Temporal And Spatial Distribution In Three Typical Mariculture Bays Of Northern Shandong Province And Biological Toxicity In Apostichopus Japonicas Of Semicarbazide

Pollutants in the ocean are harmful to marine organisms and human health.Semicarbazide is called a new type of marine pollutant currently,reaching a dynamic equilibrium between seawater and sediment and accumulated by marine organisms ultimately.Therefore,it is the prerequisite to make the content,distribution characteristics and the toxic effects certain for taking corresponding measures.In this dissertation,a high performance liquid chromatography tandem mass spectrometry method（HPLC-MS/MS）for determination of semicarbazide was established in seawater,sediment and marine organisms.The temporal and spatial distribution was studied in three typical mariculture bays of northern Shandong province,and its distribution characteristics and influencing factors were discussed to provide theoretical support for marine regulation and aquaculture.As the most economical species of aquatic products,the biological toxicity was studied in the aspects of acute toxicity,as well as effects of tissue structures,enzyme activity of oxidative stress and neurotoxicity in Apostichopus japonicas.Also with the highest rates of detection,the distribution and elimination characteristics of low content were studied to promote the sustainable development of aquaculture.1.In this dissertation,the pretreatment,liquid chromatography and mass spectrometry conditions were optimized,and HPLC-MS/MS method for determination of semicarbazide was established in seawater,sediment and marine organisms.HLB column had the advantages of good recovery in purification and enrichment.The sediment and organisms could be extracted by ethyl acetate twice and well purified by liquid-liquid extraction with n-hexane to get a good recovery.Matrix effect was negligible,and retention time and ion abundance ratio were used for qualitative analysis.The detection limits were 0.010μg/L,0.50μg/kg and 0.50μg/kg in seawater,sediment and organisms,respectively.The intra-day relative standard deviations were between 4.59%and 7.65%,and the inter-day 4.08%and 8.97%.The method above was evaluated and proved to be highly sensitive and reproducible and of good efficient in separation,accurately qualitative and quantitative.2.Taking Jincheng Bay,Sishili Bay and Western Laizhou Bay as objects,three typical mariculture bays in northern Shandong province,each 15 stations,a total of 47voyages,705 samples analyzed,the temporal and spatial distribution was studied in the seawater,and the distribution characteristics and influencing factors were discussed.The trend was as follows:contents were higher near the inlet of the rivers,and relatively lower in the middle and outside the bay.There were three typical regions,the strip-shaped region with high content,the point source with high content and the region with low content.The maximum appeared in seeding stage（March and April）and high temperature stage with higher incidence of disease（August and September）of aquatic animals,showing a bimodal change.It was suggested that the detection rates and law enforcement should be strengthened in the two periods,and the illegal use of nitrofurazone may be still existed.The effect of aquaculture activities,runoff input,seasonal variation and natural degradation on the distribution were studied.Compared with other factors,aquaculture played a dominant role.3.The distribution was studied in sediment,and influencing factors were discussed,210 samples analyzed.Semicarbazide was at an undetected level sampled from the three bays.Semicarbazide was easily dissolved in seawater,so the desorption capacity was much greater than absorption,making it difficult to migrate from water phase to sediment phase,in the end with little semicarbazide adsorbed in sediment.At the same time,the content was very low in seawater,and the maximum was only 1/759 of Chaohe River estuary,leading to less amount adsorbed in the sediment.Meanwhile,content of organic carbon was 0.2029-0.2306,so low making it difficult to distribute in the sediment.The partition behavior of semicarbazide in sediment-water showed surface adsorption was the main factor and the partition was weak.The partition coefficient of organic carbon was only 5.60,indicating it was difficult to migrate to sediment phase.It was concluded that the behaviors above ultimately made semicarbazide at an undetected level in the sediment.4.The contents,distribution characteristics and bioaccumulation factors（BAFs）were analyzed in shellfish in Jincheng Bay,Sishili Bay and Western Laizhou Bay,69samples analyzed,and all were less than the reported value specified in the task of Shandong province aquatic products quality and safety supervision and inspection（1.00μg/kg）.The BAFs were ranging from 4.47 to 18.9,meaning accumulation ability was different in different shellfish.There was a positive correlation between content and shell length.Experiments showed that bioconcentration factors were145.3-200.0 in dinoflagellate,platymonas sp.and neostigmata sp,dominant algaes in the three bays,which indicated that the algaes could accumulate semicarbazide in the environment.Shellfish could also accumulate semicarbazide by filtering the algaes.5.According to the acute toxicity,the LC₅₀ value of semicarbazide was 3.72 g/L in Apostichopus japonicas（calculated by semicarbazide hydrochloride）,and 3.43g/L-4.02 g/L with 95%confidence level.At the three concentrations(1/5 LC₅₀,1/25LC₅₀ and 1/50 LC₅₀),the effects were mainly in tissues such as respiratory tree,intestine and muscle.In 1/5 LC₅₀ group,lesion was serious in respiratory tree,intestine was disintegrated basically and smooth muscle was in disorder.In 1/25 LC₅₀and 1/50 LC₅₀ group,lesion showed a slow and acuumulation process in the three tissues.The toxic effect was excitation,and enzyme activity of antioxidant and neurotransmission were increasing and then decreasing,induced firstly and then inhibited,and was most obvious in 1/5 LC₅₀ group for poisoning reaction.The activities in 1/25 LC₅₀ group were highest,followed by 1/5 LC₅₀ group and 1/50 LC₅₀group,and all tended to be the similar levels with the control group in the end.It was concluded that it may be related to immune fatigue in Apostichopus japonicas.6.The distribution characteristics of low residues of semicarbazide were analyzed in Apostichopus japonicas,and the trend was clarified.Semicarbazide of direct exposure,was at an undetected level in the body wall at 140 d,and the average removal rate was 0.0073μg/kg/d.Semicarbazide caused by nitrofurazone was at an undetected level in the body wall at 160 d,and the average removal rate was 0.0064μg/kg/d.The half-lives of two ways,1045.7 h and 1224.2 h,respectively,semicarbazide caused by nitrofurazone longer than direct exposure.Semicarbazide was still at an undetected level after 180 d of follow-up monitoring.Although the accumulation and elimination rates were relatively faster in viscus,it took longer time to decrease to undetected because of the larger amount with stronger capacity of accumulation in viscus.The data showed that Apostichopus japonicas fed with nitrofurazone or grown in the seawater at a certain content of semicarbazide could be decreased to undetected only after purified for a long time.