Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 39 Op e n Ac c e s s F u l l L e n g t h A r t i c l e Aflatoxin Concentrations in Various Rice Varieties of Punjab, Pakistan Nasir Ishaque Tahir1, Shabbir Hussain1,*, Imran Kalim2, Amina Asghar3, Shahzada Khurram Syed4 Muhammad Amin Idrees5, Muhammad Ameen Ahmad1 1Department of Chemistry, Lahore Garrison University, Lahore, Pakistan. 2Food & Biotechnology Research Center, Pakistan Council of Scientific and Industrial Research (PCSIR) Labs, Lahore, Pakistan. 3Department of Chemistry, DSNT, University of Education Lahore, Pakistan. 4Department of Basic Medical Sciences, School of Health Sciences, University of Management and Technology, Lahore, Pakistan. 5Department of Physics, Riphah International University, Faisalabad Campus, Pakistan. A B S T R A C T Background: Mycotoxins are secondary fungal metabolites that are produced in rice, corn, nuts and several other cereals. The contamination of food stuff by mycotoxins is a cause of numerous diseases throughout the world. Aflatoxins are potent mycotoxins that have chronic and acute toxicity. Objectives: To evaluate the presence of aflatoxins B1, B2, G1 and G2 in branded and non-branded rice varieties available in Punjab, Pakistan. Methodology: Total 150 rice samples were collected in May 2019 from various cities (Islamabad, Rawalpindi, Gujar Khan, Jehlum, Kharian, Gujrat, Gujranwala, Lahore, Kasur, Okara, Sahiwal, Mian Channu, Khanewal, Multan, Bahawalpur and Rahim Yar Khan) of Pakistan. All the rice samples were subjected to physical testing methods, followed by extraction (using chloroform extraction method) and qualitative and quantitative detection of aflatoxin using Thin layer Chromatography (TLC) Technique and Enzyme linked Immunosorbent assay (ELISA). Results: No aflatoxins was detected in branded rice varieties which were stored in good packaging. The aflatoxin B1 (AFB1) was detected in 65% of poorly stored rice varieties, with maximum contamination of 8.92ppb. The lowest moisture 9.4% content was found in (branded samples) and 10.9% (non-branded samples) while highest moisture content 10.8% found in (branded samples) and 16% found in (non-branded samples). The 54 out 100 non-branded samples found to have more than 13% moisture contents which is alarming. Conclusion: With the implementation of effective strategies and special precautions during storage, harvesting, transportation and drying, we can prevent the contamination of rice product with fungi and stop aflatoxin production which is a major threat to country economy. Keywords Aflatoxins, Branded, Nonbranded, Punjab, Pakistan, Rice. *Address of Correspondence dr.shabbirhussain@lgu.edu.pk Article info. Received: May 09, 2021 Accepted: June 14, 2021 Cite this article Tahir NI, Hussain S, Kalim I, Asghar A, Syed SK, Idrees A, Ahmad Aflatoxin Concentrations in Various Rice Varieties of Punjab, Pakistan. RADS J Biol Res Appl Sci. 2021; 12(1):39-53. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. O R I G N A L A R T I C L E Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 40 I N T R O D U C T I O N The contamination of foodstuffs by mycotoxins is a cause of numerous diseases throughout the world1. Mycotoxins are secondary fungal metabolites2 that are produced in rice, corn, nuts and several other cereals3. Aflatoxins are potent mycotoxins that have chronic and acute toxicity4 comprised of closely related compounds e.g., aflatoxins B1, B2, G1, G2, M1 and M25-6. They exist ubiquitously in nature and may lead to several harmful and toxic effects on living beings5. They are produced in food commodities and animal feeds by the fungal strains (mainly by Aspergillus. parasiticus and Aspergillus. flavus; rarely by Emericella venezuelensis, Emericella astellata, Aspergillus tamarii, Aspergillus pseudotamari, Aspergillus ochraceoroseus and Aspergillus bombycis)5, 7. When animals consume aflatoxin-contaminated feed, then their meat, eggs, and milk are also