Sample Collection Sites

For analyzing tomato samples for probable presence or absence of pesticides residues, samples were collected from four markets (Hayatabad Phase 1, Chargano Chowk, Kohat Road/ Ring Road Junction, Tarnab farm) located in Peshawar. After collection, all the samples were shifted to Pesticides Residue Laboratory, Agricultural Research Institute, (ARI) Tarnab Peshawar for analysis using 5 various pesticides testing mentioned in Table 1.

Procedure for Extraction of Pesticides

Fresh tomatoes collected from various markets and at -4 ˚C were preserved. After one hour for further procedure the preserved samples were taken off. After thawing, samples were sliced with speed blender. Erlenmeyer flask of 500 ml capacity was used where 100 g from chopped vegetable sample was added in it. Followed by anhydrous sodium sulfate 20 g, NaCl 2.5 g and freshly prepared distilled ethyl acetate 70 ml was added. The use of ethyl acetate was for extraction. For an hour the flasks were shacked. For separation of aqueous material from other solid materials (vegetable parts that were not soluble in ethyl acetate) Whatman fluted filter paper No.42 (Sigma-Aldrich) was used.

Purification procedure

Purification procedure was carried out to purify tomatoes extract. Pesticide residues were passed through different purification steps as pesticide residues were soluble in ethyl acetate. By passing through active charcoals extracted samples were purified. At 105˚C for 4 hours charcoal was activated. One ml of extract was transferred in round bottom flask and pesticide residues were dissolved in acetone. Using rotatory evaporator sample was concentrated. Pesticide residues became concentrated after passing through rotatory evaporator and easy to identify. Concentrated samples were filtered through filter paper (0.2 μm) and analyzed by HPTLC.

Preparation of standards

At Residue Laboratory pesticide standards were available in powder form in different percentages. In 1% methanol standard solution was prepared (Table 2).

Techniques for High Performance Thin Layer Chromatography (HPTLC)

For the detection as well as calculation of the concentration of pesticides high performance thin layer chromatography was used, using protocol designed by 14. In brief, glass plates (0.25 mm thickness) were prepared. At 105°C for 30 minutes each plate was activated. Initially, various concentrations of standards were spotted, then after the development of plate the diameter and concentration of spot were noted. Between concentration and spot diameter a graph was drawn that was helpful in the assessment of pesticides in samples. Along with the standards the desired sample was spotted. Followed by placing plate in tank that contained ammonia and methanol as a mobile phase, to remove undesired material plate was placed in fume hood.

Development of plate

For the development of plate different techniques were followed as applying same detection procedures all pesticides are not detected. Some pesticides when observed under UV at 254 nm for 30 minutes gave florescence. But under UV light many other pesticides were not giving florescence so for the development of plate many other techniques were followed.

Potassium iodide and O-tolidine

O-tolidine and potassium iodide spray generate brown yellowish color. To note-down spot’s diameter, coloring reagent spray was preferred. Color intensity of brown yellowish varies with respect to the concentration of pesticides in standards and sample spots.

Enzyme Inhibition Method

This method is applied for the development of those pesticides on plate which produce color by and O-tolidine and potassium iodide but do not give florescence under UV light. Plate was placed for 15 minutes in bromine vapors. Plate was placed again in fuming hood for 45 minutes for removing exceeding vapors. For 30 minutes at 37°C Horse blood serum enzyme was sprayed on the plate. To eliminate the extra vapors from plate hot air steam was given. Then for developing color substrate solution was sprayed on the plate. Spraying step was done in an incubator.

Tiny blue spots appear beside the white background of the plate. Blue spots direct the existence of carbamate pesticides. The distance covered by the ethyl acetate and the pesticides spots was measured. To calculate concentration of pesticides, the area of spots was measured vertically and horizontally. Followed by the calculation of the Rf value was measured through this procedure.

It is worth mentioning that satisfactory results were generated using O-tolidine and Potassium iodide, therefore the technique of Enzyme Inhibition Method was not applied in our research.

Identification of pesticides

After developing the plate, presence of pesticide in extracted sample of vegetable was detected. If both standard retention factor and retention factor of samples pesticide were matched then it was determined that the sample was contaminated.

Quantification of pesticide

Quantification of pesticides through high performance thin layer chromatography was done for different concentrations by measuring the diameter of standards. Different samples were checked for plate development. Diameter measurement and comparison with the standards quantifies the amount and concentration of pesticide in samples.

Statistical analysis

Data was collected from three experimental replicates. Statistical calculations were carried out with the GraphPad Prism 5 (Graphpad Software, Inc.) and SPSS (Statistical Package for Social Sciences, IBM). The results are presented as mean values with standard errors ( ± ). The mean values were subjected to Duncan’s multiple range tests and Student's t-test. Values of p < 0.05 were considered as significant.

Retention factor for Pesticides residue

Retention factor of each pesticide was calculated by running the standard samples on HPTLC plate. This factor was calculated by measuring the distance travelled by the spots and solvent. Thus, it is equal to distance travelled by the spot divided by distance travelled by the solvent. The spots of Cypermethrin and Chlorpyrifos pesticides traveled more distance (8.8cm and 8.7 cm, respectively) as compare to other pesticides (Table 3).

Concentration of pesticides in tomato samples (mg/kg)

All the samples of tomatoes, obtained from various markets had residues of various pesticides sprayed against insects. The statistical analysis using F-test indicated that significant differences existed in values calculated for concentrations of various residues of pesticides. The tomato samples accumulated maximum concentration of Bifenthrin and Lambda Cyhalothrin (0.100 ± 0.009 mg/kg and 0.097 ± 0.006 mg/kg, respectively). It indicates that both, Bifenthrin and Lambda Cyhalothrin are the most used pesticides, or the tomato plant can easily up take these pesticides as compare to others (Table 4). If the concentration of pesticides is compared on the basis of area from where the samples were collected, tomatoes from vegetable market of Chargano chowk and Kohat road contained the higher amount of pesticides residues (Figure 1).

Comparison of pesticides concentration in samples with maximum residue levels (MRL) ratified by United States Department of Agriculture (USDA)

The international permissible maximum residue levels (MRL) as approved by United States Department of Agriculture (USDA) for the observed pesticides (Lambda Cyhalothrin, Cypermethrin, Chlorpyrifos, Bifenthrin and Imidacloprid) in tomato samples are given in Table 5. Lamdba Cyhalothrin and Bifenthrin are the toxic pesticides and upon exposure it causes adverse effects on human health, therefore the MRL approved by USDA for Lamdba Cyhalothrin and Bifenthrin pesticides is limited to 0.10 mg/kg and 0.20 mg/kg, respectively.

When compared with MRLs, it is clear that the detected concentrations of pesticides residues, present in tomato samples obtained from various locations in Peshawar city, are much lower than admissible level (Table 6). Though the residue of pesticide Lambda Cyhalothrin (0.097 mg/kg) is almost equal to MRL (0.10 mg/kg) (Figure 2), its use in tomato crop must be reduced so that the product remains safe for human consumption.