Toxins 13 00682

toxins
Review
Mycotoxins in Pistachios (Pistacia vera L.): Methods for
Determination, Occurrence, Decontamination
Ana Rita Soares Mateus 1,2 , Sílvia Barros 2 , Angelina Pena 1,3, * and Ana Sanches Silva 1,2,4
1 Faculty of Pharmacy, University of Coimbra, Polo III, Azinhaga de Stª Comba, 3000-548 Coimbra, Portugal;
anarsmateus@hotmail.com (A.R.S.M.); ana.silva@iniav.pt (A.S.S.)
2 National Institute for Agricultural and Veterinary Research (INIAV), I.P., Rua dos Lagidos, Lugar da
Madalena, 4485-655 Vila do Conde, Portugal; silvia.barros@iniav.pt
3 LAQV, REQUIMTE, Laboratory of Bromatology and Pharmacognosy, Faculty of Pharmacy, University of
Coimbra, Polo III, Azinhaga de Stª Comba, 3000-548 Coimbra, Portugal
4 Center for Study in Animal Science (CECA), ICETA, University of Oporto, 55142 Oporto, Portugal
* Correspondence: apena@ci.uc.pt; Tel.: +351-239-488 480
Abstract: The consumption of pistachios (Pistacia vera L.) has been increasing, given their impor-
tant benefit to human health. In addition to being an excellent nutritional source, they have been
associated with chemical hazards, such as mycotoxins, resulting in fungal contamination and its
secondary metabolism. Aflatoxins (AFs) are the most common mycotoxins in pistachio and the
most toxic to humans, with hepatotoxic effects. More mycotoxins such as ochratoxin A (OTA),
fumonisins (FBs), zearalenone (ZEA) and trichothecenes (T2, HT2 and DON) and emerging myco-
toxins have been involved in nuts. Because of the low levels of concentration and the complexity of
the matrix, the determination techniques must be very sensitive. The present paper carries out an
extensive review of the state of the art of the determination of mycotoxins in pistachios, concerning
the trends in analytical methodologies for their determination and the levels detected as a result of

its contamination. Screening methods based on immunoassays are useful due to their simplicity
and rapid response. Liquid chromatography (LC) is the gold standard with new improvements to
Citation: Soares Mateus, A.R.;
enhance accuracy, precision and sensitivity and a lower detection limit. The reduction of Aspergillus’
Barros, S.; Pena, A.; Sanches Silva, A.
and aflatoxins’ contamination is important to minimize the public health risks. While prevention,
Mycotoxins in Pistachios (Pistacia vera
L.): Methods for Determination,
mostly in pre-harvest, is the most effective and preferable measure to avoid mycotoxin contamina-
Occurrence, Decontamination. Toxins tion, there is an increased number of decontamination processes which will also be addressed in
2021, 13, 682. https://doi.org/ this review.
10.3390/toxins13100682
Keywords: mycotoxins; aflatoxins; pistachios; Pistacia vera L.; determination; analytical methods;
Received: 30 June 2021 occurrence; decontamination
Accepted: 17 September 2021
Published: 25 September 2021 Key Contribution: A review of the state of the art of analytical methodologies for determination of
mycotoxins in pistachios, their occurrence in this type of nuts and the main decontamination methods.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations. 1. Introduction
Pistachios (Pistacia vera L.) are one of the most popular nuts in the world, due to
their flavour, nutritional quality and health benefits. Consumption of nuts like hazelnuts,
almonds, walnuts, pistachios and cashew nuts is characteristic of the Mediterranean diet [1].
Copyright: © 2021 by the authors.
Worldwide, the consumption of pistachios amounted to approximately 761.71 mil tons in
Licensee MDPI, Basel, Switzerland.
2020. In five years, the consumption increased by approximately 198 mil tons. In United
This article is an open access article
States of America (USA), the per capita consumption of pistachios increased substantially
distributed under the terms and
from 0.095 kg in 2015 to 0.245 kg in 2020 [2,3]. The consumption of nuts has been increasing
conditions of the Creative Commons
in Portugal; on average, one Portuguese person consumes 6.5 kg of nuts per year [4]. This
Attribution (CC BY) license (https://
is in part related with the fact that the consumption of nuts has been associated with a
creativecommons.org/licenses/by/
4.0/).
healthy dietary pattern and recommended by health professionals, namely nutritionists,
Toxins 2021, 13, 682. https://doi.org/10.3390/toxins13100682 https://www.mdpi.com/journal/toxins

Toxins 2021, 13, 682 2 of 41
due to pistachios being low in calories, high in mono-unsaturated fatty acids and low in
saturated fatty acids. In addition, they are a good source of proteins, carbohydrates, dietary
fibers, vitamins (A, E, K, B1 and B6) and minerals (potassium, phosphorus, magnesium and
iron). About 100 g of pistachios provides 4 g of the essential amino acid tryptophan [5–7].
Pistachio is a very versatile nut, consumed as a snack (raw, roasted, salted or flavored)
and also used in ice cream and bakery goods. In 2019, the global market of pistachios was
dominated by Iran and the United States of America, which produced 337,000 tons and
335,000 tons, respectively, followed by China and Turkey [8,9].
The composition of nuts is determinant for beneficial effects. From a health point of
view, several studies indicate that pistachios reduce the risk of coronary heart disease since
there is a reduction in cholesterol levels and a decrease in blood pressure [6]. Other studies
suggest a reduction in oxidative and inflammatory stress, blood glucose control, better
appetite management and consequent weight control [7].
Similar to other nuts, pistachio contains low amounts of water after being dried,
which restricts spoilage by microorganisms. However, some fungi are able to develop,
since they require a smaller amount of water to multiply [10]. Fungal contamination
can occur along the food chain, in the development of the plant in the field, as well as
in post-harvest, drying, transport, storage and processing. Contamination may occur in
these phases following harvest or there may be an increase in previous contamination [11].
Fungal contamination is closely related to environmental conditions, such as temperature
and humidity, which must be favorable to its growth. Moreover, crop damage due to
insect infestation and improper drying of crops before storage are factors to be taken into
account [12] (Figure 1).
As a result of this contamination, mycotoxins appear in nuts. The word “mycotoxin”
is derived from the Greek word “mykes” meaning “fungus” and the Latin word “toxicum”
meaning “poison” [13]. Mycotoxins are secondary metabolites of filamentous fungi; low
mass molecules produced by multiples genera and species of fungi have in common
toxic effects in animals and humans. Mycotoxins are a heterogeneous group due to
several chemical structures, biosynthetic origins and biological effects [14]. Food may be
contaminated with several different mycotoxins because, when conditions are favorable
for fungal contamination, more than one fungal species can contaminate food, and also, a
single species of fungi can produce several toxic metabolites [11,15]. It is also important to
mention that the presence of fungi may not be related to the presence of mycotoxins. On
the one hand, not all fungi are mycotoxin producers, and on other hand, mycotoxins are
only produced under certain conditions. In fact, the occurrence of aflatoxin contamination
is sporadic and, although large populations of A. flavus infect crops, serious outbreaks are
associated with above-average temperature and below-average rainfall [11].
Mycotoxins have different adverse effects on human health, such as, carcinogenicity,
mutagenicity, teratogenicity, cytotoxicity, neurotoxicity, nephrotoxicity, immunosuppres-
sion and estrogenic effects [16]. The severity of the effects depend on amounts ingested,
duration of exposure and on individual characteristics, such as age, gender, weight, diet or
health status [17]; for example, a low variety and insufficient diet constitute a risk factor
for greater severity of negative effects of mycotoxicosis [18]. In addition, the interaction
between mycotoxins could result in antagonistic, additive or synergistic effects [19,20].
Toxins 2021, 13, 682 3 of 41
Toxins 2021, 13, x FOR PEER REVIEW 3 of 38
Figure 1. Major factors influencing mycotoxin proliferation along the food chain and main analytical methods for
Figure 1. Major factors influencing mycotoxin proliferation along the food chain and main analytical methods for screening
screening and confirmatory determination of mycotoxins in pistachios (ELISA—Enzyme-Linked Immunosorbent Assay;
and confirmatory determination of mycotoxins in pistachios (ELISA—Enzyme-Linked Immunosorbent Assay; FLD—
FLD—Fluorescence detector; HPLC—High-Performance Liquid Chromatography; HRMS—High-Resolution Mass Spec-
Fluorescence detector; HPLC—High-Performance
trometry; LC—Liquid Liquid Chromatography;
Chromatography; MS—Mass Spectrometry;HRMS—High-Resolution
MS/MS—Tandem Mass Mass Spectrometry;
Spectrometry;
LC—Liquid Chromatography; MS—Mass Spectrometry; MS/MS—Tandem Mass Spectrometry;
Q-Orbitrap—Quadrupole-orbital ion trap; QqQ- Triple Quadrupole; QqTOF—Double Quadrupole-TOF; Q-Orbitrap—Quadrupole-
orbital ion trap; QqQ-Triple
TLC—Thin-layer Quadrupole;
chromatography; QqTOF—Double Quadrupole-TOF; TLC—Thin-layer chromatography; TOF—
TOF—Time-of-flight).
Time-of-flight).
Toxins 2021, 13, 682 4 of 41
Mycotoxins are a concern for food safety, Food and Agriculture Organization of
the United Nations (FAO) estimates that 25% of foods are contaminated by mycotoxins,
with consequences on health but also leading to economic losses at all levels of the food
chain [11]. Mycotoxins are more common in developing countries, where less concern for
food safety, insufficient quality control, hot weather, inadequate production techniques
and poor crop storage conditions are suitable for the growth of fungi [21]. However,
contamination is a global concern because it is an unpredictable and inevitable problem,
one of the most challenging to food safety, even when all good practices in the food chain
are implemented. The Rapid Alert System for Food and Feed (RASFF), in 2018, reported
569 notifications for mycotoxins, predominantly in the group of dried fruits, derived from
dried fruit and seeds, such as nuts, pistachios and almonds. The most prevalent reported
group are aflatoxins, followed by ochratoxin A. The same trend is maintained in 2019, with
588 notifications for mycotoxins and 90% of notifications are from countries outside the
EU, particularly, Turkey and Argentina. In pistachio nuts, RASFF, between January 2020
and June 2021, reported 84 notifications, mostly from Turkey, Iran and the USA, related
with aflatoxins and one notification concerning ochratoxin A in pistachios.
To ensure consumer health, the occurrence of mycotoxins is monitored, and maxi-
mum levels are regulated worldwide. In the European Union, the European Food Safety
Authority (EFSA) is responsible for scientific opinions concerning risks associated with
mycotoxins and advice to the European Commission (CE), which established Regulation
no. 1881/2006 concerning the maximum levels of certain contaminants, including certain
mycotoxins. The levels of aflatoxins in foodstuffs not for direct human consumption are
higher as they will still be processed. Based on the toxicity of different aflatoxins, a limit is
provided for the total aflatoxins in food, corresponding to the sum of aflatoxin B1 (AFB1),
aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), as well as the individual
content of AFB1 since this is aflatoxin with the greatest concern given its carcinogenicity.
Peanuts and nuts available on the market for the consumer must have a content of AFB1
less than 2 µg/kg and total aflatoxin content of less than 4 µg/kg. As aflatoxins (AFs) are
carcinogenic substances, maximum levels should be imposed at a level that is as low as
reasonably achievable (ALARA), defined as “the concentration of a substance that cannot
be eliminated without seriously compromising the availability of main food nutrients” [11].
In Codex Alimentarius, maximum levels for total aflatoxins in treenuts, including almonds,
hazelnuts, pistachios and shelled Brazil nuts, for human direct consumption are 10 µg/kg
and for treenuts still to undergo further processing are 15 µg/kg. However, maximum
levels of Desoxynivalenol (DON), fumonisins (FB1 and FB2) and ochratoxin A (OTA) in
nuts are not established [22]. EFSA [23] published a scientific opinion concluding that
increasing the maximum level of AFs in pistachios, almonds and hazelnuts to 8 or 10 µg/kg
would increase aflatoxin exposure by 1%, with more impact in groups with a high level of
nut consumption, and, despite the minor effects on cancer risk, EFSA strengthens the rec-
ommendation that exposure to AFs should be as low as reasonably achievable. European
legislation covers other mycotoxins, for example, OTA, but in dried fruit other than raisins,
the maximum levels are not defined yet, and for DON, zearalenone (ZEA), FB1, FB2, and
toxins T-2 (T2) and H-T2 there is no reference to the maximum levels in nuts.
Thus, pistachios, despite their beneficial effects on human health, also have chemical
hazards and are an important source of exposure to mycotoxins, especially aflatoxins,
constituting a current public health problem. Pistachios are considered to be the ones
with the highest risk of contamination by aflatoxins, largely due to shell splitting at end
of maturation [24,25]. This shell protects the pistachio kernel and, as a consequence of
splitting, pistachios are susceptible to molds and insect invasions. For example, navel
orangeworm (NOW) (Amyelois transitella) is a common pest of pistachio nuts in the field.
This worm causes direct physical damage in pistachios due to the worm’s growth, feeding
on kernels and insect excrement [26]. However, it also causes indirect damage because
it predisposes contamination by the aflatoxin-producing fungi. In fact, a study focused
on California pistachios showed that kernel infested by NOW had substantially more
Toxins 2021, 13, 682 5 of 41
infections by Aspergillus fungi producers of AFs and OTA, A. flavus and A. niger, respectively,
and consequently AFs are more frequently found in higher levels [27].
The present paper is the result of a vast literature review performed to evaluate the
state of the art of the determination of mycotoxins in pistachios concerning the new trends
in analytical methodologies for its determination and the levels detected as a result of its
contamination. Moreover, the mitigation strategies are presented, namely regarding the
decontamination by physical, chemical or biological methods.
2. Aflatoxins
Aflatoxins (AFs) are a class of mycotoxins produced by fungi of the genus Aspergillus,
especially the species A. flavus and A. parasiticus. Fungi A. nomius, A. pseudotamari, A.
bombycis and A. ochraceoroseus are also producers of aflatoxins but found less frequently [28].
Aspergillus are distributed worldwide, but the great predominance is in countries with
subtropical climate and warm temperature. They are characteristically greenish to greyish
molds and grow in hot (15 to 40 ◦ C) and humid conditions.
in dried fruits [28], mainly produces B-series aflatoxins, while A. parasiticus produces
2.1. aflatoxins
both Physical and
B Chemical Characteristics
and G [30]. In terms of toxicity, the most toxic aflatoxin is AFB1, fol-
Aflatoxins are low molecular
lowed by AFG1, AFB2 and, the least weight
toxic,molecules,
AFG2 [31],among
while312–346
AFM1Da has[29], composed
similar of to
toxicity
carbon,
AFG1 oxygen and hydrogen atoms (Figure 2). They are highly oxygenated heterocyclic
[32].
compounds derived from
These mycotoxins aredifuranocumarinic, wherecrystals
characterized by being the difuran
that group is attached
are colorless to one
to light yel-
side of the cumarin nucleus and the pentatone ring is connected to the other
low. They present fluorescence under UV light, but UV light is instable in the presence side, in the of
case of AF-B series, or the hexagonal lactone ring, in the case of AF-G series [28]. The
extreme oxygen and pH (<3 or >10). The melting points of these molecules are between
designation of series is related to fluorescence of molecules under UV light: B series has
240 and 280 °C. AFs are soluble in organic solvents, such as chloroform and methanol,
a blue color and G series has a green color, while associated numbers are related to the
moderately soluble in water and insoluble in non-polar solvents [31].
mobility of molecules in chromatography [11].
Figure 2. Examples of the main mycotoxins’ most common determinate in foods (structures from www.chemspider.com
Figure 2. Examples of the main mycotoxins’ most common determinate in foods (structures from www.chemspider.com).
(accessed on 11 May 2021)).
2.2. Toxicokinetics
AFB1 is the best studied aflatoxin due to its relevance in human health and to its
Toxins 2021, 13, 682 6 of 41
More than 20 aflatoxins are known, but the four main ones are: Aflatoxin B1 (AFB1),
aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2), as well as the metabolites
of AFB1 and AFB2, aflatoxin M1 (AFM1) and M2 (AFM2), respectively, as they were
primarily found in animal milk [14]. It should be noted that A. flavus, more common in
dried fruits [28], mainly produces B-series aflatoxins, while A. parasiticus produces both
aflatoxins B and G [30]. In terms of toxicity, the most toxic aflatoxin is AFB1, followed by
AFG1, AFB2 and, the least toxic, AFG2 [31], while AFM1 has similar toxicity to AFG1 [32].
These mycotoxins are characterized by being crystals that are colorless to light yellow.
They present fluorescence under UV light, but UV light is instable in the presence of
extreme oxygen and pH (<3 or >10). The melting points of these molecules are between
240 and 280 ◦ C. AFs are soluble in organic solvents, such as chloroform and methanol,
moderately soluble in water and insoluble in non-polar solvents [31].
2.2. Toxicokinetics
AFB1 is the best studied aflatoxin due to its relevance in human health and to its being
the one that most frequently occurs in food, reflecting metabolisms of other AFs. AFB1 is
rapidly absorbed by the gastrointestinal tract, reaching maximum concentrations in the
bloodstream after 1 h [33]. About 95% of AFB1 and metabolites are excreted in the urine
in the first 24 h after exposure [33]. AFB1 is metabolized in the liver by the cytochrome
P450 system, by epoxidation, to an electrophilic and very reactive molecule, aflatoxin
B1-exo-8,9-epoxide (AFBO), capable of covalently binding to DNA, RNA and proteins [14].
Conjugation of AFBO with glutathione by glutathione-S-transferase is a detoxification
route since it inhibits the ability of AFBO to bind to DNA, forming an inert metabolite,
followed by biotransformation with mercapturic acid, and then excreted in urine [34]. In
addition to epoxidation, AFB1 can be metabolized by hydroxylation reaction and also by
cytochrome P450 system enzymes, resulting in several metabolites: Aflatoxin M1 (AFM1),
aflatoxin Q1 (AFQ1), aflatoxin P1 (AFP1), aflatoxicol (AFL), aflatoxicol H1 (AFH1) and
aflatoxin B2a (AFB2a). AFM1 is the predominant metabolite, most commonly found as
a consequence of AFB1 exposure, and the most carcinogenic, by a similar mechanism
concerning AFB1. Moreover, these metabolites have toxic effects on humans [34]. AFM1
and AFQ1, although toxic, are less reactive than other molecules and are eliminated directly
in urine [35].
2.3. Toxicity
Aflatoxins are the leading cause of non-infectious diseases of food origin. It is esti-
mated that 4.5 to 5.5 billion people are exposed to these mycotoxins [36]. AFs are genotoxic,
carcinogenic and hepatotoxic; therefore, there is no threshold level for their toxicity and a
tolerable daily intake is not established. The Joint FAO/WHO Expert Committee on Food
Additives (JECFA), in 1997, through epidemiological data, estimated that intake of 1 ng
AFB1/kg bw/day increases the incidence of liver cancer by 0.013 cancer cases/year per
100,000 subjects, for HBsAg-negative individuals, concerning risk assessment. In 2016,
JECFA recalculated the cancer risk associated with aflatoxin exposure and concluded that
European people and those of other developed countries had a lower cancer risk, ranging
from <0.01 to 0.1 aflatoxin-induced cancers per year and per 100,000 subjects [37].
Toxins 2021, 13, 682 7 of 41
2.3.1. Acute Toxicity

