Mycotoxins in Corn: Occurrence, Impacts, and Management

Chapter 9

Mycotoxins in Corn: Occurrence, Impacts, and Management Gary P. Munkvold*, Silvina Arias*, Ines Taschl† and Christiane Gruber-Dorninger† *Department of Plant Pathology and Microbiology, Seed Science Center, Iowa State University, Ames, IA, United States, † BIOMIN Holding GmbH, Getzersdorf, Austria

INTRODUCTION Mycotoxins are secondary metabolites of fungi that have toxic properties to animals, including humans. The broad definition of mycotoxins (Kirk et al., 2008) includes any nonenzymatic metabolite of a fungus that is injurious to another organism, but in practice, “mycotoxin” refers only to those toxins that are produced by fungi and injurious to animals other than insects. Mycotoxins can be found in a wide variety of food and animal feeds. Cereal grains, including corn, are among the most commonly contaminated commodities (CAST, 2003). Mycotoxin contamination of cereal grains occurs as a result of fungal infection or infestation of the plants during their development, harvest, or after their harvest. Mycotoxins comprise a serious, multifaceted economic problem, and corn is one of the three crops most commonly affected by mycotoxins. Economic losses in corn can be attributed to: 1) yield loss due to diseases induced by toxigenic fungi; 2) reduced crop value resulting from mycotoxin contamination; 3) losses in animal productivity from mycotoxinrelated health problems; and 4) human health costs. Additional costs associated with mycotoxins include the cost of management at all levels—prevention, sampling, mitigation, litigation, and research costs. These costs are shared by crop producers, animal producers, grain handlers and distributors, processors, consumers, and society in general (Pitt et al., 2012). Quantification of these costs is extremely difficult, but models have been proposed for estimating mycotoxin costs within limited contexts. One analysis (CAST, 2003) of the costs of mycotoxins (not including human health impacts) concluded that costs of about $0.5 to $1.5 billion/year occur in the United States. Aflatoxins undoubtedly have the largest impact in corn, and these losses were reported as $225 million/year. This estimate does not include mitigation costs: this aspect was reviewed by Robens and Cardwell (2003) who noted that testing costs alone for aflatoxins are $20–30 million/ year. Losses associated with deoxynivalenol in the United States were estimated at $655 million/year (CAST, 2003), but the majority of that figure can be assigned to losses in wheat. For fumonisins, losses were estimated only for the food industry at $11 million/year. More recent analyses indicate higher losses. A study estimating the economic impacts of aflatoxins in the USA under several climate change scenarios concluded that losses could range from $52 million to $1.68 billion annually (Mitchell et al., 2016). Using a model developed to estimate the impacts of Fusarium toxins in animal feeds in the USA, Wu (2007) concluded that fumonisins caused losses of up to $46 million annually. Taking into account higher fumonisin levels in ethanol coproducts used in animal feeds, a similar model generated estimated losses in the swine industry of up to $146 million annually due to fumonisin effects (Wu and Munkvold, 2008). Outside the USA, particularly in the developing world, human health impacts are the dominant cost associated with mycotoxins. However, health impacts of mycotoxins are the most difficult to measure and quantitative estimates are generally not available. It is clear that mycotoxins, especially aflatoxins, affect human health in developing countries. These effects are due to acute toxicoses and immunosuppression by mycotoxins, as well as chronic effects. Aflatoxins have caused numerous fatalities during the past two decades in Africa; for example, in Kenya between 2004 and 2007, 477 cases of aflatoxin poisoning were reported, with a 40% fatality rate (Daniel et al., 2011). Over 98% of individuals tested in several West African countries were positive for aflatoxin exposure (Cardwell and Miller, 1996). Diseases modulated by mycotoxins accounted for 40% of lost disability-adjusted life years in a 1993 World Bank report on human health (Miller, 1996). In Southeast Asia, the impact of aflatoxins has been estimated at $400 million/year, with most of the costs related to human health effects (Cardwell and Miller, 1996). A report from the National Academy of Science (NRC, 1996) concluded that mycotoxins probably contribute to human cancer rates, even in the United States. Corn. https://doi.org/10.1016/B978-0-12-811971-6.00009-7 Copyright © 2019 AACCI. Published by Elsevier Inc. in cooperation with AACC International. All rights reserved.

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Mycotoxins found in corn can be grouped according to the fungi that produce them. The major toxigenic fungi in corn are species of Aspergillus, Fusarium, and Penicillium. Members of each genus can produce diverse types of toxic compounds, but the most important mycotoxins in corn are aflatoxins, fumonisins, trichothecenes (especially deoxynivalenol), and zearalenone (Miller, 1995). Ochratoxins also can be significant mycotoxins in corn in some parts of the world. Tables 9.1 and 9.2 indicate which fungal species produce these and other mycotoxins. Mycotoxins probably have occurred in corn grain for as long as the crop has been cultivated. Toxigenic species of Fusarium are found in teosinte (Desjardins et al., 2000), the probable progenitor of domesticated corn (Galinat, 1988), and are closely associated with corn wherever it is grown (Marasas, 1996). It follows that mycotoxins also probably have had impacts on livestock and humans consuming corn throughout the history of its cultivation. Matossian (1989) described the first evidence of human mycotoxicoses possibly associated with corn, suggesting that epidemiological records and mortality patterns in colonial America were consistent with impacts of Fusarium mycotoxins. It is not clear, however, what impacts might have been associated with corn rather than small grains. At that time, about 60% of the starch in the diet of colonists came from corn, but most of the remainder came from rye, which is susceptible to toxigenic Fusaria (as well as ergot). The symptoms described as “throat distemper” in American colonists are consistent in many ways with poisoning by T-2 toxin, which can occur in corn and small grains. Problems with animals fed moldy corn may have been informally recognized for some time, but these impacts were not documented until fairly recently. A neurological disorder of horses (equine leucoencephalomalacia) associated with moldy feed was first described in 1850 (Graham, 1912) and it was first reproduced experimentally with moldy corn in 1902 (Butler, 1902). This problem was associated with a specific fungus in 1904 (Sheldon, 1904), but the mycotoxin itself (fumonisin B1) was not described until 1988 (Bezuidenhout et al., 1988; Gelderblom et al., 1988). Aspergillus flavus was reported to cause an ear mold of corn in 1920 (Taubenhaus, 1920), but the fungus was not recognized as toxigenic until the 1960s when it was shown to be associated with Turkey X disease (CAST, 2003). Subsequently, aflatoxins were considered to be only a storage problem, but during the 1970s it became clear that preharvest aflatoxin contamination could be widespread (Anderson et al., 1975; Lillehoj et al., 1975b). Estrogenic effects in swine due to consumption of moldy grain were reported in 1928 (McNutt et al., 1928), but the cause of this problem (zearalenone) was not described until 1967 (Mirocha et al., 1967). Trichothecene mycotoxins were not recognized as agents of mycotoxicoses until the late 1960s (Hsu et al., 1972), and the effects of deoxynivalenol (DON) on swine fed contaminated corn were not described until 1973 (Vesonder et al., 1973). Currently, several hundred potentially toxic compounds from fungi in corn are recognized, but risks associated with many of them have not been established. There are three crucial phases of corn production during which mycotoxins can develop: preharvest, harvest and drying, and postharvest (Wilson and Abramson, 1992). During each phase, environmental conditions have a strong influence on the magnitude of the mycotoxin problem. Additionally, toxigenic fungi are adapted to different phases. Some species are more aggressive as plant pathogens and can cause preharvest diseases in the field. These fungi have traditionally been described as “field fungi.” They may continue to grow and produce mycotoxins during the harvest and drying phase, but their development usually does not continue during storage because their activity is limited by low grain moisture content (Payne and Widstrom, 1992; Sauer et al., 1992; Widstrom, 1996). In corn, Fusarium species are the most common toxigenic fungi in this category. Aspergillus and Penicillium species are typically described as “storage fungi” because they are not aggressive pathogens and generally do not infect plants in the field, except when kernels are physically damaged. Infection and mycotoxin production by these fungi may be initiated during the harvest and drying phase, when kernels are subjected to physical injury. There are several other genera of storage fungi that are not known to be toxigenic, such as Mucor and Rhizopus (Sauer et al., 1992). Ability to grow at 18% grain moisture is often cited as the division between field fungi and storage fungi. These categories are not entirely clear-cut, as some species of Aspergillus and Penicillium can cause kernel rots in the field (Smith and White, 1988), notably Aspergillus flavus, which commonly causes aflatoxin contamination of corn in the field (Sauer et al., 1992). Conversely, some species of Fusarium will grow in storage if grain moisture content is not adequately controlled. Recognizing the inadequacies of describing toxigenic fungi simply as “field” or “storage” fungi, Miller (1995) defined four groups of toxigenic fungi, based on their interactions with plants at different stages of crop production: 1. Aggressive plant pathogens, such as Fusarium graminearum. 2. Opportunistic plant pathogens that grow and produce mycotoxins on senescent or stressed plants, such as Fusarium verticillioides (syn. F. moniliforme) and Aspergillus flavus. 3. Fungi that initially colonize the plant and predispose the commodity to mycotoxin contamination after harvest, such as Aspergillus flavus. 4. Fungi that are found in the soil or decaying plant material and later proliferate in storage if conditions permit, such as Penicillium verrucosum and Aspergillus ochraceous.

TABLE 9.1 Reported Mycotoxin Production for Selected Fusarium spp. That Colonize Corn Kernels

Fusarium avenaceum Fusarium boothii

X

X

X

X

X

X

X

X

X X

X

X

Fusarium culmorum

X

X

Fusarium dlamini

X

Fusarium equiseti

X

Fusarium globosum

X

Fusarium graminearum

X

X

X

X

X

X

X X

X X

X

X X

X X X

X

X

X

Fusarium poae

X

X

Fusarium proliferatum

X

Fusarium semitectum

X

X X

X

X

X

X

X X

?

X

X

X

X

b

Fusarium temperatum

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

X

?

X

X

X

X

X

?

X

?

X

X

X

X

X

X

X

? X

?

Fusarium thapsinum Fusarium verticillioides

X

X

X

X

Fusarium subglutinans

?

X

X

Fusarium solani Fusarium sporotrichioides

X

X

Fusarium meridionale

X

X

X

b

Fusarium oxysporum

X

X

?

X

?

X

X

Fusarium cortaderiae

X

X

X

b

Fusarium crookwellense

X

X

b

Fusarium chlamydosporum

Zearalenone

X

Nivalenol

Diacetoxyscirpenol

X

Deoxynivalenol

Trichothecenesa

X

T-2

X

Moniliformin

X

Fusarins

Fusaric acid

Fusaproliferin

Fumonisins

Enniatins

Cyclonerodiol

X

HT-2

Fusarium armeniacum

Culmorin

X

Chlamydosporol

X

Butenolide

Beauvericin

Fusarium acuminatum

Aurofusarin

Trichothecenes

X

X

X

X

X

X

X X

X

X

X

X

X X

a

Includes diacetoxyscirpenol, deoxynivalenol, nivalenol, HT-2, T-2, and all other known trichothecenes. Mycotoxin production has not been thoroughly investigated. Bold lettering indicates the most important mycotoxin-producing species in corn. ?¼Reports are questionable. b

From Marasas, W.F.O., Nelson, P.E., Toussoun, T.A., 1984. Toxigenic Fusarium Species. Identity and Mycotoxicology. Pennsylvania State University, University Park, 328 p; Munkvold, G., 2003b. Mycotoxins in corn-occurrence, impact, and management. In White, P.J., Johnson, L.A. (Eds.), Corn: Chemistry and Technology. second ed., American Association of Cereal Chemists, St. Paul, pp. 811–881; Desjardins, A.E., 2006. Fusarium Mycotoxins: Chemistry, Genetics, and Biology. National Center for Agricultural Utilization Research, Agricultural Research Service. US Department of Agriculture, American Phytopathological Society (APS Press), St. Paul, pp. 1–260; Leslie and Summerell, 2006; Jestoi, 2008; Logrieco et al., 1998; Goswami and Kistler, 2005; Toth et al., 2008; Moretti et al., 2007; Scauflaire et al., 2012.

TABLE 9.2 Mycotoxins Produced by Aspergillus and Penicillium Species Occurring in Corn Fungal Species

Aflatoxins

Cyclopiazonic Acid

Sterigmatocystin

Versicolorins

Citrinin

X

A. chevalieri

Penicillic Acid

Rubratoxins

X

238

A. alliaceus

Ochratoxins

X X

X

A. glaucus

Corn

A. flavus

X X

A. nomius

X a

X

A. ochraceus

A. parasiticus

X

X X

A. restrictus A. rubrobrunneus

X

A. sulphureus

X

A. sydowii

X X

A. pseudotamarii

X

X

A. ustus

X

A. versicolor

X

X

X

P. brevi-compactum P. chrysogenum

X

P. citrinum

X

P. cyclopiumb

X

P. expansum P. frequentans

X X

X

c

P. funiculosum P. oxalicum P. purpurogenumd

X

e

X

P. urticae

P. variabile P. verrucosum

X f

P. viridicatum

X

X

X

X

X

X

a

A. alutaceus var. alutaceus. P. aurantiogriseum. P. glabrum. d Includes P. rubrum. e P. griseofulvum. f Includes P. puberulum. b c

From Mislivec, P.B., Tuite, J., 1970. Temperature and relative humidity requirements of species of Penicillium isolated from yellow dent corn kernels. Mycologia 62(1), 75–88; Scott, P., 1977. Penicillium mycotoxins. In Wyllie, T. D., Morehouse, L.G. (Eds.), Mycotoxic Fungi, Mycotoxins, Mycotoxicoses, vol. 1. Marcel Dekker, New York, pp. 283–356; Sauer, D., Meronuck, R., Christensen, C., 1992. Microflora. In Sauer, D.B. (Ed.), Storage of Cereal Grains and Their Products, fourth ed., American Association of Cereal Chemists, St. Paul, pp. 313–340; Wilson, D., Abramson, D., 1992. Mycotoxins. In Sauer, D.B. (Ed.), Storage of Cereal Grains and Their Products, fourth ed., American Association of Cereal Chemists, St. Paul, pp. 341–391; Ito, Y., Peterson, S.W., Donald, T., Tetsuhisa, G., 2001. Aspergillus pseudotamarii, a new aflatoxin producing species in Aspergillus section Flavi. Mycol. Res. 105(2), 233–239; and CAST, 2003. Mycotoxins: Risks in Plant, Animal, Human Systems, Task Force Report No. 38, Ames.

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In this chapter, we summarize the occurrence of the major mycotoxins in corn, the nature and impacts of these mycotoxins, and strategies for management of mycotoxin problems in corn, with special emphasis on Fusarium mycotoxins. The information here is not comprehensive; many books have been published on the topic of mycotoxins. Other publications (Rodricks, 1976; Rodricks et al., 1977; Cole and Cox, 1981; Marasas et al., 1984; Wilson and Abramson, 1992; Miller and Trenholm, 1994; Sinha and Bhatnagar, 1998; CAST, 2003; Desjardins, 2006) are recommended for more comprehensive information about a wider range of mycotoxins, their toxicological properties, and the analytical methods used for their quantitation.

TOXIGENIC FUNGI IN CORN The genus Fusarium includes the most widespread toxigenic fungi in corn; Aspergillus species are associated with the most serious human health impacts for mycotoxin-contaminated corn, whereas species of Penicillium are secondarily important.

Fusarium Fusarium species cause two distinct diseases on corn ears in the field. Gibberella ear rot or “red ear rot” usually initiates from the tip of the ear and develops a red or pink mold covering a large proportion of the ear. It usually is caused by F. graminearum, but other closely related species such as F. culmorum or F. meridionale can cause identical symptoms and are important in some parts of the world (Bottalico, 1998; Munkvold, 2017). The common name of the disease is related to the sexual stage of F. graminearum, Gibberella zeae. Gibberella ear rot predominates in cooler areas or those with higher precipitation levels during the growing season (Bocˇarov-Stancˇi
c et al., 1997; Munkvold, 2003a, b). Fusarium ear rot typically occurs on random, groups, or on physically injured kernels and consists of a white or light pink mold. Fusarium verticillioides is the primary pathogen, but identical symptoms are caused by F. proliferatum, F. subglutinans, or F. temperatum (Munkvold, 2017). In the USA, Fusarium ear rot is the most common disease associated with corn ears and it can be found at low severity in nearly all corn fields late in the season (Kommedahl and Windels, 1981; White, 2016). Aside from causing ear-rot symptoms, F. verticillioides frequently can be isolated from symptomless kernels (Foley, 1962; Munkvold et al., 1997a, b). Fusarium ear rot and Gibberella ear rot are favored by distinctly different conditions. Fusarium ear rot is more common in warmer and drier areas, in comparison with Gibberella ear rot (Munkvold, 2003a, b). This pattern is associated with different temperature optima for F. graminearum vs. F. verticillioides. Gibberella ear rot is favored by high levels of moisture around silking, followed by moderate temperatures and high rainfall during the maturation period. Fusarium ear rot generally is favored by warm, dry weather during the grain-filling period (Marasas et al., 2000; Reid et al., 1999). Both diseases have been associated with insect injury and can be spread by insects (Dowd, 1998), but this relationship seems to be much more critical for Fusarium ear rot (Munkvold, 2003a, b). In experiments conducted in Iowa and Italy, there were very close correlations among insect injury, Fusarium ear rot, and fumonisin concentrations in corn (Munkvold, 2003a, b; Alma et al., 2005). In the Iowa experiments, correlations between insect injury and Fusarium ear-rot symptoms ranged from 0.51 to 0.92 in experiments conducted between 1996 and 2011, and correlations between insect injury and fumonisin concentrations ranged from 0.43 to 0.77 (Munkvold, 2003a, b; Bowers et al., 2013; Bowers et al., 2014). Fusarium species and their mycotoxins can develop in stored corn, but only when storage conditions are less than ideal. Generally, grain moisture of 19% or more is needed to support the development of Fusarium species in stored grain. This condition is more likely to occur in developing countries where adequate drying and storage facilities are lacking. Even in good storage facilities holding dried grain, high-moisture pockets can form as a result of rain or snow, moisture migration, or metabolic activity by insects or storage fungi that can grow at lower water activity. Effects of storage conditions on fungal growth and mycotoxins are discussed later in this chapter; also see Ominski et al. (1994). Frequent changes in taxonomy and nomenclature in the genus Fusarium have complicated efforts to document the occurrence of species. According to current taxonomic concepts, at least 22 toxigenic species of Fusarium can be found in corn (Table 9.1), many of which occur worldwide. The most important species are F. graminearum, F. proliferatum, F. subglutinans, F. temperatum, and F. verticillioides. F. verticillioides is indicated as the most common species globally, but the prevalence of F. verticillioides is affected by latitude. In tropical, subtropical, and warm temperate regions, F. verticillioides is the most common species. In temperate regions, the prevalence of F. verticillioides diminishes with increasing latitude. In the southern two-thirds of the United States, F. verticillioides dominates (Calvert et al., 1985; Leslie et al., 1990; Bullerman and Tsai, 1994; Leslie, 1995), but in the northern United States and Canada, F. subglutinans and F. graminearum are more prevalent (Munkvold and Stahr, 1994; Vigier et al., 1997). A similar latitudinal effect is evident in Europe, where F. verticillioides (and to some extent F. proliferatum) predominates in Spain and Italy (Bottalico, 1998; Castella et al., 1999), but F. subglutinans (and to some extent F. graminearum) are predominant

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in Central and northern Europe (Bottalico, 1998). Similarly, in South America, F. verticillioides is usually reported as the most common species in corn kernels (Chulze et al., 1996), but in cooler areas of Peru, F. subglutinans was the most common species (Logrieco et al., 1993). Fusarium temperatum is a more recently recognized species that previously was included within F. subglutinans. It appears to be common in N. America, Argentina, and Europe (Munkvold, 2017).

