Engineering an Epoxide Hydrolase for Chemoenzymatic Asymmetric Synthesis of Chiral Triazole Fungicide (S)- and (R)-Flutriafol.

Flutriafol, a globally utilized triazole fungicide in agriculture, is typically applied as a racemic mixture, but its enantiomers differ in bioactivity and environmental impact. The synthesis of flutriafol enantiomers is critically dependent on chiral precursors: 2,2-bisaryl-substituted oxirane [(2-fluorophenyl)-2-(4-fluorophenyl)oxirane, 1a] and 1,2-diol [1-(2-fluorophenyl)-1-(4-fluorophenyl)ethane-1,2-diol, 1b]. Here, we engineered a Rhodotorula paludigensis epoxide hydrolase (RpEH), obtaining mutant Escherichia coli/RpehH336W/L360F with a 6.4-fold enhanced enantiomeric ratio (E) from 5.5 to 35.4. This enabled a gram-scale resolution of rac-1a by E. coli/RpehH336W/L360F, producing (S)-1a (98.2% ees) and (R)-1b (75.0% eep) with 44.3 and 55.7% analytical yields, respectively. As follows, chiral (S)-flutriafol (98.2% ee) and (R)-flutriafol (75.0% ee) were easily synthesized by a one-step chemocatalytic process from (S)-1a and a two-step chemocatalytic process from (R)-1b, respectively. This chemoenzymatic approach offers a superior alternative for the asymmetric synthesis of flutriafol enantiomers. Furthermore, molecular dynamics simulations revealed insight into the enantioselectivity improvement of RpEH toward bulky 2,2-bisaryl-substituted oxirane 1a.

Understanding the Metabolism and Dissipation Kinetics of Flutriafol in Vegetables under Laboratory and Greenhouse Scenarios.

Flutriafol is a systemic triazole fungicide that is used to control diseases in various crops. A study was developed to evaluate the metabolism and dissipation of flutriafol in two different scenarios: laboratory and greenhouse conditions. Courgette and tomato samples treated with a commercial product (IMPACT® EVO) at the manufacturer recommended dose were analyzed, and courgette samples were also treated at double dose. Ultra-high-performance liquid chromatography coupled with Q-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap-MS), performing targeted and non-targeted approaches (suspect screening and unknown analysis), were used to analyze the samples. The dissipation of flutriafol was fitted to a biphasic kinetic model, with a persistence, expressed as half-life (t1/2), lower than 17 days. During suspect screening, three metabolites (triazole alanine, triazole lactic acid and triazole acetic acid) were tentatively identified. Unknown analysis led to the identification of four additional metabolites (C16H14F2N4, C16H14F2N4, C19H17F2N5O2 and C22H23F2N3O6). The results revealed that the proposed methodology is reliable for the determination of flutriafol and its metabolites in courgette and tomato, and seven metabolites could be detected at low concentration levels. The highest concentration of metabolites was found in the laboratory conditions at 34.5 µg/kg (triazole alanine). The toxicity of flutriafol metabolites was also evaluated, and some of them could be more toxic than the parent compound.

Removal of pesticides from secondary treated urban wastewater by reverse osmosis.

The residues of pesticides that reach water resources from agricultural activities in several ways contaminate drinking water resources and threaten aquatic life. This study aimed to investigate the performance of three reverse osmosis (RO) membranes (BW30-LE, SW30-XLE, and GE-AD) in rejecting four different pesticides (tributyl phosphate, flutriafol, dicofol, and irgarol) from secondary treated urban wastewater and also to elucidate the mechanisms underlying the rejection of these pesticides. RO experiments were conducted using pesticide-spiked wastewater samples under 10 and 20 bar transmembrane pressures (TMP) and membrane performances were evaluated. Overall, all the membranes tested exhibited over 95% rejection performances for all pesticides at both TMPs. The highest rejections for tributyl phosphate (99.0%) and irgarol (98.3%) were obtained with the BW30-LE membrane, while for flutriafol (99.9%) and dicofol (99.1%) with the GE-AD membrane. The increase in TMP from 10 to 20 bar did not significantly affect the rejections of all pesticides. The rejection performances of RO membranes were found to be governed by projection area as well as molecular weight and hydrophobicity/hydrophilicity of pesticides. Among the membranes tested, the SW30-XLE membrane was the most prone to fouling due to the higher roughness.

