Food and Chemical Toxicology 180 (2023) 114006

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Food and Chemical Toxicology

journal homepage: www.elsevier.com/locate/foodchemtox

Aflatoxins posing threat to food safety and security in Pakistan: Call for a
one health approach
Waseela Ashraf a, b, e, Abdul Rehman a, *, Masood Rabbani c, Waseem Shaukat d, Jia-Sheng Wang b
a
Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences Lahore, 54000, Pakistan
b
Department of Environmental Health Science, The University of Georgia, Athens, GA, USA
c
Institute of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan
d
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, T2N4N1, Alberta, Canada
e
Health Services Academy, Islamabad, 44000, Pakistan

A R T I C L E I N F O A B S T R A C T

Handling Editor: Dr. Bryan Delaney Aflatoxins are among the most important mycotoxins due to their widespread occurrence and adverse impacts on
humans and animals. These toxins and/or their metabolites cannot be destroyed with cooking or boiling
Keywords: methods. Therefore, consumption of aflatoxin-contaminated food may lead to impaired growth, compromised
Mycotoxin immunity, stomach and liver cancer, and acute toxicity. These adverse effects along with food wastage might
Commodities
have detrimental consequences on a country’s economy. Several studies from Pakistan reported a high preva­
Contamination
lence of aflatoxins in food and feed commodities (Range; milk = 0.6–99.4%, cereals, and grains = 0.38–41%,
Toxicity
Food safety animal feed = 31–100%). Notably, Pakistan reported very high figures of impaired child growth—stunted 40.2%,
Policy framework wasted 17.7% and underweight 28.9%—that could be associated with the higher aflatoxin prevalence in food
items. Importantly, high aflatoxins prevalence, i.e. 100%, 69% and 60.5%, in children has been reported in
Pakistan. Food and feed are more prone to aflatoxin contamination due to Pakistan’s hot and humid climate;
however, limited awareness, inadequate policy framework, and weak implementation mechanisms are the major
obstacles to effective control. This review will discuss aflatoxins prevalence, associated risk factors, adverse
health effects, required regulatory regime, and effective control strategies adopting the One Health approach to
ensure food safety and security.

1. Introduction
Safe and adequate food is crucial for physical and cognitive devel­
opment, immune system function and overall health. Food safety and
security are key aspects of ensuring a healthy life for humans. World
Health Organization (WHO) defined food safety as the need for people to
have food that is free of harmful organisms or toxic substances. Food
security is the accessibility, affordability, and availability of safe and
nutritious food in sufficient quantities to meet the dietary needs of all
individuals. Food safety breaches cause more than 200 diseases and food
insecurity can lead to poverty, social unrest, conflict, hunger, malnu­
trition, and stunting (Tamene et al., 2022). It can also lead to over­
consumption of low-quality, unhealthy foods, contributing to a rise of
diet-related diseases i.e., obesity, diabetes, and heart disease. There­
fore, food safety and security are of utmost importance with many
advantages to people. They help in social, political, and economic sta­
bility, protect public health, prevent foodborne disease, support inter­
national trade, build consumer confidence, and safeguard agricultural
practices (Ramirez-Hernandez et al., 2020).
Contamination of food with harmful bacteria, viruses, toxins or
foreign substances and intentional adulteration, malpresentation, or
substitution of food products sparks a significant concern. Among them,
edible contamination with toxic substances such as aflatoxins is a serious
public health issue. It seriously threatens agricultural commodities and
food in terms of nutritional value and has detrimental effects on the
economy (Kumar et al., 2017). Aflatoxin contamination is a global issue
with a relatively higher prevalence in hot and humid regions like
Pakistan. Aflatoxin poses a real threat to food safety and security and
destroys approximately 25% of the world’s food crops and every year
420,000 people die from consuming contaminated food causing an

* Corresponding author. Department of Epidemiology and Public Health, University of Veterinary and Animal Sciences, Syed Abdul Qadir Jilani Road (Out Fall
Road), 54000, Lahore, Pakistan.
E-mail address: abdul.rehman@uvas.edu.pk (A. Rehman).

https://doi.org/10.1016/j.fct.2023.114006
Received 17 March 2023; Received in revised form 26 July 2023; Accepted 26 August 2023
Available online 29 August 2023
0278-6915/© 2023 Elsevier Ltd. All rights reserved.
W. Ashraf et al.
estimated loss of 33 million healthy life years (WHO, 2022).
Aflatoxins are fungal toxins produced by members of the genus
Aspergillus (A.); among these A. flavus, A. parasiticus, A. niger and
A. nomius are predominant aflatoxin-producing species. Several
different types of aflatoxins are produced; however, four types, namely
aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), and afla­
toxin G2 (AFG2) are significantly important due to their potential impact
on human and animal health. Among these AFB1 is produced abundantly
by A. flavus and A. parasiticus (Perrone and Gallo, 2017), and is the most
potent genotoxin classified as a class 1 carcinogen by the International
Agency for Research on Cancer (IARC, 2021). These toxins are heat
resistant and are decomposed between 237 and 306 ◦ C. These envi­
ronmental contaminants are naturally present in soil and ubiquitously
distributed throughout the world. Aflatoxin-producing fungi can grow
on a wide range of substrates like soil, decaying vegetation, hay, silage,
wheat, walnut, corn, cotton, peanuts, spices, stored food, and feed (Abo
Nouh et al., 2020).
Several studies have been conducted to reveal the carcinogenic and
mutagenic abilities of aflatoxins and to understand their mode of action.
It has been observed that aflatoxins decrease protein synthesis leading to
birth defects as well as affecting the growth of young children (da Silva
et al., 2021). The upper limit of acceptable levels for aflatoxins in
various food and feed commodities is set to be a minimum in order to
reduce the exposure of aflatoxins in humans ((EFSA), 2020).
Despite the injurious effects of aflatoxins on health, Pakistani food
and feed commodities are repeatedly found to be contaminated with
these toxins that poses a serious threat to the animal and human pop­
ulation in Pakistan (Ashiq, 2015; Aslam and Wynn, 2015). This review
not only focuses on the occurrence of aflatoxins reported in various food
and feed items for human and animal consumption in Pakistan but also
discusses the adverse effects of aflatoxins on health and economy, the
important factors contributing to the increased incidence of aflatox­
icosis, and the major hurdles towards reducing the burden of aflatoxins.
Finally, it suggests a conducive policy framework required for the
effective control of aflatoxicosis with a special focus on Pakistan.
2. Background of aflatoxin
After ingestion of aflatoxin-contaminated food, AFB1 is processed by
liver cytochrome P415 enzymes. The first by-product of AFB1 is 8, 9-
epoxide which is highly reactive and can either bind with genetic ma­
terial to form guanine adducts or with serum protein to yield lysine
adducts (Taranu et al., 2020). Besides this, AFB1 is oxidized into some
other hydroxylated metabolites such as AFM1 and aflatoxin Q1 (AFQ1);
de methylated metabolic product aflatoxin P1 (AFP1), and a reduced
metabolic product aflatoxicol. After conjugation, these metabolites are
excreted through urine and bile. Chronic exposure to aflatoxins causes
the production of reactive metabolites which may stimulate or inacti­
vate various epigenetic mechanisms leading to the development of
various types of cancers. AFM1 also has high genotoxic activity and
therefore has been classified as a class 2B carcinogen (IARC, 2021).
Besides urine and feces, AFM1 is also secreted in the milk of both humans
and animals.
2.1. Consequences aflatoxin incurred
2.1.1. Effects on human health
Consumption of aflatoxin-contaminated cereals, meat, eggs, milk,
and milk products and aquaculture may cause acute or chronic toxicity
(El-Sayeda et al., 2022). At high levels of exposure, aflatoxin causes
disease (aflatoxicosis) with acute onset of abdominal pain, vomiting,
pulmonary edema, and liver necrosis or even sometimes it can be fatal
(Barac, 2019). Outbreaks of acute aflatoxicosis have been reported in
Kenya, India, Malaysia, and Tanzania (Kamala et al., 2018) with fatality
rates as high as 40% in Kenya. Chronic exposure may cause hepatocel­
lular carcinoma (HCC), suppression of the immune system, poor
2
Food and Chemical Toxicology 180 (2023) 114006
reproductive performance, growth retardation, and poor absorption of
nutrients in children (Fig. 1). Global research shows that about 150,000
cases of liver cancer annually and 3–36% of population attributable risk
of child stunting is attributed to aflatoxins alone (Rasheed et al., 2021).
Aflatoxins significantly reduce humoral as well as cell-mediated
immunity of the individual resulting in an increased risk of acquiring
various infectious diseases (Githang’a et al., 2019). If a mother is taking
an aflatoxin-contaminated diet, it can cross the placental barrier and
adversely affect the growth of the fetus. Similarly, a newborn can get
exposed to aflatoxin through breast milk resulting in poor growth such
as stunting, wasting, or being underweight (Smith et al., 2017). Several
studies from Africa have revealed a significant association between the
intake of aflatoxin-contaminated diet and poor child growth and the
authors found a significantly higher (30–40%) mean concentration of
aflatoxins’ metabolites in children who were stunted and underweight
(Rasheed et al., 2021). According to the 2018 Pakistan National Nutri­
tional Survey, half of the children under five years of age in Pakistan
suffered from stunting or wasting which is the third highest rate in the
world (UNICEF Pakistan, 2019). Although childhood stunting has
significantly dropped globally from 40% in 1990 to 21.3% in 2019,
however, in Pakistan it further deteriorated during this period, from
36.3% in 1990 to 40.2% in 2019, which is alarming and remains at a
global critical level (UNICEF, 2020; UNICEF Pakistan, 2019; Vaivada
et al., 2020). Importantly, Asia has shown a dramatic decrease during
this period, i.e. 49%–21.8% and is expected to reach 20% in 2020 (SUN,
2020). Despite multiple initiatives such as Infant and Young Children
Feeding (IYCF) and Pakistan Integrated Nutrition Strategy (PINS) by the
WHO and UNICEF to improve children’s growth and development, the
situation is not much improving. However, Pakistan is committed to
achieving the Sustainable Development Goals (SDGs) through “zero
hunger” by 2030 (Ponum et al., 2020; UnitedNations, 2018).
Continuous ingestion of aflatoxins-contaminated food is now
considered as the leading cause of liver cancer (Magnussen and Parsi,
2013). It increases the severity of liver problems in patients infected
with hepatitis B virus (HBV) and could also be associated with a higher
prevalence of cancer in developing countries. Ingestion of aflatoxins also
increases the chance of occurrence of HCC. Numerous epidemiological
studies from different parts of the globe have reported a strong associ­
ation of the presence of aflatoxin in blood with the occurrence of HCC
and liver cirrhosis (Davwar et al., 2023; Gong et al., 2016). Alarmingly,
in our recent study, we detected AFM1 in 65.5% of urine samples and
AFB1 in 100% of the serum samples collected from children attending a
tertiary care hospital in Lahore, Pakistan (Ashraf et al., 2022a; 2022b).
Another study from Pakistan found that 69% of the study participants
were positive for AFM1 (Xia et al., 2020). Notably, this high prevalence
of aflatoxicosis in Pakistani population reported by Ashraf and Xia,
relative high burden of liver diseases (Cheemerla and Balakrishnan,
2021), and aflatoxins contaminated diet in Pakistan and their comor­
bidity can exacerbate the problem, therefore special attention is needed
to design a comprehensive control strategy.
2.1.2. Effects on animal health
In ruminants, aflatoxins have adverse effects not only on the liver
and kidney function, but also cause a decrease in feed intake and milk
production, poor reproductive performance, and growth impairment in
young and lactating animals (Peles et al., 2019). In poultry, aflatoxins
can cause tissue damage, adversely impair uptake of essential nutrients,
affect all-important production parameters as well as poor egg and meat
quality and increase susceptibility to various infectious diseases by
suppressing immunity that may lead to higher mortality (Fouad et al.,
2019). Similar effects have been observed in aquaculture (Mahfouz and
Sherif, 2015; Mwihia et al., 2018) to a lesser extent, hunters usually use
poor quality grains that can be contaminated with aflatoxins and throw
them in the field which increases the contamination level and may have
adverse effects on wildlife and migratory birds (Lawson et al., 2020).
Aflatoxin contamination leads to a loss in animal productivity which
W. Ashraf et al.
Fig. 1. Sources of aflatoxins exposure and its consequences on human and animal health.
results in economic losses to the country. Currently, Pakistani livestock
yield is amongst the lowest in the world which is around 3–4 L/day per
animal. Although multiple reasons could be associated with the low milk
production, including genetics, poor nutrition, worm infestation and
various diseases and aflatoxins level in animal feed, however the effects
of later on milk production have not been assessed in Pakistani breeds
yet (Arnold and Gaskill, 2023; Jiang et al., 2021). If the animal feed is
contaminated by AFB1, approximately 0.6%–6.2% of the toxin is
metabolized by the liver and excreted into milk in the form of AFM1.
Excretion of AFM1 depends upon the genetics, stage of lactation, and
production of milk, milking process, and health status of the animal
(Sumon et al., 2021). Due to the high stability of AFM1, Ultra-high
Temperature (UHT) and pasteurization procedures remain unsuccess­
ful in destroying it (Pierezan et al., 2022). For this reason, milk
contamination can only be prevented by testing the milk and its prod­
ucts on regular basis and by providing mycotoxin-free feed to animals
which is an actual challenge both for the livestock feed industry and
livestock farmers. As milk is an essential component of our daily diet,
therefore aflatoxin-contaminated milk may lead to chronic exposure to
AFM1 which is a serious health hazard (Gong et al., 2016).
2.1.3. Economic losses
The aflatoxin-producing molds invade crops in the field and grow on
stored food/feed ingredients and deteriorate the food/feed quality that
leads to direct economic losses to the agriculture, livestock, and fisheries
sectors. These losses mainly include reduced marketable volume, low
3
Food and Chemical Toxicology 180 (2023) 114006
price, rejection of shipments, and inadmissibility in the international
market resulting in reduced revenue and heavy economic losses (Patel
et al., 2021). The productivity and nutritive value of
aflatoxin-contaminated crops are relatively lower. More importantly,
aflatoxicosis is associated with indirect losses through reduced produc­
tivity, increased incidence and severity of various diseases including
liver cancer, decreased healthy/productive life years, increased diag­
nostic and healthcare costs, and mortality in humans as well as animals
(Peles et al., 2019; UnitedNations, 2018). As there is no systematic work
measuring the impact of aflatoxins on human, animal, and plant health
and their association with aflatoxin contamination in various food and
feed items, it is hard to estimate the extent of economic and health losses
accompanied by consuming aflatoxin-contaminated food and feed.
2.2. Aflatoxin detection methods
Several methods are available for the detection of aflatoxins, how­
ever, two of them are commonly used, i.e., High-Performance Liquid
Chromatography (HPLC) and Enzyme-Linked Immunosorbent Assay
(ELISA). Although HPLC provides a complete quantification of the toxin
in question, this technique is relatively expensive and used for regula­
tory control in official laboratories, while ELISA—which is compara­
tively cheap and provides rapid detection—analyzes samples
qualitatively and semi-quantitatively and is commonly used in industrial
units. With the advancement in technology, scientists continue to
develop new methods for aflatoxin detection such as dip-stick kits,
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