Exposure to high concentrations of aflatoxins in a short period of time leads to hepato-
toxic effects, manifesting early as anorexia, malaise and low fever, and maybe progressing
to vomiting, abdominal pain and jaundice, as well as pulmonary and cerebral edema, coma
and convulsions [38]. In addition, acute exposure to a high AF content can lead to death by
hepatitis [28]. Estimated total aflatoxin intake that causes a mortality risk is >1 mg/day,
i.e., >20 µg/kg body weight/day in adults [33]. Children are a more vulnerable population
group since consumption of food by body weight is higher compared to adults; immune
and neurological systems are immature and diet is more restricted, so there is greater
susceptibility to development of complications [18]. AFB1 may cause weight loss, growth
delay or even malnutrition states in children [39]. Acute exposure to AFs is associated with
Kwashiorkor Syndrome, identified through epidemiological studies and outbreaks that
have occurred throughout history. Kwashiorkor syndrome is intermediate malnutrition
associated with high carbohydrate intake due to a lack of proteins and vitamins and occurs
mainly in children. Studies indicate that children with this syndrome are more exposed
to AFB1 by cereals consumed and have a higher frequency and higher concentration of
aflatoxicol in serum, indicating a change in AFB1 metabolism and interference in micronu-
trient absorption [34]. Some studies also indicate a relationship with Reye Syndrome,
an acute encephalopathy with visceral fat degeneration, more common in adolescents;
however, the cause–effect relationship of aflatoxins with this syndrome has not yet been
fully established [11].
2.3.2. Chronic Toxicity

Aflatoxin B1 is considered the most potent hepatic carcinogenic of aflatoxins, and the
International Agency for Research on Cancer (IARC) since 1987 classified it in group 1,
proven carcinogenic to humans, related to hepatocellular carcinomas, since there is suf-
ficient scientific evidence in both studies conducted on animals and human studies [40].
Toxicity mechanisms are related to the metabolite of AFB1. AFBO is capable of linking
to DNA, by nucleophilic addition, to nitrogen 7 (N7) of guanine base, forming AFB1-
N7-guanine adduct. The formation of this adduct in DNA leads to G-to-T transversion
during cell replication. One consequence is the AGG–AGT (Arginine–Serina) transversion,
resulting in the inactivation of the p53 tumor suppressor gene in codon 249, responsible
for cell cycle control, DNA repair and apoptosis [11,35]. In addition, AFBO can bind to
primary amine groups of amino acids (such as lysine) and proteins (namely albumin),
forming adducts found in the bloodstream [14,35].
For epidemiological studies, it was concluded that exposure to AFs constitutes a risk
factor for the development of hepatocellular carcinoma (HCC) [14]. HCC is the fourth
most common cause of cancer-related death worldwide. In addition to exposure to AFs,
alcohol, hepatitis B and C and other metabolic liver diseases are considered risk factors
for HCC [41]. Epidemiological studies conducted in Asia and Africa have indicated a
combination of AFB1 exposure and hepatitis B virus (HBV) infection increases the risk of
HCC; that is, there is a synergistic effect between AFB1 and HBV. The first clinical evidence
of this synergism occurred in China where it was found that HCC occurred in individuals
infected with HBV living in villages with high consumption of aflatoxins, with a mortality
rate 10 times higher than in individuals living in villages with lower consumption [36].
HBV infection can sensitize hepatocytes to carcinogenic effects of AFB1, explained by
different mechanisms related to mutation in codon 249. One hypothesis states that the
HBV genome is inserted in the HBV X gene, translated into the HBV X protein that inhibits
DNA repair and also contributes to uncontrolled cell proliferation. Another hypothesis
states that necrosis of hepatocytes and proliferation results in an increase of cells with
mutation. Moreover, chronic inflammatory liver disease, resulting from the HBV virus,
causes production of reactive oxygen and nitrogen species that increase oxidative stress
and can induce mutation [36]. In addition, these studies have shown that exposure to AFB1
alone was sufficient to significantly increase the risk of developing cancer [34]. Hepatitis
Toxins 2021, 13, 682 8 of 41
C virus (HCV) has also shown a correlation with the incidence of HCC, in synergy with
exposure to AFB1, but this is not yet fully established [34].
Children are chronically exposed to high levels of aflatoxins in areas where food
contamination is endemic, and this exposure begins in the uterine phase, in the fetal devel-
opment, through mother’s milk, and continues throughout life [33]. AFs are considered
a risk factor for compromising children’s growth [34]. Furthermore, studies show that
AFB1 has the ability to decrease immune system functions, with changes in immunological
parameters in populations chronically exposed to aflatoxins [33,34].
3. Ochratoxin A (OTA)
Ochratoxin A (OTA) is the second most important mycotoxin produced by fungi
Aspergillus ochraceus, A. carbonarius and Penicillium verrucosum. This occurs predominantly
in cereals and derivatives, namely flours, bread, rice, breakfast cereals and infant feed [11].
OTA have nephrotoxic effects associated with oxidative stress. In humans, epidemiological
studies demonstrate a possible association with Balkan endemic nephropathy and endemic
chronic interstitial nephropathy, but a causal link has not yet been established [38,42]. It is
classified by the IARC as possibly carcinogenic to humans and belongs to group 2B since
there is sufficient scientific evidence of carcinogenicity in animals, but human studies are
still insufficient [33,40]. Moreover, OTA is considered immunotoxic, neurotoxic, mutagenic,
teratogenic and hepatotoxic and affects development [39,42]. In 2008, JECFA reconfirmed a
provisional tolerable weekly intake (PTWI) of 100 ng OTA/kg bw from 1995, and estimated
that dietary exposures, mainly in Europe, ranging from 8 to 17 ng/kg bw per week are
below the PTWI [42].
OTA is a polypeptide derivative of dihydro-isocomarina, bound by the 7-carboxylic
group to 1-b-phenylalanine by an amide bond (Figure 2). Characterized by being a white
crystal with a melting point of 90 ◦ C, when recrystallized with benzene, it is very soluble
in polar organic solvents, moderately soluble in water and soluble in sodium hydrogencar-
bonate solutions. It presents absorption in ultraviolet to λMeOHmax (nm; ε) = 333 (6400) and
intense native fluorescence, with a maximum emission at 467 nm in 96% ethanol [43,44].
4. Fumonisins (FB1 and FB2)

