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Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels
Food and Chemical Toxicology 48 (2010) 3304–3307
Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels Zahid Hussain a, Muhammad Zargham Khan a,⇑, Ahrar Khan a, Ijaz Javed b, Muhammad Kashif Saleemi a, Sultan Mahmood c, Muhammad Rafique Asi d a
Department of Pathology, University of Agriculture, Faisalabad 38040, Pakistan Department of Physiology and Pharmacology, University of Agriculture, Faisalabad 38040, Pakistan c Department of Poultry Science, University of Agriculture, Faisalabad 38040, Pakistan d Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan b
a r t i c l e
i n f o
Article history: Received 13 May 2010 Accepted 15 August 2010
Keywords: Aflatoxin Aflatoxin B1 residues Liver Muscles Broiler Aflatoxicosis
a b s t r a c t This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 lg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 lg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Mycotoxins, the secondary metabolites of toxigenic fungi, are unavoidable contaminants in foods and feeds exerting harmful effects upon animal and human health (Zahoor-ul-Hassan et al., 2010). The most important mycotoxins in naturally tainted foods and feeds are aflatoxins, ochratoxins, zearalenone, T-2 toxin, deoxynivalenol and fumonisins (Devegowda et al., 1998; Sultana and Hanif, 2009). Aflatoxin (AF) produced by the toxigenic fungi mainly Aspergillus flavus and Aspergillus parasiticus constitute one of the major health hazard groups of naturally occurring toxicants both for human and animals. Among the four major groups of aflatoxin namely B1, B2, G1 and G2, aflatoxin B1 (AFB1) is the most toxic and a known carcinogenic. Acute or chronic aflatoxicosis in poultry birds results in decreased meat/egg production, immunosuppressant, and hepatotoxicosis (Arafa et al., 1981; Verma et al., 2004; Khan et al., 2010). AF residues may also appear in body tissues and samples collected from the commercial poultry birds showed detectable levels of AF in liver (Asim et al., 1990; Bintvihok and Davitiyananda, 2002). Poultry birds fed AF contaminated rations under experimental conditions resulted in the presence of AF residues in their edible tissues like liver and muscles. Residues of AFB1 in liver of broiler and layer birds have been reported to
⇑ Corresponding author. Tel.: +92 41 9200161x3118; fax: +92 41 9201094. E-mail address: [email protected] (M.Z. Khan). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.08.016
vary from no detection to 3.0 ng/g by feeding 250–3310 ng/g AFB1 for variable periods (Gregory et al.,1983; Chen et al., 1984; Wolzak et al., 1986; Bintvihok et al., 2002). The wide variations in the tissue AF residue concentration suggested that these levels might be influenced by different factors including dietary AF levels, duration of administration, age, type of the birds etc. However, effect of such factors upon concentration of AFB1 residues in poultry meat (liver and muscles) and clearance of AFB1 from the body tissues after withdrawal of dietary AF have not been adequately studied. Keeping in view the above mentioned concerns, this experimental study was designed to induce aflatoxicosis in broiler chicks in attempt to establish a relationship of tissue AFB1 concentration with age, and dietary AFB1 levels. 2. Materials and methods Tailoring and execution of the experiments followed national and institutional legislations regarding animal protection and welfare of laboratory animals. Moreover, the Directorate of Advanced Studies and Research Board, University of Agriculture, Faisalabad, Pakistan approved synopsis of the project.
2.1. Aflatoxin and feeds AF was produced in rice by inoculating with A. flavus (CECT 2687, Culture Collection Center University De Valencia Spain) following Shotwell et al. (1966). AFB1 concentration in the fermented rice was determined by high pressure liquid chromatography method.
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Z. Hussain et al. / Food and Chemical Toxicology 48 (2010) 3304–3307 Basal feed (22% total protein and 3000 kcal/kg metabolizable energy) was prepared without addition of toxin binder, vitamins, minerals and antibiotics. Prior to use, each batch of the basal feed was analyzed for aflatoxin, ochratoxin and zearalenone to ensure their levels below 1 lg/kg. AF contaminated feeds were prepared by incorporation of known quantities of AFB1. For this purpose fermented rice was extracted by soaking it into threefold quantity of chloroform (100:300) for overnight and filtered through cotton cloth. All the chloroform was evaporated and the concentrated residues were re-suspended into Polyethylene glycol (PEG). This suspension was evenly mixed in the required quantity of basal feed to prepare the experimental feeds containing AFB1 in concentrations of 1600, 3200 and 6400 lg/kg. 2.2. Experimental birds Two thousand, one day-old commercial broiler chicks (Hubbard Classic) were maintained on basal diet under standard conditions of brooding and management. Experimental groups were prepared from these birds by random selection as and when required. 2.3. Experimental plans Experiments 1, 2 and 3 were conducted on birds of 7, 14 and 28 days of age. In each experiment four groups of 80 broiler chicks were maintained and kept on feeds containing 0, 1600, 3200 and 6400 lg/kg AFB1 for 7 days. Afterwards the birds were switched back to basal feed till the termination of experiment. Duration of experiment 1, 2 and 3 was 36, 29 and 15 days, respectively so that age of depletion of birds in all experiments was 42/43 days. In each experiment six birds were killed from each group at days 1, 2, 3, 5, 7, 10 and 15 of the experiment. All remaining birds were killed at the end of each experiment.
