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Microbiological & Chemical Exposure Assessment Research Division Publications: 2003

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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 2003, organized by Publication Type. Your search has returned 86 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
BOOK CHAPTER Mold Pollution 10/01/2003
Haugland, R. A. AND S. J. Vesper. Mold Pollution. Richard M. Stapleton, Patricia Hemminger, Susan L. Senecah (ed.), Pollution A-Z. Thomson Gale, New York, NY, 2:52-54, (2003).
Abstract: Mold pollution is the growth of molds in a building resulting in a negative impact on the use of that structure. The negative impacts generally fall into two categories: destruction of the structure itself and adverse health impacts on the building's occupants. It is estimated that about ten percent of U.S. buildings may suffer from mold pollution. This article will discuss mold and the impacts of molds on building and their occupants.
Molds (also known as fungi) are microorganisms that generaly have thread-like bodies and reproduce by producing spores (Figure 1). Spores are generally round or ovoid single cells (but in some cases multicellular), that are dispersed from the thread-like body of the mod called mycelium (pl. mycelia). Spores can be colorless or pigmented and vary in size from about one to fifteen microns. There are about fifty to one hundred different molds that are typically found growing indoors in water damaged buildings.

Water problems in buildings are generally the result of leaks from roofs or plumbing condensation, and flooding. When building materials or furnishings such as wood, drywall, ceiling tiles or carpets get wet, molds will grown on them. The types of substrates and the amount of moisture will often determine the kinds of molds that grow. For example, some molds like Stachybotrys require a highly water-saturated substrate. For other molds such as Aspergillus, only small amounts of excess moisture are required for growth. Thus, moisture control is key to controlling mold growth and eliminating their effects on the building's structure or the building occupants.
JOURNAL Identification of a Flavobacterium Strain Virulent Againt Giardia Lamblia Cysts 10/01/2003
Rodgers, M. R., D. J. Flanigan, B. K. Kinkle, AND S. Pfaller. Identification of a Flavobacterium Strain Virulent Againt Giardia Lamblia Cysts. WORLD JOURNAL OF MICROBIOLOGY AND BIOTECHNOLOGY 19(7):703-709, (2003).
Abstract: We have isolated a bacterial strain capable of killing the cyst form of Giardia lamblia, from a Kentucky stream. This bacterium, designated Sun4, is a Gram negative, aerobic rod which produces a yellow pigment, but not of the flexirubin-type. Although true gliding motility has not been observed in Sun4, this strain does exhibit a spreading colony morphology when grown on low nutrient agar. Strain Sun4 has been identified by 16S rRNA sequencing and phylogenetic analysis as belonging to the genus Flavobacterium, and is most closely related to Cytophaga sp. strain Type 0092 and associated Flavobacterium columnare strains. Lipid analysis also identified fatty acids characteristic of the Cytophaga-Flavobacterium group of bacteria. In culture, Sun4 is able to degrade casein and cellulose, but not chitin, gelatin, starch, or agar. Degradation of Giardia cysts by Sun4 appears to require direct cellular contact as neither cell-free extracts nor cells separated from the cysts by dialysis membranes showed any activity against cysts. In addition, Giardia cysts are killed more effectively by Sun4 growing in static versus shaking cultures. Activity against Giardia cysts is rapid, with Sun4 killing over 90% of cysts within 48 hours. Strain Sun4 requires elevated levels of Ca2+ for optimal growth and degradative activity against Giardia cysts. We propose that bacterial strains such as Sun4 could be used as biological control agents against Giardia cysts in drinking water treatment systems.
JOURNAL Immunocytochemical Localization of Stachylysin in Stachybotrys Chartarum Spores and Spore-Impacted Mouse and Rat Lung Tissues 03/01/2003
Gregory, L., T. G. Rand, D. Dearborn, I. Yike, AND S. J. Vesper. Immunocytochemical Localization of Stachylysin in Stachybotrys Chartarum Spores and Spore-Impacted Mouse and Rat Lung Tissues. MYCOPATHOLOGIA 156(2):109-117, (2003).
Abstract: Stachylysin is a proteinaceous hemolytic agent that is producted by S. chartarum. Stachylysin was found, using immunohistochemistical and immunocytochemical methods, to be localized in S. chartarum spores/mycelia primarily in the inner wall suggesting that it is constitutively produced. Spores instilled in mouse or rat lung tissues resulted in granuloma formations which showed the highest stachylysin concentration in the inner wall of the spore, with less at distance indicating that it has diffused out from the spore. The in vitro high stachylysin producing strain (58-06) was also highest in vivo, based on immunohistochemistical staining. More stachylysin was observed in the lung tissue at 72 h than at 24 h indicating that production /release is a relatively slow process. The localization of stachylysin in macrophage phagolysosomes suggests that these cells may be involved with hemolysin inactivation. This would be consistent with what is know about asp-hemolysin produced by Aspergillus fumigatus.
JOURNAL Detection of Cyclospora Cayetanesis Using a Quantitative Real-Time Pcr Assay 04/01/2003
Manju Varma, M., Jeff D. Hester, F. Frank W. Schaefer III, M. Michael W. Ware, AND H. H. D. Alan Lindquist. Detection of Cyclospora Cayetanesis Using a Quantitative Real-Time Pcr Assay. JOURNAL OF MICROBIOLOGICAL METHODS 53(1):27-36, (2003).
