| Objective:Plant growth regulators,as exogenous non-nutritive chemicals,promote specific effects of plants under specific application conditions and are widely used in agricultural production.Forchlorfenuron?CPPU?is a new type of high-efficiency plant growth regulator,widely used in vegetables,food crops,fruit trees,etc.,a small amount of exposure is not harmful to the human body,but the potential impact on the use of people after repetitive heavy use is unclear.Therefore,dynamic residue research and toxicological evaluation should be carried out for forchlorfenuron,and rapid residue detection methods and safety evaluation systems should be established to promote the rational application of growth regulators.In this paper,kiwifruit was taken as an example to study the residual dynamics of forchlorfenuron during the growth period and logistics storage period of kiwifruit.The degradation of forchlorfenuron in the growth period of kiwifruit was investigated.The residual changes at the different treatment methods,different storage days and different temperatures during the logistics storage period were observed.At the same time,the single toxicity test of mice was used to determine the LD50and 95%confidence limit of forchlorfenuron administered to mice.Repetitive toxicity test in rats was used to analyze the effects of forchlorfenuron on toxicological indicators of animals,determine the non-toxic dose?NOAEL?,and evaluate the target organs and recovery.The toxicokinetics of forchlorfenuron in different age animals was studied by using forchlorfenuron in young and adult rats.The experiment also examined the effect of forchlorfenuron on precocious puberty in young animals at parental toxic doses.This study can improve the effective forchlorfenuron residue monitoring system,clarify the hazard and toxicological effects of growth regulators,and determine the exposure and safe dose of forchlorfenuron in order to provide theoretical guidance for the rational use of this growth regulator.Methods:1.Study on residual dynamics of forchlorfenuron in kiwifruit1.1 Establishment of methodology for analysis of forchlorfenuron residuesIn this test,the concentration of forchlorfenuron in kiwifruit was determined by LC-MS/MS.The chromatographic column was ACQUITY UPLC BEH C18?2.1 mm×50 mm,1.7?m?.The mobile phase is:0.1%formic acid water-acetonitrile solution?0.00-1.80 min:40:60-90:10,1.80-2.50 min:90:10,2.50-3.00 min:90:10-40:60,3.00-4.00 min:40:60?V/V.The flow rate was 0.3 m L/min,the column temperature was 35?,and the injection volume was 5?L.Using MRM detection mode,Capillary voltage is 3.50 k V,source temp is 150?;desolvation temp is 400?,collision gas flow is 0.15 m L/min,cone gas flow is 50 L/Hr;and desolvation gas flow is 700 L/h.The detection parameters of forchlorfenuron:parent ion m/z is 248.02,daughter ion m/z is 93.07,cracking voltage is 22.0 V,and collision energy is 36 e V.The sample was purified using Qu ECh ERS dispersive solid phase extraction reagent.Specificity,standard curve and quantitative range,limit of quantitation,precision and accuracy,extraction recovery,matrix effect,dilution reliability and residue were examined.1.2 Residue detection of forchlorfenuron in kiwifruit?1?The residues of forchlorfenuron in kiwifruit after spraying different concentrations?5.0,10.0,20.0,50.0 mg/L?of forchlorfenuron were determined by LC-MS/MS.?2?Investigate the residual amount and degradation of forchlorfenuron during the growth of kiwifruit.?3?Investigate the residual changes after treatment with0.05 mmol of ethylene?Eth?,degradation pattern in different storage time?0 d,15 d,30d,60 d,90 d,120 d?;and residual dynamics at different storage temperatures?2-8?,-25-15??of forchlorfenuron during kiwifruit logistics storage.2.Non-clinical safety evaluation of forchlorfenuron2.1 Single dose toxicity assessment in miceSixty qualified quarantine mice were randomly divided into six groups according to their body weight with 10 mice in half genders in each group.They were divided into solvent control group and five test groups.The dose of the test group was 1500mg/kg/day,1350 mg/kg/day,1215 mg/kg/day,1094 mg/kg/day and 985 mg/kg/day.The corresponding concentration of test solution was given by single intragastric administration,the volume was 0.4 m L/10 g,and the solvent control group was given equal volume of 0.5%CMC-Na solution.The appearance,behavioral activities,mental state,appetite,stool and urine color,fur,nose,eye and mouth abnormal secretions