6.4.1 Chemical properties
The 12,13-epoxytrichothecenes (Fig. 6.3) are a group of related and biologically active my-cotoxins produced by certain species of Fusarium, such as F. poae, F. sporotrichioides, F. culmorum and F. graminearum. They are often classified as Group A and Group B com-pounds depending on whether they have a side chain on the C-7 atom. The most commonly re-ported Group A trichothecenes include T-2 toxin, HT-2 toxin, neosolaniol, monoacetoxyscir-penol and diacetoxyscirmonoacetoxyscir-penol. These are highly soluble in ethyl acetate, acetone, chloroform, methylene chloride and diethyl ether.
Deoxynivalenol (commonly called DON or vomitoxin) is the most widely occurring Group B trichothecene, together with nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, fusarenone-X, scirpentriol and T-2 tetraol. These compounds are highly hydroxylated and thus relatively polar, being soluble in methanol, acetonitrile and ethanol.
Another group of trichothecenes that is generally more acutely toxic than T-2 toxin is known as the macrocyclic trichothecenes. These are produced by mould species such as Stachybotrys atra and include the satratoxins, verrucarins and roridins, which may be pro-duced in hay and straw stored under unsatisfactory conditions. However, there is little ev-idence that these compounds occur in human food, although the presence of macrocyclic
trichothecenes in airborne fungal spores may contribute to some forms of sick building syn-drome (Croftet al., 1986).
All trichothecenes containing an ester group are hydrolysed to their respective parent alcohols when treated with alkali. A dilute solution of potassium carbonate, sodium hydroxide or ammonium hydroxide hydrolyses T-2 toxin and neosolaniol to T-2 tetraol and diacetoxy-and monoacetoxy-scirpenol to scirpentriol. Many of the alcohols are unaffected, even by hot dilute alkali. Trichothecenes are thus chemically stable and can persist for long periods once formed. Prolonged boiling in water or under highly acidic conditions causes a skeletal rearrangement due to opening of the epoxide ring. Owing to the hindered nature of the epoxide and stability of the ring system, reactions of the trichothecenes usually proceed in a manner predictable from sound chemical principles. For example, primary and secondary hydroxyl groups are easily oxidised to the aldehyde and ketone derivatives by reagents such as CrO3–H2SO4in acetone, CrO3–pyridine and CrO3–acetic acid.
6.4.2 Analytical methods
The determination of trichothecene mycotoxins is complicated by the number of closely related compounds that can occur together and the low UV absorbance properties of the molecules. Combinations of solvents, usually acetonitrile/water and methanol/water, are used for the extraction of grain, food, and feeds. A variety of solid-phase materials such as, silica gel, florisil, cyano and C18SPE-cartridges are used for subsequent clean-up. DON and T-2 toxin immunoaffinity columns are also now available.
Methods based on TLC are still common especially where resources are limited and, sepa-ration efficiency and precision have been increased with the introduction of high-performance TLC (HPTLC) and scanning densitometers. Concentrated sulphuric acid andp-anisaldehyde and other derivatisation reagents are often used to give characteristic colours, which aid de-tection (Mirochaet al., 1977).
Several HPLC methods have been published for the determination of trichothecenes in food and cereals such as that for baby foods and animal feed (Strokaet al., 2006). However, different methods of analysis may be required for type A and B trichothecenes, e.g. HPLC with UV detection is not usually applicable to type A trichothecenes as they lack a keto group at the C-8 position. In these circumstances MS-based methods may be the preferred methods for the determination of type A trichothecenes. However, low wavelength UV detection is used in DON HPLC methods although sensitivity is poor, lack of specificity being the main problem.
The earlier GC methods are based on derivatisation procedures such as trimethylsilylation or fluoroacylation to increase volatility and sensitivity with electron capture detection or the use of mass spectrometric or tandem mass spectrometric detection (MS/MS). The choice of derivatisation reagent depends on the type of trichothecene and the method of detection.
