A subacute experiment was conducted to find out the safe concentration to carry out further experiments. The control group, vehicle control group and three treatment groups (Total 5 groups, each group n=10) were used in 14-day subacute experiments. Three experimental dose ranges (40, 90, 200umg/kgbwd) were selected based on results from the acute study. Doses were administered daily for 14 consecutive days with dose adjusted the body weight of each lizard. All signs of intoxication, other abnormal behaviour, and mortality were recorded. The maximum non-lethal dose in subacute study was chosen as the highest dose in metabolism, distribution, and bioaccumulation exposure.
2.5. Metabolism, distribution, bioaccumulation, and restoring experiment
Two experimental dose ranges (40, 200umg/kgbw) were selected based on results from the subacute study for the further experiments.
Metabolism experiment was conducted
using 54 new lizards (27 lizards for each dose). Each lizard was administered orally once. Blood samples were collected at 2, 6, 8, 12, 24, 48, 72, 120 and 168uh after lizards were dosed. Three lizards were euthanized with carbon dioxide randomly for each dose group at once.
Considering the possible death of lizards during the exposure period, Empagliflozin
distribution experiment was conducted using 10 new lizards (5 lizards for each dose). These lizards were continuously administered of TF daily for 14 days with dose adjusted according to the body weight. All lizards were euthanized with carbon dioxide after 14 days of exposure. Each lizard;s blood, brain, heart, lungs, liver, kidneys, and intestines were collected for concentration analysis of enantiomers of TF and TN.
A chronic bioaccumulation experiment was conducted using 80 new lizards (40 lizards for each dose). New lizards were continuously administered of TF daily for 35 days with dose adjusted according to the body weight. Lizards were euthanized with carbon dioxide at 1, 3, 5, 7, 10, 14, 21, 28, 35 days. Blood and liver samples were collected for concentration analysis of enantiomers of TF and TN. Three lizards were euthanized randomly from each group at once.
A restoring experiment was conducted using 50 lizards (25 lizards for each dose). These lizards were first administered of TF daily for 14 days with dose adjusted the body weight. Then these lizards were stopped exposing under TF and fed normally for 14 days. Lizards were euthanized with carbon dioxide at 1, 2, 3, 5, 7, 10, 14 days in normal feeding. Blood and liver samples were collected for concentration analysis of enantiomers of TF and TN. Three lizards were selected at randomly from each group and were simultaneously euthanized.
2.6. HPLCaMS/MS conditions
HPLC was performed using Thermo ACCELA series (Thermo Electron Corporation, Hopkinson, MA) equipped with ACCELA Autosampler, ACCELA 600 pump, a 20u?L injection loop, and a 2u?L flow cell. Enantiomers were separated on a Phenomenex Lux Cellulose-1 column (2504.6umm id, 5u?m particles), packed with CSP of CDMPC and obtained from Guangzhou FLM Scientific Instrument (Guangzhou, China). The mobile phase was a mixture of 73 percent methanol, 27 percent water with a flow rate of 0.5umL/min. Chromatographic separation
was conducted at 20uC, and the injection volume was 10u?L. TSQ QUANTUM ACCESS MAX was used for LCaMS/MS analysis (Thermo Electron Corporation, Hopkinson, MA). Quantification was achieved in positive-ion mode (ESI+). The signals were received and processed with Thermo Xcalibur 2.2 SP1.48 software. The optimized major working parameters were as follows: Spray Voltage 3200uV, Vaporizer Temperature 250uC, Sheath Gas Pressure 30upsi, Aux Gas Pressure 10 arbitrary units, Capillary Temperature 350uC, Capillary Offset 35uV, Q2 Collision Gas Pressure 1.5umTorr. Multiple reactions monitoring mode was used; the precursor and product ions of TF and TN with corresponding declustering potentials and collision energies are summarized in Supporting Information Table S1.