Fumonisins are produced by the fungi Fusarium proliferatum and F. verticillioides,
predominantly found in corn and derived products. Fumonisin B1 (FB1) is the most toxic
fumonisin, followed by fumonisin B2 (FB2) [45]. However, it has recently been discovered
that Aspergillus niger also produces FB2 [17].
Fumonisins are characterized by a long chain hydroxylated hydrocarbon, hydroxyl
groups in C14 and C15 esterified with terminal carboxylic group of tricarboxylic acid
(Figure 2) [17]. They are different molecules from other mycotoxins because they are hy-
drophilic, dissolve completely in organic solvents such as methanol and acetonitrile:water
(1:1) and do not present fluorescence [14,46]. FB1 and FB2 are structurally similar to
sphingosine and sphinganin bases. They interfere with the metabolism of sphingolipids,
competitively inhibiting the ceramide synthase enzyme, causing dysregulation in cell
cycle [17,38,47]. These mycotoxins are considered to be possibly carcinogenic to humans,
belonging to IARC Group 2B. They are associated with esophageal cancer [48]. The largest
target organs of these mycotoxins are the liver and the kidneys, and FB1 is carcinogenic,
hepatotoxic and nephrotoxic [38,48]. JECFA (2011) [49] established a provisional maxi-
mum tolerable daily intake (PMTDI) for FB1, FB2 and FB3 of 0.002 mg/kg bw, alone or in
combination.
Toxins 2021, 13, 682 9 of 41
5. Zearalenone (ZEA)
Zearalenone (ZEA) is a secondary metabolite of fungi of the genus Fusarium, mainly
of the species F. graminearum and F. culmorum [47], very common in cereals such as corn,
wheat, barley, rye and their derivatives [38].
This mycotoxin is a macrocyclic–resorcyclic acid lactone (Figure 2) [38], with a similar
structure to 17-β-estradiol, a human sex hormone, so ZEA is considered a non-steroidal
estrogenic mycotoxin [38,46,47]. Given this structural similarity, they have affinity for
estrogen receptors and, as a consequence, lead to negative effects on the reproductive
system, such as fertility problems, precocious puberty, change in serum levels of estradiol
and progesterone [17,45]. IARC categorized ZEA in group 3, not classified as carcinogenic to
humans, since studies are limited [40]. In 2000, JECFA established a provisional maximum
tolerable daily intake (PMTDI) for ZEA of 0.5 µg/kg/bw.
ZEA presents in the form of white crystals, is soluble in benzene, acetonitrile, methanol,
ethanol and acetone, is very stable for degradation up to 120 ◦ C and is stable to hydrolysis
in neutral or acid buffer solutions.
6. Trichothecenes
Trichothecenes are a group of structurally related mycotoxins produced mainly by
fungi of the genus Fusarium. These molecules consist of a 12,13-epoxytrichothene skeleton
and a double bond with several substitutions in the side chain (Figure 2). This group in-
cludes non-macrocyclic mycotoxins: Desoxynivalenol (DON), T2 toxin and HT-2 toxin [14],
all classified in group 3 of IARC, due to inadequate scientific evidence in animals and a
lack of human studies [40]. These mycotoxins are cytotoxic, interfering in synthesis of
nucleotide acids and proteins and cell division [17].
6.1. Desoxynivalenol (DON)

Desoxynivalenol is a B-type trichothecene with carbonyl group in carbon 8 (Figure 2) [14].
Mainly produced by the species Fusarium graminearum and F. culmorum, it is very common
in cereals such as wheat and corn [45]. DON is known as vomitoxin, due to its acute
exposure and is linked to gastroenteritis in humans with nausea, vomiting, abdominal pain,
headache, fever and also with immunosuppressive effects, mostly reported in Asia [50].
They deregulate the normal functioning of cells, by inhibiting protein synthesis, influence
on signaling, differentiation and cell proliferation [51]. In 2011, JECFA established PMTDI
for DON and its acetylated derivatives (3-Ac-DON and 15-Ac-DON) of 1 mg/kg bw/day,
and also established an acute reference dose (ARfD) of 8 mg/kg bw [50]. DON was later
recognized as responsible for an epidemic in Japan called “red mold poisoning” due
to consumption of maize and moldy wheat, whose symptoms were nausea, vomiting,
diarrhea and seizures [30]. DON is characterized by white needle-shaped crystals. It is
soluble in chloroform, ethanol, methanol and ethyl acetate and stable at pH 4 even at high
temperatures [46].
6.2. HT-2 Toxin and T-2 Toxin

HT-2 and T-2 toxins are A-type trichothecene, with a hydrogen or an ester group in
lateral chain in carbon-8; the difference between these two molecules is the carbon-4-bound
group: In the case of HT-2 it is a hydroxyl group, and in the case of T2 it is an acetate group
(Figure 2) [14]. These mycotoxins are produced by species Fusarium sporotrichioides and
Fusarium poae, found especially in oats and also in corn and wheat [45]. HT-2 toxin (HT2) is
a metabolite of T-2 toxin (T2). T-2 toxin has a haematotoxicity effect and is linked to food
toxic aleukia (ATA), a condition that involves irritation of gastrointestinal tract, vomiting,
diarrhea and, in the most severe cases, leukemia, anemia and even death [14,17]. Some
in vivo studies show that T2 and HT2 have anorectic effects upon short-term exposure [52].
In 2016, EFSA 2016 established a tolerable daily intake (TDI) for T2 and HT2 of 0.02 mg/kg
bw/day based on immune- and haematotoxicity of T2. The EFSA scientific report [53]
shows a high chronic exposure in lower age groups.
Toxins 2021, 13, 682 10 of 41
7. Emerging Mycotoxins
Besides common mycotoxins, there is also a group of emerging mycotoxins, defined as
“mycotoxins, which are neither routinely determined, nor legislatively regulated; however,
the evidence of their incidence is rapidly increasing” [54]. These new mycotoxins are
more usually found in cereals like wheat, maize and barley, and Mediterranean crops;
determination on pistachios and other tree nuts are rare.
Fusarium second metabolites like fusaproliferin (FUS), beauvericin (BEA), enniatins
(ENNs), and moniliformin (MON) are included in the group of emerging mycotox-
ins. Moreover, fusaric acid, culmorin, butanolide [55] and, more recently, NX-2 [56]
are Fusarium emerging mycotoxins. Moniliformin (MON) was first described by
Cole et al. [57] isolated from the Fusarium strain, initially called F. moniliforme, which
contaminated cereals like maize. MON is a small, water-soluble and very acidic molecule
that occurs in nature typically as sodium or potassium salt [58]. The toxicity of MON is
due to the inhibition of thiamine enzymes, compromising the tricarboxylic acid cycle and
resulting in cytotoxic effects for lymphocytes and cardiomyocytes. Muscle weakness,
breathing difficulties and myocardial lesions are reported symptoms resulting from
MON exposure, based on animal studies, and the heart is the main target organ [59].
However, MON is suspected to be associated with the development of Keshan’s disease,
an endemic disease reported in China characterized by myocardial insufficiency [55,58].
Beauvericin (BEA) and Enniatins (ENNs) are structurally very similar mycotoxins
found in grains and cereal based food. Fusarium species like F. proliferatum, F. subglutinans
or F. verticillioides produce BEA, primarily found in 1969, and F. avenaceum, F. poae or
F. tricinctum produce ENNs, and ENN A, A1, B and B1 are the most commonly detected in
food. F. oxysporum produces both mycotoxins. The toxicity of BEA and ENNs are based
on their ionophore proprieties; they act as transporters for mono- or divalent cations,
for example, K+ or Ca2+ , resulting in disruption of normal physiological concentrations,
inducing DNA fragmentation and apoptosis. BEA and ENNs have also been demonstrated
to inhibit acyl-CoA:cholesterol acyltransferase (ACAT) which causes the accumulation
of cholesteryl ester in atherogenesis. BEA and ENNs have no cytotoxic in vitro studies
and no mutagenicity in the Ames test. Moreover, they show pharmacological properties,
such as anticonvulsant, antineoplastic and lower cholesterol levels of blood [58]. EFSA
(2014) conclude that acute exposure to BEA and ENNs is not a concern to human health
and since there is a lack of toxicity in in vivo data, there are no conclusions concerning
chronic exposure [55,58]. Liao et al. [60] detect 1.9 µg/kg of BEA in one sample of roasted
pistachios, out of a total of ten samples.
Fusaproliferin (FUS) is one of the most recent mycotoxins, discovered in 1993 by
Randazzo et al., so very little is known about it yet. Most of the studies are in plants,
insects, and cell cultures. These studies indicate that FUS have phytotoxic properties, are
moderately cytotoxic to human B lymphocyte, interact with DNA and show teratogenic
effects [55,58]. However, toxicity and mode of action have not been comprehensively
investigated and there is still an insufficient amount of toxicity data to assess the impact on
human health.
Toxins 2021, 13, 682 11 of 41
In addition Aspergillus, Alternaria and Penicillium are fungi that produce emerging myco-
toxins. Sterigmatocystin (STC) is an Aspergillus mycotoxin, mainly produced by A. nidulans
and A. versicolor, and a structurally closely related and toxic precursor to aflatoxins [55].
Studies show mutagenicity and cytotoxic effects, with formation of DNA adducts. In 1987,
IARC classified STC in group 2B (possibly carcinogenic to humans) [61]. Alternaria mycotoxins
are mostly produced by Alternaria alternata and include alternariol, alternariol monomethyl
ether, tenuazonic acid (TeA) and altertoxins with some effects in animals.
8. Analytical Methods for the Determination of Mycotoxins

Mycotoxins are present in low concentrations, in the order of µg/kg, and nuts
represent a complex food matrix, mainly due to the lipid content (53%) [5,62]. Therefore,
sensitive analytical methods with low limits of detection and quantification and good
specificity, precision and accuracy are needed [63]. Analysis of mycotoxins, regard-
less of analytical method, follows a common protocol: Sampling, sample preparation,
extraction, with or without purification, and detection/quantification [64]. Sample
preparation is very important because this step is responsible for eliminating matrix in-
terferents and pre-concentrate mycotoxins and transferring them to an adequate solvent
for the next analytical technique [65]. High-performance liquid chromatography (HPLC)
or ultra-high pressure liquid chromatography (UHPLC) with fluorescence detection
(FLD), mass spectrometry (MS) or tandem mass spectrometry (MS/MS) are the main
analytical techniques reported in the scientific literature. Other researchers have more
recently used immunoassays, like enzyme-linked immunosorbent assay (ELISA) and
sensor methodology. Analytical methods used for mycotoxin determination in pistachio
and other related food matrices are summarized in Table 1. The most used analytical
techniques for screening and confirmatory determination of mycotoxins in pistachio
are represented in Figure 1. In screening analysis, immunoassays are the most applied
techniques, due to simple and rapid performance; and for confirmatory analysis, liquid
chromatography is the gold standard, with distinct detectors. The validation of the ana-
lytical methodology, whether it is for screening or confirmatory, is of utmost importance
in order to assure reliable data.
Toxins 2021, 13, 682 12 of 41
Table 1. Summary of analytical methodologies used for mycotoxins determination in pistachio.
Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Analytes Detector Conditions Reference
Sample Methods Extraction Column Standard (µg/kg) (µg/kg)
Sample quantity: 125 g
Sample extraction:
Mobile phase:
475 mL
H2 O/MeOH/ACN (42:29:17,
MeOH/H2 O/Hexane
v/v/v) C18
(63:16:21 v/v/v); HPLC—
AFB1; AFB2; Flow-rate: 1 mL/min 250 mm × 4.6
Pistachio IAC filtration, dilution FLD with - 0.1–0.4 - [24]
AFG1; AFG2 Temperature column: mm
with water; PCD
Injection volume: 100 µL 5 µm
IAC:10 mL PBS; 75 mL
λexcitation: 365 nm
filtrate; wash 15 mL
λemission:450 mm
H2 O, vacuum; elution
with 0.5 mL MeOH
Mobile phase:(A) H2 O with
0.1% FA
(B) ACN with 0.1%FA
Gradient program: 90% A at 0
min, 30% A at 12 min, 10% A at
17.5 min, 90% at 21 min
(t = 25 min)
Peanuts, AFB1, AFB2, Sample quantity: 25 g
Flow-rate: 0.3 mL/min
pistachio, AFG1, AFG2, Sample extration: 100 Alltima C18
Ionization: ESI source in the
wheat, maize, OTA, DON, mL ACN/H2 O (80:20 LC- 150 mm × 3.2
- positive mode - 0.5–200 1–200 [66]
cornflakes, FB1, FB2, T2, v/v), shaken 2 h, diluted MS/MS mm
Temperature column: 30 ◦ C
raisins and HT2, ZEA, 1 mL extract with 3 mL 5 µm
Injection volume: 20 µL
figs CIT, etc H2 O, filtration
positive mode
Capillary voltage: 2.5 kV
Collision gas pressure: 0.8 bar
Vaporizer temperature: 450 ◦ C
Sheath gas pressure:
Auxiliary gas flow: 600 L/h
Toxins 2021, 13, 682 13 of 41
Table 1. Cont.

Mobile phase: MeOH/ACN
(60:40, v/v):5 mM ammonium
formate (45:55)
Gradient program: After 8 min,
Dried fruits
washed with MeOH/ACN
(peanuts,
(60/40, v/v) for 2 min and
walnut,
Sample quantity: 0.5 g returned to the initial conditions
cashews, Zorbax Eclipse
Sample extraction: 1 mL in 2 min
pistachio, XD8-C8
AFB1, AFB2, MeOH:H2 O (80:20 v/v), HPLC— Flow-rate: 1 mL/min
almond, pecan SPME 150 mm × 4.6 AFM1 0.02 0.05 [67]
AFG1, AFG2 centrifugation, filtration MS Temperature column: 40 ◦ C
walnut), mm
of supernatant and Injection volume: 10 µL
cereals, 5 µm
added to in-tube SPME Ionization: ESI source in the
dehydrated
positive mode
fruits and
spices
Collision gas pressure:
Sheath gas pressure: 30 psi
Auxiliary gas flow: 13 L/min
Sample quantity: 5 g for
AFs and 10 g for OTA
Mobile phase: ACN/H2 O/acetic
Sample extraction: 30
acid (51:47:2, v/v/v) Spherisorb
mL ACN/H2 O (60:40
AFB1, AFB2, HPLC— Flow-rate: 1 mL/min ODS2
v/v), belnded 10 min, 2
Pistachios AFG1, AFG2, IAC FLD with Temperature column: 40 ◦ C 150 mm × 4.6 - 0.2 - [68]
mL extract diluted with
OTA PCD Injection volume: 100 µL mm
48 mL PBS;
λexcitation: 333 nm 5 µm
Easi-extart AF IAC for
λemission:443 mm
AFs and Ochraprep
IAC for OTA
Toxins 2021, 13, 682 14 of 41
Table 1. Cont.