electronic noses, hyperspectral imaging, molecularly imprinted poly­ and feed commodities. After the successful control, harmonization of
mers, and aptamer-based biosensors which can potentially be used in tolerance levels is taking place in some free trade zones, e.g., in the
remote areas in more convenient manners. Commonly, blood samples United States, all 50 states have the same standards of food safety
are tested for the presence of AFB1-albumin adduct (which remains in developed by the Food and Drug Administration (FDA). Similarly,
the blood for weeks to months), while urine samples are used to detect Argentina, Brazil, Paraguay, and Uruguay have harmonized their
AFM1 (which can only be detected within 24 h of exposure). Strengths food-safety regulations under MERCOSUR—a free-trade zone treaty.
and weaknesses of different detection methods are presented in Table 1 Moreover, the Association of Southeast Asian Nations (ASEAN),
(Arora, 2018; Wacoo et al., 2014). comprising 10 countries (Darussalam, Cambodia, Indonesia, Lao,
Malaysia, Myanmar, Philippines, Singapore, Thailand, and Viet Nam),
3. Global initiatives for regulatory & compliance enforcement to and six states of the Gulf Cooperation Council in the Middle East (Sau­
control aflatoxin in food & feed di-Arabia, Bahrain, Oman, Kuwait, the United Arab Emirates, and
Qatar), have also developed harmony in their food-safety standards.
Keeping food free from aflatoxins is one of the major food safety Legislation for aflatoxin control has been enforced in most parts of the
challenges. Aflatoxin limits were first set after its discovery in 1960; world (~90% of the world’s population) but some countries, including
however, setting limits is a complex procedure and involves many fac­ Pakistan, still need enforcement of this legislation (Aslam and Wynn,
tors including the demands of interested parties. World Health Organi­ 2015; Food Safety Magazine, 2019).
zation (WHO) with the support of the Food and Agriculture
Organization (FAO), Codex Alimentarius Commission (CAC), and Joint 4. Aflatoxin from Pakistan’s perspective
Expert Committee on Food Additives (JECFA) is continuously working
on food safety regulations. JECFA is an advisory body and provides the 4.1. Regulatory framework in Pakistan
information on toxicity and exposure assessment of toxins which helps
in developing standards while CAC develops international standards for In Pakistan, food safety standards are regulated by federal as well as
food and feed safety. Currently, there are 188 member countries and provincial governments. The federal government applies FDA and CAC
one-member organization of CAC (FAO & WHO, 2020). Many developed standards through the Pakistan Standard and Quality Control Authority
and developing countries around the world have successfully adopted (PSQCA) and is responsible for its enforcement in the federal territory
the guidelines and regulations for tolerable levels of aflatoxin in food and international entry/exit points including all air- and seaports.
PSQCA is a national standardization body enforced in 1996 and works
for the federal government under the Ministry of Science and Technol­
Table 1
ogy. All manufacturing and export companies operating in Pakistan
Strengths and weakness of different aflatoxin detection methods.
must be registered with PSQCA to ensure compliance with food safety
Detection Method Strengths Weaknesses laws. Being a member of CAC, PSQCA follows the same standards,
HPLC Provides complete Relatively Expensive however, for the whole as well as skim milk, it has set a different
quantification of maximum tolerable limit of AFM1 (10 μg/kg), which is 20 times higher
aflatoxins
than the CAC permissible limit of 0.5 μg/kg (Table 2) (Aslam and Wynn,
Used for regulatory Requires skilled personnel and
control in official sophisticated equipment
2015). PSQCA serves as the central collaborating body for all national,
laboratories international, and regional organizations and institutions such as ISO,
Requires sample preparation CAC, and WTO while provincial food authorities regulate standards of
and extraction food articles, their manufacturing, storage, distribution, and sale in
ELISA Comparatively cheap Prone to false positives and
respective provinces. Following the devolution to the provinces through
negatives due to cross-reactivity
and matrix inference the 18th amendment in 2010, Pakistan’s food industry is badly affected
Provides rapid detection Requires sample preparation because food safety standards are now locally regulated by the provin­
and extraction cial governments and provinces developed their own food authorities
Analyzes samples
which involve the Food Regulatory Authorities, Health, Plant Protec­
qualitatively and semi-
quantitatively
tion, and livestock and dairy development departments. For example,
Commonly used in the Punjab Food Authority (PFA), which came into force in 2011, has the
industrial units operational jurisdiction of the whole of Punjab and regulates standards
Dip-stick kits Simple, portable, and user Limited sensitivity and of food articles, their manufacturing, storage, distribution, and sale in
friendly specificity
Punjab province. However, its regulations are not synchronized with the
Can be used in remote Cannot quantify the aflatoxins
areas standards set by federal authorities. Despite the active working of PFA in
Electronic noses Fast, non-destructive, and Affected by environmental Punjab, progression towards aflatoxin control is still very slow. PFA has
cost-effective factors such as humidity and updated aflatoxins regulations in 2018, while Khyber Pakhtunkhwa
temperature Food Safety and Halal Food Authority has updated their food regulations
Can detect multiple toxins Requires calibration and
simultaneously validation
Hyperspectral Non-invasive, non- High cost of equipment and data
imaging contact, and non- processing Table 2
destructive Comparison of maximum permissible levels of aflatoxins in various food com­
Can provide spatial and Limited by sample
modities as set by Pakistan and different international organizations.
spectral information on heterogeneity and background
the sample noise Food PSQCA EFSA FDA CAC
Molecularly Highly selective and Difficult to synthesize and
Liquid milk 0.5 ppb 0.05 ppb 0.5 ppb 0.5 ppb
imprinted stable optimize
Milk powder 10 ppb 0.05 ppb 0.5 ppb 0.5 ppb
polymers Can be reused multiple Low binding capacity and slow
Food grains & roasted nuts (all) 20 ppb 2–12 ppb 20 ppb 15 ppb
times mass transfer
Animal feed 50 ppb 20 ppb 20 ppb 20 ppb
Aptamer-based High affinity and Susceptible to nuclease
biosensors specificity degradation and ppb = parts per billion.
conformational changes PSQCA=Pakistan Standards and Quality Control Authority; EFSA = European
Easy to modify and Requires sophisticated
Food Safety Authority; FDA=Food and Drug Administration; CAC=Codex Ali­
functionalize detection systems
mentarius Commission.

