Occurrence, Prevention and Limitation of Mycotoxins in Feeds

Abstract

There has been a significant concern regarding the potential health risks for humans and animals via foods and feeds that are contaminated with different agents. Particularly, mycotoxin contamination is of great importance as it is widespread and unpreventable. In both foods and feeds, molds produce secondary metabolites called mycotoxins; these are produced generally after the fungi reach their maturity. Depending on the definition used, hundreds of fungal compounds are recognized as mycotoxins. However, the attention is mainly focused on aflatoxins, ochratoxins, fumunisins, and zearalenone which are considered the most important threats for human and animal health. Mycotoxin contamination causes a fundamental problem all over the world including developed countries. Additionally, the economic impact of mycotoxins is another global concern on the agricultural markets. These concerns are based on toxicological data, which
show that naturally occurring levels of mycotoxins have adverse effects in farm and laboratory animals as well as humans. The diversity of mycotoxin structures induces various toxic effects. Owing to the significant health risks and economic impacts, considerable investigations are being performed to diminish their harmful effects and to prevent their formation. In order to limit their levels, much research has been focused on detecting the mycotoxins in contaminated food and feedstuffs. This review will focus on information about primary mycotoxins, their occurrence, related regulations, prevention and methods of detection within the light of the current literature.
Key words: Aflatoxin, Fumonisins, Mycotoxins, Ochratoxin, Zearalenone.

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Animal Nutrition and Feed Technology (2015) 15: 471-490
doi: 10.5958/0974-181X.2015.00048.7
Occurrence, Prevention and Limitation
of Mycotoxins in Feeds
M.F. Abdallah1, G. Girgin and T. Baydar*
Department of Toxicology, Faculty of Pharmacy
Hacettepe University, 90-06100, Ankara, Turkey
(Received July 25, 2014)
ABSTRACT
Abdallah, M.F., Girgin, G. and Baydar, T. 2015. Occurrence, prevention and limitation of mycotoxins
in feeds. Animal Nutrition and Feed Technology, 15: 471-490.
There has been a significant concern regarding the potential health risks for humans and animals
via foods and feeds that are contaminated with different agents. Particularly, mycotoxin contamination
is of great importance as it is widespread and unpreventable. In both foods and feeds, molds produce
secondary metabolites called mycotoxins; these are produced generally after the fungi reach their maturity.
Depending on the definition used, hundreds of fungal compounds are recognized as mycotoxins. However,
the attention is mainly focused on aflatoxins, ochratoxins, fumunisins, and zearalenone which are considered
the most important threats for human and animal health. Mycotoxin contamination causes a fundamental
problem all over the world including developed countries. Additionally, the economic impact of mycotoxins
is another global concern on the agricultural markets. These concerns are based on toxicological data, which
show that naturally occurring levels of mycotoxins have adverse effects in farm and laboratory animals
as well as humans. The diversity of mycotoxin structures induces various toxic effects. Owing to the
significant health risks and economic impacts, considerable investigations are being performed to diminish
their harmful effects and to prevent their formation. In order to limit their levels, much research has been
focused on detecting the mycotoxins in contaminated food and feedstuffs. This review will focus on
information about primary mycotoxins, their occurrence, related regulations, prevention and methods of
detection within the light of the current literature.
Key words: Aflatoxin, Fumonisins, Mycotoxins, Ochratoxin, Zearalenone.
INTRODUCTION
Mycotoxins are secondary metabolites produced naturally by about 200 recognized
filamentous fungi growing under a wide range of climatic conditions on different
agricultural stuffs. A number of fungal genera, mainly Aspergillus, Penicillium,
*Corresponding author: tbaydar@hacettepe.edu.tr
1Department of Toxicology, Faculty of Veterinary Medicine, Assiut University, Egypt.
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472
Alternaria, Fusarium, and
Claviceps produce mycotoxins
(Zollner and Mayer-Helm,
2006; Marin et al., 2013). Some
fungi have the ability of
producing more than one
mycotoxin and some mycotoxins
can be produced by more than
one mold species (Hussein and
Brasel, 2001). Mycotoxin
contamination in animal feed
and the potential transfer into
animal products to be consumed
by humans still remains a major
problem alerting the entire
world (Cheli et al., 2014).
Several outbreaks have been
reported in humans and animals
after the consumption of
mycotoxin-contaminated food
and feed. Mycotoxin production
and/or contamination in
agricultural products can take
place at different stages in food
and feed chain: pre-harvest,
harvest and post-harvest (Binder
et al., 2007). Mycotoxins are
commonly present in nuts, dried
fruits, coffee, cocoa, spices, oil
seed, dried beans, corn, wheat,
and several other cereals. Not
only foods, but also animal
feeds and products such as milk,
cheese and meat are important
exposure sources (Imperato et
al., 2011; Da Rocha et al.,
2014). As a result of mycotoxin-
contaminated animal feed
consumption, decreased feed
intake, feed refusal in some
cases, poor feed utilization,
reduced body weight gain,
increased disease susceptibility,
Table 1. Toxic effects of mycotoxins in different animals
Mycotoxin IARC†Major effects Clinical and pathological signs on most susceptible animals
classification
Aflatoxins 1 Carcinogenic, hepatotoxic and Reduced productivity; inferior egg shell and carcass quality; increased
Aflatoxin M1 2B impaired immune system susceptibility to infectious disease.
Ochratoxin A 2B Carcinogenic, nephrotoxic, hepatotoxic, Kidneys are grossly enlarged and pale due to nephrotoxicity; fatty livers in
neurotoxic and teratogenic poultry; shell decalcification/thinning.
Deoxynivalenol 3 Immunotoxic and ATA Decreased feed intake and weight gain in pigs; feed refusal and vomiting
(alimentary toxic leukopenia) at very high concentrations.
