In ternationa l Scholars Journa ls African Journal of Food Science Research ISSN 2375-0723 Vol. 6 (2), pp. 263-266, February, 2018. Available online at www.internationalscholarsjournals.org © International Scholars Journals Author(s) retain the copyright of this article. Full Length Research Paper A study of the effect of heat treatment on furazolidone residues in liver and muscle tissues of chicken Anakalo A. Shitandi 1 *, Oketch Aila 2 , Stellah Ottaro 1 , Leakey Aliong’o 1 , Grace Mwangi 1 , Harish Kumar- Sharma 3 and Matofari Joseph 1 1 Guildford institute, Egerton University, P.O. Box 536 Egerton 20107 Kenya. 2 Ministry of health, P.O. Box 42 Oyugis Kenya. 3 Food Technology Department, Sant Longowal Institute of Technology, Longowal. Sangrur-148106. Punjab, India. Accepted 02 July, 2017 This study investigated the effect of deep frying (210 o C/15 min) on furazolidone residues in liver and muscle tissues of chicken. Furazolidone was administered (2 mg/kg body weight) orally to chicken daily for five days. The hens were then sacrificed at 1, 5, 24 168 and 264 h after treatment stopped and liver and muscle tissue samples obtained. The samples were deep fried, blended with distilled water and then centrifuged at 6000 rpm for five minutes. The supernatant was analyzed for the concentration of the drug using a using the Delvotest SP microbiological assay. A detection limit of 11.0 g/ml was obtained with spiked liver tissues contaminated with Furazolidone. Furazolidone residues were detected in fried liver and muscle tissues 264 h post treatment. It was concluded that furazolidone drug residues in chicken liver and muscle tissues were not destroyed by deep frying. Key words: Furazolidone, depletion rate, delvotest test, poultry, deep frying. INTRODUCTION Various antibiotics, sulfonamides and coccidiostats are usually administered via feed or drinking water for the prevention and treatment of infectious diseases in laying hens. They also enhance feed efficiency, promote growth and improve productivity (Hermes, 2003; Gaudin et al., 2004; Bergwett, 2005). The uncontrolled and unlimited use of anticoccidial drugs may however lead to the accu- mulation of undesirable residues in the animals treated and their products. These residues may have adverse effects on both the animals and human beings (Lee et al., 2001; McCracken et al., 2005). In high income countries, birds whose residues exceed tolerance levels are removed from the distribution line (Lee et al., 2001). However, in many low income coun-tries such as Kenya, monitoring of drug residues is not done (Shitandi and Sternesjo, 2001). Hence, the risk consumers of poultry products are exposed to is not known. Furazolidone is a nitrofuran that has been used for many years for treatment of bacterial and protozoan *Corresponding author. E-mail: ashitandi@lycos.com. Phone: +254 51 62454; Fax: + 254 51 62527. infections in poultry. It was banned in USA and European countries, but it’s widely used in veterinary practice in low income countries like Kenya (Ali, 1999; Cooper et al., 2005). Furazolidone produces adverse reactions in both man and animals as it’s carcinogenic and mutagenic (Hoogenboom et al., 2002). When administered, furazoli-done metabolizes to its metabolite 3-amino-2-oxazolidi-none (AOZ). The Delvotest SP system is a broad spectrum screening test for the detection of antibiotic residues and sulphonamides in milk. This is a microbial inhibition assay that is based on the International Dairy Federation (IDF) reference method. It is the standard test method used for the detection of antibiotic residues in liquid milk at the pasteurizing plants (Fallon et al., 1995, 1996). The Delvotest SP system is dependent on rapid growth and acid production of Bacillus stearothermophilus var. calidolactis. Being a microbial assay, it can be used to test a broad spectrum of antibiotics (Food Safety / Autho-rity of Ireland / 2002). In a previous study the potential of the B. stearothermophilus var calidolactis C953 as a test organism to detect a broad spectrum of drugs residue was demonstrated in unprocessed poultry meat (Shitandi et al., 2006). The fate of drug residues during heat pro-cessing is however unclear. This