185 resultados para PAHs-degrading microorganisms
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调查了沈阳市细河河水及其沿岸地下水水质中石油类和美国环保局(USEPA)"黑名单"上的16种优先控制多环芳烃(PAHs)的有机污染现状,评价细河污染带来的生态风险。结果表明:细河河水中石油类浓度和16种PAHs总浓度范围分别为0.031~1.819mg.L-1和0.026~0.384μg.L-1,平均浓度分别为1.007mg.L-1和0.151μg.L-1;细河沿岸地下水中石油类浓度和16种PAHs总浓度范围分别为0.020~0.987mg.L-1和0.051~0.389μg.L-1,平均浓度分别为0.364mg.L-1和0.133μg.L-1;细河河水和地下水中的石油类污染严重;河水中的PAHs浓度在流经规模较大的城镇或乡镇处出现高值点,苯并(b)荧蒽和苯并(k)荧蒽在河水与地下水中的检出率较高,对水生生态系统健康构成了潜在的威胁。
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以菲、芘作为多环芳烃代表污染物,采用室内培养方法,研究了4种真菌细胞色素P450含量与多环芳烃(PAHs)浓度及降解率的相互关系。结果表明,在一定的浓度范围内(菲0~200mg·L-1、芘0~100mg·L-1),菲、芘浓度与真菌P450含量呈明显正相关,菲、芘浓度与真菌P450含量之间表现出明显的剂量-效应关系。在上述浓度内,真菌降解多环芳烃的能力与真菌P450含量之间也表现出明显的剂量-效应关系。在选用的4株真菌中,Zj1(镰刀菌)降解菲和芘的能力最强,Zj3(小克银汉)次之,Zj2(毛霉)和Zj4(青霉)降解能力较弱。
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以小麦(Triticum acstivnm)为供试植物,草甸棕壤为供试土壤,以微粒体细胞色素P450及抗氧化酶SOD、POD和CAT酶活性为指标,进行了土壤中菲、芘单一及复合胁迫响应研究。结果初步表明,菲、芘胁迫引起植物体内解毒代谢和抗氧化防御酶反应。菲、芘单一胁迫浓度为1mgkg-1时对细胞色素P450产生显著诱导;4mgkg-1时P450酶含量明显被抑制,表明低浓度菲、芘单一胁迫对植物代谢解毒系统产生损伤;而菲、芘复合1mgkg-1时P450酶含量明显被抑制,说明菲、芘复合胁迫对植物的代谢解毒具有协同毒性效应。土壤中菲、芘单一胁迫未引起SOD酶活性的明显改变,复合胁迫下SOD酶活性出现微弱下降;菲、芘单一胁迫对CAT和POD酶活性具有显著抑制作用;复合胁迫对CAT产生抑制作用,而POD酶活性并未对菲、芘复合产生增强毒性响应。研究从代谢解毒和抗氧化防御酶系统两方面,为土壤低浓度PAHs污染诊断提供了实验依据。
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In this paper, organic-inorganic hybrid material, which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)), was employed to immobilize Trichosporon cutaneum strain 2.570 cells. Cells entrapped into the hybrid material were found to keep a long-term viability. The mechanism of such a long-term viability was investigated by using confocal laser scanning microscopy (CLSM). Our studies revealed that arthroconidia produced in the extracellular material might play an important role in keeping the long-term viability of the immobilized microorganism. After the arthroconidia were activated, an electrochemical biochemical oxygen demand (BOD) sensor based on cell/hybrid material-modified supporting membrane was constructed for verifying the proposed mechanism.
