Till now, mycotoxins of concern to European legislation [88] present in cereals and their products include the four (B1, B2, G1, G2) aflatoxins (AFs), ochratoxin A (OTA), two (B1, B2) fumonisins (FBs), zearalenone (ZEN), deoxynivalenol (DON) also known as vomitoxin, Citrinin (CIT), Ergot alkaloids (EAs), and T-2 and HT-2. The above-mentioned mycotoxins have been considered dangerous, and it is encouraged that simultaneous analysis of them be performed to detect co-occurrence. Table 3 cites the major regulated or registered as carcinogenic from IARC mycotoxins and producing species in cereal grains as reported in the literature. Fumonisins B3, B4, and nivalenol (NIV) are also frequently detected in cereals, although no limits exist to regulate their allowable levels. On the other hand, patulin, detected mainly in fruits, is now present in fruit-based cereal products [89].

Table 3. Major mycotoxins and fungal species associated with their production in cereal grains.

Mycotoxins	Acronym	Fungal Species	Source
Aflatoxin B1, B2, G1, G2	AFs	A. flavus, A. nomius, A. parasiticus	[19,161,162]
Citrinin	CIT	P. expansum, A. ochraceus, P. verrucosum	[163]
Deoxynivalenol	DON	F. acuminatum, F. culmorum, F. graminearum	[19,110,161,162,164,165]
Ergot alkaloids (EA s)	EAs	C. purpurea	[166]
Fumonisin B1, B2, B3, B4	FBs	F. proliferatum, F. verticillioides, A. niger	[137,162,164,165]
HT-2	HT-2	F. langsethiae, F. poae, F. sambucinum, F. sporotrichioides	[164,165]
Nivalenol	NIV	F. cerealis, F. crokwellense, F. culmorum, F. graminearum, F. poae	[110,164,165]
Ochratoxin A	OTA	A. carbonarius, A. ochraceus, P. cyclopium, P. nordicum, P. verrucosum, P. viridicatum,	[19,161,162]
Sterigmatocystin	STC	A. flavus, A. parasiticus, A. nidulans, A. versicolor	[167]
T-2	T-2	F. acuminatum, F. equiset, F. langsethiae, F. poae, F. sambucinum, F. sporotrichioides	[161,164,165]
Zearalenone	ZEN	F. crokwellense, F. cerealis, F. culmorum, F. equiseti, F. graminearum,	[161,162,164,165]

As observed in Table 3, the most important mycotoxins in cereals and their products are produced by different species of eight genera of fungi: Aspergillus, Alternaria, Byssochlamys, Claviceps, Fusarium, Neotyphodium, Paecilomyces, and Penicillium. However, species of the Fusarium genus are responsible for the contamination of cereals with DON, FBs, HT-2, T-2, and ZEN, but also with beauvericin (BEA), asenniatins (ENNs), fusaproliferin (FUS), moniliformin (MON), NX-2 toxin, and NIV, whereas those of Aspergillus for contamination with AFs and Sterigmatocystin. Alternariol (AOH) is produced by fungi species of the genus Alternaria. On the other hand, one mycotoxin can be produced by a variety of fungi. For example, patulin is a secondary metabolite of several species of fungi of the genera Penicillium, Aspergillus, and Byssochylamys, OTA and CIT of the genera Penicillium, and Aspergillus, FBs of Fusarium and Aspergillus whereas EAs of Claviceps and Neotyphodium. On the other hand, toxigenic fungi species can produce more than one type of mycotoxin; therefore, the co-existence of more than one mycotoxin on the same substrate could usually be noticed [90].

