Effect of deep frying on furazolidone anticoccidial drug residues in liver and muscle tissues of chicken

African Journal of Food Science. Vol (2) pp. 144-148, December, 2008
Available online http://www.academicjournals.org/ajfs
ISSN 1996-0794 ©2008 Academic Journal



Ful Length Research Paper
Effect of deep frying on furazolidone anticoccidial drug
residues in liver and muscle tissues of chicken

Anakalo A. Shitandi1*, Oketch Aila2, Stellah Ottaro1, Leakey Aliong’o1, Grace Mwangi1, Harish
Kumar- Sharma3 and Matofari Joseph1

1Guildford institute, Egerton University, P.O. Box 536 Egerton 20107 Kenya.
2Ministry of health, P.O. Box 42 Oyugis Kenya.
3Food Technology Department, Sant Longowal Institute of Technology, Longowal. Sangrur-148106. Punjab, India.

Accepted 15 September, 2008

This study investigated the effect of deep frying (210oC/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
infections in poultry. It was banned in USA and European
usual y administered via feed or drinking water for the
countries, but it’s widely used in veterinary practice in low
prevention and treatment of infectious diseases in laying
income countries like Kenya (Ali, 1999; Cooper et al.,
hens. They also enhance feed efficiency, promote growth
2005). Furazolidone produces adverse reactions in both
and improve productivity (Hermes, 2003; Gaudin et al.,
man and animals as it’s carcinogenic and mutagenic
2004; Bergwett, 2005). The uncontrol ed and unlimited
(Hoogenboom et al., 2002). When administered, furazoli-
use of anticoccidial drugs may however lead to the accu-
done metabolizes to its metabolite 3-amino-2-oxazolidi-
mulation of undesirable residues in the animals treated
none (AOZ).
and their products. These residues may have adverse
The Delvotest SP system is a broad spectrum
effects on both the animals and human beings (Lee et al.,
screening test for the detection of antibiotic residues and
2001; McCracken et al., 2005).
sulphonamides in milk. This is a microbial inhibition assay
In high income countries, birds whose residues exceed
that is based on the International Dairy Federation (IDF)
tolerance levels are removed from the distribution line
reference method. It is the standard test method used for
(Lee et al., 2001). However, in many low income coun-
the detection of antibiotic residues in liquid milk at the
tries such as Kenya, monitoring of drug residues is not
pasteurizing plants (Fal on et al., 1995, 1996). The
done (Shitandi and Sternesjo, 2001). Hence, the risk
Delvotest SP system is dependent on rapid growth and
consumers of poultry products are exposed to is not
acid production of Bacil us stearothermophilus var.
known.
calidolactis. Being a microbial assay, it can be used to
Furazolidone is a nitrofuran that has been used for
test a broad spectrum of antibiotics (Food Safety / Autho-
many years for treatment of bacterial and protozoan
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
*Corresponding author. E-mail: ashitandi@lycos.com. Phone:
et al., 2006). The fate of drug residues during heat pro-
+254 51 62454; Fax: + 254 51 62527.
cessing is however unclear. This study thus investigated

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Shitandi et. al. 145






Table 1. Positive, doubtful and negative rates of
SP tube and replicated ten times (a total of 10 tubes). Fol owing the
detection for Furazolidone contaminated liver tissues.
manufacturers instructions, the Delvotest SP tubes were incubated

for thee hours in a water bath at a constant temperature of 64 ±
Conc.
%
% Positive/
%
1oC. The number of positive (Purple), doubtful (partly purple, partly
( g/ml) Positive
Negative
Negative
yel ow) and negative (yel ow) colour changes were noted for each
standard. Percentages positive, doubtful and negative were
0.1
0
30
70
calculated for each standard tissue homogenate.
0.2
10
40
50
Percent positive responses were plotted against tissue
0.4
20
30
50
homogenate drug concentration to determine the detection limit of
the Delvotest SP. The limit of detection was defined as the
0.6
30
30
40
concentration where 95% of the test results were positive. Liver and
0.8
40
30
30
muscle tissues that were free of antimicrobial drugs were used as
1.0
50
20
30
the negative control. Liver and muscle tissue spiked at 50 g/ml
2.0
60
20
20
were used as positive controls.

