重金属污染对萝卜的生态毒理效应研究硕士定稿毕业论文.doc

硕 士 学 位 论 文重金属污染对萝卜的生态毒理效应研究Ecotoxicological effects of Heavy Metals on Raphanus Sativus摘 要随着全球经济的发展，重金属通过各种方式进入土壤中，从而使得土壤的重金属污染越来越严重。被重金属污染的土壤不仅会影响到植物的生长，降低农作物的产量，而且还会导致大气环境和水环境的污染，最后可以通过食物链威胁到人类的健康。在没有受污染的土壤中，重金属大都以很少量存在。 从全球范围来看，重金属污染被认为是最主要的环境污染之一。全世界大部分范围内的土壤都在不同的程度上受到重金属的污染。世界上每年向环境中释放的重金属量近似于：汞是150万吨，铜是340万吨，铅是500万吨，锰是1500吨，镍100万吨。在中国的北部地区，一些蔬菜和作物的种植基地中的土壤受重金属污染非常严重，比如北京、天津、沈阳、济南、长春、郑州等地的种植基地。相比北方，中国南部大部分地区重金属污染较轻，但是在福州、宁波、上海、武汉、成都和一些地区重金属污染日益严重且造成了各种各样的生态问题。随着经济发展以及由此带来的生态结构破坏，自然灾害的增加，可利用的土壤已经越来越少。所以，怎样控制重金属的土壤污染，研究重金属污染的机理是非常重要的。相应地，对重金属影响下的种子萌发和植物早期幼苗生长的研究将是十分必要的。重金属是指60种金属元素中比重超过4.0或45种元素中比重超过5.0的一些金属元素砷和硒不是金属，但由于其毒性和其它性质与重金属相似，所以砷和硒也被认为是重金属污染物。由于汞、镉、铬、 铅、砷的强毒性使得它们在土壤重金属污染中占有非常重要的地位，除此之外，锌、铜、 钴、镍、硒和一些其它元素也因其对生物的毒性而特别被关注。重金属锌、铁、铜、锰等是植物生长不可缺少的微量元素，它们在很多生物酶的合成与代谢过程中起到非常重要的作用，而其它重金属如铅，镉，砷，铯，铬，铝等虽然在生物学上并不是必须的，但如果它们超过一定浓度将会对生物产生极大的毒性效应。重金属铬通常主要以三种形态存在于自然界，即单质铬、三价铬和六价铬。其中以三价铬最多，而六价铬和单质铬主要来自工业生产当中。三价铬对植物及其生长一般表现出很少的毒性，但是六价铬对植物及其生长大都表现出有很大的毒性效应。过量的镉可以在植物生长过程中产生各种各样的毒性症状，比如植物生长的衰退，光合作用的减弱等等。在含有重金属镉的物质中，硫酸盐是最常见的，它的溶液也是具有毒性的，而水中镉的来源主要分为天然源和人类生产活动源。汞是毒性很大的污染物。同时，对于生物来说，汞既不是植物细胞生长所必须元素，也不会产生一般积极的生态作用。在本实验研究中，汞对萝卜种子的萌发和早期幼苗的生长都表现出了显著的抑制作用。其中汞对萝卜根生长和茎生长的抑制能力远远大于对萝卜种子萌发的抑制力，原因可能在于是萝卜种皮对种子萌发的保护作用，当种子萌发开始生长时，萝卜的根和茎生长将不再受到种皮的保护，所以使得汞对萝卜根和茎生长的抑制力表现出最大。然而，不同重金属对植物种子萌发和幼苗早期生长抑制的影响机理是不同的。造成汞对萝卜根生长和茎生长的抑制能力远远大于对萝卜种子萌发的抑制力的原因可能是由于汞在萝卜的根和茎中的积累到一定量时造成抑制性最大。汞还能够改变生物体内蛋白质的结构，影响谷氨酸运输和生物酶的合成。除了汞、 铅、镉、铬之外，土壤中还有其它的金属元素。这些重金属元素的过量存在都会导致植物种子萌发和早期幼苗生长受的影响。另外一些重金属对植物以及生长具有更大的毒性作用，包括砷、钴、铝、硒等。锌是生物体内许多重要生物酶的组成元素，其在氮的循环、细胞的合成代谢、光合作用和生物生长素合成中起着非常重要的作用，再加上锌也是生物界所必须的营养元素之一，所以其对于植物的影响具有种类异质性，比如其毒性作用的大小与植物种类的生长年龄、生长时期以及锌本身是否可以直接被植物利用等因素有关。萝卜广泛生长于世界各地，本次实验是为了研究锌对萝卜种子萌发及其幼苗生长的影响。虽然铅对于植物来说不是必须的元素，但当其达到一定的量时将会对植物生长起到抑制作用，从而影响植物的光合作用、细胞分裂和植物对水分的吸收。铅的可溶性和不溶性的形态对于植物都具有毒性作用。铅可以通过采矿、冶炼、杀虫剂的使用、交通、煤炭等被释放到环境中，它没有积极的生物效应。许多研究表明，当铅通过植物的根和茎进入植物体内后，将在植物体内大量积累，然而其积累的量主要取决于铅本身的特性。土壤中的重金属污染源有很多。首先，一些土壤本身就含有大量重金属，不同的土壤类型包含的重金属的量不同，人类活动可以使得土壤中固有的这些重金属转移到空气中、水中和其它土壤中，造成重金属污染。虽然有些痕量元素是组成土壤的重要元素，但频繁的人类活动如：采矿、工业生产、局部地区集约的农业生产使得世界许多地区大量的有毒重金属得以积累。其次，重金属污染是随着人口数量的增加、土壤利用率和人类交通工具的增长而不断加强。污水灌溉也是重金属污染的主要来源之一。早在20世纪60年代，污水灌溉就成为农业灌溉的重要部分，特别是在中国的北方。毫无疑问，频繁用污水灌溉土壤将不断增加重金属在土壤中的含量，尤其是以下重金属：汞、镉、铬、砷、铜、锌、铅等。在中国太原市，土壤中重金属浓度不仅已超过当地的环境背景值，而且数量还在不断增加。除此之外，废弃物中也包括很多的重金属元素，不同重金属的混合使得不同废弃物对生物有着不同的毒害作用。当然，重金属的生物毒性是与其存在的形态是密不可分的，比如就汞、铅、砷、镉来说，当它们以阳离子形态存在时毒性会增加。重金属在液体中移动和其相对迁移能力主要取决于氧化还原条件、PH和有机络合物量等环境条件，在溶液中，溶液的离子强度和阳离子的饱和条件也会影响到重金属的移动，环境中大部分毒性重金属由于其非溶性而很难在维管植物中自由转移。此外，许多硫酸盐、碳酸盐和硫酸盐将会影响重金属在非质原体和共质体中的移动性，这些重金属非原质体的运输很大程度上是由很高的离子交换能力所决定，除非重金属离子是以非阳离子形式存在。大多数情况下，由于这些重金属阳离子对巯基具有很强的亲合力，从而对有机体中正常的酶活动造成很大的破坏，最后将改变酶物质的活性，产生致命性的副作用。总之，重金属毒性的大小往往是由其从在的形态所决定，毒性大的形态将被有机体的组织器官吸收后，或对神经系统造成严重的影响，或造成生理上的迫害甚至死亡。化工用品和工业“三废”的排放对土壤，空气和水源的污染也越来越严重。特别是对土壤的污染，从而污染了生长在土壤上的植物。在污染环境下生长的植物将含有重金属以及其他污染物，这必将影响到人们的健康生活水平。重金属还可通过农肥，下水道污泥，市政废弃物以及杀虫剂等进入土壤，重金属一旦进入土壤它的总量就很难减少。虽然重金属在自然环境下很难被降解，但是通过生物降解作用其在环境中的毒性可能被一定程度的降低。环境中的重金属污染往往会影响到植物的生长、发育以及种子的萌发，国内外很多学者和专家对重金属影响种子萌发生长等方面做了大量的实验研究。