A number of studies have been carried out into improving and verifying trichothecene analysis. Pettersson and Langseth (2002) carried out inter-comparisons of GC/MS methods and studied the factors that influence method performance as a pre-requisite for producing certified calibrants. Mateoet al. (2001) published a critical study of, and improvements in, chromatographic methods for the analysis of type B trichothecenes. In a study in which laboratories used their own in-house methods for the determination of theFusarium myco-toxins zearalenone and DON in common wheat and maize samples, the results were deemed acceptable for DON at the 100 μg/kg level (Josephs et al., 2001).
LC/MS with either atmospheric pressure chemical ionisation (APCI) or electrospray ion-isation or tandem mass spectrometry (MS/MS) have been successfully employed for the de-termination and identification of trichothecenes at trace levels (Biselli and Hummert, 2005;
Klotzelet al., 2005; Klotzel et al., 2006). Trace mycotoxin analysis in complex biological and food matrices using LC/MS has been comprehensively reviewed (Z¨ollner and Mayer-Helm, 2006).
A variety of test kits are now marketed for the rapid detection of individual trichothecenes, mostly for DON and T-2 toxin. These are particularly useful for screening purposes and may be set to provide a yes/no answer at a pre-set level or in ELISA formats. If necessary, samples can then be checked using fully quantitative methods. Rapid methods for DON and other trichothecenes have been reviewed (Schneideret al., 2004).
6.4.3 Occurrence in raw materials and processed foods
Trichothecenes occur in cereals and other food commodities. There are many reports of DON and nivalenol in cereals although T-2 toxin and HT-2 toxin are found much less frequently except in oats. Surveillance commonly targets DON only although other trichothecenes are likely to be present.
A collection of occurrence data forFusarium toxins was carried out in food in the EC (European Commission, 2003) and enabled a preliminary assessment of dietary intake by the population of member states to be carried out (Schothorst and van Egmond, 2004). By far most of the occurrence data were obtained for DON in wheat. Among cereals, corn (maize) showed the highest level of contamination with trichothecenes. There was a significant lack of consumption data in some countries. In particular, information on baby and children’s food was generally not available. Wheat and wheat containing products (like bread and pasta) represented the major source of intake for the four trichothecenes, DON, nivalenol, HT-2 toxin and T-2 toxin. DON and related compounds were found in a survey of Soy products in Germany using an MS method (Schollenbergeret al., 2007).
Studies have suggested that DON may sometimes be present in cereals in a bound forms and an acid solvolysis procedure may be required to release this bound mycotoxin (Liuet al., 2005b).
There are occasional reports of trichothecenes in commodities other than cereals and in view of the widespread distribution ofFusarium spp., this remains a possibility.
6.4.4 Toxicology
The acute toxicity of the trichothecenes varies considerably and LD50 values for mice (in-traperitoneal route) for some trichothecenes are given in Table 6.7 LD50 (mg/kg bw). The LD50value for DON is about ten times that for nivalenol, T-2 toxin and HT-2 toxin that are in turn about ten times greater than for the macrocyclic mycotoxins. Fortunately, these have rarely been reported in food. Acute trichothecene toxicity is characterised by gastrointestinal
Table 6.7 LD50values for mice (ip) for some trichothecenes.
Trichothecene LD50 (mg/kg bw)
Deoxynivalenol 70
Diacetoxyscirpenol 23
Neosolaniol 14.5
HT-2 toxin 9.0
T-2 toxin 5.2
Nivalenol 4.1
Verrucarin A 0.5
Table 6.8 EU Regulations for DON, EC as from 1st July 2007.
Cereal product Limit (μg/kg)
Unprocessed cereals other than durum wheat, oats and maize 1250
Unprocessed durum wheat and oats 1750
Unprocessed maize with the exception of unprocessed maize intended to be processed by wet milling
1750
Pasta (dry).
