Animal Feed Science and Technology133 (2007) 149–166
Managing the risk of mycotoxins inmodern 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 per-formance in animal production significantly. Due to the complex nature of these naturally occurringcontaminants and their elaborate analytics a risk-management concept has to be adopted in order toreduce the risk encounter to a defined and acceptable level.
Mycotoxins are secondary metabolites produced by filamentous fungi that cause a toxicresponse (mycotoxicosis) when ingested by higher animals. Fusarium, Aspergillus, andPenicillium are the most abundant moulds that produce these toxins and contaminate
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 veterinarycrisis near London, England, during which approximately 100,000 turkey poults died. Thismysterious turkey X disease was linked to a peanut meal contaminated with secondarymetabolites from Aspergillus flavus (aflatoxins) (Bennet and Klich, 2003).
Due to modern methods and to a growing interest in this field of research more than 300different mycotoxins have been differentiated to date. However for practical consideration inthe 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 occurrenceof mycotoxins, exposure to these substances is worldwide (Kuiper-Goodman, 2004).
For practical consideration in the feed manufacturing process aflatoxins, trichothecenes,zearalenone, ochratoxins, and fumonisins are of particular interest, though the extent ofharm each toxin (group) can cause is highly species-dependant.
2. Mycotoxins in feed
The most commonly known mycotoxins (Table 1) are the aflatoxins due to the fact thatthey represent one of the most potential carcinogenic substances known so far. They wererated as Class 1 human carcinogens by the IARC (International Agency for Research onCancer). Aflatoxins are produced by many strains of A. flavus and Aspergillus parasiticuson many different commodities, including cereals, figs, oilseeds, nuts, tobacco, and others(Diener et al., 1987). Aflatoxin B1 is moreover considered the main hepatocarcinogen in ani-mals, although effects vary with species, age, sex, and general nutritional conditions. Trout,ducklings and pigs are highly susceptible, ruminants being less susceptible (Weidenborner,2001).
Trichothecenes constitute a large group of mycotoxins produced by various speciesof moulds, in particular those belonging to the genus Fusarium. Approximately 170trichothecene mycotoxins have been identified to date, having a sesquiterpenoid 12,13-epoxytrichothec-9-ene ring system in common (Krska et al., 2001). Trichothecenes arepotent inhibitors of eukaryotic protein synthesis (Bennet and Klich, 2003). Epidemiolog-ical surveys have revealed that the predominant type-A and -B trichothecenes are widelydistributed in cereals as natural pollutants, whereas the macrocyclic trichothecenes occurrarely in food or feed. The most prevalent mycotoxins of these groups are deoxynivalenol(DON, vomitoxin), nivalenol (NIV), 3- or 15-acetyl-deoxynivalenol (AcDON), FusarenonX (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 com-pounds occur simultaneously (Fuchs et al., 2004) and are proven to cause synergistic effects(Weidenborner, 2001). In particular DON is prevalent worldwide in crops used for food andfeed production, and although it is one of the least acutely toxic trichothecenes, it should betreated 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 ofthem have high are acute toxicity. They may cause haematological changes and immune sup-pression, reduced feed intake and skin irritations as well as diarrhoea and haemorrhages of
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Table 1Overview of most relevant mycotoxins in animal production
Major classes ofmycotoxins
Most relevant representatives in grains andfeed
Examples of mycotoxin-producing fungi Effects observed in animals
internal tissues. An extensive toxicological evaluation of trichothecenes in animal feed wasconducted by Eriksen and Pettersson (2004). They concluded that trichothecenes are toxicto all tested species, but that sensitivity varied considerably between toxins and betweenspecies. While poultry are more sensitive to trichothecenes than ruminants, pigs seem tobe the most sensitive farm animals. Effects occurring at the lowest levels of trichotheceneswere 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-estrogeniceffects, which result in impaired fertility, stillbirths in females and a reduced sperm qualityin male animals. Due to its structural similarity to estradiol it is able to bind to estrogenreceptors in mammalian target cells, so that it is classified by some authors as nonsteroidalestrogen, mycoestrogen, or phytoestrogen. Though the biological potency of zearalenoneis high, actual toxicity is low (Shier, 1998).
