Animal Feed Science and Technology 133 (2007) 149166

Managing the risk of mycotoxins in modern feed production

Eva M. Binder
Biomin GmbH, Industriestrasse 21, A-3130 Herzogenburg, Austria

Abstract Mycotoxin-contaminated feeds impair farm operations as well as feed production in various ways: mycotoxins are invisible, odourless and cannot be detected by smell or taste, but can reduce performance in animal production signicantly. Due to the complex nature of these naturally occurring contaminants and their elaborate analytics a risk-management concept has to be adopted in order to reduce the risk encounter to a dened and acceptable level. The best control is the prevention of mycotoxins in the eld, which is supported by proper crop rotation and fungicide administration at the right time. In the case of toxin manifestation, measures are required that act specically against certain types and groups of toxins. Adsorptive compounds can be used for reduction of potency of mycotoxins in general. While adsorbents have proved to be efcient against some mycotoxin-induced toxicosis, alternative strategies such as enzymatic or microbial detoxication, have been used recently for counteracting impacts of certain fungal toxins. 2006 Elsevier B.V. All rights reserved.
Keywords: Mycotoxins; Mycotoxin analysis; Mycotoxin detoxication; Feed additives

1. Introduction Mycotoxins are secondary metabolites produced by lamentous fungi that cause a toxic response (mycotoxicosis) when ingested by higher animals. Fusarium, Aspergillus, and Penicillium are the most abundant moulds that produce these toxins and contaminate

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human foods and animal feeds through fungal growth prior to and during harvest, or during (improper) storage (Bhatnagar et al., 2004). The term mycotoxin was adopted in 1962 in the aftermath of an unusual veterinary crisis near London, England, during which approximately 100,000 turkey poults died. This mysterious turkey X disease was linked to a peanut meal contaminated with secondary metabolites from Aspergillus avus (aatoxins) (Bennet and Klich, 2003). Due to modern methods and to a growing interest in this eld of research more than 300 different mycotoxins have been differentiated to date. However for practical consideration in the feed manufacturing process only a small number of toxins is of relevance (Miller, 1995). Although there are geographic and climatic differences in the production and occurrence of mycotoxins, exposure to these substances is worldwide (Kuiper-Goodman, 2004). For practical consideration in the feed manufacturing process aatoxins, trichothecenes, zearalenone, ochratoxins, and fumonisins are of particular interest, though the extent of harm each toxin (group) can cause is highly species-dependant.

2. Mycotoxins in feed The most commonly known mycotoxins (Table 1) are the aatoxins due to the fact that they represent one of the most potential carcinogenic substances known so far. They were rated as Class 1 human carcinogens by the IARC (International Agency for Research on Cancer). Aatoxins are produced by many strains of A. avus and Aspergillus parasiticus on many different commodities, including cereals, gs, oilseeds, nuts, tobacco, and others (Diener et al., 1987). Aatoxin B1 is moreover considered the main hepatocarcinogen in animals, although effects vary with species, age, sex, and general nutritional conditions. Trout, ducklings and pigs are highly susceptible, ruminants being less susceptible (Weidenb rner, o 2001). Trichothecenes constitute a large group of mycotoxins produced by various species of moulds, in particular those belonging to the genus Fusarium. Approximately 170 trichothecene mycotoxins have been identied to date, having a sesquiterpenoid 12,13epoxytrichothec-9-ene ring system in common (Krska et al., 2001). Trichothecenes are potent inhibitors of eukaryotic protein synthesis (Bennet and Klich, 2003). Epidemiological surveys have revealed that the predominant type-A and -B trichothecenes are widely distributed in cereals as natural pollutants, whereas the macrocyclic trichothecenes occur rarely in food or feed. The most prevalent mycotoxins of these groups are deoxynivalenol (DON, vomitoxin), nivalenol (NIV), 3- or 15-acetyl-deoxynivalenol (AcDON), Fusarenon X (FUS-X) in case of B-trichothecenes, and T-2 toxin, HT-2 toxin, and diacetoxyscirpenol (DAS) of the type-A toxins. An important issue is that some of these closely related compounds occur simultaneously (Fuchs et al., 2004) and are proven to cause synergistic effects (Weidenb rner, 2001). In particular DON is prevalent worldwide in crops used for food and o feed production, and although it is one of the least acutely toxic trichothecenes, it should be treated as an important food safety issue because it is a very common contaminant of grain (Rotter et al., 1996). Different types of trichothecenes vary in their toxicity though all of them have high are acute toxicity. They may cause haematological changes and immune suppression, reduced feed intake and skin irritations as well as diarrhoea and haemorrhages of