Table 1 Aflatoxin B1 residues (ng/g) in liver and muscles of broiler chicks given different levels of dietary aflatoxins for 7 days from 7 days of age (Mean ± SD). Organ/Days of experiment
Groups (aflatoxin B1 in feed lg/kg) A (0)
B (1600)
C (3200)
D (6400)
Liver 1 2 3 5 7 10 15 35
ND* ND ND ND ND ND ND ND
ND ND 0.83 ± 0.01c 1.74 ± 0.07c 3.51 ± 0.08c 2.49 ± 0.04c 1.24 ± 0.03a ND
ND ND 2.45 + 0.08b 2.71 + 0.04b 3.74 + 0.09b 3.31 ± 0.07b 0.95 ± 0.08b ND
ND 0.89 ± 0.06a 5.23 ± 0.07a 5.75 ± 0.44a 6.97 ± 0.08a 3.57 ± 0.04a 1.13 ± 0.04a ND
Muscles 1 2 3 5 7 10 15 35
ND ND ND ND ND ND ND ND
ND ND ND 0.22 ± 0.01b 1.63 ± 0.04c 0.74 ± 0.01b ND ND
ND ND ND 0.24 ± 0.01b 1.90 ± 0.05b 0.93 ± 0.01a ND ND
ND ND 2.17 ± 0.04a 3.19 ± 0.11a 3.27 ± 0.05a 0.95 ± 0.12a ND ND
Values in each row followed by different letters are significantly different (p 6 0.05). * ND = Not detected.
2.4. Parameters studied 2.4.1. Estimation of aflatoxins in liver and muscle tissue Samples of muscles and livers of six birds at each killing in each group were randomly pooled to make three samples of each tissue and stored at 20 °C. The aflatoxins in the tissues were extracted using immune-affinity columns (Aflatest ÒWB, Vicam USA) and estimated by a HPLC-fluorescent detector method with pre-column derivatization. A modification of the methods of Chiavaro et al. (2005) and AOAC (990.33, 2000) was used as briefly mentioned below. Twenty-five grams of defrosted sample was homogenized and blended with 5 g of NaCl in 100 ml of methanol–water (80:20) for 3 min. After filtration through a paper filter, an aliquot of 10 ml of filtrate was diluted with 40 ml PBS Wash Buffer containing 0.1% Tween-20 Wash Buffer and passed through an immunoaffinity column. AFB1 was eluted with 1.0 ml of methanol in a glass vial and dried near to dryness under gentle stream of nitrogen. Pre-column derivatization was performed with trifluroacetic acid (AOAC 990.33, 2000) and a 20 ll of the derivitized extract was injected into HPLC system (Shimadzu LC-10 AS) equipped with reverse phase column (Supelco SIL LC-18, 15 cm 4.6 cm ID) and fluorescent detector (Shimadzu RF-530) at 360 nm for excitation and 440 nm inhibition wave lengths. Mobile phase consisted of water, acetonitril and methanol (60:20:20). Limit of detection (LOD) was 0.025 ng/ml and the recovery rate 89%. 2.5. Statistical analysis The data obtained in all the experiment were subjected to statistical analysis by analysis of variance. The means of different groups were compared by Duncan’s Multiple Range Test using MSTATC statistical software. The level of significance was p 6 0.05.
3. Results 3.1. AFB1 residues 3.1.1. Liver In birds fed AF contaminated feed from 7 days of age, AFB1 was detected on day 2, 3 and 3 in livers of 1600, 3200 and 6400 lg/kg AFB1 groups, respectively (Table 1). The AFB1 increased with increase in dietary levels and were highest on last day (day 7) of AF feeding being 3.51 ± 0.08, 3.74 ± 0.09 and 6.97 ± 0.08 ng/g, in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. Following withdrawal of AF from feed it decreased gradually and was below 2 ng/g on day 15 of experiment and undetectable on day 35 (42 days of age). In birds kept on AF contaminated feed from 14 days of age, AFB1 was detected on day 3 in livers of 1600, 3200 and 6400 lg/kg AFB1
groups, respectively (Table 2). These AFB1 levels on last day of AF feeding (day 7) were 3.65 ± 0.09, 3.26 ± 0.23 and 6.59 ± 0.23 ng/g, respectively in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. Following withdrawal of dietary AF, these residues decreased and were not detectable on day 10 of the experiment in1600 and 3200 lg/kg AFB1groups. In 6400 lg/kg AFB1group, AFB1 was not detected on day 15 of the experiment. In birds offered AF contaminated feed from 28 days of age, AFB1 residues were detected on day 5, 3 and 3 in liver of 1600, 3200 and 6400 lg/kg AFB1groups, respectively (Table 3). The AFB1 levels on last day of AF feeding (day 7) were at their height being 2.87 ± 0.13, 3.03 ± 0.15 and 3.48 ± 0.11 ng/g in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. After withdrawal of dietary AF these levels decreased and were not detectable on day 15 of the experiment. Table 2 Aflatoxin B1 residues (ng/g) in liver and muscles of broiler chicks of 14 days of age given different levels of dietary aflatoxins for 7 days (Mean ± SD). Organ/days of experiment
Groups (aflatoxins in feed lg/kg) A (0)
B (1600)
C (3200)
D (6400)
Liver 1 2 3 5 7 10 15 28
ND* ND ND ND ND ND ND ND
ND ND 2.67 ± 0.12b 3.60 ± 0.21b 3.65 ± 0.09b 0.96 ± 0.01b ND ND
ND ND 2.50 ± 0.44a 2.72 ± 0.21a 3.26 ± 0.23a 2.66 ± 0.03a ND ND
ND ND 4.55 ± 0.44a 5.74 ± 0.02a 6.59 ± 0.23a 2.68 ± 0.02a ND ND
Muscles 1 2 3 5 7 10 15 28
ND ND ND ND ND ND ND ND
ND ND ND 0.38 ± 0.01c 0.49 ± 0.05c 0.23 ± 0.02c ND ND
ND ND ND 0.77 ± 0.02b 0.77 ± 0.01b 0.48 ± 0.03b ND ND
ND ND ND 2.05 ± 0.04a 2.18 ± 0.00a 0.65 ± 0.01a ND ND
Values in each row followed by different letters are significantly different (p 6 0.05). * ND = Not detected.
