Aflatoxins in Rice Imported to Bushehr, A Southern Port of Iran

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1 American-Eurasian Journal of Toxicological Sciences 4 (): 3-35, 202 ISSN IDOSI Publications, 202 DOI: /idosi.aejts Aflatoxins in Rice Imported to Bushehr, A Southern Port of Iran M. Mohammadi, G.H. Mohebbi, P. Hajeb, S. Akbarzadeh and I. Shojaee Persian Gulf Research and Studies Center, Persian Gulf University, Busheher, Iran 2 Food and Drug Department, Bushehr University of Medical Sciences, Busheher, Iran 3 Department of Chemistry, School of Medicine, Bushehr University of Medical Sciences, Bushehr, Iran Abstract: Aflatoxins (B, B, G and G ) levels were surveyed in rice samples imported to Iran through a southern 2 2 port in Bushehr. Aflatoxin analysis was performed by solvent extraction, immunoaffinity clean-up and determination using high performance liquid chromatography equipped with post-column photochemical reactor and fluorescence detector. Among 52 samples analyzed, 75 % showed levels of aflatoxin B (AFB ) contamination. However, there was no sample with AFB above maximum tolerated level (MTL) of 5 ng/g assigned by Institute of Standard and Industrial Research of Iran (ISIRI). AFB concentrations in the samples was ng/g. Out of 52 samples analyzed, about 76.97% were contaminated with total aflatoxin (AFT) with the mean of 0.67 ng/g which is lower than MTL of AFT in rice (30 ng/g) assigned by (ISIRI). AFT concentration ranged from ng/g. Contamination of aflatoxins in imported rice was dissimilar between different months. The highest levels of AFB and AFT were detected in rice samples imported in September, while the lowest levels were in rice imported during November. Key words: Aflatoxins Food Safety Rice Iran INTRODUCTION while A. parasiticus produces all four aflatoxins; aflatoxin B is the most toxic and best studied of the aflatoxins [8]. Rapid population growth in developing countries Afs are the most potent carcinogens in animal and leads to an ever increasing demand for food. In the human populations [8, 9-]. production of food crops losses occur during the growth The International Agency for Research on Cancer cycle in the field, harvest and storage where up to 5% has designated Afs as a human liver carcinogen [2]. of grain weight can reduce the agricultural output []. They also interfere with the function of the immune The risk of contamination by mycotoxins is an important system [3]. A wide variety of animals including fish, food safety concern for grains and other field crops [2-4]. rodents, waterfowl, poultry, swine and cattle can be The genus Aspergillus is distributed worldwide and affected by aflatoxins [4-6]. The knowledge that contains over 80 species. It is one of the most ubiquitous AFs effect on humans and animals has led many countries and abundant of all groups of fungi and one of the most to establish maximum tolerated level (MTL) on aflatoxins studied fungal groups [5-7]. A. flavus and A. parasiticus, levels allowed in food and feed in the last decades to the two Aspergillus species of most concern in safeguard the health of humans, as well as the economical agriculture, are predominant saprotrophs with limited interests of producers and traders. Currently, worldwide parasitic ability [8]. range of limits for AFB and total AF (AFT) are -20 and Toxins produced by some Aspergillus spp. are called 0-35 ng/g, respectively [2-7, 8-20]. aflatoxins. Aflatoxins were first discovered in Europe in Rice (Oryzae sativa L.) is the most important staple animal feed. AFs are found as contaminants in various food crop in Iran and is cultivated in different areas that agricultural commodities such as maize, rice, sorghum, have sultry and rainy climate. Since the amount of rice wheat, oats, spices (black pepper, ginger) and chilli and cultivation is not enough for domestic consumption, are considered to be of greater significance for human the country imports rice from other regions such as India, beings [0]. The four major AFs that occur in crops are Pakistan, Bangladesh and Thailand which are the largest B, B, G and G. A. flavus produces aflatoxins B and B, producers of rice in the World. However some of those Corresponding Author: G.H. Mohebbi, Bushehr University of Medical Sciences, Food and Drug Deputy, Laboratory of Toxicology, Moallem ave, Bushehr, Iran. Zip Code: Tel:

