Quantification and genotyping of Cryptosporidium spp. in river water by quenching probe PCR and denaturing gradient gel electrophoresis.

A new detection method was developed for the simultaneous quantification and genotyping of Cryptosporidium spp. in river water. Several modifications made to the US EPA Method 1623 enabled high and stable recovery of Cryptosporidium from 40 L of river water (geometric mean = 35%, standard deviation = 8.7%). Quenching probe PCR (QProbe PCR) was used to quantify the 18S rRNA gene of Cryptosporidium spp. This method could successfully detect single oocysts in a sample, and the lower quantitation limit was as low as 2.5 oocysts/sample. In addition, denaturing gradient gel electrophoresis (DGGE) followed by DNA sequencing was used to identify the genotypes. These methods were applied to detect Cryptosporidium spp. in the Koyama River, Japan. The positive ratio was 69% (11/16) with the maximum concentration of 59 oocysts/100 L. Seven genotypes including two novel ones were identified. These results showed that this detection method could provide valuable information on Cryptosporidium in river water, both in the concentration and in the genotypes, which is essential for the precise assessment of waterborne risk to human health.     

Inactivation of enteric microbes in water by electro-chemical oxidant from brine (NaCl) and free chlorine.

Oxidant solutions of mostly free chlorine can be electrochemically produced on-site from brine (NaCl) solution and used to disinfect water at the household or community level. In this study electrochemical oxidant (ECO) from brine and free chlorine were evaluated under laboratory conditions for inactivation of test microbes. Purified suspensions of Escherichia coli, the rugose strain of Vibrio cholerae, Clostridium perfringens spores, MS2 coliphage and Cryptosporidium parvum oocysts were treated with 2 mg/L or 5 mg/L solutions of ECO or free chlorine at 5 degrees C and 25 degrees C and pH 6, 8, and 10 (pH 7 and 25 degrees C only for C. parvum oocysts) for contact times <60 min. Under nearly all conditions, inactivation kinetics were more rapid for E. coli, V. cholerae, C. perfringens spores and MS2 coliphage with ECO than with free chlorine. ECO reduced E. coli, V. cholerae and MS2 by >4 log10 within 30 min and C. perfringens spores by >2 log10 within 10 min at pH 8 and 25 degrees C. Contrary to previous results, however, C. parvum oocysts were not inactivated by ECO, and the reasons for this difference are uncertain. The on-site electrolytic generation of oxidants from brine provided a convenient and inexpensive disinfectant containing free chlorine that was effective against many enteric microbes, for the treatment of household and community drinking-water supplies worldwide. However, the effectiveness of such oxidants for inactivating C. parvum oocysts was variable and sometimes ineffective.     

Using ultraviolet light for disinfection of finished water.

Ultraviolet light is now recognised to be very effective for inactivation of Cryptosporidium parvum oocysts; however, its application for disinfection of finished water necessitates validation of UV reactors prior to their installation. Although reactor performance will likely be assessed using non-pathogenic microorganisms as biodosimetry surrogates, it would be prudent for the water industry to simultaneously measure Cryptosporidium oocysts inactivation in controlled bench-scale studies using the water matrix intended for disinfection. The likelihood of that occurring is dependent upon the availability of infectivity measurement procedures that are more user-friendly than the mouse infectivity assays currently used. This study describes a modified cell culture procedure that would enable reliable measurement of changes in oocysts' infectivity following their UV treatment. Also, a number of different biodosimetry surrogates were examined and one selected for comparing the UV doses delivered between bench-scale and full-scale biodosimetry studies. Impacts of UV disinfection on production of disinfection byproducts, effects of lamp ageing on effectiveness of disinfection and the costs associated with employing this technology were also examined.     

