Mycotoxins

Mycotoxins are naturally occurring toxins produced by filamentous fungi in many agricultural crops but especially in cereals and most oilseeds both in the field, after harvest, during storage, and later when processed into food, animal feed, and feed concentrates  Mycotoxins occur particularly in regions or countries with climates of high temperature and humidity or where there are poor crop harvesting and storage conditions, which encourage mould growth and mycotoxin development.  Human intake of mycotoxins occurs mainly from plant-based foods and from animal-derived foods such as milk and milk products and certain fermented meat-based products.  Mycotoxicosis is poisoning associated with exposures to mycotoxins and the symptoms depend on the type of mycotoxin, the concentration and length of exposure as well as age, health, and gender of the exposed individual. There is little published information on synergistic effects associated with other factors such as genetics, diet, and interactions with other toxins.

Human reactions to mycotoxins have been recorded for many centuries and the most significant is the disease known as ergotism. In this disease of rye, the fungus Claviceps purpurea grows in the developing seed and eventually replaces it with a hard fungal biomass.  When the rye seeds were harvested and ground into flour, the ergot was also ground up releasing a Pandora’s box of deadly poisons i.e. ergotamine (a vascoconstrictor); ergonovine (cause of spontaneous abortion); ergine and lysergic acid hydroxyethylamide  (causing convulsion).  In Europe in the Middle Ages, rye was the staple diet of the peasantry and records show that there were massive outbreaks of convulsive ergotism in the 9^th and 16^th centuries, and later, with huge levels of suffering. Thus while for many centuries rye was the staple food, it was not only “the staff of life” but also “the sceptre of death”.  It is accepted by most medical historians that throughout the Middle Ages in Europe, the ergot mycotoxins exerted a major role in restricting population expansion and only the reduced dependency on rye cereal as the staple food in the 16^th and 17^th centuries, with the introduction of wheat and potatoes, allowed the steady upward movement in population growth. Other more recent large-scale outbreaks of human mycotoxicoses, involving different mycotoxins have occurred in South East Asia, India, China, Russia, and Africa, but in all of these outbreaks, including ergotism, there has been strong correlation with the consumption of heavily contaminated food sources.

While over 300 mycotoxins have been identified, about 20 have been shown to occur naturally in foods and feeds at significant levels and frequency to be of a food safety concern. The majority of these toxins are produced by fungi of the genera, Aspergillus, Penicillium, and Fusarium.  The most commonly occurring mycotoxins are aflatoxins (B₁, B₂, G₁, G₂, M₁), ochratoxin A, patulin, citrinin, sterigmatocystin, and the fusarium toxins namely fumonisins (B₁, B₂ and B₃), T-2 and HT-2 toxins, zearalenone, nivalenol, and deoxynivalenol (Table 1).

Table 1. Some important toxigenic species of filamentous fungi and related mycotoxins

Aflatoxin is the most regulated of all the mycotoxins regulated worldwide. Aflatoxin B₁ in food and feed, is metabolised to AFM₁ in milk in ruminants and in human breast milk. Mycotoxins are, in general, low molecular weight, non-antigenic fungal secondary metabolites formed through several metabolic pathways, e.g. the polyketide route (aflatoxins), the terpene route (trichothecenes), the amino acid route (aflatoxin), and the tricarboxylic acid route (rubratoxin).  Sterigmatocystin and o-methyl sterigmatocystin are, respectively, the penultimate and ultimate precursors of aflatoxins. Although these precursors are chemically and structurally very similar to aflatoxins, their accumulation differs at the species level for Aspergilli. Aspergillus versicolor and A. nidulans produce sterigmatocystin but do not possess the enzymes necessary for the conversion of the toxin into aflatoxin (JECFA, 2016). Some mycotoxins are formed from a combination of two or more principal pathways. Although trichothecene mycotoxins are produced mainly by the genus, Fusarium (Table 1), other species e.g. Trichoderma, Stachybotrys, Trichothecium, can also form these toxins. The trichothecenes are the most chemically diverse of all the mycotoxins.

