There is a growing awareness of the serious health risks posed by specific mycotoxin contaminations of grain-based food and feed [1
]. The grain industry needs reliable information on the prevalence of the mycotoxins to develop sustainable mycotoxin management along the food and feed chains. Mycotoxins are toxic secondary fungal metabolites produced by specific fungal species that have a negative impact on global food safety and security, and may be produced when conditions are favourable in the fields during production, during storage and during grain transportation [3
]. The supply chain responsible for food safety is challenged by the fact that mycotoxin contamination is mostly found in localized “hot-spots” in a consignment [4
Survey results of mycotoxin contamination in the main staple foods, such as maize, wheat, rice, sorghum, soybeans and cassava, together with the related final food and feed products, are critically important for developing sound practices to reduce mycotoxin contamination along the value chain. Only then can these practices be effectively monitored and improved. The movement of food and feed products across the world, including mycotoxin-contaminated products, highlights the importance of worldwide as well as country- and region-specific surveys on mycotoxin occurrence. The results of such surveys have confirmed that the presence and concentration levels of specific mycotoxins vary among the grain types, production regions and year of production [7
Nowadays, mycotoxins are grouped into “regulated”, “masked” and “emerging” mycotoxins [8
], with the list of “regulated” mycotoxins and their maximum allowable levels written in legislations, although not harmonized worldwide. The “regulated” mycotoxins associated with grains generally accepted to have unfavorable health effects in humans and animals include aflatoxins (specifically aflatoxin B1
), a known carcinogen), fumonisins (fumonisin B1
), ochratoxin A (OTA), trichothecenes type A (T2-toxin, HT-2 toxin) and type B (deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15-ADON), nivalenol) and zearalenone (ZON) [8
]. There is not yet enough data on the occurrence and toxicity of the masked and emerging mycotoxins available to date, and therefore no regulations exist for these compounds.
In South Africa (SA), maize is the main grain produced and consumed as a staple food, followed by wheat [11
]. A surplus of maize is produced annually by SA commercial maize producers; the 10-year (2006–2007 to 2016–2017) average maize production in South Africa was 11,096,676 t, with the exception of the 2015–2016 production season, when a severe drought was experienced [12
]. In the 2016–2017 production season, an all-time record crop of 9,916,000 t white maize and 6,904,000 t yellow maize were delivered by the commercial maize growers [12
]. White maize is a major cereal in South Africa, consumed fresh or processed into milled, cooked or fermented products, and yellow maize is produced mainly for the animal feed industry [12
Although the 10-year average wheat production in SA is 1,826,800 t, SA is a net importer of wheat; during the 2017–2018 season 1,677,162 t of wheat were imported [13
]. Wheat contributed 79% to the total winter cereal crop production in the Western Cape winter rainfall region, and was also planted in the summer rainfall regions and irrigation areas of various regions (Figure 1
) was first reported in South Africa in homegrown maize in January 1990 by Sydenham et al. (1990) [14
] in the Transkei, Eastern Cape, where a high incidence of human esophageal cancer was reported [15
]. These results led to ongoing research projects in that area of South Africa, with emphasis on the occurrence of fumonisins (FUM) [16
], exposure and improvement of storage and cleaning practices of the rural population [17
]. However, the homegrown maize produced by subsistence farmers contributes to less than 1% of the maize produced annually in SA [12
]. Commercial maize is mainly produced in seven of the nine provinces in SA (Figure 1
), with three provinces—Free State (44%), Mpumalanga (20%) and North West (19%)—contributing approximately 83% of the annual maize production [12
), fumonisin B1
) and deoxynivalenol (DON) were reported in maize in the SA food mycotoxin studies (from 2007–2016) reviewed by Misihairabgwi et al. (2017) [20
]. The reviewed studies focused mainly on small numbers of maize samples collected either in small production areas of subsistence farming [18
] or at research sites in South Africa [22
]. In a 2008–2009 maize cultivar study, DON was reported as the most frequently observed mycotoxin, together with 15-acetyl-deoxynivalenol (15-ADON), FB1
and zearalenone (ZON) [22
]. These multi-mycotoxin results were performed in Germany. Only one study in 2011 focused on commercial maize collected at two feed companies (40 samples) [23
]. The only wheat included in the review were DON results of 23 wheat flour samples, analyzed in 2006 [20
Aflatoxins, fumonisins, deoxynivalenol, ochratoxin A and zearalenone were reported in another multi-mycotoxin survey of commercial compound feeds collected at South African feed mills in 2010–2011 (92 samples) [24
]. The more-recent annual Biomin surveys, conducted globally, reported mycotoxin results in maize collected in South Africa from 2014 to 2017 [10
], and compound feed samples (n = 74) from 2012 to 2015 [8
]. A high occurrence of deoxynivalenol, fumonisin and zearalenone, but also aflatoxins and OTA, were reported in the maize samples [10
]. With approximately 4.5 million t maize (10-year average) processed in South Africa for the animal industry annually [12
], these results clearly may not necessarily represent the status of mycotoxins in animal feed prepared with only SA-produced maize.