在本实验中，其中一些重金属（铅、镉）在低浓度时对某些种子的萌发有促进作用，但是大多重金属在超过一定的浓度范围后都会对萝卜的种子萌发产生抑制作用。本实验对萝卜种子在重金属污染环境中的萌发、早期生长、叶绿素含量、重金属累积等生理生化指标进行了初步研究。萝卜根据其不同的种类在一年中的任何时候都可以种植，所以其在中国是很常见的蔬菜。近年来，由于工业和城市化的发展中国的很多农田被重金属所污染，而萝卜往往种植于高速公路附近和矿区附近的农田中。这就说明萝卜对重金属污染有一定的忍耐性，如果已受重金属污染的萝卜被当做食物食用势必会对人体造成很大危害。如今，随着工业化、城市化的发展，污染物包括重金属进入环境的数量越来越多，各种疾病的突发率都与水果和蔬菜的食用紧密相关。重金属是造成全球作物产量减少的重要原因之一。尽管许多研究都关注于研究重金属对植物的影响上，可是很少有注重于对多种环境因素的相互作用或共同作用上。作为基础的相关研究，本实验通过Hg、Pb、Cd、Co、Cr对大部分萌发种子的抑制的研究来说明重金属对种子萌发与生长的毒害效应。实验中所用的重金属盐及浓度如下： CdCl22.5H2O (0.02、 0.2、 0.4、 0.8、 1.6 mg/l), K2Cr2O7 (0.2、0.4、0.8、1.6、3.2 mg/l), CoCl26H2O (2、 4、 8、16、32 mg/l), PbCl2 (0.2、0.4、0.8、1.6、3.2 mg/l), HgCl2 (0.2、0.4、 0.8、1.6、3.2 mg/l) and ZnCl2 (0.2、0.4、0.8、1.6、3.2 mg/l)。本实验所用萝卜种子由甘肃省农科院提供。本次实验首先筛选饱满、大小均匀的萝卜种子用做实验种子，用流水清洗培养皿3次，再用蒸馏水冲洗，烘干。种子用0.5%的次氯酸钠杀菌10-20分钟后用蒸馏水冲洗。在培养皿中放入两层的滤纸，滤纸分别用蒸馏水（对照实验）和复合重金属的不同浓度的溶液浸湿；每个培养皿中放入50粒种子，然后放入培养箱中，白天温度为25-301，晚上温度为20-251，光周期为12小时。每隔24小时测一次萌发率，72小时后测根长和径长，称鲜重； 叶绿素和类胡萝卜素测定在7天后进行，取0.08g幼芽放在研钵中，加少量石英砂和碳酸钙粉及0.8-1.21mL 95%乙醇（或80%丙酮），研成匀浆，再加乙醇4mL，继续研磨至组织变白。静置3-5分钟。然后取滤纸1张，置漏斗中，用乙醇湿润，沿玻璃棒把提取液倒入漏斗中，过滤到25 mL棕色容量瓶中，用少量乙醇冲洗研钵、研棒及残渣数次，最后连同残渣一起倒入漏斗中。用滴管吸取乙醇，将滤纸上的叶绿体色素全部洗入容量瓶中，直至滤纸和残渣中无绿色为止。最后用乙醇定容至10 mL，摇匀。把叶绿体色素提取液倒入光径1cm的比色杯内。以95%乙醇为空白，在波长665nm、649nm和470nm下测定吸光度。根和幼芽中重金属含量的测定也在7天后，重金属含量的测定用火焰原子吸收测定仪测定。取出培养皿中的样品，分离根和幼芽，然后把根和幼芽放入烤箱中，在温度为80条件下，烘干24h。称取干样（根0.05g，幼芽0.2g），再分别加入混合酸（65% HNO3:H2O2=4:1）溶解，然后用高压锅在120下消毒灭菌90分钟。冷却后用蒸馏水稀释定容至25,最后用原子吸收分光光度计测定重金属含量；试验数据源于每组浓度六次重复的平均值，数据分析应用SPSS (version16.0)软件，对数据进行单因素方差分析（one-way analysis of variance (ANOVA)）和多重比较（least significant difference (LSD)）。通过实验，得到如下结论：1. 在重金属镉、铅单独处理时，所有萝卜种子的萌发率和幼苗生长均受到明显的抑制。且随着重金属浓度的增加，抑制程度增加。 2. 重金属镉、锌单独处理时，低浓度处理促进了萝卜种子的萌发和茎的伸长，高浓度处理时，则受到抑制。 3. 萝卜种子在受所有不同浓度的重金属处理下，其根长均受到了抑制，且抑制显著。在所有重金属对萝卜种子的毒性测试下，根和茎比萌发率更准确、敏感。4. 随着重金属浓度的增大，叶绿素含量和类胡萝卜素含量降低。但总叶绿素含量比类胡萝卜素含量降低的多，叶绿素B的含量比叶绿素A的含量降低得多。5. 重金属在萝卜体内的累积与重金属浓度呈正相关，且重金属更容易累积在萝卜的根部，不易迁移至茎部。6. 在铬和钴复合处理时，低浓度表现为拮抗作用，较高浓度时，表现为协同作用。7. 在铅和汞复合处理时，汞的加入增加了铅的毒害作用，铅的加入，降低了汞的毒害作用。8. 镉和锌复合污染时对萝卜的萌发表现为协同作用。但在两种重金属低浓度污染对根和茎的生长表现为拮抗作用。关键词：重金属；种子萌发；根；茎；抑制；叶绿素；累积论文类型： 基础研究AbstractWith the development of global economy, the heavy metal pollutants enter soil by many ways too, and then the soil was polluted by heavy metals seriously. Polluted soil not only effects the plant growth and degrade the quality of food, but also threaten peoples health through the Food Chain, lead to air and water environment polluted too. Heavy metals occur naturally at low concentrations in soils. Heavy metal pollutant was regarded as very important pollutant all around the world. Now the soil around the world was polluted by heavy metals at different degree, totally the heavy metal released to the environment annually nearly as follow: Hg: 1.5million ton, Cu: 340million ton, Pb: 500million ton, Mn: 1500 ton, Ni: 100million ton . In north of China, many vegetable and food base exist some heavy metal polluted phenomenon seriously, for example, Beijing, Tianjin, Shenyang, Jinan, Changchun, Zhengzhou etc. On the other hand, South of China seemed to be a