Depletion and Distribution Studies for Oxytetracycline
in Broiler Chicken using Commercial ELISA with Subsequent Confirmatory Analysis
by HPLC-UV
Mehwish Mumtaz*, Uzma Maqbool, Muhammad Ismail Chughtai, M. Salahuddin Shah and Ghulam Hussain
Animal Sciences Division, Nuclear Institute for Agriculture and Biology
(NIAB), P.O. Box 128, Faisalabad-38000, Pakistan
*For correspondence: mehwish@niab.org.pk; mehwish.pieas@hotmail.com
Received 07 September 2021;
Accepted 22 November 2021; Published 15 December 2021
Abstract
Tetracyclines
are commonly used in livestock for the treatment of various diseases. Their
residues above the maximum residual limit-MRL (200 µg kg-1) can cause health hazard issues not only in humans but
also in animals. For this purpose, efforts were made to study distribution and
depletion profile of Oxytetracycline in broiler chicken by ELISA and HPLC.
Regarding this, an experiment was conducted on healthy chickens of average
weight 600 ± 20 g. Six broiler chickens were treated
with formulation (OXTRA L.A.) containing Oxytetracycline dehydrate @20 ppk solution (150 µL) equivalents to recommended dose 20 mg kg-1 through intramuscular route, while control birds were untreated. Treated
chickens were slaughtered at an interval of 1, 8, 16, 32,
64 and 120 h. Different tissue samples including liver, kidney, thigh and chest
muscles were collected and screened by ELISA (Cat No. 5091TC, Euro Proxima). Lowest
detection limit (IC20) was calculated as 0.12 ng mL-1 and
middle of the test (IC50) 0.5 ng mL-1 with recovery 75 to
86%. Reverse phase liquid chromatography (HPLC-UV) method was also standardized
by using range of standards from 25 to 200 µg
kg-1. Calibration curve showed good response with correlation
coefficient (R2) 0.9727 and recovery 85 to 93%. Overall, withdrawal
period (< MRL) in all tissue samples was calculated as 117 h (4.8 days) by
ELISA while 128 h (5 days) by HPLC-UV. Both validated methods can be further
utilized to generate reliable data for food safety measures in Pakistan to
enhance international trade. © 2021 Friends Science Publishers
Keywords: Oxytetracycline residues; Withdrawal period; Validation studies; ELISA; Liquid
chromatography
Abbreviation:
HPLC-UV: High Pressure
Liquid Chromatography, Ultra Violet, ELISA: Enzyme-Linked Immunosorbent Assay,
WHO: World Health Organization, MRM: Maximum
Residue Limit, FDA: Food and Drug
Administration, CODEX: Alimentarius Collection
of Food Standards, JECFA: Joint FAO/WHO Expert Committee on Food
Additives
Introduction
Food safety is of great importance these days, especially in a world
where monitoring studies is favorable in search of safe food. Local people are
unaware of ill practices used in antibiotics in livestock sector, several
efforts are being made to reduce antibiotics use (Rana et al. 2019). This
is due to non-existence of strict regulations and lack of food safety awareness
to public. Antibiotics are most commonly used in poultry but hardly examined (Khatun et al.
2018). Their
illegal use can increase the chances of food contamination instead of their
benefits, e.g., creates resistance in
human pathogens (Al-Gendy et al. 2014).
Antibiotics
are widely used in animals to control diseases as well as growth promoter to
increase meat, milk and egg production (Wang et al. 2011). The tetracyclines have
applications for the treatment of infections in poultry, cattle, sheep, and
swine. In some cases, for therapeutic treatment of large numbers of poultry
reared on commercial farms, the antibiotics are added directly to feed or water
or can be administered (Khatun et al. 2018). The presence of drug residues in animal-derived food
can cause serious health issues. Due to this reason, food safety has become a
serious concern all over the world (Biswas et
al. 2007). The inappropriate and overuse of veterinary drugs has
become a common practice in recent years (CODEX
2012).