Other trichoth- 3 Immuno-depressants, gastrointestinal Reduced feed intake; vomiting, skin, and gastrointestinal irritation; neurotoxicity;
ecenes (T-2 toxin) haemorrhaging and hematotoxicity abnormal offspring; increased sensitivity to infection; bleeding.
Zearalenone 3 Fertility and reproduction Swollen, reddened vulva, vulvovaginitis, anestrus vaginal prolapse and sometimes
(estrogenic activity) and disrupts rectal prolapse in pigs; feminization and suppression of libido; suckling piglets
endocrinesystem may show enlargement of vulvae; fertility problems.
Fumonisins 2B Carcinogenic,hepatotoxic,central nervous Equine leucoencephalomalacia (ELEM), porcine pulmonary edema,
system damage and immuno-depressants liver damage in poultry.
†International Agency for Research on Cancer. 1: carcinogenic to humans AFs; 2A: probably carcinogenic to humans; 2B: possibly carcinogenic to humans; 3: not classifiable as to its carcinogenicity
to humans; 4: probably not carcinogenic to humans.
Abdallah et al.

473
and reduced reproductive abilities are commonly observed; moreover deaths can
occur which leads to serious economic losses (Binder et al., 2007).
Mycotoxicoses are diseases caused due to mycotoxin exposure. Mycotoxins
have different toxicological effects in humans and animals. Table 1 shows toxic
effects of some mycotoxins in different domestic animals. Some of them have the
same characteristic clinical and pathological signs with differences in severity (Duarte
et al., 2011). The clinical signs may vary according to the species in addition to the
exposure dose and period (Cheli et al., 2014). Nausea, vomiting, abdominal colic, and
diarrhea are the general signs that occur as a result of contaminated food/feed stuffs
ingestion(Fung and Clark, 2004). In animals, the first observed symptoms are decreased
feed intake and growth retardation. After that, immunosuppression signs like ineffective
vaccine response and decreased drug efficacy against infectious diseases can also be
observed (Bryden, 2011). Many internal factors play a role in mycotoxin impact such
as health state of affected living organism, and also, sex, age and body weight (Steyn,
1995).
Human exposure is possible either through contaminated foods of plant origin,
mostly cereal grains, or foods of animal origin such as contaminated milk, meat, and
eggs. Another rare way is also possible through inhalation of polluted air and dust
(Bryden, 2011). There are hundreds of mycotoxins that have been isolated and chemically
described. Up to now, it has been documented that approximately 400 secondary
metabolites with toxicity potential are produced by more than 100 moulds. Most of
the researches have focused on those forms causing significant injuries to humans and
farm animals. Rate of occurrence and severity of the diseases are the detrimental
factors to identify which mycotoxins are important. Some mycotoxins with harmful
effects on animals and human health are aflatoxins (AFs), ochratoxin A (OTA),
trichothecenes (deoxynivalenol (DON) and T-2 toxin), zearalenone (ZEN) and fumonisins
(FBs) (Trucksess and Diaz-Amigo, 2011; Afsah-Hejri et al., 2013). In this review,
mycotoxins and their occurrence in foods and feeds, exposure and outcomes will be
reviewed in light of the recent literature.
Aflatoxins
Aflatoxins are difuranocoumarins mainly produced by two Aspergillus species,
A. flavus and A. parasiticus (Reddy et al., 2010). Concerning their chemical structures,
there are two main categories for AFs; the first category is difurocoumarocyclopentenone
group and includes aflatoxins B1, B2 (AFB1, AFB2) and aflatoxins M1, M2 (AFM1,
AFM2). The second category is difurocoumarolactone group involving aflatoxins G1
(AFG1) and G2 (AFG2). The four major naturally occurring aflatoxins B1, B2, G1
and G2 are ubiquitous in animal feed stuffs. The nomenclature of AFs B1 and B2
derives from the blue fluorescent color produced and visualized under UV light while
AFs G1 and G2 produce green fluorescent color (Gupta, 2011; Womack et al., 2014).
AFsM1 and M2 are the main metabolites of aflatoxins in which ‘‘M’’ refers to milk
of mammals consuming aflatoxin-contaminated feeds (Kara and Ince, 2014). In liver,
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cytochrome P450 (CYP)-associated enzymes convert AFB1 to AFM1, the major
monohydroxylated derivative. In humans, the main CYP enzymes involved in aflatoxins
metabolism are CYP3A4, 3A5, 3A7, and 1A2. However, liver is the primary target
organ, under certain conditions, lung, kidney, and colon may also be affected (Marin
et al., 2013).
Among all discovered mycotoxins, aflatoxins are the most intensively researched
group, because of their potent acute toxicological and chronic hepatocarcinogenic
effects in various susceptible animals. Consumption of aflatoxins contaminated
agricultural stuffs is the main route of exposure. Adverse effects of aflatoxins are
anorexia, decreased feed intake, immune system suppression in both animals and
humans. Immunosuppressive, hepatotoxic, carcinogenic, mutagenic, and teratogenic
effects can be observed according to animal species, sex, age and aflatoxintype,
exposure dose and period (Arslan and Essiz, 2009; Afsah-Hejri et al., 2013). It has
been detected that median lethal dose (LD50) of AFB1 is estimated to be between
0.3 and 18 mg/kg according to the administration route, animal species, age and
health condition. Poultry are more sensitive to aflatoxins than mammals. Within
poultry, ducks are the most susceptible species then turkey poults and then chicken.
Within domestic animals, the order is canine, swine, calves, cattle and sheep. Young
animals are more susceptible to AFs than matures. Nutritional deficiencies, especially
protein and vitamin E increase the susceptibility to AFs (Bryden, 2011).
