)]发光细菌法在水质综合毒性在线检测中的应用

以海洋发光细菌费氏弧菌(Vibrio fischeri)作为检测生物,采用冻干菌粉快速复苏技术,研究费氏弧菌在水质检测中的最佳测试温度和有效性测试条件,并对硫酸锌等多种毒物和几种实际水样进行发光抑制作用分析。研究表明,费氏弧菌冻干粉复苏菌液保存在2~5℃条件下能有效测试7 d,最佳测试温度为15℃,最佳测试时间为15 min。氯化汞、硫酸锌、硫酸镉等重金属和苯胺、多菌灵、甲醛等有机毒物对费氏弧菌均具有较强的光抑制作用,也即费氏弧菌对以上毒物较为敏感,并能够连续7 d保持对同一浓度硫酸锌的敏感性较为一致。对几种实际水样的测试和分析表明,以费氏弧菌为指示生物的发光细菌法能够应用于水质环境安全的综合毒性在线监测预警中。     

发光细菌冷冻干燥条件优化研究与应用

发光细菌用于水质毒性快速检测时,使用冷冻干燥制剂复苏发光细菌非常必要。配制冷冻干燥保护剂时,采用较低浓度脱脂牛奶(质量分数分别为4%、6%、8%、10%、12%)以及NaCl溶液(质量分数分别为0.5%、1.0%、1.5%、2.0%、2.5%、3.0%)进行双因素多梯度的实验,确定发光细菌的最佳冷冻干燥保护剂配方为12%脱脂牛奶以及2.0%NaCl溶液。采用复苏后的发光细菌,检测实际水样的毒性,结果与工业废水毒性评价结果相符。     

发光细菌急性毒性测试方法的优化研究

针对国内现行发光细菌急性毒性标准测试方法中存在的结果重现性不佳、准确度不高等不足,在充分借鉴国际标准化组织ISO11348-3:2007的基础上,通过对实验条件和操作步骤的优化改良,并在数据处理过程中新增了发光自然变化因子(fk)和原始发光光强(S0)2个参数,进一步减少了菌种发光稳定性差异和手工加样所带来的误差。此外,选用费氏弧菌NRRLB—11177、青海弧菌Q67、明亮发光杆菌502这3种不同来源的发光细菌对该优化方法进行了适用性实验。结果表明,在一定的发光强度自然变化范围内,明亮发光杆菌502的实验结果(线性相关系数等)均明显优于其传统推荐方法。同时,根据上述实验结果,还对《水质急性毒性的测定发光细菌法》(GB/T15441—1995)的修订工作提出了一系列参考意见。     

11种农药对淡水发光细菌青海弧菌Q67的毒性研究

以淡水发光细菌青海弧菌Q67作为测试菌剂,利用水质毒性分析仪对11种农药进行了毒性研究,获得11种农药与青海弧菌的剂量-效应关系.结果表明,作用时间为15 min时,5种可溶农药对青海弧菌Q67的毒性大小为敌敌畏＞敌百虫＞杀虫单＞乐果＞乙酰甲胺磷,6种难溶于水农药的毒性大小为甲氨基阿维菌素＞甲胺磷＞莎稗磷＞高效氯氰菊酯...     

毒性微生物传感器的研制及应用

以从土壤中分离、筛选出的X4菌株作为分子识别元件,采用夹层法制备微生物膜并与极谱型氧电极组成了呼吸抑制型毒性微生物传感器。实验确定了传感器的最佳工作条件:温度为30℃,pH范围为7.0~7.5;以HgCl2作为参比毒物时,微生物传感器标准曲线为y=1.05 x+0.21,线性相关系数为0.9933,线性范围为0.1~1.97 mg/L,相对标准偏差为1.8%。用该微生物传感器对某印染废水的毒性进行测试,该废水的毒性相对于HgCl2的浓度为1.69 mg/L,属于剧毒,与发光细菌法进行比对,测定结果具有一致性。     

