331

Journal of Food Protection, Vol. 80, No. 2, 2017, Pages 331–337

doi:10.4315/0362-028X.JFP-16-290
Copyright Ó, International Association for Food Protection

Research Paper

Dietary Exposure and Natural Occurrence of Total Aflatoxins in

Basmati Rice of Pakistan
NIGHAT SULTANA,1* IFFAT TAHIRA,1 MUMTAZ KAUSAR,2 SYED MUJAHID HASSAN,1 AND NAFEESA QUDSIA HANIF1

1Romer Labs, Pakistan, Flat No. 8, First Floor Satellite Shopping Plaza, 71-A 6th Road, Rawalpindi, Punjab 46000, Pakistan; and 2Government Post
Graduate College for Women, Rawalpindi, Punjab 46000, Pakistan

MS 16-290: Received 14 July 2016/Accepted 11 October 2016/Published Online 24 January 2017

ABSTRACT
This study presents the assessment of total aflatoxins (TAFs) in basmati rice (brown, 1,081; white, 1,170) collected from
different areas of Punjab, Pakistan, during 2010 to 2015. Due to the carcinogenicity of TAFs, daily dietary exposure is also
evaluated based on rice consumption survey data. Methodology was standardized by matrix spike recoveries at four fortification
levels (0.1, 0.5, 2.5, and 12.5 ng/g) for TAFs (aflatoxins B1 [AFB1], B2 [AFB2], G1 [AFG1], and G2 [AFG2]). The present study
reveals that 1,750 samples (77.74%) were tainted with AFB1, whereas TAFs were detected in 370 samples (16.43%). Of positive
samples, 854 brown rice samples (79%) were positive for AFB1, and 154 samples (14.24%) were contaminated with TAFs. For
white rice, 896 samples (76.58%) were contaminated with AFB1, whereas 205 samples (18.46%) were found positive for TAFs.
Study findings were used to construct a frequency distribution, and AFB1 levels were also compared against permissible levels of
TAFs (10 ng/g) as legislated by the European Commission. Results further revealed that daily dietary exposure of TAFs ranged
from 0.51 to 10.22 ng/kg of body weight per day, which exceeds the permissible limit of 1 ng/kg of body weight per day as
defined by the Joint FAO/WHO Expert Committee on Food Additives.
Key words: Brown rice; Carcinogenic; Dietary intake; Total aflatoxins; White rice

Rice (Oryza sativa), the second-largest cereal crop of
Pakistan, is the most important staple food in the country
(17). Pakistan is one of the largest producers of rice and
stands fourth among major rice exporters of the world (8).
The provinces Punjab and Sindh contribute up to 88% of
total rice production in Pakistan (11). Punjab, because of its
favorable agroclimatic conditions, produces 100% of the
basmati rice in the country. Rice, owing to its hygroscopic
nature and high carbohydrate content, may provide a
substrate for colonization of mycotoxin-producing fungi
(8). Mycotoxin contamination is an important food safety
concern for grains and other field crops. Approximately 20%
of agricultural commodities are affected by mycotoxins
annually (6). They are produced by certain types of fungi
that grow on human food and animal feed, such as
Aspergillus, Penicillium, and Fusarium. Contamination by
mycotoxins may occur during harvesting, processing,
transportation, and storage (1). The most important myco-
toxins occurring in food are aflatoxins, ochratoxin A,
zearalenone, deoxynivalenone, and fumonisins.
Among these, aflatoxins have received great attention
owing to their wide occurrence and carcinogenic nature.
Among total aflatoxins (TAFs), the most important are
aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2
(AFG2), which are mainly produced by Aspergillus spp. (15).
* Author for correspondence. Tel: (051) 4852546; Fax: (051) 4852174;
E-mail: nighatbashir77@gmail.com.
Aflatoxins exert acute and chronic effects that may lead to
death, including edema, liver damage, and hemorrhage. The
International Agency for Research on Cancer has classified
AFB1 as a class IA carcinogen for humans (9). AFB1 is
hepatotoxic in nature, and people who are suffering from
hepatitis infection are at greater risk (7, 19). Consumption of
TAF-contaminated food increases the risk of liver cancer
manyfold in people infected with hepatitis B virus (7).
In Pakistan, over the past few years, delayed rainfall
during the sowing season, heavy rains and flooding before
harvesting, and high humidity may have provided the
conditions for TAF contamination. The production and
occurrence of TAFs differ based on geography and
environmental conditions; once food is contaminated with
aflatoxin, it cannot be completely eliminated from the food
chain. In view of the abovementioned background, the present
study was planned to assess TAF contamination in basmati
rice collected from 2010 to 2015. The dietary exposure of the
Pakistani population to TAFs was also evaluated.
MATERIALS AND METHODS
Sampling. A total of 2,251 basmati rice samples (brown rice,
1,081; white rice, 1,170) were collected from Punjab during 2010
to 2015. About 500 g of each collected sample was divided into
representative subsamples after thorough mixing. Samples were
then ground with an RAS mill (Romer Labs, Inc., Union, MO) and
were analyzed.
332 SULTANA ET AL. J. Food Prot., Vol. 80, No. 2