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Table 1 Overview of most relevant mycotoxins in animal production Major classes of mycotoxins Aatoxins Most relevant representatives in grains and feed Aatoxin B1, B2, G1, G2 Examples of mycotoxin-producing fungi Aspergillus avus, Aspergillus parasiticus Effects observed in animals Liver disease (hepatotoxic, hepatocarcinogen), carcinogenic and teratogenic effects Immunologic effects, hematological changes, digestive disorders (emesis, diarrhea, reduced feed intake) dermatitis, oral lesions, hemorrhages of intestinal tissues, edema Estrogenic effects (edema of vulva, enlargement of uterus), atrophy of ovaries and testicles, abortion Nephrotoxicity, porcine nephropathy, mild liver damage, immune suppression Nervous or gangrenous syndromes Pulmonary edema, leukoencephalomalacia, nephrotoxicity, hepatotoxicity

Trichothecenes

Zearalenone

Deoxynivalenol, 3- or 15-Acetyl-deoxynivalenol, nivalenol, fusarenon X (type-B trichothecenes), T-2 toxin, diacetoxyscirpenol, HT-2 toxin (type-A trichothecenes) Zearalenone

Fusarium graminearum, Fusarium sporotrichioides, Fusarium poae, Fusarium equiseti

Fusarium graminearum

Ochratoxins Ergot alkaloids Fumonisins

a

Ochratoxin A Ergometrine, ergosine, ergotamine, clavinesa Fumonisin B1, B2, B3

Aspergillus ochraceus, Penicillium verrucosum, Penicillium viridicatum Claviceps purpurea, Claviceps paspaspali, Claviceps fusiformis Fusarium verticillioides (syn., moniliforme), Fusarium proliferatum

Ergot alkaloids comprise a large group of fungal compounds.

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internal tissues. An extensive toxicological evaluation of trichothecenes in animal feed was conducted by Eriksen and Pettersson (2004). They concluded that trichothecenes are toxic to all tested species, but that sensitivity varied considerably between toxins and between species. While poultry are more sensitive to trichothecenes than ruminants, pigs seem to be the most sensitive farm animals. Effects occurring at the lowest levels of trichothecenes were reduced feed intake and weight gain, as well as impairment of the immune system. Zearalenone is also produced by Fusarium species and has strong hyper-estrogenic effects, which result in impaired fertility, stillbirths in females and a reduced sperm quality in male animals. Due to its structural similarity to estradiol it is able to bind to estrogen receptors in mammalian target cells, so that it is classied by some authors as nonsteroidal estrogen, mycoestrogen, or phytoestrogen. Though the biological potency of zearalenone is high, actual toxicity is low (Shier, 1998). Ochratoxin A (OTA), which is produced by a number of Aspergillus and Penicillium species, has been listed as possibly carcinogenic to humans (group 2B) by the International Agency for Research on Cancer (IARC). It causes renal toxicity, nephropathy and immune-suppression in several animal species, resulting in reduced performance parameters in animal production. Ochratoxin has also been detected in blood and other animal tissues and in milk, and has been implicated in the fatal human disease Balkan endemic nephropathy (Marquardt and Frohlich, 1992). Ergot alkaloids are classied as indole alkaloids, with lysergic acid as a common structure to all representatives of this group. They are produced in the sclerotia of species of Claviceps, which are common pathogens of various grass species. Uptake of these sclerotia, or ergots, has been associated with human diseases reported in the Middle Ages, leading to ergotism or St. Anthonys re after ingestion of cereals infected with ergot sclerotia, usually in the form of bread made from contaminated our (Bennet and Bentley, 1999). While modern methods of grain cleaning have almost eliminated ergotism as human disease, it is still an important veterinary problem. The principal animals at risk are cattle, sheep, pigs, and chickens. Clinical symptoms of ergotism in animals include gangrene, abortion, convulsions, suppression of lactation, hypersensitivity and ataxia (Bennet and Klich, 2003). Data on the toxicity of individual ergot alkaloids are scarce, since under eld conditions animals are exposed to complex feed mixtures with a varying composition of ergot alkaloids depending on the fungal strain, the host plant and on environmental factors. Systematic analyses of common grains and forage grasses would be necessary to establish a correlation between exposure to ergot alkaloids and adverse effects in individual animal species. The few data available, however, do not provide any evidence that ergot alkaloids accumulate in edible tissues, including milk and eggs and thus food from animal origin is unlikely to be an important source of human exposure (EFSA, 2005). The most recently described mycotoxins with relevance in human and animal nutrition are fumonisins, which were rst reported in South Africa in 1988 (Bezuidenhout et al., 1988; Gelderblom et al., 1988). Fumonisins are produced by a number of Fusarium species, notably Fusarium verticilliodides, Fusarium proliferatum and Fusarium nygamai, as well as Alternaria sp. The most abundantly produced member of this toxin family is fumonisin B1. Fumonisins cause severe animal diseases such as equine leukoencephalomalacia (ELEM) in horses (Marasas et al., 1988), and hydrothorax and porcine pulmonary edema in swine (PPE) (Colvin and Harrison, 1992; Halloy et al., 2005). Besides their hepatotoxicity (Gelderblom

