil health. The results showed that S-(+)-ZXM preferentially dissipated in soil. ABX prolonged dissipation half-life and reduced enantioselectivity of ZXM. Soil was detected to be more acidic after long-term treatment of ZXM and ABX. Lowest soil available N, P, K were found in ZXM + SMX, ZXM + OTC and ZXM + SMX groups at 80 days, respectively. ABX had demonstrated effective promotion of catalase (S-CAT), urease (S-UE) and negative impact on dehydrogenase (S-DHA), sucrase (S-SC) activities. Bacteria Lysobacter, Sphingomonas and fungus Mortierella were identified as the most dominant genera, which possessed as potential microbial resources for removal of composite pollution from ZXM and ABX. SMX and TC, SMX, ENR, respectively, contributed to the alteration of bacteria and fungi community abundance. Soil acidity, available N and enzyme activity showed stronger correlations with bacteria and fungi compared to other environmental factors. Our findings highlighted the interactions between ZXM and ABX from the perspective of soil microenvironment changes. Moreover, a theoretical basis for the mechanism was actively provided.

Proteomic analysis of zoxamide-induced changes in Phytophthora cactorum.

In this study, the global proteomic response of Phytophthora cactorum to zoxamide was evaluated using a two-dimensional gel electrophoresis (2-DE)-based proteomic approach. Among the 21 proteins identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS), four cytoskeleton-related proteins were down-regulated upon addition of zoxamide. Five detoxification metabolism enzymes, seven sugar metabolism proteins and one mitochondria-related protein were up-regulated by more than 2-fold in response to zoxamide. Taken together, these results suggest that zoxamide can decrease the expression of cytoskeleton-related proteins of P. cactorum, resulting in cell death; however, the up-regulation of detoxification metabolism-related enzymes may protect P. cactorum against zoxamide, and the up-regulation of proteins related to sugar metabolism and mitochondria may lead to the generation of more energy for detoxification metabolism. These data also suggest that proteomics may be useful not only in exploring the mode of action of fungicides but also for gaining insight into the resistance mechanisms that pathogens employ against fungicides.

Lateral flow immunoassay based on gold nanoparticles for rapid and sensitive detection of zoxamide in grape, tomato and cucumber samples.

In the study, we discovered zoxamide hapten (ZOX-hapten) by introducing a carboxyl extension chain, combined it with protein to make a complete antigen to immunize mice, and generated a monoclonal antibody (mAb) against ZOX. To identify ZOX residues in grape, tomato, and cucumber samples, we used our anti-ZOX mAb to develop a lateral flow immunoassay (LFIA) strip. In grape, tomato, and cucumber samples, the calculated detection limit of the LFIA strip in grape, tomato and cucumber samples was 3.44, 4.78 and 3.53 ng/g, respectively. Using the LFIA strip, the recovery rate from grape samples was 96.4-106.8%, and that from tomato samples was 98.4-107.5%, while the recovery from cucumber samples was 99.4-111.3%. These results showed that our LFIA strip could be expected to achieve rapid screening of ZOX residues in fruits and vegetables.

Review of the existing maximum residue levels for zoxamide according to Article 12 of Regulation (EC) No 396/2005 and setting of an import tolerance for onions, garlic and shallots.

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 zoxamide. To assess the occurrence of zoxamide residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Regulation (EC) No 1107/2009, the MRLs established by the Codex Alimentarius Commission and the European authorisations reported by Member States and the UK (including the supporting residues data). Based on the assessment of the available data, MRL proposals were derived, and a consumer risk assessment was carried out. Although no apparent risk to consumers was identified, some information required by the regulatory framework was missing. Hence, the consumer risk assessment is considered indicative only and some MRL proposals derived by EFSA still require further consideration by risk managers. Furthermore, in accordance with Article 6 of Regulation (EC) No 396/2005, the Applicant Gowan Crop Protection Ltd, submitted a request to the competent national authority in Latvia to set an import tolerance for zoxamide in onions (extrapolated to garlic and shallots) based on the use authorised in USA. The data submitted in support of the request were found to be sufficient to derive MRL proposals for all crops under assessment. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of zoxamide according to the agricultural practices on onions, garlic and shallots is unlikely to present a risk to consumer health.

Removal of azimsulfuron and zoxamide using a tapered variable diameter biological fluidized bed combined with electrochemistry: Mass fraction division, energy metabolism activity and carbon emissions.

