Pesticide residues in grapes, wine, and their processing products

In this review the results obtained in the 1990s from research on the behavior of pesticide residues on grapes, from treatment to harvest, and their fate in drying, wine-making, and alcoholic beverage processing are reported. The fungicide residues on grapes (cyproconazole, hexaconazole, kresoxim-methyl, myclobutanil, penconazole, tetraconazole, and triadimenol), the application rates of which were of a few tens of grams per hectare, were very low after treatment and were not detectable at harvest. Pyrimethanil residues were constant up to harvest, whereas fluazinam, cyprodinil, mepanipyrim, azoxystrobin, and fludioxonil showed different disappearance rates (t(1/2) = 4.3, 12, 12.8, 15.2, and 24 days, respectively). The decay rate of the organophosphorus insecticides was very fast with t(1/2) ranging between 0.97 and 3.84 days. The drying process determined a fruit concentration of 4 times. Despite this, the residue levels of benalaxyl, phosalone, metalaxyl, and procymidone on sun-dried grapes equalled those on the fresh grape, whereas they were higher for iprodione (1.6 times) and lower for vinclozolin and dimethoate (one-third and one-fifth, respectively). In the oven-drying process, benalaxyl, metalaxyl, and vinclozolin showed the same residue value in the fresh and dried fruit, whereas iprodione and procymidone resides were lower in raisins than in the fresh fruit. The wine-making process begins with the pressing of grapes. From this moment onward, because the pesticide on the grape surface comes into contact with the must, it is in a biphasic system, made up of a liquid phase (the must) and a solid phase (cake and lees), and will be apportioned between the two phases. The new fungicides have shown no effect on alcoholic or malolactic fermentation. In some cases the presence of pesticides has also stimulated the yeasts, especially Kloeckera apiculata, to produce more alcohol. After fermentation, pesticide residues in wine were always smaller than those on the grapes and in the must, except for those pesticides that did not have a preferential partition between liquid and solid phase (azoxystrobin, dimethoate, and pyrimethanil) and were present in wine at the same concentration as on the grapes. In some cases (mepanipyrim, fluazinam, and chlorpyrifos) no detectable residues were found in the wines at the end of fermentation. From a comparison of residues in wine obtained by vinification with and without skins, it can be seen that their values were generally not different. Among the clarifying substances commonly used in wine (bentonite, charcoal, gelatin, polyvinylpolypyrrolidone, potassium caseinate, and colloidal silicon dioxide), charcoal allowed the complete elimination of most pesticides, especially at low levels, whereas the other clarifying substances were ineffective. Wine and its byproducts (cake and lees) are used in the industry to produce alcohol and alcoholic beverages. Fenthion, quinalphos, and vinclozolin pass into the distillate from the lees only if present at very high concentrations, but with a very low transfer percantage (2, 1, and 0.1%, respectively). No residue passed from the cake into the distillate, whereas fenthion and vinclozolin pass from the wine, but only at low transfer percentages (13 and 5%, respectively).     

Evolution of chlorpyrifos, fenarimol, metalaxyl, penconazole, and vinclozolin in red wines elaborated by carbonic maceration of Monastrell grapes

The influence of the different steps involved in the wine-making process on the disappearance of chlorpyrifos, fenarimol, metalaxyl, penconazole, and vinclozolin in red wines elaborated by carbonic maceration of Monastrell grapes was studied. The initial levels of the residues in grapes ranged from 0.28 mg/kg (penconazole) to 1 mg/kg (chlorpyrifos). Ten days after the beginning of maceration, the compound that had decreased least was chlorpyrifos (83% of initial value remaining), whereas metalaxyl, the least persistent of the residues, had decreased to 49% of its initial value. In the free-run juice, on the other hand, the highest percentage remaining corresponded to metalaxyl (10%) and the lowest to chlorpyrifos (0. 1%). After pressing, the percentages of initial values eliminated in pomace varied from 82.7% for chlorpyrifos to 17.7% for metalaxyl, whereas in the press juice the opposite was the case (37% metalaxyl and 2% chlorpyrifos remaining). In finished wine, there were residues of all the pesticides, with the exception of chlorpyrifos, metalaxyl (21%) being the most persistent. The percentages eliminated in the lees varied from 1.5 to 2.5% of the initial value.     