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Z. Hussain et al. / Food and Chemical Toxicology 48 (2010) 3304–3307
Table 3 Aflatoxin residues (ng/g) in liver and muscles of broiler chicks of 28 days of age given different levels of dietary aflatoxin B1 for 7 days (Mean ± SD). Organ/days of experiment
Groups (aflatoxins in feed lg/kg) A (0)
B (1600)
C (3200)
D (6400)
Liver 1 2 3 5 7 10 15
ND* ND ND ND ND ND ND
ND ND ND 2.71 ± 0.24d 2.87 ± 0.13c 1.00 ± 0.18d ND
ND ND 0.95 ± 0.04b 2.91 ± 0.11a 3.03 ± 0.15b 2.40 ± 0.20a ND
ND ND 1.27 ± 0.09a 2.77 ± 0.28ab 3.48 ± 0.11a 2.54 ± 0.04a ND
Muscles 1 2 3 5 7 10 15
ND ND ND ND ND ND ND
ND ND ND 0.25 ± 0.03c 0.41 ± 0.05ab ND ND
ND ND 0.32 ± 0.04b 0.41 ± 0.01b 0.49 ± 0.01bc ND ND
ND 1.10 ± 0.05a 1.10 ± 0.10a 1.69 ± 0.19a 1.90 ± 0.03a 0.94 ± 0.10a ND
Values in each row followed by different letters are significantly different (p 6 0.05). ND = Not detected.
*
3.1.2. Muscles In experiment 1, the AFB1 was detected in muscles on day 5, 5 and 3 in 1600, 3200 and 6400 lg/kg AFB1groups. The AFB1 residue levels were at highest levels on day 7 being 1.63 ± 0.04, 1.90 ± 0.05 and 3.27 ± 0.05 ng/g, in 1600, 3200 and 6400 lg/kg AFB1groups, respectively (Table 1). Following withdrawal of AF from feed these residue levels decreased gradually and on day 10 were was present below 1 ng levels. On day 15 these levels could not be detected in any group. In experiment 2 (Table 2), the AF levels were detected in muscles on day 5 in all experiments. These levels on last day of AF feeding (day 7) were 0.49 ± 0.05, 0.77 ± 0.01 and 2.18 ± 0.004 ng/g in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. These residues decreased and were not detectable on day 15 of the experiment in all groups. In experiment 3 (Table 3), the AFB1 residues in muscles were detected on days 5, 3 and 2 in 1600, 3200 and 6400 lg/kg AFB1, respectively. On day 7 (last day of AF feeding) these levels increased and became 0.41 ± 0.05, 0.49 ± 0.01 and 1.90 ± 0.03 ng/g in 1600, 3200 and 6400 lg/kg AFB1 groups. After with drawl of dietary AF these levels decreased and were not detectable on day 10 in 1600 and 3200 lg/kg AFB1groups and day 15 day in 6400 lg/kg AFB1group. 4. Discussion Feeding of three levels of AFB1 to broiler chicks resulted in increased tissue concentration of AFB1 with increase in its dietary levels. Most of the authors have detected aflatoxins in the tissues by offering only one level of dietary aflatoxins to the birds for variable time periods (Trucksess et al., 1983; Wolzak et al., 1986; Micco et al., 1988; Bintvihok et al., 2002; Ortatatli et al., 2005; Zaghini et al., 2005). However, two levels of dietary aflatoxins offered to broiler chicks resulted in higher tissue AFB1 levels in birds offered higher dietary aflatoxins levels (Bintvihok and Kositcharoenkul, 2006). Liver AF residue levels obtained in the present study were comparable with those reported by other authors. Zaghini et al. (2005) found 4.13 ppb AFB1 in liver tissue of laying hens following feeding of 2500 ppb AFB1 for 28 days. In the broiler chicks less than 3 ppb AF were present in tissues after feeding of 2057 ppb AFB1 plus 1323 ppb AFB2 for 35 days (Chen et al.,
1984). However, some authors have reported lower liver residual levels of AFB1 in broiler and layer birds by feeding different levels of AFB1 than those observed in the present study (Trucksess et al., 1983; Wolzak et al., 1986; Micco et al., 1988). A variation in the type of bird, dose of AFB1 and duration could be a cause for this variation. Levels of AFB1 residues in muscles were lower than those observed in the liver tissue. A lower AFB1 residual level of muscles in comparison with liver was also reported by Bintvihok et al. (2002), Begum et al. (2001) and Gregory et al. (1983). Concentration of AFB1 residues in liver and muscles increased with the duration of ingestion of the toxin and were at its highest levels on the last day (7th) of feeding of AFB1 contaminated ration. Broiler chicks of 7, 14 and 28 days of age fed same level of AFB1 had lower tissue residues of AFB1 in older birds compared with younger ones. This observation suggested that birds develop a more efficient mechanism of metabolizing AFB1 with increase in age and had a decreased retention of AFB1 in tissue. There is scarce information describing the effect of age of the birds upon retention of AFB1 in body tissues of broiler chicks. AFB1 tissue residues in all experiments of the present study appeared earlier in high dose groups (day 2 in liver and day 3 in muscle) than those low dose groups (day 3 in liver and day 5 in muscle). Aflatoxins residues in tissues of layer hens have been detected