TABLE 1. Mean recoveries of matrix spikes in brown and white ricea

Brown rice White rice

Mycotoxin Fortification level (ng/g) Observed (ng/g) Recovery (%) Observed (ng/g) Recovery (%)

Aflatoxin B1 0.1 0.096 96 0.097 97

0.5 0.48 95 0.47 93
2.5 2.32 93 2.40 96
12.5 12.25 98 11.87 95
Aflatoxin B2 0.1 0.068 68 0.069 69
0.5 0.48 95 0.44 87
2.5 2.22 89 2.30 92
12.5 11.37 91 11.62 93
Aflatoxin G1 0.1 0.088 88 0.091 91
0.5 0.45 90 0.47 93
2.5 2.32 93 2.37 95
12.5 12.12 97 11.62 93
Aflatoxin G2 0.1 0.070 69.5 0.067 66.9
0.5 0.46 91 0.48 95
2.5 2.32 93 2.32 93
12.5 12.12 97 12.01 96
a
n ¼ 3.

Spike recoveries. For TAF analysis, a method described by standard spot. The dilution factor was multiplied to get the final
Khatoon et al. (13) was modified and optimized by matrix spikes at exact concentrations of TAFs (3).
four fortification levels (0.1, 0.5, 2.5, and 12.5 ng/g) for AFB1, The concentration of TAFs in the sample was calculated by
AFB2, AFG1, and AFG2 (Table 1). the following formula:
S3Y3V
Chemicals and reagents. All the reagents and solvents used in Concentration of TAFs ðng=gÞ ¼
W 3Z
the present study were of analytical grade and were supplied by
Merck (Darmstadt, Germany). An aflatoxin mixture (2.02 lg/ml for where S is the volume of the TAFs standard, Y is the standard
AFB1 and AFG1, 0.5 lg/ml for AFB2 and AFG2) was purchased from concentration, V is the volume of solvent for dilution, W is the
Biopure (Tulln, Austria). For fortification assays, working solutions weight of the sample, and Z is the volume of sample extract spotted
for aflatoxins were prepared in toluene-acetonitrile (95:5, vol/vol). along with the standard.

Sample preparation. A 25-g portion of finely ground sample
was extracted with 100 ml of extraction solution (methanol-water,
60:40, vol/vol) for 1 h by shaking on a gyratory shaker in
duplicate. An aliquot (4 ml) of sample extract was mixed with 8 ml
of phosphate buffer (pH ¼ 7.4), and pH was maintained at 7.0 for
TAFs with 0.1 N HCl and/or NaOH. The aliquot was then loaded
on an Aflastar Immunoaffinity column (Romer Labs, Tulln/Donau,
Austria). The flow rate was adjusted at a pace of 1 ml/min. The
column was then washed with 20 ml of distilled water. TAFs were
eluted with 3 ml of methanol at a flow rate of 0.5 ml/min. Purified
extracts were transferred into capped glass vials and were
evaporated using the Romer Evap System (Romer Labs, Inc.,
Union, MO) under vacuum at approximately 608C.
Estimation of daily dietary exposure. In the present study,
daily intake of TAFs was calculated on the basis of the observed
mean value of TAFs only in white rice because there is no trend to
consume brown rice in Pakistan. Daily rice intake was calculated
by conducting a survey with approximately 1,200 people. In this
survey, volunteers were asked about daily rice consumption and
the number and weight of family members. On the basis of this
survey, it was determined that daily white rice consumption of an
adult Pakistani ranged from 23.8 to 114.28 g/day. During cooking,
it was documented that TAFs content was reduced up to 30% (18).
Therefore, for daily dietary exposure, 30% of TAFs were deducted
from the observed mean value of TAFs and calculated by the
following formula:

Daily dietary exposure ðng=kg of body weight=dayÞ

Quantification of TAFs. A dilution-to-extinction method was
used to quantify the TAFs in rice samples. For this, purified
extracts were redissolved in toluene-acetonitrile (95:5, vol/vol). To
quantify the TAFs by visual estimation, volumes of standard
solution (1, 2, 3, and 5 ll) were spotted, along with sample spots,
on silica gel 60 thin-layer chromatography plates (Merck).
Developing of TAFs was carried out in chloroform-acetone (9:1,
vol/vol). After developing, TAFs were derivatized with 10%
H2SO4 (16) and were visualized under long wavelength UV light
(365 nm). TAFs were quantified by comparing sample spots with
the standard; observations were recorded for only those samples
that showed the same fluorescence as any one standard spot.
Samples with higher fluorescences were diluted to be equal to a
½Observed mean value ðng=gÞ
30% reduction after cooking ðng=gÞ
3 Daily rice consumption ðg=dayÞ
¼
Average adult body weight ðkgÞ
Statistical analysis. All data were analyzed using Microsoft
Office Excel 2007 (Microsoft Corporation, Redmond, WA) and
Statistical Package for Social Sciences software (version 16, SPSS
Inc., Chicago, IL). Data were input for frequency distribution to
predict how many samples would lie within the safe level.
Statistical differences among different years were tested by one-
way analysis of variance, following Duncan’s multiple test. A
J. Food Prot., Vol. 80, No. 2
probability of 0.05 was used to determine the statistical
significance.
RESULTS
Precision and accuracy. In the present study, the
method was optimized, and a linear regression equation
was drawn for a calibration curve. The linear regression
equation showed good linearity because all R2 values
ranged from 0.87 to 0.99 for all the spike levels, as shown
in Figure 1. Four different spiking levels of TAFs, i.e., 0.1,
OCCURRENCE OF TOTAL AFLATOXINS IN BASMATI RICE 333
FIGURE 1 Linear regression equation of
total aflatoxins in brown and white rice.
0.5, 2.5, and 12.5 ng/g, were evaluated. Spiked samples
were analyzed in triplicate. All mean percentage spike
recoveries were observed for AFB1 (95.50 and 95.25%),
AFB2 (91.75 and 91.25%), AFG1 (92 and 93%), and AFG2
(92.5 and 93.5%) in brown and white rice, respectively.
Intraday and interday repeatability was evaluated at
fortification levels of 5 and 2 ng/g for brown and white
rice, respectively. The detection limit of the present method
was 0.1 ng/g for AFB1 and AFG1 and 0.5 ng/g for AFB2
and AFG2 (Table 2).
334 SULTANA ET AL. J. Food Prot., Vol. 80, No. 2

TABLE 2. Precision and accuracy of total aflatoxins in brown and white rice samples
Brown rice White rice

Parameters AFB1 AFB2 AFG1 AFG2 AFB1 AFB2 AFG1 AFG2

Spike level (ng/g) 5 5 5 5 2 2 2 2

Interday repeatability 13.34 10.18 13.15 15.78 8.78 11.63 14.56 13.33
Intraday repeatability 7.13 9.10 10.11 13.13 5.78 8.91 8.07 7.89
Limit of detection (ng/g) 0.1 0.5 0.1 0.5 0.1 0.5 0.1 0.5

Aflatoxins in rice samples. A total of 2,251 samples of
brown (1,081) and white (1,170) rice were collected from
2010 to 2015. The results of analyzed samples showed that,
overall, 1,750 samples (77.74%) were positive for AFB1,
whereas 370 samples (16.43%) were tainted with TAFs.
Among positive samples, 854 brown rice samples (79%)
were contaminated with AFB1 (8.98 ng/g), and 154 samples
(14.24%) were positive for TAFs (3.1 ng/g). AFB1 (5.83 ng/
g) was found in 896 white rice samples (76.58%), whereas
TAFs (3.27 ng/g) were prevalent in 216 samples (18.46%)
(Table 3). However, no samples were positive for AFG1 and
G2.
The present findings show elevated aflatoxin contam-
ination from 2010 to 2015. A higher percentage of incidence
was found for AFB1 in brown rice in 2012 (97.69%),
followed by 2014 (93.33%), 2011 (89.18%), 2015 (87.50%),
and 2010 (84.84%). The prevalence of AFB1 in white rice
was higher in 2010 (88.23%), followed by 2013 (87.6%),
2015 (76.19%), 2011 (75.01%), and 2014 (73.68%).
However, elevated mean contamination levels were ob-
served for AFB1 in brown (15.22 ng/g) and white (9.33 ng/
g) rice samples during 2015 and 2014, respectively (Table
3).
Data from the present findings were further computed
for frequency distribution with respect to permissible limits
as defined by the European Commission (EC; 2010), that is,
2 and 5 ng/g for AFB1 in white and brown rice samples,
respectively, and 4 and 10 ng/g for TAFs in white and brown
rice samples, respectively. Furthermore, owing to the fatal
potency of AFB1, the prevalence of AFB1 found in white