Determining potential indicators of Cryptosporidium oocysts throughout the wastewater treatment process

Most research on wastewater treatment efficiency compliance focuses on physicochemical and microbial indicators; however, very little emphasis has been placed so far on determining suitable indicator organisms to predict the discharge level of pathogens from treatment plants. In this study, raw wastewater, activated sludge, and the resulting final effluents and biosolids in four municipal wastewater treatment plants (WWTPs A, B, C and D) were seasonally investigated for human-virulent water-borne pathogens Cryptosporidium parvum/hominis and Giardia duodenalis, and microsporidia (e.g. Encephalitozoon hellem, E. intestinalis, and Enterocytozoon bieneusi) between 2008 and 2009. A suite of potential microbial indicators for human-virulent protozoa and microsporidia was also determined. A combination of multiple fluorescent in situ hybridization and immunofluorescent antibody assays were applied to detect Cryptosporidium oocysts, Giardia cysts, and microsporidian spores. Escherichia coli, enterococci and Clostridium perfringens spores were cultivated in selective media. Positive correlations were found between the abundance of enterococci and E. coli and abundance of Cryptosporidium oocysts (r(s) > 0.47, p < 0.01) and Giardia cysts (r(s) > 0.44, p < 0.01) at WWTPs A-D. Cryptosporidium perfringens spores were positively correlated to Cryptosporidium oocysts (r(s) = 0.40, p < 0.01) and Giardia cysts (r(s) = 0.46, p < 0.01). There was a strong positive correlation between abundance of Giardia cysts and that of Cryptosporidium oocysts (r(s) > 0.89, p < 0.01). To sum up, a suite of faecal indicator bacteria can be used as indicators for the presence of Cryptosporidium oocysts and Giardia cysts in these activated-sludge systems (WWTPs A, B and C). Overall, Giardia duodenalis was noted to be the best Cryptosporidium indicator for human health in the community-based influent wastewater and throughout the treatment process.     

上海市浦东给水厂两虫去除研究

采用EPA1623法(免疫磁分离及荧光染色法)对上海市几家典型的给水厂的水源水、出厂水、管道水中贾第鞭毛虫和隐孢子虫的分布状况进行了测定分析,并对给水厂净水过程中不同操作单元对两虫的去除效果进行了研究.研究表明:上海浦东给水厂水源水中存在贾第鞭毛虫与隐孢子虫,但其密度分别维持在0～6个/10 L和0～8个/10 L的低水平,而出厂水与管道水中均未检出两虫;给水厂净水过程中,混凝沉淀对两虫具有明显的去除效果.     

Evidence for the existence of Cryptosporidium oocysts as single entities in surface runoff.

There is uncertainty whether Cryptosporidium oocysts attach to particles or to each other under ambient water conditions. Particle size distributions of Cryptosporidium oocyst suspensions were determined over a range of ionic strengths and pHs to determine under those environmental conditions that may promote oocyst aggregation. Cryptosporidium oocysts were shown to only aggregate in high ionic strength solutions (>0.45 M) and remain largely as single entities at ionic strengths and pHs that were likely to be encountered in surface runoff. Similarly, in loam soil suspensions, rather than attaching to the soil particles the majority of oocysts also remained as single entities. Overall, oocysts are expected to remain largely unattached to either themselves or soil particles in overland runoff. This has implications for pathogen transport and modelling since oocysts that are freely suspended are more likely to be transported in runoff to surface waters than if attached to more dense soil/faecal particles.     

Quantitative evaluation and application of Cryptosporidium parvum inactivation with ozone treatment.

Despite a wealth of studies concerning the factors affecting the inactivation of Cryptosporidium parvum on ozone disinfection, practical approaches for predicting the inactivation level on ozone treatment system and determining the required ozone dose or contact time to achieve the required inactivation level remain unclear. When modified version of the "Delayed Chick-Watson model" was applied, the results show that Cryptosporidium parvum inactivation was well fitted with one unique line as "CTlag" and "inactivation slope" for several water parameters and water types. Thus, this study suggests quantitative methodology for predicting the Cryptosporidium parvum inactivation level with observation for the contact time of ozone and Cryptosporidium parvum and ozone residual.     

Evaluation of activated sludge treatment and the efficiency of the disinfection of Giardia species cysts and Cryptosporidium oocysts by UV at a sludge treatment plant in Campinas, south-east Brazil.

