Fungicide Sensitivity of Sclerotinia sclerotiorum from U.S. Soybean and Dry Bean, Compared to Different Regions and Climates.

Fungicide use is integral to reduce yield loss from Sclerotinia sclerotiorum on dry bean and soybean. Increasing fungicide use against this fungus may lead to resistance to the most common fungicides. Resistance has been reported in Brazil (Glycine max) and China (Brassica napus subsp. napus), however, few studies have investigated fungicide sensitivity of S. sclerotiorum in the United States. This work was conducted to determine if there was a difference in fungicide sensitivity of S. sclerotiorum isolates in the United States from: (i) dry bean versus soybean and (ii) fields with different frequencies of fungicide application. We further hypothesized that isolates with fungicide applications of a single active ingredient from tropical Brazil and subtropical Mexico were less sensitive than temperate U.S. isolates due to different management practices and climates. The EC50(D) fungicide sensitivity of 512 S. sclerotiorum isolates from the United States (443), Brazil (36), and Mexico (33) was determined using a discriminatory concentration (DC) previously identified for tetraconazole (2.0 ppm; EC50(D) range of 0.197 to 2.27 ppm), boscalid (0.2; 0.042 to 0.222), picoxystrobin (0.01; 0.006 to 0.027), and thiophanate-methyl, which had a qualitative DC of 10 ppm. Among the 10 least sensitive isolates to boscalid and picoxystrobin, 2 presented mutations known to confer resistance in the SdhB (qualitative) and SdhC (quantitative) genes; however, no strong resistance was found. This study established novel DCs that can be used for further resistance monitoring and baseline sensitivity of S. sclerotiorum to tetraconazole worldwide plus baseline sensitivity to boscalid in the United States.

Dietary risk evaluation of tetraconazole and bifenazate residues in fresh strawberry from protected field in North China.

Residues and dietary risk assessment of tetraconazole and bifenazate were investigated in strawberry under greenhouse conditions using high performance liquid chromatography (HPLC-DAD) after QuEChERs extraction in Beijing of China. The effects of different processing factors on the two pesticides were studied. The recoveries of tetraconazole and bifenazate were 87.0% and 89.1%, respectively. The dissipation curves of tetraconazole and bifenazate were in accordance with the first-order kinetic equation and the half-life were 5.92 d and 5.58 d, respectively. When the pre-harvest interval (PHI) was 3 d, the risk quotient (RQs) of both pesticides was less than 100%. Although soaking was a poor way to remove the two pesticides and heating at high temperatures increases the concentration of both pesticides, the residues of two pesticides can be effectively removed by washing after soaking. The results of dietary intake assessment indicated that potential dietary risk caused by tetraconazole and bifenazate in strawberry were acceptable for Chinese consumers.

Fluorescence polarization immunoassay for rapid screening of the pesticides thiabendazole and tetraconazole in wheat.

Fluorescence polarization immunoassays (FPIAs) for thiabendazole and tetraconazole were first developed. Tracers for FPIAs of thiabendazole and tetraconazole were synthesized and the tracers' structures were confirmed by HPLC-MS/MS. The 4-aminomethylfluorescein-labeled tracers allowed achieving the best assay sensitivity and minimum reagent consumption in comparison with aminofluorescein-labeled and alkyldiaminefluoresceinthiocarbamyl-labeled tracers. Measurements of fluorescence polarization were performed using a portable device. The developed FPIA methods were applied for the analysis of wheat. Fast and simple sample preparation technique earlier developed by authors for pesticides was adapted for thiabendazole and tetraconazole. The limits of detection of thiabendazole and tetraconazole in wheat were 20 and 200 µg/kg, and the lower limits of quantification were 40 and 600 µg/kg, respectively. The recovery test was performed by two methods-FPIA and HPLC-MS/MS. The results obtained by FPIA correlated well with those obtained by HPLC-MS/MS (r2 = 0.9985 for thiabendazole, r2 = 0.9952 for tetraconazole). Average recoveries of thiabendazole and tetraconazole were 74 ± 4% and 72 ± 3% by FPIA, and average recoveries of thiabendazole and tetraconazole were 86 ± 2% and 74 ± 1% by HPLC-MS/MS (n = 15). Graphical abstract ᅟ.

Enantioselective behaviour of tetraconazole during strawberry wine-making process.