Residues of spiroxamine in grapes following field application and their fate from vine to wine

Dissipation of the fungicide spiroxamine in grapes of two vine varieties, Roditis and Cabernet Sauvignon, exposed to field treatments was evaluated. Vines of a grape vineyard located in central Greece were sprayed once or twice with a commercial formulation of the fungicide at 30 g a.i./hL. Residues in grapes, must, and wine were determined by gas chromatography/IT-MS after extraction with cyclohexane-dichloromethane (9:1), with a limit of quantitation 0.02 mg/kg in grapes and 0.012 mg/kg in wine. Under field conditions, spiroxamine dissipation on grapes was faster during the first 2 weeks and then slower to the sixth week. About 7 days after application, half of the initial spiroxamine concentration remained on the grapes; the respective proportion at 42 days was about 10%. At 14 and 35 days, residues were lower than 0.44 and 0.22 mg/kg, respectively, values below the maximum residue levels set by the European Union (1 mg/kg). Spiroxamine residues transferred from grapes into the must and through the vinification process into the wine were also studied. Mean transfer factors of 0.26 and 0.55 were found from grapes into wine for the wines obtained without maceration and with maceration, respectively. Residues in wine, prepared from grapes with a spiroxamine content of 0.11-0.20 mg/kg, varied from <0.026 to 0.09 mg/kg. Spiroxamine diastereomer B was found to dissipate slower than diastereomer A in the field as well as during the vinification process.     

Fate of ochratoxin A during vinification of Semillon and Shiraz grapes

Semillon and Shiraz grapes containing ochratoxin A (OA) were obtained by inoculation of bunches on the vine with Aspergillus carbonarius. Citric acid content was greater in the inoculated grapes than in healthy grapes. Samples were collected throughout vinification of these grapes and the OA content was quantified using a stable isotope dilution liquid chromatographic-tandem mass spectrometric method. The mass of processed and waste streams during vinification was also noted. Reduction in the amount of OA in juice and wine occurred at every solid-liquid separation stage. The OA concentration (microg/kg) in white and red wine after racking was 4% and 9%, respectively, of that in crushed grapes. This corresponds to 1% and 6% of the total OA content that was initially present in the inoculated grapes. The OA content was divided between solid and liquid phases at each stage of vinification. OA did not appear to be transformed either chemically or biologically by yeast during fermentation, rather was discarded with the marc, juice lees, and gross lees.     

Fate of iprovalicarb, indoxacarb, and boscalid residues in grapes and wine by GC-ITMS analysis

The behavior in field and the transfer from grapes to wine during winemaking of iprovalicarb, indoxacarb, and boscalid was studied. The residue levels found in grapes were far below the MRLs set for grapes in EU, accounting at harvest time 0.81, 0.43, and 4.23 mg/kg for iprovalicarb, indoxacarb, and boscalid, respectively. The residue levels in the samples treated with boscalid may have residual problems (due to an accumulation effect) if repeated field treatments will be performed. Winemaking experiments showed a complete transfer of all pesticide from grapes to the must, while in wine the residues were low or negligible due to the adsorbing effect of lees and pomace. The clarification experiments showed a good removal of pesticide residues from the wine media, for all pesticides. The GC-ITMS method showed good performance with adequate recoveries ranging from 75 to 115%, and good method limits of quantitation (LOQs) and of determination (LODs) far below MRLs.     

Fungicide dissipation curves in winemaking processes with and without maceration step

The evolution of residual levels of four fungicides (cyprodinil, fludioxonil, pyrimethanil, and quinoxyfen) during the elaboration of three types of wine with maceration (traditional red wine, carbonic maceration red wine, and red wine of long maceration and prefermentation at low temperature) and two types of wine without maceration (rose and white) has been studied. The disappearance curves of each fungicide have been analyzed during the period of each winemaking process (21 days) and during the different enological steps involved in the elaborations. The residual levels of fludioxonil reduce most quickly during the winemaking processes without maceration, whereas the decrease in levels of pyrimethanil was the slowest in practically all cases (with and without maceration). During carbonic maceration winemaking, the decay constant of cyprodinil was greater than that of the other pesticides in all assays (time and steps).     

