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Microbiological & Chemical Exposure Assessment Research Division Publications: 2008

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This page lists publication titles, citations and abstracts produced by NERL's Microbiological & Chemical Exposure Assessment Research Division for the year 2009, organized by Publication Type. Your search has returned 45 Matching Entries.

See also Microbiological & Chemical Exposure Assessment Research Division citations with abstracts: 1999,  2000,  2001,  2002,  2003,  2004,  2005,  2006,  2007,  2008,  2009

Technical Information Manager: Angela Moore - (513) 569-7341 or moore.angela@epa.gov

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Presented/Published
JOURNAL Obtaining Highly Purified Toxoplasma Gondii Oocysts By a Discontinuous Cesium Chloride Gradient 11/03/2009
Staggs, S. E., M. J. See, J. Dubey, AND E. VILLEGAS. Obtaining Highly Purified Toxoplasma Gondii Oocysts By a Discontinuous Cesium Chloride Gradient. Journal of Visualized Experiments . JoVE, Somerville, MA, (33):1-2, (2009).
Abstract: Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

JOURNAL Correlation Between Ermi Values and Other Moisture and Mold Assessments of Homes in the American Healthy Home Survey 11/01/2009
VESPER, S. J., C. McKinstry, P. Ashley, D. Cox, AND G. Dewalt. Correlation Between Ermi Values and Other Moisture and Mold Assessments of Homes in the American Healthy Home Survey. JOURNAL OF URBAN HEALTH: BULLETIN OF THE NEW YORK ACADEMY OF MEDICINE. Oxford University Press, Cary, NC, 86(6):850-860, (2009).
Abstract: Objective: The objective of this study was to determine the correlation between the Environmental Relative Moldiness Index (ERMI) values in the HUD American Healthy Home Survey (AHHS) homes and either inspector reports or occupant assessments of mold and moisture. Methods: In the AHHS, moisture and mold were assessed by a trained pair of inspectors and with an occupant questionnaire. These results were compared to the ERMIresults of the Environmental Relative Moldiness Index (ERMI) values for each home. Results: Homes in the highest ERMI quartile were most often in agreement with visual inspection and/or occupant assessment. However, in 52% of the fourth quartile ERMI homes, the inspector and occupant assessment did not indicate water or mold problems. Yet the concentrations of each ERMI panel mold species detected in all fourth quartile homes were statistically indistinguishable. Conclusions: About 50% of water-damaged, moldy homes were not detected by inspection or questioning of the occupant about water and mold.

JOURNAL Cryptosporidium Propidium Monoazide-Pcr, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts 11/01/2009
Brescia, C., S. Hunt, M. W. WARE, E. VARUGHESE, A. EGOROV, AND E. VILLEGAS. Cryptosporidium Propidium Monoazide-Pcr, a Molecular Biology-Based Technique for Genotyping Viable Cryptosporidium Oocysts. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(21):6856-6863, (2009).
Abstract: Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium propidium monoazide-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to polymerase chain reaction (PCR) analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only heat-killed oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts and not dead oocysts were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof of concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical in assessing human health risks associated with this pathogen.

JOURNAL Quantitative Real-Time Pcr Analysis of Total Propidium Monazide-Resistant Fecal Indicator Bacteria in Wastewater 11/01/2009
VARMA, M., R. I. FIELD, M. K. STINSON, B. Rukovets, L. J. WYMER, AND R. A. HAUGLAND. Quantitative Real-Time Pcr Analysis of Total Propidium Monazide-Resistant Fecal Indicator Bacteria in Wastewater. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 43(19):4790-4801, (2009).
Abstract: A real-time quantitative PCR (qPCR) method and a modification of this method incorporating pretreatment of samples with propidium monoazide (PMA) were evaluated for respective analyses of total and presumptively viable Enterococcus and Bacteroidales fecal indicator bacteria. These methods were used in the analyses of wastewater samples to investigate their feasibility as alternatives to current fecal indicator bacteria culture methods for predicting the efficiency of viral pathogen removal by standard treatment processes. PMA treatment was effective in preventing qPCR detection of target sequences from non-viable cells. Concentrates of small volume, secondary-treated wastewater samples, collected from a publicly owned treatment works (POTW) under normal operating conditions, had little influence on this effectiveness. Higher levels of total suspended solids, such as those associated with normal primary treatment and all treatment stages during storm flow events, appeared to interfere with PMA effectiveness under the sample preparation conditions employed. During normal operating conditions at three different POTWs, greater reductions were observed in PMA-qPCR detectable target sequences of both Enterococcus and Bacteroidales than in total qPCR detectable sequences. These reductions were not as great as those observed for cultivable fecal indicator bacteria in response to wastewater disinfection. Reductions of PMA-qPCR as well as total qPCR detectable target sequences from enterococci and, to a lesser extent, Bacteroidales correlated well with reductions in infectious viruses during both normal and storm flow operating conditions and therefore may have predictive value in determining the efficiency at which these pathogens are removed.

JOURNAL Virulence Factor-Activity Relationships: Workshop Summary 10/01/2009
DeLeon, R. Virulence Factor-Activity Relationships: Workshop Summary. JOURNAL OF WATER AND HEALTH. IWA Publishing, London, Uk, 7(S1):S94-S100, (2009).
Abstract: The concept or notion of virulence factor–activity relationships (VFAR) is an approach for identifying an analogous process to the use of qualitative structure–activity relationships (QSAR) for identifying new microbial contaminants. In QSAR, it is hypothesized that, for new chemical contaminants, their potential acute or chronic toxicity may be reasonably estimated on the basis of structural relationships to other known toxic contaminants. Thus the parallel that is being attempted for pathogenic microorganisms is that known virulence factors may be used as predictors for identifying undiscovered pathogens and microbial causes of emerging diseases. Advances in molecular biology, genomics and proteomics have led the Committee on Drinking Water Contaminants of the National Research Council, as requested by the EPA, to recommend the VFAR approach as a potentially more systematic and scientific process for the selection of microorganisms for inclusion in the Contaminant Candidate List (CCL).