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Electrochemical measurement of respiratory chain activity is a rapid and reliable screening for the toxicity on microorganisms. Here, we investigated in-vitro effects of toxin on Escherichia coli (E. coli) that was taken as a model microorganism incubated with ferricyanide. The current signal of ferrocyanide effectively amplified by ultramicroelectrode array (UMEA), which was proven to be directly related to the toxicity. Accordingly, a direct toxicity assessment (DTA) based on chronoamperometry was proposed to detect the effect of toxic chemicals on microorganisms. The electrochemical responses to 3,5-dichlorophenol (DCP) under the incubation times revealed that the toxicity reached a stable level at 60 min, and its 50% inhibiting concentration (IC50) was estimated to be 8.0 mg L-1. At 60 min incubation, the IC50 values for KCN and As2O3 in water samples were 4.9 mg L-1 and 18.3 mg L-1, respectively. But the heavy metal ions, such as Cu2+ Pb2+ and Ni2+, showed no obvious toxicity on E. coli.
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The membraneless biofuel cell (BFC) is facile prepared based on glucose oxidase and laccase as anodic and cathodic catalyst, respectively, by using 1,1'-dicarboxyferrocene as the mediators of both anode and cathode. The BFC can work by taking glucose as fuel in air-saturated solution, in which air serves as the oxidizer of the cathode. More interestingly, the fruit juice containing glucose, e.g. grape, banana or orange juice as the fuels substituting for glucose can make the BFC work. The BFC shows several advantages which have not been reported to our knowledge: (1) it is membraneless BFC which can work with same mediator on both anode and cathode; (2) fruit juice can act as fuels of BFCs substituting for usually used glucose; (3) especially, the orange juice can greatly enhance the power output rather than that of glucose, grape or banana juice. Besides, the facile and simple preparation procedure and easy accessibility of fruit juice as well as air being whenever and everywhere imply that our system has promising potential for the development and practical application of BFCs.
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A novel type of biochemical oxygen demand (BOD) biosensor was developed for water monitor, based on co-immobilizing of Trichosporon cutaneum and Bacillus subtilis in the sol-gel derived composite material which is composed of silica and the grafting copolymer of poly (vinyl alcohol) and 4-vinylpyridine (PVA-g-P(4-VP)). Factors that influence the performance of the resulting biosensor were examined. The biodegradable substrate spectrum could be expanded by the co-immobilized microorganisms. The biosensor prepared also exhibited good reproducibility and long-term stability. Good agreement was obtained between the results of the sensor BOD measurement and those obtained from conventional BOD5 method for water samples.
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Recent research carried out at the Chinese Institute of Applied Chemistry has contributed significantly to the understanding of the radiation chemistry of polymers. High energy radiation has been successfully used to cross-link fluoropolymers and polyimides. Here chain flexibility has been shown to play an important role, and T-type structures were found to exist in the cross-linked fluoropolymers. A modified Charlesby-Pinner equation, based upon the importance of chain flexibility, was developed to account for the sol-radiation dose relationship in systems of this type. An XPS method has been developed to measure the cross-linking yields in aromatic polymers and fluoropolymers, based upon the dose dependence of the aromatic shake-up peaks and the F/C ratios, respectively. Methods for radiation cross-linking degrading polymers in polymer blends have also been developed, as have methods for improving the radiation resistance of polymers through radiation cross-linking.
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Tumor necrosis factor receptors (TNFRs) are a superfamily of proteins characterized by the unique cysteine-rich domain (CRD) and their important roles in diverse physiological and pathological events such as inflammation, apoptosis, autoimmunity and organogenesis. The first member of the molluscan TNFR family, designated as CfTNFR, was identified from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfTNFR was of 1334 bp, consisting of a 5' UTR of 17 bp, a 3'UTR of 69 by with a poly (A) tail, and an open reading frame (ORE) of 1248 by encoding a polypeptide of 415 amino acids with a theoretical isoelectric point of 8.33 and predicted molecular weight of 47.07 kDa. There were a signal peptide, a CRD, a transmembrane region and a death domain in the deduced amino acid sequence of CfTNFR, suggesting that it was a typical type 1 membrane protein. The high identities (22-40%) of CfTNFR with other TNFR superfamily members indicated that CfTNFR should be a member of TNFR superfamily, and moreover, it should be the first death domain-containing TNFR found in invertebrates. Phylogenetic analysis revealed that CfTNFR was closely related to TNFR-like proteins from Strongylocentrotus purpuratus, Drosophila melanogaster and Ciona intestinalis, and they formed a separate branch apart from vertebrate TNFRs. The spatial expression of CfTNFR transcripts in healthy and bacteria challenged scallops was examined by quantitative real-time PCR. CfTNFR transcripts could be detected in all tested tissues, including haemocytes, gonad, gill, mantle and hepatopancreas, and significantly up-regulated in the tissues of gonad, gill, mantle and hepatopancreas after Listonella anguillarum challenge, indicating that CfTNFR was constitutive and inducible acute-phase protein involved in immune defence. The present results suggested the existence of the TNFR-like molecules and TNF-TNFR system in low invertebrates, and provided new insights into the role of CfTNFR in scallop innate immune responses to invading microorganisms. (C) 2009 Elsevier Ltd. All rights reserved.