Cereals intended for direct human consumption, cereal flour, bran and germ as end product marketed for direct human consumption with the exception of foodstuffs listed below
750
Milling fractions of maize with particle size >500 micron falling within CN code 1103 13 or 1103 20 40
750
Milling fractions of maize with particle size≤500 micron falling within CN code 1102 20
1250
Bread (including small bakery wares), pastries, biscuits, cereal snacks and breakfast cereals
500
Processed cereal-based food for infants and young children and baby food 200
disturbances, such as vomiting, diarrhoea and inflammation, dermal irritation, feed refusal, abortion, anaemia and leukopenia. This group of toxins is acutely cytotoxic and strongly im-munosuppressive. The trichothecenes have not been shown to be mutagenic or carcinogenic but do inhibit DNA and protein synthesis.
DON is a common contaminant of cereals and causes vomiting in pigs at relatively low concentrations. However, pigs are very sensitive to its presence and will reject contaminated feed, effectively limiting any further toxic effects. DON is, however, immunosuppressive in low concentrations and this may be more important than its low acute toxicity. Because of the number of closely related metabolites likely to occur in combination in foods or animal feeds, the toxicology is complex with both synergistic and antagonistic effects. This has been discussed by Miller (1995).
Alimentary toxic aleukia (ATA) has probably been the most common human trichothecene mycotoxicosis. T-2 toxin is thought to have contributed to the epidemiology of ATA in Russia, which was responsible for widespread disease and many deaths. Continuous exposure to trichothecenes results in skin rashes, which may proceed to necrotic lesions.
6.4.5 Regulation and control
Legislation for DON is set in a number of countries (FAO, 2004) and the introduction of internationally agreed legislation is expected to continue. Legislation for DON in cereals that was introduced during 2006 and 2007 within the EU is summarised (Table 6.8). In addition, Statutory or Guideline limits for T-2 toxin and HT-2 toxin in cereals are expected to be introduced during 2008. Codes of practice for minimising the occurrence of DON and the trichothecenes are being developed by international bodies such as CODEX/WHO/FAO and within the EU. Some basic rules can be summarised; avoid growing maize as the previous crop, reduce crop debris on soil surface, avoid susceptible varieties, apply a T3 fungicide againstFusarium and avoid delays in drying at harvest.
6.5 ZEARALENONE
6.5.1 Chemical properties
Zearalenone (Fig. 6.4) is a phenolic resorcyclic acid lactone produced by a number of species ofFusarium including F. culmorum, F. graminearum and F. crookwellense. 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 α- and β-zearalenols into the milk of sheep, cows and pigs fed high concentrations (Mirochaet al., 1981).
Zearalenone is a white crystalline compound that exhibits blue-green fluorescence when excited by long wavelength UV light (360 nm) and a more intense green fluorescence when excited with short wavelength UV light (260 nm). In methanol, UV absorption maxima occur at 236 nm (ε= 29 700), 274 nm (ε = 13 909) and 316 nm (ε = 6020). Maximum fluorescence in ethanol occurs with irradiation at 314 nm and with emission at 450 nm.
Solubility in water is about 0.002 g/100 mL. Zearalenone 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. It appears to be very stable in many processes.
6.5.2 Analytical methods
Ground cereal samples are usually extracted using a suitable solvent such as acetonitrile, chloroform, ethyl acetate or methanol and water or acidic solutions. TLC initially provided an acceptable method in laboratories with limited instrumentation and a recent improvement has been introduced by using HPTLC (Ostry and Skarkova, 2003). Liquid–liquid partitioning or solid-phase extraction is necessary to provide clean extracts for separation and detection.
Recently commercially available clean-up columns have become a widely used clean-up medium (Visconti and Pascale, 1998; Krugeret al., 1999). An HPLC immunoaffinity col-umn method for barley, maize and wheat flour, polenta, and maize-based baby food has shown to meet required analytical criteria in an interlaboratory study (MacDonaldet al., 2005).
In a comparative study, mixtures of methanol and 1% aqueous NaCl were found to be the best extraction solvent while immunoaffinity columns were very effective for clean-up although of lower capacity than solid phase extraction columns (Llorenset al., 2002).
There have been several recent developments for the determination of low concentrations of zearalenone in foods and body liquids or tissues including an automated flow-through immunosensor method for the determination of zearalenone in pig feed (Urracaet al., 2005).