The
main group of drugs used in veterinary medicine include tetracyclines,
amphenicols, aminoglycosides, macrolides, nitrofurans, nitromidazoles, sulfonamides and quinolones (Ang
et al. 2017).
Tetracyclines are effective
against Gram-positive and Gram-negative bacteria, including some anaerobes.
Susceptible organisms include Escherichia coli, Klebsiella species, Pasteurella species, Salmonella species,
and Streptococcus species. Tetracyclines are also active against
chlamydia, mycoplasmas, some protozoan and several rickettsia. Drugs are mostly
used in broiler chickens to treat chronic respiratory disease and infectious
sinusitis, it is also suitable to enhance immune system in farm produce animals
(Shahid et
al. 2007; Wang et al. 2011).
Tetracyclines inhibit protein synthesis in both
bacterial and human cells, the first members
which were derived from the Streptomyces genus of Actinobacteria.
Oxytetracycline and chlortetracycline show moderate lipid solubilities while
doxycycline and minocycline show higher, so that they are able to traverse cell
membranes moderately or readily (Wang et al. 2011). Tetracyclines after
absorption bound to plasma protein to a limited extend, absorption percentage
of tetracycline’s in animal body is chlortetracycline 46‒51%, tetracycline 28‒41%, oxytetracycline 21‒76% and doxycycline 84‒92% (Riviere and Papich 2018).
Chlortetracycline and oxytetracycline both discovered in the late 1940s,
were the first members of the tetracycline group to be described, are commonly used in livestock sector to control diseases. If their depletion profile
studies are not conducted then their
residues will remains in edible tissues (Widiastuti
and Anastasia 2015). Overuse of these drugs can cause microbial
resistance. Hence, non-susceptible
organisms grow rapidly; this may result in colitis and severe diarrhea (Wang et al.
2011). About 20 to 90% of the drugs are
not adsorbed and excreted (EU Regulation 2010;
Muaz et al. 2018).
Local farmers are unaware of
actual withdrawal period of the antibiotics so the residues persist in the meat
beyond safe limit (Mund
et al. 2017; Xu et al. 2019). The continual use of the antibiotics in poultry also increases
resistance against antibiotics not only in animals but also in consumers (Khatun et al. 2018; Muaz et al. 2018).
This leads to rapid change in genetic variation of certain beneficial bacteria which become dangerous for the human life and their presence continued
in environment (Agha et al. 2003).
The
World Health Organization (WHO) reported public health problems emerging from
microbial resistance due to excessive use of antibiotics (Cinquina et al.
2003). The Food and Drug Administration (FDA) also set criteria for the
approval of new antibiotics to perform risk assessment (Biswas et al. 2007; Khatun et al. 2018). Codex and FAO/WHO experts set maximum residue levels for tetracyclines
are 0.2, 0.6 and 1.2 µg g-1
for poultry muscle, liver and kidney; respectively. In order to ensure the
customers food safety, plans for monitoring of contaminants in food are set by
Codex (Al-Gendy et al. 2014; CODEX 2018). Similarly, tetracyclines MRL is described as the combination of
parent compounds and its metabolites in poultry products, set as 200 ng g-1 for meat, 600
ng g-1 for liver and 400 ng g-1 for eggs. A group ADI for tetracycline, oxytetracycline, and chlortetracycline has
been allocated by JECFA. The MRL value is also set by CAC for tetracycline,
oxytetracycline and chlortetracycline applicable to cattle, sheep, pigs,
poultry and fish while for giant prawn only oxytetracycline (Wang et al.
2011).
According to EU MRL limits for
tetracyclines is set as 100 µg kg-1 for
muscles, 300 µg kg-1 for liver and 600 µg kg-1 for kidney (Commission Decision 2002; CODEX 2012). A strict surveillance system exists in the European Union by Council
Directive 96/23/EC for screening of veterinary drug residue. Rapid alerts due to residues of antibiotics in broiler chicken and its
products have been appeared in the market of Bangladesh, Indonesia, Oman and
Philippines as their consignments are rejected by EU member states. Indonesia has also sets the MRL for oxytetracycline as 100 ng g-1
for meat and 50 ng g-1 for eggs through SNI No. 01‒6366 2000 (Widiastuti and Anastasia 2015).