Effect of Flutriafol Exposure on Residue Characteristics in Pig Muscle and Fat Tissue.

This study investigated the effect of exposure to flutriafol based on residues in pigs. Pigs were exposed to different concentrations (0.313, 0.625, 3.125, 6.25, and 12.5 mg/kg bw/d, n=20) for 4 wk in different treatment groups. Serum biochemical analysis, residue levels, and histological analysis were conducted using the VetTest chemistry analyzer, liquid chromatography mass spectrometry, and Masson's trichrome staining, respectively. The body weight (initial and final) was not significantly different between groups. Parameters such as creatinine, blood urea nitrogen, alanine aminotransferase, and lipase levels were significantly different as compared to the control group. Flutriafol increased the residue limits in individual tissue of the pigs in a dose dependent manner. Flutriafol exposures indicated the presence of fibrosis, as confirmed from Masson's trichrome staining. These results suggest that flutriafol affects the morphology and serum levels in pigs. The dietary flutriafol levels can provide a basis for maximum residue limits and food safety for pork and related products.

Fungicide Sensitivity of Colletotrichum Species Causing Bitter Rot of Apple in the Mid-Atlantic U.S.A.

Apple growers in the Mid-Atlantic region of the U.S.A. have reported increased losses to bitter rot of apple. We tested the hypothesis that this increase is because the Colletotrichum population has developed resistance to commonly used single-mode-of-action (single-MoA) fungicides. We screened 220 Colletotrichum isolates obtained from 38 apple orchards in the Mid-Atlantic region for resistance to 11 fungicides in Fungicide Resistance Action Committee (FRAC) groups 1, 7, 9, 11, 12, and 29. Eleven (5%) of these isolates were resistant to FRAC group 1 with confirmed ß-tubulin E198A mutations, and two (<1%) were also resistant to FRAC group 11 with confirmed cytochrome-b G143A mutations. Such low frequencies of resistant isolates indicate that fungicide resistance is unlikely to be the cause of any regional increase in bitter rot. A subsample of isolates was subsequently tested in vitro for sensitivity to every single-MoA fungicide registered for apple in the Mid-Atlantic U.S.A. (22 fungicides; FRAC groups 1, 3, 7, 9, 11, 12, and 29), and 13 fungicides were tested in field trials. These fungicides varied widely in efficacy both within and between FRAC groups. Comparisons of results from our in vitro tests with results from our field trials and other field trials conducted across the eastern U.S.A. suggested that EC25 values (concentrations that reduce growth by 25%) are better predictors of fungicide efficacy in normal field conditions than EC50 values. We present these results as a guideline for choosing single-MoA fungicides for bitter rot control in the Mid-Atlantic U.S.A.

Effects of Flutriafol Fungicide on the Lipid Accumulation in Human Liver Cells and Rat Liver.

Flutriafol (FTF) is a triazole fungicide that can cause liver toxicity through the ingestion of its residues in food and water. However, little is known about the liver toxicity of FTF, particularly nonalcoholic fatty liver disease (NAFLD) in humans. Therefore, the purpose of this study was to investigate whether FTF induces NAFLD in human liver cells and animal liver. HepG2 cells and Sprague Dawley (SD) rats were treated with FTF at doses of 0-640 µM for 24 h and 0-150 mg/kg bw/day for 28 days, respectively. FTF (80, 160, and 320 µM) treatment to cells induced lipid accumulation. FTF (80 and 160 µM)-treated cells had higher levels of cytochrome P450 enzymes and reactive oxygen species and increased mitochondrial membrane potential loss than the control. FTF also increased the mRNA levels of antioxidant enzymes through oxidative stress and nuclear factor erythroid 2-related factor 2 pathways in HepG2 cells. However, a higher level of FTF (320 µM) induced apoptosis. The treatment of SD rats with FTF (2.5-150 mg/kg bw/day) induced fatty infiltration in the liver by impairing liver metabolism and inducing apoptosis. Therefore, our data suggest that human exposure to FTF residues may be a risk factor for liver diseases, such as NAFLD.