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et al., 2001) and nephrotoxicity (Edrington et al., 1995) they affect also the immune system (Bhandari et al., 2002; Dombrink-Kurtzman, 2003). In the mid 1980s the topic of conjugated or masked mycotoxins received attention, because in some cases of mycotoxicoses, clinical observations in animals did not correlate with the low mycotoxin content determined in the corresponding feed. The unexpected high toxicity was attributed to undetected, conjugated forms of mycotoxins that hydrolyze to the precursor toxins in the digestive tract of animals. As part of their metabolism, plants are capable of transforming mycotoxins into conjugated forms (Berthiller et al., 2005a,b; Gareis, 1994). So far, natural occurrence of a zearalenone glucoside (Schneweis et al., 2002) and deoxynivalenol glucoside (Berthiller et al., 2005a,b) have been reported. Gareis et al. (1990) demonstrated that zearalenone-4-beta-d-glucopyranoside was decomposed during digestion, releasing zearalenone into the animal gut. As zearalenone-glycoside is not detected during routine analysis, but is hydrolysed during digestion, it seems likely that masked mycotoxins may contribute to cases of mycotoxicoses. Various mycotoxins may occur simultaneously, depending on the environmental and substrate conditions (Sohn et al., 1999). Considering this coincident production, it is very likely, that humans and animals are exposed to mixtures rather than to individual compounds. Heussner et al. (2006) evaluated the interactive (synergistic) cytotoxic effects of ochratoxin A, ochratoxin B, citrinin, and patulin which are produced by a number of Penicillium and Aspergillus species. By application of a step-wise approach to test combination toxicity, using various full factorial as well as a central composite experimental design, the interactive (synergistic) cytotoxic effects of the these four toxins were assessed. The results obtained in this study conrmed a potential for interactive (synergistic) effects of citrinin and ochratoxin A and possibly other mycotoxins in cells of renal origin. Creppy et al. (2004) proved a synergistic effect of fumonisin B1 in combination with ochratoxin A in vitro, testing three different cell lines, i.e. C6 glioma cells, Caco-2 cells and Vero cells. In vivo toxicity (LD50) was consistent with the in vitro data, (IC50 values) for both toxins as well as for the combination of 10 M OTA and variable concentrations of FB1 (1050 M).

3. International regulatory standards Agreement and setting of international regulatory standards is very difcult, as not only potential health benets but also political and economical issues have to be considered. Establishment of mycotoxin limits and regulations may be inuenced by several factors, both scientic and socio-economic in nature, including: availability of toxicological data, availability of data on the occurrence in different commodities, knowledge of the distribution of mycotoxin concentration within a lot, availability of analytical methods, national legislation, and need for sufcient food supply (Egmond and Jonger, 2004a,b).

Moreover, in any scientic attempt to establish a safe level of a mycotoxin it seems essential to dene safety and criteria to be used in its establishment. It is very difcult

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to set up valid and accepted levels of performance in animal production on a worldwide basis, and it is even more difcult to produce numbers and correlations that refer directly to the impact of hazards. Should safe levels refer to lethal effects, growth depression, and pathological ndings, or rather to the grade of immunological changes, deviations of enzymatic parameters or haematological factors? What is the proper criterion for safety? Diagnosis of animal mycotoxicosis is based on experimental studies with specic toxins and specic animals, very often under well-dened toxicological laboratory conditions, so that the results of such studies can be far from real-life or natural situations. Furthermore factors such as breed, sex, environment, nutritional status, as well as other toxic entities can affect the symptoms of intoxication. Diagnosis is very much dependent on receiving a sample of feed that was ingested prior to intoxication, but also on data from another representative group of animals of the facility and the results of a post-mortem examination (Cast Report, 2003). Furthermore, legislation calls for methods of control. Reliable analytical methods will have to be available to make enforcement of the regulations possible. Tolerance levels that do not have reasonable expectation of being met are wasteful in the resources that they utilize, and they may well condemn products that are perfectly t for consumption. AOAC International and CEN (the European Standardization Committee) have a number of standardized methods of analysis for mycotoxins available that have been validated in formal inter-laboratory method validation studies (Egmond and Jonger, 2004a). A survey on worldwide limits and regulations for mycotoxins was published by the FAO recently (FAO, 2004) and provides the status as per December 2003. Approximately 100 countries have developed specic limits for mycotoxins in food and feedstuffs with the population in these countries representing 87% of the worlds inhabitants. All countries with mycotoxin regulations have at least regulatory limits for aatoxin B1 or the sum of aatoxins B1, B2, G1, and G2 in foods and/or feeds. Comparing the situation in 1995 and 2003 the number of countries having regulations increased by approximately 30% and it appears that in 2003 more mycotoxins were regulated in more commodities and products, whereas the tolerance limits generally remained the same or tended to decrease. Regulations became more diverse and detailed with newer requirements regarding ofcial procedures for sampling and analytical methodology. At the same time several regulations have been harmonized between countries belonging to economic communities, such as Australia/New Zealand, EU, MERCOSUR, or some are in the stage of harmonization (FAO, 2004). The economic costs of mycotoxins are impossible to be determined accurately, but the US Food and Drug Administration (FDA) provided estimations based on a computer model. In the US alone the mean economic annual costs of crop losses from the mycotoxins aatoxins, fumonisins, and deoxynivalenol, were estimated to be USD 932 million (Cast Report, 2003). Some countries, with the United States, Argentina and China being probably the mostly affected, have to face unavoidable economic losses impacted by tighter mycotoxin regulations. In the event that the EU proposed standards were adopted worldwide, total export losses from fumonisin in maize could exceed USD 300 million annually, threefold higher than if the less stringent US standards were adopted. Likewise estimated export losses from aatoxins in peanuts might exceed USD 400 million under EU standards, which is vefold higher than if the US standards were adopted (Wu, 2004).

