# Residual Level of Chlorpyrifos in Lettuces Grown on Chlorpyrifos-Treated Soils
## 2 Materials and Methods  
Chemicals. Chloryrifos (99.7%) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). A granule formulation (2%) for chlorpyrifos was supplied by Enbio Co. (Gunpo, Korea). A HPLC-grade of solvents such as acetone, dichloromethane (DCM), and n-hexane were purchased from Burdick and Jackson(r) (Ulsan, Korea). A florisil solid phase extraction (1 g) cartridge and filter paper (Whatman No. 2) were acquired from Phenomenex (Torrance, CA, USA) and Whatman International Ltd. (Maidstone, England), respectively. A rotary vacuum evaporator made by Heidolph Instruments GmbH & Co. (Schwabach, Germany) and a nitrogen evaporator designated as Hurricane-Lite made by Chongmin Tech (Seoul, Korea) were equipped for removing organic solvents. All other chemicals are of highest purity and reagent grade. Chloryrifos used in this study was formulated as 2% GR. Field experiments. Greenhouses were selected for the field experiments and they were located in two different cities, which were Yongin City (field 1) and Gwangju City (field 2) in Gyeonggi Province, Korea. Soil treatment of chlorpyrifos in field 1 was conducted in triplicate plots of 5 m\({}^{2}\) for a total of 15 m\({}^{2}\) for each trial. The chlorpyrifos formulation was scattered over the soil at an application rate of 1.0 mg/kg and 2.0 mg/kg. Field 2 was treated with 10.0 mg/kg and 20.0 mg/kg to 10 m\({}^{2}\) on each test plot. The lettuce seeds were sowed immediately after treatment with the pesticide. The seeding density was 20 \(\times\) 20 cm. Two environmental parameters (temperature and relative humidity) during the cultivation period were recorded daily using an electric data logger (Lascar, China). The chlorpyrifos-spiked soil was collected at five different days after treatment (DAT) as 0, 7, 14, 22, 28, and 36 DAT for field 1, and 0, 7, 14, 25, 35, 39, and 43 DAT for field 2 to assess the dissipation pattern. Soil samples were left for shadow air-dryness and sieved using a 10-mesh sieve. After sieving, dried soil samples were kept in a refrigerator under \(-20\)\({}^{\circ}\)C until chlorpyrifos analysis. Additionally, a random collection was employed for selecting crop samples from each chlorpyrifos-treated plot at 22, 25, 28, 32, and 36 DAT for field 1, and at 35, 37, 39, 41, and 43 DAT for field 2. Immediately after picking, the crop samples were washed with tap water two times and the remaining water was removed with a paper tissue to remove surface contaminants. The samples were then placed into polyethylene bags and transported with ice to the laboratory where the roots were removed and aerial samples were homogenized. Then, each sample was placed into a polyethylene bottle and frozen at \(-20\)\({}^{\circ}\)C until use for analysis. Instrumental analysis and calibration curve. A stock solution for chlorpyrifos (99.7%, 10.03 mg) was prepared in 100 mL of acetone to a concentration of 100 mg/L. A working standard solution at a concentration of 20 mg/L was made by dilution using acetone and was then serially diluted to obtain various standard solutions of 0.1, 0.5, 1.0, 5.0, and 10.0 mg/L. An aliquot of 1.0 mL was used to analyze chlorpyrifos using a gas chromatography-electron capture detector (GC-ECD). A standard calibration curve was prepared based on the peak area vs. concentration. As shown in Table 1, the residual analysis conditions were modified according to the experimental conditions previously reported [11]. Recovery of chlorpyrifos and sample analysis. For each sample (soil: 20 g; lettuce: 10 g), a standard solution of chlorpyrifos was fortified at levels of 0.2 mg/kg and 1.0 mg/kg. Then, the sample was