In animals, aflatoxicosis can also cause reduction in feed efficiency,
immunosuppression, vaccination failure, and reduced reproduction efficacy or reduced
fertility which clinically appears as decreased weight gain, rough hair coat, lowered
meat, wool, and milk yield. Similarly in poultry, severe hepatic damage, stunning
in growth, and egg production, hemorrhagic syndrome as a result of increased blood
capillary fragility are observed (Hussein and Brasel, 2001; Zain, 2011).
In humans, the plasma half-life of AFB1 is short and it is estimated that after
absorption, about 65% is removed from the blood within one and half hours (Fung
and Clark, 2004). Of the known AFs, AFB1 is a potent human carcinogen. The
International Agency of Research on Cancer (IARC) has classified AFB1 as a human
carcinogen, Group 1 while AFM1 is categorized as possible human carcinogen;
Group 2B as its carcinogenicity is 10 times less than the parent compound (Arslan
and Essiz, 2009; Kara and Ince, 2014). Carcinogenic and mutagenic effects result
from the highly electrophilic intermediate AFB1-8,9-epoxide, which is produced by
CYP mediated biotransformation of AFB1. Somatic mutation and carcinogenesis
probably occur due to depurination process of DNA molecules. Covalent bonds at the
N-7 guanine residue are formed as a result of a reaction between AFB1-epoxide and
DNA leading to carcinogenesis (Fung and Clark, 2004; Bryden, 2011).
Acute toxicity in humans is rare and generally, it includes a wide variable
clinical signs range from sudden death without signs to general unspecific symptoms
like nausea, vomiting, abdominal cramp, anorexia, diarrhea, ataxia, edema in lung,
Abdallah et al.

475
fatty liver which is manifested by jaundice, anemia, depression, and photosensitivity.
Chronic aflatoxicosis leads to moderate to severe icterus, hepatic cirrhosis, benign
hepatoma, cholangiocarcinoma, or hepatocellular carcinoma. Chronically, aflatoxins
are implicated in hepatocellular carcinoma synergistically with hepatitis B or C
virus. The highest percentage of hepatocellular carcinoma incidences occur in parts
with frequent exposure to contaminated foods and high rate of infection with hepatitis
as Eastern and South-Eastern Asia and Middle and Western Africa. AFs act as
immune modulators, causing suppression of resistance to secondary infections. They
can also affect testes and sperm quality which leads to infertility (Reddy et al., 2010;
Trucksess and Diaz-Amigo, 2011; Marin et al., 2013).
Ochratoxin A
Ochratoxin A is the most commonly encountered and toxic member of ochratoxins
group. Two genera of fungi produce OTA, Aspergillus and Penicillium. The main
OTA producing species are A. ochraceus, A. carbonarius, A. melleus, and A.
sclerotiorum, P. verrucosum, and P. nordicum. OTA nomenclature is derived from
A. ochraceus, the first fungus that was discovered to produce the toxin (Reddy et al.,
2010; Zain, 2011).
Chemically, OTA is similar to phenylalanine(Phe), the toxin involves Phe
linked by a peptide bond to an isocoumarin molecule. Because of the structural
similarity, OTA inhibits all biological processes involving Phe, particularly, Phe-
tRNA synthetase and thus, inhibits protein synthesis as well as DNA and RNA. OTA
also interferes with lipid peroxidation through impairing the endoplasmic reticulum
membrane and causes oxidative stress and mitochondrial damage, triggering cytotoxicity
(Steyn, 1995; Fung and Clark, 2004; Abrunhosa et al., 2010).
Ochratoxin A has been detected in a wide range of different animal feed
ingredients such as cereal and cereal-based products (barley, corn, wheat soy) and
also foods, including various cereal products, coffee, spices, beans and other products
of animal origin including milk (Imperato et al., 2011; Martins et al., 2012). OTA
is carcinogenic, genotoxic, immunotoxic and nephrotoxic agent. The main target
organ in OTA toxicity is kidneys. In Balkan countries, Croatia, Bulgaria, and Romania,
OTA causes high incidence of urinary tract carcinoma which is called Balkan
Endemic Nephropathy (BEN) and also, causes urothelial tumors. The main pathological
feature is tubular damage in proximal convoluted tubules. The common signs include
anemia, proteinuria, icterus, and uremia. As stated by IARC, OTA has sufficient
evidence of carcinogenicity in experimental animals but inadequate in humans, classified
as possibly carcinogenic to humans, Group 2B. At high concentrations, OTA affects
many organs including liver and brain which is manifested by multifocal hemorrhages
and may involve intravascular coagulation of heart (Richard, 2007; Duarte et al.,
2011; Afsah-Hejri et al., 2013; Marin et al., 2013).
Similar to humans, in domestic animals, chronic renal failure is the main
effect accompanied by OTA toxicity. OTA exposure leads to testicular carcinoma.
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Immunologically, OTA causes obvious immunosuppression with atrophy of immune
organs. Mono gastric animals such as dogs and pigs are more susceptible to
ochratoxicoses then chickens while ruminants are more resistant (Yiannikouris et al.,
2002; Gupta, 2011; Zain, 2011; Martins et al., 2012).
Fumonisins
Fumonisins are a group of non-fluorescent mycotoxins, mainly produced by
Fusarium moniliforme, F. proliferatum. F. napiforme, F. dlamini and F. nygamai (Lino
et al., 2007; Marin et al., 2013). Corn is mostly infected with fumonisin producing
moulds, particularly when corn is imported from humid climates (Abbas et al., 2006;
Seo et al., 2013; Scussel et al., 2014). Structurally, fumonisins contain a long
hydroxylated hydrocarbon chain having a methyl and either acetyl amino or amino
groups. Fumonisins B1, B2, and B3 are the most significant toxins among more than
12 fumonisin analogues. Fumonisins are cancer-inducing toxins due to its similarity
with sphinganine and sphingosine, the main constituent of sphingolipids (Hussein and
Brasel, 2001; Yiannikouris et al., 2002). Disruption of sphingomyelin through inhibition
of sphingolipid formation is considered as the main pathway of fumonisin toxicity
(Fung and Clark, 2004). In mammals, equines and swines are the most susceptible
species among domestic animals then ruminants. Poultry are more resistant than
mammals. In horses, fatal neurological disease, equine leukoencephalomalacia (ELEM)
is the prominent toxic effect. Massive softening and liquefaction of the white matter
of brain is the prominent post mortem lesion. ELEM is characterized by nervous
signs including ataxia, aimless moving, facial paralysis, blindness, coma, and death.