Aspergillus Several species of Aspergillus can infect corn kernels, primarily in storage, but also in the field. A. flavus and A. parasiticus are the most important in terms of mycotoxin production, and in the field, kernel-rot symptoms are usually caused by A. flavus. A. flavus appears as an olive-green, powdery mold. Under some conditions, A. flavus infection in the field can be extensive, with high levels of aflatoxins accumulating before harvest. Aspergillus species, in particular A. flavus, can be found worldwide in corn, but they are most common at latitudes between 26 degrees and 35 degrees North or South (Klich et al., 1992). The main conditions leading to A. flavus infection and subsequent aflatoxin accumulation are high temperature and drought stress. Aspergillus infection and aflatoxins in corn are a chronic problem in the southern United States, and sporadically cause significant problems in the Midwestern United States, in years with abnormally high temperatures and low rainfall (CAST, 2003). Aspergillus species are capable of growth at relatively high temperatures and low water activity, which makes them well-suited for growth in stored grain. Aspergillus ear-rot severity, A. flavus infection, and elevated aflatoxin levels also have long been associated with kernel injury due to insect feeding, in the field and in storage (Dowd, 1998; Windham et al., 1999). Damage to the husks and kernels provides easy access for fungal spores dispersed by air. Corn earworm, Helicoverpa zea, European corn borer, Ostrinia nubilalis, southwestern corn borer, Diatraea grandiosella, and fall armyworm, Spodoptera frugiperda, are caterpillar species that have been associated with elevated A. flavus infection. Additionally, several species of sap beetles (Carpophilus spp. and Glischrochilus spp.) have been implicated as vectors of A. flavus (Dowd, 1998; CAST, 2003). In tropical areas, other insects are associated with A. flavus infection. In the Republic of Benin in Africa, the ear borer, Mussidia nigrivenella, was the most important insect connected with A. flavus infection and aflatoxins, while the presence of maize weevils (Sitophilus zeamais) and sap beetles was associated with higher aflatoxins in one of the two years of the study. Corn ears with severe damage by M. nigrivenella had aflatoxin concentrations up to 514 ng/g, more than 40 times the concentrations in ears with minor insect damage (S
etamou et al., 1998). Insect damage to stored grain can be a significant cause of mycotoxin accumulation. Insect feeding physically damages the kernels and increases the temperature and moisture content of the grain, all of which make the kernels more susceptible to colonization by any toxigenic fungi. Beti et al. (1995) reported that aflatoxins were about six times higher in stored corn infested with maize weevils and A. flavus compared to A. flavus alone, and three times higher than in mechanically damaged corn. Maize weevil infestation caused an increase in grain moisture content associated with higher aflatoxin levels. Primary insect pests, including rice weevils, granary weevils, and maize weevils, develop within the corn kernel and tend to cause the most damage. Secondary pests, including flour beetles and Indian meal moth (Holscher, 2000), feed on kernels, especially broken kernels, but develop outside the kernel itself. Numerous other insects can be associated with stored grain and cause some damage, but usually they are minor compared to the primary and secondary pests. Several other species of Aspergillus can produce aflatoxins, but do so rarely (Table 9.2). Aside from A. flavus and A. parasiticus, another important toxigenic Aspergillus species is A. ochraceus, also known as A. alutaceus var. alutaceus, which produces ochratoxins, primarily in stored grains. At higher latitudes, such as in Canada, the major mycotoxin associated with Aspergillus spp. is sterigmatocystin, associated with A. versicolor (CAST, 2003).

Penicillium Penicillium species typically produce a blue-green, powdery mold on corn kernels, although the appearance differs among species. Like Fusarium, taxonomy in the genus Penicillium has undergone numerous changes and overlapping species designations exist. Several species of Penicillium are toxigenic, but the most common species found in corn are not associated with major mycotoxin problems. Several species of Penicillium infect corn in storage, but Penicillium oxalicum is the primary species known to cause an ear rot in the field (Payne, 1999). Like Fusarium and Aspergillus, infection in the field by Penicillium often occurs in kernels damaged by insects or other agents. Penicillium species such as P. aurantiogriseum and P. viridicatum can grow at low water activity and fare well in stored corn. Penicillium is more common in cooler climates than is Aspergillus. Some Penicillium species can grow at grain moisture contents of 16% to 17%. Kernels with "blue eye" have embryos infected with Penicillium. Most species of Penicillium are not associated with mycotoxin problems in corn, but Penicillium verrucosum and P. viridicatum can produce ochratoxins, cyclopiazonic

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FIG. 9.1 Molecular structures of fumonisins B1 and B2.

acid, penicillic acid, or citrinin (Mislivec and Tuite, 1970; Wilson and Abramson, 1992; Abramson, 1997; Payne, 1999) (Table 9.2). At higher latitudes, such as in Canada, P. verrucosum is the primary producer of ochratoxin A, which has received considerable attention in recent years as a potential health hazard in corn grain. Several other mycotoxins can be produced by Penicillium species, and these are covered in more detail in other publications (Wilson and Abramson, 1992; Scott, 1994; Abramson, 1997).

Stenocarpella Stenocarpella maydis, previously known as Diplodia maydis, has been associated with a neuromycotoxicosis in cattle consuming culture material of the fungus from South Africa, North, and South America (Marasas, 1978). Although this fungus is cosmopolitan, mycotoxicoses have not been reported outside South Africa and Argentina.

MAJOR MYCOTOXINS IN CORN AND THEIR IMPACTS Fumonisins Fumonisins are the most common mycotoxins found in corn, and they are found in several other crops. Fumonisins are produced primarily by Fusarium verticillioides and F. proliferatum, but several other species of Fusarium have been reported to produce fumonisins (Marasas et al., 2000). Minor amounts of fumonisin B2 can be produced by some species in Aspergillus section Nigri (Yoshizawa, 1983). The fumonisins are a family at least 28 polyketide mycotoxins with a linear 18- to 20-carbon backbone. Fumonisins B1 (Fig. 9.1), B2, and B3 are the most common and most intensively studied; fumonisin B1 is known chemically as 1,2,3-Propanetricarboxylic acid, 1,10 -[1-(12-amino-4,9,11-trihydroxy-2-methyl-tridecyl)2-(1-methylpentyl)-1,2-ethane-diyl] ester. It has the formula C34H59NO15 and also is known as macrofusine (Laurent et al., 1989). The fumonisin family consists of A, B, C, and P-series compounds. B-series fumonisins have a 20-carbon backbone with a terminal amine function, several hydroxyl functions, and two propane-1,2,3-tricarboxylate esters at various positions.

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A-series and P-series fumonisins differ due to alteration or replacement of the terminal amine group, while C-series fumonisins have a 19-carbon backbone (Eudes et al., 2000). Fumonisins were discovered fairly recently (1988), but they are now recognized as the most common mycotoxins found in corn on every continent, and research papers on fumonisins now number in the thousands. The investigations that led to the discovery of fumonisins were the result of efforts to identify the cause of high rates of esophageal cancer in some human populations of the Transkei region of South Africa (Marasas, 1996). Subsequently, fumonisins have been shown to have various toxic and carcinogenic effects on laboratory animals, and these have been reviewed in detail previously NTP (2001). The primary concerns about fumonisin exposure are related to its possible connections with human cancer and neural tube birth defects, and its acute toxicity to domestic livestock. Fatal diseases in domestic livestock are caused by fumonisins. In pigs, fumonisins cause pulmonary edema, and in horses, leukoencephalomalacia. Fumonisin B1 is hepatotoxic in all animals that have been tested, including mice, rats, equids, rabbits, pigs, and primates. Fumonisins are nephrotoxic in pigs, rats, sheep, mice, and rabbits. Fumonisin B1 is hepatocarcinogenic and nephrocarcinogenic in rats and hepatocarcinogenic in mice. This compound was the first specific inhibitor of sphingolipid metabolism to be discovered (Marasas et al., 2000). Fumonisin B1 is phytotoxic, and some evidence suggests it might be involved in pathogenicity of F. verticillioides toward corn seedlings. Fumonisins are not necessary for ear-rot infection or disease development (Desjardins et al., 2002), but some studies have indicated that fumonisin B1 contributes to symptom development in seedling disease caused by F. verticillioides (Arias et al., 2012). Fumonisins do not accumulate significantly in the tissues of animals consuming them (except for the liver and kidney) and are not excreted in milk (Marasas et al., 2000), although at least one study indicated very low levels of fumonisins in milk. Human exposure to fumonisins is common, and in the early stages of fumonisin research, a correlation with human esophageal cancer was noted. In several surveys, a very high incidence of the cancer was recorded in the Transkei region, and this was correlated with a higher incidence of F. verticillioides and fumonisin levels 20 times higher in the high-risk area compared to a nearby low-esophageal-cancer-risk area (Marasas et al., 2000). The significance of this finding was strengthened by the high level of corn consumption in these areas. Similar relationships between esophageal cancer and fumonisin exposure have been postulated for areas of China, northern Italy (Marasas et al., 2000), and Iran (Shephard et al., 2000), but the correlations for these areas are weaker, due to incomplete data or missing information. In 1993, the International Agency for Research on Cancer concluded that toxins derived from Fusarium verticillioides are possibly carcinogenic to humans (Group 2B) (Marasas et al., 2000). However, esophageal symptoms have not been experimentally reproduced (Gelderblom et al., 2001; Howard et al., 2001) and the role of fumonisins in this disease has not been definitively shown. Due to its nephrotoxic effects, JECFA has established a PMTDI for fumonisin ingestion by humans (2.0 mg total fumonisins/kg body wt/day). Another human health impact associated with fumonisin exposure is an increased incidence of neural tube birth defects. Associations between fumonisin ingestion and elevated incidence of NTDs have been observed along the US-Mexico border, in Guatemala, China, and South Africa (Marasas et al., 2004; Missmer et al., 2006). In studies with mice, fumonisins have been shown to cause neural tube defects and other similar birth defects by interfering with folate receptors in embryonic neuroepithelial cells (Marasas et al., 2004; Gelineau-van Waes et al., 2009). Therefore, there is strong evidence that maternal fumonisin exposure contributes to the incidence of NTDs in some human populations. Fumonisins cause acute and chronic effects in various livestock species. Horses and other equids are among the most sensitive animals, and the main effect of fumonisin consumption is leukoencephalomalacia, the liquefaction of portions of the brain tissue. Leukoencephalomalacia is fatal and apparently occurs only in equids. Large outbreaks of this disease occurred during the 19th century, the early 20th Century, and recently in 1989–90. Sporadic cases have occurred since that time. Liver damage also occurs in horses consuming fumonisins. Fumonisins also can be fatal to pigs. Lung edema was reported as a consequence of feeding culture material of F. verticillioides in 1981, seven years before the discovery of the fumonisins. Symptoms include the accumulation of clear yellow fluid in the pleural cavity and interstitial and interlobular areas of the lungs. Purified fumonisins have been shown in several studies to cause symptoms identical to those caused by consumption of the culture material. Pigs consuming fumonisins also show signs of liver toxicity, whether or not there are symptoms of pulmonary edema (Marasas et al., 2000). Outbreaks of pocine pulmonary edema (PPE) were common during the same period (1989–91) that equine leukoencephalomalacia (ELEM) was widespread (Marasas, 1996), and the disease continues to occur sporadically. Effects of fumonisins on lipid metabolism have been proposed as a common mechanism for carcinogenic and toxic properties, with cardiovascular effects possibly leading to the diverse acute toxicities observed among different animal species (Smith et al., 2002; Constable et al., 2003). Cattle and sheep are comparatively resistant to fumonisins, with mild liver damage occurring at feed levels above 100 mg/g in feed and moderate feed refusal and reduced growth at 200 mg/g or more. Poultry are more resistant to fumonisins than other common livestock,

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and signs of fumonisin poisoning do not occur until feed levels are above 100 mg/g for turkey poults and 200 mg/g for broiler chicks. Effects include liver damage, diarrhea, and rickets or related tibial (leg) lesions. Field outbreaks of fumonisin poisoning in poultry have not yet been reported. Table 9.3 lists some concentrations of fumonisins and other common mycotoxins associated with acute and chronic effects. Because of the hazards posed to humans and livestock by fumonisins in grains and food, many countries have imposed guidelines or regulatory limits on allowable levels of fumonisins (Table 9.4) (Egmond and Jonker, 2004).

TABLE 9.3 Expected Detrimental Feed Concentrations of the Major Mycotoxins Found in Corn Aflatoxins Animal Species

Concentration (ng/g)

Swine

200

Slow growth, reduced feed efficiency

400

Liver damage and immune suppression

400

Tissue residues

700

Mild liver damage, reduced growth and feed efficiency

1000

Moderate liver damage, weight loss

2000

Severe liver damage, jaundice, death

Feeder cattle

Dairy cows

Poultry

Horses

Effect

20

Detectable aflatoxin in milk

1500

Decreased milk production

250

Turkeys have reduced growth

210

No effect on broiler chicks

420

Broiler chicks lose weight, moderate liver damage after 3 weeks

400

Liver damage and immune suppression

Zearalenone Concentration (mg/g)

Duration

Effect

Prepubertal gilts

1–5

3–7 days

Hyperestrogenism, prolapse

Sexually mature open gilts

3–10

Mid-cycle (day 11–14)

Anestrus, pseudopregnancy

Bred sows

5–30

1st trimester

Early embryonic death, small litters

Juvenile boars

10–50

Indefinite

Reduced libido, small testicles

Mature boars

200

Indefinite

No effect

Virgin heifers

12

Open heifers

Reduced conception

Dairy cows

50

Open cows

Reduced conception

200

Indefinite

No effect

Swine

Cattle

Poultry Broilers & turkey poults

Deoxynivalenol (DON, vomitoxin) Swine Feeder pigs

1–3

1–5 days

Reduced feed intake

Feeder pigs

5–10

1–5 days

50% reduction in feed intake, vomiting Continued

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TABLE 9.3 Expected Detrimental Feed Concentrations of the Major Mycotoxins Found in Corn—cont’d Aflatoxins Animal Species Feeder pigs

Concentration (ng/g)

Effect

10–40

1–5 days

Complete feed refusal, vomiting

3.5

Gestation 1–52 days

Lower fetal weights

Feeder cattle

10

Indefinite

No effect

Dairy cows

6

6 weeks

No effect or slightly reduced feed intake

Dairy cows

12

10 weeks

No effect on milk production

50

Indefinite

No effect

Sows Cattle

Poultry Broilers and turkey poults

Fumonisins (FB1 and/or FB2) Horses

>10

30 days

Liver damage, leukoencephalomalacia, death

Swine

>25

30 days

Reduced gain and feed efficiency, mild liver damage

>50

10 days

Reduced gain and feed efficiency, moderate liver damage

>100

5 days

Severe pulmonary edema, death

>100

30 days

Slightly reduced gain, mild liver damage

>200

14 days

Reduced feed intake and gain, moderate liver damage

Turkeys

>100

7–21 days

Reduced feed intake, liver damage, diarrhea, rickets, tibial lesions

Chickens

>200

7–21 days

Reduced feed intake, liver damage, diarrhea, rickets, tibial lesions

Cattle and sheep

From Munkvold, G.P., Osweiler, G., Hartwig, N., 1997b. Corn Ear Rots, Storage Molds, Mycotoxins, and Animal Health. Iowa State University, University Extension, Ames, Extension and outreach publications 24, pp. 1–15.

Trichothecenes The trichothecene mycotoxins are sesquiterpenoid compounds produced by several Fusarium species, including F. graminearum, F. culmorum, F. sporotrichioides, and F. poae, and other fungi. A characteristic of the trichothecenes is a tetracyclic 12,13-epoxytrichothec-9-ene ring that can be chemically substituted at several positions, resulting in multiple derivatives (Wilson and Abramson, 1992). There are more than 200 trichothecenes, which have been classified into four groups (types A, B, C, and D) based on substitutions at C-8 and other positions around the core structure. Trichothecenes in corn are typically type A (T-2 and related compounds) or B (deoxynivalenol and related compounds). The best-known trichothecenes include deoxynivalenol (also known as DON or vomitoxin), T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and nivalenol (Fig. 9.2). Deoxynivalenol is 3a, 7a,15-trihydroxy-12,13-epoxytrichothec-9-en-8-one. Its formula is C15H20O6. T-2 toxin is 3a-hydroxy-4,15-diacetoxy-8a-(3-methylbutyryloxy)-12,13-epoxytrichothec-9-ene, and its formula is C24H34O9. HT-2 toxin is 3a,4b-dihydroxy-4,15-diacetoxy-8a-(3-methylbutyryloxy)-12,13epoxytrichothec-9-ene, and its formula is C22H23O8. Diacetoxyscirpenol is 3a-hydroxy-4,15-diacetoxy-12,13epoxytrichothec-9-ene, and its formula is C19H26O7. Nivalenol is 3 a,4b,7a,15-tetrahydroxy-12,13-epoxytrichothec9-en-8-one, and its formula is C15H20O7. For more detailed chemical properties of trichothecenes and other Fusarium mycotoxins, see Cole and Cox (1981), Savard and Blackwell (1994), or Ueno (1983). Trichothecenes are associated with severe mycotoxicoses in humans and animals. Acute toxicity of trichothecenes tends to be much higher than that of the fumonisins. Among the better-known trichothecenes, intraperitoneal LD50 values for mice range from 3.0 mg/kg body weight for T-2 toxin up to 77 mg/kg for deoxynivalenol. Effects of trichothecenes also encompass a wide range of symptoms (Table 9.3), including weight loss, decreased feed conversion, feed refusal, vomiting,

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TABLE 9.4 Guidelines for Safe Levels of Fumonisins in Human Food and Animal Feeds Proposed by United States Food and Drug Administration (2001) Product or Animal Species

Concentration (mg/g)

Human food Dry-milled products

4

De-germed dry-milled products

2

Corn for masa

4

Dry-milled corn bran

4

Popcorn

3

Animal feeds

Corn portion of diet

Total diet

Horses

5

1

Rabbits

5

1

Catfish

20

10

Swine

20

10

Ruminants

60

30

Mink

60

30

Poultry

100

50

Ruminant, mink, and poultry breeding stock

30

15

All others (including dogs and cats)

10

5

FIG. 9.2 Molecular structures of the most prevalent trichothecene mycotoxins deoxynivalenol, T-2 toxin, HT-2 toxin, and diacetoxyscirpenol.

