软骨藻酸直接竞争ELISA方法的建立及优化

为建立软骨藻酸(domoic acid,DA)直接竞争ELISA方法快速检测样品中的软骨藻酸,本研究利用碳化二亚胺法将辣根过氧化物酶(HRP)标记软骨藻酸(DA),成功制得偶联物DA-HRP.在前期已制得抗软骨藻酸单克隆抗体的基础上,利用酶标抗原和单克隆抗体的特异性反应,对软骨藻酸进行定量分析.通过对封闭液、封闭时间和温育温度的优化,建立标准曲线.结果表明以1%明胶作为封闭液,在37℃下封闭1 h,加入单抗37℃温育1 h是直接竞争ELISA法的最佳反应条件,方法检出限为3.58 ng.mL-1,孔间变异系数均≤15%,加标回收率为80%～120%,检测速度较快,1.5 h内可检测出一批样品,适用于现场和批量检测,具有广泛的发展前景.     

我国近海有毒微藻及其毒素的分布危害和风险评估

近年我国海域有害赤潮频发,赤潮优势种的变化显著,最近7a记录了51种引发赤潮的优势赤潮生物,其中由有毒微藻引发的赤潮增加。有毒微藻在我国海域广泛分布,至少3个株产麻痹性贝毒的亚历山大藻,5种能产生腹泻性贝毒软海绵酸毒素和鳍藻毒素、以及扇贝毒素的鳍藻在我国沿海都有分布;产生虾夷扇贝毒素的三种甲藻在北黄海常年存在。我国多种贝类中已发现麻痹性贝毒、软海绵酸毒素和鳍藻毒素、扇贝毒素、虾夷扇贝毒素和环亚胺毒素等多种微藻毒素。本文较系统的归纳综述了我国有害赤潮、优势种的变化趋势和分布特点,产生的藻毒素结构;我国双壳贝类等海洋生物中存在的微藻毒素的种类结构;首次利用风险商值法研究评估了我国贝类的健康风险。结果表明,只有春季来自福建的2个样品具有食用风险;但大连海区的贝类样品、评估值全都接近安全限值,可能表明具有某种区域性风险;在春末夏初,大窑湾的贻贝风险最大,其次是扇贝,牡蛎的风险最小;不同的贝器官,富集藻毒素的能力差别大,消化腺中的藻毒素含量远高于其它器官,不同的食用方式可能会导致不同的中毒风险。总体上,我国沿海常见种双壳贝类的麻痹性贝毒和虾夷扇贝毒素食用风险低。     

总微囊藻毒素免疫检测及其预处理方法研究

在验证广谱型微囊藻毒素酶联免疫分析试剂盒性能基础上,主要研究了基于我国与美国分别推荐的水样预处理方式所检测的地表水中微囊藻毒素浓度差异.结果表明：(1)酶联免疫分析试剂盒对微囊藻毒素-LR(MC-LR)的检出限为0.04μg·L^-1,并在0.08～1.00μg·L^-1的浓度范围内表现出良好的线性检测范围;(2)基于我国与美国分别推荐的水样预处理方式,后者测得的微囊藻毒素浓度普遍高于前者(最大差值：前者未检出,后者为(0.25±0.01)μg·L^-1),且基于两者所测试的地表水样中的微囊藻毒素浓度均低于世界卫生组织(WHO)对饮用水中MC-LR所设定的残留限值;(3)酶联免疫分析试剂盒在对上述未检出水样进行加标检测时,回收率为80%～120%,变异系数低于16%,表明上述地表水中微囊藻毒素的检测结果具有较高的准确度.     

间接竞争ELISA法测定稻田土壤中除草剂毒莠定的残留量

通过对反应体系中酶标二抗(辣根过氧化物酶标记羊抗兔IgG)和抗体的最佳稀释浓度等条件的筛选和确定,最终建立了一种快速灵敏地测定污染稻田土壤中毒莠定残留量的方法--间接竞争ELISA分析方法.结果表明,酶标二抗和抗体的最佳稀释度均为1:500(体积比),毒莠定的检出下限达到5 ng·mL-1,在0.07～0.7μg·mL-1浓度范围内有良好的线性;土壤浸出液加样的平均回收率为104.11%,变异系数在3.13%～11.13%之间,符合农药残留分析的要求;样品基质对检测结果没有干扰.     

