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Microbiological & Chemical Exposure Assessment Research Division Publications: 2005

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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 2005, organized by Publication Type. Your search has returned 68 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 Investigation of Arsenic Speciation on Drinking Water Treatment Media Utilizing Automated Sequential Continuous Flow Extraction With Ic-ICP-MS Detection 12/30/2005
CREED, P. A., C. A. SCHWEGEL, AND J. T. CREED. Investigation of Arsenic Speciation on Drinking Water Treatment Media Utilizing Automated Sequential Continuous Flow Extraction With Ic-ICP-MS Detection. JOURNAL OF ENVIRONMENTAL MONITORING. Royal Society of Chemistry, Cambridge, Uk, 7(11):1079-1084, (2005).
Abstract: Three treatment media, used for the removal of arsenic from drinking water, were sequentially extracted using 10mM MgCl2 (pH 8), 10mM NaH2PO4 (pH 7) followed by 10mM (NH4)2C2O4 (pH 3). The media were extracted using an on-line automated continuous extraction system which allowed the arsenic in each of the extraction fluids to be speciated on-line using IC-ICP-MS.
The 10mM MgCl2 preferentially extracted As(III) from each of the media. The percentage of the arsenic extracted by the MgCl2, relative to a HNO3/H2O2 digestion of the media, ranged from 0.1-2.3% for the three solids. The next sequential extraction fluid, 10mM NaH2PO4, extracted some of the residual As(III) remaining on each of the media but the predominant species extracted was As(V). The 10mM NaH2PO4 extracted 15.3 to 42.8% of the total arsenic relative to a total digested concentration for each of the media. The As(III) and As(V) stability studies conducted in these two extraction fluids indicated that conversion between As(III) and As(V) was not significant for the short extraction fluid sample contact time associated with the on-line continuous flow extraction cell.

Finally, the 10mM (NH4)2C2O4 extraction fluid was utilized in an off-line analysis mode because the Fe and As concentrations extracted from the media were not compatible with direct ICP-MS detection. The (NH4)2C2O4 extracted 2.9-29% As(III) for all three media and caused an oxidation of As(III) to As(V) during the extraction period for one of the three media.

The sum of the arsenic from each of the three extraction fluids represented 92%, 44% and 53% of the available total arsenic for the three media, respectively. The speciation results for each media were obtained by adding all the speciations results from all three extraction fluids together and the resulting distribution of As(III)/As(V) compared well with the speciation results obtained via XANES.


JOURNAL Chromatographic Separation and Identification of Products from the Reaction of Dimethylarsinic Acid With Hydrogen Sulfide 12/01/2005
FRICKE, M., M. ZELLER, H. SUN, V. W. LAI, W. R. CULLEN, AND J. A. SHOEMAKER. Chromatographic Separation and Identification of Products from the Reaction of Dimethylarsinic Acid With Hydrogen Sulfide. CHEMICAL RESEARCH IN TOXICOLOGY. American Chemical Society, Washington, DC, 18(12):1821-1829, (2005).
Abstract: The reaction of dimethylarsinic acid (DMAV) with hydrogen sulfide (H2S) is of biological significance and may be implicated in the overall toxicity and carcinogenicity of arsenic. The course of the reaction in aqueous phase was monitored and an initial product, dimethylthioarsinic acid, was observed by using LC-ICP-MS and LC-ESI-MS. Dimethylarsinous acid was observed as a minor product. A second slower-forming product was identified and the electrospray mass chromatograms for this species produced ions at m/z 275, 171 and 137 in positive mode. To aid in the identification of this slower-forming product, crystalline standards of sodium dimethyldithioarsinate and dimethylarsino dimethyldithioarsinate were prepared and re-characterized by using improved spectroscopic and structural analysis techniques. An aqueous solution of sodium dimethyldithioarsinate produced a single major chromatographic peak that matched the retention time (7.6 minutes) of the slower-forming product and contained similar molecular ions at m/z 275, 171 and 137 via LC-ESI-MS. The dimethylarsino dimethyldithioarsinate standard produced four aqueous phase species one of which co-eluted with the slower forming product. This co-eluting peak also produced the identical ESI-MS ions as the slower-forming product of DMAV + H2S. ESI-MS/MS experiments conducted on sodium dimethyldithioarsinate in deuterated water produced molecular ions at m/z 276, 173 and 137. Subsequent CAD experiments on m/z 276 did not produce a product ion at m/z 173. This data indicates that two different species are present in solution while NMR data indicates that only dimethyldithioarsinic acid exists in aqueous solutions. This discrepancy was investigated by conducting NMR studies on the acidic solution of sodium dimethyldithioarsinate after taking this solution to dryness. The resolubilized solution produced a proton NMR signal characteristic of dimethylarsino dimethyldithioarsinate. Therefore, it was concluded that the ESI-MS ion at m/z 275 associated with the slowly forming second reaction product and the sodium dimethyldithioarsinate standard is a product of the ESI desolvation process.

JOURNAL Evaluation of Quantitative Real Time Pcr for the Measurement of Helicobater Pylori at Low Concentrations in Drinking Water 11/01/2005
MCDANIELS, A. E., L. J. WYMER, C. C. RANKIN, AND R. A. HAUGLAND. Evaluation of Quantitative Real Time Pcr for the Measurement of Helicobater Pylori at Low Concentrations in Drinking Water. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 39(19):4808-4816, (2005).
Abstract: Aims: To determine the performance of a rapid, real time polymerase chain reaction (PCR) method for the detection and quantitative analysis Helicobacter pylori at low concentrations in drinking water.
Methods and Results: A rapid DNA extraction and quantitative PCR (QPCR) analysis method was evaluated for the measurement of Helicobacter pylori cells on membrane filters at concentrations that might be expected to be found in drinking water samples. A QPCR assay utilizing primers and a TaqManTM hybridization probe targeting the ureA gene of H. pylori was developed for the method. Related, non-target species were detected at approximately a 5 log10 lower level of sensitivity by this assay. A standard curve was generated for the method from analyses of filters containing known numbers of added H. pylori cells. Cell numbers on these filters were determined by staining with species specific fluorescent anti-bodies and solid phase cytometry analyses. The mean sensitivity of the method was 10 H. pylori cells per filter with a 95% confidence sensitivity of 40 cells and a 95% confidence precision interval of +/- 0.57 log10 based on duplicate analyses of individual samples. One liter drinking water samples from several diverse locations in the U.S. all tested negative by the method. Filtrates of additional one liter portions of these samples spiked with the same H. pylori cell suspensions used for the standard curve samples gave results that were consistent with the standard curve suggesting that these sample matrices produced no interference in the method.

Conclusions: The QPCR analysis method, described in this study, can reliably detect as few as 40 H. pylori cells from membrane filtrates of drinking water samples and can determine the numbers of these cells within approximately a 1 log10 interval based on analyses of individual samples.

Significance and Impact of the Study: This method may be useful for the rapid screening of drinking water in the USA for H. pylori.


JOURNAL Comparison of Populations of Mould Species in Homes in the Uk and US Using Mold-Specific Quantitative Pcr (Msqpcr) 10/06/2005
VESPER, S. J., L. J. WYMER, R. STOTT, M. RICHARDSON, AND R. A. HAUGLAND. Comparison of Populations of Mould Species in Homes in the Uk and US Using Mold-Specific Quantitative Pcr (Msqpcr). Letters in Applied Microbiology. Blackwell Publishing, Malden, MA, 41(4):367-373, (2005).
Abstract: The goal of this research was to compare the populations of 81 mold species in homes in USA and UK using mould specific quantitative polymerase chain reaction (MSQPCR) technology. Dust samples were obtained from randomly selected homes in Great Britain (n=11). The mould populations in British homes were compared with those found in typical homes (no visible mold) in the USA (in the state of Ohio, n = 45). Only thirteen of the 81 species screened showed significantly different populations in these two sets of home. Although only a small survey, the results suggest that typical mould profiles in USA (Ohio) and British homes are very similar. Analysis of 26 mould Indicator species revealed that the British homes fell into 2 clusters, tentatively identified as "atypical" and "typical" mould conditions.

JOURNAL The Development of a Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry-Based Method for the Protein Fingerprinting and Identification of Aeromonas Species Using Whole Cells 09/19/2005
DONOHUE, M. J., W. SMALLWOOD, S. L. PFALLER, M. R. RODGERS, AND J. A. SHOEMAKER. The Development of a Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry-Based Method for the Protein Fingerprinting and Identification of Aeromonas Species Using Whole Cells. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 65(3):380-389, (2006).
Abstract: This report describes the development of a method to detect the waterborne pathogen Aeromonas using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).

JOURNAL Cyanotoxins: New Generation of Water Contaminants 09/01/2005
ANTONIOU, M. G., A. A. DELACRUZ, AND D. D. DIONYSIOU. Cyanotoxins: New Generation of Water Contaminants. JOURNAL OF ENVIRONMENTAL ENGINEERING. American Society of Civil Engineers (ASCE), Reston, VA, 131(9):1239-1243, (2005).
Abstract: Cyanobacteria, more commonly known as blue-green algae, are found worldwide in various aquatic environments as well as in water distribution systems (Atikovic 2003; Carmichael 1994; Madigan et al. 2003). Blooms of cyanobacteria have recently become spatially and temporally more prevalent in the United States and worldwide as a consequence of increasing nutrient levels such as nitrates and phosphates from fertilizers and detergents. Cyanobacterial blooms impart color, odor, and taste problems in water. More importantly, such blooms produce and release toxic compounds that dramatically impair the quality of water bodies. Up to 50% of the recorded blooms can be expected to contain toxins (Carmichael 1992). These compounds have severe and sometimes irreversible effects on mammalian health. Episodes of human and animal poisoning by consumption of water contaminated with cyanobacterial toxins have been reported since the late 1800s (Carmichael 1994). Exposure to cyanobacterial toxins can affect the number and diversity of wild animal populations, cause bioaccumulation of toxins in the tissues of fish and shellfish, and indirectly affect other organisms through the food chain. Moreover, the presence of cyanobacteria and cyanobacterial toxins in sources of drinking water supply has raised major concerns. Another major issue is the lack of guidelines or regulations of cyanobacteria and cyanotoxins in terms of maximum contaminant level (MCL) and analytical detection methods. In the past few years, major research effort has been targeted toward the treatment of these toxins, especially the hepatotoxin microcystin-LR (MC-LR).

JOURNAL Comparison of Rapid Methods to Evaluate Chlorine Inactivation of the Biological Agent E. Coli 0157:H7 08/01/2005
MCDANIELS, A. E., L. J. WYMER, AND R. A. TALLEY. Comparison of Rapid Methods to Evaluate Chlorine Inactivation of the Biological Agent E. Coli 0157:H7. JOURNAL OF WATER SUPPLY: RESEARCH AND TECHNOLOGY - AQUA. IWA Publishing, London, Uk, 54(5):313-319, (2005).
Abstract: Rapid viability tests of the Catagory B agent Escherichia coli O157:H7 were evaluated after disinfection with chlorine. The metabolic activity dyes ChemChrome V6, a modified fluorescein diacetate (FDA) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) were compared to standard plate counts. ChemChrome V6 results were obtained using a solid phase cytometer and CTC results by microscopic analysis. The water-borne bacteria Legionella pneumophila and Mycobacterium avium were also tested as positive and negative controls. Under the conditions tested, CTC provided more consistency with plate count estimates of viability than did ChemChrome V6.

JOURNAL Comparison of a Chemical and Enzymatic Extraction of Arsenic from Rice and An Assessment of the Arsenic Absorption from Contaminated Water By Cooked Rice 07/15/2005
ACKERMAN, A., P. A. CREED, A N. Parks, M. FRICKE, C A. Schwegel, J T. Creed, D. T. Heitkemper, AND N. Vela. Comparison of a Chemical and Enzymatic Extraction of Arsenic from Rice and An Assessment of the Arsenic Absorption from Contaminated Water By Cooked Rice. ENVIRONMENTAL SCIENCE AND TECHNOLOGY. John Wiley & Sons, Ltd., Indianapolis, IN, 39(14):5241-5246, (2005).
Abstract: Rice represents a unique set of arsenic exposure assessment challenges in that it contains relatively high concentrations of arsenic and it absorbs about 100% of its dry weight in water during cooking. The actual arsenic exposure from rice consumption becomes difficult to calculate without knowing the arsenic concentration in rice before cooking, the arsenic concentration of the water, the percentage of arsenic absorbed by rice from water during the cooking process, and the bioavailability of the arsenic in cooked rice. While the first two unknowns are relatively straightforward to determine, the last two are unknown quantities and require further study.
Arsenic extraction efficiencies ranged from 83.7 to 103.5% for cooked rice samples using a trifluoroacectic acid (TFA) extraction procedure. Rice samples cooked in water containing no arsenic and in water containing a known concentration of arsenic were both analyzed. Chromatographic recoveries, measuring the percent of arsenic injected on the column that was determined chromatographically, ranged from 98.7 to 116.2%. Absorption of arsenic by rice from the total volume of water used in cooking was between 88.8 and 104.5% for two different contaminated drinking water samples. A detailed statistical analysis found replicate standard deviations for individual matrices to be between 2.0 and 4.0% for extraction efficiency, chromatographic recovery, and overall recovery. However, matrix standard deviation ranged between 4.6 and 6.2%, indicating that the rice matrix contributed the largest percentage of the variation. A comparison of TFA extraction to an enzymatic extraction produced overall recoveries of 96.6 and 93.3%, respectively. However, the presence of an unidentified arsenic species and an Ar40Cl35 peak in the chromatograms of the enzymatic extracts necessitated a significantly longer analysis time for those samples.

Per capita daily rice consumption rates in various countries range between 25 and 410 g, on an uncooked, dry-weight basis. The levels of inorganic of arsenic that would be found in these amounts of rice after cooking were calculated and compared to the U.S. drinking water maximum contaminant level (MCL) for inorganic arsenic. Finally, the inorganic arsenic exposure from rice was calculated based on mean consumption rates from a number of different countries. These ezposures were compared to the new drinking water MCL of 10 ppb using a 2L/day consumption rate.