In Pakistan, oxytetracycline
residues monitoring in Rawalpindi, Islamabad and Faisalabad regions was
reported as 44.8% broiler chicken samples found positive as per recommendations
of Joint FAO/WHO Committee of food additives (Shahid
et al. 2007). Punjab Food
Authority of Pakistan has also established a document for setting MRPL and MRL to control antibiotics
by regular monitoring through different ministries. Screening of antibiotics through like commercial and
in-house ELISA and subsequent confirmatory analysis of positive or selected samples by HPLC-UV is cost-effective to
screen large number of samples (Cinquina et al. 2003). The present studies
depict the procedure to determine withdrawal period of oxytetracycline in
broiler chicken and drug distribution in different body parts of broiler
chicken. The generated data can be applied to set withdrawal period of
oxytetracycline. It can further contribute in giving awareness to the farmers
and other stakeholders involved in this business as well as consumption.
Materials and Methods
Chemicals and reagents used
Trichloroacetic acid (Merck), Methanol (VWR), Oxalic acid (Sigma), nylon
membrane PTFE membrane (0.45 µm), syringe filter (4 mm), PP housing, double
distilled water, (from Cyclon Firsteem Automatic Ultrapure Water Still) Filtration Assembly, Sartorius, Ethyl acetate (VWR), n-Hexane (VWR), Polycons
(50 mL capacity, VWR), Glass test tubes (Kimax), Separatory funnels, Erlenmeyer
flask, Commercial ELISA Kits (Cat. # 5091TC, Europroxima, Netherlands), DMSO
(Sigma), Trifloroacetic acid (Sigma) and Acetonitrile (VWR).
Experimental planning and execution
Experiments were conducted to determine drug profile in vitro. Regarding this, all
requirements were fulfilled as mentioned in ARRIVE guidelines with the recommendations
of NIAB animal house committee. Special considerations were taken to minimize
the stressful conditions of the animals. A group of seven broiler chicken was grown under controlled conditions
in NIAB Animal Farm House. Injectable
Oxytetracycline products are stable as shown by the retention of more than 90%
potency for at least 24 month storage (Ang et
al. 2017). In group-I (n = 6), which served as the treated or
positive control, each bird received antibiotic-free water and feed until the
end of the experiment. In the fifth week of the chickens’ lives, a veterinary
drug containing oxytetracycline dihydrate (OXTRA L.A. E.U.P 21.978 g equivalent
to oxytetracycline 20 g-excipients q.s. 100 mL (Bologna, Italy)
was administered @ 0.15 mL per kg body weight. One bird was taken as negative
control that was non-treated. Drug was administered to each broiler chicken at right thigh and breast
side through intramuscular route. Five treated chickens were slaughtered after
1, 8, 16, 32, 64 h while 6th after one week along with control.
Selected tissue samples were collected and transferred to Food Safety Labs
(ISO/IEC 17027:2017 accredited) of NIAB Faisalabad and stored at -20°C till
further analysis.
Collection of samples
For distribution and depletion profile studies of
Oxytetracycline, different tissue organs (liver, kidney, thigh and chest) were
selected. From these selected organs, total 42 treated including 6 control
samples were collected for analysis. Detail of samples is given in Table 1:
Preparation of standards and
buffers
Stock
and working standards: Lyophilized stock standard (2 µg kg-1)
was provided with ELISA kit. Tetracycline working dilutions were prepared
including 0, 0.025, 0.125, 0.25, 0.5, 1.0 and 2.0 µg kg-1 with dilution buffer and stored at -20°C. A standard of 1000 µg kg-1
was also provided for spiking of negative control samples.