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bloody diarrhea, severe dermatitis, hemorrhage, decreased egg production, altered immune function, and death (CAST, 2003). The most susceptible organs and systems are the mucous membranes of the digestive system, the skin, and the immune system. Mycotoxicoses related to trichothecenes can be complicated to diagnose for several reasons, including the fact that F. graminearum and other species can produce multiple mycotoxins, and symptoms may not be due to a single toxin. Synergistic effects of mycotoxins, resulting in more serious effects than would be expected from observed concentrations, also are possible. Synergy among mycotoxins has been investigated and documented (Marasas et al., 2000; CAST, 2003), but still is not well understood. Deoxynivalenol is the most commonly encountered, but among the least toxic of the trichothecenes, and the most common effect of deoxynivalenol is feed refusal by swine. In fact, most animals refuse to consume high concentrations of trichothecenes and never achieve the full range of reported toxic effects. Reduced feed intake in swine begins at approximately 1 mg/g deoxynivalenol in feed and increases proportionally until complete refusal occurs at >10 mg/g, although the response is highly variable. The primary documented clinical health effects in livestock as a result of deoxynivalenol are from reduced feed intake, but other effects, especially vomiting (at about 20 mg/g for swine), altered immune function, and even death, have been demonstrated experimentally. The toxicological effects of deoxynivalenol have been reviewed (Rotter, 1996; Eriksen and Pettersson, 2004; Rocha et al., 2005; Arunachalam and Doohan, 2013). Other livestock species are much less sensitive to deoxynivalenol than swine. Feed refusal has been reported in poultry, but in at least one study, no effects were observed at concentrations of 50 mg/g. Beef cattle tolerate deoxynivalenol-contaminated feed at approximately 10 mg/g in the ration. Limited studies in dairy cows indicate 6 mg/g may slightly reduce feed intake in some cows. Exposure of humans to low levels of deoxynivalenol may be common, especially in wheat products. Low levels of deoxynivalenol have been found in meat, milk, and eggs. Human exposure data suggest that humans are probably more sensitive than swine to deoxynivalenol (Miller et al., 2001). Regulations related to deoxynivalenol exist in several countries; in the United States, the FDA has issued advisory levels for the toxin animal feeds and food products for humans (Table 9.5). Deoxynivalenol tolerances are very low or zero for brewing products, because deoxynivalenol can persist in beer and cause serious quality problems. Trichothecenes inhibit protein and DNA synthesis, and this mechanism is responsible for many of their toxic effects, including feed refusal. The more severe toxicoses associated with trichothecenes are attributed primarily to Group A trichothecenes, although they often occur in combination with Group B trichothecenes. T-2 toxin causes hemorrhaging, inflammation of the digestive tract, vomiting, diarrhea, decreases in milk production, oral lesions, edema (in chickens), feed refusal, and reduced weight gain (CAST, 2003; Eriksen and Pettersson, 2004; Rocha et al., 2005; EFSA, 2011a; Arunachalam and Doohan, 2013; EFSA, 2013a). These symptoms can be fatal in some cases. T-2 toxin was reported to cause “moldy corn toxicosis” in cattle (Hsu et al., 1972), a fatal condition characterized by bloody diarrhea and hemorrhage of the stomach, heart, intestines, lungs, bladder, and kidneys. These symptoms were likely due to a combination of trichothecenes (Mirocha, 1983). In addition to the general symptoms described above, in poultry, T-2 toxin induces oral lesions, edema of the body cavity, reduced egg production, neurotoxic effects, and death. In swine, infertility effects have been associated with T-2 toxin, in addition to aforementioned symptoms. Diacetoxyscirpenol has high acute toxicity to animals; in swine it caused vomiting, posterior paralysis, lethargy, extreme hunger, oral lesions and hemorrhagic lesions digestive tract, and frequent defecation (Mirocha, 1983; CAST, 2003). Severe diseases in humans have been attributed to trichothecene exposure, but definitive cause-and-effect evidence is lacking. Most of these outbreaks have not been associated with corn consumption. One reason is that the Group

TABLE 9.5 United States Food and Drug Administration Advisory Levels for Deoxynivalenol in Grains and Grain Products (CAST, 2003) Product

Concentration (mg/g)

Finished wheat products for human use

1 Grain portion of diet

Total diet

Grains and grain products destined for beef cattle >4 months in age

10

5

Grains and grain products destined for chickens

10

5

Grains and grain products destined for swine

5

1

Grains and grain products for all other animals

5

2

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A trichothecenes are more common in small grains such as wheat and barley. Fusarium species that produce high levels of Group A compounds (F. sporotrichioides, F. poae, F. acuminatum) more commonly infect those crops than corn. A second reason is that wheat is a staple in the diets of many more people than is corn, and in parts of the world where corn is a dietary staple, trichothecene-producing species such as F. graminearum are less common in corn than are other Fusarium species. The most widely known human disease associated with trichothecenes is alimentary toxic aleukia, known as ATA and many other names. This disease has been reported primarily from the former USSR. Symptoms are leukopenia, high fever, low white blood cell counts, necrotic angina, bleeding from the nose, throat, and gums, and a hemorrhagic rash. Outbreaks have been reported from the former USSR since 1913, with the most severe occurring 1994, when 10% of the population was affected in one district and mortality rates reached 60% in some counties (Beardall and Miller, 1994). Another wellknown human disease associated with trichothecenes is called “red-mold disease.” Symptoms are nausea, vomiting, diarrhea, abdominal pain, throat irritation, and fever, and the syndrome is associated with F. graminearum and other species in wheat, rice, and barley. Similar symptoms have been reported in China since 1961, associated with consumption of Fusarium-contaminated corn and wheat, and in India, associated with wheat. In these cases, deoxynivalenol seems to be the mycotoxin most closely associated with the symptoms. Human mycotoxicoses associated with Fusarium fungi have been reviewed (Ueno, 1983; Beardall and Miller, 1994).

Zearalenone Zearalenone is an estrogenic compound once known as F-2 toxin. It is known chemically as 6-(10-hydroxy-6-oxo-trans-1undecenyl)-b-resorcylic acid lactone (Fig. 9.3), and its formula is C18H22O5. With an LD50 higher than that of sodium chloride, zearalenone is a potent fungal estrogen, rather than a toxin (Wilson and Abramson, 1992). Hyperestrogenism in swine is the most commonly reported effect of zearalenone, and its impact is significant. Swine seem to be more sensitive than any other livestock species. A commonly reported symptom is vulvovaginitis, an inflammation of the vulva in gilts and sows, However, the use of the term “vulvovaginitis” has been questioned (Hagler Jr et al., 2001). Prolapse of the vagina or rectum, inflammation of the mammary glands, and atrophy of the ovaries are other symptoms (Prelusky et al., 1994). These and other mechanisms combine to interfere with the reproductive capabilities of sows. Zearalenone will induce clinical signs of estrus or vulvovaginitis in prepubertal gilts at dietary concentrations as low as 1–5 mg/g. With removal from toxic diets, pigs return to normal in 3–7 days. The first estrus may be delayed, or there may be continuous estrus or pseudopregnancy. Dietary zearalenone as low as 3 mg/g has caused this effect in some sows. Litter size and milk production may be reduced when zearalenone levels are high (>5 mg/g). Loss of pregnancy and increased fetal mortality have been reported, but several studies have disputed this effect (Prelusky et al., 1994). Generally, grain contaminated with zearalenone at any level is not recommended for sows or replacement gilts. Cattle are considerably more resistant to the effects of zearalenone than are swine, although field cases have been reported with symptoms similar to those in swine (Prelusky et al., 1994). In virgin dairy heifers, conception rate is reduced when dietary zearalenone is greater than 12.5 mg/g. Poultry are not affected by zearalenone except at high levels, but reduced egg production has been reported. Livestock reproductive effects of zearalenone were reviewed by Dickman and Green (1992). The impact of zearalenone on humans has not been established, but available data suggests that we have sensitivity similar to that of pigs; due to its hormonal effects, JECFA has established a PMTDI for zearalenone ingestion by humans. Zearalenone appears to be effective for treatment of postmenopausal symptoms in women, and as an oral contraceptive. Both zearalenone and zearalenol have been patented for this use (Hagler Jr et al., 2001).

FIG. 9.3 Molecular structure of zearalenone.

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Other Fusarium Toxins Moniliformin has been implicated in various field mycotoxicoses, especially in poultry. It is the potassium or sodium salt of 1-hydroxycyclobut-1-ene-3,4 dione (Fig. 9.4) and has an LD50 of 20.9 to 29.1 mg/kg body weight for intraperitoneal injection in mice (Bryden et al., 2001) and approximately 25 mg/kg body weight for oral exposure in rats ( Jonsson et al., 2013). It is produced by numerous Fusarium species. Moniliformin can cause rapid death in poultry and swine. Toxicological effects include inhibition of protein synthesis, cytotoxicity, and chromosome damage. The primary mode of action appears to be inhibition of mitochondrial respiration (Bryden et al., 2001) and damage to myocardial mitochondria, resulting, in some cases, in acute congestive heart failure. Reduced phagocytic activity has been observed in rats ( Jonsson et al., 2015) and immunosuppression also has been reported in poultry. Moniliformin is suspected of involvement in several distinct diseases in poultry, including ascites, round heart disease, and “spiking mortality syndrome.” Chinese researchers also have reported a possible connection to Keshan disease, a serious endemic heart disease in some parts of China (Bryden et al., 2001). Insect injury to corn increases contamination by moniliformin (Lew et al., 1991). Fusaric acid is a common mycotoxin produced by several Fusarium species. It is 5-(butyl)-2-pyridinecarboxylic acid (Fig. 9.5). Several derivatives also have been reported. Fusaric acid is phytotoxic (Stipanovic et al., 2011) and may be involved in the colonization of plants by Fusarium species. In rats, fusaric acid caused changes in brain weight and body weight. It also is an inhibitor of DNA synthesis. Acute toxicity of fusaric acid seems to be negligible, but it appears to enhance the activity of several other Fusarium mycotoxins, including fumonisins, zearalenone, deoxynivalenol, diacetoxyscirpenol, and T-2 toxin (Bryden et al., 2001). Thus, the greatest significance of fusaric acid as a mycotoxin may be its interactions with other, more acutely toxic compounds. Fusaproliferin is a bicyclic sesterterpene with a molecular formula of C27H44O5, and is primarily produced by F. proliferatum, F. subglutinans, and F temperatum. A deacetylated form also is a naturally occurring metabolite. Toxicity of fusaproliferin has been evaluated primarily in vitro. It is toxic to brine shrimp larvae, and cytotoxic to cell lines from an insect (Spodoptera frugiperda) and from humans (IARC/LCL 171). Fusaproliferin also is teratogenic to chicken embryos (Gruber-Dorninger et al., 2016). The role, if any, of fusaproliferin on naturally occurring mycotoxicoses is not clear, but it is a contaminant of feed samples associated with feed refusal by swine, where no deoxynivalenol or other trichothecenes could be detected (Munkvold et al., 1998). Fusaproliferin often occurs in corn together with beauvericin or fumonisins. Beauvericin is a cyclic hexadepsipeptide that is closely related to the enniatins that are produced by several fungi. Beauvericin originally was identified from the entomopathogenic fungus Beauveria bassiana. It also can be produced by several other fungal species, including F. proliferatum, F. subglutinans, and F. verticillioides (Bottalico et al., 1995; Moretti et al., 1996). Beauvericin is toxic to several insect species and cell lines of insects and humans, but toxicity to other animals has not been demonstrated. Fusarium species produce dozens of other less common or less well-known mycotoxins (Munkvold, 2017) that can be found in corn, and may have significant toxicological implications. Details of these mycotoxins are beyond the scope of this chapter, but several of these toxins were reviewed recently (Gruber-Dorninger et al., 2016).

FIG. 9.4 Molecular structure of moniliformin.

FIG. 9.5 Molecular structure of fusaric acid.

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Aflatoxins and Other Aspergillus Toxins Aflatoxins are the best-known class of mycotoxins and among the most potent in terms of acute toxicity and carcinogenic properties; they are considered the most potent naturally occurring hepatocarcinogen. Their discovery was a turning point in the diagnosis of animal health problems associated with fungi in grains and feeds. Aflatoxins are a closely related group of metabolites of Aspergillus flavus and A. parasiticus. Those toxins generally recognized are B1, B2, G1, G2, and M1 (Fig. 9.6). The most abundant member of the group present under natural contamination is aflatoxin B1 (C17H12O6), a potent carcinogen. Aflatoxin B1 has an LD50 of 6.0 mg/kg body weight for intraperitoneal dosage in rats. Other mycotoxins produced by Aspergillus species include ochratoxins, cyclopiazonic acid, versicolorins, and sterigmatocystin (Ito et al., 2001; CAST, 2003). This section will focus on the aflatoxins. The structure and chemistry of the aflatoxins have been described in detail by Bhatnagar et al. (1992), and B€uchi and Rae (1969). The main target organ for aflatoxins is the liver. These toxins can be fatal in large doses, and in smaller doses, can cause chronic toxicity or cancer, primarily of the liver (CAST, 2003). Aflatoxins also can be mutagenic, teratogenic, and immunosuppressive (Smith, 1997). Young animals are more susceptible than mature ones. The greatest concern about exposure to aflatoxins is related to their potential to cause human cancer, since they can be found in corn and other food commonly consumed by humans, including the milk of cows consuming aflatoxin-contaminated feed. Aflatoxins suppress both nucleic acid and protein synthesis. This impairment of protein synthesis and related ability to mobilize fats causes growth suppression and lesions in the liver of affected animals. Aflatoxins have been shown to lower the resistance of several animal species to infection by bacteria, fungi, and parasites. Bacterial and yeast infections were associated with Turkey X disease, the first mycotoxicosis attributed to aflatoxins. Outbreaks of salmonellosis in swine also have been associated with high concentrations of aflatoxins in the corn crop (CAST, 2003). In swine, effects following exposure to high dosages of aflatoxin reflect liver cell damage and hemorrhage. Affected animals are depressed and have reduced feed intake. Jaundice and hemorrhages become evident during this same time period. The most likely effects of aflatoxin in swine are reduced gain and feed efficiency, subtle liver lesions, and possible increases in incidence or severity of infectious diseases. Constant exposure to aflatoxin for more than 7–10 days can result in hemorrhages in a variety of organs. In acute to subacute poisoning, liver weight may be increased, but is reduced in chronic cases where the liver becomes shrunken and filled with scar tissue. In cattle, clinical signs of aflatoxicosis are reduced feed consumption, dramatic drops in milk production, weight loss, and liver damage. The major economic impacts of chronic exposure involve reduced feed efficiency, immunosuppression,

FIG. 9.6 Molecular structures of the most common aflatoxins.

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and reduced reproduction Aflatoxins also have been shown to affect rumen function in cattle (CAST, 2003). Generally, aflatoxin concentrations of 1 to 2 mg/g in mature cattle for short periods of time result in reduced gain and decreased milk production. As little as 1 mg/g aflatoxin has caused liver damage, reduced gain, and death in feedlot steers. Aflatoxins were first discovered as the cause of a fatal disorder in turkeys, and aflatoxicoses have caused major economic losses in the poultry industry. Clinical signs observed from acute aflatoxicosis include hemorrhage, jaundice, anemia, depression, poor appetite, decreased weight gain, decreased egg production, embryo toxicity, and increased susceptibility to infectious diseases and environmental stress (CAST, 2003). Horses and ponies are susceptible to aflatoxins. Clinical signs of acute poisoning include anorexia, fever, rapid heart rate, ataxia, colic, icterus, convulsions, bloody feces, and abdominal straining. Horses given 0.3 mg aflatoxin/kg body weight died within 25 to 30 days (CAST, 2003). Human health problems are associated with the consumption of aflatoxin-contaminated food. In laboratory animals, aflatoxins are among the most potent liver carcinogens known. Shortly after the discovery of the aflatoxins, it was proposed that they might be a factor in the incidence of liver cancer in humans, and several studies have established a strong relationship between dietary aflatoxin and liver cancer and an interaction between hepatitis B virus and aflatoxins in liver cancer development (Palliyaguru and Wu, 2013). Human consumption levels of aflatoxin B1 were similar to carcinogenic levels for the rat and trout. Additionally, several outbreaks of acute, fatal toxicosis in humans have been associated with aflatoxins (CAST, 2003; Lewis et al., 2005). Acute aflatoxin symptoms are manifested as acute hepatitis: jaundice, lowgrade fever, depression, anorexia, and diarrhea, along histopathological signs of liver alterations. Two human diseases of uncertain etiology, kwashiorkor and Reye’s syndrome, have been associated with aflatoxins, but evidence is not definitive (CAST, 2003). Aflatoxins are considered as known human carcinogens (Group 1) by International Agency for Research in Cancer (IARC), and evidence for potential human health impacts has convinced many governments to place regulatory limits on aflatoxin content in food and feeds. In the United States, aflatoxins are the only mycotoxins to which the FDA has assigned action levels, specifying that grain with aflatoxin concentrations higher than 20 ng/g may not enter interstate commerce. Higher levels are allowed for local use as livestock feed. The European Community has adopted stricter standards than the United States (Table 9.6). Ochratoxins can be produced by species of Aspergillus and Penicillium. They are isocoumarins with an amide linkage to L-phenyl-alanine. Ochratoxin A is the most commonly occurring member of the group. It is 7-carboxy-5chloro-8-hydroxy-3,4-dihydro-3R-methylisocoumarin-7-L-3-phenylalanine, and its formula is C20H18O6HCl (Fig. 9.7). Ochratoxin A is a potent nephrotoxin and teratogen (Wilson and Abramson, 1992). Toxicoses of swine, poultry, and ruminant animals have been associated with exposure to ochratoxins. In swine, clinical signs may be subtle with low doses, but the kidneys appear swollen and discolored, with various histological abnormalities. At higher toxin levels, clinical signs appear primarily as decreased feed intake and weight gain, excessive thirst, depression, and frequent urination (Prelusky et al., 1994; CAST, 2003), and symptoms of renal failure may appear after chronic exposure. The most significant adverse effect of ochratoxins is their connection with a human kidney disease known as Balkan endemic nephropathy. This disease was first described in the 1950s and it is now estimated that 20,000 people are affected. Approximately half the sufferers die within two years (Beardall and Miller, 1994). Symptoms are consistent with a nephrotoxin: progressive renal failure, atrophy of the kidneys, jaundice, headaches, anorexia, uremia, listlessness, and proteins in the urine. Exposure to ochratoxin A is common in Europe. Furthermore, ochratoxin A is carcinogenic to rats and mice, and many patients with Balkan endemic nephropathy have kidney tumors. Definitive evidence is lacking, but “…most evidence for the causal relationship of Balkan endemic nephropathy points toward ochratoxin A,…” (CAST, 2003). A detailed analysis of ochratoxin A in relation to Balkan endemic nephropathy can be found in Beardall and Miller (1994). Ochratoxin A persists in the meat of nonruminant animals consuming contaminated feed. Ochratoxin A is considered a possible human carcinogen by IARC. Cyclopiazonic acid is an indole-tetramic acid with the formula C20H20N2O3 (Fig. 9.8), produced by A. flavus and other species of Aspergillus and Penicillium. Its LD50 to rats by intraperitoneal injection has been reported as 2.3 mg/kg body weight. This toxin has been reported to cause anorexia, diarrhea, dehydration, fever, and weight loss in rats, dogs, chickens, and pigs (CAST, 2003). Animals dying from cyclopiazonic acid poisoning have a characteristic rigid extension of the legs. Histological signs include hemorrhage and ulceration of the digestive organs and necrotic foci in various tissues including the liver, spleen, kidneys, pancreas, and myocardium. The role of cyclopiazonic acid in naturally occurring toxicoses is not clear, but it occurs together with aflatoxin and may be involved in Turkey X disease, along with aflatoxins (Wilson and Abramson, 1992). Sterigmatocystin is a biosynthetic precursor of aflatoxin and belongs to the furofuran group of mycotoxins, along with aflatoxins. The aflatoxins contain a coumarin moiety, but sterigmatocystin contains a xanthone moiety.

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TABLE 9.6 United States Food and Drug Administration Action Levels and European Community Guidance Levels for Total Aflatoxins in Human Food and Animal Feeds (Smith, 1997; CAST, 2003) Concentration (ng/g) United States

EC

All products, except milk, designated for humans

20

2–4

Milk

0.5

0.5

Corn for immature animals and dairy cattle

20

10

Corn products for breeding beef cattle, swine, and mature poultry

100

20–50

Corn for finishing swine

200

20

Corn for finishing beef cattle

300

50

All other feedstuffs

20

10

Commodity

OH

COOH O

O

O

N

CH3 H H

H Cl Ochratoxin A

FIG. 9.7 Molecular structure of ochratoxin A.

FIG. 9.8 Molecular structure of cyclopiazonic acid.

Sterigmatocystin is known chemically as 3a,12C-dihydro-8-hydroxy-6-methoxyfuro[30 ,20 4,5]furo[3,2C]xanthene-7one: its formula is C18H12O6 (Fig. 9.9). The effects of sterigmatocystin are similar to those of aflatoxin B1, but sterigmatocystin is less potent. It is considered a toxin, carcinogen, mutagen, and teratogen (Wilson and Abramson, 1992). Its LD50 to rats by intraperitoneal injection has been reported as 60 mg/kg body weight, ten times that of aflatoxin B1 (CAST, 2003). Tumorigenicity also differs by a factor of ten between the two toxins. Like cyclopiazonic acid, the role of sterigmatocystin in naturally occurring toxicoses is not clear, but it co-occurs with aflatoxin and may contribute to the symptoms of aflatoxicoses.

252

Corn

FIG. 9.9 Molecular structure of sterigmatocystin.