JOURNAL Comment on "DERIVATION of Numerical Values for the World Health Organization Guidelines for Recreational Waters" 07/01/2005
Wymer, L J., A P. Dufour, R L. Calderon, T J. Wade, AND M. Beach. Comment on "DERIVATION of Numerical Values for the World Health Organization Guidelines for Recreational Waters". WATER RESEARCH 39(12):2774-2777, (2005).
Abstract: The subject paper describes a procedure for adjusting a risk model based upon a measure of personal exposure (the "UK personal exposure model") in order to attribute an expected rate of gastroenteritis among a group of swimmers to a mean recreational water quality value (enterococci per 100 mL). We term the resulting model for group risk the "UK ecologic exposure model." The distinction is essential to establishing recreational water quality guidelines because exposures of individual bathers are not known from a water monitoring program, the only assessment available being some form of ecologic exposure such as a mean log indicator density. While the authors of the subject paper solved the UK ecologic exposure model for only a single point (that value of mean log10 enterococcus density which is expected to result in five extra cases of gastroenteritis per 100 swimmers), we extend their model to show the entire curve over a relevant range of densities. The resulting exposure-response curve is seen to not differ substantially from the existing USEPA model for "highly credible gastrointestinal illness" in marine waters.
However, particularly since such correspondence is not guaranteed for future studies or for other existing epidemiological studies, we recommend the direct approach to evaluating ecologic exposure, such as used in the USEPA studies, rather than the indirect approach of the UK ecologic exposure model, given the number of untested assumptions that are necessary for accomplishing the latter.

JOURNAL Hemolysin, Chrysolysin from Penicillium Chrysogenum Promotes Inflammatory Response 07/01/2005
Donohue, M J., Y. Chung, M L. Magnuson, M. W. Ward, M K. Selgrade, AND S J. Vesper. Hemolysin, Chrysolysin from Penicillium Chrysogenum Promotes Inflammatory Response. INTERNATIONAL JOURNAL OF HYGIENE AND ENVIRONMENTAL HEALTH. Urban & Fischer Verlag Jena, Jena, Germany, 208(4):279-285, (2005).
Abstract: Some strains of Penicillium chrysogenum produce a proteinaceous hemolysin, chrysolysin, when incubated on sheep's blood agar at 37 �C but not at 23 �C. Chrysolysin is an aggregating protein composed of approximately 2 kDa monomers, contains one cysteine amino acid, and has an isoelectric point of 4.85. Treatment of murine macrophage cell line RAW 264.7 with purified chrysolin caused statistically significant (T-test, p<0.05) increased productionj of macrophage inflammatory protein-2 (MIP-2) in a dose dependent manner after 6 h treatment. This suggests that chrysolysin might act to promote the host's inflammatory response after P. chrysogenum exposures.

JOURNAL Dose-Dependent Allergic Responses to An Extract of Penicillium Chrysogenum in Balb/Mice 04/01/2005
CHUNG, Y., N. HAYKAL-COATES, M. E. VIANA, L. B. COPELAND, S. J. VESPER, M. K. SELGRADE, AND M. D. WARD. Dose-Dependent Allergic Responses to An Extract of Penicillium Chrysogenum in Balb/Mice. TOXICOLOGY. Elsevier Science Ltd, New York, NY, 209(1):77-89, (2005).
Abstract: Indoor mold has been associated with the development of allergic asthma. Penicillium chrysogenum, a common indoor mold, is known to have several allergens and can induce allergic responses in a mouse model of allergic penicilliosis. Our hypothesis is that soluble components of P. chrysogenum (PCE) can dose-dependently induce responses typical of allergic asthma in BALB/c mice. Mice were exposed to 10, 20, 50, or 70 µg of PCE by involuntary aspiration 4 times over a 4 week period. Serum and bronchoalveolar lavage fluid (BALF) were collected before (Day 0), and at Day 1 and 3 following the final exposure. PCE-exposed mice demonstrated dose-dependent increases in: BALF total cell numbers including eosinophil, serum and BALF total IgE levels, BALF IL-5 levels, and increased severity of histopathologic lesions. A single exposure to the highest dose of PCE resulted in edema and cellular damage but not immune responses. Four exposures to Metarhizium anisopliae crude antigen (10 µg, positive control) resulted in equivalent or greater allergic asthma-like responses than those demonstrated by multiple exposures to 50 or 70 µg of PCE. Multiple exposures to 70 µg of PCE showed increased allergen-triggered immediate respiratory responses as well as non-specific airway hyperresponsiveness to methacholine as assessed by barometric whole-body plethysmography. Taken together, repeated pulmonary challenge with P. chrysogenum extract induced dose-dependent allergic asthma-like responses in mice.

JOURNAL Dose-Dependent Allergic Responses to An Extract of Penicillium Chrysogenum in Bal/C Mice 04/01/2005
CHUNG, Y., N. HAYKAL-COATES, M. VIANA, L. B. COPELAND, S. J. VESPER, M. K. SELGRADE, AND M. D. WARD. Dose-Dependent Allergic Responses to An Extract of Penicillium Chrysogenum in Bal/C Mice. TOXICOLOGY. Elsevier Science Ltd, New York, NY, 209(1):77-89, (2005).
Abstract: Indoor mold has been associated with the development of allergic asthma. Penicillium chrysogenum, a common indoor mold, is known to have several allergens and can induce allergic responses in a mouse model of allergic penicilliosis. Our hypothesis is that soluble components of P. chrysogenum (PCE) can dose-dependently induce responses typical of allergic asthma in BALB/c mice. Mice were exposed to 10, 20, 50, or 70 µg of PCE by involuntary aspiration 4 times over a 4 week period. Serum and bronchoalveolar lavage fluid (BALF) were collected before (Day 0), and at Day 1 and 3 following the final exposure. PCE-exposed mice demonstrated dose-dependent increases in: BALF total cell numbers including eosinophil, serum and BALF total IgE levels, BALF IL-5 levels, and increased severity of histopathologic lesions. A single exposure to the highest dose of PCE resulted in edema and cellular damage but not immune responses. Four exposures to Metarhizium anisopliae crude antigen (10 µg, positive control) resulted in equivalent or greater allergic asthma-like responses than those demonstrated by multiple exposures to 50 or 70 µg of PCE. Multiple exposures to 70 µg of PCE showed increased allergen-triggered immediate respiratory responses as well as non-specific airway hyperresponsiveness to methacholine as assessed by barometric whole-body plethysmography. Taken together, repeated pulmonary challenge with P. chrysogenum extract induced dose-dependent allergic asthma-like responses in mice.

JOURNAL Comparison of Enterococcus Measurements in Freshwater at Two Recreational Beaches By Quantitative Polymerase Chain Reaction and Membrane Filer Culture Analysis 02/01/2005
Haugland, R A., S D. Siefring, L J. Wymer, K Brenner, AND A P. Dufour. Comparison of Enterococcus Measurements in Freshwater at Two Recreational Beaches By Quantitative Polymerase Chain Reaction and Membrane Filer Culture Analysis. WATER RESEARCH 39(4):559-568, (2005).
Abstract: Cell densities of the fecal pollution indicator genus, Enterococcus, were determined by a rapid (2-3 hr) quantitative PCR (QPCR) analysis based method in 100 ml water samples collected from recreational beaches on Lake Michigan and Lake Erie during the summer of 2003. Enumeration results by this method were compared with counts of Enterococcus.colony forming units (CFU) determined by filter plating on mEI-agar selective medium. The QPCR method showed a 95% confidence, minimum detection limit of approximately two Enterococcus cells per sample in analyses of undiluted DNA extracts and highly accurate quantitation of spiked lake water samples based on the geometric means of replicate sample results. Geometric means of Enterococcus densities, determined from multiple collection points during each sampling visit, showed approximately lognormal distributions at both beaches using both QPCR and culture analyses. These geometric means ranged from 10 to 8548 cells by QPCR analysis and 1 to 2499 CFU by culture analysis in Lake Michigan (N = 56) and from 8 to 8695 cells by QPCR analysis and 3 to 1941 CFU by culture analysis in Lake Erie (N = 47). Regression analysis of these results showed a high level of agreement between the two methods with an overall correlation coefficient of 0.68.

JOURNAL Mold-Specific Quantitative Pcr: the Emerging Standard in Mold Analysis 01/01/2005
Vesper, S J., D. Kahane, AND R A. Haugland. Mold-Specific Quantitative Pcr: the Emerging Standard in Mold Analysis. AMERICAN LABORATORY 37(1):10-11, (2005).
Abstract: Molds can cause health problems like infections and allergies, destroy crops, and contaminate our food or pharmaceuticals. We can't avoid molds. Molds are essential players in the biological processes on earth, but we can now identify and quantify the molds that will be most problematic and remove them in a timely manner. Through the recent development and application of the technique of mold-specific quantitative polymerase chain reaction (MSQPCR), we can identify indoor molds accurately and with such quantifying precision that predictive models are emerging that can help define abnormal mold conditions.

JOURNAL Effects of Chlorination on the Persistence of Pharmaceuticals in the Environment 01/01/2005
Glassmeyer, S AND J A. Shoemaker. Effects of Chlorination on the Persistence of Pharmaceuticals in the Environment. BULLETIN OF ENVIRONMENTAL CONTAMINATION AND TOXICOLOGY 74(1):24-31, (2005).
Abstract: In the past decade, the identification of pharmaceuticals in surface (Jones et al. 2001; Ternes 1998), ground (Sacher et al. 2001) and drinking (Heberer 2002) waters has attracted the attention of both the scientific and lay communities. Although the concentrations of these compounds are very small (typically less than 1 g/L), their presence is a reminder that many people's drinking water was once another community's wastewater. To date, the majority of the studies looking for pharmaceutical compounds have been conducted in Europe. Due to the differences in drinking and wastewater treatment technologies, as well as the dosage level and types of pharmaceuticals administered, it is difficult to estimate the concentrations of pharmaceuticals present in the waters of the United States based on European data. The United States Geological Survey (USGS) has recently conducted the first nationwide reconnaissance of pharmaceuticals (both human and veterinary) and other wastewater components in the United States (Kolpin et al. 2002). They found at least one of their target non-prescription drugs in approximately 80% of the 139 streams sampled. At least one prescription drug was found in over 30% of the streams and at least one antibiotic was found in over 50% of the sampled locations. While the sample locations were biased towards areas that were expected to yield detections (such as downstream from urban centers and areas if dense livestock populations), this study demonstrates the pervasiveness of pharmaceuticals in the aquatic environment of the United States.
Due to the low concentrations of pharmaceuticals in the environment, most of the mass spectrometry methods that have been developed only measure known target compounds. These methods only monitor selected ions, which helps to improve detection limits. However, this improvement comes at a price. Since only specified ions are monitored, in studies where the influent and effluent of waste and drinking water treatment plants were analyzed, only removal efficiencies can be reported (Ternes 1998; Ternes et al. 2002; Zwiener and Frimmel 2000). The ultimate fate of these compounds, that is whether they were degraded into something harmless, or potentially transformed into something more toxic, cannot be determined.

In the United States, chlorine is commonly used to disinfect sewage, as well as drinking water (USEPA 1999a). Although this procedure removes pathogens, the chlorine can also react with compounds in the water. As past research on disinfection byproducts in water has shown, this addition of chlorine may increase the toxicity of the compounds (Clark et al. 2001; Nieuwenhuijsen et al. 2000). Thus, if the removal of pharmaceuticals in waste and drinking water treatment plants is due to the chlorination of the compounds, the potential negative human health impacts may be increased rather than decreased. Mass spectrometry is useful in chlorination experiments, because it can easily detect the formation of new degradation products which contain chlorine atoms.

The purpose of this work was to determine via benchtop experiments the fate of pharmaceuticals during chlorination. This information can be used to focus occurrence studies on the analytes that have a higher probability of persisting through treatment (and thus being found in the environment), as well as the disinfection/ degradation byproducts that might be unknowingly produced.

NON-EPA PUBLISHED PROCEEDINGS Methods to Classify Environmental Samples Based on Mold Analyses By Qpcr 01/01/2005
Haugland, R A., T Meklin, M Varma, L Wymer, AND S J. Vesper. Methods to Classify Environmental Samples Based on Mold Analyses By Qpcr. 5th International Conference on Bioaerosols, Fungi, Bacteria, Mycotoxins and Human Health, Saratoga Springs, NY, September 10 - 12, 2003. E. Johanning (ed.), Boyd Printing Company (B Print Services, Inc.), Albany, NY 327-334, (2005).
Abstract: Quantitative PCR (QPCR) analysis of molds in indoor environmental samples produces highly accurate speciation and enumeration data. In a number of studies, eighty of the most common or potentially problematic indoor molds were identified and quantified in dust samples from homes. Among the molds identified and quanitfied are 25 species of Aspergillus and 36 species of Penicillium. Other molds in the analysis include Alternaria alternata, Stachybotrys chartarum, Chaetomium globosum, 3 species of Cladosporium, 3 species of Ulocladium, 3 species of Trichoderma and many others. The primer and probe sequences for all assays are published in US Patent No. 6,387,652. This technology is now being used commercially by nine licensed companies in the US and one in the UK. The mold data from this study were analyzed by cluster analysis based on quantitative species occurrence. Cluster analysis was also performed based on water requirements (low, medium, high) for growth of each of the molds. Finally, a weighted diversity index was developed to classify each sample. QPCR results provide a unique insight into the mold characterization of homes and may provide a way to define a home's mold condition as "outside the norm".

PRESENTATION The Application of Whole Cell Bacterial Analysis to Environmental Challenges 12/08/2005
SHOEMAKER, J. A. The Application of Whole Cell Bacterial Analysis to Environmental Challenges. Presented at ASMS Fall Workshop, San Diego, CA, December 08 - 09, 2005.
Abstract: The U.S. EPA National Exposure Research Laboratory's proteomic research efforts was presented at the American Society for Mass Spectrometry's 2005 Fall Workshop on Mass Spectrometry of Microorganisms. The U.S. EPA is investigating the potential of using this technique as a way to rapidly identify Aeromonas species in drinking water. A number of bacteria, including Aeromonas hydrophila, are listed on EPA's 1998 Contaminant Candidate List (CCL) as research needs. To assess if matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) can be used to identify species of Aeromonas, as well as predict the strain's possible pathogenicity, the project is broken down into three major objectives: 1) to create a mass spectral library of known Aeromonas strains, 2) identify unknowns isolates, and 3) identify m/z ions that may be used as biomarkers of human pathogenicity.
A library of mass spectral fingerprints of a number of Aeromonas strains was generated using MALDI-MS. In addition to investigating virulence factors, this mass spectral library was used to rapidly species/strain differentiate using MALDI-MS. Unique masses were observed in each of the Aeromonas isolates providing a means to differentiate between Aeromonas species. Mass lists were created from each strain's spectrum and cluster analysis performed using Phylogenetic Analysis Using Parsimony* (PAUP*).

