A growth chamber study was conducted at the Virginia Tech Glade Road Research Facility (GRRF) (37.23, -80.44 ) in 2020 to determine the persistence of 14 C-glufosinate and 14 Cglyphosate on dormant zoysiagrass leaves. The experiment was implemented as a completely randomized design, twofactor factorial with four replications and two temporal runs. The two factors were herbicide (glufosinate and glyphosate) and harvest time (0.2, 1, 3, 7, 14, and 21 days after treatment). Dormant Zenith zoysiagrass leaves were collected from a site mowed to a height of 7 cm. The leaves were clipped to 2.5-cm long pieces and treated with three 1-μL droplets containing 4.0 kBq 14 C-glufosinate or 14 C-glyphosate. The glyphosate (phosphonomethyl-14 C, specific activity = 50 μCi mmol -1 , purity 99%) spotting solution was converted to the isopropylamine salt by combining 200 μL 14 C-glyphosate acid with 1.6 μL isopropyl amine, then adding 0.8% v v -1 MON56164 surfactant (Monsanto Company). Radio-labeled glufosinate (glufosinate hydrochloride, specific activity = 51.8 μCi mg -1 , purity = 99%) with 0.1% v v -1 nonionic surfactant (Induce; Helena) was used for spotting solution. The treated leaves were placed in dry Petri dishes and incubated for up to 21 days in a growth chamber maintained at 30˚C day and 25˚C night temperatures, respectively, with 330 μmol m -2 s -1 photosynthetically active radiation (PAR) for 12-h photoperiod each day.
At each harvest time, leaves were vortexed for 30 s in 10 mL of deionized water to remove water-extractable radio-labeled herbicide. Then, leaf tissues were immersed in liquid nitrogen, followed by grinding of plant material with a mortar and pestle. A 2 mL extraction solution, 1:1 methanol:deionized water was added to the grounded plant tissue and further homogenized using the motor and pestle. The ground plant material and extraction solution were suction filtered using a Buchner funnel fitted with Whatman No. 1 filter paper (Whatman International Ltd.). The mortar, pestle, funnel, and filter paper were further rinsed with an additional 8 mL of extraction solution. A 0.5 mL aliquot from the resulting extract was added to 15 mL of scintillation fluid (Sciniti Verse LC Cocktail Scitanalyzed; Fisher Scientific), and total radioactivity was measured via liquid scintillation spectrometry (LSS 6500 Multipurpose Scintillation Counter; Beckman Coulter Inc.). Homogenates from previously described extraction and filtration procedures were dried in a nitrogen evaporator (N-EVAP 112; Organomation Associates Inc.) and residues were resuspended with 500 μL cold methanol. Of this resuspended solution, 100 μL was then delivered to a 20-cm by 20-cm silica gel thin layer chromatography (TLC) plate (TLC Silica gel 60G F 254 ; Millipore Sigma) and developed in a 55 mL ethanol, 35 mL H 2 O, 2.5 mL of 15 N NH 4 OH, 3.5 g trichloroacetic acid, and 2 mL of 17 N acetic acid (Sprankle et al., 1978) (link) within an airtight glass chamber. The plates were then air-dried, and radioactive positions, proportions, and corresponding R f values were determined with a radiochromatogram scanner (Bioscan, System 200 Imaging Scanner and Auto Changer 1000; Bioscan Inc.). Parent herbicide was identified by comparing it with radio-labeled standards spotted on adjacent lanes of each plate. Radioactive trace peaks were integrated with Win-Scan software (WIN-SCAN Imaging Scanner Software Version 1.6c; Bioscan Inc.) with smoothing set to 13-point cubic and background excluded
At each harvest time, leaves were vortexed for 30 s in 10 mL of deionized water to remove water-extractable radio-labeled herbicide. Then, leaf tissues were immersed in liquid nitrogen, followed by grinding of plant material with a mortar and pestle. A 2 mL extraction solution, 1:1 methanol:deionized water was added to the grounded plant tissue and further homogenized using the motor and pestle. The ground plant material and extraction solution were suction filtered using a Buchner funnel fitted with Whatman No. 1 filter paper (Whatman International Ltd.). The mortar, pestle, funnel, and filter paper were further rinsed with an additional 8 mL of extraction solution. A 0.5 mL aliquot from the resulting extract was added to 15 mL of scintillation fluid (Sciniti Verse LC Cocktail Scitanalyzed; Fisher Scientific), and total radioactivity was measured via liquid scintillation spectrometry (LSS 6500 Multipurpose Scintillation Counter; Beckman Coulter Inc.). Homogenates from previously described extraction and filtration procedures were dried in a nitrogen evaporator (N-EVAP 112; Organomation Associates Inc.) and residues were resuspended with 500 μL cold methanol. Of this resuspended solution, 100 μL was then delivered to a 20-cm by 20-cm silica gel thin layer chromatography (TLC) plate (TLC Silica gel 60G F 254 ; Millipore Sigma) and developed in a 55 mL ethanol, 35 mL H 2 O, 2.5 mL of 15 N NH 4 OH, 3.5 g trichloroacetic acid, and 2 mL of 17 N acetic acid (Sprankle et al., 1978) (link) within an airtight glass chamber. The plates were then air-dried, and radioactive positions, proportions, and corresponding R f values were determined with a radiochromatogram scanner (Bioscan, System 200 Imaging Scanner and Auto Changer 1000; Bioscan Inc.). Parent herbicide was identified by comparing it with radio-labeled standards spotted on adjacent lanes of each plate. Radioactive trace peaks were integrated with Win-Scan software (WIN-SCAN Imaging Scanner Software Version 1.6c; Bioscan Inc.) with smoothing set to 13-point cubic and background excluded