In swine, FB1 cause cardiotoxicity and acute fatal porcine pulmonary edema (PPE).
PPE is characterized pathologically by presence of pale yellow colored proteinaceous
fluid in lungs and interlobular pulmonary edema with severe respiratory distress and
cyanosis. In addition to neurotoxicity, pulmoner toxicity and cardiotoxicity, FB1 may
also exert hepatotoxicity and nephrotoxicity (Steyn, 1995; Yiannikouris et al., 2002;
Oruc et al., 2006; Voss et al., 2007; Scussel et al., 2014).
Till now, there are no specified impacts of FBs on human health. It’s suggested
that some types of esophageal and hepatic tumors and cardiac toxicity in humans are
associated with fumonisin exposure through consumption of contaminated maize
(Waskiewicz et al., 2012; Marroquin-Cardona et al., 2014). FB1, the most important
member of fumonisins, is a tumor promoter, but has no genotoxic effects. Regarding
IARC, FB1 is categorized as possible carcinogen to humans, Group 2B (Richard,
2007; Voss et al., 2007).
Zearalenone
Zearalenone is a mycotoxin with hyperestrogenic effects in animals produced
by Fusaria, mainly by F. graminearum, F. culmorum and F. sporotrichioides. Maize,
wheat, oats, barley and rye are mostly infected with ZEN producing moulds. (Saeger
et al., 2003; Trucksessand Diaz-Amigo, 2011). Furthermore, milk contamination by
zearalenone and its metabolites has been reported (Signorini et al., 2012; Huang et
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al., 2014). Structurally, ZEN is similar to 17β-estradiol and it is classified as non-
steroidal estrogen (Hussein and Brasel, 2001; Da Rocha et al., 2014). Zearalenone
is metabolized into two diastereoisomeric zearalanols, α and β-zearalanol. All ZENs
have the same estrogenic properties but their potential differs possibly due to variations
in binding affinity to estrogen receptors. It has been found that α-zearalanol is three
times more estrogenic than zearalenone itself. Reproductive problems in domestic
farm animals are the frequent disorders occurring with ZEN exposure. Although it
is not common, in humans hyperoestrogenic syndromes can be observed (Zinedine et
al., 2007; Marin et al., 2013).
In animals, ZEN is a weak estrogen and it inhibits follicle stimulating hormone
(FSH) and therefore delays the maturation of preovulatory follicle in ovaries thus
exerts reproductive toxicity. Animal susceptibility shows a variation according to
species, sex, age, and reproductive state. Swine are the most susceptible farm
animals to reproductive effects of ZEN. Prepubertal gilts are more sensitive than
mature ones. Sows in estrous cycle are more sensitive than both pregnant and non-
cycling pigs. Prepubertal gilts show a hyperestrogenism, enlargement of the mammary
glands while mature sows exhibit nymphomania and pseudopregnancy. Castrated
males may develop enlargement of the prepuce and nipples and immature boars
demonstrate reduced or loss of libido and testicular atrophy. Ruminants may exhibit
some adverse effects, reduced fertility and repeated breeding but generally are of low
clinical incidence. ZEN can be excreted into milk of pigs and cows as a result of
exposure to high doses in feed. Poultry shows some resistance to ZEN, only highly
contaminated feed stuff can lead to infertility and reduced spermatogenesis (Fung and
Clark, 2004; Richard, 2007; Zain, 2011).
In humans, toxicity is mainly chronic while acute form after oral administration
is rare. ZEN and its metabolites can effectively stimulate mammary gland cells
growth. Thus, it was suggested that ZEN may be implicated in breast cancer. There
are some reported cases of precocious puberty in adolescent girls with ZEN exposure
(Zinedine et al., 2007; Marroquin-Cardona et al., 2014). ZEN is included in non-
carcinogenic agents to humans, Group 3, according to the IARC (Afsah-Hejri et al.,
2013).
Trichothecenes
Different Fusarium species such as F. culmorum, F. sporotrichioides, F.
tricinctum, F. roseumF. graminearum, F. nivale and F. sambucinum, and some members
of Myrothecium are able to produce trichothecenes. Corn, barley, wheat, oats, rye,
soybeans, and fruits as well as animal feeds are mostly attacked by fusarium. Fungal
infection appears as a red-pink colored area, mostly at the tip of the crop (Berthiller
et al., 2005; Marques et al., 2008; Kim et al., 2014). During the last 40 years, more
than 180 trichothecene mycotoxins have been discovered. Structurally, trichothecenes
have been classified according to the difference in the functional group, hydroxyl and
acetoxy side, into four groups. Type A involves HT-2, T-2, diacetoxyscirpenol (DAS)
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and neosolaniol; type B is represented by Deoxynivalenol (DON), 3-acetyl-DON, 15-
acetyl-DON, and nivalenol (NIV); type C including crotocin; and type D or
macrocyclics. Despite so many forms, a few numbers of trichothecenes have a
toxicologic potency. The most important are DON, HT-2, and T-2. Mechanism of
toxicity is conducted through inhibition of protein synthesis by interaction with the
60S ribosomal subunit and the peptidyltransferase (Sweeney and Dobson, 1998;
Chrevatidis, 2003; Zou et al., 2012). Disruption of DNA and RNA is occurred
through peptidyltransferase enzyme inhibition. It affects the actively mitotic cells
such as intestinal epithelial cells, dermal, lymphoid and erythroid cells. Trichothecenes
affect mitochondrial functions, enhance lipid peroxidation, and stimulate cell death.
