Occurence of Mycotoxins in Animal Feed

Created by: Sara Cunha

Maize, wheat, and soybean along with their by-products are widely used in the production of animal feed. Lately, we have been witnessing a steady tendency in the increase of the global demand for these crops due to the accelerated growth and strengthening of the livestock industry. Thus, animal feed safety has gradually become more important, with mycotoxins representing one of the most significant hazard concerns. Mycotoxins comprise different classes of secondary metabolites of molds. With regard to animal feed, aflatoxins, fumonisins, ochratoxins, trichothecenes, and zearalenone are the more prevalent ones. Bearing in mind that mycotoxins can represent a serious threat to feed supply chain, animal health and, ultimately, to human health, one of the purposes of this review was to briefly discuss the constraints posed by these contaminants at economical and commercial levels, on industries related with animal production systems. Also, European Union legislation established to restrict mycotoxins levels in animal feed is discussed. Finally, the occurrence of legislated mycotoxins in feed and the main raw materials will be reviewed. Co-occurrence phenomenon will also be addressed as it poses great concern.

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1. Introduction

Feed is described by the European Commission as any substance or product, including additives, whether processed, semi-processed or unprocessed, intended to be used for oral feeding of animals [1]. It can be classified into the following four groups [2]:
  • Forages—silage made from grass or cereal crops;
  • Cereals and other home-grown crops—feeds with a high energy and/or protein content;
  • Compound feeds—manufactured mixtures of single feed materials, minerals, and vitamins;
  • Products and by-products of the human food and brewing industries—residues of vegetable processing, spent grains from brewing and malting and by-products of the baking, bread-making, and confectionery industries.
Livestock diets typically include a combination of feeds that are designed to meet not only the nutritional needs of animals with minimal costs, but also to provide everything they need for their health, welfare, and production [2,3]. However, cereals and cereal-based products are possibly the most commonly used ingredients in animal feed, supplying most of the nutrients for livestock [4,5,6,7]. In developed countries, up to 70% of the cereal harvest is used in the daily diet of animals, whereas, in developing countries this commodity is mainly used for human consumption [8]. In addition, plant protein sources, such as by-products from the extraction of oil from oilseed crops, are regularly present in animal feeding and complement the cereal grains which are usually poor in protein [4,6,9,10].
Cereals for the global feed industry include maize, wheat, barley, sorghum, and oats grains [7,9]. Essentially maize, as well as wheat, are considered key global agricultural commodities in regard to farm animal diets [4,11,12,13]. In fact, the majority of the maize production in the world (approximately 55%) goes into animal feed, because maize and products derived thereof are widely used feed raw materials [7,12,13,14,15,16]. Wheat in feedstuffs represents around 20% of the total wheat, with the remainder of the wheat used for human consumption. Nevertheless, in the European Union (EU) almost half of the wheat is used in feed [17,18]. Therefore, wheat grains and the respective by-products are also seen as suppliers of various significant materials in livestock feed [13,18,19].
Oilseed crops like soybeans, cottonseed, sunflower, sesame, and palm are also used as vegetable protein sources in the manufacturing of animal feed [9,10]. However, soybean products remain universally accepted as the most important and preferred feed commodities due to their high-quality protein content [4,10,13,20,21,22]. In fact, soybean meal, which is the by-product of oil extraction from soybeans, represents two-thirds of the total world output of protein feedstuffs [20].
The global demand for agricultural crops has been increasing over the years, with an expected growth of 84% between 2000 to 2050 [4,11,23,24,25]. This development is intended, in part, to meet the rapid growth and strengthening of the livestock industry, propelled by the rising demand for livestock products [2,10,25]. This is, in turn, driven essentially by increases in world population and urbanization rates, as well as changes in lifestyles and food preferences [10,11,23,25]. Consequently, animal feed safety has become even more of a concern for both producers and governments since feed consumption is, eventually, a potential route for hazards to reach the human food chain [10,25,26,27]. Thus, in accordance with the Directive 2002/32/EC, the quality and safety of products intended for animal feed must be assessed prior to their use in feed to ensure that they do not represent any danger to human health, animal health or the environment, or do not adversely affect livestock production [27,28]. Among the undesirable substances laid down in this Directive, mycotoxins have been increasingly targeted as becoming one of the most important dangers in the raw materials of feed, due to the verified increase in their formation [29,30]. In this review, several constraints posed by these contaminants at the economical and the commercial level will be discussed, along with the legislation established in the European Union to restrict mycotoxins levels in animal feed. In addition, the occurrence of legislated mycotoxins in the raw materials and their by-products of the feeds of interest, as well as in the feed, will be reviewed. Additionally, an overview of the different sample preparation and detection techniques reported for mycotoxin analysis will be discussed.

