2.1. Chemicals and materials
Certified pesticide standards of CLO (97%), DFN (98%), DFZ (97%), and standards of SA (99.5 %) were purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China)
Reagents: Sodium chloride (NaCl) and anhydrous magnesium sulfate (MgSO4) were purchased from Sinopharm Chemical Reagent (Beijing, China). PSA (40-63 µm) was obtained from Tianjin Bonna-Agela Technology Co., Ltd. (Tianjin, China). Acetonitrile (HPLC grade) was obtained from Fisher Chemicals (Fair Lawn, NJ, USA).
2.2. Experiment design
Plant cultivation: The seeds of cucumber were provided by the Xintai Yuyuan Seed Industry Co., Ltd (Shandong, China). Cucumber seeds were wrapped in wet gauze and placed in a petri dish to accelerate germination. When the seeds germinating, we transferred the seeds into a seedling tray and put one seed into each hole. The temperature of the greenhouse was 25 ℃ in the day and 20 ℃ at night. After growth 21 days, the young plants were used in pesticides exposure trials.
2.2.1. Pesticide exposure experiments-uptake and translocation
Hydroponic experiments:The concentration of three pesticides was set at 5 mg L-1, and the concentrations of SA were set at 1 mg L-1,10 mg L-1, and 50 mg L-1, respectively. CLO, DFN or DFZ was first dissolved in acetonitrile to form the stock standard solution, respectively. And then the standard stock solutions were diluted to the required concentration by ultrapure water. SA was dissolved and diluted with 20 % methanol-water.
After germination, the cucumber seedlings were removed from the seedling tray, and the roots were washed by deionized water before transferred to hydroponic cultivation buckets with 0.25 L modified Hoagland nutrient solution in the greenhouse. The composition of the nutrient solution can be found inTable S2 . The growth conditions were the same as the plant cultivation conditions. And a total of 12 treatments were set in the experiment with 27 cucumber seedlings per treatment, the processing information for each test can be found in Table 1 . The hydroponic cultivation buckets were covered with aluminum foil to prevent algae growth and water evaporation. The nutrient solution was supplemented to the initial volume at 11 am every day.
Control experiments: Two control groups were included: nutrient solution with cucumber seedlings without pesticides was as the treatment of C1 group; and nutrient solution with CLO, DFN and DFZ but without cucumber seedlings were as the treatments of CLO-C2, DFN-C2 and DFZ-C2 groups, respectively.
Sample collection: Cucumber seedlings were grown for 28 days, nutrient solution and plant samples were collected at 0.25, 1, 3, 5, 7, 10, 14, 21, 28 days after the transfer. The plants were divided into roots, stems and leaves for pesticide analysis. At each sampling time, three replicate samples were randomly taken. All samples were stored at -20℃ until analysis.