They also stimulate type four hypersensitivity reactions concurrently with inhibition
of suppressor T-cells. At low doses, they can interrupt glucose and calcium ions
transfer. Acute toxicity in humans shows abdominal cramps, nausea, vomiting and
bloody diarrhea. Alimentary Toxic Aleukia (ATA) is a disease caused due to long
term exposure totrichothecenes, manifested by gastrointestinal problems followed by
gastroenteritis, fever, immunosuppression, and lastly bronchial pneumonia and death.
According to IARC, both DON and T2 are classified as non-carcinogenic toxins to
human, Group 3 (Reddy et al., 2010; Mostrom and Raisbeck, 2012; Da Rocha et al.,
2014; Marroquin-Cardona et al., 2014).
Among animal species, large ruminant, small ruminant, equine, poultry and
swine are susceptible. The most sensitive are swine while cattle and birds are more
resistant. Oral exposure to trichothecenes leads to feed refusal as a primary sign even
after adding flavoring agents. Moreover, weight loss, anemia, and weakness may
occur as consequences. In poultry, decreased egg production, abnormal feathering
especially in broilers and some neurological signs are observed. Some studies suggest
that trichothecenes are able to cause limb and tail deformities (Fung and Clark, 2004;
Richard, 2007; Mostrom and Raisbeck, 2012).
Occurrence of mycotoxins in plant and animal products
Factors affecting the production and occurrence of mycotoxins in crops and
consequently, the extent of contamination in feed and food involve climate conditions
such as temperature, relative humidity; and agricultural practices such as fungicide
usage and techniques used in agriculture. Other factors may include drying, processing,
handling, packaging, storage and transport conditions. Insects play an important role
through physical damage of the grains and mechanical transmission of the
microorganisms (Chrevatidis, 2003; Abbas et al., 2006; Richard, 2007; Marroquin-
Cardona et al., 2014). Most of cereal grains, oil seeds, tree nuts, and fruits (especially
dried ones) are susceptible to fungal attack and mycotoxin formation. Agricultural
products like cereal grains and forages can be contaminated during pre-harvest (field
period), harvest, and post-harvest (storage and transportation period). Corn and other
grains used in animal feed could also be contaminated by pathogenic moulds, thereby
mycotoxins, even they may be destroyed at different rates during industrial processing
(Griessler et al., 2010; Reddy et al., 2010; Bryden, 2011; Kocasari et al., 2013).
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479
Distribution of mycotoxins varies according to fungus nature. Depending on the
geographical and climate conditions, different fungal species can invade foods and
feedstuffs. For example, aflatoxins are mostly expected in tropical areas where
climate conditions and storage practices are favorable to fungal growth and toxin
production, while ochratoxin A is frequently detected in moderate and subtropical
regions; fumonisins in subtropical and tropical locations; zearalenone and trichothecenes
are worldwide mycotoxins (Sweeney and Dobson, 1998; Afsah-Hejri et al., 2013). In
general, the crops are susceptible to contamination by the most dangerous mycotoxins
in tropical and subtropical areas with high humidity and temperature. AFs are
ubiquitous in corn-based animal feed and mostly present in groundnut meal and
cottonseed. OTA may be present in most of cereals, corn, hay, oats, and wheat, and
also in oilseed products, particularly if the products were not dried well after
harvest. Corn is a major source for ZEN particularly if it is not harvested on time.
Interestingly, ZEN has been detected in damp hay and straw. Furthermore, corn,
wheat, barley, oats, rye and other crops have been reported to contain T-2 toxin and
DON (Chrevatidis, 2003).
Direct contamination of dairy products is through colonization of mycotoxigenic
fungi especially in cheese. Mould contamination can occur either from unhygienic
manufacturing medium or fungal starter cultures used for the production of specific
dairy products. Another route for mycotoxins and their metabolites is excretion
through milk. In dairy cows, AFM1 reaches the maximum concentration in milk two
days after ingestion of AFB1 containing feed and can disappear after 4 days from
removing the contaminated feeds. The amount of AFM1 in milk represents
approximately 3-5% of AFB1 found in the animal feed stuff. AFM1 has been commonly
found in many of food stuffs including infant formulas, dried milk, cheese and
yoghurt. Not only dairy products, but also meat of swine or eggs of laying birds can
be contaminated if the animals consumed considerable amounts in their feed. It is
possible that fungi may spread from one country to another with increases in global
grain trade. In many European regions, OTA has been revealed in swine viscera,
muscle tissue, and blood (Steyn, 1995; Sweeney and Dobson, 1998; Yiannikouris et
al., 2002; Arslan and Essiz, 2009; Marin et al., 2013). A lot of investigations have
reported the contamination of mycotoxins and summarized in Table 2.
Limitation in food and feed
Beside their obvious health impacts, mycotoxins also affect the agricultural
trade among countries through decreasing livestock and crop yield production. Total
elimination of moulds and their toxins from agricultural products seems to be impossible
or impractical. The possible risk caused by mycotoxins to human and animal health
has presupposed the urgent need to control their levels. According to the geographical
and climatic variations, different limits are being set to monitor and control mycotoxin
levels (Afsah-Hejri et al., 2013; Da Rocha et al., 2014).