4.0
60
30
10

6.0
70
20
10
In vivo studies
8.0
80
10
10

Eighteen broiler chicken (Ross Breed) one day old were purchased
10.0
90
10
0
from Kims Poultry farm (Nakuru, Kenya) and reared for six weeks at
12.0
100
0
0
the poultry section of Animal Science Department, Egerton
14.0
100
0
0
University. The birds had access to water and antibiotic free broiler
20.0
100
0
0
feed ad libitum (the feed was obtained from Unga Limited, Nakuru).
The birds were gavaged with a commercial oral suspension of

NB: The responses were determined from replicates of ten
Furazolidone once daily for five days at a dosage of 2 mg/kg of
for each drug concentration.
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 col ected and frozen

at -20oC until analyzed for furazolidone residues. Thee birds served
the effect of heat treatment on furazolidone residues in
as controls and were kil ed before the treatment began. The liver
liver and muscle tissues of chicken. It further evaluated
and muscle tissues were deep fried at 210oC for 15 min. The
the depletion rate of Furazolidone so as to establish
tissues were homogenized with distil ed water at a ratio of 1:2
appropriate withdrawal period for the drug using the
(tissue: distil ed water). The homogenates were centrifuged for five
Delvotest SP system
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
MATERIALS AND METHODS
and negative responses were calculated for the tissues at the

different concentrations and plots of concentration versus
Site of experiment
percentage positive made. Semi quantification of furazolidone

residues was done by comparing the obtained growth inhibition of
The experiment was carried out at the Taton farm and Microbiology
samples and that of standards.
Laboratory of Dairy and Food Science Technology Department,

Egerton University. Al experimental animals were acquired,

retained, and used in compliance with the national laws and
Data analysis
regulations of the research institution(s) of the authors.

Experimental animals were properly housed and used in
Data was analyzed using Chi Square test at 95% confidence
accordance with the research plan.
interval. The test was used to compare depletion rates of the drug

in liver and muscle tissues.


In vitro studies


RESULTS
Preparation of standards


In vitro studies
100 mg of furazolidone (Cosmos, Kenya ltd) were weighed into a
100 ml flask and dissolved in 100 ml distil ed water to produce a

solution of 1000 g/ml (Stock solution A). Stock A was further
The results of Furazolidone contaminated liver tissues
diluted with distil ed water (Stock B) producing a solution of 100
are shown in Table 1. A detection limit of 11.0 g/ml
g/ml. Stock B (1 ml) was added to 4 ml of liver homogenate
(Figure 1) was obtained with spiked liver tissues
prepared at a dilution of 1:2 (tissue: distil ed water) producing a
contaminated with Furazolidone. 100% positive Delvotest
solution of 20 g/ml. Standards of 14, 12 10, 8, 6, 4, 2, 1, 0.8, 0.6,
response was achieved for concentrations greater than or
0.4, 0.2 and 0.1 g/ml were prepared from stock solution B using
blank tissue solutions.
equal to 11 g/ml.




Determination of limits of detection
In vivo studies


A sample of each standard homogenate was placed in a Delvotest
The Delvotest SP showed a 30% positive response at 1 h

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146 Afr. J. Food Sci.






Table 3. In vivo results for muscle tissues in Delvo test
100

Withdrawal
%
%
%
Conc
80
Hours
Positive Negative Doubtful ( g/ml)
1
40
30
30
0.8
60
5
30
50
20
0.6
24
10
80
10
0.3
40
168
10
90
0
0.3
e
a
n

p
e
r
c
e
n
t

p
o
s
i
t
i
v
e
264
10
30
60
0.2
M

20
NB: The responses were determined from replicates of ten for
each sample.

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
Table 4. Chi – square test.

Cases weighted by percent positive



Value
Df
Asymp. Sig.
Figure 1. Limit of detection of Furazolidone in liver tissues by
(2-sided)
Delvotest SP.

Pearson chi-square
7.500a 6
0.277

Likelihood ratio
7.777
6
0.255

Linear-by-Linear Association 3.104
1
0.078
Table 2. In vivo results for liver tissues in Delvo test

No. of valid cases
5



Withdrawal
%
%
%
Conc
a. 12 cel s (100%) have expected count less than 5. The minimum
Hours
Positive Negative Doubtful ( g/ml)
expected count is 20.

1
30
30
40
0.6

5
20
50
30
0.4

there is no significance (p < 0.05) difference between the
24
20
40
40
0.4
depletion rates of the drug in liver and muscle tissues.
168
10
20
70
0.2

