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Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxins and heavy metals in animal feed in Iran a
a
M.H. Eskandari & S. Pakfetrat a
Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran Accepted author version posted online: 02 Jan 2014.Published online: 21 Feb 2014.
To cite this article: M.H. Eskandari & S. Pakfetrat (2014) Aflatoxins and heavy metals in animal feed in Iran, Food Additives & Contaminants: Part B: Surveillance, 7:3, 202-207, DOI: 10.1080/19393210.2013.876675 To link to this article: http://dx.doi.org/10.1080/19393210.2013.876675
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 3, 202–207, http://dx.doi.org/10.1080/19393210.2013.876675
Aflatoxins and heavy metals in animal feed in Iran M.H. Eskandari* and S. Pakfetrat Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran
Downloaded by [Monash University Library] at 09:37 06 December 2014
(Received 14 September 2013; accepted 15 December 2013) The occurrence of aflatoxin (aflatoxin B1, aflatoxin B2, aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2)) and heavy metal (Pb, Cd, As and Hg) contamination was determined in 40 industrially produced animal feed samples which were collected from the southwest of Iran. The results indicated that 75% of samples were contaminated by four aflatoxins and the level of AFB1 and sum of aflatoxins were higher than the permissible maximum levels in Iran (5 and 20 µg kg−1, respectively) in all feed samples. A positive correlation was found between four types of aflatoxins in all the tested samples (p < 0.01) and the positive correlation between AFG1 and AFG2 was significant (r2 = 0.708). All feed samples had lead concentrations lower than the maximum EU limit, while 5%, 17% and 42.5% of feed samples had As, Cd and Hg concentrations higher than the maximum limits, respectively. Keywords: feed; aflatoxins; heavy metals; Iran
Introduction When considering human health, the role of animal feedstuff in the production of safe and healthy food is of paramount importance and the “farm-to-fork” approach needs the assessment and control of major components of the food production chain (Leeman et al. 2007). Toxic substances such as mycotoxins, heavy metals, polycyclic aromatic hydrocarbons and dioxins are present in the environment and may enter into the livestock feed. Depending on their physicochemical properties, some toxic substances (such as veterinary drugs and feed additives) are metabolised into generally harmless constituents, whereas other feed contaminants such as heavy metals and dioxins remain in animal tissues and products (Kan & Meijer 2007). Aflatoxins and heavy metals are potential environmental contaminants with the capacity of causing serious human health problems and tracing of these hazardous materials in animal feedstuffs is significant (Alkhalaf et al. 2010). Aflatoxins are a family of mycotoxins produced in agricultural products mainly by Aspergillus flavus and Aspergillus parasiticus. The major aflatoxins are found in animal feedstuffs, including aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). AFB1 is by far the most prevalent and potent natural carcinogen amongst these aflatoxins (Rai & Varma 2010). FAO has estimated that up to 25% of the world’s food crops are significantly contaminated with mycotoxins, leading to food unsafety and economic losses (Kabak et al. 2006). Human exposure to aflatoxins can result from direct consumption of contaminated commodities or from the consumption of *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
foods from animals previously exposed to aflatoxins in feedstuffs (Park & Liang 1993). Many surveys showed the occurrence of mycotoxins in different feedstuffs of plant origin in Iran (Yazdanpanah et al. 2001; Yazdanpanah 2010; Feizy et al. 2012; Karami-Osboo et al. 2012), but there is still a lack of data which show aflatoxin contamination and their co-occurrence in animal feeds in Iran (Beheshti & Asadi 2013). Soil contamination by heavy metals is important around the world, because metals are difficult to remove from the soil and are very toxic to animals (Dong et al. 2001). Heavy metal contamination may occur due to irrigation with polluted water, application of fertilisers and metal-based pesticides, industrial activities and postharvest contamination (Alkhalaf et al. 2010). Contaminated plant materials may be ingested by animals grazing the pastures or consuming conserved forage and heavy metals may subsequently pass to the human food via milk, meat or other products of animal origin (Hinton 2000). An important feature of heavy metals is that the chemical form in which they are present may change during passage through the intestine or storage in animal tissues, but they are not metabolised (Kan & Meijer 2007). Many reports indicate the presence of heavy metals in animal products (Cang et al. 2004; Vidovic et al. 2005; Tajkarimi et al. 2008; Javed et al. 2009), but heavy metal content of animal feedstuffs is poorly documented in Iran. Therefore, the aims of this study were to determine the aflatoxins occurrence and heavy metals contamination in 40 industrially produced animal feed samples which were collected from southwest Iran.