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The C1q-domain-containing (C1qDC) proteins are a family of proteins characterized by a globular C1q (gC1q) domain in their C-terminus. They are involved in various processes of vertebrates and supposed to be an important pattern recognition receptor in innate immunity of invertebrates. In this study, a novel member of C1q-domain-containing protein family was identified from Zhikong scallop Chlamys farreri (designated as CfC1qDC) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of CfC1qDC was of 777 bp, consisting of a T-terminal untranslated region (UTR) of 62 bp and a 3' UTR of 178 bp with a polyadenylation signal sequence AATAAA and a poly (A) tail. The CfC1qDC cDNA encoded a polypeptide of 178 amino acids, including a signal peptide and a C1q-domain of 158 amino acids with the theoretical isoelectric point of 5.19 and the predicted molecular weight of 17.2 kDa. The C1q-domain in CfC1qDC exhibited homology with those in sialic acid binding lectin from mollusks and C1qDC proteins from higher vertebrates. The typical 10 beta-strand jelly-roll folding topology structure of C1q-domain and the residues essential for effective packing of the hydrophobic core were well conserved in CfC1qDC. By fluorescent quantitative real-time PCR, mRNA transcripts of CfC1qDC were mainly detected in kidney, mantle, adductor muscle and gill, and also marginally detectable in hemocytes. In the bacterial challenge experiment, after the scallops were challenged by Listonella anguillarum, there was a significant up-regulation in the relative expression level of CfC1qDC and at 6 h post-injection, the mRNA expression reached the maximum level and was 4.55-fold higher than that of control scallops. Similarly, the expression of CfC1qDC mRNA in mixed primary cultures of hemocytes stimulated by lipopolysaccharides (LPS) was up-regulated and reached the maximum level at 6 h post-stimulation, and then dropped back to the original level gradually. In order to investigate its function, the cDNA fragment encoding the mature peptide of CfC1qDC was recombined and expressed in Escherichia coli BL21 (DE3). The recombinant CfC1qDC protein displayed a significantly strong activity to bind LIDS from E. coli, although no obvious antibacterial or agglutinating activity toward Gram-negative bacteria E. coli JM109, L. anguillarum and Gram-positive bacteria Micrococcus luteus was observed. These results suggested that CfC1qDC was absolutely a novel member of the C1qDC protein family and was involved in the recognition of invading microorganisms probably as a pattern recognition molecule in mollusk. (c) 2008 Elsevier Ltd. All rights reserved.
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Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.