Mycotoxins enter the human and animal digestive system by direct or indirect contamination of food commodities and animal feed (Table 2).

Table 2. Probable routes for mycotoxin contamination of foods and feeds

Animal feed plays an important role in the human food chain because of the implications for the composition and quality of products from livestock such as milk, eggs, and meat. Legislation on animal feed is harmonised at European Union level and applies to feed for farmed livestock; the legislation also covers farmed fish and others including pets and horses. The European Food Safety Authority (EFSA) have carried out risk assessment on a number of mycotoxins (AFB₁, deoxynivalenol, zearalenone, ochratoxin A, fumonisins, T-2 and HT-2) that are considered to pose health risks to human and animals.

Mycotoxin contamination of food and feeds is a world-wide problem.  The Food and Agriculture Organisation (FAO) estimated that 25 per cent of the world’s food crops are significantly contaminated with mycotoxins.  Currently, more than 100 countries have regulations regarding levels of mycotoxins in the food and feed industry. Direct economic losses resulting from mycotoxin-contaminated agricultural crops can be measured in reduced crop yields and lower quality, reduced animal performance and reproductive capabilities, and increased disease incidence.  Developed countries are less at risk from mycotoxins than developing countries since they are significantly protected by the high standards of the major food suppliers and retailers, and the regulatory controls which deter the importation of seriously contaminated material. Globalisation of food trade with regard to mycotoxins may have contributed to two major issues in the developing world.  First, stringent mycotoxin standards on exported food crops mean that these countries are likely to export their best-quality foods while keeping contaminated foods domestically, which inadvertently results in higher risk of mycotoxin exposure in those nations.  Second, a large portion of even the best quality foods produced in the developing world for export is rejected for not meeting the stringent standards, resulting in millions of dollars in losses (Wu, 2004; Wu and Gudu, 2012).

Mycotoxicology is a rapidly developing field; thus this, like most Information Statements can at best be a “benchmark”, trying to present the position at a particular point in time.  The EU works toward harmonisation of mycotoxin standards and in the UK, the Food Standards Agency’s (FSA) strategy can be divided into three major themes: (i) maintain current awareness of levels of mycotoxin contamination of foods in general and of UK in particular (UK/FSA, 2003, 2004, 2005a,b,c), (ii) identify factors contributing to the occurrence of mycotoxins and thereby provide information necessary to manage the risk of mycotoxin formation, and (iii) provide stakeholders with advice/tools on how best to manage the problem of mycotoxin contamination.  To enable stakeholders to participate in consultations on mycotoxins and other biologically-derived toxins in foods, a list of interested parties exists.  To fulfil FSA’s strategy, projects on mycotoxins fall into four broad categories:  (i) exposure assessment based on surveys of different mycotoxins in a wide range of foods; (ii) development and improvement of analytical methods; (iii) impact of agronomy and storage – a number of projects have been initiated on the impact of agronomy, the development of tools for good management of the problem, and analytical methods to detect mycotoxigenic fungi early in the toxin synthesis cycle; and (iv) impact on sampling as a key issue in making accurate determinations of mycotoxins.

The European Mycotoxin Awareness Network (EMAN, 2012, 2014) provides high quality scientific information and news about mycotoxins to industry, consumers, legislators, and the scientific community.  Another useful document is the Mycotoxins Factsheet (4th edition) published by the Joint Research Centre of the European Commission (EC, 2011). It gives details and references to the nature of mycotoxins, their occurrence and toxicities, details of useful links to other organizations, as well as having sections on training, proficiency testing, reference materials and methods of analysis.

Animals can demonstrate variable susceptibilities to mycotoxins, depending on genetic factors (species, breed, strain), physiological factors (age, nutrition, etc) and environmental factors (climatic conditions, husbandry and management).  In most developed countries the natural contamination levels of mycotoxins in animal feeds do not normally occur at levels that can cause acute or overt mycotoxicosis, e.g. hepatitis, haemorrhage, nephritis and necrosis of oral and eneteric epithelia, and even death.  Rather, the observed levels induce symptoms of chronic primary mycotoxicosis and immune suppression.  It is often difficult to observe or diagnose such manifestations of disease but certainly they represent the most common forms of mycotoxicosis in farm animals, e.g. reduced productivity, growth and reproductive efficiency, reduced feed conversion efficiency, milk yields or egg production.