2 Am-Euras. J. Toxicol. Sci., 4 (): 3-35, 202 countries have frequent and heavy rainfall and floods in micro fiber filter. Afla test was used for samples clean up. coastal areas particularly near harvest, which under this First, 0 ml phosphate buffer saline (PBS) was passed climate the development of fungi, especially species of the through the IAC. Then, 75 ml of the filtrate was passed Aspergillus and Penicillium, is a common and unresolved through the IAC at a flow rate of ml/min. The column problem [2, 22]. Thus, when the rice imported to Iran was washed with water and dried using vacuum. Finally, port, it is usually examined for aflatoxins contamination. AF was eluted with methanol using the following The Institute of Standards and Industrial Research of procedure. First, 0.5 ml methanol was applied on the Iran (ISIRI) has set minimum levels of 5 and 30 ng/g for column which passed through by gravity. After min, the aflatoxin B and the total aflatoxins, respectively [24]. second portion of 0.75 ml methanol was applied and Bushehr is the major port for importing rice (Oryza sativa) collected. The elute was diluted with water and analyzed in south west of Iran. Therefore, the aim of this using HPLC. investigation was to identify the levels of aflatoxin B and the total aflatoxins in imported rice to Bushehr port. AF Standards: After preparation of standard solutions of AFs, the concentration of each one was determined using MATERIALS AND METHODS UV spectrophotometer. These standards were used to prepare mixed working standards for HPLC analysis. Samples: One hundred and fifty two samples were collected randomly by inspectors of Food Control Offices Recovery and Limit of Detection (LOD): The in Bushehr port from May 2008 to February Samples effectiveness of the extraction procedure was confirmed were taken according to method of the Iranian National by sample fortification. The recovery of extraction method Standard of Sampling for aflatoxins analysis in agricultural was determined by fifty grams of milled rice fortified with products [23]. Then, all the samples were transferred to a solution of AF in methanol at 5 µg/ml(for B and G ) and Toxicology Labs in Food and Drug Control Laboratory. A µg/ml(for B 2 and G 2) h before extraction. The AF minimum size of 2 Kg from each sample was used for fortification solution was prepared in methanol and analysis. Samples were kept at 20 C in PE (Poly ethylene) used for quantification of the analytic recovered after bags until analysis. extraction. Sample were fortified with 0.25 ml of this solution in order to have 5 ng /g of AFB in rice, which is Reagents and Apparatus: All reagents (potassium the maximum permitted limit in cereals by the National chloride, phosphoric acid, hydrochloric acid) and solvents Standard of Iran [26]. LOD were 0.07, 0.08, 0., 0.07 and methanol, acetonitrile, propanol-2-ol, n-hexane, 0.32 ng/g for AFB, AFB 2, AFG, AFG 2 and total AFs, chloroform) used were of HPLC grade. AF standards were respectively. purchased from Sigma Chemical Company, USA. Afla test immunoaffinity columns (IAC) were purchased from Analysis of Af Using HPLC: AF was quantitified by VICAM Company, Watertown, MA, USA. reverse-phase HPLC and 2475 Multy fluorescence Apparatus characteristics were WATERS 525 binary detector with post column derivatization (PCD) involving HPLC pump and 2475 Multy fluorescence detector. bromination [27]. The waters HPLC system was applied HPLC column (C mm: 4 µm) was purchased with a Kobra cell and addition of bromide to the mobile from Waters, USA. phase. After dilution of AF elute with water, 00 µl was injected into HPLC. Mobile phase was water: methanol: Sample Preparation: To avoid the sub-sampling error due acetonitrile (600:300: 200, v/v/v) and 350 µl of nitric acid to highly heterogeneous nature of fungal distribution in 4M and 20 mg of potassium bromide with a flow rate of AF analysis, every sample was grinded with the miller