摘要
红树林是生长在热带、亚热带陆海交汇的海湾河口区潮间带、受周期性潮水浸淹的潮滩湿地木本生物群落。近年来随着人口的增长，流域工农业、沿岸城市开发及港口驳岸海运的发展，大量污染物直接或间接注入而汇集于河口海湾区，日益增加了红树林的环境压力。红树林生态系统因生产力高、富含有机质以及强还原性环境条件等特性，使之成为吸收和积累多环芳烃(Polycyclic AromaticHydrocarbons，PAHs)的重要场所，但目前有关红树林湿地中PAHs的研究大多限于分析湿地沉积物中PAHs的种类、各种组分的含量与分布比例、湿地内PAHs的来源研究以及林地内微生物对PAHs的降解作用等，而作为湿地主体核心的红树植物对环境中PAHs的直接吸收研究很少涉及，亦未对相关机理加以阐释。
本文首先利用固体表面荧光(Solid Surface Fluorimetry，SSF)分析法结合荧光显微(Fluorescence Microscopy，FM)和双光子激光共焦扫描显微(TWoPhoton Laser Confocal Scanning Microscopy，TPLCSM)技术，建立了红树林生态系统中典型PAHs环境行为原位研究的系统方法，进而利用所建方法初步开展了原位研究典型PAHs在红树植物中环境行为等工作，主要研究内容和结果包括以下几个方面：
(1)利用荧光分光光度计配合自制的固体样品架建立了直接测定吸附于白骨壤(Avicennia marina，Am)、木榄(Bruguiera gymnorrhiza，Bg)、秋茄(Kandeliacandel，Kc)和红海榄(Rhizophors stylosa，Rs)叶片上An的SSF新方法。实验结果表明：(a)在相同的暴露时间下，不同种红树叶片对An的吸附量不同，且在一定范围内，吸附于四种红树叶片上表面An的量与其SSF相对强度间有较好的线性关系，所建方法的回收率为70．2％～110．8％，可满足实际测定的需要；(b)在相同的暴露An浓度下，随An暴露时间的增加，四种红树叶片上表面富集An后测得的SSF相对强度都呈现先增大后减小的趋势，且测得吸附于四种红树叶片上表面An的SSF相对强度最高处的顺序为：Am＞Kc＞Rs＞Bg；(c)在相同的实验暴露条件下，同种红树叶片上表面吸附An后测得的SSF相对强度都明显强于下表面，但下表面对An的吸附作用亦很重要。实验还发现，吸附在四种红树叶片上表面An的SSF相对强度随吸附部位的不同都表现出较大差异，即可能存在于叶片表面的An并不是“均匀”地穿过表皮蜡质层进入到叶片内部，而是可能存在“优先”的进入位置，但具体原因尚不清楚。
(2)在上述工作研究基础上，首先，利用荧光分光光度计配合光纤附件建立了直接测定吸附于Kc、桐花树(Aegiceras corniculatum Ac)两种红树叶片上表面单组分及其混合组分An、芘(Py)的光纤同步SSF新方法。实验结果表明，在一定范围内，吸附于两种红树叶片上表面单组分及其混合组分An、Py的量分别与其同步SSF相对强度间有较好的线性关系，且所建方法的回收率为82．9％～116．5％，可满足实际测定的需要；其次，采用所建立的光纤同步SSF法原位研究了存在于活体Kc叶片上表面An的光谱行为，同时辅助GC-MS手段验证了所建方法在实际测定过程中的可行性。实验结果表明，在一定的实验条件下，随暴露时间的延长，采用两种方法测得的存在于Kc叶片表面An的量的变化趋势基本一致；最后，采用所建立的光纤同步SSF法原位研究了An、Py及其混合组分在两种红树叶片上表面的光降解情况。实验结果表明，在实验条件下：(a)吸附于两种红树叶片上表面的单组分An、Py的光降解过程均遵循一级反应动力学过程；在相同的实验条件下，吸附于红树叶片上表面PAH的含量越高，光降解的反应速率越慢，且吸附于同种红树叶片上相同物质量的Py的降解速率远小于An；(b)吸附于两种红树叶片上表面的An、Py混合组的光降解过程均遵循一级反应动力学过程。而对于处于混合组分中相同量的PAH，吸附于Kc叶片上表面PAH的光降解速率慢于吸附于Ac叶片上表面的PAH。实验结果还表明，不管是Kc叶片还是Ac叶片，与吸附于叶片表面单组分An或Py的光降解作用相比，混合组分中An的光降解速率减慢，而Py的光降解速率加快；(c)单组分及其混合组分An、Py在水溶液中的光降解过程均遵循一级反应动力学过程。对于相同量的单组分An、Py，处于水溶液中的PAH的光降解速率都快于吸附于红树叶片表面的PAH。在水溶液中，与单组分An或Py的光降解作用相比，混合组分中An的光降解速率减慢，而Py的光降解速率加快。
