Comprehensive Review of Aflatoxin Contamination, Impact on Health and Food Security, and Management Strategies in Pakistan

Aflatoxins (AFs) are the most important toxic, mutagenic, and carcinogenic fungal toxins that routinely contaminate food and feed. While more than 20 AFs have been identified to date, aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), and M1 (AFM1) are the most common. Over 25 species of Aspergillus have been shown to produce AFs, with Aspergillus flavus, Aspergillus parasiticus, and Aspergillus nomius being the most important and well-known AF-producing fungi. These ubiquitous molds can propagate on agricultural commodities to produce AFs in fields and during harvesting, processing, transportation, and storage. Countries with warmer climates and that produce foods susceptible to AF contamination shoulder a substantial portion of the global AF burden. Pakistan’s warm climate promotes the growth of toxigenic fungi, resulting in frequent AF contamination of human foods and animal feeds. The potential for contamination in Pakistan is exacerbated by improper storage conditions and a lack of regulatory limits and enforcement mechanisms. High levels of AFs in common commodities produced in Pakistan are a major food safety problem, posing serious health risks to the population. Furthermore, aflatoxin contamination contributes to economic losses by limiting exports of these commodities. In this review, recent information regarding the fungal producers of AFs, prevalence of AF contamination of foods and feed, current regulations, and AF prevention and removal strategies are summarized, with a major focus on Pakistan.

The limit between countries can differ considerably; the maximum/action limit for total AFs has been set at 20 ppb (μgkg -1 ) in the United States [97] and 4 ppb in the EU [98]. The PSQCA is responsible for developing and enforcing food standards in Pakistan and has set the maximum acceptable limit for AFs in food and food products at 20 ppb except for milk (whole and skim), which has a maximum limit of 10 ppb. Despite Pakistan's establishment of maximum tolerated levels of AFs in foods, European and other countries have banned the import of various Pakistani commodities due to poor quality related to AF levels. High levels of AF contamination were found in custard powder and peanut snacks shipped from Pakistan to South Korea and the United Kingdom, respectively. The PSQCA was criticized in both incidents for failing to monitor high AF levels in the food destined for export. For EU member states, the presence of AFs in hot chili powder from Pakistan has also been a cause for concern [99].
The Pakistan Agricultural Research Council (PARC) reported that various countries have rejected considerable quantities of food in recent years due to contamination concerns [99]. AF contamination was the most frequent cause of border rejections by the EU, US, and Australia from 2002 to 2008 (in 35% cases), followed by product composition and prohibited food additives [99]. Since AFs are significant food contaminants harming a nation's trade, national authorities should pay a special attention to AF control.
Unfortunately, strict regulations for AF control in food are not always the best option. The fact that high-quality food products are exported and those contaminated with AFs are left to the domestic population may have a negative influence on the health of local customers in Pakistan. AF exposure in low-income populations may increase because contaminated products are sold at reduced prices in local marketplaces. Pakistan, like other less developed nations, has laxer AF enforcement, putting the local population at greater risk of AF exposure from low-quality imported food products. Care must be taken to ensure that strengthening AF standards for exported products does not jeopardize the health of the local population by forcing them to eat contaminated food items that have been rejected.
The limit between countries can differ considerably; the maximum/action limit for total AFs has been set at 20 ppb (µgkg -1 ) in the United States [97] and 4 ppb in the EU [98]. The PSQCA is responsible for developing and enforcing food standards in Pakistan and has set the maximum acceptable limit for AFs in food and food products at 20 ppb except for milk (whole and skim), which has a maximum limit of 10 ppb. Despite Pakistan's establishment of maximum tolerated levels of AFs in foods, European and other countries have banned the import of various Pakistani commodities due to poor quality related to AF levels. High levels of AF contamination were found in custard powder and peanut snacks shipped from Pakistan to South Korea and the United Kingdom, respectively. The PSQCA was criticized in both incidents for failing to monitor high AF levels in the food destined for export. For EU member states, the presence of AFs in hot chili powder from Pakistan has also been a cause for concern [99].
The Pakistan Agricultural Research Council (PARC) reported that various countries have rejected considerable quantities of food in recent years due to contamination concerns [99]. AF contamination was the most frequent cause of border rejections by the EU, US, and Australia from 2002 to 2008 (in 35% cases), followed by product composition and prohibited food additives [99]. Since AFs are significant food contaminants harming a nation's trade, national authorities should pay a special attention to AF control.
Unfortunately, strict regulations for AF control in food are not always the best option. The fact that high-quality food products are exported and those contaminated with AFs are left to the domestic population may have a negative influence on the health of local customers in Pakistan. AF exposure in low-income populations may increase because contaminated products are sold at reduced prices in local marketplaces. Pakistan, like other less developed nations, has laxer AF enforcement, putting the local population at greater risk of AF exposure from low-quality imported food products. Care must be taken to ensure that strengthening AF standards for exported products does not jeopardize the health of the local population by forcing them to eat contaminated food items that have been rejected.