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In this regard, establishment of strict limitation and tolerance levels of
mycotoxins is held by national and international authorities such as the European
Commission (EC), US Food and Drug Administration (FDA) and World Health
Organization (WHO) as shown in Table 3. FDA has established the maximum
acceptable limits in food for sum of AFs (B1, B2, G1, and G2) at 20 µg/kg and AFM1
in milk at 0.5 µg/kg while the total AFs residue limit in feeds for mature and
immature animals is 100 µg/kg and 20 µg/kg, respectively (Richard, 2007; Womack
et al., 2014). According to the EC, maximum permitted level for total AFs in feed
stuffs used for animal and poultry is 50 µg/kg while this limitation in Turkey is 100
µg/kg in feeds for ruminant, swine. For immature animal and poultry feeds, the limit
is 50 µg/kg (EC, 2006; Oruc et al., 2006). Limit of AFB1 for mature animal feed
Table 2. Levels of different mycotoxins reported in some food and feed commodities
Commodity Mycotoxins Reported range Detection References
method(s)†
Corn Total aflatoxins 21-699 µg /kg LC-MS/MS Abbas et al., 2006
Aflatoxins B1, B2 2- 20 µg /kg HPLC-FLD Irama et al., 2014
Ochratoxin A 3-5 µg /kg HPLC-FLD Majeed et al. 2013
Deoxynivalenol 96- 1,790 µg /kg HPLC-FLD Marques et al., 2008
T-2, HT-2 0.8-18.3 µg /kg LC-MS/MS Berthiller et al., 2005
Zearalenone 40- 64 µg /kg HPLC-FLD Almeida-Ferreira et al., 2013
Milk Fumonisins 66-7832 µg /kg HPLC-FLD Scussel et al., 2014
Aflatoxin M1 0.008 -0.05 ng/l LC-MS/MS Kara and Ince, 2014
Aflatoxin M1 55-116 ng/l ELISA Kamkara et al., 2014
Ochratoxin A 1-50 ng/l HPLC-FLD Imperato et al., 2011
Zearalenone 0.003-45.8 ng/l UHPLC-MS/MS Huang et al., 2014
α-Zearalenol 0.009-73.5 ng/l UHPLC-MS/MS Huang et al., 2014
Zearalenone 0.016-0.469 ppb ELISA Signorini et al., 2012
Deoxynivalenol 0.049-1.396 ppb ELISA Signorini et al., 2012
Cereal Total aflatoxins 0.03-3.16 µg/kg HPLC-FLD Baydar et al., 2005
flour Ochratoxin A 0.025-10.5 µg/kg ELISA Aydin et al., 2008
Deoxynivalenol 0.0-23.0 µg/kg ELISA Manthey et al., 2004
Zearalenone 40- 64 µg/kg HPLC-FLD Almeida-Ferreira et al., 2013
Fumonisins 142-550 µg/kg HPLC-FLD Lino et al., 2007
Animal Total Aflatoxins 0.17 -0.92 µg/kg HPLC-FLD Beheshti and Asadi, 2014
feed Ochratoxin A 2-130 µg/kg HPLC-FLD Martins et al., 2012
Zearalenone 0.009-0.405 µg/kg HPLC-FLD Kim et al., 2014
α-Zearalenol 25-600 µg/kg HPLC-FLD Saeger et al., 2003
Fumonisin B1 30-14.600 ng/g LC-MS/MS Seo et al., 2013
Fumonisin B2 35-2.280 ng/g LC-MS/MS Seo et al., 2013
Fumonisins 104-2999 µg/kg LC-MS/MS Njobeh et al., 2012
Deoxynivalenol 18.5 -500 µg/kg ELISA Kocasari et al., 2013
T-2, HT-2 35-40µg/kg HPLC-FLD Griessler et al., 2010
†UHPLC-MS/MS: Ultra high performance liquid chromatography combined with electrospray ionisation triple
quadrupole tandem mass spectrometry; LC-MS/MS: Liquid chromatography tandem mass spectrometry; HPLC-
FLD: High performance liquid chromatography with fluorescence detector; ELISA: Enzyme linked immunosorbent
assay.
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Table 3. Permitted limits for mycotoxins in various species
Mycotoxins Feed stuff(s) Limit (ppb) Country / Authority
Aflatoxin B1 Maize 5 Turkey, Russia, Egypt
Maize 10 China, Korea, Japan
Animal feed 10 Egypt
Animal feed 50 Turkey
All cereals except rice and maize 2 EU
Unprocessed maize and rice 5 EU
Animal feed ingredients 20 EU
Feed stuffs for immature animal 20 FDA
Aflatoxin B1& G1 Maize 30 Brazil
Aflatoxin M1 Milk 0.5 U.S.A, Russia, Egypt
0.05 Turkey
Milk and milk products 0.05 EU
Milk 0.5 FDA
Deoxynivalenol Unprocessed cereals other than wheat, 1250 EU
oats and maize
Unprocessed wheat and oats, maize 1750 EU
Cereal products 500 EU
Cereals and cereal products for feed 8000 EU
Maize by-products for feed 12000 EU
Animal feed 100 FDA
Fumonisin B1, B2 Animal feeds except Equines 50 EU
Animal feeds for Equines 5 EU
Fumonisin B1, Animal feeds except Equines 30 FDA
B2, B3 Animal feeds for Equines 5 FDA
Fumonisins Unprocessed maize 2000 EU
Maize products for human 1000 EU
Ochratoxin A Unprocessed cereals 5 EU
Cereals and cereal products for feed 250 EU
Cereal products for food 3 EU
T-2 and HT-2 All cereals grains 100 EU
Total aflatoxin Animal feed ingredients 50 EU
Animal feed 20 Canada, Egypt, Iran
50 Brazil
Maize 10 Turkey, Egypt
30 India
Cereals feedstuffs 200 Mexico
Feedstuff (ingredient)s 20 Japan, U.S.A, Korea
All cereals except rice and maize 4 EU
Maize and rice 10 EU
Feed stuffs for mature animal 100 FDA
Zearalenone Unprocessed cereals other than maize 100 EU
EU: Europena Union. FDA: Food and Drug Administration.
