Cereals have been a vital source of human nourishment for thousands of years due to their excellent nutritional qualities and availability [104]. It is the staple food for a large portion of the world’s population [105]. Wheat provides up to 14.1% and 24.3% of the total calorie intake in America and Asia, respectively, while rice alone provides up to 28.5% of total calorie intake in Asia [106]. In the year 2022, estimated cereal production is 2799 million tons worldwide, with a high proportion of coarse grains, wheat, maize, and rice [107]. Wheat (Triticum aestivum L.), rice (Oryza sativa L.), and maize (Zea mays L.) are the major food grain crops of Pakistan. Wheat is grown on 9.2 million hectares (almost 40% of the country’s total cultivated land), with an annual production of 27 million metric tons (MMT) [107]. Rice ranks second among Pakistan’s primary food grain crops, with an annual yield of around 8.2 MMT [107]. During the summer or “Kharif” season, rice is grown on around 10% of Pakistan’s total agricultural land [107]. Pakistan is a major exporter of rice, exporting more than 4 MMT to East Africa, Europe, the Middle East, and China each year [108]. Rice exports are a major source of foreign exchange earnings, accounting for 6.11% of total agricultural value and 1.4% of total GDP [109]. Maize is the third-most important crop after wheat and rice in Pakistan, with an annual production of over 7.8 MMT [107] and accounting for 0.5% of the GDP of Pakistan [110]. It is a multipurpose crop in Pakistan, used as food and animal feed [111].

Peanut (Arachis hypogaea L.), also called ‘The King of Oilseeds’, is one of the most important leguminous oilseed crops grown in Pakistan. Its kernel is rich in oil (43–55% of total content) and protein (25–28%) [140]. Pakistan is the leading peanut producer in the world, with an annual production of 6.10 million tons in 2017, cultivated in an area of approximately 1.33 million hectares (ha). The Pothohar plateau in Punjab province is famous for peanut cultivation, with total annual exports of over 0.1 million tons (USD 15 million) [141]. Peanuts are at high risk for contamination with mycotoxins, particularly AFs, because they are prone to fungal attacks when drying in the field after uprooting [142]. High concentrations of AFs are found in oilseed crops and edible oil products. Mean total AF levels from local peanut oils from two Pakistan markets were 14.52 and 8.59 ppb [144,145], with 35% of samples in the Peshawar market having total AF levels exceeding the EU’s MTL.

Sesame (Sesamum indicum L.) is another important oilseed crop of Pakistan, cultivated in an area of approximately 176,000 ha, yielding an annual production of approximately 35,000 metric tons [109]. Sesame seed is also an important source of edible oil that is largely used as a seasoning [103]. In Pakistan, sesame seeds are a common part of cuisine, used regularly in bakeries, confectioneries, and Unani herbal medicines. The sesame crop is vulnerable to a wide range of infectious plant pathogens that damage the plant and facilitate fungal infection (Fusarium, Alternaria, Penicillium, and Aspergillus) and mycotoxin production [151]. Only limited studies have reported levels of AF contamination in sesame seeds grown in Pakistan (see Table 2). Ajmal et al. [150] reported high contamination levels for AFB₁ in sesame seeds from rainfed and irrigated zones of Punjab, Pakistan. In samples from the rainfed zones, 20 ppb were found in 20% of fresh and 100% of stored seeds samples, while in samples from the irrigated zones, 28% of fresh and 60% of stored samples contained AFB₁ levels more than 20 ppb. Such levels surpass the maximum limits for human consumption assigned by the U.S. FDA and the Food and Agriculture Organization of the United Nations. In another study, Ajmal et al. [152] confirmed that most (72.31%) of 260 isolates of A. flavus from sesame seeds grown in Pakistan were aflatoxigenic.

Nuts and dried fruits are widely grown and processed in Pakistan. With a total production of 455,990 metric tons (MTs) of dates and 2889.38 MTs of nuts in 2019, Pakistan was placed 6th and 50th in the world, respectively, in their production [153]. Among the dried fruits produced in Pakistan, the government has explicitly prioritized dates as a subsistence crop in vast desert areas [154]. Due to its ideal climatic conditions and very fertile plains, the Gilgit-Baltistan (GB) of the Khyber Pakhtunkhwa (KPK) province is a favorable region to produce dried fruits. In GB, dried fruit is prepared and preserved by removing the original water content by sun drying. The fruits are commonly contaminated by molds during the drying process, which can lead to subsequent AF contamination during storage. However, due to the challenging terrain and remoteness of this region, there is little investment in adequate storage or processing facilities, and 40% of samples of dried fruits from this region showed AFB₁ levels exceeding the EU’s MTL [155]. 