2. Mycotoxins Classes and Toxicity

Mycotoxins are a relatively large and chemically diverse group of toxic secondary metabolites of low molecular weight. They are typically produced by filamentous fungi, especially those belonging to the genus AspergillusPenicilliumAlternaria, and Fusarium, although Claviceps and Stachybotrys are also important mycotoxins producers. Approximately 300 to 400 mycotoxins have been identified and reported so far [5,31,32]. However, regarding their prevalence in feeds and their known effects on livestock health, only a few groups of mycotoxins are considered to be a safety and economic concern, namely, aflatoxins (AFs), fumonisins (FMs), ochratoxins (OTs), trichothecenes (TRCs), and zearalenone (ZEN) [5,27,33]. Other mycotoxins, such as patulin, citrinin, and other emerging mycotoxins are beyond the scope of this review. With these relevant classes in mind, a brief introduction about each one will be provided along with the associated toxicological effects.

2.1. Aflatoxins

Aspergillus flavus and A. parasiticus are the main species of aflatoxin-producing fungi, although A. nomius and A. pseudotamarri are known to produce them, as well. The AFs group encompasses several different toxins, however, only the following four types are most abundant: aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2(AFG2) (Figure 1) [32,34,35]. The metabolic products derived from AFs are aflatoxin M1 (AFM1) and M2 (AFM2) which are also referred to as important contaminants of this class [32,36,37].
AFs represent the group of fungal toxins of greatest concern in terms of human toxicity. Their toxic effects advert from their entry in the human food chain in two ways: (i) First, directly, after human exposure by consumption of contaminated crops or finished processed food products, since aflatoxins are very stable and may resist food processing operations. (ii) Secondly, indirectly from tissues, eggs, milk, and dairy products of animals fed with aflatoxin-contaminated feeds, through excretion of the hydroxylated derivative of AFB1 and AFM1. Actually, AFB1 is the most commonly occurring aflatoxin and most potent hepatocarcinogen, classified by the International Agency for Research on Cancer (IARC) as a human carcinogen (group 1) and AFM1 as possibly carcinogenic to humans (group 2B) [33,38,39,40,41,42]. Concerning livestock health, AFs are also a major problem causing acute death to chronic disease. Clinical signs of animal intoxication include gastrointestinal dysfunction, anemia, jaundice, hemorrhage, and an overall decrease in productive parameters, such as reduction in weight gain, lower feed efficiency, decreased egg or milk production, inferior carcass quality, and increased susceptibility to environmental and microbial stressors [32,41,42,43]. Ultimately, prolonged exposure to low dietary levels of AFs can result in extensive functional and structural liver lesions, including cancer. It is important to note that nursing animals, as well, are exposed to the AFB1 toxic metabolite secreted in milk [32,41,42,43].
Figure 1 – Chemical structure of AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2.