264
10
10
80
0.2


DISCUSSION
NB: The responses were determined from replicates of ten for each

sample.
The Delvotest SP system is a broad spectrum screening


test for the detection of antibiotic residues and sulpho-

namides in milk (IDF, 1991; Krol , 1999). The present
post treatment and 10% at 264 h post treatment. The
research suggests that in addition to detecting antibiotics
30% positive response did correspond to a Furazolidone
in milk, the Delvotest SP system is could also be capable
concentration of 0.6 ug/ml in deep fried liver tissues at 1
of detecting the Furazolidone residues in fried chicken
h after treatment. The concentration of furazolidone in
tissues. The in vitro studies revealed positive responses
liver tissues declined steadily between 1h and 264 h post
of the system to Furazolidone residues greater than or
treatment. More doubtful samples appeared 5 h post
equal to 11 g/ml in liver homogenates. However, the
treatment at 50%.The least concentration determined in
response of the Delvotest SP system to liver homoge-
liver was 0.2 g/ml 264 h post treatment (Table 2). The
nates was unpredictable between 2 and 10 g/ml, but
results indicate the presence of residues 11 days post
concentrations below 2 g/ml were not detected. These
treatment. The results were also presented graphical y in
levels of detection seem to be higher than those for
figure 1
another B. stearothermophilus based test, the two tube
The Delvotest SP detected a maximum furazolidone
test which showed much lower detection levels in
concentration of 0.8 g/ml in deep fried muscle tissues at
experiments carried out in the same laboratory (Shitandi
1h after treatment. The concentration of furazolidone in
et al., 2006).
muscle tissues declined rapidly between 1h and 24 h
In the in vivo experiment, the Delvotest SP system
post treatment. Afterwards the concentration remained
detected a maximum furazolidone concentration of 0.6
relatively constant between 24 and 168 h post treatment.
ug/ml in deep fried liver tissues at 1 h after treatment.
This was fol owed by a decline to 0.2 g/ml at 264 h post
The concentration of furazolidone in deep fried liver tis-
treatment (Table 3). Like for liver tissues, the muscles al-
sues declined steadily between 1 h and 264 h post treat-
so had residues 11 days post treatment.
ment. The least concentration determined in deep fried
The depletion rate of the drug in liver and muscle tiss-
liver was 0.2 g/ml 264 h post treatment. The system
ues were compared using chi – Square test which gave a
detected a maximum furazolidone concentration of 0.8
p value of 0.227 (Table 4). The findings suggest that
g/ml in deep fried muscle tissues at 1 h after treatment.

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Shitandi et. al. 147






The concentration of furazolidone in deep fried muscle
furazolidone in their daily feed (McCracken et al., 2001).
tissues declined rapidly between 1h and 24 h post
The side chain accounts for the long lasting residues
treatment. Afterwards the concentration remained rela-
detected in deep fried chicken tissues such as muscle
tively constant between 24 and 168 h post treatment.
and liver.
This was fol owed by a decline to 0.2 g/ml at 264 h post
The general public is becoming increasingly aware of
treatment. The concentration detected after 1h post treat-
food safety issues and the potential for chemical and
ment in deep fried muscle tissue was higher than that
microbiological hazards in foods. There is a possibility of
detected in deep fried liver tissue. However, the depletion
health risk due to protein bound metabolites of drugs that
rates were not significantly different at 95% confidence
are formed during metabolism as they are characterized
interval.
by long half lifes (Hoogenboom et al., 2002). Considering
The Delvotest SP system microbial assay contains a
the withdrawal time of ten days for furazolidone in
pre-defined number of B. stearothermophilus spores in
chicken as prescribed by the manufacturer, residues can
agar wel s which enables the test to be carried out in 2 h
be expected to persist in treated animals even after cook-
30 min (at the time the negative control has been
ing, indicating the consumers are not given adequate
changed to yel ow). The growth of the spores at 64°C
protection.
initiates an acidification process which causes the turning
From the present research, it was apparent that furazo-
of a pH indicator from purple to yel ow. The presence of
lidone drug residues in chicken liver and muscle tissues
antibacterial substances wil cause delay or inhibition of
were not destroyed by deep frying. This study further
the spores, depending on the concentration of the
demonstrated that the depletion rates in both tissues
residues. In the presence of residues the spores wil not
were not significantly different at 95% confidence interval.
multiply and the pH indicator wil remain purple (Food
The depletion studies suggests that the withdrawal
Safety Authority of Ireland, 2002).
periods for furazolidone in liver and muscle tissues may
The bioavailability and effect of cooking on AOZ was
need to be adjusted above the manufacturer’s recom-
studied by McCracken and Kennedy (1997). There were
mended periods. It may be of future interest to also
no significant effects on total concentration of AOZ due to
investigate the possible elution of the drug residues into
gril ing, micro waving or frying. Humans may be exposed
oil that has been used to deep fry chicken liver and
to AOZ if animals treated with Furazolidone have no
muscle tissues.
adequate withdrawal periods before slaughter. Drug

residues in food animals being raised for human con-

sumption may pose a public health concern. In low
ACKNOWLEDGMENTS
income countries such as Kenya, monitoring of drug

residues is not done (Shitandi and Sternesjo, 2001) and
We are grateful to the Food Science and Technology
hence the risk consumers of animal products are
department, Egerton University for financial support
exposed to is not known. Consumer protection can be
through the internal research funds. We appreciate the
ensured by screening such animals for residues (Anadon
animal science department for support and advice in
and Martinez, 1998; Kozarova and Mate, 2000). Most
rearing the chicken used in this project, in particular Mr.
food containing drug residues is consumed after cooking
Maritim, Mr. Kibe and Mr. Amwayi.
or processing, yet surveil ance for these residues is

almost always conducted on raw tissue (Rose et al.,

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