Advances in analytical techniques and equipment allowed the determination of many toxic fungal metabolites contaminating cereals and their derived products. Mycotoxins such ENNs, BEA, MON, FUS, alternariol (AOH), sterigmatocystin (STC), and NX-2 toxin, are receiving increased attention due to their high frequency of occurrence and levels of contamination in cereals [55,56,57]. These toxins, although reported in the literature more than one decade ago [91,92,93,94,95], are not routinely determined, and not legislatively regulated, and are still considered “emerging mycotoxins” even though literature reports increasing incidence in the cereals. On the other hand, the small number of investigative studies dealing with the occurrence of these mycotoxins and/or the lack of toxicity data impede risk assessment and the dietary exposure of humans to these mycotoxins [96,97,98].

Some mycotoxins are produced by fungi that colonize the host plant (fungal metabolite) and are released into the cereals, while others are modified mycotoxins, either plant-made metabolites or process-made, through the chemical reaction in the food matrix during food processing. The plant-made metabolites can be divided into two groups, involving the free (extractable) and the bound to other molecules form of modified mycotoxins recognized as “masked mycotoxins” (or conjugated). Most of these modified mycotoxins are considered “emerging mycotoxins”, and awareness about them is increasing.

Although alternariol (AOH) is the most prominent mycotoxin produced by the genus Alternaria, it can produce a wide variety of toxic metabolites that are now getting the attention of scientists. They can be classified into five different structural groups. The first is the group of the dibenzopyrone derivatives to which the AOH belongs, alternariol monomethyl ether (AME), and altenuene (ALT). The second group includes the perylene derivative, including altertoxins I, II, and III (ATX-I, ATX-II, and ATX II), alterperylenol (ALTP), and stemphyltoxins (STE), and the third is a tetramic acid derivatives group that comprises tenuazonic acid (TeA) and iso-tenuazonic acid (iso-TeA). The fourth group comprises TA1, TA2, TB1, and TB2 toxins (AAL TA1, TA2, TB1, and TB2) of A. alternata f. sp. lycopersici, and in the fifth group, arecyclic tetrapeptide toxins tentoxin (TEN), iso-tentoxin (iso-TEN), and dihydrotentoxin (DHT) [99,100,101]. Among them, AOH, AME, ALT, and TeAwere most frequently studied. Tebele et al. [102] reported the presence of AME and TeA in cereals. whereas Gotthardt et al. [103] reported the presence of AOH, AME, TEN, ATX-I, ALTP, and TeA in cereal food for infants and young children. Of them, AOH and AME have been recognized as genotoxic in mammalian cells in vitro [104]. According to EFSA’s opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food, taking into consideration AOH, AME, ALT, TEN, TeA, altertoxins, STE, and Alternaria alternata f. splycopersici toxins, there is need for additional toxicity and occurrence data [105,106]. Besides the aforementioned mycotoxins produced by Alternaria spp. in cereals, other toxic metabolites, such as macrosporin and radicinin, are observed [107].

Fusarium mycotoxins, frequently detected in cereals and cereal-based products, are predominantly zearalenones, trichothecenes, and fumonisins.

Zearalenone (ZEN) is the main mycotoxin of the group of zearalenones, considered possibly carcinogenic (IARC Group 3), present in cereals and has estrogenic effects [108]. Moreover, its derivatives, α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL), have also been detected in cereals [102].

Trichothecenes present in cereals, on the other hand, are classified in the Type A and Type B groups. Type B trichothecenes group are the most frequently occurring mycotoxins in cereals and include DON and NIV (both considered Group 3 according to IARC) [108] and their acetylated derivatives 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol), and 4ANIV (4-acetylnivalenol), respectively. On the other hand, the new type, A trichothecenes group besides T-2 toxin (Group 3, [108]), HT-2 toxin, neosolaniol (NEO), and diacetoxyscirpenol (DAS) toxins, comprises NX-2, NX-3, NX-4, NX-5, and NX-6 that can also be produced in cereals [109,110]. Varga et al. (2015) [109] reported the production of NX-2trichothecene mycotoxin in rice cultures and its deacetylated form NX-3 in wheat. They tested the toxicity of these mycotoxins and noted that NX-3 inhibits protein biosynthesis to the same extent as deoxynivalenol, while NX-2 is far less toxic, similar to 3-ADON. Although detected in low amounts in cereals and their products, under continuous changes in climate and agronomic practices, their presence should no longer be regarded as negligible [111]. Moreover, F. culmorum strains are able to simultaneously produce NX-2 with 3-ADON and DON or NIV [112]. New, less-toxic toxins belonging to A trichothecenes group, named NX2-M1, and the related acetylated compounds (NX3-M1 and NX4-M1), representing the degradation products encountered during cereal processing, have also been isolated in processed cereals recently [102]. The group of Type-B trichothecenes that are present in cereals and their products [113,114] comprises fusarenon-X (FUS-X), considered Group 3 by IARC [108]. Moreover, 4,15-diacetoxyscirpenol (DAS), neosolaniol (NEO), and verrucarol (VOL) are other mycotoxins of type-A trichothecene group produced by several Fusarium species reported in cereal grains and cereal-based products [115,116].