4
W. Ashraf et al.
in 2017 but they lack a setting of aflatoxin permissible level (KPKFood,
2018). Similarly, Sindh Food Authority updated its regulations in 2016
which are silent on aflatoxin permissible levels (Sindh, 2016). The food
industry in Pakistan is facing a problem of the growing divergence be­
tween food standards across the country and there is a dire need to
harmonize the standards (Kowalska and Manning, 2021).
4.2. Factors contributing towards the increased incidence of aflatoxin in
Pakistan
Pakistan is located in the sub-tropical arid zone, with high temper­
atures averaging 23.9 ◦ C and a double maximum rainfall pattern (489
mm). These hot and humid climatic conditions are quite favorable for
the production of AFs (Asghar et al., 2014). The average temperature for
the growth of A. parasiticus ranges between 12 and 42 ◦ C, whereas
A. flavus is more thermotolerant with a temperature range of 12–55 ◦ C
(Wikipedia, 2019). Aspergillus flavus growth occurs at different moisture
levels in different crops. The optimal moisture level for the growth of
A. flavus in starchy cereals is 13–13.2%, for soybean, it is 11.5–11.8%,
while for the other crops, it is 14%. Notably, A. flavus has been reported
as the most common fungal species in Pakistan (Table 3).
Continually climate change is causing an increase in heat and hu­
midity in Pakistan which will lead to longer summers and provide a
favorable environment for fungi production. According to Pakistan
Meteorological Department, a rising trend of environmental tempera­
ture by 0.47 ◦ C was observed from 1961 to 2007 (Chaudhry, 2017) that
directly influenced the expression levels of regulatory genes (aflR and
aflS) and hence aflatoxin production by A. flavus and A. parasiticus which
are common contaminants in the agriculture sector. Climate change also
directly influences host susceptibility which favors fungal colonization
resulting in increased aflatoxin production (Liu et al., 2017). With the
consequential rise in the prevalence of aflatoxin due to favorable cli­
matic conditions, many products used for human food and animal feed
are at risk of aflatoxin contamination.
Besides this, several factors related to poor crop management prac­
tices also contribute to the increased prevalence of aflatoxins. In the pre-
harvest period, the presence of aflatoxin-producing fungi could be
influenced and potentiated by different factors such as plant genetics as
some varieties of plants are resistant to aflatoxins while others are not
(Daou et al., 2021). Sometimes, during the growing and harvesting
stages, toxin production and its evolution get enhanced by wrong agri­
cultural practices such as the application of poor quality fungicides and
pesticides, the use of open-pollinated varieties for cultivation, the con­
tact with aflatoxin-producing fungi or their spores, stress due to weather
conditions and climate during planting and growing and, finally,
wounding of developing crops by insect, birds, mammals or mechanical
harm which all increases the susceptibility of plant for fungal infection.
Delayed harvest is also associated with an increased concentration of
aflatoxins. Post-harvest conditions of plant stress due to insect
Table 3
Fungal species isolated from different food/feed commodities in Pakistan.
Fungal species Isolated Commodity Reference
A. flavus, A. niger, A. oryzae, A. fumigatus, Chickpea Tania et al. (2022)
A. ochraceous, A. nidulenes seed
A. flavus, A. niger Nuts (Mustansir Abbas
et al., 2019)
Aspergillus species Chili (Iqbal et al., 2011)
A. flavus Chili Paterson (2007)
A. niger, A. ochraceous Maize Saleemi et al. (2012)
Aspergillus.flavus Maize Shah et al. (2010)
A. flavus, A. niger, A. parasiticus, A. Poultry Feed Sana et al. (2019)
fumigatus, A. terreus
A. flavus Poultry Feed Iram et al. (2019)
A. flavus Poultry Feed Rashid et al. (2012)
A. parasiticus Poultry Feed (M. Afzal et al.,
1979)
5
Food and Chemical Toxicology 180 (2023) 114006
infestation and periods of drought, soil conditions like high organic
content and soil moisture, use of inappropriate chemical agents, and
poor storage conditions are the important factors that produce a favor­
able environment for the growth of molds (Kumar et al., 2021).
Above all, inadequate knowledge among the general public espe­
cially the food and feed producers, people involved in the supply chain,
and consumers about the aflatoxin contamination is one of the most
important factors contributing to the higher prevalence of aflatoxicosis
in humans as well as animals. Lack of awareness among farmers led to
poor pre-harvest, post-harvest, and storage crop management practices.
In Pakistan, it is a common practice that many farmers add wasted
pieces of bread contaminated heavily with molds as well as aflatoxins to
concentrated feed such as cotton seed cake which not only affects animal
health but also contaminates the meat and milk of those animals who
consume such feed. Furthermore, many food products are not properly
packed and labeled especially when it comes to the date of expiry which
is a big challenge in reducing aflatoxin contamination in the food items.
Another important factor is the public behavior and affordability as most
of them prefer to buy open products as compared to packed items due to
the difference in prices, freshness, and lack of knowledge about the
possibility of contamination. Moreover, the majority of the people who
have some knowledge about food contamination think that all the
contaminants are destroyed when they cook food which is not true, at
least, for aflatoxins. However, in the past, this issue could not gain
enough attention from responsible authorities resulting in inadequate
facilities in the laboratories—equipment/consumables as well as skilled
laboratory workers—for the detection of aflatoxins.
4.3. Major hurdles to reduce aflatoxin in Pakistan
The problem of aflatoxin contamination of food is not properly
placed on the food security and safety agenda in Pakistan’s health and
livestock policy (MNFSR, 2015). One of the major problems is limited
awareness about aflatoxin contamination in food and feed and its po­
tential impact on health and economics. The main reason for a very low
level of awareness among the general public and medical practitioners is
that aflatoxins, mostly, do not cause acute problems and therefore could
not get the attention of the food regulatory and health departments.
Secondly, there is a deficiency in the surveillance system and testing
facilities due to the high costs involved in the detection of aflatoxins.
Thirdly, there is a scarcity of local systematic research showing the
adverse impacts of aflatoxin consumption on the health of the Pakistani
population leading to a lack of adequate attention by the authorities.
Although childhood stunting and liver problems have been very com­
mon in Pakistan for many years and aflatoxin presence in different food
commodities has been reported in many studies, yet researchers have
not tried to find out the association and possible causality between these
two. In absence of such evidence-based research, aflatoxins continue to
be a non-significant issue for the policymakers. Lastly, deficiencies in
law enforcement by the responsible authorities against aflatoxin
contamination have further exacerbated this problem.
Milk quality testing at different levels of supply chain—starting from
production to the sale points/retail shops—is the most ignored area in
Pakistan. Many of the large milk processing companies did not routinely
test samples for the presence of aflatoxins in the past, though this trend
has been changing for the last few years (Aslam and Wynn, 2015). Also,
aflatoxin testing is mostly targeted toward export quality products and
there is very limited quality control testing for local consumers (Yunus
et al., 2020). Furthermore, there are no clear rules and guidelines to
handle and treat the wasted and rejected food items contaminated with
aflatoxins as these rejected items can be incorporated into the local
markets for human and/or animal consumption (Ahmad and Syed,
2017).
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

5. Prevalence of aflatoxin among various food commodities in
Pakistan
During the last two decades, several studies have reported aflatoxins
contamination in various types of food and feed commodities in most
parts of the country (Hassan et al., 2020; Khatoon et al., 2012; Manzoor
et al., 2018; Razis et al., 2020; Saleemullah et al., 2006).
5.1. Milk
Pakistan is the third-largest milk-producing country in the world and
milk is one of the main sources of nutrition for the Pakistani population
(Akbar et al., 2020). Since milk is an essential food item and is being
consumed daily in every household, therefore, safe milk is an absolute
requirement for public health. Compromised milk quality due to the
presence of adulterants (Lateef et al., 2009), aflatoxins (Sadia et al.,
2012), and antimicrobial residues (Khaskheli et al., 2008), may have
serious implications on consumers’ health. Aflatoxin-contaminated milk
may pose serious public health implications. The primary source of af­
latoxins in the milk is aflatoxin-contaminated feed offered to the ani­
mals. The AFB1 and AFB2 present in the feed that animals consume are
metabolized within the animal body and are converted to AFM1 and
AFM2, respectively, which are then secreted through the mammary
glands in the milk. Although several researchers have reported a high
prevalence of aflatoxin contamination in milk and milk products in
Pakistan (Ahmad et al., 2019; Asghar et al., 2018; Gill et al., 2022;
Hussain and Anwar, 2008; Iqbal et al., 2014a,b), however, there are
hardly any studies that have explicitly investigated the direct impact of
aflatoxins on human health in Pakistan (Table 4). This is one of the main
reasons why this issue has not been able to acquire adequate attention
from the policymakers in Pakistan despite its significant public health
relevance.
The dairy supply chain in Pakistan is predominantly comprised of
loose milk whereby the share of processed and packaged milk and milk
products is merely 3% of the national milk production (Akbar et al.,
2020), while the remaining 97% of the milk is delivered and consumed
in raw form. The lack of traceability in the dairy value chain is also one
of the major issues that hinder the control of aflatoxins and other con­
taminants. Various studies have been conducted in Pakistan in different
regions to estimate the prevalence of aflatoxins in different types of milk
and milk products. Raw milk is more contaminated with AFM1
compared to processed milk as various studies in different cities have
reported that a significant proportion of raw milk samples were
exceeding the CAC limit, e.g. 6.3% in Lahore, 7.1% in Multan (Ahmad
et al., 2019), 17% in Jhang (Younus et al., 2013), 32.7% in Karachi
(Asghar et al., 2018), 45% in Hyderabad (Jawaid et al., 2015), 87.2% in
Kasur, Okara and Pakpattan (Aslam et al., 2016), 91.9% in Raw­
alpindi/Islamabad (Yunus et al., 2019), etc. Another study has reported
that 3% of the raw milk samples collected from 14 districts of Punjab
exceeded the CAC limit while 99.4% of samples exceeded the European

