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Artyku³ przegl¹dowy Review
The term mycotoxins was first used in 1962 during a veterinary crisis in the London area that witnessed the death of nearly 100,000 turkey poults. Turkey disease X was identified after the secondary metabolites (afla-toxins) of the mold Aspergillus flavus had been detec-ted in peanut meal (14). The growing interest in myco-toxins, observed particularly in recent years, and the progress in laboratory technology has enabled scientists to identify more than 300 compounds produced by toxic fungi. Yet from the practical point of view those achievements have little bearing for the production of high-quality food and feedstuffs. Despite some geo-graphic and climatic variations, mycotoxins occur globally (23). The resistance of mycotoxins, including aflatoxins, ochratoxins, trichothecenes, zearalenone (ZEA) and fumonisins (individually or in combination), in process water used in the production of food and feedstuffs (26) is a very important consideration, although not all compounds have a significant effect on the nutritional and economic value of the end product (5).
The objective of this study was to discuss the find-ings relating to the presence of mycotoxins, in particular ZEA, in potable water and water used for feedstuff production, and to investigate the consequences thereof.
Mycotoxins
Mycotoxins are secondary metabolites of fungi, capable of producing acute toxic, carcinogenic, muta-genic, teratomuta-genic, immunogenic and estrogenic effects in humans and animals. Fungi of the genera Fusarium, Aspergillus and Penicillium are the main source of mycotoxins contaminating plant material which is used in the production of food and feedstuffs. Fungi of the genera Fusarium, Aspergillus and Penicillium are the main source of mycotoxins contaminating plant material used in the production of food and feedstuffs. Fungal infection takes place during the growing season, the harvest and storage of grains, semi-processed products (flour) and end products, such as bread, pasta and feed-stuffs (4). Fungal strains are characterized by varied levels of aggression towards the host plant (41). The ability to produce mycotoxins is one of the most im-portant indicators of the aggressiveness of pathogenic strains. The presence of mycotoxins produced by the above fungi lowers the quality of cereal grains by posing a threat to human and animal health (11, 15). In humans and animals, poisoning with toxic fungi is referred to as mycotoxicosis. For many years, mycotoxicoses were very difficult or impossible to diagnose, and they remained neglected diseases until the early 1960s.
Fusarium is the predominant fungal genus in Europe. It produces various metabolites with toxic effects for humans, animals and plants. Every fungal species
Threats resulting from the presence
of zearalenone in water*
)
MAGDALENA GAJÊCKA, £UKASZ ZIELONKA, MICHA£ D¥BROWSKI, MACIEJ GAJÊCKI
Department of Veterinary Prevention and Feed Hygiene, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13/29, 10-718 Olsztyn, Poland
Gajêcka M., Zielonka £., D¹browski M., Gajêcki M.
Threats resulting from the presence of zearalenone in water Summary
Water samples collected randomly from animal farms and feed mills using process water have confirmed the presence of zearalenone (ZEA). ZEA is a mycotoxin (undesirable substance) produced by fungi of the genus Fusarium, showing estrogenic properties. It has a significant effect on the development of the male and female reproductive system and fertility in humans and animals. Previous research results indicate that long-term exposure to low doses of ZEA may lead to pathological states in humans and animals. In view of the above, regulatory standards for the maximum allowable ZEA concentrations in water should be established as soon as possible, since this mycotoxin poses serious health risks to humans and animals.
Keywords: plant material, zearalenone, water
*) Financial support from the State Committee for Scientific Research
(Pro-ject No. PB KBN N N308 242635) and the Ministry of Scientific Research and Information Technology of Poland.
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produces a unique combination of mycotoxins, mostly trichothecene compounds (deoxynivalenol, nivalenol, T-2, HT-2), fumonisin and ZEA.
ZEA
ZEA is a mycotoxin synthesized by several fungi, including Fusarium graminearum (known in its sexual stage as Gibberella zeae), Fusarium culmorum, Fusa-rium cerealis, FusaFusa-rium equiseti and FusaFusa-rium semi-tectum (3, 24). Fungi of the genus Fusarium may infect grain during flowering, but they also contaminate stored plant materials. Grain kernels infected with Fusarium spp. fungi may contain mycotoxins, including ZEA. This mycotoxin is frequently observed in maize, but since Fusarium spores are ubiquitous it has also been found in barley, oat, wheat, rice, sorghum and soybeans in both warm and moderate climate zones (43). ZEA (also known as F2 toxin) and its metabolites (á- and â-zearalenol, á- and â-zearalanol) are resorcinolic acid lactones chemically described as 6-(10-hydroxy-6-oxo-trans-1--undecenyl)-beta-resorcylic acid lactone. ZEA, a myco-toxin and a phytoestrogen, is also a resorcylic acid lactone (RAL) that demonstrates estrogenic properties with a high affinity for estrogen receptors (10). RALs are considered undesirable substances in food and feed. According to a most recent hypothesis, the potential source of RAL compounds are surface waters drained from farm fields, agricultural runoffs in periods of heavy precipitation, inadequate slurry management (sprinkling) and ploughless soil tillage (41).
