Acute Toxicity And Genetic Toxicity Of Crude Oil And No.0Diesel Oil On Marine Microalgae

With the increasing production and transportation of crude oil at sea, oil spillsoccur frequently, and the impact on marine environment is intensified, the assessmentof the toxicity of crude oil contaminants is particularly important. At the lowest levelof the marine food chain, marine microalgae are sensitive to the toxic substance. Inthis thesis, Heterosigm akashiwo was selected preferably as a model organism, theacute toxicity of crude oil pollutants was detected by fluorescence technology, thegenetic toxicity of crude oil pollutants was detected by the single cell gelelectrophoresis（SCGE）, respectively. An assessment method of the acute toxicity andgenetic toxicity of oil spills was established based on H.akashiwo, the acute toxicityof two kinds of crude oil and No.0diesel oil was evaluated and the genetic toxicity ofO^#diesel oil was also evaluated. The prime results are as follows:1. H.akashiwo was selected preferably as a model organism, and an assessmentmethod of the acute toxicity of crude oil pollutants was established based onfluorescence technology. First, the WAF （1:50, water accommodated fraction） of twokinds of crude oil and No.0diesel was prepared, and the principal components weredetermined by GC-MS. Second, the sensitivity of Skeletonema costatum, Chaetocerosgracilis, and H.akashiwo to the WAF （1:50） was investigated, the chlorophyll afluorescence intensity at the wavelength of680nm was measured using fluorescencespectrometer. The relative inhibitory rate was calculated using the chlorophyll afluorescence intensity of microalgae without toxictants as control, and then the EC50values of different time were obtained, among which the96h-EC50was used toevaluate the acute toxicity of crude oil pollutants. the sensitivity order was as below:H.akashiwo> S.costatum> C.gracilis. Thus, H.akashiwo was selected as the modelmicroalgae to test the acute toxicity of the oil pollutants.2. An assessment method of the genetic toxicity was established usingH.akashiwo as test organisms, and this method based on the modified single-cell gel electrophoresis technique. First, sensitive and suitable microalgea was creened amongfive microalge species using H₂O₂as a standard toxicant, and it turned out that onlyH.akashiwo proved feasible in the single cell gel electrophoresis experiments.Furthermore, through condition experiments, the traditional single cell gelelectrophoresis technique was modified by omitting the cell lysis step, theelectrophoresis of slide was conducted with an electrophoresis buffer for20min at23V and300mA after unwinding for20min, the modified technique was moreapplicable to H.akashiwo. Subsequently, tail length, tail moment and Olive tailmoment were used as the index for evaluating the DNA damage of H.akashiwo.3. Using H.akashiwo as a model organism, the acute toxicity and genetic toxicityof the No.0diesel fuel WAF （1:9） were evaluated by fluorescent technology and themodified single cell gel electrophoresis. Firstly, the No.0diesel fuel WAF （1:9） wasprepared and analyzed by the GC-MS, the concentration of alkane was14.9mg/L andthe concentration of PAHs was1.2mg/L, the UV analysis showed that thevolatilization of PAHs concentration with the time is negligible. Next, the acutetoxicity of the WAF（1:9） was evaluated by fluorescence technology, obviousinhibition was observed at the lowest concentration0.81mg/L, the genetic toxicity ofthe WAF（1:9） was evaluated by the modified single cell gel electrophoresis, DNAdamage had significant difference （P<0.01） at the concentration of5%（0.81mg/L）compared with the control group after12h exposure when tail moment was used asthe index of the DNA damage, the DNA damage increased with the concentration.When the exposure time was24h, the DNA damage of the lowest concentration nolonger had significant difference, but the DNA damage of the concentration10%（1.62mg/L） and20%（3.24mg/L） also had significant difference, that showed thatH.akashiwo had self-repair process at the low concentration of WAF, The situationwas similar after96h exposure. Furthermore, H.akashiwo was transferred to cleanwater for24h after exposing to the WAF（1:9）for12h, DNA damage was fullyrepaired at the concentration of5%and10%, but not repaired completely at theconcentration of20%.In sum, this thesis, for the first time, adopted H.akashiwo as a model organism and evaluated the acute toxicity of three kinds of oil and the genetic toxicity of No.0diesel oil using fluorescence technology and modified single cell gel electrophoresis.