腹泻性贝类毒素标准样品研制

为解决贝类产品检测实验室严重缺乏贝类毒素标准样品的现状,保证小鼠生物法及酶联免疫法等腹泻性贝类毒素检测结果的准确,可靠和可比,本研究制备出腹泻性贝类毒素标准样品。将腹泻性贝类毒素阳性的新鲜贝类去壳后取中肠腺,匀浆,冷冻干燥等步骤制备冻干粉,均匀性和稳定性检验结果表明该样品均匀,稳定,满足标准样品的要求,以多家实验室定值的方式对标准样品进行定值,定值结果采用标准值±扩展不确定度的方式表示:即(8.83±0.60)×10-6(k=2)。该标准样品可用于腹泻性贝类毒素检测过程的方法验证与质量控制。     

一种可商业化生产的胶体金快速免疫层析卡:检测大田软海绵酸及其衍生物(DTX1, DTX2)

建立一种胶体金快速免疫层析检测贝类体内腹泻性贝毒主要成分软海绵酸及其衍生物鳍藻毒素的方法。合成偶联抗原OA-OVA,将适当浓度的偶联抗原和HRP标记的羊抗鼠IgG分别包被在试纸条的检测线和控制线上,胶体金标记的抗OA单克隆抗体包被金标垫,优化检测线的最佳包被浓度等条件以得到最低检出限。结果表明:该试纸条的检出限为16 ng/mL（OA,DTX1 & DTX2）,检测时间为10 min。本研究所建立的胶体金免疫层析检测贝类中大田软海绵酸及其衍生物鳍藻毒素的方法可以满足规定的160 μg/kg贝肉的安全阈值,可以应用于实际贝类样品的腹泻性贝毒的半定量检测。     

赤潮毒素研究进展

近年来,赤潮是全球最严重的海洋生态灾害之一,在我国也尤为突出.本文就赤潮毒素麻痹性贝毒、腹泻性贝毒、记忆缺失性贝毒、神经性贝毒,以及两加鱼毒的分布特征、产毒藻类、化学组分与结构、毒素转化、致毒机理、食用标准、检测方法等国内外研究进展,进行系统的论述,为我国的赤潮监测和灾害防治提供参考.     

麻痹性贝毒的表面等离子体共振快速检测方法研究

采用表面等离子体共振(SPR)技术研究了赤潮毒素麻痹性贝毒(PSP)的快速检测方法,分别采取固定化PSP抗体分子和PSP分子的方法,进行直接和间接检测溶液中PSP的含量,检测下限分别为70.2μg.L-1(~235 nmol.L-1)和0.5μg.L-1(~1.67 nmol.L-1),相对标准偏差(RSD)为4.2%~7.7%,回收率为101%~105%。检测方法的选择性好,样品、标准物和抗体的用量少,检测成本低,单个样品的测试时间不到10 min,能够满足赤潮毒素的快速检测需求。     

中国南部沿海近岸西加鱼毒素研究

为了查清我国南部沿海珊瑚礁鱼类体内的西加鱼毒素状况,首次在海南三亚、琼海、广西涠洲岛、珠海担杆列岛、广东徐闻县灯楼角和福建东山岛收集渔民从附近珊瑚礁海域捕捞的野生鱼类,采用酶联免疫法、小白鼠生物法和高效液相色谱质谱联用法进行毒素测定.结果表明,6个调查海区均检测出染毒鱼类,总阳性检出率达50%,其中福建东山岛(77.8%) >广东徐闻县灯楼角(66.7%) >广东珠海担杆列岛(55.6%) >广西涠洲岛(37.5%) >海南三亚(37.5%) >海南琼海(16.7%),与海域环境质量可能存在一定的关联.鱼组织毒素含量为0~169ng P-CTX-1/kg肉,8个样品 (占总样17.4%) 超过100ng P-CTX-1/kg肉,达到可致人中毒水平.染毒鱼种类繁多,主要包括蝴蝶鱼科(Chaetodontidae)、鹦嘴鱼科(Scaridae)、鳂科(Holocentridae)、笛鲷科(Lutjanidae)和鮨科(Serranidae).酶联免疫法与小鼠生物法对鱼毒定性检测结果基本一致,但仅在福建东山岛2个鱼样中检测到P-CTX-1成分.染毒鱼类的毒素含量和鱼体重/体长没有相关性,也与其食性无关.     

