| Luminescent bacteria test(LBT) is widely used in the toxicity detection of environmental pollutants in recent years. The level of toxicity was determined qualitatively or quantitatively by the increase or decrease of luminescence when the bacteria were exposed to different toxicants. However, most luminescent bacteria were only applied in the detection of the acute toxicity of fresh water sample. The method for the detection of genotoxicity of marine pollutants has not been established yet. In this study, we developed and optimized a LBT method based on Acinetobacter sp. Tox2 and Acinetobacter sp. RecA to apply in the quick detection of toxicity of marine pollutants, and this method is applied in the toxicity evaluation of Bohai Sea sewage samples and contaminated seawater in Huangdao oil spill.Firstly, the culture condition and toxicity detection apparatus of the two luminescent bacteria were optimized. The bacterial liquid of Acinetobacter sp. Tox2 for the detection of acute toxicity could be prepared within 8 h of incubation, while the bacterial liquid of Acinetobacter sp. RecA for the detection of genotoxicity could be prepared within 16 h of incubation. Both the two bacteria could be applied in the samples with salinity of 30 and the optimum detection temperature is 30 ℃. The acute toxicity detection method could be done within 30 min, while the genotoxicity detection method could be done within 3 h.Secondly, the two toxicity detection method were also used to determine the artificially polluted seawater samples containing total petroleum hydrocarbons(TPH), each of four heavy metals(Hg2+、Zn2+、Cu2+、Cd2+), and each of four PAHs(phenanthrene, pyrene, fluoranthene, and benzo[a]pyrene). The toxicity detection limits of TPH by the two methods were as low as 0.001 mg/L. The two luminescent bacteria exhibited different levels of sensitivity towards four heavy metals. The EC50 of HgCl2, ZnSO4, CuSO4, and CdCl2 were 0.108 mg/L, 46.0 mg/L, 0.816 mg/L, and 14.2 mg/L in the acute toxicity detection method, respectively. In addition, Acinetobacter sp. Tox2 is sensitive to the acute toxicity of PAHs, the EC50 of phenanthrene and pyrene were 1.46 mg/L and 0.266 mg/L, respectively.Finally, toxicity of 12 Bohai Sea sewage samples were evaluated by these two luminescent bacteria. The acute toxicity of most sewage samples belongs to inferior level except BH3, which the equivalent HgCl2 concentration is 0.193 mg/L. The equivalent HgCl2 concentration of most stations was less than 0.02 mg/L. However, genetic toxicity of most sewage samples belongs to high-level and among them the equivalent MMC concentration of BH4 is up to 2.41 mg/L. The biotoxicity also showed a certain geographic distribution which the toxicity of samples from Shandong Peninsula is higher than samples from Liaodong Peninsula and Beijing-Tianjin-Hebei. In the second applicated example, genetic toxicity of the seawater has been detected by RecA after the Huangdao "11.22" oil spilling. The chemical composition of petroleum hydrocarbons in the seawater samples collected from five sites ranging from 1 km to 15 km away from the oil leakage point during a time course of 90 days were first analyzed by fluorescence spectrophotometer. The petroleum hydrocarbon contents of 3 samples from sites S1, S2, and S3 within 1 km away from the oil leakage point were from 0.55 mg/L to 1.04 mg/L. The corresponding genetic toxicities were equivalent to 0.60 mg/L to 1.78 mg/L of MMC. The genetic toxicity level decreased as the petroleum hydrocarbon content gradually decreased to <0.02 mg/L over time. The samples from sites S4 and S5 which were over 5 km away from the oil leakage point had low petroleum hydrocarbons content(<0.02 mg/L) and exhibited low or no genetic toxicity during the 90 days.In summary, two luminescent bacteria Acinetobacter sp. Tox2 and Acinetobacter sp. RecA could be applied to quickly and accurately detect the biotoxicity of contaminated sea water samples. These two methods have great potential in the establishment of standards for the quick detection of biotoxicity of marine pollutants, which is promising in evaluation of sea water quality in the emergency response of marine pollution. |
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