little better, but in Fuzhou, Ningbo, Shanghai, Wuhan, Chengdu and some other place, heavy metal pollution has caused serious ecological problems. With the economic development and ecosystem degrade the structure change and nature disaster etc, the sources of soil will be decreased too, so how to control the heavy metal pollution and investigate how the heavy metal environment is very important and some measures should be done. Heavy metal means those 60 element that density exceed to 4.0 or those 45 element that density exceed to 5.0, As and Se are not metal element, but because of their toxicity and other characteristics are much nearly to heavy metals, so we put As and Se as heavy metal pollutant. Because of the bio-toxicity of Hg, Cd, Cr, Pb, and As, they have been taken the most important heavy pollutants in the environment, also Zn, Cu, Co, Ni，Se and some other pollutant were regarded very toxic. Heavy metals like Zn, Fe, Cu, Mn are essential for plant growth and important constituents of many enzymes of metabolic importance. Other metals like Pb, Cd, As, Se, Cr and Al are biologically non-essential and toxic above certain threshold levels. It is found in the environment in three major states: chromium (0), chromium (III), and chromium (VI). Chromium (III) occurs naturally in the environment, while chromium (VI) and chromium (0) are generally produced by industrial processes. Cr (III) showed no toxicity effect to plant, but Cr (VI) has very serious toxicity to plant. Excess Cd causes a number of toxic symptoms in plants i.e., growth retardation, inhibition of photosynthesis. It forms a number of salts, of which the sulfate is most common; Cadmium and solutions of its compounds are toxic. Cadmium can enter surface waters from the natural sources and from a variety of manufacturing operations. Mercury is not essential to living cells and performs no known biological function. Hg has inhibitory effect on the germination and seedling growth of the test species.The root and shoot length were inhibited much more than germination in this study, may be its because of the seed coat is able to reduce the amount of Hg entering the seed, but after the seed germination, root and shoot have no barrier to protect. Some other studies have the same conclusion, and the different metals may inhibit seed germination and growth by different mechanism, may be its because of holding and accumulating the mercury in the root and shoot, bring about the result that the growth of the root and shoot growth were affected much more. Mercury also causes alterations in protein structure, alterations in calcium transport, along with the inhibition of glucose transport and enzyme function. Except Hg, Pb, Cd, Cr, there exist many other heavy metals in the environment and soil, nearly all the heavy metals have negative effect to seed germination and seedling growth when the concentration exceeded to some degree, and some heavy metal has the more toxicity effect