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Microbiological & Chemical Exposure Assessment Research Division Publications: 2008
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This page lists publication titles, citations and abstracts produced by NERL's Microbiological & Chemical Exposure Assessment Research Division for the year 2008, organized by Publication Type. Your search has returned
45 Matching Entries.
See also Microbiological & Chemical Exposure Assessment Research Division citations with abstracts:
1999,
2000,
2001,
2002,
2003,
2004,
2005,
2006,
2007,
2008
Technical Information Manager: Angela Moore - (513) 569-7341 or moore.angela@epa.gov
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Presented/Published |
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Complementary Molecular and Elemental Detection of Speciated Thioarsenicals Using Esi-MS in Combination With a Xenon-Based Collision-Cell ICP-MS With Application to Fortified Nist Freeze-Dried Urine |
04/01/2008
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ELLIS, J., C. GALLAWA, J. CARUSO, J. T. CREED, S. CONKLIN, P. A. CREED, AND A. R. YOUNG. Complementary Molecular and Elemental Detection of Speciated Thioarsenicals Using Esi-MS in Combination With a Xenon-Based Collision-Cell ICP-MS With Application to Fortified Nist Freeze-Dried Urine. Analytical and Bioanalytical Chemistry. Springer, New York, NY, 390(7):1731-1737, (2008).
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The simultaneous detection of arsenic and sulfur in thio-arsenicals was achieved using xenonbased collision cell ICP-MS in combination with HPLC. In an attempt to minimize the 16O16O+ interference at m/z 32, both sample introduction and collision cell experimental parameters were optimized. Low flow rates (0.25mL/min) and a high methanol concentration (8%) in the mobile phase produced a four fold decrease in the m/z 32 background. A plasma sampling depth change from 3mm to 7mm produced a two fold decrease in background at m/z 32, with a corresponding
four fold increase in the signal associated with a high ionization surrogate for sulfur. The quadrupole and octopole bias were used as a kinetic energy discriminator between background and analyte ions, but a variety of tuning conditions produced similar ( |
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| JOURNAL |
Higher Environmental Relative Moldiness Index (Ermism) Values Measured in Detroit Homes of Severely Asthmatic Children |
05/01/2008
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VESPER, S. J., C. MCKINSTRY, R. A. HAUGLAND, L. M. NEAS, E. E. HUDGENS, B. HEIDENFELDER, AND J. E. GALLAGHER. Higher Environmental Relative Moldiness Index (Ermism) Values Measured in Detroit Homes of Severely Asthmatic Children. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 394(1):192-196, (2008).
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Sieved vacuum bag dust from the homes of 143 children in Detroit was analyzed by mold specific quantitative PCR (MSQPCR) and the Environmental Relative Moldiness Index (ERMIsm) was calculated for each home. Children living in these homes were categorized into non-asthmatic (n=83), moderately asthmatic (n=28) and severely asthmatic (n=32) based on prescription medication usage for their asthma management (none, occasional and daily, respectively). The mean ERMI for each group of homes was 6.2 for non-asthmatic, 6.3 for moderately asthmatic and 8.2 for severely asthmatic children. The ERMI values in the homes of severely asthmatic children were significantly greater compared to the non-asthmatics (p = 0.04 in Wilcoxon Rank-sum test). Aspergillus niger and Aspergillus unguis were the primary mold species that distinguished severely asthmatic children’s homes and non-asthmatic children’s homes (p < 0.05; Wilcoxon Rank-sum test). The determination of the home’s ERMI values may aid in prioritizing home remediation efforts, particularly in those children who are at increased risk for asthma exacerbation.
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Molecular Characterization of a Functional Type Vi Secretion System from a Clinical Isolate of Aeromonas Hydrophilia |
04/01/2008
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Suarez, G., J. C. Sierra, J. Sha, S. Wang, T. E. Erova, A. A. Fadl, S. M. Foltz, A. J. Horneman, AND A. K. Chopra. Molecular Characterization of a Functional Type Vi Secretion System from a Clinical Isolate of Aeromonas Hydrophilia. Microbial Pathogenesis. Elsevier BV, AMSTERDAM, Netherlands, 44(4):344-361, (2008).
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Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.
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| JOURNAL |
Cold Shock Exoribonuclease R(vacb) Is Involved in Aeromonas Hydrophila Virulence |
05/01/2008
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Erova, T. E., V. G. Kosykh, A. A. Fadl, J. Sha, A. J. Horneman, AND A. Chopra. Cold Shock Exoribonuclease R(vacb) Is Involved in Aeromonas Hydrophila Virulence. JOURNAL OF BACTERIOLOGY. American Society for Microbiology, Washington, DC, 190(10):3467-3474, (2008).
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In this study, we cloned and sequenced a virulence-associated gene (vacB) from a clinical isolate SSU of Aeromonas hydrophila. We identified this gene based on our recently annotated genome sequence of the environmental isolate ATCC 7966T of A. hydrophila and the vacB gene of Shigella flexneri. The A. hydrophila VacB protein contained 798 amino acid residues, had a molecular mass of 90.5 kDa, and exhibited an exoribonuclease (RNase R) activity. The RNase R of A. hydrophila was a cold-shock protein and was required for bacterial growth at low temperature. The vacB isogenic mutant, which we developed by homologous recombination using marker exchange mutagenesis, was unable to grow at 4°C. In contrast, the wild-type (WT) A. hydrophila exhibited significant growth at this low temperature. Importantly, the vacB mutant was not defective in growth at 37°C. The vacB mutant also exhibited reduced motility, and these growth and motility phenotype defects were restored after complementation of the vacB mutant. The A. hydrophila RNase R-lacking strain was found to be less virulent in a mouse lethality model (70% survival) when given by the intraperitoneal route at as two 50% lethal doses (LD50). On the other hand, the WT and complemented strains of A. hydrophila caused 80 to 90% of the mice to succumb to infection at the same LD50 dose. Overall, this is the first report demonstrating the role of RNase R in modulating the expression of A. hydrophila virulence.
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| JOURNAL |
Mold Species in Dust from the International Space Station Identified and Quantified By Mold Specific Quantitative Pcr Mceard |
07/01/2008
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VESPER, S. J., W. Wong, C. M. Kuo, AND D. L. Pierson. Mold Species in Dust from the International Space Station Identified and Quantified By Mold Specific Quantitative Pcr Mceard. Research in Microbiology. ELSEVIER, AMSTERDAM, Holland, 159(6):432-435, (2008).
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Dust was collected over a period of several weeks in 2007 from HEPA filters in the U.S. Laboratory Module of the International Space Station (ISS). The dust was returned on the Space Shuttle Atlantis, mixed, sieved, and the DNA was extracted. Using a DNA-based method called mold specific quantitative PCR (MSQPCR), 39 molds were measured in the dust. Potential opportunistic pathogens Aspergillus flavus and A. niger and potential moderate toxin producers Penicillium chrysogenum and P. brevicompactum were noteworthy. No cells of the potential opportunistic pathogens A. fumigatus, A. terreus, Fusarium solani or Candida albicans were detected.
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| JOURNAL |
Use of (1-3)-Β-D-Glucan Concentrations in Dust as a Surrogate Method for Estimating Specific Mold Exposures |
05/01/2008
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VESPER, S. J., C. McKinstry, R. A. HAUGLAND, L. M. NEAS, E. E. HUDGENS, B. HEIDENFELDER, AND J. GALLAGHER. Use of (1-3)-Β-D-Glucan Concentrations in Dust as a Surrogate Method for Estimating Specific Mold Exposures. SCIENCE OF THE TOTAL ENVIRONMENT. Elsevier Science Ltd, New York, NY, 394(1):192-196, (2008).
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Indoor exposure to fungi has been associated with respiratory symptoms, often attributed to their major cell wall component, (1-3)-β-D-glucan (DG). This and the ease and low cost of performing DG analysis rather than cultivation or microscopic counting of mold spores, has prompted many to use DG as a surrogate for mold exposure. The aim of this study was to examine which indoor mold species predict DG concentration in field dust samples, and thus whether DG can be used as a surrogate for total mold or specific mold genera exposures.
We used the quantitative polymerase chain reaction (QPCR) method to analyzed 36 indoor fungal species in 297 indoor dust samples, which were also simultaneously analyzed for DG concentration. Linear regression analysis, followed by factor analysis and structural equation modeling, were utilized in order to identify fungal species that mostly contribute to the DG concentration in field dust samples.
