Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 1, 57–62, http://dx.doi.org/10.1080/19393210.2013.843205
Aflatoxins and ochratoxin A in maize of Punjab, Pakistan Wajiha Irama, Tehmina Anjuma*, Mateen Abbasb and Abdul Muqeet Khanb a
Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan; bQuality Operating Laboratory (QOL), University of Veterinary and Animal Sciences, Lahore, Pakistan (Received 7 June 2013; accepted 8 September 2013) Aflatoxin and ochratoxin levels were determined in maize samples collected from store houses of 15 districts belonging to three agro-ecological zones of Punjab, Pakistan. Toxins were extracted by Aflaochra immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC). Mean moisture content of maize kernels was recorded above the safe storage level of 15%. Results indicated that aflatoxin B1 and B2 contamination was found in 97.3% and 78.9% of the collected samples, respectively. Aflatoxin G1, aflatoxin G2 and ochratoxin A were not detected in any sample. Among positive samples, 77.3% contained aflatoxin B1 and 28% aflatoxin B2, exceeding the legal limits as set by the European Union (EU) and the United States Food and Drug Administration (USFDA). It was concluded that a significant number of samples contained aflatoxin B1 and B2 above the legal limits. Keywords: aflatoxins; moisture; maize; HPLC; immunoaffinity
Introduction Maize is Pakistan’s third most important cereal crop after wheat and rice. The average productivity of maize in Pakistan is 2850 kg ha–1, which is the highest among all cereals grown in the country (Tariq & Iqbal 2010). Maize kernels are processed primarily for livestock feed (78%) and to some extent for human consumption (13%) (Lgawa et al. 2007). Maize crop is considered to be most susceptible for mould infection and production of harmful mycotoxins which possess a serious health hazard to animals and humans. Among these mycotoxins, aflatoxins and ochratoxins have significance due to their deleterious effects on human beings, poultry and livestock (Sur & Celik 2003; Wild & Montesano 2009). Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus that occur in cereal crops and nuts. These compounds have a high acute toxicity, as well as immune-suppressive, mutagenic, teratogenic and carcinogenic activities and are classified as group-1 carcinogens by International Agency for Research on Cancer (International Agency for Research on Cancer 2002). Ochratoxins are polyketide-derived fungal secondary metabolites with nephrotoxic, immune-suppressive, teratogenic and carcinogenic properties. Ochratoxin producing fungi include A. ochraceus, A. carbonarius and Penicillium verrucosum, which may contaminate a wide variety of food commodities (e.g. cereals, cocoa, dried fruits, nuts, spices, legumes, coffee beans, etc.) in the field (pre-harvest spoilage), during storage (post-harvest spoilage) or during processing (Varga et al. 2010).
*Corresponding author. Email: [email protected] © 2013 Taylor & Francis
It has been reported that about 5–10% of agricultural products in the world are spoiled by mould contamination to the extent that they cannot be consumed by humans or animals (Topal 1993). Due to poor drying and storage facilities, post-harvest losses are quite substantial in developing countries such as Pakistan. Moulds are distributed widely as environmental contaminants and under favourable conditions of temperature and humidity, grow on commodities like maize (Brera et al. 1998) and produce mycotoxins. In Pakistan, few studies have reported on mycotoxin contamination in local varieties of maize during the last three years (Ahsan et al. 2010; Shah et al. 2010; Ishrat et al. 2012; Khatoon et al. 2012). Furthermore, recent domestic surveys report the presence of mycotoxins in chillies (Iqbal et al. 2009) fruits and vegetables (Sahar et al. 2009) and poultry feed (Saleemi et al. 2010; Anjum et al. 2011; Rashid et al. 2012). Mycotoxins attract worldwide attention because of the significant economic losses associated with their impact on human health, animal productivity and both domestic and international trade (Wu 2004). The Food and Agriculture Organization (FAO) estimate that world losses of foodstuffs due to mycotoxins are in the range of 1000 million tons per year (Bhat et al. 2010). Health issues related to mycotoxin contamination of foodstuffs are more problematic in developing countries where no proper food safety regulation has been established. The present study aimed to evaluate to which extent aflatoxins and ochratoxin A occur in maize, grown in one of the most fertile regions of Pakistan and under local storage conditions.