AFB₁ occurrence in dried raisins, figs, and dates were reported by Alghalibi and Shater [157]. Asghar et al. [158] evaluated 624 samples of dried fruits for the presence of AFs and reported that 165 (26%) samples were contaminated, with levels ranging from 0.22 to 30.11 ppb and a mean level of 0.85 ppb. They stated that 28 (4%) of the samples were found to exceed the EU limit (4 ppb). Other research revealed that there were significant amounts of total AFs in dried fruit products, with a mean level of total AFs of 2.90 ppb in watermelon seed samples [159]. Masood et al. [160] examined 307 samples of Pakistani edible nuts and dried fruits and found that 132 (43%) of the samples were contaminated with AFB₁ and total AFs.

Roots, bulbs, rhizomes, stems, bark, leaves, and seeds are all utilized to make spices. Spices have substantial economic value and are a common component of many people’s daily diets around the world. Chilies constitute 16% of the global spice trade, ranking the second among spices [161]. Pakistan is the world’s fourth-largest chili producer, after India, China, and Mexico, with an annual production of 141,500 tons cultivated on an area of approximately 157,800 acres. Pakistan ranks 6th worldwide in the export of chilies, exporting 25,000 tons which contribute 1.5% to the country’s total GDP [153]. Chilies, both in fresh and dried form, are considered a basic ingredient of everyday food in Pakistan and are, therefore, used throughout the year. However, they are grown seasonally, harvested in mid-July up to the end of November.

Due to their plant origin, spices can become contaminated by microbes before, during, and after harvest [162]. In Pakistan, spices are typically harvested, dried, stored, and processed using substandard methods in warm and wet environments that promote the growth of molds. Aspergillus, Penicillium, and Rhizopus are the most common genera of fungi found in spices [163,164,165]. AF contamination of spices is an important problem in Pakistan.

Additionally, the spice markets’ hygienic standards are exceedingly poor in Pakistan, especially in the Karachi district of Sindh. Most of Karachi’s population is from a lower socioeconomic background and is ignorant of AF contamination in spices. When kept in moist, humid conditions for a long time, unpackaged ground spices are a favorable medium for the growth of fungi. Packaging can also influence AF contamination; hot pepper samples in jute bags (common in Pakistan) were reported to be more vulnerable to AF contamination than samples that were packed in polyethylene bags [175]. Loosely packaged compound spices sold in wholesale markets may be easily contaminated by dust, sewerage, and animal or human excrement [176]. Akhund et al. [169] reported the level of AFs in red chilies from the Sindh province, Pakistan. They examined AF levels using TLC and HPLC and demonstrated that 67% of samples were tainted with AFB₁, with a range of 1.2–600 ppb and a mean level of 131.7 ppb. In another study, spices from different markets of Peshawar were reported to have AF levels ranging from 1.86 to 7.46 ppb. Coriander, omam seed, and turmeric samples contained high levels of AF [102].

Different feed ingredients in Pakistan are susceptible to mold growth due to inadequate harvesting, handling, storage, and processing conditions, so the feedstuff can be contaminated with AFs [177]. As a result of the negative impacts of tainted animal feed, the livestock sectors suffer substantial economic losses. AFB₁ levels in poultry feed samples from Rawalpindi’s Poultry Research Institute and from West Central Pakistan were reported to be above the safe limit (20 ppb) by Bhatti et al. [178] and Rashid et al. [179], respectively. AFB₂ was found in poultry diets and feed ingredient samples from Punjab at concentrations ranging from 10.80 to 39.20 ppb [180]. Alam et al. [181] examined 216 samples of chicken feed ingredients (maize, wheat, rice, and cottonseed meal) gathered in the summer, winter, autumn, and spring seasons of 2007/2008 from Swat, Peshawar, and D. I. Khan districts of KPK, Pakistan. Contaminations levels of AFB₁, AFB₂, AFG₁, and AFG₂ were up to 191.65, 86.85, 167.82, and 89.90 ppb, respectively. The highest concentrations of AFB₁, AFB₂, and AFG₂ were found in the summer samples, while the highest concentration of AFG₁ was found in the autumn samples. Approximately 61% of samples of poultry feed were tested positive for AFB₁ by Khan et al. [182], and 47% by Anjum et al. [183]. High levels of AF contamination were found in bakery trash (724.6 ppb) and cottonseed cake (600.8 ppb), as reported in Yunus et al. [184]. 

Fungal infestation is highly affected by the season [137]. In Pakistan, variations in feed AF contamination have been linked to persistent relative humidity and the rainy season, particularly the hot monsoon season, which typically lasts from June to September [179,183,196]. Humidity is strongly linked to the production of AFs in feed during the winter, spring, and summer seasons [191,197,198]. Crops, particularly corn and cotton, are most impacted by AF contamination during the rainy season (June to September) [191]. Corn harvested during the rainy season had a higher level of AFB₁ (66.4 ppb) than corn harvested during the dry season (37 ppb; Tangendjaja et al. [199]), although these findings are slightly different from those reported by Chauhan et al. [200], which found the highest AFB₁ levels from June to November and the lowest from December to May.