2.2. Fumonisins

FMs are commonly classified as Fusarium toxins since they can be produced by several species of this genus, with F. verticillioides (previously classified as F. moniliforme) and F. proliferatum as the main producing species. However, A. niger was recently found to also produce FMs [36,42,44]. Within the 16 fumonisin analogues known to date, the B-series FMs (FBs), which compromise fumonisin B1, B2, B3, and B4, are the most important ones  (Figure 2) [36,42,45].
Fumonisin B1 (FB1) is reported as the predominant and most toxic member of the FMs family and has been recognized as a possible human carcinogen (group 2B) [38,42,46]. Fumonisin B2 (FB2) is also toxicologically significant. Apparently, the carcinogenic character of FBs is not related to direct DNA damage, but rather it is associated with the disruption of sphingolipid biosynthesis due to structural similarities of these toxins with the backbone precursors of sphingolipids [36,40,41]. In animals, ingestion of feed contaminated with FBs can cause significant disease in horses, swine, and rabbits which are considerably more sensitive than cattle and poultry [32,41,47]. Leukoencephalomalacia syndrome appears mainly in horses triggering primary symptoms like lethargy, blindness, and decreased feed intake, and ultimately, convulsions and death. In pigs, FB1 is associated with pulmonary oedema whose clinical signs typically include reduced feed consumption, dyspnea, weakness, cyanosis, and death [36,40,41]. In addition, these mycotoxins have also shown hepatotoxicity [32,40].
 Figure 2 – Chemical structure of FB1 and FB2 

2.3. Ochratoxins

Production of the OTs, ochratoxin A (OTA) and ochratoxin B (OTB), occurs essentially by fungi belonging to the genus Aspergillus and Penicillium, namely by the species A. ochraceusA. carbonariusP. verrucosum, and P. nordicum [32,36,37,48].
OTA (Figure 3) are linked with potent nephrotoxic effects in animals as a consequence of exposure to naturally occurring levels in feed, since the kidneys are the major target organ [32,40,41,46]. In fact, OTA have been associated with endemic nephropathy in swine [36,46]. High dietary doses of this toxin may cause liver damage and necrosis of intestinal and lymphoid tissue [32,40]. Regarding humans toxicity, OTA have been implicated in a fatal kidney disease typical in the Balkan countries (Balkan endemic nephropathy) and have been classified as possibly carcinogenic (group 2B) [32,38,41,46]. Additionally, there has been a public health concern with respect to the transfer of OTA to animal-derived food [42].
Figure 3 – Chemical structure of OTA.

2.4. Trichothecenes

TRCs are produced to a great extent by Fusarium species, although not exclusively, since some CephalosporiumMyrotheciumStachybotrys, and Trichoderma species also produce these mycotoxins. This is a large class of fungal metabolites with more than 150 structurally related compounds, which are chemically divided into four types (A to D) [32,41,43]. TRCs from type A and B are the most important. Type A-TRCs comprises mainly HT-2 and T-2 toxins (HT-2 and T-2) (Figure 4), while type B-TRCs are frequently represented by deoxynivalenol (DON) (Figure 5), its derivatives 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON) and nivalenol (NIV) [49,50].
HT-2 and T-2, although not being very prevalent, are the most toxic members of type A-TRCs [40,41,42,43]. They were found to inhibit protein and DNA synthesis and weaken cellular immune responses, in animals [40,42]. Symptoms include decreased feed intake and weight gain, bloody diarrhea, hemorrhaging, oral lesions, low egg and milk production, abortion, and death in some cases [40,41,42,43].
DON is one of the least acutely toxic TRCs, however, as it is highly incident, it is considered very relevant in animal husbandry [32,40,42,51]. Exposure to DON more severely affects monogastric animals, especially swine, and may cause feed refusal, vomiting, and anorexia, as well as the symptoms described previously for HT-2 and T-2 [32,41,43]. Overall, ingestion of low to moderate levels of this mycotoxin by animals leads to increased susceptibility to pathogens and to a poor performance [32,41]. DON was categorized by IARC as not classifiable with respect to its carcinogenicity to humans (group 3) [38].
Figure 4 – Chemical structure of HT-2 and T-2. Figure 5 – Chemical structure of DON.

2.5. Zearalenone

ZEN is a Fusarium mycotoxin produced particularly by F. graminearum and also by F. culmorumF. cerealisF. equiseti, among others, and it has α-Zearalenol (α-ZEL) and β-Zearalenol (β-ZEL) as derivatives [36,41,52]. Once ZEN has structural similarities to the female sex hormone, estradiol, it is classified commonly as a nonsteroidal estrogen. This chemical characteristic gives it the capability of binding to estrogen receptors, causing adverse effects associated with reproductive disorders and hyperestrogenism, both in humans and breeding animals [32,36,37,42]. According to IARC, ZEN belongs to group three, which means it is not classifiable regarding its carcinogenicity to humans [38].
Figure 6 - Chemical structure of ZEN.