and death were recorded for 7 days.The single toxicity LD50 and 95%confidence limit of mice were calculated.2.2.Repetitive dose toxicity assessment in rats120 eligible rats in half genders were selected.They were randomly divided into high dose group?600 mg/kg/day?,medium dose group?300 mg/kg/day?,low dose group?150 mg/kg/day?and solvent control group.Rats in each group were given the corresponding concentration of the drug solution once a day.The volume of the solution was 10 m L/kg,and the solvent control group was given the same volume of 0.5%CMC-Na solution.Every day,the rats were observed after the administration.The observation included the appearance,physical signs,behavioral activities,urine and fecal characteristics,hair color,eye,mouth and nose secretions.The reaction,posture and respiratory status of the rats were also observed.The body weight was weighed twice a week,and the food intake was weighed once a week for 48 hours.Ten animals per group were anaesthetized after one month of administration,and ten animals per group were anaesthetized after the recovery period.Then blood was collected from abdominal aorta,and whole blood,plasma and serum were tested for hematology,coagulation function and blood biochemistry.After the animals were sacrificed,the general anatomical examination was carried out to observe the abnormal changes of the main visceral organs with visual observation.The main visceral organs were weighed and the visceral organ coefficients were calculated.The femur was exfoliated,and a bone marrow smear was prepared by smear method for bone marrow examination.The organs of solvent control group,forchlorfenuron group?600 mg/kg/day?and autopsy abnormal animal were taken,paraffin-embedded,sectioned and stained with HE for histopathological examination.2.3.Study on toxicity of the development of reproductive system of forchlorfenuron in lactating ratsRats aged 10-12 weeks were mated after feeding for a period of time.Female rats were administered forchlorfenuron?300 mg/kg/day?on the 15th day of pregnancy.Sixty-four lactating rats,weighing 45-55 g,half male and half female,were randomly divided into four groups:high dose group?300 mg/kg/day?,medium dose group?100mg/kg/day?,low dose group?30 mg/kg/day?and solvent control group?16 rats in each group?.While PND21,rats in each group were intragastrically administered with the corresponding concentration of the drug solution at a dose of 10 m L/kg every day.The solvent control group was given an equal volume of 0.5%CMC-Na solution once a day for 4 weeks.Observe the appearance signs and behavioral activities,feces,urine traits,hair color,mouth,eyes,nose and secretions and so on.The reaction,respiratory state,and posture in animal were observed after administration.The body weight was weighed and recorded on two days every week.Vaginal smears of female rats were used to observe changes in estrous cycle.The rats were weighed and killed after 2 weeks,3weeks,4 weeks of administration and 4 week after withdrawal respectively.The animals were anesthetized,blood was collected,and gross anatomy was performed to observe the abnormal changes of the main visceral organs with visual observation.Heart,liver,spleen,lung,kidney,adrenal,pituitary,uterus,ovary?testis,epididymis,prostate?,and hypothalamus was picked and weighed.Then,visceral organ coefficient was calculated.The biochemical indicators were tested,and perm Vitality in male animals was detected.Serum was used to detect the concentration of testosterone T,female estradiol,luteinizing hormone,follicle stimulating hormone,progesterone.After administration,the uterus of female rats and testis of male rats were pathologically sliced and the thickness of uterine wall was measured.The purpose of the experiment is to investigate the effect of forchlorfenuron on precocious puberty in young rats.2.4.Study on the toxic exposure of forchlorfenuron in animals of different ages2.4.1 Establishment of a bioanalytical method for forchlorfenuron in plasma of ratsThis experiment used LC-MS/MS to detect the concentration of forchlorfenuron in rat plasma.The chromatographic column was ACQUITY UPLC BEH C18?2.1 mm×50mm,1.7?m?.The mobile phase is 0.1%formic acid water-acetonitrile solution?0.00-2.00 min:90:10-5:95,2.00-3.50 min:5:95,3.50-4.00 min:5:95-90:10,and4.00-5.00 min:90:10?V/V.The flow rate was 0.3 m L/min,the column temperature was30?,and the injection volume was 7?L.Using MRM detection mode,Capillary voltage was 3.00 k V,source