FACT SHEET - www.micotoxinas.com.br
Richard Lawley, Leatherhead Food International, Randalls Road, Leatherhead, Surrey KT22 7RY, England
Zearalenone is described chemically as a phenolic resorcyclic acid lactone and can be produced by a number of species of Fusarium including F. culmorum, F. graminearum and F. crookwellense. These species are known to colonise cereals and tend to develop particularly during cool, wet growing and harvest seasons. The main concern is that zearalenone is a naturally occurring oestrogen that is well recognised as causing hormonal effects in animal special such as the pig.
It may be produced in wheat, barley, rice, maize, and other cereals and in some other food crops and can survive into consumer products. In cereals and animal feeds, closely related compounds or conjugated products are known to also occur. Some of these are considered to be more potent oestrogens than the parent compound. Because of these complex interactions some of these zearalenone-related chemicals may enter the food chain in animal products such as meat and milk. Attempts are now being made to determine the extent of this secondary source of contamination.
Chemical and Physical Properties
Zearalenone is a white crystalline compound, which exhibits blue-green fluorescence when excited by long wavelength UV light (360) and a more intense green fluorescence when excited with short wavelength UV light (260 nm). In methanol, UV absorption maxima occur at 236 (e =29,700), 274 (e =13,909) and 316 (e =6,020). Maximum fluorescence in ethanol occurs with irradiation at 314nm and with emission at 450nm. Solubility in water is about 0.002g/100ml. It is slightly soluble in hexane and progressively more so in benzene, acetonitrile, methylene chloride, methanol, ethanol and acetone. It is also soluble in aqueous alkali. In fungal cultures a number of closely related metabolites are formed, but there is only limited evidence that these occur in foodstuffs, although there is experimental evidence for some transmission of zearalenone and a - and b -zearalenols into the milk of sheep, cows and pigs fed high concentrations.
Toxicity and Importance
The most important effect of zearalenone is on the reproductive system. In New Zealand, zearalenone in pasture is a recognised cause of infertility in sheep although its acute toxicity is low. Animal studies show that zearalenone is fairly rapidly absorbed following oral administration and can be metabolised by intestinal tissue in pigs and possibly also in humans with the formation of a - and b -zearalenols, which are subsequently conjugated with glucuronic acid. The proportions of these various products have been shown to vary considerably between species.
The ability of zearalenone to cause hyperestrogenism, particularly in swine has been known for many years. Several of a number of closely related metabolites of zearalenone produced by Fusarium spp also possess similar properties, although few have been proven to occur naturally. It is reported that swine fed a diet containing 50 mg/kg of pure zearalenone suffered abortion and stillbirths, while levels above 10 mg/kg reduced the litter size and reduced the weight of piglets. Trial feeding of female pigs demonstrated that a concentration of 0.25 mg/kg or less, produced distinct redness and swelling of the vulva, slight swelling of the mammae with numerous vesicular follicles and some cystic follicles on the ovaria. It was also found that lower levels of zearalenone in swine fed naturally contaminated feed could produce these effects. Although swine have been found to be the most sensitive domestic animal to zearalenone, calves have been reported to show earlier sexual maturity, dairy cows have been reported to have vaginitis, prolonged oestrus and infertility and sheep are reported to become sterile. The effective dose for sheep may be approximately 1 mg/kg.
Sub-acute and sub-chronic toxicity studies, of up to 14 weeks duration have been completed using several species and results showed that most effects were due to the oestrogenic effects of zearalenone. The oestrogenic potency of zearalenone has been compared the with other plant derived oestrogens in MCF-7 or T-47D breast cancer cells and this suggested that in comparison with 17-b -oestradiol, it is one of the most potent natural xenoestrogens.
