Tetraconazole alters the methionine and ergosterol biosynthesis pathways in Saccharomyces yeasts promoting changes on volatile derived compounds.

The influence of antifungal tetraconazole residues (either as an active substance or as a commercial formulation product) on the fermentative activity of Saccharomyces cerevisiae yeast was evaluated in pasteurized Garnacha red must by using laboratory-scale fermentation assays. The presence of this fungicide promoted a slight decrease in glucose consumption. Volatile fermentative-derived compounds were evaluated in deep. Statistically significant changes were found in methionol (with a mean decrease of around 24%), fatty acids (with increments ranged from 23% to 66%), and ethyl esters (with increases ranged from 23% to 145%) contents when grape musts were enriched with the commercial formulation at both contamination levels assayed. Based on protein mass fingerprinting analysis, it was possible to relate these variations on volatiles content with changes in the activity of several enzymes (Met3p, Met14p, Adh2p, Hmg1p, Erg5p, Erg6p, Erg11p, and Erg20p) involved in the secondary metabolism of yeasts.

Mepanipyrim residues on pasteurized red must influence the volatile derived compounds from Saccharomyces cerevisiae metabolism.

The impact of mepanipyrim (Mep) and its corresponding commercial formulation (Mep Form) on Saccharomyces cerevisiae metabolites was assessed, separately, by using laboratory-scale wine fermentation assays on pasteurized red must. The presence of Mep did not alter the fermentation course. With regard to volatiles formed at the intracellular level by fermenting yeast cells, Mep residues affected mainly the acetate and ethyl ester biochemical pathways. In particular, the target acetates showed a notorious increment, >90%, in presence of commercial Mep Form at the higher dose assayed. The addition of Mep and Mep Form, at both tested levels, highly increased ethyl caprylate (between 42 and 63%) and ethyl caprate (between 36 and 60%) contents as the same as their respective fatty acid precursors. No important effects were observed on colour and non-volatile pyranoanthocyanins, probably due to the low anthocyanin content characteristic of pasteurized musts.

Effects of hydrochemistry variables on the half-life of mancozeb and on the hazard index associated to the sum of mancozeb and ethylenethiourea.

Mancozeb is a dithiocarbamate non-systemic agricultural fungicide with multi-site, protective action. It helps to control many fungal diseases in a wide range of field crops, fruits, nuts, vegetables, and ornamental plants. We have investigated the stability profiles of mancozeb in aqueous solutions to determine the effect of pH, temperature and light on the degradation process of mancozeb. In addition, the toxicological risk for humans associated with the joint intake of mancoze7b and its final degradation product, ethylenethiourea (ETU), was calculated and modelled as a function of the experimental conditions. Stability study results showed a very low stability profile of mancozeb in all the aqueous solutions with rapid degradation that varied with experimental conditions. The process followed first order kinetics. The study of the degradation kinetics showed a significant effect of pH*temperature interaction on the degradation process. The results also expressed that light has a greater impact on the stability of mancozeb and the formation of ETU. The current study concludes that mancozeb is unstable in aqueous solutions, particularly at an acid pH, in addition to presenting both severe light and lower temperature sensitivity. The toxicological risk associated with mancozeb degradation increases with time and temperature, being higher at basic pH and in absence of light.

A critical review on the applications of artificial neural networks in winemaking technology.

Since their development in 1943, artificial neural networks were extended into applications in many fields. Last twenty years have brought their introduction into winery, where they were applied following four basic purposes: authenticity assurance systems, electronic sensory devices, production optimization methods, and artificial vision in image treatment tools, with successful and promising results. This work reviews the most significant approaches for neural networks in winemaking technologies with the aim of producing a clear and useful review document.

Dissipation kinetics of pre-plant pesticides in greenhouse-devoted soils.

