| Large accidental spills of crude oil in the sea focus great public attention,the number of oil spills have fallen over year.However,most oil entered the sea chronically as a result of numerous small and less visible leakages.Oil spills and related petroleum products can be seriously damaged the marine ecosystem health.After oil spills,the water accommodated fractions(WAF)of petroleum hydrocarbon components will have long-term toxic effects on aquatic organisms.Microalgae are the primary producer of marine ecosystems,and play an important role in maintaining the stability of ecosystem.Therefore,marine microalgae are an effective biomarker for the assessment of the toxicity of oil spill pollution.In the past decades,a number of studies have been conducted to investigate the toxic effects of oil spill,and mainly have focused on physiological and biochemical indicators,such as the cell growth rate,chlorophyll and half maximal effective concentration(EC50),stress proteins,reactive oxygen species and various enzymes.However,limited information is available on the transformation of relative compounds.Compound-specific stable isotope analysis(CSIA)can insight into the molecular-level changes of compounds.The isotopic compositions of individual amino acids(?13CAAs)is the first time to evaluate the acute toxicity mechanism of petroleum hydrocarbon in microalgae.In the present study,three widespread microalgae were used as target organisms,eg.Nitzschia closterium(N.closterium),Heterosigma akashiwo(H.akashiwo)and Platymonas helgolandica(P.helgolandica).They were exposed to WAF of#180 fuel oil for an acute toxicity test of 96 h.Specific research results are as follows:(1)Determine and analyze the toxic components of WAF of 180#fuel oil,and explore the trends of microalgae cell density,chlorophyll a content and inorganic nitrogen nutrients under different WAF concentrations.Result show that the phenanthrene,anthracene,and fluoranthene were detected as the predominant congeners of polycyclic aromatic hydrocarbons(PAHs)in the WAF,and may be the main toxic components.The chlorophyll a content of microalgae decreased with increasing WAF concentrations at the same culture time,and the highest inhibition rate of chlorophyll a in P.helgolandica was 48.10%with 10%WAF concentration.The N.closterium and H.akashiwo promoted growth on 1%and 3%WAF concentrations,whereas the P.helgolandica promoted growth on only 1%WAF concentration.In addition,the study also found that microalgae could still absorb and assimilate the NO3-N and release of NO2-N and NH3-N under the WAF stress,the nitrogen cycle in microalgae was in the equilibrium status.(2)On the basis of the above,to explore the trends of ?13C and ?15N under WAF stress,revealing the effects of the WAF on C and N fixation capacity on microalgae.Result show that the ?13C value of microalgae was significantly lower than that of the control group,indicated that carbon was limited in WAF stress.The ?15N value in microalgae was no significantly different with the increasing of WAF concentrations at the same culture time.Because of the nitrogen cycle in microalgae was in the equilibrium status,the effect of carbon on growth of microalgae was more prominent than nitrogen.By further the ?13C value of microalgae appeared peak valley at 48 h in control group,corresponding to the enhanced capacity in carbon fixation during microalgae photosynthesis.The physiological acclimation capacity of microalgae was revealed by the occurrence time when the ?13C value was in peak valley,and thus the physiological acclimation capacity of microalgae decreased in the order of N.closterium>H.akashiwo>P.helgolandica.Finally,combined statistical analysis methods,it was found that the ?13C value of microalgae was completely separated under low and high concentrations,indicating that the ?13C value in order to verify the "hormesis" phenomenon in microalgae.(3)The relative abundance of AAs were used to explore the effect of WAF stress in microalgae.Result show that three kind of microalgae have similar AAs composition,and the relative abundance of 6 AAs,alanine,proline,aspartic acid,cysteine,lysine and histidine are relatively high.Under WAF stress,the relative abundance of Ala in N.closterium and P.helgolandica was positively correlated with culture time,whereas the relative abundance of Cys was negatively correlated with culture time.The Ala abundance of in H.akashiwo was negatively correlated with the cultivation time,while Cys was positively correlated.Under the low WAF concentrations(1%and 3%),the relative abundance of His in N.closterium decreased with the increasing of culture time,while variation trend was opposite in the relative abundance of Tyr in P.helgolandica.In the high WAF concentrations(5%,7%and 10%),the His abundance of in N.closterium decreased first and then increased with the culture time,the abundance of Lys in P.helgolandica was no significantly different with the control group.These findings indicated that the synthesis pathways of AAs in microalgae cells were affected by the WAF.(4)The CSIA were used to explore the changes of AAs in microalgae under the action of WAF stress,thus revealing the toxicological effects of petroleum hydrocarbons on marine microalgae.The change of ?13CAla and ?13CMet values with culture time as well as WAF concentrations were barely noticeable respectively,since the synthesis pathwaysof these AAs were complex,and interconversions with other AAs might occured.The similar variation tendency was observed for ?13CLeu and ?13CPhe in N.closterium and H.akashiwo,because Leu and Phe are synthesized from the same precursor(phosphoenolpyruvate,PEP).The ?13CLeu value of P.helgolandica was significantly higher than the control group,whereas the opposite of ?13CPhe and ?13CTyr value.It may be the phenylalanine pathway preferentially enriched in 13C skeletons.With the WAF stress,?13CCys value of N.closterium and H.akashiwo was significantly higher than that of the control group.However,the ?13CLys values in P.helgolandica was higher in low WAF(1%and 3%)concentrations group than that of control group,whereas decreased in the high WAF(5%,7%and 10%)concentrations group.Under WAF stress,the ?13CHis values of three microalgae was significantly higher than the control group,mainly because of histidine may be attributed to its single synthesis pathway.So the?13CHis can be used as a biomarker to evaluate the degree of petroleum hydrocarbon stress in microalgae. |
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