The study revealed that Cladosporium and Aspergillus species were the main DG contributors followed by Epicoccum nigrum, Wallemia sebi and Penicillium brevicompacatum. Another interesting finding of the study is that the species that contribute most to the DG concentration are also the ones that are most prevalent in indoor environments. However, Alternaria alternata, the third most common fungal species in indoor dust, did not seem to be a significant source of DG. We can speculate whether this was due to low extraction efficiency, or that allergic effects of Alternaria are not determined by the (1-3)-β-D-glucan content.
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| PRESENTATION |
Investigation of the Biotransformation of a Dma in Mouse Cecum Samples Using Ic-ICP-MS and LC-Esi-MS/MS Detection |
01/06/2008
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KUBACHKA, K., S. CONKLIN, J. T. CREED, AND D. J. THOMAS. Investigation of the Biotransformation of a Dma in Mouse Cecum Samples Using Ic-ICP-MS and LC-Esi-MS/MS Detection. Presented at 2008 Winter Conference on Plasma Spectrochemistry, Tennecula, CO, January 06 - 12, 2008.
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Recent arsenic metabolism studies have begun to indicate the presence of sulfur analogs of the more common arsenic oxides in biological systems. An emerging area of research is how and where these arsenic species are formed in the metabolic pathway. The authors have previously indicated that the microflora in the cecum of a mouse is able to biotransform an arsenosugar oxide (a tri-alkyl substituted arsenic oxide) to its corresponding sulfide [1]. The initial mechanism of the aforementioned conversion theorized that the bacteria produced H2S as a waste product which then facilitated the conversion. Subsequent research using H2S in an ideal solution indicated that the rate of conversion was influenced by the degree of substitution of the arsenic oxide. For instance, the reaction rate for tetramethylarsine oxide (TMAO, tri-alkyl substituted) was faster than dimethylarsinic acid (DMA, di-alkyl substituted) which was faster than monomethylarsinic acid (MMA, mono-alkyl substituted) [2]. The conversion of DMA to dimethylthioarsinic acid (DMTA) in these ideal solutions raised the question if a similar conversion would be observed in the anaerobically incubated cecal samples from a mouse.
In this experiment, DMA was added at various concentrations to the cecum contents of a mouse, which were then incubated at 37 °C. The DMA spiked cecum samples were incubated for various periods and then flash frozen at -80 °C to minimize additional unwanted activity and subsequent transformation. An outline of the study design is given below:
Cecum Samples
(cecum + buffer) Time
(Hours at 37 °C)
Arsenic (ng/g) 0 1 6 18 24
0 X X X
20 X X X X X
200 X X X X X
1000 X X
Controls
(cecum, no buffer) Time
(Hours)
Arsenic (ng/g) 0 24
0 X X
20 X X
200 X X
1000 X X
The fortified samples, blanks and controls were analyzed for arsenic metabolites via speciation with IC-ICP-MS, utilizing arsenic specific detection at m/z 75. Molecular characterization was also conducted using LC-ESI-MS/MS. The metabolites detected were: DMA, DMTA, dimethyldithioarsinic acid (DMDTA), and trimethylarsine sulfide (TMAS). Both elemental and molecular data will be presented to support these identifications. The presence of TMAS was not expected and thorough analysis of controls and blanks lead the authors to believe it is not an artifact of sample contamination. Its presence could possibly be explained by DMA being methylated to TMAO, which could then be sulfurylated to TMAS. Finally, the conversion from DMA to the corresponding sulfur analogs will be presented with respect to incubation times. This data set indicates that sulfur analogs of arsenic oxides may be produced in the gastrointestinal tract. The data set shows evidence of novel biotransformation pathways of DMA in the gastrointestinal tract, and should prove useful in further understanding the arsenic metabolism pathways.
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| PRESENTATION |
Rna Extraction Methods for Reverse Transcriptase Real-Time Pcr and Microarray Analysis of Cryptosporidium and Toxoplasma Gondii Oocysts |
03/29/2008
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See, M. J., M. W. WARE, J. P. Dubey, AND E. VILLEGAS. Rna Extraction Methods for Reverse Transcriptase Real-Time Pcr and Microarray Analysis of Cryptosporidium and Toxoplasma Gondii Oocysts. Presented at ASM Annual Meeting, Muncie, IN, March 28 - 29, 2008.
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The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion processes or about their survival after disinfection treatment. Microarrays and reverse transcriptase real-time PCR (RT-qPCR) are useful techniques that are amenable to identifying genes involved in regulating such mechanisms. In order to effectively perform these analyses, high quality RNA must be isolated. Here, we evaluated four RNA extraction methods (Ambion RiboPure, Qiagen RNeasy, Epicentre Master Pure and TRIzol LS reagent) in their ability to extract high quality RNA from C. parvum and T. gondii oocysts. RNA quality was measured and expressed as an RNA Integrity Number (RIN), with a 9.0 or above indicating minimal degradation, while RNA purity was determined using RT-qPCR. Preliminary results indicated high quality RNA (RIN 8.3 – 9.8) was extracted from C. parvum oocysts using all four extraction methods; however, genomic DNA (gDNA) was present in all samples. DNase I treatment effectively removed gDNA contaminants only in Ambion and Qiagen extracted RNA, but the quality was compromised (RIN 5.5 - 7.7). RNA extracted from T. gondii oocysts proved to be more difficult and the quality/purity of RNA extracted was variable. Nevertheless, all RNA extraction methods used in this study were effective in quantitating hsp-70 and β-tubulin expression in C. parvum oocysts and SporoSAG expression in T. gondii oocysts using RT-qPCR. These studies will now enable us to use RT-qPCR, and perhaps microarray analysis, for identifying novel genes essential for oocyst development, survival in the environment, and establishment of infection in the host.
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| PRESENTATION |
Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary By Real-Time Quantitative Pcr |
06/01/2008
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CHERN, E. C., M. VARMA, M. Byappanahali, R. Whitman, R. Zepp, AND R. A. HAUGLAND. Analysis of Enterococci and Bacteriodales Fecal Indicator Bacteria in a Lake Michigan Tributary By Real-Time Quantitative Pcr. Presented at 108th General Meeting of the American Society for Microbiology, Boston, MA, June 01 - 05, 2008.
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The Salt Creek watershed in northwest Indiana drains into Lake Michigan near several heavily used recreational beaches. This study aimed to investigate the levels of fecal indicator bacteria, enterococci and Bacteroidales, in Salt Creek using real-time quantitative PCR (qPCR) analysis and to obtain a preliminary assessment of the contribution of these organisms in effluent discharged by a publicly owned treatment works (POTW) into this stream. Bacterial concentrations were estimated from qPCR results as calibrator cell equivalents (CCE) at 1 site upstream and 5 sites downstream from the POTW as well as in the POTW effluent. Additionally, enterococci were enumerated on mEI media to compare CCE with culturable results. Our data indicated that the qPCR CCE/100 ml geometric mean over an 8 week collection period in the summer of 2007 ranged from 1.0 x 103 to 3.5 x 103 enterococci and from 3.6 x 104 to 2.3 x 105 total Bacteroidales at the 6 different stream sites. Analysis of Bacteroides thetaiotaomicron resulted in levels ranging from 8.9 x 102 to 9.0 x 103 at the same sites. The geometric mean concentrations of enteroccoci and Bacteroidales CCE in the effluent samples were approximately 1 log greater than the respective mean concentrations in the creek. Culture counts revealed that the geometric mean enterococci at these stream sites ranged from 5.0 x 102 to 2.6 x103 CFU/100 ml. Comparison of the trend between enterococci culture and qPCR data showed that the only major divergence was in the treated effluent where the mean CCE estimate was 3.1 x 104 CCE/100 ml and the mean culture concentration was 3.4 x 101 CFU/100 ml. Indicator levels in the stream samples collected right above and below the POTW were not significantly different for qPCR determined enterococci but were significantly greater below the outfall for Bacteroidales and above the outfall for culturable enterococci. Further studies are needed to evaluate the relationships between qPCR and culturable indicator levels in POTW effluents and receiving waters.
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| PRESENTATION |
Molecular Genotyping of Viable Cryptosporidium Oocysts |
03/28/2008
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Brescia, C., M. W. WARE, S. L. BARTELT-HUNT, A. EGOROV, AND E. VILLEGAS. Molecular Genotyping of Viable Cryptosporidium Oocysts. Presented at ASM Annual Meeting, Muncie, IN, March 28 - 29, 2008.