and ml/min. The fluorescence detector was operated at an collected in plastic bag and finally 50 g of test portion excitation wavelength of 365 nm and emission wavelength from the ground samples were taken for analysis. of 435 nm [27,28]. The calibration curve for each individual AF including AFB, AFB 2, AFG and AFG 2 was used to Extraction and Clean Up: Samples were analyzed using a check for the linearity and quantification of AF in rice high performance liquid chromatography (HPLC) samples. following AOAC [24] and ISIRI method [25]. Samples were extracted with methanol: water: hexane (240:60:00, v/v/v). RESULTS AND DISCUSSION The mixture was shaken for 30 minutes on a mechanical shaker. The solution was left to sediment and filtered Among 52 samples analyzed, 38 samples (25 %) were through a Whatmann filter No.. After filtration, the not contaminated with AFB (<LOD). A high proportion extract was diluted with water and filtered through glass of samples (75 %) were positive to AFB contamination 32

3 Am-Euras. J. Toxicol. Sci., 4 (): 3-35, 202 Fig. : Maximum levels (ng/g) of aflatoxin B (AFB ) and total aflatoxins AFT detected in the examined rice samples in different months Table : Mean and standard deviation of aflatoxin B (AFB) and total aflatoxins (AFT) (ng/g) in the examined rice samples Month N ND AFBI (ng/g) AFT(ng/g) May ± ±.27 Jun ± ±0.03 Jul ± ±.087 Aug ± ±0.509 Sep ± ±.294 Oct ± ±0.33 Nov ± ±0.37 Dec Jan ± ±0.202 Feb ± ±0.58 (Table). Mean of AFB in the samples was 0.46 ng/g. However, there was no sample with AFB above MTL of 5 ng/g assigned by Institute of Standard and Industrial Research of Iran (ISIRI) [26] (Figure ). Maximum level of AFB in rice samples was 3.3 ng/g. Among 52 samples analyzed, 35 samples (23.03 %) did not show any AFT contamination. However, 76.97% of samples were contaminated with AFT with the mean of 0.67 ng/g which were lower than MTL of AFT in rice in Iran (30 ng/g) [26] (Table). Maximum level of AFT in rice samples was 4.27ng/g (Figure ). Levels of aflatoxins in rice samples were different between different months. The highest levels of AFB and AFT detected were in rice samples imported in September The lowest levels were detected in rice imported during November 2009 (Figure ). In a similar study from Iran by Mazaheri [28] among 7 rice samples analyzed, AFB was detected in 59 samples (83% of the total). The mean of AFB was.89 ng/g for all samples. Total AF was detected in 83% of samples. Mean of AFT was 2.09 ng/g. AFB levels in two samples (2.8%) were above the maximum tolerable level (MTL) of AFB in Iran (5ng/g). Another survey conducted by Food Standards Agency, UK in 2002 [29] to determine the levels of mycotoxins in rice showed that their levels ranged from 0.2 to.8 mg/kg. All levels found were below the EC ( European Commission) legislative limits of 2 mg/kg aflatoxin B and 4 mg/kg total aflatoxin in cereal products for direct human consumption [30, 3]. Sales and Yoshizawa [32] reported that the incidence of AFB in rice from the Philippines ranged from to.0 mg/kg. In another study the AFB contamination was detected in 37 samples of rice grains from China. They found that 92% of the samples were positive to AFB [33]. Toteja et al. [34] reported the presence of AFB in parboiled rice collected from states in India; 38.5% of the samples were positive to AFB. It has been reported that from 200 samples from India, 67.8% were positive to AFB. The highest level of AFB found in the samples was 38.5 mg/kg [35, 36]. Prasad et al. [37] tested 56 samples of stored rice and found that 2 were positive for aflatoxin. Levels of aflatoxins ranged from 84 to 2830 mg/kg. Jayaraman and Kalyansundaram [38] reported that 35% of the samples of raw rice bran and parboiled rice bran showed the presence of aflatoxin B. It was shown that bran of parboiled rice supports higher aflatoxin production than bran of raw rice. In a study carried by Bandara et al. [39] who reported that in almost all the samples of parboiled rice, AFB and AFG contents were significantly higher than the raw milled rice. Cultivar differences in the amount of 33

