” Phytosanitary treatment of vines: residues “
I’m going to give you a summary of my thesis, the subject of which is the study of aromatic sulfur compounds in red wines, particularly compounds whose origin may be due to pesticide residues. First, I’ll give an overview of sulfur metabolism and the research already carried out by other authors. The largest quantities of sulfur compounds are found in amino acids, peptides, tripeptides (gutathione) and proteins. Glutathione is a reserve of thiol groups (SH groups), which are necessary in the biological redox system. Glutathione represents 40% of total cysteine. Glutathione increases in the grape berry from the onset of ripening, and rapidly disappears as soon as the must is released during grape crushing, reacting with quinones.
Sulfur is essential for yeast growth. Most yeasts take sulfur from sulfates, which are transported across the yeast membrane by transport systems. These reactions require energy. Sulfate uptake parallels yeast growth. Yeast growth is enhanced if we add methionine and glutathione to the medium. As for the sulfur-containing amino acids methionine and cysteine, their uptake by yeast is different. While methionine is 100% assimilated, being a direct competitor of sulfates as a source of sulfur in yeast, cysteine is more rarely used by yeast.
Two other points in the metabolism of souffres are the biosynthesis of cysteine and methionine, necessary for protein production. The nitrogen composition of the must, and in particular the level of easily-assimilable nitrogen (NH4+), can modify the yeast’s uptake of these compounds. Yeast require biotin and thiamine for fermentation. We can see that sulfur metabolism in yeast is linked to nitrogen metabolism. The processes involved in H2S formation during fermentation depend on these reactions.
Among sulfur compounds, we can differentiate between those that are highly volatile, which we call “light” sulfur compounds, and those that are less volatile, which we call “heavy” (boiling point above 90″ C). The methods of analysis are different, the light ones are analyzed with the “headspace” method, while for the “heavy” ones an extraction is required. The main light sulfur compounds are H2S, CS2, methane thiol, ethane thiol, and their corresponding disulfides. The main “heavy” sulfur compound is methionol. There are many references in the literature on light sulfur compounds, sometimes contradictory in terms of their formation and the levels found in wine. On the other hand, there is very little data on heavy sulfur compounds.
Recently, studies on these fairly important compounds have been carried out mainly on white wines, but there is little data on red wines. The search for and identification of new “heavy” sulfur compounds has led us to study certain sulfur pesticide residues. This study is complemented by an evaluation of the organoleptic impact of sulfur-based residues that may exist in wine. H2S is the main compound formed from treatment residues in wine. These reaction compounds are studied in wine, since they are as important as the wine itself.
For many years, it was assumed that sulfur-containing pesticide residues were the cause of reduction defects in wines. Some authors have shown that yeasts can use ethylene bisthiocarbamates to produce sulfur compounds such as H2S and CS2. The mechanism for degrading dithiocarbamates to ethylene diamine and CS2 or to ethylene thio-uramide, which is then converted to ethylene thio-urea and CS2, has been demonstrated. Thiram is a fungicide with antibotrytis action. Numerous studies have focused on the release of sulfur compounds from dithiocarbamates into wine.
These molecules can degrade into isothiocyanate and H2S, ethylene diamine and CS2, and ethylenethiourea and CS2. Another defect in wines is due to acephate residues (the active ingredient in orthene). Acephate is hydrolyzed to methane thiol and other metabolites. This reaction depends on the wine’s pH, time and storage temperature. Residues of methomyl in must, an insecticide widely used in viticulture, lead to the formation of methane thiol, ethane thiol, diethyl sulfide and diethyl disulfide. These compounds, once present in the wine, impart odors of cooking cauliflower and wet wool.
Sulfur, used in the fight against powdery mildew, is also transformed by the yeast into H2S. I’ll now show you the results of our own research. We’re going to look at the problem arising from the use of dithiocarbamates, in particular thiram, as well as sulfur. We’ll be looking at specific cases where late treatment can be the cause of defects in wines. We will also study the organoleptic impact of the substances formed. We have found a sulphur residue, a heavy sulphur compound, in some wines.