3. Mycotoxins Economic and Commercial Implications

Animal consumption of mycotoxin-contaminated crops may cause adverse health effects which include occult conditions (for example, growth retardation, impaired immunity, and decreased disease resistance), chronic to acute disease, and even death. Basically, these hazards affect animal performance to a great extent, representing a global concern for the livestock industry [5,32,46]. Therefore, a threat, such as mycotoxins, to the safety of the feed supply chain becomes a significant constraint to animal production systems [5,53]. These metabolites cause perturbations in the feed industry due to the decrease in the quality of commodities which may even lead to the rejection and disposal of highly contaminated crops [5,32,46]. Naturally, large costs on the economy of these industries arise from mycotoxin contamination. Apart from the aforementioned problems, economic losses may be associated with increased costs for health care, finding alternative feed sources, prevention strategies, investment in testing methods, and for regulations [5,8,32,33]. Additionally, mycotoxins presence may impact on international commodity trade, propelled by increasing globalization [32,34].
In an attempt to avoid the adverse effects and implications discussed above, several worldwide institutions and organizations have restricted the accepted levels of certain mycotoxins in animal feeds, since truly mycotoxin-free feedstuffs are impossible to guarantee. Naturally, the limits and the mycotoxins targeted by legislation vary from country to country since different scientific, economic, and political factors influence this decision-making process [26,32,33,43].
Particularly, in the EU, legislation (regulation or recommendation) established so far cover AFs, FBs, OTA, some types of A and B TRCs, and ZEN, in different feeding matrices. Directive 2002/32/EC specifies maximum content for AFB1 in products intended for animal feed [28]. Guidance values for DON, FBs, OTA, and ZEN contamination were set in the Commission Recommendation 2006/576/EC [54].