Sample extraction:100
mL H2 O + 4 g NaCl, Mobile phase:
Pistachios, 150 mL MeOH, H2 O/MeOH/ACN (42:29:17,
walnuts, filtration, 5 mL filtrate + v/v/v) Luna C18
HPLC—
cashews, 25 mL PBS; Flow-rate: 1 mL/min 25 cm × 4.6
AFB1 IAC FLD with - 0.2 0.6 [69]
almonds, IAC:10 mL PBS, 30 mL Temperature column: 40 ◦ C mm,
PCD
peanuts, seeds, filtrate, wash 15 mL Injection volume: 5 µm
etc. H2 O, elution 0.5 mL λexcitation: 362 nm
MeOH, 1 mL H2 O; λemission:456 mm
filtration if solution
not clear.
Sample extraction: 33%
Euroclon kit
MeOH, filtration, 500 ELISA - - - -
Absorbance at 450 nm [70]
µL filtrate + 500 µL
33% MeOH
Almonds, Sample quantity: 10 g
walnuts, Sample extraction: 1 g
sunflower AFB1, AFB2, NaCl + 40 mL
seeds, sesame AFG1, AFG2, IAC Mobile
MeOH/H2 O (80:20 v/v)
seeds, peanuts, AFM1, phase:ACN/MeOH/H2 O
+ 20 mL n-hexane,
pistachios, AFM2 (17:29:54, v/v/v) Hichrom ODS
blended for 3 min, HPLC-
hazelnuts and Flow-rate: 1 mL/min 250 mm × 4.6
eliminate n-hexane FLD with - 0.05–0.42 0.19–1.4
cashews Temperature column: mm
phase, filtration; 7 mL PCD
Injection volume: 20 mL 5 mm
filtrate + 43 mL PBS;
IAC: 10 mL PBS, 50 mL
λemission:435 nm
filtrate, wash 20 mL
H2 O, dried with air,
elution 2 mL MeOH
Toxins 2021, 13, 682 15 of 41
Table 1. Cont.

Mobile phase:(A) H2 O with 0.3%
FA and 5 mM ammonium
Sample quantity: 2 g formate, (B) MeOH with 0.3% FA
Sample extration: 8 mL and 5 mM ammonium formate
H2 O + 10 mL ACN: 5% Gradient program: 0 min:5% B; 1
Dried fruits FA; 4 g MgSO4 + 1 g min:50% B; 2 min:72% B; 4
(peanuts, NaCl + 1 g sodium min:80% B; and 6 min:90% B,
AFBI, AFB2
almonds, citrate + 0.5 g disodium finally back to 5 B in 0.2 min and
AFG1, AFG2, Zorbax Eclipse
walnuts, hydrogen citrate maintained for 1.8 min for
OTA, FB1, Plus RRHD
pistachios, sesquihydrate, UHPLC— column equilibration
FB2, T-2, QuEChERS 50 mm × 2.1 - 0.17–9.68 0.57–32.6 [71]
hazelnuts) and centrifugation; MS/MS Flow-rate: 0.4 mL/min
HT-2, STE, mm
seeds DLLME for AFs: 2 mL Temperature column: 35 ◦ C
CIT, DON, 1.8 um
(sunflower, supernatant: Injection volume: 5 µL
ZEN
pumpkin, pine evaporation and 1 mL Ionization: ESI source in the
nuts) MeOH/H2 O (50:50), 4 positive mode
mL H2 O, 0.21 g NaCl; Capillary voltage: 5 kV
injection 950 µL ACN + Collision gas pressure: 30 psi
620 µL chloroform Vaporizer temperature: 500 ◦ C
Auxiliary gas flow:
Sample extraction: 5 g
Mobile phase:
NaCl + 125 mL
H2 O/ACN/MeOH
Walnuts, MeOH/H2 O (60:40 v/v), Spherisorb
(6:3:1, v/v/v)
pistachios, blended for 1min, HPLC— ODS C18
AFB1, AFB2, Flow-rate: 1 mL/min 0.273–
hazelnuts, IAC filtration; 20 mL filtrate FLD with 150 mm × 4.5 - 0.9–1.8 [72]
AFG1, AFG2 Temperature column: 0.536
cashews, + 20 mL H2 O; PCD mm
Injection volume: 20 µL
almonds IAC: 10 mL filtrate 5 µm
diluted, wash 10 mL
λemission:440 mm
H2 O, elution 1 mL
MeOH
Toxins 2021, 13, 682 16 of 41
Table 1. Cont.

Mobile phase:(A) H2 O with 0.3%
FAand 5 mM ammonium
formate, (B) MeOH with 0.3% FA
and 5mM ammonium formate
Sample quantity: 1 g Gradient program: 100% A at 0
Sample extraction: 5 mL min, increase to 100% B at 8 min,
AFBI, AFB2 ACN/H2 O (85:15 v/v), until 12 min, then, return to 100%
AFG1, AFG2, shaking for 30 min in A in 8.5 min, equilibration for 5.5
Cereals and Hypersil
OTA, OTB, higher speed with min (t = 18 min) Isotope
nuts (almond, UHPLC— GOLD aQ
T-2, HT-2, - pulsation, Flow-rate: 0.3 mL/min labeled - - [60]
peanut, MS 100 × 2.1 mm
STE, CIT, centrifugation, 500 µL Temperature column: 35 ◦ C 13 C
pistachio) -
DON, ZEN, extract + 20 µL ISs + Injection volume: 5 µL
etc. 480 µL 20 mM Ionization: ESI source in the
ammonium formate, positive mode
vortex and filtration. Capillary voltage: 4 kV
Pistachios,
Sample extraction:
peanuts and
AFB1, AFB2, 50 mL 33% MeOH, Clone total AF ELISA test kit,
walnuts (raw - ELISA - - - - [73]
AFG1, AFG2 filtration, Absorbance at 450 nm
and roasted
dilution 1:2 with
with salt)
33% MeOH
Toxins 2021, 13, 682 17 of 41
Table 1. Cont.

Sample extraction: 5 g
NaCl + 125 mL
Almond, MeOH/H2 O (60:40 v/v), Mobile
hazelnuts, blended with hight phase:H2 O/MeOH/ACN
peanuts, speed 1 min, sediment, (64:23:13, v/v/v) C18
HPLC—
pistachio, AFB1, AFB2, filtration of supernatant; Isocratic program 150 mm × 4.6
IAC FLD with - - 0.4–1.3 [74]
walnuts, brazil AFG1, AFG2 20 mL filtrate + Flow-rate: 1 mL/min mm
PCD
nuts, 20 mL PBS; Injection volume: 100 µL 5 µm
chestnuts and IAC:20 mL diluted λexcitation: 364 nm
apricot filtrate, wash λemission: 440 mm
MeOH/H2 O (25:75 v/v),
elution 2 mL MeOH +
3 mL H2 O
Mobile phase: (A) H2 O with 0.1%
FA, (B) ACN with 0.1% AF
Gradient program: 0–5 min 4% B,
Sample extraction:10
5–20 min 100% B, 20–24 min
mL H2 O, 10 mL
100% B, 24–28 min 2% B and this
ACN:FA 0.1%; 4 g
latest rate was maintained for
MgSO4 + 1 g NaCl + 1 g
AFBI, AFB2, 10 min (t = 38 min)
sodium citrate + 0.5 g Easy-Spray
AFG1, AFG2, Flow-rate: 200 nL/min
disodium hydrogen PepMap C18
Peanuts, OTA, FB1, Ionization: ESI source in the
citrate sesquihydrate, nano
almonds and FB2, T-2, QuEChERS HPLC-MS positive mode - - 0.05–5 [75]
centrifugation; d-SPE 150 mm × 75
pistachios HT-2, STE, Temperature column: 25 ◦ C
with EMR-lipid: µm
CIT, DON, Injection volume: 100 nL
activation with 5ml 3 µm
ZEN, etc. Ionization: ESI source in the
H2 O + 5 mL extrat,
positive mode
centrifugation, 5 mL
supernatant + 0.4 g
NaCl + 1.6 g MgSO4 ,
centrifugation
Auxiliary gas flow:
Toxins 2021, 13, 682 18 of 41
Table 1. Cont.

Sample quantity: 2.5 g
Sample extration: 10
mL ACN + 10 mL H2 O
Mobile phase:(A) H2 O
with 0.2% FA, rotation
Gradient program:
for 30 min; 4 g MgSO4 +
1 g NaCl + 1 g sodium
Temperature column: 30 ◦ C
citrate + 0.5 g disodium
Injection volume: 4 µL ODS C18
Raw peanuts AFB1; AFB2; hydrogen citrate
LC— Ionization: ESI source in the 150 mm × 2.1
and roasted AFG1; AFG2; QuEChERS sesquihydrate, - 0.05–0.10 0.08–0.30 [76]
MS/MS positive mode mm
pistachios OTA centrifugation, follow
Capillary voltage: 5 µm
by 2 extraction with
Collision gas pressure: 25 psi
20 mL hexane;
d-SPE: supernatant +
150 mg C18 + 900 mg
MgSO4 , centrifugation,
wash 2 × with
5 mL ACN
FA, (B) ACN with 0.1% FA
Gradient program: 25% A
increased to 100% in 3.75 min,
reduction to 25% A in 6 min (t =
7.5 min)
Sample extraction: 10
Almonds, mL ACN/H2 O (80:20
Ionization: ESI source in the C18,
hazelnuts, AFB1, AFB2, v/v), rotation for 20 min,
UHPLC— negative mode 100 mm × 2.1
peanuts, AFG1, AFG2; QuEChERS 4 g Na2 SO4 + 1 g NaCl, - - 0.5–1.0 [77]
MS/MS Temperature column: 25 ◦ C mm,
pistachios, ZEA centrifugation;
Injection volume: 5 µL 1.8 µm
walnuts d-SPE: 3 mL supernant
+ 100 mg C18,
positive mode for AFs
centrifugation
Collision gas pressure: 45 psi
Toxins 2021, 13, 682 19 of 41
Table 1. Cont.