Penicillium Toxins Several of the important mycotoxins produced by Penicillium species, such as ochratoxins and cyclopiazonic acid, also are produced by Aspergillus species and have been discussed already. A summary of mycotoxin production by Penicillium species is shown in Table 9.2. Toxicity of several Penicillium toxins such as citrinin, penicillic acid, and rubratoxins has been demonstrated in laboratory studies, but the role of these compounds in naturally occurring toxicoses is not clear. Citrinin is (3R-trans-)-4,6-dihyfdro-8-hydroxy-3,4,5-trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid, and its formula is C13H14O5. The LD50 for citrinin is reported as 35 mg/kg body weight for mice and 67 mg/kg for rats by subcutaneous or intraperitoneal injection (Wilson and Abramson, 1992). This compound in corn is due primarily to P. citrinum, P. viridicatum, and P. expansum. Penicillic acid is 3-methoxy-5-methyl-4-oxo-2,5-hexadienoic acid, and its formula is C8H10O4. It has an LD50 of 70 mg/kg body weight for intraperitoneal injection in mice. Penicillic acid is carcinogenic to rats and mice and enhances the nephrotoxicity of ochratoxin A (Scott, 1977; Wilson and Abramson, 1992). Histological signs of penicillic acid activity include lesions of the liver, kidney, and thyroid. Rubratoxins include at least two related compounds, rubratoxins A and B, produced by P. purpurogenum (P. rubrum). Studies have shown hepatotoxicity, nephrotoxicity, and congestion and hemorrhage in several organs of pigs, horses, and goats consuming cultures containing rubratoxins. Intravenous injection produced symptoms of hepatotoxicity, hemorrhage, and death (Scott, 1977; CAST, 2003). Citreoviridin has been isolated primarily from rice, but also occurs in corn. It has an LD50 of 7.2 mg/kg body weight for intraperitoneal injection in mice. In Japan, this mycotoxin has been associated with an acute disease in humans known as cardiac beriberi and similar symptoms have been induced in animals. It is a fatal disease characterized by disturbance of the central nervous system, paralysis, cardiovascular damage, and respiratory failure. The disease, once common, nearly disappeared from Japan after the initiation of quality control measures in rice commerce in the 1910s and 1920s (Scott, 1977; CAST, 2003). Numerous other mycotoxins can be produced by species of Penicillium and Aspergillus. More details can be found in Scott (1977, 1994).

OCCURRENCE OF MYCOTOXINS IN CORN AND DDGS In this section, information is presented from mycotoxin surveys, including an annual survey conducted by BIOMIN (Taschl et al., 2017), in which the occurrence of mycotoxins in corn and DDGS (Dried Distillers’ Grains and Solubles) collected worldwide were investigated for the years 2014–17. In this study, mycotoxin concentrations were determined using enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In addition, we highlight important recent publications by other authors about mycotoxin occurrence and co-occurrence of the major mycotoxins in corn, corn-based products, and corn silage.

Global Mycotoxin Occurrence in Corn The concentrations of aflatoxins, zearalenone, deoxynivalenol, T-2 toxin, fumonisins, and ochratoxin A were analyzed in 11,237 corn samples collected in 75 countries from January 2014 to June 2017 (Table 9.7, Fig. 9.10). Fumonisins were the most prevalent, detected in 82% of samples at a median level of 1.4 mg/g. A maximum level as high as 171.9 mg/g was detected in a sample from Chile. In total, 9% of samples exceeded the most stringent guidance level for fumonisins issued by the Food and Drug Administration (FDA) for corn intended to be used in animal feed (5 mg/g for corn destined for equids

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TABLE 9.7 Occurrence of Mycotoxins in 11,237 Corn Samples Collected From 75 Countries From January 2014 to June 2017 All Regions

Afla

ZEN

DON

T-2

FUM

OTA

Number of samples tested

8885

9061

8013

4088

6563

3369

Median of Positive (ng/g)

3

76

581

24

1374

2

Average of Positive (ng/g)

22

239

1388

53

2536

17

1352

16,495

43,770

976

171,920

889

Maximum (ng/g)

Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

FIG. 9.10 Prevalence of mycotoxins in 11,237 corn samples collected from 75 countries from January 2014 to June 2017. Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

and rabbits). Aflatoxins were detected in 24% of samples at a median level of 3 ng/g. In one sample from Brazil, an aflatoxin concentration as high as 1.4 mg/g was found. In total, 4% of samples exceeded the action level of 20 ng/g established by the FDA for corn destined for immature animals and dairy animals. Zearalenone and deoxynivalenol were present in 49% and 72% of samples with median levels of 76 ng/g and 581 ng/g, respectively. Maximum levels as high as 16.5 mg/g and 43.8 mg/g were detected in samples from Austria and Brazil in case of zearalenone and deoxynivalenol, respectively. In case of deoxynivalenol, 4% of samples exceeded the most stringent advisory level (5 mg/g) issued by the FDA for livestock feed. Of the tested samples, 92% contained at least one mycotoxin. The majority of samples was co-contaminated with multiple mycotoxins. In total, 68% of the samples tested for  3 mycotoxins were contaminated with at least 2 mycotoxins.

Mycotoxins in North American Corn Concentrations of aflatoxins, zearalenone, deoxynivalenol, T-2 toxin, fumonisins, and ochratoxin A were determined in 1013 corn samples taken from North America from January 2014 to June 2017 (Table 9.8, Fig. 9.11). Deoxynivalenol and fumonisins were most prevalent and occurred in 66% and 61% of samples, respectively. Deoxynivalenol and fumonisins reached maximum concentrations of 24.8 mg/g and 154.0 mg/g and median concentrations of 460 ng/g and 900 ng/g, respectively. In case of deoxynivalenol and fumonisins 1% and 7% of samples exceeded the most stringent advisory level (5 mg/g) and guidance level (5 mg/g), respectively, issued by the FDA for animal feed. Zearalenone was detected in 27% of samples at a median level of 100 ng/g and a maximum level as high as 13.7 mg/g. In total, 7% of samples contained aflatoxins with a median concentration of 9 ng/g and a maximum concentration as high as 980 ng/g. The action level of 20 ng/g issued by the FDA for corn products destined for immature animals and dairy animals was exceeded in 2% of the samples. In total, 84% of samples contained at least one mycotoxin and 50% samples that were tested for  3 mycotoxins were co-contaminated with at least 2 mycotoxins.

Mycotoxins in Corn in Countries Outside USA A survey of the occurrence of relevant mycotoxins in corn samples from South America, Central and South Europe, North, South-East and South Asia, and Oceania was performed from January 2009 until December 2011 (Rodrigues and Naehrer,

254

Corn

TABLE 9.8 Occurrence of Mycotoxins in 1013 Corn Samples Collected From North America From January 2014 to June 2017 North America

Afla

ZEN

DON

T-2

FUM

OTA

Number of samples tested

952

958

893

855

945

912

Median of Positive (ng/g)

9

100

460

19

900

3

Average of Positive (ng/g)

39

246

814

48

3169

25

Maximum (ng/g)

980

13,700

24,792

200

154,000

200

Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

FIG. 9.11 Prevalence of mycotoxins in 1013 corn samples collected from North America from January 2014 to June 2017. Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

2012). The corn contamination pattern of aflatoxins (Afla), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FUM), and ochratoxin A (OTA A) varied among geographic regions. FUM was the first or second most prevalent mycotoxin in corn for all regions. DON was also detected with a high frequency, except in South America and South Asia. However, while all the maximum levels detected for FUM exceeded 5 mg/g, only two of the DON maximum concentrations (Central Europe and North Asia) exceed the risk thresholds for livestock. Remarkable concentrations of Afla, with median concentrations of 38 and 96 ng/g were registered in SE Asia and South Asia, respectively. The prevalence of mycotoxins in South America, according the percentage of positive samples, was FUM (92%), ZEN (43%), Afla (25%), DON (17%), and OTA (12%). FUM reached high maximum levels of 53,700 ng/g and median concentrations of 2008 ng/g for the 807 samples analyzed. In Central Europe, DON was detected in 72% of samples, followed by FUM (60%). In South Europe, like in South America, FUM was the main contaminant (90% positive samples) at median level of 1407 ng/g for the 48 samples tested. Corn sourced in North Asia followed the same pattern as Central Europe, with DON being the main contaminant (present in 92% of tested samples) at median levels of 15,073 ng/g. In the South and South-East part of Asia, a clear predominance of Afla was found, with 82% and 71% positive samples, respectively. The highest levels were 2230 and 6105 ng/g, respectively. However, the presence of FUM and DON in these regions cannot be ignored. Despite the relatively low number of samples analyzed from Oceania, quite high average levels of FUM (average of positive: 2823 ng/g) were present in 64% of analyzed samples. In a 2009 survey, a total of 63 maize samples from Asia and Africa were analyzed for Afla, ZEN, FUM, B-Trichothecenes (nivalenol, Niv; deoxynivalenol, DON and acetyldeoxynivalenol, Ac-DON), and Type A trichothecenes (diacetoxyscirpenol, DAS; HT-2 toxin; HT-2 and T-2 toxin, T-2) (Rodrigues et al., 2011). FUM was the most frequent mycotoxin, found in 84% of tested samples. The mean level was 987 ng/g and the median of positive was 1116 ng/g. A corn sample from South Africa presented the highest contamination level for this mycotoxin (4398 ng/g). Likewise, the maximum level found for Type B trichothecenes was detected from the same country (3035 ng/g). The highest level for Afla (343 ng/g) was found in Nigeria, and a maize sample from Ghana presented the highest ZEN contamination (310 ng/g). Other authors’ work (Garrido et al., 2012; Kos et al., 2013; Schatzmayr and Streit, 2013; Streit et al., 2013; Pereira et al., 2014; Pleadin et al., 2014; Leggieri et al., 2015; Matumba et al., 2015; Anjorin et al., 2016; Hove et al., 2016; Misihairabgwi et al., 2017; Oliveira et al., 2017) are there for more detail information on mycotoxins in corn surveys.

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Multi-Mycotoxin Analysis of North American Corn Samples The occurrence of more than 400 mycotoxins, masked mycotoxins, and other fungal secondary metabolites in corn samples taken from North America was investigated using Spectrum 380®, a multi-analyte liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based method. In total, 66 corn samples collected from January 2014 to June 2017 were analyzed (Fig. 9.12). The Fusarium mycotoxins moniliformin and aurofusarin were the most frequently detected analytes. Moniliformin and aurofusarin were found in 95% and 92% of samples at a median level of 163 ng/g and 254 ng/g and a maximum level of 2.7 and 8.4 mg/g, respectively. The most prevalent main mycotoxin was the Fusarium mycoestrogen zearalenone, detected in 85% of samples with an average of 55 ng/g and a maximum of 544 ng/g. Furthermore, trichothecenes were prevalent in samples analyzed in this survey. Deoxynivalenol was detected in 82% of samples, with a median concentration of 397 ng/g and a maximum concentration of 4.1 mg/g. Interestingly, the masked mycotoxin deoxynivalenol-3-glucoside was detected in an approximately equivalent fraction of samples (85%) at lower levels (median: 41 ng/g; maximum: 325 ng/g), indicating a high incidence of glycosylation of deoxynivalenol in corn. Fig. 9.13 depicts the prevalence of main groups of mycotoxins in the North American corn samples. All samples contained Fusarium mycotoxins. Likewise, the 10 most frequently detected metabolites were Fusarium metabolites (Fig. 9.12). Type B trichothecenes were present in 94% of samples with a median level of 357 ng/g. Zearalenone or its metabolites occurred in 88% of samples with a median level of 15 ng/g. In case of deoxynivalenol and fumonisins, 2% and 7% exceeded 5 mg/g. Ochratoxins were not detected in any sample. Aflatoxins were detected in one sample at a concentration of <1 ng/g. Other metabolites of Aspergillus spp. were detected in 77% of samples. In total, 86% of the North American corn samples contained between 10 and 39 fungal metabolites (Fig. 9.14). More than 30 metabolites were detected in 32% of the samples. Only 3% of the samples contained less than 10 metabolites. Numerous other studies have reported co-occurrence of multiple mycotoxins in corn from many parts of the world (Ferreira et al., 2012; Souza et al., 2013; Pereira et al., 2014; Zachariasova et al., 2014; Kovalsky et al., 2016; Oliveira et al., 2016). These publications are recommended for additional information about trends in mycotoxin occurrence in specific countries. Studies using multi-mycotoxin analysis are creating a new understanding of the complexity of mycotoxin contamination of agricultural products. The identification of co-occurring mycotoxin combinations should be useful in estimating possible synergistic or additive effects in contaminated food or feed.

Global Mycotoxin Occurrence in Corn DDGS From January 2014 to June 2017, the concentrations of aflatoxins, zearalenone, deoxynivalenol, T-2 toxin, fumonisins, and ochratoxin A were analyzed in 416 Dried Distillers Grains with Solubles (DDGS) samples from 31 countries (Table 9.9, Fig. 9.15). Highly prevalent mycotoxins were deoxynivalenol, zearalenone, and fumonisins, which were found in 92%, FIG. 9.12 Prevalence of mycotoxins and fungal secondary metabolites in 66 corn samples from North America. Metabolites that were present in over 50% of samples are shown.

256

Corn

120% % positive samples

100%

100%

94%

88%

82%

80%

77%

68%

67%

61%

60% 32%

40%

18%

20% 2%

0%

M et ab ol ite s llu s M et ab Fu ol sa ite riu s m M et Pe ab ol ni ite ci lliu s m M et ab ol ite s

Al te rn ar ia

En ni a

As pe rg i

in Be au ve ric

al oi ds tin s

an d

ns

in s

ox i

Er go ta lk

O ch ra t

on is Fu m

ho th ec en es

BTr ic

ho th ec en es

bo lit

ATr ic

Ze ar al

en on e

m

et a

Af la

to xi

es

ns

0%

FIG. 9.13 Prevalence of main groups of fungal secondary metabolites in 66 corn samples from North America.

FIG. 9.14 Co-occurrence of fungal secondary metabolites in 66 corn samples from North America.

TABLE 9.9 Occurrence of Mycotoxins in 416 Corn DDGS Samples Collected From 31 Countries From January 2014 to June 2017 All Regions

Afla

ZEN

DON

T-2

FUM

OTA

Number of samples tested

357

381

375

260

351

260

Median of Positive (ng/g)

3

182

1596

34

908

3

Average of Positive (ng/g)

13

432

2414

82

2784

4

Maximum (ng/g)

277

7279

64,588

914

28,605

22

Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

82%, and 82% of samples at median levels of 1.6 mg/g, 182 ng/g, and 908 ng/g, respectively. In case of deoxynivalenol and fumonisins, 8% and 13% of samples exceeded 5 mg/g. Deoxynivalenol and fumonisins were found at maximum levels as high as 64.6 mg/g and 28.6 mg/g in samples from China and the United States, respectively. In case of aflatoxins, 28% of samples were contaminated with a median concentration of 3 ng/g. A maximum aflatoxin level of 277 ng/g was detected in a sample from China. In total, 3% of samples exceeded the action level of 20 ng/g issued by the FDA. In total, 99% of samples contained at least one of the 6 major mycotoxin types and 90% of samples tested for 3 mycotoxins were co-contaminated with at least 2 mycotoxins.

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FIG. 9.15 Prevalence of mycotoxins in 416 corn DDGS samples collected from 31 countries from January 2014 to June 2017. Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

Mycotoxins in North American Corn DDGS The occurrence of aflatoxins, zearalenone, deoxynivalenol, T-2 toxin, fumonisins, and ochratoxin A was investigated in 128 DDGS samples collected in North America from January 2014 to June 2017 (Table 9.10, Fig. 9.16). Deoxynivalenol, fumonisins, and zearalenone were highly prevalent and occurred in 93%, 90%, and 81% of samples, respectively. Deoxynivalenol was detected at a median concentration of 1.6 mg/g and a maximum concentration of 5.8 mg/g. In total, 2% of samples exceeded 5 mg/g. Fumonisins were detected at a median concentration of 908 ng/g and a maximum concentration of 28.6 mg/g. The most stringent guidance level (5 mg/g) issued by the FDA for animal feed was exceeded in 18% of samples. Zearalenone was detected at a median concentration of 218 ng/g and a maximum concentration of 666 mg/g. In total, 15% of samples contained aflatoxins with a median concentration of 3 ng/g and a maximum concentration of 37 ng/g. The action level of 20 ng/g was exceeded in 2% of the samples. At least one mycotoxin was detected in 99% of the samples. Most samples were co-contaminated with multiple mycotoxins. More than 97% of DDGS samples that were tested for 3 mycotoxins were co-contaminated with at least 2 mycotoxins. Occurrence of important mycotoxins in DDGS was previously reported by Khatibi et al. (2014). In this study, 141 DDGS lots were collected from 78 ethanol plants located in 12 US states in 2011. Levels of ZEN, DON, 15-ADON, 3-ADON, and NIV were analyzed. DON was detected in a range <0.50 to 14.62 mg/g and 3% of the samples contained >10 mg/g of this toxin. 15-ADON ranged between <0.10 and 7.55 mg/g and ZEN ranged from <0.10 to 2.12 mg/g. NIV and 3-ADON were not detected in any of the samples. Yanhong, Zhang and Caupert (2012) also investigated the presence of mycotoxins in corn DDGS from ethanol plants from 2009 to 2011. In this survey, DON was detected in the 67 DDGS samples and 12% of the corn DDGs contained levels higher than the minimum advisory level by FDA. Rodrigues and Naehrer (2012) evaluated corn DDGS contamination outside the United States. Levels of mycotoxins (Afla, ZEN, DON, FUM, and OTA) sourced in North and South-East Asia and Oceania were reported from 2009 to 2011. DON, ZEN, and FUM were the main contaminants in the Asian regions. Data from this study reveals high average levels for contaminated samples, especially for DON (3618 and 2866 ng/g). In contrast, there was no evidence of Afla contamination in South-East Asian samples and low incidence in North Asia. For all 22 Oceanian samples tested, DON, FUM, and OTA were identified in 20% of the corn DDGS, whereas ZEN was only found in 10% and aflatoxins were not detected.

TABLE 9.10 Occurrence of Mycotoxins in 128 DDGS Samples Collected From North America From January 2014 to June 2017 North America

Afla

ZEN

DON

T-2

FUM

OTA

Number of samples tested

122

125

121

119

124

120

Median of Positive (ng/g)

3

218

1641

13

908

2

Average of Positive (ng/g)

7

236

1811

25

3474

5

Maximum (ng/g)

37

666

5778

53

28,605

22

Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T-2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

258

Corn

FIG. 9.16 Prevalence of mycotoxins in 128 DDGS samples collected in North America from January 2014 to June 2017. Afla, aflatoxins; ZEN, zearalenone; DON, deoxynivalenol; T2, T-2 toxin; FUM, fumonisins; OTA, Ochratoxin A.

OCCURRENCE OF MYCOTOXINS IN CORN-BASED FOOD Mycotoxin analysis of corn-based food has been conducted around the world. The European Food Safety Authority (EFSA) reported occurrence data of T2 and HT-2 (EFSA, 2011a) and nivalenol (NIV) (EFSA, 2013a) in food and feed, and zearalenone (ZEN) (EFSA, 2011b) and deoxynivalenol (DON) (EFSA, 2013b) in cereal products from European countries. Additionally, the Joint FAO/WHO Expert Committee (FAO/WHO, 2012) published information on levels and patterns of contamination of fumonisins and co-occurrence with other mycotoxins. The data set contained analytical results for food and feed samples collected from 59 countries from 2001 to 2011. Details can be found in the published results of these studies. Co-occurrence of two or more mycotoxins including masked and emerging toxins has become an emphasis in mycotoxin surveys. In addition, developments in mycotoxin analysis continue, with emphasis on novel methods for a wide range of metabolites. In Africa, four recent surveys were reported using a LC-MS/MS multitoxin method. Warth et al. (2012) determined 63 metabolites in 122 samples of mainly maize and groundnuts from Burkina Faso and Mozambique. Njumbe Ediage et al. (2011) analyzed simultaneously 25 mycotoxins in maize flour, peanut cake, and cassava samples obtained from local markets in Benin. Fumonisin B1, B2, and B3 were detected in all the maize flour samples and beauvericin was detected in two of the maize samples at maximum concentrations of 25 mg/kg. In Nigeria, a total of 18, 21, and 32 metabolites were quantified in the groundnut-, groundnut/maize-, and maize-based snacks, respectively (Kayode et al., 2013). Geary et al. (2016) collected 101 maize-based porridge samples from different areas of Tanzania, where 82% of samples were co-contaminated with more than one group of mycotoxins. This finding indicates a potential increase in the risk of exposure and significant health implications in children. In Malaysia, Soleimany et al. (2012) determined the occurrence of mycotoxins in 100 commercial cereal samples including maize meal. 80% of the maize samples were positive for at least one mycotoxin. Meanwhile, in Spain, occurrence of mycotoxins in ethnic food and gluten-free food was researched by the classic HPLC methodology. FBs and DON were widely found in several corn-based food (Cano-Sancho et al., 2012). Pereira et al. (2015) analyzed 12 trichothecenes in processed cereal-based baby food commercialized in Portugal by GC-MS. Four out of 12 trichothecenes were found: DON, 15AcDON (15-acetyl-deoxynivalenol), T2-Tetrol, and NEO (Neosolaniol). DON was the most commonly detected, in some cases at significant levels (29–270 mg/kg). Cunha and Fernandes (2010) evaluated the occurrence of five selected mycotoxins (ZEN, DON, NIV, FUS, and 15-AcDON) in samples of breakfast cereals and flours commercialized in Portugal; DON and ZEN were the most predominant.

