Standardization And Application Of Luminescent Bacteria Test In The Biotoxicity Detection Of Marine Pollutants

Biotoxicity test of marine pollutant is an important part of the marine environmental protection. Luminescent bacteria test（LBT） can be used to confirm the relationship between the concentration of toxicants and reaction of bacteria, and it can reflect the integrated toxic effects of pollutants exerted on the environment. In this study, we used two genetically engineered luminescent bacteria, Acinetobacter sp. Tox2 and Acinetobacter sp. Tox2, to evaluate the standardization of LBT. Firstly, we assessed the accuracy of LBT using these two bacteria in the detection of artificial seawater, then evaluated the biotoxicity of sediment samples collected from Huangdao oil spill-impacted marine beach. Finally, we evaluated the freeze-dried method of these two bacteria, which aim to optimize this standardization. The main research contents are summarized as follows:（1） We used Acinetobacter sp. Tox2 to detect the acute toxicity of 6 typical marine pollutants, including 4 kinds of heavy metals and 2 kinds of oil water accommodated fractions（WAFs）, and the results was assessed by fish exposure experiment using Mugilogobius chulae. The EC50 of Hg²⁺, Zn²⁺, Cu²⁺, Cd²⁺ and crude oil WAF in LBT were 0.12 mg/L, 71.81 mg/L, 0.68 mg/L, 43.67 mg/L and 2.01 mg/L, respectively. The LC50 of the above five toxicants by M. chulae were 0.14 mg/L, 107.73 mg/L, 3.23 mg/L, 39.83 mg/L, and 2.69 mg/L, respectively. In addition, the LC50 of diesel oil WAF by M. chulae was 4.96 mg/L. The results showed that these two methods for acute toxicity detection fitted well with each other. And LBT have the advantage of rapid detection since it could determine the acute toxicity within 30 minutes, while fish exposure experiment need 96 hours at least.（2） We have got the optimum pre-treatment of sediments which can be used in the biotoxicity testing based on the orthogonal combining with the chemical analysis of petroleum hydrocarbons. The optimum pre-treatment is that sediments could be extracted for about 0.5 h in artificial seawater at 25 ℃ and the ratio between sediments and seawater is 1:4. Then the biotoxicity of sediments collected from Huangdao oil spill was evaluated using LBT. The results showed that both sediment samples collected from remediation zone and non-remediation zone exhibited no acute toxicity, while these sediments exhibited genotoxicity of different degree. In general, the genotoxicity of sediments decreased over time, and the genotoxicity of sediments collected from remediation zone decreased more rapidly. The genotoxicity of remediation zone sample was at high level after the oill spill,and was equivalent to 1.65 mg/L of MMC. The genotoxicity decreased to medium level, and was equivalent to 0.17 mg/L of MMC after 14 days. The samples collected from remediation zone exhibited no genotoxicity after 30 days（NMMC<0）, while the genotoxicity of non-remediation zone decreased much more slowly than its counterpart. It took 60 days and 120 days for the genotoxicity of the non-remediation zone sample to decrease from high level to medium level and none tocicity, respectively. In addition, the genotoxicity decreased along with the decreasing concentration of PAHs in the sediments.（3） To study the optimum freeze-dried conditions of Acinetobacter sp. Tox2 and Acinetobacter sp. RecA, skim milk and LB culture containing NaCl, which can be used as protective agent, were employed in the two-factor and multi-gradient experiments. The optimum content of lyoprotectant for Tox2 is 24% skim milk, 0.5% NaCl, and the optimum content of lyoprotectant for RecA is 24% skim milk, LB culture containing 0.5% NaCl. Freeze-dried Tox2 and RecA could recover its luminous intensity by 88.58% and 85.13% compared with the corresponding fresh bacterial liquid formula, respectively. Both the fresh bacterial liquid and freeze-dried bacteria were used to detect the the acute toxicity of HgCl₂ or genotoxicity of MMC. The result have shown that the luminous curves of these two strains were almost identical before or after freeze-drying treatment. The EC50 of HgCl2 by both fresh bacterial liquid and freeze-dried bacterial powder of Tox2 are 0.11 mg/L. The EC50 of MMC by fresh bacterial liquid and freeze-dried bacterial powder of Rec A is 0.92 mg/L and 1.02 mg/L, respectively. The freeze-dried bacteria could be preserved in long-term, and the availability of freeze-dried bacteria would not affect the biotoxicity evaluation.