A blind study was done using 51 isolates from a collection of 205 isolates obtained from drinking water distribution systems. Of the 51 isolates used in this study, traditional biochemical analyses were only able to confidently identify 25 isolates, and tentatively identify the other 26 isolates. Species identification for the isolates was accomplished by comparing mass spectral fingerprints of the isolates against well characterized Aeromonas ATCC strains using the PAUP software. The MALDI-MS analysis of the water distribution samples correlated 100% with the biochemical identification of the 25 confirmed isolates. Of the 26 isolates that were only tentatively identified, MALDI-MS analysis was able to identify, with a 95% confidence, 15 of the 26 isolates.

Animal studies are being conducted to identify virulent and avirulent strains of the isolates obtained from drinking water distribution systems. This research, along with the MALDI-MS analysis of the isolates, will be used to identify potential biomarkers of human pathogenicity. Once these biomarkers are identified, further research will be conducted to identify the proteins unique to virulent Aeromonas using MALDI-MS mass spectral fingerprints. Electrospray (ESI)-MS/MS will be used then to sequence the virulent proteins.

PRESENTATION US Environmental Protection Agency's Pathogen Research 12/08/2005
GRIMM, A. US Environmental Protection Agency's Pathogen Research. Presented at Global Water Research Coalition, Paris, FRANCE, December 08 - 09, 2005.
Abstract: Overview of the Pathogen Rsearch Program

PRESENTATION Evaluating in Vitro Infectivity for Measuring UV Disinfection of Cryptosporidium Parvum Oocysts in Finished Water 11/06/2005
BUKHARI, Z., D. M. HOLT, M. W. WARE, AND F. W. SCHAEFER. Evaluating in Vitro Infectivity for Measuring UV Disinfection of Cryptosporidium Parvum Oocysts in Finished Water. Presented at Water Quality Technology Conference, Quebec, QC, CANADA, November 06 - 10, 2005.
Abstract: UV technology to inactivate Cryptosporidium parvum oocysts has become well established in the US. The challenge now is to effectively demonstrate UV reactor performance and disinfection capacity with various finished water matrices and under different operational conditions. In such evaluations, surrogate organisms such as MS2 bacteriophage are finding acceptance in the US; however, it remains imperative that reactor validation studies continue to include parallel bench scale UV disinfection studies using C. parvum oocysts in finished water. In this manner, the impact of chemicals constituents, used in the abatement of taste and odor or in the enhancement of filtration and/or coagulation, on the effectiveness of UV light for inactivation of C. parvum oocysts can be monitored. With this information the appropriate applied UV dose needed to achieve a target disinfection credit can be determined.
Currently the neonatal mouse infectivity assays are considered the method of choice for measuring oocyst inactivation. These procedures require specialized animal experimentation licenses and animal care facilities and usually require 7 to 10 days to generate results. In addition to the time consuming nature of these procedures, the cost of animal experimentation makes their routine use prohibitive. Alternative methods, such as in vitro viability assays using fluorogenic vital dyes or in vitro excystation assays, perform poorly in predicting inactivation of C. parvum oocysts following UV disinfection. In addition, currently only anecdotal evidence exists for the effectiveness of in vitro infectivity assays in accurately predicting inactivation of UV treated C. parvum.

The utility of the in vitro infectivity assay was evaluated with "blind" spike doses of C. parvum oocysts that were enumerated by flow cytometry. At the American Water laboratories, these unknown spike doses of C. parvum oocysts were administered to a cell culture assay utilizing human adenocarcinoma (HCT-8) cells and were detected by immunofluorescence (IFA) microscopy. Data from these "blind" trials indicated that the optimized cell culture-IFA procedure yielded highly reproducible and accurate results, thereby confirming the findings reported by Bukhari and LeChevallier (2003). The next phase of these "blind" trials is to evaluate this assay for measuring the infectivity of UV treated C. parvum oocysts. The successful completion of these trials will help to ensure that a standardized procedure is available for the water industry to use as a tool for conducting UV disinfection studies on C. parvum oocysts suspended in different finished water matrices.

PRESENTATION Protecting Health With Same Day Water Quality Monitoring Results for Bathing Beaches 11/02/2005
WADE, T. J., R. L. CALDERON, M. J. BEACH, E. A. SAMS, K. P. BRENNER, AND A. P. DUFOUR. Protecting Health With Same Day Water Quality Monitoring Results for Bathing Beaches. Presented at Annual Meeting, Great Lakes Beach Association, Green Bay, WI, November 02 - 03, 2005.
Abstract: Current US Environmental Protection Agency guidelines recommend the use of cultural methods for E. coli and enterococci to monitor beach water quality. The guidelines recommend a single sample value or a geometric mean value from at least five samples. The single sample guideline is used by many authorities who wish to monitor bathing beach waters on a daily basis. Methods used to monitor beach waters on a daily basis, however, produce results in 24 hours, long after swimming exposures occur. This shortcoming in current monitoring practices for measuring beach water quality has led EPA to develop new analytical technology and indicators that will provide rapid measurement of beach waters.

PRESENTATION Persistence of Pharmaceuticals and Other Wastewater Related Compounds: Utility as Indicators of Human Fecal Contamination 10/31/2005
GLASSMEYER, S., E. L. FURLONG, D. L. KOLPIN, J. D. CAHILL, S. D. ZAUGG, S. L. WERNER, AND D. D. KRYAK. Persistence of Pharmaceuticals and Other Wastewater Related Compounds: Utility as Indicators of Human Fecal Contamination. Presented at 2005 Sustainable Beaches Conference, St. Petersburh, FL, October 31 - November 02, 2005.
Abstract: The quality of drinking and recreational water is currently ascertained using indicator bacteria. The tests to analyze for these bacteria require a considerable length of time to complete, and do not discriminate between human and animal fecal material sources. To shorten the time needed to test water quality and distinguish the fecal pollution source, chemicals found in human wastewater, such as pharmaceuticals, surfactants, and fecal sterols, can be used as tracers of human fecal pollution. These chemicals would have the advantage of requiring shorter analysis times, and can be selected to be human specific. At ten locations, water samples were collected upstream, and at two successive points downstream from a wastewater treatment plant. A treated effluent sample was also collected at each location. This longitudinal sampling scheme was used to determine the persistence of the target compounds in streams. The water samples were extracted using either solid phase or liquid-liquid extraction and were analyzed using either liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry. Of the 110 chemical analytes investigated in this project, 78 were found in at least one sample. Seventeen of the 110 compounds were classified as either a prescription or non-prescription pharmaceutical. Of these, nine were found in at least 50 % of the samples and thirteen were found in at least 10 % of the samples. While most concentrations of all of the compounds were in the range of 0.1 to 1.0 ?g/ L, in some of the more highly contaminated samples, concentrations were in the range of 5-20 ?g/ L. The concentrations of the majority of the chemical compounds present in the samples generally followed an expected trend: 1) they were non-existent or at only trace levels in the upstream samples, 2) had their maximum values in the wastewater effluent samples, and 3) declined in the two downstream samples. This work indicates that these chemical analytes do have utility as tracers of human wastewater discharge.

PRESENTATION The Empact Beaches Project a Study of the Parameters That Affect Microbiological Monitoring 10/31/2005
BRENNER, K. P. The Empact Beaches Project a Study of the Parameters That Affect Microbiological Monitoring. Presented at 2005 Sustainable Beaches Conference, St. Petersburg, FL, October 31 - November 02, 2005.
Abstract: The current U. S. Environmental Protection Agency (EPA) water sample collection method for recreational water was suggested by the Federal Water Pollution Control Agency in 1968 as part of the fecal coliform guideline. Although improvements have been made in the indicator bacteria detection methods and health guidelines, the old method of collecting samples continues to be used. The objective of the EMPACT (Environmental Monitoring for Public Access and Community Tracking) Beaches Study, carried out by EPA and the EMPACT cities and laboratories, was to determine the best way to monitor recreational water (i.e., how many samples to collect, where and when the samples should be collected, and how the data should be analyzed) to assist beach managers in developing site-specific monitoring protocols for their beaches. The water from five representative beaches was examined using an extensive sample collection protocol and the currently approved indicator methods for Escherichia coli (2 freshwater beaches) and Enterococci (3 marine and/or estuarine water beaches). Ancillary measurements, such as pH, turbidity, total suspended solids, rainfall, weather conditions, tides and/or currents, number of bathers and animals in the water and on the beach, debris, and boats near the beach, were also recorded. The results showed that the greatest single influence on beach water sampling was the distance from the shoreline at which the sample was taken. Time of day and environmental factors, such as sunshine, rain, wind and tides, were also found to affect water quality and must be considered in the interpretation of monitoring results. The information from this study will be used by the EPA Office of Water to develop a set of official monitoring guidelines that will assist the beach managers to develop their own site-specific monitoring protocols and to make time-relevant, understandable water quality data information available to the public.

PRESENTATION Children's Dietary Exposures to Chemical Contaminants 10/30/2005
MELNYK, L. J. AND C. E. BERNARD. Children's Dietary Exposures to Chemical Contaminants. Presented at ISEA 2005 Annual Conference, Tucson, AZ, October 30 - November 03, 2005.
Abstract: The Food Quality Protection Act of 1996 requires EPA to more accurately assess children's aggregate exposures to environmental contaminants. Children have unstructured eating behaviors which cause excess exposures as a result of their activities. Determining total dietary intake is an important factor in assessing children's aggregate exposures. A deterministic model was developed to more accurately estimate dietary intake of a chemical contaminant by young children. The sum of three terms determine total intake: the original contaminant residue on food, surface-to-food contamination, and surface-to-hand-to-food contamination. Transfer of the contaminant from surfaces (including hands) to food and the activity level of the child are the dominant factors of exposure for food consumed in highly-contaminated environments. Pesticide transfer from surfaces to foods has been measured to establish relationships between different pesticide classes, surface types, and contact duration. Transfer efficiencies from hardwood flooring surfaces were 30 to 57% of applied pesticide concentrations. The corresponding transfer efficiencies from carpet were much less. Transfer to foods was greater with both increased contact force and duration. Limited data are available for assessing factors associated with children?s dietary activities. Videotaping allows frequencies and durations of hand and food contacts to be recorded and translated by a computerized software program. However, video analysis is labor intensive and new approaches to define children's activity patterns are being evaluated including accelerometers, handling of a standard food, and questionnaires. Research directed at developing measurement approaches for evaluating children?s dietary exposures, including factors associated with additional contamination caused by surface contacts and eating activities, will be presented.

PRESENTATION Recreational Water Quality and Swimming Associated Health Effects 10/07/2005
DUFOUR, A. P. Recreational Water Quality and Swimming Associated Health Effects. Presented at 22nd Annual NCASM Fall Meeting, San Ramon, CA, October 07 - 08, 2005.
Abstract: The U.S. EPA's National Epidemiological and Environmental Assessment of Recreational Water study is currently underway with the goal of determining if new rapid methods for measuring water quality can be used to predict illness in swimmers. This lecture will provide a historical perspective on the evolution of water quality standards and the rationale for risk-based regulations for recreational waters.

PRESENTATION Recovery and Assay of Viruses from Biosolids 09/20/2005
FOUT, G. Recovery and Assay of Viruses from Biosolids. Presented at Pathogen Equivalency Committee Retreat, College Corner, OH, September 20 - 21, 2005.
Abstract: In a review of EPA's rule on sewage sludges, the National Research Council recommended that EPA develop improved methods for pathogens using modern technologies. They specifically mentioned viral pathogens that cannot be cultured and those that are difficult to culture. As a result of this recommendation, EPA funded work under Contract # 68-C-00159 with the University of Cincinnati to develop "Improved Methods for Virus Detection in Biosolids." This talk describes the areas and status of the research that is being conducted under the work assignment.

PRESENTATION Cyanobacterial Toxins and 2005 Isochab Exposure Assessment Workgroup 09/11/2005
DELACRUZ, A. A. Cyanobacterial Toxins and 2005 Isochab Exposure Assessment Workgroup. Presented at Association of Analytical Communities (AOAC) International Meeting, Orlando, FL, September 11 - 15, 2005.
Abstract: The US EPA, Office of Research and Development, in collaboration with other US federal agencies, is leading the organization of an International Symposium on Cyanobacterial Harmful Algal Blooms on 6-10 September, 2005. The goal of this symposium is to develop a comprehensive national research program on cyanobacteria to meet the mandates of the Harmful Algal Bloom and Hypoxia Research and Control Act (HABHRCA) and to assist the US EPA, Office of Water in making regulatory determinations on cyanobacteria and cyanobacterial toxins. The Agency has yet to publish guidelines or issue regulatory determinations on cyanobacteria or cyanobacterial toxins due to limited exposure and risk assessment data. The symposium is divided into six session topics: Toxins; Exposure Assessment; Occurrence; Effects; Causes, Prevention and Mitigation; and Risk Assessment. This presentation will focus on exposure assessment which deals with methods to detect cyanobacteria and cyanobacterial toxins in various matrices. Advances in field methods, conventional laboratory methods, emerging high-throughput analysis methods as well as sampling and sample processing methods will be highlighted.