Enantioselective Effect of Flutriafol on Growth, Deoxynivalenol Production, and TRI Gene Transcript Levels in Fusarium graminearum.

In recent years, deoxynivalenol (DON) has frequently been detected in wheat grains and their products. The enantioselective impact of flutriafol on the growth and DON biosynthesis of Fusarium graminearum was investigated in relation to water activity (αw, 0.97 and 0.99) and temperature (20, 25, and 30 °C) on the wheat-based medium. R-(-)-flutriafol exhibited higher bioactivity than S-(+)-flutriafol and Rac-flutriafol under the above conditions. Flutriafol enantiomers reduced or stimulated DON biosynthesis depending on αw. DON levels were negligible after 14 or 7 days of incubation times under 0.97 and 0.99 aw, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that the expression levels of trichothecene biosynthetic (TRI) genes of F. graminearum under 0.97 aw were significantly higher than those under 0.99 aw. In addition, R-(-)-flutriafol can induce more TRI gene expression than S-(+)-flutriafol. Taken together, this study indicated that aw and temperature play important roles in regulating DON biosynthesis in F. graminearum with flutriafol enantiomers.

Evaluation of confirmatory data following the Article 12 MRL review and setting of an import tolerance for flutriafol in cucurbits (inedible peel).

The applicant Cheminova A/S submitted a request to the competent national authority in the United Kingdom to evaluate the confirmatory data for the active substance flutriafol that were identified in the framework of the maximum residue level (MRL) review under Article 12 of Regulation (EC) No 396/2005. The applicant provided residue trials on rice representative for an adjusted Good Agricultural Practice (GAP), metabolism studies in goats and storage stability data in tissues of animal origin addressing the data gaps identified in the MRL review. For the nature of residues in processed commodities, relevant to confirm the MRLs on pome fruits and wine grapes, the applicant referred to the information provided in a previous MRL application and already assessed by EFSA. The data gaps on the authorised uses on beetroots, melons and watermelons assessed in the MRL review were not addressed. At the same time, the applicant submitted a request to the United Kingdom to modify the existing maximum residue level (MRL) for flutriafol in cucurbits with inedible peel according to Article 6 of Regulation (EC) No 396/2005. The data submitted were found to be sufficient to derive an MRL proposal for these crops. EFSA concluded that the proposed use of flutriafol on the crops under assessment will not result in a consumer exposure exceeding the toxicological reference values for flutriafol and therefore is unlikely to pose a risk to consumers' health. Regarding the triazole derivative metabolites (TDMs), the conclusions reached in a previously issued assessment for another triazole fungicide are still valid. An update of the indicative consumer risk assessment for each individual metabolite is not necessary since the residues expected in the concerned commodities are covered.

Electrochemical treatment of flutriafol wastewater using a novel 3D macroporous PbO2 filter: Operating parameters, mechanism and toxicity assessment.