It has been concluded that there is limited evidence in experimental animals for the carcinogenicity of zearalenone while the evidence for genotoxicity has been contradictory although it has suggested that zearalenone is genotoxic in mice. However, these effects may be species dependant and further studies are required to confirm whether zearalenone should be considered as a potential human mutagen or carcinogen. Although the association between zearalenone exposure and human diseases remains speculative at present, it was considered as a possible causative agent in the outbreaks of precocious pubertal changes in thousands of young children in Puerto Rico and has been suggested to have a possible involvement in human cervical cancer.
A risk assessment of the mycotoxin was carried out some time ago and the authors concluded that no adverse human health effects would be anticipated from zearalenone contamination of corn in Canada, but expressed concern that other unidentified sources might add to the oestrogenic burden. Since the publication of that work, there has been increasing awareness of the potential effects of such natural compounds and more recently zearalenone was evaluated in 2000 by JECFA.
Products affected and Natural Occurrence
Zearalenone has been shown to occur in almost every agricultural products and a variety of food-grade grains and foods have been found to contain this mycotoxin including corn and corn products, breakfast cereals, corn beer, wheat flour, bread and walnuts and in animal feed products. As zearalenone is metabolised by yeasts during brewing, mainly to b -zearalenol, this metabolite should be looked for in beer. The presence of zearalenone in whole plants and parts of maize used for silage making was investigated in Germany during 1989-1990 and zearalenone was detected at concentrations up to 300 µg/kg mainly accumulating at the end of the ripening process, with subsequent contamination of the silage. Because zearalenone and its metabolites are produced by the same fungi that give rise to deoxynivalenol and nivalenol it is not surprising that mixtures of all these mycotoxins can occur in the same sample.
Sampling and Analysis
In common with other mycotoxins sampling food commodities for zearalenone must be carried out to obtain samples representative of the consignment under test. Commonly used extraction solvents are aqueous mixtures of methanol, acetonitrile or ethyl acetate followed by a range of different clean-up procedures that depend in part on the food and on the detection method in use. TLC methods have been used but are now superseded by HPLC with UV or fluorescence detection, GC/ECD, GC/MS and HPLC/MS. Use of immunoaffinity clean up columns with HPLC is becoming used increasingly. Depending on the method used, concentrations down to and below 5 µg/kg can be measured. In addition ELISA can be used but is currently often less sensitive and can only measure one toxin at a time. A more comprehensive coverage of analytical methods for zearalenone is given in the analytical series of fact sheets.
Stability and Persistence
Zearalenone is only partly decomposed by heat. Approximately 60% of zearalenone remained unchanged in bread while about 50% survived the production of noodles. In dry milling of maize, concentrations in the main food producing fractions including flour and grits, were reduced by 80-90% although increased concentrations were found in bran and germ. Extrusion cooking in spiked food grade corn grits has been shown to result in significant reduction of zearalenone with higher reductions of ZEN at 120 -140 ºC than at 160 ºC although the effect of moisture level on the reduction of ZEN was not significant. Wet milling of maize concentrates zearalenone in the gluten fraction by a factor between 2 and 7. Metabolism of zearalenone to related oestrogenic compounds can be caused by enzymic action or within livestock.
Legislation and Control
Zearalenone was evaluated by JECFA in June 2000 and the Committee concluded that its safety could be evaluated on the basis of the dose that had no hormonal effect in pigs, the most sensitive species. It established a temporary TDI of 0.2 µg/kg of body weight per day. This decision was based on the NOEL of 40 µg/kg of body weight obtained in a 15 day study with pigs and the lowest observed effect level of 200 µg/kg of body weight per day in this study. A safety factor of 200 was also applied. Estimates of average dietary intakes for some European consumers were of the order of 0.02 µg/kg of body weight per day suggesting a significant margin of safety based on current knowledge. However further Research is required on hormonal effects in pigs, potential genotoxicity, long term carcinogenicity tests and blood levels in humans. Maximum levels for zearalenone in cereals have not yet been set by the EC although in a review published in 1997, nine countries had set guidelines or maximum tolerable levels in food (mainly cereals) ranging from non detectable to 1000 µg/kg.