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Cryptosporidium is a chlorination-resistant protozoan parasite that causes a self-limiting diarrheal disease in the immunocompetent or severe chronic diarrhea in the immunocompromised. Two species, C. parvum and C. hominis, cause most cases of cryptosporidiosis in humans, while C. muris infects primarily rodents. The current US EPA standard methods for the detection of Cryptosporidium oocysts in water, Methods 1622 and 1623, are labor intensive and cannot determine the species or distinguish viable from nonviable oocysts. The goal of this project is to develop a rapid molecular method that specifically detects viable waterborne Cryptosporidium oocysts and that determines the species present in a sample. This alternative technique employs propidium monoazide (PMA) staining in conjunction with conventional PCR detection and genotyping. PMA is a DNA intercalating dye that only penetrates dead cells. Using traditional DNA extraction procedures, only the genomic DNA from live cells, which is not intercalated with PMA, is retained and detected in subsequent PCR analysis. To date, PMA has only been used to differentiate live/dead bacteria and fungi. It has not yet been applied to protozoan parasites. Preliminary results revealed that heat killed oocysts treated with PMA were not detected while live oocysts treated with PMA were detected using conventional PCR. This method was then paired with Cryptosporidium PCR-based genotyping methods to allow for species discrimination. When live C. parvum and dead C. muris oocysts were mixed at a 1:9 ratio, only live C. parvum oocysts were detected. These results indicate that PMA treatment is very specific at detecting DNA from live oocysts in the presence of a higher concentration of dead oocysts. We are currently investigating the application of this method in environmental water matrices.
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| PRESENTATION |
Development of a Microarray Detection Method for Waterborne Pathogens Mceard/Barb |
04/08/2008
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BRINKMAN, N., E. VILLEGAS, R. Francisco, F. W. SCHAEFER, T. NICHOLS, D. Roberts, AND P. Schaudies. Development of a Microarray Detection Method for Waterborne Pathogens Mceard/Barb. Presented at Joint EPA and DHS Conference on Real-World Applications and Solutions for Microbial Risk Assessment, Bethesda, MD, April 08 - 10, 2008.
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Presentation at the Joint EPA and DHS Conference on Real-World Applications and Solutions for Microbial Risk Assessment in Bethesda, MD, April 8-10, 2008
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| PRESENTATION |
Mra Research Activities to Support Drinking Water Standards |
04/08/2008
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ASHBOLT, N. Mra Research Activities to Support Drinking Water Standards. Presented at The Joint US.EPA and Department of Homeland Security Conference on Real-World Applications and Solutions for Microbial Risk Assessment, Bethesda, MD, April 08 - 10, 2008.
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Slide Presentation for the Joint U.S. EPA and Department of Homeland Security Conference on Real-World Applications and Solutions for Microbial Risks Assessment, Bethesda, MD, April 8-10, 2008
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Improving Lab Sample Management Pos/Mceard |
04/23/2008
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BASSETT, M. AND M. BINFORD. Improving Lab Sample Management Pos/Mceard. Presented at 2008 EPA 2008 Conference on Managing Environmental Quality Systems, Seattle, WA, April 21 - 24, 2008.
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"Scientists face increasing challenges in managing their laboratory samples, including long-term storage of legacy samples, tracking multiple aliquots of samples for many experiments, and linking metadata to these samples. Other factors complicating sample management include the need to share samples amongst team members and dealing with multiple sample storage units. To address these issues, the National Exposure Research Laboratory in Cincinnati has an ongoing project to increase scientist’s productivity and improve the condition under which their samples are stored. This talk will provide details of the project, including the development of a sample handling policy and the deployment of an electronic sample tracking system."
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| PRESENTATION |
Feasibility of Community Food Item Collection for the National Children's Study |
04/16/2008
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Jordan, K. C., M. Knuth, B. Sherwood, R. R. Larson, L. J. MELNYK, S. McNutt, J. J. QUACKENBOSS, S. M. Viet, AND L. J. Moyer-Mileur. Feasibility of Community Food Item Collection for the National Children's Study. Presented at Utah Dietetic Association - University of Utah Research Conference, Salt Lake City, UT, April 16 - 18, 2008.
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The National Children’s Study proposes to investigate the role of contaminants on health outcomes in pregnant women and children. A specific area of concern is contaminant exposure through the ingestion of solid foods. National food contaminant databases may miss environmental exposures unique to specific communities. Therefore, alternative sampling procedures are proposed to capture contributing environmental exposures on a household level, such as storage, preparation, and consumption in the residence.
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| PRESENTATION |
Quantitative Microbial Risk Assessment of Drinking Water the Next Step |
04/16/2008
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ASHBOLT, N. Quantitative Microbial Risk Assessment of Drinking Water the Next Step. Presented at Meeting at the Technische Universiteit Delft, Holland, NETHERLANDS, April 16, 2008.
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Presentation given at the Technische Universiteit Delft, Holland, The Netherlands, April 16, 2008
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| PRESENTATION |
Microbial Risk Assessment: Underpinning Research Needs for Recycled Water |
05/05/2008
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ASHBOLT, N. Microbial Risk Assessment: Underpinning Research Needs for Recycled Water. Presented at 12th Annual Water Reuse and Desalination Research Conference, Denver, CO, May 05 - 06, 2008.
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Slide Presentation to be presented at the 12th Annual Water Reuse and Desalination Research Conference, Denver, Colorado, May 5-6, 2008
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| PRESENTATION |
Emerging Contaminants in the Drinking Water Cycle Mceard |
01/29/2008
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GLASSMEYER, S. Emerging Contaminants in the Drinking Water Cycle Mceard. Presented at Presentation at Wooster College, Wooster, OH, January 29, 2008.
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In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-g/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50 % wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the complex mixtures of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. In the source water of one conventional drinking water facility, 45 out of 113 monitored chemicals were detected at least once, with 21 chemicals still detectable in the finished drinking water. This documents the incomplete removal of such chemicals during treatment. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.
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| PRESENTATION |
Analysis of Arsenical and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS |
05/21/2008
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KUBACHKA, K., C. GALLAWA, P. A. CREED, J. T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Analysis of Arsenical and Their Sulfur Analogs Using Hplc With Collision Cell ICP-MS and Esi-MS. Presented at 2nd International Congress: Arsenic in the Environment, Valencia, SPAIN, May 21 - 23, 2008.
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Slide presentation at the 2nd International Congress: Arsenic in the Environment, Valencia, Spain, May 21-23, 2008
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| PRESENTATION |
Detection of Mycobacterium Avium Subsp. Paratuberculosis in Drinking Water and Biofilms Using Quantitative Pcr |
06/05/2008
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Beumer, A., D. N. KING, AND S. L. PFALLER. Detection of Mycobacterium Avium Subsp. Paratuberculosis in Drinking Water and Biofilms Using Quantitative Pcr. Presented at American Society for Microbiology General Meeting, Boston, MA, June 01 - 05, 2008.
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Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s disease in domestic animals and has been implicated in Crohn’s disease in humans. Cows infected with Johne’s disease shed large quantities of MAP into soil. Further, MAP has been isolated from surface water, is resistant to chlorine and able to form biofilms; therefore, it is likely to colonize drinking water distribution systems and be found in drinking water. Occurrence of MAP in drinking water has never been demonstrated. Because MAP is extremely difficult to isolate from water quantitative PCR (QPCR) assays have been developed to identify the presence and estimate the number of MAP. We used primer and probe sets designed to target IS900 sequences in the MAP genome, and we confirmed our IS900 results using Target 251 which is known to be specific to MAP. Drinking water was collected from 31 cold water faucets in southwestern Ohio. Two one liter samples were collected: a first pull sample, taken when the water was first turned on, and a standard method sample taken after running the water two minutes. The first pull is intended to collect cells from household pipes and potentially cells sloughed from biofilms; the standard method sample is intended to collect cells primarily from the water column. Biofilms were collected by swabbing the entire faucet grating, followed by vortexing in sterile water to remove attached biofilm. Water and biofilm samples were filtered, DNA was extracted and analyzed using QPCR (IS900 and Target 251 primers). Eighty four percent of first pull samples were positive for MAP, 92% of standard methods samples were positive, and 89% of biofilm samples were positive for MAP. Numbers of MAP (assuming 18 copies IS900 per organism) ranged from 0 to 29,000; however 82% of samples had less than 500 organisms per liter or faucet biofilm. There does not appear to be any statistical difference between presence in the drinking water distribution system and biofilm or first pull samples. This study shows that MAP is common in drinking water distribution systems, residential pipes and residential biofilms.