The fate of tetraconazole enantiomers in strawberries during wine-making process was studied. The residues were determined by ultra-performance convergence chromatography tandem triple quadrupole mass spectrometry after each process steps. Results indicated that there was significant enantioselective dissipation of tetraconazole enantiomers during the fermentation process. And (-)-tetraconazole degraded faster than (+)-tetraconazole. The half-lives of (-)-tetraconazole and (+)-tetraconazole were 3.12, 3.76 days with washing procedure and 3.18, 4.05 days without washing procedure. The processing factors of strawberry wine samples after each step were generally less than 1. In particular, the processing factors of the fermentation process were the lowest. The results could help facilitate more accurate risk assessments of tetraconazole during wine-making process.

Dissipation behaviour, residue distribution and dietary risk assessment of tetraconazole and kresoxim-methyl in greenhouse strawberry via RRLC-QqQ-MS/MS technique.

20% commercial suspension emulsion (SE) of (8% tetraconazole + 12% kresoxim-methyl), as a pre-registered product in China, was firstly investigated under Chinese greenhouse-field conditions. A MWCNTs-based QuEChERS method for simultaneous determination of tetraconazole and kresoxim-methyl in strawberry was developed and validated via RRLC-QqQ-MS/MS. On basis of this method, the dissipation behaviours, residue distributions and dietary risk probability of these fungicides in strawberry were further investigated for food safety. The dissipations of tetraconazole and kresoxim-methyl followed first-order kinetics with the half-lives of 8.0-18.2 days. The highest residues (HRs) of these fungicides in the supervised trials at the pre-harvest interval (PHI, 3 days) were below 0.8970mgkg-1. The total national estimated daily intake (NEDI) of tetraconazole and kresoxim-methy in strawberry at the PHI 3day was 0.2784mg and 0.4031mg, respectively, based on Chinese dietary pattern and terminal residue distributions under good agricultural practices (GAP) conditions. The risk quotients (RQs) of tetraconazole and kresoxim-methy at PHI 3 days were below 82.7% and 1.6%, respectively, showing that the evaluated strawberry exhibited an acceptably low dietary risk to consumers. The current study could not only guide reasonable usage of the formulation, but also facilitate the setting of maximum residue limits (MRLs) of tetraconazole in strawberry.

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction.

Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.

Cross-resistance to fluconazole induced by exposure to the agricultural azole tetraconazole: an environmental resistance school?

This study aimed to investigate the influence of tetraconazole and malathion, both used in agricultural activities, on resistance to fluconazole, itraconazole and voriconazole in Candida parapsilosis ATCC 22019. The susceptibility to tetraconazole, malathion, fluconazole, itraconazole and voriconazole, through broth microdilution. Then, 12 independent replicates, were separated and exposed to four treatment groups, each one containing three replicates: G1: tetraconazole; G2: malathion; G3: fluconazole (positive control); G4: negative control. Replicates from G1, G2 and G3, were exposed to weekly increasing concentrations of tetraconazole, malathion and fluconazole, respectively, ranging from MIC/2 to 32 × MIC, throughout 7 weeks. The exposure to tetraconazole, but not malathion, decreased susceptibility to clinical azoles, especially fluconazole. The tetraconazole-induced fluconazole resistance is partially mediated by the increased activity of ATP-dependent efflux pumps, considering the increase in antifungal susceptibility after the addition of the efflux pump inhibitor, promethazine, and the increase in rhodamine 6G efflux and CDR gene expression in the G1 replicates. Moreover, MDR expression was only detected in G1 and G3 replicates, suggesting that MDR pumps are also involved in tetraconazole-induced fluconazole resistance. It is noteworthy that tetraconazole and fluconazole-treated replicates behaved similarly, therefore, resistance to azoles of clinical use may be a consequence of using azoles in farming activities.

Response of microbial communities from an apple orchard and grassland soils to the first-time application of the fungicide tetraconazole.