study thus investigated the effect of Shitandi et. al. 263 Table 1. Positive, doubtful and negative rates of detection for Furazolidone contaminated liver tissues. Conc. % % Positive/ % ( g/ml) Positive Negative Negative 0.1 0 30 70 0.2 10 40 50 0.4 20 30 50 0.6 30 30 40 0.8 40 30 30 1.0 50 20 30 2.0 60 20 20 4.0 60 30 10 6.0 70 20 10 8.0 80 10 10 10.0 90 10 0 12.0 100 0 0 14.0 100 0 0 20.0 100 0 0 NB: The responses were determined from replicates of ten for each drug concentration. heat treatment on furazolidone residues in liver and muscle tissues of chicken. It further evaluated the depletion rate of Furazolidone so as to establish appropriate withdrawal period for the drug using the Delvotest SP system MATERIALS AND METHODS Site of experiment The experiment was carried out at the Taton farm and Microbiology Laboratory of Dairy and Food Science Technology Department, Egerton University. All experimental animals were acquired, retained, and used in compliance with the national laws and regulations of the research institution(s) of the authors. Experimental animals were properly housed and used in accordance with the research plan. In vitro studies Preparation of standards 100 mg of furazolidone (Cosmos, Kenya ltd) were weighed into a 100 ml flask and dissolved in 100 ml distilled water to produce a solution of 1000 g/ml (Stock solution A). Stock A was further diluted with distilled water (Stock B) producing a solution of 100 g/ml. Stock B (1 ml) was added to 4 ml of liver homogenate prepared at a dilution of 1:2 (tissue: distilled water) producing a solution of 20 g/ml. Standards of 14, 12 10, 8, 6, 4, 2, 1, 0.8, 0.6, 0.4, 0.2 and 0.1 g/ml were prepared from stock solution B using blank tissue solutions. Determination of limits of detection A sample of each standard homogenate was placed in a Delvotest SP tube and replicated ten times (a total of 10 tubes). Following the manufacturers instructions, the Delvotest SP tubes were incubated for thee hours in a water bath at a constant temperature of 64 ± 1 o C. The number of positive (Purple), doubtful (partly purple, partly yellow) and negative (yellow) colour changes were noted for each standard. Percentages positive, doubtful and negative were calculated for each standard tissue homogenate. Percent positive responses were plotted against tissue homogenate drug concentration to determine the detection limit of the Delvotest SP. The limit of detection was defined as the concentration where 95% of the test results were positive. Liver and muscle tissues that were free of antimicrobial drugs were used as the negative control. Liver and muscle tissue spiked at 50 g/ml were used as positive controls. In vivo studies Eighteen broiler chicken (Ross Breed) one day old were purchased from Kims Poultry farm (Nakuru, Kenya) and reared for six weeks at the poultry section of Animal Science Department, Egerton University. The birds had access to water and antibiotic free broiler feed ad libitum (the feed was obtained from Unga Limited, Nakuru). The birds were gavaged with a commercial oral suspension of Furazolidone once daily for five days at a dosage of 2 mg/kg of body weight. After the last treatment, the birds were sacrificed by decapitation in groups of thee at intervals of 1, 5, 24, 168 and 264 h. The liver and breast muscle samples were collected and frozen at -20 o C until analyzed for furazolidone residues. Thee birds served as controls and were killed before the treatment began. The liver and muscle tissues were deep fried at 210 o C for 15 min. The tissues were homogenized with distilled water at a ratio of 1:2 (tissue: distilled water). The homogenates were centrifuged for five minutes at 6000 rpm to eliminate tissue debris which appeared to inhibit diffusion of the drug into the medium. Supernatant from the homogenates were analyzed for residues using the Delvotest SP system as earlier described. The percentages of positive, doubtful and negative responses were calculated for the tissues at the different concentrations and plots of concentration versus percentage positive made. Semi quantification of furazolidone residues was done by comparing the obtained growth inhibition of samples and that of standards. Data analysis Data was analyzed using Chi Square test at 95% confidence interval. The test was used to compare depletion rates of the drug in liver and muscle tissues. RESULTS In vitro studies The results of Furazolidone contaminated liver tissues are shown in Table 1. A detection limit of 11.0 g/ml (Figure 1) was obtained with spiked liver tissues contaminated with Furazolidone. 