Dilution buffer: Prepared from 4X concentrate
buffer by adding three times double distilled water.
Rinsing buffer: Prepared from 20X concentrate by
dilution of 2 mL of buffer with 38 mL of double distilled water.
Sample dilution buffer: Prepared by adding 2 mL 100%
methanol in 18 mL dilution buffer (mix and store at 4˚C).
Enzyme-linked
conjugate: Concentrate centrifuged at 1000 rcf for 1 min before use, diluted with
dilution buffer in proportion (10 µL:1 mL) and kept in dark until use.
Preparation of tissue samples
A sample weighing 1 g + 0.01 g was taken in 50 mL polycon tube by using
analytical balance (
Assay development
As per layout plan (Table 2),
ELISA was performed in the polystyrene 96 well microtiter plates (provided
along the kit). For assay preparation, 50 µL
of each standard and/or samples and enzyme conjugate were added. After rocking and mixing, sealed the microtiter plate
and incubate for 1 h at 20‒25şC. After incubation, microtiter plates were washed three times with
washing buffer using Microplate ELISA Washer (BioTek, ELx50). After washing,
added 100 µL of substrate solution in
all wells and incubate for 30 min at 20‒25şC in dark to develop colour. After incubation, added 100 µL stop solution (provided along kit) in
each well, mixed gently by rocking the plate manually on top of the working
bench and optical density was measured at 450 nm using Microplate ELISA Reader
(BioTek. ELx808).
Calculations
The
relative absorbance (RA) was calculated for both standards and samples by using
the formula given below and Microsoft Excel was used to construct standards
curve point by point. The RA of unknown samples was interpolated in standards
curve to calculate the concentration of unknown samples.
Confirmatory analysis
An HPLC system (HITACHI L-2000 series) equipped with UV-Vis detector
(L-2420), column oven (L-2300) and auto-sampler (L-2200) was used for
standardization and validation of oxytetracycline detection in tissue for
analysis of selected positive tissue samples.
HPLC conditions
Agilent Zorbax Rx C8 Column (5 µm,
4.6 mm and 150 mm) was used with mobile phase 0.1% TFA: ACN (75:25 v/v and
filtered by filtration assembly using filter AG 0.45 µm, Sartorius) at 252 nm wavelength and 1.0 mL per min flow rate.
Column oven temperature was set at 26şC.
Extraction and cleanup of
samples
Sample weighing 5 g + 0.05 g muscle/tissue was transferred in 50 mL
centrifuge tube and homogenized by for 30 s at 1000 rpm. Added 20 mL of 5%
Trichloroacetic acid (TCA, Sigma), mixed it at 120 rpm for 20 min in shaker
(Julabo, SW(22), Germany) and centrifuged at conditions 4000 rpm, 25şC, 10 min.
Took the supernatant without disturbing the tissue and filtered it using
cellulose filter paper, repeated extraction in twice using 10 mL of 5% TCA.
Supernatant from 3 extractions were collected and filtered through Table
1: Details of Oxytetracycline treated selected tissue
samples
No.