JOURNAL Rooftop Runoff as a Source of Contamination: A Review 10/01/2009
LYE, D. J. Rooftop Runoff as a Source of Contamination: A Review. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 407(20):5429-5434, (2009).
Abstract: Scientific reports concerning chemical and microbiological contaminant levels of rainwater runoff from rooftop collection in both urban and rural areas are reviewed. This alternative source of water has been documented to often contain substantial amounts of contaminants. Studies describing levels of heavy metal contamination specific to runoff from rooftop catchment areas containing exposed metal surfaces are discussed. Depending upon the intended use, scientific evidence is also accumulating that various treatments and disinfections will be required prior to release of roof runoff water either into surface waters or for more direct consumer usage. For microbial contamination, current proposed standards and guidelines regarding this type of water source are shown to vary widely worldwide. Scientific literature reveals a lack of clarity regarding water quality guidelines and health related standards for certain types of rooftop runoff. Studies suggests that rainwater collection systems which are properly designed, maintained, and treated may provide a valuable supplement to existing water supplies by reducing demand on community water supplies/infrastructure costs, enhancing effective management of storm water runoff, and increasing restoration of underground reservoirs through controlled infiltration

JOURNAL Concentrating Toxoplasma Gondii and Cyclospora Cayetanensis from Surface Water and Drinking Water By Continuous Separation Channel Centrifugation 10/01/2009
Borchardt, M., S. Spencer, P. D. Bertz, M. W. WARE, J. P. Dubey, AND H. LINDQUIST. Concentrating Toxoplasma Gondii and Cyclospora Cayetanensis from Surface Water and Drinking Water By Continuous Separation Channel Centrifugation. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 107(4):1089-1097, (2009).
Abstract: Aims: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. Methods and Results: Ready-to-seed vials with known quantities of Toxoplasma gondii and Cyclospora cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l-1 into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64-100%, with the lowest recoveries in the most turbid waters. Method precision was usually between 10% and 20% coefficient of variation. Conclusion: Toxoplasma gondii and Cyclospora cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation. Significance and Impact of the Study: Waterborne transmission of Toxoplasma gondii and Cyclospora cayetanensis has been documented, presenting another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from an ambient water sample, otherwise false negatives may result. While several concentration methods are available, validation data specific to Toxoplasma gondii and Cyclospora cayetanensis recoveries are limited. Using continuous separation channel centrifugation, oocysts are recovered with high efficiency and precision, the method attributes required for accurately assessing the risk of waterborne transmission.

JOURNAL Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of C-Jun and C-Fos in Human Tissue Culture Cells 09/01/2009
HAYES, S. L., M. Waltmann, M. J. DONOHUE, D. J. LYE, AND S. J. VESPER. Predicting Virulence of Aeromonas Isolates Based-on Changes in Transcription of C-Jun and C-Fos in Human Tissue Culture Cells. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 107(3):964-969, (2009).
Abstract: Aims: To assess virulence of Aeromonas isolates based on the change in regulation of c-jun and c-fos in the human intestinal tissue culture cell line Caco-2. Methods and Results: Aeromonas cells were added to Caco-2 cells at approximately a one to one ratio. After 1, 2 and 3 hours incubation at 37oC, mRNA was extracted from the cells and gene expression of two host genes, c-jun and c-fos, quantified. Aeromonas isolates which were virulent in the neonatal mouse model demonstrated up-regulation of c-jun and c-fos compared to avirulent isolates. Conclusions: Human cell culture results showed that c-jun and c-fos were predictive of Aeromonas virulence. Significance and Impact of the Study: An Aeromonas relative virulence scale (ARVS) is proposed for use in the testing of Aeromonas drinking water isolates.

JOURNAL Quantitative Pcr for Genetic Markers of Human Fecal Pollution 09/01/2009
SHANKS, O. C., C. A. KELTY, M. SIVAGANESAN, M. VARMA, AND R. A. HAUGLAND. Quantitative Pcr for Genetic Markers of Human Fecal Pollution. APPLIED AND ENVIRONMENTAL MICROBIOLOGY. American Society for Microbiology, Washington, DC, 75(17):5507-5513, (2009).
Abstract: Assessment of health risk and fecal bacteria loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantificationapproach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface associated genes. Both assays exhibited a range of quantification from 10 to 1x106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker was measured in primary effluent wastewater samples collected from 20 geographically distinct locations and compared to quantities estimated by real-time PCR assays specific for ribosomal RNA gene sequences from total Bacteroidales and enterococci fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

JOURNAL The Role of Biofilms and Protozoa in Legionella Pathogenesis: Implications for Drinking Water 08/01/2009
LAU, H. Y. AND N. ASHBOLT. The Role of Biofilms and Protozoa in Legionella Pathogenesis: Implications for Drinking Water. JOURNAL OF APPLIED MICROBIOLOGY. Blackwell Publishing, Malden, MA, 107(2):368-378, (2009).
Abstract: Current models to study Legionella pathogenesis include the use of primary macrophages and monocyte cell lines, various free-living protozoan species and murine models of pneumonia. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization and parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles in drinking water distribution systems. Moreover, the implications of these environmental niches for drinking water exposure to pathogenic legionellae are poorly understood. This review summarizes the known mechanisms of Legionella spp. proliferation within Acanthamoeba and mammalian cells and advocates the use of the amoeba model to study Legionella pathogenicity because of their close relationship with Legionella spp. in the aquatic environment. The putative role of biofilms and amoeba in the proliferation, development and dissemination of potentially pathogenic Legionella spp. is also discussed. Elucidating the mechanisms of Legionella pathogenicity development in our drinking water systems will aid in elimination strategies and procedural designs for drinking water systems in controlling exposure to Legionella spp. and similar pathogens.

JOURNAL The Determination of Pesticidal and Non-Pesticidal Organotin Compounds in Water Matrices By in Situ Ethylation and Gas Chromatography With Pulsed Flame Photometric Detection 07/01/2009
EVANS, O. M., P. KAUFFMAN, A. M. PAWLECKI-VONDERHEIDE, L. J. WYMER, AND J. N. MORGAN. The Determination of Pesticidal and Non-Pesticidal Organotin Compounds in Water Matrices By in Situ Ethylation and Gas Chromatography With Pulsed Flame Photometric Detection. Microchemical Journal. Elsevier BV, AMSTERDAM, Netherlands, 92(2):155-164, (2009).
Abstract: The concurrent determination of pesticidal and non-pesticidal organotin compounds in several water matrices, using a simultaneous in situ ethylation and liquid-liquid extraction followed by splitless injection mode capillary gas chromatography with pulsed flame photometric detection, is described. The speciation analysis of nine organotin compounds includes low molecular weight - low boiling (non-pesticidal) and high molecular weight - high boiling analytes (pesticidal) of significant environmental interest. The minimum time for sodium tetraethylborate alkylation, using mechanical agitation, is determined to be fifteen minutes in order to ensure the complete derivatization of the complete list of analytes. The utilization of a “hot needle” and a rapid injection rate is shown to be an efficacious means to eliminate “mass” or “needle” discrimination when determining the mixture of organotin compounds. Method detection limits are calculated to be in the low ng L-1 range. The final method is applied to various water samples; storm water from the Cincinnati area demonstrated low native levels of three of the organotin compounds.