As a result of their diverse chemical structures and differing physical and biochemical properties, mycotoxins exhibit a wide array of biological effects on mammalian systems and individual mycotoxins can be genotoxic, mutagenic, carcinogenic, embryotoxic, teratogenic or oestrogenic. Although acute aflatoxicosis in humans is rare, several outbreaks have been reported.  In 1967, 26 people in a Taiwanese farming community became ill with suspected food poisoning; 19 were children and 3 of them died. No post-mortem was performed but rice from the affected households contained 200 µg aflatoxin B₁/kg which was probably responsible for the outbreak.  In 1974, an outbreak of hepatitis in India affected 400 people resulting in 100 deaths. The deaths were caused by aflatoxins found in corn heavily contaminated with A. flavus containing up to 15mg of aflatoxins per kg (Montville and Matthews, 2008).  In 2004, one of the largest aflatoxicosis outbreak occurred in rural Kenya, resulting in 317 cases and 125 deaths.  Contaminated maize was responsible for the outbreak and officials found aflatoxin B₁ levels as high as 4,400 parts per billion (ppb), 220 times higher than the Kenyan regulatory limit for food (Lewis et al, 2005).

Throughout the world there are many advisory bodies concerned with food safety, including the World Health Organisation (WHO), Codex Alimentarius Joint Expert Committee for Food Additives and Contaminants (JECFA), and the European Food Safety Authority (EFSA). They regularly assess the risk from mycotoxins and advise on controls to reduce consumer exposure.   

In the UK, the Food Standards Agency (FSA) is responsible for ensuring mycotoxin safety (UK/FSA, 2002).  The UK legislation on mycotoxins is harmonised with the European Union. In the EU, regulatory limits for mycotoxins permitted in food and animal feed are set by a range of directives and Commission regulations.

Table 3.  Regulatory limit of aflatoxins in EU and some countries

Table 3 shows regulatory limits of aflatoxins in the EU and other countries. Commission Regulation (EC) No. 466/2001 sets maximum permitted levels for mycotoxins in food and feed and new limits are introduced by amendments to this legislation.  For example, EC Regulation No. 1881/2006 set limits for aflatoxins.  The current EU limits for mycotoxins are as follows:

        •   Maximum limits for oilseeds 2 ppb (µg/kg) for aflatoxin B₁ and 4 ppb for total aflatoxin; EFSA was asked to deliver a scientific opinion regarding the effect on public health of a possible increase of the maximum level (ML) for aflatoxin total from 4 to 10µg/kg in peanuts and processed products thereof. EFSA assessed the toxicology of aflatoxins in 2007 and the most recent evaluation was carried out by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2016. Given the short deadline of this request, no new toxicological evaluation was performed by the CONTAM Panel, the CONTAM Panel therefore decided to use the cancer potencies estimated by JECFA (2016) for risk characterisation. The cancer potencies for an AFB₁ exposure of 1 ng/kg body weight (bw) per day was established.

• Oilseeds for further processing with limits of 8 ppb and 15 ppb for aflatoxin B₁ and total aflatoxin respectively (these limits do not apply to oilseeds including groundnuts for crushing for refined vegetable oil production);
• 0.1 ppb aflatoxin B₁ in baby food and 0.05 ppb aflatoxin M₁ in milk.  The limit for aflatoxins in rice for direct consumption is 2 ppb for aflatoxin B₁ and 4 ppb for total aflatoxin.
• The maximum limit for OTA is 10 ppb in dried vine fruits (currants, raisins, sultanas) and in soluble coffee, 5 ppb in roasted coffee, 3 ppb in all products from cereals intended for direct human consumption, 2 ppb for wines, grape juice, grape must and grape must concentrate, and 0.5 ppb for baby foods processed cereal-based foods for infants and young children. 