Research into the phytosanitary treatments used in the vineyards where they treat the grapes used to make wine shows that two products with active ingredients containing sulfur atoms had been used, product 1 (active ingredient thiram Iprodione) and product 2 (active ingredient zinc methyram). Product 2 is an anti-mildew and was used in early April, while product 1 is used to combat botrytis, and was last used on August 5, about a month and a half before the harvest date.
We have analyzed these two active ingredients using the method used for heavy sulfur compounds, and in neither of these products do we find the residue cited. Analysis of the headspace of these products reveals a lot of CS2. These same products are added to a wine. We analyzed the wine after three days. The residue is also absent in the case of both active ingredients. We performed the same experiment with must. We took two Erlenmeyer flasks, each containing 1 liter of must, to which we added the two products separately, and set the samples at 25°C for 5 days. In no case was any residue observed.
Finally, the musts were inoculated with the addition of these substances and dry yeast, and the alcoholic fermentation process began. Once alcoholic fermentation was complete, the wines were analyzed. In the case of the wine obtained after addition of product 1 (Thiram), the appearance of this residue was demonstrated. In the case of product 2 (Zinc methiram), this substance was absent. We have concentrated our research on the study of product 1, and yet thiram. We analysed this commercial product in greater depth. Analysis of the product using the headspace technique shows that, in addition to CS2, traces of H2S are present in this preparation.
Another case study: We analyzed the wines of two Bordeaux châteaux, from two different appellations, but owned by the same proprietor, who, according to his oenologists, presented certain organoleptic characteristics that, for them, gave the wines a lack of clarity. We studied the phytosanitary treatments used in recent years, in all cases using dithiocarbamates, thiram, methyram, zinc and others. We analyzed wines from 1989 to 1994. The sulfur compounds we analyzed were thiram residues and CS2, a compound recognized as being produced by dithiocarbamate residues.
We can observe relationships between residue levels in certain years. Residue levels are similar in 1994, 1992 and 1991, while in 1993 and 1990 the results are very different. Unfortunately, we do not know the exact dates of treatment or the doses of plant protection products used, except for the two châteaux in 1994. In the absence of precise data, we can imagine that high residue concentrations correspond to rather late treatments. For the 1994 vintage, we analyzed wines from a third château, which underwent the same treatments. In all cases, the amount of residue is similar, and quite high.
Treatments using thiram were as follows:
– Pure thiram: June 30
– Thiram + Iprodione: August 5.
It was probably the August 5th treatment that produced this residue. In all the châteaux, we find a fairly high level of residue. We can’t guarantee that a treatment carried out on that date will produce residue in the wine, because rain, for example, could wash it out. As far as CS2 is concerned, there are wines from certain vintages with fairly high concentrations, while others have zero. We have correlated residue and CS2 levels. We find no relationship between the amount of CS2 and the amount of residue. In fact, it seems easy to explain, since CS2 comes not only from thiram, but also from the other dithiocarbamates. It is also possible that fermentation temperature and other factors can vary the level of CS2 in wine, a highly volatile substance.
Another case of Beaujolais wines made from Gamay grapes. Several treatment products were used at different times. In addition to thiram in early August, another treatment product, methomil, was suspected of leaving residues in the wine used in early July. The wines are made using the typical Beaujolais technique of carbonic maceration. Samples are analyzed once malolactic fermentation is complete. We found that neither CS2 nor thiram residues were present in the control treatment trials, nor in those treated with Méthomyl. In wines whose grapes were treated with thiram, CS2 is still present in varying concentrations, as is the residue.
Another case from the Saint-Emilion region, involving three wines from different châteaux belonging to the same owner. The problem was that of these three wines, only one had been awarded the label, the others having been rejected by the tasting panel. In principle, the vines underwent the same phytosanitary treatments. Among the products used were two treatments with thiram on July 6 and August 11. We found that the wines that failed to obtain the label had high concentrations of CS2 and residues, far higher than the wine that had no defects (60 times in the case of residues). Distillation of a wine with this sulphur compound leads to degradation of the compound and formation of CS2.