Food Additives & Contaminants: Part B Materials and methods
Downloaded by [Monash University Library] at 09:37 06 December 2014
In total, 40 samples (each sample weighing approximately 1 kg or more) including dairy cattle and poultry feeds were collected between January and June 2012 from different animal feed factories in southwest Iran. Feed samples were taken according to the methods of sampling and analysis for the official control of feed of the European Union (2009). The whole sample was finely ground in a laboratory mill (Retsch GmbH, Model SK1, Haan, Germany) to pass through a 1-mm sieve. The sieved sample was mixed and collected in a suitable clean, dry container fitted with an airtight stopper. The sample was mixed again, immediately before weighing out the amount for analysis.
Aflatoxins analysis Materials and reagents Aflatoxin standards (AFB1, AFB2, AFG1 and AFG2) were supplied by Sigma-Aldrich (St Louis, MO, USA). Working standard solutions were prepared by diluting the stock standard solutions with acetonitrile-methanole (50/50, v/v). All solvents of the mobile phase were HPLC grade and purchased from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Plus apparatus from Millipore (Milford, MA, USA). Phosphate-buffered saline (PBS) was prepared by dissolving Sigma-Aldrich PBS tablets in distilled water. The immunoaffinity columns for aflatoxins (NeoColumnTM Test systems) were purchased from Neogen (Neogen Europe Ltd, Scotland, UK).
Extraction and Immunoaffinity clean-up The animal feed samples were extracted and cleaned-up according to AOAC official method 999.07 (AOAC 2000). Fifty grams of feed samples were extracted with 5 g NaCl and 300 ml methanol-water (8/2, v/v) using a blender at high speed for 3 min. The extract was filtered through Whatman No. 4 filter paper. Then, 10 ml of clear filtrate was diluted with 60 ml PBS, passed through the immunoaffinity column. The column was washed twice with 10 ml ultrapure water and dried with air. Finally aflatoxins were eluted with methanol (0.5 + 0.75 ml) that was applied on the column. The eluate was diluted with 1.75 ml ultrapure water, after which 100 µl was injected to HPLC.
HPLC-FD analysis HPLC analysis was performed with an Agilent Technologies 1200 series consisted of a quaternary pump (Agilent, G1311A), a degasser (Agilent, G1322A) and column oven (Agilent thermostative column compartment, G1316A, Agilent Technologies, Palo Alto, CA, USA), coupled to an Agilent fluorescence detector at 360 nm (excitation) and
203
455 nm (emission). Chromatographic separations were performed using a reversed phase symmetry C18 column (250 × 4.6 mm i.d. and 3.5 mm particle size) supplied by GL Sciences Inc. (Tokyo, Japan). The isocratic LC mobile phase was water/acetonitrile/methanol (60:20:30, v/v/v) containing 0.12 gl−1 potassium bromide and 350 µl l−1 nitric acid (4 M), pumped at a flow rate of 1.0 ml min−1. Post-column derivatisation was carried out electrochemically generated bromine in Kobra cell (Coring System Diagnostics GmbH, Gernsheim, Germany) using a reaction tube of 340 × 0.5 mm i.d. PTFE to enhance the fluorescence intensity of AFB1 and AFG1. Analytical quality parameters Each working day, the linearity was estimated by constructing six-point calibration curves over the concentration ranges of 0.5–20 ng ml−1 for AFB1 and AFB2, 0.1–6 ng ml−1 for AFG1 and AFG2. Calibration curves were derived by plotting concentrations as a function of peak area of each aflatoxin. For recovery experiments, noninfected feed samples were spiked in six replicates at three concentration levels (1, 2 and 5 µg kg−1 for AFB1 and AFG1, 0.2, 0.5 and 2 µg kg−1 for AFB2 and AFG2. Both spiked and unspiked materials were analysed by the same operator according to the method protocol previously described. The recovery rates of AFB1, AFB2, AFG1 and AFG2 were 97%, 103%, 101% and 98% respectively. Aflatoxins levels were corrected for recoveries. Limits of detection (LODs) for aflatoxins peaks were defined as three times baseline signal and were 0.05 µg kg−1 for AFB1 and AFG1, 0.03 µg kg−1 for AFG2 and 0.015 µg kg−1 