One-step Extraction of Multiresidue Pesticides in Soil by Microwave-assisted Extraction Technique

MATERIALS AND METHODS

The present study was carried out during the year of 2007 in the Laboratory of the Soil Sciences Department, King Saud University, Saudi Arabia.

Chemicals and reagents: Pesticide standards (Dimethoate, Chloroneb, Methomyl, Oxamyl, Toxaphen, DDT, DDE, Monocrotophos, Chlorpyrifos, Diazinon, Cypermethrin and Lindane) were provided by Chemservice, USA. All pesticide standards were of 98-99% purity. All the solvents (hexane and acetone) used were residue analysis grade. The stock working solutions were prepared in acetone.

Preparation of spiked soil samples: Spiked samples were prepared by adding an appropriate volume of spiking solution to 5 g of soil. The soil was spiked with a stock solution of the pesticides (5 mg L^-1) prepared in Acetone. The spiked samples were prepared just before analysis, waiting approximately 30 min until solvent evaporation. Soil samples were extracted by two techniques, MAE and Soxhlet extraction (Barriada-Pereira et al., 2003; Sporring et al., 2005). The main purpose of this step was to calculate the average of the recovery percent of investigated pesticides by both extraction techniques.

Table 1:	Optimum conditions of MAE technique for soil samples extraction

Minimum detection limit (MDL): To determine the MDL and perform the GC-MS quantification using a four-point calibration curve plotting peak area versus ppm concentration of 12 pesticides using the dilution levels ranged from 0.0001-5.00 ppm (this sentence needs to be re-written).

Extraction of pesticide residues by MAE: The proposed EPA Method 3540C (USEPA, 1996) and CEM application note No. E003 (CEM Corporation, 1994) was modified and used while conducting the present study. A Microwave Assisted Extraction (MAE) system model MES-1000 (CEM Corporation, Matthews, NC, USA) with Lined Extraction Vessels (LEV) was used. This system consists of a 950 watt microwave instrument which has been specifically designed for use with organic solvents. Extraction vessels are double-walled vessels specifically adapted for use with organic solvents. Preliminary studies were performed to evaluate MAE efficiency for the effects of temperature, microwave power, extraction time and solvent volume (Table 1) in extracting different groups of pesticide residues from soil and water samples. For this purpose, 5.0 g of soil was weighed in a tetrafluoromethaxil (TFM) microwave extraction vessel with addition of 1 mL of an aqueous spiking solution containing mixture of the pesticide standard solution. Samples were equilibrated by shaking for 1 h before microwave extraction. Finally, all samples were extracted under different conditions as showed in the results to obtain the optimal MAE conditions with this procedure. The optimum Extraction conditions with high recovery was conducted with 5.0 g soil samples. The extraction solvent was 35 mL of Acetone : Hexane (3:2), pressure was 80 psi, microwave power was 60%, temperature was 120°C and time of extraction 20 min. After extraction, soil extracts were filtered and evaporated to dryness. The residues were re-dissolved and directly analyzed by Gas Chromatography- Mass Spectrometer (GC-MS) and clean chromatograms were obtained without any additional cleanup step.

Extraction of pesticide residues by soxhlet: The extraction was done by Soxhlet method (Barriada-Pereira et al., 2003; Sporring et al., 2005). A 5.0 g soil sample was extracted with Acetone: Hexane (3:2) for 8 h. After extraction, soil samples were filtered and evaporated to dryness. The residues were re-dissolved in 1 mL of acetone and directly analyzed by Gas Chromatography-Mass Spectrometer (GC-MS). Clean chromatograms were obtained without any additional cleanup step.

Gas Chromatography-Mass Selective (GC-MS) analysis: Both extracts of Pesticide residues in soil samples extract were performed and analyzed by a HP 5890 series II plus GC coupled to an HP 5972 Mass Selective Detector. The GC columns were a DB-5 fused silica capillary column (30 mx0.32 mm i.d., 1 μm film thickness; J and W Scientific, Folsom, CA). One micro liter of the soil sample extract was injected split less injector temperature of 250°C, on the GC-MS for analysis. The temperature program for the GC was as follows: isothermal for 1 min at 100°C, increased at a rate of 10°C min^-1 to 240°C and isothermal for 15 min. Helium was used as a carrier gas (1.2 mL min^-1). Quantification was performed using a four-point calibration curve plotting peak area versus ppm concentration. The result was expressed in percentage recovery of pesticide.