The aim of the study was to assess the impact of the triazole fungicide tetraconazole applied at the field rate (FR) and at ten-fold the FR (10FR) on microorganisms in orchard soil with a long-term history of fungicides application and in grassland soil that had not previously been treated with pesticides. To ascertain this impact, the microbial activity determined by fluorescein diacetate (FDA) hydrolysis, the culturable number of bacteria, fungi and tetraconazole-resistant fungi, and the phospholipid microbial biomass and the structural and functional biodiversity assessed by the PLFA and Biolog approaches, respectively, were examined under laboratory conditions during 28-day experiment. The response of soil microorganisms to the fungicide tetraconazole, which had never been used before in these soils, depended on the management of the soils. In apple orchard soil that had been treated with FR or 10FR tetraconazole, a decrease in microbial activity was still observed on the 28th day after the application of the fungicide. In contrast, a significant impact of tetraconazole on the number of bacteria was still observed at the end of experiment in grassland soil. Results of principal component analysis (PCA) indicated that the application of tetraconazole significantly changed the structure of the microbial communities in the orchard soil. In addition, analysis of the Biolog profiles revealed a decrease in the catabolic activity of the microbial communities in grassland soil that had been treated with tetraconazole at both rates over time. The evaluation of the structural and functional diversity of microbial communities using PCA appears to be the most valuable monitoring tool for assessing the impact of tetraconazole application on soil microorganisms.

Non-target impact of fungicide tetraconazole on microbial communities in soils with different agricultural management.

Effect of the fungicide tetraconazole on microbial community in silt loam soils from orchard with long history of triazole application and from grassland with no known history of fungicide usage was investigated. Triazole tetraconazole that had never been used on these soils before was applied at the field rate and at tenfold the FR. Response of microbial communities to tetraconazole was investigated during 28-day laboratory experiment by determination of changes in their biomass and structure (phospholipid fatty acids method-PLFA), activity (fluorescein diacetate hydrolysis-FDA) as well as changes in genetic (DGGE) and functional (Biolog) diversity. Obtained results indicated that the response of soil microorganisms to tetraconazole depended on the management of the soils. DGGE patterns revealed that both dosages of fungicide affected the structure of bacterial community and the impact on genetic diversity and richness was more prominent in orchard soil. Values of stress indices-the saturated/monounsaturated PLFAs ratio and the cyclo/monounsaturated precursors ratio, were almost twice as high and the Gram-negative/Gram-positive ratio was significantly lower in the orchard soil compared with the grassland soil. Results of principal component analysis of PLFA and Biolog profiles revealed significant impact of tetraconazole in orchard soil on day 28, whereas changes in these profiles obtained for grassland soil were insignificant or transient. Obtained results indicated that orchards soil seems to be more vulnerable to tetraconazole application compared to grassland soil. History of pesticide application and agricultural management should be taken into account in assessing of environmental impact of studied pesticides.

Does farm fungicide use induce azole resistance in Aspergillus fumigatus?

Azole resistance of Aspergillus fumigatus isolates has been reported worldwide and it would appear to be mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which is the target enzyme for azoles. The mutation has been confirmed in isolates from patients who received long-term itraconazole (ITZ) therapy and from agricultural fields where high levels of azole fungicides were employed. However, the relationship between farm environments and azole-resistant A. fumigatus has not been fully studied. In this investigation, 50 isolates of A. fumigatus were obtained from a farm where tetraconazole has been sprayed twice a year for more than 15 years. The mean minimum inhibitory concentration (MIC) of isolates was 0.74 (0.19-1.5) mg/L against ITZ, which was below the medical resistance level of ITZ. The sequence of CYP51A from isolates indicated no gene mutations in isolates from the farm. Antifungal susceptibility of isolates to tetraconazole showed that spraying with tetraconazole did not induce resistance to tetraconazole or ITZ in A. fumigatus.

Optimization of supercritical fluid extraction method for detection of fluquinconazole and tetraconazole in soil using gas chromatography and confirmation using GC-MS: application to dissipation kinetics.

The aim of this study was to establish an analytical method to detect fluquinconazole and tetraconazole in soil using supercritical fluid extraction (SFE) and gas chromatography (GC). The optimal extraction conditions for SFE were: temperature, 60 °C; pressure, 280 kg/cm(2) ; extraction time, 50 min; and a 10% modifier ratio. The linearity of the calibration curves was good and yielded a determination coefficient (R(2) ) ≥ 0.995. The soil samples were fortified with known quantities of the analytes at three different concentrations (0.01, 0.02 and 0.1 µg/g for fluquinconazole; 0.05, 0.1 and 0.5 µg/g for tetraconazole), and the recoveries ranged between 83.7 and 94.1%. The intra- and inter-day relative standard deviations were 1.3-10.6 and 2.2-11.9% for fluquinconazole and tetraconazole, respectively. The limit of detection and limit of quantitation were 0.002 and 0.01 µg/g for fluquinconazole and 0.01 and 0.05 for tetraconazole, respectively. The method was successfully applied to the analysis of soil residues collected from an onion field. The results show that a combination of SFE and GC can be used as an environmentally friendly technique to detect fungicides in soil.

