Preliminary Research On Residues Regularity And Cytotoxicity Of Trichlorfon On Crucian Carp

Trichlorfon is a organophosphorus compound that is often used as an insecticide with low toxicity and low residue.Trichlorfon is relatively stable in neutral and weakly acidic solutions,which forms more toxic dichlorvos in alkaline solutions.The high concentration of trichlorfon not only affects the growth and reproduction of aquatic organisms,but also can endanger human health through the food chain.Not only can it affect the growth,development and reproduction of organisms,but it can also cause oxidative stress at the cellular and molecular levels,promote the production of free radicals,affect the expression of related genes and even cause cell death.(1)Accumulation and elimination of trichlorfon in Carassais auratus gibebio tissue were studied here.Three different detection methods for trichlorfon including high performance liquid chromatograph,gas chromatograph-mass spectrometer and liquid chromatograph/mass spectrometer were compared for their detection sensitivity.The results showed that maximum sensitivity of liquid chromatograph/mass spectrometer was 0.1ng/g.The concentration of the muscle were detected after immersion of 0,0.2mg/Land 0.5mg/L at 20±2℃,trichlorfon concentration-time data in the muscle of Carassais auratus gibebio was described as one-compartment model with first-order absorption.The peak time of immersion and oral gavage was 5.74 d and 5.650 d,the Cmax was 0.163μg/mLand 0.502μg/mL respectively,the proposed withdrawal time in crucian carp should not be less than 5 d.The concentration of the muscle were detected after oral gavage of 0.5 mg/g、1 mg/g and 2 mg/gat 20±2℃,trichlorfon concentration-time data in the muscle of Carassais auratus gibebio was described as one-compartment model with first-order absorption.The peak time of oral gavage was 5.188 h,5.038 h and 5.997 h respectively,the Cmax was 0.199μg/g、0.261μg/g and 0.422μg/g,the proposed withdrawal time in crucian carp should not be less than 4d.The text aimed to compare the way of immersion and oral gavage of trichlorfon residues in the muscle of Carassais auratus gibebio,the way of usingtrichlorfon provided scientific basis for prevention and treatment for aquatic animal parasite disease.(2)Even though some successful results have been reported with bath treatments of trichlorfon to control parasites in aquaculture.However,the use of trichlorfon on a large scale has caused increasingly serious environmental pollution.The aim of this study is to investigate the metabolism,hematotoxicity and hepatotoxicity of trichlorfon in Carassius auratus gibelio after a single oral administration.In this study,Carassius auratus gibelio was oral gavaged to various concentrations of trichlorfon(0.5 g/kg,1 g/kg,2 g/kg).The trichlorfon concentration in plasma and liver tissue was quantified using LC–MS/MS at indicated time points.At the beginning of oral exposure,the uptake of trichlorfon in plasma and liver tissue was fast,and then the trichlorfon was rapidly eliminated to a low level within 24 h.Besides,AChE activity,SOD,CAT and GSH-ST activity in plasm and liver tissue changed significantly after trichlorfon exposure.Additionally,vacuolar degeneration,necrosis,congestion of central vein was observed in liver after trichlorfon exposure by HE staining.(3)Trichlorfon has been widely used in the treatment of diseases caused by parasite infection in grass carp.To explore the toxicity of trichlorfon on grass carp,this study investigated the mechanism of apoptosis induced by trichlorfon in grass carp liver cell(L8824)lines.The L8824 cells were treated with different concentrations of trichlorfon and then the activity of cells,apoptosis rate,caspase-3/7activity and expression changes of apoptosis-related genes caspase-3 and caspase-9were detected.The results indicated that different concentrations(10μg/mL,50μg/mL,100μg/mL,500μg/mL and 1000μg/mL)of trichlorfon exposure caused the decrease of cell activity,when the concentration of 1000μg/mL of trichlorfon,The lowest level is46.9%.At the same time,the apoptosis rate increased.the highest apoptotic rate was32.3% when the concentration of trichlorfon was 1000 μg/mL.With the increase of trichlorfon concentration,the activity of caspase-3/7 increased and apoptosis-related genes increased significantly.When the concentration of trichlorfon was 500 μg/mL,the cells appeared distorted and broken.the cells formed apoptotic bodies and showed significant TUNEL positive signals.After adding three different inhibitors(Vc,ALC and Z-VAD-FMK),it was found that different concentrations of the caspase inhibitor Z-VAD-FMK had a greater effect on the cells.After treating with different concentrations of inhibitor Z-VAD-FMK,the L8824 cell s’ activity were increased,the apoptosis rate was decreased,the expression of apoptosis-related genes wasdown-regulated and the activity of caspase-3/7 was increased with the increase of inhibitor concentration.In this study,different detection methods were used to detect trichlorfon,and the rule of muscle residue of trichlorfon in soaking and oral was compared and the drug withdrawal period was established.Tissue metabolism,hematotoxicity,and hepatotoxicity of trichlorfon in carassius auratus gibelio after a single oral administration indicate that high concentrations of trichlorfon solutions cause damage to the body.Trichlorfon-induced apoptosis of grass carp is mediated through the death receptor pathway in liver cells.The treatment of cells with trichlorfon eventually leads to apoptosis by altering the expression of caspase-3 and caspase-9.