RESULTS AND DISCUSSION

Screening and optimum extraction factors of MAE: The application of MAE techniques based on modified extraction approaches were demonstrated by its application in the extraction of 12 pesticide residues with a broad range of physico-chemical properties from agricultural soil samples. Comparative study was carried out by Soxhlet extractor. The evaluation of the extraction efficiency of MAE was affected by 6 factors. These factors were defined to evaluate their contribution to pesticides extraction efficiency of spiked soil samples. The results of this part show the optimum extraction conditions (Table 1) with the average of high recovery ranging from 98.00±2.25 to 102.44±2.73 for all tested pesticides and low MDL.

Minimum Detection Limits of pesticide residues (MDL): The MDL of all tested pesticide residues extracted by modified MAE technique compared with Soxhlet extractor and analyzed by GCMS were determined to evaluate the efficiency and availability of both extraction methods. The averages of MDL ranged from 0.0001 to 0.004 and from 0.002 to 0.012 mg kg^-1 for MAE and Soxhlet, respectively as shown in Table 2.

Recovery (%) of pesticide residues in spiked soil samples: Recovery studies to validate MAE extraction techniques were carried out with spiked soils obtained by adding a low volume of spiking pesticide solution in acetone to compare with Soxhlet extractor.

Table 2:	MDL of tested pesticides in spiked soil samples extracted by MAE and Soxhlet and determined by GC-MS

Table 3:	Pesticide recovery (%) and relative standard deviation (RSD, %) of spiked soil samples extracted by MAE and Soxhlet and determined with GC-MS
RSD relative standard deviation

From the comparison of both methods, it was found that higher recoveries were obtained by modified MAE than Soxhlet for all tested pesticides. On the other hand, the recoveries by Soxhlet were still good for Lindane, Toxaphen, DDT and Dimethoate under their respective optimum conditions. Therefore, MAE are suitable techniques for extracting all tested pesticides from spiked soil with recovery, % (±RSD) ranging from 98.00±2.25 to 102.44±2.73 and 93.11±2.08 to 97.09±2.36 % for Soxhlet extractor as shown in Table 3.

Monitoring of pesticide residues in soil samples: By applying MAE in multiresidues, which we thus developed to obtain the optimum conditions, it was possible to extract 12 pesticides in soil samples (fresh, control or spiked) in 20 min. GC-MS was applied as analysis technique with high sensitivity of all 12 detected pesticides in this study (Al-Turki et al., 2009).

Table 4:	Averages of pesticide residues in soil samples (mg kg-1 soil) extracted by MAE and determined with GC-MS

The results of studied pesticides was shown in Table 4 and showed that: Dimethoate was detected in 51.85% of total soil samples followed by Methomyl as 39.63%, Chloroneb as 34.44%. The most important notice in the results of soil analysis was the detection of the residue of DDT and one of their derivatives DDE. Both pesticide residues were detected in 4 regions namely, Al-Qatif, Al-Ahsa, Wadi Al-Dawaser and Gizan. Dimethoate was detected as the highest contaminant in Abha (0.002-1.220 mg kg^-1) followed by DDT (0.960 mg kg^-1) in Wadi Al-Dawaser. Meanwhile, Diazinon was detected as a lower contaminant. All detected pesticide residues were over the Maximum Residue Limits (MRLs) i.e., 0.1 mg kg^-1 except Monocrotophos and Diazinon. These results are in agreement with those reported in previous studies for the soil samples analyzed by MAE (Coscolla et al., 2009; Carvalho et al., 2008; Schreck et al., 2008; Oldal et al., 2006; Shegunova et al., 2006; Westbom et al., 2008; Chen, 2010).

CONCLUSIONS

The described method is efficient and fast to determine multi-residue pesticides in soils. Most of the compounds studied were recovered in good yields with Relative Standard Deviations (RSDs) below 3.5%. The averages of MDL ranged from 0.0001 to 0.004 and 0.002 to 0.012 mg kg^-1 for MAE and Soxhlet techniques, respectively. MAE technique had more extraction advantages than Soxhlet such as short time, did not require refluxing large volumes of solvent, cleanup and permitting the simultaneous extraction of several samples. The modified MAE method was successfully applied to the extraction of 12 pesticide residues namely, Dimethoate, Chloroneb, Methomyl, Oxamyl, Toxaphen, DDT, DDE, Monocrotophos, Chlorpyrifos, Diazinon, Cypermethrin and Lindane from soils which were considered an appropriate index to establishing its applications.

ACKNOWLEDGMENT

The authors would like to thank the Deanship of Scientific Research at King Saud University for financial support through Project # DSR- AR- 2- (14).