The study was undertaken in the pesticide analytical laboratory, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur during 2008-2009 seasons. Many types of chemicals were used in the analysis of the insecticides. All the chemicals (acetone, n-hexane, cyclohexane, methanol, acetonitrile, silicagel 60, sodium sulphate) and insecticide standards (quinalphos, diazinon, malathion, fenitrothion, acephate, cypermethrin) were procured from Sigma-Aldrich Laborchemikalien, Gmbh P. O. Box-100262, D-30918, Seelze, Germany via Bangladesh Scientific Pvt. Ltd. Dhaka, Bangladesh. Standards of insecticides contained 99.6% purity.
2.1. Collection of cauliflower samples from farmers field A total of seventy five samples of cauliflower were collected from farmer’s field of three different locations such as Jessore, Gazipur and Rangpur. In selecting fields, standing crops and marketable size of cauliflower at harvest were the main criteria and samples were collected according to regulation made in the ''Guidelines for the control of pesticide residues in foods" (Anon. 1996), which incorporate the EU directive (Anon.1979) and Codex recommendations (Anon.1993) regarding sampling. The size of the sample of cauliflower was 1kg. Field collected samples were kept in "Chilled box" and carried to the laboratory at the quickest time and preserved in refrigerator.
2.2. Extraction, separation and clean up of cauliflower samples Collected samples (250g) were chopped by knife on white board and mixed well. A sub sample of 20g was taken into a wide mouth jar then 100 ml of hexane was added to it. Sodium Sulphate (Na2SO4) was also added with sample until water was removed from the sample. The mixture was then macerated with high-speed homogenizer (Ultra-turrax, IKA T18 basic, Germany) for 2 minutes. The homogenized material was then poured into 250 ml conical flask and placed into the shaker (Refrigerated Shaker, Rexmed, Sweden) for 12 hrs continuous shaking. After shaking, the slurry was filtered through whatman filter paper no.40 and a Buchner funnels with suction. The flask and filter cakes were rinsed with 25 ml of hexane each. The filtrate was then transferred into 250 ml round bottom flask and was dried to around 5-7 ml by evaporation using a rotary vacuum evaporator (Laborota-4001, Heidolph, Germany). Then, the concentrate filtrate was collected in a centrifuge tube adjusted at 10 ml volume which was then centrifuged at 16500 rpm for 10 minutes with Laboratory Refrigerated Centrifuges, Sigma-3K30, Germany. After centrifuge, supernatant was collected and cleaned up by Super Phase Extraction (SPE) cartridge. Then the final volume was kept in 10 ml volumetric flask. Before injection, this volume was again cleaned up by High Performance Liquid Chromatography filter (0.2 PTFE) which was ready for injection.
2.3. Detection and quantification of insecticide residue in cauliflower samples The condensed extracts were subjected to analysis by GC-2010 (Shimadzu). Flame Thirmionic Detector (FTD) was used for the detection of quinalphos, diazinon, malathion, fenitrothion and acephate and Electron Capture Detector (ECD) for cypermethrin. The capillary column used in FTD was ATTM-1, 30 m in length, 0.25 mm inner diameter (ID) and 0.25 µm film thickness and in case of ECD it was Optima-1 and length, ID and film thickness were the same. Nitrogen was used as carrier and make up gas in ECD and in FTD it was Helium. The instrument parameters for detecting organophosphorus insecticides and cypermethrin were as follows.
Foregoing to the injection of the sample extract, standard solutions of different concentrations of five insecticides were prepared and injected with the above instrument parameters. The samples were calibrated (retention time, peak area etc.) against four pointed calibration curve of standard solution of concerned insecticide. Each peak was characterized by its retention time. Sample results were expressed in mgkg-1 automatically by the GC software which represented the concentration of the final volume injected. From this value the actual amount of insecticide residue present in the sample was determined.
Out of 25 analyzed cauliflower samples of each location, the contaminated samples were almost same in Gazipur, Jessore and Rangpur location. In Gazipur 9 samples of cauliflower were contaminated with insecticides only in single product residue. Malathion, diazinon and fenitrothion could not be detected in cauliflower. The detected quinalphos residue was remained below MRL but the detected residue levels of acephate was doubled the MRL. The residue of 0.605 mgkg-1 of cypermethrin was above MRL only in one sample and the remaining 5 samples showed residue level below MRL. Multiple product residues were not found in the contaminated samples in this location.
Both single and multiple products were detected in the cauliflower in Jessore region where 8 contaminated samples showed single insecticide in the residue. Malathion was detected but remained below MRL. Cypermethrin was detected in 7 samples with two samples above MRL and its rest of the samples were below MRL. Two samples of multiple insecticides detected in cauliflower revealed three insecticides. Fenitrothion, quinalphos and acephate were present in one multiple product sample where the first two (fenitrothion and quinalphos) insecticides were above MRL. The second sample of multiple products contained diazinon, acephate and quinalphos, latter being remained above MRL and diazinon and acephate at low residual level.
The contaminated samples of cauliflower from Rangpur location presented single product and multiple products residue. In 8 single product samples, cypermethrin was detected in 6 samples (frequency 6). Two samples showed 0.961-0.867 mgkg-1 concentration of cypermethrin which were above MRL and rest of the samples were below MRL. Both acephate and diazinon were detected in single product sample, but were below MRL. Malathion was not detected in cauliflower in this location. There were 3 insecticides in one multiple product sample and two insecticides in other sample. All these insecticides remained above MRL in these two samples.