PRESENTATION Toxicity Assessment of Paralytic Shellfish Poisons (Psps) Using Quantitative Structure-Activity Relationships 09/11/2005
DELACRUZ, A. A., B. BOUTIN, AND R. VENKATAPATHY. Toxicity Assessment of Paralytic Shellfish Poisons (Psps) Using Quantitative Structure-Activity Relationships. Presented at AOAC Annual Meeting, Orlando, FL, September 11 - 15, 2005.
Abstract: The most significant harmful algal bloom (HAB) toxin in terms of public health is commonly known as paralytic shellfish poisons (PSPs, "red tides" toxins). PSPs are neurotoxins produced by marine dinoflagellates and some cyanobacteria. PSPs comprise of over 21 natural toxins with varying toxicity and are broadly classified into saxitoxins, neosaxitoxins, gonyautoxins, decarbamoyl gonyautoxins and decarbamoyl saxitoxins, depending upon the functional groups R1, R2, R3 and R4 (see Figure below). Humans potentially may be exposed to PSP poisoning through drinking water, recreational water and hemodialysis treatment. Consumption of fish and shellfish has been implicated in human outbreaks of PSP in the US and worldwide. Humans have been reported to exhibit characteristic neurological symptoms, nausea, vomiting, diarrhea and sometimes death within 2-12 hours. Standard mouse bioassay data are available in the literature for some PSPs. The mouse toxicity model is labor-intensive, time-consuming and expensive. Routine use of animal models to detect and quantify PSP toxicity may be circumvented using Quantitative Structure-Activity Relationships (QSARs). Currently, there are no QSARs in the literature for predicting the toxicity of PSPs. USEPA is investigating the use of QSAR models to support risk assessment of chemical toxicity. The objective of the study is to develop a QSAR model that can provide quantitative estimates of the PSP dose that causes toxicity in mice. To meet the objective, known PSP mouse bioassay data were collected from the literature. The molecular structures of the PSPs were optimized to their global minimum using the CONFLEX module in CAChe (Computer Aided Chemistry) software. The descriptor generator programs Dragon, Molconn-Z, CAChe and AMPAC/CODESSA were used to generate the descriptors for the QSAR model. Initially, a QSAR model for PSP toxicity was developed using 2-dimensional descriptors from Dragon as independent variables. The QSAR models had a high degree of predictability using the Dragon descriptors. However, the models predicted similar toxicities for some of the isomeric PSPs and conformers as the 2-dimensional descriptors could not differentiate between their respective chemical structures. The models were redeveloped with 3-dimensional descriptors from CAChe and AMPAC/CODESSA. The results of the QSAR model using 2-dimensional and 3-dimensional descriptors will be presented and discussed.

PRESENTATION Identifying Chemical Compounds in the Environment 08/23/2005
GLASSMEYER, S., E. L. FURLONG, D. L. KOLPIN, J. D. CAHILL, S. D. ZAUGG, S. L. WERNER, M. T. WERNER, AND D. D. KRYAK. Identifying Chemical Compounds in the Environment. Presented at U.S. EPA Workshop on Pharmaceuticals in the Environment, Las Vegas, NV, August 23 - 25, 2005.
Abstract: The quality of drinking and recreational water is currently ascertained using indicator bacteria. The tests to analyze for these bacteria require a considerable length of time to complete, and do not discriminate between human and animal fecal material sources. To shorten the time needed to test water quality and distinguish the fecal pollution source, chemicals found in human wastewater, such as pharmaceuticals, surfactants, and fecal sterols, were evaluated as tracers of human fecal pollution. These chemicals would have the advantage of requiring shorter analysis times than traditional microbiological techniques, and can be selected to be human specific. At ten locations, water samples were collected upstream, and at two successive points downstream from a wastewater treatment plant. A treated effluent sample was also collected at each location. This longitudinal sampling scheme was used to estimate the persistence of the target compounds in streams. The water samples were extracted using either solid phase or liquid-liquid extraction and were analyzed using either liquid chromatography/mass spectrometry or gas chromatography/mass spectrometry. Of the 110 chemical analytes investigated in this project, 78 were found in at least one sample. Seventeen of the 110 compounds were classified as either a prescription or non-prescription pharmaceutical. Of these, nine were found in at least 50 % of the samples and thirteen were found in at least 10 % of the samples. While most concentrations of all of the compounds were in the range of 0.1 to 1.0 mg/ L, in some of the more highly contaminated samples, concentrations were in the range of 5-20 mg/ L. The concentrations of the majority of the chemical compounds present in the samples generally followed an expected trend: 1) they were non-existent or at only trace levels in the upstream samples, 2) had their maximum values in the wastewater effluent samples, and 3) declined in the two downstream samples. This work indicates that these chemical analytes do have utility as tracers of human wastewater discharge. The correlation between the concentration wastewater chemicals and incidence of illness is currently being investigated via a recreational water epidemiological study. The results of this study will determine if wastewater chemicals can be used as indicators of water quality.

PRESENTATION Evidence for the Macrophage Inducing Gene in Mycobacterium Intracellulare 07/23/2005
PFALLER, S. L. Evidence for the Macrophage Inducing Gene in Mycobacterium Intracellulare. Presented at Microbes in a Changing World, Joint Meeting of the 3 Divisions of the International Union of Microbiological Societies, San Francisco, CA, July 23 - 28, 2005.
Abstract: Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-described virulence factor identified in MAC. The gene codes for a secreted protein putatively involved in the metabolism of fatty acids, and is induced when cells are phagocytized by human macrophages. A previous analysis of MAC strains for the presence of mig found the gene exclusively in MA and not in MI. The goal of this study was to develop mig PCR as a tool to rapidly distinguish between strains of MA and MI in the laboratory.
Methods: MAC isolates were obtained from Olive-View UCLA Medical Center in Sylmar, California. The collection contained 18 MA and 58 MI isolates obtained from patients and drinking water. A 372 bp fragment of mig was amplified using primers specific for a coding region of the gene. Products were verified by sequencing. Sequences were aligned with mig sequences obtained from reference strains and GenBank and analyzed using phylogenetic methods.

Results: mig was detected in 94% (17/18) of MA strains and 95% (42/44) of MI strains. Sequencing and phylogenetic analysis of mig revealed distinct species differences.

Conclusions: Previous studies with MAC isolates obtained from across the United States found mig present in MA and absent in MI. In contrast, mig PCR was unable to discriminate between MAC species in this study. Interestingly, the strains in our collection were isolated from a relatively small geographic region (Los Angeles, California). The divergence between MA and MI mig sequences suggests the gene was derived from a common ancestor and was not acquired by MI through horizontal gene transfer. It has been demonstrated that mig is associated with the virulence of MA, however it remains to be seen if mig is expressed or involved with the virulence of MI.


PRESENTATION Determination of Pesticides in Composite Beverage Samples 07/17/2005
HIEBER, T. E., P. KAUFFMAN, J. BRISBIN, AND J. N. MORGAN. Determination of Pesticides in Composite Beverage Samples. Presented at 2005 Florida Pesticide Residue Workshop, St. Petersburgh, FL, July 17 - 20, 2005.
Abstract: USEPA's National Exposure Research Laboratory conducts research to measure the exposure of individuals to chemical pollutants through the diet, as well as other media. In support of this research, methods are being evaluated for determination of pesticides in composite dietary samples. Existing methods for pesticides generally have been developed for regulatory purposes and often do not have sufficiently low detection limits for exposure studies. Consequently, there is a need to improve the performance of these methods for analysis of pesticides in composite dietary samples.
In previous work, pressurized fluid extraction (PFE) followed by diatomaceous earth and C18 reversed phase chromatography was used in the measurement of organophosphate pesticides in composite solid food samples. PFE followed by diatomaceous earth and alumina column chromatography was used for a diverse mix of organochlorine and other pesticides. The current study was designed to evaluate the application of these and other techniques to determination of pesticides in low fat (1% of calories from fat) and high fat (13% of calories from fat) composite beverage samples.

Initial beverage method development efforts utilized milk fortified with organophosphate pesticides as a surrogate for composite beverages. Milk was applied directly to a diatomaceous earth column and extracted with various combinations of solvent. Recovery of organophosphate pesticides was generally unsatisfactory. Subsequently, PFE methods with diatomaceous earth and C18 (for organophosphate pesticides) and alumina (for organochlorine and other pesticides) column cleanup were optimized for composite beverage samples. Detection limits were generally within the sub-ppb to low ppb range. Recoveries were typically within the range of 60 - 140%. Finally, matrix solid phase dispersion (MSPD) techniques were evaluated. Traditional MSPD utilizing column chromatography was compared to MSPD performed in a pressurized fluid extractor. Recoveries were acceptable (60-140%) for most pesticides.


PRESENTATION Assessing Microbial Water Quality of Rainwater Catchment Systems in Ohio and Kentucky 07/14/2005
LYE, D. J. Assessing Microbial Water Quality of Rainwater Catchment Systems in Ohio and Kentucky. Presented at North American Rainwater Harvesting Conference, Seattle, WA, July 14 - 16, 2005.
Abstract: There is no abstract for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Detection of Cryptosporidium EPA Method 1623: A Summary of the Method and Method Validation 06/28/2005
WARE, M. W. Detection of Cryptosporidium EPA Method 1623: A Summary of the Method and Method Validation. Presented at Developing a plan to identify Cryptosporidum sources in the Potomac River Workshop, Rockville, ME, June 28, 2005.
Abstract: Slide presentation discussing the method evaluation criteria, lab validation criteria, overview of the method and the summary of the results of EPA Method 1623

PRESENTATION The Use of Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry for the Identification of Aeromonas Isolates Obtained from Water Distribution Systems 06/15/2005
DONOHUE, M. J., W. SMALLWOOD, J. BEST, J. A. SHOEMAKER, AND M. R. RODGERS. The Use of Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry for the Identification of Aeromonas Isolates Obtained from Water Distribution Systems. Presented at 8th International Symposium on Aeromonas and Plesiomonas, Halifax, NS, CANADA, June 15 - 18, 2005.
Abstract: Matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) has long been established as a tool by which microorganisms can be characterized and identified. EPA is investigating the potential of using this technology as a way to rapidly identify Aeromonas species found in drinking water. A number of bacteria, including Aeromonas hydrophila, are listed on the Agency's 1998 Contaminant Candidate List (CCL) as research needs. The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, MALDI-MS was used to develop a library of proteomic signatures for seventeen species of Aeromonas. These seventeen species were represented by thirty-two strains, which included type, reference and clinical isolates. This library was then used to rapidly identify strains of Aeromonas obtained during two surveys conducted by EPA during 2000 and 2001. In these two blind studies, forty survey strains, previously assigned to a species based on biochemical test results, were analyzed by MALDI-MS. The proteomic signatures of the survey strains were then compared against the library of "known" proteomic signatures to determine the identity of the Aeromonas isolates. In the first blind study, the identification by the MALDI-MS analysis correlated 100% with the biochemical identification. In the second blind study, eighteen of the twenty strains analyzed by MALDI-MS correlated with the biochemical identification. These data demonstrate that MALDI-MS analysis can rapidly and accurately identify species of the genus Aeromonas, making it a powerful tool especially suited for environmental monitoring and detection of biological hazards.

PRESENTATION Virulence Relationships of Aeromonas Species as Determined By Exposures to Immunocompromised Mice 06/15/2005
LYE, D. J., B. BERTKE, M. R. RODGERS, AND G. N. STELMA. Virulence Relationships of Aeromonas Species as Determined By Exposures to Immunocompromised Mice. Presented at 8th International Symposium on Aeromonas and Plesiomonas, Halifax, NS, CANADA, June 15 - 17, 2005.
Abstract: Our laboratory is currently determining the virulence of opportunistic pathogens reported in treated drinking water and drinking water sources. Aeromonas hydrophila is currently on the EPA's Contaminant Candidate List (CCL) and is an example of those types of bacteria that contain both opportunistic pathogens and non-pathogenic strains within a species. Virulence relationships of thirty-five Aeromonas strains covering species of hydrophila, veronii, caviae, enchelia, allosaccharophila, and salmonicida were characterized using an intraperitoneal exposure method in mice immunocompromised by cyclophosphamide injection. Exposures of immunocompromised animals to opportunistic bacteria through intraperitoneal injection is a promising virulence detection procedure useful for a wide range of disparate bacterial types.

PRESENTATION Method Development for N-Nitrosodimethylamine and Other Nitrosamines in Drinking Water 06/14/2005
MUNCH, J. W. Method Development for N-Nitrosodimethylamine and Other Nitrosamines in Drinking Water. Presented at Louisville Chemistry Conference, Louisville, KY, June 14, 2005.
Abstract: There is no abstract for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.

PRESENTATION Elemental Speciation in Environmental Exposure Assessment Matrices 06/14/2005
CREED, JOHN T., P. A. CREED, A. PARKS, C. A. SCHWEGEL, M. FRICKE, A. ACKERMAN, S. CONKLIN, D. HEITKEMPER, N. P. VELA, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Elemental Speciation in Environmental Exposure Assessment Matrices. Presented at The Louisville Chemistry Conference, Louisville, KY, June 14, 2005.
Abstract: This is an overview slide presentation of the Arsenic Research Program.

PRESENTATION Recreational Water Quality and Swimmer Health Can Faster Methods of Measuring Recreational Water Help Prevent Swimming Associated Illness? 06/13/2005
WADE, T. J., R. L. CALDERON, M. J. BEACH, K. P. BRENNER, E. A. SAMS, AND A. P. DUFOUR. Recreational Water Quality and Swimmer Health Can Faster Methods of Measuring Recreational Water Help Prevent Swimming Associated Illness? Presented at International Workshop sponsored by the Sao Paulo State Company for Sanitation and Technology, Sao Paulo, BRAZIL, June 13 - 16, 2005.
Abstract: Evidence from various sources around the world indicate that there is a relationship between gastroenteritis in swimmers and the quality of the bathing water as measured with bacterial indicators of fecal contamination. Current EPA guidelines recommend the use of cultural methods for E. coli and enterococci to measure beach water quality. These methods produce results in 24 hours creating the pointless guidance, we can tell you tomorrow, what you swam in today. This shortcoming in current practice for measuring beach water quality has led EPA to consider new technology and indicators that will provide rapid (2 hours or less) measurement of beach waters.