Effects of clarification and filtration processes on the removal of fungicide residues in red wines (var. Monastrell)

The effects of six clarification agents (egg albumin, blood albumin, bentonite plus gelatin, charcoal, PVPP, and silica gel) on the removal of residues of four fungicides (cyprodinil, fludioxonil, pyrimethanil, and quinoxyfen) applied directly to a racked red wine, elaborated from Monastrell variety grapes from the D.O. region of Jumilla (Murcia, Spain), are studied. The clarified wines were filtered with 0.45 microm nylon filters to determine the influence of this winemaking process in the disappearance of fungicide residues. Analytical determination of cyprodinil, fludioxonil and pyrimethanil was performed by gas chromatography with an alkaline thermoionic detector (NPD), whereas that of quinoxyfen using an electron captor detector (ECD). In general, and for all of the fungicides except quinoxyfen, blood albumin has proved to be the most effective clarifying agent in the removal of residues, whereas silica gel proved to be ineffective against all of the pesticides with the exception of fludioxonil. Quinoxyfen is the least persistent fungicide in the clarified wines and that which appears with highest frequency in the lees. In general, filtration is not an effective step in the elimination of wine residues. The greatest elimination after filtration is obtained in wines clarified with charcoal and the lowest in those clarified with PVPP.     

Pear distillates from pear juice concentrate: effect of lees in the aromatic composition

Pear juice obtained from pear concentrate was fermented at room temperature using Saccharomyces cerevisiae (BDX, ENOFERM, France) as the fermentation microorganism. During the fermentation process, total sugars were measured. High performance liquid chromatography analyses were used to monitor the fermentation process and to characterize the pear wine. The pear wine obtained was distilled with its lees using three different equipments: a glass alembic (a glass pot still coupled to a glass column), a copper alembic, and a glass alembic with the addition of 5 g/L of copper shavings to the pot still. The same distillations were repeated with the wine without its lees (separated by decanting). Several distillation fractions were collected, up to a total of 500 mL of distillate. Gas chromatography was used to identify and quantify the volatile compounds in each fraction, and the methanol and ethanol contents. Based on these results, the heart fraction was defined. ANOVA tests were performed on the heart fractions to determine quantitative differences between some volatile compounds depending on the equipment used and the presence or absence of the wine lees. From this series of ANOVA tests, it can be concluded that the concentrations of the compounds that are considered to have a negative effect on the quality of the distillates (methanol, ethyl acetate, furfural) decrease or do not change when they are distilled in the presence of lees and in the copper alembic. In addition, the concentrations of the positive compounds (ethyl decanoate and ethyl-2-trans-4-cis-decadienoate) increase in the presence of lees for all of the equipment tested. So, it can be assumed that the distillation of pear wine with its lees in copper alembic leads to a better quality product.     

Determination of ethylenethiourea in grapes and wine

Residues of ethylenethiourea (ETU) in grapes and wine were determined by capillary gas chromatography and paper chromatography, without a cleanup step, and after derivatization to S-benzyl-ETU. The detection limit was 0.0002 mg/kg for flame ionization detection, 0.008 mg/kg for paper chromatography with photodensitometric evaluation of the detected spot. Results were compared with a generally used GC method specifying electron capture detection of trifluoroacetylated S-benzyl-ETU. The recoveries of ETU in grapes and wine at different concentration levels were determined. ETU residues were determined in treated grapes but no residues were detected in wine.     

