STUDY OF AFLATOXINS CONTAMINATION IN WHEAT AND MAIZE FROM ALBANIA

Original scientific paper UDC 633.11-248.212.4(496.5) 633.15-248.212.4(496.5) STUDY OF AFLATOXINS CONTAMINATION IN WHEAT AND MAIZE FROM ALBANIA Afërdita Shtëmbari 1, Dritan Topi 2* 1 Department of Industrial Chemistry, Faculty of Natural Sciences, University of Tirana, Blvd. Zogu 1, No 25, Tirana, 1009, Albania 2 Department of Chemistry, Faculty of Natural Sciences, University of Tirana, Blvd. Zogu 1, No 25, Tirana, 1009, Albania *email: dritan.topi@unitir.edu.al Abstract Mycotoxins are toxic secondary metabolites produced by certain fungi that can infect various agricultural commodities in the field and/or during storage. Mycotoxins have various acute and chronic effects on humans and animals depending on species, susceptibility, sex and age. The major classes of mycotoxins are aflatoxins, Fusarium toxins, ochratoxin A, and ergot alkaloids. Aflatoxins, the most distinguished of them are produced by Aspergillus fungi, mainly A. flavus and A. parasiticus. Four major aflatoxins - B1, B2, G1, and G2 are natural contaminants of foods and feeds. They contaminate mainly maize and groundnuts, and are more distributed to warm climate regions, under 40 o geographic latitudes north and south to equator. Acute exposure of consumers to aflatoxins may cause the disease called acute aflatoxicosis, while chronic exposure to AFB1 may cause hepatocellular carcinoma-liver cancer. In this study we have analyzed the aflatoxins contamination in cereals harvested during 2014 in different regions in Albania. Random samples (n = 20): winter wheat (14 samples) and maize (6 samples) were collected according to EU regulation 401/2006. The wheat and maize samples were collected in the Lushnja - Fieri region (n = 14) and Korça region (n = 6), according to their production distribution in the country. The objective of this study was to assess the risk of mycotoxin exposure posed to humans and animals. The samples were analyzed for for AFB1 contamination by High Pressure Liquid Chromatography (HPLC) coupled with Cobra Cell detector. Aflatoxin B1 was detected in 7 samples, and the levels varied from 1.47 to 143.55 μg/kg. The results of the infected samples with aflatoxin B1 were: 1.642 μg/kg to 74.804 μg/kg in maize samples, and 1.467 μg/kg to 143.55 μg/kg in wheat samples. Analytical data give indication that five of the analyzed samples, belonging to, wheat and maize contained aflatoxin B1 more than maximal values established by Commission Regulation 165/2010 amending EU Regulation 1881/2006 [11], AFB1 2mg/kg for wheat and 5 mg/kg maize commodities. The wheat and maize samples collected directly after their harvesting season avoided the post-harvest contamination stage, which indicate that AFB1 origin of contamination to two cereals is of pre-harvest stage. Further investigation in other natural aflatoxins presence will give a clear picture of exposure to population groups. Key words: Mycotoxins, Alfatoxin B1, Wheat, Maize, Albania. 1. Introduction The term mycotoxin for the first time was coined in 1962 in an unusual veterinary event in England, where approximately 100,000 turkey poults died. Immediately was figured out the origin of this mysterious turkey X disease, the contaminated meal peanut with secondary metabolites (aflatoxins) from Aspergillus flavus originating from Brazil. It sensitized scientists to the possibility that other occult mold metabolites might be deadly (Blout, [4]). Mycotoxins are toxic secondary metabolites produced by certain fungi that can infect various agricultural commodities in the field and/or during their storage. The most important ones associated with human and animal diseases, include aflatoxins, Fusarium toxins (fumonisins, trichothecenes and zearalenone), ochratoxin A, citrinin and ergot alkaloids. 44