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4. Assessment and management of mycotoxin contamination 4.1. Mycotoxin testing Requirements on test results can be very different. Results created by rapid test systems can often be satisfactory, while under certain conditions validated chromatographic methods might be necessary. Based on continuous testing of ingredients as well as nished feeds, the on-site situation of a feed or animal production facility should be continuously monitored. It has been mentioned earlier that different fungi can produce different mycotoxins on the same commodity. Unfortunately there is nothing like a leading toxin as it was believed in the early days of research in this eld. Therefore a clear assessment can be made only if all the major naturally occurring toxins are tested. Testing for mycotoxins involves in general three different steps: sampling, sample preparation and the analytical procedure. Sampling comprises the selection of a sample of a given size from a bulk lot, grinding and taking a representative sub-sample of ground material. Because contaminated particles may not be distributed uniformly throughout the lot, the sample should be an accumulation of small portions taken from many different locations (Schmitt and Hurburgh, 1989). Sample preparation consists of several processes, i.e. the test samples are usually ground and sub-sampled, the mycotoxin is solvent-extracted from the sub-sample, and the extract is puried before the mycotoxin in the solvent is quantied. Some methods, such as antibody-based rapid test systems or more elaborate methods such as liquid-chromatography with (multiple) mass detection, might not require clean-up in case of simple and/or thoroughly validated matrices. The mycotoxin value, measured in the analytical step is eventually used to estimate the lot concentration or is compared to a maximum limit in order to classify the lot as acceptable or unacceptable. This means that a very small quantity of the lot is used in the nal quantication step to estimate the mycotoxin concentration of the entire lot. Because of the associated uncertainty the true mycotoxin concentration of a bulk lot cannot be determined with 100% certainty, or can 100% of lots sampled be correctly classied into good or bad categories (Whitaker, 2006). An example for the dimensions involved is as follows. If the original lot is 25 tonnes, the bulk lot sample is probably 25 kg, which means that the quantity inspected is reduced by a factor of 1000. Usually a sub-sample of 250 g is taken for further quantication, thus reducing the inspected product by a factor of 100,000. In practice about 1 g of product is represented in the solvent mixture which is directly used for quantication, so that in this example only 1 g out of the original 25,000,000 g is used to estimate the mycotoxin contamination of the entire lot (Whitaker, 2003). The variation associated with a mycotoxin test procedure is the sum of sampling, sample preparation and analytical variances, with sampling being usually the largest source of error (Whitaker, 2006). It accounts for up to 82% of variability and occurs mainly because usually only a small percentage of the kernels is contaminated, and with small sample sizes it is difcult to obtain a representative amount of contaminated kernels into the analytical sample. It is easier to select a representative sample from a moving stream of product than from a static lot such as trucks or rail cars (Whitaker, 2003). This principle is used in subsampling mills such as the Romer Series II mill or the RAS mill (Romer Labs. Inc., MO),