for AFB2. In order to show that the results are reliable the validation of this method based on the LOD, limit of quantification, linearity and precision were evaluated in terms of intraday between day and interday results as relative standard deviation (RSD)%. The values of RSD ranged from 1.7% to 9.4% for intraday precision tests (n = 6) and 2.3% to 10.8% for interday precision tests (n = 8). Statistical analysis Correlation between aflatoxin contents were calculated using SPSS (Version 10.0.6, SPSS Inc, Chicago, IL, USA). Differences were considered highly significant at the p < 0.01 level. Heavy metal determination Animal feed samples were analysed for lead, cadmium, arsenic and mercury contents using atomic absorption spectroscopy (method 968.08, AOAC 2000). Five grams of each feed sample was placed in well-glazed porcelain. Feed samples were ashed in furnace for 4 h at 550°C until a white or grey ash residue was obtained. The residue was dissolved in 10 ml of HNO3 10% (v/v) and covered
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Table 1. Aflatoxins in 40 feed samples in southwest of Iran. Parameters
AFB1
AFB2
AFG1
AFG2
Sum
Percentage of positive samplesa (%) Percentage of samples above Iranian standard limitations (%) Range (µg kg−1) Mean value (µg kg−1) Mean of positive samplesb (µg kg−1)
100 100 15–35 26.4 26.4
100 0 5–25 15.4 15.4
95 0
Food Additives & Contaminants: Part B: Surveillance Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tfab20
Aflatoxins and heavy metals in animal feed in Iran a
a
M.H. Eskandari & S. Pakfetrat a
Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran Accepted author version posted online: 02 Jan 2014.Published online: 21 Feb 2014.
To cite this article: M.H. Eskandari & S. Pakfetrat (2014) Aflatoxins and heavy metals in animal feed in Iran, Food Additives & Contaminants: Part B: Surveillance, 7:3, 202-207, DOI: 10.1080/19393210.2013.876675 To link to this article: http://dx.doi.org/10.1080/19393210.2013.876675
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Food Additives & Contaminants: Part B, 2014 Vol. 7, No. 3, 202–207, http://dx.doi.org/10.1080/19393210.2013.876675
Aflatoxins and heavy metals in animal feed in Iran M.H. Eskandari* and S. Pakfetrat Department of Food Science and Technology, College of Agriculture, Shiraz University, Shiraz, Iran
Downloaded by [Monash University Library] at 09:37 06 December 2014
(Received 14 September 2013; accepted 15 December 2013) The occurrence of aflatoxin (aflatoxin B1, aflatoxin B2, aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2)) and heavy metal (Pb, Cd, As and Hg) contamination was determined in 40 industrially produced animal feed samples which were collected from the southwest of Iran. The results indicated that 75% of samples were contaminated by four aflatoxins and the level of AFB1 and sum of aflatoxins were higher than the permissible maximum levels in Iran (5 and 20 µg kg−1, respectively) in all feed samples. A positive correlation was found between four types of aflatoxins in all the tested samples (p < 0.01) and the positive correlation between AFG1 and AFG2 was significant (r2 = 0.708). All feed samples had lead concentrations lower than the maximum EU limit, while 5%, 17% and 42.5% of feed samples had As, Cd and Hg concentrations higher than the maximum limits, respectively. Keywords: feed; aflatoxins; heavy metals; Iran
Introduction When considering human health, the role of animal feedstuff in the production of safe and healthy food is of paramount importance and the “farm-to-fork” approach needs the assessment and control of major components of the food production chain (Leeman et al. 2007). Toxic substances such as mycotoxins, heavy metals, polycyclic aromatic hydrocarbons and dioxins are present in the environment and may enter into the livestock feed. Depending on their physicochemical properties, some toxic substances (such as veterinary drugs and feed additives) are metabolised into generally harmless constituents, whereas other feed contaminants such as heavy metals and dioxins remain in animal tissues and