Aflatoxin Contamination of Agricultural Products and Foods in Pakistan
In Pakistan, agriculture contributes approximately 21.8% to the GDP (gross domestic product), a substantial portion of the country's overall economy. More than 65-70% of the population in Pakistan depends on agriculture for its livelihood. The warm, humid environmental conditions of Pakistan are very favorable for the invasion of mycoflora such as Aspergillus that can produce AFs as secondary metabolites [100]. High humidity and insufficient ventilation in agricultural commodity storage areas are also problems in Pakistan and are key contributors to Aspergillus growth and the production of AFs in foods and feeds [101]. Pakistani crops are, therefore, prone to contamination by AFs, with improper agronomic and storage practices by farmers and processors exacerbating the problem [102,103].

AF Contamination in Cereals
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].
Many studies from Pakistan have reported contamination of AFs in cereal crops, as summarized in Table 1. Cereals are particularly vulnerable to AF contamination. Cereals such as rice and maize are usually grown during hot weather and harvested during the humid summer in Pakistan, conditions generally favorable for AF production. Cereal grains and their flours are hygroscopic and require careful moisture control during storage to prevent AF contamination [112,113]. As shown in Table 1, AF contamination was found in 2~53% of Pakistani wheat samples in five studies, with between 0% and 45.8% of AF contaminated samples having AF levels exceeding EU maximum tolerated levels (MTLs). Even higher percentages of rice were contaminated, with up to 95.4% of samples exhibiting AF contamination. Levels of contamination varied substantially across studies. Previous studies reported that AFB 1 and AFB 2 were present at the highest levels in broken rice from Punjab, followed in order by brown rice, white rice, and parboiled rice. Similar results were reported by Lutfullah and Hussain [114]. In a study conducted by Iqbal et al. [115], AF contamination was found in 50% of broken rice samples, a sign that broken rice may be more vulnerable to AF contamination. However, across the studies referenced in Table 1, such trends were not consistently observed.

AF Contamination in Edible Oilseed Crops
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 as shown in Table 2 [143]. 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. Ajmal et al. [150] Abbreviations (AFB 1 = Aflatoxin B 1 , AFB 2 = Aflatoxin B 2 , AFG 1 = Aflatoxin G 1 , AFG 2 = Aflatoxin G 2 , AFTS = Total aflatoxins, TLC = thin-layer chromatography, HPLC = high-performance liquid chromatography, LC = liquid chromatography, KPK = Khyber Pakhtunkhwa, LOD = limit of detection, ppb = parts per billion, EU = European Union, MTL = maximum tolerance limit). (%) * = Percentage of the samples out of contaminated samples exceeding the maximum tolerance limits (MTL) recommended by European Union [98]. Value * exceeding the exceeding the MTL recommended by FAO and FDA [97].
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 AFB 1 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 AFB 1 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.

AF Contamination in Nuts and Dried Fruits
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 AFB 1 levels exceeding the EU's MTL [155]. Some studies reported high AF contamination levels of dried fruits in the range of 0.05-50.5 ppb, as shown in Table 3. Abbreviations (AFB 1 = Aflatoxin B 1 , AFTs = Total aflatoxins, TLC = thin-layer chromatography, HPLC = highperformance liquid chromatography, LC = liquid chromatography, KPK = Khyber Pakhtunkhwa, LOD = limit of detection, ppb = parts per billion, EU = European Union, MTL = maximum tolerance limit). (%) * = Percentage of the samples out of contaminated samples exceeding the maximum tolerance limits (MTL) recommended by European Union [98]. Value * exceeding the exceeding the MTL recommended by FAO and FDA [97]. AFB 1 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 AFB 1 and total AFs.

