Sampling: Five most popular dry fish samples namely bombay duck (loittya), ribbon fish (chhuri), shrimp (chingri), Chinese pomfret (rupchanda) and Indian salmon (lakhua) were collected from Asadgonj (whole sell market of dry fish) of Chittagong, Bangladesh in July 2008. Total number of samples were 20. The control samples of five different fishes were collected from drying yards of Sonadia island (a fish processing zone of Bangladesh) that are known sample treated with no insecticides and taken into account as blank. Apparatus: Mincer fish chopper (Weisser No. 81 K), soxhlet extractor, chromatographic tube (20 mm i.d. 50 cm long), sample concentrator (Techne dry block DB.3) and gas chromatograph (GC-14B, Shimadzu). Reagents: Acetone, diethyl ether, dimethylformamide saturated with petroleum ether, n-hexane, petroleum ether (30-60°C), petroleum ether (30-60°C) saturated with dimethylformamide, 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 10 H2O), 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, cold and stored in tightly stoppered container, prior to use heated for at least 5 hours at 130°C, cold and add 5% w/w water, shake this mixture for at least 20 min and stored in a container for at least 10 hours) and cotton wool. All the solvents used for the analysis purchased from Merck, Germany. DDT and heptachlor standards were obtained from Sigma Chemicals, USA. Sample preparation: All the samples were finely comminuted in a mincer; heating of the samples during comminuting is avoided by briefly chopping several times (Peter and Zeumer, 1987). Each comminuted sample of dry fishes was divided into four parts. 1st part was prepared as without washing (normal); 2nd part was prepared after vigorous cold water washing (cold washing); 3rd part was prepared after vigorous hot water washing (hot washing) and 4th part was prepared after washing followed by 10 min boiling (boiling treatment). 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 dimethylformamide (Peter and Zeumer, 1987). Clean up: Clean up was done in two steps (Peter and Zeumer, 1987)
a) Dimethylformamide-petrolium ether partition: Transfer the solution (dissolved in 25 mL petroleum ether saturated with dimethylformamide) to 250 mL separatory funnel. Rinse the flask with a small portion of a previously measured amount of 75 mL dimethylformamide. Then add the remainder of the dimethylformamide to the separatory funnel, and shake vigorously for 1 min. Drain the dimethylformamide phase, and again extract the petroleum ether phase with 10 mL dimethylformamide. 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 subsequent partition. Then extract successively with a 40 mL portion and three 25 mL portions 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.
b) 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 approximately 2 cm layer of sodium sulphate. Drain the supernatant solvent to the top of the column packing. Pipette the sample solution onto 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 rinsings to the column, and also 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. Concentrate the eluate near to dry and add 5 mL n-hexane to the eluate. Again concentrate the eluate to 1 mL.
Sample analyses: The DDT and heptachlor residues were analyzed by GC-14B, Shimadzu with an electron capture detector (ECD), a manual sampler and GC solution software. A column of 3.1 m x 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 chromatographic separation of insecticides. The temperature was fixed for the injector at 250°C, column at 280°C and 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 was identified by injecting a high concentration of the standard (0.5 and 0.3 ppm) and the retention time for DDT and heptachlor were determined. Then calibration was done at 3 points (25, 50 and 100 ppb) by a composite stock standard solution. GC system was calibrated using the external standard technique. Individual standard stock solution (100 mg/L) was prepared by weighing appropriate amounts of active ingredients in a brown bottle with a Teflon-lined screw cap and dissolving the weighed standard in HPLC grade hexane. The stock standard solution was used to prepare primary dilution standards. The appropriate volume of each individual stock solution was taken in a volumetric flask and mixed solutions to obtain a composite stock standard solution.
Analytical quality control: Gas chromatograph equipped with ECD was checked for linearity. The instrumental limit of detection for GC-ECD was 1.0 μg/L for organochlorine pesticides. An aliquot of dry fish samples was collected as blank and treated exactly as a sample including exposure to all glassware, equipment, solvents and reagents used with the sample matrix. No analyte peak was detected in the laboratory reagent blank. An aliquot of fortified samples matrix was 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 a level of 25 ppb were 96-100% for heptachlor and 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 and heptachlor. 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.