This work was conducted to study the distribution of methyl isothiocyanate (MITC) in greenhouse soils treated with the fumigant dazomet (DZ) from the formulated product "Basamid Granular(®)", but also of 1,3-dichloropropene (1,3-D) and chloropicrin (CP) from the fumigant "Agrocelhone NE(®)". In order to achieve this aim, several methods for the determination of fumigants residues in soils, but also pepper fruits were optimized and characterized. With independence of the soil depth, no residues of MITC, 1,3-D and CP above the detection limits were observed in soils covered with a polyethylene (PE) film (0.04 mm thick) after 27, 13 and 8 days of treatment, respectively. Liberation and dissipation curves of MITC in soil in presence of a PE film (0.04 mm) used to limit volatilization losses were also obtained. According to the results, the rate of decomposition of DZ into MITC have a half-life of 3.7 days in the surface horizon (5-10 cm) of the soil while in the subsurface horizon (15-20 cm), MITC formation rate is slightly slow (half-life of 3.2 days). With respect to the dissipation process, half-lives lower than 1 day were obtained for both depths (0.8 and 0.9 for the surface and the subsurface horizon, respectively). In the case of 1,3-D and CP in soil, the dissipation half-life of 1,3-D on soils was a bit higher than for CP (2 days vs. 1). In addition, the presence of residues of the fumigants on green pepper fruits grown on the treated soils was not detected as expected.

Thermodynamics of sodium dodecyl sulphate-salicylic acid based micellar systems and their potential use in fruits postharvest.

Micellar systems have excellent food applications due to their capability to solubilise a large range of hydrophilic and hydrophobic substances. In this work, the mixed micelle formation between the ionic surfactant sodium dodecyl sulphate (SDS) and the phenolic acid salicylic acid have been studied at several temperatures in aqueous solution. The critical micelle concentration and the micellization degree were determined by conductometric techniques and the experimental data used to calculate several useful thermodynamic parameters, like standard free energy, enthalpy and entropy of micelle formation. Salicylic acid helps the micellization of SDS, both by increasing the additive concentration at a constant temperature and by increasing temperature at a constant concentration of additive. The formation of micelles of SDS in the presence of salicylic acid was a thermodynamically spontaneous process, and is also entropically controlled. Salicylic acid plays the role of a stabilizer, and gives a pathway to control the three-dimensional water matrix structure. The driving force of the micellization process is provided by the hydrophobic interactions. The isostructural temperature was found to be 307.5 K for the mixed micellar system. This article explores the use of SDS-salicylic acid based micellar systems for their potential use in fruits postharvest.

Combined determination and confirmation of ethylenethiourea and propylenethiourea residues in fruits at low levels of detection.

In this work, a new method for the determination of ethylenethiourea (ETU) and propylenethiourea (PTU) in fruits and vegetables is presented. Different extraction and purification techniques, including matrix solid phase dispersion (MSPD) and solid-liquid extraction (SLE), followed by a clean-up step by solid phase extraction (SPE), were compared. The determination of ETU and PTU was performed by high performance liquid chromatography with diode array detection (HPLC/DAD) or by gas chromatography with mass spectrometry detection (GC/MS). The effect of several parameters on the extraction, separation and detection was studied. The proposed method based on solid-liquid extraction with acetonitrile, clean-up with Envicarb II/PSA cartridges and subsequent analysis by HPLC/DAD was characterised and applied to the analysis of fruits and vegetables from different countries. Analytes recoveries were between 71% and 94% with relative standard deviations (RSDs) ranging from 8% to 9.5%. Quantification limits obtained for ETU and PTU with the HPLC/DAD method were 7 and 16 µg kg⁻¹ in strawberries (fresh weight), respectively. For apples, they were 11 and 25 µg kg⁻¹, respectively.

Influence of the adjuvants in a commercial formulation of the fungicide "Switch" on the adsorption of their active ingredients: cyprodinil and fludioxonil, on soils devoted to vineyard.

The objective of this work was to assess the effect of adjuvants in the sorption in soils of the fungicides, cyprodinil and fludioxonil, usually applied together in a mixture commonly called 'Switch'. Water suspensions of a commercial formulation of Switch were used in phase partition experiments for a set of selected soils from vineyards. A clean-up procedure of the supernatant was developed for the phase separation in presence of the adjuvants prior to quantification of cyprodinil and fludioxonil. The maximum sorption on the solid phase (which includes soil and other solids from the commercial formulation of Switch) was 2000 mg kg(-1) for fludioxonil and 3000 mg kg(-1) for cyprodinil after incubation with 800 mg L(-1) of Switch. However, adsorption to soil particles were lower; fludioxonil concentrations adsorbed in soils range from 50 to 80 mg kg(-1) of soil and cyprodinil concentrations range from 120 to 260 mg kg(-1) of soil. Adjuvants increased the solubility of fludioxonil in pure water at 25 °C up to 5 times that of the pure substance (from 1.8 to 9 mg L(-1) in control samples), and show a strong influence on the adsorption in soil. Soil pH, effective cation exchange capacity and copper content due to past anti fungal copper-based sprays, have also influence on the adsorption of the active ingredients in presence of adjuvants.

