Toxic Effects Of Calcein Injection On Juvenile Silver Carp (Hypophthalmichthys Molitrix) And Feeding On Juvenile Spinibarbus Sinensis & The Marking Effects Of Calcein Immersion

Due to these advantages such as save time&cost,simple operation and less stimulation to fish,fluorescent labeling technology has been widely used for labeling Osteichthyes and Elasmobranchii.Calcein（CAL）,as a fluorescein complex,is a small molecule fluorescent dye,which could form fluorescent labeling on calcareous and bony structures of various aquatic animals.The application methods of fluorescent marking could be summed up to three approaches,i.e.,immersion,feeding and injection.Generally,the marking effects of the fluorescent labeling will be increased with theconcentration or dosage of the fluorescent dye.The fluorescent labeling could be maintained for months,and even years.According to related studies,excessive CAL could also cause toxicity to fish.However,there were few studies on the toxicity of fluorescent dyes.Therefore,in order to explore the safety dosage of CAL intramuscular injection in juvenile sliver carp and improve the safe assessments of different labeling methods for CAL,the toxic effects of CAL intramuscular injection in juvenile sliver carp Hypophthalmichthys molitrix was studied on the basis of previous studies.Moreover,the marking effects of CAL immersion and the toxic effects of CAL feeding on juvenile Spinibarbus sinensis were studies.The main results were as follows:（1）The 96 h LD₅₀ of juvenile silver carp after CAL intramuscular injection was 263.2mg/kg.When the injection dosage of CAL was more than 50 mg/kg,the daily mortality of juvenile silver carp was significantly higher than the control group（P<0.01）during 1-12 days.The activities of serum Alkaline phosphatases（AKP）,glutamate-oxaloacetate transaminase（AST）and glutamate-pyruvate transaminase（ALT）in 100 and 200 mg/kg groups were significantly higher than those in the control group（P<0.05）after injection 1 d.From 4 d to 6d after injection,the activities of Superoxide dismutase（SOD）and Glutathione peroxidase（GPX）in hepatopancreas of juvenile silver carp were significantly increased compared with the control group.Except for 12 h and 2 d,SOD activity in gill of juvenile silver carp had little change during the whole experiment,and there was no significant difference between experimental group and the control group（P>0.05）.In addition,the contents of malondialdehyde（MDA）in gill and hepatopancreas of juvenile silver carp from 100 and 200mg/kg groups was significantly increased（P>0.05）after injection 1 d.To sum up,CAL intramuscular injection could induce the oxidative stress reaction of juvenile silver carp,and produce excessive oxygen free radicals to destroy the antioxidant stress system,even cause damages to the gills and hepatopancreas of juvenile silver carp.（2）In order to mark juvenile S.sinensis,0,50,100,150 and 200 mg/L CAL were used for immersion marking.The results showed that CAL immersion for 24h could produce fluorescent labeling on the calcareous structure（including otolith,scale and fin ray）of juvenile S.sinensis.The optimal labeling concentration was 200 mg/L,and the retention time of fluorescent mark was more than 90 days.When the concentration of CAL was more than 100 mg/L,150-200mg/L,and 150-200 mg/L,clear marks（marking quality≥2）on the fin rays,scales,and otoliths could be produced,respectively.In addition,the results of 24 h immersion and 90 d growth experiment clearly showed that 0-200 mg/L CAL immersion had no significant effects on growth and mortality of juvenile S.sinensis（P>0.05）.（3）Juvenile S.sinensis were fed with diets,which contained 0,2,8 and 32 g/kg CAL（CAL weight/feed weight）,respectively.The experiment included two parts,i.e.,toxicity accumulation part（last for 16 d）and toxicity elimination part（last for 32 d）.The results showed that the mortality of juvenile S.sinensis was 0%throughout the experiment.From 1 d to 16 d of the toxicity accumulation part,the activities of serum enzymes（e.g.,AKP,AST and ALT）in all treatment groups were significantly higher than those in the control group（P<0.01）.From1 d to 32 d of toxicity elimination part,the serum enzyme activities showed a downward trend.The activity of SOD,GPX and GST and the content of MDA in the hepatopancreas and kidney decreased at the beginning,and then increased with the dosage of CAL.The activity of GST increased from 1 d to 8 d in the toxicity accumulation part,and decreased at 16 d.The contents of MDA in hepatopancreas of all treatment groups at 8 d and 16 d and in kidneys of 8 g/kg treatment group at 4 d and 8 d returned to normal level in the toxicity elimination part.In conclusion,CAL intramuscular injection was not suitable for marking juvenile H.molitrix.Even if intramuscular injection was used,the dosage should be strictly controlled as≤50 mg/kg.The otoliths,scale,and fin ray of juvenile S.sinensis could be successfully marked by CAL immersion for 24 h.The optimal concentration for immersion labeling was 200 mg/L,and the labeling retention time was more than 90 d.In addition,when CAL（dosage of CAL was≤32 g/kg）was used for feeding mark juvenile S.sinensis,the feeding time should be strictly controlled for less than 8 days in order to reduce the damages to kidneys and hepatopancreas of experimental fish,and the marked juvenile S.sinensis should be maintained for more than 32days before released.