Donna S. Francy

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

ABSTRACT The protozoan parasite Cryptosporidium parvumis known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvumoocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.

Comparative effectiveness of membrane bioreactors, conventional secondary treatment, and chlorine and UV disinfection to remove microorganisms from municipal wastewaters

Log removals of bacterial indicators, coliphage, and enteric viruses were studied in three membrane bioreactor (MBR) activated-sludge and two conventional secondary activated-sludge municipal wastewater treatment plants during three recreational seasons (May-Oct.) when disinfection of effluents is required. In total, 73 regular samples were collected from key locations throughout treatment processes: post-preliminary, post-MBR, post-secondary, post-tertiary, and post-disinfection (UV or chlorine). Out of 19 post-preliminary samples, adenovirus by quantitative polymerase chain reaction (qPCR) was detected in all 19, enterovirus by quantitative reverse transcription polymerase chain reaction (qRT-PCR) was detected in 15, and norovirus GI by qRT-PCR was detected in 11. Norovirus GII and Hepatitis A virus were not detected in any samples, and rotavirus was detected in one sample but could not be quantified. Although culturable viruses were found in 12 out of 19 post-preliminary samples, they were not detected in any post-secondary, post-MBR, post-ultraviolet, or post-chlorine samples. Median log removals for all organisms were higher for MBR secondary treatment (3.02 to >6.73) than for conventional secondary (1.53-4.19) treatment. Ultraviolet disinfection after MBR treatment provided little additional log removal of any organism except for somatic coliphage (>2.18), whereas ultraviolet or chlorine disinfection after conventional secondary treatment provided significant log removals (above the analytical variability) of all bacterial indicators (1.18-3.89) and somatic and F-specific coliphage (0.71 and >2.98). Median log removals of adenovirus across disinfection were low in both MBR and conventional secondary plants (no removal detected and 0.24), and few removals of individual samples were near or above the analytical variability of 1.2 log genomic copies per liter. Based on qualitative examinations of plots showing reductions of organisms throughout treatment processes, somatic coliphage may best represent the removal of viruses across secondary treatment in both MBR and conventional secondary plants. F-specific coliphage and Escherichia coli may best represent the removal of viruses across the disinfection process in MBR facilities, but none of the indicators represented the removal of viruses across disinfection in conventional secondary plants.

Occurrence and distribution of microbiological indicators in groundwater and stream water

A total of 136 stream water and 143 groundwater samples collected in five important hydrologic systems of the United States were analyzed for microbiological indicators to test monitoring concepts in a nationally consistent program. Total coliforms were found in 99%, Escherichia coli in 97%, and Clostridium perfringens in 73% of stream water samples analyzed for each bacterium. Total coliforms were found in 20%, E. coli in less than 1%, and C. perfringens in none of the groundwater samples analyzed for each bacterium. Although coliphage analyses were performed on many of the samples, contamination in the laboratory and problems discerning discrete plaques precluded quantification. Land use was found to have the most significant effect on concentrations of bacterial indicators in stream water. Presence of septic systems on the property near the sampling site and well depth were found to be related to detection of coliforms in groundwater, although these relationships were not statistically significant. A greater diversity of sites, more detailed information about some factors, and a larger dataset may provide further insight to factors that affect microbiological indicators.

Effects of Seeding Procedures and Water Quality on Recovery of Cryptosporidium Oocysts from Stream Water by Using U.S. Environmental Protection Agency Method 1623

ABSTRACT U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the methods recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from <2 to <215 oocysts per 10 liters. Recoveries of oocysts ranged from 2.0 to 61% for viable oocysts and from 3.0 to 59% for modified oocysts. The recoveries between the two seeding procedures were highly correlated (r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

Comparison of methods for determining Escherichia coli concentrations in recreational waters