A review on the fate of pesticides during the processes within the food-production Chain.

Only the intake of toxicologically-significant amounts can lead to adverse health effects even for a relatively toxic substance. In the case of residues in foods this is based on two major aspects--first, how to determine quantitatively the presence of a pollutant in individual foods and diets, including its fate during the processes within the food production chain; and second, how to determine the consumption patterns of the individual foods containing the relevant pollutants. The techniques used for the evaluation of the fate of pesticides during food processing have been critically reviewed in this paper to determine those areas where improvements are needed or desirable. Options for improvements are being suggested, including, for example, the development of a pan-European food composition database, activities to understand better effects of processing on individual food pesticides, and harmonization of food consumption survey methods with the option of a regular pan-European survey. The ultimate aim is to obtain appropriate estimations for the presence and quantity of a given chemical in a food and in the diet in general. Existing pragmatic approaches are a first crude step to model food pollutant intake. It is recommended to extend, refine, and validate this approach in the near future. This has to result in a cost-effective exposure-assessment system to be used for existing and potential categories of pollutants. This system of knowledge (with information on sensitivities, accuracy, etc.) will guide future data collection.

Sorption of penconazole applied as a commercial water-oil emulsion in soils devoted to vineyards.

The objective of this work was to assess the effect of surfactants and oils of a commercial formulation on the potential mobility of penconazole in agricultural soils that have been subjected to a high rate of application of agricultural chemicals. Soil-water partition tests on a commercial water-oil emulsion formulation of penconazole (WOEP) in 0.01 M CaCl(2) containing 35 mg L(-1) penconazole, incubated for 24 h, showed a maximum retention of approximately 250-300 mg penconazole kg(-1) soil. Approximately 70% of the total penconazole retained by the solid phase was sorbed on the soil (175-200 mg kg(-1)). The other 30% was retained by the adjuvants present in the commercial formulation. The formulation also influenced the water-soil partition, increasing the sorption in tests on batch studies using technical-grade penconazole (TGP). Soils with high total copper and organic matter had the greatest affinity for penconazole when added as WOEP. Additionally, adsorption of penconazole followed an S-type isotherm, whose behavior was consistent with the ability of the technical-grade penconazole to form aggregates. In the case of the WOEP, the S-type behavior could be attributed to the surfactant present in the formulation, which could be adsorbed onto soil as hemimicelles, which in turn may facilitate adsorption of penconazole.

Improved ultrasonic-based sample treatment for the screening of anabolic steroids by gas chromatography/mass spectrometry.

A rapid sample treatment procedure for the gas chromatography/mass spectrometry (GC/MS) determination of anabolic steroids in human urine has been developed. The new procedure makes use of ultrasonic energy to reduce reaction times and increase the overall sensitivity. The following variables affecting the performance of the ultrasonic treatment were optimised: (i) time, (ii) device, (iii) frequency, and (iv) temperature. It was found that, under an ultrasonic field, the hydrolysis of conjugated steroids with beta-glucuronidase from Escherichia coli K12 was possible with a treatment time of 10 min. The accuracy and precision of the ultrasonic method were found to be in agreement with those achieved with the conventional thermal conductivity procedure (Student's t-test; p = 0.05, n = 10). After the enzymatic hydrolysis, the derivatisation of the target compounds with trimethylsilyl (TMS) reagent, methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)/NH(4)I/dithioerythritol (DTE) (1000:2:4, v/w/w), was also accelerated using ultrasonic energy. In order to test the applicability of the use of ultrasonic energy in the acceleration of the derivatisation reaction with TMS, the classic method of thermal conductivity was applied for comparative purposes to a pool of 35 androgenic anabolic steroids (AAS) and/or their metabolites. The results demonstrated that after 3 min of sonication in a Sonoreactor device (50% amplitude), 19 of the 35 compounds studied showed similar reaction yield to those obtained with the classic procedure requiring 30 min (Student's t-test; p = 0.05, n = 5); 13 increased to higher silylation yields; and for the steroids 1-testosterone, danazol and etiocholanolone-D5, the same results were obtained using a sonication time of 5 min.The overall applicability of the ultrasonic-based sample treatment method is shown by the analysis of five urine samples. The results are similar to those achieved by the routine procedure. The new method is fast, robust, and allows high sample throughput.