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| PRESENTATION |
Interferon Gamma as a Biomarker of Exposure to Enteric Viruses |
10/12/2008
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LI, L. AND A. P. DUFOUR. Interferon Gamma as a Biomarker of Exposure to Enteric Viruses. Presented at 2008 Joint ISEE-ISEA Conference, Pasenda, CA, October 12 - 16, 2008.
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Interferon gamma (IFN-γ) was selected as a biomarker for viral exposure. Twelve-week-old BALB/c mice were intraperitoneally injected with Coxsackievirus B3 or B4 diluted in phosphate-buffered saline (PBS). Control mice were injected with PBS only. Four months after viral infection, T lymphocytes were isolated from mouse thymus and spleen. T lymphocyte release of IFN-γ was examined after in vitro incubation with viral antigens, a mitogen, PHA and PBS respectively. The level of IFN-γ released by T lymphocytes was examined by antibody-capture chemiluminescent ELISA. A marked increase in level of IFN-γ was observed when T cells from Coxsackievirus B3-infected mice were incubated with B3 virus but not B4 or PBS. This indicated that Coxsackievirus B3- sensitized T cell receptor recognized only T cell epitope from B3 virus not B4. Coxsackievirus B4 primed mouse T cells did not release IFN-γ when they were incubated with B3 virus or PBS. Our results showed that IFN-γ produced by primed memory T cells is virus-specific. The data also show that memory T cells can distinguish very structurally related Coxsackievirus B3 and B4 when the sensitized T cells were directly stimulated by the viruses in vitro. The results of this study may be extended to human exposure studies related to microbial pathogens.
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| PRESENTATION |
A New Electropositive Filter for Concentrating Enterovirus and Norovirus from Large Volumes of Water Mceard |
06/01/2008
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Karim, M., E. RHODES, N. BRINKMAN, L. J. WYMER, AND G. FOUT. A New Electropositive Filter for Concentrating Enterovirus and Norovirus from Large Volumes of Water Mceard. Presented at ASM Annual Meeting, Boston, MA, June 01 - 05, 2008.
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| Abstract:
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The detection of enteric viruses in environmental water usually requires the concentration of viruses from large volumes of water. The 1MDS electropositive filter is commonly used for concentrating enteric viruses from water but unfortunately these filters are not cost-effective for routine viral monitoring. In this study, an inexpensive electropositive cartridge filter, the Nanoceram® filter, was evaluated for its ability to concentrate enterovirus and norovirus from large volumes of water. In initial experiments, one hundred liters of deionized water was seeded with 105 PFU of poliovirus 1 and concentrated using the same adsorption-elution procedure used for the 1 MDS filter. The mean retention of seeded poliovirus by Nanoceram® filters was 84 percent. To optimize the elution procedure, six protocols, each comprised of two successive elutions with varying lengths of filter emersion, were evaluated. There was a significant difference among the elution procedures tested (p=0.025). The highest virus recovery (77%) was obtained by immersing the filters in beef extract for 1 min during the first elution and 15 minutes during the second elution. The recovery efficiencies of poliovirus, coxsackie B5, and echovirus 7 from 100 liters of seeded tap water were 54%, 27%, and 32%, respectively. Finally, virus recoveries by Nanoceram® filters were compared with 1MDS filters using tap water and Ohio River water. The mean recoveries of poliovirus from 100L of tap water using Nanoceram® and 1MDS filters were 51% and 67%, respectively, and the recoveries from Ohio River water were 38% and 36%, respectively. The recoveries of Norwalk virus by Nanoceram® filters from tap and river water was higher than the recoveries by 1MDS filters. These data suggest that Nanoceram® filters can be used as an inexpensive alternative of 1MDS filters for routine viral monitoring of water.
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| PRESENTATION |
Visible Light-Activated Tio2 Photocatalytic Films; Synthesis, Characterization and Environmental Application for the Destruction of Microcystin-Lr |
08/17/2008
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Pelaez, M. A., A. A. DELACRUZ, AND D. D. Dionysiou. Visible Light-Activated Tio2 Photocatalytic Films; Synthesis, Characterization and Environmental Application for the Destruction of Microcystin-Lr. Presented at 236th American Chemical Society National Meeting, Philadelphia, PA, August 17 - 20, 2008.
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| Abstract:
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Titanium dioxide (TiO2) photocatalysis has become one of the most effective advanced oxidation technologies (AOTs) for the treatment of persistent organic contaminants. To generate hydroxyl radicals, a non-selective, reactive oxidizing species and responsible for the oxidation of pollutants, conventional TiO2 must be activated under UV light irradiation (<400nm). This represents a limitation for the use of sustainable technologies with renewable energy sources such as solar light, where only 5% of the total spectrum includes UV irradiance. Several techniques have been established and demonstrated some success to doped TiO2 with dye, metals and non-metals to shift the absorption spectrum towards the visible light [1, 2]. More recent studies dealing with anionic-doped TiO2 (i.e nitrogen-doped TiO2) catalyst have reported an improvement in the photocatalytic activity in the visible region (<500 nm) [3, 4]. A particular technique, sol-gel method, has shown promising results for the formation of nitrogen doped TiO2 from environmentally friendly molecular precursors via room temperature procedures [4]. However, most of the studies so far on the nitrogen doped TiO2 materials were not directed towards its application in engineered water treatment processes. For instance, an emerging group of contaminants of primary concern worldwide are the cyanobacteria toxins. These are released from cyanobacterial harmful algal blooms (Cyano-HABs) and are considered a serious health risk due to their high solubility in water, toxicity (i.e., hepatotoxicity, neurotoxicity) and chemical stability [5]. Microcystin-LR (MC-LR) is one of the most commonly detected cyanotoxin in Cyano-HABs and the most toxic derivative of the group of microcystins [6]. High degradation rates of MC-LR were recently obtained with nitrogen-doped TiO2 nanoparticles synthesized by sol-gel based methods [4]. An implication of using suspended TiO2 nanoparticles is their possible mobility and transport in the environment, representing a health risk and requiring an additional filtration step for removal in processed water. This study investigates immobilized TiO2 catalyst with visible light sensitization with enhanced structural properties using a novel sol-gel process. Results on the evaluation of the films on the destruction of MC-LR will be presented.
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| PRESENTATION |
Cryptosporidium Source Tracking in the Potomac River Watershed |
05/28/2008
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Wang, W., P. Chen, E. VILLEGAS, R. B. LANDY, C. KANETSKY, V. Cama, T. Dearen, C. Schultz, K. G. Orndorff, G. J. Prelewwicz, M. H. Brown, AND K. R. Young. Cryptosporidium Source Tracking in the Potomac River Watershed. Presented at 10th International Workshop on Opportunistic Protists, Boston, MA, May 28 - 31, 2008.
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| Abstract:
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To better characterize the presence of Cryptosporidium in the Potomac River watershed, a PCR-based genotyping tool was used to analyze 64 base-flow and 28 storm-flow samples from five sites within the watershed. These sites included two water treatment plant intakes as well as three upstream sites, each associated with a different type of land use. Each of these uses, urban/wastewater, agricultural (cattle)/wastewater, and agricultural (cattle), posed different risks with regard to the potential contribution of Cryptosporidium oocysts to the source water. In the study, Cryptosporidium was detected in 27 base-flow water samples and 23 storm-flow water samples. The most frequent genotype detected was C. andersoni (detected in 41 samples), while 14 other species/genotypes, almost all wildlife-associated, were occasionally detected. The two common human-pathogenic species, C. hominis and C. parvum, were not detected. Although C. andersoni was common at all four sites under agricultural influence, it was largely absent at the urban/wastewater site. There were very few positive samples by EPA Method 1623 at any site; only eight of 90 samples (9%) analyzed were positive for Cryptosporidium by microscopy. The genotyping results suggest that much of the Cryptosporidium that is present in the water treatment plant source waters may not pose a significant human health risk.
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| PRESENTATION |
Rna Extraction Methods for Real-Time Pcr and Microarray Analyses of Cryptosporidium and Toxoplasma Gondii Oocysts 2nd Presentation |
05/28/2008
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See, M. J., M. W. WARE, J. P. Dubey, AND E. VILLEGAS. Rna Extraction Methods for Real-Time Pcr and Microarray Analyses of Cryptosporidium and Toxoplasma Gondii Oocysts 2nd Presentation. Presented at 10th International Workshop on Opportunistic Protists, Boxton, MA, May 28 - 31, 2008.