58
W. Iram et al.
Material and methods
Determination of moisture content
Survey and sample collection For sampling purposes, Punjab Province of Pakistan was divided into three agro-ecological zones, depending upon agro-climatic conditions (Amjad 2008). Samples comprised of stored maize grains with different storage periods, which were randomly collected from storehouses in each agro-ecological zone of Punjab, i.e. the Southern irrigated zone (Zone 1), the Northern irrigated zone (Zone II) and the Arid (rain-fed) zone (Zone III). Fifteen districts, mentioned in Table 1, were randomly selected for sampling with five districts from each agro-ecological zone. To avoid sampling errors due to the highly heterogeneous nature of fungal distributions, each 3 kg composite sample collected from one storehouse was a mixture of 15 sub-samples of 200 g each. Sub-samples were formed by combining five increments. The 15 subsamples were collected from five diagonal points on each of the upper, middle and lower layers of each storehouse and then mixed together to form one sample. These samples were labelled, packaged in sterile polythene bags, taken to the laboratory and kept at 4°C for further analysis.
Moisture content of the maize samples was determined using the standard oven method (AOAC International 1999). The samples were dried at 100°C to constant weight, and the mean moisture content was calculated as a percentage on wet basis.
Chemicals and standards High-performance liquid chromatography (HPLC) grade methanol, acetonitrile, sodium chloride (analytical grade) and trifluoroacetic acid (TFA, which acts as a derivatising agent to enhance fluorescence) were obtained from Merck (Darmstadt, Germany). Immunoaffinity columns (AflaOchra-Test, VICAM, Watertown, MA, USA) and standards ochratoxin A and an aflatoxin mix kit containing AFB1, AFB2, AFG1 and AFG2 (Sigma-Aldrich, St. Louis, MO, USA) were used.
Table 1. Validation data for aflatoxins and ochratoxin A in maize (n = 6). Aflatoxin B1 B2 G1 G2 Ochratoxin A
Spike (µg kg−1) 2 5 10 0.08 0.75 4 2 5 10 0.08 0.75 4 0.01 0.5 5
Recovery (%) 85 87 90 84 86 88 84 88 89 85 84 86 85 88 90
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.15 0.13 0.17 0.11 0.14 0.12 0.15 0.13 0.17 0.11 0.14 0.12 0.15 0.17 0.13
RSD (%) 0.17 0.14 0.18 0.13 0.16 0.13 0.17 0.15 0.19 0.13 0.17 0.14 0.18 0.19 0.14
Extraction and analysis Collected maize samples were ground by a laboratory mill, homogenised and 25 g was taken for extraction in an Erlenmeyer flask (250 ml), and 2.5 g sodium chloride water–acetonitrile (15:85 v/v) was added. The flask was shaken on a water bath for 2 h, the extract was filtered through filter paper (Whatman, Inc., Clifton, NJ, USA) and 10 ml filtrate was diluted in 40 ml of distilled water. Immunoaffinity columns were conditioned with double-distilled water. Then, the filtrate was passed through the column in a solid phase extraction assembly. Toxins were slowly eluted from the column with 1 ml methanol in a glass vial. For ochratoxin A analysis, the elute was analysed directly by HPLC, while for aflatoxin analysis, a derivatisation was carried out as follows: the elute was evaporated to dryness with a gentle stream of nitrogen, redissolved in 200 µl nhexane, vortexed, and 50 µl of TFA was added. Then 950 µl of acetonitrile-water (1:9) was added and filtered by using a syringe filter assembly. The filtrate was analysed by HPLC. Liquid chromatographic analysis A HPLC system (Agilent 1100 series, Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column (Merck, Darmstadt, Germany), and a fluorescence detector was used for quantification. For aflatoxins analysis, a mobile phase consisting of water:methanol:acetonitrile in the volume ratio 60:20:20, at a flow rate of 1 ml min–1 was applied, and aflatoxin was detected at excitation and emission wavelengths of 360 nm and 440 nm, respectively. For ochratoxin A, the mobile phase was