Pulsed electric field-assisted vinification of aglianico and piedirosso grapes

Pulsed electric field (PEF) treatments were applied to increase the polyphenolic content of fresh red wines made from Aglianico and Piedirosso grapes. Prior to the fermentation/maceration step, the grape skins were treated at different PEF intensities (field strengths from 0.5 to 1.5 kV/cm and energy inputs from 1 to 50 kJ/kg), with their permeabilization being characterized by electrical impedance measurements. Furthermore, the release kinetics of the total polyphenols and anthocyanins were characterized during the maceration stage by spectroscopic and Folin-Ciocalteu colorimetric methods, respectively. Finally, the fresh wine, obtained after pressing, was characterized for total acidity, pH, reducing sugar, color intensity, total polyphenols, anthocyanins content, antioxidant activity, and volatile compound composition. PEF treatment on Aglianico grapes induced a significantly higher release of polyphenols (+20%) and anthocyanins (+75%), thus improving the color intensity (+20%) and the antioxidant activity of the wine (+20%) while preserving the other organoleptic characteristics. In contrast, there was only a minor impact on the polyphenolic release kinetics of Piedirosso grapes, despite the significant degree of cell membrane permeabilization.     

Optimizing the process of making sweet wines to minimize the content of ochratoxin A

During the drying process of raisins, the grapes are subjected to climatic variations that can result in heavy infections of some fungal species that produce ochratoxin A (OTA), a powerful toxic metabolite, whose maximum permitted content is set by the European Union at 2.0 μg/L for grapes, wine and other drinks derived from the grape. The aim of this paper is to optimize the process of making sweet wines in order to minimize the content of ochratoxin A. The results reflect a reduction of the OTA content in grapes dried under controlled conditions in a climatic chamber up to 24% compared to those sunned in the traditional way. A decrease of the concentrations of OTA is also observed during the processes of vinification. Those wines with prefermentative maceration reached a higher OTA content than the wines without maceration, but unexpectedly were not those preferred from a sensorial point of view. In addition, the process of aging in oak casks has been shown to serve as a natural method for reducing the OTA content of these wines.     

Determination of 24 pesticide residues in fortified wines by solid-phase microextraction and gas chromatography-tandem mass spectrometry

The present work describes a solid-phase microextraction (SPME) gas chromatography-tandem mass spectrometry (MS/MS) method to quantify 24 pesticides in fortified white wine and fortified red wine. In this study "fortified wine" refers to a wine in which fermentation is arrested before completion by alcohol distillate addition, allowing sugar and alcoholic contents to be higher (around 80-100 g/L total sugars and 19-22% alcohol strength (v/v)). The analytical method showed good linearity, presenting correlation coefficients (R(2)) ≥ 0.989 for all compounds. Limits of detection (LOD) and quantitation (LOQ) in the ranges of 0.05-72.35 and 0.16-219.23 μg/L, respectively, were obtained. LOQs are below the maximum residue levels (MRL) set by European Regulation for grapes. The proposed method was applied to 17 commercial fortified wines. The analyzed pesticides were not detected in the wines tested.     

Effect of different winemaking technologies on phenolic composition in Tinta Miúda red wines.

The influence of different types of winemaking technology on the contents of catechins, proanthocyanidins, and anthocyanins in Tinta Miúda red wines was studied. The Tinta Miúda red wines were made by fermentation with carbonic maceration, fermentation with stem contact, and fermentation without stem contact, respectively. The analysis of individual catechins, procyanidins, and anthocyanins in these wines was performed by HPLC, and quantification of total catechins, total oligomeric proanthocyanidins, total polymeric proanthocyanidins, and total anthocyanins was carried out by spectrophotometric methods. The wine made by carbonic maceration contained the highest amounts of both catechins and oligomeric and polymeric proanthocyanidins, followed by the wine made by fermentation with stem contact, whereas the wine made by fermentation without stem contact contained the lowest of these compounds. On the other hand, the concentrations of total anthocyanins and nearly all individual anthocyanins in the carbonic maceration wine were lower than those in the wines made by fermentation with stem contact and fermentation without stem contact. These results indicated that, although the carbonic maceration technique could retain higher amounts of catechins and proanthocyanidins in wine, it did not favor retaining or stabilizing anthocyanins in wine.     