AF Contamination in Spices
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. Previous studies reported high contamination of AFs in spices from Pakistan, with a range of 2.86-243 ppb as shown in Table 4. Sahar et al. [166] reported that high AF contamination levels were attributed to the drying process rather than pre-harvest conditions. On-farm sun drying is a common practice in Pakistan for various commodities, including chilies, which are typically spread out in open fields for sun drying, then stored under subpar conditions that allow the growth of molds and the production of mycotoxins [167]. Sun drying of the harvested chilies reduced the average moisture content from 69.70% to 9.87%, but also led to a gradual increased level of AFs. Abbreviations (AFB 1 = Aflatoxin B 1 , AFB 2 = Aflatoxin B 2 , AFG 1 = Aflatoxin G 1 , AFG 2 = Aflatoxin G 2 , AFTS = Total aflatoxins, TLC = thin-layer chromatography, HPLC = high-performance liquid chromatography, LC = liquid chromatography, LOD = limit of detection, ppb = parts per billion, EU = European Union, MTL = maximum tolerance limit). (%) * = Percentage of the samples out of contaminated samples exceeding the maximum tolerance limits (MTL) recommended by European Union [98]. Value * exceeding the exceeding the MTL recommended by FAO and FDA [97].
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 AFB 1 , with a range of 1.2-600 ppb and a mean level of 131.7 ppb, as shown in Table 4. 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].

AF Contamination in Animal Feeds
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. AFB 1 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.  [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]. A summary of recent studies reporting AF contamination in animal feeds from Pakistan is found in Table 5. Summia et al. [185] reported the highest mean total AF levels in bovine feed collected from Lahore was 229.7 ppb. The mean AFB 1 level was reported in a range of 3.04-214.9 ppb, as shown in Table 5. Shar et al. [190]  Abbreviations (AFB 1 = Aflatoxin B 1 , AFB 2 = Aflatoxin B 2 , AFTS = Total aflatoxins, TLC = thin-layer chromatography, HPLC = high-performance liquid chromatography, LCMS = liquid chromatography mass spectroscopy, MS = mass spectroscopy, LOD = limit of detection, ppb = parts per billion, EU = European Union, MTL = maximum tolerance limit). (%) * = Percentage of the samples out of contaminated samples exceeding the maximum tolerance limits (MTL) recommended by European Union [98]. Value * exceeding the exceeding the MTL recommended by FAO and FDA [97].
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 AFB 1 (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 AFB 1 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 AFB 1 contamination in animal feed. The direct link between AFB 1 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 AFB 1 at levels ranging from 21 to 135 ppb. Similarly, Anjum et al. [183] reported that 61% of maize AFB 1 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 AFM 1 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].

AF Contamination of Milk
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 AFM 1 contamination [216].
AFs such as AFB 1 are bio-transformed into AFM 1 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]. AFM 1 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]. AFM 1 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. AFM 1 contamination screening of the milk from areas within Punjab found that the average AFM 1 contamination levels were above US and EU regulatory limits [223] Table 6. Similarly, 99% of fresh milk samples in India (which neighbors Pakistan) exceeded the Codex limits [205].
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 AFM 1 [206,207], and various studies have reported significantly higher AFM 1 contamination levels in milk during the autumn or winter season in Pakistan [134,186,[224][225][226]. High AFM 1 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 AFM 1 contamination in November and the lowest in May. From March to August, the AFM 1 contamination levels of the milk samples rapidly declined in all locations. AFM 1 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 AFM 1 is found in all local milk samples. The most and least contaminated samples were found in the months of February and July, respectively [216].   Asi et al. [207] Raw Abbreviations (AFM 1 = Aflatoxin M 1 , LC = liquid chromatography, HPLC = high-performance liquid chromatography, KPK = Khyber Pakhtunkhwa, LOD = limit of detection (i.e., 0.004 µg/kg), ppt = parts per trillion, µg/L = microgram per liter, ng/L = nanogram per liter, µg/kg = microgram per kilogram, ng/kg= nanogram per kilogram, ng/mL = nanogram per milli liter, EU = European Union, MTL = maximum tolerance limit). (%) * = Percentage of the samples out of contaminated samples exceeding the maximum tolerance limits (MTL) recommended by European Union [98]. Value * exceeding the exceeding the MTL recommended by FAO and FDA [97].