100% positive Delvotest response was achieved for concentrations greater than or equal to 11 g/ml. In vivo studies The Delvotest SP showed a 30% positive response at 1 h 264 Afr. J. Food Sci. Res. 100 p o s it iv e 80 60 p e rc e n t 40 M e a n 20 0 .2 .4 .6 .8 1.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 20.0 concentration in ug/ml Cases weighted by percent positive Figure 1. Limit of detection of Furazolidone in liver tissues by Delvotest SP. Table 2. In vivo results for liver tissues in Delvo test Withdrawal % % % Conc Hours Positive Negative Doubtful ( g/ml) 1 30 30 40 0.6 5 20 50 30 0.4 24 20 40 40 0.4 168 10 20 70 0.2 264 10 10 80 0.2 NB: The responses were determined from replicates of ten for each sample. post treatment and 10% at 264 h post treatment. The 30% positive response did correspond to a Furazolidone concentration of 0.6 ug/ml in deep fried liver tissues at 1 h after treatment. The concentration of furazolidone in liver tissues declined steadily between 1h and 264 h post treatment. More doubtful samples appeared 5 h post treatment at 50%.The least concentration determined in liver was 0.2 g/ml 264 h post treatment (Table 2). The results indicate the presence of residues 11 days post treatment. The results were also presented graphically in figure 1 The Delvotest SP detected a maximum furazolidone concentration of 0.8 g/ml in deep fried muscle tissues at 1h after treatment. The concentration of furazolidone in muscle tissues declined rapidly between 1h and 24 h post treatment. Afterwards the concentration remained relatively constant between 24 and 168 h post treatment. This was followed by a decline to 0.2 g/ml at 264 h post treatment (Table 3) . Like for liver tissues, the muscles al- so had residues 11 days post treatment. The depletion rate of the drug in liver and muscle tiss- ues were compared using chi – Square test which gave a p value of 0.227 (Table 4). The findings suggest that Table 3. In vivo results for muscle tissues in Delvo test Withdrawal % % % Conc Hours Positive Negative Doubtful ( g/ml) 1 40 30 30 0.8 5 30 50 20 0.6 24 10 80 10 0.3 168 10 90 0 0.3 264 10 30 60 0.2 NB: The responses were determined from replicates of ten for each sample. Table 4. Chi – square test. Value Df Asymp. Sig. (2-sided) Pearson chi-square 7.500 a 6 0.277 Likelihood ratio 7.777 6 0.255 Linear-by-Linear Association 3.104 1 0.078 No. of valid cases 5 a. 12 cells (100%) have expected count less than 5. The minimum expected count is 20. there is no significance (p < 0.05) difference between the depletion rates of the drug in liver and muscle tissues. DISCUSSION The Delvotest SP system is a broad spectrum screening test for the detection of antibiotic residues and sulpho- namides in milk (IDF, 1991; Kroll, 1999). The present research suggests that in addition to detecting antibiotics in milk, the Delvotest SP system is could also be capable of detecting the Furazolidone residues in fried chicken tissues. The in vitro studies revealed positive responses of the system to Furazolidone residues greater than or equal to 11 g/ml in liver homogenates. However, the response of the Delvotest SP system to liver homoge- nates was unpredictable between 2 and 10 g/ml, but concentrations below 2 g/ml were not detected. These levels of detection seem to be higher than those for another B. stearothermophilus based test, the two tube test which showed much lower detection levels in experiments carried out in the same laboratory (Shitandi et al., 2006). In the in vivo experiment, the Delvotest SP system detected a maximum furazolidone concentration of 0.6 ug/ml in deep fried liver tissues at 1 h after treatment. The concentration of furazolidone in deep fried liver tis-sues declined steadily between 1 h and 264 h post treat-ment. The least concentration determined in deep fried liver