Among a number of LC/MS methods, Pallaroni and von Holst (2003) determined zear-alenone in wheat and corn using pressurised liquid extraction and liquid chromatography–
electrospray mass spectrometry, a method based on LC-APCI-MS was developed for the determination of zearalenone and its metabolites in urine, plasma and faeces of horses (Songsermsakul et al., 2006) and for bovine milk by LC/MS/MS (Sorensen and Elbaek, 2005).
Molecular imprint technology produced by developing a polymer template that binds the zearalenone molecule has been used to provide a clean-up method for zearalenone and alpha-zearalenol in cereal and swine feed sample extracts (Urracaet al., 2006). Krska et al. (2005) has reviewed recent advances in zearalenone methodology.
6.5.3 Occurrence in raw materials and processed foods
Zearalenone occurs in cereal grains especially maize and can survive processing into cereal products (Williams, 1985) such as maize beer (Lovelace and Nyathi, 1977), wheat flour (Tanakaet al., 1985). It has also been reported in walnuts (Jemmali and Mazerand, 1980).
The presence of zearalenone in whole plants and parts of maize used for silage making has been investigated in Germany (Oldenberg, 1993). In that work, zearalenone was detected at concentrations up to 300 μg/kg and mainly formed at the end of the ripening process, with subsequent contamination of the silage. In a survey in Germany, zearalenone has been found in small amounts in vegetables, fruits, oilseeds and nuts (Schollenbergeret al., 2005).
Information on the occurrence of zearalenone within the EU has been collated and summarised (Gareiset al., 2003).
Zearalenone is only partly decomposed by heat. Approximately 60% of zearalenone re-mained unchanged in bread while about 50% survived the production of noodles (Matsuura and Yoshizawa, 1981). In dry milling of maize, concentrations in the main food-producing fractions, including flour and grits, were reduced by 80–90% although increased concentra-tions were found in bran and germ (Bennettet al., 1976). Its distribution in wet-milled corn products is reviewed by Bennett and Anderson (1978).
6.5.4 Toxicology
The most important effect of zearalenone is on the reproductive system, its acute toxicity being low. The ability of zearalenone to cause hyperestrogenism, particularly in swine, has been known for many years. Two comprehensive reviews of this have been published by Mirochaet al. (1971) and Mirocha and Christensen (1974). In New Zealand, zearalenone in pasture grass is a recognised cause of infertility in sheep (Towers and Sprosen, 1992).
Trial feeding of female pigs demonstrated that a concentration of 0.25 mg/kg, or even less, produced changes in the reproductive organs (Baueret al., 1987).
In a review by the International Agency for Research on Cancer (IARC, 1993), it was concluded that there was limited evidence in experimental animals for the carcinogenicity of zearalenone. Evidence for genotoxicity has been contradictory but Pfohl-Leskowiczet al.
(1995) showed that zearalenone is genotoxic in mice.
A risk assessment of the mycotoxin has been carried out (Kuiper-Goodmanet al., 1987) and that paper provides a comprehensive review of the literature up to that date. A recent comprehensive review of the toxicity, occurrence, metabolism, detoxification, regulations and intake of zearalenone has been published (Zinedineet al., 2006). An opinion on zearalenone was published in 2000 (European Commission, 2000) and for animals in 2004 (EC, 2006b).
6.5.5 Regulation and control
Initial legislation for zearalenone in cereals was introduced from July 2006 within the EU.
Prior to this legislation existed in about 12 countries worldwide (FAO, 2004). These limits were modified and introduced in July 2007 (EC, 2007). In summary, these limits range from 100 μg/kg in unprocessed cereals other than maize, to 350 μg/kg in maize, 75 μg/kg in cereals intended for direct human consumption, cereal flour, bran and germ marketed for direct human consumption, down to 20 μg/kg in processed cereal foods for infants and young children. The reader should consult the EC document above for full details of these limits.
Because zearalenone often occurs along with otherFusarium mycotoxins codes of practice to prevent its formation are similar, e.g. to DON.