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| Abstract:
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The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion processes or about their survival after disinfection treatment. Microarrays and reverse transcriptase real-time PCR (RT-qPCR) are useful techniques that are amenable to identifying genes involved in regulating such mechanisms. In order to effectively perform these analyses, high quality RNA must be isolated. Here, we evaluated four RNA extraction methods (Ambion, RiboPure, Qiagen RNeasy, Epicentre Master Pure and TRIzol LS reagent) in their ability to extract high quality RNA from C. parvum and T. gondii oocysts. RNA quality was measured and expressed as an RNA Integrity Number (RIN), with a 9.0 or above indicating minimal degradation, while RNA purity was determined using RT-qPCR. Preliminary results indicated high quality RNA (RIN 8.3 – 9.8) was extracted from C. parvum oocysts using all four extraction methods; however, genomic DNA (gDNA) was present in all samples. DNase I treatment effectively removed gDNA contaminants only in Ambion and Qiagen extracted RNA, but the quality was compromised (RIN 5.5 - 7.7). RNA extracted from T. gondii oocysts proved to be more difficult and the quality/purity of RNA extracted was variable. Nevertheless, all RNA extraction methods used in this study were effective in quantitating hsp-70 and β-tubulin expression in C. parvum oocysts and SporoSAG expression in T. gondii oocysts using RT-qPCR. These studies will now enable us to use RT-qPCR, and perhaps microarray analysis, for identifying novel genes essential for oocyst development, survival in the environment, and the establishment of infection in the host.
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| PRESENTATION |
Loss of Virus-Specific Memory T. Cells in Coxsackievirus B3 and B4 Infected Mice |
06/05/2008
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LI, L. AND A. P. DUFOUR. Loss of Virus-Specific Memory T. Cells in Coxsackievirus B3 and B4 Infected Mice. Presented at FOCIS Meeting, Boston, MA, June 05 - 09, 2008.
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| Abstract:
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There are two major types of enteroviruses: polioviruses and non-polio enteroviruses. While vaccines have effectively eliminated poliovirus infections, no vaccine is currently available for the non-polio enteroviruses. Generation of long-term pathogen specific memory cells is critical for the development of a good vaccine. To investigate whether long-term memory T cells are produced in response to infection by non-polio enteroviruses, coxsackieviruses B3 (CVB3) and coxsackieviruse B4 (CVB4) were intraperitoneally inoculated into adult BABL/c mice. Virus-specific memory T cells were assayed for proliferation and release interferon gamma (IFN-γ) under ex vivo stimulation with the viral pathogens. The level of IFN-γ was measured by antibody-capture chemiluminescent ELISA. Four days after stimulation, there were no detectable changes in the levels of IFN-γ from mice 17-19 months post-exposure to CVB3 and CVB4 compared with negative control mice inoculated only with sterilized PBS buffer. In contrast, the levels of IFN-γ were markedly increased after stimulation with specific viral pathogens in mice 2½ months post-infection with CVB3 and CVB4. These results suggest that non-polio enteroviruses might generate only short-lived virus-specific memory T cells. This would imply that vaccines for non-polio enterviruses might not provide long term protection against these viruses.
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| PRESENTATION |
Evaluation of a Real-Time Quantitative Pcr Method With Propidium Monazide Treatment for Analyses of Viable Fecal Indicator Bacteria in Wastewater Samples |
06/01/2008
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VARMA, M., R. FIELDS, M. K. STINSON, B. Rukovets, E. C. CHERN, L. J. WYMER, AND R. A. HAUGLAND. Evaluation of a Real-Time Quantitative Pcr Method With Propidium Monazide Treatment for Analyses of Viable Fecal Indicator Bacteria in Wastewater Samples. Presented at 2008 American Society for Microbiology General Meeting, Boston, MA, June 01, 2008 - June 05, 2208.
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| Abstract:
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The U.S. EPA is currently evaluating rapid, real-time quantitative PCR (qPCR) methods for determining recreational water quality based on measurements of fecal indicator bacteria DNA sequences. In order to potentially use qPCR for other Clean Water Act needs, such as updating criteria for disinfected POTW effluents, the ability to distinguish live organisms would be desirable. Propidium monoazide (PMA) has been shown to be selective in penetrating the cellular membranes of dead microorganisms where, upon exposure to light, it renders the DNA sequences in these cells unavailable for PCR detection. Therefore only sequences from live organisms should be measured by qPCR following PMA treatment. In this study a qPCR method incorporating pretreatment of samples with PMA was evaluated using pure cultures and wastewater samples collected from 3 different POTWs at 4 treatment stages including the chlorine-disinfected effluents. Treatment of heat-killed, cultured Enterococcus faecalis and Bacteroides thetaiotaomicron cells with PMA resulted in ~3-4 log reductions in QPCR-detectable target sequences compared with levels detected from live cells exposed to PMA or killed cells that were not exposed to PMA. Similar results were seen with cell concentrates from 1 ml buffer and wastewater samples spiked with E. faecalis, however, an inhibitory effect on PMA activity was indicated when 10ml of the wastewater samples were processed. Analyses of wastewater samples collected from the POTWs during normal dry weather operation showed that culturable Enterococcus and fecal coliform bacteria counts were more closely associated with QPCR-determined levels of target sequences from Enterococcus and Bacteroidales in PMA treated samples than with those in samples without PMA treatment. This trend was not as evident in samples collected from the same plants during wet weather operation where blending of primary and secondary-treated samples occurred prior to disinfection. Further studies are needed to determine the efficacy of PMA treatment for distinguishing viable organisms in different wastewater and surface water matrices by qPCR analysis.
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| PRESENTATION |
In-Vitro Cell Culture and Real-Time Reverse Transcriptase Pcr-Based Assays to Detect Infective Toxoplas Gondii Oocysts |
06/01/2008
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AUGUSTINE, S. J., L. F. VILLEGAS, M. W. WARE, S. L. HAYES, M. J. See, J. P. Dubey, AND E. VILLEGAS. In-Vitro Cell Culture and Real-Time Reverse Transcriptase Pcr-Based Assays to Detect Infective Toxoplas Gondii Oocysts. Presented at 2008 American Society for Microbiology Annual Meeting, Boston, MA, June 01, 2008.
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| Abstract:
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Toxoplasma gondii is an obligate intracellular, apicomplexan parasite that infects humans. It is ubiquitous in nature and seroprevalence in the United States and in Europe ranges from 25->70%. Although typically associated with causing foodborne outbreaks, recent studies in Canada
and Brazil have shown this parasite to also cause waterborne outbreaks of toxoplasmosis, suggesting its presence and persistence in drinking and recreational waters. To date, little is known about the prevalence of T. gondii oocysts in water and its resistance to disinfection. In this
study, we developed a quantitative reverse-transcriptase PCR (RT-qPCR) assay using four constitutive and inducible gene targets (gra7, act1, sporosag, and tgowp genes) to detect and quantify viable T. gondii oocysts. Additionally, an in vitro cell culture assay was also developed to
determine viability of T. gondii. Preliminary results revealed that although sporosag is specifically expressed in the infective sporulated oocyst stage, levels of sporosag mRNA did not correlate with viability. Similar results were obtained with gra7, act1, and Tgowp gene targets. In contrast, our in vitro cell culture assay provided a sensitive and quantitative assay to measure disinfection treatment efficacies commonly used to inactivate waterborne parasites. Initial results indicated that oocysts treated with 10% formalin or heat inactivation for 1 hour at 80oC were effectively killed. Other treatments that had marked effects include sodium hypochlorite, Wescodyne, ethanol, and UV. This study reveals that mRNA-based PCR viability assay using gra7, act1,
sporosag, and tgowp genes may not be useful gene candidates to measure potential infectivity of T. gondii oocysts. Other mRNA species that have a shorter half-life may prove more useful. We also present a novel in vitro cell culture assay that can be used to quantify treatment efficacies commonly used in drinking and wastewater industries.
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| PRESENTATION |
The Role of Biofilms in Drinking Water Exposure to Potentially Pathogenic Legionella Spp. |
05/20/2008
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LAU, H. Y. The Role of Biofilms in Drinking Water Exposure to Potentially Pathogenic Legionella Spp. Presented at 2008 EPA Science Forum, Washington, DC, May 20 - 22, 2008.