It became important for the wheat and maize supply chain in South Africa to gain insight into the occurrence and prevalence of at least all the “regulated” mycotoxins in SA commercially produced crops. The industries realized that multi-mycotoxin results of a representative selection of the annual crop quality survey samples would give a complete overview of the multi-mycotoxin prevalence in SA maize and wheat, post-harvest before storage. For these surveys, approximately 1000 maize samples and 350 wheat samples were collected annually when the producers delivered their harvest to the grain storage facilities [12
When this study was commenced in 2014, only aflatoxin B1
was regulated (since 2004) in SA, in various commodities for human consumption [25
]. For farm feeds, maximum levels are written in SA legislation for aflatoxin B1
, deoxynivalenol, fumonisin B1
, ochratoxin A and zearalenone [26
]. Because only AFB1
was regulated for human consumption of grains, aflatoxin testing was often presented as the only necessary mycotoxin test to approve grains fit for human consumption. For this monitoring study, the decision was made to include at least the mycotoxins with maximum levels listed for grains in the Codex general standard for contaminants and toxins in food and feed [27
], and those with regulated or guidance values in the European Union [28
]. As pointed out by Stroka et al. (2016) [30
], social considerations may both help and hinder the compound selection process. The compound 15-acetyl deoxynivalenol (15-ADON), considered as a co-contaminant with DON [31
], was of interest to local research groups [22
A total of 13 mycotoxins were identified to be monitored in the SA maize and wheat: aflatoxins (B1, B2, G1, G2) DON and 15-ADON, fumonisins (B1, B2, B3), OTA, T2-toxin, H-2-toxin and ZON. Fortunately, by that time, the development of LC-MS/MS instrumentation provided a robust routine means for the simultaneous analysis of organic compounds at ng levels in complex food commodities. This allowed commercial analytical laboratories to develop LC-MS/MS methods for the analysis of multi-mycotoxins in different food commodities. The establishment of a test facility in SA conducting multi-mycotoxin analyses was necessary to obtain a long-term, representative picture of the multi-mycotoxin status of maize and wheat produced in SA.
The extraction methods used for the determination of single compounds or groups of toxins with HPLC techniques were initially considered for the development of multi-mycotoxin extraction methods in grains, cereals and feed products [32
]. It was soon realized that the choice of solvents to be used for extraction was complicated by the wide range of polarities, the concentration ranges of the mycotoxins and the complex product matrices. Marschik et al. (2013) [34
] compared the yield of fumonisins in various naturally contaminated, unprocessed and processed maize matrices with five different multi-mycotoxin-based extractants, and concluded that the extraction solvent mixture—methanol/acetonitrile/water (1:1:2) with a 15-min extended extraction time—was the most appropriate for fumonisins in maize. Our laboratory compared three extraction methods: (i) methanol/water (4:1, v/v) extraction (1 min blend); (ii) double-extraction first with acetonitrile/water/formic acid (80:19.9:0.1, v/v/v) (60 min shake) followed by acetonitrile/water/formic acid (20:79.9:0.1, v/v/v) (30 min shake) [35
]; and (iii) methanol/acetonitrile/water (1:1:2) (1 min blend, 15 min shake) [34
]. It was concluded that complete extraction of FB1
and DON in highly contaminated maize was achieved with longer extraction times and an increased amount of water added to the extraction solvent mixture. The mycotoxin results of the methanol/acetonitrile/water (1:1:2) and the double extraction method compared well, but a 90-min total extraction time in a routine laboratory conducting large numbers of samples in the main grain season is not justifiable if it is not of the utmost necessity to report correct results.