contaminated8, 9. Aflatoxins have mutagenic, carcinogenic and immunosuppressive effects10, 11. The presence of aflatoxins in diet severely affect kidneys, liver, growth rate, and reproduction, and can cause serious illness and even death. This problem is more common in developing countries5. Uganda has higher cases of liver cancer due to aflatoxin-contaminated foods12. In Kenya in 1981, twenty (20) people complained about stomach trouble and mild fever due to consumption of aflatoxin-contaminated food later they were diagnosed of livers damaged and 12 individuals were reported died1 3 . Aflatoxins gain entry into food products when proper storage conditions (e.g., little moisture content and low temperature) are not applied14,15. The moisture and a hot climatic conditions are important factors contributing to toxin production16,17. Plants are most valuable sources of food and food products throughout the world18-20. Rice is one of the most important food crops21 having an excellent nutritional value22. It is the most popular staple food and an important source of vitamins, minerals, energy, fiber, and other biomolecules23. Rice exports are the second highest source of income in Pakistan24. Rice is consumed worldwide as a food and food additive due to its unique fragrance, cooking advantages and taste. The diverse rice varieties include kernel, basmati, superkernel, super basmati, super shaheen, long- grain white and brown, guard supreme basmati, guard super kernal basmati, zainab basmati rice, falak premium basmati rice, mughal badshah rice, aroma super basmati rice, aroma world longest basmati rice, falak daily basmati rice, fine life super kernal basmati rice, kalaar basmati steam rice, zarafa super kernal basmati rice, ream awami basmati rice, hashmi super kernal basmati 1121, honeymoon basmati rice, shan shahi jalwa awami long grain rice family super kernel basmati rice, hassan 1121 basmati rice, khyber basmati rice, kausar basmati rice, naubhar nayab silla basmati rice, mehran basmati daily rice, Jazaa premium basmati rice, motidana steamed 1121 basmati rice, al badar classic super kernal basmati and anmol super kernal basmati rice etc. These varieties are commonly used in Asian dishes2 , 5 . After wheat, the rice family is Pakistan’s second-largest food26. It is the third most cultivated crop in Pakistan after wheat and cotton27. Rice is a major export of Pakistan and a main cash crop and a source of income for the Pakistan, accounting for approximately 5.9% of total agricultural value-added and 1.3% of GDP27,28. The contamination in this costly crop create a huge impact on Pakistan economy. In addition the existence of Aflatoxins in rice-based products may cause severe toxicity especially in human genetic material i.e. DNA and RNA29. The impact of aflatoxins on human health is an important challenge today30,31; that is why the aflatoxin related research is catching attraction world wide32. The present study was conducted to evaluate the presence of aflatoxins B1, B2, G1 and G2 in branded and non- branded rice varieties of Punjab, Pakistan. M A T E R I A L A N D M E T H O D S All the experimental work was performed in PCSIR laboratories complex, Lahore, Pakistan. AFB1, AFB2, AFG1 and AFG2 standards were imported from the Trilogy Analytical Laboratory (870 Vossbrink Dr, Washington, MO 63090, USA), Acetone (Fisher Scientific, USA), Anhydrous Ether (Merck USA), Chloroform (Sigma- Aldrich UK) and benzene acetonitrile (Sigma-Aldrich UK). TLC plates were procured from Merck (290 Concord Road Billerica Massachusetts USA). Elisa kit (Model 680) BIO-RAD USA, Reader grinding mill (Romer Series Mill) Romer Lab Singapore, Wrist action shaker (Model 75) Burrell Scientific Singapore, Hot Plate (Hot Plate 180 PCSIR Pakistan), Water bath (water bath Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 41 AL/16, PCSIR Pakistan) and UV spectrophotometer (UV reader/280) Redicom Austria, were used. COLLECTION OF RICE SAMPLES