In many cases, mycotoxins are formed in the field during the growing season; however, they also are formed or increased in levels during harvest, drying, and storage stages. Most important in the process of mycotoxin production is the water availability and air temperatures for growth of the producing fungus. Thus, when the interaction of the plant and the fungus takes place, moisture and temperature greatly affect plant growth and health, with the competitiveness of the mycotoxigenic fungi. During the grain storage, the factors of water activity, substrate aeration and temperature, inoculum concentrations, microbial interactions, mechanical damage, and insect infestation play key role in further mycotoxin contamination (CAST, [6]). Mycotoxins are ubiquitous substances in food and feed commodities that originate from all regions of the world. Despite that during second half of the XX century, the measures implemented as: Good Agriculture Practices, different legislations, Directives and recommendations, against mycotoxin protection in developed countries have ameliorated the situation on consumer health protection, while in developing countries, many individuals are not only food insecure, but also are chronically exposed to high levels of mycotoxins in their diet. Food security exists when all people, at all times, have physical and economic access to sufficient, safe and nutritious food to meet their dietary needs and food preferences for an active and healthy life. Food safety results when microbial contaminants and chemical toxicants are present below tolerance levels in foods (FAO, [12]). Mycotoxins are accompanied with various acute and chronic effects on humans and animals depending on species susceptibility, sex and age. Acute toxicity generally has a rapid onset and an obvious toxic response which may result in fatal cases, while chronic toxicity is characterized by low-dose exposure over a long time period, resulting in cancers and other generally irreversible effects. Almost certainly, the main human and veterinary health burden of mycotoxin exposure is related to chronic exposure (e.g., cancer induction, kidney toxicity, immune suppression). However, the best-known mycotoxin episodes are manifestations of acute effects (e.g., turkey X syndrome, ergotism, stachybotryotoxicosis) (Bennett and Klich [3]). Aflatoxins (AFs) are toxic secondary metabolites produced by species of Aspergillus, especially A. flavus, A. parasiticus and A. nomius. The four major aflatoxins are called B 1, B 2, G 1, and G 2 ( shown in Figure 1) in relation with their light fluorescence under UV light (blue or green) and relative chromatographic mobility during thin-layer chromatography. A. flavus produces aflatoxin B1 and B2, while the two other species produce both aflatoxin B and G. Aflatoxin is associated with both toxicity and carcinogenicity in human and animal populations (Diener et al., [7]). The diseases caused by aflatoxin consumption are loosely called aflatoxicoses. AFs are acute toxic, immunosuppressive, mutagenic, teratogenic and carcinogenic factors. Liver is the most affected organ by carcinogenic and toxic AFs. AFB1 is the most known potential hepatocarcinogenic in mammals and it is classified by the International Agency of Research on Cancer (IARC) as Group 1 carcinogen (IARC [14]). Aflatoxicosis primarily attacks the liver causing necrosis, cirrhosis and carcinomas, as well as attributed to other health effects. Acute symptoms include vomiting, abdominal pain, pulmonary edema, convulsions, coma, and cerebral edema (USDA, [18]). They occur in farm animals, both as a chronic disease characterized by an impairment of resistance and immune responsiveness, which results in a reduction in growth rate and feed efficiency and, as acute poisoning, characterized by severe clinical disease, liver tumors and death (Logrieco et al., [15]). Figure 1. Common natural aflatoxins B1, B2, G1, G2 and M1, chemical formula, and molecular weight Many substrates support growth and aflatoxin production by aflatoxigenic molds. Natural contamination of cereals, figs, oilseeds, nuts, tobacco, and a long list of other commodities is a common occurrence (CAST, [6]). Products can also serve as an indirect source of aflatoxin. When cows consume aflatoxin-contaminated feeds, they metabolically biotransform into rumen the aflatoxin B 1 ( AFB1) into a hydroxylated form called aflatoxin M 1 (Figure 1), (Esmaeilishirazifard, and Keshavarz,, [19]). AFs are difuranocoumarin derivatives produced by a polyketide pathway by fungi and their production depends on factors such as: water stress, high-temperature stress, insect s damage to the host plant, susceptible crop growth stages, poor soil fertility, high crop density, and weed competition (Bruns, [5]). Thus, the extent of aflatoxin contamination varies with geographic location, agricultural and agronomic practices, and the susceptibility of cultivars to fungal invasion during pre-harvest, storage, and/or processing stages. Aflatoxin can enter into the food chain mainly by ingestion through the dietary channel of humans and animals. 45