to plant, include As, Co, Al, Se etc. Zinc is also a constituent of metal enzyme or a cofactor for several enzymes such as anhydrases, dehydrogenases, oxidases and peroxidases 104 and plays important role in regulation the nitrogen metabolism, cell multiplication, Photosynthesis and auxin synthesis in plants,also it plays an important role as an essential trace element in all living systems from bacteria to humans .The toxic of zinc and most zinc-content in plants is species specific; it is dependent on the age, vegetation state of the plant as well as on zinc availability . Radish are widely growth around the world; the aim of the study was to compare the effect of Zn2+ to seed germination, root and plumue growth of radish. Lead is not essential for plants, and excessive amounts can cause growth inhibition, as well as reduced photosynthesis, mitosis, and water absorption.Both lead and dissoluble lead are poisonous, Pb can be entered the environment by mine, melting, pesticide, transportation, coal and industry related to Pb, Pb was regarded as no positive bio-effect, many experiment and analysis have proved that Pb can enter the tissue of plant through root and shoot, and then enriched in plant, and the enrich degree compared to its habitat. The source of heavy metal pollutant in soil have many approaches, firstly, some soil itself contain heavy metal inbeing, different soil type contain different heavy metal pollutant, in another, because of humans activity, heavy metal pollution were spread out to the air, water and soil.Although at trace levels heavy metals are natural components of soils, activities such as mining, industry, and localized intensive agriculture have contributed to undesirable accumulations of toxic metals in many areas around the world. Furthermore, heavy metal pollution has increased with the population density; soil used ratio and motor vehicles density. Sewage irrigation be used in China since 1960s, especially in north of China. Its inevitable to increase heavy metal concentration in soil by using sewage irrigation, especially those toxic metals (Hg、Cd、Cr、As、Cu、Zn、Pb), take Taiyuan for example, the heavy metal concentration in the soil has exceeded to local background much, and the concentration are increasing yearly. The rejectamenta that contains heavy metals has plenty of types, differential types has differential harm effect. Heavy metal toxicity arises when a number of certain heavy metal species, such as mercury, lead, arsenic and cadmium for example, occur as cations. The behavior of heavy metals in fluids and their relative mobility in the environment varies strongly according to oxidation-reduction conditions, to the pH of the surrounding environment, and whether there are organic complexing compounds present. When in solution the ionic strength of the solution or saturation levels of cations in the solution, also influences the behavior of heavy metals.Most environmentally hazardous metals