Table 4
Prevalence of aflatoxin contamination in milk and milk products in different regions of Pakistan (Permissible level = 0.5 ppb).
Area covered n Sampling Source Type Method Max. Conc. Positive Reference
(ng/L) (%)

Lahore 120 Local shops, dairy farm Butter, cream, cheese, HPLC 2.17 22.5 Gill et al. (2022)
yogurt
Islamabad, Karachi, Lahore, Quetta, 372 Peri-urban dairy farms Raw milk ELISA 15994 85.2 Yunus et al.
Peshawar, Muzaffarabad, and Gilgit (2020)
a
Different regions of Punjab 240 Dairy farms Raw milk ELISA 0.51 53 Akbar et al.
(2020)
Islamabad, Rawalpindi 252 Markets UHT, Pasteurized, ELISA 1.536 48.95 Yunus et al.
Raw, Powder (2019)
Lahore, Multan 210 NP Raw and processed ELISA 0.554 91 Ahmad et al.
(Lahore) (2019)
92
(Multan)
Different areas of Karachi 156 Local markets shops Raw Milk ELISA 3.090 32.7 Asghar et al.
(2018)
Kasur, Okara, Pakpattan 485 Informal marketing chains Raw milk ELISA 7.28 87.2 Aslam et al.
(2016)
Different areas of Hyderabad 84 Not mention Branded and non- ELISA 0.76 29.7 Jawaid et al.
branded (2015)
Major Districts Punjab 107 Urban and rural Farmhouses Raw HPLC 0.98 11.5 Iqbal et al.
(2014a,b)
Chakwal, Sargodha, Jhang, Sahiwal, 546 Different milking sites, Dairy Raw HPLC 0.845 58 § Iqbal et al.
Faisalabad, Sheikhupura, Gujranwala farmhouses (2013a)
Jhang N/ Urban milk shops, Household and Raw ELISA NP 17 Younus et al.
A Dairy farms near drain (2013)
Central areas of Punjab 188 Milking sites, small and large Raw Milk, UHT HPLC 0.89 24.4 § (Shah et al.,
dairies and dairy farmhouses 2010)
Chakwal, Sargodha, Jhang, Sahiwal, 546 Different milking sites, dairy Products HPLC 0.6158 31.2 § Iqbal et al.
Faisalabad, Sheikhupura, Gujranwala farmhouses (2014a)
Central areas of Punjab 245 Milking sites, small and large Yogurt, Butter, Ice HPLC 0.88 20 § Iqbal et al.
dairies Cream (2013a)
36 Districts Punjab 138 Local shops, household and large- Sweet (Burfi) HPLC 1.5 78 § Sadia et al.
scale farms (2012)
36 Districts Punjab 232 Local shops, household and large- Raw HPLC 1.9 32.3 § Sadia et al.
scale farms (2012)
Punjab, KPK (Major cities) 593 Farmhouses Raw IAC, 0.35 16.3 § (Shah et al.,
HPLC 2010)
Major cities Punjab 356 Milking sites and farmhouses Raw HPLC 0.98 37 Asi et al. (2012)
14 Districts of Punjab 168 Different dairies Raw IAC, 0.7 99.4 Hussain and
HPLC Anwar (2008)
Central areas of Punjab 480 Milking sites at urban, semi urban Raw IAC, NP 0.6 Hussain et al.
and rural areas HPLC (2008)
a
Mean values are presented, NP = Not provided, § followed EU standard.

6
W. Ashraf et al.
limit (Hussain and Anwar, 2008). One more study found that 8% and
15% of the raw milk samples from rural and urban dairy farms in Punjab
respectively exceeded the CAC limit. In comparison to this, when UHT
milk was assessed for AFM1 contamination relatively lower prevalence
was reported, for example, none of the 60 samples of UHT milk and UHT
tea whitener from Punjab were found to exceed the CAC limit for AFM1
(Ahmad et al., 2019) while 12.9% and 22.2% of the UHT milk samples
from Islamabad (Yunus et al., 2019) and Hyderabad (Jawaid et al.,
2015) respectively exceeded CAC limit. Similarly, 41% of the pasteur­
ized milk samples from Islamabad were reported to have exceeded the
permissible limit by CAC (Yunus et al., 2019).
Some studies have also compared the prevalence of AFM1 in milk in
different seasons and have reported contradictory findings. For example,
various studies have reported that AFM1 contamination was higher in
winters compared to summers (Asi et al., 2012; Iqbal et al., 2014a,b)
while others reported that milk samples were more contaminated with
AFM1 in the summer season compared to winter (Asghar et al., 2018).
Furthermore, a comparison between raw milk samples from rural dairy
farmers compared to peri-urban dairy farmers has revealed higher
contamination of milk produced at peri-urban farms compared to rural
dairy farms in various studies (Hussain and Anwar, 2008). This may be
attributed to the higher use of (low quality) concentrates in peri-urban
dairy farming compared to rural dairy farming where green forage
constitutes the higher component of the overall animal feed. Aflatoxin
contamination in dairy products has also been reported in some studies.
For example, 29%, 46%, 43%, and 23% samples of yogurt, white cheese,
cheese cream, and butter respectively were found to contain AFM1
above European standards, however, it remains unclear as to what
proportion of these samples exceeded CAC limit for AFM1 (Iqbal et al.,
2014a,b).
Furthermore, the consumers of raw milk are exposed to much higher
AFM1 levels compared to processed milk consumers—11.9 vs 4.5 ng per
kg body weight per day (Yunus et al., 2019). This study also revealed
that the mean level of AFM1 in UHT milk, pasteurized milk, and raw
milk samples was 254.9, 939.5, and 1535 ng/L, respectively. A more
recent and comprehensive study assessing prevalence of AFM1 in milk
originating from peri-urban dairy farms in big cities of all provinces of
Pakistan reported that majority of milk samples from Karachi (100%),
Lahore (96.4%), Islamabad (97.1%), Peshawar (85.5%), Quetta (91.1%)
and Muzaffarabad (100%) exceeded the CAC limit for AFM1 (Yunus
et al., 2020). However, only 7.7% of samples from Gilgit exceeded this
limit according to this study. The worst part of this exacerbating situa­
tion is that the mean AFM1 levels in milk samples from these cities were
3–10 times higher than the upper tolerable CAC limit for AFM1, i.e. 500
ng/L, e.g. Karachi (2435.9 ± 1740.3), Lahore (4842.6 ± 4310.5),
Islamabad (4935.3 ± 3468.7), Peshawar (1930.8 ± 1626.9), Quetta
(1623.5 ± 1242.5), Muzaffarabad (4436.1 ± 4068.2) and Gilgit (92.5
± 178.6) (Yunus et al., 2020). This is an alarmingly high level of AFM1
contamination in the milk being supplied to the urban consumers of
these cities. It is worth-mentioning that majority of the urban consumers
in these large metropolitan cities rely on milk supplies from these
peri-urban dairy farms which reveals the significance of these findings in
terms of public health implications. In a nutshell, the higher level of
AFM1 contamination in milk and milk products is leading to a higher
daily intake of aflatoxin by consumers which may pose a serious public
health concern.
Several attempts have been made over the last decade to reduce
AFM1 contamination in milk in Pakistan through reducing AFB1
contamination in feed for animal consumption. Notably, the dairy pro­
cessors, such as Nestle Pakistan Limited and FrieslandCampina Engro
Pakistan Limited, have introduced incentives to the farmers for pro­
ducing and supplying milk in compliance with the tolerable AFM1 levels.
Also, they have aggressively worked with the feed suppliers and other
stakeholders to create awareness and improve the quality of feed con­
cerning AFB1 level compliance (Personal communication with dairy
sector stakeholders). In addition to this, Punjab’s Livestock & Dairy
7
Food and Chemical Toxicology 180 (2023) 114006
Development Department (L&DDD) has incorporated the Punjab Ani­
mals Feed Stuff and Compound Feed Act 2016; (GOP, 2016) under
which Punjab Animals Feed Stuff and Compound Feed Rules 2017;
(L&DDD, 2017) have been framed and are currently in-place. After the
introduction of this act, for the first time in the history of Pakistan, it has
been made mandatory for the commercial feed producing companies to
get them registered with the L&DDD and obtain a license on an annual
basis. Subsequently, feed producing companies are required to meet the
standards as prescribed in the Act and Rules for the feed products pro­
duced and supplied in the market including compliance with the AFB1
level. Though all these efforts have made some improvements towards
the use of good quality feed in dairy production, the systematic assess­
ment of the improvement remains unexplored.
5.2. Nuts and dry fruits
Dry fruit varieties having high nutritional values are a part of the diet
in most parts of the country, however, due to the favorable environ­
mental conditions (e.g., temperature and relative humidity) in Pakistan,
these food commodities are at higher risk of aflatoxin contamination.
This has been reported in a recent study from Southern Punjab where the
authors collected different types of edible nuts including melon seeds,
watermelon seeds, cantaloupe seeds, and pumpkin seeds from retail
shops and local markets during the summer and winter seasons. The
authors found that 27.2% of samples exceeded the EU permissible limits
for aflatoxins with a higher prevalence in the winter season (43.5%) as
compared to the summer season (33.4%) (Table 5). The study further
reported that the dietary intake of aflatoxins was higher in females
(6.30 μg/kg/day) than in males and it was mainly associated with the
consumption of pumpkin seeds (Razis et al., 2020). Similarly, previous
studies from Punjab and KPK provinces have reported 43% (Masood
et al., 2015) and 40% (Iqbal et al., 2018) overall prevalence of aflatoxins
in edible nuts. Both studies found that the highest concentration of af­
latoxins was in peanut samples without shells (mean levels: 7.89 ± 0.99
μg/kg and 7.30 ± 1.80 μg/kg), while the lowest in watermelon seeds
with shells (mean levels: 2.45 ± 0.11 μg/kg and 2.90 ± 1.50 μg/kg,
respectively).
A study comparing samples from production units and local markets
revealed that the aflatoxin contamination was much higher in dry fruit
samples collected from local markets. These findings indicate that
improper transportation and poor storage conditions might have
increased the levels of aflatoxin contamination (Mustansir Abbas et al.,
2019). Likewise, another study from KPK reported that the concentra­
tion of aflatoxin in various dry nuts was increased by 50% during a
storage period of 18 months (Saleemullah et al., 2006). These findings
suggest that the aflatoxin content of stored food should be monitored
regularly to ensure food safety.
A wide range of aflatoxin prevalence has been reported in nuts and
dry fruits in different parts of the country. A study from the Pothohar
region of Pakistan reported that 70% of peanut samples had aflatoxin
beyond the permissible limit as set by CAC. The study also found that
among three common types of peanuts (roasted, salty, and unripe),
roasted peanuts were the most contaminated product (40%) and AFB1
was the most common aflatoxin (Mateen Abbas et al., 2012). Several
other studies found that nuts and dry fruits without shells had higher
percentage positivity as well as aflatoxin contamination levels.
Conversely, a study focusing on aflatoxin levels in export quality nuts
and dry fruits from different parts of the country could not even detect a
single sample beyond the permissible limit as set by the EU.
5.3. Maize
Maize is ranked 3rd after wheat and rice in Pakistan’s multipurpose
crops and 8th in Asia with 5.2 Mt per annum production (Afzal et al.,
2017). A wide range of maize products, such as corn flakes, corn flour,
gluten, and starch, are consumed by humans and are also used in animal
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