products (Kan & Meijer 2007). Aflatoxins and heavy metals are potential environmental contaminants with the capacity of causing serious human health problems and tracing of these hazardous materials in animal feedstuffs is significant (Alkhalaf et al. 2010). Aflatoxins are a family of mycotoxins produced in agricultural products mainly by Aspergillus flavus and Aspergillus parasiticus. The major aflatoxins are found in animal feedstuffs, including aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2). AFB1 is by far the most prevalent and potent natural carcinogen amongst these aflatoxins (Rai & Varma 2010). FAO has estimated that up to 25% of the world’s food crops are significantly contaminated with mycotoxins, leading to food unsafety and economic losses (Kabak et al. 2006). Human exposure to aflatoxins can result from direct consumption of contaminated commodities or from the consumption of *Corresponding author. Email: [email protected] © 2014 Taylor & Francis
foods from animals previously exposed to aflatoxins in feedstuffs (Park & Liang 1993). Many surveys showed the occurrence of mycotoxins in different feedstuffs of plant origin in Iran (Yazdanpanah et al. 2001; Yazdanpanah 2010; Feizy et al. 2012; Karami-Osboo et al. 2012), but there is still a lack of data which show aflatoxin contamination and their co-occurrence in animal feeds in Iran (Beheshti & Asadi 2013). Soil contamination by heavy metals is important around the world, because metals are difficult to remove from the soil and are very toxic to animals (Dong et al. 2001). Heavy metal contamination may occur due to irrigation with polluted water, application of fertilisers and metal-based pesticides, industrial activities and postharvest contamination (Alkhalaf et al. 2010). Contaminated plant materials may be ingested by animals grazing the pastures or consuming conserved forage and heavy metals may subsequently pass to the human food via milk, meat or other products of animal origin (Hinton 2000). An important feature of heavy metals is that the chemical form in which they are present may change during passage through the intestine or storage in animal tissues, but they are not metabolised (Kan & Meijer 2007). Many reports indicate the presence of heavy metals in animal products (Cang et al. 2004; Vidovic et al. 2005; Tajkarimi et al. 2008; Javed et al. 2009), but heavy metal content of animal feedstuffs is poorly documented in Iran. Therefore, the aims of this study were to determine the aflatoxins occurrence and heavy metals contamination in 40 industrially produced animal feed samples which were collected from southwest Iran.
Food Additives & Contaminants: Part B Materials and methods
Downloaded by [Monash University Library] at 09:37 06 December 2014
In total, 40 samples (each sample weighing approximately 1 kg or more) including dairy cattle and poultry feeds were collected between January and June 2012 from different animal feed factories in southwest Iran. Feed samples were taken according to the methods of sampling and analysis for the official control of feed of the European Union (2009). The whole sample was finely ground in a laboratory mill (Retsch GmbH, Model SK1, Haan, Germany) to pass through a 1-mm sieve. The sieved sample was mixed and collected in a suitable clean, dry container fitted with an airtight stopper. The sample was mixed again, immediately before weighing out the amount for analysis.
Aflatoxins analysis Materials and reagents Aflatoxin standards (AFB1, AFB2, AFG1 and AFG2) were supplied by Sigma-Aldrich (St Louis, MO, USA). Working standard solutions were prepared by diluting the stock standard solutions with acetonitrile-methanole (50/50, v/v). All solvents of the mobile phase were HPLC grade and purchased from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Plus apparatus from Millipore (Milford, MA, USA). Phosphate-buffered saline (PBS) was prepared by dissolving Sigma-Aldrich PBS tablets in distilled water. The immunoaffinity columns for aflatoxins (NeoColumnTM Test systems) were purchased from Neogen (Neogen Europe Ltd, Scotland, UK).