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Legionellosis is a bacterial infection caused by species of the genera Legionella and is the most common waterborne disease reported in the United States. This type of infection has two clinically distinct forms: Legionnaire's disease, a severe type of infection, which includes pneumonia, and Pontiac fever, a milder self-limiting illness. Legionella spp. are unbiquitous in nature with water being the major reservoir for these organisms. Exposure to Legionella spp. occurs via the inhalation of contaminated aerosols from devices such as cooling towers, showers and faucets. In these artificial water systems, Legionella spp. growth is detected almost exclusively in nutrient-rich biofilms covering the inerior of pipe walls, in-premises plumbing fixtures and heating, ventilation, and air-conditioning systems. The types of disinfectant residuals in drinking water systems have been shown to directly influence both the composition of biofim communities and on biofilm development. Thus drinking water systems not only serve as a reservoir for potential pathogens entering the system, their interactions and development ini biofilm communities has the potential to select for stains that are more fit to thrive in this type of environment. Therefore, understanding how Legionella spp. propagates and persists in man-made water systems, and the role biofilms play in that process, is critical to the development of strategies that can more effectively control their occurrence in drinking water.
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| PRESENTATION |
The Enterococci Qpcr Method for Recreational Water Quality Testing: Background and Single Lab Performance |
05/13/2008
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HAUGLAND, R. A. The Enterococci Qpcr Method for Recreational Water Quality Testing: Background and Single Lab Performance. Presented at Scientific Meeting on Development, Validation and Implementation of qPCR and PCR Methods for Use in Recreational Waters, Cincinnati, OH, May 13, 2008.
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| Abstract:
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Slide Presentation given at the Scientific Meeting on Development, Validation and Implementation of qPCR and PCR Methods for Use in Recreational Waters
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| PRESENTATION |
Methods for Measuring Occurrence and Exposure from Viruses in Drinking and Recreational Water |
03/25/2008
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FOUT, G., N. BRINKMAN, M. Karim, E. RHODES, A. EGOROV, AND S. L. BARTELT-HUNT. Methods for Measuring Occurrence and Exposure from Viruses in Drinking and Recreational Water. Presented at Future Develiopment Director for Safe Drinking Water Management International Seminar, Incheon, SOUTH KOREA, March 25, 2008.
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| Abstract:
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The United States Environmental Protection Agency (EPA) has an active research program to develop and improve methods for detecting human enteric viruses in recreational, source, and drinking waters. EPA is also developing methods to measure exposure to waterborne viruses and applying these methods to studies to assess health benefits associated with EPA drinking water rules. This paper describes EPA’s recent research on three methodologies in these areas. First, in an effort to reduce the costs associated with virus occurrence studies, EPA evaluated the NanoCeram™ filter, a new positively charged cartridge filter for concentrating viruses from water. EPA’s results showed that this filter is equivalent to the Virosorb® 1MDS cartridge filter that is specified for use in the standardized total culturable virus assay from EPA’s Information Collection Rule. Second, EPA conducted a proof of concept study to demonstrate the usefulness of a generic microarray format for virus identification. This study showed that the generic microarray can accurately type noroviruses. Third, EPA has developed a microbead immunoassay for detection of salivary antibodies against potential waterborne pathogens, including noroviruses, rotaviruses, Cryptosporidium, Toxoplasma gondii, and Helicobacter pylori.
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| PRESENTATION |
Legionella - (re-)awakening to the Amoeba-based Pathogens of Distribution System Biofilm |
07/15/2008
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ASHBOLT, N. Legionella - (re-)awakening to the Amoeba-based Pathogens of Distribution System Biofilm. Presented at Center for Biofilm Engineering, Bozeman, MT, July 15, 2008.
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| Abstract:
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Fecal pathogens have long been the focus of concern in the distribution of drinking waters. Yet today, with distribution system ‘failures’ accounting for the majority of waterborne outbreaks in the USA, there is growing realization that pathogens endemic to aquatic biofilms may also be of concern, particularly to susceptible sub-populations. These concerns have focused on strains of Legionella spp., Mycobacterium avium complex and Helicobacter pylori, as raised in EPA’s contaminant candidate lists (CCL-1, 2 & 3). Such pathogens, however, may only be the tip of the ‘iceberg’. Turning to biofilm ecology, it is clear from studies on Legionella that biofilm amoeba play an important ‘Trojan Horse’ role by introducing, amplifying and possibly up-regulating virulence in Legionella and other pathogens. However, there are very few studies of Legionella spp. pathogenesis aimed at associating the role of biofilm colonization, parasitization of biofilm microbiota and release of virulent bacterial cell/vacuoles into drinking water distribution systems. Moreover, the implications of these environmental niches for drinking and reuse water exposures to pathogenic legionellae are poorly understood. On-going research at EPA’s Cincinnati laboratories into the putative role of biofilms and amoeba in the proliferation, development and dissemination of potentially pathogenic Legionella and other intracellular pathogens will be discussed. The goal of this research is to aid in identifying control strategies and to provide key data to enable quantitative microbial risk assessments of biofilm-associated pathogens.
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| PRESENTATION |
Laboratory Evaluations of the Enterococcus Qpcr Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements |
06/01/2008
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HAUGLAND, R. A., S. SIEFRING, M. VARMA, AND L. J. WYMER. Laboratory Evaluations of the Enterococcus Qpcr Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements. Presented at 108th General Meeting of the American Society for Microbiology, Boston, MA, June 01 - 05, 2008.
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| Abstract:
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The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for enterococci that meets the requirements for rapid, risk-based water quality assessments at beaches. However, questions
remain over how this method should be used in water quality criteria. These questions relate in part to the lack of a clear understanding of the
performance characteristics of the method such as detection limits and precision under the influence of mixed sources of variation. In the present study, single laboratory results are reported that further
define these attributes. The 95% confidence method detection limit (95% MDL) was estimated at ~100 calibrator cell equivalents (CE)/sample from analysis results of replicate 100 ml buffer samples spiked with 10-1000 cultured E. faecalis cells. This CE value can be
translated to a 95% MDL of 3230 target sequence copies (TSC)/sample based on the recovery efficiency of 32 TSC/cell determined for these
samples or 5.4 TSC/reaction based on the use of 0.166% of the sample extracts for PCR analysis. Pure method precision, expressed as log10 variance, was estimated as 0.018, 0.054 and 0.114 from spikes of
1000, 333 and 100 cells, respectively. Analyses of diverse surface water samples indicated that the influence of non-interfering matrices on log10
variance was negligible. Based on analyses of different sets of beach water samples from 2003-2005 U.S. EPA epidemiological studies where
the geometric means for ambient enterococci by qPCR were within 100 to 1000 CE, the 90% prediction interval was estimated as 170 to 650 for a true value of 333 CE. These analyses further indicated that the major source of the measurement uncertainty was associated with sample variability. Accurate characterization of beach water quality by this method is therefore highly dependent upon the analysis of multiple samples.
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| PRESENTATION |
Development of a Gas Chromatography/Mass Spectrometry Method for the Analysis of the Solvent Stabilizer 1,4-Dioxane in Drinking Water |
08/11/2008
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GRIMMETT, P. AND J. W. MUNCH. Development of a Gas Chromatography/Mass Spectrometry Method for the Analysis of the Solvent Stabilizer 1,4-Dioxane in Drinking Water. Presented at 24th Annual National Environmental Monitoring Conference, Washington, DC, August 11 - 15, 2008.
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| Abstract:
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The solvent stabilizer 1,4-dioxane was named to the latest draft Drinking Water Contaminant Candidate List (CCL3) in February 2008 by the United States Environmental Protection Agency (USEPA). To collect occurrence data under the Unregulated Contaminant Monitoring Regulation (UCMR) program, a standardized method that exhibits ruggedness, accuracy, and precision is needed. Analysis of
1,4-dioxane has proved challenging because its volatility and miscibility with water make the compound a poor candidate for traditional extraction and concentration techniques. USEPA’s National Exposure Research Laboratory (NERL) has developed a new method, employing an activated carbon solid phase extraction, with quantitation performed by gas chromatography/mass spectrometry (GC/MS) in selected ion monitoring (SIM) mode. Using the method parameters, 1,4-dioxane (1.0 µg/L) was recovered from groundwater, surface water, and surface water high in total organic carbon (TOC) at efficiencies of 96%, 99%, and 102%, respectively, using 500-mL drinking water samples. Relative standard deviations (RSD) were less than 6% for all drinking water sources (n = 7). Small-scale extractions using 100-mL water samples yielded comparable results. Method detection limits (MDL) calculated from fortified water samples analyzed at three laboratories were 0.012 µg/L, 0.020 µg/L, and 0.021 µg/L, with lowest concentra¬tion minimum reporting level (LCMRL) values of 0.013 µg/L, 0.036 µg/L, and 0.080 µg/L. Drinking water samples preserved with sodium bisulfate, dechlorinated with sodium sulfite, and stored under refrigeration were stable for 28 days.
