RECOGNIZING THE CHARACTERISTICS EFFECTS OF SULFUR DIOXIDE (SO2)

AND

TOXICOLOGICAL

Dr. Vincenzo Longo, Director of the Pisa Section of the National Research Centers Biology and Agricultural Biotechnology Institute

WHAT IS SULFUR DIOXIDE? SO2 is a heavy gas that can be obtained by the combustion of sulfur in air according to the equation: S + O2 ---> SO2. It is a colorless gas with an irritant odor, which is non-inflammable, toxic, should not be breathed in and is very soluble in water and in numerous organic compounds. Given that it is heavier than air it tends to stratify in the lower zones. It represents the atmospheric pollutant par excellence, being the most widespread, one of the most aggressive and dangerous and by far the one that is most studied and also emitted in the largest quantity by anthropogenic sources. Its olfactory threshold is 0.3 ppm (780 mg/m3). From the oxidation of sulfur dioxide (SO2) comes sulfur trioxide (SO3) which, when it reacts with water, both in the liquid and vaporous states, turns rapidly into sulfuric acid, responsible in large part for the phenomenon of acid rain that damages forests and buildings. SO2 gas also damages paint and metals. SOURCES OF POLLUTION: THEIR SPREAD AND LEGISLATIVE LIMITS Natural emissions of sulfur dioxide are principally the result of volcanic activity. Anthropogenic emissions represent more than 150 million metric tons a year and are mainly due to processes of combustion of fossil and liquid fuels such as coal, petroleum and diesel oil. Also relevant are the emissions in the production processes of sulfuric acid, in the manufacture of many plastic materials, in the desulfurization of natural gas, in the roasting of pyrites and in the incineration of refuse. On the other hand, the contribution to pollution created by emissions from various means of transport is negligible. The basic concentration was valued at around 0.2-0.5 icrograms per cubic meter, while in urban areas it can reach 50 icrograms per cubic meter. Sulfur dioxide remains in the atmosphere for 1-4 days, undergoing chemical reactions such as oxidation to become sulfuric acid that then falls in the form of acid fog or rain. At night sulfur oxides are also absorbed by the droplets of water present in the air creating an aerosol effect that gives a morning mist. Because of the great reactivity of sulfur dioxide, its concentration in indoor spaces is generally very low (at least half of that in outdoor ones). In addition, in the
RECOGNIZING THE CHARACTERISTICS AND TOXICOLOGICAL EFFECTS OF SULFUR DIOXIDE (SO2)
Dr. Vincent Longo, Responsible of the Section in Pisa of the Institute of Biology and Agrarian Biotechnology, CNR

winter months, when the concentration levels outside tend to increase because of the greater use of domestic heating, houses remain closed because of the cold and therefore indoor concentrations are lower. In recent years, due to the steps that have been taken to enhance the quality of fuels and because of the use of methane gas in heating systems, the emissions of sulfur oxides in the urban areas of countries in the Western world have been significantly reduced. Based on Italian Government Decrees. no. 351 of 4.8.99 and no. 60 of 2.4.2002, the level of sulfur dioxide that should not be surpassed more than 24 times in a calendar year is 350 g/m3 as an hourly average, while the daily average must not go over the value of 125 g/m3 more than three times a year. The regulations also impose a limit, which is already in force, for the protection of vegetation. This limit is 20 g/m3 as an annual and winter average.

SULFUR DIOXIDE IN FOODSTUFFS AND PARTICULARLY IN WINE Sulfur dioxide is used, in spite of its high level of toxicity, as an additive in all areas of the food sector, and in particular in enology. One finds it for example in: dried salt cod, shrimp and preserves, fresh or frozen shellfish, nuts, products preserved in vinegar and in oil, jams and jellies, vinegar, wines and drinks based on fruit juice. Sulfur dioxide has several properties that amply justify its use in the production of wine: Antiseptic: the two principal antiseptic properties are the action of selecting micro-flora in musts and antimicrobial action during wine storage. Antioxidant: in the presence of catalysts it combines with dissolved oxygen. This process develops according to a slow chemical reaction: SO2 + O2 ---> SO3. This reaction protects wines from oxidation of a chemical nature, such as oxidation of some of the polyphenols and of some of the aromatic substances; Antioxidasic: it inhibits the effect (and sometimes it determines their destruction) of oxidasic enzymes in the must. It thus protects the must from pre-fermentation oxidation; Solubilizing: sulfur dioxide in contact with the grape skins favors the spread of the not very substantially polymerized coloring substances contained in the vacuoles, by means of small holes in the cell walls, thus favoring the release of the anthocyanins; Combining: a correct use of SO2 improves the olfactory and gustatory qualities of wines, in that this preservative combines with some pungent smell and flavor components, such as acetaldehyde and pyruvic acid, rendering them undetectable by the taster; Clarifying: SO2 also has a slight clarifying action, in that it favors the coagulation of colloidal substances, thus increasing the phenomenon of spontaneous precipitation of the lees.