of samples |
Identification
code |
Sample
quantity |
|
Left
Thigh Muscle |
07 |
CHKLT-18-001
to CHKLT-18-007 |
50
g |
Kidney |
07 |
CHKK-18-001
to CHKK-18-007 |
50
g |
Liver |
07 |
CHKL-18-001
to CHKL-18-007 |
20
g |
Right
Thigh Muscle |
07 |
CHKRT-18-001
to CHKRT-18-007 |
50
g |
Right
Breast Muscle |
07 |
CHKRB-18-001
to CHKRB-18-007 |
50
g |
Left
Breast Muscle |
07 |
CHKLB-18-001
to CHKLB-18-007 |
50
g |
RT = Right Thigh, RB
= Right Breast, LT = Left Thigh, LB = Left Breast, L= liver, K = kidney, SP =
Spiked Broiler chicken
Table 2: Microtiter plate layout for tetracycline
analysis by ELISA
|
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
A |
B |
B |
CK-1RT |
CK-1RT |
CK-1RB |
CK-1RB |
CK-1LT |
CK-1LT |
CK-1LB |
CK-1LB |
CK-1L |
CK-1L |
B |
S0 |
S0 |
CK-2RT |
CK-2RT |
CK-2RB |
CK-2RB |
CK-2LT |
CK-2LT |
CK-2LB |
CK-2LB |
CK-2L |
CK-2L |
F |
S0.0625 |
S1 |
CK-3RT |
CK-3RT |
CK-3RB |
CK-3RB |
CK-3LT |
CK-3LT |
CK-3LB |
CK-3LB |
CK-3L |
CK-3L |
D |
S0.125 |
S2 |
CK-4RT |
CK-4RT |
CK-4RB |
CK-4RB |
CK-4LT |
CK-4LT |
CK-4LB |
CK-4LB |
CK-4L |
CK-4L |
E |
S0.25 |
S3 |
CK-5RT |
CK-5RT |
CK-5RB |
CK-5RB |
CK-5LT |
CK-5LT |
CK-5LB |
CK-5LB |
CK-5L |
CK-5L |
F |
S0.5 |
S4 |
CK-6RT |
CK-6RT |
CK-6RB |
CK-6RB |
CK-1K |
CK-1K |
CK-4K |
CK-4K |
CK-6L |
CK-6L |
G |
S1.0 |
S5 |
SP-6RT |
SP-6RT |
SP-6RB |
SP-6RB |
CK-2K |
CK-2K |
CK-5K |
CK-5K |
SP-6L |
SP-6L |
H |
S2.0 |
S2.0 |
SP-6LT |
SP-6LT |
SP-6LB |
SP-6LB |
CK-3K |
CK-3K |
CK-6K |
CK-6K |
SP-6L |
SP-6L |
cellulose filter paper in the separatory funnel. To remove fat from
supernatants, mixed with n-hexane (VWR), then transfer lower layer in
Erlenmeyer flask (Pyrex) and kept for sample loading. SPE cartridge assembly
(Phenomenex) was used for cleanup of samples using cartridges, lower layer was
passed over C18 SPE Cartridges (Strata C18-E, (55 µm, 70A, Phenomenex, 500 mg/6 mL). Cartridges were conditioned with
20 mL methanol and 20 mL double distilled water, the combined supernatant
(treated sample) were loaded and desired analyte were eluted with 4 mL of 0.01 M Oxalic acid (Merck) in MeOH (VWR). The
sample was dried using Evap. system having capacity 24 samples at a time (Romer
labs, USA) at 35‒40°C under vacuum. Re-constitute extract with 400 µL mobile phase 0.1% TFA: ACN (75:25) v/v Vortex and filter with
0.45 µm nylon membranes (PTFE membrane syringe filters, 4 mm, 0.45 µm) in sample injection vial (VWR) for
injection to Amber glass vial 1.5 mL VWR HPLC grade for determination of
oxytetracycline residues.
Preparation of calibration curve
Different oxytetracycline working standards (above and below the EU MRL)
including 25, 50, 100 and 200 µg kg-1
were used for standardization and validation studies. Calibration curve was prepared between concentration and their respective areas to
determine limit of detection (LOD) and limit of quantification (LOQ) for
method.
Results
Temperature and weight of each
animal was recorded twice a day throughout the experimental duration (one
month). When animals gained average weight of 600 ± 20 g, then they were
considered for the recommended dose as desired in experiment. Applied drug was
also standardized with HPLC method with provided conditions and good response
was observed. The declared period for Oxytetracycline in injectable
(Hydrochloride salt, 20 ppk) dose was 5 days before slaughtering.