Total 150 rice samples were collected in May 2019 from various cities (Islamabad, Rawalpindi, Gujar Khan, Jehlum, Kharian, Gujrat, Gujranwala, Lahore, Kasur, Okara, Sahiwal, Mian Channu, Khanewal, Multan, Bahawalpur and Rahim Yar Khan) of Punjab, Pakistan. It is worth mentioning that rice crop planted from May-June and harvested in October-December in Pakistan. SAMPLE PREPARATION AND TESTING Physical Testing Homogenous rice samples (100g each) were collected from each rice variety, marked and stored for analysis. The defective, broken/smashed, other contaminations and rice weevils were removed before sample collection. Grinding A total of 50g of each sample was carefully mixed for 10min. It was ground in a sample grinder (ILP, FBRC/AL/05) and then passed through 20 mesh sieve to obtain a uniform (1mm particle size) and descriptive sample. The ground samples were stored in a sealed plastic pack to keep them safe before the experimental use. These samples were finally used for aflatoxins assessment. Extraction of Aflatoxins Various methods of extraction and analysis are utilized for diverse types of raw materials because of their different chemical behaviors. Researchers follow numerous extraction procedures depending upon their resources. In current studies, we have chosen the chloroform extraction method which has been considered an appropriate method for the aflatoxin extractions. Extraction technique was applied by utilizing 50g of milled sample of rice in a 500ml Erlenmeyer flask. Twenty five (25ml) of water and 150ml of chloroform was added in the allocated flask. The Erlenmeyer flask was mounted on a wrist shaker and shaken for 30min. The sample solution was then filtered by Whatman Grade No. 1 filter paper. Extract (50ml) was positioned on a hot plate for evaporation33. It was finally subjected to analysis for the presence of aflatoxins B1, B2, G1 and G2. Aflatoxin Determinations by Thin Layer Chromatography Technique (TLC) Dilutions were done up to per microliters for TLC spot applications. Then 25µl of the test solution was applied on a thin layer chromatography plate, using a microscope syringe. Total 5μl and 10μl concentrations of AFB1, AFB2, AFG1 and AFG2 were also applied as internal standards for aflatoxins on this plate. The TLC plate was placed in “First Mobile Phase” (Anhydrous Ether) in TLC container No. 1, until the solvent was reached on an average height. Then TLC plate was taken out and dried before it was placed in the “Second Mobile Phase” tank containing acetone and chloroform (2:1). When the mobile phase was reached to the required height, the TLC plate was taken out from tank 2 and then dried. The test plate was carefully observed under 365nm ultraviolet light for the presence/absence of aflatoxins. Aflatoxins were evaluated by careful observation of fluorescence brightness and comparing the results with those of the aflatoxin standards applied on the TLC plates along with the samples34. The test solution was completely dried for quantitative determination. Benzene and acetonitrile (98:2) were mixed to the test solution. Then 3.5, 5.0 and 6.5µl spots (all of the same size) of the test solutions were put on the TLC plates. Spots of the identical size of the standards were also put on the TLC plate. The tested and standard solutions were compared to identify the similarities between the two. fluorescent light used to examine the spots. When the sample spot and standard spot were superimposed, this indicates the presence of aflatoxin in the sample. In such situation, the color of the sample and Rf values of the samples were corresponded to the aflatoxin standards35. Calculations The concentration of aflatoxins in a sample can be calculated by the following equation36: Concentration of aflatoxins (mg/kg) = S x V x Y W x Z S: Volume (ml) of aflatoxin standard of equivalent intensity to Z = ml of sample V: Volume (ml) of solvents required for dilution of the final extract Y: Concentration (mg/ml) of aflatoxin standard Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 42 W: Weight (g) of the original sample contained in the final extract Z: Volume (ml) of sample extract required to produce fluorescence intensity comparable to that of S = ml of aflatoxin standard. Enzyme linked Immuno Sorbent Assay (ELISA) Method Sample preparation was preformed according to the instructions of the test kit manual of RIDASCREEN Aflatoxin B1 30/15 (R-Biopharm GmbH, 2010)37. Twenty- five milliliter (25ml) mixture of methanol: water (70:30) was added to 5g of rice followed by its vigorous stirring for 3min manually. The obtained extract was fil`tered through a filter paper and diluted with distilled water (1:1). At last, 50ml of the diluted filtrate was used for each well in a test. Aflatoxin level was determined quantitatively by an ELISA test procedure (RIDASCREEN Aflatoxin B1 30/15 (Art No.: 1211) test kit manual) reported earlier38. To each well of a microtiter plate, 50ul of a solution (test solution and a standard solution) was added in duplicate followed by addition of 50ul each of the enzyme conjugate and the anti- aflatoxin antibody to every well. The resultant solution was mixed gently and incubated at 20-25°C for 30min. Then the wells were tapped upside down vigorously for the removal of their liquids into an absorbent paper. Each empty well was then washed twice by 250ul washing buffer. Subsequently, there was addition of 100ul of substrate/chromogenic solution to each well followed by its gentle mixing and incubation at 20-25°C for 15min in a dark place. Then there was addition of 100ul of 1N H2SO4 (stop solution) to each well. Finally, the absorbance was noted in ELISA plate reader at 450nm38. Moisture Analysis A known amount of fine and ground sample was taken in a well dried (by oven) petri dish. The petri dish was then heated in the oven at 100ºC overnight to ensure the effective removal of all the moisture. The dish was placed in a desiccator and cooled; it was heated again in an oven for 2hours again and weighed was taken to ensure the maximum removal of moisture from the sample. Heating and weighing was continued until the constant reading of petri dish and sample was achieved39. % Moisture = Weight of the dried rice sample x 100 Weight of the original sample R E S U L T S A N D D I S C U S S I O N The rice samples were collected and analyzed (qualitatively and quantitatively) for the aflatoxin contents by Thin Layer Chromatography (TLC) and Enzyme Linked Immunosorbent Assay (ELISA). The prepared samples were run through TLC plates and finally observed with a UV spectrophotometer. The results for TLC tests are compared in Fig. 1 and 2 for branded (packed) and non- branded (open or poorly stored) samples, respectively. AFLATOXIN CONCENTRATION IN OPEN RICE SAMPLES No aflatoxins were detected in all 50 branded rice samples which had their packaging according to the standard requirements. The corresponding TLC results are shown in Fig. 1. The obtained data are displayed in Table 1 and summarized in Table 2. Table 1. Aflatoxin Concentration in Branded Rice Varieties Available in Local Market (Each Rice Sample = 500g). S. No. City and Location from where the rice sample was picked Company name & Rice Variety Packaging conditions Time in shelf (Months) H2O Content (%age) Aflatoxin conc. (ppb) 1 Kechery Chowk, Rawalpindi Guard supreme basmati Good 2 10 Not Detected 2 Kechery Chowk, Rawalpindi Guard Super Kernal Basmati Good 4 9.8 Not Detected 3 Tulsa Rd, opposite Lalazar, Rawalpindi Zainab Basmati rice Good 5 10.2 Not Detected Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 43 4 Tulsa Rd, opposite Lalazar, Rawalpindi Falak Premium Basmati rice Good 7 10.5 Not Detected 5 GT Road, Kotha Kalan Morgah, Rawalpindi Mughal Badshah rice Good 3 9.5 Not Detected 6 GT Road, Kotha Kalan Morgah, Rawalpindi Guard Super KernalBasamati rice Good 8 9.8 Not Detected 7 Islamabad, Islamabad Capital Territory, Pakistan Aroma