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Oxytetracycline Residue in Tilapia

A. K. A. Barman
Department of Fisheries Technology and Quality Control, Sylhet Agricultural University, Bangladesh

M. M. Hossain*
Department of Fisheries Technology and Quality Control, Sylhet Agricultural University, Bangladesh

M. M. Rahim
Institute of Food science and Technology, Bangladesh Council of Scientific and Industrial Research, Bangladesh

M. T. Hassan
Institute of Food science and Technology, Bangladesh Council of Scientific and Industrial Research, Bangladesh

M. Begum
Institute of Food science and Technology, Bangladesh Council of Scientific and Industrial Research, Bangladesh

Abstract/ Executive Summary:

The present study was conducted to determine the persistence of oxytetracycline residue in Tilapia (Oreochromis niloticus) available in local fish markets of Sylhet Sadar Upazila. To carry out this experiment, 24 fish samples were randomly collected from four (4) local fish markets under study area from March 2016 to August 2016. Fish samples were analyzed by using High Performance Liquid Chromatography (HPLC) method to detect amount of residues of oxytetracycline. In this study, detectable oxytetracycline residues were observed in five (5) samples of Tilapia ranged between 23.77-39.94 ppb (mean 38.88±2.99 ppb). Oxytetracycline residues less than limit of detection were also found in 19 (79.17%) samples. The detected residues of oxytetracycline in these fish samples did not exceed the maximum residue limit (MRL) 100 ppb recommended by the European Commission. However, long term persistence of high level oxytetracyclines could be a potential hazardous for public health. For this reason supervision of antibiotic uses and monitoring of optimum MRL in Tilapia are utmost needed for farmed fish species.

Keywords: Oxytetracycline; Tilapia; Residues; HPLC; MRL

Location: Sylhet Sadar Upazila

Start Date: 00-03-2016

End Date: 00-08-2016

Program Area: Animal Health and Management

Commodity: Tilapia

Objectives:

Present study is aimed to determine the presence of oxytetracycline residues in Tilapia (Oreochromis niloticus) available in local fish markets of Sylhet Sadar Upazila of Bangladesh.

Methodology:

Study area and collection of fish samples Four (4) fish markets of Sylhet Sadar Upazila were selected for the purpose of collection of fish samples. The selected fish markets were Kazir Bazar, Baluchar Noya Bazar, Mejor Tilah Bazar and Tuker Bazar. Twenty four (24) samples of Tilapia (Oreochromis niloticus) were collected from March 2016 to August 2016. The fish samples were collected individually from the selected fish markets in separate marked polythene bags. Then collected samples were kept in an ice box with sufficient amount of ice. After collection, samples were transported to Microbiology Laboratory of Department of Fisheries Technology and Quality Control, Sylhet Agricultural University, Sylhet and kept in a deep refrigerator. For analyzing purpose, the samples were transported to Food Toxicology Laboratory of Bangladesh Council of Scientific and Industrial Research, Dhaka with icing condition and kept in a deep refrigerator at - 20°C for promoting analysis. HPLC analysis of oxytetracycline residue in fish samples HPLC system Agilent Liquid chromatography consisting of i. Agilent : Solvent delivery system series 1100 (Isocratic pump) ii. Agilent series 1100 Column oven iii. Agilent 1200 series Fluorescence detector for HPLC iv. Manual injector capable of injection volumes up to 50 micro liters v. Software : Chem Station Rev A. 10.02 vi. Column: Phenomenex – Gemini 5u C18 110A (250 ´4.60 mm) Chemicals The following chemicals were used. Oxytetracycline hydrochloride (Sigma Aldrich), Methanol – HPLC grade (Merck), Magnesium Acetate (Extra Pure, BDH), Citric Acid – Monohydrate (Merck), Sodium Hydrogen phosphate – anhydrous (Merck), EDTA - disodium dehydrate (Scharlu), Acetic acid (Riedel de Haein), Imidazole (Merck), n-Hexane (Merck), HPLC grade water. Solution The following chemicals were prepared. Mcllvaine Buffer, Mcllvaine Solution: (Mcllvaine Buffer/0.1 M EDTA), Extraction Solution, Imidazole Buffer (1M), Mobile Phase. Preparation of calibration curve Calibration curve was prepared from injecting corresponding concentrations of oxytetracycline standard solutions of 25, 50, 75, 100, 125 and 150 ppb. The linear t curve obtained using, y = mx + b; = 0.0132368x+ 0.04568 Where, y = peak area and x = concentration of oxytetracycline (ppb) and the correlation coefficient (r²) = 0.99687. The detection limit for oxytetracycline was 23.62 ppb. The mean retention times (RT) of the oxytetracyclines were found between 4.031 to 4.25 minutes. Sample preparation and extraction After adequate thawing, few grams of muscle sample were collected from fish and minced using chopping board and knife. Then weighed 5.0 g of partially thawed intact samples was taken separately into 50 mL polypropylene centrifuge tubes. Then 20 mL extraction solution was added to each sample and homogenized by using Ultra Turrax until samples were uniformly blended (15- 30 seconds). After rinsing probe with 4 mL of extraction solution, rinses were added to centrifuge tube. Tubes were capped and shaken 10 minutes on a flatbed shaker at speed. Contents of tubes were centrifuged at a minimum 8000 rpm for 20 minutes at approximately 15°C. Supernatants were poured into a second centrifuge tube carefully for not allowing any transfer of tissue. Five (5) mL n-Hexane was added to solution and briefly shaked. Upper layer was removed. A single Whatman #1 filter paper was placed into a 5.5 cm Bucher filtering funnel and attached to a 250 mL sidearm ?ask with vacuum condition. Centrifuge tubes were rinsed with 4 mL extraction solution and filtered into a flask.An SPE cartridge was attached to an SPE vacuum manifold. The cartridge was conditioned with 10 mL methanol followed by 15-20 mL distilled water at approximately 1.5-2.5 mL/minute with vacuum as required. The elute were discarded. A 75 mL reservoir was connected to the cartridge. The filtered sample extracts were added to the SPE reservoir. The flask was rinsed with approximately 4 mL buffer solution and was added to the rinses to the reservoir. Extract was drained through the column by gravity. The sidearm flask was rinsed with 20 mL distilled water and added to reservoir. After draining under – 10 mm Hg vacuum cartridges were allowed to go dry after the water rinse is completed, and continue to draw air through the cartridge for at least 2 minutes. Then Elute was discarded. A 15 mL graduated centrifuge tube was placed in the vacuum apparatus to serve as a collection vessel and elute oxytetracycline from the cartridge with 6 mL elution solution. Vacuum condition was applied to initiate flow continue elution. Once flow stops, vacuum applied to remove residual solvent from the cartridge. Tubes were removed from vacuum manifold and vortex was done. The tube containing elute were placed in the sample concentrator at the temperature at 40-50°C to reduce volume of the elute to 0.5-0.25 mL under a stream of dry nitrogen. Final volume was adjusted to1 mL with methanol + water (1:1) and briefly vortexing. Then approximately 1.0 mL extract were drawn into a 3 mL syringe and was filtered through a syringe into an HPLC vial (1.5 mL). The remaining extract was store at -20° C. HPLC parameters for analysis of oxytetracycline residues The concentrate extract were subjected to analysis by Agilent 1100 series HPLC system. Mobile phase: Buffer: Methanol = 70:30; Injection volume: 20 mL; Flow rate: 1 mL/min, Column temperature: 30°C; Detector: Fluorescence detector (Agilent 1200 series); Excitation wavelength: 380 nm; Emission wavelength: 520 nm and Run time: 12 minutes. Recovery evaluation The precision of the method was determined as recoveries of oxytetracycline spiked blank samples. For this two replicate oxytetracycline free fish samples were spiked with 150 ppb oxytetracycline standard just before test. Statistical analysis For preliminary processing of raw data obtained from this study was analysed by using the computer software like Microsoft Excel, SPSS etc.

Source of Information: Bangladesh J. Sci. Ind. Res. 53(1), 41-46, 2018

Article Link (Online Journal):

Funding Source:

1.

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From the results and findings obtained in the present investigation it can be concluded that small portion of Tilapia (Oreochromis niloticus) available in local fish markets of Sylhet Sadar Upazila are contaminated with oxytetracycline residues. It is a positive sign that in most samples oxytetracycline residues were below the detection limit and detected oxytetracycline residues in the positive samples did not exceed the maximum residue limit recommended by the European Commission. However, some remedial and precaution measures are also necessary to ensure disease free fish for public health safety. Oxytetracycline and other antibiotics should only be used for treatment of fishes in prescribed doses. The withdrawal period of antibiotics should be taken into consideration before marketing of fish. Awareness build up programs and regular residue monitoring of marketed fish should be performed by government authorities such as Department of Fisheries, Fish Inspection and Quality Control etc. Finally investigation on antibiotic resistance bacteria in the intestine of fish and sediments of farming areas and antibiotics residue monitoring study on other species of fishes are utmost needed.

Knowledge Management: Journal