acetonitrile:water:acetic acid, 99:99:2 v/v/v, and it was detected at 335 nm and 465 nm as excitation and emission wavelengths, respectively. For HPLC method validation, calibration curves were drawn using a series of calibration solutions in methanol. Each standard solution was chromatographed in duplicate. Statistical analysis Measurements were statistically analysed by one-way analysis of variance (ANOVA). Results and discussion The HPLC method was validated by testing linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ). Results for recovery are given in Table 1. The data for the calibration curves are given in Table 2 and
Food Additives & Contaminants: Part B Table 2. Linear regression parameters for aflatoxins B1, B2, G1, G2 and ochratoxin A. Mycotoxin
µg kg−1
Slope
Intercept
R2
Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 Ochratoxin
0.5–12 0.05–5 0.5–12 0.05–5 0.005–6
1.0907 2.6638 1.0907 2.6638 5.5622
0.042 0.0388 0.042 0.0388 0.8973
0.9996 0.9993 0.9996 0.9993 0.9995
showed good linearity. LOD and LOQ were 0.5 and 1 µg kg−1 for AFB1 and AFG1, respectively, and 0.05 and 0.1 µg kg−1 for AFB2 and AFG2, respectively. Results (Table 3) showed that aflatoxin B1 contamination was found in 97.3% of maize samples tested, 78.7% were contaminated with AFB2, while AFG1, AFG2 and ochratoxin A were not detected in tested samples. Out of the total 75 samples, 27 samples had an aflatoxin B1 content above the limit of 20 µg kg−1, as set by United States Food and Drug Administration (USFDA), while 31 samples had levels of AFB1 above the European Union (EU) statutory limit, i.e. 2 µg kg−1 for aflatoxin B1 and 4 µg kg−1 for total aflatoxins. Also, among all tested samples, 21 samples contained AFB2 concentrations above the limit as set by the EU. High aflatoxin B1 contamination has been observed in samples obtained from districts of rain-fed arid zone (Zone 3). The highest AFB1 level was detected in samples taken from the Attock district (mean 240.6 μg kg−1). After Attock, Chakwal had a mean of 148.7 µg kg−1. However, in Rawalpindi, Jhelum and Khushab districts of the same zone, mean AFB1 levels were 5.0, 29.0 and 77.7 µg kg−1, respectively (Table 3), whereas aflatoxin B2 was
59
detected in 22 samples with an average concentration of 2.0 µg kg−1 (Table 4). Out of 25 contaminated samples of the arid zone, 28% and 36% contained AFB1 and AFB2, respectively, with a concentration range of 2–16 µg kg−1; 8% contained AFB1 in a range of 16–20 µg kg−1, and more than 20 µg kg−1 AFB1 was found in 48% of samples (Table 5). In the northern irrigated zone (Zone 2), 25 samples were contaminated with AFB1. The highest level (mean 104.3 µg kg−1) was detected in samples taken from Rahim yar khan. However, mean concentrations of AFB1, i.e. 29.0 µg kg−1, 17.5 µg kg−1, 14.8 µg kg−1 and 12.2 µg kg−1 were recorded in samples collected from Bahawalpur, Lodra, Bahawalnagar and Multan, respectively (Table 3). In Zone 2, 24 samples were contaminated with aflatoxin B2, but its average concentration was below the limits, i.e. 1.3 µg kg−1. Table 5 illustrates that 64% and 28% of Zone 2 samples contained aflatoxin B1 and B2 in the concentration range 4–16 µg kg−1. Only 4% of the samples showed levels in the range 16–20 µg kg−1, while 32% of the samples contained AFB1 exceeding the legal limits of 20 µg kg−1. In the southern irrigated zone (Zone 1), aflatoxin content was lower than in arid and northern zone. Among five districts of Zone 1, a higher level of AFB1 was only found in samples taken from district Sahiwal (116.2 µg kg−1). However, mean AFB1 concentrations of 20.3 µg kg−1, 1.8 µg kg−1, 1.2 µg kg−1 and 0.7 µg kg−1 were observed in samples collected from Sialkot, Faisalabad, Lahore and Chiniot, respectively. AFB2 concentration was 1.1 µg kg−1 (Table 4). Table 5 shows that in Zone 1, 20% of the samples contained aflatoxin B1 and B2 in the range of 2–16 µg kg−1, while 28% contained AFB1 levels above 20 µg kg−1.