PRESENTATION Identification of Microcystin Toxins from a Strain of Microcystis Aeruginosa 06/07/2005
DIEHNELT, C., S. M. PETERMAN, N. DUGAN, AND W. L. BUDDE. Identification of Microcystin Toxins from a Strain of Microcystis Aeruginosa. Presented at 53rd Annual Conference American Society for Mass Spectrometry, San Antonio, TX, June 07, 2005.
Abstract: Microcystin toxins are cyclic heptapeptides produced by several genera and species of cyanobacteria that are responsible for the "green scum" frequently observed on eutrophic surface waters. These toxins, which are a million times more toxic than cyanide ion, have caused deaths of farm animals, humans, pets, and wildlife through exposures from drinking and recreational waters. Over 65 microcystin structural variants are known, and all of them contain some standard and some unusual bacterial amino acid residues. The toxins produced by a strain of Microcystis aeruginosa that had not been investigated previously were separated and identified by microbore liquid chromatography (LC)/mass spectrometry (MS), LC/MS/MS, LC/MS/MS/MS, and exact m/z measurements. This research could be considered an example of "environmental proteomics".

PRESENTATION New Target and Control Assays for Quantitative Polymerase Chain Reaction (Qpcr) Analysis of Enterococci in Water 06/05/2005
SIEFRING, S., L. J. WYMER, AND R. A. HAUGLAND. New Target and Control Assays for Quantitative Polymerase Chain Reaction (Qpcr) Analysis of Enterococci in Water. Presented at American Society for Microbiology General Meeting, Atlanta, GA, June 05 - 09, 2005.
Abstract: Enterococci are frequently monitored in water samples as indicators of fecal pollution. Attention is now shifting from culture based methods for enumerating these organisms to more rapid molecular methods such as QPCR. Accurate quantitative analyses by this method requires highly specific primer and probe assays and the inclusion of effective controls to detect and adjust for influences of the sample matrix on both DNA template recovery and PCR efficiency. A new primer and probe set targeting a conserved rDNA sequence in Enterococcus species and with high predicted selectivity against related non-Enterococcus species was developed. A second highly discriminatory primer and probe set, targeting exactly the same rDNA region of the related species Lactococcus lactis, was also developed to allow known additions of this organism to water sample filtrates to be used for assessing variability in DNA recovery. Finally, an easy to perform method was devised for the generation of an artificial PCR template with extensive nucleotide sequence similarity to the same rDNA region but again with specific primer and probe target sequences. This template was designed for addition as a positive control to DNA extracts to test for PCR inhibition. Preliminary tests confirmed that there was no cross-reactivity between the three assays and that their amplification efficiencies were similar (~0.9). Different extraction conditions, designed to alter the efficiency of cell lysis, resulted in DNA recoveries from Lactococcus and Enterococcus that were strongly correlated (r = 0.85) as determined by the respective QPCR assays. Extracts of different water sample filtrates were spiked with Enterococcus, Lactococcus and positive control template DNAs and analyzed to determine the relative sensitivities of the respective assays to PCR inhibition. The three assays showed highly correlated changes in results in response to varying amounts of PCR inhibitory substances in the different water extracts as evidenced by r values of 0.9 or higher. This system shows great promise for improving the accuracy of enterococci measurements in water samples by QPCR.

PRESENTATION Viral Pathogens and Microbiological Indicators in Ground Water from Small Public Water Supplies in Southeastern Michigan 06/05/2005
FRANCY, D. S., R. N. BUSHON, E. J. LUZANO, E. BERTKE, AND G. FOUT. Viral Pathogens and Microbiological Indicators in Ground Water from Small Public Water Supplies in Southeastern Michigan. Presented at American Society for Microbiology Annual Meeting, Atlanta, GA, June 05 - 09, 2005.
Abstract: Thirty-eight public ground-water-supply wells serving less than 3,300 people were sampled from July 1999 through July 2001 in southeastern Michigan to determine (1) occurrence of viral pathogens and microbiological indicators, (2) whether indicators are adequate predictors of the presence of viruses, and (3) the factors that affect the presence of viruses. Samples were analyzed for enteric viruses by reverse transcriptase-polymerase chain reaction (RT-PCR), for culturable viruses by cell culture, and for the indicators total coliforms, E. coli, enterococci, and F-specific and somatic coliphage. Ancillary environmental and water-quality data also were collected.
A total of 169 regular samples and 32 replicate pairs were collected. Each well was sampled from one to five times. By use of RT-PCR, enterovirus was found in four wells (10.5%) and hepatitis A virus (HAV) in five wells (13.2 %). Culturable viruses were found once in two wells (5.9%), and neither of these wells was positive for viruses by use of RT-PCR. Nine of the 38 wells (23.7%) were positive for viruses by either RT-PCR or cell culture. One or more indicators were found in 18 of 38 wells. Total coliforms, E. coli, enterococci, and F-specific and somatic coliphage were found in 34.2, 10.5, 15.8, 5.9, and 5.9%, respectively, of the wells tested. Five out of nine (55.6%) virus-positive wells were also found to be positive for an indicator.

More virus-positive samples were found at sites served by septic systems than those served by sewerlines. Statistically significant relations were found between total coliforms and dissolved organic carbon, iron, or chloride concentrations. Presence of nitrate was related to presence of E. coli, enterococci, coliphage, or enteric viruses but not to total coliforms.

This study provides evidence for fecal contamination of ground water at small public-supply wells and shows the importance of collecting multiple samples at each site. The study also suggests that the collection of site-characteristic data (e.g., population density, land usage, hydrogeology, well construction), data on multiple water-quality parameters (e.g., mineral and organic carbon concentrations) and data on microbiological indicators is important for making reliable predictions of the presence of enteric viruses in small public water systems.


PRESENTATION A Critical Evaluation of a Flow Cytometer Used for Detecting Enterococcus Faecium and Enterococcus Faecalis in Recreational Waters 06/05/2005
STANG, D., K. P. BRENNER, AND M. R. RODGERS. A Critical Evaluation of a Flow Cytometer Used for Detecting Enterococcus Faecium and Enterococcus Faecalis in Recreational Waters. Presented at 2005 American Society of Microbiology General Meeting, Atlanta, GA, June 05 - 09, 2005.
Abstract: The current U. S. Environmental Protection Agency-approved method for Enterococci (Method 1600) in recreational water is a membrane filter (MF) method that takes 24 hours to obtain results. If the recreational water is not in compliance with the standard, the risk of exposure to enteric pathogens will occur before the water is identified as hazardous. Because flow cytometry combined with specific fluorescent antibodies has the potential to be used as a rapid detection method for microorganisms, this technology was evaluated as a rapid, same-day method to detect Enterococci in bathing beach waters. A comparison of the viable MF counts with flow cytometry counts of pure Enterococci cultures in phosphate buffer and sterile-filtered recreational water showed fairly good agreement between the two methods. However, when the flow cytometer was used with spiked and unspiked natural water samples, the counts were several orders of magnitude higher than the MF counts with no correlation to Enterococcus spike concentrations, and the unspiked sample controls frequently had higher counts than the samples spiked with Enterococci. Particles within the spiked water samples were probably counted as target cells by the flow cytometer because of autofluorescence or non-specific adsorption of antibody, and carryover to subsequent samples occurred often. Carryover can cause acceptable water samples to exceed the recreational water Enterococci limits and result in unnecessary beach closings. In order to eliminate this problem, several steps were added to the manufacturer's method to clean the system between samples. This significantly decreased the number of samples that could be processed in one day. For these reasons, this technology may not be suitable for Enterococci detection in recreational waters.

PRESENTATION The Identification of the Water-Borne Pathogen Aeromonas Using Whole Cell Analysis By Matrix Assisted Laser Desorption/Ionization-Mass 06/05/2005
DONOHUE, M. J., W. SMALLWOOD, D. J. LYE, J. A. SHOEMAKER, AND M. R. RODGERS. The Identification of the Water-Borne Pathogen Aeromonas Using Whole Cell Analysis By Matrix Assisted Laser Desorption/Ionization-Mass. Presented at 53rd American Society for Mass Spectrometry Conference, San Antonio, TX, June 05 - 09, 2005.
Abstract: MALDI-MS has long been established as a tool by which microorganisms can be characterized and identified. The U.S. Environmental Protection Agency (EPA) is investigating the potential of using this technique as a way to rapidly identify Aeromonas species in drinking water. A number of bacteria, including Aeromonas hydrophila, are listed on the EPA's 2005 Contaminant Candidate List (CCL) as requiring research.
To assess if MALDI-MS can be used to identify species of Aeromonas, as well as predict the strains possible pathogenicity, the project is broken down into three major objectives: 1) to create a mass spectral library of known Aeromonas strains, 2) identify unknowns isolates, and 3) identify m/z ions that may be used as biomarkers of human pathogenicity.

Methods and Instrumentation:

Bacteria growth

Aeromonas reference strains, representing the 17 recognized species, were obtained from several culture collections. The Aeromonas environmental isolates were collected from a pilot study of water distribution systems (WDS), between the years of 2000-2001, using EPA Method 1605. Over 205 were obtained from the surveyed distribution centers.

Both reference strains and WDS isolates (stored at -20 0C) were used to inoculate tryptic soy agar plates (Difco, Detroit, MI, USA). Individual colonies were carefully removed from the agar surface with a sterile loop to inoculate 5 mL of tryptic soy broth (TSB). The cultures were incubated overnight at 370C. One-hundred microliters of the TSB broth were lawned onto 5% sheeps blood agar plates and incubated overnight at 350C. Five milliliters of sterile saline were then used to harvest bacteria from the plates.

Sample Preparation

The harvested cells were centrifuged at 4,000 RPM. The supernatant was removed and the pellet was washed three times with water. The dried cells were then overlaid with a 1.0
MALDI-MS analysis

All mass spectra were generated on a Biflex III (Bruker Daltonics, Billerica, MA, USA) MALDI-TOF mass spectrometer, operated in positive linear mode. The spectra were obtained by summing four scans of 100 shots each. The summed spectra were smoothed and calibrated externally using the [M+H]+ ions of insulin and myoglobin. Blank controls were analyzed in parallel with the bacteria samples to verify that the protein signals were not from contamination introduced during sample preparation. Peak lists were created from each strains spectrum and later analyzed by Phylogenetic Analysis Using Parsimony* (PAUP*).

Results and Discussion:

The long range goal of this project is to identify novel virulence factors for Aeromonas using MS techniques. A library of mass spectral fingerprints of a number of Aeromonas strains was generated using MALDI-MS. In addition to investigating virulence factors, this mass spectral library can be used to determine if rapid species/strain differentiation is possible using MALDI-MS. Unique masses were observed in each of the Aeromonas isolates, providing a means to differentiate between Aeromonas species by MALDI-MS.

A blind study was done using 51 isolates from a collection of 205 isolates obtained from drinking water distribution systems. Of the 51 isolates used in this study, traditional biochemical analyses were only able to definitively identify 25 isolates, and tentatively identify the other 26 isolates. Species identification was accomplished by comparing mass spectral fingerprints of the isolates against the ATCC strains using the PAUP software. The MALDI-MS analysis of the water distribution samples correlated 100% with the biochemical identification of the 25 confirmed isolates. Of the 26 isolates that were only tentatively identified, MALDI-MS analysis was able to identify, with a 95% confidence, 15 of the 26 isolates. In the cases where a clear identity was not determined, research is on-going to ascertain whether the isolate is a mixture of species.

Concurrently with the above research, animal studies were conducted to identify virulent and avirulent strains of the isolates obtained from drinking water distribution systems. This research, along with the MALDI-MS analysis of the isolates, will be used to identify potential biomarkers of human pathogenicity. Once these biomarkers are identified, further research will be conducted to identify the proteins unique to virulent Aeromonas using MALDI-MS mass spectral fingerprints. Electrospray (ESI)-MS/MS will then be used to sequence the virulent proteins.

PRESENTATION Fecal Indicator Bacteria Measurements By Quantitative Polymerase Chain Reaction (Qpcr) Analysis in Fresh Archived Dna Extract of Water Sample Filtrates 06/05/2005
VARMA, M., L. J. WYMER, AND R. A. HAUGLAND. Fecal Indicator Bacteria Measurements By Quantitative Polymerase Chain Reaction (Qpcr) Analysis in Fresh Archived Dna Extract of Water Sample Filtrates. Presented at American Society for Microbiology 105th General Meeting, Atlanta, GA, June 05 - 09, 2005.
Abstract: The U.S. EPA has initiated a new recreational water study to evaluate the correlation between illness rates in swimmers and Enterococcus concentrations determined by the mEI agar membrane filter (MF) method and several new technologies including QPCR analysis. Results of this study in 2003 showed a significant positive correlation between QPCR and MF method measurements of Enterococcus concentrations (Haugland et al. Water Research, 2005). A potential benefit of molecular methods such as QPCR is that they may be able to provide occurrence data for new indicator organisms by analyses of freezer-archived DNA extracts with new assays. To test whether QPCR analyses of archived DNA extracts provide comparable measurements to those of the fresh extracts, DNA extracts from the 2003 study were reanalyzed in 2004 and the results of the two data sets compared. The 2004 analyses were also performed with a newly developed QPCR primer and probe assay for enterococci (Entero2) that provides greater selectivity against related non-Enterococcus species. Fresh DNA extracts of cultured Enterococcus cells were used as calibration standards for measuring Enterococcus cell equivalents in the test samples in both sets of analyses and gave similar results. The overall mean of Enterococcus calibrator cell equivalents per sampling visit (N = 47) from the 2004 analyses was approximately five-fold lower than that from the initial 2003 analyses. The overall mean from the Entero2 assay was 28% lower than that from the original assay. In contrast, correlations between log10-transformed Enterococcus colony forming units (CFU), determined in 2003 by the MF method, and the 2003 and 2004 QPCR measurements were the same (r = 0.76). Correlations between CFUs and the 2004 Entero2 assay results were similar (r = 0.77). These results indicate that archived DNA extracts may undergo some deterioration that will bias absolute measurements of indicator bacteria, however, the changes in different samples are relatively consistent. Measurements of new indicators in archived DNA extracts by QPCR may therefore be useful for comparisons with epidemiological health data.