Fumonisins (FBs), produced mainly by the species F. verticillioides and F. proliferatum, have been divided into four categories (A, B, C, and D), with B containing, among others, the most toxic compounds. B-group fumonisins comprise fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3). FB1 and FB2 are considered potential human carcinogens (Group 2B) IARC [108] and IARC [117], respectively. Since these toxins can be present contemporarily in cereal commodities, the regulated limits in the EU include the sum of them (FB1 + FB2).

Fusarins are another group of mycotoxins produced by Fusarium fungi, such as F. avenaceum, F. culmorum, F. fujikuroi, F. graminearum, Fusarium oxysporum, Fusarium poae, Fusarium sporotrichioides, and Fusarium venenatum [118]. Among the different fusarins (A, B, C, D), fusarin C is the most isolated and identified type in cereals. It is biosynthesized by several Fusarium fungi species that contaminate cereals and is classified as possibly carcinogenic Group 2B by IARC [108].

There are also Fusarium species that do not produce zearalenones, trichothecenes, fumonisins, or fusarins but produce instead enniatins (ENs), beauvericin (BEA), and moniliformin (MON). These mycotoxins, together with fusaproliferin (FUS), may contaminate cereals [119]. Fusarium species F. acuminatum, F. arthrosporioides, F. avenaceum, F. tricinctum, F. torulosum, F. kyushuense, F. poae, F. sporotrichioides, F. venenatum, F. compactum, F. proliferatum, F. subglutinans, F. verticillioides, F. temperatum, and F. ramigenum are reported to produce ENs in cereals [120]. Fusarium species that produce beauvericin are reported to be the following: F. subglutinans, F. bulbicola, F. denticulatum, F. lactis, F. phyllophillum, F. pseudocircinatum, and F. succisae [121,122]. Several Fusarium species, such as F. avenaceum, F. proliferatum, F. subglutinans, F. oxysporum, F. chlamydosporum, and F. anthophilum produce MON, whereas, although named after it, only a few of the strains of F. moniliforme can produce it [123]. MON is one of the main Fusarium toxins in cereal and is less toxic than the T-2 toxin [124].

ENN and BEA that belong to the group of cyclic hexadepsipeptides are detected in food and unprocessed grains [119,125,126]. In cereals, only seven enniatins (enniatins A, A1, B, B1, B2, B3, and B4) have been detected, although naturally, they exist in a much higher number. Of them, most frequently, only four enniatins (A, A1, B and B1) have been detected [97], with ENB as the most detected enniatin [114,127]. Eniatins A, A1, B, and B1 in cereals are produced mainly by F. avenaceum, F. tricinctum, and F. poae [128]. On the other hand, in cereals is present FUS [119,125], a toxic bicyclic sesterterpene produced by F. proliferatum, F. subglutinans, F. antophilum, F. begoniae, F. bulbicola, F. circinatum, F. concentricum, F. succisae, and F. udum [129,130,131]. According to [96], beauvericin, enniatins, and moniliformin regularly co-occur in cereal grains with other Fusarium toxins, such as deoxynivalenol and fumonisins. Other less-known Fusarium mycotoxins include metabolites equisetin and butanolide [132].