(3)在人工模拟实验条件下，选取An为模型化合物并将其均匀地涂抹于活体Kc叶片的上表面，经过96h的暴露后，首先借助于所建的SSF方法，确定了存在于Kc叶片上表面的目标污染物为An，其次借助于FM技术，直接观察发现，随着An暴露时间的增加，存在于活体Kc叶片上表面An的量逐渐减少，一定时间后，存在于Kc叶片上表面的An会与大气中的An达到挥发和再沉降的“相对平衡状态”，且随着An暴露时间的延长，存在于Kc叶片上表面的An会通过上表面的蜡质层进入到第一、第二内皮层、上栅栏组织，从大气再沉降至Kc叶片下表面的An会通过下表面的气孔进入到下皮层、下栅栏组织，并最终都分别到达海绵组织。实验结果还表明，经过24h的暴露后，在Kc叶片的上、下栅栏组织和海绵组织内部都会观察到一些“淡蓝色”的光斑，很可能是An的代谢产物。同时实验还发现，在相同An暴露时间(24h后)下，存在于叶片“中央”An的量会少于存在于叶片“边缘”An的量，即随着暴露An时间的延长，存在于叶片“中央”An的量减少的“更快”。此研究结果可能暗示了一条重要的信息，即可能存在于红树叶片表面的An并不是“均匀”地穿过表皮蜡质层进入到叶片内部，而是可能存在“优先”的进入位置。
(4)在人工模拟实验条件下，利用TPLCSM技术，原位观察了典型PAHs从水培液迁移进入活体红树植物幼苗的根部并向其地上组织传输的路径，及其在内部组织中的最终分布情况。实验结果表明：(a)经过20d的培养，萘(Na)、菲(Phen)、An、Py都从水培液迁移进入至Kc幼苗的根部，并在蒸腾拉力的作用下传输至茎、叶等地上组织，且四种PAHs迁移进入并在Kc幼苗内部传输的路径及其最终存在位置类似，且四种PAHs在Kc幼苗内部组织中传输的速率顺序为Na＞An和Phen＞Py。四种PAHs胁迫对Kc幼苗根的生长都产生了抑制作用，且其抑制作用大小的顺序为Na＞An和Phen＞Py，其中An和Phen胁迫对Kc幼苗根生长的抑制作用差别不显著，而Na对Kc幼苗根生长的抑制程度最为明显。(b)经过20d的培养，三种PAHs(Na、Phen、Py)都从水培液迁移进入至Ac幼苗的根部，并在蒸腾拉力的作用下传输至茎部，且三种PAHs迁移进入并在Ac幼苗内部传输的路径及其最终存在位置类似，且三种PAHs在Ac幼苗内部组织中传输的速率顺序为Na＞Phen＞Py。三种PAHs胁迫对Ac幼苗根的生长都产生了抑制作用，且其抑制作用大小的顺序是Na＞Phen＞Py，其中Na对Ac幼苗根生长的抑制程度最为明显。
本文首次结合了SSF分析法、FM和TPLCSM技术研究了典型PAHs在红树植物中的环境行为，为PAHs在红树林生态系统中的相关研究提供了新的手段、方法和研究思路。通过研究不同种类红树植物的叶、茎、根对典型PAHs的吸收、转运、降解等过程，初步揭示了红树植物对典型PAHs的植物修复、污染控制的过程、机制等。也为进一步研究PAHs污染胁迫下不同种类红树植物生长情况、生理生态及生化效应等奠定了研究方法基础。
Mangroves are the intertidal plant formations growing along the coastlines oftropical and subtropical regions.Due to an increase in population,and expansion ofindustrial agricultural areas and port,a large number of pollutants are dischargingdirectly or indirectly into offshore marine areas.Human activities have seriously led tothe damage of many mangroves.The unique features of mangrove ecosystems such ashigh primary productivity,abundant detritus,rich organic carbon and anoxic conditionsmake themselves a reservoir of polycyclic aromatic hydrocarbons (PAHs)fromanthropogenic inputs.However,the published research on PAHs in the mangrovewetland has mainly focused on the analysis of concentrations of individual PAH andtotal PAHs compounds in the sediment;the determination of the distribution andproportion of PAHs in different tissues of many mangrove species;the evaluation of thepossible sources of PAHs in the wetland;and the microcosm studies concerning theremoval of PAHs from surface-or bottom-contaminated sediments.As a result,research on the direct uptake of PAHs by mangrove plants as the core of the wetland israrely involved,nor are the related mechanisms being explained.