PRESENTATION Mceard Cyanobacteria and Their Toxins 05/18/2005
HUDNELL, K., A. A. DELACRUZ, B. BOUTIN, G. PEROVICH, N. DUGAN, AND P. RUBLEE. Mceard Cyanobacteria and Their Toxins. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.
Abstract: Harmful algal blooms (HAB) of cyanobacteria, also known as blue-green algae, have recently become more spatially and temporally prevalent in the US and worldwide. Waterborne cyanobacteria and their highly potent toxins are a significant hazard for human health and the ecosystem. Currently, the US has no guidelines or regulations on cyanobacteria or cyanotoxins. Cyanobacteria and their toxins are on the Agency's Unregulated Contaminant Monitoring Regulation List 3 (UCMR), and the Contaminant Candidate List. The Office of Water (OW) is unable to promulgate regulations or produce guidelines at this time due to the lack of sensitive, specific, and field-ready analytical methods; data on the occurrence of cyanobacterial HABs; proven prevention and mitigation technologies; and human-health risk estimates.
To date, OW has assembled an interactive database of over 3,000 scientific articles on cyanobacteria, produced documents on the state of science on cyanobacteria and their toxins a summary for cyanotoxins in drinking water. Scientists in the Office of Research and Development (ORD) are reviewing the literature on cyanobacterial toxicology, identifying data needed to assess the risk of exposure to four cyanotoxins, and assessing the plausibility of modeling quantitative structural-activity relationships (QSAR) for cyanotoxins. A number of scientists are refining analytical methods to detect selected cyanotoxins in water to support the OW UCMR monitoring study. Several investigators have assessed the efficacy of cyanobacteria removal and cyanotoxin destruction by conventional water treatment processes and are currently exploring advanced oxidation technologies and sonolysis to inactivate and/or destroy cyanotoxins in water. The behavioral and developmental effects of cyanotoxins are being studied. The feasibility of an epidemiologic study on the health effects of repeated, low-level exposure to cyanotoxins in drinking water is also being assessed. These tasks are being carried out through collaborations within the Agency, and with academia and stakeholders.

The Agency's National Center for Environmental Research (NCER) is supporting cyanobacterial research by funding five research grants, two student fellowships, and two small business initiatives. Research studies include the development of microarrays to detect cyanobacteria and cyanotoxins and the relationships between nutrient loading and cyanobacterial dominance.

The ORD will sponsor an international symposium on cyanobacteria in September, 2005. Invited speakers and workgroup members will identify and prioritize cyanobacteria research needs. The products of this symposium will enable ORD management to assess the need for an integrated research program on cyanobacteria to support source water protection programs and CCL decisions.

PRESENTATION Evaluation of Wastewater Chemicals as Indicators of Human Fecal Contamination 05/16/2005
GLASSMEYER, S., E. L. FURLONG, D. L. KOLPIN, AND L. B. BARBER. Evaluation of Wastewater Chemicals as Indicators of Human Fecal Contamination. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.
Abstract: The quality of drinking and recreational water is currently ascertained using indicator bacteria. The traditional tests that analyze for these bacteria require approximately 24 hours to complete, and do not discriminate between human and animal sources. One solution is to use human wastewater chemicals, which would require shorter analysis times and are human specific. However, questions about the presence, persistence, fate and transport of wastewater compounds must be answered before their utility as indicators of human fecal contamination can be determined. This information is being gathered through a series of projects, each designed to further the understanding of the behavior of these chemicals. The first steps were to determine what chemicals survive wastewater treatment, and obtain information about their environmental occurrence and approximate persistence. This was accomplished by collecting an effluent sample and water samples up- and down-stream from ten wastewater treatment plants. To further refine the persistence estimates, a dye tracer study was performed at two locations to determine the time it takes for a given parcel of water to travel from a wastewater treatment plant to successive downstream sampling points. By knowing the time of travel, the kinetics of the changes in concentration can be calculated. Knowing the environmental occurrence and persistence, the next step was to participate in the National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study to determine if there is a link between these chemicals and negative health impacts associated with exposure to waterborne pathogens. At the successful completion of this research effort, the US EPA will have supplementary tools to monitor water for human wastewater contamination. The speed and specificity of the chemical analysis methods will protect humans from exposure to pathogens. In addition, occurrence information for numerous unregulated wastewater compounds will be obtained.
This effort compliments concurrent research within the National Exposure Research Laboratory (NERL) focused on developing rapid microbial detection methods. This project has relied heavily on both internal and external collaboration. The U.S. Geological Survey has been extensively involved in the collection and analysis of the surface samples for the chemical analytes. NEEAR is a joint effort between NERL and the National Health and Environmental Effects Research Laboratory (NHEERL).

Although this work was reviewed by EPA and approved for publication, it may not necessarily reflect official Agency policy.

PRESENTATION Evaluation of a Cryptosporidium Internal Standard for Determining Recovery With Environmental Protection Agency Method 1623 05/16/2005
WARE, M. W., D. S. FRANCY, E. J. GRANGER, F. W. SCHAEFER, M. D. SOBSEY, AND O. D. SIMMONS. Evaluation of a Cryptosporidium Internal Standard for Determining Recovery With Environmental Protection Agency Method 1623. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.
Abstract: The current benchmark method for detecting Cryptosporidium oocysts in water is the U.S. Environmental Protection Agency (U.S. EPA) Method 1623. Studies evaluating this method report that recoveries are highly variable and dependent upon laboratory, water sample, and analyst. Therefore, appropriate quality assurance/quality controls are vital for interpreting results. A key control is the matrix spike that is used to determine the percent recovery for a given water sample. To determine the percent recovery using the matrix spike procedure, paired water samples are taken, one sample is processed and analyzed, and the second sample is spiked with a known number of live Cryptosporidium oocysts, processed, and analyzed. This is very time consuming and expensive.
To improve the current method, scientists at the U.S. EPA, USGS, and UNC investigated the possibility of using an internal control as the matrix spike. A commercial product, ColorSeed, contains a known number of killed and tagged oocysts, which allows for the differentiation of spiked and naturally occurring oocysts in only one water sample. This study evaluated the recoveries of ColorSeed and live oocysts in 20 stream samples from all over the United States in matrix spike samples. The results indicate that recoveries obtained with ColorSeed oocysts were not significantly different from live oocysts. Therefore, the use of ColorSeed as an internal standard allows for the analysis of only one sample to determine recovery rather than the two samples required for live oocysts.


PRESENTATION Global Water Research Coalition: An International Collaboration on Water Research 05/16/2005
HAUCHMAN, F. S., S. GLASSMEYER, S. L. PFALLER, D. TILLMAN, AND H. M. SHUKAIRY. Global Water Research Coalition: An International Collaboration on Water Research. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.
Abstract: The research needs for drinking water far exceed the ability of any one research organization to fully address by itself. For this reason, the U.S. Environmental Protection Agency's (U.S.EPA) Office of Research and Development (ORD) historically has sought opportunities to leverage funding and expertise with other national and state governmental research organizations, academia, and the water industry. The establishment of a formal partnership between the U.S. EPA and the Global Water Research Coalition (GWRC) enables the ORD to collaborate and jointly fund research with the most important water research organizations in the world. The GWRC is a formal nonprofit organization that is dedicated to promoting international cooperation and collaboration on water-related research. It is comprised of 12 of the most prominent water and wastewater research organizations from the U.S., the United Kingdom, France, Germany, Australia, the Netherlands, and South Africa. Established in 2002, the GWRC provides a forum for member organizations to coordinate research strategies, address issues of mutual interest, and avoid duplication of effort. It also promotes the sharing and dissemination of water research information on a global scale.
Priority issues include such topics as newly identified waterborne pathogens and chemicals, distribution systems, innovative treatment technologies, and renewable water resources. Research collaborations between GWRC members are underway or are being planned in a number of areas. Several specific collaborative efforts have already been conducted, including the development of GWRC research strategies and/or research needs reports on endocrine disruptors, pharmaceuticals and personal care products, water quality in the distribution system, and algal toxins. Collectively, these activities represent important steps toward achieving the GWRC's mission of cooperation for worldwide water knowledge, innovation, and progress.

PRESENTATION A Collaborative Effort to Identify the Causative Agent of Two Waterborne Outbreaks of Gastroenteritis in Wyoming 05/16/2005
CASHDOLLAR, J., G. FOUT, D. R. DAHLING, AND S. PARSHIONIKAR. A Collaborative Effort to Identify the Causative Agent of Two Waterborne Outbreaks of Gastroenteritis in Wyoming. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.
Abstract: Two outbreaks of acute gastroenteritis were reported to the Wyoming Department of Health in 2001. The first was reported in February from recent vacationers of a snowmobile lodge. The second was in October among diners of a tourist saloon. The duration and type of symptoms exhibited by the individuals affected during the outbreaks suggested that noroviruses could be the causative agent. A collaborative investigation by individuals from key federal and state agencies, as well as academic institutions, was conducted in order to determine the causative agent or agents responsible for each outbreak. Both projects had a three-pronged approach: (1) an epidemiological investigation that was performed to identify any common routes of exposure among those afflicted with gastroenteritis, (2) an environmental survey that was done on the site of each outbreak in order to determine possible sources of contamination, and (3) a laboratory analysis that was performed on well water and fecal samples.
With each outbreak, epidemiological studies revealed a close association between water consumption and illness. In addition, environmental surveys in both outbreaks determined that the water supply was vulnerable to fecal contamination. Furthermore, water samples were tested for coliform bacteria using standard methods and for enteric viruses using molecular assays based on reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing techniques. In addition, stool samples were analyzed for the presence of noroviruses. Well water samples in both cases were positive for coliforms, which are indicators of fecal pollution, while molecular assays confirmed the presence of noroviruses. In each outbreak, a single norovirus strain was identified in the well water and fecal samples, thereby linking the water contamination with the gastroenteritis.

The partnerships that were formed to complete these investigations allowed for a timely and thorough study. Additionally, the work performed by the project partners created an atmosphere that will allow for future collaborative efforts. These investigations demonstrate that the U.S. Environmental Protection Agency's viral concentration and molecular methods, in conjunction with epidemiological and environmental analyses, are very useful in outbreak studies. The methods used in this study can be performed in most laboratories with trained personnel and appropriate equipment, and their use would allow for routine monitoring of enteric viruses in drinking water.

PRESENTATION Understanding Children's Total Dietary Exposure to Pesticides 05/16/2005
MORGAN, J. N., C. E. BERNARD, AND L. J. MELNYK. Understanding Children's Total Dietary Exposure to Pesticides. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.
Abstract: Recent residential monitoring studies have demonstrated that significant portion of total exposure of infants and children to environmental contaminants can result from contamination of food in the home. Children's foods become contaminated through handling and contact with surfaces. Though a series of collaborations with industry, academia and other federal agencies, EPA is gaining better understanding of the influence of eating activities on children's total dietary exposure to pesticides.

PRESENTATION Quantifying Indoor Molds 05/11/2005
VESPER, S. J. Quantifying Indoor Molds. Presented at Product Expo Region 7, Kansas City, KS, May 11, 2005.
Abstract: There is growing awareness that indoor molds/fungi may be connected to such conditions as asthma, allergies, hemorrhaging, chronic rhinosinusitis, memory loss, and a symptom complex called sick-building-syndrome. In addition, molds cause frequently fatal nosocomical infections. The US EPA has developed a mold specific quantitative PCR (MSQPCR) process that relies of the unique DNA signature of each mold species to do the identification and quantification. Assays are now available for more than 100 of the most common and relevant molds. These assays are standardized, specific, and sensitive (usually down to a single spore). The analysis can be completed in 2 to 3 hours. An automated platform can be used to perform these analyses. Examples of the use of the technology will be described in the presentation.

PRESENTATION The Persistence of Mycobacterium Avium in a Drinking Water System After the Addition of Filtration 05/11/2005
HILBORN, E. D., T. C. COVERT, M. YAKRUS, S. F. DONNELY, E. W. RICE, S. TONEY, S. BAILEY, AND G. N. STELMA. The Persistence of Mycobacterium Avium in a Drinking Water System After the Addition of Filtration. Presented at International Meeting on Microbial Epidemiological Markers, Victoria, BC, CANADA, May 11 - 14, 2005.
Abstract: Drinking water is increasingly recognized as a major source of pathogenic nontuberculous mycobacteria (NTM) associated with human infection. Our goal was to determine if the prevalence of NTM would decrease after the addition of filtration treatment to an unfiltered surface water source. We sampled water from two initially unfiltered surface drinking water treatment plants over a 29-month period. One plant received the addition of filtration after six months of sampling. At each plant, we sampled treatment plant influent and effluent, distributed water, and cold water taps (point-of-use (POU) sites) in public or commercial buildings located within the distribution systems. Water samples collected at POU sites yielded the majority of NTM with a prevalence of greater than 50%; overall prevalence was 25%, with no reduction in the frequency of isolation of NTM from water samples despite the addition of filtration. Specific clones of Mycobacterium avium, as determined by pulsed field gel electrophoresis, were found to persist at POU sites for up to 27 months. Our data indicate that water derived from POU cold water taps was persistently colonized despite the addition of filtration to the drinking water plant. Evidence of this ability of clonal strains of M. avium to persist within drinking water distribution systems is useful, and may be applied during the investigation of environmental sources of human M. avium infection using molecular methods.

PRESENTATION Real Time Pcr Analysis of Indoor Molds: Principles, Procedures and Applications 04/29/2005
HAUGLAND, R. A. Real Time Pcr Analysis of Indoor Molds: Principles, Procedures and Applications. Presented at Invited Seminar Presentation for Environmental Research Group, Atlanta, GA, April 29, 2005.
Abstract: This presentation will endeavor to present an overview of the real time polymerase chain reaction method developed for indoor mold detection and quantification by the EPA. It will begin with a brief discussion of the PCR technology that provides the basis for this method and how it is used to obtain quantitative results. The complete step by step method will then be illustrated followed by a discussion of the methodology used for designing specific PCR assays for different fungi. The presentation will conclude with two illustrations of how this method has been applied for the analyses of fungi in buildings and a summary of the perceived advantages of this method over conventional culture-based mold analyses.