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| PRESENTATION |
Legionella - (re-) awakening to the amoeba-based pathogens of distribution system biofilms |
07/24/2008
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ASHBOLT, N. Legionella - (re-) awakening to the amoeba-based pathogens of distribution system biofilms. Presented at With Environmental Research Laboratory, Tucson, AZ, July 24, 2008.
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| Abstract:
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Presentation to be given at the Environmental Research Laboratory, University of Arizona, Tucson, Arizona, July 24, 2008
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| PRESENTATION |
Reconfiguration of Water-Energy Systems for Healthy and Sustainable Urban Water Management |
12/01/2008
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ASHBOLT, N. Reconfiguration of Water-Energy Systems for Healthy and Sustainable Urban Water Management. Presented at International Conference on Water Scarcity, Global Changes, and Groundwater Management Responses, Irvine, CA, December 01 - 05, 2008.
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| Abstract:
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Presentation at the the International Conference on Water Scarcity, Global, Changes, and Groundwater Management Responses
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| PRESENTATION |
Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time Qpcr Analysis of Enterococci in Recreational Waters |
09/15/2008
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SIEFRING, S., R. A. HAUGLAND, AND L. J. WYMER. Evaluation of Commercial Cell Preparations as Sources of Calibration Standards for Real-Time Qpcr Analysis of Enterococci in Recreational Waters. Presented at Great Lakes Beaches Association Annual Meeting, Porter, IN, September 15 - 17, 2008.
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| Abstract:
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In response to the Beach Act, the U.S. EPA has developed a quantitative PCR (qPCR) method for enterococci that meets requirements for rapid, risk-based water quality assessments of recreational waters. Widespread implementation of this method will require that different laboratories obtain consistent quantitative results. An important prerequisite will be the availability of reproducible, stable and sustainable standards. In this study two commercial sources of enumerated and lyophilized Enterococcus cells, BioBallTM (BTF, Ltd.) and EpowerTM (MicroBioLogics, Inc.) were used to prepare calibration samples for the qPCR method. The precision and recovery of target sequences from multiple samples prepared from each these cell sources and from currently used laboratory cultured cells were determined from qPCR cycle threshold (CT) results and by comparisons with a standard curve from known target sequence quantities. Unlike cultured cells, samples prepared from both of these products required filtration to remove PCR-inhibitory materials before DNA extraction. The ratios of variances in target sequence recoveries from replicate Epower and BioBallTM samples were 2.48 (p = 0.068) and 1.10 (p = 0.433), respectively, compared to the cultured cell samples. Lower precision obtained from the BioBallTM samples may be related to the lower cell quantities in these samples (~550 CFU vs. ~10^5 CFU in the EpowerTM and cultured cell samples). Absolute recoveries of target sequences per CFU from the three cell sources were similar. Each of these products has potential advantages in either cost (EpowerTM) or ease of use and more appropriate cell numbers for quantifying recreational water sample results (BioBallTM).
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| PRESENTATION |
From Source-to-Tap; Understanding the Ecology of Environmental Pathogens for Sustainable Urban Water Use |
08/20/2008
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ASHBOLT, N. From Source-to-Tap; Understanding the Ecology of Environmental Pathogens for Sustainable Urban Water Use. Presented at The Water Cycle ISME, Cairns, AUSTRALIA, August 20, 2008.
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| Abstract:
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Presentation given at The Water Cycle ISME, Cairns, Australia, August 20, 2008
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| PRESENTATION |
Emerging Contaminants in US Drinking Water: Do You Know Where Your Water's Been? |
10/03/2008
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GLASSMEYER, S., E. Furlong, D. Kolpin, R. J. MILTNER, AND S. Werner. Emerging Contaminants in US Drinking Water: Do You Know Where Your Water's Been? Presented at Society of Environmental Toxicology and Chemistry Ohio Valley Chapter Annual Meeting, Bloomington, IN, October 03, 2008.
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| Abstract:
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The drinking water and wastewater cycles are integrally linked. Chemicals that are present in household wastewater may be sufficiently mobile and persistent to survive on-site or municipal wastewater treatment and post-discharge environmental processes. Such compounds have the potential to reach surface and ground waters. These downstream / down gradient waters are typically the source for another community’s drinking water. To determine which wastewater chemicals persist through drinking water treatment, a joint USEPA / USGS study examined source and finished waters for nine drinking water treatment plants from across the United States known to be impacted by wastewater. All water samples were analyzed for 96 different emerging contaminants, including 35 pharmaceuticals, at sub-ug/L levels. The sample collection was designed to account for residence time within the plant in order to match waters before and after treatment. The investigated utilities used varying source waters (surface or ground water), disinfectants (chlorine, chlorine dioxide, chloramine, ozone or UV), and produced different volumes of treated water per day (2.3 to 200 mgd). Thirty-nine chemicals were detected at least once, with 32 chemicals detected in the source waters and 26 chemicals detected in the finished waters. Overall, the most frequently detected chemicals were aspirin (60 %), bupropion (60 %), caffeine (40 %), carbamazepine (40 %), tri(2-chloroethyl) phosphate (40 %) and venlafaxine (40 %). The greatest number of chemicals detected in a single source water sample was 15; the greatest number detected in a single finished water was 11. In general, the results from locations that used conventional treatment (coagulation, clarification, filtration, and chlorination) showed negligible removal of the monitored emerging contaminants. The results from those sites that used more advanced treatment (granular activated carbon adsorption, ozonation or UV irradiation) showed greater removal percentages.
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| PRESENTATION |
International Use of Risk Assessment/Risk Management to Direct Waterborne Pathogen Research |
09/04/2008
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ASHBOLT, N. International Use of Risk Assessment/Risk Management to Direct Waterborne Pathogen Research. Presented at Pathogens-In-Groundwater Research Consortium National Workshop, Calgary, AB, CANADA, September 04 - 05, 2008.
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Presentation to given at the Pathogens-In-Groundwater Research Consortium National Workshop, Delta Bow Valley, Calgary, Canada, September 4-5, 2008
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| PRESENTATION |
Sustainable Urban Waterfutures: A Vision |
10/16/2008
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ASHBOLT, N. Sustainable Urban Waterfutures: A Vision. Presented at Federal Subcommittee on Water Availability and Quality Executive Office of the President, Washington, DC, October 16, 2008.
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Background:
Urban growth is seriously limited by water scarcity on every continent, and trying to house more people that aspire to current developed region water services is simply impossible due to lack of available water, let alone the cost. Furthermore, traditional water/sanitation services are net users of energy (e.g. 10% of US energy production is used in drinking and wastewater services), whereas the biodegradable organic matter within wastewater contains over nine times as much energy as typically used to treat wastewater in developed regions. However, an often overlook third key point here is the finite nature of agriculturally-required phosphorus (P, mined from rock phosphate reserves) that is expected to be fully exploited in 60-150 years time (variability depending on global food/biofuel production increases). Hence, it should be obvious that ‘wastewater’ nutrients, and in particular P need to be recycled for agriculture to be sustainable, not to mention the energy savings that would result from co-recycling nitrogen and potassium.
Why do we have unsustainable water services?
Despite our centralized water services being engineered to meet public health protection, there has been a series of misinformed decisions that provide us with today’s legacy – starting in the mid 1800’s when London’s engineer Sir Joseph Bazalgette instituted major sewers systems on the basis that bad air (miasma) resulting from human wastes fouling the Thames River were the causes of cholera and typhoid. The word Sewer means "seaward" in Old English, and that was the start of the ‘solution to pollution was dilution’ instituted by Queen Victoria and used by many sanitary engineers to this day. The large-scale introduction of the flushing toilet from the 1890’s only exacerbated the need for larger sewers and waterworks, with the latter primarily articulated throughout cities for fire fighting (=reduced house insurance premiums), with only some 10% required for drinking water purposes. Also lost was the concept of ‘night soil’ for nutrient recycling to agriculture.