Maize and cotton seed cake are important feed ingredients in Pakistan, either directly or as a component of concentrate feed. Rainfall and the accompanying hot and humid conditions during harvest time increases the likelihood of contaminated feed ingredients. Additionally, contamination of these feed materials with AFs can occur when they are kept in storage for subsequent use. Maize crops can be contaminated with a variety of different AFs [201]. Anjum et al. [183] and Bhatti et al. [178] reported the highest contamination of AFs in corn. These results are consistent with those of Kamkar et al. [202], who found that increased moisture content and delayed storage caused increased AFB₁ contamination in animal feed. The direct link between AFB₁ contamination and storage period was reported in numerous previous research [203,204,205]. Reddy and Salleh [206] reported that 23% of animal feed samples were contaminated with AFB₁ at levels ranging from 21 to 135 ppb. Similarly, Anjum et al. [183] reported that 61% of maize AFB₁ contaminated samples exceeded the permissible limits. Additionally, farmers feed scraps of bread, a significant source of AF contamination, to animals. According to Asi et al. [207], animals fed on bread pieces and concentrates produced more AF in their milk.

Due to the shortage of available feed during the winter, farmers supplement animal feed with compound feed. Compound feed is typically made from leftover grains, making it especially vulnerable to rises in AF levels during storage. According to Asi et al. [207], animals in Pakistan that were typically fed with compound feed showed higher concentrations of AFM₁ in their milk than animals that grazed or were fed fresh green feed. To ensure the highest milk production during the winter, when fresh pasture or fresh feed is not available, farmers feed animals the highest possible amounts of corn, cotton seeds or cotton seed cake, raw rice bran/rice polish, wheat bran, and gluten. These components support a high level of milk production but are most susceptible to fungal infestation and AF production [208,209].

Crop harvesting time is another factor that has been connected to increased levels of AF contamination in winter. In Punjab, Pakistan, the corn-harvesting time is October, while the cotton-harvesting time is August to September, so corn and cotton seed become a primary, economically efficient source of feed in these months. Dairy farmers grow these crops and feed animals without knowing whether the feeds are contaminated with AFs [207,210,211,212].

The livestock sector is important for the economic development of all countries. It plays a crucial role in reducing the poverty of rural areas by providing food and income [213]. Milk is a rich source of nutrition for all age groups that contributes to the optimal growth of newborns and children. Pakistan produces over 60 billion liters of milk annually, making it the world’s fourth-largest milk producer [214]. It contributes 46.8% of agriculture revenue, where 10–25% of the income is generated by rural people [215]. As the demand for milk rises, it becomes more difficult for the dairy industry in developing nations to maintain a uniform and standardized quality. In Pakistan, milk demand is fulfilled primarily (94%) by informal, nonindustrial supplies, whereas the packed milk sector bridges only 6% of the gap. Milk in this informal supply chain is expected to have high levels of AFM₁ contamination [216].

AFs such as AFB₁ are bio-transformed into AFM₁ in dairy animals’ livers and subsequently excreted into milk, eventually reaching the humans who consume the milk [217,218]. Animals fed on AF-contaminated feed exhibit decreased growth rate, decreased milk production, and lowered milk quality, in addition to compromised immunity against infections [219].

AFM₁ in raw milk cannot be destroyed by pasteurization, heat processing, or other simple methods [220]. Recent research has emphasized significant human health risks connected to the consumption of milk tainted with AFs [221,222]. AFM₁ contamination of milk is a global issue, especially in developing countries. The Punjab province of Pakistan is the major cash-crop-producing and livestock-keeping area. AFM₁ contamination screening of the milk from areas within Punjab found that the average AFM₁ contamination levels were above US and EU regulatory limits [223] 

In Pakistan, particularly in Punjab, the months of December to March are associated with increased rates of milk contamination [191]. In the winter, when green fodder is scarce, farmers are forced to use stockpiled feed sources [191]. As a result, milk from cows that consume stored feed is positively associated with AFM₁ [206,207], and various studies have reported significantly higher AFM₁ contamination levels in milk during the autumn or winter season in Pakistan [134,186,224,225,226]. High AFM₁ contamination levels in raw milk samples were reported during the winter and autumn seasons, with average values of 54.24 and 34.92 ppt (parts-per trillion: ng/L), respectively, according to studies by Shokri and Torabi [212]. Akbar et al. [216] reported that milk samples had the highest range of AFM₁ contamination in November and the lowest in May. From March to August, the AFM₁ contamination levels of the milk samples rapidly declined in all locations. AFM₁ trends were nearly identical across all locations in Pakistan, i.e., AFM1 levels in milk produced from mid-April to August typically meet the US MRL. Following August, a marked rise in the concentration of AFM₁ is found in all local milk samples. The most and least contaminated samples were found in the months of February and July, respectively [216].