It is quite possible that during bottle ageing, this molecule is degraded, resulting in the appearance of a sulphurous taste. We studied the organoleptic impact of CS2. First, we calculated the perception threshold in water. According to the tasters, the dominant aromas are sulfur, rubber and burnt, reduced for CS2. The perception threshold for CS2 in water, according to our tastings, is 20f-lg/L. We have also calculated the perception threshold of this substance in wine, with tasters recognizing it at concentrations of around 150 6 ~lg/L. However, at lower concentrations, of the order of 50 pg/L, the aromatic nuance of the wine changes. It seems to act as an aroma mask for certain fruity odors, or on the contrary, it can potentiate certain defects, such as musty odors or those of volatile phenols. The wine seems to lack clarity.
Among all the wines analyzed, a large number contained CS2 concentrations far in excess of the levels we obtained, which could give rise to wine defects. We tried to eliminate CS2 from the wine. The only treatment shown to be useful enough to get rid of it is bubbling. Laboratory tests to remove CS2 have been successfully carried out. Wine containing fairly high concentrations of CS2 was bubbled with nitrogen at 150 ml/min for 5 or 10 min. CS2 was observed to disappear. With 5 minutes bubbling, the concentration compared with the control is 50%; with 10 minutes we find barely 20%. On tasting, the wines subjected to bubbling are clearer from an olfactory point of view, with a more complex aroma.
This experiment shows that CS2 is quite volatile. During alcoholic fermentation, a large proportion escapes; however, fermentation conditions (temperature, pumping-over) determine the final CS2 level, independently of the level of pesticide residue. Sulfur is a phytosanitary product that has long been used to treat powdery mildew (Uncinula Necator). In Spain, powdery mildew is a widespread disease, and it’s quite common to have late attacks, close to harvest time, especially on certain grape varieties such as Tempranillo. To curb the disease, a sulfur treatment, generally in high doses, is applied at this stage. It can be used in powder form (sublimable or flower) or dissolved (wettable sulfur).
For a very long time, sulfur-based treatments were the main means of combating this disease. In the 80s, synthetic products were used. The mode of attack of these products was to inhibit the synthesis of powdery mildew sterols (DMI). After years of treatment, it was discovered that there were resistant strains of powdery mildew against these products. This led to fairly late attacks of the disease. As IBS had no effect, they reverted to the use of sulfur. Concentrations of sulfur residues found in the berries are still below 3pg/g of grapes, equivalent to 3.4 mg/L of sulfur in the must, for treatments of 13 Kg/Ha of sulfur in the vineyard.
The amount of residue does not vary if one or both sides of the vine row are treated. Residue levels decrease during the two weeks following treatment. If treatments are carried out before veraison, as they should be, the residues found range from 0.9 to 1.7 pg/L. H2S is formed in wine by sulfur reduction. Experiments with fermentations to which concentrations of between 0 and 3.4 mg/L sulfur were added to musts fermented with different yeast strains showed no relationship between residual sulfur and the amount of H2S formed. Depending on the yeast strain, H2S production from sulfur may vary. The dose at which we use this yeast can also vary the concentration found. Fermentation temperature and pumping-over with aeration in red winemaking obviously also vary the H2S level in the wine.
Poor treatment with sulfur, which causes residues in the grapes, is one of the causes of H2S production in wine. This phenomenon is exacerbated when the grapes are machine-harvested, as the sulfur in the grapes is washed away. The wines analyzed that showed an olfactory defect came from grapes treated three weeks before the harvest date. The dose used is of the order of 20 Kg/Ha. We did not analyze the musts, but they suggest that residue levels exceed 3.4 mg/L. The wines analyzed were machine-picked Cabernet Sauvignon. According to the tasters, the wines showed an olfactory defect of the sulphurous type. After analyzing the “light” sulfur compounds, we found that there was a high concentration of H2S, as well as small concentrations of methane thiol and ethane thiol. These two thiols are formed by the reaction of H2S with either ethanol or methanol, according to Professor Maujean. According to other authors, methane thiol can also be formed by reaction between H2S and ethanal, via a cyclic thioacetaldehyde intermediate. In no case do we find dimethyl disulfide or diethyl disulfide.
In these wines with reduction defects, H2S is the predominant compound, followed by small quantities of methane thiol and ethane thiol. We have carried out experiments to eliminate these odors. As H2S is a highly reactive compound, it’s important to get rid of it as soon as possible. Mercaptans are the most difficult to eliminate by aeration, as their boiling point is higher than H2S (methane thiol=6°C, ethane thiol=35°C and H2S -60°C). It is therefore important to get rid of H2S as soon as possible, before other sulfur compounds are formed.
