The probability of finding only one mycotoxin contaminating raw materials or feed is extremely low. Worldwide, the incidence of co-contamination is high. The global monitoring reported that 72% of the samples of feed and raw materials were contaminated with more than one mycotoxin [27]. The same authors [28] detected 83 samples of feed and raw materials contaminated with 7 to 69 mycotoxins per sample, having analysed 169 different compounds.

The occurrence of mycotoxin co-contamination in Europe tends to follow the same pattern. Several studies have revealed the simultaneous presence of mycotoxin co-contamination in samples from European countries, finding a high percentage of feed samples contaminated with trichothecenes (DON, acetyldeoxynivalenol (AcDon), T2, and HT2) and FUM, as well as with ZEN [29,30,31,32,33,34,35,36]. In Germany, [37] maize was found simultaneously contaminated with 14 Fusarium mycotoxins, such as DON and its acetylated forms, ZEN, Moniliformin (MON), Beauvericin (BEA), Nivalenol (NIV), Eniantins (ENNs), FBs, and HT-2 Toxin. Recently, data have been published regarding fodder mycotoxin co-contamination, which showed all silage samples positive for at least one mycotoxin, and 61% of samples contained five or more mycotoxins simultaneously. According to [5], an average of 30 mycotoxins and their metabolites per sample were found and 87% of the samples have 10 or more mycotoxins or metabolites.

The most frequently detected toxins were DON, NIV, ZEA, enniatins, and BEA, although the levels of these toxins were relatively low [38]. Co-contamination is a great concern, as it may exert adverse effects on animals due to the additive/synergistic interactions of the mycotoxins, the complexity of which varies according to the animal species, the level and type of mycotoxin contamination, the toxicity of the compound ingested, body weight, age and animal physiological condition, compound action mechanism, the presence of other mycotoxins, and the length of exposure. In general, in most cases, there are additive or synergic effects [39,40]. Many authors have highlighted this additivity, synergy, or enhancement. Although most results reveal the additive or synergic effects of mycotoxins, it should be noted that antagonistic effects could also be seen [41,42,43]. The co-occurrence between regulated, modified, and emerging toxins and their interactions are still little known.

The European Food Safety Authority (EFSA) refers to “modified mycotoxins” as all forms that have been structurally modified in relation to their “parental compound” or the free mycotoxins [44,45]. Plants and certain microorganisms, such as yeasts, filamentous fungi, and bacteria, are capable of transforming mycotoxins into conjugated forms (biologically modified mycotoxins), reducing their toxicity [46]. In fact, plant metabolites have been identified for DON, NIV, fusarenon-X, T-2 toxin, HT-2 toxin, ZEN, OTA, destruxins, and fusaric acid, while modified fumonisins have been detected in cereal commodities, such as corn, wheat, and barley. Toxicological data on modified mycotoxins, including those of processing origin (chemically modified mycotoxins), are still limited [47,48]. However, recent advances in modified mycotoxin occurrence and toxicity have suggested that mycotoxins conjugates have a reduced toxicity potential due to the lower absorption in the gastrointestinal tract [6]. These modified mycotoxins differ in their structure, solubility, polarity, and molecular mass; furthermore, they can be formed during the processing of foods from contaminated raw materials and can be reconverted to the original toxin during the human and animal metabolism [49,50,51,52]. Free mycotoxins co-occur with modified mycotoxins [49,50], and the modified mycotoxin concentration exceeds the level of free form. The possibility of modified mycotoxin conversion to its free form may involve risks for human and animal health. The conversion of modified to free form can result in increased bioavailability of mycotoxin [53,54]. It is necessary to set up and validate affordable methods for the detection of modified mycotoxin as well as to study their stability during the processing of feeds, their outcome in the animal digestive system, and their toxicokinetic and toxicodynamic properties [55]. In addition, the knowledge of their formation process and their structure and molecular mass may resolve analytical and technological gaps [56,57].