Aflatoxin contamination affects the economic value of the crops and reduces the efficiency of animal production, resulting in higher costs incurred by all sectors from production to consumption. The intake of AFB1 over a long period of time, even at very low concentration, may be highly dangerous. It has been shown that pre-harvest contamination requires drought climatic conditions of 30-50 days and a mean soil temperature in the interval of 29-31 0 C. Infection and aflatoxin contamination in cereals can be related to the occurrence of soil moisture stress during pod-filling where soil temperature is optimal for A. flavus. This observation can provide the basis for a decision support system (DSS) that can be used by crop scientist to predict infection and contamination in the field in environments where aflatoxin is a serious problem [1]. 2. Materials and Methods 2.1 Materials 2.1.1 Sample characterization Winter wheat and maize was sampled after respective harvesting seasons from three different regions of Albania: Korça (wheat and maize), Lushnja and Fieri. Selection of these regions was based on the statistics for agriculture production in country (INSTAT, [13]). As the distribution of mycotoxins is not uniform throughout the matrix, the sampling procedure was carried out according to Commission Regulation (EC) No 401/2006 (European Commission, [9]) on sample collection. 2.2 Methods 2.2.1 Mycotoxins analysis Apparatus Linear shaker IKA HS 501 digital (IKA Labortechnik, Germany) was used for the extraction. For liquid chromatography, the system Waters Alliance 2690 with the fluorescence detector Waters 474 (Waters, MA, USA) equipped with columns Prodigy 5μ ODS (2) was used for aflatoxin B1 detection. For the post column derivatisation, either Kobra cell (Rhône diagnostics, Scotland) was mounted between chromatographic column and fluorescence detector. Chemicals and reagents Standard solutions of aflatoxin B1 was purchased from Biopure (Tulln, Austria). For sample clean-up, immunoaffinity columns (R-Biopharm Rhône) were used. Reagents purchased at Merck (Darmstadt, Germany) and Supelco (PA, USA) were of analytical or chromatography grade purity. Analytical procedure Aflatoxin B1 was determined following the instruction for use enclosed to immunoaffinity columns and the B1 standard (R-Biopharm Rhône, 2003). Mycotoxins were extracted from samples with appropriate solvents. After sample clean-up, mycotoxins were determined by liquid chromatography. Before the detection, aflatoxin B1 was derivatised with bromine in Kobra cell. The limit of detection (LOD) and the limit of quantification (LOQ) for aflatoxin B1 were 0.4 and 1.0 μg/kg, respectively. The results of were calculated according to sample moisture content of 12%. 3. Results and Discussion Two cereal grains, wheat and maize, are the most important cereal crop in terms of cultivated area, production, and consumption in the world as well in Albania. Owing to the abundant production and the main role of wheat and its flour products in the diet of humans and animals, they can play a very important role in endangering human health in case of contamination with health-threatening factors. In the farm and the warehouse, wheat can be invaded by different microorganisms, especially fungi (Saari et al., [17]). Critical and high AFs risk indexes corresponding to high aflatoxin risks were highlighted in some areas of southern European countries (i.e. in Greece, southern Italy, Bulgaria, Albania) under the climate change scenario that is the most favorable to A. flavus (2 0 C scenario). Although these countries do not belong to the