Levels of stilbene oligomers and astilbin in French varietal wines and in grapes during noble rot development

Phenolics from grapes and wines can play a role against oxidation and development of atherosclerosis. Stilbenes have been shown to have cancer chemopreventive activity and to protect lipoproteins from oxidative damage. A method for the direct determination of stilbene oligomers (viniferin and pallidol) as well as astilbin in different types of wine using high-performance liquid chromatography with UV detection is described. In a survey of 21 commercial wines from the south of France, levels of pallidol and viniferin are reported for the first time in different types of wines. Viniferin was found to be present only in red and botrytized sweet white wines with levels between 0.1 and 1.63 mg/L; pallidol was not found in dry and sweet white wines but only in wines made by maceration with stems, with levels between 0.38 and 2.22 mg/L. Highest levels of astilbin were found in Egiodola (15.13 mg/L), Merlot (11.61 mg/L), and Cabernet Sauvignon (8.24 mg/L) for red wines and in Sauvignon (5.04 mg/L) for white varietal wines. Astilbin levels are highest for recent vintages, but pallidol is not found in older vintages. During noble rot development in Sauvignon or Sémillon grapes from the Sauternes area, levels of trans-astringin, trans-resveratrol, trans-piceid, and pallidol are quite low (<0.5 mg/kg of grapes). Viniferin and astilbin levels become optimum at 2 and 30 mg/kg, respectively, during spot grape and speckle grape stages.     

Effect of oak extract application to Verdejo grapevines on grape and wine aroma

Volatile compounds from a commercial aqueous oak extract application to white Verdejo grapevines at veraison have been studied. Treated grapes under two types of formulation (25% and 100%) have been analyzed at the optimum maturation time, and winemaking was then subsequently carried out. The volatile compounds were analyzed by stir bar sorptive extraction-gas chromatography-mass spectrometry. The results suggest that after the grapevine treatments, grapes store the volatiles in the form of nonvolatile precursors, and some of the volatiles are released during the winemaking process, especially six months after the alcoholic fermentation. The sensory analysis shows that wines maintain the typical aroma properties of Verdejo wines at the end of fermentation; but after six months, the wine color is greener and more astringent, and, in terms of aroma, it has wooden notes as if the wine has been aged in oak barrels.     

Postharvest UV-C-irradiated grapes as a potential source for producing stilbene-enriched red wines

The use of postharvest controlled irradiation by UV pulses is proposed as a potential method to produce stilbene-enriched red wine. "Monastrell" grapes were UV-C-irradiated to increase stilbene content. The main inducible stilbenes were resveratrol and piceatannol, which are molecules with reported health-beneficial activities. The evolution of both compounds was followed in the different steps of an "analytical" traditional maceration wine-making process. The final wine made from UV-C-irradiated grapes was enriched about 2- and 1.5-fold in resveratrol and piceatannol, respectively, when compared to the control wine. In addition, no difference was detected regarding the standard enological parameters (color, acidity, etc.). It is strongly suggested that, with the use of more susceptible wine grapes to induce bioactive stilbenes upon UV-C irradiation, the stilbene-enrichment of wine can be much higher.     

GC-ITMS determination and degradation of captan during winemaking

Captan and its metabolite tetrahydrophthalimide (THPI) were determined in grapes, must, and wine by GC-ITMS. Pesticides were extracted with acetone/petroleum ether (50:50 v/v). Because of the high selectivity of the ITMS detector, no interferent was found and cleanup was not necessary. Recoveries from fortified grapes, must, and wines ranged between 90 and 113% with a maximum coefficient of variation of 11%. Limits of quantitation were 0.01 mg/kg for both compounds. In model systems, captan and its metabolites, THPI, cis-4-cyclohexene-1,2-dicarboxylic acid, and 1,2,3,6-tetrahydrophthalamic acid, were determined by HPLC. The degradation of captan during winemaking was studied. Captan degraded in must, giving 100% THPI, and at the end of fermentation, only THPI was found in wine. The degradation of captan to THPI was due to the acidity in must and wine. This metabolite was present at low levels on grapes, and, unlike captan, it had no negative effect on the fermentative process. Model systems showed that the mechanism of disappearance of captan in grapes was due to photodegradation and codistillation.     