4 Am-Euras. J. Toxicol. Sci., 4 (): 3-35, 202 aflatoxin B and G were showed by Sinha and Dubey [40]. 9. Jackson, C.R. and D.K. Bell, 969. Diseases of peanut A survey on the prevalence of aflatoxin B (AFB ) in rice (groundnut) caused by fungi. Georgia Agricultural bran in coastal and interior districts of Tamil Nadu and Experiment Station Bulletin, 56: 37. Andhra Pradesh, India revealed that 62% of the samples 0. Goyal, R.K., 989. Prevention and control of contained AFB and the levels far exceeded the mycotoxins in food grains in India/ permissible limit of 50 mg/kg [4]. docrep/x5036e/x5036e7.htms. Our investigation demonstrated that market rice. Ito, Y., S.W. Peterson, D.T. Wicklow and T. Goto, represents a significant source of exposure to aflatoxin Aspergillus pseudotamarii, a new aflatoxin These data suggest that public health efforts to producing species in Aspergillus section flavi. interrupt aflatoxin exposure must include both an Mycology Res., 05: assessment of aflatoxin contamination and the 2. IARC (International Agency for Research on Cancer), replacement of contaminated rice in accordance with the 993. Monographs on the Evaluation of strict laws on permissible limits of aflatoxin levels in food the Carcinogenic Risk of Chemicals to Human: and feed products. Some Naturally Occurring Substances. Food Items and Constituents, Heterocyclic Aromatic REFERENCES Amines and Mycotoxins (Lyon: IARC), 56: Compton, J.A.F., P.S. Tyler, P.S. Hindmarsh, P. Golob, 3. Hsieh, D., 988. Potential human health hazards of R.A. Boxall and C.P. Haines,993. Reducing losses in mycotoxins. In: Natori, S., K. Hashimoto and Y. Ueno, small farm grain storage in the tropics. Tropical Sci., (Eds.), Mycotoxins and Phytotoxins. Third Joint 33: Food and Agriculture Organization/ W.H.O./United 2. Bhat, R.V. and S. Vasanthi, Food Safety in Nations Environment Program International Food Security and Food Trade: Mycotoxin Food Conference of Mycotoxins. Elsevier, Amsterdam, Safety Risk in Developing Countries. Vision 2020 the Netherlands, pp: for Food, Agriculture and the Environment, Focus 4. Robinson, R.M., A.C. Ray, J.C. Reagor and 0 / http: // / focus/ focus0/ L.A. Holland, 982. Waterfowl mortality caused by focus0-03.pdfs aflatoxicosis in Texas. J. Wildlife Disease, 3. Bryden, W.L., Mycotoxins in the food chain: 8: human health implications. Asia Pacific Journal of 5. Smith, J.E. and M.O. Moss, 985. Mycotoxins: Clinical Nutrition, 6: Formation, Analysis and Significance. Wiley, New 4. CAST (Council for Agricultural Science and York, pp: 48. Technology), Mycotoxins: Risks in Plant, 6. Higgins, K.F., R.M. Barta, R.D. Neiger, Animal and Human Systems, I.A. Ames, Task G.E. Rottinghaus and R.I. Sterry, 992. Mycotoxin Force Report, pp: 39. occurrence in waste field corn and ingesta of wild 5. Dyer, P.S., Sexual reproduction and significance geese in the northern Great Plains. Prairie Natural, of MAT in the Aspergilli. In: Sex in Fungi: Molecular 24: Determination and Evolutionary Implications, Eds., 7. FAO, (Food and Agriculture Organization of the G. Heitman, J.W. Kronstad, J.W. Taylor and United Nations) Worldwide regulations for L.A. Casselton ASM Press, Washington, DC, mycotoxins in food and feed in FAO Food and pp: Make references like this style. Nutrition Paper, No. 8. FAO, Rome. 6. Kurtzman, C.P., B.W. Horn and C.W. Hesseltine, 8. Hussein, S. and J.M. Brasel, 200. Toxicity, 987. Aspergillus nomius, a new aflatoxin-producing metabolism and impact of mycotoxins on humans and species related to Aspergillus flavus and Aspergillus animals. Toxicology, 67: tamarii. Antonie van Leeuwenhoek, 53: INSPQ (Institut National de sante publique 7. Speijers, G.J.A. and M.H.M. Speijers, Que bec). 2002, Les risques a`la sante associe e a` la Combined toxic effects of mycotoxins. Toxicol. Lett., pre sence de moisissures en milieu inte rieur, 53: Que bec. 8. Payne, G.A., 998. Process of contamination by 20. Massey, T.E., R.K. Stewart, J.M. Daniels and L. Ling, aflatoxin-producing fungi and their impact on crops Biochemical and molecular aspects of In: Sinha, K.K.S., Bhatnagar, D. (Eds.), Mycotoxins in mammalian susceptibility to aflatoxin B Agriculture and Food Safety. Marcel Dekker, Inc., carcinogenicity. Proceeding Society Experimental New York, pp: Biology and Medicine, 208:

