Toxic Effects Of Lead On The Development Of Embryos And Larvae Of The Chinese Giant Salamander

Lead(Pb)is a kind of harmful element distribute in nature widely,which mainly exists in the form of inorganic salts,such as lead chloride(PbCl2),lead sulfate(PbSO4)or lead nitrate(Pb(NO3)2).In our daily life,Pb and compounds have been widely applied to the industry,agriculture including gasoline,paint,antiknock storage batteries,fuel and ammunition.Pb plays an important role in the industrial and agricultural processes,but it will pollute environment since it is a toxic.Pb poisoning can cause damage of many aspects of the body such as nerve,immunity and reproduction and can occur when containing Pb is inhaled or swallowed.With the pollutions increasing dumped into water-body,the concentration of Pb in an aqueous system have exceeded the low value of environmental standards.At present,Pb concentrations reach up to 3.1 mg/L in the polluted water,which greatly increase the opportunity of aquatic organisms exposed to Pb.Bufo gargarizans are used as one of important monitoring species in aquatic environment,which are extremely sensitive to heavy metal pollution.Therefore,the Pb exposure tests were carried out in B.gargarizans embryos and larvae to evaluate the toxicological effect of Pb on amphibians.Firstly,the LC50 value was determined after 96 h.Secondly,embryos were exposed to different concentrations of Pb2+(10,50,100,500,1000 and 2000 ?g/L)from Gs 3 to Gs 25 for 11 days.Finally,larvae also were exposed to 10-2000 ?g/L Pb2+ from Gs 25 to Gs 46 during a 60-day.The main results of the thesis are as follows:1.In acute toxicity test,B.gargarizans embryos at Gs 3 were randomly assigned to Pb2+ at different concentrations(10,20,30,40,50,60,70 and 80 mg/L).The death number was recorded,and the 96h median lethal concentration(LC50)and safety concentration(SC)was determined.After 96-h exposure,96-h LC50 and SCwas 26.6 mg/L and 266.3 ?g/L,respectively.2.Since the trial ended,we obverted embryonic malformation like edema,axial flexures,stunted growth and tissue hyperplasia in 500,1000 and 2000 ?g/L groups.In addition,scanning electron microscopy showed that Pb at high levels can cause hyperplasia of epidermal cell and keratinization in B.gargarizans embryos.3.After 5 days and 7 days of of exposure,significant increase in the growth parameters(total length,weight and developmental stage)was observed in 10 and 50?g/L Pb groups relative to untreated control,but significant reduction in development was observed in 2000 ?g/L Pb group.Our research suggested that low concentration of Pb(10 and 50 ?g/L)facilitated growth and development of embryos while high concentration of Pb exposure(2000 ?g/L)shown repress action.4.Using real-time PCR,we investigated these genes expression(SOD,GPX,HSP90,Dio2,Dio3,TR? and TR?)in B.gargarizans embryos(Gs 22).Pb at 2000 ?g/L down-regulated Dio2,TRa and TR? mRNA levels but up-regulated Dio3 mRNA level.However,Pb at 50 ?g/L increased TRa mRNA level but decreased Dio3 mRNA level.The regulation of genes(SOD,GPX and HSP90)related to oxidative stress also change after Pb exposure.5.Chronic toxicity test was conducted in B.gargarizans larvae and our result showed that 2000 ?g/L Pb concentration increased the mortality and induced deformities.6.In chronic toxicity experiment,tadpoles were collected on day 10 and 20 to weighed,staged(Gs),and measured for total length.A statistically significant inhibition of growth was observed at the highest exposure concentration(2000 ?g/L Pb),whereas the accelerated effect of Pb first became statistically significant at 50 ?g/L Pb in the present study.7.At the climax of metamorphosis(Gs 42),a highly significant increase in metamorphosis percent was observed at 10 and 50 ?g/L Pb.On the contrary,Pb at concentration of 2000 ?g/L significantly delayed metamorphosis time and decreased metamorphosis percentage in B.gargarizans larvae.In addition,exposure to 2000 ?g/L Pb significantly reduced body size(total length)and inhibited the metamorphosis,while Pb at concentration of 50 ?g/L significantly accelerated metamorphosis and size at metamorphosis(total length,HLL).8.Tadpoles were collected for thyroid measurement and histological examination at Gs 33,38 and 42 respectively.In our study,morphological lesions including colloid depletion in thyroid follicles and follicular epithelial cells with irregular arrangement were found in the thyroid gland of tadpoles exposed to different Pb concentrations.9.The double-stained method with aleian blue and alizarin red were used to investigate the skeletal development of the larvae exposed to Pb at Gs 42 and 46.The most conspicuous effect of Pb-contaminate was that low dosage of Pb(50 ?g/L)stimulated bone ossification and high dosage of Pb(1000 and 2000 ?g/L)retarded ossification in the present study.10.At Gs 42,we measured relative Dio2,Dio3,TR? and TR? mRNA transcript levels in livers,hind-limbs and tail.There is a rapid rise in Dio3 and a sharp drop in Dio2,TR? and TR? at 2000 ?g/L Pb concentration in three tissues.Interesting,abundances of transcripts of Dio2 and TR? were significantly increased,whereas abundances of transcripts of Dio3 were decreased exposed to 50 ?g/L Pb.Overall,Pb exposure has a potential effect on the growth and development in B.gargarizans embryos and larvae.High concentrations of Pb ions caused deformity,retarded growth and bone development.But Pb at low concentration could accelerate the growth and development to a certain extent.We suspected that toxicology effect of Pb may be related to interference of oxidative stress and thyroid hormones(THs)pathway.