The Study On The Toxicity And Immersion Marking Effect Of Calcein To Juvenile Silver Carp(Hypophthalmichthys Molitrix)

Fluorescent labeling technology is an important method for evaluating the effects of fish enhancement and releasing.As a labeling method that combines specific internal and external markers,it has been widely evaluated in both Osteichthyes and Elasmobranchii.Calcein(CAL),as a fluorescein complex,is a small molecule fluorescent dye.As a simple and effective chemical marker,it can form fluorescent labeling on aquatic animal calcium and bone structures of various aquatic animals.In addition,the intensity of the fluorescent labeling will increase with the increase of the concentration of the fluorescent dye or the immersion-marking time,and the fluorescent labeling on the fish body can be maintained for 100 to 360 days.However,according to related research,too high concentration of CAL often causes some toxicity to the fish.In order to better improve and popularize this marker technology,this study took juvenile silver carp Hypophthalmichthys molitrix as the research object,and carried out the effect of CAL immersion marking of juvenile silver carp,effects of CAL on the levels of antioxidant enzymes and lipid peroxidation in juvenile silver carp,and study on the recovery of antioxidant enzyme levels in juvenile silver carp after CAL exposure.This study aim to exploring the appropriate marking time and concentration of CAL-dyeing,and provide data support for the temporary rearing management of the labeled fish.The research results are helpful for establishing safe and effective monitoring methods of fish resources,and have certain theoretical and practical significance for promoting scientific management of fish resources and biodiversity protection in China.The main results of this study include:(1)When the concentration of CAL solution was used to immersion marking the juvenile silver carp,it could produce a stress response,and with the extension of the immersion-marking time,CAL caused different degrees of damage to gills and liver of juvenile silver carp.Among them,the activities of AST and ALT were significantly increased compared with the control(48 h),but at 96 h,the AST activities of the CALtreated groups were significantly decreased(P <0.05).In addition,the activities of ALP in serum of all CAL-treated groups were significantly increased at 24 h,48 h,72 h,and 96 h(P <0.05).In the early stage of immersion test,the activities of SOD,CAT,GPx and GST in gills and liver of juvenile silver carp were up-regulated compared with the control.With the extension of the immersion marking time,the activities of antioxidant enzymes(such as SOD,CAT and GST)in the gills and liver of juvenile silver carp were significantly down-regulated compared to the control.In addition,compared with the control,the contents of MDA in the juvenile silver carp liver showed an up-regulated trend at 48 h(P < 0.05),72 h(P < 0.01)and 96 h(P < 0.01).(2)When the concentrations of CAL solution with ?200 mg/L,it can be used to juvenile silver carp and produce a good marking effect.In addition,it no significant effect on their growth condition(total length and wet mass)(P >0.05),and also no significant effect on mortality during immersion and holding period(P >0.05).Detectable fluorescent markers were produced in the calcareous structure of juvenile silver carp otoliths(sagittae),cycloid scales(at and above the lateral line),and fin rays(dorsal,pectoral,ventral,anal,and caudal).Among them,when the concentration of the CAL solution is 150-200 mg/L,it can produce a good marking effect on the sagittae,scales and fins(the marking-quality ? 2).In addition,the marking effect of scales and fins were better than that of sagittae.The best mark quality was the lateral line scales of juvenile silver carp with 200 mg/L CAL-treatments.(3)When the concentrations of CAL solution with ?300 mg/L,the labeled juvenile silver carp would produce a stress response,but all the antioxidant enzyme activities in serum,gills and liver of the treated groups of could returned to normal levels compared with the control(P > 0.05)after 4 days of holding.Among them,the time required for the antioxidant enzyme activity to return to the normal levels was shorter in the low concentration(100 mg/L)of CAL immersion treatments,that is,the activities of SOD and CAT in the juvenile silver carp gill and liver returned to the normal level at 24 h compared with the control(P > 0.05).In addition,the effect of CAL on the antioxidant enzyme levels in the liver of juvenile silver carp was greater than that of the gills.The SOD activities in liver of the juvenile silver carp with the 100 mg/L CAL immersion treatment returned to the normal level at 6 h,and GPx activities in the juvenile sliver carp liver of all CAL-treated groups also returned to normal levels at 24 h.To sum up,when using fluorescent dye CAL to immersion-marking juvenile silver carp,the suitable conditions for immersion marking juvenile silver carp with fluorescent dye CAL were as follows: immersion-marking time was 24 h,immersion-marking concentration range was 150-200 mg/L,and the labeled juvenile silver carp should be kept for more than 4 days before being released into rivers or lakes.