| Marine sediments are often a final sink for numerous anthropogenic contaminants and may impose serious effects on benthic organisms and ecosystem. Pollutants in sediments may act as a pollution source via spreading from the sediment to water, resuspending when sediments are disturbed, or being absorbed by benthic organisms (bioaccumulation). As a consequence contaminated sediments can have serious effects on benthic organisms and ecosystem. Because many contaminants accumulate in food chains, they may also affect human health. So it is important to use in vitro bioassays to assess the potential toxicity of marine sediments.First, we collected the marine sediments from Li Cang River (LC), Zhan Qiao (ZQ) and Olympic Sailing Center (OSC) from Qingdao coastal areas. The nonvolatile organic ingredients of all the above sediments were extracted by a combined sonication-shaking procedure. Sixteen EPA (U.S Environmental Protection Agency) priority PAHs in the three marine sediment samples were analyzed according to the method of GC-MS. The results showed that the three sites, LC sediment extracts exhibited the highest level of total PAHs (4032.62 ng/g dry weight), followed in order by ZQ sediment extracts (1154.06 ng/g dry weight) and OSC sediment extracts (407.75 ng/g dry weight). The 5-6 rings PAH in LC and ZQ samples were significantly higher than that of OSC sediments. This indicated LC is the high contamination area and OSC is the low contamination area.Second, we used FG cells to evaluate the cytotoxicity of the three samples. The MTT assay rusults showed that all the three sediment extracts exhibited cytotoxicity to the FG cells via inhibiting their growth in a dose-dependent cytotoxic fashion. After preliminary experiments, it was found that the lowest observed effective concentrations (LOEC) and 24-h IC50 for LC, ZQ and OSC sediments were about 1.5,12.5 and 125 mg dry weight/ml and 44.87,109.14 and 1080.19 mg dry weight/ml respectively. The cytotoxicity orders of the three sampling sites were LC>ZQ>OSC.Third, we used comet assay to evaluate the genotoxicity of the three samples. The rusuts showed that all the three sediment extracts were able to induce DNA damage in exposed FG cells in a dose-dependent manner. The LOEC for LC, ZQ and OSC extracts were 0.5,10 and 200 mg dry weight/ml, respectively. The corresponding comet assay scores (au) for LC, ZQ and OSC extracts were 26-211,22-157 and 18-99. Thus the genotoxicity order of these sediment extracts in FG cells turned out to be LC>ZQ>OSC, which is consistent with their cytotoxicity order. Based on the cytotoxicity assay above, it was found that 5,20 and 200 mg dry weight/ml of LC, ZQ and OSC sediment extracts induced closely similar cytotoxicity in FG cells after 24 hrs exposure. Therefore, these doses were chosen in the time-dependent genotoxicity test. The results indicated that the three sediment extracts at the doses inducing closely similar cytotoxicity did not induce similar genotoxicity, with the genotoxicity of LC and ZQ extracts being markedly higher than that of OSC extracts. This indicated that cytotoxicity is simply incomparable with genotoxicity.Finally, we used zeabrafish embryo test to evaluate the teratogenicity of the LC, ZQ and OSC sediment extracts. When the zebrafish embryos were exposed to varied concentrations of LC, ZQ and OSC sediment extracts for 24 hrs. The LOEC (p<0.05) of LC, ZQ and OSC sediment extracts were 5,10, 300 mg dry weight/ml, respectively, and their 24-h-LC50 were 22.08,48.32 and 704.25 mg dry weight/ml, individually. Thus the teratogenicity order of these sediment extracts in FG cells turned out to be LC>ZQ>OSC. Based on the cytotoxicity assay above, we also selected 5,20 and 200 mg dry weight/ml of LC, ZQ and OSC sediment extracts to evaluate the time-dependent teratogenicity. However, the sediment extracts at the doses inducing closely similar cytotoxicity didn't induced similar teratogenicity, with the teratogenicity of LC and ZQ sediment extracts being markedly higher than that of OSC sediment extracts, indicating that cytotoxicity is simply incomparable with teratogenicity.At 9 hrs post fertilization (hpf), about 95.0±2.8% control embryos were in the stage of 90% epiboly, but only 68.0±7.6%,72.0±7.6% and 87.0±5.8% embryos treated with LC, ZQ and OSC sediment extracts reached the same stage, with the remaining embryos at about 50% to 75% epiboly. For the embryos treated with LC, ZQ, and OSC sediment extracts, the total percentages of malformed and dead embryos at 24 hpf were 30.0±5.0%,25.0±5.0% and 10.0±7.6% as well as 20.0±5.0%,12.0±7.6% and 3.0±2.8%, respectively, contrasting to those of 5.0±1.2% and 3.0±2.8% in control. At 48 hpf, The total percentage of malformed embryos then was 45.0±5.0%,30.0±2.8% and 20.0±5.0%, respectively, for the embryos treated with LC, ZQ and OSC sediment extracts, which were significantly higher (p<0.05) than 5.0±1.2% of control embryos Moreover, by this time (48 hrs), 25.0±10.0%,20.0±5.0% and 8.0±2.8% embryos exposed to LC, ZQ and OSC sediment extracts were recorded dead (no heart beat). At 72 hpf, the hatching rate of control embryos was 92.0±5.8%, but it dropped markedly to 27.0±7.6%, 43.0±7.6% and 80.0±5.0% (p<0.05), respectively, for LC, ZQ and OSC sediment extracts-exposed embryos.In summary, this study highlights the applicability of both in vitro fish cell assay and comet assay as well as zebrafish embryo test to marine sediment extracts. Both cytotoxicity and genotoxicity as well as teratogenicity orders of the three sampling sites were LC>ZQ>OSC. However, the sediment extracts at the doses inducing closely similar cytotoxicity didn't induced similar genotoxicity and teratogenicity, with the genotoxicity and teratogenicity of LC and ZQ sediment extracts being markedly higher than that of OSC sediment extracts, indicating that cytotoxicity is simply incomparable with genotoxicity and teratogenicity. With all the three assays yielding closely comparable LOECs suggesting the presence of a similar sensitivity. It is clear that the sediment toxicities are related to PAH components and concentrations, although PAHs are surely not the only cause of toxicity.
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