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where sample grinding and continuous sub-sampling are done in parallel, thus providing a good prole of the total sample. The sample preparation error, which accounts for to up to 9% of the total variability of a test procedure, can further be reduced by increasing the sub-sample size and grinding into ner particles (Whitaker, 2003). Analytical procedures for the determination of mycotoxins have improved continuously over the past years. Chromatographic methods have been used widely, including thin-layer chromatography (TLC), gas chromatography (GC) with electron capture detection (ECD) or mass selective detection (MS) as well as high-performance liquid chromatography (HPLC) with UV, uorescence detection, and, described in more recent publications, also with (multiple) mass spectrometry (Berger et al., 1999). The results of the most sophisticated chromatographic procedures depend on the efciency of the prior sample preparation, in particular on sampling, extraction and the further treatment of the extract, including any purication. As a large number of interfering compounds present in samples may contaminate the primary sample extract, these components must be removed as completely as possible for most method applications (Krska, 1998). Commonly used purication methods employ column chromatography, liquidliquid extraction, solid phase extraction columns (SPE), as well as immuno-afnity columns (IAC), and one-step multifunctional clean-up columns (MFC, Mycosep ), which in particular offer advantages of speed, simplicity, solvent efciency, and, in some cases increased recovery and lower cost (Trucksess et al., 1994; Fuchs et al., 2004). Without any rinsing steps being required, sample purication takes only 1030 s. This one-step purication process represents a very rapid and efcient alternative to conventional solid phase extraction (SPE) or immunoafnity (IAC) methods since both require usually three to four steps: precondition columns, retain extracted substances on packing material of the column, wash undesirable compounds, and elute analytes of interest (Fuchs et al., 2004). The major advantage of IACs is that purication is highly specic due to the antibodymycotoxin interaction principle, resulting in minimal interference in subsequent chromatography and allowing low detection levels. In contrary, a variety of immunological detection and quantication methods such as immuno sorbent assays (ELISAs) or radio immune assays (RIAs) are available, which require usually no further sample purication but have the major disadvantage that only one toxin can be determined by each test, referring to the specity of the antibodies. ELISA test kits are favored as high throughput assays with low sample volume requirements and testing times of less than an hour, some even less than 15 min. However, although the antibodies have the advantage of high specity and sensitivity to their mycotoxin target molecule, compounds with similar chemical groups may also interact with the antibodies. This so-called matrix effect is especially evident in cases of high complexity of the test material, which is in particular found with nished feed, and can lead to overestimates, underestimates, or even false negative or false positive results. Therefore it is critical that ELISAs are extensively studied on their accuracy and precision over a wide range of commodities. ELISA results of a certain material can be taken as trustworthy only if the kit is validated for the respective commodity (Zheng et al., 2004). Validation studies of ELISA test kits cover accuracy, precision, ruggedness and limit of detection in different commodities, which are also carried out in comparison to reference methods such as HPLC or GC, and include accelerated stability tests, as market conditions require usually a test shelf life of 1 year. Examples of validated ELISA kits are the Agraquant total aatoxin test and

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ochratoxin test, which uses HPLC with uorescence detection as a reference procedure (Zheng et al., 2005a,b); HPLC with a diode array detector (DAD) for the DON kit, while validation of the Agraquant T-2 toxin kit is achieved with HPLC-MS as a reference test system (Ung et al., 2004). Test kits based on the ELISA principle are not necessarily only in the microtiter format, thus requiring exact pipettes and photometric readers allowing quantitative determination. They can also be supplied in qualitative formats such as small cups (e.g. Aacup), where an enzymatic color reaction visualizes within 5 min if aatoxins are present or not at a certain cut-off level. This refers in general to so-called ow-through assays, where mycotoxinantibodies are coated to a membrane surface, which is soaked with the sample extract. Mycotoxins and mycotoxin-conjugates compete for the limited antibody binding sites, so that after washing and addition of a substrate solution a color reaction can be observed, indicating whether higher or lower levels than the proposed cut-off level are present in the sample. More recently immunochromatographic assays, which are also called lateral ow tests or strip tests, have received increasing market attention in particular for eld testing as they are not only inexpensive but also fast (5 min testing time) and very user-friendly. The principle of the AgraStripTM , which was validated and approved by USDA/GIPSA and is the only available kit for determination of total aatoxins at cut-off levels which are based on major international regulatory levels, i.e. 4, 10, and 20 g/kg, is that an antibodyparticle complex is dissolved in the assay diluent and mixed with sample extract und applied to the test strip. After a 5 min reaction time a positive sample containing total aatoxins above the respective cut-off level will result in no visual line in the test zone, while a negative sample containing aatoxins below the cut-off level will form a visible line in the test zone. The line in the control zone will appear always, regardless of the presence and concentration of aatoxins, as it indicates the validity of the test procedure (Zheng et al., 2006). One question is which is the analytical system of choice for a practical feed mill operation. Performance characteristics and features of quantitative methods and screening tests were described and discussed in detail by Krska et al. (2001, 2005) and Schneider et al. (2004). The most commonly used systems for rapid testing are without doubt the antibody-based test systems, with ELISAs as the fastest and most cost effective system, in case of high sample throughput and quick results requirements. Strip or cup formats are used for onsite testing processes which require inexpensive, very fast and simple-to-use applications but do not require exact quantitative results (Zheng et al., 2006). For larger operations an HPLC could be feasible, though the time factor for clean-up, chromatography, and result calculation needs to be considered and in particular evaluated against the high costs for equipment involved, particularly when mycotoxin analysis is its sole application. The advantage of HPLC is that the shortcoming of single analysis is overcome by parallel tests of the dened analytes. Very often the same extracts or puried sample solution can be used for the determination of the most relevant toxins or toxin groups within one chromatographic procedure (Berthiller et al., 2005b; Fuchs et al., 2004). As most analytical procedures are complex procedures involving several steps in which errors can occur, increasing the number of measurements made on the sub-sample extract can reduce these analytical errors (like conducting two replicates in ELISA testing), as well as using analytical methods with superior technology for regular checks on analytical