Ochratoxin A (OTA), which is produced by a number of Aspergillus and Penicilliumspecies, has been listed as possibly carcinogenic to humans (group 2B) by the Interna-tional Agency for Research on Cancer (IARC). It causes renal toxicity, nephropathy andimmune-suppression in several animal species, resulting in reduced performance param-eters in animal production. Ochratoxin has also been detected in blood and other animaltissues and in milk, and has been implicated in the fatal human disease Balkan endemicnephropathy (Marquardt and Frohlich, 1992).
Ergot alkaloids are classified as indole alkaloids, with lysergic acid as a common struc-ture to all representatives of this group. They are produced in the sclerotia of species ofClaviceps, 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 toergotism or St. Anthony’s fire after ingestion of cereals infected with ergot sclerotia, usu-ally in the form of bread made from contaminated flour (Bennet and Bentley, 1999). Whilemodern methods of grain cleaning have almost eliminated ergotism as human disease, itis 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 field conditions ani-mals are exposed to complex feed mixtures with a varying composition of ergot alkaloidsdepending on the fungal strain, the host plant and on environmental factors. Systematicanalyses of common grains and forage grasses would be necessary to establish a correlationbetween exposure to ergot alkaloids and adverse effects in individual animal species. Thefew data available, however, do not provide any evidence that ergot alkaloids accumulatein edible tissues, including milk and eggs and thus food from animal origin is unlikely tobe an important source of human exposure (EFSA, 2005).
The most recently described mycotoxins with relevance in human and animal nutritionare fumonisins, which were first 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 asAlternaria sp. The most abundantly produced member of this toxin family is fumonisin B1.Fumonisins cause severe animal diseases such as equine leukoencephalomalacia (ELEM) inhorses (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
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 correlatewith the low mycotoxin content determined in the corresponding feed. The unexpectedhigh toxicity was attributed to undetected, conjugated forms of mycotoxins that hydrolyzeto the precursor toxins in the digestive tract of animals. As part of their metabolism, plantsare 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. Gareiset al. (1990) demonstrated that zearalenone-4-beta-d-glucopyranoside was decomposedduring digestion, releasing zearalenone into the animal gut. As zearalenone-glycoside isnot detected during routine analysis, but is hydrolysed during digestion, it seems likely thatmasked mycotoxins may contribute to cases of mycotoxicoses.
Various mycotoxins may occur simultaneously, depending on the environmental andsubstrate conditions (Sohn et al., 1999). Considering this coincident production, it is verylikely, that humans and animals are exposed to mixtures rather than to individual compounds.Heussner et al. (2006) evaluated the interactive (synergistic) cytotoxic effects of ochratoxinA, ochratoxin B, citrinin, and patulin which are produced by a number of Penicillium andAspergillus 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 inthis study confirmed a potential for interactive (synergistic) effects of citrinin and ochratoxinA and possibly other mycotoxins in cells of renal origin. Creppy et al. (2004) proveda synergistic effect of fumonisin B1 in combination with ochratoxin A in vitro, testingthree 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 thecombination of 10 �M OTA and variable concentrations of FB1 (10–50 �M).
3. International regulatory standards
Agreement and setting of international regulatory standards is very difficult, as not onlypotential health benefits but also political and economical issues have to be considered.
Establishment of mycotoxin limits and regulations may be influenced by several factors,both scientific 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 sufficient food supply (Egmond and Jonger, 2004a,b).
Moreover, in any scientific attempt to establish a safe level of a mycotoxin it seemsessential to define safety and criteria to be used in its establishment. It is very difficult
to set up valid and accepted levels of performance in animal production on a world-wide basis, and it is even more difficult to produce numbers and correlations that referdirectly to the impact of hazards. Should safe levels refer to lethal effects, growth depres-sion, and pathological findings, or rather to the grade of immunological changes, devia-tions of enzymatic parameters or haematological factors? What is the proper criterion for“safety”?
Diagnosis of animal mycotoxicosis is based on experimental studies with specific toxinsand specific animals, very often under well-defined toxicological laboratory conditions, sothat the results of such studies can be far from real-life or natural situations. Furthermorefactors such as breed, sex, environment, nutritional status, as well as other toxic entitiescan affect the symptoms of intoxication. Diagnosis is very much dependent on receivinga sample of feed that was ingested prior to intoxication, but also on data from anotherrepresentative 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 willhave to be available to make enforcement of the regulations possible. Tolerance levelsthat do not have reasonable expectation of being met are wasteful in the resources thatthey utilize, and they may well condemn products that are perfectly fit for consumption.AOAC International and CEN (the European Standardization Committee) have a numberof standardized methods of analysis for mycotoxins available that have been validated informal inter-laboratory method validation studies (Egmond and Jonger, 2004a).