MYCOTOXINS IN CORN SILAGE Most mycotoxin-producing fungi that occur in corn kernels also can colonize other parts of the plant, and Fusarium mycotoxins can be found in corn cobs and stalks in the field (Mirocha et al., 1979; Di Menna et al., 1997), which can contribute to the occurrence of mycotoxins in corn silage. Under anaerobic conditions, these fungi are not active, but if contaminated corn plant material is ensiled under less-than-ideal conditions, additional fungal growth, and mycotoxin production can occur. The main fungal genera typically isolated from corn silage are Fusarium, Penicillium, and Aspergillus (Alonso et al., 2013), with a high incidence of potentially toxigenic species such as A. flavus, A. parasiticus, A. fumigatus, F. verticilloides, F. graminearum, and P. roqueforti, among other species (Alonso et al., 2013). The most common Fusarium mycotoxins found in silage were deoxynivalenol (DON) and zearalenone (ZEA) (Eckard et al., 2011; Van Asselt et al., 2012; Cheli et al., 2013). Incidence of Fusarium species and mycotoxins from 20 Swiss silage samples were analyzed by Eckard et al. (2011). In this survey, F. sporotrichioides, F. verticillioides, and F. graminearum were the

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dominant species and DON ranged from 780 to 2990 mg/kg. The occurrence of masked fumonisins was studied in silage samples from F. verticillioides-contaminated corn plants (Latorre et al., 2015). The authors showed that the 124 samples analyzed contained hidden fumonisins in significant amounts, even higher than in their free forms. Toxigenic fungi other than Fusaria, such as Penicillium roquefortii, also can sometimes be found in silage (Sumarah et al., 2005). In a study conducted by Mansfield et al. (2008), 75% of the maize silage samples collected from 30 Pennsylvania farms were contaminated with at least one Penicillium mycotoxin and 10% of the samples were co-contaminated with four mycotoxins: roquefortine C, mycophenolic acid, cyclopiazonic acid, and patulin. Roquefortine C was the most frequently detected toxin (60%), followed by mycophenolic acid (42%). Silage contamination with Aspergillus toxins was reported in samples from France (Garon et al., 2006; Richard et al., 2009) and Argentina (Gonza´lez Pereyra et al., 2011). Silage microflora may be capable of degrading some mycotoxins (Camilo et al., 2000), but hazardous levels are sometimes found, and proper management of silage must continue to be emphasized as a way to avoid potential mycotoxin problems.

MYCOTOXIN ANALYSIS Management of mycotoxins cannot be accomplished without an understanding of when, where, and to what extent they occur. Therefore, detection and quantitation of mycotoxins are crucial components of their management, required for surveillance, diagnosis and mitigation, and research. The only way to accurately assess the mycotoxin levels in a grain lot is to identify specifically the mycotoxins in question. Safety of grain or feed cannot be determined by the presence or absence of mold. Mold counts, cultures, spore counts, and other methods of determining mold infestation do not prove that mycotoxins are present. They should be used only to show that conditions for mycotoxin production exist. The “black light” or UV light test that is often used in screening for aflatoxins is based on the bright greenish-yellow fluorescence of kojic acid, not aflatoxins. Therefore, this test is not specific for aflatoxin. The only accurate way to estimate mycotoxin contamination is to conduct a specific biochemical or immunological test. Choice of analytical method is often a compromise between accuracy and precision vs. cost and convenience. There is a pressing need for quick and inexpensive methods that do not require technical skill so that they can be performed in field settings. For these quick screening tests, qualitative or semiquantitative are acceptable. Numerous such tests are now available and approved for official use. More precise determinations usually require more expensive and time-consuming laboratory procedures. Several organizations are involved in the review and certification of methods for mycotoxin analysis, including Association of Official Analytic Chemists International (AOAC), the International Union for Pure and Applied Chemistry, the American Association of Cereal Chemists, and the American Oil Chemists Society (CAST, 2003). AOAC International administers’ collaborative studies to validate methods and after a method has been approved has been published as an “Official” method (AOAC, 2016). Continuing progress is reported annually in the Journal of AOAC (Trucksess and Zhang, 2016; Zhang et al., 2016).

General Procedures Determination of mycotoxin levels in feed and food samples is usually accomplished by methods that include certain common steps: sampling, milling and homogenization, extraction followed by a cleanup, and finally the detection and quantitation, which is performed by many instrumental and noninstrumental techniques (Fig. 9.17) (CAST, 2003; Alshannaq and Yu, 2017).

Sampling Accurate determination of mycotoxins in grain and feeds is a complicated matter, and one of the most important aspects of assessing mycotoxin content is obtaining a representative sample. This is challenging because mycotoxins are not uniformly distributed among grain loads or within fields, usually a small percentage of kernels is contaminated, and the level of contamination of a single kernel can be very high. A single kernel can contain up to 400,000 ng/g of aflatoxins (CAST, 2003). It is possible to reach a concentration of 20 ng/g (the regulatory limit in the United States), with only a few contaminated kernels in 10,000 ( Johansson et al., 2000b). Because tolerances for aflatoxins are so low and the distribution of aflatoxins in samples is positively skewed, it is very challenging to conduct a sampling scheme with a sufficiently high level of precision. There are several sources of variability involved in the quantification of mycotoxins, and sampling is usually the largest source of error. Johansson et al. (2000a) tested a lot of shelled corn with 20 ng/g aflatoxins and determined that 77.8% of the total variance in aflatoxin estimation was due to sampling error. Sample preparation accounted for 20.5% of the variance, and analytical variance was only 1.7% of the total. Sampling variance can be reduced by increasing

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1

2

3

4

5

6

7

Sampling

Grinding and homogenization

Extraction

Filtration

Clean-up

Concentration

Analysis

FIG. 9.17 Analytical scheme for the determination of mycotoxins.

sample and subsample sizes and decreasing particle size (Whitaker, 2006). Sampling methods and guidance to mitigate the effects of heterogeneous distribution of mycotoxins have been described (Whitaker, 2006). In the United States, the Federal Grain Inspection Service (FGIS), a section of the USDA Grain Inspection, Packers, and Stockyards Administration (GIPSA), has established standards for sampling corn for mycotoxins and grade determination (USDA, 2013). Corn for export from the United States must be inspected by FGIS, and domestically marketed corn can be inspected voluntarily or at the discretion of FGIS. Even when grain is inspected outside the FGIS system, following the FGIS recommendations provides a desirable level of reliability to the results. Innovatively, the FAO has developed a mycotoxin sampling tool (http://www.fstools.org/mycotoxins) (FAO, 2014) that provides support in analyzing the performance of sampling plans for 24 mycotoxin commodity combinations and determining the most appropriate plan to meet the defined goals. The European Union (EU) also has official methods of sampling and analysis of mycotoxins in food ( New European Commission Regulation (EC) No. 401/2006) and sampling of groundnuts (Regulation (EC) No. 882/2004), which are rules on the importation of food and feed into the EU, implemented by Commission Regulation ( EC) No. 669/2009. There are many situations under which sampling grain for mycotoxin determination is desirable, and those involving grain commerce typically develop quality assurance schemes or management systems in which sampling is done at critical times and locations (Coker, 1998; CAST, 2003). Circumstances under which sampling might be done can be grouped into three general scenarios: in the field, in static grain lots, or in dynamic or moving grain lots. Each scenario has its own unique challenges, but the same basic principles apply. The goal is to obtain a sample representative of the whole field or grain lot. For this to occur, the sample ideally should be random, so that every kernel has an equal and unrelated probability of being sampled. Therefore, the sampling method must provide access to the entire bulk of the grain. In practice, various constraints preclude the possibility of a 100% random sample; most sampling strategies emphasize obtaining a representative sample. Sampling in the field for mycotoxins is probably the least frequently utilized scenario and it offers unique constraints; nevertheless, such sampling can be desirable to speed decision making and avoid a crisis situation after harvest. Access to all the grain is possible but time-consuming, and therefore, costly. Because the corn is still on the cob, each kernel does not have an independent probability of being sampled. Spatial distribution of mycotoxins among the kernels is very unlikely to be uniform because there is no opportunity for mixing. Ideally, the approach should include collecting random individual ears from all parts of the field, for a bulk (shelled) sample of at least 5 kg for a field up to 15 ha. The sample size should be larger for larger fields. After shelling, the kernels should be mixed thoroughly and either immediately dried or frozen, unless grain has already dried in the field to moistures too low for further mycotoxin development. After harvest, sampling may be required from a static grain lot. This includes grain that is in a truck, barge, railcar, or storage bin. It is more difficult to obtain a representative sample from a static grain lot than from a moving grain lot because access to all portions of the lot may not be equally possible. A probe usually is used to take samples from static grain lots; probes may be handheld or mechanical. Systematic sampling patterns are established for probing various types of grain

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compartments (USDA, 2013). If the grain lot is believed to be essentially uniform in condition, a single bulk sample can be made from the various probes. If obvious pockets of inferior grain exist, a sample of the entire grain lot should be taken, but separate samples should be taken of the inferior and remaining portions (USDA, 2013). Static grain lots also may be stored in sacks. This presents additional constraints on obtaining a representative sample. Standard sample size for grading corn is 2.5 kg, but a larger sample (4.5 kg or about 10 lb) is recommended for determination of aflatoxins or other mycotoxins, due to the high variability in aflatoxin compared to other grain characteristics (USDA, 2016). The best conditions for obtaining a representative sample exist when the grain lot is moving; this facilitates access to the entire grain lot. The approach is to take small increments of the grain lot at predetermined time intervals (CAST, 2003; Whitaker, 2006). The most reliable and accurate method is a mechanical diverter sampler, which collects a sample by drawing a diverter through the moving grain stream at a programmed time interval. Sample size is a function of the diverter cup size, sampling time interval, and grain flow rate. Other methods for sampling a moving grain stream are the Ellis cup and the pelican sampler. Sampling frequencies for each type of sampler are established (USDA, 2013), based on a 2.5-kg sample for each 254.2 tons (10,000 bu) of corn, but more frequent sampling (i.e., a larger sample size) is recommended for mycotoxin determination. Guidelines published by the USDA (2016) suggest a 4.54-kg (10 lb) sample for each 254.2 tons equivalent to 10,000 bu (a sampling intensity of about 0.002%), but other authors have recommended a larger sample size of 0.5 kg per 1000 kg of corn (sampling intensity of 0.05%) (Whitaker et al., 1991). Much research has gone into the theoretical and practical aspects of obtaining accurate estimates of aflatoxin contamination in corn and other commodities. The goal of this research is to design sampling plans for industry that strike a balance between accuracy and precision and the cost of obtaining the estimate and to balance the risk of a biased result between the buyer and seller. A sampling plan can be described by its operating characteristic (OC) curve, which is a plot of the probability of the sampling plan accepting a lot vs. the mean concentration of a given mycotoxin in the lot. The shape of the curve is affected by sample size, the degree of comminution of the samples, the size of subsamples analyzed, the number of subsamples analyzed, and the maximum aflatoxin concentration permitted (Coker, 1998). Changing the acceptable limit affects the cost of a mycotoxin-testing program and the balance of risk between buyer and seller. In general, increasing any of the factors other than the permissible limit decreases the variability and decreases the buyer’s and seller’s risks, but increases the cost of the program. OC curves for 35 sampling programs were constructed by FAO (1993), based on 10-kg corn samples, comminution by a hammer mill, and extraction of 50-g subsamples; a general operating characteristic curve is shown in Fig. 9.18. The seller’s risk is the probability that a lot will be rejected even though its mean concentration is below the acceptable limit. The buyer’s risk is the probability that the lot will be accepted even though its mean concentration is above the acceptable limit. These ideas are discussed in more detail by Coker (1998), and the construction of OC curves is demonstrated by Johansson et al. (2000a) and FAO (1993).

Grinding and Homogenization Corn or feed samples must be prepared for analysis by grinding or comminuting in a mill to obtain small, easily extracted particles. In order to maximize precision, the entire test sample as described above must be milled. Different mills result in differing particle sizes and this will influence the results. Smaller particle size is superior because it will result in a more homogeneous sample. A homogeneous sample is crucial because the entire sample is rarely analyzed. Smaller subsamples

1

Seller’s risk

Acceptance probability

0.9 0.8 0.7 0.6 0.5 0.4 0.3

Buyer’s risk

0.2 0.1

Critical concentration

0 Lot aflatoxin concentration

FIG. 9.18 General operating characteristic curve for mycotoxin sampling plans. Adapted from Whitaker, T., Dickens, J., Giesbrecht, F., 1991, Testing animal feedstuffs for mycotoxins: sampling, subsampling and analysis. In Smith, J.E., Henderson, R.S. (Eds.), Mycotoxins and Animal Foods. CRS Press, Boca Raton, pp. 153–164.

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must be removed from the original test sample in a representative manner. Several mills commonly used for sample preparation will divide the sample into representative subsamples. Subsample variability can be reduced by increasing the size or number of subsamples and by increased comminution of the sample (Whitaker, 2006).

Extraction Most methods require that mycotoxins be extracted from the solid material into a liquid phase, which is accomplished through the use of organic solvents or mixtures of solvents and water. This step is critical, because only the mycotoxins extracted can be quantified; therefore the final results are greatly affected by extraction efficiency. The choice of the extraction solvent depends on chemical properties of mycotoxins as well as on properties of the matrix. Generally, mycotoxins are extracted with organic solvents such as methanol, acetonitrile, chloroform, and acetone. These solvents may be used in combination with water, dilute acid, or aqueous solution of salts to aid in the breaking of weak electrostatic bonds, which bind some mycotoxins to other substrate molecules (e.g., proteins). The selection of solvent for the extraction and redissolution is critical because it affects the recovery of the mycotoxin (Stroka et al., 1999). Thereby, the polarity of the toxin and solvent is one of the main properties to take account in the extraction step. Solvents are mixed with solid samples either in a high-speed blender (3–5 min) or in shaker (10–30 min); blending is quicker but less amenable to large numbers of samples (Rahmani et al., 2009). After mycotoxin extraction, filtration and centrifugation are important steps to remove any interfering particles before performing further cleanup steps.

Cleanup Extracts from corn grain or other products are contaminated with impurities that can interfere with mycotoxin detection. Usually, these extracts will be cleaned using various methods. This is not required for all determination methods, particularly qualitative screening techniques. Laboratory methods using combinations of chromatographic and mass spectrometric methods also can be used with little cleanup. Most of the common quantitative procedures, however, require cleanup of the extracts. There are two main pretreatment strategies of the sample: solid phase extraction (SPE) columns and immunoaffinity columns (IACs). SPE or C18 reversed-phase columns are porous silica, with a surface modified to selectively adsorb either the mycotoxin or the impurities. Impurities pass through columns that adsorb the mycotoxin, and then a different rinse solution is employed to remove the mycotoxin from the column surface. IACs are a more precise type of cleanup tool. IACs are packed with activated solid phase bound to a specific antibody for a given mycotoxin(s). When the extract passes through the column, the mycotoxin binds selectively to the antibodies, while other matrix components will be removed by a washing step. The mycotoxin is then eluted with a miscible solvent such as methanol (Alshannaq and Yu, 2017). IACs are available for AFs, OTA, ZEA, FUMs, DON, and T-2 toxin. In addition to high levels of specificity, IACs can provide highly sensitive detection by concentrating mycotoxins from large amounts of sample. Other advantages include speed, low solvent use, and possibility of automation. The major disadvantage is the cost of the columns. However, methods of regenerating commercial IACs for reuse have been reported (Nakajima, 2004). Recently, an alternative to IACs has been developed based on the idea of mimicking antibodies. Molecularly imprinted polymers (MIPs) with high affinity and specific binding sites for the toxins have been configured into MIP-SPE columns. Functional monomers are polymerized using the toxin as a template and selective toxin recognition sites of the imprinted polymers are formed (Trucksess and Zhang, 2016). Bryła et al. (2013) used a commercial MIP-SPE to isolate FB1, FB2, and FB3 from corn-based and wheat-based products. MIPs can be a highly selective matrix for separation and enrichment of mycotoxins offering a viable alternative to classical sample cleanup procedures.

Concentration Concentration is usually performed on a rotary evaporator at below 50°C in a water bath under reduced pressure. When the extracts are finally dried up, evaporation under a stream of nitrogen on an aluminum block heated to below 50°C is recommended.

Analysis The final separation and detection of compounds of interest is usually achieved either by immunochemical methods or by chromatographic techniques followed by various detection methods. The methodology to be chosen for the identification and quantification of the mycotoxins will depend on the objectives of the analysis.

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Conventional Methods: Chromatographic Analysis Chromatography is a group of methods most commonly used for the measure of mycotoxin in food and feed (Alshannaq and Yu, 2017) because they are accurate, selective, and sensitive. The purpose of all types of chromatography is the separation of the analyte of interest (mycotoxin) of a complex mixture (food or feed). The food extract is dissolved in a solvent called the mobile phase, which flows over or through another material called the stationary phase. A substance with more affinity for the mobile phase moves rapidly through the system. On the contrary, an analyte with more affinity for the stationary phase spends more time immobilized in that phase and takes a longer time to pass through the system. Therefore, it separates from the first substance. Different combinations of mobile and stationary phases give rise to the types of chromatography used in analysis. In the liquid chromatography (LC), specific compounds of a sample are separated by a liquid (mobile phase) flowing through a column (stationary phase). In gas chromatography (GC), the components are separated based on interactions between a column (stationary phase) and a mobile phase (inert gas). Thin-layer chromatography (TLC) uses a planar surface as stationary phase. For mycotoxin analysis, the separation using TLC and high-performance liquid chromatography (HPLC) is widely employed as most mycotoxins are nonvolatile compounds. When the nonvolatile mycotoxins are separated by GC, they have to be modified to volatile compounds for derivatization with silylation reagents before the analysis. In recent years, the advances made in coupling mass spectrometry (MS) to HPLC allowed the development of methods to simultaneously determine co-occurring mycotoxins of regulatory significance. These multitoxin methods meet the need of monitoring for many toxins within a sample (Trucksess and Zhang, 2016).

Thin-Layer Chromatography Thin-layer chromatography (TLC) was the first method used for detection of mycotoxins and still is widely used. This method has been developed for most of the major mycotoxins in corn. AOAC international has approved several TLC methods for AFs in groundnuts and corn as well as ZEA in corn (Pitt et al., 2012). Chromatography is carried out using a planar surface. The sample is transported over a solid surface such as cellulose or silica gel, coated on a plate (stationary phase). The sample components move over the surface by the solvent system (mobile phase), which travels through the adsorbent layer by capillary action. Some mycotoxins can be detected by examination of TLC plates under ultraviolet light. Other mycotoxins give a visible spot after spraying with chemical reagent. Differences in solubility and adsorbency among compounds result in separation, and comparison with standards allows identification of individual spots. These procedures are more accurate and reliable if carried out with automatic TLC samplers and optical readers (Pitt et al., 2012). TLC methods can be improved by proper cleanup of samples using C18 SPE cartridges and IACs. For example, the use of IAC columns to purify corn for FB1 determination increases the sensitivity of TLC (Preis and Vargas, 2000). TLC is a simple, rapid, and cheap method. In addition, a number of samples can be run simultaneously. However, low precision and the lack of sensitivity for some toxins are the main disadvantages of this technique.