The performance of the combination system of tapered variable diameter biological fluidized bed (TVDBFB) with electrochemistry (EC) was evaluated for removing azimsulfuron and zoxamide under different temperatures and influent concentrations. Maximum removal efficiency of azimsulfuron and zoxamide could reach 94% and 98% under higher influent concentration (about 780 mg/L). As temperature decreased from 32 ℃ to 8 ℃, the mSe increased from 48% to 56%, and the mSo and mSxv decreased from 30% to 22% and 27% to 24%, respectively. As the influent COD equivalent concentration of azimsulfuron and zoxamide enhanced from 260 mg/L to 780 mg/L, the Kd increased from 0.06 d-1 to 0.23 d-1. Temperature and influent concentration were main influencing factors of DHA, ATP and ETS. Increasing aeration in TVDBFB and HRT in EC under shock conditions could improve azimsulfuron and zoxamide removal efficiency, however, it was also accompanied by higher carbon emissions.

Zoxamide accumulation and retention evaluation after nanosuspension technology application in tomato plant.

BACKGROUND: Low water solubility of pesticide requires formulations with high levels of stabilizers and organic solvents. Moreover, only 0.1% of the applied pesticides formulation reaches the target, while 99.9% spreads in the surrounding environment. Therefore, there is the need for more efficient and environmentally sustainable alternatives. RESULTS: Zoxamide (ZO) nanosuspension was prepared through a media milling technique by using the stabilizer polysorbate 80. The thin and acicular crystals obtained, showed particle size of 227 nm, polydispersion index of 0.247 and zeta potential of -28 mV. Dimensional data and morphology of ZO nanocrystals alone, on tomato leaves and berries, were confirmed by scanning electron microscopy. The reduction in size for ZO crystals obtained after the milling process increased pesticide water solubility till 39.6 mg L-1 , about 1.6 the solubility obtained with a conventional commercial formulation. Field and dip contamination trials performed on tomato plants showed the nanosuspension's ability to increase ZO deposition and accumulation versus a coarse ZO suspension and commercial formulation, respectively. CONCLUSIONS: The nanoformulation proposed, resulted in low cost and was easy to make. Moreover, the organic solvent-free composition together with a low surfactant addition assured a minor environmental impact. Finally, the increased retention and deposition of the fungicide can reduce the amounts of ZO formulation applied to tomatoes. © 2021 Society of Chemical Industry.

A systematic evaluation of zoxamide at enantiomeric level.

Zoxamide is a recently discovered chiral fungicide that applied to agricultural production, but the potential environmental risk may be underestimated because the risk posed by either enantiomer has not been adequately assessed. Therefore, systemic evaluation of zoxamide was first carried out at the enantiomeric level. Enantioselective bioactivity against target pathogens (Phytophthora capsici Leonian, Alternaria solani, Botryis cinerea, Colletotrichum gloeosprioides Penz, Phytophthora sojae Kaufmann & Gerdemann) was explored, and the order of the bioactivity was R-zoxamide >Rac-zoxamide >S-zoxamide, with a 9.9- to 140.0-times difference between two enantiomers. Molecular docking simulation was utilized to clarify the mechanism underlying the observed differences in enantioselective bioactivity, and the result indicated that a difference of Van der waals force between R/S-zoxamide and the specific receptor gave rise to the different antifungal activity. The enantioselective toxicity result demonstrated that R-zoxamide had 4.9- to 10.8- times greater acute toxicity to Selenastrum capricornutum and Daphnia magna than S-zoxamide. S-zoxamide degraded faster under aerobic condition in all three types of soils, giving rise to an enrichment of high-risk R-enantiomer. Under anaerobic condition, however, no significant difference in dissipation rate was observed between two enantiomers. R-zoxamide was 1.5- to 3.5-times more bioactive and 1.1- to 1.5-times more toxic than Rac-zoxamide, which means developing R-zoxamide instead of racemate is a potential way to reduce pesticide dosage without loss of efficacy against target organisms and that an inactive isomer would no more be released to the environment. This study may have implications for better practical application and environmental risk assessment of zoxamide enantiomers.

The fate and enantioselective behavior of zoxamide during wine-making process.