no earlier than 3 days of feeding of 8000 ng/g AFB1contaminated feed (Trucksess et al., 1983). No author has reported the effect of dietary AFB1 levels upon the time drift between AF exposure and its detection in the tissues of broiler chicks. An age related effect was also observed upon the time drift between AF exposure and its detection in tissues in present study. AFB1 administered at 1600 ng/g dietary levels was detected in liver earlier (day 3) in birds intoxicated at 7 days of age than those intoxicated at 28 days of age (day 5). Also scanty data is available in the literature described the effect of age upon the time lapse between AF exposure and its detection in the body tissues. Elimination of AFB1 from the tissue following withdrawal of AFB1 contaminated diets in the present study occurred earlier in older birds compared with young ones. Birds given 1600 and 3200 lg/kg AFB1for 7 days at 28 days of age had no detectable AFB1 levels in liver and muscles after 3 and 8 days of withdrawal of AF contaminated feed whereas on same withdrawal periods AFB1 residues were still present in birds fed AFB1 from day 7 of age in experiment 1. Some authors have reported elimination of aflatoxins from tissues after withdrawal of aflatoxin contaminated feed within 4 days in broilers (Chen et al., 1984) and 7–8 days in layers (Trucksess et al., 1983; Wolzak et al., 1986). However, no worker reported an earlier elimination of AFB1 from the body tissues in older birds compared with the younger ones given same dietary AFB1 levels. Dietary contamination of aflatoxins pose a big risk to human health including acute aflatoxicosis, Hepatocellular carcinoma, hepatitis B virus infection, growth impairment in different regions of the World particularly Asian and African countries (Wild and Gong, 2010). European community and many other countries have imposed 2 ng/g AFB1 as maximum tolerance level in human food products (Anonymous, 2004). Birds fed Aflatoxins, following ingestion are rapidly metabolized into nontoxic substances in the body (Trucksess et al., 1983; Chen et al., 1984). A rapid decrease in AFB1 residues below the tolerance limits from the muscles and liver within 3 and 7 days of withdrawal of dietary AFB1 in the present study confirm the rapid metabolism of aflatoxins in the body of chicken and that it may not become a significant human health risk. However, in areas with no regulatory limits on AFB1 levels of poultry feed, the secondary exposure to aflatoxins through consumption of chicken liver and meat derived from the poultry fed AF contaminated feed may pose a risk to consumers health.
Z. Hussain et al. / Food and Chemical Toxicology 48 (2010) 3304–3307
Conflict of Interest The authors declare that there are no conflicts of interest. Acknowledgement Research grant was provided by Agriculture Linkage Program, Pakistan Agricultural Research Council Pakistan. References Anonymous, 2004. Worldwide regulations for mycotoxins in food and feed in 2003. FAO Food and Nutrition Paper 81. Food and Agriculture Organization, U.N. Rome, pp. 48–126. AOAC (Association of Official Analytical Chemist), 2000. Official method of Analysis. No. 990.33: Natural Toxins, vol. 2, 17th ed. Association of Official Analytical Chemist, Gaithersburg, MD, USA, pp. 20–22. Arafa, A.S., Bloomer, R.J., Wilson, H.R., Simpson, C.F., Harms, R.H., 1981. Susceptibility of various poultry species to dietary aflatoxin. Br. Poult. Sci. 22, 431–436. Asim, A., Khan, K.N.M., Cheema, A.H., Mir F.A., Afzal, M., 1990. Occurrence of aflatoxins in poultry liver and associated pathological changes. Pak. Vet. J. 2, 51–54. Begum, F., Rehman, A., Maliha G., Nuzhat, J., 2001. Distribution of aflatoxin B1 from poultry feed to different body tissues of broilers. Pak. Vet. J. 21, 121–123. Bintvihok, A., Davitiyananda, D., 2002. Aflatoxins and their metabolites residues in chicken tissues from 5 parts (10 provinces) of Thailand. Thai. J. Health Res. 16, 37–50. Bintvihok, A., Thiengnin, S., Doi, K., Kumagai, S., 2002. Residues of aflatoxins in the liver, muscle and eggs of domestic fowls. J. Vet. Med. Sci. 64, 1037–1039. Bintvihok, A., Kositcharoenkul, S., 2006. Effect of dietary calcium propionate on performance, hepatic enzyme activities and aflatoxin residues in broilers fed a diet containing low levels of aflatoxin B1. Toxicon. 47, 41–46. Chen, C., Pearson, A.M., Coleman, T.H., Gray, J.I., Pestka, J.J., Aus, S.D., 1984. Tissue deposition and clearance of aflatoxins from broiler chickens fed a contaminated diet. Food Chem. Toxicol. 22, 447–451. Chiavaro, E., Cacchioli, C., Berni, E., Spotti, E., 2005. Immunoaffinity clean-up and direct fluorescence measurement of aflatoxins B1 and M1 in pig liver:
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Contents lists available at ScienceDirect
Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox
Residues of aflatoxin B1 in broiler meat: Effect of age and dietary aflatoxin B1 levels Zahid Hussain a, Muhammad Zargham Khan a,⇑, Ahrar Khan a, Ijaz Javed b, Muhammad Kashif Saleemi a, Sultan Mahmood c, Muhammad Rafique Asi d a
Department of Pathology, University of Agriculture, Faisalabad 38040, Pakistan Department of Physiology and Pharmacology, University of Agriculture, Faisalabad 38040, Pakistan c Department of Poultry Science, University of Agriculture, Faisalabad 38040, Pakistan d Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan b
a r t i c l e
i n f o
Article history: Received 13 May 2010 Accepted 15 August 2010
Keywords: Aflatoxin Aflatoxin B1 residues Liver Muscles Broiler Aflatoxicosis
a b s t r a c t This study describes the effect of dietary levels of aflatoxin B1 (AFB1) and age of the birds upon the residue level in liver and muscles of broiler chicks. In three different experiments broiler chicks of 7, 14 and 28 days of age were kept for 7 days on contaminated rations having 1600, 3200 and 6400 lg/kg AFB1. AFB1 residues were detected earlier in younger birds and those fed high AFB1 dietary levels. The highest residue levels in liver and muscles of young chicks fed 6400 lg/kg AFB1 was 6.97 ± 0.08 and 3.27 ± 0.05 ng/g, respectively. Maximum residue concentration was high in birds of young age and those kept on high AFB1 ration. After withdrawal of AF contaminated rations, residues clearance was slow and AFB1 was detectable in liver and muscles of birds for longer duration in younger birds and those fed high AFB1 dietary levels. AFB1 residues in poultry tissues may buildup to high levels in areas with no regulatory limits on AFB1 levels of poultry feed and may pose a risk to consumers health. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Mycotoxins, the secondary metabolites of toxigenic fungi, are unavoidable contaminants in foods and feeds exerting harmful effects upon animal and human health (Zahoor-ul-Hassan et al., 2010). The most important mycotoxins in naturally tainted foods and feeds are aflatoxins, ochratoxins, zearalenone, T-2 toxin, deoxynivalenol and fumonisins (Devegowda et al., 1998; Sultana and Hanif, 2009). Aflatoxin (AF) produced by the toxigenic fungi mainly Aspergillus flavus and Aspergillus parasiticus constitute one of the major health hazard groups of naturally occurring toxicants both for human and animals. Among the four major groups of aflatoxin namely B1, B2, G1 and G2, aflatoxin B1 (AFB1) is the most toxic and a known carcinogenic. Acute or chronic aflatoxicosis in poultry birds results in decreased meat/egg production, immunosuppressant, and hepatotoxicosis (Arafa et al., 1981; Verma et al., 2004; Khan et al., 2010). AF residues may also appear in body tissues and samples collected from the commercial poultry birds showed detectable levels of AF in liver (Asim et al., 1990; Bintvihok and Davitiyananda, 2002). Poultry birds fed AF contaminated rations under experimental conditions resulted in the presence of AF residues in their edible tissues like liver and muscles. Residues of AFB1 in liver of broiler and layer birds have been reported to
⇑ Corresponding author. Tel.: +92 41 9200161x3118; fax: +92 41 9201094. E-mail address: [email protected] (M.Z. Khan). 0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.08.016
vary from no detection to 3.0 ng/g by feeding 250–3310 ng/g AFB1 for variable periods (Gregory et al.,1983; Chen et al., 1984; Wolzak et al., 1986; Bintvihok et al., 2002). The wide variations in the tissue AF residue concentration suggested that these levels might be influenced by different factors including dietary AF levels, duration of administration, age, type of the birds etc. However, effect of such factors upon concentration of AFB1 residues in poultry meat (liver and muscles) and clearance of AFB1 from the body tissues after withdrawal of dietary AF have not been adequately studied. Keeping in view the above mentioned concerns, this experimental study was designed to induce aflatoxicosis in broiler chicks in attempt to establish a relationship of tissue AFB1 concentration with age, and dietary AFB1 levels. 2. Materials and methods Tailoring and execution of the experiments followed national and institutional legislations regarding animal protection and welfare of laboratory animals. Moreover, the Directorate of Advanced Studies and Research Board, University of Agriculture, Faisalabad, Pakistan approved synopsis of the project.