Table 3. Aflatoxin B1 and B2 levels (µg kg−1) in maize, collected from southern, northern irrigated zone and arid (rain-fed) zone (n = 25). Mean aflatoxin concentration Commodity Maize
Area
District
Zone 1 (Southern irrigated zone)
Lahore Faisalabad Chiniot Sialkot Sahiwal Bahawalpur Bahawalnagar Multan Rahim yar khan Lodhra Attock Chakwal Khushab Jhelum Rawalpindi
Zone 2 (Northern irrigated zone)
Zone 3 (Arid (rain-fed) zone)
Coordinates (latitude and longitude) 31.5758° 31.2500° 31.7167° 32.5000° 30.5833° 29.9833° 29.9969° 30.1917° 28.4167° 29.5333° 33.8936° 32.9303° 32.3000° 32.9333° 33.6000°
N, N, N, N, N, N, N, N, N, N, N, N, N, N, N,
74.3269° 73.0500° 72.9667° 74.5333° 73.3333° 73.2667° 73.2511° 71.4654° 70.3000° 71.6333° 72.2414° 72.8556° 72.3400° 73.7333° 73.0500°
E E E E E E E E E E E E E E E
AFB1
AFB2
1.2 1.8 0.7 20.3 116.2 29.0 14.8 12.2 104.3 17.5 240.6 148.7 77.7 29.0 5.0
0 0.01 0.0 1.7 3.7 1.7 1.0 0.4 2.1 1.3 4.3 2.5 1.3 1.6 0.2
60
W. Iram et al.
Table 4. Aflatoxin B1 and B2 (µg kg−1) between maize varieties in three zones of Punjab. Aflatoxin B1 Variety Agatii 2002 Agaitii- 85 Afgoye Cargle-D-MS- 3937 Desi FH-810 hybrid FH-963 hybrid FH-793 hybrid Golden 85 Hybrid 80y80 HY-1898 hybrid HY-1921 hybrid ICI 8464 ICI 984 Moncento 6525 Moncento 919 Moncento 6142 Moncento 979 MMRI yellow Neelum NK-278 Pioneer 30y87 Pioneer 8711 Pioneer 32B41 Pioneer 32B33 Pioneer 6339 Pioneer 8441 Pioneer 31R88 R-886 Sahiwal 2002 Sanchanta NK-6621 Sanchanta NK 6615 Supra Sadaf 2166 2278 AS 6789 4881 Mixed Variety 1 Variety 2 Contaminated samples (%) Average
Aflatoxin B2
Zone 1
Zone 2
Zone 3
Zone 1
Zone 2
Zone 3
238.5 – 64.1 – – – – – – – – –
Aflatoxins and ochratoxin A in maize of Punjab, Pakistan Wajiha Irama, Tehmina Anjuma*, Mateen Abbasb and Abdul Muqeet Khanb a
Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan; bQuality Operating Laboratory (QOL), University of Veterinary and Animal Sciences, Lahore, Pakistan (Received 7 June 2013; accepted 8 September 2013) Aflatoxin and ochratoxin levels were determined in maize samples collected from store houses of 15 districts belonging to three agro-ecological zones of Punjab, Pakistan. Toxins were extracted by Aflaochra immunoaffinity columns and analysed by high-performance liquid chromatography (HPLC). Mean moisture content of maize kernels was recorded above the safe storage level of 15%. Results indicated that aflatoxin B1 and B2 contamination was found in 97.3% and 78.9% of the collected samples, respectively. Aflatoxin G1, aflatoxin G2 and ochratoxin A were not detected in any sample. Among positive samples, 77.3% contained aflatoxin B1 and 28% aflatoxin B2, exceeding the legal limits as set by the European Union (EU) and the United States Food and Drug Administration (USFDA). It was concluded that a significant number of samples contained aflatoxin B1 and B2 above the legal limits. Keywords: aflatoxins; moisture; maize; HPLC; immunoaffinity
Introduction Maize is Pakistan’s third most important cereal crop after wheat and rice. The average productivity of maize in Pakistan is 2850 kg ha–1, which is the highest among all cereals grown in the country (Tariq & Iqbal 2010). Maize kernels are processed primarily for livestock feed (78%) and to some extent for human consumption (13%) (Lgawa et al. 2007). Maize crop is considered to be most susceptible for mould infection and production of harmful mycotoxins which possess a serious health hazard to animals and humans. Among these mycotoxins, aflatoxins and ochratoxins have significance due to their deleterious effects on human beings, poultry and livestock (Sur & Celik 2003; Wild & Montesano 2009). Aflatoxins are secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus that occur in cereal crops and nuts. These compounds have a high acute toxicity, as well as immune-suppressive, mutagenic, teratogenic and carcinogenic activities and are classified as group-1 carcinogens by International Agency for Research on Cancer (International Agency for Research on Cancer 2002). Ochratoxins are polyketide-derived fungal secondary metabolites with nephrotoxic, immune-suppressive, teratogenic and carcinogenic properties. Ochratoxin producing fungi include A. ochraceus, A. carbonarius and Penicillium verrucosum, which may contaminate a wide variety of food commodities (e.g. cereals, cocoa, dried fruits, nuts, spices, legumes, coffee beans, etc.) in the field (pre-harvest spoilage), during storage (post-harvest spoilage) or during processing (Varga et al. 2010).