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materials is 20 µg/kg, the same value as EC (EC, 2006; Arslan and Essiz, 2009).
Up to date, the major source of food and feed all over the world is cereal grains.
As a result of their health implications and increasing knowledge of health hazards,
regulations for major mycotoxins in commodities exist in at least 100 countries.
Limitation of some authorities are summarized in Table 3 (Egmond and Jonker, 2003;
EC, 2006; EC, 2007; Cheli et al., 2014).
Detection of mycotoxins in food and feed
The growing concern over food and feed safety has led to development of
several methodologies for mycotoxins detection. Toxicity of mycotoxins may occur
at very low concentrations, therefore sensitive and reliable methods for their detection
are required. Proper sampling, homogenization, extraction, and concentration of
samples are generally the most common steps in many analytical procedures. Detection
methods can be categorized into qualitative and quantitative (Berthiller et al., 2005;
Trucksess and Diaz-Amigo, 2011). Thin layer chromatography (TLC) methods can be
used as a preliminary test for AFs, ZEN, and ochratoxin, but for fumonisins and
some member of the trichothecenes it is not a useful method (Zinedine et al., 2007;
Bryden, 2011). Recently, for a rapid specific screening determination of mycotoxin
type, immunological methods such as enzyme-linked immunoassay (ELISA) and
radioimmunoassay (RIA) are the best approaches because they depend on specific
antibodies beside their relatively low cost, easy application and their results could
be comparable with those obtained by other conventional methods such as TLC and
high-performance liquid chromatography (HPLC) (Fung and Clark, 2004; Zheng et
al., 2006; Berthiller et al., 2007).
There is no doubt that correct detection needs a correct extraction and clean-
up methods, these include liquid-liquid extraction, supercritical fluid extraction, and
solid phase extraction (Turner et al., 2009). Several chromatographic techniques are
used as quantitative methods for a massive number of samples; high-performance
liquid chromatography (HPLC), gas chromatography mass spectrometry (GC-MS) or
liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-
MS-MS), capillary electrophoresis, and fluorometric assay (Zheng et al., 2006;
Berthiller et al., 2007; Turner et al., 2009). The most appropriate analytical method
differs according to the nature of detected mycotoxin, e.g., for AFs, ZEN, OTA,
HPLC-fluorescence andLC-MS/MS are commonly used, while for trichothecenes,
GC-MS is mainly preferred (Saeger et al., 2003; Berthiller et al., 2005; Zollner and
Mayer-Helm, 2006; Njobeh et al., 2012; Waskiewicz et al., 2012; Seo et al., 2013).
Prevention
Elimination of mycotoxins is the main goal of food and feed safety programs.
Unfortunately, mycotoxins are very stable compounds and their detoxification during
food and feed processing is difficult. Therefore, their contents in food and feedstuffs
must be controlled to a minimum level. Strict regulations are important to decrease
the risk of mycotoxin contamination which can be achieved by rigorous monitoring
Abdallah et al.

483
including strategies to decrease the mycotoxin production in feed stuffs before and
after harvest, and systematic protocols to decrease their exposure and modulate the
metabolism of the toxins to reduce toxicity. This requires a better understanding of
the ecology of mycotoxin producing organisms, animal production regimes, and feed
chain systems (Jarday et al., 2011; Trucksess and Diaz-Amigo, 2011; McCormick,
2013; Womack et al., 2014).
Pre-harvest precautions include efficient agricultural practice which involves;
the wise use of fungicides and insecticides to prevent fungal and insect invasion;
irrigation to avoid moisture stress; harvesting of plants in maturity when moisture
content is lowest. Improvement of plant genes to resist fungal attack is based on
genetic engineering, effective breeding programs, and using of biocompetitive fungi
(Bryden, 2011; Jarday et al., 2011; Trucksess and Diaz-Amigo, 2011).
A novel way has been used, in which non-toxigenic fungi are cultivated in the
field to substitute naturally occurring toxic fungi. This approach gives considerable
results for aflatoxins in some agricultural products, for instance, peanuts and maize.
Ultimately, a combination of strategies using biocompetitive fungi and enhancement
of host-plant resistance may be needed to adequately prevent mycotoxin contamination
in the field. There are two important parameters in controlling the fungal activity
in stored agricultural stuffs, temperature and moisture which are affected by
geographical location and other circumstances such as drying, aerating, turning of the
grains and transport. A direct link between mycotoxin contamination and improper
post-harvest storage conditions has long been recognized. Therefore, it’s important to
keep storage equipment and transporters free of insect and other vector activities,
water condensation, and water leakages to prevent fungal invasion (Cleveland et al.,
2003; Trucksess and Diaz-Amigo, 2011; Womack et al., 2014).
A variety of chemical, biological, and physical approaches have been developed
to control mycotoxin contamination as shown in Table 4. Moreover, many studies
have been carried out on adsorbent materials, organic and inorganic binders. These
compounds are added to the feed to bind the toxin during digestion process resulting
in reduction of toxin bioavailability. Examples for inorganic adsorbents are bentonites,
zeolites, diatomaceous earth, clays, modified clays, and activated charcoal. For
organic adsorbents different substances have been examined such as fibers from plant
sources like alfalfa, oat fibers, extracted cell wall fraction of Saccharomyces cerevisiae
and recently, beta-D-glucan fraction of yeast cell wall. These dietary additives offer
one of the greatest potentials for preventing toxicity in a stable digestive tract where
the bounded toxins can be excreted via urine or feces. Additionally, other physical
approaches such as washing with water or sodium carbonate, dehulling, sorting of
contaminated grains, heating at high temperature, milling, and irradiation treatment
(UV, X-rays or microwave irradiation) have been employed to reduce mycotoxin
contamination post-harvest (Young et al., 1987; Fandohan et al., 2005; Jouany, 2007;
Isman and Biyik, 2009; Bocarov-Stancic et al., 2011).