The fate of zoxamide and its enantiomers were evaluated in detail during wine-making process. The enantiomers of zoxamide were separated and determined by ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) after each processing procedure including washing, peeling, fermentation and clarification. Significant enantioselectivity was observed in all three treatments with the half-lives of R-zoxamide and S-zoxamide estimated to be 45.6 and 52.9 h in Group A, 45.0 and 52.1 h in Group B, 56.8 and 70.7 h in Group C, respectively. The results indicated that R-zoxamide degraded faster than S-zoxamide during the fermentation process. The processing factors (PFs) of each procedure were generally less than 1, and the PF of the overall process ranged from 0.019 to 0.051, which indicated that the whole process can reduce the zoxamide residue in red and white wine obviously. The results could help facilitate more accurate risk assessments of zoxamide during wine-making process.

The application of chiral ultra-high-performance liquid chromatography tandem mass spectrometry to the separation of the zoxamide enantiomers and the study of enantioselective degradation process in agricultural plants.

In this study, an effective and sensitive chiral analytical method was developed to detect zoxamide enantiomers in vegetables, fruits and environmental matrices using ultra-high-performance liquid chromatography-tandem mass spectrometry. Optimal separation conditions were achieved with Lux Amylose-2 chiral column using acetonitrile/water (70:30v/v) as mobile phase with a flow rate and column temperature of 0.5mL/min and 25°C. The absolute configuration, optical rotation and elution order were confirmed for the first time. The average recoveries in all matrices at four spiking levels (0.5, 5, 50, 250µg/kg) ranged from 89.7 to 117.4%, with relative standard deviations being less than 10.9% for two enantiomers. The enantioselective dissipation of zoxamide in tomato showed that (-)-R-zoxamide was preferentially degraded leading to an enrichment of (+)-S-isomer, with half-lives of 3.80 d and 5.17 d, respectively. Inversely, (+)-S-zoxamide degraded faster than (-)-R-zoxamide in pepper (1.95day and 2.28day, respectively) and grape (2.03day and 2.87day). No significant enantioselectivity was observed in cucumber. The results of this study could help facilitate more accurate risk assessments of zoxamide in the future.

Peer review of the pesticide risk assessment of the active substance zoxamide.

The conclusions of EFSA following the peer review of the initial risk assessments carried out by the competent authorities of the rapporteur Member State, Latvia, and co-rapporteur Member State, France, for the pesticide active substance zoxamide are reported. The context of the peer review was that required by Commission Implementing Regulation (EU) No 844/2012. The conclusions were reached on the basis of the evaluation of the representative uses of zoxamide as a fungicide on wine and table grapes, and potatoes. The reliable end points, appropriate for use in regulatory risk assessment, are presented. Missing information identified as being required by the regulatory framework is listed. Concerns are identified.

C239S Mutation in the ß-Tubulin of Phytophthora sojae Confers Resistance to Zoxamide.

Zoxamide is the sole ß-tubulin inhibitor registered for the control of oomycete pathogens. The current study investigated the activity of zoxamide against Phytophthora sojae and baseline sensitivity was established with a mean EC50 of 0.048 µg/ml. The data is critical for monitoring changes in zoxamide-sensitivity in the field. Three stable resistant mutants with a high resistance level were obtained by selection on zoxamide amended media. Although the development of resistance occurred at a low frequency, there were no apparent fitness penalty in the acquired mutants in terms of growth rate, sporulation, germination and pathogenicity. Based on the biological profiles and low mutagenesis rate, the resistance risk of P. sojae to zoxamide can be estimated as low to medium. Further investigation revealed all the zoxamide-resistant mutants had a point mutation of C239S in their ß-tubulin. Zoxamide also exhibited high activity against most species from the genus Pythium in which only Pythium aphanidermatum was found naturally resistant to zoxamide and harboring the natural point mutation S239 in the ß-tubulin. Back-transformation in P. sojae with the mutated allele (S239) confirmed the C239S mutation can induce resistance to zoxamide, and the resistance level was positively related to the expression level of the mutated gene. In contrast, the overexpression of the wild type gene was unable to cause zoxamide resistance. It is the first report on the resistance molecular mechanism of zoxamide in oomycetes. Based on our study, C239 is supposed to be a key target site of zoxamide, which distinguishes zoxamide from benzimidazoles and accounts for its low resistance risk. The result can provide advice on the design of new ß-tubulin inhibitors in future.