*Corresponding author. Email: [email protected] © 2013 Taylor & Francis
It has been reported that about 5–10% of agricultural products in the world are spoiled by mould contamination to the extent that they cannot be consumed by humans or animals (Topal 1993). Due to poor drying and storage facilities, post-harvest losses are quite substantial in developing countries such as Pakistan. Moulds are distributed widely as environmental contaminants and under favourable conditions of temperature and humidity, grow on commodities like maize (Brera et al. 1998) and produce mycotoxins. In Pakistan, few studies have reported on mycotoxin contamination in local varieties of maize during the last three years (Ahsan et al. 2010; Shah et al. 2010; Ishrat et al. 2012; Khatoon et al. 2012). Furthermore, recent domestic surveys report the presence of mycotoxins in chillies (Iqbal et al. 2009) fruits and vegetables (Sahar et al. 2009) and poultry feed (Saleemi et al. 2010; Anjum et al. 2011; Rashid et al. 2012). Mycotoxins attract worldwide attention because of the significant economic losses associated with their impact on human health, animal productivity and both domestic and international trade (Wu 2004). The Food and Agriculture Organization (FAO) estimate that world losses of foodstuffs due to mycotoxins are in the range of 1000 million tons per year (Bhat et al. 2010). Health issues related to mycotoxin contamination of foodstuffs are more problematic in developing countries where no proper food safety regulation has been established. The present study aimed to evaluate to which extent aflatoxins and ochratoxin A occur in maize, grown in one of the most fertile regions of Pakistan and under local storage conditions.
58
W. Iram et al.
Material and methods
Determination of moisture content
Survey and sample collection For sampling purposes, Punjab Province of Pakistan was divided into three agro-ecological zones, depending upon agro-climatic conditions (Amjad 2008). Samples comprised of stored maize grains with different storage periods, which were randomly collected from storehouses in each agro-ecological zone of Punjab, i.e. the Southern irrigated zone (Zone 1), the Northern irrigated zone (Zone II) and the Arid (rain-fed) zone (Zone III). Fifteen districts, mentioned in Table 1, were randomly selected for sampling with five districts from each agro-ecological zone. To avoid sampling errors due to the highly heterogeneous nature of fungal distributions, each 3 kg composite sample collected from one storehouse was a mixture of 15 sub-samples of 200 g each. Sub-samples were formed by combining five increments. The 15 subsamples were collected from five diagonal points on each of the upper, middle and lower layers of each storehouse and then mixed together to form one sample. These samples were labelled, packaged in sterile polythene bags, taken to the laboratory and kept at 4°C for further analysis.
Moisture content of the maize samples was determined using the standard oven method (AOAC International 1999). The samples were dried at 100°C to constant weight, and the mean moisture content was calculated as a percentage on wet basis.
Chemicals and standards High-performance liquid chromatography (HPLC) grade methanol, acetonitrile, sodium chloride (analytical grade) and trifluoroacetic acid (TFA, which acts as a derivatising agent to enhance fluorescence) were obtained from Merck (Darmstadt, Germany). Immunoaffinity columns (AflaOchra-Test, VICAM, Watertown, MA, USA) and standards ochratoxin A and an aflatoxin mix kit containing AFB1, AFB2, AFG1 and AFG2 (Sigma-Aldrich, St. Louis, MO, USA) were used.
