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.

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 mycotoxins 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). 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.

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.

Spike recoveries. For TAF analysis, a method described by Khatoon et al. (13) was modified and optimized by matrix spikes at four fortification levels (0.1, 0.5, 2.5, and 12.5 ng/g) for AFB1, AFB2, AFG1, and AFG2 (Table 1).

TABLE 1.

Mean recoveries of matrix spikes in brown and white ricea

Mean recoveries of matrix spikes in brown and white ricea
Mean recoveries of matrix spikes in brown and white ricea

Chemicals and reagents. All the reagents and solvents used in the present study were of analytical grade and were supplied by Merck (Darmstadt, Germany). An aflatoxin mixture (2.02 μg/ml for AFB1 and AFG1, 0.5 μg/ml for AFB2 and AFG2) was purchased from Biopure (Tulln, Austria). For fortification assays, working solutions for aflatoxins were prepared in toluene-acetonitrile (95:5, vol/vol).

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 = 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 60°C.

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 μl) 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 standard spot. The dilution factor was multiplied to get the final exact concentrations of TAFs (3).

The concentration of TAFs in the sample was calculated by the following formula:

formula

where S is the volume of the TAFs standard, Y is the standard concentration, V is the volume of solvent for dilution, W is the weight of the sample, and Z is the volume of sample extract spotted along with the standard.

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:

formula

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 probability of 0.05 was used to determine the statistical significance.

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, 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).

FIGURE 1.

Linear regression equation of total aflatoxins in brown and white rice.

FIGURE 1.

Linear regression equation of total aflatoxins in brown and white rice.

Close modal
TABLE 2.

Precision and accuracy of total aflatoxins in brown and white rice samples

Precision and accuracy of total aflatoxins in brown and white rice samples
Precision and accuracy of total aflatoxins in brown and white rice samples

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.

TABLE 3.

Natural occurrence of total aflatoxins in basmati brown and white rice from 2010 to 2015a

Natural occurrence of total aflatoxins in basmati brown and white rice from 2010 to 2015a
Natural occurrence of total aflatoxins in basmati brown and white rice from 2010 to 2015a

The present findings show elevated aflatoxin contamination 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 observed 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 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).

TABLE 4.

Frequency distribution of brown rice samples with respect to EC legislation (4)

Frequency distribution of brown rice samples with respect to EC legislation (4)
Frequency distribution of brown rice samples with respect to EC legislation (4)
TABLE 5.

Frequency distribution of white rice samples with respect to EC legislation (4)

Frequency distribution of white rice samples with respect to EC legislation (4)
Frequency distribution of white rice samples with respect to EC legislation (4)
TABLE 6.

Daily intake of total aflatoxins for adults at mean contamination level for white rice

Daily intake of total aflatoxins for adults at mean contamination level for white rice
Daily intake of total aflatoxins for adults at mean contamination level for white rice

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 contamination in 2014 (9.33 ng/g), 2015 (7.92 ng/g), and 2013 (6.03 ng/g ± 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 39°C), 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 28°C 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 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 experienced 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 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 recognized 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 mycotoxins. 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.

The authors are greatly thankful to Naseem Traders International for their financial support.

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