PRESENTATION Mold Specific Quantitative Pcr: the Emerging Standard in Mold Analysis 04/13/2005
VESPER, S. J. Mold Specific Quantitative Pcr: the Emerging Standard in Mold Analysis. Presented at Real Time PCR Conference, Cherry Hill, NJ, April 13 - 14, 2005.
Abstract: Today I will talk about the use of quantitative or Real time PCR for the standardized identification and quantification of molds. There are probably at least 100,000 species of molds or fungi. But there are actually about 100 typically found indoors. Some pose a threat to humans and animals and others don't. We need to know what molds are present indoors and their concentrations. The older methods of culturing molds on media or counting spores are slow, laborious and subjective. Quantitative or Real time PCR is objective because it is based on the DNA sequences unique to the different mold species.

PRESENTATION Development of An Integrated Cell Culture/Rt-Pcr Method for the Detection of Enterovirus in Water 03/23/2005
FOHL, L. AND G. FOUT. Development of An Integrated Cell Culture/Rt-Pcr Method for the Detection of Enterovirus in Water. Presented at US EPA Post-doc Poster Session, Cincinnati, OH, March 30, 2005.
Abstract: Virus contamination in environmental samples is believed to be underestimated due to the limitations in the methods available for detection. A major detection method is based upon the formation of cytopathic effect (CPE) in cell culture. The main limitations to this method are the amount of time that is required to detect CPE formation and the fact that not every virus produces CPE or is culturable. Molecular assays have been developed for the detection of virus genomic material; however, the presence of virus RNA does not indicate that infectious virus is present. Also, water samples often contain substances that inhibit molecular assays.
One method which will overcome the major limitations of the CPE-based and molecular assays is integrated cell culture-RT-PCR (ICC/RT-PCR). This work describes a method for doing ICC-RT-PCR on water samples of 200L and greater. This involves using 1MDS filters to collect the virus from water, eluting the virus from 1MDS filters with beef extract, and concentrating the sample using EPAs celite method. Secondary concentration is performed by ultracentrifugation and the resulting pellet is resuspended and placed on a cell monolayer. Cell lysates are collected and used as template for RT-PCR and quantitative real time RT-PCR (qRT-PCR) assays for the detection of virus RNA.

This method has resulted in an 89.8 and 66.4% total recovery after the ultracentrifugation step on two independent trials. Once these pellets were inoculated onto cell monolayers, virus RNA was detected in 24 hours using both qRT-PCR and RT-PCR. CPE was not detected in these samples until day 3 and was not present in all the samples until day 4. This study was performed using spiked distilled water and will be validated using environmental samples.

PRESENTATION Comparison of Mycobacterium Avium Isolates from Drinking Water and from the Population Served By the System 03/19/2005
HILBORN, E. D., T. C. COVERT, M. A. YAKRUS, S. HARRIS, S. F. DONNELY, E. W. RICE, M. T. SCHMITT, S. TONEY, S. BAILEY, AND G. N. STELMA. Comparison of Mycobacterium Avium Isolates from Drinking Water and from the Population Served By the System. Presented at International Conference on Emerging Infectious Diseases, Atlanta, GA, March 19 - 22, 2005.
Abstract: Background: Current evidence suggests that drinking water, soil, and produce are potential sources of Mycobacterium avium infections, a pathogen not known to be transmitted person-to-person.
Methods: We sampled water during 2000 - 2002 from a large municipal drinking water system in which surface source water is used. We cultured and isolated environmental M. avium from water samples. In addition, we collected clinical isolates identified as M. avium complex that were derived from patients served by the drinking water system and isolated during 1999 - 2002. We recorded the anatomical site from which the isolate was collected and the patient's residential zip code. Environmental and patient isolates were compared for genetic similarity using multilocus enzyme electrophoresis (MEE) as a screening tool. Isolates with similar MEE patterns were then compared more specifically by using pulsed-field gel electrophoresis (PFGE) analysis.

Results: M. avium was isolated from 73% (30/41) of water samples collected at all four point-of-use tap (POU) sample sites located in public and commercial buildings. Eight percent (9/113) of patient isolates were identified as being related by electrophoretic group to environmental isolates. Two of these related isolates displayed geographic proximity between POU sample site and patient zip code.

Conclusions: Given the large amount of genetic heterogeneity known to occur among M. avium strains, our results suggest that these 9 patient isolates were associated with exposure to municipal water. Molecular techniques are useful to identify specific environmental sources of human M. avium infections.


PRESENTATION Quantifying Indoor Molds 03/14/2005
VESPER, S. J. Quantifying Indoor Molds. Presented at Seminar for the Environmental Institute, Atlanta, GA, March 14, 2005.
Abstract: The US EPA has patented a mold ID technology (#6,387,652) licensed by 15 companies in the US and EU. This technology is based upon DNA sequences. In conjunction with HUD, this technology will be used in a National Survey of Homes.

PRESENTATION Water Quality and Swimming-Associated Health Effects 03/02/2005
DUFOUR, A. P. Water Quality and Swimming-Associated Health Effects. Presented at University of Hawaii, Honolulu, HI, March 02, 2005.
Abstract: Evidence from various sources around the world indicate that there is a relationship between gastroenteritis in swimmers and the quality of the bathing water as measured with bacterial indicators of fecal contamination. Current EPA guidelines recommend the use of cultural methods for E. coli and enterococci to measure beach water quality. These methods produce results in 24 hours creating the conundrum, Awe can tell you tomorrow, what you swam in today.@ This shortcoming in current practice for measuring beach water quality has led EPA to consider new technology and indicators that will provide rapid (2 hours or less) measurement of beach waters.
The EPA is currently conducting a multi-year research project to determine the relationship between swimming-associated health effects and water quality measured with methods that will provide results in 2 hours or less. This long-term study will include 9-11 marine and freshwater beaches in the United States. Approximately 5000-8000 persons from each beach will be surveyed to determine swimming exposure and risk factors for illness. Follow-up interviews conducted 10 to 12 days later will reveal illnesses possibly related to the beach visit. The water quality will be measured during the swimmer exposure using the currently recommended cultural method for enterococci as well as a quantitative PCR method. The latter test can produce results in 2 hours or less using enterococci and Bacteroides sp. as the analyte. The analysis will focus on water quality parameters and their association with increased prevalence of swimming-related health effects. Early results from the fresh water studies will be discussed with regard to the relationship between water quality, as measured with new rapid methods, and swimming-associated health effects.


PRESENTATION Elemental Speciation in Environmental Exposure Assessment Matrices 02/27/2005
Creed, J T., P A. Creed, A N. Parks, C A. Schwegel, M. Fricke, A H. Ackerman, D. T. Heitkemper, AND N. Vela. Elemental Speciation in Environmental Exposure Assessment Matrices. Presented at Pittcon 2004, Orlando, FL, February 27-March 4, 2005.
Abstract: Arsenic and tin are two trace metals where exposure assessments have moved towards a speciation based approach because the toxicity is very chemical form dependent. This toxicity difference can be one of many factors which influence the formulation of certain regulations. For arsenic, the drinking water maximum contaminant level is influenced by a wide variety of factors including best available treatment technology, analytical monitoring capability and risk assessments based on health data and relative exposure source estimates. The relative exposure source estimate is used to assess arsenic exposure from all possible routes. Target foods for speciation based exposure assessment can be determined by evaluating food groups high in total arsenic. Two of the key target foods are seafood and rice. Data will be presented on the analysis of these foods with an emphasis on the identification of unknowns and chromatography limitations.
A second concern in formulating drinking water regulation is treatment technology, since the removal efficiency of As(V) is greater than As(III) for most treatment options. Data will be presented using a speciation based analysis to investigate the As(III)/As(V) distribution on drinking water treatment related solids.

Organotins are a potential drinking water issue as PVC pipes (which contain organotins) are increasingly being used to replace the older pipe sections in drinking water distribution systems. Preliminary studies have indicated that PVC pipe initially releases a large percentage of its organotins immediately after being placed into service and the concentration drops off with continued use. This data raises two issues: initial exposure severity and long-term low dose exposure effects of organotins. GC-ICP-MS may provide the means to estimating the low dose exposure to organotins.

PRESENTATION Quality Assurance for Pcr 02/15/2005
FOUT, G. Quality Assurance for Pcr. Presented at Major Accomplishments and Future Directions in Public Health Microbiology Workshop, Columbus, OH, February 15 - 18, 2005.
Abstract: The U.S. Environmental Protection Agency (EPA) held a workshop in January 2003 on the detection of viruses in water using polymerase chain reaction (PCR)-based methods. Speakers were asked to address a series of specific questions, including whether a single standard method could be developed and used for regulatory purposes. Key conclusions of the workshop were that 1) there will be an increasing need for PCR and other molecular methods in the analyses of environmental samples, especially for agents of regulatory importance which cannot be cultured, but 2) the lack of standardization of QA/QC practices reduces the value of PCR-based data. As a result of these conclusions, EPA's Office of Water and Office of Research and Development developed and published a guidance document in October 2004 on quality assurance procedures entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples" (EPA publication number EPA 815-B-04-001). This presentation will present an overview of this guidance, focusing on the key areas facility design, workflow and quality controls. The guidance recommends that PCR facilities use at least three laboratories: 1) a reagent preparation room, 2) a sample preparation room and 3) an amplification and product room. The characteristics of each room will be described. Workflow should proceed from the reagent preparation room to the sample preparation room to the amplification and product room, and never in reverse. It is recommended that four types of positive and two types of negative controls be routinely used. These consist of a "PCR Positive Control," which is used to verify that reagents have been prepared properly; an "Inhibition Positive Control," which is used to show that interfering constituents of the environmental matrix have been removed adequately and do not interfere with the PCR portion of the method; a "Method Positive Control," which is used to demonstrate that the entire method (sampling, concentration, inhibitor removal, PCR, confirmation) is working; and a "Matrix Spike," which gives confidence that the matrix is not interfering with recovery and detection of target organisms over the entire method. The negative controls consist of a "PCR Negative Control," which shows that the PCR reagents do not contain contaminating substances; and a "Method Blank," which verifies that contamination has not been introduced throughout the processing and assay steps. Procedures for reducing QC failures and for taking corrective actions also will be described.

PRESENTATION Environmental Factors and Chemical and Microbiological Constituents Related to the Presenece of Viruses in Ground Water from Small Public Water Supplies in Southeastern Michigan 02/15/2005
FRANCY, D. S., R. N. BUSHON, E. J. LUZANO, AND G. FOUT. Environmental Factors and Chemical and Microbiological Constituents Related to the Presenece of Viruses in Ground Water from Small Public Water Supplies in Southeastern Michigan. Presented at Major Accomplishments and Future Directions in Public Health Microbiology Workshop, Columbus, OH, February 15 - 18, 2005.
Abstract: Thirty-eight public ground-water-supply wells serving fewer than 3,300 people were sampled from July 1999 through July 2001 in southeastern Michigan to determine (1) the occurrence of viral pathogens and microbiological indicators, (2) the adequacy of indicators as predictors of the presence of viruses, and (3) the factors that affect the presence of viruses. Samples were analyzed for enteric viruses by reverse transcriptase-polymerase chain reaction (RT-PCR), for culturable viruses by cell culture, and for the following indicators: total coliforms, E. coli, enterococci, and F-specific and somatic coliphage. Ancillary environmental and water-quality data also were collected.
A total of 169 regular samples and 32 replicate pairs were collected from 38 wells. Each well was sampled from one to five times. By use of RT-PCR, enterovirus was found in four wells (10.5 percent) and hepatitis A virus (HAV) in five wells (13.2 percent). In two of these wells, investigators found both enterovirus and HAV, but on different sampling dates. Analyses for Norwalk virus, reoviruses, and rotaviruses by RT-PCR in a subset of samples gave negative results. Culturable viruses were found once in two wells (5.9 percent), but neither of these wells was positive for viruses by RT-PCR on any sampling date. Of the 38 wells, 9 (23.7 percent) were positive for viruses by either RT-PCR or cell culture.

One or more indicators were found in 18 of 38 wells. Total coliforms, E. coli, enterococci, and F-specific and somatic coliphage were found in 34.2, 10.5, 15.8, 5.9, and 5.9 percent, respectively, of the wells tested. Only three of the indicator-positive wells were positive for an indicator on more than one date at the same well. Five out of nine (55.6 percent) virus-positive wells were also found to be positive for an indicator. Two wells with detections of viruses had a detection of total coliforms, one well had a detection of E. coli, one of enterococci, and one of F-specific coliphage.

More virus-positive samples were found at sites served by septic systems than those served by sewerlines. Sampling condition (ground water or a mixture of tank and ground water), distance to septic system, type of and distance to nearest surface-water body, well characteristics, or land use were not related to the presence of viruses or indicators. Statistically significant relations were found between total coliforms and dissolved organic carbon, iron, or chloride concentrations. Presence of nitrate was related to presence of E. coli, enterococci, coliphage, or enteric viruses but not to total coliforms. The data indicated that chloride-to-bromide (Cl:Br) ratios may be useful as a screening tool for total coliforms and enteric viruses but not for E. coli, enterococci, and F-specific and somatic coliphage.

This study provides evidence for fecal contamination of ground water at small public-supply wells and shows the importance of collecting multiple samples at each site. The study also suggests that the collection of site-characteristic data (e.g., population density, land usage, hydrogeology, well construction), data on multiple water-quality parameters (e.g., mineral and organic carbon concentrations) and data on microbiological indicators is important for making reliable predictions of the presence of enteric viruses in small public water systems. Future data collection toward this end could include repeat sampling several times a year for different indicators, measuring dissolved organic carbon, nitrate plus nitrite, and/or chloride concentrations, or determining Cl:Br ratios.


