Ecotoxicological Effects Of Several Heavy Metals And Organic Pollutants On The Hard Clam Meretrix Meretrix

At present, heavy metal and organic pollution have become serious problems in marine environment of China, which results in the deterioration of bivalve resources. Toxic effects of these pollutants on different life stages of Meretrix meretrix, especially the early development stages, were assessed, and the responding mechanism of the larvae to the heavy metal-stress was investigated. In addition, the impacts of these pollutants on the reproduction and immune function of hemocyte were also examined. The results discussed the effects of heavy metals and organic pollutants on recruitment amount of bivalve resources, and could provide data for establishing population-dynamic model. Moreover, these results also provide basis for marine organism protection and marine environment restoration. The details are as follows:1. The impacts of Hg2+, Cd2+ and Pb2+ on embryogenesis, survival, growth and metamorphosis of larvae were investigated. The EC50 for embryogenesis was 5.4μg/L for Hg2+, 1014μg/L for Cd2+ and 297μg/L for Pb2+, respectively. The 96 h LC50 for D-shaped larvae was 14.0μg/L for Hg2+, 68μg/L for Cd2+ and 353μg/L for Pb2+, respectively. Growth was significantly retarded at 18.5μg/L for Hg2+, 104μg/L for Cd2+ and 197μg/L for Pb2+ respectively. The EC50 for metamorphosis, similar to 48 h LC50, was higher than 96 h LC50. The heavy metals were consistently ranked in the following order from highest to lowest toxicity: Hg2+ >Pb2+> Cd2+.2. The impacts of benzo[a]pyrene (Bap) and Aroclor1254 on embryogenesis and larval development were investigated using static laboratory toxicity tests at nominal concentrations of 6.25–1600μg/L. Even 596μg/L Bap and 984μg/L Aroclor1254 only caused minor reductions in embryo development rates. The 96 h LC50 values for D-shaped larvae were 156μg/L for Bap and 132μg/L for Aroclor1254, respectively. The most sensitive toxicity endpoint in this study was metamorphosis, with an EC50 value of 20μg/L for Bap and 35μg/L for Aroclor1254.3. A new MT (designated MmMT) gene was identified and cloned from M. meretrix. The full length cDNA of MmMT consisted of an open reading frame (ORF) of 390 bp encoding a protein of 129 amino acids, with 23 cysteine residues and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys at the C-terminus. The deduced amino acid sequence of MmMT showed about 57%~84% identity with previously published MT sequences of mussels and oysters. Real-time PCR was conducted to analyze the expression level of MT mRNA in M. meretrix larvae under Cd2+ (25μg/L) exposure. Results showed that Cd2+ could induce the mRNA expression of MmMT in the larvae, and the expression level increased 5.04-fold and 3.99-fold in D-shaped larvae and pediveligers respectively. Immunolocalization of MmMT in the stressed larvae revealed that MmMT was synthesized in almost all the soft parts of the stressed larvae at trochophore and postlarva stage, while it was only synthesized in the velum and epidermis of the stressed larvae at D-shaped larva and pediveliger stages. Additionally, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities were also measured in M. meretrix larvae. The results showed the increase of enzymatic activities mainly in D-shaped larvae and pediveligers under cadmium stress.4. The gonad development of M. meretrix was retarded after exposure to 5μg/L and 50μg/L Bap for 30 days. 100μg/L Cd2+ and 50μg/L Hg2+ accelerated gonad degeneration of female clams respectively after exposure for 15 days, while they had little effect on male gonad development. 50μg/L Bap not only had obvious damage to membrane structure of organelles, but also hindered vitellogenin synthesis resulting in yolk granule deformation. 50μg/L Bap and 50μg/L Hg2+ induced a decrease of the glutathione (GSH) content in gonad. Bap (all concentration) and 100μg/L Cd2+ exposure increased GST activity in gonad, while 50μg/L Hg2+ inhibited GPx activity.5. The ultrastructure damages of heamocyte exposed to Bap were described as follows: the pseudopod stretching singularly, mitochondrion becoming swollen, and the number of organelles decreasing. There were also vacuolization and lysis on the edge of some heamocytes. The neutral red retention time (NRRT) of lysosome in heamocytes decreased as exposure time prolonged. After exposure to 5μg/L and 50μg/L Bap for 6 days, NRRTs of these groups were significantly lower than those of control group. Micronucleus (MN) frequency and total nucleus abnormality (TNA) frequency in heamocytes also enhanced as exposure time increased. The MN frequency and TNA frequency of 50μg/L group increased significantly after 6-day exposure.6. The effects of cadmium and Bap on immune function of heamocytes were examined by a flow cytometer. The results indicated that 5μg/L and 50μg/L Bap inhibited phagocytosis and increased the mortality of heamocytes, while 50μg/L Bap could enhance reactive oxygen species (ROS). 10μg/L and 100μg/L Cd2+ significantly suppressed phagocytosis of heamocytes and increased mortality of heamocytes, while 100μg/L Cd2+ stimulated ROS in hemocytes.