【作者】 张聿柏；

【导师】 唐学玺；

【作者基本信息】 中国海洋大学 ， 生态学， 2013， 博士

【摘要】 石油烃导致的海洋环境污染是全球性的重大环境问题之一，海洋生态系统不可避免会受到石油烃毒性作用的明显影响。海洋微藻是海洋食物链的基础，提供了海洋的主要初级生产力，它不仅驱动着整个海洋生态系统的能量流动和物质循环，而且对调节全球气候变化起着重要的作用。海洋微藻是海洋浮游生物的主要组成部分，海洋微藻种群和群落的动态变化一旦受到破坏将会危及其它海洋生物及整个海洋生态系统。大量研究表明，石油类污染物对海洋微藻的生长有显著影响，但现有的资料主要侧重于细胞密度、初级生产力以及少量的生理生化指标的探讨，在种群动态和种间关系定量化、光合作用特征和细胞过氧化及抗氧化能力研究方面，尚缺乏足够的数据说明石油烃对海洋微藻的作用机理。因此，研究石油烃胁迫下不同海洋微藻种群动态和种间竞争关系对短期急性毒性和长期亚急性毒性响应差异及其作用机制是十分必要的。本文以三种海洋微藻：绿藻门的饵料藻青岛大扁藻（Platymonas helgolandicavar. tsingtaoensis）、金藻门的饵料藻球等鞭金藻（Isochrysis galbana）、硅藻门的赤潮微藻小新月菱形藻（Nitzschia closterium f. minutissima）为受试生物，在实验室条件下研究了石油烃对三种微藻急性和亚急性毒性效应，并探讨了石油烃胁迫下海洋微藻种间竞争关系、光合作用特征和细胞过氧化、抗氧化酶活性及抗氧化物质含量的变化，初步探明了海洋微藻对石油烃胁迫的响应机制。结果如下：1石油烃对海洋微藻的急性毒性作用研究（1）石油烃对海洋微藻的生长有明显的抑制作用。三种海洋微藻的石油烃胁迫96h EC50值分别为：青岛大扁藻13.84mg/L，球等鞭金藻18.83mg/L，小新月菱形藻6.73mg/L。对石油烃毒性敏感性大小顺序为：小新月菱形藻＞青岛大扁藻＞球等鞭金藻。（2）三种海洋微藻的叶绿素a含量受到不同程度的抑制。小新月菱形藻的叶绿素a含量受石油烃抑制的敏感性较青岛大扁藻和球等鞭金藻高。（3）三种海洋微藻的叶绿素荧光参数受到石油烃急性作用的不同影响。除青岛大扁藻0.5mg/L石油烃处理组qP值显著增加外，三种微藻的Fv/Fm，ETR，qP等参数均受到了不同程度的抑制。（4）在较低浓度石油烃胁迫下，除球等鞭金藻的Fv/Fm和qP参数外，三种微藻叶绿素荧光参数受石油烃抑制的敏感性明显高于同一处理组的叶绿素a含量的变化，因此，叶绿素荧光参数更适合作为石油污染急性毒性海洋浮游植物初级生产力监测的参考依据。2石油烃对海洋微藻种群动态的影响（1）亚急性毒性剂量石油烃胁迫对三种海洋微藻的逻辑斯蒂模型有显著的影响。青岛大扁藻和球等鞭金藻处理组的环境承载力K值比对照组间显著降低，且随着石油烃浓度的增大而减小，瞬时最大增长率r值均比对照组显著降低。两者三个处理组生长曲线达到拐点时间均比对照组推迟。（2）在相同的相对石油烃浓度胁迫下，小新月菱形藻的环境承载力下降最多，敏感性最高。而从三种微藻的逻辑斯蒂模型参数及其生长曲线拐点出现时间来看，小新月菱形藻比青岛大扁藻和球等鞭金藻能更早更快地进入种群稳定期，存在占据种群相对优势的能力。3石油烃对海洋微藻种间竞争关系的影响（1）混合培养对两种微藻种群生长的逻辑斯蒂模型参数及其生长曲线拐点出现时间有显著影响。青岛大扁藻-小新月菱形藻和球等鞭金藻-小新月菱形藻混合培养后的三种海洋微藻环境承载力K值均有显著降低。但小新月菱形藻在两个组合中均表现出瞬时最大生长率r值和生长曲线拐点出现时间Tp上的优势性。（2）不同起始生物量比例对两种海洋微藻种间竞争关系有显著的影响。本研究中，在空白对照试验组中，起始生物量上的优势，不是长期培养下海洋微藻成为优势种的决定性因素。但相对起始生物量较高的种群，能够在石油烃毒性胁迫下占据一定的优势。4石油烃对海洋微藻光合作用的影响（1）三种海洋微藻叶绿素a含量受到亚急性毒性剂量石油烃的显著影响，叶绿素a含量显著降低，且随石油烃浓度的提高和作用时间的延长而降低。小新月菱形藻的叶绿素a含量变化受到石油烃的影响最为显著。（2）青岛大扁藻、球等鞭金藻和小新月菱形藻的各项叶绿素荧光参数受到石油烃的不同影响。从Fm/Fv、Yield、ETR、qP、NPQ参数的变化来看，青岛大扁藻和球等鞭金藻受石油烃胁迫程度比小新月菱形藻弱。（3）小新月菱形藻的RuBPCase活性在整个试验周期各石油烃处理浓度均比对照组显著降低。球等鞭金藻和青岛大扁藻在实验初期RuBPCase活性变化不明显，后期显著降低。（4）小新月菱形藻在石油烃胁迫下，光合作用各项参数反映出的受害程度明显大于青岛大扁藻和球等鞭金藻，这可以考虑是其相对敏感性高，种群环境承载力受影响较大的原因。5石油烃对海洋微藻的过氧化及抗氧化酶和抗氧化物质的影响（1）三种微藻MDA含量受石油烃胁迫后显著升高，石油烃对海洋微藻的脂质过氧化作用显著。青岛大扁藻MDA含量上升最多，小新月菱形藻相对较少。（2）石油烃胁迫下的三种海洋微藻SOD、GPx、GR活性均出现波动，呈先升高后降低的趋势，说明石油烃胁迫作用下，三种海洋微藻抗氧化酶的保护作用被激活，但在长期胁迫作用下会抗氧化酶活力下降，三种微藻均受到石油烃的伤害。（3）低浓度石油烃对青岛大扁藻和小新月菱形藻的GSH含量影响不显著，1/596h EC50石油烃处理组的球等鞭金藻GSH含量在实验中期上升最明显。（4）长期亚急性毒性剂量石油烃对三种海洋微藻的细胞过氧化、抗氧化酶和抗氧化物质有显著影响，海洋微藻在接触石油烃的开始阶段，抗氧化酶活性普遍升高，抗氧化物质含量下降，中后期抗氧化酶活性降低，抗氧化物质含量出现波动，脂质过氧化物含量随实验的进行不断上升。小新月菱形藻在MDA含量、SOD活性指标优于青岛大扁藻，而与球等鞭金藻相当，这可能是其在青岛大扁藻-小新月菱形藻培养组合中取得优势地位或保持竞争共存，而在球等鞭金藻-小新月菱形藻组合中被球等鞭金藻取代而失去优势的原因之一。更多还原

【Abstract】 Petroleum hydrocarbon caused marine environment pollution is one of the mostimportant global environment problems; petroleum hydrocarbon toxicity affect on themarine ecosystem is obviously. Marine microalgae are the base of marine food chainand are the main marine primary productivity. Marine microalgae drive the marineecosystem energy and matter cycle and global environmental change. The dynamicchange of marine phytoplankton populations and communities once damaged willendanger the other sea creatures and the whole marine ecosystem. Many studiesplaced emphasis on cell density data show that the oil pollutants have significantlyeffects on marine microalgae growth, data to illustrate the mechanism of thepetroleum hydrocarbons