PRESENTATION Using Today's Data to Close the Beach Today. Quantitative Polymerase Chain Reaction (Qpcr) Rapid Beach Closings Tool 02/08/2005
HAUGHLAND, R. A. Using Today's Data to Close the Beach Today. Quantitative Polymerase Chain Reaction (Qpcr) Rapid Beach Closings Tool. Presented at ORD Product Expo at Region 9, San Francisco, CA, February 08, 2005.
Abstract: Recreational beaches are an important economic and aesthetic asset to communities, states and the nation as a whole. Considerable resources are expended each year in the measurement of fecal indicator bacteria concentrations in the water at these beaches to determine whether these waters are safe for public use. These measurements are presently performed by culture-based methods such as membrane filtration that require at least 24 hr before results are obtained. As a result, these methods often may not allow notification of potential health risks to swimmers until after exposures have occurred. Several molecular microbial analysis technologies have the capability of circumventing this deficiency by providing results in shorter time periods. One particularly rapid technology is the quantitative polymerase chain reaction (QPCR). Water analyses using this technology can provide results in approximately 2 hours. This technology has now been adapted for the measurement of the EPA-recommended fecal indicator organisms, Eschericia coli and Enterococcus, in surface water samples. In the summer of 2003, studies were initiated by the USEPA, Office of Research and Development, at two Great Lakes beaches, to determine the correlation between QPCR and EPA Method 1600 (membrane filtration) measurements of enterococci in water samples and swimmer illness rates. Positive correlations were observed between illness rates and the results of both methods and continuing evaluations are being performed in 2004. This presentation will provide an overview of the principles of the QPCR method, describe its application for beach water quality analysis and provide current data on the relationship between results obtained by this method and membrane filtration.

PRESENTATION Investigation of the Stability of An Arsenosugar in Mouse Cecum Samples Using Ic-ICP-MS and LC-Esi-MS/MS Detection 01/30/2005
CONKLIN, S. D., M. W. FRICKE, A. ACKERMAN, P. A. CREED, JOHN T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Investigation of the Stability of An Arsenosugar in Mouse Cecum Samples Using Ic-ICP-MS and LC-Esi-MS/MS Detection. Presented at 2005 Winter Conference on Plasma Spectrochemistry, Budapest, INDIA, January 30 - February 03, 2005.
Abstract: Arsenosugar exposures occur mainly through seafood and seaweed ingestion, and the major metabolite present in urine is DMA. Understanding the pathway of this conversion is the goal of this ongoing study. Preliminary studies indicated that very little of the arsenosugar degraded to DMA in a synthetic stomach or in pH adjusted control samples. The data presented will examine the stability of an arsenosugar, As(392), in anaerobic cultures of the microflora isolated from the ceca of laboratory mice. These data are relevant in assessing the role of the gastrointestinal microflora in degradation of ingested arsenosugars.
Specifically, data will be presented which indicate that the arsenosugar is converted primarily to its sulfur analog in the small intestine. This conversion appears to take place quickly at 370C. IC-ICP-MS data indicate a good mass balance between the spiked arsenosugar and the resulting by-products. In addition, LC-ESI-MS/MS spectra of the sulfur containing arsenosugar in the cecal samples compare well with those of a synthetic standard. Details regarding the chromatographic conditions used in the characterization of the standard will also be reported.


PRESENTATION Identification of Dimethylthioarsinic Acid By ICP-MS and Ic-Esi-MS/MS in Rice Samples 01/30/2005
FRICKE, M., A. ACKERMAN, P. A. CREED, C. A. SCHWEGEL, AND JOHN T. CREED. Identification of Dimethylthioarsinic Acid By ICP-MS and Ic-Esi-MS/MS in Rice Samples. Presented at 2005 Winter Conference on Plasma Spectrochemistry, Budapest, HUNGARY, January 30 - February 03, 2005.
Abstract: Recently, sulfur analogs of well known arsenicals have been identified in biological and dietary matrices. In this presentation, the detection and identification of dimethylthioarsinic acid (DMTA) will be reported in rice samples after an enzymatic extraction. The enzymatic extraction is a synthetic two stage extraction; the first simulates the stomach and the second stage simulates the environment of the small intestine. The DMTA confirmation includes the co-elution of a DMTA standard and the DMTA in the rice extracts using two chromatographic separations and ICP-MS detection. In addition, the structure of this arsenical was confirmed using IC-ESI-MS/MS with a molecular ion of m/z 153 and fragment ions of m/z 138, 123, 105 (negative mode). Preliminary information will be provided with respect to the production of a standard and the stability of that standard in chromatographic mobile phase eluents. This identification of DMTA in rice and the report by other researchers of DMTA in sheep wool and urine may indicate a more widespread occurrence in nature.

PUBLISHED REPORT Method 535: Measurement of Chloroacetanilide and Chloroacetamide Herbicide Degradates in Drinking Water By Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS) 05/17/2005
Shoemaker, J A. AND M Bassett. Method 535: Measurement of Chloroacetanilide and Chloroacetamide Herbicide Degradates in Drinking Water By Solid Phase Extraction and Liquid Chromatography/Tandem Mass Spectrometry (LC/MS/MS). U.S. Environmental Protection Agency, Washington, DC, 2005.
Abstract: Over the past several years, ethanesulfonic acid (ESA) and oxanilic acid (OA) degradation products of acetanilide/acetamide herbicides have been found in U.S. ground waters and surface waters. The substitution of the sulfonic acid or the carbonic acid for the chlorine atom greatly increases the water solubility of degradates relative to the parent compound and contributes to the increased potential for leaching into groundwater. As a result, alachlor ESA and other acetanilide degradation products were listed on the 1998 Safe Drinking Water Act Contaminant Candidate List (CCL). One acetamide and five acetanilde herbicides are currently registered for agricultural use in the U.S. The next step in the CCL-process is to collect data on the concentrations and occurrence of these compounds in the nation's drinking water supplies. However, the existing analytical methods for measuring chloroacetanilide degradates do not address issues specific to analyzing these compounds in drinking water. Because many of the methods were developed for ground water, dechlorination was not addressed nor was the method tested in all types of drinking water matrices. In addition, existing methods do not address all twelve ESA and OA degradates of the six U.S. registered acetanilide/acetamide herbicides. Therefore, the focus of this research was to develop a sensitive and specific analytical method for the analysis of alachlor ESA and other chloroacetanilide degradates in drinking water.

PUBLISHED REPORT Method 521: Determination of Nitrosamines in Drinking Water By Solid Phase Extraction and Capillary Column Gas Chromatography With Large Volume Injection and Chemical Ionization Tandem Mass Spectrometry (MS/MS) 05/17/2005
Munch, J W. Method 521: Determination of Nitrosamines in Drinking Water By Solid Phase Extraction and Capillary Column Gas Chromatography With Large Volume Injection and Chemical Ionization Tandem Mass Spectrometry (MS/MS). U.S. Environmental Protection Agency, Washington, DC, 2005.
Abstract: NDMA is an emerging drinking water contaminant that is of interest to EPA and the environmental community. Its presence in drinking water is a potential health concern, because the EPA's IRIS data base lists the concentration of NDMA required to result in a one in one million lifetime cancer risk to be only 0.7 ng/L. NDMA is produced by industrial sources, such as the manufacture of rocket fuel, but has also been identified as a potential disinfection by-product. The focus of NERL's research was to develop an improved analytical method for the analysis of NDMA in drinking water that was sensitive, specific and cost-effective. Existing methods for measuring NDMA in water are expensive and labor intensive, and some require the use of very large amounts of toxic solvents such as methylene chloride. In addition, the sensitivity of some existing methods is insufficient for conducting low-level monitoring. The method was developed for inclusion in EPA's Unregulated Contaminant Monitoring Rule, scheduled for proposal in 2005, in order for the EPA Office of Water to collect nationwide occurrence data on nitrosamines in drinking water and make a regulatory determination. If NDMA or other nitrosamines become regulated drinking water contaminants in the future, the method could also be used for compliance monitoring.Research Approach

PUBLISHED REPORT Method 332.0: Determination of Perchlorate in Drinking Water By Ion Chromatography With Suppressed Conductivity and Electrospray Ionization Mass Spectrometry 05/17/2005
HEDRICK, E. J., R. SLINGSBY, AND D. J. MUNCH. Method 332.0: Determination of Perchlorate in Drinking Water By Ion Chromatography With Suppressed Conductivity and Electrospray Ionization Mass Spectrometry. U.S. Environmental Protection Agency, Washington, DC, 2005.
Abstract: This method is applicable to the identification and quantitation of perchlorate in raw and finished drinking waters. The approach used is ion chromatography with suppressed conductivity and electrospray ionization mass spectrometry (IC-ESI/MS)

PUBLISHED REPORT Method 529, Determination of Explosives and Related Compounds in Drinking Water By Solid Phase Extraction and Capillary Column Gas Chromatography/Mass Spectrometry 05/17/2005
Munch, J W. Method 529, Determination of Explosives and Related Compounds in Drinking Water By Solid Phase Extraction and Capillary Column Gas Chromatography/Mass Spectrometry. U.S. Environmental Protection Agency, Washington, DC, 2005.
Abstract: Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a military explosive which is known to have contaminated groundwater on and near military installations where it has been used and stored. Historical disposal practices such as open burning and detonation have contributed to environmental contamination. EPA has classified RDX as a possible human carcinogen, and has established a Lifetime Health Advisory of 2 g/L. The project focus was to develop a sensitive and specific analytical method for the analysis of RDX in drinking water. Existing analytical methods for measuring RDX are sensitive but not very specific. The need for this project arose from the 1998 Contaminant Candidate List (CCL) which was developed in response to the 1996 amendments to the Safe Drinking Water Act. The CCL, published by EPA'S Office of Ground Water and Drinking Water, identifies potential drinking water contaminants that might be regulated by EPA at some future date. RDX was identified as a CCL chemical on the 1998 list. The analytical method developed will be proposed for the collection of nationwide occurrence data for RDX, and possibly other explosives and related chemicals, in the next Unregulated Contaminant Monitoring Rule (UCMR) scheduled for 2004. In the event that a decision is made to regulate RDX in drinking water, the method could also be used for compliance monitoring.
Research

Approach The research goal was to develop a method for measuring RDX and up to 16 additional explosives, degradation products, and manufacturing by-products. The initial method detection limit goal for RDX was 0.5 g/L, well below the Lifetime Health Advisory of 2 g/L. As the project progressed, new health information indicated that additional sensitivity may be necessary. The approach involved developing procedures for (1) sample collection, preservation, shipping and handling, (2) extraction and concentration of the target analytes from an aqueous solution, (3) separation of the analytes chromatographically, and (4) detection and quantification. Solid phase extraction techniques were investigated for the extraction of explosives and related compounds from aqueous samples. Gas chromatography/mass spectrometry (GC/MS) was investigated for the chromatographic separation and detection steps, because of its specificity.

Results and Implications

The analytical method developed from this research can be used to measure RDX and 13 additional explosives and related chemicals in drinking water samples. The method detection limit for RDX ranges from 0.006 - 0.12 g/L depending upon the various options selected within the method protocol. The most sensitive procedure employs Selected Ion Monitoring (SIM) GC/MS. These concentrations are expected to be below those needed for drinking water monitoring, based upon currently available health effects information. This method is an improvement over other published methods for RDX in the following ways: (1) use of the mass spectrometer as the detector provides positive identification of all method analytes without the use of additional confirmatory techniques, and (2) the sample preservation protocol ensures sample stability between the time of sample collection and analysis. These improvements will significantly enhance the use of this methodology in the collection of nationwide occurrence data to support regulatory decision making for chemicals on the CCL.

This research project directly supports ORD's research under the Government Performance and Results Act (GPRA) Goal 2- Clean and Safe Water, Objective 2 - Ensure Safe Drinking Water and Recreational Waters, Sub-objective 7 - Safe Drinking Water Act Research. The results of this project address GPRA annual performance goal (APG) 27 ["Produce scientific reports on unregulated drinking water contaminants, in support of the development of the next list of chemicals and pathogens for potential regulatory action (i.e., Contaminant Candidate List #2). These reports will help ensure that future drinking water regulations address the contaminants of greatest public health concern."], and annual performance measure (APM) 78 ["Improved methods for CCL related chemicals in drinking water for use in the Unregulated Contaminant Monitoring Rule"].

PUBLISHED REPORT Method 415.3 Measurement of Total Organic Carbon, Dissolved Organic Carbon and Specific UV Absorbance at 254 Nm in Source Water and Drinking Water 05/17/2005
Potter, B B. AND J. C. Wimsatt. Method 415.3 Measurement of Total Organic Carbon, Dissolved Organic Carbon and Specific UV Absorbance at 254 Nm in Source Water and Drinking Water. U.S. Environmental Protection Agency, Washington, DC, 2005.
Abstract: 2.0 SUMMARY OF METHOD
2.1 In both TOC and DOC determinations, organic carbon in the water sample is oxidized to form carbon dioxide (CO2), which is then measured by a detection system. There are two different approaches for the oxidation of organic carbon in water samples to carbon dioxide gas: (a) combustion in an oxidizing gas and (b) UV promoted or heat catalized chemical oxidation with a persulfate solution. Carbon dioxide, which is released from the oxidized sample, is detected by a conductivity detector or by a nondispersive infrared (NDIR) detector. Instruments using any combination of the above technologies may be used in this method.

2.2. Setteable solids and floating matter may cause plugging of valves, tubing, and the injection needle port. The TOC procedure allows the removal of settleable solids and floating matter. The suspended matter is considered part of the sample. The resulting water sample is then considered a close approximation of the original whole water sample for the purpose of TOC measurement.

2.3. The DOC procedure requires that the sample be passed through a 0.45 um filter prior to analysis.

2.4. The TOC and DOC procedures require that all inorganic carbon be removed from the sample before the sample is analyzed for organic carbon content. If the inorganic carbon (IC) is not completely removed, significant error will occur. The inorganic carbon interference is removed by converting the mineralized IC to CO2 by acidification and sparging with an inert gas to remove the generated CO2. The sample, which is now free from IC interference, is then injected into a TOC instrument system. The orgnaic carbon is oxidized to CO2 which is released from the sample, detected, and repoted as mg/L or ppm TOC or DOC.

2.5. The UVA procedure requires that the sample be passed through a 0.45 um filter and transferred to quartz cell. It is then placed in a spectrophotometer to measure the UV absorbance at 254 nm and reported in cm -1.

2.6. The SUVA procedure requires both the DOC and UVA measurement. The SUVA is then calculated by dividing the UV absorbance of the sample (in cm -1) by the DOC of the sample (in mg/L) and then multiplying by 100 cm/M. SUVA is reported in units of L/mg-M. The formula for the SUVA may be found in Section 12.2.

 

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