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Cystatins are a superfamily of proteins as reversible inhibitor of cysteine proteinases which play essential roles in a spectrum of physiological and immunological processes In this study, a novel member of Cystatin superfamily was identified from Chinese mitten crab Enocheir sinensis (designated EsCystain) by expressed sequence tag (EST) analysis and rapid amplification of cDNA ends (RACE) approaches The full-length cDNA of EsCystatin was of 1486 bp, consisting of a 5'-terminal untranslated region (UTR) of 92 bp, a 3' UTR of 1034 bp with a polyadenylation signal sequence AATAAA and a polyA tail, and an open reading frame (ORF) of 360 bp encoded a polypeptide of 120 amino acids with the theoretical isoelectric point of 548 and the predicted molecular weight of 13 39 kDa. A signal Cystatin-like domain (Gly(25) to Lys(112)) was found in the putative amino acid sequences of EsCystatin Similar to other Cystatins, the conserved central Q(70)VVSG(74) motif was located in the Cystatin-like domain of EsCystatin But EsCystatin lacked of signal peptide and disulphide bond. The EsCystatin exhibited homology with the other known Cystatins from invertebrates and higher vertebrates, and it was clustered into Cystatin family 1 in the phylogenetic tree. The mRNA transcripts of EsCystain were mainly expressed in hemolymph, gill, hepatopancreas, gonad and muscle, and also marginally detectable in heart After Listonella anguillarum challenge, the relative expression level of EsCystatin in hemolymph was down-regulated to 0 6-fold (P < 0.05) at 3 h post-challenge. Subsequently, it was up-regulated to 3.0-fold (P < 0.01)at 24 h Afterwards. EsCystatin mRNA transcripts suddenly decreased to original level. After Pichia pastoris GS115 challenge, its mRNA expression level in hemolymph was up-regulated to the peak at 3 h (2 8-fold of that in blank (P < 0 01)) The cDNA fragment encoding the mature peptide of EsCystatin was recombined and expressed in Escherichia coli Rosetta-gami (DE3). The recombinant EsCystatin displayed a promoter inhibitory activity against papain When the concentration of EsCystatin protein was of 300 mu g mL(-1), almost 89% of papain activity could be inhibited. These results collectively suggested that EsCystatin was a novel member of protein in Cystatin family, was a potent inhibitor of papain and involved in immune response versus invading microorganisms. (C) 2010 Elsevier Ltd All rights reserved.
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Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.
