Evaluation of the OECD activated sludge,respiration inhibition test

The OECD Activated Sludge, Respiration Inhibition Test was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured for a series of reactions set under identical conditions, and the variability in IC50 values estimated for three reference compounds. Several statistical procedures were also evaluated for analyzing activated sludge inhibition data. The test method was subsequently used to examine the effects of a wide variety of inorganic and organic chemicals on activated sludge respiration rates.     

活性污泥系统神经网络建模与仿真研究

采用神经网络技术对松江污水厂污水处理活性污泥系统进行建模试验研究，在对实际运行数据按机理准则和范围准则剔除异常数据后，将样本数据随机分成训练样本、检验样本和测试样本。用试凑法确定合理的神经网络隐层节点数，以避免采用过大或过小的网络结构，在训练过程中用检验样本实时监控从而避免“过训练”现象的影响，较好地解决神经网络方法建模的两大难题，从而建立可靠、有效的活性污泥系统神经网络模型。并应用建立的网络模型对活性污泥系统的运行情况进行了仿真研究。建模研究表明，神经网络技术能较好地应用于活性污泥系统的建模，模型具有较好的泛化能力，有很好的实用价值。     

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 effect of heavy metals on the activated sludge process and its microbial community analysis using 16S ribosomal DNA

The effect of heavy metal shock loading on biological treatment systems was studied by traditional methods and molecular biological techniques. Two kinds of SBR (sequence batch reactor) operation units, unacclimated and acclimated activated sludge systems, were studied. The addition of special nutrients and powdered activated carbon (PAC) to stimulate heavy metal uptake and recovery were studied. The kinetic constants could be used to describe the effect of the inhibition of substance utilisation. The results showed that heavy metal shock loading had a greater effect on the unacclimated activated sludge system than on the acclimated one. The special nutrients greatly enhanced the uptake of copper, and the PAC improved sludge settling and decreased the turbidity of the effluent. The variation of dominant species and the diversity of the bacterial community were analysed using 16S ribosomal DNA. Compared with the slight change of dominant species during acclimation by copper, there was a great change in the acclimated system shocked by a high concentration of copper. The results confirmed that the acclimation could improve the resistance of microorganisms to heavy metal toxicity.     

Degradability of aged aquatic suspensions of C60 nanoparticles

In this study, aged aqueous suspensions of C(60) (nC(60)) were investigated in the respirometric OECD test for ready biodegradability. Two suspensions of nC(60) were prepared by stirring and aged under indirect exposure to sunlight for 36 months. ATR-FTIR analyses confirmed the presence of C(60)-structures in the suspensions. Samples of the nC(60) suspensions (20mg/l) were inoculated with activated sludge (30 mgTSS/L) and incubated in a mineral medium under aerobic conditions. Since no mineralisation of nC(60) was observed after 28 days of incubation, 5mg/l sodium acetate was added to the media. After additional 20 days, no mineralisation of nC(60) was observed. However, within a few days sodium acetate was completely mineralised, showing that the biomass was not inhibited by the presence of nC(60). Based on results from this simple approach, aged nC(60) can be classified as not ready biodegradable according to the standard OECD test procedure.     

生物降解实验中的接种物标准化研究

针对降解实验中接种物采集困难、活性差异大、实验可重复性及结果可比性差的问题展开接种物标准化研究。对多来源的接种物在实验室可控条件下进行驯养,驯养后生长状态良好的活性污泥分别固定于灭菌麸皮和藻酸钙凝胶等2种微生物载体上,形成2种固定化接种物(分别简称为Bran及Ball)。对上述固定化接种物进行微生物活性、适应性、降解能力以及稳定性研究:通过2种固定化接种物与商品化接种物Polyseed对葡萄糖-谷氨酸(GGA)五日生化需氧量(BOD5)的结果与活菌细胞数的关系,确认3种接种物在降解实验中的用量可控制在相同数量级;比较不同批次及保存时间的2种固定化接种物、相同批次不同保存时间的Polyseed以及污水处理厂曝气池污泥为接种物对苯胺及二甘醇的快速生物降解实验(OECD301F)结果,发现Ball及Bran较Polyseed及新鲜采集活性污泥的微生物适应性及降解能力更佳。此外,在较长保存期内2种固定化接种物活性相对稳定。2种固定化接种物初具成为标准化接种物的必要条件。     