A survey on worldwide limits and regulations for mycotoxins was published by the FAOrecently (FAO, 2004) and provides the status as per December 2003. Approximately 100countries have developed specific limits for mycotoxins in food and feedstuffs with thepopulation in these countries representing 87% of the world’s inhabitants. All countrieswith mycotoxin regulations have at least regulatory limits for aflatoxin B1 or the sum ofaflatoxins B1, B2, G1, and G2 in foods and/or feeds. Comparing the situation in 1995 and2003 the number of countries having regulations increased by approximately 30% and itappears that in 2003 more mycotoxins were regulated in more commodities and products,whereas the tolerance limits generally remained the same or tended to decrease. Regulationsbecame more diverse and detailed with newer requirements regarding official proceduresfor sampling and analytical methodology. At the same time several regulations have beenharmonized between countries belonging to economic communities, such as Australia/NewZealand, 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 USFood and Drug Administration (FDA) provided estimations based on a computer model. Inthe US alone the mean economic annual costs of crop losses from the mycotoxins aflatoxins,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 mostlyaffected, have to face unavoidable economic losses impacted by tighter mycotoxin regu-lations. In the event that the EU proposed standards were adopted worldwide, total exportlosses from fumonisin in maize could exceed USD 300 million annually, threefold higherthan if the less stringent US standards were adopted. Likewise estimated export losses fromaflatoxins in peanuts might exceed USD 400 million under EU standards, which is fivefoldhigher than if the US standards were adopted (Wu, 2004).
4. Assessment and management of mycotoxin contamination
4.1. Mycotoxin testing
Requirements on test results can be very different. Results created by rapid test systemscan often be satisfactory, while under certain conditions validated chromatographic methodsmight be necessary. Based on continuous testing of ingredients as well as finished feeds, theon-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 onthe same commodity. Unfortunately there is nothing like a “leading toxin” as it was believedin the early days of research in this field. Therefore a clear assessment can be made only ifall the major naturally occurring toxins are tested.
Testing for mycotoxins involves in general three different steps: sampling, sample prepa-ration and the analytical procedure. Sampling comprises the selection of a sample of a givensize 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, thesample should be an accumulation of small portions taken from many different locations(Schmitt and Hurburgh, 1989). Sample preparation consists of several processes, i.e. thetest samples are usually ground and sub-sampled, the mycotoxin is solvent-extracted fromthe sub-sample, and the extract is purified before the mycotoxin in the solvent is quanti-fied. Some methods, such as antibody-based rapid test systems or more elaborate methodssuch as liquid-chromatography with (multiple) mass detection, might not require clean-upin case of simple and/or thoroughly validated matrices. The mycotoxin value, measuredin the analytical step is eventually used to estimate the lot concentration or is comparedto a maximum limit in order to classify the lot as acceptable or unacceptable. This meansthat a very small quantity of the lot is used in the final quantification step to estimate themycotoxin concentration of the entire lot. Because of the associated uncertainty the truemycotoxin concentration of a bulk lot cannot be determined with 100% certainty, or can100% of lots sampled be correctly classified 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 reducedby a factor of 1000. Usually a sub-sample of 250 g is taken for further quantification, thusreducing the inspected product by a factor of 100,000. In practice about 1 g of productis represented in the solvent mixture which is directly used for quantification, so that inthis example only 1 g out of the original 25,000,000 g is used to estimate the mycotoxincontamination of the entire lot (Whitaker, 2003).
The variation associated with a mycotoxin test procedure is the sum of sampling, samplepreparation and analytical variances, with sampling being usually the largest source oferror (Whitaker, 2006). It accounts for up to 82% of variability and occurs mainly becauseusually only a small percentage of the kernels is contaminated, and with small sample sizesit is difficult to obtain a representative amount of contaminated kernels into the analyticalsample. It is easier to select a representative sample from a moving stream of product thanfrom a static lot such as trucks or rail cars (Whitaker, 2003). This principle is used in sub-sampling mills such as the Romer® Series II mill or the RAS mill (Romer® Labs. Inc., MO),
where sample grinding and continuous sub-sampling are done in parallel, thus providinga good profile of the total sample. The sample preparation error, which accounts for to upto 9% of the total variability of a test procedure, can further be reduced by increasing thesub-sample size and grinding into finer particles (Whitaker, 2003).