浙江近岸有害赤潮发生区麻痹性贝毒素研究

用小白鼠生物检测法和高效液相色谱法对采自浙江舟山海域和浙江中南部海域的麻痹性贝毒素进行了调查分析。结果表明:浙江舟山海域六横岛的西格织纹螺含有贝毒,其毒素含量为3.7 Mu/g,舟山海域有毒贝类检出率为3.1%。浙江中、南部海域南麂列岛的棒锥螺含有贝毒,其毒素含量<1.7 Mu/g,浙江中、南部海域有毒贝类检出率为9.1%。     

Effects of two sludge application on fractionation and phytotoxicity of zinc and copper in the soil

The potential harm of heavy metals is a primary concern in application of sludge to the agricultural land. A pot experiment was conducted to evaluate the effect of two sludges on fractionation of Zn and Cu in soil and their phytotoxicity to pakchoi. The loamy soil was mixed with 0%, 20%, 40%, 60% and 80% (by weight) of digested sewage sludge (SS) and composted sludge (SC). The additions of both sludges caused a significant raise in all fractions, resulting in that exchangeable (EXCH) and organic bound (OM) became predominance of Zn and organic bound Cu occupied the largest portion. There was more available amount of Zn and Cu in SS treatments than SC treatments. During the pot experiment, the concentration of Zn in EXCH, carbonate (CAR) and OM and Cu in EXCH and OM fractions decreased in all treatments, so their bioavailability reduced. Germination rate and plant biomass decreased when the addition rate was high and the best yield appeared in 20% mixtures at the harvest of pakchoi. The two sludges increased tissue contents of Zn and Cu especially in the SS treatments. Zn in pakchoi was not only in relationship to ΔEXCH and ΔCAR forms but also in ΔOM forms in the sludge-soil mixtures. Tissue content of Cu in pakchoi grown on SC-soils could not be predicted by ΔEXCH. These correlation rates between Zn and Cu accumulation in pakchoi and variation of different fractions increased with time, which might indicate that sludges represented stronger impacts on the plant in long-term land application.     

Intraspecific differences in effects of co-contamination of cadmium and

A hydroponic experiment was carried out to study intraspecific differences in the effects of different concentrations of cadmium (Cd)(0-10 mg/L) and arsenate (As(V)) (0-8 mg/L) on the growth parameters and accumulation of Cd and As in six wheat varieties Jing-9428, Duokang-1, Jingdong-11, Jing-411, Jingdong-8 and Zhongmai-8. The endpoints of wheat seedlings, including seed germination,biomass, root length and shoot height, decreased with increasing the Cd and As concentrations. Significant differences in seed germination, biomass, root length, shoot height and the accumulation of Cd and As were observed between the treatments and among the varieties (p ＜ 0.05). The lethal dosage 50% were about 20, 80, 60, 60, 80 and 20 mg As/L for Jing-9428, Duokang-1, Jingdong-11,Jing-411, Jingdong-8 and Zhongmai-8, respectively, and the corresponding values for Cd were about 30, 80, 20, 40, 60 and 10 mg Cd/L, respectively. Among the six varieties, Duokang-1 was found to be the most resistant to Cd and As toxicity, and Zhongmai-8 was the most sensitive to Cd and As co-contamination. The resistance of the six varieties was found dependant on the seedling uptake of Cd and As. Duokang-1 was the most suitable for cultivation in Cd and As co-contaminated soils.     

Toxic effects of acetochlor, methamidophos and their combination on nifH gene in soil