Purification of the extracts with clean-up columns is essential for HPLC analysis, but these clean-up procedures were not applicable for the simultaneous analyses of different groups of mycotoxins. Most of the LC-MS/MS multi-mycotoxin methods followed the so-called “dilute-and-shoot” approach, because with the high sensitivity of the LC-MS/MS it is possible to inject diluted sample extracts with a 20-fold smaller sample load (compared to HPLC analyses) and still reach the required limit of quantitation defined in Section 5
For the calibration of the LC-MS/MS, matrix-matched standards and stable isotope-labelled standards were considered for the accurate quantification of mycotoxins in various commodities [35
]. The unavailability and costs of all the isotope-labelled compounds and the challenges to import them into SA were carefully considered. To keep the monitoring costs as low as possible without compromising the quality of the test results, a decision was made to use matrix-matched standards for the method validation, as described in Section 5
The comprehensive data sets of 13 mycotoxins reported in this four-year monitoring project of SA-produced maize and wheat collected after harvest from the 2013–2104 to 2016–2017 maize production seasons and the 2014–2015 to 2017–2018 wheat production seasons are presented in this study. In total over four years, 1400 maize and 160 wheat samples were analyzed in the test facility that was successfully established in SA to routinely conduct multi-mycotoxin analyses on grains and related grain-based products and feeds. The results cover all the commercial maize and wheat production areas in SA.
This is the first comprehensive study that reports on the multi-mycotoxin occurrence in commercially produced wheat, white maize and yellow maize in South Africa. The study was conducted over four consecutive seasons in all the production provinces in South Africa by collecting samples at grain storage facilities when delivered by producers. The establishment of an accredited test facility for the multi-mycotoxin analyses, with a capacity to analyze 350 maize and 40 wheat samples every year at the end of the harvest seasons, enabled the present study, and will be of utmost value for future related projects. A validated LC-MS/MS method (ISO 17025 accredited) with the aim of providing an affordable, fast and sustainable commercial service in South Africa, was successfully developed for the analysis of the 13 main mycotoxins in grain. The accuracy of the method was confirmed with international proficiency tests and interlaboratory comparisons requested by European companies involved in the grain industry in SA.
The results showed for the first time:
the presence of only deoxynivalenol (at low concentrations) in SA wheat;
the absence of aflatoxin B1 in maize produced commercially in SA in all the regions;
the concentrations, regional variation and seasonal trends of deoxynivalenol, fumonisins and zearalenone in white and yellow maize;
the low prevalence of fumonisin-contaminated maize above the 4000 µg/kg regulated value for unprocessed maize for human consumption;
an increase in the percentage of white maize produced with DON above the 2000 µg/kg regulated maximum level.
The outcome of the study underwrites various aspects of the Mycotoxin Charter launched in 2018 as part of the European Union funded project MycoKey [40
]. The safety of the domestic consumers of maize and wheat products was improved. Based on the results of the first two seasons, SA expanded the regulated mycotoxins in grain to include maximum allowable levels for fumonisins in maize and DON in cereal grains intended for further processing, as well as maximum levels in related processed food products ready for human consumption [36
]. The enforcement of this amended regulation was possible only after the successful establishment of the analytical test facility in SA.
The maize and wheat producers in South Africa are now well positioned (with ongoing monitoring) to arrive at well-informed decisions, and to suggest solutions for mycotoxin problems as they arise in fields. The trends reported will guide specific research to establish rational decisions/solutions for addressing potential mycotoxin problems in fields. Grain storage facilities may develop new strategies for the correct handling and mixing-in of contaminated maize based on the occurrence and trend results.
Mycotoxins will always be the unwanted needle in the haystack, and in spite of all the information available about the negative health effects caused by mycotoxin-contaminated staple foods, reoccurring outbreaks of food and feed poisoning in Africa do occur. This study confirmed that it is possible for countries in Africa to establish an affordable test facility to monitor the staple grains produced in order to protect their consumers.