A more sustainable path for water services:
Using the sustainability principle of source-separation of waste streams, there is a range of options with household sanitary streams for nutrient and energy recovery (Figure 1). However, such changes require different incentives and institutional structures/relationships to flourish. For example, peoples’ recent enthusiasm for hybrid/electric cars is sadly not for altruistic reasons, but simply due to the sudden increase in fuel costs. In many situations, more decentralized water systems (possibly still centrally managed) may not only provide the means for local energy recovery ‘stations’, but also household greywater reuse that would reduce drinking water demand by over 70% and reduce the need for large water mains, sewers and pumping stations. Hence, incentives need to be abundant to aid in this translation to more sustainable urban water systems. Experience from projects in Sweden and Australia that are based on participatory, iterative processes of agreement for more sustainable systems will be discussed.
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| PRESENTATION |
Analysis of Arsenicals and Their Sulfur Analogs in Biological Samples Using Hplc With Collision Cell ICP-MS and Esi-MS/MS |
09/28/2008
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GALLAWA, C., K. KUBACHKA, P. A. CREED, J. T. CREED, M. J. KOHAN, K. HERBIN-DAVIS, AND D. J. THOMAS. Analysis of Arsenicals and Their Sulfur Analogs in Biological Samples Using Hplc With Collision Cell ICP-MS and Esi-MS/MS. Presented at 35th FACSS, Reno, NV, September 28 - October 02, 2008.
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Recent arsenic speciation studies have indicated that the sulfur analogs of the more common arsenic oxides are present in environmental and biological systems. This discovery was previously impeded due to the strong affinity of these arsenic-sulfides for the stationary phases typically used in arsenic oxide based speciation studies. The presence of thiolated arsenicals adds to the chromatographic resolution requirements essential for elemental-based detection (ICP-MS), mainly because misidentifications of an unknown can be made when relying solely on retention time matching when using element specific detection systems. The need for complementary molecular based information using techniques, like LC-ESI-MS/MS, have proven beneficial for assigning molecular structures to unknown arsenicals within complex matrices. The need is also especially important in indentifying the thiolated arsenicals, because primary standards of the neat materials are not commercially available.
The detection of thiolated arsenicals as urinary metabolites in both animals and humans has raised questions regarding how and when these species are produced within the metabolic pathway of arsenic. The authors have previously indicated that the microflora in the cecum of a mouse are able to biotransform an arsenosugar oxide (a tri-alkyl substituted arsenic oxide) to its corresponding sulfide [1]. This biotransformation raises questions regarding the conversion of structurally similar arsenic oxides, such as dimethylarsinic acid (DMA, di-alkyl substituted arsenic oxide). This presentation will summarize the analytical data from both LC-ICP-MS and LC-ESI-MS/MS that confirms the production of dimethylthioarsinic acid (DMTA) in the cecum of a mouse. The presentation will also report on the use of an isotopically enriched sulfur label to distinguish between proposed metabolic pathways. Finally, some preliminary research on the conversion of inorganic arsenic to its sulfur analogs will be presented.
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| PRESENTATION |
Emerging Contaminants in the Drinking Water Cycle |
10/07/2008
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GLASSMEYER, S. Emerging Contaminants in the Drinking Water Cycle. Presented at AWWA Alabama-Mississippi Regional Meeting, Montgomery, AL, October 06 - 07, 2008.
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In the past decade, the scientific community and general public have become increasingly aware of the potential for the presence of unregulated, and generally unmonitored contaminants, found at low concentrations (sub-µg/L) in surface, ground and drinking water. The most common pathway for the introduction of these chemicals is from an upstream direct discharge of wastewater effluent. In the US, there are more than two dozen communities that draw their drinking water from streams that consist of more than 50% wastewater during low flow conditions. The US Geological Survey (USGS) and US Environmental Protection Agency (USEPA) have been working on a series of collaborative research projects to determine the identity of chemicals that are commonly present in wastewater effluent, the persistence of these chemicals in surface and ground waters, the removal of these chemicals during drinking water treatment, the formation of by-products during their chlorination and the presence of these chemicals in finished drinking water. In effluents collected at eleven wastewater treatment plants (WWTPs) across the US, 72 out of 110 monitored chemicals were detected at least once, documenting incomplete removal during wastewater treatment. Downstream of the WWTPs, the chemicals exhibited varying environmental persistence. In the source water of one conventional drinking water facility, 45 out of 113 monitored chemicals were detected at least once, with 21 chemicals still detectable in the finished drinking water. This documents the incomplete removal for some chemicals during treatment. In companion laboratory studies on the effects of chlorination, eight of the 14 chemicals investigated were oxidized by the disinfectant, two of which were at least partially chlorinated. Taken as a whole, these studies demonstrate that to understand the comprehensive environmental impact of emerging contaminants, their persistence, removal efficiencies during waste and drinking water treatment, as well as the potential for by-product formation, must be known.
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| PRESENTATION |
Apples: A Standard Food for Determining Potential Residential Pesticide Transfers |
10/12/2008
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MELNYK, L. J., C. Stewart, T. E. HIEBER, J. N. MORGAN, AND A. M. PAWLECKI-VONDERHEIDE. Apples: A Standard Food for Determining Potential Residential Pesticide Transfers. Presented at International Society of Exposure Analysis, Pasenda, CA, October 12 - 16, 2008.
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Children’s unstructured eating behaviors lend themselves to potential
dietary exposures to synthetic pyrethroid pesticides applied in the
home. To determine the potential for excess dietary exposure of children
from handling food during consumption, a standard food has been developed to be used in the field as a measure of transfers of pesticides.
The standard food of choice was a raw Red Delicious apple because of its consistent transfer efficiencies for a range of pesticides and its ease of analysis. Since this type of apple may not be available everywhere, various types of apples were studied to determine if the type impacted the transfer of pesticides from contaminated Formica®. Also, in the
field, preparation of the apple slices could vary. The cutting technique and time lapse between cut and use of the slice of raw apple were investigated for their impact on the transfer of pesticides from a surface. The goal was to develop a standard operating procedure to be used in the field for residential monitoring of potential excess dietary exposures of children.
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| PRESENTATION |
Influence of Activity on Transfer of Pesticides from Treated Formica ® in Foods |
10/12/2008
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MELNYK, L. J., T. E. HIEBER, T. Turbeville, J. N. MORGAN, AND A. M. PAWLECKI-VONDERHEIDE. Influence of Activity on Transfer of Pesticides from Treated Formica ® in Foods. Presented at International Society of Exposure Analysis, Pasenda, CA, October 12 - 16, 2008.
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The children’s dietary intake model (CDIM) has been developed to predict total dietary intake of a child incorporating excess exposures due to handling of food prior to consumption (Akland et al., 2000; Melnyk et
al., unpublished). The model includes three Terms added together to estimate intake of a single contaminant. Term 1 is the pesticide residue in the food; Term 2 is the contamination due to surface-to-food contacts; and Term 3 is surface-to-hand-to-food contamination. The data for Term 1 are obtained through duplicate diet analysis or generated from residue and consumption information from available databases. The second and third input parameters may be generated from laboratory and field studies and the measurement procedure involves contaminant transfers to foods from surfaces and the frequency and duration of activities resulting in the contacts. Previous studies, conducted in the laboratory, determined transfer efficiencies between certain food and surface combinations (Rohrer et al., 2003; Vonderheide et al., 2008). The impact of activity on the transfer of contaminants to foods is largely unknown or has been estimated based on multiple touches of a single food item (Melnyk et al., unpublished). Additional laboratory studies to determine the influence of activities on the transfer of contaminants to foods were performed in simulation experiments in which food was exposed to treated Formica® under specific conditions. The objectives of the experiments were to measure the impact of the activities to properly utilize the activity factor within the CDIM. This information will allow for better predictions by CDIM for children’s total dietary exposure which, in turn, will lead to more accurate assessments for risk analysis.
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| PRESENTATION |
Verification of Cryptospporidium Removal By a Full-Scale Water Treatment Plant Using a Yeast Challenge and Ramifications for Risk Assessments |
09/26/2008
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ASHBOLT, N. Verification of Cryptospporidium Removal By a Full-Scale Water Treatment Plant Using a Yeast Challenge and Ramifications for Risk Assessments. Presented at The Greater Cincinnati Water Works Advisory Committee, Cincinnati, OH, September 26, 2008.
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Presentation at the Greater Cincinnati Water Works Advisory Commmittee, Cincinnati, September 26, 2008
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