JOURNAL The Determination of Pesticidal and Non-Pesticidal Organotin Compounds By in Situ Ethylation and Capillary Gas Chromatography With Pulsed Flame Photometric Detection 07/01/2009
EVANS, O. M., P. KAUFFMAN, A. M. PAWLECKI-VONDERHEIDE, AND L. J. WYMER. The Determination of Pesticidal and Non-Pesticidal Organotin Compounds By in Situ Ethylation and Capillary Gas Chromatography With Pulsed Flame Photometric Detection. Microchemical Journal. Elsevier BV, AMSTERDAM, Netherlands, 92(2):155-164, (2009).
Abstract: The concurrent determination of pesticidal and non-pesticidal organotin compounds in several water matrices, using a simultaneous in situ ethylation and liquid-liquid extraction followed by splitless injection mode capillary gas chromatography with pulsed flame photometric detection, is described. The speciation analysis of nine organotin compounds includes low molecular weight - low boiling (non-pesticidal) and high molecular weight - high boiling analytes (pesticidal) of significant environmental interest. The minimum time for sodium tetraethylborate alkylation, using mechanical agitation, is determined to be fifteen minutes in order to ensure the complete derivatization of the complete list of analytes. The utilization of a “hot needle” and a rapid injection rate is shown to be an efficacious means to eliminate “mass” or “needle” discrimination when determining the mixture of organotin compounds. Method detection limits are calculated to be in the low ng L-1 range. The final method is applied to various water samples; storm water from the Cincinnati area demonstrated low native levels of three of the organotin compounds.

JOURNAL A Mouse Model for Characterization of Gastrointestinal Colonization Rates Among Environmental Aeromonas Isolates 05/01/2009
LYE, D. J. A Mouse Model for Characterization of Gastrointestinal Colonization Rates Among Environmental Aeromonas Isolates. CURRENT MICROBIOLOGY. Springer, New York, NY, 58(5):454-458, (2009).
Abstract: The colonization rates of ten different environmental isolates of Aeromonas were determined using a novel mouse-streptomycin pre-treatment method. A novel streptomycin pre-treatment prepared animals with a transient alteration in colon flora that allowed colonization by Aeromonas which would not occur in the presence of normal competitive host bacteria. Colonization rates of Aeromonas salmonicida, Aeromonas enchelia, and Aeromonas allosaccharophila were low and occurred randomly with respect to concentrations of the dosage consumed by the animals. In contrast, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae exhibited relatively high rates of colonization of mouse colon tissues.

JOURNAL Solar Radiation Disinfection of Drinking Water at Temperate Latitudes: Inactivation Rates for An Optimized Reactor Configuration 02/01/2009
Davies, C. M., D. J. Roser, A. J. Feitz, AND N. ASHBOLT. Solar Radiation Disinfection of Drinking Water at Temperate Latitudes: Inactivation Rates for An Optimized Reactor Configuration. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 43(3):643-652, (2009).
Abstract: Solar radiation-driven inactivation of bacteria, virus and protozoan pathogen models was quantified in simulated drinking water at a temperate latitude (34°S). The water was seeded with Enterococcus faecalis, Clostridium sporogenes spores, and P22 bacteriophage, each at ca 1 x 10,sup>5 mL-1, and exposed to natural sunlight in 30-L reaction vessels. Water temperature ranged from 17 to 39 °C during the experiments lasting up to 6 h. Dark controls showed little inactivation and so it was concluded that the inactivation observed was primarily driven by non-thermal processes. The optimised reactor design achieved S90 values (cumulative exposure required for 90% reduction) for the test microorganisms in the range 0.63–1.82 MJm-2of Global Solar Exposure (GSX) without the need for TiO2 as a catalyst. High turbidity (840–920 NTU) only reduced the S90 value by <40%. Further, when all S90 means were compared this decrease was not statistically significant (prob. > 0.05). However, inactivation was significantly reduced for E. faecalis and P22 when the transmittance of UV wavelengths was attenuated by water with high colour (140 PtCo units) or a suboptimally transparent reactor lid (prob. < 0.05). S90 values were consistent with those measured by other researchers (ca 1–10 MJm-2) for a range of waters and microorganisms . Although temperatures required for SODIS type pasteurization were not produced, nonthermal inactivation alone appeared to offer a viable means for reliably disinfecting low colour source waters by greater than 4 orders of magnitude on sunny days at 34°S latitude.

JOURNAL Screening Tools to Estimate Mold Burdens in Homes 01/01/2009
VESPER, S. J., C. McKinstry, K. D. BRADHAM, P. Ashley, D. Cox, G. Dewalt, AND K. Lin. Screening Tools to Estimate Mold Burdens in Homes. JOURNAL OF OCCUPATIONAL AND ENVIRONMENTAL MEDICINE. Lippincott Williams & Wilkins, Philadelphia, PA, 51(1):80-86, (2009).
Abstract: Objective: The objective of this study was to develop screening tools that could be used to estimate the mold burden in a home which would indicate whether more detailed testing might be useful. Methods: Previously, in the American Healthy Home Survey, a DNA-based method of analysis called mold specific quantitative PCR was used to measure 36 molds in standard protocol dust samples. This resulted in a national index called the Environmental Relative Moldiness Index (ERMI). In this current study, two possible screening methods were considered: use of the vacuum cleaner bag dust rather than the standard protocol dust samples and reducing the number of molds needed to be quantified resulting in the creation of a simpler mold burden scale. Results: Comparison of vacuum bag and standard dust samples from 157 of the same homes demonstrated that most molds had higher detection rates in vacuum bag dust compared to athe standard dust samples but the ERMI values were still related to each other. The second approach to simplifying the screening for mold burdens produced a correlated (ρ=0.80) index to the ERMI called the American Relative Moldiness Index (ARMI) which requires the analysis of only 12 species. Conclusions: Vacuum bag dust sample ERMI values were predictive in placing a home into the lower or upper 50% of homes on the ERMI scale. If it is not possible to obtain athe standard dust sample, the vacuum cleaner bag dust may be a useful screening tool for estimating mold burdens in homes. If the standard sample is available and a simpler screening test is sought to estimate the mold burden in homes, the ARMI scale might be useful.