Super Basmati rice Good 6 10.1 Not Detected 8 Islamabad, Islamabad Capital Territory, Pakistan Aroma world Longest Basmati rice Good 2 10.5 Not Detected 9 G.T. Rd, Islamabad, Islamabad Capital Territory Falak Daily Basmati rice Good 3 10.7 Not Detected 10 G.T. Rd, Islamabad, Islamabad Capital Territory Fine Life Super Kernal Basmati rice Good 5 9.4 Not Detected 11 National Hwy 5, Sector F DHA Phase II, Islamabad. Kalaar Basmati Steam rice Good 6 10.1 Not Detected 12 National Hwy 5, Sector F DHA Phase II, Islamabad Zarafa Super Kernal Basmati rice Good 3 9.5 Not Detected 13 G.T. Rd, Islamabad Guard Super Kernal Basmati Good 6 10.8 Not Detected 14 G.T. Rd, Islamabad Guard Super KernalBasamati rice Good 4 10 Not Detected 15 G.T. Rd, Gujar Khan, Rawalpindi Ream Awami Basmati rice Good 7 9.9 Not Detected 16 G.T. Rd, Gujar Khan, Rawalpindi Mughal Pure Basmati rice Good 3 10.8 Not Detected 17 Grand Trunk Rd, Jada, Jhelum Hashmi Super Kernal basmati 1121 Good 8 9.7 Not Detected 18 Grand Trunk Rd, Jada, Jhelum Hashmi Super Kernal basmati 1121 Good 4 10.5 Not Detected 19 Grand Trunk Rd, Panjan Kasana Kharian Honymoon Basmati rice Good 8 10.8 Not Detected 20 Grand Trunk Rd, Panjan Kasana Kharian Honymoon Basmati rice Good 4 9.9 Not Detected 21 Grand Trunk Rd, Ali Chak, Gujrat Shanshahi Jalwa Awami long grain rice Good 6 10.2 Not Detected Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 44 22 Grand Trunk Rd, Ali Chak, Gujrat Shanshahi Jalwa Awami long grain rice Good 2 10.5 Not Detected 23 Askari Homes Rahwali Cantonment, Gujranwala Falak Easy cook Daily Basmati rice Good 7 10.6 Not Detected 24 Askari Homes Rahwali Cantonment, Gujranwala Falak Easy cook Daily Basmati rice Good 3 9.9 Not Detected 25 Hussain Chowk, Block B3, Gulberg III, Lahore Aroma World Longest Basmati rice Good 9 10.4 Not Detected 26 Hussain Chowk, Block B3, Gulberg III, Lahore Aroma World Longest Basmati rice Good 7 10.6 Not Detected 27 Fortress Stadium, Lahore Family Super kernel basmati rice Good 8 10.1 Not Detected 28 Fortress Stadium, Lahore Family Super kernel basmati rice Good 4 10.4 Not Detected 29 Aziz Bhatti Road, 4 st, Cantt, Lahore Falak select Super Kernal basmati rice Good 3 9.4 Not Detected 30 Aziz Bhatti Road, 4 st, Cantt, Lahore Guard Super Kernal Basmati Good 4 9.5 Not Detected 31 PAF Market, Cantt, Lahore Hassan 1121 Basmati rice Good 5 9.9 Not Detected 32 Ahmed Bukhsh Rd, R A Bazaar Cantt, Lahore Zainab Basmati rice Good 4 10.4 Not Detected 33 Walton Road, Super Town Lahore Falak Daily Basmati rice Good 8 10.6 Not Detected 34 Walton Road, Super Town Lahore Fine Old basmati rice Good 11 10.3 Not Detected 35 Walton Road, Madina Colony, Lahore Hassan 1121 Basmati rice Good 5 10.2 Not Detected 36 Walton Road, Madina Colony, Lahore Mughal Super Sella Basmati rice Good 4 9.8 Not Detected 37 Walton Road, Pir Colony, Lahore Guard Extreme Basamati rice Good 4 10.4 Not Detected 38 Model Town Circular Road, Model Town, Lahore Khyber Basmati rice Good 7 10.7 Not Detected 39 Model Town Circular Road, Model Town, Lahore Kausar basmati rice Good 6 9.4 Not Detected 40 Model Town Link Rd, Lahore Guard Super Kernal Basamati rice Good 4 10.2 Not Detected Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 45 41 Model Town Link Rd, Lahore Falak Extreme Basmati rice Good 8 9.5 Not Detected 42 Cash & Carry, Airport, Lahore Kalaar Basmati Steam rice Good 6 10.4 Not Detected 43 Cash & Carry, Airport, Lahore Mughal Pure Basmati rice Good 8 9.8 Not Detected 44 Cash & Carry, Airport, Lahore Naubhar Nayab Silla basmati rice Good 9 9.4 Not Detected 45 Cash & Carry, Airport, Lahore Mehran Basmati daily rice Good 3 9.9 Not Detected 46 Y-Block, DHA Phase 3, Lahore Jazaa Premium Basmati rice Good 4 9.6 Not Detected 47 Y-Block, DHA Phase 3, Lahore Motidana Steamed 1121 Basamti rice Good 7 9.8 Not Detected 48 Street 5, H-Block, DHA Phase 4, Lahore Al badar Classic Super Kernal basmati Good 6 10.4 Not Detected 49 Abdul Haque Rd, Johar Town, Lahore Anmol Super Kernal