The maximum levels for infants and young children according to Directive 2006/125/EC and Directive 2006/141/EC are:

• Aflatoxin B₁: 0.10 µg/kg
• Aflatoxin M₁: 0.025 µg/kg;
• Ochratoxin A: 0.5 µg/kg and the same maximum level for dietary foods for specific medical purposes, specifically for infants;
• Patulin: 10 µg/kg;
• Deoxynivalenol: 200 µg/kg;
• Zearalenone: 20 µg/kg and the same level for maize-based formulae for infants and young children;
• Fumonisins: 200 µg/kg for maize-based formulae for infants and young children.

Since EC regulation No. 1881/2006 was issued there have been several amendments to the permitted levels of various mycotoxins in miscellaneous food products. These amendments are listed in more detail below:

Commission Regulation (EU) No. 105/2010.

Setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A.

Commission Regulation (EU) No. 165/2010.

Setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins.

Commission regulation (EU) No. 594/2012.

Amending regulation (EC) No. 1881/2006 as regards the maximum levels of the contaminants ochratoxin A, non dioxin-like PCBs and melamine in foodstuffs.

Agricultural intervention to reduce mycotoxins is considered at pre- and post- harvest levels. Mycotoxins primarily enter into the human and animal food chains through agricultural products, mainly cereals, nuts and oilseeds or from products derived from them. Various potential mitigation approaches for the prevention and control of mycotoxins have been proposed and these include crop rotation, mixed crops, harvesting time, farminig techniques, plant resistance breeding, detoxifying agents (enzymes and binders) and genetic modification.

Mycotoxin contamination of seeds is mostly caused by inadequate storage conditions of harvested crops, however pre-harvest contamination of the seeds can also occur especially with Fusarium spp. producing zearalenone, trichothecenes, and fumonisins, while other fungal contamination can produce ergot alkaloids, tremorgen mycotoxins, and aflatoxins. Pre-harvest interventions include production of genetically enhanced resistant crop, good agronomic practices, harvesting crop at the optimum stage of development, biocontrol methods (e.g. use of atoxigenic A. flavus), chemical methods.

Post-harvest contamination by mycotoxigenic fungi usually occurs during storage and transportation and is normally caused by improper drying or re-wetting of the crop from condensation or rain.  Post-harvest control measures include physical methods e.g. improved storage conditions, transportation, sorting and chemical methods such as ammoniation.  Correct drying of seeds followed by efficient storage and monitoring is an effective post-harvest management technique to prevent mould growth.  Other techniques are used based on colour or visual appearance of decay or damage to separate out contaminated seeds, particularly oilseeds.  The possibility for sorting and physical treatment in case of non-compliance is limited to the cases of consignment, not complying with EU legislation but containing levels below the worldwide permitted level established for AFB₁ and total aflatoxin.  Nuts labelled for direct human consumption found with levels of total aflatoxins above those for direct human consumption or as an ingredient and below the worldwide highest level established for AFB₁ (20 µg/kg) and/or for aflatoxin total (35 µg/kg), can be re-labelled and sorted or undergo a physical treatment to reduce aflatoxin content under official control.  This requires that the transfer to the processing plant, the process and the sampling and analysis have to be performed under the official control of the competent authority.  After sorting and/or physical treatment, an official sampling and analysis must be performed to demonstrate that the nuts are compliant with the limits set for direct human consumption or use as an ingredient.  Similarly, nuts labelled for further processing found with levels above those set in legislation but below the worldwide highest level established for AFB₁ (20 µg/kg) and/or for aflatoxin total (35 µg/kg)  (FAO, 2003), can be re-labelled and also be further sorted or undergo a physical treatment under official control as above.  However, Article 19 (2) (a) of Regulation (EC) No. 882/2004 provides that if the official control indicates that a consignment is injurious to human or animal health or unsafe, the competent authority shall place the consignment in question under official detention pending its destruction or any other appropriate measure necessary to protect human and animal health.  The appropriate measures could be a) destruction of the goods under official control and the costs are borne by the food business operator, b) use under official control for industrial purposes (non feed /non food uses), c) use under official control for oil extraction provided the resulting oil is refined to reduce any aflatoxin which may be present to acceptable levels and use under official control of the residual cake/meal for animal feeding after an appropriate treatment (detoxification) d) re-dispatch to the country of origin. 