Analytical procedures for the determination of mycotoxins have improved continuouslyover the past years. Chromatographic methods have been used widely, including thin-layerchromatography (TLC), gas chromatography (GC) with electron capture detection (ECD) ormass selective detection (MS) as well as high-performance liquid chromatography (HPLC)with UV, fluorescence detection, and, described in more recent publications, also with(multiple) mass spectrometry (Berger et al., 1999). The results of the most sophisticatedchromatographic procedures depend on the efficiency of the prior sample preparation, inparticular on sampling, extraction and the further treatment of the extract, including anypurification. As a large number of interfering compounds present in samples may con-taminate the primary sample extract, these components must be removed as completely aspossible for most method applications (Krska, 1998). Commonly used purification methodsemploy column chromatography, liquid–liquid extraction, solid phase extraction columns(SPE), as well as immuno-affinity columns (IAC), and one-step multifunctional clean-upcolumns (MFC, Mycosep®), which in particular offer advantages of speed, simplicity, sol-vent efficiency, and, in some cases increased recovery and lower cost (Trucksess et al., 1994;Fuchs et al., 2004). Without any rinsing steps being required, sample purification takes only10–30 s. This one-step purification process represents a very rapid and efficient alternativeto conventional solid phase extraction (SPE) or immunoaffinity (IAC) methods since bothrequire usually three to four steps: precondition columns, retain extracted substances onpacking material of the column, wash undesirable compounds, and elute analytes of inter-est (Fuchs et al., 2004). The major advantage of IACs is that purification is highly specificdue to the antibody–mycotoxin interaction principle, resulting in minimal interference insubsequent chromatography and allowing low detection levels.
In contrary, a variety of immunological detection and quantification methods such asimmuno sorbent assays (ELISAs) or radio immune assays (RIAs) are available, whichrequire usually no further sample purification but have the major disadvantage that onlyone toxin can be determined by each test, referring to the specifity of the antibodies. ELISAtest kits are favored as high throughput assays with low sample volume requirements andtesting times of less than an hour, some even less than 15 min. However, although theantibodies have the advantage of high specifity and sensitivity to their mycotoxin targetmolecule, 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 testmaterial, which is in particular found with finished feed, and can lead to overestimates,underestimates, or even false negative or false positive results. Therefore it is critical thatELISAs are extensively studied on their accuracy and precision over a wide range of com-modities. ELISA results of a certain material can be taken as trustworthy only if the kit isvalidated for the respective commodity (Zheng et al., 2004). Validation studies of ELISAtest kits cover accuracy, precision, ruggedness and limit of detection in different commodi-ties, 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 lifeof 1 year. Examples of validated ELISA kits are the Agraquant® total aflatoxin test and
ochratoxin test, which uses HPLC with fluorescence detection as a reference procedure(Zheng et al., 2005a,b); HPLC with a diode array detector (DAD) for the DON kit, whilevalidation of the Agraquant® T-2 toxin kit is achieved with HPLC-MS as a reference testsystem (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. Aflacup), where anenzymatic color reaction visualizes within 5 min if aflatoxins are present or not at a certaincut-off level. This refers in general to so-called flow-through assays, where mycotoxin-antibodies are coated to a membrane surface, which is soaked with the sample extract.Mycotoxins and mycotoxin-conjugates compete for the limited antibody binding sites, sothat 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 thesample.
More recently immunochromatographic assays, which are also called lateral flow testsor strip tests, have received increasing market attention in particular for field testing asthey are not only inexpensive but also fast (5 min testing time) and very user-friendly. Theprinciple of the AgraStripTM, which was validated and approved by USDA/GIPSA and isthe only available kit for determination of total aflatoxins at cut-off levels which are basedon major international regulatory levels, i.e. 4, 10, and 20 �g/kg, is that an antibody–particlecomplex is dissolved in the assay diluent and mixed with sample extract und applied to thetest strip. After a 5 min reaction time a positive sample containing total aflatoxins above therespective cut-off level will result in no visual line in the test zone, while a negative samplecontaining aflatoxins below the cut-off level will form a visible line in the test zone. Theline in the control zone will appear always, regardless of the presence and concentration ofaflatoxins, 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 weredescribed 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-basedtest systems, with ELISAs as the fastest and most cost effective system, in case of highsample throughput and quick results requirements. Strip or cup formats are used for on-site testing processes which require inexpensive, very fast and simple-to-use applicationsbut do not require exact quantitative results (Zheng et al., 2006). For larger operationsan HPLC could be feasible, though the time factor for clean-up, chromatography, andresult calculation needs to be considered and in particular evaluated against the high costsfor equipment involved, particularly when mycotoxin analysis is its sole application. Theadvantage of HPLC is that the shortcoming of single analysis is overcome by parallel tests ofthe defined analytes. Very often the same extracts or purified sample solution can be used forthe determination of the most relevant toxins or toxin groups within one chromatographicprocedure (Berthiller et al., 2005b; Fuchs et al., 2004).
