| In recent years, marine environmental pollution is seriously increasing, which is harmful to halobios. The character of heavy metal pollution is persistent, non-degradable and relatively stable. Polycyclic aromatic hydrocarbons (PAHs) derived from petroleum are known to be highly mutagenic and carcinogenic and benzo[a]pyrene (B[a]P) shows the strongest carcinogenicity. Heavy metals and their interactions with PAHs frequently occur, and organisms are usually exposed to chemicals in combinations. The studies on combination toxicity effects of metals and PAHs on marine animals are limited. In the present study, Ruditapes philippinarum was exposed to Cd, B[a]P and a mixture of Cd and B[α]P in order to evaluate the relationship of acute toxicity, tissue Cd and B[α]P accumulation, metabolic parameters activities and gene expressions and the combination toxicity mechanism of metals and PAHs was discussed. The following results were obtained.1 Study on single and combination effects of Cd2+ and B[a]P on acute toxicity of R. philippinarumThe combination effects of Cd2+ and B[a]P on acute toxicity of R. philippinarum were studied. The results indicated that the 24,48,72 and 96h LC50 for Cd2+ to R. philippinarum were 119.23,50.41,24.12,14.68mg/L. The single toxic sequence of B[α]P were LC50>10mg/L. The joint toxicity of Cd2+ and B[a]P was antagonistic at 24h, synergistic at 24-96h. It was concluded that significant effects of acute toxicity of Cd2+ and B[α]P combined pollution on R. philippinarum and effects was related to component, concentration of combined pollutant and time.2 Study on single and combination pollution of Cd2+ and B[a]P on tissue accumulation of R. philippinarumThe single and combination effects of Cd2+ and B[α]P bioaccumulation in gills, digestive glands, adductor muscles and somatic tissues of R. philippinarum. were studied. The results indicated that In Cd and Cd+B[α]P exposed clams, Cd concentration increased progressively in the gills, digestive glands and somatic tissues during the experimental period (P<0.05). However, the exposure to Cd+ B[a]P significantly increased Cd accumulation compared to the treatment with Cd alone or to the control or B[α]P exposed clams (P<0.05). In control and Cd exposed tissues, B[a]P levels were no significant difference (P>0.05), whereas in B[a]P and Cd+ B[a]P treated clams, B[a]P markedly increased, especially in combinations (P<0.05). Meanwhile, concentrations of Cd or B[a]P in digestive glands were consistently higher than those in gills, and adductor muscles pollutants levels were no significant difference (P>0.05). What can be concluded is that the ability of bioaccumulation of Cd and B[α]P in clam is mighty.3 Study on single and combination effects of Cd2+ and B[a]P on metabolic parameters activities of R. philippinarumThe single and combination effects of Cd2+ and B[a]P on metabolic parameters activities of gills and digestive glands of R. philippinarum were studied. The results indicated tha MT contents of the Cd2+ and Cd2+ +B[a]P groups showed peak changes within 12h, reached the peak value at 1h or 3h, then still induced and stable (P<0.05). Meanwhile, metallothionein (MT) content after Cd+B[a]P exposed was suppressed compared to Cd only exposed (P<0.05). aryl hydrocarbon hydroxylase (AHH) activities of the B[a]P and Cd2++B[a]P treatments showed obviously increasing trend in 6d and reached the maximum at 1h or 3h, then still induced and stable (P<0.05). However a significant inhibition of AHH activity in organisms co-exposed to Cd and B[α]P (.P<0.05). Reduced glutathione(GSH) contents of the single experimental groups showed peak changes within 12h, reached the peak value at lh or 3h, then recovered to control level (P>0.05) or be induced (P<0.05) after 12h. However, those in combined experimental groups (except Cd+B[α]P,15μg/L+0.0μg/L) were restrained all the time and became stable after 12h. Subsequently, glutathione S-transferase (GST) and superoxide dismutase (SOD) activities showed obviously increasing trend (P<0.05) and reached the maximum at 1h or 3h. After 12h GST activities had no significant difference with the control group (P>0.05), but SOD activities were still induced (P<0.05) and stable. Meanwhile, in Cd+B[α]P treatments, GST and SOD activities were significantly higher while GSH contents were lower compared to treatment exposed to the single chemical. No significant difference in any of the parameter was observed in the control group. It was concluded that there were good linear correlations and dose-time effect relationship between the indexes and Cd2+ and B[a]P. GSH, SOD in Digestive gland is the monitoring of heavy metals and PAHs pollution in the marine environment potential biomarker. 4 Study on single and combination effects of Cd2+ and B[α]P on gene expression of metabolic enzyme of R. philippinarumThe single and combination effects of Cd2+ and B[α]P on GST-pi and Cu,Zn-SOD gene expressions of gills and digestive glands of R. philippinarum were studied. The results indicated that the mRNA transcription levels of GST-pi and Cu,Zn-SOD gene in clams solely exposed to Cd or B[α]P significantly increased (P<0.05) and showed peak changes within 6h, reached the peak value at 1h or 3h. And a significantly higher expressions were observed for GST-pi and Cu,Zn-SOD gene of co-exposures than those of single groups (P<0.05). Cu,Zn-SOD gene expression maintained at high level and remarkable different from control values (P<0.05) until the end of the experiment, while GST-pi gene expression recovered to the control level after day 6 (P>0.05). The consistency of enzyme and gene expression of GST-pi and Cu,Zn-SOD indicated the enzyme-level was intervened by gene regulated. It also can be drawn from the experiment that the essentiality and inseparable relationship between biotransformation enzymes and antioxidant enzymes.
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