TABLE 3. Natural occurrence of total aflatoxins in basmati brown and white rice from 2010 to 2015a
Brown rice White rice

Sampling year Parameters Aflatoxin B1 Total aflatoxins (B1þB2þG1þG2) Aflatoxin B1 Total aflatoxins (B1þB2þG1þG2)

2010 No. of samples 264 264 170 170

Positive (%) 128 (84.84) 32 (12.12) 150 (88.23) 32 (13.38)
Mean (ng/g) 10.38 C 2.65 B 4.07 A 6 0.69 3.68 A 6 0.73
Range (ng/g) 0.2–43.5 0.6–45 0.1–37.5 0.75–5.75
2011 No. of samples 222 222 160 160
Positive (%) 201 (89.18) 32 (12.16) 120 (75.01) 43 (18.29)
Mean (ng/g) 8.07 B 2.69 B 4.2 AB 2.24 A
Range (ng/g) 0.1–45 0.11–49.50 0.6–22.50 4.07–39.19
2012 No. of samples 260 260 245 245
Positive (%) 254 (97.69) 25 (9.43) 155 (67.34) 30 (12.24)
Mean (ng/g) 10.44 C 2.69 B 3.39 A 1.86 A
Range (ng/g) 0.1–45 21.25–49.5 0.25–21 10.13–24
2013 No. of samples 105 105 195 195
Positive (%) 90 (85.71) 20 171 (87.6) 21 (10.76)
Mean (ng/g) 4.52 A 7.8 C 6.03 B 1.98 A
Range (ng/g) 0.95–10.99 2.2–12.49 0.18– 21.9 7.6–23.4
2014 No. of samples 150 150 190 190
Positive (%) 140 (93.33) 30 (20.01) 140 (73.68) 60 (31.57)
Mean (ng/g) 5.15 A 2.08 B 9.33 D 7.68 B
Range (ng/g) 0.60–13.66 0.11–16.66 0.60–64.05 1.37–69.30
2015 No. of samples 80 80 210 210
Positive (%) 70 (87.50) 40 (50) 160 (76.19) 30 (14.28)
Mean (ng/g) 15.22 D 13.47 D 7.92 D 6 0.71 2.55 A
Range (ng/g) 0.6–44.37 9.93–51.87 0.6–42.42 4.58–25.20
Overall No. of samples 1,081 1,081 1,170 1,170
Positive (%) 854 (79) 154 (14.24) 896 (76.58) 216 (18.46)
Mean (ng/g) 8.98 3.1 5.83 3.27
Range (ng/g) 0.95–45 0.11–49.50 0.1–64.05 1.37–69.3
a
Values followed by different letters in a column differ significantly at P , 0.05.
J. Food Prot., Vol. 80, No. 2 OCCURRENCE OF TOTAL AFLATOXINS IN BASMATI RICE 335

TABLE 4. Frequency distribution of brown rice samples with respect to EC legislation (4)
Aflatoxin B1 (EC limit ¼ 5 ng/g) Aflatoxin B1 (EC limit ¼ 10 ng/g) Total aflatoxins (EC limit ¼ 10 ng/g)

,EC limit .EC limit ,EC limit ,EC limit ,EC limit ,EC limit

Positive Range Positive Range Positive Range Positive Range Positive Range Positive Range
Year (%) (ng/g) (%) (ng/g) (%) (ng/g) (%) (ng/g) (%) (ng/g) (%) (ng/g)