As most analytical procedures are complex procedures involving several steps in whicherrors can occur, increasing the number of measurements made on the sub-sample extractcan reduce these analytical errors (like conducting two replicates in ELISA testing), aswell as using analytical methods with superior technology for regular checks on analytical
procedures (i.e. reference methods). Quality assurance in mycotoxin analysis has become animportant and critical issue. The importance of calibrant purity, check sample programmes,proficiency testing by means of laboratory intercomparison tests and the use of certifiedreference materials are the basis for proper quality management in the laboratory (Egmondand Jonger, 2004a,b; Josephs et al., 2001; Krska et al., 2001). The proficiency testingoffered by the British FAPAS® programme follows the International Harmonised Protocolfor Proficiency testing of Chemical Analytical Laboratories as well as the ISO guide andis probably the most elaborated and acknowledged system in mycotoxin testing. Certifiedreference materials (CRM) of major mycotoxins and their carrier commodities are offeredby the Standard, Measurements and Testing Program of the European Commission, whichstarted in the early 1980s (Cast Report, 2003). An extensive overview on reference materialsfor trichothecenes and method validation was given by Josephs et al. (2004). In case ofcontracting out analytical services to a private laboratory it is important to ensure that it isa laboratory that conducts mycotoxins on a routine basis and uses the correct confirmatorymethod.
Unfortunately there is no multi-toxin rapid test format in the market that suits the practi-cal conditions of a feed mill. Therefore routine testing needs to be established for the mostlikely occurring toxins out of a specific area. That is only viable when the origin of ingredietsis known, i.e. when the cereals are procured directly from the producing area. In the case oftraded goods it is known by experience that different commodities are more often contam-inated with certain mycotoxins. An empirical testing plan can then be suggested, based oncomprehensive testing of the major traded goods from a certain area as a precondition. Butas terms of trade change quickly so must the chart for the testing. Continuous testing andadjustment may lead to the development of an efficient testing plan according to the specialneeds of an operation. An overview about feedstuffs and their associated mycotoxins wasgiven by Pettersson (2004).
A mycotoxin testing plan is also defined by the mycotoxin test procedure (sample size,sample preparation method, and analytical method) and the respective accept/reject limit,i.e. the predefined threshold that separates acceptable lots from unacceptable lots. Because ofthe variability associated with each step of the mycotoxin test procedure, the true mycotoxinconcentration of a bulk lot cannot be determined with 100% certainty. As a result, some lotscan still be misclassified by the sampling program and the magnitude of the risk associatedwith misclassification is directly related to the magnitude of the variability associated withthe mycotoxin test procedure (Whitaker, 2003).
5. Prevention of mycotoxins
Management practices to maximize plant performance and decrease plant stress candecrease 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 contaminationin 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,
which develop powerful inocula as temperatures increase in spring. Airborne release ofspores might peak during and after rainy periods, distributing the fungal sources over widedistances, and causing epidemics. During harvest it is important to prevent excess damage tokernels, which may predispose them to infection during storage. Too high a moisture contentis likewise a high risk factor for mycotoxin infestation, with the final “safe” moisture contentdepending 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 asbeing suitable. It should be mentioned that when conditions are generally favorable forfungal 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 temperatureand moisture levels. Due to these possible interactions of several fungal species, grainmay 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 mycotoxincontamination, provides valuable information on prevention strategies and good agriculturalpractices (GAP).
For post-harvest mycotoxin control prevention of conditions that favor fungal growthand subsequent toxin production needs to be considered, i.e. factors such as water activityof stored products, temperature, grain condition, gas composition of the intergranular air,microbial interactions, and presence of chemical or biological preservatives (Shapira andPaster, 2004).