In order to break the high operating cost bottleneck of electrochemical treatment of aqueous flutriafol (FTF), an emerging fungicide, a novel three-dimensional ordered macroporous PbO2 (3DOM-PbO2) filter was designed to facilitate mass transfer. The effects of operating parameters, including current density, flow rate and initial concentration on FTF electrooxidation performance were investigated using conventional flat Ti/PbO2 (F-Ti/PbO2) and 3DOM-PbO2 filters, with primary objective being the development of appropriate parameters for FTF treatment. The results indicated that the FTF removal efficiency on 3DOM-PbO2 filter was improved by 2.8 times compared to that on F-Ti/PbO2 at 5 mA cm-2, 10 ml s-1 and 100 mg L-1 FTF. The corresponding electrical energy consumption was reduced by 2.7 times, ` TOC removal and mineralization current efficiency were enhanced by 4.9 and 4.8 times, respectively. Furthermore, aromatic intermediates, nitrogenous compounds and carboxylic acids were identified as main byproducts using experimental method combined with quantum chemical calculations. Then, a possible pathway of FTF degradation on 3DOM-PbO2 was proposed. Finally, the acute toxicity results showed that toxicity of the byproducts first increases and then decreases through the proposed route. LC50,48 h value of FTF wastewater increased 35%-70% on the 3DOM-PbO2 filter, indicating a significant biodegradability enhancement.

Setting of import tolerance for flutriafol in hops.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Cheminova A/S submitted a request to the competent national authority in Spain to set an import tolerance for the active substance flutriafol in hops. The data submitted in support of the request were found to be sufficient to derive an import tolerance proposal of 20 mg/kg for crops under consideration. Adequate analytical methods for enforcement are available to control the residues of flutriafol in plant matrices under consideration. Based on the risk assessment results, EFSA concluded that the short-term and long-term intake of residues resulting from the use of flutriafol according to the reported agricultural practice is unlikely to present a risk to consumer health. However, as flutriafol belongs to the triazole chemical group, EFSA recommends that a separate risk assessment should be performed for triazole derivative metabolites (TDMs) as soon as the confirmatory data requested for triazole compounds in the framework of Regulation (EC) No 1107/2009 have been evaluated and a general methodology on the risk assessment of triazole compounds and their TDMs is available.

Flutriafol and pyraclostrobin residues in Brazilian green coffees.

The aim of this work was to monitor flutriafol and pyraclostrobin residues in Brazilian green coffees. More than 10,000 samples were analyzed. The pesticides were extracted using the QuEChERS method and analyzed by LC-MS/MS. The validated method is fast, with 5 min runs, and efficient, as precision and accuracy showed RSD no greater than 5% and recoveries within the 88-119% range. LOQ for flutriafol and pyraclostrobin were 0.005 mg/kg. The results of the analyzed samples showed that the percentage of nonconformities regarding flutriafol increased throughout the years, with over 1200 samples (11.8%). On the other hand, just 15 samples (0.15%) presented residues above 10 µg/kg for pyraclostrobin. Considering that flutriafol is a toxic and carcinogenic pesticide, as well as the increase in the number of irregularities throughout the years, it becomes important to implement public actions to assure consumer safety.

Study on the stereoselective degradation of three triazole fungicides in sediment.

The stereoselective degradation behaviors of chiral triazole fungicides (hexaconazole, flutriafol and tebuconazole) in sediment were investigated under laboratory conditions. The enantiomers were completely separated by high-performance liquid chromatography on a cellulose tris(3-chloro-4-methylphenylcarbamate) (Lux Cellulose-2) column. The mean recoveries of hexaconazole, flutriafol and tebuconazole in sediment ranged from 86.7% to 105.9%. The methods were successfully applied for the enantioselective degradation analysis of fungicides in sediment. The results showed that the dissipation of hexaconazole, flutriafol and tebuconazole stereoisomers in sediment followed first-order kinetics (R(2)>0.95). The degradation rate of the enantiomers was different in sediment, and the (-)-enantiomer (t(1/2) was 86 days for hexaconazole, 139 for flutriafol and 136 for tebuconazole) degraded faster than the (+)-enantiomer (t(1/2) was 94 days for hexaconazole, 144 for flutriafol and 151 for tebuconazole) in native condition. The fungicides were degraded slowly, and no significant enantioselective degradation were observed under sterilized conditions. The results may hold promising implications for the environmental and ecological risk assessment of three important chiral triazole fungicides.