Table 1. Validation data for aflatoxins and ochratoxin A in maize (n = 6). Aflatoxin B1 B2 G1 G2 Ochratoxin A
Spike (µg kg−1) 2 5 10 0.08 0.75 4 2 5 10 0.08 0.75 4 0.01 0.5 5
Recovery (%) 85 87 90 84 86 88 84 88 89 85 84 86 85 88 90
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.15 0.13 0.17 0.11 0.14 0.12 0.15 0.13 0.17 0.11 0.14 0.12 0.15 0.17 0.13
RSD (%) 0.17 0.14 0.18 0.13 0.16 0.13 0.17 0.15 0.19 0.13 0.17 0.14 0.18 0.19 0.14
Extraction and analysis Collected maize samples were ground by a laboratory mill, homogenised and 25 g was taken for extraction in an Erlenmeyer flask (250 ml), and 2.5 g sodium chloride water–acetonitrile (15:85 v/v) was added. The flask was shaken on a water bath for 2 h, the extract was filtered through filter paper (Whatman, Inc., Clifton, NJ, USA) and 10 ml filtrate was diluted in 40 ml of distilled water. Immunoaffinity columns were conditioned with double-distilled water. Then, the filtrate was passed through the column in a solid phase extraction assembly. Toxins were slowly eluted from the column with 1 ml methanol in a glass vial. For ochratoxin A analysis, the elute was analysed directly by HPLC, while for aflatoxin analysis, a derivatisation was carried out as follows: the elute was evaporated to dryness with a gentle stream of nitrogen, redissolved in 200 µl nhexane, vortexed, and 50 µl of TFA was added. Then 950 µl of acetonitrile-water (1:9) was added and filtered by using a syringe filter assembly. The filtrate was analysed by HPLC. Liquid chromatographic analysis A HPLC system (Agilent 1100 series, Agilent Technologies, Santa Clara, CA, USA) with a reversed-phase C18 column (Merck, Darmstadt, Germany), and a fluorescence detector was used for quantification. For aflatoxins analysis, a mobile phase consisting of water:methanol:acetonitrile in the volume ratio 60:20:20, at a flow rate of 1 ml min–1 was applied, and aflatoxin was detected at excitation and emission wavelengths of 360 nm and 440 nm, respectively. For ochratoxin A, the mobile phase was acetonitrile:water:acetic acid, 99:99:2 v/v/v, and it was detected at 335 nm and 465 nm as excitation and emission wavelengths, respectively. For HPLC method validation, calibration curves were drawn using a series of calibration solutions in methanol. Each standard solution was chromatographed in duplicate. Statistical analysis Measurements were statistically analysed by one-way analysis of variance (ANOVA). Results and discussion The HPLC method was validated by testing linearity, recovery, limit of detection (LOD) and limit of quantification (LOQ). Results for recovery are given in Table 1. The data for the calibration curves are given in Table 2 and
Food Additives & Contaminants: Part B Table 2. Linear regression parameters for aflatoxins B1, B2, G1, G2 and ochratoxin A. Mycotoxin
µg kg−1
Slope
Intercept
R2
Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2 Ochratoxin
0.5–12 0.05–5 0.5–12 0.05–5 0.005–6
1.0907 2.6638 1.0907 2.6638 5.5622
0.042 0.0388 0.042 0.0388 0.8973
0.9996 0.9993 0.9996 0.9993 0.9995
showed good linearity. LOD and LOQ were 0.5 and 1 µg kg−1 for AFB1 and AFG1, respectively, and 0.05 and 0.1 µg kg−1 for AFB2 and AFG2, respectively. Results (Table 3) showed that aflatoxin B1 contamination was found in 97.3% of maize samples tested, 78.7% were contaminated with AFB2, while AFG1, AFG2 and ochratoxin A were not detected in tested samples. Out of the total 75 samples, 27 samples had an aflatoxin B1 content above the limit of 20 µg kg−1, as set by United States Food and Drug Administration (USFDA), while 31 samples had levels of AFB1 above the European Union (EU) statutory limit, i.e. 2 µg kg−1 for aflatoxin B1 and 4 µg kg−1 for total aflatoxins. Also, among all tested samples, 21 samples contained AFB2 concentrations above the limit as set by the EU. High aflatoxin B1 contamination has been observed in samples obtained from districts of rain-fed arid zone (Zone 3). The highest AFB1 level was detected in samples taken from the Attock district (mean 240.6 μg kg−1). After Attock, Chakwal had a mean of 148.7 µg kg−1. However, in Rawalpindi, Jhelum and Khushab districts of the same zone, mean AFB1 levels were 5.0, 29.0 and 77.7 µg kg−1, respectively (Table 3), whereas aflatoxin B2 was