Basmati rice Good 4 10.4 Not Detected 50 Abdul Haque Rd, Johar Town, Lahore Falak Easy cook Sella Good 8 10.6 Not Detected Table 2. Summary of Results for the Branded Rice Varieties. Area Rice Brand Time in Shelf Moisture Content Aflatoxin Level Islamabad, Rawalpindi, Gujar Khan, Jehlum, Kharian, Gujrat, Gujranwala, Lahore Guard supreme basmati, Gurad Super Kernal Basmati, Zainab Basmati Rice, Falak Premium Basmati rice, Mughal Badshah Rice, Aroma Super Basmati Rice, Aroma world Longest Basmati Rice, Falak Daily Basmati rice, Fine Life Super Kernal Basmati Rice, Kalaar Basmati Steam rice, Zarafa Super Kernal Basmati rice, Ream Awami Basmati Rice, Hashmi Super Kernal basmati 1121, Honymoon Basmati Rice. Shanshahi Jalwa Awami long grain rice. Faimly Super kernel basmati Rice, Hassan 1121 Basmati Rice, Khyber Basmati Rice, Kausar basmati Rice, Naubhar Nayab Silla basmati rice, Mehran Basmati daily Rice, Jazaa Premium Basmati rice, Motidana Steamed 1121 Basamti rice, Al badar Classic Super Kernal Basmati, Anmol Super Kernal Basmati Rice 2-11 Months 9.4-10.8 Not Detected Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 46 Figure 1. TLC Results for the branded rice Varieties. Figure 2. TLC results of open rice (poorly stored samples) (Left lanes-Samples: 69-73, 75; Right lanes: 1ppb, 2ppb, 4ppb-Standards). Aflatoxin Concentration in Open Rice Samples with Poor Storage Conditions Rice varieties which were collected from different cities and towns of Punjab had shown the presence of aflatoxins. The corresponding TLC results are shown in Fig. 2. Out of 100 non-branded samples, the presence of aflatoxins was detected in 65 samples while the remaining 35 samples did not show the presence of aflatoxins. Out of 65 total contaminated samples, 23 rice verities had shown the existence of aflatoxins below the limits recommended by the European authorities (2-4ppb) whereas 24 samples had shown aflatoxins in the recommended limits of 2-4ppb. 18 rice samples showed aflatoxin concentration above the recommended limits. The highest value of aflatoxin (8.92ppb) for observed for the rice sample 40 (NIAB IR-9). Table 3 displays the aflatoxin concentration in the poorly stored rice samples whereas the final data are summarized in Table 4 and Fig. 3. Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 47 Table 3. Aflatoxins and Moisture content analysis of Local Rice Varieties. S. No. City and Location from where the Rice Samples Picked (Minimum 500g each) Variety of Rice Time in a Shelf (Months) H2O Content (%age) Aflatoxin Concentration (ppb) 1 Kechery Chowk, Rawalpindi Basmati 370 4 12 Not Detected 2 Bilal Street, Morgah, Rawalpindi Basmati Pak 9 12.4 Not Detected 3 Adamjee Street, Rawalpindi KS-282 12 14 1.86 4 G.T Road, Rawalpindi Basmati 385 11 14.2 2.96 5 Soan Garden Extension, Islamabad Basmati 2000 15 15 3.56 6 Shalimar Rd, Block E Soan Garden, Islamabad Basmati Pak 6 11.8 Not Detected 7 Phase 1 Jinnah Garden, Islamabad Super Basmati 2 12 Not Detected 8 GT Rd, Rawat, Islamabad Basmati 515 11 15.5 4.53 9 Chamber More Rawat, Islamabad Shaeen Basmati 6 11.5 Not Detected 10 Service Rd, Rawat, Islamabad NIAB IR-9 9 15 2.62 11 Bagga Shaikhan, Rawalpindi, Islamabad KSK-133 6 15.1 1.73 12 Banth, Rawalpindi, Punjab, Pakistan Basmati 2000 5 12 Not Detected 13 Grand Trunk Rd, Mandra, Rawalpindi Basmati 370 12 14.6 2.55 14 Tharjiyal Rd, Mandra, Rawalpindi Super Basmati 10 15.4 1.79 15 Tharjiyal Rd, Rawalpindi KS-282 6 10.9 Not Detected 16 ChehariKalyal, Rawalpindi Basmati 515 8 12.4 Not Detected 17 Gujar Khan, Rawalpindi Basmati 370 4 11.8 Not Detected 18 Sandal Road Ward 14، Gujar Khan, Rawalpindi Super Basmati 10 15.4 8.78 19 Housing Scheme 2, Gujar Khan, Rawalpindi KSK-133 4 15.5 3.64 20 W7 Gujar Khan, Pakistan Basmati 2000 6 15.2 1.62 21 Bhai Khan, Gujar Khan, Rawalpindi NIAB IR-9 10 11 Not Detected 22 Sohawa City, Jhelum KS-282 10 11.5 Not Detected 23 Ehsan Butt St, DomeliMohallah, Dina Basmati 515 9 14.9 2.48 24 Mangla Rd, Dina, Jhelum