2010 37.87 0.2–4.60 62.12 6–43.50 59.84 0.1–10 40.15 10.5–43.5 90.90 0.6–9 9.09 15.75–45
2011 54.05 0.1–4.5 45.94 5.25–45 71.62 0.1–9 28.37 10.5–45 93.24 0.11–9.5 6.75 25.5–49.5
2012 55.11 0.1–4.50 44.68 5–45.0 67.30 0.1–7.9 33.69 11–32 48.01 5.01–9.18 52.0 13.19– 49.5
2013 52.38 0.95–3.75 47.61 6–10.99 95.23 0.95–89 4.76 10.3–10.99 95.23 0.12–8.6 4.76 12.49
2014 60.00 0.6–4.54 40.00 5.36–13.63 80.01 0.6–9.18 20.00 10.5–13.63 86.66 0.1–1 13.33 14.38–16.66
2015 37.50 0.1–1.83 62.50 9.18–44.37 50.1 0.6–9.18 49.9 15.3–44.37 50 0.6–9.18 50 15.3–44.37

and brown rice was also compared with EC limits of 4 and
10 ng/g to predict how many samples were safe, according
to TAF legislation. During 2010 to 2015, 37 to 60% of
brown rice samples showed low AFB1 contamination levels
as per EC 2010 legislation (5 ng/g) (4). On the other hand,
50 to 95.23% of samples had AFB1 levels below the EC
permissible limit (10 ng/g). Similarly, 38 to 71% of white
rice samples had safe AFB1 levels, as per EC regulations (2
ng/g), over a period of 6 years (Tables 4 and 5). The findings
of the present study were also evaluated for dietary exposure
to aflatoxins based on rice consumption and observed mean
value, based on a permissible limit of 4 ng/g. Daily intake of
TAFs was found to range from 0.51 to 10.22 ng/kg (Table 6)
of body weight, which exceeds the limits as defined by the
Joint FAO/WHO Expert Committee on Food Additives, i.e.,
1 ng/kg (10).
DISCUSSION
Present findings highlighted the prevalence of TAFs in
basmati brown and white rice. TAF contamination in brown
and white rice varies from year to year. The average mean
values for AFB1 found in brown and white rice, 8.98 and
5.83 ng/g, respectively, were beyond the level considered
permissible by the EC (4), i.e., 5 and 2 ng/g. Similarly, the
mean values detected for TAFs in brown and white rice (3.1
and 3.27 ng/g, respectively) were in accordance with EC
2010 legislation, i.e., 10 and 4 ng/g (4). Results further
demonstrated that the prevalence of AFB1 in brown rice was
high in 2012, 2014, 2013, and 2011, with .90% samples
found to be contaminated; for white rice, AFB1 levels were
high during 2010 (88.23%) and 2013 (87.6%). Furthermore,
mean values for AFB1 in brown rice during 2010 (10.38 ng/
g), 2011 (8.07 ng/g), 2012 (10.44 ng/g), and 2015 (15.22 ng/
g) were far beyond the EC permissible limit (5 ng/g).
However, white rice samples had higher AFB1 contamina-
tion in 2014 (9.33 ng/g), 2015 (7.92 ng/g), and 2013 (6.03
ng/g 6 0.43), exceeding the EC limit (2 ng/g) (Table 3).
Contrarily, percentage incidence and mean contamination
were generally found to be below the legislation levels for
TAFs in brown and white rice compared with AFB1.
Pakistan has a subtropical climate, and the prevalence of
TAFs in agricultural commodities is relatively high (13).
The warm, humid tropical and subtropical climate favors the
growth of fungi, leading to TAF production. Production is
influenced by a number of factors, including type of
substrate, moisture content, relative humidity, temperature,
and insect infestation (22). Rice is a good substrate for
fungal contamination because of its hygroscopic nature and
carbohydrate content (2). In Pakistan, rice is grown as
‘‘Kharif’’ crop and is cultivated in June. During the sowing
season, the climate is subhumid, with a monsoon rainfall of
400 to 700 mm in July and August. In monsoon season,
there is high relative humidity (67 to 98%) and temperature
(26 to 398C), which provides suitable conditions for
aflatoxin-producing fungi (25). Occurrence of TAFs in
crops is strongly influenced by the weather during and after
the growing season, which increases the risk of TAF
contamination (20). TAF production in rice is optimum at 12
to 288C and at a moisture content of 8 to 35% (5). TAFs are
produced by Aspergillus parasiticus, whereas Aspergillus
flavus is responsible only for the production of AFB1 and