Toxic effects of two agrochemicals on nifH gene in agricultural black soil were investigated using denaturing gradient gel electrophoresis （DGGE） and sequencing approaches in a microcosm experiment. Changes of soil nifH gene diversity and composition were examined following the application of acetochlor, methamidophos and their combination. Acetochlor reduced the nifH gene diversity （both in gene richness and diversity index values） and caused changes in the nifH gene composition. The effects of acetochlor on nifH gene were strengthened as the concentration of acetochlor increased. Cluster analysis of DGGE banding patterns showed that nifH gene composition which had been affected by low concentration of acetochlor （50 mg/kg） recovered firstly. Methamidophos reduced nifH gene richness that except at 4 weeks. The medium concentration of methamidophos （150 mg/kg） caused the most apparent changes in nifH gene diversity at the first week while the high concentration of methamidophos （250 mg/kg） produced prominent effects on nifH gene diversity in the following weeks. Cluster analysis showed that minimal changes of nifH gene composition were found at 1 week and maximal changes at 4 weeks. Toxic effects of acetochlor and methamidophos combination on nifH gene were also apparent. Different nifH genes （bands） responded differently to the impact of agrochemicals： four individual bands were eliminated by the application of the agrochemicals, five bands became predominant by the stimulation of the agrochemicals, and four bands showed strong resistance to the influence of the agrochemicals. Fifteen prominent bands were partially sequenced, yielding 15 different nifH sequences, which were used for phylogenetic reconstructions. All sequences were affiliated with the alpha- and beta-proteobacteria, showing higher similarity to eight different diazotrophic genera.     

Arsenic uptake by arbuscular mycorrhizal maize （Zea mays L.） grown in an arsenic-contaminated soil with added phosphorus

The effects of arbuscular mycorrhizal (AM) fungus (Glomus mosseae) and phosphorus (P) addition (100 mg/kg soil) on arsenic (As) uptake by maize plants (Zea mays L.) from an As-contaminated soil were examined in a glasshouse experiment.Non-mycorrhizal and zero-P addition controls were included.Plant biomass and concentrations and uptake of As,P,and other nutrients,AM colonization,root lengths,and hyphal length densities were determined.The results indicated that addition of P significantly inhibited root colonization and development of extraradical mycelium.Root length and dry weight both increased markedly with mycorrhizal colonization under the zero-P treatments,but shoot and root biomass of AM plants was depressed by P application.AM fungal inoculation decreased shoot As concentrations when no P was added,and shoot and root As concentrations of AM plants increased 2.6 and 1.4 times with P addition,respectively.Shoot and root uptake of P,Mn,Cu,and Zn increased,but shoot Fe uptake decreased by 44.6%,with inoculation, when P was added.P addition reduced shoot P,Fe,Mn,Cu,and Zn uptake of AM plants,but increased root Fe and Mn uptake of the nonmycorrhizal ones.AM colonization therefore appeared to enhance plant tolerance to As in low P soil,and have some potential for the phytostabilization of As-contaminated soil,however,P application may introduce additional environmental risk by increasing soil As mobility.     

Degradation of metabolites accumulated of benzo[a]pyrene by coupling Penicillium Chrysogenum with KMnO4

Several main metabolites of benzo[a]pyrene （BaP） formed by Penicillium chrysogenum, Benzo[a]pyrene-1,6-quinone （BP 1,6- quinone）, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene （BP 7,8-diol）, 3-hydroxybenzo[a]pyrene （3-OHBP）, were identified by high-performance liquid chromatography （HPLC）. The three metabolites were liable to be accumulated and were hardly further metabolized because of their toxicity to microorganisms. However, their further degradation was essential for the complete degradation of BaP. To enhance their degradation, two methods, degradation by coupling Penicillium chrysogenum with KMnO4 and degradation only by Penicillium chrysogenum, were compared; Meanwhile, the parameters of degradation in the superior method were optimized. The results showed that （1） the method of coupling Penicillium chrysogenum with KMnO4 was better and was the first method to be used in the degradation of BaP and its metabolites; （2） the metabolite, BP 1,6-quinone was the most liable to be accumulated in pure cultures; （3） the effect of degradation was the best when the concentration of KMnO4 in the cultures was 0.01% （w/v）, concentration of the three compounds was 5 mg/L and pH was 6.2. Based on the experimental results, a novel concept with regard to the bioremediation of BaP-contaminated environment was discussed, considering the influence on environmental toxicity of the accumulated metabolites.     