Liquid Chromatographic Methods Most official methods are based on high-performance liquid chromatography (HPLC) (Pitt et al., 2012). Coupled with a variety of detectors, practically all mycotoxins have been separated and detected by HPLC (Krska et al., 2005). Also, this method is frequently used both for routine analyses and as a confirmatory method for novel or screening techniques. Mycotoxins of interest in food and feed contamination are relatively small polar compounds, ideally suited to separation by reversed-phase HPLC. The characteristic of the reverse mode is to employ a polar mobile phase and a nonpolar column (stationary phase). In general, the mobile phase is water, methanol or acetonitrile, or mixtures of these components. In the HPLC, small portion of the extract is injected into a stream of solvent (mobile phase). The equipment has a pump that allows the solvent go through a column of adsorptive matrix (stationary phase). The components of the sample are separated based on interactions between stationary and mobile phases. After the extract compounds are separated, the mycotoxins have to be detected. Common HPLC detectors, such as ultraviolet spectrophotometer (UV) and fluorophotometer (FL), have found widespread application (Shephard, 2011). DON and its acetylated derivatives can be routinely quantified by reverse-phased HPLC and UV detection with good accuracy and precision (MacDonald et al., 2005). HPLC coupled to fluorescence detection stage (FL) use the mycotoxin fluorescence properties to quantify them. However, if the toxin does not have this property or, if necessary, to improve the sensitivity of the technique, derivatization with a fluorophore is required. For aflatoxin quantification, although AFs possess natural fluorescence, pre- or post-column derivatization is needed in the aqueous mixtures used for reversed-phase chromatography because the fluorescence of AFB1 and AFG1 is significantly quenched (Shephard, 2011). Trifluoroacetic acid (TFA) is generally used for pre-column derivatization, whereas

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bromination methods have found wide applicability as a post-column derivatization (Brera et al., 2007). For fumonisins, non-fluorescing mycotoxins, several fluorescent derivatives have been used, including fluorescamine, o-phthaldialdehyde (OPA), 4-fluoro-7-nitrobenzofurazan (NBDF), and others. The OPA method is the most widely used, and two methods for the determination of fumonisins in maize using OPA derivatives were validated by collaborative study and achieved AOAC International official status (Sydenham et al., 1996; Visconti et al., 2001).

Mass Spectrometry A mass spectrometry is a technique that determines the mass of the substance after its ionization. Mass analysis in combination with HPLC or gas chromatography is a reliable method for the confirmation of the identity of the mycotoxin in positive samples. The specificity of mass spectrometry (MS) or tandem mass spectrometry (MS/MS) also has been used to avoid purification steps and is an important component of multitoxin methods (Pitt et al., 2012). Different liquid chromatographic (LC) and mass spectrometry (MS) interfaces have been used. The interface facilitates the transition of separated components from the LC column at atmospheric pressure to high vacuum conditions needed at the MS analyzer. In the interface, the LC solvent is removed and the analyte molecules are ionized. Currently, most extensively applied LC-MS interfaces are based on atmospheric pressure ionization (API) strategies like electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), and atmospheric pressure photo-ionization (APPI). APCI is mainly used for analysis of hydrophobic compounds, while ESI can ionize compounds of high polarity, such as proteins, peptides, and small molecular weight substances like drugs, mycotoxins, and food additives. In addition, there are many types of mass analyzers such as quadrupole, time-of-flight (TOF), ion-trap, and Fourier transform-ion cyclotron resonance (FT-ICR). ESI, triple quadrupole, and TOF have been used extensively for mycotoxin analysis (Alshannaq and Yu, 2017). The triple quadrupole instruments have the limitation that ion settings have to be made before analysis, so what is searched for has to be known beforehand. To investigate unknown or unexpected compounds, it would be better to apply full scan mass spectrometry. For this purpose, TOF is the first option (Spanjer, 2011). The increasing availability of LC-MS (/MS) methods provides relevant data about the co-occurrence of the mycotoxins in food and feed. Sulyok et al. (2006) were the first researchers to publish a validated method for the determination of 39 mycotoxins in maize and wheat using a single extraction step followed by LC/ESI-MS/MS without cleanup of the samples. More recently, Liao et al. (2013) validated a multi-mycotoxin LC-MS/MS method for the simultaneous determination of 26 common mycotoxins in maize, rice, wheat, almond, peanut, and pistachio products. Because no official/ standard methods for mycotoxins are based on LC-MS, an international Proficiency Testing (PT) was carried out (De Girolamo et al., 2017). In the study, the performance of LC-MS/MS for multi-mycotoxin determination in maize and wheat was evaluated for 18 laboratory participants from 10 countries. The authors reported an improvement of the method’s performances in corn after comparing the PT results with previous studies (2011–12). The positive result was mainly attributed to the management of factors that affect reliability such us matrix effect and a simplification of sample preparation protocols. In addition, researchers at the FDA’s Center for Food Safety and Applied Nutrition (FDA/CFSCAN) have been evaluating modern LC-MS techniques with the aim of improving the efficiency of mycotoxin determination (Zhang et al., 2016).

Rapid Methods for Mycotoxin Determination: Immunochemical Analysis The need for rapid decisions within the food and feed industry has led to a number of new screening methods. The term ‘rapid method’ generally refers to a method much faster than respective reference or conventional ones (Zheng et al., 2006). They usually provide sensitive detection and are very cost-effective. In addition, most of these easy-to-use kit tests can be conducted under field conditions. The rapid methods in mycotoxin analysis are based on immunochemical principles because they employ specific antibodies against particular mycotoxins. The most widespread tests are the enzyme-linked immunosorbent assay (ELISA), lateral flow tests, and, more recently, biosensors. Another alternative is ROSA (Rapid One Step Assay) system that provides rapid and quantitative results. The main drawbacks of many rapid screening methods are their limited capacity for mycotoxin quantification and cross-reactivity that may yield false-positive results. Positive results should, therefore, be confirmed with the reference methods to avoid misinterpretations (Kralj Cigi
c and Prosen, 2009).

ELISA (Enzyme-Linked Immunosorbent Assay) The ELISA operating principle has been developed for most economically relevant mycotoxins such as AFs, OTA, T-2 toxin, ZEA, DON, and FUMs in ready-to-use ELISA kit formats. The Official Methods of Analysis of AOAC International has adopted several commercial ELISA kits, as well as USDA´s Grain Inspection and Packers and Stockyards

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FIG. 9.19 Principle of a direct competitive ELISA for mycotoxin analysis.

Administration (GIPSA). Most of the ELISA kits employ a competitive ELISA format (Fig. 9.19) because mycotoxins are low molecular substances. That is, the immunoassays are often based upon the competitive binding between an enzymelabelled and unlabelled toxin for a limited number of antibodies on an ELISA plate (solid phase) (Fig. 9.19A). After incubation, unbound compounds are removed by washing and chromogenic substrate is added (Fig. 9.19B). The resulting colored product is measured optically. The enzymatic activity in each well is inversely proportional to the mycotoxin concentrations in the sample. Quantitative results are achieved in approximately 10–20 min when using a reader. Some advantages of using ELISA include time efficiency, relatively low cost, and convenience. A disadvantage is that any factor that interferes with the antigen-antibody binding will result in an inflated estimate of mycotoxin in the sample or false positives. If the assay is sufficiently sensitive, the matrix interference could be resolved simply by diluting the sample before the assay. Thus, the ELISA methods need the cleanup step in some samples prior to application and reanalysis of positive samples by another method, such as HPLC (Nakajima, 2006). Also, faster and more straightforward immunoassaybased tests are preferentially used for applications where on-site use is necessary (Krska and Molinelli, 2009).

Immunochomatographic Strip Test or Lateral Flow Immunoassay Immunochromatographic strip tests are a convenient one-step method for first-level screening, which does not require instrumentation and reagents. Immunochromatographic tests for the determination of AFs, FUMs, DON, OTA, and ZEA have been developed and commercialized. Many commercial devices have been validated by the USDA-GIPSA (Anfossi et al., 2013). A test strip mainly consists of a porous nitrocellulose membrane with three reaction regions in which the test solution serves as a mobile phase (Fig. 9.20). The first region contains the specific antibodies labelled with colloidal gold. After the liquid sample is added, antibody-antigen complex is formed. Then, the immunocomplex migrate in the solution along the membrane to the second region where the analyte is captured with a second antibody and a visible line is formed. Excess of antibody migrate to the third region where it is captured with a third antibody (anti-species-specific antibodies, Anti-IgG). The appearance of a color at the second control line is necessary and it indicates the success of the test (Nakajima, 2006). The results obtained are visually evaluated. In this context, the subjective perception of the person who interprets particular results is one of the main sources of error. As a rule, in the development of noninstrumental tests for the determination of mycotoxins, attempts are made to reach a detection limit that corresponds to the legislatively defined maximum permissible concentration of a given mycotoxin in the analyzed product (Goryacheva and De Saeger, 2011). Among the drawbacks of this simple and rapid technique is the loss of sensitivity and problems with reproducibility and reliability with different matrices (Krska and Molinelli, 2009). Extremely good antibodies will remain a

FIG. 9.20 Schematic illustration of a lateral flow test.

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TABLE 9.11 Comparison of Different Rapid Formats Test Conditions

ELISA

Lateral Flow Strips-QL

Lateral Flow Strips-QT

96 wells

1 assay

1 assay

Lab

on-site

Lab

½ h–3 h

Minutes

Minutes

Detection levels

mg-pg/kg level

mg/kg level

mg/kg level

Main application

Screening of raw materials

Pre and postharvest control

Fast pre-screening

Number of samples Localization Time

QL, qualitative; QT, quantitative. Adapted from Krska, R., Molinelli, A., 2009. Rapid test strips for analysis of mycotoxins in food and feed. Anal. Bioanal. Chem. 393(1), pp. 67–71.

major requirement for easy-to-use assays. Another challenge is the reduction of the matrix effect. These tests are usually combined with a simple extraction with the mix water/buffer: organic solvent (20%–40%: 80%–60%). As we mentioned for ELISA, the dilution of this extract can reduce the matrix effect and decrease the organic solvent concentration (Goryacheva and De Saeger, 2011). Most lateral flow immunoassays are qualitative, indicating a positive result when mycotoxin concentration is above a predetermined threshold. However, quantitative results are possible with some assays. Rapid One Step Assay (ROSA) is a lateral flow quantitative method that combines the ease of use of lateral flow strips with the precision of a quantitative method over a defined concentration range. The results are processed by special portable devices (readers) (Salter et al., 2006). The use of these devices makes it possible to avoid the subjective perception of color and to improve the sensitivity of the determination (Goryacheva and De Saeger, 2011). There are a variety of ROSA strips to detect mycotoxins (AFs, DON, FUMs, OTA, T2/HT2, and ZEA) in feed and grain. All ROSA mycotoxin tests follow a similar assay format and can be read on the same equipment. In relation with the strip test, recently available devices (i.e., smart phones app as a new alternative to a reading device) seem to allow fast and reliable measurements for mycotoxin determination after proper calibration. Table 9.11 summarizes characteristics of different rapid testing formats for mycotoxins.

Emerging Techniques Masked Mycotoxins Masked mycotoxins are compounds that are undetectable by routine assays due to a structural modification of the toxin mainly caused by detoxification mechanisms of the plant. Plants transform compounds like mycotoxins into more polar metabolites to be easily conjugated or transported into vacuoles for storage (Berthiller et al., 2013). As Fusarium infection usually occurs in the field, the Fusarium mycotoxins DON, FB1, NIV, ZEA, T-2, and HT-2 are the most noticeable targets for conjugation (Berthiller et al., 2015). However, technological process such as milling, cleaning, sorting, and thermal treatment can also transform and/or degrade the mycotoxins because of mechanical or thermal energies (Suman and Generotti, 2015). Masked mycotoxins usually occur as a result of enzymatic reactions involving conjugation of the parent compounds with sugars or amino acids. Zearalenone-14-glucoside (ZEN14G) and deoxynivalenol-3-glucoside (DON3G) along with the two acetylated derivatives of DON (15-acetyldeoxynivalenol, 15ADON and 3-acetyldeoxynivalenol, 3ADON) have been detected from naturally infected cereals such as maize, wheat, and barley (Cirlini et al., 2012). With regard to FUMs, there is evidence for their compartmentalization in plants, which can be determined only after application of a hydrolysis step. Occurrence of these hidden forms has been reported in raw corn as well as in derived food (Dall’Asta et al., 2009; Berthiller et al., 2013). The occurrence of masked mycotoxins generally implies a possible underestimation of the mycotoxin contamination level of a certain commodity. LC-MS/MS and immunochemical methods have been applied for the detection of masked mycotoxins. One limitation of these methodologies is the availability of appropriate analytical standards. In addition, masked mycotoxins can be quantified indirectly by hydroxylation to their parent mycotoxins using enzymes or acidic/ alkaline conditions (Berthiller et al., 2014; Berthiller et al., 2015). One important point about the analysis of modified toxins is the level of toxicity in relation to their precursors. The masking phenomenon may lead to altered toxicity; however, very little is known so far about the potential bioactivity of most masked mycotoxins and this requires further investigation.

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Biosensors The use of biosensors as rapid screening tests has the advantage of being simple, portable, quick, and cost-effective. In addition, the analysis of food and feed samples can be done on-site such as on the farm or at processing or manufacturing facilities with potential for reuse (Tothill, 2011). Biosensors are characterized by a biological element (recognition element) that interacts with a target analyte. In the case of immunosensors, the recognition element is an antibody or an antibody fragment. However, in some recently developed technologies, the recognition element is synthetic (not a biological derivative). For example, MIPs, aptamers, synthetic peptides, and so forth are not considered biosensors (Maragos, 2016). Once the binding event occurs, this is transformed into a physical signal that is usually quantified by an electrochemical or optical detector. One example of an electrochemical device is the well-known glucose biosensor based on a screenprinted amperometric disposable electrode. An ELISA-based assay conducted on the electrode surface is the most frequently used technique for identification and quantification of different mycotoxins. In this system, the antibody (or antigen) is labelled with an enzyme. The enzyme catalyzes an added substrate and produces an electroactive species, which is then detected on the sensor surface (Tothill, 2011). Applications of electrochemical immunosensors for the detection of mycotoxins in corn and corn products have included determination of AFB1 ((Piermarini et al., 2007, 2009), OTA (Liu et al., 2009), FB1 and FB2 (Abdul Kadir and Tothill, 2010), ZEA (Panini et al., 2010), ZEA, and a,b-ZOL (Zougagh et al., 2008). Concerning the optical biosensors, the transducers include optical fibers, fluorescence, spectroscopy of optical waveguides, surface plasmon resonance (SPR), and microarrays. The principle of these devices is to measure changes in amplitude, phase, frequency, or polarization of light (Narsaiah et al., 2012). Meneely et al. (2010a) described advances using an optical (SPR) biosensor for the detection of DON in maize, maize-based baby food, durum wheat, and wheat products where the sample extraction procedure requires no cleanup. A SPR screening assay also was reported for the combined detection of T-2 and HT-2 toxins in naturally contaminated cereals (Meneely et al., 2010b). Today, it is desirable not only to complete analyses as quickly as possible, but also to detect multiple toxins within a single assay. The array-based approach for detecting multiple mycotoxins was recently described by Plotan et al. (2016). This study applied biochip array technology to the semiquantitative determination of over 20 mycotoxins from all the main groups in feed samples including raw cereals by using miniaturized simultaneous immunoassays applied to the semiautomatic analyzer.

MANAGEMENT OF MYCOTOXINS IN CORN Management of mycotoxins involves practices that are employed preharvest, during harvest and drying, and postharvest (including detoxification approaches). Postharvest management can include not only the practices directed at reducing mycotoxin levels in the product, but also the management of mycotoxin-related risk through utilization strategies, or regulation. The regulatory status of mycotoxins in corn is covered elsewhere in the chapter.

Preharvest Preharvest strategies primarily consist of tactics designed to reduce infection by ear-rot fungi. In general, available ear-rot management strategies are not adequate, but the risk of ear-rot development can be reduced by some preharvest strategies. Preharvest strategies include: 1. Genetic resistance and other corn hybrid characteristics 2. Cultural practices (planting date, tillage practices, crop rotation, plant population, irrigation) 3. Crop protection chemicals (fungicides and insecticides). Breeding for resistance to ear-rot pathogens has been challenging, and high levels of resistance have been elusive. In general, major genes for resistance have not been identified. However, hybrid selection during the breeding process usually eliminates very susceptible genotypes, and significant progress has been made using molecular markers for partial resistance. Moderately resistant hybrids are widely grown, especially in areas prone to ear-rot problems. For Gibberella ear rot, major genes for resistance were identified in the 1990s (Reid et al., 1994); inbreds and hybrids incorporating this resistance demonstrated very low severity of Gibberella ear rot and greatly reduced levels of deoxynivalenol (DON) compared to susceptible genotypes. For this pathogen, resistance to infection through the silks and resistance to spread of the fungus among the kernels are under separate genetic control (Chungu et al., 1996). Since the early 2000s, numerous studies have been published reporting quantitative trait loci (QTL) for resistance to Gibberella ear rot

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(Giomi et al., 2016; Kebede et al., 2016). Molecular breeding techniques such as transcriptome profiling (Kebede et al., 2018), genotyping-by-sequencing (Kebede et al., 2016), and genome-wide association studies (GWAS) (Han et al., 2018) have greatly aided in identification and characterization of markers and candidate genes for resistance to this disease. Commercial hybrids with high levels of resistance to Gibberella ear rot are available, but the sporadic nature of the disease can be a disincentive to prioritizing resistance during hybrid selection. Genetic resistance to Aspergillus infection and aflatoxin accumulation has been studied extensively and reported in the literature. During the 1970s, hybrids were evaluated for differences in aflatoxin accumulation (Lillehoj et al., 1975a, 1976; Widstrom et al., 1978); and sources of resistance were reported by the mid- to late-1980s (Widstrom et al., 1987; Scott and Zummo, 1988). Studies of inheritance of resistance also were conducted since the 1980s. There are several well-characterized sources for resistance to A. flavus infection or aflatoxin production (Brown et al., 1999). Resistance in some of these sources has been linked to one or more kernel proteins that inhibit either fungal growth or aflatoxin production. A 14-kDa trypsin-inhibiting protein is involved with resistance in several of the sources, and it may serve as a useful marker to assist breeding efforts. Utilization of germplasm identified in these studies has been hampered by its lack of adaptation to the corn belt (Campbell and White, 1995) and difficulties in incorporating polygenic resistance into elite germplasm. Molecular breeding strategies have partially overcome these obstacles (Warburton et al., 2009, 2011). Recent advances in breeding and biotechnology contributions to A. flavus/aflatoxin resistance have been reviewed (Bhatnagar-Mathur et al., 2015). Currently, corn hybrids with improved resistance to A. flavus and aflatoxins are being used, but the level of resistance is not yet adequate to prevent unacceptable aflatoxin concentrations in some fields. Sources of resistance to Fusarium ear rot were identified as early as the 1940s, but they typically have been polygenic and were difficult to incorporate into high-yielding hybrids. Breeding efforts through the 1970s and 1980s met with some success, but the genetic basis for resistance was not well-understood and methods for evaluating genotypes were unsatisfactory. Current evaluation methods rely on artificial inoculation or manipulation of locations and planting dates to maximize disease pressure. Understanding of the genetics of resistance has made tremendous progress, first through the identification of molecular markers (QTL or single-nuleotide polymorphisms, SNPs) and use of marker-assisted selection (Robertson-Hoyt et al., 2006; Eller et al., 2008), and then through gene expression studies using microarray or RNASeq technologies (Lanubile et al., 2014). These studies have revealed that resistance to fungal infection and suppression of fumonisin levels may be under separate genetic control in the plant (Robertson et al., 2006), but some markers are associated with low levels of infection and low fumonisin concentrations. Detailed information about sources of resistance and their molecular bases can be found in Lanubile et al. (2014) and Lanubile et al. (2017). In general, the level of resistance to Fusarium ear rot in commercial hybrids has greatly improved over the past decade, but resistance in many popular highyielding hybrids is not adequate to prevent unacceptable fumonisin concentrations in some fields. An attractive alternative to conventional breeding is to employ genetic transformation or gene editing technologies to achieve resistance to mycotoxigenic fungi or to suppress mycotoxin formation in plants. Duvick (2001) outlined a number of strategies toward reducing fumonisin contamination that could be pursued. Infection may be reduced by enhancing the expression of, or introducing, novel genes to express antifungal proteins or secondary metabolites, such as hydroxamic acids, phenolics, and stilbenes. Inducing existing plant defense pathways also might be effective in fending off infection. Alternatively, inhibition of fumonisin accumulation could be achieved by engineering plants to produce or fail to produce signaling compounds that are involved in fumonisin biosynthesis or enzymes that degrade fumonisins. Similar strategies could be applied to achieve resistance to other mycotoxin-producing fungi and their corresponding mycotoxins. In one example, corn plants were engineered to express enzymes that degrade fumonisins. A yeast, Exophiala spinifera, is capable of metabolizing fumonisins in vitro (Blackwell et al., 1999). A fumonisin esterase gene and an amine oxidase gene from the yeast have been identified, cloned, and expressed in corn plants, demonstrating reduced levels of fumonisins in kernels (Duvick, 2001). Because trichothecenes are believed to be involved with pathogenicity of fungi such as F. graminearum, engineering plants for resistance to trichothecenes may protect them from disease as well. Several enzymes have been identified for trichothecene degradation (e.g., Okubara et al., 2002) and they may be effective if they can be expressed in plants. Biosynthesis of aflatoxins has been studied extensively with the goal of identifying targets for developing transgenic resistance (Yu, 2012). This strategy has recently come to fruition with the report of aflatoxin suppression in maize kernels obtained by transforming maize plants with a kernel-specific RNA interference (RNAi) gene cassette targeting the aflC gene, which encodes a key enzyme in the aflatoxin biosynthetic pathway (Thakare et al., 2017). This study demonstrated the feasibility of using genetic engineering for aflatoxin suppression, although the limit of detection for the thin-layer chromatography method was 93ng/g, well above acceptable limits. As in the previous example of fumonisin degradation through genetic engineering, this approach has limitations; although the grain was low in mycotoxins, fungal infection was not impaired, and it is not clear whether postharvest mycotoxin development would be affected (Gressel and Polturak, 2018).