2.1. Aflatoxin and feeds AF was produced in rice by inoculating with A. flavus (CECT 2687, Culture Collection Center University De Valencia Spain) following Shotwell et al. (1966). AFB1 concentration in the fermented rice was determined by high pressure liquid chromatography method.
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Z. Hussain et al. / Food and Chemical Toxicology 48 (2010) 3304–3307 Basal feed (22% total protein and 3000 kcal/kg metabolizable energy) was prepared without addition of toxin binder, vitamins, minerals and antibiotics. Prior to use, each batch of the basal feed was analyzed for aflatoxin, ochratoxin and zearalenone to ensure their levels below 1 lg/kg. AF contaminated feeds were prepared by incorporation of known quantities of AFB1. For this purpose fermented rice was extracted by soaking it into threefold quantity of chloroform (100:300) for overnight and filtered through cotton cloth. All the chloroform was evaporated and the concentrated residues were re-suspended into Polyethylene glycol (PEG). This suspension was evenly mixed in the required quantity of basal feed to prepare the experimental feeds containing AFB1 in concentrations of 1600, 3200 and 6400 lg/kg. 2.2. Experimental birds Two thousand, one day-old commercial broiler chicks (Hubbard Classic) were maintained on basal diet under standard conditions of brooding and management. Experimental groups were prepared from these birds by random selection as and when required. 2.3. Experimental plans Experiments 1, 2 and 3 were conducted on birds of 7, 14 and 28 days of age. In each experiment four groups of 80 broiler chicks were maintained and kept on feeds containing 0, 1600, 3200 and 6400 lg/kg AFB1 for 7 days. Afterwards the birds were switched back to basal feed till the termination of experiment. Duration of experiment 1, 2 and 3 was 36, 29 and 15 days, respectively so that age of depletion of birds in all experiments was 42/43 days. In each experiment six birds were killed from each group at days 1, 2, 3, 5, 7, 10 and 15 of the experiment. All remaining birds were killed at the end of each experiment.
Table 1 Aflatoxin B1 residues (ng/g) in liver and muscles of broiler chicks given different levels of dietary aflatoxins for 7 days from 7 days of age (Mean ± SD). Organ/Days of experiment
Groups (aflatoxin B1 in feed lg/kg) A (0)
B (1600)
C (3200)
D (6400)
Liver 1 2 3 5 7 10 15 35
ND* ND ND ND ND ND ND ND
ND ND 0.83 ± 0.01c 1.74 ± 0.07c 3.51 ± 0.08c 2.49 ± 0.04c 1.24 ± 0.03a ND
ND ND 2.45 + 0.08b 2.71 + 0.04b 3.74 + 0.09b 3.31 ± 0.07b 0.95 ± 0.08b ND
ND 0.89 ± 0.06a 5.23 ± 0.07a 5.75 ± 0.44a 6.97 ± 0.08a 3.57 ± 0.04a 1.13 ± 0.04a ND
Muscles 1 2 3 5 7 10 15 35
ND ND ND ND ND ND ND ND
ND ND ND 0.22 ± 0.01b 1.63 ± 0.04c 0.74 ± 0.01b ND ND
ND ND ND 0.24 ± 0.01b 1.90 ± 0.05b 0.93 ± 0.01a ND ND
ND ND 2.17 ± 0.04a 3.19 ± 0.11a 3.27 ± 0.05a 0.95 ± 0.12a ND ND
Values in each row followed by different letters are significantly different (p 6 0.05). * ND = Not detected.
2.4. Parameters studied 2.4.1. Estimation of aflatoxins in liver and muscle tissue Samples of muscles and livers of six birds at each killing in each group were randomly pooled to make three samples of each tissue and stored at 20 °C. The aflatoxins in the tissues were extracted using immune-affinity columns (Aflatest ÒWB, Vicam USA) and estimated by a HPLC-fluorescent detector method with pre-column derivatization. A modification of the methods of Chiavaro et al. (2005) and AOAC (990.33, 2000) was used as briefly mentioned below. Twenty-five grams of defrosted sample was homogenized and blended with 5 g of NaCl in 100 ml of methanol–water (80:20) for 3 min. After filtration through a paper filter, an aliquot of 10 ml of filtrate was diluted with 40 ml PBS Wash Buffer containing 0.1% Tween-20 Wash Buffer and passed through an immunoaffinity column. AFB1 was eluted with 1.0 ml of methanol in a glass vial and dried near to dryness under gentle stream of nitrogen. Pre-column derivatization was performed with trifluroacetic acid (AOAC 990.33, 2000) and a 20 ll of the derivitized extract was injected into HPLC system (Shimadzu LC-10 AS) equipped with reverse phase column (Supelco SIL LC-18, 15 cm 4.6 cm ID) and fluorescent detector (Shimadzu RF-530) at 360 nm for excitation and 440 nm inhibition wave lengths. Mobile phase consisted of water, acetonitril and methanol (60:20:20). Limit of detection (LOD) was 0.025 ng/ml and the recovery rate 89%. 2.5. Statistical analysis The data obtained in all the experiment were subjected to statistical analysis by analysis of variance. The means of different groups were compared by Duncan’s Multiple Range Test using MSTATC statistical software. The level of significance was p 6 0.05.