Impact of atrazine and nitrogen fertilizers on the sorption of chlorotoluron in soil and model sorbents

Sorption of chlorotoluron in ammonium sulfate, urea and atrazine multi-solutes system was investigated by batch experiments. The results showed application of nitrogen fertilizers to the soil could affect the behavior of chlorotoluron. At the same concentration of N, sorption of chlorotoluron decreased as the concentration of atrazine increased on the day 0 and 6 in soil, respectively. The sorption of chlorotoluron increased from 0 to 6 d when soils were preincubated with deionized water, ammonium sulfate and urea solution for 6 d. That indicated incubation time was one of the most important factors for the sorption of chlorotoluron in nitrogen fertilizers treatments. The individual sorption isotherms of chlorotoluron in rubbery polymer and silica were strictly linear in single solute system, but there were competition sorption between pesticides or between pesticides and nitrogen fertilizers. That indicated the sorption taken place by concurrent solid-phase dissolution mechanism and sorption on the interface of water-organic matter or water-mineral matter.     

Distribution and ecological effect of mercury in Laogang landfill, Shanghai, China

Laogang landfill near Shanghai is the largest landfill in China, and receives about 10000 t of daily garbage per day, Samples of topsoil and plants were analyzed to evaluate mercury pollution from the landfill. For topsoil samples, there were significant correlations among total mercury （HgT）, combinative mercury （Hgc） and gaseous mercury （HgG）, and content of total organic carbon （TOC）, but, no significantly relationship was found between Hg content and filling time. Hg content changes in vertical profiles with time showed that the average Hgv of profiles 1992, 1996, and 2000 was similar, but their average HgG was quite different. HgT was significantly correlated with Hgc in profile 1992 and 2000, and Hgv was significantly correlated with Hg6 in profile 1996. HgG/Hgv ratio in profile samples decreased in the order of （HgG,/HgT）1992〉（HgG/HgT）1996〉〉（HgG/HgT）2000. A simple outline of Hg release in landfill could be drawn： with increasing of filling time, degradation undergoes different biodegradation, accordingly, gaseous mercury goes through small, more, and small proportion to total mercury. Distribution of Hg in plants was inhomogeneous, following the order of leaf〉root〉stem. The highest value of leaf may be associated with higher atmospheric Hg from landfill. Ligneous plants （e.g. Phyllostachys glanca, Prunus salicina and Ligustrum lucidum） are capable of enriching more Hg than herbaceous plants.     

EDTA-enhanced phytoremediation of lead contaminated soil by Bidens maximowicziana

Phytoremediation is a potential cleanup technology for the removal of heavy metals from contaminated soils.Bidens maximowicziana is a new Pb hyperaccumulator,which not only has remarkable tolerance to Pb but also extraordinary accumulation capacity for Pb.The maximum Pb concentration was 1509.3 mg/kg in roots and 2164.7 mg/kg in overground tissues.The Pb distribution order in the B. maximowicziana was:leaf>stem>root.The effect of amendments on phytoremediation was also studied.The mobility of soil Pb and the Pb concentrations in plants were both increased by EDTA application.Compared with CK(control check),EDTA application promoted translocation of Pb to overground parts of the plant.The Pb concentrations in overground parts of plants was increased from 24.23-680.56 mg/kg to 29.07-1905.57 mg/kg.This research demonstrated that B.maximowicziana appeared to be suitable for phytoremediation of Pb contaminated soil,especially,combination with EDTA.     

Decomposition of alachlor by ozonation and its mechanism

Decomposition and corresponding mechanism of alachlor, an endocrine disruptor in water by ozonation were investigated. Results showed that alachlor could not be completely mineralized by ozone alone. Many intermediates and final products were formed during the process, including aromatic compounds, aliphatic carboxylic acids, and inorganic ions. In evoluting these products, some of them with weak polarity were qualitatively identified by GC-MS. The information of inorganic ions suggested that the dechlorination was the first and the fastest step in the ozonation of alachlor.     

Enzymatic oxidation of aqueous pentachlorophenol

The influence of the nonionic surfactant Tween 80 on pentachlorophenol （PCP） oxidation catalyzed by horseradish peroxidase was studied. The surfactant was tested at concentrations below and above its critical micelle concentration （CMC）. Enhancement of PCP removal was observed at sub-CMCs. The presence of Tween 80 in the reaction mixture reduced enzyme inactivation which occurred through a combination of free radical attack and sorption by precipitated products. A simple first-order model was able to simulate time profiles for enzyme inactivation in the presence or absence of Tween 80. At supra-CMCs, the surfactant caused noticeable reductions in PCP removal, presumably through micelle partitioning of PCP which precluded the hydrophobic PCP molecule from interacting with the enzyme.