JOURNAL Method Development for the Analysis of 1,4-Dioxane in Drinking Water Using Solid Phase Extraction and Gas Chromatography/Mass Spectrometry 01/01/2009
GRIMMETT, P. AND J. W. MUNCH. Method Development for the Analysis of 1,4-Dioxane in Drinking Water Using Solid Phase Extraction and Gas Chromatography/Mass Spectrometry. Journal of Chromatographic Science. Preston Publications Incorporated, Niles, IL, 47(1):31-39, (2009).
Abstract: 1,4-Dioxane has been identified as a probable human carcinogen and an emerging contaminant in drinking water. The National Exposure Research Laboratory (NERL) has developed a method for the analysis of 1,4-dioxane in drinking water at ng/L concentrations. The method consists of an activated carbon solid-phase extraction of 500-mL or 100-mL water samples using dichloromethane as the elution solvent. The extracts are analyzed by gas chromatography-mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode. In the NERL laboratory, recovery of 1,4-dioxane ranged from 94-110% in fortified laboratory reagent water (LRW), and recoveries of 96-102% were demonstrated for fortified drinking water samples. The relative standard deviations for replicate analyses were less than 6% at concentrations exceeding the minimum reporting level.

PRESENTATION Water Sample Filtration Methods Using Viradel Procedures 11/15/2009
CASHDOLLAR, J. Water Sample Filtration Methods Using Viradel Procedures. Presented at Water Quality Technology Conference, Seattle, WA, November 15 - 19, 2009.
Abstract: To be presented at WQTC, November 15-19, Seattle, Washington

PRESENTATION Quantitative Microbial Risk Assessment (Qmra) as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches 11/09/2009
SCHOEN, M. E. AND N. ASHBOLT. Quantitative Microbial Risk Assessment (Qmra) as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches. Presented at 137th Annual APHA Meeting , Philadelphia, PA, November 09 - 11, 2009.
Abstract: The US EPA and WHO have set recreational water quality standards based on epidemiologic studies to protect human health at beaches. These studies have largely been limited to sewage-impacted sites and resources are unlikely to be available to assess the myriad of other impacted sites. Here we describe how quantitative microbial risk assessment (QMRA) can be used to assess unstudied pathogen sources in a systematic way to describe risk uncertainty. To illustrate the proposed QMRA comparison an illustrative example is provided focusing on the non-sewage example sources of seagulls and human shedders.

PRESENTATION Qmra as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches 11/07/2009
SCHOEN, M. E. Qmra as a Compliment to Epidemiologic Studies Estimating Bather Risk at Recreational Beaches. Presented at American Public Health Association Annual Meeting, Philadelphia, PA, November 07 - 11, 2009.
Abstract: The US EPA and WHO have set recreational water quality standards based on epidemiologic studies to protect human health at beaches. These studies have largely been limited to sewage-impacted sites and resources are unlikely to be available to assess the myriad of other impacted sites. Here we describe how quantitative microbial risk assessment (QMRA) can be used to assess unstudied pathogen sources in a systematic way to describe risk uncertainty. A QMRA was constructed based on a recreational beach primarily impacted by seagull feces, assumed to contribute only Campylobacter and Salmonella. The pathogen dose distribution was derived from the concentration of the fecal indicator in the water column using an uncertain ratio of indicator to pathogen concentrations. The probability of gastrointenteritis was calculated using dose-response relationships from the literature. All uncertain model parameters were represented by probability distributions and sampled in a Monte Carlo analysis, allowing subsequent parameter importance analysis. Based on the high uncertainty of human-infectious pathogens in gull feces, the predicted probability of infection from gulls is of potential concern when the water quality indicator is near the single sample water quality standard (104 enterococci / 100 mL). This pathogen uncertainty is common with many animal sources of fecal contamination; hence, it is important that future research focus on specifying pathogen densities to allow comparison of risk estimates from epidemiologic studies with QMRA, which may ultimately allow risk characterization from unstudied sources of fecal contamination at recreational beaches.

PRESENTATION Detection of Quantification of Mycobacterium Avium Complex Organisms in Drinking Water 11/03/2009
PFALLER, S. L. Detection of Quantification of Mycobacterium Avium Complex Organisms in Drinking Water. Presented at International Society for Exposure Science, Minneapolis, MN, November 01 - 05, 2009.
Abstract: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and others. MAC are listed on the U. S. Environmental Protection Agency’s Contaminant Candidate List 2 (CCL2) due to their association with human disease and occurrence in public drinking water systems. Current methods for detecting MAC organisms in drinking water are culture-based. However evidence suggests that culture-based methods have severe limitations including long incubation periods, loss of target due to overgrowth of background organisms, up to 70% loss of target due to harsh decontamination techniques, and inability to recover MAC in a viable-but-non-culturable state. Because of these drawbacks and the need for more accurate and comprehensive occurrence data, we have developed real-time QPCR assays for detection and quantification of MA, MI, and MA subsp. paratuberculosis (MAP) in drinking water. Real-time QPCR assays can be used in combination with culture-based methods in order to confirm viability of MAC in samples, as well as provide isolates for further characterization. We are currently evaluating these methods for use on actual drinking water samples in order to generate a more complete understanding of MAC occurrence in drinking water.

PRESENTATION Surveillance Systems for Waterborne Protozoa Past, Present and Future 11/01/2009
VILLEGAS, E. Surveillance Systems for Waterborne Protozoa Past, Present and Future. Presented at International Society of Exposure Sciences, Minneapolis, MN, November 01 - 05, 2009.
Abstract: OVERVIEW I. Brief introduction to waterborne Cryptosporidium Historical perspective on detecting Cryptosporidium Current detection methodologies II. US EPA’s waterborne protozoan research program Detecting, typing, and tracking sources of Cryptosporidium contamination III. The future of the “Microbial Detection Toolbox”

PRESENTATION Using Propidium Monoazide as An Enrichment Step to Remove Extraneous Dma and Non-Viable Organisms for Cryptosporidium Genotyping 10/05/2009
VILLEGAS, E. Using Propidium Monoazide as An Enrichment Step to Remove Extraneous Dma and Non-Viable Organisms for Cryptosporidium Genotyping. Presented at Global Conference on Waterborne Microbial Contaminants, Singapore, SINGAPORE, October 05 - 08, 2009.
Abstract: Cryptosporidium is an important protozoan parasite that continues to cause waterborne disease outbreaks worldwide. Current methods to monitor for Cryptosporidium oocysts in water are microscopy-based USEPA Methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium propidium monoazide-polymerase chain reaction (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis. The purpose of the treatment was to prevent the amplification of DNA that was extraneous or in inactivated oocysts. Our results demonstrated that PMA effectively removes naked DNA present in environmental samples. In addition, this CryptoPMA-PCR assay can specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, only live oocysts were detected in raw waste or surface water samples that were spiked with either live or dead Cryptosporidium oocysts. This proof of concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical in assessing human health risks associated with this pathogen.