Screening of samples through
ELISA
ELISA kit was standardized by
calculating inhibition conc. IC20 and IC50 (criteria for
test performance), relative absorbance found inversely proportional to the
concentration of the tetracycline. A linear regression was obtained (y =
-20.86ln(x) +34.762) with correlation (r2) of 0.9996 as shown by
Fig. 1. To check criteria for test performance, lowest detection limit (IC20)
and middle of the test (IC50) were found to be 0.12 µg kg-1 and 0.5 µg kg-1, respectively which
is well below the maximum residue limit for tissue (200 µg kg-1). According to Fig. 2, average concentrations of oxytetracycline in
poultry tissues decreased gradually with time and after 117 h, residues were
found well below the MRL value. The detectable residue concentrations were calculated as
4870, 4062, 3354, 3142, 1556 and 101 μg kg-1 at 1, 8, 16, 32, 64 and 120 h intervals, respectively. Results showed that declared period in proportion to our findings. These are not in accordance to
Bangladesh due to may be difference in environmental conditions (Khatun et al.
2018).
In order to check the drug distribution profile, it was found that
concentration of the left sides (breast/thigh) was lowered than right side of
broiler chicken as shown by Table 3. Most important is that the treated samples
were compared with non-treated and concentration was negligible and declared as
negative control. It was cleared that the residues found in kidney and liver
was more than other parts of body, this is because organs of metabolism and
excretion were expected to have higher concentrations of these residues than
breast and thigh tissue. In order to calculate recovery (%), known negative
samples (tissue, kidney and liver) were spiked with different oxytetracycline
concentrations below and above the MRL. Overall results indicated that recovery
was calculated from 75 to 86% as shown in Table 4.
Standardization and validation by HPLC-UV
Table 3: Oxytetracycline residue depletion in treated
poultry by ELISA (n = 3)
Withdrawal
time (h) |
Animal
No. |
Concentration (µg
kg-1) in different chicken tissues |
|||||
RB |
LB |
RT |
LT |
Kidney |
Liver |
||
1 |
1 |
2120 |
1680 |
2304 |
24 |
2020 |
2480 |
8 |
2 |
22000 |
2256 |
2200 |
2340 |
2600 |
2400 |
16 |
3 |
2200 |
1680 |
2400 |
2000 |
2600 |
2200 |
32 |
4 |
1500 |
1640 |
1632 |
1540 |
2160 |
1440 |
64 |
5 |
288 |
298 |
500 |
750 |
560 |
1481 |
120 |
6 |
150 |
97 |
101 |
144 |
540 |
580 |
Fig. 1: Standard curve used
for Oxytetracycline detection in chicken tissue by ELISA
Fig.
2: Withdrawal period of
Oxytetracycline in broiler chicken through ELISA
Before confirmatory analysis,
standardization was done on at four different days by using HPLC UV-Vis detector. Good linearity of calibration curve was found through regression equation y = mx + C with ccorrelation coefficient (R2) 0.9724 using different oxytetracycline working standards including 25, 50, 100
and 200 µg kg-1 (Fig. 3). Retention time was found as 1.85 ± 0.03 min and peaks area was found
directly proportional to the standard concentration 25, 50, 100 and 200 µg kg-1 (Fig. 4). After 128 h, oxytetracycline residues were found less than MRL i.e., 200 µg kg-1 as shown by Fig. 5 and reported values are in
Table 5.
Discussion
This study relates to the public
safety and awareness to the local farmers for safe handling of drugs. Oxytetracycline is being used as
drug to enhance the growth of the poultry (Bosha
et al. 2019). In order to meet the validation criteria, all parameters were followed
as per 657/2002/EC guidelines (GL49 2015). The usage of the commercial drug
in the animal then their residues still retained the body even till 120 h. The HPLC validated method employed for analysis is accurate and capable
of determining the tetracyclines precisely over the concentration range 25 to
250 ppb.
