In 2021, Mars Incorporated brought together a Food Safety Coalition of experts from industry, academia, and international organizations to drive food safety insights and standard practices at pace, starting with aflatoxins, due to the serious health threat they pose. Work was undertaken in four areas: sampling and testing, risk assessment and communication, prediction, and risk communication. This publication forms part of the work focused on sampling.

Mycotoxins are naturally occurring toxins [1] and are of major concern in the food industry worldwide, as contamination occurs regularly in food and feed commodities [2]. Approximately 25% of the global food supply is significantly contaminated [2]. Many hundreds of different mycotoxins have been identified, including aflatoxin(s) (AF(s) [3]. AFs are secondary metabolites, produced by fungi such as Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius, that grow on agricultural products, including cereals, peanuts, rice, and dried fruit [4]. The four main AFs that pose a particular risk to humans, include aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and aflatoxin G2 (AFG2). Among these toxins, AFB1 is considered the most harmful and prevalent in corn [2]. AF contamination occurs during crop development and maturation, and thus increases due to inadequate post-harvest conditions, including insufficient storage and drying treatments [4]. AF contamination is increasing markedly due to the impact of climate change [5]. Climate change causes variation in environmental temperatures and water activity (aw), and therefore affects fungal growth and AF production in crops [5]. Aspergillus flavus is highly adaptable to climate change, and consequently dominates various non-toxic fungal species [5]. Moreover, AFs are heat stable, and thus it is difficult to completely eradicate AF contamination in crops [5]. Decontamination processes including thermal processing have reduced contamination [6]. Moreover, novel-processing methods (pulsed light) have shown significant advances in AFs’ degradation [6].AF exposure results from either direct consumption of AF-contaminated food or indirectly from food-producing animals, which have consumed AF-contaminated feed [7]. AF consumption can lead to serious health implications, as they are carcinogenic and highly toxic [8]. High AF exposure through grain consumption may cause immunosuppression, liver cirrhosis, and acute aflatoxicosis; a condition depicted by liver damage, which can possibly result in death [9]. Low levels of AF exposure over a long period of time can cause impaired growth in children [9]. Furthermore, AF consumption in animals can result in toxic effects such as chronic diseases [10], including liver damage and immunosuppression [10].The toxic potential of AF consumption highlights the importance of testing and monitoring AF contamination in the food supply chain. Robust mycotoxin sampling procedures, coupled with the fit-for-purpose mycotoxin analysis, are essential for complying with established food safety standards and regulatory limits to confirm food is safe for trade, and human and animal consumption [11]. Many countries have established common regulations and maximum levels for AFs, and these must be supported by reliable testing data [12]. The development of effective sampling and testing methods for AF analysis is a continuing issue, as it is highly challenging to estimate the true AFs’ concentration in a batch lot due to the diverse nature of AF contamination within corn kernels [11].The challenges associated with sampling include the fact that AFs’ concentration distributions are generally highly heterogeneous throughout a batch of bulk kernels [13]. Therefore, bulk sampling of corn may not represent the true AF contamination across an entire lot [2]. Hence, obtaining a sample that is representative of the entire batch lot is extremely difficult [2]. Variances can also occur during sampling, as minimal portions of kernels are highly contaminated, whereas the majority of the lot can be mycotoxin-free or contain negligible levels of AF contamination [13]. This can cause serious discrepancies of AF contamination being reported, including false positives and false negatives in terms of maximum residue limit (MRL) breaches, resulting in the misclassification of corn batches [2]. Therefore, these ambiguities within sampling variances threaten food safety and international trade [13].An effective sampling procedure has a crucial role in minimizing the impact of the heterogeneous distribution of AFs in corn [11]. The key steps to obtaining an accurate measurement of AFs’ content in a lot consists of incremental sampling, sample preparation, and sample analysis via detection methods [14]. The importance of the sampling procedure is highly underestimated, but it is the most crucial component of managing AF-contaminated food safety risks [13]. Various sampling strategies have been proposed, including random and stratified [9]. Randomized sampling is more extensively utilized; however, the effects of this method in obtaining a representative sample are limited to theoretical analysis, failing to consider the heterogeneous and spatial clustering of AF contamination [9]. However, as further discussed in this review, obtaining a larger number of incremental samples at various random locations within a lot can minimize the impact of AFs’ heterogeneity, reduce sample variation, increase the reliability of results, and provide a more accurate analysis of AFs’ contamination of corn.This review aims to provide a comprehensive overview of sampling procedures for aflatoxin analysis that have been published by governmental, non-governmental sources, and businesses (Table 1), in addition to the regulatory limits set for aflatoxins in different countries. The main themes identified from the literature reveal that official formal sampling procedures focus on sample size and frequency, thus addressing regulatory legislation and trade requirements. “Formal sampling procedures obtain samples, which are taken and analysed according to all the relevant legislation” [15]. However, informal sampling procedures focus on sampling mechanics, thus managing mycotoxin risk assessment, to ensure safe food consumption. “An Informal sampling procedure is not for enforcement purposes, but mainly a surveillance exercise to ensure food safety” [16]. Although there is an array of sampling procedures available, there is no clear indication as to which method is regarded as the best approach or has been optimized for accurate AF measurements. The overall aims of this review are to summarize the range of sample procedures identified, and critique which factors best contribute to obtaining an effective and adequate sample procedure, to produce truly representative samples. Sampling procedures have been compared and divided into informal and formal protocols. A model framework template of the standard practice has been devised for both formal and informal sampling procedures to accurately determine levels of AF contamination in corn.