PRESENTATION The Birds Did It! 09/29/2009
SCHOEN, M. E. AND N. ASHBOLT. The Birds Did It! Presented at Great Lakes Beach Association Conference, Milwaukee, WI, September 28 - October 01, 2009.
Abstract: The US EPA and WHO have set recreational water quality standards based on epidemiologic studies to protect human health at beaches. These studies have largely been limited to sewage-impacted sites, and resources are unlikely to be available to assess the myriad of other impacted sites. Here we describe how quantitative microbial risk assessment (QMRA) can be used to assess unstudied pathogen sources in a systematic way to describe risk uncertainty. A QMRA was constructed based on a recreational beach primarily impacted by seagull feces, assumed to contribute only Campylobacter and Salmonella. The pathogen dose distribution was derived from the concentration of the fecal indicator in the water column using an uncertain ratio of indicator to pathogen concentrations. The probability of gastrointenteritis was calculated using dose-response relationships from the literature. All uncertain model parameters were represented by probability distributions and sampled in a Monte Carlo analysis, allowing subsequent parameter importance analysis. Based on the high uncertainty of human-infectious pathogens in gull feces, the predicted probability of infection from gulls is of potential concern when the water quality indicator is near the single sample water quality standard (104 enterococci / 100 mL). This pathogen uncertainty is common with many animal sources of fecal contamination; hence, it is important that future research focus on specifying pathogen densities to allow comparison of risk estimates from epidemiologic studies with QMRA, which may ultimately allow risk characterization from unstudied sources of fecal contamination at recreational beaches.

PRESENTATION Contaminants of Emerging Concerns 08/20/2009
GLASSMEYER, S., E. T. Furlong, D. W. Kolpin, R. J. MILTNER, AND S. L. Werner. Contaminants of Emerging Concerns. Presented at ASIWPCA Webinar, Cincinnati, OH, August 20, 2009.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-ug/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. To determine which wastewater chemicals persist through drinking water treatment, a follow-up study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 84 different emerging contaminants, including 24 pharmaceuticals. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-five chemicals were detected at least once, with 28 chemicals detected in the source waters and 23 chemicals detected in the finished waters. The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Peak Health and the Need for More Sustainable Urban Water Systems 08/18/2009
ASHBOLT, N. Peak Health and the Need for More Sustainable Urban Water Systems. Presented at World City Water Forum 2009, Incheon, SOUTH KOREA, August 18 - 21, 2009.
Abstract: Large centralized urban water services in developed countries like the USA still provide significant environmental impact via loss of ecological water services, energy use, loss of nutrients from agricultural production, and eutrophication issues. Current climate models predict that many regions will generally be increasingly more water stressed as well as prone to intense storm events, further exacerbating the negative effects of centralized water services. As a consequence of many interacting factors based around energy/water use and social inequity, Peak Health may have already been reached in the USA, with life expectancies equal to that of Cuba (75 years). From a global perspective, rapidly developing regions, most of which are in water scarce regions, make water-based sanitation unsustainable if not impractical. Hence there is a need to rethink how water services can be obtained for the health of developed and developing regions by lowering our environmental footprint as well as empowering individuals/communities to control their water/sanitation services in a health-promoting environment. Examples include net energy production from organic components along with nutrients returned to agriculture, particularly phosphorus that has known stores of available rock phosphate to only last 60-150 years. From a public health perspective, aging water mains and their vulnerability to intrusions by fecally-contaminated waters is a rising issue; all the more reason to consider an alternative approach to water distribution and handling of associated wastewater streams rather than rebuilding more of the same problem. Most interesting is that the single largest cause of waterborne illness identified in the US (legionellosis) is not of fecal origin, nor currently regulated in most parts of the world. Legionellosis is due to the growth of a pathogen (Legionella pneumophila), which may largely be an in-premise (building) issue rather than the distribution system per se. Hence Legionella and other similar indigenous pathogens that grow in pipe biofilms are not necessarily the responsibility of the distribution system provider. As we move to greater reliance on reclaimed waters, fit-for-purpose, the long-term ramifications of fecal and indigenous pathogen issues should be considered within a broader sustainability assessment if we are to further improve public health. A framework for a way forward is described.

PRESENTATION Environmental Perspectives and Issues 07/26/2009
ASHBOLT, N., R. BASTIAN, AND R. GOO. Environmental Perspectives and Issues. Presented at Decentralized Water Systems Workshop, Nags Head, NC, July 26 - 29, 2009.
Abstract: Slide presentation

PRESENTATION Community-Scale and Household-Scale Decentralized Reuse Experiences on Two Continents 07/26/2009
ASHBOLT, N. Community-Scale and Household-Scale Decentralized Reuse Experiences on Two Continents. Presented at Decentralized Water Systems Workshop, Nags Head, NC, July 26 - 29, 2009.
Abstract: Slide presentation

PRESENTATION Analysis of Arsenicals and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS 06/07/2009
KUBACHKA, K., C. GALLAWA, P. A. CREED, J. T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Analysis of Arsenicals and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS. Presented at International Symposium on Metallomics, Covington, KY, June 07 - 13, 2009.
Abstract: Recent metabolic and exposure assessment studies have found sulfur analogs (thioarsenicals) of common oxoarsenicals in environmental and biological systems. The occurrence of thioarsenicals raises questions regarding their origin and transport, and their roles in metabolism of arsenic. Definitive answers to these questions require speciation based methods that can separate and quantify oxo- and thioarsenicals in complex mixtures. Various LC methods are often necessary to separate such complex mixtures. Because thioarsenicals are often present in samples at low ng/g concentrations, elemental detection via ICP-MS is most commonly used for quantitation. Additionally, LC-ESI-MS/MS techniques have proven valuable for complementary verification of standards and confirmation of suspect peaks, because many arsenic standards and their sulfur analogs are not commercially available. An emerging area of arsenic metabolic research focuses on understanding of origin of thioarsenicals that are detected as excreted metabolites in many higher organisms, including humans. We have used an in vitro assay to examine the role of the anaerobic microbiota of mouse cecum in the metabolism of thio- and oxoarsenicals1-3. These studies have shown that there is considerable preabsorption metabolism of these arsenicals that is mediated by the anaerobic microbiota of mouse cecum. This presentation will summarize results from our studies on the conversion of dimethylarsinic acid and trimethylarsine oxide with an emphasis on utilizing ESI-MS/MS for identification. In addition, preliminary research on the role of the anaerobic microbiota in conversion of inorganic arsenic and monomethylarsonic acid to thioarsenicals will be presented.