FA, (B)MeOH with 0.1% FA
Gradient program: 0% B for 1
min, 95% B for 1.5 min, 75% B for
2.5 min, decrease to 60% in 1 min,
Sample quantity: 1 g back to 0% B in 0.5 min and held
AFBI, AFB2 Sample extraction: 5 mL for 1.5 min (t = 8 min)
AFG1, AFG2, H2 O, 5 mL ACN with Flow-rate: 0,4 mL/min Luna Omega
Almonds, OTA, OTB, 0,1% FA; 0.5 g NaCl + Ionization: ESI source in the Polar C18,
UHPLC—
hazelnuts and T-2, HT-2, QuEChERS 2 g MgSO4, posiive and negative mode 50 mm × 2.1 - - 0.2–0.78 [78]
MS
pistachios STE, CIT, centrifugation; Temperature column: 30 ◦ C mm,
DON, ZEN, d-SPE: 1.5 mL Injection volume: 5 µL 1.6 µm
etc. supernatant + 50 mg Ionization: ESI source in the
C18, centrifugation positive and negative mode
Capillary voltage: ± 4 kV
Auxiliary gas flow:
ACN—acetonitrile; ADONs—Sum of 3-acetyl and 15-acetyl-deoxynivalenol; AFB1—Aflatoxin B1; AFB2—Aflatoxin B2; AFG1—Aflatoxin G1; AFG2—Aflatoxin G2; AFM1—Aflatoxin M1; AFM2—Aflatoxin
M2; CIT—Citrinin; ELISA—Enzyme-Linked Immunosorbent Assay; ESI—electrospray ionization; FA—formic acid; FB1/FB2—fumonisins; HPLC- FLD—High performance liquid chromatography with
Fluorescence Detection; IAC—Immunoaffinity columns; LC—MS—Liquid Chromatography Mass Spectrometry; OTA—Ochratoxin A; OTB– Ochratoxin B; MeOH—methanol; PCD—post column derivatization;
PBS—phosphate buffer saline; QuEChERS—Quick, Easy, Cheap, Effective, Rugged and Safe; STE—sterigmatocystin; UHPLC-MS—Ultra-High-Performance Liquid Chromatography tandem Mass Spectrometry;
SPME—Solid-Phase Microextraction; T-2/HT-2—Trichothecenes; ZEA—Zearalenone8.1. Sample Preparation.
Toxins 2021, 13, 682 20 of 41
Mycotoxins are distributed heterogeneously and may only occur in a fraction of sam-
ples [79]. Thus, sampling and preparation of a sample are crucial steps in the determination
of these chemical contaminants, to ensure representativeness.
For the determination of AFs in treenuts “ready-to-eat”, Codex Alimentarius recom-
mend a sample of 10 kg of pistachio in-shell nuts or 5 kg shelled nuts, and the sample should
be finely ground and mixed thoroughly using a process, to reduce particular size and dis-
perse the contaminated particles evenly throughout the sample, ensuring homogenization,
since distribution of aflatoxin and other mycotoxins is extremely non-homogeneous [22].
During sample preparation, it is important to keep samples away from sunlight and also
control temperature and humidity in order to not favor mold growth and aflatoxin forma-
tion [22]. Pre-treatment of a sample is considered a fundamental and indispensable step in
almost all analytical procedures, especially for analysis in complex food matrices [15].
In Europe, sampling and analysis methods for the official control of the mycotoxins in
food are established in Regulation No. 401/2006. To analyze AFB1 and AFs in pistachios
an overall sample of 30 kg is recommended, resulting from 10 to 100 elementary samples
collected from different points of one lot, depending on the lot’s weight. This sample is
mixed and divided into two or three equal samples for a laboratory with ≤10 kg before
crushing. Then, each laboratory sample is separately finely ground and carefully mixed
to ensure complete homogenization. In the case of lots in retail packaging, each package
could be considered as one sample for analysis when it is less than 300 g.
The first step in sample preparation is extracting mycotoxins from the solid matrix to
a liquid phase, separating them from other components. The extraction solvent is a mixture
of an organic solvent with water, where the presence of water favors penetration of organic
solvents into a matrix, and, in some cases, acids are used to break the bond of mycotoxins
to other components, increasing the effectiveness of extraction [15]. The extraction solvent
is chosen according to the characteristics of mycotoxins and matrices [80], and acetonitrile
(ACN) is the organic solvent extraction more applied, followed by methanol (MeOH).
Moreover, sodium chloride (NaCl) and n-hexane are usually added, in addition to solvent
methanol:water [70], due to the higher fat content of pistachio.
The second step is a clean-up to remove interferers and impurities from the extract,
such as lipids, proteins and other small molecules, to ensure sensitivity and selectivity.
Solid–liquid extraction techniques are often used, namely solid phase extraction (SPE),
solid phase micro-extraction (SPME) and solid phase matrix dispersion (MSPD). However,
in the pistachio nuts, researchers use immunoaffinity chromatographic columns (IACs)
and the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) method.
IAC is a very sensitive and selective technique because specific antibodies are used for
mycotoxins. Affinity of antibody and reversibility of binding are very important because
the aflatoxin–antibody complex has to be dissociated to release mycotoxins in the elution
phase. The complex has to be stable enough for the washing steps [81]. In a simplified
way, the sample is applied into a column with anti-mycotoxin antibodies; then, the column
washes, and the final step is the elution of mycotoxins. The eluate is evaporated until
dryness to reduce volume and concentrate mycotoxin in the extract. Finally, the residue is
redissolved into the mobile phase to follow chromatography analysis [82].
The QuEChERS method, in a simplified way, is divided into two extraction stages.
The first extraction step is based on the salting-out effect, with an organic phase in the
presence of salts for extraction. Acetonitrile (ACN) is the most used extraction solvent,
applicable to a wide range of organic compounds, without co-extraction of interferent
molecules from the matrix [83] and easily parts from water in the second phase [84]. To
increase efficiency, acidification with formic acid (FA) [62,71,75], acetic acid [85,86] or citric
acid [87] is frequently applied. In the case of mycotoxins, a combination of magnesium
sulfate (MgSO4 ) with sodium chloride (NaCl) in a 4:1 ratio is the most applied extraction
salt. Magnesium sulfate allows the best salting-out of ACN and the best overall recoveries
especially of polar analytes; however, MgSO4 contributes to the remaining parts of water in
the acetonitrilic layer, so it helps to control the polarity of the extraction solvents and thus
Toxins 2021, 13, 682 21 of 41
increases the selectivity of extraction [88]. In a second phase, the extract is cleaned with
adsorbents to remove interferers. Generally, dispersive Solid Phase Extraction (d-SPE) is
applied with primary secondary amine (PSA), octadecyl silica (C18) or graphitized carbon
black (GCB) [84]. More recently, new adsorbents have been available on the market, for
example, EMR-Lipid and Z-Sep+ . Alcántara-Dúran et al. [75] compare two adsorbents:
(1) EMR-lipid, remove lipids based on hydrophobic interactions and exclusion by size
between long aliphatic chains of lipids and adsorbent [84]; (2) PSA, which is useful for
removing lipids, namely fatty acids, sugars, organic acids and some pigments; and (3) C18,
which is recommended for the removal of high lipid content [84]. The authors concluded
that EMR-lipid presented the best results, with better percentage of recovery and lower
matrix effect. Cunha et al. [62] performed a clean-up with Z-sep+ and C18. Z-sep+ is
composed of C18 and zirconia oxide bound to the same silica particle, removing fatty
acids and pigments [84]. Some authors select immunoaffinity chromatography for sample
cleaning [85,86].
QuEChERS has numerous advantages like reduction of the steps; simple and easy
implementation; separation of a wide range of analytes and several samples in a short
time; and use of a smaller volume of samples and solvents, according to the principles of
green chemistry [15,62,84]. In addition, QuEChERS is also used in multiclass analysis with
simultaneous analysis of multi-mycotoxin and multi-pesticide residues, for example, in
cereals [89–91].
8.1. Detection and Quantification

8.1.1. Chromatographic Techniques
Chromatographic methods are based on the physical interaction between a mobile and
a stationary phase. Analytes are differently distributed between two phases, depending
on their characteristics, resulting in different speed movements in the column, causing
separation [35]. Thin-layer chromatography (TLC), gas chromatography (GC) and liquid
chromatography (LC) are used for analysis of mycotoxins. TLC is more used for specific
identification of mycotoxins. GC was abandoned because it needs a derivatization step
due to most mycotoxins being nonvolatile and polar substances.
In the case of confirmatory identity and quantitative determination of mycotoxins,
namely in nuts, liquid chromatography (LC) is the most common technique, given its high
precision, high sensitivity and low detection limit [65]. While reversed-phase elution and
C18 columns are mostly used, LC mycotoxin analysis is a flexible technique; it can use
different elution modes, different column sizes, different particular sizes and different
mobile phase compositions in order to improve mycotoxin separation. In recent years,
different approaches have been applied to LC mycotoxin analysis, improving efficiency
and resolution, making it faster and cheaper. For example, the reduction of particle size
or column diameter results in ultra-high liquid pressure chromatography (UHPLC) and
capillary/nano-LC, respectively. Moreover, coupling two or more separation columns or
using enrichment/extraction first column to online sample preparation are new strategies [65].
Previously, LC was combined with ultraviolet-visible detector (UV-Vis) and fluores-
cence detector (FLD) for AF analysis due to their fluorescent properties (AFB1 and AFB2
exhibit fluorescence at 425 nm, AFG1 and AFG2 exhibit fluorescence at 450 nm); however,
quenching occurred due to the mobile phase, hindering detection of AFs at lower concentra-
tions, requiring derivatization [92]. For AF determination with FLD, the derivatization step
(pre- or post-column) is needed to promote sensitivity and resolution. Chemical derivati-
zations involve chemical reaction between AF and acid (trifluoroacetic acid) or halogen
(bromine or iodine) molecules to improve fluorescence. Photochemical derivatization is
based on derivatization of AF with UV radiation generated by a photochemical reactor,
and there is no need to add any chemical reagents, which is more advantageous [15,35,92].
Photochemical derivatization is the most reported PCD for the determination of AFs in
pistachios [68,70,72,74], although some previous studies use bromination [24,69]. How-
ever, this derivatization step, especially with chemical derivatization, added complexity to
Toxins 2021, 13, 682 22 of 41
analysis. In addition, other mycotoxins do not have these fluorescence proprieties, so this
detection method is not suitable for multi-mycotoxin determination.
More recently, mass spectrometry (MS) was coupled as a detector, resulting in LC-
MS based on a separation of analytes by LC and subsequent analysis of mass to charge
(m/z) of ions in the gas phase, obtaining structural information that identifies molecules
based on molecular weight [92,93]. Nowadays, LC-MS is the most suitable technique
recommended by the guidelines for identification, quantification and confirmation of
multi-class mycotoxins, being highly sensitive and specific and one of the best options
for this type of analytical determination in complex food matrices [92]. LC-tandem mass
spectrometry (LC-MS/MS) is a powerful technique for mycotoxins because of its ability to
detect multiple regulated, unregulated and emerging mycotoxins, with a need of precursor
ions to correct identification and quantification [65]. LC-MS can be performed employing
different MS analyzers to increase detection abilities, and provide different information
and data treatment and emerging LC-High Resolution Mass Spectrometry (HRMS). For
example, there are classical, like triple quadrupole (QqQ) and time-of-flight (TOF), or hybrid
modern detectors such as QqTOF (double quadrupole-TOF) or Q-orbitrap (quadrupole-
orbital ion trap) [65]. While exhibiting high sensitivity, selectivity and mass accuracy, LC-
HRMS is a very high-cost technique, and needs recurrent maintenance and to be regularly
calibrated to maintain the high mass accuracy and resolution. In addition, its application
depends on the training of users and data file storage because, when using HRMS in
full scan mode for large numbers of samples, lots of information must be processed and
stored [92].
Recently, multi-mycotoxin methods have been developed to determinate a greater
number of mycotoxins in a single chromatographic run. This progress is relevant since one
food item may be contaminated by a fungus that produces different mycotoxins or can
be contaminated by more than one species of fungus, resulting in co-occurrence [63,94].
However, one of the challenges is the matrix effect; the signal is often suppressed due to
co-elution with matrix components. Matrix-matched calibration, the addition of standard
or use of internal standard are some of the solutions. Matrix-matched calibration uses
calibration standards for fortifying “blank” samples (without mycotoxins of interest), with
the addition of known mycotoxin concentration, and it is expected that the impact of the
matrix effect on the response of mycotoxins is similar in calibration and samples [93]. The
internal standard (IS) allows greater flexibility in extraction techniques and conditions
since it has previously been added to the sample. Moreover, IS allows correction of
signal variations, measuring the relative response ratio between a mycotoxin and IS and
associated recovery of method to final result [95]. Some of the most commonly used ISs
in AF determination are isotopes, such as 13 C-aflatoxin, and deuterated aflatoxin, since
they will have characteristics similar to AF [92]. However, for correct analyses concerning
multi-mycotoxins, a labelled compound for every single mycotoxin of interest should be
used. Zearalanone (ZAN) is also an internal standard widely used [16,66,96], with chemical
structure and chemical behavior during extraction and analysis similar to mycotoxins, but
there is a risk of natural contamination of samples.
In the scientific literature (Table 1), the widely used analytical column is C18 with
150 × 4.6 mm, and particle size of 5 µm. Most recent studies with UHPLC used sub-2 µm
diameter particles and permitted the reduction of LC column length to 100 × 2.1 mm [60,77,86]
and 50 × 2.1 mm [71,78,97]. Towards the mobile phase, the most used solvents are water,
acetonitrile and methanol, with the addition of formic acid, acetic acid or ammonium formate,
in different proportions and mixtures. Regarding LOD, the methods just for AFs present
lower LODs, as Nonaka et al. [67], with the lower LOD of 0.02 µg/kg, also Reza et al. [70] and
Alsharif et al. [76] with 0.05 µm/kg. Concerning multi-mycotoxins methods, the lowest LOD
is 0.17 µg/kg from Arroyo–Manzanares et al. [71], and the lowest LOQ is 0.05 µg/kg [75,86]
but the minor range of LOQ is in Bessaire et al.’s study (0.05–0.25 µg/kg) [86].
Toxins 2021, 13, 682 23 of 41
Chromatographic techniques have been commonly used in the determination of

aflatoxins and other mycotoxins, with good results, in particular excellent sensitivity
and the ability to detect multiple analytes in low levels in complex matrices, but require
expensive equipment and trained personnel and high maintenance costs, and may not
be a technique accessible to all countries and/or laboratories [35,98]. There is a need to
develop faster, cheaper and simpler methods [79] to improve and facilitate the control of
mycotoxins in order to ensure food safety.
8.1.2. Immunoassays
Immunochemical methods are emerging as new methods for the determination of
mycotoxins, based on the specific and high affinity reaction between the antigen (the
target (bio)analyte) and antibody [98]. Enzyme-linked immuno-sorbent assay (ELISA) is
one of the immunoassays with antibodies fixed on a solid base, able to distinguish the
three-dimensional structure mycotoxins, causing the specific bond [79]. This technique
requires antibodies produced by immunizing animals with mycotoxins, including rabbits
and goats. However, mycotoxins with low molecular weight do not produce immune
responses by themselves. Therefore, mycotoxins are conjugated with a carrier protein
or polypeptide before immunization in order to stimulate immunological response and
production of antibodies [98]. Conjugation depends on the chemical structure and func-
tional groups of mycotoxins. AFs do not have a reactive group, so a carboxylic group
is primarily introduced [98] and later conjugation with bovine serum albumin-BSA [99].
Cross-reactivity of antibodies, that is, the ability of antibodies to react with other anti-
bodies, influences the accuracy of the assay [64]. For example, Leszczynska et al. [100]
demonstrated that all antibodies used to determine total aflatoxins tested positive for
cross-reactivity (aflatoxin B1 100%, AFB2 200%, AFG1 15%, AFG2 16%, AFM1 63%). Most
monoclonal antibodies produced against aflatoxins are highly specific to AFB1 and have a
partial cross-reaction with AFG1 [101]. Other compounds with similar chemical groups can
also interact with antibodies, due to low molecular weight, resulting in underestimations
or overestimates [79].
ELISA has two main steps: (1) Reaction between antibody and antigen and (2) enzy-
matic reaction between enzyme and substrate. The assay occurs in a well of a test plate,
which contains antibodies selective to antigens of interest immobilized in a solid phase.
Then, another antibody, conjugated with an enzyme, binds to immobilized antigens. The
enzyme substrate is added, and a reaction occurs that involves color change measured and
compared with calibration curves, allowing quantification of antigens [93].
There are variations of this assay, depending on the characteristics of the antigen and
matrix. Competitive ELISA assay is based on competition for antibody binding sites [64].
There are two versions of competitive ELISA: Direct and indirect (Figure 3).
Direct ELISA uses a mycotoxin-enzyme conjugate that competes for the available
spaces on the coating antibody layer, while indirect ELISA involves a protein–mycotoxin
conjugate immobilized on the microplate that competes with mycotoxin present in the
sample [64,93]. The most commonly used enzyme is horseradish peroxidase (HRP) and
alkaline phosphatase (AP) [35,79]. In direct competitive ELISA, the sample solution or
mycotoxin standards are mixed with a mycotoxin coupled enzyme and are added to
wells coated with antibody. Thus, there is competition of mycotoxins with mycotoxin
conjugated by binding to the antibody. This is followed by a washing step to remove
any unbound enzyme conjugate. After that, an enzymatic substrate is added; enzyme
converts substrate into a color product. The reaction is interrupted by adding a stop
solution and color intensity is measured spectrophotometrically with an absorbance filter
of 450 nm [79,93,102]. In indirect competitive ELISA, antibody is added with sample
solution containing mycotoxins. Next, the solution is added to wells coated with protein–
mycotoxin conjugate, and the remaining free antibodies bind to mycotoxins in wells. After
washing, a second antibody labelled with an enzyme detected the first antibody [79,93,102].
Then, the enzymatic substrate is added, and the enzyme converts the substrate into a color
Toxins 2021, 13, 682 24 of 41
product. In these assays, color intensity is inversely proportional to the concentration of