was 0.2 g/ml 264 h post treatment. The system detected a maximum furazolidone concentration of 0.8 g/ml in deep fried muscle tissues at 1 h after treatment. Shitandi et. al. 265 The concentration of furazolidone in deep fried muscle tissues declined rapidly between 1h and 24 h post treatment. Afterwards the concentration remained rela- tively constant between 24 and 168 h post treatment. This was followed by a decline to 0.2 g/ml at 264 h post treatment. The concentration detected after 1h post treat- ment in deep fried muscle tissue was higher than that detected in deep fried liver tissue. However, the depletion rates were not significantly different at 95% confidence interval. The Delvotest SP system microbial assay contains a pre-defined number of B. stearothermophilus spores in agar wells which enables the test to be carried out in 2 h 30 min (at the time the negative control has been changed to yellow). The growth of the spores at 64°C initiates an acidification process which causes the turning of a pH indicator from purple to yellow. The presence of antibacterial substances will cause delay or inhibition of the spores, depending on the concentration of the residues. In the presence of residues the spores will not multiply and the pH indicator will remain purple (Food Safety Authority of Ireland, 2002). The bioavailability and effect of cooking on AOZ was studied by McCracken and Kennedy (1997). There were no significant effects on total concentration of AOZ due to grilling, micro waving or frying. Humans may be exposed to AOZ if animals treated with Furazolidone have no adequate withdrawal periods before slaughter. Drug residues in food animals being raised for human con- sumption may pose a public health concern. In low income countries such as Kenya, monitoring of drug residues is not done (Shitandi and Sternesjo, 2001) and hence the risk consumers of animal products are exposed to is not known. Consumer protection can be ensured by screening such animals for residues (Anadon and Martinez, 1998; Kozarova and Mate, 2000). Most food containing drug residues is consumed after cooking or processing, yet surveillance for these residues is almost always conducted on raw tissue (Rose et al., 1997). According to the manufacturer’s instructions (Cosmos, Kenya limited), the animals to which the drug has been administered should be consumed after ten days (240 h), which is considered the safe withdrawal period. However it is apparent from the present research that even after eleven days (264 h), the drug still persists in the deep fried tissues. The fact that the Delvotest system was able to detect furazolidone residues in deep fried liver and muscle tissue for longer periods of time may reflect the ability of these tissues to retain Furazolidone or its metabolites for long periods. Furazolidone is rapidly metabolized in vivo (Cooper et al., 2005). However, protein bound metabolites are formed and are detectable several weeks after administration McCracken et al.,1997; Okeeffe et al., 2004). The side chain residues (AOZ) persist longer than intact parent compounds. Furazolidone residues have been shown to accumulate in eggs from birds administered therapeutic dose of furazo- furazolidone in their daily feed (McCracken et al., 2001). The side chain accounts for the long lasting residues detected in deep fried chicken tissues such as muscle and liver. The general public is becoming increasingly aware of food safety issues and the potential for chemical and microbiological hazards in foods. There is a possibility of health risk due to protein bound metabolites of drugs that are formed during metabolism as they are characterized by long half lifes (Hoogenboom et al., 2002). Considering the withdrawal time of ten days for furazolidone in chicken as prescribed by the manufacturer, residues can be expected to persist in treated animals even after cook- ing, indicating the consumers are not given adequate protection. From the present research, it was apparent that furazo- lidone drug residues in chicken liver and muscle tissues were not destroyed by deep frying. This study further demonstrated that the depletion rates in both tissues were not significantly different at 