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Effects of Oxytetracycline Residues in Thai Koi (anabas Testudineus Bloch) Collected from Sylhet, Bangladesh

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

Md. Motaher Hossain
Department of Fisheries Technology and Quality Control, Sylhet Agricultural University, Sylhet, Bangladesh

Md. Golam Rasul
Department of Fisheries Technology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Bhaskar Chandra Majumdar
Department of Fisheries Technology, Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur-1706, Bangladesh

Md. Matiur Rahim
Institute of Food science and Technology, Bangladesh Council of Scientific and Industrial Research, Dhaka, Bangladesh

Abstract/ Executive Summary:

The present study was conducted to determine the levels of oxytetracycline residues in Thai Koi (Anabas testudineus Bloch) collected from some local fish markets of Sylhet Sadar Upazilla, Bangladesh during March to August, 2016. For the purpose of the study, 24 fresh Thai Koi fish samples were randomly collected from four (4) local markets of Sylhet Sadar Upazilla. Concentration of oxytetracycline residues in collected fish samples were determined by High Performance Liquid Chromatography (HPLC) at the Food Toxicology Laboratory of Institute of Food Science and Technology, Bangladesh Council of Scientific and Industrial Research, Dhaka. The results showed that 9 (37.50%) samples with detectable amount of oxytetracycline residues (mean = 42.30±3.00 ppb) were found in Thai Koi. Oxytetracycline residues level below detection limit was also reported in 15 (62.50%) samples of Thai Koi during this study. The detected residues of oxytetracycline in this fish sample did not exceed the maximum residue limit (MRL) 100 ppb recommended by the European Commission. Though residue level of oxytetracycline is lower than the MRL but long term exposure could be hazardous for human health. For that reason, control of antibiotic usage in aquaculture, regular residue monitoring, legislations and regulations for the use of antimicrobials in aquaculture and food safety education are utmost things in this regard.

Keywords: Anabas testudineus, High Performance Liquid, Chromatography, Oxytetracycline, Recommended level

Location: Sylhet Sadar Upazilla

Start Date: 00-03-2016

End Date: 00-08-2016

Program Area: Resource Development and Management

Commodity: Koi/Climbing

Objectives:

To determine the occurrence of oxytetracycline residues in Thai koi fish which are marketed in local fish markets of Sylhet Sadar region of Bangladesh.

Methodology:

This study was carried out in Sylhet Sadar Upazilla, the southeastern part of Bangladesh. The Experimental fish were collected from four (4) selected fish markets of Sylhet Sadar Upazilla. The selected fish markets were Kazir Bazar, Baluchar Noya Bazar, Mejor Tilah Bazar and Tuker Bazar under the study area. For analysis purposes 24 samples of Thai Koi (Anabas testudineus) were collected between the periods March to August, 2016. At first fish samples were collected individually from the selected fish markets in separate marked polythene bags and were kept in an ice box. After collection of fish samples, they 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 then 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 prior to analysis. Agilent (1100 series) solvent delivery system (Isocratic pump) connected Fluorescence detector with C18 (Phenomenex – Gemini 5μ, 250 ´4.60 mm) column were used as HPLC device. Manual injector capable of injection volumes was up to 50 microliters. Laboratory grade 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) and HPLC grade water were used as necessary chemicals. Mcllvaine Buffer, Mcllvaine Solution (Mcllvaine Buffer/0.1 M EDTA), Extraction Solution, Imidazole Buffer (1M) and Mobile Phase (Buffer and Methanol ratio of 70:30 v/v) were also utilized as various solutions. Calibration curve was prepared from injecting corresponding concentrations of oxytetracycline standard solution of 25, 50, 75, 100, 125 and 150 ppb. The linear fit 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. After adequate thawing of frozen fish, about 20 gm of muscle sample were collected from the dorsolateral area of fish and minced using chopping board and knife. Then weighed 5.0 gm minced muscle samples were taken 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 care- fully not to transfer any 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 flask with vacuum condition. Centrifuge tubes were rinsed with 4 mL Ex- traction solution and filtered into a flask. An SPE (Solid Phase Extraction) 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 necessary. 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 to 1 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. The concentrate extract were subjected to analysis by Agilent 1100 series HPLC system. For analyzing purpose 20 mL extract volume were injected to HPLC system. Flow rate was maintained 1 mL/min and column temperature was 30°C. Excitation wavelength and emission wavelength were 380 nm and 520 nm, respectively. 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. For preliminary processing of raw data obtained from this study was analysis by using the Microsoft Excel, SPSS software etc.

Source of Information: Archives of Agriculture and Environmental Science 3(2): 174-179 (2018)

Article Link (Online Journal): DOI: 10.26832/24566632.2018.0302011

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Results and findings of the present study shown that small portion of Thai Koi (Anabas testudineus) available in local fish markets of Sylhet Sadar Upazilla contaminated with oxytetracycline residues. About nine numbers of positive samples with detectable level of oxytetracycline residues were found during the study period. This residue occurrence indicates abuse of antibiotics in commercial fish production in this area without following specific rule and also indicates lack of farmer’s aware- ness about antibiotic residue at the root level. Though, oxytetracycline residues found in the positive samples did not exceed the maximum residue limit recommended by the European Commission but it is an important indicator of antibiotic contamination in fish. Fish is considered as a major source of protein in Bangladesh and in these consequences some remedy measures should be taken to ensure better public health. That’s why, arising of public awareness about antibiotic use and its withdrawal period, antibiotic residue monitoring of cultured fish species by governmental authority like Department of Fisheries (DoF), Fish Inspection and Quality Control (FIQC) and monitoring of antibiotic resistance bacteria from fish and farming environment are needed.

Knowledge Management: Journal