In this study,a systematic method was established for in situ study on theenvironmental behaviors of typical PAHs in the mangrove wetland utilizing the solidsurface fluorimetry (SSF)and fluorescence microscopy (FM)as well as thecombination of two photon laser confocal scanning microscopy (TPLCSM).And theenvironmental behaviors of typical PAHs in mangrove plants were studied in situ withthe established methods.Main results were summarized as followings:
(1)A SSF approach combining with a laboratory-made solid substrate holder anda fluorescence spectrophotometer was established for direct determination ofanthracene (An)adsorbed on the leaves of four mangrove species (Avicennia marinaAm,Bruguiera gymnorrhiza Bg,Kandelia candel Kc,Rhizophors stylosa Rs).Theexperimental results were as follows:(a)With the same exposure time (90min)of An, the leaves of the four mangrove species showed different capacities for adsorbing An,and within a certain range,the relative SSF intensities of the An adsorbed on theupper sides of leaves of the four mangrove species increased linearly with incrementsin the exposure quantity of An.The recoveries for the four species were between70.2% and 110.8%,which met the requirement of practical applications.(b)In thesame exposure concentration of An,the relative SSF intensities of leaves of the fourmangrove species increased first and then decreased as the time progressed.And themaximum relative SSF intensities of the four species were in the orderAm＞Kc＞Rs＞Bg.(c)In the same experimental conditions,the relative SSF intensitiesof the adsorbed An on the upper side of the same mangrove leaves were muchstronger than those adsorbed on the lower side,which has many stomata.Therefore,in addition to the uptake of PAH via waxy cuticle,the stomatal pathway was alsoimportant for the PAH entering into the mangrove leaves.Further studies showed thatthe relative SSF intensities in different regions of the same leaf differed from eachother,and it provided us with very important information that the adsorbed An on themangrove leaf surface might not“equally”penetrate the epidermal wax layer into theinternal leaf tissues,but might have“priority”locations.However,the reasons werenot clear.
(2)Based on the established SSF method,a synchronous solid surface fluorimetry(S-SSF)method combining with an optical fiber and a fluorescencespectrophotometer was established firstly for direct determination of the An andPyrene (Py)which were adsorbed on the leaves of two mangrove species insingle-component or in mixtures.The results indicated that the quantity of An and Pyadsorbed on the mangrove leaves in single-component or in mixtures linearlycorrelated with their S-SSF intensities.And the recoveries of this S-SSF method forthe leaves of two mangrove species were between 82.9% and 116.5%,which met therequirement of practical applications;Secondly,the distribution of An adsorbed onthe living Kc leaves were studied in situ utilizing the established S-SSF approach,and the GC-MS was also used to verify the accuracy of the S-SSF method.Theexperimental results demonstrated that in a certain period of exposure time of An,thetrends of the quantity of adsorbed An on the upper side of Kc leaves determined bythe two methods were similar.Thus,the S-SSF could be used as a novel in situmethod for further studies.Finally,the photolysis of An and Py on the leaves of twomangrove species in single-component or in mixtures were directly studied by theS-SSF,and the results were as follows:(a)The processes of photolysis of An and Pyin single-component adsorbed on the leaves of two mangrove species followedfirst-order reaction kinetics.And in the same experimental conditions,the higher thequantity of adsorbed PAH on the mangrove leaves,the slower the reaction rate of thephotolysis of the PAH,moreover,the raction rate of the adsorbed Py on the leaves ofthe same mangrove species was much slower than that of the same amount ofadsorbed An.(b)The processes of photolysis of An and Py in mixtures adsorbed onmangrove leaves also followed first-order reaction kinetics.And to the same amountof PAH in mixtures,the raction rate of PAH adsorbed on Kc was slower than thatadsorbed on Ac.The experimental results also revealed that compared with the Anand Py in single-component mode adsorbed on the same kind of mangrove leaves,thereaction rate of An in mixtures slowed down,while the reaction rate of Py in mixturesspeeded up.(c)The processes of photolysis of An and Py in single-component or inmixtures,and in water followed first-order reaction kinetics.And to the same amountof PAH in single-compont mode,the reaction rate of PAH in water was faster thanthat adsorbed on mangrove leaves.Compared with An and Py in single-componentmode in water,the reaction rate of An in mixtures slowed down,while the reactionrate of Py in mixtures speeded up.