TABLE 5. Frequency distribution of white rice samples with respect to EC legislation (4)
Aflatoxin B1 (EC limit ¼ 2 ng/g) Aflatoxin B1 (EC limit ¼ 4 ng/g) Total aflatoxins (EC limit ¼ 4 ng/g)

,EC limit .EC limit ,EC limit ,EC limit ,EC limit ,EC limit

Positive Range Positive Range Positive Range Positive Range Positive Range Positive Range
Year (%) (ng/g) (%) (ng/g) (%) (ng/g) (%) (ng/g) (%) (ng/g) (%) (ng/g)

2010 70.5 0.1–0.9 29.41 2.85–37.5 82.35 0.1–4 17.64 4.01–37.5 43.75 2.10–3.75 57 4.13–10.98
2011 43.75 0.6–1.5 56.25 2.25–22.5 75 0.6–3.75 25 6–22.50 25.58 2–3.67 74.42 4.7– 39.19
2012 65.38 0.25–1.8 34.69 2.1–21 69.38 0.1–3 30.61 5.25–21 87.75 0.1–2.01 12.24 10.13–24
2013 38.46 0.18–1.8 61.53 2.25–21.9 49.23 0.1–3.75 50.76 4.50–21.9 89.23 0.1–1.19 10.76 7.6–23.4
2014 42.10 0.6–1.83 57.89 3.03–64.05 63.15 0.6–3.75 36.84 6.12–64.05 85.70 0.1–1 14.28 4.58–25.2
2015 38.09 0.6–1.22 61.90 3.06–42.42 47.61 0.6–3.06 52.83 4.59–42.42 73.68 0.1–1.37 26.35 11.34–69.3
336 SULTANA ET AL.
TABLE 6. Daily intake of total aflatoxins for adults at mean contamination level for white rice
30% reduction Final mean Rice consumption (g/day)a
Mean TAFs after cooking value after 30%
Year (ng/g) (TAFS) (ng/g) reduction (ng/g) Minimum
2010 3.68 1.10 2.6 23.8
2011 2.24 0.67 1.56
2012 1.86 0.55 1.30
2013 1.98 0.59 1.38
2014 7.68 2.3 5.37
2015 2.55 0.76 1.78
a
Daily consumption (grams per day) of rice was based on a survey of 1,200 people.
b
Average adult body weight ¼ 60 kg.
AFB2. Present findings revealed a higher incidence of
aflatoxins in brown and white rice samples during different
years. During 2010 and 2011, rice growing areas experi-
enced devastating flood spells that ruined the fields, with
aftereffects that persisted for a long time. Areas that were out
of reach of flood threats were challenged with greater than
70% humidity, which was an important factor for TAF
production.
Data from the present study were used for frequency
prediction, to indicate how many samples of brown and
white rice were within safe levels. The results show that
60% of brown rice samples in 2014 had AFBI levels that
were permissible according to EC regulations, and that in
2010, 93% of brown rice samples had TAF levels that were
in accordance with EC regulations (10 ng/g). However, in
2010, 70.5% of white rice samples had AFBI levels that fell
within permissible limits of EC (2 ng/g), whereas 89.23% of
white rice samples in 2013 had TAF levels that were found
safe for human consumption according to legislation (4 ng/
g).
The findings of the present study further revealed that
the overall mean levels of AFB1 and TAFs in brown rice
(8.98 and 3.1 ng/g, respectively) were higher than in white
rice (5.83 and 2.55 ng/g, respectively). One study (22)
reported that aflatoxins are produced at the site of fungal
growth and that the bran layer acts as an obstacle that
prevents the entry of mycelia into rice seed. TAFs were
heterogeneously accumulated in the bran layer, and high
contamination levels for TAFs were observed in the external
surface of bran. However, lower levels were detected in the
inner layer, such as white rice. They observed that brown
rice contained high levels of aflatoxins compared with white
rice, which supports the results of the present findings. Rice
milling and dehusking might reduce the risk of exposure to
consumers of white rice.
Rice is uniquely important because it is a primary
source of carbohydrates for humans (21). It is the second-
largest cereal crop of Pakistan and contributes more than 2
million tons to our national food production. It is also used
in manufacturing snacks, pasta, beverages, etc. (14). Based
on the importance of rice in our daily diet, dietary intake was
calculated on the basis of high levels of TAFs detected in
basmati white rice. A value of 1 ng of TAFs per kg of body