Potential effects of individual and combined exposure to tetraconazole and cadmium on zebrafish from the perspective of enantioselectivity and intestinal microbiota.

As the wide use of pesticides, they could form combined pollution with heavy metals, which would affect their environmental behaviors and toxic effects. Particularly, the effects would be more intricate for chiral pesticides. In this study, the accumulation and dissipation trends of tetraconazole enantiomers in zebrafish were investigated by individual and combined exposure of cadmium (Cd) and tetraconazole (including racemate and enantiomers) after confirming the absolute configuration of tetraconazole enantiomer. For the enantiomer treatments, Cd enhanced the accumulation of S-(+)-tetraconazole, but declined the concentrations of R-(-)-tetraconazole in zebrafish. The dissipation half-lives of tetraconazole enantiomers were extended by 1.65-1.44 times after the combined exposure of Cd and enantiomers. The community richness and diversity of intestinal microbiota were reduced in all treatments, and there were significant differences in R + Cd treatment. There was synergistic effect between Cd and S-(+)-tetraconazole for the effects on the relative abundances of Fusobacteria, Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. For R-(-)-tetraconazole, Cd mainly exhibited antagonistic effects. In the combined exposure of Cd and S-(+)-tetraconazole, the relative abundance changes of Cetobacterium (Fusobacteria, increase) and Edwardsiella (Proteobacteria, decrease) might affect the carbohydrate metabolism and energy metabolism, and led to the increase of S-(+)-tetraconazole bioaccumulation concentration. In the combined exposure of Cd and R-(-)-tetraconazole, Cd could increase the relative abundance of Edwardsiella (Proteobacteria), and affect the amino acid metabolism, which might reduce the bioaccumulation concentration of R-(-)-tetraconazole. This study reported for the first time that the abundance of intestinal microbiota in zebrafish might affect the bioaccumulation and dissipation of tetraconazole enantiomers, and would provide new insight for the study of combined pollutions.

High contamination of a sentinel vertebrate species by azoles in vineyards: a study of common blackbirds (Turdus merula) in multiple habitats in western France.

Azoles represent the most used family of organic fungicides worldwide and they are used in agriculture to circumvent the detrimental impact of fungi on yields. Although it is known that these triazoles can contaminate the air, the soil, and the water, field data are currently and dramatically lacking to assess if, and to what extent, the use of triazoles could contaminate non-target wild vertebrate species, notably in agroecosystems. In this study, we aimed to document for the first time the degree of blood contamination of a generalist wild bird species by multiple azoles which are used for plant protection and fungi pest control in various habitats. We deployed passive air samplers and captured 118 Common blackbirds (Turdus merula) in an agroecosystem (vineyard), a protected forest, and a city in western France. We collected blood and analyzed the plasma levels of 13 triazoles and 2 imidazoles. We found that a significant percentage of blackbirds living in vineyards have extremely high plasma levels of multiple azoles (means (pg.g-1); tebuconazole: 149.23, difenoconazole: 44.27, fenbuconazole: 239.38, tetraconazole: 1194.16), while contamination was very limited in the blackbirds from the protected forest and absent in urban blackbirds. Interestingly, we also report that the contamination of blackbirds living in vineyard was especially high at the end of Spring and the beginning of Summer and this matches perfectly with the results from the passive air samplers (i.e., high levels of azoles in the air of vineyards during June and July). However, we did not find any correlation between the levels of plasma contamination by azoles and two simple integrative biomarkers of health (feather density and body condition) in this sentinel species. Future experimental studies are now needed to assess the potential sub-lethal effects of such levels of contamination on the physiology of non-target vertebrate species.

Review of the existing maximum residue levels for tetraconazole 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 tetraconazole. To assess the occurrence of tetraconazole residues in plants, processed commodities, rotational crops and livestock, EFSA considered the conclusions derived in the framework of Directive 91/414/EEC, as well as the import tolerances and 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, with the exception of the MRL proposal for kaki, all MRL proposals derived by EFSA still require further consideration by risk managers. Regarding triazole derivative metabolites (TDMs), separate indicative exposure assessments were performed and no risk to consumers was identified for what regards these metabolites individually. However, TDMs may be generated by several pesticides belonging to the group of triazole fungicides, and a comprehensive risk assessment has thus to be performed that covers all existing European uses for all pesticides belonging to the class of triazole fungicides. EFSA recommended to elaborate together with risk managers a strategy to ensure that the required data are made available to finalise the overall risk assessment for triazole fungicides.

