Sampling: Samples were collected from Chittagong New Market, Asadgonj, Reajuddin Bazaar, Cox’s Bazar Sadar, Moheshkhali and Teknaf. Four commercially avail-able species of dry fishes namely Ribbon fish (Lepturacanthus savala), Sin Croaker (Johnius dussumieri), Bombay duck (Harpodon nehereus) and Shrimp (Mixed species) were collected from each market. These dry fish species were selected for this experiment due to their greater market demand and availability. Total numbers of samples were 24. The control samples of three different fishes were collected from drying yards of Moheshkhali Island that were known sample treated with no insecticides and taken into account as blank.
Apparatus: Mincer fish chopper (Weisser No. 81 K), Soxhlet extractor, separatory funnels (500 ml and 200 ml), chromatographic tube (20 mm I.D 50 cm long), sample concentrator (Techne dry block DB.3), round bottomed flask (500 ml and 100 ml), volumetric flask (50 ml and 10 ml), gas chromatograph (GC-14B, Shimadzu), syringe (10 μl, Hamilton Co.).
Reagents: Acetone, diethyl ether, dimethyl formamide saturated with petroleum ether, nhexane, petroleum ether (30°C - 60°C), petroleum ether (30°C - 60°C) saturated with dimethyl formamide, eluting mixture I (petroleum ether + diethyl ether 94:6 v/v), standard solutions, eosin solution (2 mg in 100 ml), sodium sulfate solution (2 g/100 ml NaSO4·10H2O), sodium sulfate anhydrous (heated for at least 2 hours at 550°C), florisil 60 - 100 mesh (heated for at least 2 hours at 550°C, cool and stored in tightly stopper container, prior to use heated for at least 5 hours at 130°C, cool and add 5% w/w water, shake this mixture for at least 20 min and stored in a container for at least 10 hours), cotton wool. All the solvents used for the analysis purchased from MERCK, Germany. Dichlorodiphenyltrichloroethane (DDT) and heptachlor standards were obtained from Sigma Chemicals.
Sample Preparation: All the samples were finely comminuted in a mincer; heating of the samples during comminuting was avoided by briefly chopping several times.
Extraction: Triturate a sample of 25 g, with sodium sulfate to dry, powdery mixture, with the aid of an extraction thimble; extract the mixture exhaustively with Petroleum Ether in Soxhlet apparatus. Concentrate just to dryness the extract solution by a concentrator and dilute to 25 ml with petroleum ether saturated with dimethyl formamide. Cleanup was done in two steps according to the procedure followed by Hans and Zeumer (1987).
Dimethyl Formamide-Petrolium Ether Partition: Transfer the solution (dissolved in 25 ml petroleum ether saturated with dimethyl formamide) to 250 ml separatory funnel. Rinse the flask with small portion of a previously measured amount of 75 ml dimethyl formamide. Then add the remainder of the dimethyl formamide to the separatory funnel, and shake vigorously for 1 min. Drain the dimethyl formamide phase, and again extract the petroleum ether phase with 10 ml dimethyl forma- mide. Transfer the combined dimethyl formamide phases to a 500 ml separatory funnel, and add 200 ml sodium sulfate solution. Add a few drops of eosin solution to achieve better recognition of phase separation in the sub- sequent partition. Then extract successively with a 40 ml portion and three 25 ml potions of petroleum ether for 1 min each time. Wash the combined petroleum ether phases with 10 ml water, dry on sodium sulfate, filter through a cotton wool plug, add 5 ml n-hexane, and concentrate to approximately 5 ml.
Florisil Column Chromatography: About half filled a chromatographic tube with petroleum ether, and sprinkle with 30 g florisil in small portions through a funnel with stopcock open, tapping the column in the process. Cover the florisil with an approx. 2 cm layer of sodium sulfate. Drain the supernatant solvent to the top of the column packing. Pipette the sample solution on to the column. Let the solution percolate to a level of 1 - 2 mm above the top of the column. Then rinse the flask with small portions of eluting mixture I, add the rinsing to the column, and let them percolate to a level of 1 - 2 mm above the top of the column. Next eluate the column with the remainder of the total 200 ml amount of eluting mixture I, at a flow rate of about 5 ml/min. Add 5 ml n-hexane to the eluate, concentrate the eluate to 5 ml and dilute with n-hexane to 10 ml.
Sample Analyses: The DDT residues were analyzed by GC-14B, Shima- dzu with an electron capture detector (ECD), a manual sampler and GC solution software. A column of 3.1 m × 3.2 mm; I.D glass spiral; stationary phase silicon OV-17, 5%, aging 300°C, support chromosorb-W-AW-DMCS, mesh 80/100, 1 μm film thickness was used for the chro- matographic separation of insecticides. The temperature was fixed for the injector at 250°C, column at 280°C, detector at 280°C. The carrier gas was nitrogen with a 60 ml/min-flow rate. 1.0 μl sample was injected for each run and the running time was 25 min. Standards’ peak were identified by injecting high concentration of the standard (0.5 ppm and 0.25 ppm) and the retention time for DDT was determined. Then calibration was done at 3 points (25 ppb, 50 ppb and 100 ppb) by composite stock standard solution. GC system was calibrated using external standard technique. Individual standard stock solution (100 mg/L) was prepared by weighting appropriate amounts of active ingredients in a brown bottle with a Teflon-lined screw cap and dissolving the weighed standard in HPLC grade hexane. Stock standard solution was used to prepare primary dilution standards. Appropriate volume of each individual stock solution was taken in a volumetric flask and mixed the solutions to obtain com- posite stock standard solution.
Analytical Quality Control: Gas chromatograph equipped with ECD was checked for linearity. Instrumental limit of detection for GC-ECD was 1.0 μg/L for organochlorine pesticides. An aliquot of dry fish samples which were collected as blank and treated exactly as a sample including exposure to all glassware, equipment, solvents and reagents used with the sample matrix. No analytic peak was detected in laboratory reagent blank. An aliquot of fortified samples matrix were prepared to which known quantities of the pesticides were added in the laboratory in ppb range. This laboratory fortified matrix was analyzed exactly like the sample. Extraction and clean up were done as mentioned and the recoveries from untreated control samples of dry fish fortified with the analyzed compounds at level of 25 ppb was 98% - 100% for DDT. Prior to injection of the first sample solution, a standard solution was injected at least three times to check the operating conditions and the constancy of the detector signals. Further linearity of the ECD signal was checked by injecting serial dilutions of DDT. A standard solution injected after at least every other sample solution so that any alterations of the gas chromatographic system recognized due to column contamination.