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Although significant research advances have been published and development efforts undertaken in the private sector, there are many obstacles to development and commercialization of genetically engineered resistance to fungi, and corn hybrids with genetically engineered resistance to mycotoxigenic fungi are not available on the market. Genetic engineering or gene editing for direct protection from mycotoxigenic fungi and their mycotoxins is likely to be a valuable management strategy in the future, but complicated intellectual property and regulatory issues need to be resolved. Most corn hybrids grown in the U.S. and several other countries express transgenes for protection against Lepidopteran insects that damage corn ears. Transgenic insect protection indirectly protects the kernels from infection by mycotoxigenic fungi, especially those that cause Fusarium ear rot. Because of the correlation between insect injury and incidence of Fusarium ear rots (Lew et al., 1991; Dowd, 1998; Munkvold et al., 1999), transgenic insect control can reduce ear-rot occurrence and fumonisin concentrations (Munkvold et al., 1998, 1999). Bt corn hybrids have been shown to be at a much lower risk for fumonisin contamination than conventional hybrids (Masoero et al., 1999; Clements et al., 2001; Hammond et al., 2004; Delgado and Wolt, 2011; Bowers et al., 2013, 2014). Similar results have been reported on a limited basis for DON in Bt hybrids (Schaafsma et al., 2002), although the relationship between insect damage and DON is much weaker than that for fumonisins. Aflatoxins are correlated with insect damage, but transgenic insect protection has demonstrated inconsistent effects on aflatoxin levels. Aside from direct genetic resistance to infection or mycotoxin production, hybrid selection can contribute to mycotoxin reduction in other ways. Hybrids are developed with tolerances to specific stresses that may occur in local environments. Plants under stress are generally more susceptible to toxigenic fungi. Shelby et al. (1994) reported fumonisin concentrations in a group of hybrids planted in 17 locations ranging from Georgia to Nebraska and Wisconsin. Hybrids grown outside their adapted range appeared to be more susceptible to fumonisin accumulation, and there was a negative correlation between fumonisins and latitude. Tolerance to specific environmental stresses, such as drought, has been suggested as a way to reduce vulnerability of corn hybrids to aflatoxins (Tubajika and Damann, 2001). Some ear and kernel characteristics, such as pericarp thickness or husk coverage, may provide physical barriers to ear-rot infection or insect vectors of fungi. Cultural practices can be employed to reduce infection by mycotoxigenic fungi. Most of the fungi causing ear rots survive in crop residue. Reducing the amount of residue through crop rotation or tillage can reduce the risk of some ear rots, but may not significantly affect mycotoxins levels in corn. Head scab of wheat, caused by F. graminearum, and associated DON concentrations were significantly affected by crop rotation; DON levels were more than twofold higher when wheat followed corn in the rotation (Schaafsma et al., 2001). Similarly, head scab and DON were reduced in wheat by plowing under crop residues (Dill-Macky and Jones, 2000). The same pathogen causes Gibberella ear rot of corn and crop residues are clearly the most important source of inoculum (Sutton, 1982). Managing crop residue (through rotation or tillage) is suggested as a control measure for this disease (Payne, 1999; Sutton, 1982), but there is little direct evidence for the success of this approach. In South Africa, tillage practices did not affect the incidence of ear rot caused by F. verticillioides or F. graminearum (Flett and Wehner, 1991; Flett et al., 1998). Studies on the survival of A. flavus and Fusarium species that cause Fusarium ear rot suggest that tillage and crop rotation are unlikely to affect these diseases and their mycotoxins (Cotten and Munkvold, 1998; McGee et al., 1996). Planting dates can be manipulated in some cases to reduce the risk of severe ear rot; in general, earlier planting dates in temperate areas result in a lower risk, but this effect can be affected greatly by annual fluctuations in weather. The effects of several cultural practices on aflatoxin development in corn have been investigated (Jones and Duncan, 1981; Jones et al., 1981; Lillehoj, 1983; Payne et al., 1986). Earlier planting and harvest, along with irrigation or deep tillage to alleviate drought stress all reduced infection and aflatoxin levels. A study conducted in Mexico demonstrated that a combination of cultural practices (early planting, reduced plant population, and irrigation), hybrid selection, and insect control reduced aflatoxin concentrations down to 0 to 6 ng/g, compared to 63 to 167 ng/g in late-planted, nonirrigated corn at a higher plant population without insect control (Rodriguez-delBosque, 1996). Elevated aflatoxin levels have been associated with fertility- and weed-related stresses (Lisker and Lillehoj, 1991). Jones and Duncan (1981) reported that a higher rate of nitrogen fertilization consistently resulted in reduced aflatoxin levels. Plants receiving 145.7 kg/ha N had aflatoxins ranging from 19 to 2000 ng/g, while those receiving only 11.2 kg/ha N ranged from 64 to 4875 ng/g aflatoxins. Fungicidal control of ear rots typically is not recommended because standard application methods do not result in adequate coverage of silks and ears. Although some fungicides are effective in vitro against ear-rot pathogens, efficacy in the field has not been well-demonstrated and further development of effective application methods is needed. The relatively greater economic impact of Fusarium head blight of wheat has stimulated a great deal of research on fungicidal control of F. graminearum in that crop, and this is now a common management tactic for Fusarium head blight in wheat (Paul et al., 2010; Caldwell et al., 2017; Machado et al., 2018). Choice of active ingredient is important because some fungicides used for foliar disease management in wheat can enhance mycotoxin production (D’mello et al., 1998). Because of the economic and application technology barriers, it is unlikely that fungicides will play a substantial role in control of ear-rot diseases of corn.

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Biological control of mycotoxigenic fungi, particularly A. flavus, has been successfully demonstrated in corn grown in North America and Africa. By treating fields with non-aflatoxigenic strains of A. flavus, researchers were able to reduce aflatoxin levels in kernels; the atoxigenic strains are able to out-compete endemic, toxigenic strains (Weaver et al., 2017). The effectiveness of this approach in Africa, as well as a discussion of some potential barriers to its implementation, are discussed by Bandyopadhyay et al. (2016). Because of the correlation between insect injury and incidence of Fusarium ear rots (Smith and White, 1988; Dowd, 1998; Munkvold et al., 1999), insect control with insecticides can reduce ear-rot occurrence (Farrar and Davis, 1991; Blandino et al., 2010) and fumonisin concentrations (Munkvold, 2014). Economically feasible insecticide programs, however, may not be adequate to substantially reduce ear-rot severity. Similarly, although insecticides can significantly reduce Aspergillus ear-rot severity and aflatoxins (Rodriguez-del-Bosque, 1996), this may not be achievable with an economically feasible insecticide program. Another approach that may contribute to preharvest management decisions is to predict mycotoxin concentrations based on seasonal weather and other risk factors. Many studies have reported correlations or other mathematical relationships between weather factors and fungal growth and/or mycotoxin levels (Marı´n et al., 1999a; Stewart et al., 2002), but translating these relationships into accurate predictions has been challenging. Laboratory studies have generated detailed information about environmental impacts on expression of mycotoxin biosynthesis genes that should prove useful in further development of predictive models (Magan and Medina, 2016). Models have been developed to assess the risk of aflatoxin contamination of preharvest corn in southern Europe (Battilani et al., 2013, 2016) and Kenya (Chauhan et al., 2015), which take different modeling approaches (empirical vs. mechanistic) to account for the effects of temperature, rainfall, and other factors. Similarly, models for use in the U.S. have been tested (Widstrom et al., 2000), but not widely used. Models also have been developed to predict occurrence of Fusarium toxins in both wheat and corn. Wheat models are well-developed and commercially used in the U.S. and elsewhere in the world (see Landschoot et al., 2013), but models for corn have been more difficult because of the more complicated epidemiology of mycotoxigenic Fusaria in the corn crop (Schaafsma and Hooker, 2007). The utility of predicting mycotoxin levels in wheat is primarily related to decision making for fungicide applications in the field. Until fungicides become a more efficient tool for mycotoxin management in corn, there is less incentive to develop predictive models.

Harvest and Drying Once the crop is nearing maturity, the key to preventing mycotoxin problems is addressing mold problems early, in the field and in the bin. If there is little ear-rot disease, if insect activity is not a serious problem, and weather conditions are favorable for grain drying, it is generally safe to allow field drying to proceed to desirable moisture levels for harvest. However, in fields with a significant amount of ear-rot symptoms, harvest timing can have a major impact on the ultimate level of mycotoxin accumulation (Jones et al., 1981). Moisture content of corn kernels after physiological maturity can remain high enough to allow continued development of fungi for several weeks, especially if conditions are poor for dry-down. Insects may continue to feed on corn in the field late in the season, enhancing the ability of fungi to attack the kernels. Under these conditions, delayed harvest allows for more prolonged periods of fungal growth and mycotoxin accumulation prior to drying. To assess the need to harvest early, scouting in the field is necessary. Unfortunately, late-season scouting is an often-neglected activity. During harvest and transportation operations, grain can be physically damaged, and this damage contributes to the potential for fungal colonization and mycotoxin development. Mechanical combines that are used to harvest the vast majority of corn grain can cause superficial damage or breakage of kernels. This damage, and the vulnerability of the grain to toxigenic storage fungi, can be reduced by adjusting the combine cylinder speed and the clearance between the cylinder and concave (Herum, 1987). Furthermore, harvested grain quality can be improved by increasing the combine fan speed so that the low-density, moldy kernels are discarded out the back of the combine. This strategy obviously has limitations; discarded grain is an economic loss, so it is undesirable to discard more than a low percentage of the grain. After harvest, reducing grain moisture by artificial drying is a critical way to arrest fungal development and mycotoxin production. Corn drying strategies are described in detail in Chapter 5. Prior to the advent of the combine harvester, natural drying in the field or crib was the norm. Contemporary practices for corn usually involve some artificial drying, although it is economically desirable to allow grain to dry in the field as much as possible. The objective of grain drying is to reduce moisture content to the extent that molds, both toxigenic and otherwise, are not able to grow or remain physiologically active. For corn, recommended maximum moisture content is 15% for storage through the winter, 14% for storage through the following summer, and 13% or less for storage of a year or longer (Brook, 1992). These recommendations are based on the ability of some fungi to grow at relatively low grain moistures (Table 9.12), taking into consideration that some

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TABLE 9.12 Temperature and Relative Humidity Conditions for Growth of Mycotoxigenic and Other Storage Fungi in Corn Relative Humidity

Temperature, °C

Grain Moisture

Fungal Species

Lower Limit

Optimum

Upper Limit

Lower Limit

Lower Limit

Aspergillus flavus

10–15

37–39

45–48

82

16.0–16.5

Aspergillus glaucus

0–5

30–35

40–45

73

14.0–14.5

Aspergillus restrictus

5–10

30–35

40–45

70

13.5–14.5

Fusarium verticillioides

4

28–30

36

91

19.0–20.0

Gibberella zeae (Fusarium graminearum)

4

28

32

94

20.0–21.0

Penicillium brevi-compactum

2

20–24

30–32

81

16.0–16.5

Penicillium cyclopium

2

20–24

30–32

81

16.0–16.5

Penicillium oxalicum

8

31–33

35–37

86

17.0

Penicillium funiculosum

8

31–33

35–37

91

19.0–20.0

Penicillium viridicatum

2

20–24

34–36

81

16.0–16.5

Adapted from Watson, S., 1987. Measurement and maintenance of quality. In Watson, S.A., Ramstad, P.E. (Eds.), Corn, Chemistry and Technology. American Association of Cereal Chemists, St. Paul, pp. 125–183. From: Mislivec, P.B., Tuite, J., 1970. Temperature and relative humidity requirements of species of Penicillium isolated from yellow dent corn kernels. Mycologia 62(1), 75–88; Watson, S., 1987. Measurement and maintenance of quality. In Watson, S.A., Ramstad, P.E. (Eds.), Corn, Chemistry and Technology. American Association of Cereal Chemists, St. Paul, pp. 125–183, Klich, M., Tiffany, L., Knaphus, G., 1992. Ecology of the aspergilli of soils and litter. In Bennett, J.W., Klich, M.A. (Eds.), Aspergillus: Biology and Industrial Applications. Butterworth Heineman, Boston, pp. 329–354; Marı´n, S., Magan, N., Belli, N., Ramos, A., Canela, R., Sanchis, V., 1999a. Two-dimensional profiles of fumonisin B1 production by Fusarium moniliforme and Fusarium proliferatum in relation to environmental factors and potential for modelling toxin formation in maize grain. Int. J. Food Microbiol. 51(2–3), 159–167 and Marı´n, S., Sanchis, V., Sanz, D., Castel, I., Ramos, A.J., Canela, R., Magan, N., 1999b. Control of growth and fumonisin B1 production by Fusarium verticillioides and Fusarium proliferatum isolates in moist maize with propionate preservatives. Food Addit. Contam. 16(12), 555–563; Reid, L., Nicol, R., Ouellet, T., Savard, M., Miller, J., Young, J., Stewart, D., Schaafsma, A., 1999. Interaction of Fusarium graminearum and F. moniliforme in maize ears: disease progress, fungal biomass, and mycotoxin accumulation. Phytopathology 89(11), 1028–1037; Stewart, D., Reid, L., Nicol, R., Schaafsma, A., 2002. A mathematical simulation of growth of Fusarium in maize ears after artificial inoculation. Phytopathology 92(5), 534–541.

variability will exist within the grain mass. Most research on grain moisture in relation to toxigenic fungi has considered only the effects of moisture on fungal growth, not on mycotoxin production. The implicit assumption is that mycotoxin production will not occur unless there is sufficient moisture for fungal growth and metabolism. Research on fumonisin production by F. verticillioides and F. proliferatum has supported that assumption, and indicated that fumonisin production ceases around water activity of about 0.90 (Marı´n et al., 1999a). Indeed, it appears that in many cases, mycotoxin production occurs in more restricted range of temperature and water activity than fungal growth (Ominski et al., 1994). Artificial drying can be achieved through the use of natural gas burners, which provide high temperatures (50–82°C) and rapid drying, or ambient or low-temperature drying, which involves drawing outside air through a bin of grain, drying the grain slowly. In general, high-temperature drying is less risky in terms of mold problems. Corn with significant ear-rot symptoms from the field should be dried at high temperature as quickly as possible to minimize the risk of mycotoxin development. The lower the moisture content in storage, the lower the risk of mycotoxin development.

Postharvest Management Even after grain is dried to proper moisture levels, vigilance toward the possibility of mycotoxin problems must continue. Mold development can arise in storage due to moisture variability within the grain mass or because of moisture migration that results from rapid grain cooling along the bin walls. Therefore, a postharvest management strategy for mycotoxins must be employed. This primarily involves the maintenance of proper storage conditions, including insect control. Stored corn management is covered in detail in Chapter 5. Most grain in the United States is stored in closed bins with aeration systems, and these allow for the best management of the grain in storage. Other open or closed structures are frequently used, especially in developing countries, and their tendency to promote mycotoxin problems depends on the extent to which grain moisture and temperature can be maintained at low levels. Inadequate storage facilities are a major cause of mycotoxin

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problems in corn grown in developing countries (Hell et al., 2000). Storage facilities should be thoroughly cleaned before the new crop is stored, because grain residue will often harbor large populations of storage molds. Other practices, such as mold inhibitors and detoxification methods, may be employed in some cases. Additional management decisions are related to the utilization of mycotoxin-contaminated grain either with or without storage. Storage temperature is the most critical factor in managing potential mycotoxin problems in dried grain (Midwest Plan Service, 1987; Watson, 1987). Ideally, grain should be cooled after drying and maintained at 1°C to 4°C for the duration of storage. At this temperature, fungal metabolism is minimal. During the summer months, grain temperature can be maintained between 10°C and 15°C. Table 9.12 lists the moisture and temperature conditions required for growth and development of common toxigenic fungi and storage molds. Temperature control is achieved by aerating the grain when outside air temperature is within the desired range and humidity is low. Aeration is essential for maintaining grain quality in storage, by controlling temperature and evaporating moisture that has migrated and condensed in the bin. There is some unpredictability associated with stored grain, no matter how carefully it is dried and aerated. For this reason, frequent observations are necessary to head off developing problems with molds and mycotoxins. Sophisticated digital monitoring systems have been implemented in many storage operations, providing continuous data collection and early-warning capabilities to prevent mold and mycotoxin problems that result from changing storage conditions. Postharvest management of mycotoxins in high-moisture corn or silage also depends on proper storage conditions. These materials are stored in sealed storage facilities, where the goal is to establish anaerobic conditions. Under these conditions, mycotoxigenic fungi are not able to grow and produce mycotoxins, although preformed mycotoxins will persist. As with dried grain, variable conditions in the storage facility can lead to mycotoxin development in silage or high-moisture corn. If this occurs, it can be more difficult to manage than problems with stored grain, because toxigenic fungal development may not be visually evident, and problems may not become evident until symptoms are observed in livestock. There are few options for utilizing or disposing of contaminated high-moisture corn or silage. Antifungal agents can be applied to grain to inhibit toxigenic mold growth in storage. In relation to chemical control of stored grain fungi, Tuite and Foster (1979) commented, “This area has attracted the unscrupulous and the naı¨ve.” The comment stems from the fact that numerous products have been marketed for this purpose without an adequate research base. Some materials, however, are effective. Propionic acid has been among the most effective and widely used materials. It is used primarily to preserve grain at high-moisture contents. Propionic acid is applied as a spray inside a small auger (Sauer et al., 1992). Tuite and Foster (1979) and Sauer et al. (1992) provide more detailed accounts of the use of propionic acid and other organic acids as mold inhibitors, but little of this work has reported specifically on the inhibition of mycotoxin accumulation. Marı´n et al. (1999b, 2000) reported that propionic acid reduced fungal growth and fumonisin production, especially by Fusarium proliferatum, on irradiated maize. On natural stored maize, however, propionic acid did not result in lower fumonisin concentrations, but reduced populations of Penicillium spp. Anhydrous ammonia also has been used as a mold inhibitor in grain during low-temperature drying (Sauer et al., 1992). A major disadvantage of chemical control of storage molds is that the grain cannot be marketed through normal commercial channels. Grain preservatives such as propionic acid do not reduce toxins already formed. Insect activity in stored grain promotes the development of toxigenic fungi, so controlling insects will help reduce the risk of molds and mycotoxins. Insect control in stored grain involves an integrated approach, including sanitation, good control of grain moisture and temperature, frequent monitoring, and chemical methods. Sanitation includes cleaning the grain and the empty bin, to remove fines, broken kernels, and other debris that provide breeding sites and food for storage insects. The area around the bin also should be kept clean and free of vegetation (Holscher, 2000). Monitoring for insect problems can be achieved in several ways, by visual inspection or other monitoring devices (Pedersen, 1992). Empty bins may be treated with a residual insecticide or fumigated. Additionally, grain-protectant insecticides may be applied to the grain itself, as it is placed in the bin, as a “top-dressing,” or in a fumigation process. More details about stored grain insects and their control can be found in Reed (2006), Harein and Davis (1992), and Chapter 8 of this volume. Recent changes in insecticide registrations will complicate the issue of insect management in stored grain (Holscher, 2000). When preventative measures have failed, and grain has become contaminated with mycotoxins either in the field or in storage, there are management options for utilization of this grain. Ideally, problems occurring in the field should be detected before harvest and management decisions made accordingly. After harvest, mycotoxin problems can worsen quickly if delays result from indecision. Ideally, moldy or mycotoxin-contaminated grain should not be fed to livestock—mycotoxins may occur for which there are no documented effects or means of identification. But very moldy grain is a poor candidate for storage, and often the best strategy is to sell the grain immediately or feed it to less sensitive livestock species (if toxin analyses indicate safe levels). Another option is to attempt to reduce the impact of the mycotoxins through remediation or detoxification procedures. There are many limitations, however, to this approach.