Enantioselective bioactivity, acute toxicity and dissipation in vegetables of the chiral triazole fungicide flutriafol.

The enantioselective bioactivity, acute toxicity and stereoselective degradation of the chiral triazole fungicide flutriafol in vegetables were investigated for the first time using the (R)-, (S)- and rac-flutriafol. The order of the bioactivity against five target pathogens (Rhizoctonia solani, Alternaria solani, Pyricularia grisea, Gibberella zeae, Botrytis cinerea) was found to be (R)-flutriafol>rac-flutriafol>(S)-flutriafol. The fungicidal activity of (R)-flutriafol was 1.49-6.23 times higher than that of (S)-flutriafol. The (R)-flutriafol also showed 2.17-3.52 times higher acute toxicity to Eisenia fetida and Scenedesmus obliquus than (S)-flutriafol. The stereoselective degradation of flutriafol in tomato showed that the active (R)-flutriafol degraded faster, resulting in an enrichment of inactive (S)-form, and the half-lives were 9.23 d and 10.18 d, respectively. Inversely, the (S)-flutriafol, with a half-life of 4.76 d, was preferentially degraded in cucumber. In conclusion, the systemic assessments of the triazole fungicide flutriafol stereoisomers on the enantioselective bioactivity, acute toxicity and environmental behavior may have implications for better environmental and ecological risk assessment.

Green and sensitive supercritical fluid chromatographic-tandem mass spectrometric method for the separation and determination of flutriafol enantiomers in vegetables, fruits, and soil.

A green and sensitive chiral analytical method was developed to determine flutriafol enantiomers in vegetables (tomato, cucumber), fruits (apple, grape), and soil by supercritical fluid chromatography-tandem mass spectrometry. The enantioseparation was performed within 3.50 min using Chiralpak IA-3 column with CO2/methanol (88:12, v/v) as the mobile phase at a 2.2 mL/min flow rate. The postcolumn compensation technology provided with 1% formic acid/methanol greatly improved the ionization efficiency of mass spectrometry. Column temperature, auto back pressure regulator pressure, and flow rate of compensation solvent were optimized to 30 °C, 2200 psi, and 0.1 mL/min, respectively. The simple and fast QuEChERS pretreatment method was adopted. Mean recoveries for flutriafol enantiomers were 77.2-98.9% with RSDs ≤ 9.6% in all matrices. The limits of quantification ranged from 0.41 to 1.18 µg/kg. Well-applied to analyze authentic samples, the developed method could act as a versatile strategy for the analysis of flutriafol enantiomers in food and environmental matrices.

Selective and simultaneous determination of trace bisphenol A and tebuconazole in vegetable and juice samples by membrane-based molecularly imprinted solid-phase extraction and HPLC.

Nanofibrous molecularly imprinted membranes (nano-MIMs) with multi-analyte selectivity were prepared by encapsulating two types of molecularly imprinted polymer nanoparticles (MIP-NPs) into electrospun polyvinyl alcohol nanofibers. The obtained nano-MIMs maintained high molecular selectivity offered by each of the MIP-NPs. Nano-MIM embedding BPA-imprinted nanoparticles and TBZ-imprinted nanoparticles together showed the highest binding selectivity for acid bisphenol A (BPA) and basic tebuconazole (TBZ). This nano-MIM was used as affinity material of membrane-based molecularly imprinted solid-phase extraction (m-MISPE) to extract trace BPA and TBZ in vegetables and juices simultaneously. The recoveries of BPA and TBZ from different samples were higher than 70.33% with RSDs lower than 9.57%. m-MISPE gave better HPLC separation efficiencies and higher recoveries than conventional SPE based on C18/SCX. Multi-analyte selective m-MISPE combined with HPLC realized selective and simultaneous determination of several trace analytes with opposite charges/polarities in different food samples.