6.6 FUMONISINS
6.6.1 Chemical properties
The fumonisins (Fig. 6.4) are a group of at least 15 closely related mycotoxins that often occur in maize and less commonly in other products, the most important compound being fumonisin B1. They are polar metabolites formed by several species ofFusarium, but mainly F. verticilliodes (Sacc.) Nirenberg (= F. moniliforme Sheldon). Their structures are based on a long hydroxylated hydrocarbon chain. Two hydroxyl groups are esterified to two propane-1,2,3-tricarboxylic acids. Fumonisin B1 differs from fumonisin B2 in that it has an extra hydroxyl group at the 10-position.
Fumonisins contain four free carboxyl groups and an amino group, which accounts for their solubility in water and some polar organic solvents. Fumonisin B1is stable in acetonitrile–
water (1:1) over a 6-month period at 25◦C (Viscontiet al., 1993) and in methanol if stored at –18◦C but steadily degrades at 25◦C and above. Fumonisin B1is hydrolysed under some conditions and may bind to food constituents. The pure substance is a white hygroscopic powder. The insolubility in many organic solvents partly explains the difficulty in their original identification.
A review of the occurrence and toxicity of the fumonisins is that of Dutton (1996) while the history and discovery of this group of mycotoxins has been described (Marasas, 2001).
6.6.2 Analytical methods
TLC, HPLC, MS/MS and immunochemical methods have been reported for the fumonisins.
Lack of a suitable chromophore in the molecule means that fumonisins must be derivatised with reagents such asp-anisaldehyde, fluorescamine or o-phthaldialdehyde to allow detection by TLC or HPLC. Currently, HPLC methods, mainly employing fluorescence detection together with sample pre-treatment, are the methods of choice for routine analysis, and an overview has been presented for the determination of the fumonisin mycotoxins in various matrices (Arranzet al., 2004). Reliable immunoaffinity clean-up columns are available and becoming used widely.
The first collaborative study of a method for liquid chromatographic determination of fu-monisins B1, B2and B3in corn was a joint AOAC–IUPAC collaborative trial (Sydenhamet al., 1996). Viscontiet al. (2001b) presented an in-house and inter-laboratory validation study for the determination of fumonisin B1and B2in corn-based foodstuffs including cornflakes by HPLC using immunoaffinity clean-up.
Stability and problems in the recovery of fumonisins added to corn-based foods has been known to be a problem for many years (Scott and Lawrence, 1994; Avantaggiatoet al., 2003).
The analysis of heat-processed corn foods such as cornflakes, corn-based breakfast cereals, tortilla chips and corn chips can in addition present further special problems in analysis as hydrolysed fumonisin B1as well as protein- and total-bound fumonisin B1can be present as well as the parent fumonisins. Bound (hidden) fumonisins cannot be detected by conventional analysis and improved methods for the determination of bound fumonisin B1were developed (Parket al., 2004). A number of studies have been carried out recently developing LC/MS methods (e.g. Cavaliereet al., 2005; Faberi et al., 2005; Paepens et al., 2005).
Determination of total fumonisins in corn by competitive direct ELISA (Birdet al., 2002) and commercial rapid test kits are suitable for use in monitoring situations.
6.6.3 Occurrence in raw materials and processed foods
In the years since fumonisins were first identified there have been many surveys reporting fumonisins in harvested maize sometimes in very high concentrations. Worldwide occurrence of fumonisins in maize has been reviewed (Shepherdet al., 1996) while a more recent review of their occurrence in foods has been carried out (Soriano and Dragacci, 2004). While maize is by far the most important commodity in which fumonisins occur, there have been reports of their occurrence in other commodities, usually in much lower concentrations. These include wheat, barley and soybean in Spain (Castellaet al., 1999) and other warm growing areas.
However, a recent re-examination of wheat from South Africa claimed to contain fumonisins as high as 1.7 mg/kg failed to find any evidence of their presence (Shepherdet al., 2005).
Fumonisins have been reported to be quite stable and not broken down even by moderate
Fumonisins have been reported to be quite stable and not broken down even by moderate