Comparison of bioluminescence and nitrification inhibition methods for assessing toxicity to municipal activated sludge

The aim of this study was to compare two alternative toxicity assessment methods to determine wastewater toxicity and predict treatment plant process upsets. The toxicity of two synthetic organic compounds (triclosan and 4-n-nonylphenol), which are commonly detected in municipal wastewater, and municipal and industrial wastewaters with different heavy metals content were evaluated by the nitrification inhibition assay and bioluminescence toxicity test. Comparison between both assays confirmed that Vibrio fischeri is generally more sensitive than autotrophic bacteria, and, if not calibrated, the bioluminescence method tends to overestimate toxic effects on activated sludge biomass. The nitrification inhibition assay appears to predict plant process upsets more accurately. Both methods showed a significant toxicity decrease through treatment that could be partially attributed to the significant heavy metals removal obtained by primary and secondary treatment. A good correlation for the two assays was obtained, as indicated by a high correlation coefficient (r2 = 0.80).     

Comparison of sewage sludge toxicity to plants and invertebrates in three different soils

Understanding the effect of soil type on the overall toxicity of sewage sludge is one of the most important issues concerning environmental risks associated with the sewage sludge land application. The aim of the study was to determine the influence of different soils (sandy, loamy and OECD soil) on sewage sludges toxicity in relation to plants (Lepidium sativum, Sorghum saccharatum, Sinapis alba) and an invertebrate species (Heterocypris incongruens). The most evident negative influence of sewage sludges on root growth was observed in the case of OECD soil. The EC(50) values determined on the basis of the root growth inhibition of all tested plants were in the range 0.1-6.4%, 0.03-9.4% and 6.6-22.1% (% of sewage sludgekg(-1) soil) for OECD, sandy and loamy soil, respectively. Soil type also affects the sewage sludge toxicity in relation to H. incongruens. The LC(50) (mortality) values ranged from 0.26% to 11.5% depending on the sludge tested. For EC(50) (growth inhibition) values ranged from 10.7% to 36.2%.     

Incomplete aerobic degradation of the antidiabetic drug Metformin and identification of the bacterial dead-end transformation product Guanylurea

Active pharmaceutical ingredients as well as personal care products are detected in increasing prevalence in different environmental compartments such as surface water, groundwater and soil. Still little is known about the environmental fate of these substances. The type II antidiabetic drug Metformin has already been detected in different surface waters worldwide, but concentrations were significantly lower than the corresponding predicted environmental concentration (PEC). In human and mammal metabolism so far no metabolites of Metformin have been identified, so the expected environmental concentrations should be very high.To assess the aerobic biodegradability of Metformin and the possible formation of degradation products, three Organisation of Economic Cooperation and Development (OECD) test series were performed in the present study.In the Closed Bottle test (OECD 301 D), a screening test that simulates the conditions of an environmental surface water compartment, Metformin was classified as not readily biodegradable (no biodegradation). In the Manometric Respiratory test (OEDC 301 F) working with high bacterial density, Metformin was biodegraded in one of three test bottles to 48.7% and in the toxicity control bottle to 57.5%. In the Zahn-Wellens test (OECD 302 B) using activated sludge, Metformin was biodegraded in both test vessels to an extent of 51.3% and 49.9%, respectively.Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass spectrometry (HPLC-MS(n)) showed in the tests vessels were biodegradation was observed full elimination of Metformin and revealed Guanylurea (Amidinourea, Dicyandiamidine) as single and stable aerobic bacterial degradation product. In another Manometric Respiratory test Guanylurea showed no more transformation. Photodegradation of Guanylurea was also negative.A first screening in one of the greatest sewage treatment plant in southern Germany found Metformin with high concentrations (56.8 μg L⁻¹) in the influent (PEC = 79.8 μg L⁻¹), but effluent concentration was much lower (0.76 μg L⁻¹) whereas Guanylurea was detected in a low influent and high effluent concentration (1.86 μg L⁻¹). These data support the experimental findings in the OECD tests and analytical results of other studies, that Metformin under aerobic conditions can bacterially be degraded to the stable dead-end transformation product Guanylurea.     