氧化锌吸收-空气氧化法烟气脱硫实验研究

采用次氧化锌配制成的悬浮浆液脱除吸收模拟烟气中的SO2，吸收产物亚硫酸锌浆液再通过鼓风湿式氧化成硫酸锌溶液，以验证氧化锌吸收-空气氧化法烟气脱硫的效果。通过改变气体流量、SO2浓度、浆液温度及pH值等工艺参数，探索吸收和氧化过程的最佳控制条件。结果表明，氧化锌浆液对含SO2浓度从5800～8600mg／m^3的烟气脱除效率均可达到90％以上，吸收容量为3．9gSO2／15g次氧化锌；氧化过程控制吸收终点浆液pH值为5．0以及浆液温度在38℃．氧化率可达95％。     

BOD微生物传感检测仪中高效微生物膜的研究

为了制备高效微生物膜，从污水中筛选菌种（QT2061），经BIOLOG鉴定为枯草芽孢杆菌。并以聚乙烯醇为包埋剂制作BOD生物传感器微生物膜，通过对国家标准样品及几种典型水样的检测，结果表明，该微生物传感器的响应时间为8 min，可连续稳定测试10 d以上；对国家标准溶液的测试结果准确度较高，相对误差≤5%；对实验中几种水样均有响应，测试结果与5日法的测试结果有良好的相关性（相对偏差≤5%）。     

氧化锌吸收-空气氧化法烟气脱硫实验研究

采用次氧化锌配制成的悬浮浆液脱除吸收模拟烟气中的SO2,吸收产物亚硫酸锌浆液再通过鼓风湿式氧化成硫酸锌溶液,以验证氧化锌吸收空气氧化法烟气脱硫的效果。通过改变气体流量、SO2浓度、浆液温度及pH值等工艺参数,探索吸收和氧化过程的最佳控制条件。结果表明,氧化锌浆液对含SO2浓度从5800～8600mg/m3的烟气脱除效率均可达到90%以上,吸收容量为39gSO2/15g次氧化锌;氧化过程控制吸收终点浆液pH值为50以及浆液温度在38℃,氧化率可达95%。     

液液萃取—气相色谱法同时测定地下水中16种有机氯农药

地下水是水资源的重要组成部分,地下水中有机氯农药(OCPs)的测定十分必要。通过优化衬管、进样口温度、载气流速、升温程序和检测器温度等条件,建立了一种液液萃取—气相色谱法同时测定地下水中16种OCPs的方法。结果表明,16种OCPs可以在27min内完全分离,在1~60μg/L线性关系良好(R20.995 0),检出限低至0.6~1.5ng/L,回收率达到80.1%~109.0%,相对标准偏差为2.1%~11.2%,能够满足实际地下水水样的测定。其中最佳色谱条件为选用4mm内径中空超高惰性不分流衬管;进样口温度250℃;载气流速0.65mL/min;升温程序:从120℃开始以10℃/min升至220℃,保持16min,继续以10℃/min升至250℃,保持2min;检测器温度320℃。     

粉煤灰/聚合硫酸铁复合材料处理炼钢连铸冷却喷淋水

炼钢连铸冷却喷淋水中的高含油量易形成黏性油泥团,堵塞管道和设备,导致处理后的水难以达到回用水质的要求,除油降浊是其处理稳定达标的关键环节。为了寻求对连铸含油冷却喷淋水处理工艺简单、投资少、费用低、效率高的絮凝/吸附处理工艺及方法,实验制备了粉煤灰/聚合硫酸铁复合材料(PFA),表征了其结构、形貌,研究了其除油降浊的最佳pH值、反应温度、最佳配比、最佳投加量等,并从处理效率、投药成本、除浊率等方面考虑,确定最佳运行条件:pH为7~8,反应温度为50℃,搅拌300 r·min~(-1)左右,反应3 min,静置10 min,粉煤灰/聚合硫酸铁的配比为100∶8~100∶18,投加量为80~220 mg·L~(-1)。处理后水样含油浓度为低于5 mg·L~(-1)、循环水处理成本低于0.1元·(t水)-1,除浊率为89.5%,满足实际生产使用要求,达到炼钢废水循环利用的目的。     

A comparison of five rapid direct toxicity assessment methods to determine toxicity of pollutants to activated sludge