Dietary Exposure to Aflatoxins
Two methods are commonly used to determine the human exposure to AFs. The first and most popular approach combines data on food consumption with AF levels found in food samples [241]. By dividing the total AFs consumed by the average human body weight, the exposure is further standardized and represented as nanograms per kilogram of body weight per day (ngkg -1 b.w./day) [242]. The European Food Safety Authority (EFSA) has developed detailed guidelines for dietary data collection and processing for risk and exposure assessment purposes [243]. Measuring AF biomarkers in human bodily fluids is an alternate approach that is thought to be a direct and more precise method of determining human AF exposure [244]. Biomarkers of AFs such as the AF-N7-guanine adduct excreted in urine and AFM 1 present in breast milk are used to determine short-term exposure to AFB 1 , while AF-albumin adduct levels in plasma or serum are used for chronic exposure assessment [245].
Although AFs are genotoxic carcinogens, and regulations regarding their presence in food have mostly focused on its carcinogenic consequences, international risk assessment agencies such as the Joint Expert Committee on Food Additives (JECFA) have never defined a TDI for AF [246]. However, daily exposure to levels as low as 1 ngkg -1 b.w./day is considered dangerous to human health [247]. Turna and Wu [246] estimated a range of TDIs for AF-related immune impairment is 0.017-0.082 µg/kg b.w./day.
In Pakistan, average adults consume approximately 171-239 g of rice daily. Abdullah et al. [248] reported the estimated AF intake for typical rice consumers ranged between 19.1 and 26.6 ngkg -1 b.w./day. Iqbal et al. [127] reported the average estimated dietary intake (EDI) of AFs for adults in Pakistan was 22.2-ngkg −1 b.w./day. These intakes are much higher than 1 ngkg -1 b.w./day that is considered unsafe and pose considerable health risks to people in Pakistan [249]. Majeed et al. [123] also reported that the estimated AFB 1 and AFB 2 intake in Pakistani adults and children through rice consumption was above the recommended limits. More recently, Xia et al. [116] estimated the average probable daily intake (PDI) and EDI for AFB 1 to be 30.3 ngkg -1 b.w./day and 3.5 ngkg -1 b.w./day, respectively, in the rural population of Pakistan. In contrast, Raad et al. [250] reported a lower level of dietary exposure to AFB 1 , i.e., 0.63-0.66 ngkg -1 b.w./day for Pakistani adults.
According to Ismail et al. [234], the people of Pakistan are also at high risk of health issues related to AFM 1 in milk. The estimated daily intake (EDI) of AFM 1 during various seasons of the year for various age groups was found in the range of 0.22-5.45 ngkg -1 b.w./day, with infants in the highest risk group. Male babies were found to have the highest EDI values for AFM 1 during the winter (5.45 ngkg -1 b.w./day), whereas adult females had the lowest values during the summer (0.22 ngkg -1 b.w./day). Children were reported to be more susceptible to AFM 1 because of higher milk intake levels and lower body weight. EDI values for AFM 1 decreased with age, with adults having the lowest levels [234]. According to Xia et al. [251], the rural Pakistani population had a high prevalence of AFM 1 in urine. AFM 1 prevalence in human breast milk samples is a sign that mothers' diets are contaminated with AFs. AFM 1 contamination in breast milk samples is ultimately a reflection of AF contamination of Pakistani food products. The range of AFM 1 in milk samples from women in Southern Punjab was reported to be between 0.001 and 0.044 µg/L by Khan et al. [252], with 6.4% of samples exceeding the EU's maximum limit (0.025 µg/L).
A recent study in a rural Pakistan population used urinary biomarkers to estimate AFB 1 exposure and calculate its contribution to liver cancers. Urinary AFM 1 (a marker of AFB 1 exposure) was found in 69% of urine samples (mean levels of 0.023 ng/mL, with a maximum level of 0.393 ng/mL) [116]. Compared to the average rates of liver cancer in Pakistan (7.6 and 2.8 cases per 10 5 individuals each year for males and females, respectively), AFB 1 exposure calculated via urinary marker data contributes significantly to the total risk (0.514 cases per 10 5 individuals annually) [253]. This risk was significantly higher than the risk attributed to AFB 1 exposure from rice alone (estimated by food frequency questionnaires) in Pakistan; 0.07 to 0.122 cancer cases per 10 5 individuals per year [123].