Emerging mycotoxins became a major issue due to their high occurrence in cereals, feed, and food commodities [58,59,60]. They are lesser-known or newer forms of mycotoxins that, by definition, are neither routinely determined nor legislatively regulated. The most prevalent emerging mycotoxins are Fusarium toxins, such as ENNs, BEA, MON, fusaproliferin (FP), fusidic acid (FA), culmorin (CUL), and butenolide (BUT) [61,62]. The presence of emerging mycotoxins in feed and food commodities, such as Aspergillus toxins (sterigmatocystin (STE) and emodin (EMO)), Penicillium toxins (mycophenolic acid (MPA)), Alternaria toxins (alternariol (AOH), monomethyl alternariol ether (AME), altenuene (ALT), altertoxin (ATX), and tenuazonic acid (TeA)), ergot alkaloids, and citrinin, is equally common [58,61,62]. Research indicates that these emerging toxins are rapidly becoming prevalent co-contaminants in feed and food such as grains (corn, wheat, barley, etc.) showing greater occurrence when other Fusarium mycotoxins are present. In an extensive review on co-occurrence of regulated, modified, and emerging mycotoxins in finished feed and maize, emerging mycotoxins, such as ENNs, MON, and BEA, were found to be ubiquitous in analysed samples [48]. According to [5], the raw materials most frequently presenting emerging toxins are maize and animal compound feed. In particular, corn presented 93% of MON and 83% of Aurofusarium, while the finished feed presented 97% and 93% for Bauvericin, and Enniantin B and B1.

However, these data have to be considered with caution. Indeed, although thanks to certain modern analytical methods we are able to detect hundreds of “new” different fungal metabolites in a variety of food and feed samples, we have to consider that many of these compounds are irrelevant in terms of food and feed safety [62]. Their limited risk, however, can change in the future: climate change, commodities origin, and processing as well as several others environmental factors can alter both toxicity and occurrence of these compounds. Thus, for an adequate risk assessment and in order to prevent future food and feed safety crisis, it is also important to start collecting information (occurrence, acute vs. chronic toxicity, distribution, commodities, etc.) about these fungal metabolites.

3. Mycotoxin Risk in the Feed Supply Chain: Need for a Management Plan

HACCP for Feed Mycotoxin Contamination

In an integrated management system, prevention is key; the risks related to mycotoxin hazards should be minimized at each production phase. Mycotoxin contamination is best dealt with in the pre-harvest phase, but when contamination occurs, the related dangers can be handled through post-harvest techniques, applying the corrective actions reported in the HACCP plan.

The HACCP system is designed to decrease the risk of feed safety exposure by identifying the hazards and by monitoring controls [74,75]; it provides a scientific quality control methodology. In addition, HACCP system can be designed and used in combination with other quality systems. The most crucial source of mycotoxin intake can be found in agro-commodities (FAO/WHO 2014); for this reason, an effective preventative strategy could be represented by an approach spanning the entirety of the commodity supply chain. The application of an HACCP system aimed at improving food safety, from the fields to farm animals, can control mycotoxin contamination of raw material [76,77]. To ensure that the product has acceptable mycotoxin levels, an integrated HACCP approach in the pre-harvest stage can be used. Effective integrated management programmes cover agro-products mycotoxin prevention/detoxification as well as routine surveillance, updating national and international regulatory measures, information, education, and communication activities.

The key points for an effective HACCP plan are hazard identification and analysis and the record keeping procedures [78,79,80]. The text “Manual on the application of the HACCP system in mycotoxin prevention and control” was published [77] and is a reference for the HACPP plan drafting, but there is a lack of HACCP plans specific for the feed chain in this text. The most crucial points of HACCP for mycotoxin control in the feed chain are reported in Figure 3.

Figure 3. Crucial HACCP tasks for mycotoxin control in the feed chain (modified from FAO, 2001).