Besides the aflatoxins, ochratoxins (OTA), patulin, and sterigmatocystin (STC), the fungi of the Aspergillus genus can also produce other toxins in cereals. Aspergillus mycotoxins AFs (B1, B2, G1, G2) got great attention due to their potent toxicity (Group 1, [117]). Patulin is classified as a Group 3 carcinogen according to IARC [133]. On the other hand, sterigmatocystins are considered a penultimate precursor of aflatoxins B1 and G1 [134,135] and are defined as a possible human carcinogen (Group 2B) according to the International Agency for Research on Cancer classification [133]. Because of climate change, this mycotoxin is considered a high risk of exposure for consumers [136]. According to EFSA CONTAM Panel [97], there is limited data about STC occurrence in food to assess human dietary exposure. It was noticed that certain strains of A. niger, a very important industrial microorganism, produce fumonisin B2, whereas others can produce both ochratoxin A and fumonisins, contaminating foods with both types of carcinogenic mycotoxins [137]. Besides STC, ochratoxin B (OTB) and cyclopiazonic acid (CPA) are also present in cereals [17] but have received much less attention. CPA, ochratoxins, and citrinin (CIT) (Group 3, [133]) have been reported to be produced in cereals by several fungus species of two genera, Aspergillus and Penicillium [123,138,139]. It was recognized that the negative effects of the simultaneous presence of aflatoxins and CPA were cumulative in most cases [135,139]. Among ochratoxins, ochratoxin A (OTA) (Group 2B according to IARC) [108] occurs more frequently in cereals and is considered ten times more toxic than OTB, while ochratoxin C (OTC) is less than OTB [138]. Gliotoxín is another mycotoxin produced by several species of Aspergillus (i.e., A. fumigatus) in cereals. It is also associated with the presence of fungi from species of other genera, such as Trichoderma and Penicillium [140,141,142].

Mohammed et al. [107], in their study conducted on sorghum grains reported, the presence of a high number of less-known toxic metabolites; methoxysterigmatocystin, versicolorin C, averufin, 8-O-methylaverufin, kojic acid, 3-nitropropionic acid, asperflavine, asperfuran, asperloxine A, aspochracin, sydonic acid, viomelleinemodin produced by Aspergillus species and mycophenolic acid, mycophenolic acid IV, 1-deoxypebrolide, 7-hydroxypestalotin, barceloneic acid, chanoclavin, cycloaspeptide A, cyclopenin, cyclopenol, dechlorogriseofulvin, dehydrogriseofulvin, F01 1358-A, flavoglaucin, griseofulvin, NP1793, O-methylviridicatin, penicillic acid, quinolactacin A, quinolactacin B, PF 1163, rugulovasine A from Penicillium species.

Fungi of the fungal genus Claviceps, which causes ergot disease in plants, are recognized to produce toxic ergot alkaloids in cereal crops [143]. EAs are produced by the fungi C. purpurea, C. fusiformis, and C. africana of the genus Claviceps. Based on the data collected, EFSA CONTAM Panel [143] suggested monitoring some of the C. purpurea EAs. In addition to ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (mixture of α- and β- isomers), and ergocornine, the biologically inactive corresponding -inine epimers were suggested to be monitored because, at different processing conditions, interconversion could occur.

In addition to all the aforementioned mycotoxins, the presence of other, less common fungus genera metabolites was observed in cereals, such as Abscisic acid, Cytochalasin B, Destruxin A, Monocerin, Preussin, Terphenyllin, Terrein, and Trichodermamide C [107].