RECOGNIZING THE CHARACTERISTICS AND TOXICOLOGICAL EFFECTS OF SULFUR DIOXIDE (SO2)

Dr. Vincent Longo, Responsible of the Section in Pisa of the Institute of Biology and Agrarian Biotechnology, CNR

The use of sulfur dioxide and its salts is a well-established technique that has been used for many years in winemaking and is allowed in all wine-producing countries. In spite of its usefulness at a technical level, it is well known that sulfur dioxide has a toxic effect on humans that limits its use. The World Health Organization includes sulfur dioxide among preservatives (E220) and indicates that the Daily Permissible Dose is 0.7 mg/kg of body weight. The LD50 (Lethal Dose 50%) is 1.5 g/kg of body weight. There are various disturbances attributed to excess consumption of sulfur dioxide, such as migraine headaches. In addition to its toxic effects, sulfur dioxide also has an allergenic action. For this reason, since 25th November 2005, when European Directive EC no. 89/2003 (the allergen directive) came into force, it has became obligatory to state the presence of sulfites and of sulfur dioxide in wine and in all other foodstuffs when the concentration is higher than 10 mg/L or 10 mg/kg, expressed as SO2. One must also remember that large doses of sulfur dioxide can damage a wine from the point of view of quality, interfering with the aroma and provoking an attenuation of the perfumes. The current legal limits for sulfites in wines are fixed by national and EU legislation. For the Italian regulations, the maximum limit is 200 mg/L of total sulfur dioxide for all wines. The European rules place maximum limits of 160 mg/L for reds and 210 mg/L for whites, with derogations that permit the member states to raise the maximum value by 40 mg/L in difficult vintages. The current regulations fix the maximum limits for Italy at 160 milligrams per liter for red wines, 210 for whites and 400 for sweet wines. TOXICOLOGICAL EFFECTS ON HUMAN HEALTH Sulfur dioxide is easily absorbed by the mucous membranes in the nose and in the upper respiratory tract and its highly reactive nature makes it an extremely irritating compound. At low concentrations, the effects of sulfur dioxide are principally linked to pathologies of the respiratory system such as bronchitis, asthma and tracheitis and to irritations of the skin, eyes and mucous membranes. When dissolved in water, sulfur dioxide transforms itself into sulfite and bisulfite, which are mainly used as additives in foodstuffs, and particularly in wine. Bisulfite can be oxidized by a peroxidase to form a powerful radical anion (whose chemical formula is *SO3-). Recently a work was published that shows the powerful toxic effect of sulfur dioxide. The authors drew attention to the formation of the radical anion *SO3from sulfur dioxide and its ability to start off a process of oxidative damage that might be the basis of allergic reactions caused by SO2 and its derivatives (Ranguelova et al., Environ Health Perspect 118(7):970-5, 2010). Sulfur dioxide can provoke changes in the metabolism of some amino acids and of vitamin B1. In particular, the principal negative effect of sulfur dioxide, in

RECOGNIZING THE CHARACTERISTICS AND TOXICOLOGICAL EFFECTS OF SULFUR DIOXIDE (SO2)