5 Am-Euras. J. Toxicol. Sci., 4 (): 3-35, Reddy, C.S., K.R.N. Reddy, N. Raja Kumar, G.S. Laha 3. FAO., FAO Food and Nutrition Paper, No. 8. and K. Muralidharan, Exploration of aflatoxin FAO, Rome. Food Standards Agency, Food contamination and its management in rice. J. Survey information sheet no. 2/02. Mycology and Plant Pathol., 34(3): foodstandards.gov.uk/multimedia/ pdfs/2nuts.pdf 22. Van Egmond, H.P. and M.A. Jonker, Sales, A. and T. Yoshizawa, Updated profile of Worldwide Regulations for Mycotoxins in Food and aflatoxin and Aspergillus flavi contamination in rice Feed in Food and Agriculture Organizations of and its byproducts from the Philippines. Food the United Nations, Rome. Additives and Contaminants, 22(5): ISIRI 258 (Institute of Standard and Industrial 33. Liu, Z., J. Gao and J. Yu, Aflatoxins in stored Research of I.R. Iran), 998. Sampling of maize and rice grains in Liaoning Province, China. Agricultural Product for Aflatoxins. National Journal of Stored Products Res., 42: Standard, pp: Toteja, G.S., A. Mukherjee, S. Diwakar, P. Singh, 24. AOAC OFFICIAL METHODS Aflatoxin B B.N. Saxena, K.K. Sinha, A.K. Sinha, N. Kumar, and Total Aflatoxins in peanut Butter, Pistachio K.V. Nagaraja, B. Girija, C.A.Krishna Prasad, S. Paste, Fig Paste and Paprika Powder Immunoaffinity Vanchinathan, R. Rajat and S. Sarkar, Aflatoxin Column Liquid Chromatography with Post-Column B contamination of parboiled rice samples collected Derivatization, Official Methods of Analysis of from different states of India: a multi center study. th AOAC International 7 Edition Volum Ï, Chapter 49 Food Additives and Contaminants, 23(4): Natural Toxins. 35. Reddy, K.R.N. and C.S. Reddy, Detection of 25. ISIRI 6872 (Institute of Standard and Industrial Aspergillus spp. and aflatoxin B in rice in India. Research of I.R. Iran), Foodstuffs, Food Microbiol., 26: Determination of Aflatoxins by HPLC and Clean up 36. Reddy, K.R.N., C.S. Reddy and K. Muralidharan, by IAC.National Standard, pp: Potential of botanicals and biocontrol 26. ISIRI 5925 (Institute of Standard and Industrial agents on growth and aflatoxin production by Research of I.R. Iran), Maximum Tolerated Aspergillus flavus infecting rice grains. Food Limits of Mycotoxins in Foods and Feeds. National Control., 20(2): Standard, pp: Prasad, T., R.K. Sinha and P. Jeswal, 987. Seed 27. Stroka, J., E. Ankle, U. Jorissen and J. Gilbert, mycoflora of cereals and aflatoxin contamination Immunoaffinity column clean up with liquid under storage systems. J. Indian Botanic Society, chromatography using post-column bromination for 66: determination of aflatoxins in peanut butter, pistachio 38. Jayaraman, P. and I. Kalyansundaram, 990. Natural paste, fig paste and paprika powder: collaborative occurrence of toxigenic fungi and mycotoxins in rice study. J. AOAC International, 83: bran. Mycopathologia, 0: Mazaheri, M., Determination of aflatoxins in 39. Bandara, J.M.R.S., E.K. Vithanega and G.A. Bean, imported rice to Iran. Food and Chemical Toxicology, 99. Occurrence of aflatoxins in parboiled rice in Sri 47: Lanka. Mycopathologia, 6: UK Food Standards Agency Survey of retail 40. Sinha, R.K. and A. Dubey, 99. Aflatoxin production rice for a range of mycotoxins. Food Survey in different rice varieties of paddy and wheat by Information Sheet no , Food Standards Aspergillus parasiticus NRRL Indian J. Agency, London, UK. Mycology and Plant Pathol., 2: European Commission, 998. Commission Directive 4. Elangovan, T.V.P., K. Indira and I. Kalyanasundaram, 98/53/EC of 6 July, 998, laying down the sampling 999. Prevalence of aflatoxin B in rice bran and some methods and the methods for analysis for the official associatedfactors.indian Phytopathol., 52: control of the levels for certain contaminants in foodstuffs. Official J. European Community, L20,

ANALYTICAL METHODS IN DETERMINING AFLATOXIN B1 IN FOOD

1 International Journal of Advance Research, IJOAR.org Volume 1, Issue 4, April 2013, ISSN 2320-9186 ANALYTICAL METHODS IN DETERMINING AFLATOXIN B1 IN FOOD MATERIALS Melvin Samuel. S * * School of Biosciences