There are several treatments available to remove these sulfur compounds:
– Filtration
– Aeration: on the one hand, sulfur compounds evaporate, on the other, H2S oxidizes to sulfur (TANNER (1969)): 2H2S+02=2H20+3S
– With a treatment containing S02: 2H2S+S02=2H20+3S.
In the wines analyzed, filtration was not sufficient to completely remove these compounds. With strong aeration, H2S disappeared almost entirely, and traces of methane thiol remained. Compounds with a thiol group have the property of precipitating with copper salts. This treatment is used in oenology to remove H2S odors. Copper sulfate was used at doses of 0.01 and 0.03 g/hl. H2S disappeared completely, even at the lowest dose, while methane thiol and ethane thiol decreased but did not disappear completely. The analysis was carried out immediately after treatment. The rate of reaction of methane thiol and ethane thiol is probably much slower than that of H2S.
If the reduction defect is very strong, we recommend combining these two treatments, aeration and treatment with copper sulfate. In conclusion, the best way to prevent these defects is to adopt a good strategy in the fight against powdery mildew. On the other hand, copper in anti-mildew treatments is a good palliative for the appearance of these false tastes. Unfortunately, in some regions, this treatment is rarely used, as the climate is such that the risk of mildew is almost nil. Other sulfur compounds are formed from H2S. H2S can react with carbon dioxide to produce carbonyl sulfide, a highly toxic and volatile compound (boiling point -50 OC). The reaction between H2S and ethanal produces 2,4,6-trimethyl-s-trithiane and ethane thiol.
As far as ethane thiol is concerned, it is never found in wines without reduction defects. In some wines we have analyzed with very high H2S levels, estimated at 20 ~lg/L, ethane thiol concentrations are always very low. Concentrations found in some reduced whites are higher than those found here for red wines. The chemical composition of white and red wines is very different (especially in terms of phenolic compounds), which could explain why we don’t find the same results. We found methane thiol in many wines, with or without a reduction defect. MAUJEAN (1984) demonstrates the formation of methane thiol and dimethyl disulfide from methionine in the presence of riboflavin in what he calls the “taste of light”. This compound is found in many of the wines we have analyzed.
We measured methane thiol in 42 wines from Bordeaux grands crus, as well as other wines of recognized quality. In a large number of these wines, we found methane thiol at maximum concentrations of 1.2 ~lg/L. On average, the value we found is quite low, but above the threshold for perception of this substance in synthetic solution. Some wines have no methane thiol, and none of them contain H2S or ethane thiol. At such concentrations, we believe that this substance can contribute to the somewhat reduced, closed aging bouquet of most old wines. Although the main subject of my thesis was the study of red wines, we were also interested in studying certain white wines from the Chardonnay grape variety, where we found aromatic notes reminiscent of the smell of methane thiol.
We analyzed 13 wines made from the Chardonnay grape variety, from Burgundy. These included Pouilly fumés, Chassagne Montrachet Grand Cru and other wines of recognized quality. Average concentrations exceeded 6 times the perception threshold for methane thiol in synthetic solution. In no case did these wines present an olfactory defect for the tasters, methane thiol contributing rather to a somewhat heavy, melon-like odor possessed by most Chardonnay wines. There is only one wine where the concentration found is relatively high. This wine is one of the most highly rated in the series, and is noted at tasting as the most typical Chardonnay. We don’t know whether methane thiol is a compound typical of this grape variety, or whether it’s a by-product of Burgundian winemaking. The precursor of methane thiol in these wines remains to be elucidated. Note also that neither H2S nor ethane thiol are found in these wines. Other compounds, such as trithiolannes and trithiannes, may appear in wine as a result of the reaction of H2S with ethanal, but only under very specific conditions.
CONCLUSION
First of all, it must be said that it is necessary to respect the processing times of the various products, especially if sulfur atoms are present in the molecule. We have shown that they can be degraded by yeast, forming other sulfur compounds that are themselves precursors of more sulfur compounds. Another important point, which I haven’t mentioned, is the dose and the way in which treatments are carried out, with regard to the final residues in the grapes.