Table 5
Aflatoxin contaminations in food and feed commodities reported from different regions of Pakistan.
Food/Feed Location n Sampling source Method Positive (%) Max. Conc. (μg/kg) Reference
Commodity used

Rice Selected villages from Sahiwal, Kasur, 62 Local shops and household UPLC 66 AFB1 = 5.04a (For Xia et al. (2020)
Bahawalpur, Rahim Yar Khan consumers)
Rawalpindi, Chakwal, Faisalabad, Lahore, 180 Small shops, wholesale, and LC-MS/MS AFB1=56 AFs = 7.75a (All Majeed et al.
Sahiwal, Muzaffargarh, Multan, supermarkets AFB2=48 varieties for (2018)
Bahawalpur, Rahim Yar Khan, Gujranwala consumers)
Lahore, Narowal, Faisalabad, Multan 48 Local markets HPLC 56§ AFB1=>60 (Super Mukhtar et al.
kernel basmati for (2016)
consumers)
PCSIR Labs Lahore 2679 Export companies TLC 13.31§ All NP (Stored rice) Nisa et al.
varieties (2015)
PCSIR Labs Karachi 262 Various vendors across TLC 0.38 §
AFB1 = 24.65 Asghar et al.
Pakistan (Brown rice for (2014)
consumers)
Karachi 2047 Export rice vendors TLC 0.0y AFB1 = 6.91 Asghar et al.
(Basmati rice) (2013)
Rice Research Institute, Kala Shah Kaku NP Export Quality HPLC 0.0 §
AFs = 4.27 (Brown Shabbir et al.
rice) (2013)
Lahore 1025 Selected areas TLC & 6.4§ AFB1 = 32.9 (Super Firdous et al.
HPLC kernel basmati) (2014)
Various cities of Punjab 413 Retail markets, processing HPLC 27.8§ AFs = 16.35 (Paddy (Shahzad Iqbal
units, field rice) et al., 2012)
Punjab 519 Exporters HPLC 5.58§ AFB2 = 16.65 Firdous et al.
(Basmati rice) (2012)
PCSIR labs Peshawar 40 Local Markets LC 7.5§ AFs = 10.8 (For Lutfullah and
consumers) Hussain (2012)
PCSIR Labs Peshawar 40 Rice producing areas & major TLC 20§ AFB1 = 18.0 Hussain et al.
stores (Stored rice) (2011)
Faisalabad, Samundari, Gojra, Toba Tek NP Local Markets HPLC NP AFs = 15.26 (For Qamar et al.
Singh, Jhang, Chiniot consumers) (2008)
KPK, Peshawar NP Stored Grains TLC NP AFs = 23.3 (For Saleemullah
consumers) et al. (2006)
Wheat Selected villages from Sahiwal, Kasur, 195 Household UPLC 3 AFB1 = 0.04a (For Xia et al. (2020)
Bahawlpur, Rahim Yar Khan consumers)
Islamabad, Rawalpindi, Gujrat, Sialkot, 333 Export quality product, PCSIR TLC NP ND Ahmad and
Lahore, Sheikhupura, Gujranwala, Lahore Syed (2017)
Sargodha, Faisalabad, Jhang, Toba Tek
Singh, Pirmahal, Kamaliya, Sahiwal,
Multan, Bahawalpur, Muzaffargarh, Dera
Ismail Khan, Abbottabad, Peshawar,
Quetta, Pishin, Karachi, Hyderabad,
Sukhar
Different areas of Pakistan 185 NP HPLC 2.7§ AFs = 5.26 (For Asghar et al.
consumers) (2016b)
Major districts Punjab 147 Local Markets (Wheat-based HPLC 22 §
AFB1 = 49.2 Iqbal et al.
products) (Lasagna) (2014a,b)
PCSIR labs Peshawar 20 Local Markets LC 10§ AFs = 15.5 (For Lutfullah and
consumers) Hussain (2012)
Sindh 60 Farmers, Field, Godowns, Mills TLC, ELISA ND ND (For Sahar et al.
consumers) (2009)
KPK, Peshawar NP Stored Grains TLC NP AFs = 22.3 (For Saleemullah
consumers) et al. (2006)
Maize Faisalabad 46 Experimentally Cultivated HPLC 89 AFs = 362.8y Hassan et al.
Samples (Ayub-1/26 maize) (2020)
Multan, Vehari, Bahawalpur, Raim Yar 72 NP HPLC 69.4§ AFs = 70.7a Manzoor et al.
Khan, Rawalpindi, Chakwal, Khushab, (Yellow maize) (2018)
Attock, Lahore, Faisalabad, Sargodha,
Gojar Khan
Southern, Northern, and Arid zones 15 75 Stored Grains HPLC 41§ AFB1 = 409.3 Iram et al.
districts of Punjab (Sadaf variety) (2014)
Lahore 100 Selected areas TLC & 96§ AFB1 = 1405.4 Firdous et al.
HPLC (Popcorn) (2014)
Muree, Peshawar, Sawat, Rawalpindi, 65 Open markets and Farmers HPTLC 27.6 AFs = 850 (For Khatoon et al.
Multan, Sahiwal, Faisalabad consumers) (2012)
PCSIR labs Peshawar 15 Local Markets LC 20§ AFs = 18.5 (For Lutfullah and
consumers) Hussain (2012)
Faisalabad 90 Markets, suppliers & HPLC NP AFB1 = 1374 Saleemi et al.
manufacturers (2012)
KPK, Swat Valley 36 Local Markets HPLC 36§ AFB1 = 30.92 Shah et al.
(Stored grains) (2010)
Faisalabad 40 Wholesale & supermarket, HPLC 84§ AFB1 = 46 (For Ahsan et al.
grocery shop consumers food & (2010)
feed)
(continued on next page)

8
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

Table 5 (continued )
Food/Feed Location n Sampling source Method Positive (%) Max. Conc. (μg/kg) Reference
Commodity used

Faislabad, Samundari, Gojra, Toba Tek NP Local Markets HPLC NP AFB1=>68 (For Qamar et al.
Singh, Jhang, Chiniot consumers) (2008)
KPK, Peshawar NP Stored Grains TLC NP AFs = 50.2 (For Saleemullah
consumers) et al. (2006)
Peshawar, Malakand, Dera Ismail Khan, 12 Local Markets TLC 41.6 No guide AFB1 = 8333 (For Munir et al.
Rawalpindi, Multan, Bahawalpur, followed consumers) (1989)
Sargodha, Lahore, Karachi, Sukkur, AJK
Nuts Bahawalpur, Dera Ghazi Khan, Multan, 60 Local markets HPLC 86.7 AFs = 24.2 Naeem et al.
Rahim Yar Khan (Walnuts) (2022)
Multan, Bhakkar, Layyah, Muzaffargarh 779 Local markets, Open shops, HPLC 27.2§ AFs = 20.9a Razis et al.
Superstores (Watermelon seed (2020)
without shell)
Major cities of Punjab and KPK 320 Retail markets, super stores, HPLC 59.6§ AFs = 7.30a (Shahzad Zafar
farms, and local shops (Peanuts without Iqbal et al.,
shell) 2018)
Different areas of Pakistan 624 Retail shops and local markets HPLC 4§ AFs = 30.11 Asghar et al.
(Pistachio) (2017)
Different areas of Pakistan 170 Export vendors HPLC 0§ AFs = 2.58 Asghar et al.
(Raisins) (2016a)
KPK, Northern areas 307 Retail markets and local shops HPLC 24§ AFs = 7.89a Masood et al.
AFB1 (Peanuts without (2015)
13§ shell)
AFs
Attock, Chakwal, Jhelum, Rawalpindi 72 Different localities HPLC 70y AFB1 = 98.8a (Mateen Abbas
(Roasted peanuts) et al., 2012)
Lahore Faisalabad 125 Local food stores HPLC 21 §
AFB1 = 1.24 Mushtaq et al.
(Peanuts (Nimko)) (2012)
KPK (PCSIR Peshawar) 180 Retail shops, local markets IAC 13.3§ AFs = 14.5 Luttfullah and
(Peanuts with Hussain (2011)
shell)
KPK, Peshawar (see the effect of storage) NP Stored Grains TLC NP AFs = 30.0 Saleemullah
et al. (2006)
PCSIR labs Karachi (experimented NP Vendors and shop TLC NP AFB1 = 183 (Mobeen et al.,
microwave heat) n.d.)
Chilies Islamabad, Faisalabad, Lahore, Multan 252 Dhaba, local food restaurants HPLC AFB1= 44.8y AFs = 26.6 (Red (Shahzad Zafar
Gojra and fast-food outlets AFs = 42.1y chili sauce) Iqbal et al.,
2021)
Multan 120 Open markets ELISA non branded AFB1 = 19.8a (Non- Akhtar et al.
47§ branded red chili) (2020)
branded 41§
Lahore, Sheikhupura, Multan, Faisalabad, 312 Residential areas, open HPLC 56.4 AFs = 15.2a (Shahzad Zafar
Gojra, Islamabad, Rawalpindi markets and food restaurants Iqbal et al.,
2017)
Kunri Sindh(aflatoxin reduction by manual 70 Farms TLC NP AFs = 148.75 Khan et al.
sorting technique) (2013)
Faisalabad, Lahore, Jhang, Toba tek singh 170 Open market, food restaurants HPLC AFs = 27.1 §
AFs = 27.5a (Chili (Shah et al.,
AFB1=21.7§ powder) 2010)
PCSIR Labs Peshawar 18 Markets TLC 67 AFs = 7.46 (Omum Hussain et al.
seed) (2012)
Central cities of Punjab 156 Herbal Shops, growing areas HPLC 22§ AFs = 81.5 (Whole (Shah et al.,
chili) 2010)
Faisalabad (experimental study) NP Grown HPLC NP AFs = 1.5 Iqbal et al.,
2011
Faisalabad 87 Markets, Shops, chili growing HPLC 46.5 AFs = 95.9 (Shah et al.,
areas 2010)
Different areas of Punjab 40 NP HPLC 57.5§ AFs = 34.6 (Longi (Shah et al.,
variety) 2010)
Faisalabad 44 Markets, Herbal shops, and HPLC 79.5 AFB1 = 96.3 Iqbal et al.
chili growing areas (Whole chili) (2010a)
Rural, Semi-rural, and Urban areas of 132 Representative samples HPLC 56.1§ AFB1 = 67.33 Iqbal et al.
Punjab (Ground chili) (2010a)
Karachi, Islamabad, Faisalabad, Lahore, 13 Markets HPLC NP AFB1 = 93.0 Paterson (2007)
Peshawar (Ground chili)
Karachi 176 Stores TLC 66 AFB1 = 25.0 (For Shamsuddin
consumers) et al. (1995)
Hazara, Multan, Sargodha, Karachi, 12 Local Markets TLC 25 AFB2 = 41.67 (For Munir et al.
Quetta, Bahawalpur, Kalat, Rawalpindi consumers) (1989)
Chickpea Rawalpindi, Chakwal, Khushab 50 Local Markets 12.9y AFB1 = 29.0 (For Tania et al.
seeds consumers) (2022)
Fruits & (Nawabshah, Sukkur) Sindh 56 Open market and exporters TLC ND ND Sahar et al.
Vegetables ELISA (2009)
Barely, PCSIR labs Peshawar 10 Local Markets LC 20 AFs = 12.6 Lutfullah and
Sorghum 10 10 AFs = 9.4 Hussain (2012)
Beans 65 6.1 AFs = 10.5
(continued on next page)