Dr. Vincent Longo, Responsible of the Section in Pisa of the Institute of Biology and Agrarian Biotechnology, CNR

individuals not affected by hypersensitivity, is connected to its degrading action on vitamin B1 (thiamine), a deficiency of which in man can provoke significant alterations in the metabolism of sugars. Studies conducted on a cell line have shown the toxicity that sulfites can exert on nutrients such as vitamins and folic acid. (Stammati et al., Food Addit Contam, 9(5):551-60, 1992). Sulfites are very reactive molecules and are eliminated and detoxified by the sulfite oxydase enzyme that transforms them into sulfates. The conversion of sulfites into sulfates occurs during the passage through the digestive system. In the stomach, where the pH is very low during the digestive phase, oxidation is very slow, while it is more rapid in the intestine and in the blood (more alkaline pH). Gastric irritation depends on the fact that the sulfites, which react in a decidedly acidic way, free sulfur dioxide, which provokes a painful sensation accompanied at times by vomiting if the dose of sulfur dioxide ingested is above a certain threshold. The famous headache that one can experience after ingesting sulfur dioxide would appear to be linked to the action of the sulfite oxydase enzyme, which by using - if only to a limited degree oxygen in the formation of sulfates, limits the flow of oxygen to the brain, which reacts with this painful symptom. In a study conducted on erythrocyte cells, it was seen how the sulfite acted as an oxidant. In fact, it provokes in the erythrocytes an increase in the endogenous antioxidant system that the cell activates in order to defend itself against the damage caused by the sulfites. (Ozturk et al., J Physiol Biochem, June 2010). Epidemiological studies have shown an association between the inhalation of SO2 and ischemic cerebral damage ( Sang et al., Toxicol Sci, 114(2):226-36, 2010). A study on a Korean population was published which demonstrated that, especially in smokers, sulfur dioxide causes a reduction in pulmonary function (Min et al., J. Toxicol Environ Health A, 71(5):301-3, 2008). Studies were conducted on the effects of a mixture of sulfites and bisulfites derived from sulfur dioxide on cells in different organs of mice. The results demonstrated that the above compounds caused damage to the DNA of various organs and not only to the respiratory system. Taken together, these results suggest that SO2 and its derivatives are, at certain doses, toxic agents for all the organs of the body. (Meng et al., Mutagenesis 19: 465-8, 2004). The same authors have reported that exposure to SO2 can cause damage to the reproductive system of mammals, where this chemical molecule was seen to increase the oxidative stress and deplete the antioxidant system. (Meng and Bai, Environ Res, 96(3):298-304, 2004). Moreover, SO2 and its derivatives can have a toxic effect on cytochrome P450 enzymes, that is to say an enzymatic system designed for the metabolism and elimination of a whole series of chemical molecules that find their way into our organisms. This alters the detoxifying potential of our organisms. (Oin and Meng, Inhal Toxicol 18(8):581-8, 2006). LET US REFLECT AND REDUCE THE QUANTITY OF SULFUR DIOXIDE IN OUR WINE

RECOGNIZING THE CHARACTERISTICS AND TOXICOLOGICAL EFFECTS OF SULFUR DIOXIDE (SO2)

Dr. Vincent Longo, Responsible of the Section in Pisa of the Institute of Biology and Agrarian Biotechnology, CNR

When one considers the numerous studies on the toxicological effects of sulfur dioxide and its derivatives, one must work to reduce the level of these substances in wine to an absolute minimum. At the moment, the application of vinification using no sulfur dioxide whatsoever is complex but not impossible: a reduction in the employment of SO2 is a goal to be pursued, by means of rational and planned use during the vinification process. This reduction in the use of sulfur dioxide can take place as the result of intervening at various stages of vinification. These interventions can be summarized as follows: interventions in the vineyard; picking of the grapes; selection and quality of the grapes; selection and use of yeasts; oxygenation of the musts; alternatives to SO2 for preventing malolactic fermentation; use of selected bacteria; use of products that can help reduce the doses of SO2 (natural antioxidants); use of inert gases; and winery hygiene. These and other techniques can help us to reach our final goal, which is to produce wines without added sulfur dioxide: salubrious wines, which benefit the health of the consumer.
This document was published by

www.freewine.eu September 2011 Freewine is a set of guidelines, to be put into effect by following a non-binding technical protocol. The aim of these guidelines is to reduce considerably the quantity of sulfites added to the wines produced by the companies taking part in the project.

RECOGNIZING THE CHARACTERISTICS AND TOXICOLOGICAL EFFECTS OF SULFUR DIOXIDE (SO2)

Dr. Vincent Longo, Responsible of the Section in Pisa of the Institute of Biology and Agrarian Biotechnology, CNR