Evolution of phenolic composition of red wine during vinification and storage and its contribution to wine sensory properties and antioxidant activity

The objective of this work was to study the evolution of the phenolic composition of red wine during vinification and storage and its relationship with some sensory properties (astringency and bitterness) and antioxidant activities. Thus, red wine was made by a classic vinification method with Castela?o and Tinta Miu?da grapes (Vitis vinifera L.) harvested at maturity (3:2; w/w). Samples were taken at 2 and 7 days of maceration, at second racking, at the time of bottling and at 6 and 14 months after bottling. The total polyphenols extract (TPx) in each sample was isolated by column chromatography. The phenolic composition (anthocyanins and proanthocyanidins), in vitro antioxidant activity, and sensory property (astringency, bitterness) of the isolated TPx from different winemaking stages were evaluated through high-performance liquid chromatography-diode array detection, 1,1-diphenyl-2-picrylhidrazyl radical test, ferric reducing antioxidant power assay, total phenolic index, MWI (polyphenol molecular weight index), TSA (tannin specific activity), and sensory panel tasting. The results showed that the phenolic composition of red wine varied significantly during winemaking. The intensity of astringency (IA) and the intensity bitterness (IB) of the isolated TPx from different winemaking stages increased from 2 days of maceration until second racking and then decreased. Furthermore, MWI and TSA are positively correlated with IA and IB. The in vitro antioxidant activity of the isolated TPx from different winemaking stages maintained unchanged after alcoholic fermentation, which was independent of the variation of phenolic composition and sensory properties.     

Relationship of changes in rotundone content during grape ripening and winemaking to manipulation of the 'peppery' character of wine

Biosynthesis of the sesquiterpene rotundone in Vespolina grapes during berry ripening was investigated over two consecutive seasons, revealing that the compound accumulates from veraison to harvest and reaches relatively high concentrations (up to 5.44 μg/kg). Rotundone levels up to 1.91 μg/kg were also found in clones of Gruener Veltliner, a white grape variety known to give 'peppery' wines. These concentrations are higher than those reported for Syrah grapes and are similar to the levels found in some plants. Rotundone was shown to accumulate almost exclusively in berry exocarp, suggesting that skin contact during winemaking could be used to modulate the peppery character of red wine. However, rotundone yield after the winemaking process was relatively low. Indeed, only 10% of the rotundone present in grapes was extracted during fermentation, and only 6% was recovered in bottled wine. The results presented in this work provide key knowledge for manipulation of the peppery character of wine in order to optimize the intensity of this characteristic wine aroma.     

Interactions between yeast lees and wine polyphenols during simulation of wine aging. II. Analysis of desorbed polyphenol compounds from yeast lees

In the first part of this work, the analysis of the polyphenolic compounds remaining in the wine after different contact times with yeast lees during simulation of red wine aging was undertaken. To achieve a more precise view of the wine polyphenols adsorbed on lees during red wine aging and to establish a clear balance between adsorbed and remnant polyphenol compounds, the specific analysis of the chemical composition of the adsorbed polyphenolic compounds (condensed tannins and anthocyanins) after their partial desorbtion from yeast lees by denaturation treatments was realized in the second part of the study. The total recovery of polyphenol compounds from yeast lees was not complete, since a rather important part of the initial wine colored polyphenols, especially those with a dominant blue color component, remained strongly adsorbed on yeast lees, as monitored by color tristimulus and reflectance spectra measurements. All anthocyanins were recovered at a rather high percentage (about 62%), and it was demonstrated that they were not adsorbed in relation with their sole polarity. Very few monomeric phenolic compounds were extracted from yeast lees. With the use of drastic denaturing treatments, the total recovery of condensed tannins reached 83%. Such tannins extracted from yeast lees exhibited very high polymeric size and a rather high percentage of galloylated residues by comparison with initial wine tannins, indicating that nonpolar tannins were preferentially desorbed from yeast lees by the extraction treatments.