9
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

Table 5 (continued )
Food/Feed Location n Sampling source Method Positive (%) Max. Conc. (μg/kg) Reference
Commodity used

Chocolate Lahore 200 Branded and local from HPLC 83 AFs = 3.48 Naz et al.
markets (2017)
Animal Feed Mansehra, Oghi, Balakot 40 Poultry farms ELISA 92.5 AFs = 86.2 Naveed et al.
(2022)
Karachi 204 Farms ELISA 31 y
AFs = 72.27 Iram et al.
(2019)
Rawalpindi 186 Farms & production sites ELISA NP AFs = 165 Fareed et al.
(2014)
Charsadda, Chitral, Mansehra, Mardan, 396 Farms HPLC NP AFs = 99.5 Khan et al.
Sawabi, Sawat, PCSIR Peshawar (2014)
Quetta 487 Farmers, feed Samplers PRI TLC 40 AFB1=166 Rashid et al.
(2012)
KPK 182 Feed Mills, Domestic Industries TLC NP AFB1=191.65 Alam et al.
(2012)
Punjab, Sindh, KPK, Baluchistan, GB 100 Commercial Feed mills 100 y
AFB1=78 Anjum et al.
(2012)
Rawalpindi, Islamabad, Abbottabad, 1021 Poultry farms, feed production HPLC NP AFB1=13.11 Hanif et al.
Mansehra, Lahore, Gujranwala, (2006)
Faisalabad, Sumundari, Kamaliya, Multan,
Rahim Yar Khan, Thatta
Punjab, Lahore 14 Local Markets TLC NP AFB2=39.2 (Anjum et al.,
2011)
Punjab 24 City market and feed depots TLC NP AFB1=156 Khan et al.
(2011)
KPK Peshawar 54 Poultry and animal feed TLC NP AFB1=266 Ali et al. (2010)
industry
All over the country 215 Samples received in a ELISA 44.2 AFB1=210.07 Chohan et al.
commercial testing lab (2016)
Karachi and Hyderabad 220 Retailer and wholesaler shops ELISA and 84 AFB1=150 Shar et al.
IAC-HPLC- (2020)
FLD
Islamabad, Karachi, Lahore, Quetta, 429 Peri-urban dairy farms Dry ELISA NP AFs = 724.6 Yunus et al.
Peshawar, Muzaffarabad, and Gilgit feedstuff including (Bakery waste) (2020)
concentrates, waste bread, AFs = 600.8a
legumes, oil seed cakes, grains, (Cottonseed cake)
etc.
Different regions of Punjab 240 Dairy farms ELISA 86.6 AFB1=41 Akbar et al.
(2020)

a
Mean values are presented, NP=Not provided, ND=Not Detected, § followed EU standard, y followed FDA standard, ⌠ followed CAC standard.

feed. However, this crop is highly susceptible to fungal growth resulting
in toxin production (Trung et al., 2008). A previous study from Pakistan
has isolated different fungi from maize samples collected from various
levels of the supply chain and found that 56% maize and 75% maize
gluten meal were contaminated. The study also assessed the aflatoxin
production potential of these fungi and reported that A. parasiticus
(26.68–1374.23 μg/kg) had higher potential than A. flavus
(12.7–635.50 μg/kg) (Saleemi et al., 2012).In popcorns, a wide range of
aflatoxin prevalence has been estimated in Pakistan with the highest in
Faisalabad (85%, max. 62 μg/kg) and lowest in Lahore (14.7%, max
AFB1 = 1405.4 μg/kg) (Firdous et al., 2014).
Natural occurrences of aflatoxin were found about 69.4% (mean =
70.8 μg/kg) from 12 districts of Punjab (Manzoor et al., 2018) followed
by 18% (ranged 3.0–18.5 μg/kg) in Peshawar (Lutfullah and Hussain,
2012), 85% (max. 62 μg/kg) in Faisalabad (Ahsan et al., 2010), 36%
(max. 30.92 μg/kg) in upper and lower Swat regions of KPK (Shah et al.,
2010) and 41%(max 8333 μg/kg) in samples from different districts of
Pakistan (Munir et al., 1989). Additionally, 14.7% (max AFB1 = 1405.4
μg/kg popcorns) in Lahore (Firdous et al., 2014), 41% of AFB1 (max.
409.3 μg/kg) and 28% of AFB2 from 15 districts of Punjab revealed
levels exceeding the EU legislation. In contrast, in an experimental study
from Faisalabad researcher cultivated different varieties of maize in a
completely randomized block design and evaluated 89% of contami­
nation in samples (n = 46, max. 362.8 μg/kg) (Hassan et al., 2020).
Variations in temperature, water activity, and physical and storage
conditions all these factors strongly contributed to the incidence of af­
latoxins in maize (Shah et al., 2010). For this reason, topographical and
ecological conditions on freshly harvested and stored grains were eval­
uated with higher aflatoxin (mean 226 μg/kg) levels from freshly har­
vested grains as compared to stored grains (mean 96 μg/kg). It was due
to the differences in mean moisture levels of both stored (12.74%) and
freshly (14.6%) harvested grains, showing a relation between moisture
content and aflatoxin (Khatoon et al., 2012). Correspondingly, southern
and northern irrigated zones had less aflatoxin contamination due to a
lower moisture content (Iram et al., 2014).
Similarly, maize, wheat and rice samples were tested from two
different areas in Faisalabad division to find out the association of
contaminated maize with liver cancer. The contamination levels of af­
latoxins were higher in Toba Tek Singh District (AFB1 >68 μg/kg)
compared to Jhang (AFB1 >19 μg/kg) where the prevalence of liver
cancer was low, indicating that the high aflatoxin exposure could be
associated with the occurrence of liver cancer. The study also found the
higher mycotoxin contamination levels were possibly due to the inva­
sion of fungus during harvesting season and higher moisture level of the
storage place (Qamar et al., 2008).
To see the effect of storage, 18 months of stored maize samples from
KPK showed an increase of 45.7–50.2 μg/kg in aflatoxin revealing
contamination was dependent on storage condition and duration
(Saleemullah et al., 2006). Likewise, the performance of Purdue
Improved Crop Storage (PICS) was evaluated over polypropylene and
jute bags. Higher levels of aflatoxin contamination were observed for
polypropylene bags (AFB1 = 0.53 ng/g, AFG2 = 1.13 ng/g) than PICS
(AFB1 = 0.08 ng/g, AFG2 = 0.55 ng/g) additionally propylene bags had
higher insect damage and weight loss (35% in polypropylene as