weight per day was used as a reference in the risk
assessment of TAF exposure (10). Results show that dietary
exposure to TAFs ranged from 0.51 to 10.22 ng/kg with
J. Food Prot., Vol. 80, No. 2
Aflatoxin daily intake (ng/kg of body weight)b
Maximum Minimum Maximum
114.28 1.03 4.92
0.61 2.98
0.51 2.47
0.54 2.63
2.13 10.23
0.69 3.39
respect to body weight (Table 6). Detected aflatoxin
exposure exceeds the limit of 1 ng/kg of body weight per
day (10), posing a significant health risk for consumers.
AFB1 is a naturally occurring genotoxic carcinogen, and
chronic exposure to AFB1 has been reported to increase the
risk of liver cancer, especially when it is associated with
hepatitis B or C (12). AFB1 has been classified as
carcinogenic to humans (group 1) by the International
Agency for Research on Cancer (9).
Food contamination by mycotoxins has been recog-
nized as a public health threat (10). Mycotoxins have
been included as priority food contaminants by the Global
Environment Monitoring System/Food Contamination
Monitoring Assessment Programme of the World Health
Organization (24). Aflatoxins, ochratoxin A, zearalenone,
T-2, and deoxynivalenol are considered potent mycotox-
ins. They occur in staple foods such as cereals, spices,
pulses, dried fruits, etc. (23). The present findings
highlight only the prevalence of aflatoxins in rice.
Different foods may be contaminated with more than
one type of mycotoxin, increasing the likelihood of
exposure to different mycotoxins through consumption of
different food items.
The results of the present findings illustrate the high
levels of TAFs in white and brown rice, levels beyond the
permissible limit of the EC. Rice is an important component
of the Pakistani diet, but consumption of aflatoxin-
contaminated food might be lethal for humans. The dietary
exposure observed in the present study was higher than
levels defined as safe by the Joint FAO/WHO Expert
Committee on Food Additives (1 ng/kg). The situation
depends on whether the data collection and survey of dietary
habits is statistically representative. Analysis of rice for
aflatoxins is considered during trade and export activities.
Screening for TAFs should be mandatory prior to marketing
rice and rice-based products. Moreover, in Pakistan there is
no legislation that defines permissible levels of TAFs in food
items, and we have to rely on European and FDA legislation.
Therefore, it is currently necessary to establish and
implement regulatory limits for food items. Prevention is
the best control strategy for mycotoxin contamination.
ACKNOWLEDGMENT
The authors are greatly thankful to Naseem Traders International for
their financial support.
J. Food Prot., Vol. 80, No. 2 OCCURRENCE OF TOTAL AFLATOXINS IN BASMATI RICE
REFERENCES
1. Asghar, M. A., J. Iqbal, A. Ahmed, Z. A. Shamsuddin, and M. A.
Khan. 2013. Incidence of aflatoxins in export quality rice collected
from areas of Pakistan. Sci. Technol. Dev. 32:110–119.
2. Brooker, D. B., F. W. Bakker-Arkema, and C. W. Hall. 1992. Rice
drying, p. 68. In Drying and storage of grain and oil seeds. AVI Book,
New York.
3. Castegnaro, M., D. C. Hunt, E. B. Sansone, P. L. Schuller, M. G.
Siriwardana, G. M. Telling, H. P. van Egmond, and E. A. Walker
(ed.). 1980. Laboratory decontamination and destruction of aflatoxins
B1, B2, G1, G2 in laboratory wastes. International Agency for
Research on Cancer (IARC) publication no. 37. IARC, Lyon, France.
4. European Commission. 2010. Commission Regulation (EC) No 165/
2010 of 26 February 2010 amending Regulation (EC) No 1881/2006
setting maximum levels for certain contaminants in foodstuffs as
regards aflatoxins. Off. J. Eur. Union L 50:8–12.
5. Firdous, S., N. Ejaz, T. Aman, and N. Khan. 2012. Occurrence of
aflatoxins in export-quality Pakistani rice. Food Addit. Contam.
5:121–125.