main European maize producers, it has to be considered that the locally produced maize might go directly to local consumers exposing both human and animal populations to a continuous AFs risk. Fungal infections in the field may be an important source of mycotoxin production which may occur and increase in the subsequent supply chain stages. Fungi can be active during the post-harvest period and AFs contamination may increase dramatically if the drying and storage phases are poorly managed. Therefore, the highlighted predicted risks could be used by risk managers to take proper control measures to mitigate AFs risks (EFSA, [8]). In this study we have analyzed the aflatoxins contamination in cereals harvested during 2014 in different regions in Albania. Random samples (n = 21) = winter wheat (14 samples) and maize (7 samples) were collected according to EU regulation 401/2006. The wheat and maize samples collected directly after their harvesting season avoided the post-harvest contamination stage, which indicate that AFB1 origin of contamination to two cereals is of pre-harvest stage. The wheat and maize samples were collected in the Lushnja - Fieri region (n = 14) and Korça region (n = 7), according to their production distribution in the country. The objective of this study was to assess the risk of mycotoxin exposure posed to humans and animals. The samples were analyzed for AFB1 contamination by High Pressure Liquid Chromatography (HPLC) coupled with Cobra Cell detector (LOD = 1 μg/kg). 46

AFB1 was detected in 7 samples where in maize samples vary 1.64 to 74.80 μg/kg while in wheat samples 1.47 to 143.55 μg/kg. Analytical data indicate that in five samples, both wheat and maize, the AFB1 value was above the Maximum Residue Level established by Commission Regulation 165/2010 amending EU Regulation 1881/2006 [11] amending Commission regulation 1881/2006 [10], for wheat (2 μg/kg) and maize (5 μg/kg) commodities intended for food consumption. Table 1. Levels of Aflatoxin B1 in wheat samples in Lushnja and Korçë regions in Albania Code Regions Aflatoxin B1 (µg/kg) 1 Lushnja < LOD 2 Lushnja < LOD 3 Lushnja < LOD 4 Lushnja < LOD 5 Lushnja < LOD 6 Lushnja < LOD 7 Lushnja < LOD 8 Korçë 1.47 9 Korçë < LOD 10 Korçë < LOD 11 Korçë < LOD 12 Korçë < LOD 13 Lushnja 32.01 14 Lushnja 143.55 Mean value / positive samples 59.01 Min. value 1.47 Max. value 143.55 Incidence of positive samples 21.4% (3/14) Above MRL (%) 14.3% (2/14) Table 2. Levels of Aflatoxin B1 in maize samples in Lushnja, Fieri and Korçë regions in Albania Code Regions Aflatoxin B1 (µg/kg) 1 Fieri 50.33 2 Fieri < LOD 3 Fieri 74.80 4 Korçë < LOD 5 Korçë < LOD 6 Lushnja 1.64 7 Lushnja 3.17 Mean value / positive samples 32.49 Min. value 1.64 Max. value 74.80 Incidence of positive samples 57.1% (4/7) Above MRL (%) 28.6% (2/7) All European countries, those in the southern part included, normally are not characterized by warm and dry weather during spring season where winter crops are grown and harvesting in early Summer. In certain areas and years it happens, and Aspergillus section Flavi can found suitable conditions for their growth and AFs production. Surveys carried out to quantify AFs contamination in cereals in Europe showed that maize grain was the most investigated commodity followed by wheat. In particular, in Italy 3,607 maize samples have been analyzed between 1982 and 2007. During these years, the maximum level (233 μg/kg) was found both in 2004 and in 2006 with mean values of 21 and 14 μg/kg, respectively (Battilani et al., [2]). A high AFB1 contamination (maximum level 155 μg/kg) was recorded in 2003 (Piva et al., [16]), a year characterized by very high temperatures during the whole growing season of maize and an extraordinary drought from early May to September. In particular, EU countries with the more representative maize contamination are Italy and Romania. Among EU countries, the maximum level (233 μg/kg) was found in Italy, even if the highest maize contamination reported was in Turkey (431.9 μg/kg). Aflatoxins presence in wheat is not a main concern in most of the countries, but it is of interests in Romania (EFSA, [8]). 