Among many waterborne diseases the giardiasis and cryptosporidiosis are of particular public health interest, because Giardia cysts and Cryptosporidium oocysts can persist for long periods in the environment, and both pathogenic protozoa have been implicated as the cause of many outbreaks of gastroenteritis in the last 25 years. In order to evaluate the efficiency of cysts and oocysts' removal by the activated sludge process, and by UV reactor in inactivating cysts and oocysts in one wastewater treatment plant (WWTP) of Campinas, three sampling points were selected for study: (1) influent, (2) treated effluent without UV disinfection and (3) treated effluent with UV disinfection. Giardia spp. cysts prevailed with higher density in the three different sample types. Cryptosporidium spp. oocysts were observed in only two samples of influent and just one sample of treated sewage with UV disinfection. In the animal infectivity assay for Giardia spp, one mouse of the UV treated group revealed trophozoites in intestinal scrapings. The results of the present study indicate that treatment by activated sludge process delivered a reduction of 98.9% of cysts and 99.7% of oocysts and UV disinfection was not completely efficient regarding the inactivation of Giardia cysts in the case of the WWTP studied.     

Evaluation of the purification capacity of nine portable, small-scale water purification devices.

A test was performed to evaluate the microbial and chemical purification capacity of nine portable, small-scale water purification filter devices with production capacity less than 100 L/h. The devices were tested for simultaneous removal capacity of bacteria (cultured Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae and Enterobacter cloacae), enteric protozoans (formalin-stored Cryptosporidium parvum oocysts), viral markers (F-RNA bacteriophages) and microcystins produced by toxic cyanobacterial cultures. In general, the devices tested were able to remove bacterial contaminants by 3.6-6.9 log10 units from raw water. Those devices based only on filtration through pores 0.2-0.4 microm or larger failed in viral and chemical purification. Only one device, based on reverse osmosis, was capable of removing F-RNA phages at concentrations under the detection limit and microcystins by 2.5 log10. The present study emphasised the need for evaluation tests of water purification devices from the public safety and HACCP (Hazard Analysis and Critical Control Point) points of view. Simultaneous testing for various pathogenic/indicator microbes and microcystins was shown to be a useful and practical way to obtain essential data on actual purification capacity of commercial small-scale drinking-water filters.     

Aerobic bacterial spores as process indicators for protozoa cysts in water treatment plants.

The possibility of using aerobic spores as indicators (surrogates) of water treatment efficiency for the removal of Giardia cysts and Cryptosporidium oocysts was evaluated in a water treatment plant that supplies the Barcelona area of Spain. The water treatment consists of pre-chlorination, flocculation-sedimentation, double filtration (sand and granular activated carbon, GAC) with intermediate ozonation and post-chlorination. Aerobic spores significantly increased after GAC filtration, which indicated an active propagation of aerobic spore-formers. However, anaerobic (Clostridium) spores could be a good surrogate for Cryptosporidium oocysts, especially if their detection in samples at low concentrations was improved.     

Development of a sensitive polymerase chain reaction method for the detection of Toxoplasma gondii in water.

Toxoplasma gondii is becoming a potential threat for public water supplies worldwide, as demonstrated by the occurrence of waterborne toxoplasmosis outbreaks in developing countries as well as industrialised countries. The aim of the present study was to develop a sensitive molecular approach (PCR) for the detection of Toxoplasma oocysts in water. Sporulated and unsporulated T. gondii oocysts (strains DX and AHC1) were isolated from faeces of laboratory-infected cats. After purification and enumeration, oocysts were spiked into 1 -L water replicates and concentrated using centrifugation, Al2(SO4)3 or Fe2(SO4)3 flocculation. DNA was extracted from the concentrated pellets, and a universal primer and a T. gondii-specific primer were selected to amplify a region at the small subunit ribosomal RNA gene. A theoretical detection limit of 0.1 oocysts was achieved for samples that had been concentrated using centrifugation or Al2(SO4)3 flocculation. No PCR products were generated for samples that had been pre-treated using Fe2(SO4)3 flocculation. The final target would be the development of a complete technique able to work as a diagnostic tool for the detection of Toxoplasma in environmental and drinking water.     