6. Food and Agriculture Organization of the United Nations (FAO).
2005. Worldwide regulations for mycotoxins in food and feed in
2003. FAO Food and Nutrition Paper 81. FAO, Rome.
7. Groopman, J. D., T. W. Kensler, and C. P. Wild. 2008. Protective
interventions to prevent aflatoxin-induced carcinogenesis in develop-
ing countries. Rev. Public Health 29:187–203.
8. Husain, A., J. Ali, and Shafqatullah. 2011. Studies on contamination
level of aflatoxins in Pakistani rice. J. Chem. Soc. Pak. 33:481–484.
9. International Agency for Research on Cancer (IARC). 2002. IARC
monographs on the evaluation of carcinogenic risk to humans, vol. 56.
Some naturally occurring substances: food items and constituents,
heterocyclic aromatic amines and mycotoxin. IARC, Lyon, France.
10. Joint FAO/WHO Expert Committee on Food Additives (JECFA).
1998. Safety evaluation of certain food additives and contaminants:
aflatoxins. World Health Organization (WHO) Food Additive series
40, 49th Meeting of JECFA. International Program on Chemical
Safety, WHO, Geneva.
11. Junejo, M. A., C. L. Rohra, and H. A. Kanasro. 2007. Analyzing the
impact of Sindh rice industries on economy of Pakistan. Aust. J. Basic
Appl. Sci. 1:853–859.
12. Kew, M. C. 2003. Synergistic interaction between aflatoxin B1 and
hepatitis B virus in hepatocarcinogenesis. Liver Int. 23:405–409.
337
13. Khatoon, S., N. Q. Hanif, I. Tahira, N. Sultana, K. Sultana, and N.
Ayub. 2012. Natural occurrence of aflatoxins, zearalenone and
trichothecenes in maize grown in Pakistan. Pak. J. Bot. 44:231–236.
14. Kim, E.-K., P. M Scott, B. P.-Y. Lau, and D. A. Lewis. 2002.
Extraction of fumonisins B1 and B2 from white rice flour and their
stability in white rice flour, cornstarch, corn meal and glucose. J.
Agric. Food Chem. 50:3614–3620.
15. Lereau, M., G. Doriane, and S. Villar. 2012. Interaction between
hepatitis B virus and aflatoxin B1: effects on p53 induction in
HepaRG cells. J. Gen. Viriol. 93:640–650.
16. Lin, L., J. Zhang, P. Wang, Y. Wang, and J. Chen. 1998. Thin-layer
chromatography of mycotoxins and comparison with other chromato-
graphic methods. J. Chromatogr. A 815:3–20.
17. Nasir, M. 16 July 2010. Pakistan to lead global rice exports in 2010:
FAO. The Express Tribune. Available at: http://tribune.com.pk/story/
28117/pakistan-to-lead-global-rice-exports-in-2010-fao/. Accessed 7
November 2011.
18. Park, J. W., S. Y. Choi, H. J. Hawang, and Y. B. Kim. 2005. Fungal
mycoflora and mycotoxins in Korean polished rice destined for
humans. Int. J. Food Microbiol. 103:305–314.
19. Qian, G. S., R. K. Ross, M. C. Yu, J. M. Yuan, Y. T. Gao, B. E.
Henderson, G. N. Wogan, and J. D. Groopman. 1994. A follow-up
study of urinary markers of aflatoxin exposure and liver cancer risk in
Shanghai, People’s Republic of China. Cancer Epidemiol. Biomark-
ers Prev. 3:3–10.
20. Reddy, K. R. N., C. S. Reddy, H. K. Abbas, C. A. Abel, and K.
Muralidharan. 2008. Mycotoxigenic fungi, mycotoxins and manage-
ment of rice grains. Toxin Rev. 27:287–317.
21. Reiter, E. V., F. Vouk, J. Bohm, and E. Razzazi-Fazeli. 2010.
Aflatoxins in rice—a limited survey of products marketed in Austria.
Food Control 21:988–991.
22. Sales, A., and T. Yoshizawa. 2005. Updated profile of aflatoxin and
Aspergillus section flavi contamination in rice and its byproducts from
the Philippines. Food Addit. Contam. 22:429–436.
23. Weidenborner, M. 2008. Mycotoxins in foodstuffs. Springer, New
York.
24. World Health Organization. 2002. List of priority contaminants and
commodity combinations. Available at: apps.who.int/iris/bitsteam/
1066542562/1/9241562196.pdf. Accessed August 2013.
25. Yunus, A. W., M. K. Nasir, T. Aziz, and J. Böhm. 2009. Prevalence
of poultry diseases in district Chakwal and their interaction with
mycotoxicosis: 2. Effects of season and feed. J. Anim. Plant Sci.
19:1–5.