4. Conclusions - After analysis performed by HPLC, some of the wheat and maize samples resulted being contaminated with aflatoxin B1. It is noticed that the incidence of positive samples in maize samples were bigger than in wheat samples. - It is worth noting that in both samples, wheat and maize, we found samples containing higher AFB1 values than maximum limits as defined by national and European legislations. From this experimental work, we conclude that the risk of the pre-harvest contamination of aflatoxin in cereals, in Albania, is high, especially for the maize produced during 2014. - Considering all the problems that causing contaminated cereals with mycotoxins (especially AFB1) in human and animal health, it is recommended to make systematic analyses, because cereals are the basic food of population in Albania. Being the basic food of the population, increases the possibility of exceeding the daily limits that everybody (human and animals) should consume. 5. References [1] Bankole, S. A. and Mabekoje O. O. (2004). Occurrence of aflatoxins and fumonisins in pre-harvest maize from south western Nigeria. Food Additives and Contaminants, 21, pp. 251-255. 47

[2] Battilani P., Barbano C., and Piva G. (2008). Aflatoxin B1 contamination in maize related to the aridity index in North Italy. World Mycotoxin Journal, 1, pp. 449-456. [3] Bennett J. W., and Klich M. (2003). Mycotoxins. Clinical Microbiology Reviews, pp. 497-516. [4] Blout W. P. (1961). Turkey X disease. Turkeys, 9, pp. 52, 55-58, 61, 77. [5] Bruns H. A. (2003). Controlling aflatoxins and fumonisins in maize by crop management. Journal of Toxicology: Toxin Reviews, 22, pp. 153-173. [6] CAST, (2003). Mycotoxins: Risks in Plant, Animal and Hu man Systems, Report No. 139, Council for Agricultural Science and Technology, Ames, Iowa, USA. pp. 217. [7] Diener U. L., Cole R. J., Sanders T. H., Payne G. A., Lee, L. S., and Klich M. A. (1987). Epidemiology of aflatoxin formation by Aspergillus flavus. Annual. Reviews in Phytopathology, 25, pp. 249-270. [8] EFSA. (2012). Modelling, predicting and mapping the emergence of aflatoxins in cereals in the EU due to climate change. Scientific report submitted to EFSA, pp. 1-72. [9] European Commission. (2006). Commission Regulation (EC) No 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union, L 70, pp. 12-34. [10] Commission Regulation. (2006). Regulation No 1881/2006 setting maximum levels for certain contaminants in foodstuffs. Official Journal of European Union, L 364, pp. 5-24. [11] European Commission. (2010). Commission Regulation (EU) No 165/2010 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards aflatoxins. Official Journal of European Union, L 50/8, pp. 1-5. [12] FAO. (1996). Rome Declaration on World Food Security and World Food Summit Plan of Action. FAO, Rome, Italy. [13] INSTAT. (2014). Statistical Annual of Ministry of Agriculture. Agriculture-Structure of area in field crops in Ha, Table 1, Sheet 38. <URL: http://www.instat.gov.al/al/themes/ agriculture,-forestry-and-fishery.aspx?tab=tabs-4. Accessed May 19, 2015. [14] International Agency for Research on Cancer. (1993). Aflatoxins. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Scientific Publication, No. 56, IARC. Lyon, France. [15] Logrieco A., Bottalico A., Mule G., Moretti A., and Perrone G, (2003). Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops. European Journal of Plant Pathology, 109, pp. 645-667. [16] Piva G., Battilani P., and Pietri A. (2006). Emerging issues in southern Europe: Aflatoxins in Italy. In: The mycotoxin factbook: Food & feed topics, Barug D., Bhatnagar D., Egmond P. H., Kamp W. J., Osenbruggen A. W., Visconti A. (Eds.), Wageningen Academic Publishers, The Netherlands, pp. 139-153. [17] Saari E. E., Young, H. C., Kernkamp M. F. (1968). Infection of North American Thalictrum spp. with Pucciniareconditaf sp. tritici. Phytopathology, 58, pp. 939-943. [18] USDA. (2006). Grain fungal diseases and Mycotoxin reference. United States Department of Agriculture, 36, pp. 36. [19] Esmaeilishirazifard E. & Keshavarz T. (2014). Aflatoxin Occurrence. In: Aflatoxins- Food Sources, Occurrence and Toxicological Effects. Ed. A. G. Faulkner, Nova Publisher, New York, USA, pp. 35-63. 48