Control Strategies for Aflatoxins Contamination
AF contamination in the field is difficult to control due to several factors, including temperature, insect infestation, humidity, soil moisture, and mineral deficiencies [254]. Various strategies can be used before or after harvest or during storage to prevent their contamination with AFs [255]. At the pre-harvest stage, AF contamination can be reduced by proper agronomic techniques and using resistant cultivars. In addition, insect and mechanical damages to plants should be minimized during the preharvest stages [256].
An effective biological method to prevent AF contamination in the field and during storage is the introduction of naturally occurring non-aflatoxigenic strains of A. flavus and A. parasiticus to competitively exclude aflatoxigenic strains. A conidial suspension of a non-aflatoxigenic strains is administered to the soil before planting or directly to the seedlings [257]. Successful management of AF production in the field by using nonaflatoxigenic Aspergillus also helps prevent AF contamination during later storage. This strategy was first used by Cotty and Bayman [258] and it has been subsequently employed for the control of AFs around the globe (Figure 2). While biopesticides are not yet used in Pakistan, AflaPak TM , a regionally named native biocontrol product, is in development as a public-partnership between the USDA, the Centre for Agriculture and Biosciences International (CABI), Rafhan Maize, and the National Agricultural Research Council (NARC) (https://www.cabi.org/projects/ AF -control-in-pakistan/ accessed on 15 August 2022). AflaPak TM is being developed for the control of AF in maize. CABI is currently evaluating the efficacy of AflaPak TM in seven maize growing districts of Punjab province in Pakistan, hopefully leading to the commercialization and adoption of this biocontrol product in Pakistan. Once registered, AflaPak TM will be the first registered native biocontrol product of a fungal nature in Pakistan, opening opportunities for more green technologies to be adopted in Pakistan.

PEER REVIEW
20 of 36 public-partnership between the USDA, the Centre for Agriculture and Biosciences International (CABI), Rafhan Maize, and the National Agricultural Research Council (NARC) (https://www.cabi.org/projects/ AF -control-in-pakistan/ accessed on 15 August, 2022). AflaPak TM is being developed for the control of AF in maize. CABI is currently evaluating the efficacy of AflaPak TM in seven maize growing districts of Punjab province in Pakistan, hopefully leading to the commercialization and adoption of this biocontrol product in Pakistan. Once registered, AflaPak TM will be the first registered native biocontrol product of a fungal nature in Pakistan, opening opportunities for more green technologies to be adopted in Pakistan. Some biopesticide (non-aflatoxigenic strains) fungal strains do not produce AFs because of a deletion in the AF biosynthetic gene cluster. The AF36 strain, on the other hand, does not produce AFs due to a SNP (single nucleotide polymorphism) that triggers a stop codon in a key gene involved in the polyketide biosynthesis necessary for AF production [267]. In addition to Aspergillus, other fungal species such as yeast, Trichoderma, and Penicillium have been demonstrated to reduce AFs in the field [268,269]. Globally, some of the microorganisms are currently at the experimental stage against fungi that produce AFs (Figure 3). Some biopesticide (non-aflatoxigenic strains) fungal strains do not produce AFs because of a deletion in the AF biosynthetic gene cluster. The AF36 strain, on the other hand, does not produce AFs due to a SNP (single nucleotide polymorphism) that triggers a stop codon in a key gene involved in the polyketide biosynthesis necessary for AF production [267]. In addition to Aspergillus, other fungal species such as yeast, Trichoderma, and Penicillium have been demonstrated to reduce AFs in the field [268,269]. Globally, some of the microorganisms are currently at the experimental stage against fungi that produce AFs (Figure 3).  At the post-harvest stage, the dry chain technique has been used to minimize AF contamination of crops in Pakistan. The basic principle of the dry chain is to dry grains to reduce moisture content and to maintain this dryness throughout the supply chain [275]. A dry chain is an economically viable and environmentally friendly method to reduce the chances of AF contamination. Bakhtavar et al. [276] reported that insect populations, associated storage losses, and deterioration of maize seed quality with respect to loss of germination, food reserves, and increased AF contamination can be controlled by maintaining the dry chain through hermetic storage of maize grain at 8% and 10% seed moisture content.