temp was400?,desolvation temp was 350?,collision gas flow was 0.15 m L/min,cone gas flow was 50 L/h,and desolvation gas flow was 800 L/h.The detection parameters of forchlorfenuron:parent ion m/z was 248.02;daughter ion m/z was 93.07,cracking voltage was 22.0 V,and collision energy was 36 e V.Curcumin was selected as the internal standard,and its detection parameters:parent ion m/z was 369.09;daughter ion m/z was 176.99,cracking voltage was 27.0 V,and collision energy was 22.0 e V.The sample was subjected to protein precipitation treatment using a 3-fold methanol solution?containing 100 ng/m L curcumin internal standard solutions?.Specificity,standard curve and quantification range,quantification limit,precision and accuracy,matrix effect,extraction recovery,stability,dilution reliability and residue were examined.2.4.2 Study on toxic exposure of forchlorfenuron in animals of different agesSixty qualified quarantine SD rats were randomly divided into six groups with 10mice in half genders in each group.Adult animals?180-230 g?and young animals?90-130 g?were administered forchlorfenuron with 150,300 and 600 mg/kg/day doses,respectively.Blood was collected by Tail-cutting after intragastric administration at 0 min,15min,30 min,1 h,2 h,3 h,8 h,24 h,and 48 h.Plasma samples were tested for the concentration of forchlorfenuron in plasma using a validated LC-MS/MS assay.All measured data are collected and processed by Mass Lynx software,calculated and processed by Microsoft Excel.The kinetic parameters were calculated by the statistical moment method with DAS 3.0 software.The blood concentration peak time Tmax and the peak concentration Cmax were measured values.Toxic exposure parameters include AUC?0-t?,Cmax,Tmax,etc.,The purpose of the experiment is to compare the toxic exposure between adult and young animals.3.Safety risk assessment of forchlorfenuronBased on the NOAEL obtained from toxicological and related animal experiments,the allowable daily intake of forchlorfenuron?ADI?was deduced according to the uncertainty factor?safety factor,UF?recommended in the Guidelines for Establishing Daily Allowable Intake of Pesticides.The EDI,Risk ratio and safe concetration of forchlorfenuron in fruits was calculated according to 350 g of edible fruit recommended in the Dietary Guidelines for Chinese Residents.The risk was assessed according to the residual forchlorfenuron detected in the experiment.Results:1.Study on residual dynamics of forchlorfenuron in kiwifruit1.1 Establishment of methodology for analysis of forchlorfenuron residuesThis experiment used LC-MS/MS to detect forchlorfenuron in kiwifruit,and the kiwifruit matrix did not interfere with forchlorfenuron detection.The correlation coefficient?r?of the forchlorfenuron standard curve was greater than 0.99?r=0.997195?,and the quantitative range was 0.5-500 ng/m L.The intra-batch precision of forchlorfenuron control samples?0.8,40.0,400 ng/m L?ranged from 1.25%to 2.61%,the inter-batch precision ranged from 2.94%to 8.58%,and the intra-batch accuracy ranged from-4.17%to 2.61%,the inter-batch accuracy ranged from-7.44%to 2.29%.The recovery of extraction ranged from 93.52%to 99.69%.The matrix effect ranged from 86.09%to 91.33%.The concentration of forchlorfenuron was reliabile under dilution of 50 times,and the residues of samples were accepted.1.2 Determination of forchlorfenuron residue in kiwifruit?1?To investigate the residual amount and degradation rate of forchlorfenuron in kiwifruit after 90 days of application during kiwifruit growing season.After kiwifruit is sprayed with forchlorfenuron at concentrations of 0,5.0,10.0,20.0,50.0 mg/L,on the day of application,the residual concentration of forchlorfenuron was over 11562?g/kg.However,after 90 days of growth,the concentration of forchlorfenuron in the kiwifruit peel has degraded to 13.1-92.1?g/kg,and the degradation rate is greater than 98.6%.The concentration of forchlorfenuron in the kiwifruit pulp is degraded to 3.0-20.4?g/kg,and the degradation rate is greater than 99.6%.?2?The changes of forchlorfenuron residues in kiwifruit treated with ethylene during logistics storage were investigated.After treatment with 0.05 mmol/L Eth,,the content of forchlorfenuron in kiwifruit peel all decreased under the same storage days.The degradation rate of forchlorfenuron sprayed at different concentrations was7.4-61.8%,while the content of forchlorfenuron in the kiwifruit pulp increased,the rate of increase was 8.3-66.7%.After 30 days