95% confidence interval. The depletion studies suggests that the withdrawal periods for furazolidone in liver and muscle tissues may need to be adjusted above the manufacturer’s recom- mended periods. It may be of future interest to also investigate the possible elution of the drug residues into oil that has been used to deep fry chicken liver and muscle tissues. ACKNOWLEDGMENTS We are grateful to the Food Science and Technology department, Egerton University for financial support through the internal research funds. We appreciate the animal science department for support and advice in rearing the chicken used in this project, in particular Mr. Maritim, Mr. Kibe and Mr. Amwayi. REFERENCES Ali BH (1999). Pharmacological, Therapeutic and Toxicological Properties of Furazolidone: Some Recent Research. Vet. Res. Commun. 23: 343-360. Anadon A, Martinez-Larranaga MR (1998). Residues of Antimicrobial Drugs and Feed Additives in Animals products: Regulatory Aspects. Livestock Prod. Sci. 59: 183-198. Bergwett AA (2005). Rapid Assays For Detection Of Residues Of Veterinary Drugs. In: Rapid Methods For Biological and Chemical Contaminants in Food And Feed. Wangeningen Academic Publishers. pp. 28-34. Cooper M, Mulder J, Van Rhijn A, Kovacsics L, McCracken J, Young B, Kennedy G (2005). Depletion of four Nitroforan Antibiotics and their Tissue-bound Metabolites in Porcine Tissues and Determination using LC-MS/MS and HPLC-UV. Food Addit. Contamn. 22: 406-414 Food Safety Authority of Ireland (2002). Rapid test kits for the detection of antibiotics and sulfonamides in milk. pp.12-18. Gaudin V, Maris P, Fusetier R, Ribouchon C, Cadieu N, Rault A (2004). Validation of a Microbiological Method: The Star Protocal, A Five Plate Test for Screening of Antibiotic Residues in Milk. Food Addit. Contam. 21(5): 422-433. Hermes C (2003). Avoiding Residues in Small Poultry and Game Flocks. Pacific Northwest Extension Publication Oregon State Univer- 266 Afr. J. Food Sci. Res. sity. 6-9. Hoogenboom L, Van Bruchem D, Sonne K, Enninga C, Van Rhijn A, Heskamp H, Huveneers- Oorsprong M, Van der Hoeven M, Kuiper A (2002). Absorption of a Mutagenic Metabolite Released from Protein Bound Residues of Furazolidone. Environ. Toxic. Pharma. 11: 273- 287. International Dairy Federation (1991). Detection and confirmation of inhibitors in milk and milk products. International Dairy Federation International Dairy Federation, Brussels, Belgium. Bulletin 258. Kozarova I, Mate D (2000). Evaluation of the sensitivity of individual test organisms to residual concentrations of selected types of anticoccidial drugs. Bull. Vet. Inst. Pullawy. 44: 187-192. Kroll S (1999). Suitability of rapid test methods for the detection of residues of betalactam antibiotics in milk. Dissertation, University of Munchen, Germany. 42-46. Lee H, Lee J, Rys D (2001). Public Health Risks: Chemical and Antibiotic Residues- Rev. Asian–Aust. J. Anim. Sci. 14(3):402-413. McCracken J, Kennedy D (1997). The Bioavailability of Residues of the Furazolidone Metabolite 3-amino-2-oxazolidinone in Porcine Tissues and the Effect of Cooking upon Residue Concentration. Food Addit. Contam. 14: 507-513. McCracken J, McCoy A, Kennedy G (1997). The Prevalence and Possible Causes of Bound and Extractable Residues of the Furazolidone Metabolite 3-amino-2-oxazolidinone in rcine Tissues. Food Addit. contamn. 14: 287-294. McCracken J, Van Rhijn A, Kennedy G (2005). Transfer of Nitrofuran Residues from Parent Broiler Chickens to Broiler Progeny British Poultry Sci. 46: 287-292. McCracken J, Spence E, Floyd R, Kennedy G (2001). Evaluation of the Residues of Furazolidone and its Metabolite, AOZ in Eggs. Feed Addit. Contamn. 18: 954-959. O’Keeffe A, Horne E, Cadogan A, Coyle T (1999). Protein Bound Veterinary Drug Residues. pp.3-12. Rose D, Rowley L, Shearer G, Farrington H (1997). Effect of Cooking on Veterinary Drug Residues in Food. J. Agric food chem. 45: 927- 930. Shitandi A, Sternesjo A (2001). Detection of Inhibitory Substances in Kenyan Milk. J. Food Safety, 21: 205-214. Shitandi A, Oketch A, Mahungu M (2006). Evaluation of a Bacillus stearothermophilus Tube Test as a Screening Tool for Anticoccidial Residues in Poultry. J. Vet. Sci 2006. 7(2):177-180.