4. Mycotoxin Occurrence

Various factors are known to influence the incidence of mycotoxins, despite their unavoidable and unpredictable nature. Their production can start in the field throughout the crop growing cycle and continue during harvesting, drying, processing, and storage steps, strongly depending on various environmental conditions. These comprehend not only climatic factors, such as temperature and moisture content which are the main aspects modulating fungal growth and mycotoxins production, but also pH, bioavailability of micronutrients, and insect damage, for example [32,33,37,46,50,55]. Others factors like geographic location, agricultural practices, harvest year, and the length and conditions of storage affect the extent of the contamination of a particular commodity [32,33,56,57]. However, the substrate susceptibility to fungal invasion plays a major role in mycotoxin production [58]. Moreover, due to the climate changes across the globe, some changes in the distribution and cycles of the molds are expected, since every mold species has its own optimum conditions of temperature and water activity for growth and formation of toxic metabolites.
In order to understand the mycotoxin prevalence and contamination levels in the main raw materials of feed that are the subject of this work, global mycotoxin occurrence data was gathered in Table A1Table A2 and Table A3. These tables represent an overview of contamination in maize, wheat, and soybeans and their by-products, respectively, collected by several authors, in the last three years (2016–2018) through searches in PubMED and ScienceDirect. Globally, maize and wheat are by far the most studied matrices, while soybean is the least studied, which is in agreement with previous reports [59]. In all substrates, the raw ingredients themselves were more subjected to mycotoxin contamination surveys than the respective by-products, perhaps because the last ones are usually more complex matrices.
Considering Table A1, it can be pointed out that in 2016 there was an increase in the samples of maize and the derived by-products in which mycotoxins were researched. This may be because this crop is among the most susceptible to mycotoxigenic fungi infection, and also since its production is growing from year to year the need to target these impurities has also raised [12]. The fact that maize by-products are increasingly used in animal diets may also explain the larger number of assayed samples of these feedstuffs [58]. In maize, most studies focused on ZEN and type A-TRCs, followed by the occurrence of AFs and FMs. According to FAO [15,27], maize is especially linked with these two contaminants, having a relevant role in economic losses in maize production [60]. Regarding the levels found, AFB1 was the mycotoxin that exceeded the EU legislative level more often, with a maximum value of 1137.4 µg/kg in a sample of raw-cereal from Kenya [61]. ZEN, T-2, and HT-2 have also been reported to exceed the EU legislative levels in some cases, as reported in Table A1.
Inversely, the wheat samples examined decreased from 2016 to 2018 (Table A2). Concerning the mycotoxins targeted in the reports reviewed, DON was by far the most searched, probably because it is frequently associated with this grain [44]. Nevertheless, ZEN and AFs were also studied in this matrix, and the last one exceeded the EU legislative level in three studies (Table A2).
From Table A3 it is possible to observe the mycotoxins that were studied more and these were AFs, followed by ZEN, and DON. Additionally, fewer samples of soybeans and its by-products were analyzed as compared with maize or wheat, and the by-products were studied more than the raw leguminous. Generally, this substrate is not considered a relevant problem in terms of mycotoxin contamination which may be because of its low moisture content and composition (high protein/carbohydrate ratio) that inhibit fungi growth, and also the better conditions used in the storage of this commodity due to the high price of soybean [37,62]. Nevertheless, the mycotoxin contamination verified in the studies reported was remarkable. In the future, more research on this commodity is needed, especially if this trend of production growth continues, in order to better understand which mycotoxins are most commonly associated with soybean and their by-products and whether contamination levels are of concern.
Overall, it seems that the common association between certain raw materials and a specific mycotoxin contamination profile has led researchers to favor the determination of these same contaminants. However, in addition to the fact that mycotoxins formation is a complex and multifactor phenomenon, worldwide contamination and distribution patterns of fungi and their secondary metabolites are predicted to be affected significantly by climate change scenarios, as a result of the appearance of favorable environmental conditions for fungal proliferation in uncommon places [33,46,53]. Therefore, mycotoxins presence is unpredictable and multi-mycotoxins surveys end up being more realistic and preferred.
Safety complications arising in the feed manufacturing process include aspects like the practice of mixing different batches of distinct raw ingredients, which creates a new matrix with an entirely new risk profile, and the fact that the majority of mycotoxins are stable compounds that are not destroyed during the storage, milling or high-temperature feed manufacturing process [63]. For these reasons, the knowledge of the occurrence and distribution of mycotoxins in animal feeds is of extreme importance, and it provides the opportunity to determine the direct risk posed to animals. Therefore, occurrence data of these toxins in animal feed, collected by several authors from various countries, from 2016 to 2018 was gathered in Table A4. Globally, AFs, DON, and ZEN were the mycotoxins most studied, but the determination of AFs and ZEN derivatives experienced a great increase, from 2016 to 2018. In this late year, the number of samples analyzed was far less than in 2016. The incidence of the mycotoxin, AFB1, most exceeded the EU legislative level in this kind of samples (Table A4).
Once formed, most mycotoxins are very stable during harvesting and storage. This draws attention to the need for prevention and control strategies such as hazard analysis and critical control point (HACCP) approaches, good agricultural and manufacturing practices (GAP and GMP), and quality control from the field through to the final product [64,65]. However, contaminated feed may be redirected to less vulnerable animal species, or, ultimately, detoxification methods can be used which involve the addition of feed additives “that can suppress or reduce the absorption, promote the excretion of mycotoxins or modify their mode of action” [30,66,67]. These substances have to be authorized under the feed additive Regulation 1831/2003 amended by Regulation 386/2009 [68]. In this way, the hygienic and nutritional quality of feed is guaranteed, ensuring the safety and productivity of the farm animals [30,65].
It is important to mention that when constructing Table A1Table A2Table A3 and Table A4, only papers that quantitatively determined mycotoxins were included and the ones that mentioned explicitly the use of the raw materials for human consumption were excluded. Moreover, year-to-year variations were reduced to the maximum because this parameter is beyond the scope of this review, and whenever the results permitted, the percentage and the average of positive samples was calculated. In addition, since all the information was obtained from different methodologies of analysis with distinct sensitivity and accuracy, the quantitative comparison might be quite complex.