ZEA has the following physical and chemical cha-racteristics: empirical formula C18H22O5; molar mass [g/mol] 318.36; water solubility [mg/l]a max. 20;
solubility at various pH levels [mg/l]b 4.51 at pH 2,
4.81 at pH 7, 40.15 at pH 10; hydrophobicity [log Kow]b
3.66; dissociation constant pKab 7.62; dipole
moment Dc 2.2. Zearalenone is a white crystalline
compound with a melting temperature of 164-165°C, and it is unstable in an alkaline environment. This hydrophobic organic molecule is practically insoluble in water, but it is soluble in alkaline solutions and orga-nic solvents (ether, chloroform, methyl chloride, ethyl acetate, acetonitrile, alcohol and acetone). Its maximum UV absorption from methanol is determined at ë = 236 and 274 nm (green fluorescence) and ë = 316 nm (blue--green fluorescence). Maximum fluorescence in etha-nol is reported at excitation of ëEx = 314 and emission of ëEm = 450 nm. ZEA occurs naturally in E configura-tion, and it contains a double bond between C-1 and C-2. When exposed to intensive UV radiation, ZEA changes its configuration to Z (14).
ZEA is a diphenolic compound with a dissociation constant of pKa = 7.62, which suggests that at pH 3,
ZEA is mainly neutral, at pH 7 the phenol anion is present in the solution, and at pH 9 ZEA exists nearly entirely in anionic form. The toxic effects of ZEA and its metabolites are attributed mainly to their estrogenic properties, including structural resemblance to natural
estrogen, estron and estriol, as well as interactions with estrogen receptors that compete with 17â-estradiol. ZEAs estrogenic potential is several folds higher in comparison with other environmental estrogens (15).
The nature and intensity of damage caused by ZEA in porcine and canine reproductive systems (10, 11, 31) varies subject to age (physiological condition of the body) and the phase of the reproductive cycle. The above is a very important consideration in animal breeding. ZEA often affects the reproductive system by comple-mentarily binding to estrogen receptors (12) and mo-dulating their transcription (1). ZEA molecules contain a phenol ring which supports the competition for binding sites with the estrogen receptor of target cells. The above affects ovulation, and it explains ZEAs influence on the animal myometrium (12, 30, 36, 37, 42, 43).
Fusarioses
The most predominant pathogenic fungi affecting cereal crops include Fusarium ssp., Pseudocerco-sporella spp. and Rhizoctonia spp. Ear and stem-base diseases caused by fungi of the genus Fusarium, mainly F. culmorum, F. nivale, F. poae, F. avenaceum and F. graminearum (Gibberella zeae), pose a particularly high risk to wheat kernels.
Fusarium fungi are observed in all climate zones. They cause pre-emergence and post-emergence rot, stem-base rot and ear blight. Fungi infect various types of plant material, and they often produce mycotoxins that contaminate 20-30% of crops on average. Wheat germ infections lower yield by 7-17%. Root infections at later development stages decrease yields by 10-30%. Fusarium ear blight may lead to crop losses as high as 30-70%. Fungicidal treatment reduces the incidence and spread of diseases, but yield losses and deterioration of grain quality cannot be completely eliminated. Fungi of the genus Fusarium easily adapt to changing envi-ronmental conditions. F. culmorum can grow and form spores even if the soil water potential is low. The greatest damage is caused by F. culmorum and F. gra-minearum. Fungi of the genus Fusarium may also attack wheat heads, thus contributing to a decrease in yield and grain quality. The severity of fusarioses in the field may vary due to the presence of pathogenic species marked by different spore-forming ability, infection intensity and growth rates (41).
Mycotoxins significantly decrease the thousand grain weight, the Hagberg falling number and the sedimenta-tion value as well as the total protein content of grain. Ear blight is caused by fungi of the genus Fusarium in the period from ear formation to late flowering. The optimal temperature for fungal growth falls in the range of 15-20°C. Drizzling rain and dew create the most supporting environment for fungal infections of wheat and other cereals. Heavy precipitation throughout the growing season may contribute to the elution of ZEA from plant tissue into the soil, surface and ground water (6, 19).
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Prolonged wheat flowering also contributes to fungal infections, including fusarioses. Fungi of the genus Fusarium may grow on after-harvest residues of cereals, maize and monocotyledonous weeds, which become a source of infection for the successive crop wheat. Wheat growing after other cereals and maize poses a particularly high threat of yield decrease and grain quality deterioration.
Presence of ZEA in aqueous environments
The presence of ZEA is more and more often reported in potable water (18, 34) and water used for feedstuff production (6, 19). RALs (in particular ZEA and its metabolites) are determined in surface water, treated wastewater and industrial effluents at concentrations exceeding 60 ng/l (6, 19).