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procedures (i.e. reference methods). Quality assurance in mycotoxin analysis has become an important and critical issue. The importance of calibrant purity, check sample programmes, prociency testing by means of laboratory intercomparison tests and the use of certied reference materials are the basis for proper quality management in the laboratory (Egmond and Jonger, 2004a,b; Josephs et al., 2001; Krska et al., 2001). The prociency testing offered by the British FAPAS programme follows the International Harmonised Protocol for Prociency testing of Chemical Analytical Laboratories as well as the ISO guide and is probably the most elaborated and acknowledged system in mycotoxin testing. Certied reference materials (CRM) of major mycotoxins and their carrier commodities are offered by the Standard, Measurements and Testing Program of the European Commission, which started in the early 1980s (Cast Report, 2003). An extensive overview on reference materials for trichothecenes and method validation was given by Josephs et al. (2004). In case of contracting out analytical services to a private laboratory it is important to ensure that it is a laboratory that conducts mycotoxins on a routine basis and uses the correct conrmatory method. Unfortunately there is no multi-toxin rapid test format in the market that suits the practical conditions of a feed mill. Therefore routine testing needs to be established for the most likely occurring toxins out of a specic area. That is only viable when the origin of ingrediets is known, i.e. when the cereals are procured directly from the producing area. In the case of traded goods it is known by experience that different commodities are more often contaminated with certain mycotoxins. An empirical testing plan can then be suggested, based on comprehensive testing of the major traded goods from a certain area as a precondition. But as terms of trade change quickly so must the chart for the testing. Continuous testing and adjustment may lead to the development of an efcient testing plan according to the special needs of an operation. An overview about feedstuffs and their associated mycotoxins was given by Pettersson (2004). A mycotoxin testing plan is also dened by the mycotoxin test procedure (sample size, sample preparation method, and analytical method) and the respective accept/reject limit, i.e. the predened threshold that separates acceptable lots from unacceptable lots. Because of the variability associated with each step of the mycotoxin test procedure, the true mycotoxin concentration of a bulk lot cannot be determined with 100% certainty. As a result, some lots can still be misclassied by the sampling program and the magnitude of the risk associated with misclassication is directly related to the magnitude of the variability associated with the mycotoxin test procedure (Whitaker, 2003).

5. Prevention of mycotoxins Management practices to maximize plant performance and decrease plant stress can decrease mycotoxin contamination substantially. This includes planting adapted varieties, proper fertilization, weed control, necessary irrigation, and proper crop rotation (Ewards, 2004). But even the best management strategies cannot eliminate mycotoxin contamination in years favorable for disease development. Some fungi, such as several Fusarium species, are widespread colonizers of crop residues, where the pathogen survives during winter. Thus wheat stubble, maize stalks and rice stubble can be major sources of these moulds,

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which develop powerful inocula as temperatures increase in spring. Airborne release of spores might peak during and after rainy periods, distributing the fungal sources over wide distances, and causing epidemics. During harvest it is important to prevent excess damage to kernels, which may predispose them to infection during storage. Too high a moisture content is likewise a high risk factor for mycotoxin infestation, with the nal safe moisture content depending on the crop and the climatic conditions under which the commodity is stored, although drying to 150 g moisture content per kilogram or below is widely recognized as being suitable. It should be mentioned that when conditions are generally favorable for fungal contamination it is not uncommon for more than one type of fungus to be involved. During storage grain is often colonized by a succession of fungi, depending on temperature and moisture levels. Due to these possible interactions of several fungal species, grain may be contaminated with a number of different mycotoxins (Cast Report, 2003). CODEX: CAC/RCP 51-2003 (2005), covering the practice for prevention and reduction of mycotoxin contamination, provides valuable information on prevention strategies and good agricultural practices (GAP). For post-harvest mycotoxin control prevention of conditions that favor fungal growth and subsequent toxin production needs to be considered, i.e. factors such as water activity of stored products, temperature, grain condition, gas composition of the intergranular air, microbial interactions, and presence of chemical or biological preservatives (Shapira and Paster, 2004). Some common physical methods employed are mechanical separation of broken kernels, density segregation, color sorting, and screening. Electronic and hand sorting, density segregation and combinations thereof have been reported for removal of aatoxin contamination in peanuts (Cast Report, 2003). Simple washing procedures using water or sodium carbonate solution resulted also in some reduction of mycotoxins in maize and other grains. The use of mould inhibitors or preservation by acids can only reduce the amount of mould but does not inuence the content of mycotoxins generated prior to treatment. If mycotoxins have been produced earlier they will not be affected in any form by mould inhibitors or acid mixtures, as they are very stable compounds. Thus these toxic compounds remain in the formerly infected commodity even if no further mould can be seen or detected. The only way to really assess the quality of ingredients is the specic testing of mycotoxins or certain groups thereof.