Abstract Seventy water samples were collected from three Lake Erie beaches to compare recoveries of Escherichia coli ( E. coli ) using the USEPA-recommended method for recreational waters (mTEC) to recoveries using three alternative methods (MI, modified mTEC, and Colilert). Statistical tests showed no differences in recoveries of E. coli between MI and mTEC; however, statistically-significant differences were found between modified mTEC or Colilert and mTEC. The MI agar method provided the most similar assessment of recreational water quality to mTEC among the three alternative methods tested. The range of differences between Colilert and mTEC was widest among the three alternative methods. In a sample group with a range of values near the single-sample bathing-water standard, recoveries of E. coli were statistically lower using modified mTEC than mTEC; however, MI and Colilert compared well to mTEC in this range. Because samples were collected in a small geographic area, more work is necessary to test within-method variability of the modified mTEC, MI, and Colilert methods and to evaluate these methods as substitutes for the mTEC method in a variety of recreational waters.

Comparison of filters for concentrating microbial indicators and pathogens in lake-water samples

ABSTRACT Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

Comparison of traditional and molecular analytical methods for detecting biological agents in raw and drinking water following ultrafiltration

Aims:  To compare the performance of traditional methods to quantitative polymerase chain reaction (qPCR) for detecting five biological agents in large‐volume drinking‐water samples concentrated by ultrafiltration (UF).

Use of predictive models and rapid methods to nowcast bacteria levels at coastal beaches

The need for rapid assessments of recreational water quality to better protect public health is well accepted throughout the research and regulatory communities. Rapid analytical methods, such as quantitative polymerase chain reaction (qPCR) and immunomagnetic separation/adenosine triphosphate (ATP) analysis, are being tested but are not yet ready for widespread use. Another solution is the use of predictive models, wherein variable(s) that are easily and quickly measured are surrogates for concentrations of fecal-indicator bacteria. Rainfall-based alerts, the simplest type of model, have been used by several communities for a number of years. Deterministic models use mathematical representations of the processes that affect bacteria concentrations; this type of model is being used for beach-closure decisions at one location in the USA. Multivariable statistical models are being developed and tested in many areas of the USA; however, they are only used in three areas of the Great Lakes to aid in notifications of beach advisories or closings. These “operational” statistical models can result in more accurate assessments of recreational water quality than use of the previous days Escherichia coli (E. coli) concentration as determined by traditional culture methods. The Ohio Nowcast, at Huntington Beach, Bay Village, Ohio, is described in this paper as an example of an operational statistical model. Because predictive modeling is a dynamic process, water-resource managers continue to collect additional data to improve the predictive ability of the nowcast and expand the nowcast to other Ohio beaches and a recreational river. Although predictive models have been shown to work well at some beaches and are becoming more widely accepted, implementation in many areas is limited by funding, lack of coordinated technical leadership, and lack of supporting epidemiological data.

Section 5. Procedures for Developing Models To Predict Exceedances of Recreational Water-Quality Standards at Coastal Beaches

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Predictive models for Escherichia coli concentrations at inland lake beaches and relationship of model variables to pathogen detection.

ABSTRACT Predictive models, based on environmental and water quality variables, have been used to improve the timeliness and accuracy of recreational water quality assessments, but their effectiveness has not been studied in inland waters. Sampling at eight inland recreational lakes in Ohio was done in order to investigate using predictive models for Escherichia coli and to understand the links between E. coli concentrations, predictive variables, and pathogens. Based upon results from 21 beach sites, models were developed for 13 sites, and the most predictive variables were rainfall, wind direction and speed, turbidity, and water temperature. Models were not developed at sites where the E. coli standard was seldom exceeded. Models were validated at nine sites during an independent year. At three sites, the model resulted in increased correct responses, sensitivities, and specificities compared to use of the previous days E. coli concentration (the current method). Drought conditions during the validation year precluded being able to adequately assess model performance at most of the other sites. Cryptosporidium, adenovirus, eaeA (E. coli), ipaH (Shigella), and spvC (Salmonella) were found in at least 20% of samples collected for pathogens at five sites. The presence or absence of the three bacterial genes was related to some of the model variables but was not consistently related to E. coli concentrations. Predictive models were not effective at all inland lake sites; however, their use at two lakes with high swimmer densities will provide better estimates of public health risk than current methods and will be a valuable resource for beach managers and the public.