Evaluation of the toxicity of substances to be assessed for biodegradability

The ability to distinguish between true non-biodegradability, and inhibition of biodegradability due to the toxic effects of the compound tested present problems. It results in possible false identification of substances as non-biodegradable.Six chemicals were evaluated in a variety of tests to assess their toxicity to aerobic sludge organisms. The tests employed were: BOD₅ and Closed Bottle inhibition tests, inhibition of respiration of activated sludge, growth inhibition of activated sludge, light emission from (MICROTOX test) and the repetitive die-away test (RDA). Results from these tests were compared with results obtained from a number of ready biodegradability tests using the compounds listed above at anticipated non-toxic and toxic concentrations.No test method evaluated consistently forecast toxicity due to the chemicals tested. The MICROTOX and nitrification inhibition tests were too sensitive. A combination of sludge respiration rate and/or growth tests seem most appropriate. To avoid toxicity to the sludge, biodegradability testing should be made at 10% of the EC₅₀ value. Compounds with an EC₅₀ value greater than 300 mg/l are unlikely to be toxic in ready biodegradability tests. Compounds with an EC₅₀ value of less than 20 mg/l may pose problems necessitating the use of the stringent closed bottle test, or the use of C¹⁴ labelled test materials. Compounds with an intermediate EC₅₀ that is between 20–300 mg/l, need to be evaluated at a range of concentrations in biodegradability tests, or may need to be evaluated carefully to define the precise no-effect level. Procedure recommendations to distinguish between the inhibition of biodegradability and inertness of the test substance are made.     

菌源对多环芳烃降解菌的筛选及降解性能的影响

高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。分别以石油污染土壤和焦化废水活性污泥为菌源,分离出芘降解菌和混合PAHs(菲、荧蒽和芘)降解菌共14株并对其降解性能进行对比研究。结果表明,筛选得到的菌株分别属于9个菌属,其中2种菌源共有的菌属为Mycobacterium sp.、Ralstonia sp.和Shinella sp.。芘和PAHs的高效降解菌(CP16和CM32)均属于分支杆菌属(Mycobacterium),来源于焦化废水活性污泥;菌株CP16对芘(50mg/L)的7 d降解率为74.99%,CM32对PAHs(菲50 mg/L、荧蒽和芘各10 mg/L)的7 d降解率为100%。因此,以焦化废水活性污泥为菌源更有利于获得高效的多环芳烃降解菌。     

Model development and application for surfactant biodegradation in an acclimatising activated sludge system

A model for the biodegradation of non-ionic surfactants in an activated sludge system during acclimatisation was developed, based on respirometric and titrimetric experimental data. The data were obtained in a sequencing batch reactor (SBR) using a non-ionic surfactant as sole carbon source and sludge previously acclimatised to a different surfactant. The model was successfully applied to successive SBR-cycles of sludge acclimatisation processes subjected to two ethoxylated surfactants. The model was validated using the corresponding total organic carbon data. The evolution of the model parameters along the acclimatisation process was examined. An acclimatisation model was developed using the evolution of two of these parameters (affinity constant and inhibition constant), supported by two independently calculated acclimatisation indicators. This acclimatisation model was then applied to determine an optimal surfactant concentration sequence to feed non-acclimatised sludge during a period of 41 days, in order to induce pre-acclimatisation to this surfactant before it replaces another one in the wastewater. The model was also useful in the economical assessment of this and alternative procedures to cope with frequent changes in activated sludge feed composition.     