Behaviour of metalaxyl as copper oxychloride-metalaxyl commercial formulation vs. technical grade-metalaxyl in vineyards-devoted soils.

The objective of this work is to asses the sorption of metalaxyl applied as a copper oxychloride (CO)-metalaxyl formulation, for a set of selected soils devoted to vineyards. The method involved batch incubation of soils suspended with a commercial copper oxychloride-metalaxyl-based fungicide in 0.01M CaCl(2). Afterwards, the metalaxyl concentration remaining in solution was determined by high-performance liquid chromatography (HPLC). The amount of dissolved metalaxyl in the fungicide suspension depends mainly on the soil pH, its potential acidity, and the cation exchange capacity. Of the approx. 20% metalaxyl retained by the solid colloids, the effect of organic matter colloids in soils (15-20 mg kg(-1)) had a poor contribution (six times lower) than the copper oxychloride colloids (40%, w/w) in the commercial fungicide formulation (100-130 mg kg(-1)). When comparing these retention data with the behaviour of metalaxyl used as a technical grade fungicide of about 100% purity (10-15 mg kg(-1) in solids), it is clear that the commercial formulation increases a 30% retention of metalaxyl by soil (15-20 mg kg(-1) in solids). The overall effect of the metalaxyl formulation plus soil show values of 10 times higher retention than technical grade-metalaxyl plus soil. Commercial formulation can decrease the mobility of soluble metalaxyl in agricultural soils with regard to the expected values obtained from batch studies using analytical grade-metalaxyl. Therefore, the effect of surfactants should be considered in the assessment of water contamination by the pesticides used in agriculture.

Comparative study of matrices for their use in the rapid screening of anabolic steroids by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

New data on sample preparation and matrix selection for the fast screening of androgenic anabolic steroids (AAS) by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) is presented. The rapid screening of 15 steroids included in the World Anti-Doping Agency (WADA) prohibited list using MALDI was evaluated. Nine organic and two inorganic matrices were assessed in order to determine the best matrix for steroid identification in terms of ionisation yield and interference by characteristic matrix ions. The best results were achieved for the organic matrices 2-(4-hydroxyphenylazo)benzoic acid (HABA) and trans-3-indoleacrylic acid (IAA). Good signals for all the steroids studied were obtained for concentrations as low as 0.010 and 0.050 microg/mL on the MALDI sample plate for the HABA and IAA matrices, respectively. For these two matrices, the sensitivity achieved by MALDI is comparable with the sensitivity achieved by gas chromatography/mass spectrometry (GC/MS), which is the conventional technique used for AAS detection. Furthermore, the accuracy and precision obtained with MALDI are very good, since an internal mass calibration is performed with the matrix ions. For the inorganic matrices, laser fluences higher than those used with organic matrices are required to obtain good MALDI signals. When inorganic matrices were used in combination with glycerol as a dispersing agent, an important reduction of the background noise was observed. Urine samples spiked with the study compounds were processed by solid-phase extraction (SPE) and the screening was consistently positive.

Ultrasonic-assisted matrix solid-phase dispersion as an improved methodology for the determination of pesticides in fruits.

An ultrasonic-assisted matrix solid-phase dispersion (UA-MSPD) method has been developed for extracting and cleaning-up 15 organophosphorus pesticides (OPPs) and 9 triazines in fruits. Using reversed phase octasilyl-derivatised silica (C8) as dispersant and ethyl acetate as extraction solvent, two sonication devices, an ultrasonic bath and a sonoreactor, were tested for speeding and increasing the efficiency of the MSPD process. A standard MSPD and a heating-assisted MSPD procedure were also done. Gas chromatography-mass spectrometry was used for analyte determination. 1-min sonication with the sonoreactor at 50% amplitude provided the best results, with reproducibilities below 15% for the pesticides studied. The general low detection limits (1-42microgkg(-1)) ensure proper determination at maximum allowed residue levels set in current legislations, except for dimethoate and disulfuton. The analytical performance of the method was evaluated for apples, pear and apricot, showing little or no matrix effect.

Ultrasonic-assisted enzymatic digestion (USAED) for total elemental determination and elemental speciation: a tutorial.