STUDY ON THE POLLUTION OF URBAN SCENIC WATER BODY BY MUNICIPAL DRAINAGE IN FLOOD SEASON AND ITS CONTROL PLANNING

In this article, based on river quality simulation and system optimization, a water quality model was established for scenic river after rainfall discharge in flood season, with the target of making water pollutants meet the standard in priority and saving expenditure on pollution control. With the principle of reducing sewage from combined sewage pumping station and heavily polluted initial rainwater, a mathematical multiobjective planning model was constructed for rain sewage pollution control in flood season, and one scenic river in a northern city was taken for simulation example. The results show that： the optimization result meets the requirements of planning, among which, sewage reduction from the combined pumping station accounts for 17.38% in the total reduction of rain sewage, and the reduction in the heavily polluted rain water accounts for 77.24% in the total reduction of rainwater pumping station. The planning scheme can provide theoretical basis for pollution control of scenic river in flood season, and for rational reconstruction and layout of outfalls along two banks of the river.     

Giardia and Cryptosporidium in source waters of S?o Paulo State, Brazil.

Giardia lamblia and Cryptosporidium parvum are two protozoan intestinal parasites responsible for many drinking-water-related disease outbreaks in recent years. They are very resistant to conventional water treatment processes, can persist for long times in the environment and are, therefore, of great concern for public health. This work aimed to evaluate the presence of Giardia and Cryptosporidium in water sources from S?o Paulo State, Brazil, as part of the "Evaluation of Inland Waters from S?o Paulo State" project from CETESB. Over a period of 19 months, 278 water samples from 28 sites located in 10 watersheds were analysed. The immunofluorescence assay was used after concentration of the samples by the calcium carbonate flocculation technique. Thermotolerant (faecal) coliforms, faecal streptococci and Clostridium perfringens were also determined in order to verify the existence of correlation between these bacterial indicators and the protozoa. Giardia and Cryptosporidium were detected in 27% and 2.5% of the samples, respectively, a lower figure compared with the results reported by other authors, especially for Cryptosporidium. A Spearman rank correlation test demonstrated a significant correlation between Giardia and faecal indicator concentrations. According to the American Regulation of Monitoring (ICR), treated water from 16 of these 28 collection sites should also be analysed to evaluate whether the treatment process could remove the parasites. Some technical deficiencies of these methods still limit the utilisation of the monitoring results for public health decisions, but the data here reported will help to improve the quality of drinking water in S?o Paulo State.     

长江流域入河排污口监督管理问题的探讨

入河排污口监督管理办法包括对入河排污口的设置、变更的审批原则、审批程序、监管体制及制度等方面.新水法颁布实施前,入河排污口的监督管理是水资源保护工作的一个盲区,但<长江片水资源保护规划>及<长江片水功能区划>规划水质及总量管理目标的实现,入河污染物控制量及削减量等指标最后都要落实在入河排污口上,所以对入河排污口有效的监督管理是规划及区划实施的重要保障.依照新水法授权,对入河排污口进行监督管理是水利部门没有系统开展过的开创性工作,没有现成的理论和方法可以遵循,在今后的工作中要逐步建立完善其监督管理的法制建设、科技标准建设、执法队伍建设,形成系统完整、公正高效的监督管理体系.     

Seasonal profiles of human noroviruses and indicator bacteria in a wastewater treatment plant in Tokyo, Japan.