The LOD value is minimum amount of
the analyte detected, calculated as Xn+3SD under optimal conditions. Similarly, the LOQ is smallest
quantity of analyte that can be measured with acceptable accuracy and precision
(95% confidence level). The LOQ was found by calculating recovery and precision
data i.e., CV, and was defined as the
lowest validated spike level meeting the requirements of a recovery was found
from 85 to 93% by spiked samples with oxytetracycline by HPLC as shown by Table
6. LOD and LOQ of the method were found to be 16 and 25 µg kg-1 respectively. Signal to noise ratio was found to
be 3:1 in acceptable range.
Table 4: Recovery calculations of different
Oxytetracycline concentrations in known negative samples (n = 3)
Matrix |
Spiking Conc. (µg kg-1) |
Analysis by ELISA |
Analysis by HPLC |
||
Recovery (%) |
CV |
Recovery (%) |
CV |
||
Liver |
100 |
75 |
5.4 |
85 |
3.8 |
150 |
82 |
3.8 |
87 |
2.8 |
|
200 |
85 |
4.5 |
92 |
3.8 |
|
Kidney |
100 |
79 |
3.5 |
87 |
2.8 |
150 |
85 |
5.4 |
86 |
6.7 |
|
200 |
82 |
6.5 |
93 |
8.6 |
|
Muscle |
100 |
82 |
3.2 |
86 |
3.8 |
150 |
86 |
2.4 |
92 |
3.9 |
|
200 |
75 |
1.5 |
85 |
5.4 |
CV:
Coefficient of variation
Table 5: Oxytetracycline residues depletion in
treated poultry by HPLC (n = 3)
Withdrawal
time (h) |
Animal
No. |
Concentration (µg
kg-1) in different chicken tissues |
|||||
RB |
LB |
RT |
LT |
Kidney |
Liver |
||
1 |
1 |
7164 |
5844 |
7195 |
5150 |
1076 |
2786 |
8 |
2 |
5139 |
6095 |
4736 |
4820 |
763 |
2819 |
16 |
3 |
3619 |
2927 |
5783 |
4526 |
620 |
2848 |
32 |
4 |
1425 |
4421 |
4702 |
4341 |
995 |
1800 |
64 |
5 |
680 |
179 |
2800 |
280 |
890 |
1785 |
120 |
6 |
210 |
21 |
235 |
80 |
838 |
1741 |
Fig.
3: Standard curve used for
Oxytetracycline detection in chicken tissue by HPLC
Most important findings are that
the concentrations of the tetracycline were varied between slaughtered
samplings. The possible reason is the reabsorption or recirculation of OTC in
the birds’ body to other organs or parts. As a target tissue bone tissue (breast/chest) were collected along with lever and kidney, detected OTC
concentrations in treated broiler chicken bone tissue. It is also being
reported that this were a more complex link takes place between the tissue and
the rings of the basic tetracycline structure (GL49
2015; Odore et al. 2015). Most important is that the
treated samples were compared with non-treated and concentration was negligible
and declared as negative control (Khatun et al. 2018). It was cleared that the residues found in
kidney and liver was more than other parts of body, this is because organs of
metabolism and excretion were expected to have higher concentrations of these
residues than breast and thigh tissue.
Further, it is very important to
consider the interaction of the drug with the body, OTC is lipophilic with a
large distribution volume, because of this reason the concentration in edible
tissue was found higher (Mestorino et al. 2007). Further, it can be
evaluated that by passage of time concentration in the body parts decreases but
definitely it is continuously deposited in the manure/droppings. Literature
provides evidence that droppings may be a route of contamination and
dissemination of OTC residues in the environment indeed a great health risk for
the soil microflora. Poultry birds were free to move in specified place and as
a result dust in the air can result in the dissemination of OTC from treated to
untreated birds. The long half-life of OTC and the use of broiler droppings to
fertilize soil also present a risk for the transfer and persistence of OTC. It
is a threat to the food cycle that excretion of Oxytetracycline bind to soil (Carballo et al.
2016; Pokrant et al. 2021).