Detoxification of Aflatoxins
Once a food or food product has been contaminated, AF detoxification is needed. Detoxification involves removing or minimizing the poisonous effects of AF, and can involve physical, chemical, or biological methods. Any detoxification procedure used on human food, according to the FAO, must inactivate, destroy, or remove AFs; not create or leave toxic, carcinogenic, or mutagenic residues on the treated substrates; maintain the nutritional, sensory, or other quality attributes of the food product; and be able to get rid of fungal spores or mycelium that could multiply and produce mycotoxins [277].

Physical Detoxification
Thermal and ultrasonic treatments, solvent extraction, mechanical sorting, adsorption, UV and solar irradiation, density gradient, ozone gas, flotation, gamma rays, electronic eye sorting, roasting, and microwave heating are some of the physical techniques used to inactivate or reduce AF contamination [254]. Damaged and fragmented grains contain higher concentrations of mycotoxins; therefore, removing them lowers the overall contamination [278]. Hand sorting and segregation of grains based on physical characteristics can be beneficial in lowering AF levels in agricultural products, but not on a large Biological control of AF contamination with microorganisms currently under development [270][271][272][273][274][275].
At the post-harvest stage, the dry chain technique has been used to minimize AF contamination of crops in Pakistan. The basic principle of the dry chain is to dry grains to reduce moisture content and to maintain this dryness throughout the supply chain [275]. A dry chain is an economically viable and environmentally friendly method to reduce the chances of AF contamination. Bakhtavar et al. [276] reported that insect populations, associated storage losses, and deterioration of maize seed quality with respect to loss of germination, food reserves, and increased AF contamination can be controlled by maintaining the dry chain through hermetic storage of maize grain at 8% and 10% seed moisture content.

Detoxification of Aflatoxins
Once a food or food product has been contaminated, AF detoxification is needed. Detoxification involves removing or minimizing the poisonous effects of AF, and can involve physical, chemical, or biological methods. Any detoxification procedure used on human food, according to the FAO, must inactivate, destroy, or remove AFs; not create or leave toxic, carcinogenic, or mutagenic residues on the treated substrates; maintain the nutritional, sensory, or other quality attributes of the food product; and be able to get rid of fungal spores or mycelium that could multiply and produce mycotoxins [277].