Given that, worldwide, the highest level established for AFB₁ is 20 µg/kg and for aflatoxin total 35 µg/kg these levels are considered as being upper limits above which consignments must be rejected.  These levels do also apply to other foodstuffs imported into the EU e.g. spices, melon seeds, sesame seeds.

The Hazard Analysis Critical Control Point (HACCP) is an internationally recognised food safety management system, however, application of HACCP principles to mycotoxin control is a relatively new approach, but is based on some very sound reasoning according to the European Mycotoxin Awareness Network (EMAN, 2012). In 1993, the CODEX published guidelines on application of HACCP. Now there are some very good reasons for the application of HACCP to prevent mycotoxin formation in the first place.

Most foods not of animal origin can be imported into the EU without any further controls except for the fact they must comply with all the EU regulations appropriate to the food. However when a specific food from a specific country is shown to be exhibiting excess levels of contaminants, including aflatoxin, then additional controls are applied before it is given a Common Entry Document and allowed into the EU.

These additional controls are defined in Commission Regulation (EC) No. 669/2009 which further strengthens the requirements of No. 882/2004 as regards the increased level of official controls on importation of certain food and feed of non-animal origin from countries outside of the EU. 

The system is comprehensive in that only certain ports with sufficient facilities to sample and test the consignments are designated as points of entry. The annex to this regulation is revised every 3 months (at the time of writing No. 1295/2014 of 4th December 2014 is in force). Only when a food has undergone additional checks and the contaminant found to be below the EU limits is the consignment provided considered to be within compliance.

In addition foods potentially containing aflatoxins are also controlled by Regulation No. 884/2014. This regulation is made under Article 53 of the General Food Regulation No. 178/2002 as a safeguard measure to protect the health of the EU citizens. In this case a similar system to the above is in place although with more stringent requirements; foods identified under this regulation gain entry to the EU via through designated ports of import. Special conditions governing the import of certain feed and food from certain third countries due to contamination risk by aflatoxins and repealing Regulation (EC) No 1152/2009 can be found in regulation EC 884/2014.

The additional controls are that each consignment has to be accompanied by a certificate of analysis performed by the competent authority of the exporting country together with a completed health certificate. The analysis has to be carried out in accordance with Regulation No. 401/2006.

Chemical preservatives such as organic acids (sorbic, propionic, acetic, benzoic) have been used to restrict the growth of mycotoxigenic fungi.  Reduction in the levels of aflatoxins has also been achieved with the use of ammonia either in solution or in gaseous form.  Studies have been reported on the use of phyllosilicate clays (hydrated sodium calcium alumino-silicate) to chemi-sorb aflatoxin in aqueous suspensions including milk.  Clinical and/or dietary intervention for aflatoxins include binding agents (chlorophyll, chlorophyllin), phase II enzyme inducers (sulphoraphane found in cruciferous and other vegetables, triterpenoids, Oltipraz), inflammation reducers (NSAIDs, green tea poly phenols) Hepatitis B vaccine which blocks insertional mutagenesis (Galvano et al, 2001, Groopman et al, 2008).

The influence of climate on the distribution of mycotoxins in food crops and feeds is now receiving some attention. Compounds in cereals such as nivalenol, ergot alkaloids, aflatoxins and fumonisins in maize are now appearing in areas and locations that had not been seen before due to changes in climatic patterns. These challenges require further surveillances and more refined exposure assessments.

The awareness of the toxic role of mycotoxins in the human diet and animal feeds would be greatly improved by the wider teaching and dissemination of mycotoxicology in courses in food science, analytical chemistry, food safety, nutrition, microbiology and environmental health.