Extraction and Immunoaffinity clean-up The animal feed samples were extracted and cleaned-up according to AOAC official method 999.07 (AOAC 2000). Fifty grams of feed samples were extracted with 5 g NaCl and 300 ml methanol-water (8/2, v/v) using a blender at high speed for 3 min. The extract was filtered through Whatman No. 4 filter paper. Then, 10 ml of clear filtrate was diluted with 60 ml PBS, passed through the immunoaffinity column. The column was washed twice with 10 ml ultrapure water and dried with air. Finally aflatoxins were eluted with methanol (0.5 + 0.75 ml) that was applied on the column. The eluate was diluted with 1.75 ml ultrapure water, after which 100 µl was injected to HPLC.
HPLC-FD analysis HPLC analysis was performed with an Agilent Technologies 1200 series consisted of a quaternary pump (Agilent, G1311A), a degasser (Agilent, G1322A) and column oven (Agilent thermostative column compartment, G1316A, Agilent Technologies, Palo Alto, CA, USA), coupled to an Agilent fluorescence detector at 360 nm (excitation) and
203
455 nm (emission). Chromatographic separations were performed using a reversed phase symmetry C18 column (250 × 4.6 mm i.d. and 3.5 mm particle size) supplied by GL Sciences Inc. (Tokyo, Japan). The isocratic LC mobile phase was water/acetonitrile/methanol (60:20:30, v/v/v) containing 0.12 gl−1 potassium bromide and 350 µl l−1 nitric acid (4 M), pumped at a flow rate of 1.0 ml min−1. Post-column derivatisation was carried out electrochemically generated bromine in Kobra cell (Coring System Diagnostics GmbH, Gernsheim, Germany) using a reaction tube of 340 × 0.5 mm i.d. PTFE to enhance the fluorescence intensity of AFB1 and AFG1. Analytical quality parameters Each working day, the linearity was estimated by constructing six-point calibration curves over the concentration ranges of 0.5–20 ng ml−1 for AFB1 and AFB2, 0.1–6 ng ml−1 for AFG1 and AFG2. Calibration curves were derived by plotting concentrations as a function of peak area of each aflatoxin. For recovery experiments, noninfected feed samples were spiked in six replicates at three concentration levels (1, 2 and 5 µg kg−1 for AFB1 and AFG1, 0.2, 0.5 and 2 µg kg−1 for AFB2 and AFG2. Both spiked and unspiked materials were analysed by the same operator according to the method protocol previously described. The recovery rates of AFB1, AFB2, AFG1 and AFG2 were 97%, 103%, 101% and 98% respectively. Aflatoxins levels were corrected for recoveries. Limits of detection (LODs) for aflatoxins peaks were defined as three times baseline signal and were 0.05 µg kg−1 for AFB1 and AFG1, 0.03 µg kg−1 for AFG2 and 0.015 µg kg−1 for AFB2. In order to show that the results are reliable the validation of this method based on the LOD, limit of quantification, linearity and precision were evaluated in terms of intraday between day and interday results as relative standard deviation (RSD)%. The values of RSD ranged from 1.7% to 9.4% for intraday precision tests (n = 6) and 2.3% to 10.8% for interday precision tests (n = 8). Statistical analysis Correlation between aflatoxin contents were calculated using SPSS (Version 10.0.6, SPSS Inc, Chicago, IL, USA). Differences were considered highly significant at the p < 0.01 level. Heavy metal determination Animal feed samples were analysed for lead, cadmium, arsenic and mercury contents using atomic absorption spectroscopy (method 968.08, AOAC 2000). Five grams of each feed sample was placed in well-glazed porcelain. Feed samples were ashed in furnace for 4 h at 550°C until a white or grey ash residue was obtained. The residue was dissolved in 10 ml of HNO3 10% (v/v) and covered
204
M.H. Eskandari and S. Pakfetrat
Table 1. Aflatoxins in 40 feed samples in southwest of Iran. Parameters
AFB1
AFB2
AFG1
AFG2
Sum
Percentage of positive samplesa (%) Percentage of samples above Iranian standard limitations (%) Range (µg kg−1) Mean value (µg kg−1) Mean of positive samplesb (µg kg−1)
100 100 15–35 26.4 26.4
100 0 5–25 15.4 15.4
95 0