Physical Detoxification
Thermal and ultrasonic treatments, solvent extraction, mechanical sorting, adsorption, UV and solar irradiation, density gradient, ozone gas, flotation, gamma rays, electronic eye sorting, roasting, and microwave heating are some of the physical techniques used to inactivate or reduce AF contamination [254]. Damaged and fragmented grains contain higher concentrations of mycotoxins; therefore, removing them lowers the overall contamination [278]. Hand sorting and segregation of grains based on physical characteristics can be beneficial in lowering AF levels in agricultural products, but not on a large scale. Treatment with UV light and ionization can prevent AF contamination in food and extend food shelf life because such treatments degrade the fungal cells [279]. Thermal treatments can also be very effective. Depending on the thermal treatment used and the commodity being treated, amounts of AFs can be reduced by 9% to 100%. For example, when fruits and other species are autoclaved at 120 • C for 30 min, amounts of AFs can be reduced by 9~39%; however, when peanuts are autoclaved at 1.5 atm for 90 min, the amount of AF can be reduced by up to 100% [280]. However, because AFs are heat resistant and not entirely degraded at temperatures commonly used in food processing (80-121 • C), typical cooking procedures such as frying, boiling, or pasteurization are unlikely to result in a significant reduction in AF levels [280]. In addition to thermal processing, non-thermal processes such as cold plasma can be utilized to reduce the AF levels in certain grains and nuts by up to 95% [281].
One of the most effective methods to reduce exposure to AFs such as AFB 1 is the addition of non-nutritive adsorbent in contaminated animal feed to decrease the bioavailability of AFs in the gastrointestinal tract. However, the non-specificity of adsorbents and their high costs are major drawbacks for such a method. In addition, due to their non-degradable nature, which is necessary to survive the gastrointestinal tract, deposition of absorbents in the environment when excreted from animals also limits their use [282].
In Pakistan, some studies have reported detoxification of AFs by using physical methods. A study by Khan et al. [283] reported a 78% reduction in AFs in red chilies by removing midget/dwarfed, damaged, broken, dusty, and dirty chilies. Khan and Zahoor [284] effectively detoxified AFB 1 in poultry feed by using a novel adsorbent that was prepared from bagasse. Saleemi et al. [285] investigated the effect of a locally produced mycotoxin binder in commercial broiler chicks. This study showed that local toxin binder (bentonite clay 58%, baker yeast 40%, and silymarin 2%) was a more cost-effective way to mitigate AFB 1 in poultry feed than commercial toxin binders. Summia et al. [286] reported AF reduction of up to 58% by heating poultry feed samples at 250 • C for 10 min. Awan et al. [287] detoxified AFB 1 in pine nuts by using UV irradiation and reported 50%, 70%, and 90% detoxification after exposure to UV for 20, 40, and 60 min, respectively. Some of these physical procedures, while effective, are expensive and may have an impact on key nutrients or sensory characteristics in the commodities. Gillani et al. [288] reported the use of 15 kGy gamma irradiation for 24 h, sunlight-drying for 20 h, and UV irradiation for 12 h almost completely degraded AFs in maize. Microwave heating for 120 s resulted in 9-33% degradation of AFs in maize. Moreover, the treatment of maize grain extracts with activated charcoal (5% w/w) removed 96% of total AFs and AFB 1 . The use of bentonite at the same rate removed total AFs and AFB 1 by 73% and 92%, respectively [288].

Chemical Detoxification
A variety of chemicals have been used to detoxify AFs, including oxidizing and reducing reagents, acids, bases, and chlorinating agents. The efficacy of AF detoxification is increased when chemical agents are utilized in conjunction with physical approaches. More than 100 chemical compounds have been found to inhibit or reduce mold development, resulting in a reduction in AF levels [289]. In Pakistan, Iqbal et al. [141] reported that AFs were reduced by 44% in samples of corn with the addition of a single step of rinsing the corn with water. Nazir et al. [122] reported 80.02% degradation of AFB 1 in rice and cattle feed by treating the contaminated samples with NaHCO 3 and washing them with distilled water three times. According to Summia et al. [286], 0.5% hydrochloric acid treatment of feed samples lowered AF levels by up to 58.4%.
Chemical treatments to detoxify AF-contaminated agricultural goods have been effectively used in a variety of contexts, process parameters, and food products, but they may leave some toxic residues in the treated substrate. Detoxification of AFs by using natural substances may be more acceptable to consumers than using synthetic chemicals. In Pakistan, several studies reported detoxification of AFs by using natural substances. Nazir et al. [122] reported that black seed oil reduced AF contamination up to 100% in rice, maize, and corn. Anjum et al. [289] used a water-based extract of Acacia nilotica to achieve 86-90% detoxification of both AFB 1 and AFB 2 in maize when incubated for 72 h at 60 • C and pH 10, and 82-83% detoxification at 30 • C and pH 8. Nazir et al. [122] detoxified AFs in food by using natural compounds such as sodium bicarbonate, citric acid, extract of Allium sativum, and black seed oil. Reduction in AFs was reported to be 63~100%. The same study also reported the 63.59% to 90% reduction in AFB 1 levels by treating contaminated samples with lemon juice. Awan et al. [287] reported that Zingiber officinale powder detoxified 90% of AFs in dry fruits.
While detoxification of AFs has been studied extensively, little is known about the potential of food processing to increase or hide AF exposure. Chemical and physical treatments applied to food may release AFs from masked forms and make them bioavailable or convert into forms not detectable by conventional analytical methods [290], while retaining their toxic potential [291] or stimulating fungi to produce AFs, e.g., during steeping of barley. Analytical tools for mycotoxins transformed by processing by structural modification or binding to the food matrix need to be developed.