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484
Table 4. Detoxification or degradation of some mycotoxins
Process Effect References
Chemical process
Ozone Total degradation of ZEA. No detected McKenzie et al., 1997
ZEA orZEA-like products.
Reductions of 80% and 93% AFB1 Inan et al., 2007
Ammonium hydroxide Decrease fumonisin B1 content by 30-45% Norred et al., 1991
Bisulfite solutions At 80°C for 18h can convert 85% of Young et al., 1987
DON into a DON-sulfonate conjugate
Ammonium persulfate Reduction of AFB1 by 53-87% Burgos-Hernandez
et al., 2002
Ammonia At 40-50°C for 48 h decrease AFB1 Chen et al., 2013
from 1280 ppb to 10 ppb
NaNO2Deamination of fumonisin B1 Lemke et al., 2001b
HCl Reduce AFB1 levels by 19.3% Doyle et al., 1982
within 24 h (pH 2.0)
Physical process
Activated carbon Binding 100% ZEA (pH 3 and 7.3) Bueno et al.,2005
Activated carbon Reduction of AFM1 (76%) Rao and Chopra, 2001
Sodium bentonite Reduction of AFM1 (67%)
Bentonite, diatomite and zeolite Binding of 95% AFB1 Bocarov-Stancic
(pH 3.0 and 6.9) et al., 2011
Binding of 25% DON in case of diatomite
and 50% DON for the others (pH 3.0)
Binding of 12.2% to 37% ZEA
(pH 3.0 and 6.9)
Binding of 16.7% to 33.33% T-2 toxin
(pH 3.0 and 6.9)
Diatomite Binding of 66.67% OTA (pH 3.0)
Sorting and/or washing Efficient with respect to Fusarium sp. Fandohan et al., 2005
Thermal treatment Fumonisin B1 and B2 losses exceed 70% Scott & Lawrence, 1994
in maize after heating at 190°C for
60 min and reached 100% when heated
at 220°C for 25 min
Dehulling Induced a 48% reduction of DON Fandohan et al., 2005
and ZEN levels
Modified clays Effective in sorbing the estrogenic ZEN Lemke et al.,2001a
UV Reduction of total aflatoxins by 25% Isman and Biyik, 2009
Gamma irradiation A dose 5 kGray inactivates the growth of Aziz and Moussa, 2004
Fusarium & mycotoxin formation in seeds
Reduction of total AFs by 34-40% Herzallah et al., 2008
and AFB1 by 33-43%
Microwave Reduction of total AFs by 21-33%
and AFB1 by 23-32%
Table 4. Contd. ...
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485
Various chemicals (acids, bases, oxidizing agents, different gases) have been
examined for detoxification of mycotoxins, but most of them cause reduction in the
nutritive value and palatability of the feed. Generally, they are very expensive and
time consuming due to their need for additional cleaning treatments. Furthermore,
toxic by-products may be produced. Many organic acid compounds, especially propionic
acid, inhibit fungal growth and form the core stone of many commercial antifungal
agents used in animal feed industry. Extensive research has been done to consider
ozonation as a practical method for decontamination of mycotoxins, especially aflatoxins
(Norred et al., 1991; McKenzie et al., 1997; Burgos-Hernandez et al., 2002; Inan et
al., 2007; Jouany, 2007; Womack et al., 2014).
Biological approach for mycotoxins decontamination by using microorganisms
such as Eubacterium and certain types of isolated yeasts have been used successfully
for the management of mycotoxins in particular aflatoxins and ochratoxin A in food
as well as animal feeds. Recent studies indicate that molecular approaches may offer
insight into the interactions of mycotoxin-producing fungi and other organisms including
mycotoxin-degrading microbes (El-Nezami et al., 1998; Molnar et al., 2004; Abrunhosa
et al., 2010; Zou et al., 2012; McCormick, 2013). However, none of the approaches
individually fulfills the required efficacy, safety and cost needed for the removal of
mycotoxins from contaminated agricultural products. Under all circumstances,
detoxification processes should eliminate or inactivate mycotoxins, generate no toxic
products, guarantee the nutritional value of the feed and induce no modification to
the technological properties of the product.
Process Effect References
Biological process
Mixed bacterial culture Total degradation of ZEA. Megharaj et al., 1997
No detected ZEA orZEA-like products.
Trichosporon mycotoxinivorans Degradation of ZEA to Molnar et al., 2004
non-toxic metabolites
Lactobacillus rhamnosus strains 80% AFB1 removed El-Nezami et al., 1998
(GG and LC-705)
Lactobacillus plantarum Reduction of DON and T-2 toxins by Zou et al., 2012
strain-102 physical binding and biotransformation
F420-dependent reductases Reduction of total AFs through biotrans- Lapalikar et al., 2012
(FDR-A and FDR-B) formation of Mycobacterium smegmatis
Aspergillusniger Degradation of OTA by lipase Abrunhosa et al., 2010
Gram negative bacterium Deamination of fumonisin B Heinl et al., 2011
ATCC 55552 by aminotransferase
Table 4. Detoxification or degradation of some mycotoxins. Contd. ...
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486
CONCLUSION
Despite all the efforts to prevent mycotoxin contamination and related outcomes,
outbreaks of mycotoxicosis or untoward effects due to mycotoxin exposure are still
possible. Not only human but animal exposure should also be considered by authorities.
Awareness of mycotoxin properties, limiting their presence in the environment,
preventing exposure above toxic levels will help to maintain both human and animal
welfare. Countries should have their own national policies and limits to save public
health from toxic outcomes.
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