Plants could decrease the toxicity of certain mycotoxins by utilizing their enzymatic and/or hormone potential to bind them with specific moieties, transforming them biologically. This modification is realized in plants that have developed a defensive mechanism to protect themselves from the deleterious nature of mycotoxins [144,145]. Plants can metabolize mycotoxins utilizing their metabolism following three phases. Phase I comprises the enzymatic transformation of mycotoxins through oxidation, reduction, or hydrolysis; during phase II, there are observed processes such as sulfatation, glucosidation, and glucuronidation [146]; and during phase III (detoxification), the compounds conjugated to glucose or glutathione are confined/attached to the plant cells [147].

Following a series of processes, the mycotoxins’ structure is changed and stabilized by conjugation with glucoside, acetyl, sulfate, and/or glutathione or other macromolecular substances [148]. These modified forms can be both covalently or not covalently bound and are not only restricted in the kernels of cereals but are proven to occur with mild temperature exposure as well as in thermally treated cereal products [149,150]. Mycotoxins after structure transformation are referred to as ‘’modified”, “masked”, or “conjugated” mycotoxins. The transformed mycotoxins are permanently stored in the plant tissue rather than excreted.

The literature reports many mycotoxins such as deoxynivalenol, zearalenone, fumonisins, nivalenol, fusarenon-X, T-2 toxin, HT-2 toxin, ochratoxin A, and patulin to be metabolized or bind by the plants [147,148]. Besides DON and its biologically transformed form, the deoxynivalenol-3-glucoside (DON-3G) [126,147] has been detected. Moreover, α- and β -zearalenone-14-β-D-glucopyranoside (ZEN-14-Glc) is the plant metabolite of zearalenone (ZEA). Another risk in cereals can arise from cis-ZEN, the isomerized form of ZEN, which can be produced as a result of exposure to daylight and can be found as a natural food contaminant [64] together with its cis-form retaining significant estrogenic activity [151]. Streit et al., have detected the presence of zearalenone-4-sulfate in their samples [126]. Beccaccioli et al., suggested that fumonisins produced by F. verticillioides alter maize lipid metabolism in order to adapt fungal growth to a relatively harmless destructive form and protect themselves [152]. Fumonisins undergo modification in cereal plants conjugating with fatty acid esters forming fatty acid esters of FBs, thus affecting their analytical detection [149]. These mycotoxins that are not screened routinely in foods are not regulated by legislation. EU Commission [153] recommends, as appropriate, analyzing T-2 and HT2 toxins and their masked mycotoxins, particularly the mono- and di-glycosylated conjugates of T-2 and HT-2 toxins.

The modified mycotoxins have raised the concern of scientists because, inside the human metabolism, they are hydrolyzed to their initial much higher toxic form [154]. Although present in food, they are not detected during routine determinations due to their physicochemical behavior that depends on their different chemical structure.

Fusarium species are strongly related to the production of mycotoxins and the contamination of cereals in the field while at the post-harvest stage depending on the storage conditions, species of Aspergillus and Penicillium are predominant [19,155]. The conditions that favor mycotoxin production include moisture (expressed as either relative humidity (RH) or water activity (a_w)), temperature, pH, fungal species, substrate, drought stress, insect damage, and mechanical stress of the plants [156,157]. Therefore, a holistic approach should be implemented involving every stakeholder in the food chain to minimize mycotoxin contamination [158].

Due to the modifications in plants and/or during processing, mycotoxin detection is strongly affected by several factors in the experimental setup, resulting in an altered final compliance assessment. Besides the already legislated mycotoxins, the new mycotoxins, and the masked ones, should not be ignored since combined toxicity may be higher than predicted from individual effects. Thus, the cumulative risk assessment must consider each mycotoxin, its derivatives, and its modified forms present in the same sample [159,160].

Of “new emerging toxins” and “masked mycotoxins”, only a few have been identified as toxicologically relevant for public food safety, however, currently, there are no regulations on most of the toxins contaminating cereals in Europe or other regions of the world. Acute exposure to some of these mycotoxins may not indicate concern for human health, but chronic exposure can represent a concern that needs to be investigated. This fact, together with the high consumption of cereals and their products, makes the detection and study of these mycotoxins a primary necessity for food safety.