Due to its potential as sample treatment for Analytical Chemistry, the Ultrasonic-Assisted Enzymatic Digestion (USAED) for total elemental determination and elemental speciation is described under the most recent achievements published in literature, focusing on the variables that critically affect the performance of this relatively new sample treatment, such as the type of enzymes or the type of ultrasonic system used for the acceleration of the solid-liquid extraction process. Moreover, analytical chemists are aware of common errors produced in data interpretation concerning USAED. In addition, a guide for the rapid application of this methodology is also provided along with detailed explanations. Finally, future trends regarding USAED are also given and commented.

Ultrasonic energy as a new tool for fast isotopic 18O labeling of proteins for mass spectrometry-based techniques: preliminary results.

Preliminary results regarding fast isotopic labeling of proteins with (18)O in conjunction with matrix assisted laser desorption ionization time of flight mass spectrometry technique are presented. Similar (16)O/(18)O isotopic labeling ratios were found for the overnight procedure (12h) and the new fast ultrasonic one (30 min) for the BSA, ovalbumin and alpha-lactalbumin proteins. The procedure, however, failed to promote double (18)O isotopic labeling for the proteins, ovalbumin and alpha-lactalbumin. Two different sonication frequencies, 35 and 130 kHz, were studied at two different sonication times of 15 and 30 min, being best results obtained with the procedure at 130 kHz of sonication frequency and 30 min of sonication time. For comparative purposes the overnight isotopic (18)O labeling procedure was done. In addition, the new fast isotopic labeling procedure was also studied without ultrasonication, in a water bath at 60 degrees C.

Influence of the protein staining in the fast ultrasonic sample treatment for protein identification through peptide mass fingerprint and matrix-assisted laser desorption ionization time of flight mass spectrometry.

The influence of the protein staining used to visualize protein bands, after in-gel protein separation, for the correct identification of proteins by peptide mass fingerprint (PMF) after application of the ultrasonic in-gel protein protocol was studied. Coomassie brilliant blue and silver nitrate, both visible stains, and the fluorescent dyes Sypro Red and Sypro Orange were evaluated. Results obtained after comparison with the overnight in-gel protocol showed that good results, in terms of protein sequence coverage and number of peptides matched, can be obtained with anyone of the four stains studied. Two minutes of enzymatic digestion time was enough for proteins stained with coomassie blue, while 4 min was necessary when silver or Sypro stainings were employed in order to reach equivalent results to those obtained for the overnigh in-gel protein protocol. For the silver nitrate stain, the concentration of silver present in the staining solution must be 0.09% (w/v) to minimize background in the MALDI mass spectra.

Ultrasonic assisted protein enzymatic digestion for fast protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sonoreactor versus ultrasonic probe.

Two different ultrasonic energy sources, the sonoreactor and the ultrasonic probe, are compared for enzymatic digestion of proteins for protein identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) using the peptide mass fingerprint (PMF) procedure. Variables such as (i) trypsin/protein ratio; (ii) sonication time; (iii) ultrasound amplitude; and (iv) protein concentration are studied and compared. As a general rule, the trypsin/protein ratio and the minimum protein concentration successfully digested are similar with both ultrasonic energy sources. Results showed that the time needed to digest proteins was shorter with the ultrasonic probe, 60s versus 120s, for the same amplitude of sonication, 50%. However, lower standard deviations and cleaner MALDI-TOF-MS spectra were obtained with the sonoreactor. In addition, the sonoreactor device provided higher sample throughput (6 samples for the sonoreactor versus 1 sample for the ultrasonic probe) and easier sample handling for lower sample volumes (25 microl). Finally, a comparison of both methodologies for the specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the procedure.

Improving sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Three ultrasonic energy sources were studied to speed up the sample treatment for in-solution protein identification by peptide mass fingerprint using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein reduction, alkylation, and enzymatic digestion steps were done in 15 min. Nine proteins, including zinc resistance-associated protein precursor from Desulfovibrio desulfuricans strain G20 and split-soret cytochrome c from D. desulfuricans ATCC27774 were successfully identified with the new protocol.

Sonoreactor-based technology for fast high-throughput proteolytic digestion of proteins.

Fast (120 s) and high-throughput (more than six samples at once) in-gel trypsin digestion of proteins using sonoreactor technology has been achieved. Successful protein identification was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS. Specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the methodology. The new sample treatment is of easy implementation, saves time and money, and can be adapted to online procedures and robotic platforms.