6. Application of a hazard analysis critical control point (HACCP) system in mycotoxin control Implementation of an HACCP concept with emphasis on fungal toxins can be outlined as follows (Table 2) (see also, FAO, 2001): The rst critical point of action is to conduct a hazard analysis, preparation of a list of steps in the process where mycotoxin or mould infestation could occur and description of preventive measures. One could be the purchasing of ingredients. Many contracts do not mention mycotoxins at all and that is the rst point of action, for example by adding a clause with maximum acceptable levels of mycotoxin contamination to the contract.

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Table 2 A hazard analysis critical control point (HACCP) concept with focus on fungal toxins (based on FAO, 2001) Principles of a HACCP plan 1 Hazard analysis Exemplary measures Identify potential hazards, i.e. points where mycotoxin or mould infestation could occur, assess the risks associated and describe preventive measures Dene materials or processes, that have to be monitored for fungal contaminants Determine maximum tolerable toxin levels, that are acceptable within an operation Establish procedures for monitoring of critical control points, e.g. for sampling, sample preparation, analytical testing, etc. Establish a procedure for corrective actions, when monitoring at a critical control point indicates a deviation from an established critical limit, e.g. plan measures to prevent fungal infestation, introduce proper maintaining and sanitation procedures and develop strategies for detoxication (if applicable) Establish procedures for verication to conrm the effectiveness of the HACCP plan, e.g. an audit plan, sampling and testing plans Set up documentation of all procedures and records appropriate to these principles and their application

2 3 4 5

Critical control points Critical limits Monitoring procedures Corrective actions

6 7

Verication procedures Documentation and record keeping

The second step in a HACCP system is to determine the critical control points, i.e. determination of materials, products or production steps that have to be monitored for fungal contaminants. One rule of the thumb could be the ratio of tests conducted on ingredients versus tests done on nished products, which is for example 9 to 1. The third step is to establish critical limits, i.e. to determine the maximum tolerable toxin levels. What is the internal risk prole that is acceptable within an operation? Step number four is the establishment of procedures for monitoring the critical control points. This can include procedures for sampling, sample preparation and testing itself, or the out-sourcing of parts of or even the total analytical process. Step ve covers the establishment of corrective actions, which could comprise the introduction of certain cleaning procedures for silos, bins, hoppers, and elevators into the maintaining plan, as repeated contamination could originate from bins containing materials like wheat bran that have never been cleaned so that contamination might originate from and spread within the same operation and not only from purchased ingredients. Step six comprises the verication procedures. Step seven comprises the documentation and record keeping procedures.

7. Management of mycotoxin contamination in the daily operation In the case that mycotoxin manifestation is evident, the rst and most practical approach to date has been redirection into feed for less-susceptible animal species or blending of non-contaminated material with material above the limits thus lowering the average contamination levels to the accepted standards. Where that is not possible or is prohibited by law (as in Europe) other methods need to be applied.

