Sampling
Six most popular dry fish samples namely Bombay duck (Loittya), Ribbonfish (Chhuri), Shrimp (Chingri), Hilsha shed (llish), Chinese pomfret (Rupchanda) and Indian salmon (Lakhua) were collected from Asadgonj (whole sell market for dry fish) of Chittagong, Bangladesh at winter season (December) and another six samples of same species from the same place were collected at rainy season (July). The total number of samples was twelve. The control samples of six 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, separatory funnels (500 ml & 200 ml ), Chromatographic tube (20 mm i.d. 50 cm long), sample concentrator (Techne dry block DB.3), round bottomed flask (500 ml & 100 ml ), volumetric flask (50 ml & 10 ml ), Gas Chromatograph (GC-14B, Shimadzu), syringe(10 ul, Hamilton Co.).
Reagents
Acetone, Diethyl ether, Dimethyl Formide desaturated with petroleum ether, n-hexane, petroleum ether (30-60°C ), petroleum ether (30-60°C ) saturated with Dimethyl Formide de, an eluting mixture I ( petroleum ether + diethyl ether 94:6 v/v), standard solutions, eosin solution (2 mg in 100 ml ), sodium sulphate solution (2 g/ 100 mina2S04 10 H20), sodium sulphate anhydrous ( heated for at least 2 h at 550°C ), Florisil 60-100 mesh (heated for at least 2 h at 550°C, cool & stored in the tightly stoppered container, prior to use heat for at least 5 h at 130°C, cool & add 5% w/w water, shake this mixture for at least 20 min and stored in a container for at least 10 h), cotton wool. HPLC grade solvents purchased from MERCK, Germany and DDT (98.3%) & Heptachlor (97.7%) standards obtained from Sigma, USA were used for the analysis.
Sample preparation: All the samples are finely comminuted in a mincer; heating of the samples during comminuting is avoided by briefly chopping several times.
Extraction
Triturate a sample of 25 g, with Sodium Sulphate 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 Formide de.
Clean up
Clean up was done in two steps
a) Dimethylformide de-petroleum ether partition
Transfer the solution (dissolved in 25 ml Petroleum Ether saturated with Dimethyl Formide de) to a 250 ml separatory funnel. Rinse the flask with a small portion of a previously measured amount of 75 ml Dimethyl Formide de. Then add the remainder of the Dimethyl Formide de to the separatory funnel, and shake vigorously for 1 min. Drain the Dimethyl Formide de phase, and again extract the Petroleum Ether phase with 10 ml Dimethyl Formide de. Transfer the combined Dimethyl Formide de phases to a 500 ml separatory funnel, and add a 200 ml sodium sulphate 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 potions of petroleum ether for 1 min each time. Wash the combined petroleum ether phases with 10 ml water, dry on sodium sulphate, filter through a cotton wool plug, add 5 min-hexane and concentrate to approx. 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 approx. 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 min- hexane to the eluate. Again concentrate the eluate to 1 ml.
Sample analyses: The DDT & 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%, ageing 300°C, support chromosorb-W-AW-DMCS, mesh 80/100, lum film thickness was used for the chromatographic separation of insecticides. The temperature was fixed for the injector at 250°C, column at 280°C, a detector at 280°C. The carrier gas was nitrogen with a 60 ml/min flow rate. 1.0 ul sample was injected for each run and the running time was 25 min. Standards' peak were identified by injecting a high concentration of the standard ( 0.5 mg kg-1 & 0.25 mg kg-1) and the retention time for DDT & Heptachlor were determined. Then calibration was done at 3 points (25 ug kg-1, 50 ug kg-1 and 100 ug kg-1) by a composite stock standard solution. GC system was calibrated using external standard technique. Individual standard stock solution (100mg/1) 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
A gas chromatograph equipped with ECD was checked for linearity. The instrumental limit of detection for GC-ECD was 1.0 ug/1 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. Nine fortified sample matrixes (three for each concentration) were prepared to which known quantities of the pesticides (25 ug kg-1, 50 ug kg-1 and 100 ug kg-1) were spiked in the laboratory. These laboratory fortified matrixes were analyzed exactly like the samples. 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 ug kg-1, 50 ug kg-1 and 100 ug kg-1 were 96-100% for Heptachlor and 98-100% for DDT. Prior to injection of the first sample solution, a standard solution followed by a blank 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 & 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.