Table 6: Recoveries of
Oxytetracycline spiked matrices collected from control poultry (n = 3)
Spiking Concentration (µg kg-1) |
Analysis by ELISA |
Analysis by HPLC |
|||
Matrix |
Recovery (%) |
CVa (%) |
Recovery (%) |
CVa (%) |
|
Liver |
100 |
75 |
5.4 |
85 |
3.8 |
150 |
82 |
3.8 |
87 |
2.8 |
|
200 |
85 |
4.5 |
92 |
3.8 |
|
Kidney |
100 |
79 |
3.5 |
87 |
2.8 |
150 |
85 |
5.4 |
86 |
6.7 |
|
200 |
82 |
6.5 |
93 |
8.6 |
|
Muscle |
100 |
82 |
3.2 |
86 |
3.8 |
150 |
86 |
2.4 |
92 |
3.9 |
|
200 |
75 |
1.5 |
85 |
5.4 |
a Coefficient of variation
Fig. 4: Oxytetracycline chromatographs of standards
and sample (a) 50 µg kg-1 (b) 100 µg kg-1
(c) 200 µg kg-1 and (d)
Oxytetracycline treated tissue
Fig. 5:
Withdrawal period of Oxytetracycline in broiler chicken through HPLC
Withdrawal period reported by Bangladesh research article is not
resembled to our findings, it is due to difference in environmental conditions. In comparison to Bangladesh, the withdrawal periods for
oxytetracycline in poultry birds were found greater throughout the study period
on all visited farms. That’s why; they set the different MRP value to control
illegal use of oxytetracycline. This means residue value of oxytetracycline can
be used in marketed poultry which was found in the build-up relationship study
as shown by the research article (Khatun et al. 2018). Regression equation was found to be y=339866x=E+07 for
confirmatory analysis, R2=0.9724 as shown in Fig. 3 and
chromatograms Fig. 4. Our findings are in accordance with other studies (Cinquina et al.
2003).
So, in our experiment maximum oxytetracycline residue
values in marketed poultry were found after one hour followed by drug
administration. The present finding is agreed by researchers (Agha et al.
2003). According to FARAD digest, withdrawal period in egg laying
poultry for tetracycline were approximately more than 5 days in Ireland,
Canada, US and Australia (Marmulak et al. 2015). Our results are in
accordance with Mund et al. (2017) as withdrawal period was observed 5
days. However, only trace concentrations of OTC were
detected in droppings and litter from sentinel birds. These findings establish
the first evidence that there is a low likelihood of the transfer of OTC
residues from treated birds to the environment and to untreated birds in
adjacent or separate pens, which needs to be further studied.
Conclusion
This study provides the
depletion and distribution of oxytetracycline in edible parts of broiler
chicken like muscles, liver and kidney. Withdrawal period of oxytetracycline
was calculated 117 h by ELISA while 128 h by HPLC. It is of great concern
especially for the farmers to wait until the residues of the injected dose
becomes less than the MRL. According to the study, it is strongly suggested that
the need for more stringer regulatory authorities for the use of antibiotics in
the poultry farms, as well as the survey of chicken for drug residues prior to
marketing.
Acknowledgments
We are grateful to the International Atomic Energy
Commission (IAEA) for providing commercial ELISA kits under Technical
Cooperation Project RAS 5078 entitled “Enhancing food safety laboratory capabilities and establishing a network
in Asia to control veterinary drug residues and related chemical components” to
develop validated protocols for monitoring veterinary drug
residues in different food matrices.
Author
Contributions
MM
planned experiments and conducted
sampling, analysis, interpretation of results, and write up. UM
contributed in planning of experiment. MIC and MSS contributed in the experiment
execution and write up. GH helped during collection of samples and analysis.
Conflicts of Interest
All authors declare no conflict of interest.
Data Availability
Data presented in this study
will be available on a fair request to the corresponding author.
Ethics Approval
Experiment
was conducted at Animal House, NIAB with proper handling and as per ARRIVE
guidelines.
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