are too insoluble to move freely in the vascular system of the plant. Many form sulfate, carbonate or phosphate precipitates immobilizing these metals in apoplastic and symplastic compartments. Apoplastic transport of these metals is further limited by the high cation-exchange capacity of cell walls, unless the metal ion is transported as a noncationic metal chelate. The strong affinity for these cations to form complexes with sulphur interferes with normal enzymatic functions within humans. Numerous enzymes which function as metabolic regulators contain sulphur within sulfhydryl groups, and it is these groups which attract, or are attracted to, the metal cations. This consequently alters the capacity of the enzyme to function properly and thus causes often lethal side-effects. Whilst toxicity may differ according to the metal species, the most potent forms are those which can be absorbed into the tissues of organisms often causing serious inhibitive effects on the nervous system, physical damage or even death, Heavy metals are known to be toxic metal that can cause severe damage to plants and animals. Heavy metals-induced oxidative stress involves induction of lipid peroxidation in plants that causes severe damage to cell membranes. Oxidative stress induced by heavy metals initiates the degradation of photosynthetic pigments causing decline in seed germination and seedling growth. High heavy metals concentration can disturb the chloroplast ultra-structure thereby disturbing the photosynthetic process. Heavy metals can affect antioxidant metabolism in plants bioprocess.Through the test, we concluded that the study isnt very well designed too; still there exist many problems to be improved, for example: the concentration should be changed a little for some species of this test; before the study, some necessary knowledge must be understand well, some other test should be done to make the test much more perfect.The development of chemical industry and the three waste of industry given of seriously polluted the soil, air and water, which the plant lived on. The planted under polluted environment, content heavy metal and other pollutants, these affected people health and lives. Metallic contaminants may enter soil from farm manures, sewage sludge, municipal solid wastes, and pesticides. Metallic contaminants in feedstock are not lessened quantitatively during composting.Heavy metals are difficult to be decomposed under natural environment, however, the bioavailability of metallic contaminants may be lessened by composting. Those heavy metals must take some negative effect to environment and ecosystem, a lot of domestic and international scholars have made a large number of experiment research about the influence of heavy metals to seed germination. Consequently, the study of heavy metal pollution on plants and the seed is urgently important.Many results suggested that when at low density some heavy metals may be accelerate seed