Novel, Environment-Friendly Cellulose-Based Derivatives for Tetraconazole Removal from Aqueous Solution.

In this study, cellulose-based derivatives with heterocyclic moieties were synthesized by reacting cellulose with furan-2-carbonyl chloride (Cell-F) and pyridine-2,6-dicarbonyl dichloride (Cell-P). The derivatives were evaluated as adsorbents for the pesticide tetraconazole from aqueous solution. The prepared adsorbents were characterized by SEM, TGA, IR, and H1 NMR instruments. To maximize the adsorption efficiency of tetraconazole, the optimum conditions of contact time, pH, temperature, adsorbent dose, and initial concentration of adsorbate were determined. The highest removal percentage of tetraconazole from water was 98.51% and 95% using Cell-F and Cell-P, respectively. Underivatized nanocellulose was also evaluated as an adsorbent for tetraconazole for comparison purpose, and it showed a removal efficiency of about 91.73%. The best equilibrium adsorption isotherm model of each process was investigated based on the experimental and calculated R2 values of Freundlich and Langmuir models. The adsorption kinetics were also investigated using pseudo-first-order, pseudo-second-order, and intra-particle-diffusion adsorption kinetic models. The Van't Hoff plot was also studied for each adsorption to determine the changes in adsorption enthalpy (∆H), Gibbs free energy (∆G), and entropy (∆S). The obtained results showed that adsorption by Cell-F and Cell-P follow the Langmuir adsorption isotherm and the mechanism follows the pseudo-second-order kinetic adsorption model. The obtained negative values of the thermodynamic parameter ∆G (-4.693, -4.792, -5.549 kJ) for nanocellulose, Cell-F, and Cell-P, respectively, indicate a spontaneous adsorption process. Cell-F and Cell-P could be promising absorbents on a commercial scale for tetraconazole and other pesticides.

Enantioselective effects of the chiral fungicide tetraconazole in wheat: Fungicidal activity and degradation behavior.

Tetraconazole, a chiral triazole fungicide, is widely used for the prevention of plant disease in wheat fields. However, the chirality of pesticides like tetraconazole can cause diverse biological responses. Therefore, it is important that research is conducted to investigate the enantioselective effects of chiral enantiomers in this regard. The absolute configurations of two tetraconazole enantiomers were initially confirmed by ECD (Electrostatic circular dichroism). The bioassay test showed that the fungicidal activity of (R)-(+)-tetraconazole against two pathogens (R. cerealis and F. graminearum) was approximately 1.49-1.98 times greater than that for (S)-(-)- tetraconazole. Following recovery experiments, a modified QuEchERS (Quick, easy, cheap, effective, rugged, safe) method was established using UPLC-MS/MS (ultra-performance liquid chromatography tandem mass spectrometry). The mean recoveries from plant and soil sample ranged from 78.9% to 100.5% with intraday relative standard (RSDr) values of 0.8%-6.9% and interday relative standard (RSDR) values of 3.0%-5.2% respectively. The stereoselective degradation of tetraconazole in wheat meant that (S)-(-)-tetraconazole was more rapidly degraded than (R)-(+)-tetraconazole. Conversely, (R)-(+)-tetraconazole was preferentially degraded in wheat soil. These results will provide us with a greater understanding when assessing future environmental risk assessments and strategies that invoke pesticide reduction.

Impact of mepanipyrim and tetraconazole in Mencía wines on the biosynthesis of volatile compounds during the winemaking process.

The impact of fungicides mepanipyrim (Mep) and tetraconazole (Tetra) and their corresponding commercial formulations (Mep-Form and Tetra-Form) on the aroma composition of wines was assessed. Fungicide residues can affect the biotransformation of aroma precursors from grapes and/or the yeast metabolism. The results confirmed that both maximum residue levels (MRL and 2xMRL) of Mep promoted benzyl alcohol and 4-vinylguaiacol contents; while MRL and 2xMRL of Mep-Form promoted benzene derivatives (benzyl alcohol, benzaldehyde, and trans-isoeugenol), 2-phenylethanol and γ-nonalactone. The addition of Tetra (2xMRL) and Tetra-Form (MRL and 2xMRL) release higher contents of cis-3-hexen-1-ol and ethyl vanillate and affected yeast metabolism related to phenylacetaldehyde, 2-phenylethanol, methionol, capric acid, ethyl 2-methylbutyrate, ethyl isovalerate, ethyl monosuccionate, diethyl succinate and γ-butyrolactone production. Fungicide residues did not display higher variations in global odour activity values with respect to control wines, although some variations on the "floral", "fruity", "spicy" and "lactic" nuances could be sensed.