Some common physical methods employed are mechanical separation of broken ker-nels, density segregation, color sorting, and screening. Electronic and hand sorting, densitysegregation and combinations thereof have been reported for removal of aflatoxin contam-ination in peanuts (Cast Report, 2003). Simple washing procedures using water or sodiumcarbonate 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 ofmould but does not influence the content of mycotoxins generated prior to treatment. Ifmycotoxins have been produced earlier they will not be affected in any form by mouldinhibitors or acid mixtures, as they are very stable compounds. Thus these toxic compoundsremain 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 specific testing of mycotoxinsor certain groups thereof.
6. Application of a hazard analysis critical control point (HACCP) system inmycotoxin control
Implementation of an HACCP concept with emphasis on fungal toxins can be outlinedas follows (Table 2) (see also, FAO, 2001):
• The first critical point of action is to conduct a hazard analysis, preparation of a list ofsteps in the process where mycotoxin or mould infestation could occur and descriptionof preventive measures. One could be the purchasing of ingredients. Many contracts donot mention mycotoxins at all and that is the first point of action, for example by addinga clause with maximum acceptable levels of mycotoxin contamination to the contract.
Table 2A hazard analysis critical control point (HACCP) concept with focus on fungal toxins (based on FAO, 2001)
Principles of a HACCP plan Exemplary measures
1 Hazard analysis Identify potential hazards, i.e. points where mycotoxin or mouldinfestation could occur, assess the risks associated and describepreventive measures
2 Critical control points Define materials or processes, that have to be monitored for fungalcontaminants
3 Critical limits Determine maximum tolerable toxin levels, that are acceptablewithin an operation
4 Monitoring procedures Establish procedures for monitoring of critical control points, e.g.for sampling, sample preparation, analytical testing, etc.
5 Corrective actions Establish a procedure for corrective actions, when monitoring at acritical control point indicates a deviation from an establishedcritical limit, e.g. plan measures to prevent fungal infestation,introduce proper maintaining and sanitation procedures anddevelop strategies for detoxification (if applicable)
6 Verification procedures Establish procedures for verification to confirm the effectiveness ofthe HACCP plan, e.g. an audit plan, sampling and testing plans
7 Documentation and recordkeeping
Set up documentation of all procedures and records appropriate tothese principles and their application
• 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 forfungal contaminants. One rule of the thumb could be the ratio of tests conducted oningredients versus tests done on finished products, which is for example 9 to 1.
• The third step is to establish critical limits, i.e. to determine the maximum tolerable toxinlevels. What is the internal risk profile that is acceptable within an operation?
• Step number four is the establishment of procedures for monitoring the critical controlpoints. 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 five covers the establishment of corrective actions, which could comprise the intro-duction of certain cleaning procedures for silos, bins, hoppers, and elevators into themaintaining plan, as repeated contamination could originate from bins containing mate-rials like wheat bran that have never been cleaned so that contamination might originatefrom and spread within the same operation and not only from purchased ingredients.
• Step six comprises the verification 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 first and most practical approachto date has been redirection into feed for less-susceptible animal species or blending ofnon-contaminated material with material above the limits thus lowering the average con-tamination levels to the accepted standards. Where that is not possible or is prohibited bylaw (as in Europe) other methods need to be applied.
Although certain treatments have been found to reduce the levels of specific mycotoxins,no single method has been developed that is equally effective against the wide variety ofmycotoxins which may occur together in various commodities (Shapira and Paster, 2004).The most commonly used strategy of reducing exposure to mycotoxins is the decrease intheir 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 targetorgans. Various substance groups have been tested and used for this purpose, with aluminiumsilicates, in particular clay and zeolitic minerals, as the most commonly applied groups.Froschl et al. (2000) investigated the aflatoxin-binding capacities of a large number ofdifferent aluminium silicates in relation to their physico-chemical properties. The testedmaterials were classified as bentonites (calcium bentonites, sodium bentonites, organophilic(modified) bentonites, acid-treated bentonites, as well as some special forms), zeolithes,diatomites, and vermiculites. Most minerals tended to show higher adsorption of aflatoxinsat higher pH levels, with sorption by vermiculites and zeolites being the most sensitive topH alteration. No correlation could be found between the cation exchange capacity andsorption of aflatoxins, while high specific surface and micro-pore volumina seemed tobe related to better binding properties. Criteria considered important in the evaluation ofpotential mycotoxin binders are the stability of the sorbent-toxin bond, in order to preventdesorption of the toxin, as well as their effectiveness within a broad pH level since a productmust work throughout the gastro-intestinal tract.