10
W. Ashraf et al.
compared to 3% in PICS) upon two month storage (Afzal et al., 2017).
5.4. Chilies and spices
Pakistan is ranked sixth in exporting chili and chili stands as one of
the most revenue-generating commodities with 7.2% contribution to
global chili production and adds value of 1.5% to the national GDP in
Pakistan (Iqbal et al., 2011a–d; Q. Iqbal et al., 2011; S. Z. Iqbal et al.,
2011, 2021; Khan et al., 2013).
Despite having importance as a cash crop, this commodity is not
saved from fungal attacks. An alarming amount of aflatoxin contami­
nation was reported from locally used chili and chili-based products. A
very recent study reported 75.4% (mean Afs = 26.6 μg/kg, mean AFB1
= 11.7 μg/kg) aflatoxin contamination in chili sauce (red, green and
garlic) samples collected from dhabas, local food restaurants and fast
food outlets reporting one of the highest dietary intake (AFs = 3.82 μg/
kg and AFB1 = 3.67 μg/kg = AFB1) (Iqbal et al., 2021). These findings
were comparable with Multan’s open market’s samples of branded
(mean AFs = 8.04 μg/kg) and non-branded (AFs = 11.36 μg/kg) chilies
with an overall (AFs + AFB1) 38.3% (max AFB1 = 44.32, max AFs =
30.45) samples exceeding the EU standard and highest dietary intake of
non-branded spices in females (3.29 ng per kg of body weight per day)
(Akhtar et al., 2020). A similar trend was followed by more studies
where dietary levels of 3.26 mg/kg for AFB1 and 3.52 mg/kg for AFs in
the whole chili, chili powder, crushed chili, and chili sauce (with a
positive percentage of 56.4% and max mean AFs = 15.2 μg/kg) (Iqbal
et al., 2017) and 3.3 mg/kg for AFs in chili sauce, crushed chili, and
powdered chili was evaluated. Also, samples from the open market
(mean AFs = 27.5 μg/kg) were found to be more contaminated than food
restaurant samples (mean AFs = 21.1) (Iqbal et al., 2013a). Further­
more, the researcher found exceeding EU toxin levels in ground chilies
as 56% for AFB1 (max. 67.3 μg/kg) and 28% for AFs (73.6 μg/kg) in
rural areas as compared to samples from cities with a contamination
level of 22.5% (Iqbal et al., 2010a,b). The maximum levels found for
AFB1 were 81.5 μg/kg and 84.8 μg/kg for whole and ground chili,
respectively (Shah et al., 2010). Similarly, the mean concentration in
powdered chilies (32.20 μg/kg) was higher statistically than in whole
chilies (24.69 μg/kg) in Faisalabad (Iqbal et al., 2010a). Also when 3
different varieties (Longi, Wonder hot and Skyline 1) were evaluated,
Longi variety had the highest (AFs = 34.6 μg/kg) contamination with
60% samples being positive (Iqbal et al., 2011a) followed by another
25% (max. 41.67 μg/kg) from 8 districts, 66% (max. >25 μg/kg) from
stores in Karachi and 61.5% (max.93 μg/kg) from 5 major cities of
Pakistan (Munir et al., 1989; Paterson, 2007; Shamsuddin et al., 1995).
Considering the factors which contribute to the aflatoxin contami­
nation, the effect of seasonal variation was analyzed in a study and the
authors found that the contamination was higher in summer (53%) as
compared to winter (49%) with total aflatoxin content also being higher
in summer (43.5%) than in winter (40.5%) in chili (46.5% exceeding EU
legislation) (Shah et al., 2010). While looking for storage conditions,
chili stored in jute bags was found more contaminated (higher than
75%) in contrast to polythene bags because jute bags were more aerated
as compared to polythene bags, so this factor contributes to favorable
fungal growth with the significance of temperature upon toxin
contamination. Higher temperature (25–30 ◦ C) upon storage increased
the contamination by 61%, which was a marked increase, as compared
to chili stored at a relatively lower temperature (20 ◦ C) (Iqbal et al.,
2011). Therefore, both these factors should be considered in reduction
of aflatoxin contamination. One more finding that found sorting tech­
nique was effective in reducing aflatoxin by 78%, reported 148.75 μg/kg
maximum level of AFs in the study sample (Khan et al., 2013).
5.5. Wheat
Wheat is the main staple crop of Pakistan with an agriculture added
value of 10%. However, aflatoxin presence in wheat and wheat products
11
Food and Chemical Toxicology 180 (2023) 114006
(e.g. spaghetti, macaroni, noodles) was reported from 2.7%–22% (with
maximum range 5.26–49.2 μg/kg) from different areas of Pakistan with
predominant species A. falvus (14%) isolated in wheat (Asghar et al.,
2016b; Iqbal et al., 2014a; Xia et al., 2020). In contrast, aflatoxin was
reported within the prescribed limit of PSQCA and FDA in export quality
wheat grains and flour samples from Punjab and Sindh (Ahmad and
Syed, 2017; Sahar et al., 2009). Additionally, an experimental study
indicated that prolonged storage conditions in KPK were not sufficient
(an increase of 18.4–22.3 μg/kg in aflatoxin in 18 months) for wheat
stock storage (Saleemullah et al., 2006).
5.6. Rice
Rice is 3rd largest crop of Pakistan in terms of area sown after wheat
and cotton, with an agriculture added value of 3.1%. Punjab‘s eastern
belt is famous for the production of rice and around 60% is exported
annually. Besides human consumption, about 40–100% of broken rice is
used in poultry and animal feed. This commodity is an ideal substrate for
aflatoxin-producing fungi after maize (Cheemerla and Balakrishnan,
2021). So, researchers from Punjab and KPK reported 2.3–66% (mean
AFB1=5.04–68.3 μg/kg) aflatoxin contamination in different types of
local and export quality rice with a higher estimated intake of 19.1–26.6
ng/kg body weight per day (Asghar et al., 2013; Firdous et al., 2014;
Hussain et al., 2011; Shahzad Iqbal et al., 2012; Mukhtar et al., 2016;
Nisa et al., 2015; Xia et al., 2020). However, the studies from Karachi
and Kala Shah Kaku reported higher aflatoxin contamination (95.4%)
but within the prescribed limit of PSQCA and FDA in export quality rice
(Asghar et al., 2014; Shabbir et al., 2013). Inappropriate storage con­
ditions had a bad impact on the quality of rice as the aflatoxin content
could be increased (from 17.7 to 23.3 μg/kg) during the storage (Sale­
emullah et al., 2006). But washing and cooking can potentially degrade
aflatoxin content. A study from Punjab reported that washing can reduce
up to 15% and cooking degrade AFB2 up to 63% and AFB1 up to 51% in
rice samples (Majeed et al., 2018). As stated earlier for liver cancer
prevalence, the content of aflatoxin was found higher in rice (AFB1
15.26 μg/kg) with more number of liver cancer cases as compared to an
area with a low number of cases accompanied with low aflatoxin content
(AFB1 10.36 μg/kg) in rice (Qamar et al., 2008).
5.7. Animal feed
Natural occurrences of aflatoxins have been studied in various types
of poultry and livestock feed throughout the country. A wide range
(18.5%–86.84%) of various feed ingredient samples have been found to
be contaminated with aflatoxin in Pakistan (Naveed et al., 2022; Afzal
et al., 1979; Anjum et al., 2011; Fareed et al., 2014; Hanif et al., 2006;
Khan et al., 2011; Maqbool et al., 2004). In an analysis of AFB1, various
factors have been evaluated, and the results revealed that the sample
from Peshawar was higher in AFB1 concentration (191.65 ng/g) than
that of Swat and DI Khan during summer. The reason could be due to
variations in temperature, humidity, the physical condition of the grain,
transportation, and storage conditions (Alam et al., 2012). Seasonal
variations in terms of fungal growth are also well documented. For
example, 64.7% samples of home mixed layer poultry feed in Karachi
were found to have fungal growth in a year (A. flavus) (Iram et al., 2019).
However, large seasonal variations were found within these results, e.g.,
92.5% samples in summer (June to August) had fungal growth
compared to 40% in winter (December to February). Another study
found that 44.7% samples of commercial poultry feed were positive for
A. flavus, therefore, the researcher recommended the regular monitoring
of compound feed to avoid mycological contamination (Rashid et al.,
2012).
Aflatoxin content was determined higher in poultry grower feed than
starter feed. The maximum levels for AFB1, AFB2, AFG1, AFG2 were
found to be 99.5, 22.1, 18.5, and 11.3 μg/kg respectively (Khan et al.,
2014). The researcher explored the moisture contents of 14 samples
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

from 7 different local feed companies of Peshawar that were positively Table 6
correlated with aflatoxin contamination (Ali et al., 2010). Different control methods used to reduce aflatoxin contamination in various
Acute exposure to the higher level of aflatoxins has been associated food items.
with high mortality and morbidity in animals. Such an incident was Product Methods AFs Reference
reported in Karachi Landhi Cattle Colony in 2007, where approximately Reduction
500 animals died and more than 1000 suffered from acute aflatoxicosis. Biological Control
Subsequent investigations revealed that these animals were exposed to Maize & Ground Alfasafe-Nontoxigenic 80–99% Atehnkeng et al.
high aflatoxin-contaminated feed having 200 ppb T-2 toxin, 118.2 ppb Nut strains of A. flavus (2014)
Maize Inoculation of AF 71% (Alaniz Zanon
B1 and 20.2 ppb B2 (Ilyas, 2007). A recent study from Sindh province has
competitive exclusion et al., 2013)
reported that 58.2% samples of cotton seed (69 μg/kg) and 64.5% strain A. flavus AFCHG2
samples of cotton seed cake (89 μg/kg) exceeded the permissible limit of Wheat flour Bioplastic-based 90% (Accinelli et al.,
aflatoxins for animal feed, i.e., 20 μg/kg) (Shar et al., 2020). Another during bread- formulation with A. flavus 84–100% 2014; Saladino
study that analyzed various feed items reported that 64%, 21.7%, making NRRL 30797 et al., 2016b)
Lactobacillus,
33.3%, and 12.5% samples of cattle feed (Vanda), silage, TMR, and hay, Bifidobacterium,
respectively, exceeded the permissible limit of 20 μg/kg (Chohan et al., Propionibacterium, and
2016). The authors further reported that 32% of samples of maize and Lactococcus
68% of cotton seed cake were contaminated with aflatoxins above the Sorting
Groundnut grain Size and visual 85.3% Aoun et al.
permissible limit. The study also found that the prevalence of aflatoxin
(2020)
contamination was highest in August (65.6%), followed by June (60%), Image-Based Sorting
and July (52.8%), however, it was lowest during the month of April Grains maize Hyperspectral imaging 92.3% Wang et al.
(9.1%). kernels (HSI) (2016)
Another recent and comprehensive study of aflatoxin contamination Figs UV light coupled with the 98% Özlüoymak
color detection system (2014)
in dairy value chain across all provinces reported that cottonseed cake
Electromagnetic radiation treatment
samples from Karachi, Lahore, Islamabad, Peshawar, Quetta, Muzaf­ Maize Gamma (γ) radiation 35–95% Markov et al.
farabad and Gilgit had a mean aflatoxin level of 595.9 μg/kg which is (2015)
substantially higher than the permissible limit of 20 μg/kg (Yunus et al., Skim milk Ultraviolet (UV) irradiation 50% Nguyen et al.
(2022)
2020). More importantly, 64.7% of the samples peri-urban dairy farms
Rough rice Pulsed light (PL) 75% Wang et al.
(n = 448) in these cities used cottonseed cake at their farms at an (2016)
average inclusion rate of 9.3% of the total dry matter fed. It was found Highland Oil Radio frequency (RF) N/A Vearasilp et al.
that the quantity of cottonseed cake fed per animal per day was posi­ Seed (2015)
tively associated with AFM1 contamination in milk (Yunus et al., 2020). Chemical Control Agents
Stored Maize Combination of 2, 6-di (t- 90% Barra et al.
This explains why milk supplied to urban consumers of major metro­
butyl)-p-cresol (BHT) and (2015)
politans in all provinces of Pakistan is highly contaminated with AFM1. Purpureocillium lilacinum
Soybean media Soaking in Tartaric acids 95% Lee et al. (2015)
5.8. Fruits and vegetables Peanuts, Piadina 1 g oriental mustard flour/ 65–100% Hontanaya et al.
50 g sample (2015)
Milk 1.21% calcium 68% Maki et al.
There is a lack of research focusing on the detection of aflatoxin montmorillonite clay (2016)
contamination in fruits and vegetables in Pakistan. A study from Sindh Any food item 3 g/day calcium 44–54% Awuor et al.
province showed that the fruits and vegetables were free from afla­ used for human montmorillonite clay, (2017)
toxins; however, the authors tested a small number of samples (n = 56) consumption ACCS 100
Wheat, Maize, 20% neem powder NP Sultana et al.
from exporters and an open market that might have influenced the Rice (2015)
findings (Sahar et al., 2009). Moreover, as the fruits and vegetables are Dried fruits, Essential oils of different NP Salas et al.
mostly used fresh and, therefore, the chances of fungal growth and Spices, and aromatic plants (2016)
aflatoxin contamination are less. However, during the recent past, stored Areca nut
fruit and vegetable items have been increased in the market due to high NP=Not Provided.
prices in the off seasons. Therefore, further large-scale studies are
required to confirm the absence of aflatoxin contamination in fruits and aflatoxin control such as using resistant plant varieties, reducing plant
vegetables in Pakistan. stress by irrigation and pest management, avoiding late harvest, crop
rotation, use of a biological and chemical agent, and introduction of
6. Aflatoxin control and prevention methods nontoxigenic fungal strains. Nontoxigenic strains when high in number
compete with toxigenic strains and replace them from the environment,
As stated earlier, aflatoxins are carcinogenic, therefore exposure to that has a carryover effect to protect storage contamination (Atehnkeng
animals and humans should be minimized as much as possible. The et al., 2014) The competitive exclusion strain of A. flavus gave a prom­
growth of mold in various food items is mainly affected by climatic ising result in reduction of aflatoxins in Argentinian ground nuts (Alaniz
factors—temperature and humidity—moisture levels in the food item. Zanon et al., 2013).
Many control strategies have been adopted by several developed and Sorting is based on the difference in color, size, density, and visible
developing countries to prevent and mitigate the effects of aflatoxins identification of fungal growth, size and visual separation could reduce
(Mutegi et al., 2018). Policies and procedures should be developed for 85.3% of aflatoxins in ground nuts (Aoun et al., 2020) but manual
pre-harvest, post-harvest, and storage stages of crop management, sorting is laborious, inefficient, and impractical for in-line measure­
which will effectively save the product from contamination. Special ments. Hyperspectral Imaging detects 92.3% aflatoxin in maize kernels
emphasis should be given to developing indigenous technologies for (Wang et al., 2016). Ultraviolet irradiation detects aflatoxins effectively
cost-effective detoxification of aflatoxin-contaminated feed and food 50–100% in figs (Özlüoymak, 2014). Iqbal et al. (2014a) used gamma
through provision of technology development grants/funds etc. Various irradiation to destroy the aflatoxins in red chilies, gamma irradiation is
methods used to reduce aflatoxin contamination in different food and an effective tool for preserving and destroying microorganisms by
feed products are listed (Table 6). damaging DNA (Markov et al., 2015). Ultraviolet irradiation is a
Several strategies can be taken at the pre-harvest stage of crops for