storage,the content of forchlorfenuron in the kiwifruit pulp was less than 23?g/kg.?3?The residues dynamics of forchlorfenuron in kiwifruit at different storage days?0 d,15 d,30 d,60 d,90 d,and 120 d?were investigated during logistics storage period.As the storage time increases,the residual amount of forchlorfenuron gradually decreases.After 120 days,the storage degradation rate in the kiwifruit peel reached47.3?89.3%,and the storage degradation rate in the kiwifruit pulp reached 35.4?74.3%.The residual amount of forchlorfenuron during storage was gradually degraded by first-order reaction kinetics mode.?4?The residual changes of forchlorfenuron in kiwifruit under different storage temperatures?2-8?,-25--15??were investigated during logistics storage period.As the storage temperature decreases,the residual elimination rate of forchlorfenuron is slowed down.The lower temperature,the less residual degradation under the same storage days.2.Non-clinical safety evaluation of forchlorfenuron2.1.Single dose toxicity assessment in miceThe abnormal reactions of 1500 mg/kg,1350 mg/kg,1215 mg/kg,1094 mg/kg and985 mg/kg groups were mainly due to the reduction of voluntary activity,dyspnea,abdominal breathing,apnea,tremors,prone,disappearance of righting reflexes,and clonic convulsions,etc.Surviving animals return to normal within two hours.Death occurred after administration of forchlorfenuron above 1094 mg/kg,and most animals died within 3 hours after administration.The phenomenon of diffuse and strip bleeding in the stomach and intestines of the animals,gastrointestinal dilation,a large amount of transparent liquid inside,bleeding in the serosal layer of the stomach wall,intestinal dark red were showed in the anatomical mice.The body weight of the animals in the test group first decreased and then increased.The body weight of the forchlorfenuron test animals decreased slightly compared with the solvent control group,but there was no significant difference?P?0.05?.The mortality rates of mice intragastrical administered with CPPU at 1500 mg/kg,1350 mg/kg,1215 mg/kg,1094 mg/kg and 985mg/kg were 100%,90%,60%,40%,and 0%,respectively.The LD50 value calculated by the Bliss method was 1164.7 mg/kg,and its 95%confidence limit was 1102.6-1230.3mg/kg.2.2.Repetitive dose toxicity assessment in ratsAfter 4 weeks of administration of forchlorfenuron in SD rats,on the first day of drug withdrawal,compared with the solvent control group,urea nitrogen and creatinine in male rats were significantly increased at 300 mg/kg and above dose groups of forchlorfenuron?P<0.05?,and blood glucose was also increased in the 600 mg/kg/day group?P<0.05?.The renal coefficient of male and female rats with 300 mg/kg and above of forchlorfenuron was significantly increased?P<0.05?,The liver coefficient of female rats in 300 mg/kg group of forchlorfenuron was significantly increased?P<0.01?.Histopathological change was found in the kidney pathological biopsy,and mainly manifested as mild renal tube atrophy,leukocyte tubular type,clear tubular type,interstitial inflammatory cell infiltration,and renal tubular dilatation.On the 4 weeks of withdrawal,the symptoms of toxicity were restored.It indicated that SD rats have a mild renal toxicity at a dose of 300 mg/kg/day after continuous intragastric administration of forchlorfenuron for 4 weeks,the main toxic target organ is the kidney,and no significant toxicity was observed at a dose of 150 mg/kg/day.2.3.Study on toxicity of the development of reproductive system of forchlorfenuron in lactating ratsDuring the experiment,there was no change in blood biochemistry,visceral organ coefficient,pathological tissue of female rat uterus and male rat testis,thickness of uterine wall of female rats,and sperm motility of male animals.Slight depilation which could be gradually recovered after drug withdrawal was observed in rats in high dose group?300 mg/kg/day?.The average body weight of 300 mg/kg/day female rats of forchlorfenuron was significantly decreased after 4 weeks of administration?P<0.01?,and recovered slightly after 4 week of recovery.After administered 300 mg/kg/day for 3weeks,Bun,CREA and GLU increased significantly in male rats?P<0.05?,and the abnormality remained unchanged after 4 weeks of administration until the recoveryperiod ended.Bun index of female rats increased significantly after 4 weeks of 300mg/kg/day administration?P<0.05?,and recovered at the end of convalescent period.The estrus cycle of female animals