抗生素类污染物对活性污泥生物毒性测定方法的筛选与评价

尽管衡量化学物质生物毒性的标准方法很多,但关于环境中抗生素类污染物的生物毒性及其对污泥活性影响的科学数据较少。以磺胺和四环素两类作用机理和作用谱带不同的抗生素为研究对象,分别用发光细菌法、脱氢酶活性法、生长抑制法及呼吸速率法进行了抗生素类污染物生物毒性测定方法的筛选与评价。结果表明,发光细菌标准方法中30min的作用时间太短,延长作用时间不仅导致各种抗生素的EC50值大幅度降低,同时毒性排列顺序也发生了改变;以活性污泥为对象的脱氢酶活性和呼吸速率抑制实验的EC50值与抗生素类物质对敏感致病菌的MICs相比异常高,不适宜于作为单独方法准确评估抗生素类污染物的生物毒性。生长抑制实验中,活性污泥混合菌种增殖生长对磺胺类抗生素敏感,而假单胞杆菌对四环素类抗生素敏感。不同方法测定抗生素毒性的灵敏度顺序是发光细菌(24 h)>生长抑制(7 h)>呼吸速率(24 h)>脱氢酶活性(24 h)。用标准方法评价抗生素类污染物的生物毒性,可能导致对抗生素排放到水环境中所带来的风险估计不足。     

Automated color correction method for Vibrio fischeri toxicity test. Comparison of standard and kinetic assays.

We demonstrate in this study that the toxicity of solid and highly colorful samples can be measured with kinetic bioassay using luminescent bacterium Vibrio fischeri. The Flash assay, named after the test protocol, is performed with a tube luminometer. In this method, each sample acts as a reference for itself, and therefore, the color correction is possible with minimal hands-on-time. The bacteria are dispensed into the sample and the signal is recorded continuously. The maximum signal received after immediately dispensing is compared to the signal after an incubation period. With many chemicals, the toxic effects are obtained after a very short contact time. However, different chemicals have different modes of toxicity. Thus, kinetic data from sample analyses after 15 or 30 min for this bacterium gives an additional dimension for obtaining reliable results. The performance of the test was compared to the standardized photobacteria test protocol with reference chemicals. The repeatability of the test was excellent. The coefficient of variation was normally below 1% with 10 replicates.     

Development of a three-stage system for wastewater toxicity monitoring: a design and feasibility study.

A three-stage system was developed to automate a batchwise toxicity testing protocol designed for assessing wastewater toxicity to activated sludge. The three-stage system used the luminescent bacterium Shkl. The three stages were cell storage, cell activation, and continuous toxicity testing. Shkl cells were stored in a bioreactor at 4 degrees C when the system was not in use and activated in another bioreactor for use in toxicity tests conducted in a continuous manner. The system could quickly be switched between the "off" and "on" modes, and operation of the system was easy. The stability of the system, in terms of cell density and bioluminescence in the storage and activation bioreactors, and the response of the activated cells to a metal and an organic toxicant were studied. The feasibility of the system design was demonstrated by simulating zinc toxicity episodes in synthetic wastewater. The needs for further modifications and improvements of the system were discussed.     

絮凝剂对高速厌氧反应器污泥颗粒化的强化作用

以考察絮凝剂对厌氧反应器中活性污泥性能的影响为目的,将聚合氯化铝(PAC)、聚丙稀酰胺(PAM)、聚季铵盐3种不同类型絮凝剂以不同投加量添加到反应器中,采用生物化学甲烷势(BMP)和厌氧毒性测定(ATA)分析方法,评价了3种絮凝剂的厌氧生物可降解性以及3种絮凝剂对厌氧污泥产甲烷活性、沉降性能等方面的促进或抑制作用。确定以聚季铵盐作为厌氧污泥颗粒化促进剂,建议采用多次投加方式,反应器中聚季铵盐质量浓度以10-50mg／L为宜。     

Acute toxicity of chlorophenols to earthworms using a simple paper contact method and comparison with toxicities to fresh water organisms

An acute toxicity test of chlorophenols on earthworms (Eisenia fetida) was performed using a simple paper contact method proposed by OECD testing guideline no. 207, that were applied as an earthworm toxicity test. The median lethal concentration, EC50, had significant correlation with logP(ow) (1-octanol/water partition coefficient) of the chemicals. The toxicity of chlorophenols on E. fetida was compared with toxicities for other species: an algae (Selenostrum capricornutum), a crustacean (Daphnia magna), and a fish (Oryzias latipes). It was found that the toxicity of chlorophenols was almost same for E. fetida and for fresh water organisms. These results suggest the possibility of drawing correlations between the effects of pollutants on living things in different environments, fresh water and soil.     