PRESENTATION Population Based Exposure Assessment of Bioaccessible Arsenic in Carrots 06/07/2009
Yathavakilla, S. K., A. R. Young, S. E. Lenhof, M. Mantha, C. GALLAWA, P. A. CREED, J. XUE, AND J. T. CREED. Population Based Exposure Assessment of Bioaccessible Arsenic in Carrots. Presented at International Symposium on Mettalomics, Covington, KY, June 07 - 13, 2009.
Abstract: The two predominant arsenic exposure routes are food and water. Estimating the risk from dietary exposures is complicated, owing to the chemical form dependent toxicity of arsenic and the diversity of arsenicals present in dietary matrices. Two aspects of assessing dietary exposure risk, which are often overlooked in speciation analysis, are producing a bioaccessibility estimate and the need to collect samples which support a population based exposure assessment. In an attempt to address these shortcomings, the authors have applied an enzymatic extraction to carrots which were collected from various geographical locations based on harvest demographics. The extracts were speciated by IC-ICP-MS utilizing collision cell technology to address the high chloride concentrations characteristic of in-vitro assays designed to mimic the gastrointestinal tract. The distribution of the inorganic arsenic concentration in carrots was then combined with the distribution associated with carrot consumption in the US using a probabilistic based model. The model randomly selects an arsenic concentration and consumption rate from the distributions and through an iterative process generates a population based exposure profile for arsenic in carrots. This approach to population based exposure assessment is especially applicable to arsenic because over 90% of the exposure can be attributed to 4-5 commonly consumed foods. This limited number of foods produces a relatively robust estimate without having to independently estimate the contribution of all other foods to the cumulative exposure. This presentation will discuss the limitations associated with the IC-ICP-MS approach and outline the application of the model to this type of exposure assessment.

PRESENTATION Role of Waterborne Pathogens in the Food Supply Chain: Implications to Risk Management With Local and Global Perspectives 06/06/2009
ASHBOLT, N. Role of Waterborne Pathogens in the Food Supply Chain: Implications to Risk Management With Local and Global Perspectives. Presented at IFT Conference, Anaheim, CA, June 06 - 09, 2009.
Abstract: Microbial risk assessment (MRA) in the food industry is used to support HACCP – which largely focuses on bacterial pathogen control in processing foodstuffs 􀂴 Potential role of microbially-contaminated water used in food production is not as well understood 􀂴 Emergence of Quantitative MRA as a tool for assessing & informing waterborne pathogen risks internationally WHO: water safety plans (2004), wastewater reuse (2006) 􀂴 Primary point of this paper is to highlight potential risks from water in food production 1

PRESENTATION Overview of Challenges for Water Quality Protection and Conservation Across the Food Supply Chain and Tools for Assessing Sustainability 06/06/2009
ASHBOLT, N. Overview of Challenges for Water Quality Protection and Conservation Across the Food Supply Chain and Tools for Assessing Sustainability. Presented at IFT Conference, Anaheim, CA, June 06 - 09, 2009.
Abstract: To be presented at the IFT Conference in Anaheim, CA, 06/6-9/2009

PRESENTATION Diversity of Free-Living Amoebae in a Dual Distribution (Potable and Recycled) Water System 06/01/2009
Thomas, J., M. Storey, R. Stuetz, S. Kjelleberg, AND N. ASHBOLT. Diversity of Free-Living Amoebae in a Dual Distribution (Potable and Recycled) Water System. Presented at IWA Health-Related Water Microbiology Biannual Conference, Naxos, GREECE, June 01 - 04, 2009.
Abstract: Free-living amoebae are known to facilitate the growth of water associated pathogens. This study, for the first time, explored the diversity of free-living amoebae in a dual distribution (potable and recycled) water system in Rouse Hill NSW, Australia. Water and biofilm samples were taken from the tertiary recycled water treatment plant and from within the two distribution systems (using a Modified Robbins Device). Amoebae were not detected entering the distribution systems from the tertiary recycled water treatment plant over a 6 week sampling period. However, amoebae were isolated within the recycled water distribution system (0.1 amoebae/ml). Furthermore, amoebae were isolated from within the potable water distribution system (0.2 amoebae/ml). Chlorine concentrations appear to explain the different amoebae numbers. More research is required to determine the relationship between these free-living amoebae and water associated pathogens within this water distribution system.

PRESENTATION Molecular-Based Detection Systems for Cryptosporidium Oocysts 05/20/2009
VILLEGAS, E. Molecular-Based Detection Systems for Cryptosporidium Oocysts. Presented at 2009 USEPA STAR Grants Workshop on Innovative Approaches to Detecting Microorganisms and Cyanotoxins in Water, Philadelphia, PA, May 20 - 21, 2009.
Abstract: The presentation describes on-going studies in collaboration with US EPA Region 2, 3, and the CDC on identifying sources of Cryptosporidium oocyst contamination in source waters using conventional and real-time PCR approaches.

PRESENTATION The Virtual Environmental Microbiology Center a Social Network for Enhanced Communication Between Water Researchers and Policy Makers 05/17/2009
Mistry, J. H., F. S. HAUCHMAN, M. R. RODGERS, AND N. ASHBOLT. The Virtual Environmental Microbiology Center a Social Network for Enhanced Communication Between Water Researchers and Policy Makers. Presented at American Society for Microbiology 109th General Meeting, Philadelphia, PA, May 17 - 21, 2009.
Abstract: Effective communication within and between organizations involved in research and policy making activities is essential. Sharing information across organizational and geographic boundaries can also facilitate coordination and collaboration, promote a better understanding of technical and policy issues, and avoid duplication of effort. To enhance communication within the environmental microbiology community, the U.S. Environmental Protection Agency (EPA) developed the Environmental Science Connector (ESC) (http://portal.epa.gov/ESC ), a web-based communication tool that can bring together people with diverse interests and expertise to share information on a wide range of environmental microbiology topics. It also provides a convenient means for EPA scientists to manage projects, interact with project collaborators through message boards, and participate in real-time web based seminars. The ESC is being used to bring the environmental microbiology community together to share information on a variety of topics. Using the ESC as a platform, a virtual seminar series has been launched to allow subject matter experts in the fields of environmental microbiology and microbial ecology to openly discuss state-of-the-science research topics with network members, further fostering a sense of community and exchange of ideas. In addition, the ESC is providing a focal point for intra-governmental communication and collaboration on several specific research topics, including sample preparation for waterborne pathogens, the development of graywater guidance and microbial risk assessment.