3. Results 3.1. AFB1 residues 3.1.1. Liver In birds fed AF contaminated feed from 7 days of age, AFB1 was detected on day 2, 3 and 3 in livers of 1600, 3200 and 6400 lg/kg AFB1 groups, respectively (Table 1). The AFB1 increased with increase in dietary levels and were highest on last day (day 7) of AF feeding being 3.51 ± 0.08, 3.74 ± 0.09 and 6.97 ± 0.08 ng/g, in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. Following withdrawal of AF from feed it decreased gradually and was below 2 ng/g on day 15 of experiment and undetectable on day 35 (42 days of age). In birds kept on AF contaminated feed from 14 days of age, AFB1 was detected on day 3 in livers of 1600, 3200 and 6400 lg/kg AFB1
groups, respectively (Table 2). These AFB1 levels on last day of AF feeding (day 7) were 3.65 ± 0.09, 3.26 ± 0.23 and 6.59 ± 0.23 ng/g, respectively in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. Following withdrawal of dietary AF, these residues decreased and were not detectable on day 10 of the experiment in1600 and 3200 lg/kg AFB1groups. In 6400 lg/kg AFB1group, AFB1 was not detected on day 15 of the experiment. In birds offered AF contaminated feed from 28 days of age, AFB1 residues were detected on day 5, 3 and 3 in liver of 1600, 3200 and 6400 lg/kg AFB1groups, respectively (Table 3). The AFB1 levels on last day of AF feeding (day 7) were at their height being 2.87 ± 0.13, 3.03 ± 0.15 and 3.48 ± 0.11 ng/g in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. After withdrawal of dietary AF these levels decreased and were not detectable on day 15 of the experiment. Table 2 Aflatoxin B1 residues (ng/g) in liver and muscles of broiler chicks of 14 days of age given different levels of dietary aflatoxins for 7 days (Mean ± SD). Organ/days of experiment
Groups (aflatoxins in feed lg/kg) A (0)
B (1600)
C (3200)
D (6400)
Liver 1 2 3 5 7 10 15 28
ND* ND ND ND ND ND ND ND
ND ND 2.67 ± 0.12b 3.60 ± 0.21b 3.65 ± 0.09b 0.96 ± 0.01b ND ND
ND ND 2.50 ± 0.44a 2.72 ± 0.21a 3.26 ± 0.23a 2.66 ± 0.03a ND ND
ND ND 4.55 ± 0.44a 5.74 ± 0.02a 6.59 ± 0.23a 2.68 ± 0.02a ND ND
Muscles 1 2 3 5 7 10 15 28
ND ND ND ND ND ND ND ND
ND ND ND 0.38 ± 0.01c 0.49 ± 0.05c 0.23 ± 0.02c ND ND
ND ND ND 0.77 ± 0.02b 0.77 ± 0.01b 0.48 ± 0.03b ND ND
ND ND ND 2.05 ± 0.04a 2.18 ± 0.00a 0.65 ± 0.01a ND ND
Values in each row followed by different letters are significantly different (p 6 0.05). * ND = Not detected.
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Table 3 Aflatoxin residues (ng/g) in liver and muscles of broiler chicks of 28 days of age given different levels of dietary aflatoxin B1 for 7 days (Mean ± SD). Organ/days of experiment
Groups (aflatoxins in feed lg/kg) A (0)
B (1600)
C (3200)
D (6400)
Liver 1 2 3 5 7 10 15
ND* ND ND ND ND ND ND
ND ND ND 2.71 ± 0.24d 2.87 ± 0.13c 1.00 ± 0.18d ND
ND ND 0.95 ± 0.04b 2.91 ± 0.11a 3.03 ± 0.15b 2.40 ± 0.20a ND
ND ND 1.27 ± 0.09a 2.77 ± 0.28ab 3.48 ± 0.11a 2.54 ± 0.04a ND
Muscles 1 2 3 5 7 10 15
ND ND ND ND ND ND ND
ND ND ND 0.25 ± 0.03c 0.41 ± 0.05ab ND ND
ND ND 0.32 ± 0.04b 0.41 ± 0.01b 0.49 ± 0.01bc ND ND
ND 1.10 ± 0.05a 1.10 ± 0.10a 1.69 ± 0.19a 1.90 ± 0.03a 0.94 ± 0.10a ND
Values in each row followed by different letters are significantly different (p 6 0.05). ND = Not detected.