Basmati 370 8 15.1 2.93 Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 48 25 Main Bazaar, Dina, Jhelum KS-282 6 16 3.73 26 Grand Trunk Rd, Burha Jungle, Dina, Jhelum NIAB IR-9 9 14.7 1.97 27 Kala Gujran, Jhelum, Punjab, Pakistan Basmati 370 5 12.3 Not Detected 28 G.T Road, OppFauji Mill Ground، Kala Gujran, Jhelum KSK-133 10 15.4 3.47 29 Nawab Colony, Jhelum KS-282 6 11.6 Not Detected 30 Sultan St, Jada, Jhelum NIAB IR-9 10 14.6 1.21 31 Chak Jamal Rd, Jada, Jhelum Basmati 2000 8 15.7 4.63 32 Tufail Rd, Jhelum Cantt, Jhelum Super Basmati 6 12.4 Not Detected 33 Jhelum Cantt, Jhelum 8 11.8 Not Detected 34 Arain Street, Eidgah, Jhelum Basmati 2000 8 14.3 0.92 35 Pinddadan Khan - Jhelum Rd, Jhelum Cantt, Jhelum Basmati 515 6 16 3.85 36 Arsal Town Jhelum Cantt, Pinddadan Khan - Jhelum Rd KSK-133 10 15.4 6.43 37 Bagga, Jhelum Pinddadan Khan - Jhelum Rd KS-282 8 12.9 Not Detected 38 Nougran Link Rd, Pinddadan Khan - Jhelum Rd, Jhelum Basmati 370 6 14.7 1.42 39 Nougran, Jhelum Pinddadan Khan - Jhelum Rd Super Basmati 14 13.2 2.76 40 Ghous Plaza, Grand Trunk Rd, Sarai Alamgir, Gujrat NIAB IR-9 12 15.7 8.92 41 JalilPura, SaraiAlamgir, Gujrat Basmati 2000 6 11.7 Not Detected 42 Main Bazaar, Mohalla Shaheedan, Sarai Alamgir, Gujrat Basmati 370 10 12.5 Not Detected 43 N5, Mohalla Shaheedan, Sarai Alamgir, Gujrat Super Basmati 4 14.7 2.96 44 G.T. Road, Sarai Alamgir, Gujrat KS-282 5 14.4 5.32 45 Bani Mohalla Kharian, Guliana Road, Gujrat KSK-133 7 12.6 Not Detected 46 Panjan Kasana Village, Gujrat NIAB IR-9 2 14.5 1.62 47 Grand Trunk Rd, Thikrian, Lala Musa, Gujrat Basmati 370 4 14.7 6.12 Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 49 48 Asghar Town Haji Asghar Town, Lalamusa, Gujrat Basmati 2000 6 12.8 Not Detected 49 Karimpura, Lalamusa, Gujrat KSK-133 6 15.5 3.76 50 Chota Bazar Road, Lalamusa, Gujrat KS-282 10 15.2 1.62 51 Service Rd W, MeherMohalla, Lalamusa, Gujrat Basmati 385 5 15.3 5.74 52 Grand Trunk Rd, Ellahi Colony, Gujurat Super Basmati 11 15.4 3.63 53 Kalra Khasa, G.T Road, Gujrat NIAB IR-9 6 14.6 0.75 54 Muslim Town, Rahwali Cantonments, Gujranwala Basmati 2000 10 15.1 8.75 55 Meher Colony, Rahwali Cantonments, Gujranwala Basmati 370 5 12.1 Not Detected 56 Service Rd, Shaheenabad, Gujranwala KS-282 4 15.4 1.52 57 Safdar & Asif Rice Trader, Mohalla Raitanwala Krishan Nagar, Gujranwala Basmati 515 9 12.5 Not Detected 58 Civil Lines, Gujranwala Super Basmati 6 14.9 5.73 59 G.T. Rd, Near Jama Masjid Bilal, Eminabad, Gujranwala KSK-133 5 15.4 1.55 60 Mandiala Rd, Kamoke, Gujranwala NIAB IR-9 10 15.5 2.73 61 Canal Road, Sadhoke, Gujranwala Basmati 2000 6 14.9 1.7 62 Mohalla Faiz-e-Madina, Muridke, Sheikhupura Super Basmati 5 12.4 Not Detected 63 Ravi Rayon, Kala Shah Kaku, Sheikhupura KS-282 4 14.4 1.22 64 Main Bazar, Rana Town Ferozewala, Sheikhupura Basmati 370 6 15.7 6.86 65 G.T. Road, Shahdara Town, Shahdara, Lahore Basmati 2000 9 15.4 3.76 66 Ahmed Bukhsh Rd, R A Bazaar Cantt, Lahore KSK-133 6 11.6 Not Detected 67 Akram Park Gurumangat Gulberg III, Lahore KS-282 3 14.4 1.65 68 Main Bazar Canal Park, Block O Gulberg 2, Lahore Basmati 515 5 12.5 Not Detected 69 Chahgallanwala, Imran St, Rasool Park, Lahore Basmati 370 4 12.6 Not Detected 70 Link Ferozepur Rd, New Islamia Park Islamia Park, Lahore Super Basmati 6 14.8 1.76 Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 50 71 Multan Rd, Maraka Village, Lahore NIAB IR-9 10 11.4 Not Detected 72 Canal Bank Rd, Chimbran wali Jhuggi, Lahore Basmati 2000 6 15.1 4.93 73 Hospital Road, Manga - Raiwind Road, Manga Mandi, Lahore KSK-133 2 12.4 Not Detected 74 Sherpur Road, Phool Nagar, Kasur KS-282 6 14.4 1.92 75 Malik Colony, Phool Nagar, Kasur Super Basmati 7 15.4 5.76 76 Jamber, Lahore Multan Road, Kasur NIAB IR-9 5 11.8 Not Detected 77 Megha Rd, Pattoki, Bilal Colony, Kasur Basmati 2000 4 15.2 3.72 78 Habibabad, Habibabad Road, Okara Basmati 370 3 15.7 1.47 79 G.T. Rd, RenalaKhurd, Okara Basmati 2000 1 15.4 5.92 80 Multan Okara Rd, Sheikh Basti, Okara