Natural contamination of raw ingredients and feeds with an array of mycotoxins is a frequent scenario around the world, which can be explained by the ability of molds to simultaneously produce different kinds of mycotoxins and because commodities may be concurrently, or in rapid succession, infected with different fungal species. In addition, composite feed is made up of a mixture of several raw ingredients, making it particularly vulnerable to multiple mycotoxins contamination [5,33].
When it comes to the reports considered for this review, several ones described this phenomenon only within the regulated mycotoxins. In maize and products derived thereof, Chen et al. [69] found co-contamination with AFB1and ZEN. Chilaka et al. [70] reported that 60% of maize samples were infected with at least two mycotoxins and FBs co-existing with DON in 11% of samples. ZEN and DON were simultaneously found by Dagnac et al. [71], who reported a frequent co-contamination of more than one mycotoxins in the samples under analysis. Jovaišienė et al. [72] found DON and ZEN co-occurring in all samples of silage and DON, ZEN, and T-2/ HT-2 co-occurred in all fermented silage samples. Kamala et al. [73] detected that 33% of the samples were contaminated with both AFs and FB1 + FB2. Kosicki et al. [15] frequently identified this phenomenon in their study, with the combination of DON and ZEN being the most prevalent, however, the co-occurrence of DON, T-2 and HT-2; ZEN, T-2 and HT-2; DON, T-2, HT-2, and ZEN; and DON and FMs were commonly found in maize. While in maize silage, apart from these groups, the co-existence of DON and OTA; DON, OTA and ZEN; ZEN and OTA; and T-2, HT-2, and OTA were also detected. Mngqawa et al. [74] reported the occurrence of a wide variety of mycotoxins in their samples with relevance to AFs and FBs. Finally, Murugesan [75] verified that 50% of samples were contaminated with more than one these analytes. In wheat, co-occurrence between ZEN and DON was found by Calori-Domingues et al. [76], in several samples. Already in soybeans and derived by-products, Egbuta et al. [77] showed that there was simultaneous occurrence of AFs and FB1. Regarding animal feed, Hu et al. [78] concluded that combinations of two mycotoxins were more frequent than three but highlighted the presence of AFB1, OTA, and ZEN. Kongkapan et al. [79] detected that AFs co-occurred with ZEN and AFB1 with DON. Kosicki et al. [15] frequently identified this phenomenon with combinations of DON and ZEN; DON, T-2 and HT-2; ZEN, T-2 and HT-2; DON, T-2, HT-2, and ZEN; DON and FMs; DON and OTA; DON, OTA, and ZEN; ZEN and OTA; and also T-2, HT-2 and OTA. Lastly, a high incidence of co-contamination was reported by Kovalsky et al. [43]. Globally, these results are in line with the statements that combinations of two mycotoxins occur more frequently [32,33]. It was verified that DON and ZEN along with AFB1 and FBs were commonly reported as co-existing in the reviewed samples, followed by DON and FBs as well as DON, ZEN, and HT-2/T-2.
Multiple mycotoxin contaminations pose great concerns since it is completely stated that adverse effects on animal health and performance can be additive and/or synergistic, which means that the overall toxicity is not only the sum but the multiple of the individual toxicities of the mycotoxins [5,80]. This means that the study of just one of these toxins provides insufficient information about the risk associated with a respective feedstuff and that attention toward mycotoxin co-occurrence should be increased [42,81]. Additionally, the use of raw materials from different types and origin contributes to increase the likelihood of multi-mycotoxin contamination, including nonregulated compounds, usually called “emerging mycotoxins”. The presence of conjugated mycotoxins, sometimes in amounts similar or even higher than the corresponding free mycotoxins, is another issue that deserves detailed attention. However, the potential for biological effects remains, and the toxicological potential can be substantial enhanced.
Currently, legislation over the world, including in Europe, only considers mycotoxin mono-exposure data and does not address relevant mycotoxin combinations, which is considered a loophole that should be taken into account in the future.