mycotoxins in the sample [79]; that is, the higher concentration of mycotoxin, the lower
signal generated, since there is less mycotoxin conjugated with the enzyme or less second
antibody labelled with an enzyme.
Figure 3. 3.
Figure Schematic illustration
Schematic ofof
illustration Immunoassays:
Immunoassays:(a)(a)Direct
Directcompetitive
competitiveELISA;
ELISA;(b)
(b)Indirect
Indirect Competitive
Competitive ELISA
ELISA and
and (c)
Chemiluminescence Enzyme Immunoassay.
(c) Chemiluminescence Enzyme Immunoassay.
Direct
While ELISA uses auses
direct ELISA mycotoxin-enzyme
a single conjugate,conjugate
requiresthat
one competes for the
less incubation available
step and,
consequently,
spaces one lessantibody
on the coating washinglayer,
step [101],
whileindirect
indirectELISA
ELISAisinvolves
more sensitive and flexible
a protein–mycotoxin
since more
conjugate than one second
immobilized on theantibody can be
microplate bound
that per primary
competes antibody [93].
with mycotoxin On the
present in the
sample [64,93]. The most commonly used enzyme is horseradish peroxidase (HRP)inand
market, ELISA kits based on the direct competitive assay for the test of aflatoxins
differentphosphatase
alkaline food matrices(AP)
are already
[35,79].available,
In directincluding in nuts.
competitive In pistachio
ELISA, nuts, ELISA
the sample is or
solution
used for rapid methods for mycotoxin detection. Lee et al. [103] developed rapid direct
mycotoxin standards are mixed with a mycotoxin coupled enzyme and are added to
competitive ELISA for monitoring aflatoxin AFB1 at 10 µg/kg in pistachio and other nuts
wells coated with antibody. Thus, there is competition of mycotoxins with mycotoxin
and cereals. Bensassi et al. [104] studied the contamination of pistachio nuts in two years of
conjugated by binding to the antibody. This is followed by a washing step to remove any
storage, screening levels of AFB1 by ELISA combined with an immunoaffinity step. Some
unbound
biosensorsenzyme conjugate.
based on After that, an
indirect competitive enzymatic substrate
immunoassay is added;
for the detection enzyme
of AFB1 havecon-
verts substrate into a color product. The reaction is interrupted by adding
been developed for different matrices, like cereals [105,106], and peanuts [105,107]. a stop solution
and color intensity is measured spectrophotometrically with an absorbance filter of 450
nm [79,93,102]. In indirect competitive ELISA, antibody is added with sample solution
containing mycotoxins. Next, the solution is added to wells coated with pro-
tein–mycotoxin conjugate, and the remaining free antibodies bind to mycotoxins in wells.
After washing, a second antibody labelled with an enzyme detected the first antibody
[79,93,102]. Then, the enzymatic substrate is added, and the enzyme converts the sub-
strate into a color product. In these assays, color intensity is inversely proportional to the
concentration of mycotoxins in the sample [79]; that is, the higher concentration of my-
cotoxin, the lower signal generated, since there is less mycotoxin conjugated with the
Toxins 2021, 13, 682 25 of 41
Several studies have compared the determination of mycotoxins by ELISA and HPLC
method, since HPLC is considered a reference method and widely used [35]. For example,
Azer and Cooper (1991) analyzed 178 food samples for total aflatoxins, including nut
and nut products, obtaining a correlation coefficient of 0.999, i.e., there is a high degree
of agreement between the two methods. It should also be noted that the ELISA method
demonstrated a high degree of precision, useful for rapid testing, in a concentration range
of 15 to 50 µg/kg [108]. Moreover, Reza et al. [70] and Ostadrahimi et al. [73] used
the ELISA method to determine AFs in pistachio and other nuts, and the results were
favorably confirmed by HPLC. Contrary to HPLC, ELISA is not useful in multi-mycotoxin
determination because it requires different assays with different antibodies specific to each
mycotoxin [103,104] or group of mycotoxins [109], becoming more expensive and more
time consuming.
8.1.3. Biosensors
The chromatographic methods are expensive and require trained personnel and the
procedures are, in general, complex and slow for multiclass residues. For these reasons,
a new technology is necessary to simultaneously detect different compounds including
mycotoxins.
In general (bio) sensors provide fast, reliable screening, with good sensitivity and
selectivity, and low detection limits and are relatively economic, especially if applied to a
large number of routine analyses. The detection of mycotoxins by biosensors mostly relies
in two types of detection methods: Optical and electrochemical [15]. The current trend is the
optical biosensors based on chemiluminescent methods, which can be divided into CLIA
(chemiluminescent immunoassay) and CLEIA (chemiluminescent enzyme immunoassay)
(Figure 3).
CLIA detection is the result of a very selective (bio)chemical reaction between the anti-
gen (the target (bio)analyte) and an antibody specific to detection of the target (bio)analyte.
The reaction mechanism is based on oxidation and reduction reactions that yield changes
in chemiluminescence, depending on the amount of target analyte that can be monitored
by optical detection methods. The most commonly utilized chemiluminescent (CL) com-
pound in aqueous solution is luminol or isoluminol. In the presence of a catalyst (enzyme,
metal-containing molecule or metal), luminol interacts with hydrogen peroxide in alkaline
solution to produce 3-aminophthalate in an excited electronic state, which returns to the
ground state with the emission of light. The signal is then detected by an optical detec-
tion system. To increase the lifetime and the amplitude of the signal, a substance known
as an “enhancer” (for example, 4-iodophenol) is added to the reaction medium. At the
end of an immunoenzymatic experiment, this luminous reaction can be used to detect
antigen–antibody binding [110].
CLEIA (combines chemiluminescence (CL) and enzyme immunoassay) detection
techniques are currently the most sensitive in immunoassay research. CLEIA is becoming
increasingly popular for the detection of trace compounds due to its great qualities of
high specificity, lower limit of detection, good linearity range and environmental friendli-
ness [111]. The main two label enzymes used in CLEIA are horseradish peroxidase (HRP)
and alkaline phosphatase (ALP). Due to the low cost and the ease of access, horseradish per-
oxidase is considered the most used. While the luminescence efficiency of the horseradish
peroxidase system can be increased by using a suitable enhancer [111], it is quite poor
when compared to the ALP system [111]. In any case, CL substrates, such as the lu-
minol/peroxide/enhancer system for horseradish peroxidase (HRP) or dioxetane-based
substrates for alkaline phosphatase, can efficiently detect enzyme labels.
Toxins 2021, 13, 682 26 of 41
One of the advantages of the CLEIAS is the possibility of application of advanced

nanotechnology. For example, Freitas, Barros, Brites, Barbosa and Silva (2019) used the
Evidence Investigator Biochip Array Technology (BAT) (Randox, Crumlin, UK) in a semi-
quantitative methodology in the analysis of mycotoxins in maize [112]. In this case, Biochips
were used, composed of 9 mm square-shaped solid substrate with a panel of discrete test
regions (DTR) where each DTR consists of different antibodies or other reactive species
specific (multiplexing) to each assay. The advantage of being able to detect and semi-
quantify, in a single analysis, multiple analytes, makes CLEIA a powerful screening tool in
several matrices.
While (bio)sensors are a trend and numerous have been developed during the last
years, there is a lack of application of this methodology to determine mycotoxins, especially
in pistachios. Kumaniaris et al. [113] developed an electrochemical immunosensor for the
determination of AFB1 in pistachio based on the immobilization of the AFB1 antibodies
on the surface of gold screen printed electrodes. This method presented good sensitivity
(LOD = 1 ng/mL) showing potential as a screening method, but also as a quantitative method
since it successfully determines AFB1 concentrations in the range of 4.56–50.86 ng/mL in
unknown pistachio samples.
Spectroscopy techniques have been applied for rapid and real-time analysis for mycotoxins,
with little or no sample preparation, without destroying the sample [114]. Paghaleh et al. [115]
developed a method based on the laser induced fluorescence spectroscopy, using a UV laser
(λ = 308 nm) for in line measurement of the concentration of AFs in pistachio nuts, without
sample preparation, and results are in agreement with the HPLC method. Wu and Xu [116]
developed a multiplexing fiber optic laser induced fluorescence spectroscopy for detection
of AFB1 in pistachios, using five wavelengths between 440 and 564 nm because physical
and chemical characteristics of pistachios at different positions of contaminated products
are unequal or nonuniform. Results show an accuracy of 97% and low levels of AFB1
(50 ppb). Valasi et al. [117] used diffuse reflectance infrared Fourier transform spectroscopy
with chemometrics for screening AFs in pistachios using four spectral regions to classify AF-
contaminated from non-contaminated pistachios and results show that this methodology
correctly separated 80% of test samples.
9. Occurrence of Mycotoxins in Pistachios