KSK-133 5 11.6 Not Detected 81 Kot Liaquat Hayat, Okara Basmati 2000 2 12.4 Not Detected 82 Wan Bazar, Sahiwal, Sahiwal NIAB IR-9 6 15.1 3.65 83 Liaqat Chowk, Sadman Town Sahiwal, Sahiwal KS-282 6 15.4 2.73 84 Block 5, Chichawatni, Sahiwal Basmati 370 4 15.1 6.57 85 Multan Road, Iqbal Nagar Super Basmati 6 14.8 5.7 86 Multan-Mian Channu Road, Mian Channu Basmati 370 5 12.6 Not Detected 87 Ayoub Chowk, Ayoub Road, Khanewal Basmati 2000 6 15.4 3.72 88 Old Kachehri Rd, Main Bazar, Kabirwala Basmati 515 4 15.1 1.75 89 T Block, New Multan Colony, Multan KS-282 6 11.4 Not Detected 90 Gulshan Town, Multan NIAB IR-9 7 12.4 Not Detected 91 Chak Raas, Multan Basmati 370 5 16 3.75 92 Raiway Road, Shujaabād, Multan KSK-133 4 15 3.42 93 Permit Road, Jalalpur Pirwala, Multan Super Basmati 6 15.4 1.77 94 Alipur Road, Uch Sharif, Bahawalpur Basmati 2000 4 14.6 2.63 95 Liaqat Pur Rd, Janpur, Rahim Yar Khan KS-282 7 15.1 6.83 96 Khan Bela, Rahim Yar Khan Basmati 370 5 14.4 1.65 Contd… Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 51 97 N5, Fateh Pur Kamal, Rahim Yar Khan KSK-133 6 15 2.45 98 Jamia Masjid Chowk Zahir Pir, nearby, Khanpur Rd, Rahim Yar Khan NIAB IR-9 5 15.4 1.42 99 Sardargarh, Rahim Yar Khan KS-282 4 15.1 6.94 100 Main Road, Gulshan e Usman, Rahim Yar Khan Super Basmati 6 12.4 Not Detected Table 4. Summary of Open Rice Samples with Poor Storage. Aflatoxin Total No. of Samples Contaminated Samples Uncontaminated Samples Moisture Content %age Max ppb EU limits for contamination B1 in Rice sample with poor Storage 100 65 35 10.9 - 16 8.92 2-4ppb Figure 3. Comparison of aflatoxin concentration in open rice samples. Aflatoxin B1 (AFB1) which is most common aflatoxin, was detected (0.75-8.92 ppb) in all the 65 rice samples having poor storage. AFB2, AFG1 and AFG2 were not detected in anyone of the tested samples. The recommended limit of aflatoxin B1 by European Union (EU) is 2ppb for rice and other edible foods for humans; it is 4ppb for total aflatoxins. However, according to the Pakistan Standards and Quality Control Authority (PSQCA), Food and Agriculture Organization (FAO), Food and Drug Administration (FDA), the allowed concentration of AFB1 is 10ppb. So the observed AFB1 concentration in the rice samples was found within the recommended limits of PSQCA, FAO and FDA. However, it was found beyond the limits of EU indicating a dire need to overcome aflatoxin contamination in rice. C O N C L U S I O N In the current study, detailed analysis has been conducted to monitor the presence of aflatoxins B1, B2, G1 and G2 in branded and non-branded rice varieties from various cities of Punjab, Pakistan. Interestingly, all the branded rice samples were totally free form aflatoxins, however, 65% of non-branded and poorly stored rice samples had shown the aflatoxin contamination (0.75 to 8.92 ppb). Moreover, in the remaining 35% samples, aflatoxins were found below the limits recommended by the European authorities (2-4ppb). Highest concentration of aflatoxin was observed 8.92 ppbin few rice sample. In the branded rice samples the lowest moisture content detected were 9.4 and highest moisture content was 10.8%. Non-branded (54%) rice samples have shown the moisture contents more than 13%, which is alarming. It is recommended that Pakistani authorities should review periodically and analyze rice samples to ensure the absence of aflatoxins in foods. Special precautions must be taken during storage, harvesting, transportation, drying and harvesting to stop aflatoxin production in cereals. If aflatoxins are present in Aflatoxin Concentrations in Rice Varieties of Punjab Vol. 12 (1), June 2021 ISSN (Print): 2305 – 8722 ISSN (Online): 2521 – 8573 R A D S J . B i o l . R e s . A p p l . S c i . 52 food, their concentration should be minimized or eradicated by recommended de-toxifying technologies. C O N F L I C T S O F I N T E R E S T None. F U N D I N G S O U R C E None. 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