Biodegradability of the X-ray contrast compound diatrizoic acid, identification of aerobic degradation products and effects against sewage sludge micro-organisms

Pharmaceuticals and contrast media have been detected in hospital effluents, sewage treatment plants, surface water, and ground water. Only little is known about their elimination during sewage treatment and effects of possible biotransformation products against bacteria. The modified Zahn-Wellens test (ZWT, OECD 302 B) and a test simulating biological sewage treatment (modified OECD 303 A test) were used to assess the biodegradability of the widely used ionic iodinated contrast agent diatrizoic acid (diatrizoate). Effects against sewage sludge bacteria were studied in the two test systems by monitoring the biomarkers quinones, polyamines, phospholipids and adenosine triphosphate. Diatrizoate was biotransformed into 2,4,6-triiodo-3,5-diamino-benzoic acid in the ZWT. 2,4,6-Triiodo-3,5-diamino-benzoic acid was stable under the test conditions of the ZWT. Diatrizoate was not eliminated in the OECD 303 A simulation test. It was not adsorbed by the sewage sludge. No effects of the test compound or its aerobic transformation products against the bacteria present in the sewage sludge were detected using phospholipids, quinones, polyamines, and adenosine triphosphate as biomarkers.     

Effects of different methods of DNA extraction for activated sludge on the subsequent analysis of bacterial community profiles

The effect of different DNA extraction protocols on activated sludge DNA yield and bacterial community composition was evaluated by temperature gradient gel electrophoresis (TGGE). Nine different procedures to extract DNA were compared-sonication (30s), sonication (40s), sonication (50s), freezing-thawing, bead milling, sodium dodecyl sulfate (SDS)-lysozyme, SDS-proteinase K, SDS-lysozyme-proteinase, and a commercial extraction kit. It was found that the TGGE profiles and the DNA band numbers made significant differences via various extraction methods. The yield and purity of DNA extracted by sonication and other physical methods were not satisfactory, while the DNA purity extracted by SDS and other chemical-biological methods were better. Crude DNA extracts isolated by sonication and other physical methods passed the polymerase chain reaction, despite the absence of purification and acquired affluent DNA bands in TGGE. The affluence of bands in TGGE was not consistent with the yield and purification of DNA, but was correlative with extraction protocols. To analyze the activated sludge bacterial community by TGGE fingerprint, it is necessary to make a synthesis of the TGGE fingerprint profiles of chemical and physical DNA extraction methods to overcome the representative bias.     

Reduction of toxicity of antimicrobial compounds by degradation processes using activated sludge,gamma radiation,and UV

The occurrence and persistence of pharmacologically active compounds in the environment has been an increasingly important issue. The objectives of this study were to investigate the decomposition of aqueous antimicrobial compounds using activated sludge, γ-irradiation, and UV treatment, and to evaluate the toxicity towards green algae, Pseudokirchneriella subcapitata, before and after treatment. Tetracycline (TCN), lincomycin (LMC) and sulfamethazine (SMZ) were used as target compounds. Gamma (γ)-irradiation showed the highest removal efficiency for all target compounds, while UV and activated sludge treatment showed compound-dependent removal efficiencies. TCN and SMZ were well degraded by all three treatment methods. However, LMC showed extremely low removal efficiency for UV and activated sludge treatments. Overall, the algal toxicity after degradation processes was significantly decreased, and was closely correlated to removal efficiency. However, in the case of γ-irradiated TCN, UV and activated sludge treated LMC as well as sludge treated SMZ, the observed toxicity was higher than expected, which indicates the substantial generation of byproducts or transformed compounds of a greater toxicity in the treated sample. Consequently, γ-radiation treatment could be an effective method for removal of recalcitrant compounds such as antibiotics.