Modification of the existing maximum residue levels for tetraconazole in kaki/Japanese persimmon, linseeds and poppy seeds.

In accordance with Article 6 of Regulation (EC) No 396/2005, the applicant Isagro S.p.A submitted a request to the competent national authority in Italy to modify the existing maximum residue levels (MRLs) for the active substance tetraconazole in various crops and animal commodities. The data submitted in support of the request were found to be sufficient to derive MRL proposals for tetraconazole in kaki/Japanese persimmon, linseeds and poppy seeds. Adequate analytical methods for enforcement are available to control MRL compliance for parent compound tetraconazole on the commodities under consideration and in animal products. The proposed use of tetraconazole on crops under assessment will not result in a dietary exposure to residues of parent tetraconazole exceeding the toxicological reference values for tetraconazole. For triazole derivative metabolites (TDMs), only an indicative exposure assessment was performed considering only the crops under assessment; the results showed that the expected exposure to TDMs in the three assessed commodities is well below the toxicological reference values derived for the TDMs. The proposed use of tetraconazole on crops under assessment is therefore unlikely to pose a risk to consumers' health. TDMs may be generated by several pesticides belonging to the group of triazole fungicides, a comprehensive risk assessment has thus to be performed that covers all existing EU uses and import tolerances for all pesticides belonging to the class of triazole fungicides. EFSA recommended to elaborate together with risk managers a strategy to ensure that the required data are made available to finalise the overall risk assessment for triazole fungicides.

Enhanced Dissipation of Triazole and Multiclass Pesticide Residues on Grapes after Foliar Application of Grapevine-Associated Bacillus Species.

Disease management in vineyards with fungicides sometimes results in undesirable residue accumulations in grapes at harvest. Bioaugmentation of the grape fructosphere can be a useful approach for enhancing the degradation rate and reducing the residues to safe levels. This paper reports the in vitro and in vivo biodegradation of three triazole fungicides commonly used in Indian vineyards, by Bacillus strains, namely, DR-39, CS-126, TL-171, and TS-204, which were earlier found to enhance the dissipation rate of profenophos and carbendazim. The strains utilized the triazoles as carbon source and enhanced their in vitro rate of degradation. Myclobutanil, tetraconazole, and flusilazole were applied in separate vineyard plots at field doses of 0.40 g L(-1), 0.75 mL L(-1), and 0.125 mL L(-1), respectively. Residue analysis of field samples from the treated fields reflected 87.38 and >99% degradations of myclobutanil and tetraconazole, respectively, by the strain DR-39, and 90.82% degradation of flusilazole by the strain CS-126 after 15-20 days of treatment. In the respective controls, the corresponding percent degradations were 72.07, 58.88, and 54.28, respectively. These Bacillus strains could also simultaneously degrade the residues of profenofos, carbendazim, and tetraconazole on the grape berries and can be useful in multiclass pesticide residue biodegradation.

Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices.

Tetraconazole is currently used as a fungicide in fruit and vegetables. The aim of this work was the development of immunochemical techniques based on recombinant antibodies for the screening of tetraconazole residues in fruit juices. Recombinant antibodies were produced from a hybridoma cell line secreting a monoclonal antibody specific for tetraconazole and from lymphocytes of mice hyperimmunised with tetraconazole haptens conjugated to bovine serum albumin. From these antibodies, enzyme-linked immunosorbent assays in the conjugate-coated format were developed, which were able to detect tetraconazole standards down to 1ng/mL. From recovery studies with spiked samples, these immunoassays determined tetraconazole in orange and apple juices with acceptable reproducibility (coefficients of variation below 25%) and recoveries (ranging from 78% to 145%) for a screening technique. The analytical performance of RAb-based immunoassays was fairly similar to that of the MAb-based immunoassays. Due to their simplicity and high sample throughput, the developed recombinant-based immunoassays can be valuable analytical tools for the screening of tetraconazole residues in fruit juices at regulatory levels.