Five rapid direct toxicity assessment methods were used in three European partner countries to determine the toxicity of single toxicants, mixed toxicants and real industrial wastes. The final aim was to protect microbial degradation of organic wastes in biological treatment processes and hence enhance the quality of treated effluents to be discharged to the environment. Nitrification inhibition, Respirometry, Adenosine triphosphate luminescence and Enzyme inhibition were tested utilising activated sludge as the testing matrix. The Vibrio fischeri toxicity test was used as a surrogate to compare the various microbial bioassays. The IC50 (toxicant concentration eliciting a 50% inhibitory effect) was determined for a number of pollutants including single toxicants Cd, Cr, Cu, Zn, 3,5-dichlorophenol, toluene and linear alkylbenzenesulphonate (LAS); a standard mixture of metals and LAS; a standard mixture of organics and LAS, and 16 industrial effluents. The V. fischeri bioassay was also chosen in order to assess quality control of toxicant preparation during testing in the different laboratories of the partner countries. Comparisons of sensitivity, cost of implementation, cost per test, relevance, and ease of use were made. The most sensitive bioassays were V. fischeri and Nitrification inhibition, however, this depended in the main on the pollutant and mixtures tested. It is recommended that during assessment of wastewater toxicity a suite of tests be used rather than reliance on one particular test.     

Ecotoxicological testing with new kinetic Photorhabdus luminescens growth and luminescence inhibition assays in microtitration scale

Miniaturized luminescence and growth inhibition assays in microtitration plates with the terrestric enthomopathogenic nematode symbiont Photorhabdus luminescens (DSMZ 3368T) are presented and compared with standardized tests with Vibrio fischeri (DSMZ 7151/NRRLB-11177) and Pseudomonas putida (DSMZ 50026). Toxicological parameters (EC and G values) of selected reference toxicants (e.g. heavy metals and environmental samples) were obtained at different temperatures and without sodium chloride supplementation. Kinetic data recordings were compared with results of a cuvette test protocol using integral and endpoint calculation. Growth inhibition experiments with reference samples reveal similar or higher sensitivities as for the Vibrio or Pseudomonas spp. The luminescence inhibition assay shows reduced sensitivities to most of the reference samples compared with the V. fischeri standard assay. But G values obtained with other standardized aquatic assay systems with daphnids, algae and growth inhibition assays with V. fischeri and Ps. putida correspond more closely to data observed with Ph. luminescens. The test procedures are easily to perform and to evaluate and seem to be reliable alternatives to the established protocols at low osmolarities. The sample specifity of the toxic responses of the marine and the terrestric strain recommends to employ both assays to determine the toxic potential of environmental samples. This will support to reduce false positive results in future investigations.     

Toxicity of metals and organic chemicals evaluated with bioluminescence assays

The development of a bioluminescent sensor organism (Shk1) that was created for assessing wastewater toxicity was reported several years ago. In order to establish a test battery to better characterize wastewater toxicity, additional luminescent sensor organisms were later created. The present study focused on one promising candidate (PM6), a Pseudomonas spp. strain, because of its high level of luminescence compared to that of other newly created organisms. Using a batch toxicity testing protocol, the toxicity of 7 metals and 25 organic compounds was evaluated with the PM6 and Shk1 assays. Results indicated that the toxicity data of the PM6 and the Shk1 assays were correlated, and no assay appeared to be particularly more sensitive to a group of toxicants than the other assay. The results of the PM6 and Shk1 assays were further evaluated by comparing with the results of the Vibrio fischeri luminescence inhibition assay and activated sludge inhibition assays. Data suggested that PM6 and Shk1 more closely represented activated sludge organisms than V. fischeri. The suitability of using PM6 and Shk1 for assessing wastewater toxicity on activated sludge, both individually and in a test battery, was discussed.     

The toxicity of antibiotic agents to the luminescent bacterium Vibrio fischeri.