In pistachio nuts, AFs are the most frequently found mycotoxins (Table 2). The
occurrence of AF contamination is sporadic and very dependent on environmental condi-
tions [11]. In nuts, FAO indicates that Aspergillus flavus and A. parasiticus do not grow or
produce aflatoxins at temperatures below 10 ◦ C, relative humidity below 70% and water
activities (aw) lower than 0.7 [118]. According to Baazeem et al. [119], A. flavus grows in
pistachio when incubated at between 25 and 35 ◦ C and with aw ranging from 0.95 to 0.98,
in in vitro and in situ studies, but AFB1 was optimally produced at 30 ◦ C and aw >0.98.
These mycotoxins are predominant in Africa, Asia and North and South America, where
environmental conditions are more favorable. However, due to globalization and climate
change, AFs can be found all over the world [120]. Beside nuts, AFs occur in various
other foods, namely cereals (corn, rice, wheat), spices (pepper, turmeric, ginger), oilseeds
(peanuts, soybeans, sunflower) and legumes, among others [39].
Toxins 2021, 13, 682 27 of 41
Table 2. Occurrence of mycotoxins in pistachios worldwide.
Average
Number Nº Positive % Positive Min-Max
Reference Country Mycotoxin Concentration
Samples Samples Samples (µg/kg)
(µg/kg)
AFB1 3699 37 5.9 -
[24] Iran 10,068
AFs 2852 28 7.3 -
AFs 2 6 - 0.4–0.7
[68] Algeria 31
OTA 1 3 170 -
[121] Spain 70 OTA 2 3 0.228 0.134–0.321
AFB1 - 9.5–43.8
AFB2 - 0.9–9.4
[70] Iran 32 17 53
AFG1 - n.d.–19.7
AFG2 - n.d.–7.1
[122] Spain 70 AFs 14 20 8.9 n.d.–108
53 AFS 18 34 16.6 -
AFB1 - 1.9–411
[72] Saudi Arabia AFB2 - n.d.–10.7
9 9
AFG1 - n.d.–4.6
AFG2 - n.d.–0.8
AFs 0 0 - -
H-T2 0 - - -
[25] Austria 8 OTA 1 13 <LOQ -
T2 0 - - -
ZEA 0 - - -
AFB1 2 20 - 0.5–1.2
AFB2 1 10 0.9 -
AFG1 1 10 0.5 -
AFG2 0 - 0.0 -
DON 0 - - -
[60] USA 10
FB1 0 - - -
FB2 0 - - -
OTA 3 30 1.4 1.0–6.6
T2 0 - - -
ZEA 0 - - -
AFB1 50 31.9 (median) 8.2–354.5
[74] Italy 8 4
AFs 50 33.9 (median) 8.8–387.3
AFB1 4 40 7.10 5.30–10.15
AFB2 3 30 2.18 1.46–3.47
[76] Malaysia 10 AFG1 4 40 2.45 1.90–3.31
AFG2 2 20 0.86 0.81–0.90
OTA 0 - - -
[123] Turkey 50 OTA 2 4 0.527 0.198–0.850
AFs—Aflatoxins (AFB1, AFB2, AFG1 and AFG2); AFB1—Aflatoxin B1; AFB2—Aflatoxin B2; AFG1—Aflatoxin G1; AFG2—Aflatoxin G2;
FB1 and FB2—Fumonisins; OTA—Ochratoxin A; DON—Desoxynivalenol; T-2/HT-2 –Trichothecenes; ZEA—Zearalenone; LOQ—limit of
quantification; n.d.—not defined.
AFs were first identified in England, in the 1960s, where an outbreak arose, known
as “Turkey X disease”, which caused the death of more than 100,000 turkeys due to con-
sumption of peanut flour contaminated by fungi, namely species such as Aspergillus flavus
and aflatoxins [13]. The first outbreak of aflatoxicosis in humans occurred in 1974 in India
and caused 106 deaths due to consumption of contaminated maize from environmental
causes that occurred before harvest [32]. In Kenya, in 2004, one of the largest and most
severe outbreaks of aflatoxicosis occurred in humans, which caused the death of 125 people
due to liver failures due to consumption of contaminated maize, with more than 300 cases
of abdominal pain, pulmonary edema and liver necrosis [124]. This outbreak was due to
Toxins 2021, 13, 682 28 of 41
incorrect storage of maize in a humid and hot environment, providing for the growth of
fungi, combined with a poor diet among the low socio-economic population and also a
lack of medical resources [125].
The vast majority of studies summarized in Table 2 present high values of positive
samples; however, sampling is reduced and may not be representative of the global market.
Cheraghali et al.’s study [24] comprises a greater number of samples, collected between
March 2002 and February 2003 in Iran; 37% of samples were contaminated with AFB1 and
11.8% were above maximum levels in the country (5 µg/kg), higher than that legislated in
Portugal and Europe. About 28% of the samples were contaminated with all AFs. AFB1
is the most frequently found and most concentrated. In some samples, the maximum
levels were exceeded, constituting a risk to the health of the population, particularly in the
study by Diella et al. [74], which presented the highest levels of AFB1 and sum of AFs,
El Tawila et al. [72], Ali Alsharif et al. [76] and Cheraghali et al. [24]. El Tawila et al. [72]
showed that AFB1 content in pistachio nuts has the highest amplitude, ranging from
1.9 to 411 µg/kg, and in the study by Diella et al. [74] values of AFs are between 8.8 and
387.3 µg/kg.
In Europe, the occurrence data on food as submitted to EFSA, resulting from samples
collected between 2003 and 2018 to reflect the current contamination levels in European
countries, show that the food category “Legumes, nuts and oilseeds” is one of the greatest
contributors to dietary exposure to AFs and AFB1, and the highest AF mean concentrations
are in pistachios, peanuts and other seeds [37]. Previously, pistachios also had the highest
level of AFs compared with other tree nuts [23]. In Iran, the main producing country, the
mean concentrations of AFT in pistachio was 54 µg/kg and considering the maximum level
of 4 µg/kg and 20 µg/kg, 40 and 60% of pistachio samples were rejected, respectively [126].
JECFA conclude that pistachios were the main contributor to dietary AF exposure from
tree nuts, ranging from 0.2 to 0.8 ng/kg bw per day [127]
Few studies have evaluated AFs in nuts and derivatives with different types of
processing. Ostadrahimi et al. [73] determined AFs in raw pistachios and roasted with
salt, demonstrating that samples toasted with salt contained higher AFs than unprocessed
samples, in the order of 22.02 µg/kg. This fact may be due to prolonged storage time with
conditions suitable for fungal growth in addition to thermoresistance and stability of AFs
at processing temperatures. AF occurrences were sometimes different from study to study
depending on the characteristics of the samples analyzed. Some studies have not detected
AFs in the food matrices [67], but other studies reported high levels of contamination. This
is justified by the different origins of products (not mentioned in many cases), different
storage conditions or type of processing.
Several studies evaluated the OTA levels in pistachio nuts [25,60,76,122,124,128] and
the results show low percentage of positive samples for OTA contamination. In the study
by Liao et al. [60], three of the pistachio samples were contaminated with OTA, between
1.0 and 6.6 µg/kg. Moreover, Varga et al. [25] and Zinedine et al. [128] noticed the presence
of OTA, but at levels lower than LOQ.
Fernane et al. (2010) conclude that pistachio can be highly contaminated with aflatoxin-
or ochratoxin-producing isolates but the presence of mycotoxins is not high. In fact, out
of 31 samples, only two samples were contaminated with AFs and only one sample had
OTA [68].
None of the studies in this review mentioned the presence of other mycotoxins, such
as DON, FB1, FB2, ZEA, T2 or HT2, in pistachios. In summary, the available data is still
scarce in pistachio nuts and the evaluation of the effect of processing is lacking.
Regarding emerging mycotoxins, few studies have evaluated the presence of these
mycotoxins in pistachios. Tolosa et al. [129] surveyed the occurrence of ENNs and BEA in nuts
and dried fruits in Spain, studying three samples of pistachio. Results show that no presence
of BEA and ENNs in pistachio fruit is detected, but, in pistachio shell, ENA, ENA1 and
ENB are found at concentrations of 0.326 µg/kg, 0.015 µg/kg and 0.209 µg/kg, respectively,
explained by the protective effect of the shell. FUS is produced by Fusarium proliferatum,
Toxins 2021, 13, 682 29 of 41
F. subglutinans and F. verticillioides and occurs in grains and grain-based foodstuff [55]. STC,
an Aspergillus mycotoxin, mainly occurs in grain, green coffee beans, spices, nuts and cheese,
but information is still limited. Concerning Alternaria mycotoxins, TeA is the most frequently
found in nuts like almonds, hazelnuts, peanuts and pistachio [68] and are probably associated
with negative effects on protein biosynthesis [60].
10. Biomonitoring
Biomonitoring is an important method for assessing the real exposure to aflatoxins by
humans, determining concentrations of mycotoxins, their metabolites or reaction products
in biological fluids [130]. It involves the collection of biological samples from individuals,
such as blood, urine, saliva, breast milk, as well as hair and nails. To do this, it is necessary
to have knowledge of toxicokinetics, especially biotransformation, to identify possible
biomarkers of exposure. Biomonitoring is currently an area under development; determina-
tion of AFs in food does not constitute a true assessment of exposure since individuals are
exposed to multiple food sources with aflatoxins, in addition to other routes of exposure,
such as inalatory and dermal [47].
In the case of AFs, biomonitoring can be performed by analyzing the presence of AFB1
metabolites in blood, milk and urine. In addition, excreted DNA and protein adducts in
blood can also be monitored [14]. AF metabolite evaluation in biological fluids is usually
performed through liquid chromatography coupled with tandem mass spectrometry (LC-
MS/MS). Based on studies in humans and animals, adduct AFB1-N7-guanine in urine
represents the most reliable biomarker for exposure to aflatoxin, but reflects only recent
exposure. In addition, AFB1-albumin adduct is also considered a biomarker for prolonged
blood exposure to AFB1 due to the half-life time of albumin of 20 days. AFM1 can be found
in human breast milk, which can be considered as a biomarker of maternal and infant
exposure to AFB1. It is also excreted in urine and can be considered a biomarker, however,
only for recent exposure to aflatoxins [14,47].
In Portugal, Martins et al. [130] evaluate the exposure of the population to mycotoxins,
between 2015 and 2016, through analysis of 37 biomarkers in 24 h urine samples and first
morning urine, to estimate probable daily intake and perform risk characteristics. From this
study, it was concluded that the Portuguese population is more exposed to six mycotoxins:
Deoxynivalenol, zearalenone, ochratoxin A, alternariol, fumonisin B1 and citrinin. The
levels are above safety limits, representing a public health problem. In this study, exposure
to aflatoxins was not evaluated.
11. Prevention and Control

Due to the risks of aflatoxins to human health and economic losses, strategies have
been developed to reduce Aspergillus and AFs contamination. Prevention of fungal contam-
ination is the most effective and preferable measure.
Reducing levels of AFs in pre-harvest begins with plant selection, planting and har-
vesting dates, plant density and crop rotation, as well as soil treatments, irrigation and pest
management [49]. AF contamination can be prevented using seeds genetically modified
to be resistant to Aspergillus infection and/or environmental stress [34]. However, plant
breeding could not be effective because resistance is conferred by multiple genes and
environmental pressure is an uncontrollable factor [131].
Another biological pre-harvest strategy to reduce AFs is using non-toxigenic/atoxigenic
A. flavus isolates to competitively exclude aflatoxin-producing strains during crop coloniza-
tion or physical displacement. To ensure efficacy, atoxigenic fungi must be (1) selected from
local environments, (2) highly competitive and (3) predominant relative to the toxigenic
strain in agricultural environments. This strategy shows a reduction in AF contamination
of between 70 and 90% in cotton, maize and peanuts, and it has also been implemented
in pistachios with reductions ranging from 20 to 45% [131–133]. Several of the atoxigenic
A. flavus strains have been developed into biopesticides for the management of AF con-
tamination and they are already used in the USA in pistachios, such as AF36 coated into
Toxins 2021, 13, 682 30 of 41
a carried sterile grain [134]. However, there are uncertainties regarding their use, mainly
(1) the impact of the addition of biocontrol strains in Aspergillus population, like a decrease
of A. flavus followed by an increase in A. niger [135] and (2) the possibility of atoxigenic
strains reverting back to toxin producers [136]. Some Aspergillus strains could be also
effective in post-harvest AF mitigation [132].
While the pre-harvest contamination with AFs is more common in tree nuts than post-
harvest contamination [131], in post-harvest, control of moistures, temperature, mechanical
damage, insect damage and aeration can prevent mycotoxin contamination.
Another prevention strategy is predictive modelling, using large volumes of data
and various correlate environmental factors with the potential for A. flavus growth and
consequently aflatoxin production in the entire food chain [136]. In pistachio storage,
Marín et al. [137] applied models to predict the growth of A. flavus and AF production as
functions of moisture and temperature and results show that the model correctly predicts
the presence of A. flavus in 90% of cases and AFs in 89% of cases. Aldars-García et al. [138]
also attempted to model growth and AFB1 production by A. flavus in storage and transport
in order to support decisions on ventilation timing and refrigeration adequation, respec-
tively; the model correctly predicted the presence of AFB1 in 70 to 81% of cases. While
post-harvest modelling is more developed, preventing contamination in pre-harvest is also
a good perspective. Kaminiaris et al. [139] developed a mechanistic model considering a
tree’s phenology and meteorological data which correctly predicted 75% of AFB1 contami-
nation in pre-harvest; the authors suggested that this model could indicate the appropriate
time for harvest, supporting agricultural systems, and also the pistachios with the highest
risk of contamination due to the prediction of field conditions. Predictive modelling has
also been applied to OTA in pistachio by Marín et al. [140], who built a probability model
function of moisture and temperature, correctly predicting 90% of the cases.
Recently, metabolomics was applied to future prevention of mycotoxins. This new
science analyzes metabolomes, all the low molecular weight metabolites in biological
samples, as a result not only of the cell’s genome but also of the environment interaction.
Metabolomics is useful for understanding the chemical interactions between plant, toxi-
genic fungus and microbiota, and the influence of biotic and abiotic stress in biosynthesis
of mycotoxins and modified forms of mycotoxins as a result of biological or chemical
modifications, such as food processing. The knowledge of determinants and factors that
govern fungus infection and mycotoxin production allows the development of new efficient
strategies to mitigate the occurrence of mycotoxin in food [141].
12. Decontamination
In order to reduce or eliminate AF contamination and to ensure food safety, decon-
tamination methods can be physical, chemical or biological. The effectiveness depends on
several factors, such as the chemical stability of mycotoxins, the nature of the process, the
type and the interaction with the food matrix and the interaction with multiple mycotox-
ins [142]. It should always be taken into account that these methods should: (1) Inactivate,
destroy or remove the toxin; (2) not be able to produce or leave toxic residues; (3) necessarily
maintain the nutritional value of the food; (4) not change the acceptability or technological
properties of the food; and (5), if possible, destroy fungal spores, preventing the prolifera-
tion and production of new mycotoxins [142]. Table 3 presents the outcomes from studies
on aflatoxin B1 decontamination by physical, chemical and biological methods.
Toxins 2021, 13, 682 31 of 41
Table 3. Summary of studies using decontamination methods to degrade AFB1 in pistachio nuts.
Reduction
Method Treatment Assay Conditions Reference
AFB1
Heat/Roasting 150 ◦ C for 30 min 63% [143]
Physical
Gamma radiation 10 kGy 68% [144]
Ozonation 0.9 mg/L for 420 min 23% [145]
Seed extract
37 ◦ C for 24 h 91% [146]
Trachyspermum ammi
Leaf extract
Chemical 37 ◦ C for 24 h 96% [147]
Adhatoda vasica
Leaf extract
30 ◦ C for 72 h 95% [148]
Corymbia citriodora
Leaf extract
30 ◦ C for 72 h 90% [149]
Ocimum basilicum
Kefir-grains 30 ◦ C for 6 h 97% [150]
Biological Bacillus subtilis UTBSP1 35 ◦ C for 5 days 95% [151]
Saccharomyces cerevisiae - 40–70% [152]
15 mL lemon juice
Others Heat + Acidification 6 g citric acid 49% [153]
120 ◦ C for 1 h
12.1. Physical Decontamination