in marine microalgae on relationship between populationdynamics and species of quantitative, photosynthetic characteristics and cellper-oxidation and antioxidant research the was lack. Research on marine microalgaeshort-term acute toxicity and long-term sub-acute toxicity effects response differencesand mechanism of action under the condition of petroleum hydrocarbon is necessary.In lab, short-term acute toxicity and long-term sub-acute toxicity affects onPlatymonas helgolandica var. tsingtaoensis, Isochrysis galbana and Nitzschiaclosterium f. minutissima, competitive relation between the marine microalgae,photosynthesis characteristics, cell per-oxidation, antioxidase enzyme activities andantioxidant content changes were studied, in which the marine microalgae responsemechanism under the condition of petroleum hydrocarbon was found out.1Petroleum hydrocarbon acute toxicity effects on marine microalgae（1）Petroleum hydrocarbon had significant inhibition of the marine microalgaegrowth, only the0.5mg/L petroleum hydrocarbon had inapparent stimulated effect.96h EC50of the Platymonas helgolandica var. tsingtaoensis was13.84mg/L（12.11-18.14mg/L）, Isochrysis galbana was18.83mg/L（17.22-22.39mg/L）,Nitzschia closterium f. minutissima was6.73mg/L（2.76-14.11mg/L）. The level ofPetroleum hydrocarbon sensitivity was Nitzschia closterium f. minutissima＞Platymonas helgolandica var. tsingtaoensis＞Isochrysis galbana. （2）Chlorophyll-a content was inhibited. Isochrysis galbana content changes weremore sensitive than Platymonas helgolandica var. tsingtaoensis and Nitzschiaclosterium f. minutissima.（3）Petroleum hydrocarbon acute toxicity effects on chlorophyll fluorescenceparameter were different. Platymonas helgolandica var. tsingtaoensis’s in0.5mg/Lincreased significantly. Fv/Fm，ETR and qP were inhibited.（4）In the lower concentration （0.5mg/L） condition, chlorophyll fluorescenceparameter changes sensitive was more obvious than the chlorophyll-a content’s,except the Isochrysis galbana Fv/Fm and qP. Chlorophyll fluorescence parameter ismore suitable for oil pollution acute toxicity biological monitoring.2Petroleum hydrocarbon effects on marine microalgae populations dynamic（1）Petroleum hydrocarbon sub-acute toxicity had significant effect on Logisticmodel. In Platymonas helgolandica var. tsingtaoensis and Isochrysis galbana groupsenvironment capacity K decreased with the petroleum hydrocarbon concentrationincreasing was obvious lower than in the blank control, and so was the instantmaximum growth r. They needed more time than in the blank control to arrive at thegrowth curve inflexion point.（2） In the same petroleum hydrocarbon concentration condition, Nitzschiaclosterium f. minutissima K decreased the most. The Logistic model and the growthcurve inflexion point showed that Nitzschia closterium f. minutissima could arrive atthe population stability earlier and more quickly than the Platymonas helgolandicavar. tsingtaoensis and Isochrysis galbana, and which was the basis of populationrelative dominance.3Petroleum hydrocarbon effects on interspecific competition（1）Mixed cultivation had obvious effects on the Logistic mode and growth curveinflexion point. K decreased significantly in the condition of mixed cultivation ofPlatymonas helgolandica var. tsingtaoensis-Nitzschia closterium f. minutissima andIsochrysis galbana-Nitzschia closterium f. minutissima. In the two groups theNitzschia closterium f. minutissima instant maximum growth and growth curveinflexion point privilege over the other.