suffered early after administration,and the high-dose group?300 mg/kg/day?can be advanced about 2?3 days.Vaginal opening time was significantly also shortened?P<0.05?.Serum test results showed that the female estradiol index increased significantly with increasing dose after 4 weeks of administration?P<0.001?.It was shown that 300 mg/kg dose of forchlorfenuron can affect the estrous cycle and sex hormone levels in young rats.2.4.Study on the toxic exposure of forchlorfenuron in animals of different ages2.4.1 Establishment of a bioanalytical method for forchlorfenuron in plasma of ratsThis test uses LC-MS/MS to detect forchlorfenuron in rat plasma,and there was no interference in the detection of forchlorfenuron and curcumin.The correlation coefficients?r?of standard curve of forchlorfenuron were all greater than 0.98?r=0.9988?,and the quantitative range was 20-2000 ng/m L.The intra-batch and inter-batch precision of quality control samples?60,1000,1600 ng/m L?of forchlorfenuron were between 0.8%-2.5%and 0.4%-1.8%,respectively,the intra-batch accuracy is between 97.7%and 107.5%,and the inter-batch accuracy is between 92.5%and 102.3%.The extraction recovery rate is between 82.5%and 93.1%.The matrix effect is between 90.5%and 98.1%.The forchlorfenuron plasma sample remained stable when placed in an ice box for 12 hours,placed at-15-25?for 14 days,repeated freezing and thawing for 3 times,and placed in the autosampler for 24 hours.The concentration of forchlorfenuron was reliabile under dilution of 20 times,and the residues of samples were accepted.2.4.2 Study on toxic exposure of forchlorfenuron in animals of different agesAfter a single intragastric administration of forchlorfenuron in SD rats,AUC?0-t?and Cmax exposure to forchlorfenuron in adult and young SD rats increased with the increase of dose in the dosage range of 150-600 mg/kg/day.Compared with adult rats,the AUC?0-t?and Cmax of forchlorfenuron in young rats increased after administration of150,300,600 mg/kg/day forchlorfenuron.The female AUC?0-t?increased by 12.1,25.2,and 45.8%,respectively,and the male AUC?0-t?increased by 16.0,15.1,and 42.5%,respectively.The female Cmax increased by 16.3%,23.5%,and 37.2%,respectively,and the male Cmaxincreased by 28.3,42.2,and 50.7%,respectively.3.Safety risk assessment of forchlorfenuronAs the single toxicity,repetitive toxicity,toxicokinetics and the effect of precocious puberty in young rats were investigated,it is preliminarily speculated that the No-observed-adverse-effect-level?NOAEL?of forchlorfenuron was 100 mg/kg.According to the Guidelines for Establishing Daily Allowable Intake of Pesticides,the uncertainty factor?safety factor,UF?is generally 100.At the same time,combined with toxicological test data,the extrapolation from 4-week toxicity test to chronic toxicity test needs to be magnified 10 times.Thus,the uncertainty factor was 1000.The allowable daily intake?ADI?of forchlorfenuron was calculated,and the value is 0.10mg/kg.According to 350 g of edible fruit per day recommended in the Dietary Guidelines for Chinese Residents?60 kg of adult weight?,Risk ratio was calculated to be 0.1%,and the safe concentration of forchlorfenuron in fruits was calculated to be 25mg/kg,it is far lower than the forchlorfenuron residue of kiwifruit.The results showed that the kiwifruit sprayed with forchlorfenuron?5-50 mg/L?was safe to eat while logistics storage.Conclusion:The degradation rate of kiwifruit was more than 99.6%after 90 days'spraying with forchlorfenuron in the growing season.With the prolongation of storage time,the degradation of forchlorfenuron residues increased,and the lower storage temperature,the less degradation.After ethylene treatment,the content of forchlorfenuron in kiwifruit pulp increased,but it was still lower than the national standard limit of 50?g/kg.The single toxicity,repetitive toxicity,toxicokinetics and the effect of precocious puberty in young rats were investigated.It was inferred that the NOAEL of forchlorfenuron was 100 mg/kg.The calculated allowable daily intake of forchlorfenuron?ADI?is 0.10 mg/kg,risk ratio is 0.1%,and the safe concentration of forchlorfenuron in fruit is 16.4 mg/kg,it is far more than the forchlorfenuron residue of kiwifruit,indicating that forchlorfenuron has good safety.The subject can provide relevant scientific basis for the rational use of forchlorfenuron,and also improve the toxicological data of forchlorfenuron. |
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