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REMEDIATION AND DETOXIFICATION Detoxification methods have been extensively researched in relation to aflatoxins and some Fusarium toxins. Several reviews have been published (Charmley and Prelusky, 1994; Basappa and Shantha, 1996; Sinha, 1998). Remediation/ detoxification approaches can be grouped in several different ways, but they are commonly separated into physical, chemical, and biological methods. Table 9.13 summarizes the physical and chemical processes that are applicable to corn and corn products to mitigate several common mycotoxins (Karlovsky et al., 2016). Food and feed processing may employ combinations of physical and chemical, or even biological detoxification, either as a side effect of standard processing, or as a dedicated detoxification effort. Processing potentially has a greater role in products destined for human food use, because corn destined for animal feed is not likely to undergo extensive processing.

Physical Methods Physical methods include various types of separation processes, such as sorting, sieving (screening), density separation using a gravity table or flotation procedure, or optical sorting. The practice of blending moldy or mycotoxin-contaminated grain with good-quality grain is a physical method that is continuously employed, as untested lots of grain with varying levels of mycotoxins are mixed together on the farm or at the elevator. Knowingly blending contaminated grain with healthy grain, however, is not recognized by the United States FDA as an acceptable means for utilization of contaminated grain and is specifically banned in the European Community. With respect to food, it has been illegal in some countries for years (Karlovsky, 1999). Although the procedure can be effective, there is a risk that insufficient dilution will result in unacceptable contamination of an even greater amount of grain; and in general, it is undesirable to add mycotoxins to otherwise high-quality grain. Standard grain-handling procedures can result in physical separation of moldy kernels, and specific steps can be modified to further improve grain quality. Screening and aspirating grain removes many broken and diseased kernels, which are frequently among the highest in mycotoxins; corn screenings are notoriously high in fumonisins and have been associated with the worst outbreaks of fumonisin toxicity in livestock. Removing fines also improves grain storability by facilitating better aeration, reducing its vulnerability to both storage insect and mold problems. With the use of a grain sieve and gravity separator to segregate moldy kernels, broken kernels, and fines, a reduction in fumonisin concentrations of 66% was achieved in a lot of grain with an initial concentration of 7.9 mg/g (J.L. Richard, personal communication). Grain also may be separated by density through a flotation procedure. Starting with corn at 1.7 or 6.0 mg/g deoxynivalenol, removal of kernels buoyant in water or 30% sucrose led to a 53 to 77% reduction in deoxynivalenol concentration, and aflatoxin concentrations also were significantly reduced (Karlovsky et al., 2016). Optical sorting machines have been available for several decades; early models could effectively detect and remove only the obviously discolored kernels, and their capacity was limited. With current technology, it is feasible to rapidly sort large quantities of grain, and instruments using multiple wavelengths can identify and remove contaminated kernels that may not show visible symptoms (Karlovsky et al., 2016). This approach resulted in >80% reductions in both aflatoxins and fumonisins in corn (Pearson et al., 2004). Dry-milling of corn separates the grain into particulate components that primarily originate from different anatomical parts of the kernel (see Chapter 15). This process has the potential to segregate the grain into more-contaminated and lesscontaminated components. Aflatoxins are found primarily in the germ and hull fractions (CAST, 2003). Dehulling alone can significantly reduce aflatoxin contamination (Grenier et al., 2014). Similar results have been reported for fumonisins and other Fusarium toxins in dry-milling fractions, with the highest concentrations in the bran followed by the germ (Schollenberger et al., 2008; Pietri et al., 2009). Starch fractions tend be low in mycotoxins; this distribution is due to the mostly superficial colonization of intact corn kernels by toxigenic fungi. In damaged kernels, however, the endosperm is much more likely to be colonized, resulting in higher mycotoxin levels in the starch fractions. Washing or steeping contaminated corn can reduce mycotoxin levels, especially for the more water-soluble toxins. Solubility of some toxins such as zearalenone can be greatly increased by increasing the pH of the solution with sodium carbonate (Grenier et al., 2014). In wet-milling studies, the highest amounts of aflatoxins, fumonisins, DON, ochratoxin A, T-2 toxin, and zearalenone have been found in the steep water (CAST, 2003). In various studies, the percentage of the original toxin content in the steep water varied: 22% for fumonisins, 39% to 42% for aflatoxins, 43% for ochratoxin A, and 67% for deoxynivalenol (Lopez-Garcia and Park, 1998). The high value for deoxynivalenol is due to its solubility in water. In fact, reductions of up to 69% in deoxynivalenol and 61% in zearalenone have been achieved by washing methods using distilled water (Sinha, 1998). These reductions reached 74% and 87% when a sodium carbonate solution was used instead of distilled water. With fumonisins and T-2 toxin, process water from the wet-milling process also was found to contain

TABLE 9.13 Summary of Physical and Chemical Processes Applicable to Corn for Partial Mitigation of Contamination by Important Mycotoxins Physical Processes Mycotoxins

Chemical Processes

Sorting

Sieving

Flotation

Washing

Dehulling

Steeping

Milling

Heating

UV

Gamma

Plasma

Acid

Alkaline

Oxidation

Reduction

Ammonia

Trichothecenes

Y

P

Y

P

Y

Y

Y

Y

X

P

P

N

Y

P

N

Y

Ochratoxin A

P

P

N

N

N

Y

N

Y

P

P

P

P

P

P

X

Y

Aflatoxins

Y

P

Y

P

Y

Y

Y

Y

Y

Y

P

Y

Y

Y

Y

Y

Fumonisins

Y

P

Y

P

N

Y

Y

Y

X

Y

P

P

P

P

X

X

Zearalenone

P

P

N

P

N

Y

Y

Y

X

P

P

P

Y

Y

X

Y

Y ¼ Yes (has been demonstrated experimentally). P ¼Possible (partially successful or scientifically conceivable). N ¼Not enough information or not applicable. X ¼ Not possible or not compatible. Adapted from Karlovsky, P., Suman, M., Berthiller, F., De Meester, J., Eisenbrand, G., Perrin, I., Oswald, I.P., Speijers, G., Chiodini, A., Recker, T., 2016. Impact of food processing and detoxification treatments on mycotoxin contamination. Mycotoxin Res. 32(4), 179–205.

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significant portions of the mycotoxins. Concentrations of aflatoxin were also high in the fiber, but lower in the gluten and germ. Zearalenone was not found in the starch of wet-milled corn; the highest concentrations were in the gluten and soluble fractions, which contained 75% of the zearalenone (Hagler Jr et al., 2001). For fumonisins, the concentrations were higher in the gluten than in the fiber or germ, but concentrations were lower in each fraction than in the original corn, due to removal of fumonisins in the steep water (Bennett et al., 1996). Concentrations of T-2 were higher in the germ than in the other fractions. In general, wet-milling removes most mycotoxins from the desired fraction (the starch); therefore, wet-milling can be useful in reducing mycotoxin concentrations significantly below those found in the raw grain (CAST, 2003). Gamma or UV irradiation of mycotoxin-contaminated products has been attempted with some positive results. This strategy has recently been reviewed (Calado et al., 2014). Gamma irradiation of corn, wheat, and soybeans did not destroy aflatoxins, but doses above 7.5 kGy caused significant reductions in T-2 toxin, deoxynivalenol, and zearalenone. Aflatoxins, on the other hand, are sensitive to UV radiation and several studies have reported detoxification through exposure to sunlight or UV light (Sinha, 1998). The effects of ethanol fermentation on mycotoxins also have been studied. Generally, ethanol distilled from fermented products does not contain mycotoxins. After distillation, however, the solids (DDGS) typically contain higher concentrations of mycotoxins than the original grain. In one study, a large proportion (54%) of the fumonisins was extracted into the whole stillage, while the fermented mash contained low levels of fumonisins (1.8–2.7 mg/g) compared to the whole grain (15 mg/g). Fumonisins were concentrated in the distillers dried grains (19.2–25.3 mg/g). Corn screenings with a high level of fumonisin contamination (36 mg/g) produced less ethanol than the less-contaminated corn, probably as a result of degraded starch in the more moldy screenings (Bothast et al., 1992). Although fumonisins at very high concentrations (up to 1400 mg/ g) can be toxic to fermentation yeast (Sosa et al., 2013), concentrations normally found in grain probably do not inhibit ethanol production. Pure fumonisin B1 spiked into an ethanol fermentation at a concentration equivalent to 37.4 mg/g had no effect on fermentation rate or ethanol yield (Bowers & Munkvold, 2014). The mycotoxin enrichment factor between grain and DDGS is variable, but a threefold enrichment is typical and would be expected due to the usual ratio (1/3) of DDGS yield to original grain mass, assuming all the mycotoxins in the original grain remain intact. Comparing fumonisin concentrations in grain samples and DDGS derived from the grain, Bowers & Munkvold (2014) found that the enrichment factor was not significantly different from 3.0 in 50 of 57 comparisons. A threefold enrichment factor also has been reported for DON in DDGS (Schaafsma et al., 2009). These results suggest that DDGS, a valuable by-product of ethanol fermentation, may not be suitable as a feed material if they are derived from mycotoxin-contaminated grain. Several mycotoxins from corn (aflatoxins, deoxynivalenol, fumonisins, ochratoxin A) have been detected in beer, suggesting that fermentation does not degrade most mycotoxins. Conversely, some reports indicate that yogurt fermentation and bread fermentation can detoxify aflatoxins (Karlovsky, 1999). Processing methods that use high temperatures can affect mycotoxins, but the effects are very dependent on the specific mycotoxin and temperature involved. Even for a given mycotoxin, there are conflicting results regarding heat stability. Temperatures achieved in typical food preparation are unlikely to affect mycotoxins, but higher temperatures used in roasting, frying, and extrusion may reduce mycotoxin contamination (Sinha, 1998; Bullerman and Bianchini, 2007; Karlovsky et al., 2016). In general, fumonisins are resistant to thermal degradation. However, thermally processed corn products tend to have somewhat lower fumonisin concentrations than the original corn. This result must be viewed with caution however, since heating may cause structural changes that block detection of fumonisins, but do not decrease biological activity. Nixtamalization, an alkaline cooking and steeping process used to make tortillas (see Chapter 17), decreased detectable concentrations of zearalenone by 59% to 100%, deoxynivalenol by 72% to 82%, and 15-acetyl deoxynivalenol by 100% (Charmley and Prelusky, 1994). The process also reduced detectable fumonisin concentrations, but hydrolyzed fumonisins were in the end product, which was equally toxic to rats as the original contaminated corn (Murphy et al., 1996). Reductions in detectable mycotoxins through thermal treatments always involve transformation reaction, and care must be taken to understand the nature of the resulting compounds. Reduced bioavailability of mycotoxins through the use of mycotoxin-binding agents (adsorbents) is a physical process for mitigation of toxic effects in food or feed. The most well-known binding agent is hydrated sodium calcium aluminosilicate (HSCAS); commercial formulations of this product are available. It has reduced the effects of aflatoxin when fed to swine, cattle, lambs, rats, or poultry (Phillips et al., 1995). Data suggest that HSCAS tightly binds aflatoxin and can reduce absorption of aflatoxin from the gastrointestinal tract. HSCAS added to feed, however, has not been consistently effective in reducing the effects of deoxynivalenol or other Fusarium toxins. Various other materials have been tested or patented for use as mycotoxin adsorbents including zeolite, bentonite, polyvinylpyrrolidone (Charmley and Prelusky, 1994), activated charcoal, molecularly imprinted polymers, and micronized yeast biomass or plant fibers (Zhu et al., 2016).

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Chemical Degradation Chemical treatments to detoxify mycotoxins have been widely studied, but there are few examples of implementation. Corn products destined for human consumption generally cannot be subjected to dedicated chemical detoxification treatments. Regulatory approval is required for the use of any specific chemical treatment performed for mycotoxin degradation. As with thermal treatments, chemical treatments transform mycotoxins into altered compounds that must be assessed for toxicity. Chemical alterations also may occur as side effects of common processing practices, often in combination with physical processes, as already described. Chemical treatments that have been studied for mycotoxin degradation include ammoniation, treatments with acids or bases, treatment with oxidizing agents (such as ozone) or reducing agents (such as sodium bisulfite), and enzymatic degradation. The best-known example of chemical treatment for mycotoxins is the use of ammonia or ammonium hydroxide. This method has been studied in relation to corn and other aflatoxin-contaminated products. The process can be carried out at high temperatures and pressures, which is quick but requires specialized equipment, or at ambient temperature and pressure (Hammond, 1991). The process requires several weeks if the ammonia treatment is at ambient temperature. Reductions in aflatoxin of up to 99% have been reported with ammoniation (Bothast, 1991). Reductions in measured aflatoxin concentration have been confirmed by a lack of toxicity to domestic animals fed the decontaminated corn. Ammoniation has been adapted to use with a fermentation process to make ethanol from aflatoxin-contaminated grain (Bothast, 1991). By integrating ammoniation and fermentation, aflatoxins in the DDGS were drastically reduced. Ammoniation has found success with other mycotoxins as well. In another study, a 79% reduction in fumonisins occurred with exposure to 2% ammonia (Charmley and Prelusky, 1994). A major obstacle to the use of ammoniation is the lack of approval by FDA, due to incomplete knowledge of the composition and toxicities of the end products of ammoniation. Currently, ammonia-treated corn cannot be sold for interstate shipment, but must be used on-farm or locally. Calcium or sodium hydroxide, as well as other alkaline reagents, have been shown to decontaminate feeds containing aflatoxins, T-2, diacetoxyscirpenol, or zearalenone. The efficacy ranged from reductions of 45% to 99% and varied significantly among toxins and feed moisture content (Charmley and Prelusky, 1994; Karlovsky et al., 2016). Sodium bisulfite is effective at reducing deoxynivalenol concentrations in corn, but this process is believed to be suitable only for corn destined for animal feed, due to alterations in flour characteristics. Numerous other chemicals have been tested with some success in reducing deoxynivalenol or zearalenone concentrations in wheat or corn. Ozone and chlorine gases were effective in corn, but not wheat (Young, 1986; Young et al., 1986). Charmley and Prelusky (1994) reported the effects of 13 different chemical treatments on zearalenone in corn or corn grits. Formaldehyde and ammonium hydroxide were effective, but formaldehyde also is likely to be unsuitable for human food products. Enzymatic treatment for inactivation of mycotoxins offers several advantages over other chemical processes because of the potential for specificity and predictability of end-product composition. Several reviews are available summarizing the potential for enzymatic degradation of mycotoxins, either through the direct use of enzymatic treatments, or microbial treatments that rely on enzymatic mechanisms (Karlovsky, 2014; Vanhoutte et al., 2016; Loi et al., 2017). There are several commercial products on the market for biotransformation of mycotoxins, such as Mycofix, FUMzyme, Biomin BBSH 797, and Biomin MTV (Biomin Holding GmbH, Getzersdorf, Austria). Most of these products employ a combination of mechanisms, but FUMzyme (for fumonisin degradation) is the only one that consists of a purified enzyme, fumonisin esterase. Ten or more enzymes, derived from plants or microbes, have been reported to be effective for aflatoxins, including several laccases and peroxidases (Loi et al., 2017). Although detoxification of fumonisins has been extensively studied, only a few specific enzymes have been identified for fumonisin degradation, including esterases and an aminotransferase, derived from a yeast (Spinifera exophiala) and a bacterium (Sphingomonas sp.). UDP-glycosyltransferase has been reported for detoxification of certain trichothecenes, and several enzymes including laccases are reportedly effective for zearalenone (Karlovsky et al., 2016; Loi et al., 2017). Exploitation of enzymatic treatments for mycotoxin degradation holds a great deal of promise for the future. In general, chemical detoxification (including enzymatic treatments) has not been widely adopted because of difficulties and expense in applying laboratory-scale successes to commodity-scale operations, and because of the need for extensive safety testing of chemical additives and “detoxified” end products. No chemical or enzymatic treatments have been approved in the European Union for mycotoxin degradation in products intended for human consumption (Karlovsky et al., 2016).

Biological Degradation Biological degradation can include enzymatic treatments derived from microorganisms, or the direct use of microorganisms. In most cases, activity of specific microorganisms to degrade mycotoxins can be traced to specific enzymatic

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activity, blurring the distinction between biological and chemical degradation of mycotoxins. Microbial degradation of mycotoxins has been studied for many years. A strain of Flavobacterium aurantiacum capable of degrading aflatoxins was identified only a few years after the discovery of aflatoxins. There have been many reports of mycotoxin-degrading microorganisms; in some cases, the degradation products are still toxic, but in other cases there seems to be true detoxification. Some studies report inactivation of mycotoxins with microbial cocktails in which specific organisms were not identified. For example, incubating feed with intestinal microflora of chickens was shown to improve feed intake, feed efficiency, and weight gain of pigs and reduced the concentration of deoxynivalenol by 54%–56% (Charmley and Prelusky, 1994). On the other hand, at least one specific microorganism has been identified that can degrade each of the major mycotoxins in corn through enzymatic activity (Zhu et al., 2016). This activity derives from a range of microorganisms, but the majority are bacteria, especially Bacillus spp. The most promising developments related to biological detoxification are likely to be the expression of microbial genes in transgenic plants. This aspect is covered elsewhere in this chapter.

CONCLUSIONS Corn is susceptible to infection by numerous mycotoxigenic species of fungi in the genera Fusarium, Aspergillus, and Penicillium. The most important mycotoxins in corn are fumonisins, trichothecenes, zearalenone (all produced by Fusarium spp.), and aflatoxins (produced by Aspergillus spp.). The occurrence of these fungi and their associated mycotoxins in corn is widespread, but varies by geographical location. Mycotoxins have serious impacts on human and animal health, as well as significant economic impacts in corn production, livestock production, food and feed processing, human health care, and lost productivity. A range of methods is available for the detection and quantitation of mycotoxins in corn grain and cornbased products; some methods are portable and designed to provide rapid results, while others require advanced laboratory instrumentation, but can provide precise results and measurement of multiple toxins simultaneously. Mycotoxins can be managed to safe levels in corn and corn products through an integrated approach that involves preharvest and postharvest interventions; however, this can be challenging, especially in developing countries where corn is a staple food, and conditions are very favorable for mycotoxin contamination. Climate change threatens to result in the expansion of mycotoxin problems globally in the coming years.

ACKNOWLEDGMENT Thanks go to Anne McDermott for assistance in preparing some of the figures.

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