PRESENTATION Utilization of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (Maldi-MS) to Identify Environmental Strains of Mycobacterium Complex 05/17/2009
DONOHUE, M. J., J. H. Mistry, AND S. L. PFALLER. Utilization of Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (Maldi-MS) to Identify Environmental Strains of Mycobacterium Complex. Presented at American Society for Microbiology Annual Meeting, Philadelphia, PA, May 17 - 21, 2009.
Abstract: Species within the Mycobacterium avium Complex (MAC) group are found to be both prevalent and persistent in drinking water distribution systems. The MAC is composed of two predominant species: M. avium and M. intracellulare. These species have the ability to survive drinking water disinfection treatments and to thrive in distribution systems via biofilms. For these reasons, the US Environmental Protection Agency (EPA) has listed MAC on the Contaminant Candidate List (CCL) as a potential contaminant that may require regulation if it is proven to have a significant health burden to the public. A major challenge associated with environmental MAC isolates is the ability to rapidly identify the isolate to the species level. The tools currently available for identification/speciation can be time-consuming, as well as give ambiguous results due to their inability to clearly differentiate species. The purpose of this study was to evaluate Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) as a means to rapidly speciate MAC environmental isolates. The research presented will demonstrate the use of MALDI-MS to speciate environmental isolates based on their m/z signature. Initially, a database of m/z signatures was constructed using MAC reference and type strains. Next, m/z signatures from environmental isolates were compared to the database of known m/z signatures. Additionally, DNA sequence analyses of genes coded for the heat shock protein 65 (hsp65), RNA polymerase subunit B (rpoB), and 16S rRNA were used to validate the MALDI-MS determination. MALDI-MS analysis is ideal for the identification/speciation of environmental isolates. This is primarily due to the minimal sample preparation involved (i.e., the ability to go from culture plate directly to analysis), as well as the short analysis time (<2 min) before a determination can be made. These traits make MALDI-MS a powerful tool suited for environmental monitoring and identification of microbial hazards in drinking water.

PRESENTATION U.S. Environmental Protection Agency and Emerging Contaminants 05/13/2009
GLASSMEYER, S. U.S. Environmental Protection Agency and Emerging Contaminants. Presented at New Jersey institute of Technology Workshop, Newark, NJ, May 13, 2009.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-ug/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. To determine which wastewater chemicals persist through drinking water treatment, a follow-up study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 84 different emerging contaminants, including 24 pharmaceuticals. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-five chemicals were detected at least once, with 28 chemicals detected in the source waters and 23 chemicals detected in the finished waters. The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Surveillance Systems for Waterborne Protozoa: Beyond Method 1623 04/24/2009
VILLEGAS, E. Surveillance Systems for Waterborne Protozoa: Beyond Method 1623. Presented at Greater Cincinnati Water Works Science Advisory Board Meeting, Cincinnati, OH, April 24, 2009.
Abstract: 1. Brief introduction to waterborne Cryptosporidium and Giardia Historical perspective on detecting Cryptosporidium and Giardia Current detection methodologies 2. US EPA’s waterborne protozoan research program Building a “Protozoan Detection Toolbox” 3. Perspectives on the future of the “Protozoan Detection Toolbox” Future directions Factors to consider for developing a pathogen specific detection method

PRESENTATION Comparison of Enterococcus Qpcr Analysis Results from Fresh and Marine Water Samples on Two Real-Time Instruments 04/20/2009
HAUGLAND, R. A., M. VARMA, R. OSHIRO, J. Parr, AND M. Doolittle. Comparison of Enterococcus Qpcr Analysis Results from Fresh and Marine Water Samples on Two Real-Time Instruments. Presented at National Beaches Conference 2009, Huntington Beach, CA, April 20 - 22, 2009.
Abstract: EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method’s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM and StepOnePlusTM series instruments that can retail for under $20 K and provide 48 or 96 sample analysis capacity. In this study we compared the results obtained on a StepOnePlusTM 96 well instrument with those obtained on the Cepheid Smart Cycler® which has been the primary source of the method’s results to date. Analyses were performed simultaneously on DNA extracts from multiple, replicate filter retentates of 12 marine and 12 freshwater samples from diverse locations using study and data analysis designs from EPA's microbial alternate test procedure protocol. Precision among log10 target sequence copy (TSC) estimates in the samples from the two instruments were compared with no significant difference (p > .05) based on the one-way ANOVA of Levene's Test for Homogeneity of Variance. Three–way ANOVA with fixed factors: instrument, matrix, instrument*matrix; and random factors: sample (nested in matrix) and inst*sample (nested in matrix) was used to compare the mean log10 TSC estimates with no significant difference seen between the instruments (p > .05). Given the wide variety of qPCR instruments that are already available and the likelihood that additional advances will occur in instrument technology, this study may provide a useful model for the design and implementation of additional comparative studies in the future.

PRESENTATION What Is the Relative Health Risk to Swimmers from California Seagull Feces Compared to Bather Shedders? 04/20/2009
Schoen, M. AND N. ASHBOLT. What Is the Relative Health Risk to Swimmers from California Seagull Feces Compared to Bather Shedders? Presented at National Beach Conference, Huntington Beach, CA, April 20 - 22, 2009.
Abstract: Estimated infection risks to swimmers from California seagull and bather sources of fecal contamination at a beach in Southern California were compared using quantitative microbial risk assessment (QMRA). The risk to swimmers of gastro-intestinal infections was estimated from Campylobacter jejuni, Cryptosporidium hominis, and Norovirus from human bathers and Campylobacter jejuni from Californian seagulls over the observed range of surfzone enterococci (ENT) concentration during normal summer conditions at Dohney Beach. A beta-Poisson dose-response model was utilized with pathogen specific parameters to calculate the probability of infection using Monte Carlo analysis of the uncertain input variables. Overall, the individual risks from C. jejuni and Norovirus were greater than that from C. hominis. Specific predictions of risk remain uncertain due to large uncertainty in model parameters; particularly the proportion of Campylobacter strains that are human infectious. If the proportion of infectious strains from gulls is low, near 0.01, then human infection risk from accidental ingestion of bathing water containing gull feces is only greater than the risk from bather shedders when gull fecal matter in the bathing waters exceeds that from humans by more than 20 times; that reduces to four times should the proportion of infectious C. jejuni gull strains be 0.05. The best estimate model results indicated that gull fecal-derived enterococci counts contribute less of a health threat to swimmers than human sources; however there remains large uncertainty in prediction due to the remaining uncertainty in human infectious campylobacter species in gull feces, their unknown environmental persistence and the level of bather shedding of human pathogens.