Despite their common use the fate and effects of antibiotics in the environment are largely unknown. These compounds may enter the environment through different pathways, resulting in the contamination of waste water or fresh water, where bacteria are most likely the primarily affected organisms. In this paper the toxicity of several drugs, reflecting the most important groups of antibiotics and chemotherapeutics, towards Vibrio fischeri are presented. The chronic bioluminescence inhibition assay with Vibrio fischeri is shown to be sensitive against many of the high volume antibiotics used for veterinary purposes and in aquaculture. Thus the assay may be a valuable tool for an effects assessment and biomonitoring of these xenobiotics. The available data for both parts of the risk assessment procedure--exposure assessment and effects assessment--have to be regarded as insufficient for most antibiotics. When the available data about environmental concentrations of antibiotics are compared with their toxicity towards Vibrio fischeri, direct effects on natural microbial communities are to be expected.     

Toxicity testing with luminescent bacteria – Characterization of an automated method for the combined assessment of acute and chronic effects

The luminescent bacteria test according to EN ISO 11348 is frequently applied in (eco) toxicity testing and is applicable for a huge variety of environmental and industrial samples. A big disadvantage of this method is the very short exposure time, which is expressed in a low sensitivity in regard to substances with a delayed effect. Chronic effects, i.e. interference with cell growth, cannot be assessed with this conventional standard method. The goal of this research was to develop an automated testing system for long term toxicity towards the luminescent bacteria Vibrio fischeri by implementing microtitration-based instrumentation. The optimized method, hereinafter referred to as “kinetic luminescent bacteria test”, can be described as a miniaturized combination of the conventional short-term luminescence inhibition test according to EN ISO 11348 and the Photobacterium phosphoreum growth inhibition test (DIN 38412-37). The validation procedure included the evaluation of six reference compounds (3,4-Dichloroaniline, 3,5-Dichlorophenol, Chloramphenicol, Streptomycin sulfate, Potassium dichromate, Zinc sulfate heptahydrate) and three different endpoints that are acute luminescence inhibition (acute LI) after 30 min, chronic luminescence inhibition (chronic LI) after 24 h and growth inhibition (GI) after 14 h. The optimized method allows the assessment of acute and chronic effects within one test, by what a misinterpretation of the toxicity of substances with delayed bacterial toxicity can be prevented, without abandoning most of the advantages of the conventional short-term test. Therefore, the kinetic luminescent bacteria test is exceptional as an initial screening test for environmental samples or substances with unknown (eco) toxicological characteristics.     

Applicability of the bioluminescence inhibition test in the 96-well microplate format for PAH-solutions and elutriates of PAH-contaminated soils

The use of conventional plastic microplates for a miniaturised luminescent bacteria test may result in an underestimation of the toxicity for poorly water soluble highly adsorbing toxicants such as PAHs. In this study, the suitability of microplates for testing elutriates of PAH-contaminated soils was investigated. The LUMIStox test was performed as the standard test in the miniaturised format using contaminated soil elutriates and aqueous solutions of four selected PAHs (viz. naphthalene (NAP), acenaphthene (ACE), fluorene (FLU), and phenanthrene (PHE)). For the aqueous PAH-solutions, we observed reduced light inhibition values for the miniaturised bioassay when using black microplates made of polypropylene (PP) and polystyrene (PS) compared to the standard LUMIStox test. This phenomenon was most likely due to adsorption of toxicants to the microplate surfaces with PAHs of lower water solubility being significantly more affected; however, after minimizing the exposure of samples to plastic surfaces, polystyrene microplates revealed equivalent performance (>80% 'relative' light inhibition) to the standard glass cuvette test system. For soil elutriates, black microplates again exhibited slightly lower light inhibition values while white plates made of PS and Barex resulted in a pronounced overestimation of toxicity for a coloured soil elutriate. In general, microplates were applicable for testing elutriates of PAH-contaminated soils. In cases where samples are coloured or turbid, the application of black microplates is recommended.     