Gromadzka et al. (17) determined ZEA concentra-tions in samples of surface water, ground water and wastewater in Poland. The cited authors reported that low molecular weight organic micro-substances that contaminate water act as ZEA carriers. The ZEA con-tent of the collected water samples ranged from 0 to 43.7 ng L1.
A study by Paterson (32) has demonstrated that ZEA can be produced in water by F. graminearum, and a methodology is proposed for determining the concen-trations of this fungus in process lines that use water for the production of food and feedstuffs. The above study is the first ever report documenting the presence of mycotoxins in potable and process water. The study indicates that the presence of ZEA in water samples from water distribution devices contaminated by F. gra-minearum is very easy to determine.
Fusarium graminearum is one of the most predomi-nant fungal species that produces mycotoxins in aqueous environments. The strain produces deoxynivalenol, nivalenol and ZEA. ZEA has estrogenic properties owing to a broad tropism within the organs of its human and animal hosts (ovaries, uterus, mammary gland, duo-denum, large intestine, bone marrow and the central nervous system 2, 8, 21, 25, 28, 29, 39), and it may lead to poisoning even in children (13). The above can be attributed to high estrogen (and even xenoestrogen) concentrations in water, as has been documented by various published sources and water status reports (38). Although it has been researched extensively in the past decade, the problem of fungal development in water is not addressed in practical applications (7, 22).
According to Kelley et al. (22), ergosterol, a lipid component of fungal cell membranes, may be used to determine the presence of fungi in water and develop fungal concentration standards for an aqueous environ-ment. The presence of fungal odor is also used in the process of identifying those toxins (16).
The discussed fungi produce aurofusarin, a characte-ristic red pigment with toxic properties which is trans-ferred to the culture medium and infected grain (27). Unfortunately, this pigment is produced in very small
quantities, and it is difficult to use for analytical pur-poses. Nonetheless, fungi are a rich source of various pigments.
The production of ZEA by the above fungi in aqueous environments and the resulting consequences remain poorly documented in literature (34, 35). It can be presumed that ZEA concentrations increase rapidly in environments that support fungal growth, as well as in the presence of stress factors. Heavy precipitation may lead to the elution of mycotoxins from plant tissue into surface and ground water which reaches food and feed-stuff processing plants. Fungal mycelia and spores may travel the same route to water supply systems where they find an environment supporting the growth, deve-lopment and production of e.g. ZEA.
Justification for legal regulations
Recent research findings show (29) that mycotoxins exert a significant influence on human and animal health. These observations have led to the introduction of legal acts regulating allowable mycotoxin concen-trations in food products and feedstuffs (Commission Regulations (EC) No. 856/2005, No. 1881/2006 and No. 1126/2007 setting maximum levels for certain con-taminants in foodstuffs, and Commission Recommen-dation 576/2006 regulating mycotoxin levels in feed), but not in water. The first legislative acts were develo-ped in highly industrialized countries, and they mainly addressed the problem of aflatoxin contamination of plant materials. Those regulations were introduced as guidelines rather than prescriptive standards. By 2005, nearly 100 countries (9), including EU member states, had developed their own mycotoxin regulations (40). Most of them set the allowable concentrations of 13 individual mycotoxins or mycotoxin groups in foods and feedstuffs, but not in water.
Legal acts are developed and implemented in view of the following factors (5): (i) availability of toxico-logical data for selected mycotoxins, (ii) the risk of scientific data disclosure, (iii) data regarding the dis-tribution and concentrations of mycotoxins in plant material, semi-processed products and end products, (iv) availability of approved analytical methods (20, 33), (v) legislation in countries that are trade partners (40), (vi) demand for food products and feedstuffs. The first two considerations provide information necessary for estimating the risk of data disclosure. The following two factors are the key principles of risk evaluation. Risk assessment is a scientific evaluation of the proba-bility that the consumption of food products and feed-stuffs will deliver known (predictable) and unknown (unpredictable) health consequences (5). Those are the basic scientific premises for developing and implemen-ting legal regulations. The subsequent two factors regu-late sample collection and analytical procedures (20, 33). The last two considerations have social and economic implications, and they play an important role during the development of mycotoxin standards (allowable values)
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for food products, feedstuffs, water administered to animals as well as water used for food and feedstuff production. The latter has become a serious considera-tion only in recent years (6, 19).
Conclusions
In view of the facts and arguments contained in this report, and recent advances in the analytical methods employed to determine ZEA levels in water (17), rese-archers should be encouraged to further investigate the consequences of ZEA presence in water administered to animals as well as water used for feedstuff produc-tion. The obtained results could provide a basis for developing and implementing a pioneer legal act regu-lating the maximum allowable ZEA concentrations in water, aimed at improving food safety.
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Authors address: Magdalena Gajêcka, DVM, Department of Veterinary Prevention and Feed Hygiene, Faculty of Veterinary Medicine University of Warmia and Mazury in Olsztyn, Oczapowskiego 13/01, 10-718 Olsztyn; e-mail: mgaja@uwm.edu.pl