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对虾病害在世界范围内的广泛传播,给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明,当对虾等甲壳动物受到外界病原刺激时,其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢,引起呼吸爆发,产生多种活性氧分子。另外,受到病原侵染的对虾还会产生其他多种免疫反应,这些免疫反应将消耗大量的能量(ATP),产能的呼吸链会加速运转,由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物,但同时由于活性氧分子反应的非特异性,它们也会对宿主的细胞、组织和器官造成严重伤害,进而导致对虾生理机能的损伤和免疫系统的破坏。所以,消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力,提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶(mMnSOD)、胞质型超氧化物歧化酶(cMnSOD)、过氧化氢酶(Catalase)和过氧化物还原酶(Peroxiredoxin)等四种与免疫系统相关的抗氧化酶基因,分析了它们的分子结构特征,组织分布及应答不同病原刺激的表达变化模式,并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶(SOD)基因,通过序列比对分析发现,其中一个为mMnSOD基因,另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基,其中开放阅读框为660个碱基,编码220个氨基酸,其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明,该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示,对虾感染病毒3 h时,该基因在血细胞和肝胰脏中的转录水平显著升高。此外,通过构建原核表达载体,本研究对该基因进行了体外重组表达,并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基,其中开放阅读框为861个碱基,编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示,cMnSOD基因在对虾被检测的各个组织中均有表达。 另外,半定量RT-PCR结果表明,对虾感染病毒23h时,该基因在肝胰脏中的转录上升到正常水平的3.5倍;而感染后59 h时,该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物,结合RACE技术,从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段,片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现,该基因在肝胰脏、鳃、肠和血细胞中表达水平较高,在卵巢、淋巴器官和肌肉中的表达水平相对较弱;感染病毒23 h和37 h时,对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息,结合SMART-RACE技术,从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因(Peroxiredoxin), 该基因的cDNA全长为942个碱基,其中开放阅读框为594个碱基,编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da,pI为5.17。Northern blot结果表明,Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示,弧菌感染后,该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外,对该基因进行了体外重组表达,并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明,复性后的重组蛋白能在DTT存在的条件下还原H2O2。
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海洋沉积环境中多氯联苯(PCBs)和多环芳烃(PAHs)的研究对于揭示其污染历史、来源途径、迁移转化以及评价其对环境的潜在生态风险都有重要的科学意义和应用价值,本研究选择我国典型近海中比较开阔的南黄海和受人为影响严重的渤海湾沉积物中的PAHs和PCBs作为主要研究对象,结合对生态环境对应关系的剖析,系统研究了沉积物中PAHs和PCBs的地球化学分布特征、影响控制因素、演变趋势、潜在生态风险等,获得了以下系统的认识: 1.南黄海表层沉积物中多环芳烃和多氯联苯的分布与沉积类型及模式相一致,受控于“沉积类型-动力过程-来源途径”。PCBs含量(范围:518~5848 pg/g,平均值:1715 pg/g)低于受人为影响严重的长江口、珠江口和渤海,分布具有中部海区>东部海区>西部海区的特征;PCBs随着沉积物粒径的减小和粘土含量的增加而增加,且与总有机碳(r=0.61,p<0.01)含量呈显著线性正相关,表明PCBs在沉积物中的分布受控于被水动力过程原动力控制的沉积类型与沉积模式。 2. 1914~2004年间,南黄海沉积物中PAHs和PCBs的变化比较显著,在时间序列上经历了三个明显的不同阶段。近90年来,PAHs和PCBs在柱状样中垂直分布随深度的增加而降低,即近年南黄海沉积物中PAHs和PCBs的残留水平比上世纪初明显增加。其中1914~1932年间,PAHs和PCBs保持在较低的水平;1932~1962年间,PAHs和PCBs的含量发生急剧的变化,在1932~1944和1956~1962年两个时间段,PAHs和PCBs的含量达到峰值;自1962年至今,PAHs和PCBs呈稍有增加趋势。PAHs的组成和特征组分比值分析显示,1920~1944年间PAHs主要来自石油产品泄漏,1944~1980年间,主要来自草/木材/煤燃烧,1980年至2004年则显示出石油和燃烧产物混合来源的特征。 3.渤海湾沉积物中的PAHs、PCBs、DDTs和HCHs的分布模式不同,反映了这四种污染物的地球化学行为存在着明显的差异性。PAHs、PCBs、DDTs和HCHs的含量范围为149.0~393.4 ng/g,360.8~1728.3 pg/g,462.2~2007.3 pg/g和4.31~33.8 ng/g。马颊河口、海河口和黄河口附近的海区的沉积物中PAHs和PCBs的含量显著高于渤海湾内其它站位,DDTs在湾外沉积物中的含量大于湾内,在海河口附近站位测得HCHs含量的最高值,在其它站位其浓度变化不大。PAHs特征成分的比值显示渤海湾沉积物中PAHs主要来源于草/木材/煤燃烧的产物经过大气的输运过程进入水体;DDTs和HCHs的组成显示,在DDTs和HCHs被禁用后仍有新的输入源。 4.南黄海沉积物POPs总体水平不高,其环境污染危害和潜在生态风险不大,从沉积物POPs的角度来说南黄海的环境质量较好。潜在生态危害指数评价表明,渤海湾沉积物中芴可能会产生潜在的生态风险,DDTs和HCHs的含量低于一类沉积物质量标准值,总体而言,其沉积物质量良好,潜在生态风险较低。 论文的创新性点在于:1)首次研究了近百年南黄海沉积物中多环芳烃和多氯联苯的演变趋势,判断了其来源并对近百年二者的潜在生态风险进行评价。2)系统剖析了南黄海及渤海湾的生态环境与PAHs和PCBs的耦合关系,对阐明POPs的毒理效应有重要的科学意义。3)系统解析了渤海湾沉积物中PAHs,DDTs和HCHs的污染现状,来源和迁移途径,可为科学开发和利用渤海海域提供重要的理论依据。