In order to deal with the preliminary stages in commodity flow, an effective team, made of microbiologist; mycologist; and experts in farming, storage, distribution, and trading should be formed. In a holistic approach to mycotoxin control, there is a need for a commodity flow diagram (CFD), which includes all aspects of primary production, drying, storage, transport, and final processing steps (Figure 1). The modern commodity supply chain complicates the creation of this document due to the fact that products move between several owner groups (farmers, traders, transporter, and processors). The commodity and the final product type, climatic zone, and production country will affect the drafting of this report.

Control parameters for the manufacturing of mycotoxin susceptible commodities involve harvesting time, temperature, storage and transportation moisture level, selection prior to processing of agricultural feedstuffs, decontamination environments, and final product storage and transportation [81].

An HACCP plan to manage mycotoxin hazard in feed chain would guide experts at every stage of the supply chain, for which a guide model for the feed chain is reported in Table 2. It is paramount to define critical the control points, hazards, control limits, preventive actions, monitoring strategies (measurable parameters, methods, control frequency, and responsible figure), corrective actions, records, and finally the verifications for each feed chain step. These parameters are certainly specific for each operation and depend on the kind of risk involved.

Table 2. Managing mycotoxins in the feed chain: guide model of a HACCP plan.

CCP	Hazard	Critical Limit	Preventive Action	Monitoring				Corrective Action	Records	Verification
				Parameter	Method	Frequency	Responsable
Pre-harvest	Low soil moisture/plant stress	Lower limit of critical Aw	Irrigate	Soil moisture value		Weekly on Tuesday morning	Agronomist	Supplementary irrigation	Soil moisture	State of plants
	Insufficient soil nutrients	N,P,K applications	Fertilise	Fertilizer application		As recommended for hybrid	Agronomist	Additional fertilizer	Amounts and type of fertilize	State of plants
	Insect attack	Insect population within accettable limits	Integrated pest management plan	Visual inspection and sample		Weekly	Agronomist	Apply pestice in accordance with IPM plan	Results of the monitoring	State of plants
Harvest	Damage kernel	Moisture content <14%	Harvest when kernels are dry	Measure grain moisture		Prior to harvest	Farmer/ Agro-mechanical	Postpone harvest till kernels properly dried	Grain moisture	Visual inspection/analyses of raw materials
Storage	Excessive moisture content	Moisture content <14%	Do not store until kernels dry	Measure grain moisture		Immediately prior to storage	Commodity quality assistant	Dry mechanically	Grain moisture	Analysis
	Insect attack	Inspection protocols show no evidence of insect or rodent infestation	IPM plan	Visual inspection		Weekly	Mill operators	Follow IPM plan for pest control method	Visual inspection	Analysis results
	High humidity and temperature	Temperature and humidity within limits recommended in industry literature	Aerate grain to control temperature and humidity	Measure humidity, temperature and airflow		Daily during storage	Mill operators	Adjust aeration- time, or airflow to achieve desidered temperature and humidity	Humidity, temeperature and airflow	Authomatic monitoring systems
Feed mill	Increase of myctoxin levels in mixer phase	mixer cleaning mycotoxin levels	Controlling mixer cleaning and way of frequency	ppb	ELISA and UV	Before every mixing process	Feed quality assistant	Changing the time and method of cleaning	Cleaning and disinfection register form	Cleaning, analysis results
	Increased myctoxin levels in Cooler	The heat of feed should be at most 5 °C more than environment heat	Increasing empting time of the cooler; decreasing the capacity of pellet; controlling the heat levels of the cooler	°C	Thermometer	Daily	Foremen	Mixing with cold feed, keeping a backup cooler	Cooler heat follow form	Measuring heat during cooling process
Livestock production	Increase of mycotoxins levels	Temperature, cleaning	Feeding silo cleaning, climate and insect/rodent control	°C, ppb	Thermometer, ELISA	Before every entering livestock, daily, weekly	Farmers, Livestock keeper	Dietary manipulation, on-farm management strategies, use of binding agents	Live activity form	Cleaning and disinfection results, analises of animal products