The seasonal profiles of microorganisms in raw sewage, secondary-treated sewage, and final effluent at a wastewater treatment plant in Tokyo, Japan, were quantitatively determined each month for one year, from July 2003 to June 2004. Human noroviruses, which were determined by real-time PCR, in raw sewage varied from 0.17-260 copies/mL for genotype 1 and from 2.4-1900 copies/mL for genotype 2, showing much higher values in winter, the epidemic season. The concentration of total coliforms, Escherichia coli, or F-specific phages in raw sewage was almost constant throughout the year. Human noroviruses of genotype 2 were removed most effectively (3.69 log10 on average) at the wastewater treatment plant, followed by E. coli (3.37 log10), total coliforms (3.05 loglo), F-specific phages (2.81 log10), and human noroviruses of genotype 1 (2.27 log10). The removal ratio of human noroviruses was almost constant, independent of the initial concentration of the viruses in raw sewage, which led to the increasing concentration of human noroviruses in final effluent in winter. None of the tested bacteria was judged to be a reliable indicator of human noroviruses in final effluent.     

浑太河流域水环境容量分配研究

为实现浑太河流域经济社会可持续发展、排污口排污权最大程度优化、工业及农业合理布局,通过区域水环境容量优化配置,分区段对浑河上游主要排污口、太子河流域生活排污口的交易情况进行优化计算,采用"排污口(直排口)—子流域(支流)—流域/干流"的分配过程对陆域、入河排污口、污染源的水环境容量总量进行分配。结果表明:(1)在考虑安全余量的情况下,对流域COD、氨氮水环境容量总量进行初次分配,分配总量分别为141 343 t、13 636 t,削减量分别占污染物应削减量的44.4%、47.7%,污染物治理总投入为2.18亿元,基本上能够实现流域污染物削减50%的控制目标。(2)通过加大污水处理厂建设力度以及污水达标处理力度进行二次削减,COD、氨氮排放总量分别为208 832 t、17 177 t,分别是流域水环境容量的1.48倍、1.26倍。(3)借助流域水量调整和下泄方式转变对流域污染物最大允许排放情况进行优化控制,得到COD、氨氮的最大允许排放量分别为158 313 t、14 796 t。重新分配后的水环境容量能够保证污染物入河后的水体浓度达标。     

黄河流域入河排污口情况调查

根据黄河流域入河排污口调查资料，对入河排污口的分布废水排放方式、排污口入河废水量、污染物量、河流纳污量、流域水质污染情况进行了分析。流域内共调查入河排污口１７０８个，其中连续掌上排放的１４８１个，间断排放的有２２７个；排污方式以明渠漫流排放为主，占排污口总数的６９．７％；从排放的污水性质看，以工业废水为主。全流域废污 入河量约为４１．７亿ｍ＾３，并且主要集中在一些大中型排污口；全流域调查的入河排污     

南京河西水系引江冲污改善水环境方案研究

针对城市河道的水环境污染日益严重的问题,采用引水冲污方式,可提高河道的水环境净化能力,有效改善城市水环境。以南京市河西水系水质现状为例,建立了一维河网水量水质模型,根据从长江引水的线路、方式及规模,利用所建立的模型,分析了南京市河西水系各河流引水冲污改善水环境的效果。分析结果表明,引水冲污能使城市河道污染浓度快速下降,有效改善城市河道水环境。     

Pathogen monitoring offers questionable protection against drinking-water risks: a QMRA (quantitative microbial risk analysis) approach to assess management strategies.

Risk mitigation provided by human monitoring and control over a water supply system has been consistently overlooked when estimating pathogen exposure to consumers. The Systems-Actions-Management (SAM) framework lends itself neatly to Quantitative Microbial Risk Assessment (QMRA) as one way to establish this link. The general premise is that an organisational protocol will influence how a human controller behaves, in turn influencing the system performance. For illustrative purposes, the framework was applied to a hypothetical water supply system to quantify the risk reduction offered by routine Cryptosporidium monitoring and the response to oocyst 'detects'. Our findings suggest that infrequent direct pathogen monitoring may provide a negligible risk barrier. The practice of sampling treated water to verify microbiological integrity is also dubious: oocyst densities were largely under-estimated, in part due to the spatial dispersion of oocysts in the waterbody, but predominantly from imperfect detection methods. The development of 'event-driven' monitoring schemes with barrier performance-based treatment verification methods, as promoted in new guidelines, is supported as a pressing issue to reduce the likelihood of undetected pathogen passage through a treatment plant.