Biological Detoxification
Biological methods, in comparison to other methods of AF detoxification, are thought to be less aggressive, environmentally safe, and cost-effective. Such methods involve using microbes and their products to eliminate AFs from food or feed by surface adsorption, degradation into nontoxic chemicals, or binding to inhibit bioavailability [292]. For example, Flavobacterium aurantiacum effectively eradicates AFB 1 from a variety of foods, including oil, milk, peanut butter, maize, and peanuts, without producing any harmful byproducts [293]. Biodegradation technology has provided an appealing alternative for controlling or eliminating AFs while maintaining food and feed quality and safety. Customers are becoming increasingly opposed to the use of chemical and synthetic compounds in their foods; therefore, the employment of biological agents provides a more "natural" appeal [294]. Azeem et al. [295] assessed the in vitro binding of AFB 1 by probiotic Lactobacilli by incubating with a standard amount of AFB 1 in phosphate-buffered saline at 37 • C for 2 h. The isolates' levels of AFB 1 binding ranged from 28 to 65%. Four isolates (PDP 10, PDP 24, PL 120, and PL 149) resulted in complete binding of AFs.
The use of plant-derived nanoparticles (NPs) is an emerging technology with a potential to reduce AF contamination levels. Various plants leaf extracts such as Gongronema latifolium, Moringa oleifera, bitter leaf (Veronica amygdalina), and Psidium guajava have been utilized for the biosynthesis of metal NPs [296,297]. In Pakistan, some studies have reported antifungal and AF inhibitory activities of green synthesized nanoparticles. Asghar et al. [298] prepared iron (Fe), copper (Cu), and silver (Ag) nanoparticles (NPs) from a Syzygium cumini leaf extract. The obtained metal NPs showed excellent antimicrobial activities against A. flavus and A. parasiticus. Furthermore, production of AFs was also significantly inhibited; no AFs (100% inhibition) were produced when treated with 100 µgmL −1 of Ag-NPs in both strains in vitro, while 43−49% and 76−80% reductions were achieved using Fe-and Cu-NPs, respectively.

Conclusions
This review summarizes AF-producing fungi, agricultural and food products prone to contamination with AFs, exposure and risk assessment of AFs, related legislations, and current AF prevention and mitigation options, with a focus on AF problems encountered in Pakistan. AFs are a significant problem in Pakistan due to the nation's hot and humid climate and the substantial role of agriculture in its economy. In addition, the high rates of hepatitis viral infections in Pakistani people means that the population is more vulnerable to AF-related cancers. Numerous studies have found agriculture crops and food products produced in Pakistan were contaminated with AFs at levels above the US and EU permissible limits. Exporting food products with high levels of AFs results in trade restrictions. If food goods planned for export have been found to contain high levels of AFs, it can be safely assumed that local consumers are at danger because of the availability of highly contaminated products in local markets that have not been tested for AFs. There is a critical need to raise awareness among the population regarding the occurrence of these carcinogenic toxins in food commodities and their potential risks to humans and animals. Consumption of AF-contaminated feed has a negative impact on livestock performance, including decreased productivity and weight gain. As a result, the livestock industries in Pakistan experience substantial economic losses. Most of the AF-related studies have been conducted in the Punjab province of Pakistan. More comprehensive nationwide investigations are required to properly assess the AF contamination issues more broadly. To compete on the international market, Pakistan must improve compliance to its standards in food quality control related to AFs. The regulatory authorities should take this issue of AF contamination into account for consumer safety and the nation's economy, and proper control measures should be implemented and the food quality control system upgraded.