(3)Under the artificial simulating conditions,the An selected as the model PAHwas applied as a homogeneous layer to the upper side of the living Kc leaves.Duringthe exposure time of 96 hours,the SSF was firstly utilized for direct determination ofthe adsorbed An on the Kc leaves,then the FM was used to visualize how An was taken up into Kc leaves and where it was located in the inner leaf tissues.The resultsdemonstrated that the overall amount of An residing on the Kc leaf surface decreasedgradually as time progressed.And after a certain period of exposure time,equilibration of the An might be achieved between the leaf surface and theatmosphere.And as the time passed,the An existing on the upper side of Kc leaveswas observed moving from the upper epicuticular wax into the first and secondhypodermis and upper palisade tissue,and the An existing on the lower side of Kcleaves moving from the stomata into the lower hypodermis and the lower palisadetissue,then ultimately into the spongy mesophyll.The results also showed that afterexposed for 24 hours,there were several light blue dots in the upper and lowerpalisade tissues and spongy mesophyll,which might be the autofluorescence of Anmetabolites.Further studies also revealed that the residual quantity of An at the centerwas much less than that remaining at the edges of the same leaf surface after exposureof 24 hours,and this suggested that as time passed,the quantity of An retaining at thecenter of the leaf surface decreased more quickly,which might provide important newinformation about how An enters into Kc leaves.
(4)Under the artificial simulating conditions,the uptake,transportation anddistribution of PAHs were in situ visualized in mangrove plants using TPLCSM,some results were as follows:(a)After 20 days of culture in contaminated solutions,the Naphthalene (Na),An,Phenanthrene (Phen)and Py had been observed enteringinto the roots of Kc seedlings and being transmitted to the stem and leaf due totranspiration.The pathways of the four PAHs from the culture solutions into theplants were similar,and the results also indicated that the transmission rates of thefour PAHs in Kc seedings were in the order Na＞An and Phen＞Py.The growth of theroot of Kc seedlings was inhibited by the PAHs and the inbibitional effect was in theorder Na＞An and Phen＞Py.In addition,the inhibition of An and Phen to the rootgrowth was not significant difference,and the inhibition of Na was the most obvious.(b)After 20 days of culture in contaminated solutions,the three PAHs (Na,Phen,Py) had been observed entering into the roots ofAegiceras corniculata (Ac)seedlings andbeing transmitted to the stem due to transpiration.The pathways of the three PAHsfrom the culture solutions into the plants were similar,and the results also indicatedthat the transmission rates of the three PAHs in Ac seedlings were in the orderNa＞Phen＞Py.The growth of the root of Ac seedlings were inhibited by the PAHs andthe inbibitional effect was in the order Na＞Phen＞Py,and the inhibition of Na was themost obvious.
In this study,the SSF,FM and TPLCSM were firstly combined for in situ studyon the environmental behaviors of typical PAHs in the mangrove plants.It providednovel means,methods,and research idea for the related studies of PAHs in mangroveecosystems.Through studying the processes of uptake,transportation and degradationof typical PAHs in different kinds of mangrove leaves,stem and roots,we revealedthe processes and mechanisms on phytoremediation for typical PAHs and pollutioncontrol of mangrove plants.Moreover,it layed a methodological foundation forfurther study of the growth,ecophysiology and biochemical effects of different kindsof mangrove plants under the pollution stress of PAHs.
引文
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