| To evaluate the effect of oil spill on the marine phytoplankton,on the basis of compound specific isotope analysis(CSIA),taking water accommodated fraction(WAF)of fuel oil as toxic substance,single-celled marine microalgae as target subjects,fatty acids are selected in microalgae as the object of study,due to easier the Gas chromatography(GC)analysis.Through the analysis of changes of stable carbon isotopic composition of fatty acids(δ13CFAs)in microalgae cell under the stress of oil WAF of different content to determine the degrees of sublethal cells damage,and extract the appropriate "fatty acids-isotope" biomarkers.First of all,optimization of pretreatment of fatty acid extraction in microalgaeTo determine whether(e)and(f)in "in situ" methanol esterification of fresh microalgae samples suitable for the analysis of δ13CFAs,and on the condition of whether dry,whether applying extraction,using hydrochloric acid or sulfuric acid as catalyst,a total of eight ways were designed:(a)drying the sample followed by direct methyl esterification using HCl-CH3OH;(b)drying the sample followed by direct methyl esterification using H2SO4-CH3OH;(c)drying the sample by ultrasonic extraction and methyl-esterification using HCl-CH3OH;(d)drying the sample by ultrasonic extraction and methyl-esterification using H2SO4-CH3OH;(e)freshing sample followed by direct methyl-esterification using HCl-CH3OH;(f)freshing sample followed by direct methyl-esterification using H2SO4-CH3OH);(g)freshing sample with ultrasonic extraction followed by methyl-esterification using HCl-CH3OH and(h)freshing sample with ultrasonic extraction followed by methyl-esterification using H2SO4-CH3OH.The results of variance analysis(δ13CFAs)show that there exist significant difference(s)between method(f)and the other seven methods,but no significant difference between method(e)and traditional method(crude fat extraction,and then methanol esterification),so method(e)is suitable for δ13CFAs analysis of microalgae.Secondly,the concentration effect of the oil spills on the microalgae δ13CFAsTo evaluate the effect of oil WAF of different amount on δ13CFAs of microalgae,different kinds of microalgae cultivation under different oil WAF amount are designed.The results summarized δ13CFAs changes with oil WAF amount show that δ13C of some fatty acids(mostly unsaturated fatty acid)in some microalgae there exist relationship of"oil WAF content-δ13CFA" similar to the relationship of "Dose-Response",which comply with the law of "low-dose stimulation and high dose inhibition" from Calabrese(2002)."Fatty acids-isotope" biomarkers from different kind of microalgae are different,δ13C16:1,δ13C18:4 can be used as markers for toxic effect of oil spill on I.zhanjiangenisis,δ13C20:5,δ13C16:4 are suitable for P.helgolandica,and δ13C16:1 is suitable for N.closterium.Finally,the time effect of the oil spills on the microalgae δ13CFAsTo evaluate long-term effects of the oil WAF on δ13CFAs of microalgae,different kinds of microalgae(I.zhanjiangenisis,N.closterium,P.helgolandica and C.gracilis)cultivation under different oil WAF amount are designed,to sample and analyze at different point of time.The results demonstrate the universal law that "oil WAF makesδ13CFAs of microalgae rise in short-term and decline in long-term" compared with control group,and δ13CFAs changes are more sensitive than the changes of fatty acid content.Under the stress of low amount of oil WAF,the methods of cell damage assessment are put forward.The δ13CFAs increased with oil WAF amount during exponential growth phase,the stronger the early stimulation,the greater degree of later cell damage,and δ13C181 can show obvious changes by the effect of toxicity of oil spill on the marine microalga C.gracilis.During relative growth decline period,to evaluate the damage degree of microalgae through the correlation of algal cell density and δ13CFAs(by linear regression equation),the lower the slope shows that the worse the cell damage.Through the contrast of the fractionation degree between different lipid class,the greater degree of fractionation manifests the larger cell damage. |