Physical processes of decontamination include separation of the density-contaminated
fraction and the reduction/inactivation of AFs by cooking, boiling, toasting, microwave
heating or irradiating contaminated food. However, AFs are highly heat stable and are
not easily destroyed, so it is necessary to heat at high temperatures to effectively decrease
the levels of aflatoxins [34], depending on time, temperature and moisture content [13].
Some studies indicate that roasting aflatoxin-contaminated pistachios at 150 ◦ C for 30 min
reduced AFB1 levels by 63%; when the same process was performed, for 120 min, more
than 95% of the AFB1 was degraded, but changes were caused in the appearance and taste
of the pistachios [12,143]. Ostadrahimi et al. (2014) determined AFs in raw and roasted
with salt pistachios, demonstrating that the samples roasted with salt contained higher
content of AFs (mean: 22.02 µg/kg) than the unprocessed samples (mean: 0.48 µg/kg) [73].
This fact, according to the authors of the study, may be due to the prolonged storage time
in conditions suitable for fungal growth. According to Yazdanpanah et al. (2005), the effect
of toasting on the reduction of AFs in pistachios was evaluated to define the optimum
conditions [143]. It was found that the treatment of samples at 150 ◦ C for 30 min significantly
reduced the levels of AFs, without alteration of organoleptic characteristics. Heat treatment is
widely applied in the food industry, for biscuits, pasta, cereals, snacks, etc. [154].
Recently, non-thermal processes like Cold Plasma treatment, electron beam irradiation
and pulsed electric field have been applied to reduce mycotoxin contamination with good
results in different foodstuffs [155–158]. These techniques are processed at near room
temperature, and so do not significantly affect the nutritional status or the organoleptic
properties, constituting alternatives to the conventional decontamination techniques for
pistachio [159–161]. Cold plasma treatment (CP) is an interesting tool to reduce mycotoxins
due to both fungi reduction and mycotoxin degradation in all food chains [162,163]. CP
was already applied by Tasouji et al. [164] in pistachio nuts to reduce Aspergillus flavus and
results showed a reduction of 67% with 10 min of irradiation time, without alteration of the
texture. The CP technique was also performed to reduce AFB1 in hazelnuts and reduced
70–73% of spiked AFB1 [165]. This decontamination process is possible for industrial
implementation because it is eco-friendly, energy efficient, low cost and fast. Gamma
(γ) and ultraviolet (UV) radiation are also applied for destroying AFS because they are
photosensitive [13]. Ghanem et al. [144] studied the effect of gamma radiation on the
inactivation of AFB1 and concluded that at a dose of 10 kGy there was a reduction of 68.8%
Toxins 2021, 13, 682 32 of 41
and 84.6% in shelled and in-shell pistachios, respectively, and degradation was positively
correlated with the increase of dose. There is a significant difference between shelled and
in-shell pistachios; the authors explain this due to the fact that in in-shell pistachios the
fungal growth was limited to the surface of the peel and limited Aspergillus entrance into
the kernel itself. These techniques are applicable to different food matrices. However, due
to the associated risks to human health, more studies are needed [34].
Currently, mechanical separation based on size and density by gravity systems re-
moves small and shriveled pistachios. Additionally, manual sorting of stained shells,
discolored shells and defective pistachios is also applied in industry. Both methods are
applied to reduce AF contamination in pistachios [154,166–168]. Several studies indicate
a positive correlation between physical properties (size, color, shape, density and fungal
damages) and AF contamination. Doster and Michailides [169] reported that pistachio
nuts with oily shells, crinkled shells or shell discoloration had more kernel decay and
NOW infestation, and consequently more AF contamination. Shakerardekani et al. [170]
concluded that pistachios with yellowish-brown and dark-greyish stains have the highest
levels of AFs, and, after removing those stained nuts, there is a contamination reduc-
tion of between 95 and 99%, depending on pistachio cultivar. Manual sorting is a more
time-consuming and tedious task, so sorting using new technologies has been studied.
McClure and Farsaie [171] reported the elimination of pistachios contaminated with AFs by
fluorescence sorting. Özlüoymak and Güzel [172] develop an image processing technique
to measure and analyze color by irradiating pistachios at a wavelength of 365 nm and the
contaminated pistachios exhibited bright-greenish yellow fluorescence. This method can
be applied at a new real-time determination and separation system.
Furthermore, some pistachios could look healthy on the outside but have necrotic
spots resulting from stigmatomycosis disease, which has a positive correlation with higher
aflatoxin contamination. In addition, Yanniotis et al. [173] developed a method based on
X-ray imaging for the detection of necrotic spots in pistachios and rejecting these nuts
results in a reduction of AFs of 60%. This methodology could be applied in an automatic
separation machine at industrial levels to reduce AF contamination.
12.2. Chemical Decontamination

Chemical decontamination methods use chemical compounds that degrade the struc-
ture of AFs. These methods may result in toxic degradation products that may harm
the consumer’s health, and/or unacceptable changes in the quality of the final product,
both nutritional and sensory [11]. Within the chemical methods, three are highlighted:
Acidification, ammonization and ozonation.
Acidification is a way to prevent fungal growth or inactivate AFs. Lactic acid, citric
acid, tartaric acid or hydrochloric acid are used more frequently and the use of salicylic,
benzoic, boric, oxalic or propionic acids has been shown to be effective in reducing the
content of AFs. In the case of AFB1, the use of acids results in the conversion to AFB2,
AFB2a, AFD1 and less toxic forms [13,29,31,154]. This is a simple method, without the
need for equipment or specialized people, and only requires the contact of the food matrix
with acid for a certain period of time [34], and is a low-cost technique.
Ammonization is the most efficient technique, with a reduction of about 99%, but more
common in animal feed decontamination. Ammonium is used in gaseous or hydroxide
form, degrading AFB1 in AFD1 in alkaline, and consequently reducing mutagenicity. This
technique requires more complex infrastructure [34].
Ozonation is one of the most promising methods, using gaseous ozone, a potent
oxidant, for short periods of time. It is very effective in different types of food matrices and
is accepted to be used at the industrial level [34,154]. Akbas and Ozdemir (2006) study the
efficiency of ozone in the degradation of AFs in pistachios, and the results indicated that
ozonation at 0.9 mg/L for 420 min reduced AFB1 and AFs by 23% and 24%, respectively,
which indicated that AFB1 is more sensitive to this method than the other aflatoxins (AFB2,
Toxins 2021, 13, 682 33 of 41
AFG1 and AFG2). In addition, no significant changes occur in color, fatty acid composition
or organoleptic properties of pistachios [145].
Another method for decontamination is the association of two or more types of
processes. Rastegar et al. (2017) use physical and chemical methods through roasting 50 g
of pistachio nuts at 120 ◦ C for 1 h with 15 mL lemon juice and/or 6 g of citric acid to remove
AFB1. The level of AFB1 was reduced by 49% without a noticeable change in the desired
appearance of pistachios. The reduction was higher (93%) using 30 mL lemon juice but
desired physical properties were altered [153].
It is also worth mentioning the use of aqueous extracts of plants, since they are rich in
bioactive compounds such as tanines, terpenoids, alkaloids and flavonoids, with antifungal
properties [34,154]. Several studies have indicated the high efficiency in the degradation of
AFs with the use of plant extracts. The authors indicate that detoxification is related to the
modification of the lactone ring structure of the AFs [148,151]. The extracts will have high
molecular weight compounds, be soluble in water and be thermolabile [146,147]. In the case
of A. vasica extract, alkaloides appear as a principle of detoxification of aflatoxins [147]. All
studies reveal a reduction in the contents of other aflatoxins, especially AFB2 [146,147,149].
While this technique is more time consuming, it is simple since the sample is incubated
with plant extract in specific time and temperature conditions, it has high efficacy and,
because it is considered “natural”, it is more acceptable to the consumer. These extracts
have compounds that are biodegradable, environmentally friendly, safe and low cost,
constituting an alternative to other synthetic chemical compounds [154]. However, the
standardization of the extract activity must be assured, since the composition of the extract
is influenced by edapho-climatic conditions, and different cultivars. A drawback is the
influence on the organoleptics characteristics of the foodstuffs that can be overcome by
micro- or nanoencapsulation [147,174,175].
12.3. Biological Decontamination

Biological methods use bacteria, yeasts or enzymes to degrade or inactivate AFs, or,
in some cases, for adsorption of mycotoxins. Lactic acid bacteria, such as Lactobacillus,
and Saccharomyces cerevisiae, are among the most studied for this process, especially in
fermented products and beverages. The food is inoculated with the microorganism, so
this method is more complex and time consuming, because it requires the growth of the
microorganism. The enzyme peroxidase decomposes hydroperoxides and free radicals
are generated that react with aflatoxins [29]. For example, kefir-grains are a symbiotic
association of microorganisms and Ansari et al.’s (2015) study indicated a 96.8% reduction
of AFG1 in pistachio with kefir grains pre-treated at 70 ◦ C and incubated for 6 h at
30 ◦ C [150]. Bacillus subtilis UTBSP1 have the ability to reduce AFB1 by 95% as shown
in Farzaneh et al.’s (2012) study, resulting from incubation at 35–40 ◦ C for five days, and
its degradation activity was likely due to the extracellular enzymes [151]. Yeasts are also
studied for decontamination, for example, Saccharomyces cerevisiae has the ability to surface
binding aflatoxin in 40% and 70%, depending on the initial AF concentrations (10 ppb and
20 ppb, respectively) and the study also showed that acid and heat treatments increase
this ability to 60–73% and 55–75%, respectively. This treatment had no effect on qualitative
characteristics of pistachio nuts, such as color and texture [152].
13. Conclusions
Food fungal contamination is a hot topic receiving considerable attention by re-
searchers and the food industry. While it is important to understand the mechanisms
and conditions that favor the production of mycotoxins by fungi in foods, it is also im-
portant to monitor the levels of contamination of highly consumed food products such
as nuts, to ensure consumers’ health. Pistachio is considered a “healthy food”, due to its
nutritional level and its health benefits, so research is of utmost importance to predict the
reduction of mycotoxin contamination. The development of analytical methods, screen-
ing and confirmation is of utmost importance in nuts, where the extraction procedures
Toxins 2021, 13, 682 34 of 41
are very complex, mainly because of the high fat content. Most published studies agree
that QuEChERS extraction followed by HPLC-MS/MS for detection/quantification is the
most suitable analytical method to carry out multi-mycotoxin determination in pistachios
allowing achieving low detection levels with higher sensitivity, selectivity and specificity.
In fact, multi-target methods are the most useful due to the co-occurrence of mycotoxins
in food. The detection of multiple mycotoxins employing HRMS hybrid detectors as
QqTOF or QOrbitrap will have the most impact in the near future, due to their potential for
high-throughput analysis and more accurate mass measurement. Moreover, new methods
should be targeted to research emerging mycotoxins due to their negative effects on human
health and lack of information available concerning the occurrence in nuts. However, the
previous use of screening methods is of great importance due to their simplicity and rapid
throughput.
The information presented here suggests that the number of monitored mycotoxins in
pistachios increases with a vast range of physicochemical properties and proper research
on the means of decontamination should be conducted to reduce the number of nuts that
are rejected due to mycotoxin contamination. Heat treatment is widely applied in the food
industry but the use of plant extracts has aroused more interest because it is more “natural”
and has fewer health effects. However, there are difficulties related to standardization:
Availability varies throughout the year and there is variability in composition among, for
example, different cultivars and diverse edaphoclimatic conditions.
Author Contributions: Conceptualization, A.S.S. and A.P.; methodology, A.R.S.M. and S.B.; formal
analysis, A.R.S.M.; investigation, A.R.S.M. and S.B.; resources, A.R.S.M. and A.S.S.; data curation,
A.R.S.M. and S.B.; writing—original draft preparation, A.R.S.M.; writing—review and editing, A.P.,
A.S.S. and S.B.; visualization, A.R.S.M.; supervision, A.P. and A.S.S.; funding acquisition, A.S.S. and
A.P. All authors have read and agreed to the published version of the manuscript.
Funding: The work was supported by UIDB/00211/2020 with funding from FCT/MCTES through
national funds.
Informed Consent Statement: Not applicable because the work was based on the literature review.
Data Availability Statement: Not applicable because the work was based on the literature review.
Acknowledgments: Data sharing is not applicable to this article.
Conflicts of Interest: The author declares no conflict of interest and that this work is original and
has not been published anywhere before.
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toxins

Toxins 2021, 13, 682. https://doi.org/10.3390/toxins13100682 https://www.mdpi.com/journal/toxins

2.3.1. Acute Toxicity

2.3.2. Chronic Toxicity

4. Fumonisins (FB1 and FB2)

6.1. Desoxynivalenol (DON)

6.2. HT-2 Toxin and T-2 Toxin

8. Analytical Methods for the Determination of Mycotoxins

Table 1. Summary of analytical methodologies used for mycotoxins determination in pistachio.

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

Type of Clean-Up Procedure of Analytical Internal LOD LOQ

8.1. Detection and Quantification

Chromatographic techniques have been commonly used in the determination of

product. In these assays, color intensity is inversely proportional to the concentration of

One of the advantages of the CLEIAS is the possibility of application of advanced

9. Occurrence of Mycotoxins in Pistachios

Table 2. Occurrence of mycotoxins in pistachios worldwide.

11. Prevention and Control

12.1. Physical Decontamination

12.2. Chemical Decontamination

12.3. Biological Decontamination

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