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Although certain treatments have been found to reduce the levels of specic mycotoxins, no single method has been developed that is equally effective against the wide variety of mycotoxins which may occur together in various commodities (Shapira and Paster, 2004). The most commonly used strategy of reducing exposure to mycotoxins is the decrease in their bioavailability by the inclusion of various mycotoxin binding agents or adsorbents, which leads to a reduction of mycotoxin uptake and distribution to the blood and target organs. Various substance groups have been tested and used for this purpose, with aluminium silicates, in particular clay and zeolitic minerals, as the most commonly applied groups. Fr schl et al. (2000) investigated the aatoxin-binding capacities of a large number of o different aluminium silicates in relation to their physico-chemical properties. The tested materials were classied as bentonites (calcium bentonites, sodium bentonites, organophilic (modied) bentonites, acid-treated bentonites, as well as some special forms), zeolithes, diatomites, and vermiculites. Most minerals tended to show higher adsorption of aatoxins at higher pH levels, with sorption by vermiculites and zeolites being the most sensitive to pH alteration. No correlation could be found between the cation exchange capacity and sorption of aatoxins, while high specic surface and micro-pore volumina seemed to be related to better binding properties. Criteria considered important in the evaluation of potential mycotoxin binders are the stability of the sorbent-toxin bond, in order to prevent desorption of the toxin, as well as their effectiveness within a broad pH level since a product must work throughout the gastro-intestinal tract. An extensive review on the prevention of toxic effects of mycotoxins by non-nutritive adsorbent compounds was presented by Ramos et al. (1996). Galvano et al. (2001) reviewed dietary strategies to counteract the effects of mycotoxins, covering additional aspects such as antiodixants or plant ingredients as possible protectants. Among all aluminosilicates tested with regard to mycotoxin adsorption, hydrated aluminosilicate (HSCAS) have been the most extensively studied and described, in particular because of their promising aatoxin binding capacity (Phillips et al., 1988; Harvey et al., 1989; Kubena et al., 1990b). Studies on the elimination of mycotoxins other than aatoxins (e.g. trichothecenes, zearalenone, ochratoxins or fumonisins) from contaminated feedstuffs by the use of adsorbents show somewhat controversial results. While some authors suggest a positive inuence caused by the addition of glucomannans (Raymond et al., 2003; Swamy et al., 2004), or clays (Dakovic et al., 2005; Lemke et al., 1998; Tomasevic-Canovic et al., 1996; Carson and Smith, 1983), others observed no or only slight reduction of toxic effects (Kubena et al., 1990a, 1998; Huff et al., 1992; Galvano et al., 1998; Santin et al., 2002; Swamy et al., 2003; Bursian et al., 2004; D ll et al., 2005; Diaz et al., 2005). Avantaggaito et o al. (2004, 2005) performed extensive in vitro screening tests to evaluate the efcacy of various adsorbent materials in binding Fusarium mycotoxins. Most of the commercially available mycotoxin-binders failed in sequestering Fusarium mycotoxin in vitro. Only a small number of adsorbent materials possessed the ability to bind more than one mycotoxin. Cholestyramine was proven to be an effective binder for fumonisins and zearalenone in vitro, which was conrmed for zearalenone in experiments using a dynamic gastrointestinal model and for fumonisins in in vivo experiments. No adsorbent materials, with the exception of activated carbon, showed relevant ability in binding deoxynivalenol and nivalenol. The in vitro efcacy of activated carbon toward fumonisins was not conrmed in

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vivo by the biomarker assay. D ll et al. (2005) suggested the general necessity for a critical o verication of detoxifying agents in vivo. As it is known that in the case of trichothecenes the 12,13-epoxide ring is responsible for their toxic activity and that removal of this epoxide group entails a signicant loss of toxicity, research has focused on the identication of natural processes in which this reaction occurs. Several authors described this de-epoxidation bioreaction of ruminal or intestinal ora (Kollarczik et al., 1994; He et al., 1992; Swanson et al., 1987; King et al., 1984; Yoshizawa et al., 1983). A pure bacterial isolate of bovine rumen uid, which was able to bio-transform the epoxide group of trichothecenes into a diene, was identied as a new species of the genus Eubacterium. Inclusion of bacteria in feed contaminated with trichothecenes counteracted mycotoxin-induced performance decrease in piglets and broilers (Binder et al., 2001; Fuchs et al., 2002; Diaz et al., 2005). Recently, a novel yeast strain was isolated and characterized which has the capability of degrading ochratoxin A and zeralenone. On the basis of the yeasts afliation to the genus of Trichosporon and to its main property to degrade OTA and ZON (lat. voraredegrade), this strain was named Trichosporon mycotoxinivorans (MTV) (Schatzmayr et al., 2003, 2004, 2006; Molnar et al., 2004). A feeding trial which tested the efcacy of T. mycotoxinivorans to suppress ochratoxicosis could prove that the dietary inclusion of this yeast blocks ochtratoxin-induced immune suppression in broiler chicks (Politis et al., 2005).

8. Summary Mycotoxins are a chemically diverse group of fungal metabolites that have a wide variety of toxic effects. An internal risk prole has to be established in order to manage the risk of mycotoxins in a feed mill operation. Based on continuous testing of ingredients as well as nished product, the situation should be monitored continuously. Where action is necessary to detoxify contaminated materials, the choice depends on a consideration of the mycotoxins and species involved, in order to secure safety and performance of the farm animals in general and the whole food chain in particular. Blending with noncontaminated feed ingredients, re-routing contaminated grain to less susceptible animal species, or the addition of feed additives, based on adsorptive and, more recently, enzymatic modes of action, are widely used strategies to reduce mycotoxin-induced performance impairment.

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