An extensive review on the prevention of toxic effects of mycotoxins by non-nutritiveadsorbent compounds was presented by Ramos et al. (1996). Galvano et al. (2001) revieweddietary strategies to counteract the effects of mycotoxins, covering additional aspectssuch as antiodixants or plant ingredients as possible protectants. Among all aluminosil-icates tested with regard to mycotoxin adsorption, hydrated aluminosilicate (HSCAS) havebeen the most extensively studied and described, in particular because of their promis-ing aflatoxin binding capacity (Phillips et al., 1988; Harvey et al., 1989; Kubena et al.,1990b).
Studies on the elimination of mycotoxins other than aflatoxins (e.g. trichothecenes, zear-alenone, ochratoxins or fumonisins) from contaminated feedstuffs by the use of adsorbentsshow somewhat controversial results. While some authors suggest a positive influencecaused by the addition of glucomannans (Raymond et al., 2003; Swamy et al., 2004), orclays (Dakovicı et al., 2005; Lemke et al., 1998; Tomasevic-Canovic et al., 1996; Carsonand Smith, 1983), others observed no or only slight reduction of toxic effects (Kubenaet al., 1990a, 1998; Huff et al., 1992; Galvano et al., 1998; Santin et al., 2002; Swamyet al., 2003; Bursian et al., 2004; Doll et al., 2005; Diaz et al., 2005). Avantaggaito etal. (2004, 2005) performed extensive in vitro screening tests to evaluate the efficacy ofvarious adsorbent materials in binding Fusarium mycotoxins. Most of the commerciallyavailable mycotoxin-binders failed in sequestering Fusarium mycotoxin in vitro. Only asmall number of adsorbent materials possessed the ability to bind more than one myco-toxin. Cholestyramine was proven to be an effective binder for fumonisins and zearalenonein vitro, which was confirmed for zearalenone in experiments using a dynamic gastroin-testinal model and for fumonisins in in vivo experiments. No adsorbent materials, withthe exception of activated carbon, showed relevant ability in binding deoxynivalenol andnivalenol. The in vitro efficacy of activated carbon toward fumonisins was not confirmed in
vivo by the biomarker assay. Doll et al. (2005) suggested the general necessity for a criticalverification of detoxifying agents in vivo.
As it is known that in the case of trichothecenes the 12,13-epoxide ring is responsiblefor their toxic activity and that removal of this epoxide group entails a significant lossof toxicity, research has focused on the identification of natural processes in which thisreaction occurs. Several authors described this de-epoxidation bioreaction of ruminal orintestinal flora (Kollarczik et al., 1994; He et al., 1992; Swanson et al., 1987; King etal., 1984; Yoshizawa et al., 1983). A pure bacterial isolate of bovine rumen fluid, whichwas able to bio-transform the epoxide group of trichothecenes into a diene, was identifiedas a new species of the genus Eubacterium. Inclusion of bacteria in feed contaminatedwith trichothecenes counteracted mycotoxin-induced performance decrease in piglets andbroilers (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 ofdegrading ochratoxin A and zeralenone. On the basis of the yeast’s affiliation to the genusof Trichosporon and to its main property to degrade OTA and ZON (lat. vorare—degrade),this strain was named Trichosporon mycotoxinivorans (MTV) (Schatzmayr et al., 2003,2004, 2006; Molnar et al., 2004). A feeding trial which tested the efficacy of T. myco-toxinivorans to suppress ochratoxicosis could prove that the dietary inclusion of thisyeast 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 varietyof toxic effects. An internal risk profile has to be established in order to manage the riskof mycotoxins in a feed mill operation. Based on continuous testing of ingredients aswell as finished product, the situation should be monitored continuously. Where actionis necessary to detoxify contaminated materials, the choice depends on a considerationof the mycotoxins and species involved, in order to secure safety and performance ofthe farm animals in general and the whole food chain in particular. Blending with non-contaminated feed ingredients, re-routing contaminated grain to less susceptible animalspecies, or the addition of feed additives, based on adsorptive and, more recently, enzymaticmodes of action, are widely used strategies to reduce mycotoxin-induced performanceimpairment.
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