*
3.1.2. Muscles In experiment 1, the AFB1 was detected in muscles on day 5, 5 and 3 in 1600, 3200 and 6400 lg/kg AFB1groups. The AFB1 residue levels were at highest levels on day 7 being 1.63 ± 0.04, 1.90 ± 0.05 and 3.27 ± 0.05 ng/g, in 1600, 3200 and 6400 lg/kg AFB1groups, respectively (Table 1). Following withdrawal of AF from feed these residue levels decreased gradually and on day 10 were was present below 1 ng levels. On day 15 these levels could not be detected in any group. In experiment 2 (Table 2), the AF levels were detected in muscles on day 5 in all experiments. These levels on last day of AF feeding (day 7) were 0.49 ± 0.05, 0.77 ± 0.01 and 2.18 ± 0.004 ng/g in 1600, 3200 and 6400 lg/kg AFB1 groups, respectively. These residues decreased and were not detectable on day 15 of the experiment in all groups. In experiment 3 (Table 3), the AFB1 residues in muscles were detected on days 5, 3 and 2 in 1600, 3200 and 6400 lg/kg AFB1, respectively. On day 7 (last day of AF feeding) these levels increased and became 0.41 ± 0.05, 0.49 ± 0.01 and 1.90 ± 0.03 ng/g in 1600, 3200 and 6400 lg/kg AFB1 groups. After with drawl of dietary AF these levels decreased and were not detectable on day 10 in 1600 and 3200 lg/kg AFB1groups and day 15 day in 6400 lg/kg AFB1group. 4. Discussion Feeding of three levels of AFB1 to broiler chicks resulted in increased tissue concentration of AFB1 with increase in its dietary levels. Most of the authors have detected aflatoxins in the tissues by offering only one level of dietary aflatoxins to the birds for variable time periods (Trucksess et al., 1983; Wolzak et al., 1986; Micco et al., 1988; Bintvihok et al., 2002; Ortatatli et al., 2005; Zaghini et al., 2005). However, two levels of dietary aflatoxins offered to broiler chicks resulted in higher tissue AFB1 levels in birds offered higher dietary aflatoxins levels (Bintvihok and Kositcharoenkul, 2006). Liver AF residue levels obtained in the present study were comparable with those reported by other authors. Zaghini et al. (2005) found 4.13 ppb AFB1 in liver tissue of laying hens following feeding of 2500 ppb AFB1 for 28 days. In the broiler chicks less than 3 ppb AF were present in tissues after feeding of 2057 ppb AFB1 plus 1323 ppb AFB2 for 35 days (Chen et al.,
1984). However, some authors have reported lower liver residual levels of AFB1 in broiler and layer birds by feeding different levels of AFB1 than those observed in the present study (Trucksess et al., 1983; Wolzak et al., 1986; Micco et al., 1988). A variation in the type of bird, dose of AFB1 and duration could be a cause for this variation. Levels of AFB1 residues in muscles were lower than those observed in the liver tissue. A lower AFB1 residual level of muscles in comparison with liver was also reported by Bintvihok et al. (2002), Begum et al. (2001) and Gregory et al. (1983). Concentration of AFB1 residues in liver and muscles increased with the duration of ingestion of the toxin and were at its highest levels on the last day (7th) of feeding of AFB1 contaminated ration. Broiler chicks of 7, 14 and 28 days of age fed same level of AFB1 had lower tissue residues of AFB1 in older birds compared with younger ones. This observation suggested that birds develop a more efficient mechanism of metabolizing AFB1 with increase in age and had a decreased retention of AFB1 in tissue. There is scarce information describing the effect of age of the birds upon retention of AFB1 in body tissues of broiler chicks. AFB1 tissue residues in all experiments of the present study appeared earlier in high dose groups (day 2 in liver and day 3 in muscle) than those low dose groups (day 3 in liver and day 5 in muscle). Aflatoxins residues in tissues of layer hens have been detected no earlier than 3 days of feeding of 8000 ng/g AFB1contaminated feed (Trucksess et al., 1983). No author has reported the effect of dietary AFB1 levels upon the time drift between AF exposure and its detection in the tissues of broiler chicks. An age related effect was also observed upon the time drift between AF exposure and its detection in tissues in present study. AFB1 administered at 1600 ng/g dietary levels was detected in liver earlier (day 3) in birds intoxicated at 7 days of age than those intoxicated at 28 days of age (day 5). Also scanty data is available in the literature described the effect of age upon the time lapse between AF exposure and its detection in the body tissues. Elimination of AFB1 from the tissue following withdrawal of AFB1 contaminated diets in the present study occurred earlier in older birds compared with young ones. Birds given 1600 and 3200 lg/kg AFB1for 7 days at 28 days of age had no detectable AFB1 levels in liver and muscles after 3 and 8 days of withdrawal of AF contaminated feed whereas on same withdrawal periods AFB1 residues were still present in birds fed AFB1 from day 7 of age in experiment 1. Some authors have reported elimination of aflatoxins from tissues after withdrawal of aflatoxin contaminated feed within 4 days in broilers (Chen et al., 1984) and 7–8 days in layers (Trucksess et al., 1983; Wolzak et al., 1986). However, no worker reported an earlier elimination of AFB1 from the body tissues in older birds compared with the younger ones given same dietary AFB1 levels. Dietary contamination of aflatoxins pose a big risk to human health including acute aflatoxicosis, Hepatocellular carcinoma, hepatitis B virus infection, growth impairment in different regions of the World particularly Asian and African countries (Wild and Gong, 2010). European community and many other countries have imposed 2 ng/g AFB1 as maximum tolerance level in human food products (Anonymous, 2004). Birds fed Aflatoxins, following ingestion are rapidly metabolized into nontoxic substances in the body (Trucksess et al., 1983; Chen et al., 1984). A rapid decrease in AFB1 residues below the tolerance limits from the muscles and liver within 3 and 7 days of withdrawal of dietary AFB1 in the present study confirm the rapid metabolism of aflatoxins in the body of chicken and that it may not become a significant human health risk. However, in areas with no regulatory limits on AFB1 levels of poultry feed, the secondary exposure to aflatoxins through consumption of chicken liver and meat derived from the poultry fed AF contaminated feed may pose a risk to consumers health.
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