germination, but when exceeding certain density range nearly all heavy metals inhibit seed germination. This study investigated the possible involvement of Seed germination, seedling growth, photosynthetic pigment production and metal accumulation response to heavy metal stress. Radish (Raphanus Sativus) is a popular vegetable in China. This vegetable has a large number of varieties and can be cultured throughout the year depending on the varieties. In recent years a relatively large area of farmland has been contaminated by heavy metals because of industrialization and urbanization in China. This vegetable often is grown on farmland near highways, in mining areas where soils are usually contaminated by heavy metals. This suggests that the plant can tolerate some extent of heavy metal contamination and that as a food supply it could create a health risk to consumers. With the developing of the economy and urbanization, polluted material including heavy metals entered the environment more and more seriously, an increased frequency in the number of outbreaks of illness associated with the consumption of fresh fruits and vegetables .For the test, seeds of Radish (Raphanus Sativus) were offered by Gansu Academy of Agricultural Sciences.Heavy metals are the main environmental factor accountable for decreasing crop productivity in many geographic areas. Despite the enormous studies that have investigated the effects of heavy metals on plants, only few have embraced the reality of multiple, interacting environmental factors. In order to offer the basis for relevant research, a summary has been done about heavy metals such as Hg、Pb、Cd、Co and Cr influence seed germination, drawing the conclusion that heavy metals inhibit the sprouting of most tested seeds.We studied the effects of several doses of CdCl22.5H2O (0.02, 0.2, 0.4, 0.8, 1.6 mg/l), K2Cr2O7 (0.2, 0.4, 0.8, 1.6, 3.2 mg/l), CoCl26H2O (2, 4, 8, 16, 32 mg/l), PbCl2 (0.2, 0.4, 0.8, 1.6, 3.2 mg/l), HgCl2 (0.2, 0.4, 0.8, 1.6, 3.2 mg/l) and ZnCl2 (0.2, 0.4, 0.8, 1.6, 3.2 mg/l) on different seeds germination, seedling growth, chlorophyll and the carotenoid content and the heavy metals content.This study investigated the possible involvement of Seed germination, seedling growth, photosynthetic pigment production and metal accumulation response to heavy metal stress. Firstly filter the seeds, choose those satiation and slick seeds for the after using, wash the test Petri with tap water for three times, then wash another time with distilled water, and then put the Petri under sunlight until dry; Seeds were surface sterilized in 0.5% sodium hypochlorite solution for10-20 min and washed thoroughly with distilled water. The seeds were germinated in Petri dishes with double layer of filter paper soaked in distilled water (control) and different heavy metal solutions.50 seeds were put in each Petri. The seeds were set under a photoperiod of 12h, and 25-301/20-251 day/night temperature. The germ