Study And Application Of Gill Filament And Hepatic Erod And Bile Facs Biomarkers From Sebastiscus Marmoratus

In laboratory, using 7-ethoxyresorufin as substrate and the water soluble fraction (WSF) of the crude oil obtained from Beibu Gulf, GuangXi Zhuang Autonomous Region as inducer the optimal conditions for the measurement of gill filament EROD activity were studied in controlled and exposed groups of Sebastiscus marmoratus, then a gill filament-based assay for EROD acticity was established for the species of fish. Moreover, after dose-response and time-course experiments by the WSF the minimal doses and times were determined for significant induction of the EROD activities from gill filament and liver supernatant, as well as bile FACs including naphthalene-, phenanthrene-, pyrene- and BaP–type metabolites from Sebastiscus marmoratus. In field, the above two types of 6 biomarkers were used for monitoring oil pollution in Xiamen West Waters combining with chemical determination of the oil contents in the sea water. Correlation between the biomarkers and the oil contents in the sea water was revealed by biological statistical analysis. Tthe result indicated that the gill filament EROD activity was proved to be a biomarker for monitoring oil pollution as effective as hepatic supernatant EROD and bile FACs in Sebastiscus marmoratus. Meanwhile, the bio-effect of oil pollution was evaluated in Xiamen West Waters. The major results were shown as follow:1. For the gill filament EROD activity from Sebastiscus marmoratus, the optimal conditions for the enzyme reaction system were pH 7.8 and 35℃, respectively; the optimal total length of gill filament was 18.8±1.9mm (about 6 no-cut gill filaments); the gill filament EROD with higher activity at the beginning 50min of reaction; no significant change for the EROD activity was observed for the total length about 20mm gill filaments either by cut into 2mm or not.2. The result of dose-response experiment showed that, exposed to WSF of crude oil at 10μg/L～100μg/L for 5 days, gill filament and hepatic supernatant EROD activities were significantly induced at the concentration of 50μg/L, compared to the controlled group. Then, gill filament EROD activity increased continually and significantly with the rise of oil concentration, while hepatic supernatant EROD decreased into the controlled level at 75～100μg/L. The correlation coefficient of the gill filament EROD activity and the oil contents of sea water was 0.9599. The contents of 4 kinds of PAHs metabolites in bile were increased with the increment of oil concentration at 10～100μg/L of crude oil WSF. One-way ANOVA analysis showed that the concentrations of naphthalene- and BaP-type metabolites in bile were significantly raised compared to the controlled group.3. The result of time-course experiment showed that, exposed to crude oil WSF at 40μg/L for 2～10 days, the gill filament and hepatic supernatant EROD activities reached the peaks in the 4th day simultaneously, both of them were significantly elevated compared to the controlled level. The gill filament EROD activity remained at a high level with a significant difference compared to the controlled group, but hepatic supernatant EROD returned to the controlled level after 6 days of exposure. Naphthalene-type metabolites in bile was significantly increased during the experiment and showed two peaks after the exposure of 2 and 10 days, respectively, while the other kinds of bile FACs had 2 peaks separately after 4 and 10 days of exposure. One-way ANOVA analysis showed that the FACs concentrations in these peak days were significantly increased compared to the controlled group.4. The results of two filed monitoring experiments indicated that after 2 weeks of exposure, the correlation coefficients between the gill filament EROD activities and oil contents of the sea water was 0.9332 and 0.9321, respectively, better than the results after the exposure of 4 or 5 weeks in field. Hepatic supernatant EROD activities and the concentrations of 3～5 rings of PAHs metabolites showed a stable and good positive correlation to the water oil contents (R2=0.7830～0.9991). The results indicated that the gill filament EROD activity of Sebastiscus marmoratus was also an effective biomarker for the monitoring marine oil pollution, and further supported the fact that hepatic supernatant EROD activity and bile FACs were available biomarkers for monitoring organic pollution.5. After 2 weeks of exposure twice in field, the results demonstrated a good positive correlation between the gill filament and hepatic supernatant EROD activities (R2 = 0.9767 and 0.9971, respectively). A good positive correlation were observed between the gill filament EROD activity and the 4 kinds of PAHs metabolites in biles as well (R2= 0.6534～0.9321). However, the correlation between the gill filament EROD activity and the other kinds of biomarkers declined when the monitoring time prolonged to 4 or 5 weeks. However, the hepatic supernatant EROD activity revealed a stable and good positive correlation to 3～5 rings of PAHs metabolites in bile (R2= 0.7134～0.9616 for most of the results). The results of 2 field monitoring experiments indicated that 4 kinds of PAHs metabolites in bile represented a stable and good positive correlation to each another (R2= 0.7187～0.9996).6. Results of the contents of 1-OH pyr and pyrene-type metabolites in bile measured by SFS and FF respectively showed that the correlation coefficient for both of the metabolites were above 0.9931 during the two filed monitoring experiments (including two 2 weeks and two 4/5 weeks)。Most of experimental results indicated that 1-OH pyr was the major metabolite of pyrene and a typical metabolite of FACs in bile. Compared with SFS, measurement of PAHs metabolites in bile by FF was a simplified and fast method.