（2）Different had significant effects on interspecific competition. In the blank controlgroups, the initial biomass advantage was not the determine factor of dominance inlong term marine microalgae cultivation. In the condition of petroleum hydrocarbon toxicity, the higher proportion of initial biomass privilege over the other.4Petroleum hydrocarbon effects on photosynthesis（1）Chlorophyll-a content reduced for the petroleum hydrocarbon of sub-acutetoxicity concentration, and chlorophyll-a content changes were largely in line with thetrend of the higher petroleum hydrocarbon concentration and the longer of the time,the lower chlorophyll-a content. Nitzschia closterium f. minutissima chlorophyll-awas effected the most.（2） Petroleum hydrocarbon effects on the Platymonas helgolandica var.tsingtaoensis, Isochrysis galbana and Nitzschia closterium f. minutissima chlorophyllfluorescence parameters were different. The Fm/Fv、Yield、ETR、qP、NPQ changesshowed that the Platymonas helgolandica var. tsingtaoensis and Isochrysis galbanawere inhibited smaller than the Nitzschia closterium f. minutissima.（3）In the Petroleum hydrocarbon condition, Nitzschia closterium f. minutissimaRuBPCase activity was lower than in the blank control. Isochrysis galbana andPlatymonas helgolandica var. tsingtaoensis RuBPCase activity changes were notobvious in the beginning and decreased significantly in the late.5Petroleum hydrocarbon Petroleum hydrocarbon effects on per-oxidation, antioxidantenzyme and antioxidant content（1）Petroleum hydrocarbon had effects on MDA content increase and lipidperoxidation. The Platymonas helgolandica var. tsingtaoensis MDA content rose themost, and Nitzschia closterium f. minutissima MDA content rose the lest in the threekinds.（2）In the condition of petroleum hydrocarbon the SOD, GPx and GR activityshowed a trend of decrease after the first rise in roughly. It showed the antioxidantenzyme protection was activated and antioxidant enzyme activity was destroyed in thelong term petroleum hydrocarbon toxicity condition, microalgae were hurt by thepetroleum hydrocarbon.（3）Lower Petroleum hydrocarbon concentration had little effects on Platymonashelgolandica var. tsingtaoensis and Nitzschia closterium f. minutissima GSH content.In the condition of1/596h EC50, Isochrysis galbana GSH content increasedobviously during the experimental middle.（4）In the long-term and sub-acute toxicity petroleum hydrocarbon concentrationcondition, the marine microalgae cell per-oxidation; antioxidase and antioxidant were significantly affected. The antioxidase activity increased and the antioxidant contentdecreased in the beginning, the antioxidase activity decreased and the antioxidantcontent fluctuated in the late, and the lipid peroxide content increased with theexperiment. Nitzschia closterium f. minutissima MDA and SOD indexes were betterthan the Platymonas helgolandica var. tsingtaoensis, and were similar to theIsochrysis galbana, which was the cause of the interspecific dominance.更多还原