12
W. Ashraf et al.
non-thermal economical technology for aflatoxin destruction in
different food products (Nguyen et al., 2022).
Mutagenic activity of AFB1 and AFB2 of A. flavus is destroyed by
pulse light technology in rough rice (Wang et al., 2016). A. niger and
A. flavus are highly inhibited by radio frequency (Vearasilp et al., 2015).
With the combination of 2, 6-di (t-butyl)-p-cresol (BHT) and Pur­
pureocillium lilacum highest reduction of AFB1 in stored maize was
observed (Barra et al., 2015). Tartaric acid also reduces high amounts of
AFB1(Lee et al., 2015). The use of 1 g oriental mustard flour/50 g sample
is effective against inhibiting aflatoxins effectively by reducing the
growth of A. parasiticus (Hontanaya et al., 2015; Saladino et al., 2016a).
Neem leaves were effective for up to 9 months of storage of grains for all
types of aflatoxins (Sultana et al., 2015).
Essential oils inhibit aflatoxins in dried fruits, spices, and Areca nuts
(Salas et al., 2016). Feed supplementation with modified yeast cell wall
extract reduces the absorption of AFB1 in ruminants and decreases the
chance of chronic exposure. Calcium montmorillonite clay used in feed
reduced the AFM1 content in milk without affecting the properties of
milk (Maki et al., 2016).
The inclusion of calcium silicate 100 in the human diet reduced the
bioavailability by binding aflatoxins and decreased the risk of aflatox­
icosis in toxin-prone areas (Awuor et al., 2017).
Physical and chemical detoxification methods have some disadvan­
tages, such as loss of nutritional value, altered organoleptic properties,
and undesirable effects on the products as well as high cost of equipment
and practical difficulties making them infeasible, particularly for lower-
income countries (Ahlberg et al., 2015).
The weather conditions in Pakistan are highly favorable for the
growth of mold, therefore, a lack of precautionary measures while
storing various food items can lead to the growth of mold and hence
contamination with aflatoxins. On a large scale, grains can be stored in
crop storage bags; a three-layered bag that effectively reduces the
chances of oxygen entrance from the environment and escape of carbon
dioxide from the bag which prevents mold growth and insect infestation.
Metal or cement bins and treatment with ozone and gamma irradiation
are also effective preventive measures. Purdue Improved Crop Storage
(PICS) bags successfully suppressed the development of A. flavus, they
absorb less moisture than other bags. PICS has triple bagging hermetic
technology with two inner liners made of high-density polyethylene and
an outer layer is woven polypropylene. PICS reduce oxygen influx and
limit the escape of carbon dioxide (Williams et al., 2014).
Household products should be stored in airtight containers to avoid
air exposure to food. Containers should be used after proper cleaning
and the government should take measures to discourage the prolonged
storage of food items at the household level.
Pakistan must work a lot to control aflatoxins, not only setting a limit
is the solution but also there are many areas to cover. Innovative solu­
tions suitable to local geographic conditions must be explored and
tested.
7. Recommendations
A national aflatoxin task force should be formed to develop and
implement a comprehensive and integrated approach for the prevention
and control of aflatoxins from farm to fork using a One Health approach
involving the agriculture, livestock, health, trade, and environmental
sectors. Harmonized standards and regulatory measures should be
applied uniformly across the country. Hence, a federal level agency such
as PSQCA should be responsible to set standards while provincial bodies,
such as food authorities of respective provinces, may ensure enforce­
ment and compliance of the set standards so that inter-provincial uni­
formity with regards to food production, processing, distribution, and
consumption may be maintained. Aflatoxin control policy should be
included in the national nutrition action plan, while detection of afla­
toxicosis should be included in the national nutrition survey. Moreover,
intensive surveillance programs should be developed through regulated
13
Food and Chemical Toxicology 180 (2023) 114006
inspection services for generating data on population exposure to afla­
toxins and their adverse effects on health. Since aflatoxin contamination
is a highly neglected issue, therefore, extensive awareness campaigns
should be launched among the public—producers as well as consum­
ers—and health practitioners about possible sources of aflatoxin
contamination and their control.
Furthermore, there is a need for capacity building of field and lab­
oratory staff for inspection, sampling, and testing of products for
detecting aflatoxins. Similarly, it is suggested to develop standard
operating procedures for pre-harvest, post-harvest, and storage stages of
crop management such as strict monitoring of cataloging operations,
application of functional bin systems, and improved store management
practices. Detoxification methods should be available to the producers
and applied if aflatoxin contamination occurs after pre-harvest and post-
harvest control to reduce losses of commodities. Special emphasis
should be given to developing indigenous technologies for cost-effective
detoxification of aflatoxin-contaminated food and feed through the
provision of technology development funds, etc. Special attention
should be given to monitor livestock feed quality especially in peri-
urban dairy farming to improve the quality of raw milk (with regards
to aflatoxins) being supplied to the urban consumers from these peri-
urban farms. Testing of aflatoxins in all types of processed and fresh
milk should be mandated to the respective food authorities and done
regularly. The regulatory bodies should update their websites regularly
and ensure the availability of information and policy guidelines to the
public. Moreover, consumer awareness using different types of media,
especially social/digital media, should be prioritized.
8. Conclusion
Aflatoxicosis is a global issue with a higher prevalence in hot and
humid regions like Pakistan. Every year 420,000 people die globally by
consuming aflatoxin-contaminated food. Global research shows that
approximately 150,000 cases of liver cancer annually and 35% of child
stunting cases are attributed to aflatoxins alone. Climate change is
causing an increase in environmental temperature and humidity in
Pakistan with a consequential rise in the prevalence of aflatoxin,
therefore several products used for human food and animal feed are at
risk of aflatoxin contamination. Generally, aflatoxins lead to chronic
illnesses such as stunting, wasting, and poor growth in children, various
types of cancers, liver toxicity, and immunosuppression, however, they
may also cause acute health problems including mortality. Alarmingly,
several studies have reported a very high prevalence of aflatoxin
contamination in various food items including milk, dry fruits, cereals,
etc., and animal feed including poultry feed, compound cattle feed, feed
ingredients, silage, and hay. Despite the high public health and eco­
nomic significance, it is not properly placed on the food security and
safety agenda in Pakistan’s health and livestock policies. As there is no
systematic research work assessing the impact of aflatoxins on human,
animal, and plant health in Pakistan and its association with aflatoxin
contamination in various food and feed items, it is hard to estimate the
extent of economic and health losses due to consuming aflatoxin-
contaminated food and feed. Limited awareness among producers,
consumers as well as medical professionals, unavailability of cost-
effective diagnostic tests, limited implementation of aflatoxin control
regulations, and inadequate policy framework for aflatoxins control are
the main hurdles to tackling the challenge. Hence, there is a dire need to
develop a comprehensive control strategy adopting a One Health
approach at the national level to reduce the aflatoxin burden and its
adverse effects on food safety, security and public health.
Funding
This research received no specific funding.
W. Ashraf et al. Food and Chemical Toxicology 180 (2023) 114006

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