Removal of PAHs from laboratory columns simulating the humus upper layer of vertical flow constructed wetlands

Removal of three polycyclic aromatic hydrocarbons or PAHs (fluoranthene, pyrene and benzo(k)fluoranthene) from two types of PAH-contaminated effluents was investigated using four laboratory columns filled with two different organic media: a green compost and a layer coming from the first stage of vertical flow constructed wetlands. Synthetic runoff polluted by polycyclic aromatic hydrocarbons were fed through the columns during a period of two months. After a period of hydrodynamic stabilisation, the results showed a great adsorption of PAHs (>95%) on the solid media due to their large adsorption capacities. Leaching of these compounds by water was monitored. The concentrations of PAHs in leaching samples indicated that PAHs were strongly adsorbed on organic substrates and that lixiviation was limited. Fluoranthene metabolites were also investigated. Accumulation of metabolites was transitory and located in the first few cm of the media, as was observed for PAH concentrations. A toxicity test of leachates based on the inhibition of the bioluminescence of luminescent bacteria Vibrio fischeri indicated a low inhibition which can be enhanced by metal traces.     

Optimisation of a microbial bioassay for contaminated soil monitoring: bacterial inoculum standardisation and comparison with Microtox assay

This work represents the first step to set up a toxicity testing procedure and to evaluate the sensitivity of the test microorganism to several classes of environmental pollutants. First, three different techniques were employed to standardise the microbial inoculum, then two different toxicity assessment protocols have been compared: Microtox and a dehydrogenase (DHase) activity inhibition test. The main goal was the optimisation of a microbial bioassay based on the dehydrogenase activity (DHase) inhibition in Pseudomonas fluorescens bacterial strain ATCC 13525. Triphenyl tetrazolium chloride (TTC) was used as electron acceptor and its reduction produces Triphenyl formazane (TPF). The P. fluorescens DHase inhibition bioassay was investigated for being a reliable and rapid method for assessing toxicity. The optimisation of the operating conditions resulted in a repeatable bioassay. Then, P. fluorescens and Vibrio fischeri sensitivity were firstly compared by testing Zn++, one of the reference compounds for Microtox test. In addition, other compounds (Ni++, Cd++, Cu++, phenol) were also tested with both bioassays. A high statistical significance of data was obtained with the logistic curve. The present work has demonstrated that P. fluorescens is as sensitive as Microtox culture (V. fischeri), for some of the metal ions. With reference to organic compounds, the lower sensitivity of P. fluorescens to phenol makes its use difficult in organic polluted samples.     

Cr(VI) reduction into Cr(III) as a mechanism to explain the low sensitivity of Vibrio fischeri bioassay to detect chromium pollution

Vibrio fischeri bacteria, used as a biological target in either acute or chronic toxicity tests, display a low sensitivity to Cr(VI). This phenomenon could be due to the capacity of these bacteria to reduce Cr(VI) into Cr(III). This reducing capacity was found to depend on culture medium composition, pH value, incubation time and the presence of a carbon source. It also depends on the nature of the carbon source, glucose being more efficient than glycerol. This is probably related to differences in bacterial metabolism when given either glucose or glycerol. The thermostable Cr(VI)-reducing activity found in the supernatants of V. fischeri cultures grown on glucose suggests that, under these conditions, the bacteria release non-proteic reducing substances which have not been identified yet.     

Comparative toxic and genotoxic effects of chloroacetanilides,formamidines and their degradation products on Vibrio fischeri and Chironomus riparius

Toxic and genotoxic effects of alachlor, metolachlor, amitraz, chlordimeform, their respective environmentally stable degradation products 2,6-diethylaniline, 2-ethyl-4-methylaniline, 2,4-dimethylaniline, and two other related compounds, 3,4-dichloroaniline and aniline were compared. Acute toxicity tests with Chironomus riparius (96 h) and Vibrio fischeri (Microtox) and genotoxicity tests with a dark mutant of V. fischeri (Mutato) were carried out. Our results demonstrate that toxicity and genotoxicity of the pesticides are retained upon degradation to their alkyl-aniline metabolites. In the case of the herbicides alachlor and metolachlor, the toxicity to V. fischeri was enhanced upon degradation. Narcosis alone explains toxicity of the compounds to the midge, but not so for the bacteria suggesting a disparity in the selectivity of the test systems. All compounds showed direct genotoxicity in the Vibrio test. but amitraz and its metabolite were genotoxic at concentrations 10(3)-10(5) lower than all the other compounds. The observations indicate that stable aniline degradation products of the pesticides may contribute considerably to environmental risks of pesticides application and that genotoxic effects may arise upon degradation of pesticides.