PRESENTATION Qpcr Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches 04/20/2009
CHERN, E. C., K. P. Brenner, L. J. WYMER, AND R. A. HAUGLAND. Qpcr Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches. Presented at EPA National Beaches Conference 2009, Huntington Beach, CA, April 20 - 22, 2009.
Abstract: The use of real-time qPCR to determine fecal indicator bacteria (FIB) densities is currently being investigated by the U.S. EPA. The present recreational water quality guidelines, based on culturable FIB, prevent same day determinations of water quality whereas results from the qPCR method can be available within several hours. Epidemiological studies at POTW-impacted freshwater beaches have shown a strong correlation between qPCR determined Enterococcus densities and swimming-related illness rates. This study provides an initial assessment of qPCR estimated Enterococcus, Bacteroidales, E. coli and Clostridium densities in marine water from two recreational beaches sampled over one summer. The estimated geometric mean cell densities per 100 ml of marine water from both beaches across sampling visits were 3.28 x 10 1, 1.71 x 103, 7.37 x 102, and 9.26 x 102 for Enterococcus, Bacteroidales, E. coli and Clostridium, respectively. These cell equivalent density estimates, determined using whole cell calibrator samples by a comparative cycle threshold (CT) approach, did not correspond with the relative target sequence density estimates of the different FIB in the samples which gave geometric means of 1.28 x 103, 2.35 x 104, 3.04 x 102, and 1.03 x 104 for Enterococcus, Bacteroidales, E. coli and Clostridium, respectively. This discrepancy was determined to be attributable to differences in recovery of target sequences from cells of the different organisms. QPCR analyses using whole cell calibrator samples provides a simple approach for comparing both total cell and target sequence density estimates of different FIB groups in water samples.

PRESENTATION Emerging Contaminants in the Drinking Water Cycle. 04/15/2009
GLASSMEYER, S. Emerging Contaminants in the Drinking Water Cycle. Presented at Region 3 Emerging Contaminants Webcast, The Web, Newark, NJ, April 15, 2009.
Abstract: In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-g/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. To determine which wastewater chemicals persist through drinking water treatment, a follow-up study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 84 different emerging contaminants, including 24 pharmaceuticals. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-five chemicals were detected at least once, with 28 chemicals detected in the source waters and 23 chemicals detected in the finished waters. The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.

PRESENTATION Postmodern Urban Infrastructure Pathogens and Other Scum Associates 03/11/2009
ASHBOLT, N. Postmodern Urban Infrastructure Pathogens and Other Scum Associates. Presented at Johns Hopkins, Washington, DC, March 11, 2009.
Abstract: To be presented at Johns Hopkins, Washington, DC, March 11, 2009

PRESENTATION An Investigation of Bioaccessibility of Arsenic in Rice Using Ic-ICP-MS 03/08/2009
Young, A. R., H. Trenary, S. K. Yathavakilla, P. A. CREED, J. T. CREED, AND J. XUE. An Investigation of Bioaccessibility of Arsenic in Rice Using Ic-ICP-MS. Presented at 2009 Pittcon , Chicago, IL, March 08 - 13, 2009.
Abstract: Arsenic exposure occurs mainly through drinking water and food; therefore, both aspects should be incorporated into any aggregate exposure assessment. Drinking water exposures are predominately inorganic arsenic while dietary exposures are made up of a diverse set of arsenicals with widely varying toxicities. Rice collected throughout the world has been shown to be relatively high in total arsenic. In fact, the FDA’s market basket survey supports this observation and for this reason rice is a target food group for dietary speciation studies. Arsenic speciation has been conducted on rice grown in endemic areas and in the U.S. In these studies, inorganic arsenic and DMA are the predominant arsenicals found in rice; however, the variation associated with the distribution of these two arsenicals is far from a constant. Therefore, a quantitative exposure assessment for rice needs to incorporate not only species specific information, but also the variation associated with the distribution of the arsenicals, in order to improve the exposure risk estimate for rice. Another factor which needs to be considered in the exposure assessment is the biologically relevant arsenic dose associated with rice consumption. Information on bioaccessible arsenic, the fraction of analyte which is solubilized by the gastrointestinal (GI) tract, could provide a means to estimating the biologically relevant dose. Ideally, this bioaccessibility term would also include the GI tract induced biotransformations associated with the ingested arsenicals. Currently, the species specific rice data are mainly comprised of chemically based extractions. These extractions are relatively simple and quantitative, but it is not known how accurately these extractions correlate with bioaccessibility. Therefore, an assay that can estimate the species specific bioaccessibility and capture the biotransformation in the GI tract should improve the exposure estimate. An application of this type of assay should provide data essential to estimating biologically relevant exposures in target foods. This presentation will attempt to estimate the bioaccessible fraction associated with U.S. consumed rice using a synthetic gastrointestinal extraction technique prior to speciation via ICICP-MS. Finally, the potential for biotransformation of the extracted arsenicals will be evaluated by using an in vitro technique in which a gastrointestinally extracted rice sample is incubated in the cecum content of a mouse.

PRESENTATION Rapid Methods for the Detection of General Fecal Indicators 02/11/2009
HAUGLAND, R. A. AND K. OSHIMA. Rapid Methods for the Detection of General Fecal Indicators. Presented at Gulf of Mexico Alliance, Microbial Source Tracking (MST) & Pathogens Detection Workshop, St. Petersburg, FL, February 11, 2009.
Abstract: Specified that EPA should develop: appropriate and effective indicators for improving detection in a timely manner of pathogens in coastal waters appropriate, accurate, expeditious and cost-effective methods for the timely detection of pathogens in coastal waters

PRESENTATION Surveillance Systems for Waterborne Cryptosporidium: US EPA Method 1523 and Beyond 02/08/2009
VILLEGAS, E. Surveillance Systems for Waterborne Cryptosporidium: US EPA Method 1523 and Beyond. Presented at 2009 USDA-CSREES National Water Conference, St. Louis, MO, February 08 - 12, 2009.
Abstract: Waterborne cryptosporidiosis remains a significant public health concern in countries around the world. Many species and genotypes of Cryptosporidium contaminate drinking water sources, but C. parvum and C. hominis remain the two predominant species known to cause waterborne disease outbreaks in humans. To improve human health and reduce risks posed by these pathogens, the US EPA promulgated the Long Term 2 Enhanced Surface Water Treatment Rule (LT2 Rule). This rule requires drinking water utilities to monitor for Cryptosporidium oocysts in their source waters using US EPA Method 1622 or 1623. These methods are designed to enumerate oocysts by microscopy and although very useful in determining concentration levels of Cryptosporidium oocysts in various drinking water sources throughout US, the methods are time consuming, labor intensive and cannot distinguish animal from human specific species or determine if the oocysts are infectious to humans. Because many Cryptosporidium spp. oocysts are morphologically similar and can contaminate drinking water supplies, the development of more specific detection and typing approaches for this parasite are essential to better understand the impact of this protozoan in source waters. The data generated by these methods will provide additional information that will be useful for future source water management strategies and for human health risk assessments related to Cryptosporidium oocyst contamination. Current research activities focused on developing new and more rapid molecular-based approaches (e.g., quantitative real-time PCR, microarrays, etc.) that can detect and determine the infectious potential of oocysts will be described. Advantages and inherent limitations of these approaches and their potential application(s) as alternative methods to current Cryptosporidium surveillance systems will be discussed.

 

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