59
detected in 22 samples with an average concentration of 2.0 µg kg−1 (Table 4). Out of 25 contaminated samples of the arid zone, 28% and 36% contained AFB1 and AFB2, respectively, with a concentration range of 2–16 µg kg−1; 8% contained AFB1 in a range of 16–20 µg kg−1, and more than 20 µg kg−1 AFB1 was found in 48% of samples (Table 5). In the northern irrigated zone (Zone 2), 25 samples were contaminated with AFB1. The highest level (mean 104.3 µg kg−1) was detected in samples taken from Rahim yar khan. However, mean concentrations of AFB1, i.e. 29.0 µg kg−1, 17.5 µg kg−1, 14.8 µg kg−1 and 12.2 µg kg−1 were recorded in samples collected from Bahawalpur, Lodra, Bahawalnagar and Multan, respectively (Table 3). In Zone 2, 24 samples were contaminated with aflatoxin B2, but its average concentration was below the limits, i.e. 1.3 µg kg−1. Table 5 illustrates that 64% and 28% of Zone 2 samples contained aflatoxin B1 and B2 in the concentration range 4–16 µg kg−1. Only 4% of the samples showed levels in the range 16–20 µg kg−1, while 32% of the samples contained AFB1 exceeding the legal limits of 20 µg kg−1. In the southern irrigated zone (Zone 1), aflatoxin content was lower than in arid and northern zone. Among five districts of Zone 1, a higher level of AFB1 was only found in samples taken from district Sahiwal (116.2 µg kg−1). However, mean AFB1 concentrations of 20.3 µg kg−1, 1.8 µg kg−1, 1.2 µg kg−1 and 0.7 µg kg−1 were observed in samples collected from Sialkot, Faisalabad, Lahore and Chiniot, respectively. AFB2 concentration was 1.1 µg kg−1 (Table 4). Table 5 shows that in Zone 1, 20% of the samples contained aflatoxin B1 and B2 in the range of 2–16 µg kg−1, while 28% contained AFB1 levels above 20 µg kg−1.
Table 3. Aflatoxin B1 and B2 levels (µg kg−1) in maize, collected from southern, northern irrigated zone and arid (rain-fed) zone (n = 25). Mean aflatoxin concentration Commodity Maize
Area
District
Zone 1 (Southern irrigated zone)
Lahore Faisalabad Chiniot Sialkot Sahiwal Bahawalpur Bahawalnagar Multan Rahim yar khan Lodhra Attock Chakwal Khushab Jhelum Rawalpindi
Zone 2 (Northern irrigated zone)
Zone 3 (Arid (rain-fed) zone)
Coordinates (latitude and longitude) 31.5758° 31.2500° 31.7167° 32.5000° 30.5833° 29.9833° 29.9969° 30.1917° 28.4167° 29.5333° 33.8936° 32.9303° 32.3000° 32.9333° 33.6000°
N, N, N, N, N, N, N, N, N, N, N, N, N, N, N,
74.3269° 73.0500° 72.9667° 74.5333° 73.3333° 73.2667° 73.2511° 71.4654° 70.3000° 71.6333° 72.2414° 72.8556° 72.3400° 73.7333° 73.0500°
E E E E E E E E E E E E E E E
AFB1
AFB2
1.2 1.8 0.7 20.3 116.2 29.0 14.8 12.2 104.3 17.5 240.6 148.7 77.7 29.0 5.0
0 0.01 0.0 1.7 3.7 1.7 1.0 0.4 2.1 1.3 4.3 2.5 1.3 1.6 0.2
60
W. Iram et al.
Table 4. Aflatoxin B1 and B2 (µg kg−1) between maize varieties in three zones of Punjab. Aflatoxin B1 Variety Agatii 2002 Agaitii- 85 Afgoye Cargle-D-MS- 3937 Desi FH-810 hybrid FH-963 hybrid FH-793 hybrid Golden 85 Hybrid 80y80 HY-1898 hybrid HY-1921 hybrid ICI 8464 ICI 984 Moncento 6525 Moncento 919 Moncento 6142 Moncento 979 MMRI yellow Neelum NK-278 Pioneer 30y87 Pioneer 8711 Pioneer 32B41 Pioneer 32B33 Pioneer 6339 Pioneer 8441 Pioneer 31R88 R-886 Sahiwal 2002 Sanchanta NK-6621 Sanchanta NK 6615 Supra Sadaf 2166 2278 AS 6789 4881 Mixed Variety 1 Variety 2 Contaminated samples (%) Average
Aflatoxin B2
Zone 1
Zone 2
Zone 3
Zone 1
Zone 2
Zone 3
238.5 – 64.1 – – – – – – – – –