Abstract: Cyclosporal cayetanensis, a coccidian parasite of humans, has been recognized worldwide as an emerging pathogen in both immunocompromised (Ortega et al.1993) and immunocompetent individuals (Berlin et al.1994). Presently, humans apear to be the primary host for this parasite (Eberhard et al.2000). Clinical manifestations associated with C. cayetanensis can include prolonged diarrhea, nausea, abdominal cramps, anorexia, weight loss, and other symptoms of gastronenteritis. The transmission form of C. cayetanensis is an environmentally resistant, 8-10 um spherical oocyst that contains two ovoid sporocysts each of which contains two sporoxoites. Cyclospora cayetanensis oocysts shed in the feces of an infected host are not infectious until they become sporulated. Depending on a variety of environmental factors such as temperature and humidity, sporulation occurs after approximately two weeks outside the host.
A striking feature of this parasite is its seasonality. In some areas, during the rainy season, infection rates are high, decreasing to undetectable levels during the dry season. While the mode of transmission has not been completely elucidated, most cases of cyclosporiasis in the United States have been associated with consumption of fruits and vegatables that may have become contaminated after contact with tainted water (Berline et al.1994; Herwaldt and Ackers 1997). Since the epidemiology of this disease is still in doubt, it would be valuable to have sensitive and specific tools to detect and quantify the presence of this organism in the environment. These tools might also lead to the development of more sensitive and specific clinical diagnostic methods.
JOURNAL A Method to Detect Viable Helicobacter Pylori Bacteria in Groundwater 06/01/2003
Flanigan, D. J. AND M. R. Rodgers. A Method to Detect Viable Helicobacter Pylori Bacteria in Groundwater. ACTA HYDROCHIM 31(1):45-48, (2003).
Abstract: The inability to detect the presence of viable Helicobacter pylori bacteria in environmental waters has hindered the public health community in assessing the role water may playin the transmission of this pathogen. This work describes a cultural enrichment method coupled with an H. pylori-specific PCR to identify these bacteria in water. While far from perfected at the present time, this represents an exciting new approach to studying the significance of water as a transmission mechanism for H. pylori. Evidence is presented that indicates culturable H. pylori bacteria were found using this enrichment/PCR method in a local groundwater source.
JOURNAL Transfer Efficiencies of Pesticides from Household Flooring Surfaces to Foods 11/01/2003
Rohrer, C. A., T. E. Hieber, L. J. Melnyk, AND M. R. Berry Jr. Transfer Efficiencies of Pesticides from Household Flooring Surfaces to Foods. JOURNAL OF EXPOSURE ANALYSIS AND ENVIRONMENTAL EPIDEMIOLOGY 13(6):454-464, (2003).
Abstract: The transfer of pesticides from household surfaces to foods was measured to determine if excess dietary exposure potentially occurs when children's foods contact contaminated surfaces prior to being. Three common household surfaces (ceramic tile, hardwood flooring, and carpet) were contaminated with an aqueous solution of commercially available pesticides (diazinon, helptachlor, malathion, chlorpyrifos, isofenphos, and cis- and trans-permetrhin) frequently found in residential environments. A surface wipe method was used to measure the pesticides available on the surfaces as a basis for calculating transfer efficiency to the foods. Three foods (apple, bologna, and cheese) routinely handled by children before eating were placed on the contaminated surfaces and transfers of pesticides were measured after 10 min contact. Other contact durations (1 and 60 min) and applying additional contact force (1500g) to the foods were evaluated for their impact on transferred pesticides. More pesticides transferred to the foods from the hard surfaces tested, i.e., ceramic tile and hardwood flooring, than from carpet. Mean transfer efficiencies for all pesticides to the three foods ranged from 24 to 40% from ceramic tile and 15 to 29% from hardwood, as compared to mostly nondetectable transfers from carpet. Contact duration and applied force notably increased pesticide transfer. The mean transfer efficiency for the seven pesticides increased from around 1% at 1 min to between 55 and 83% when contact duration was increased to 60 min for the three foods contacting hardwood flooring. Mean transfer efficiency for 10-min contact increased from 15% to 72% when a 1500-g force was applied to bologna placed on hardwood flooring. Contamination of food occurs from contact with pesticide-laden surfaces, thus increasing the potential for excess dietary exposure of children who ingest foods that have contacted such surfaces.
JOURNAL Germination, Viability and Clearance of Stachybotrys Chartarum in the Lungs of Infant Rats 03/01/2003
Yike, I., S. J. Vesper, J. F. Tomashefski Jr., AND D. G. Dearborn. Germination, Viability and Clearance of Stachybotrys Chartarum in the Lungs of Infant Rats. MYCOPATHOLOGIA 156(2):65-67, (2003).
Abstract: The fungus Stachybotrys chartarum has been associated with many adverse health effects including the condition known as idiopathic pulmonary hemorrhage in infants. In order to gain some insight into possible mechanisms, viable conidia of S. chartarum were instilled into the lungs of 4 and 14 day-old rat pups. Germination was observed in the lungs by 24 h in the 4 day old but not until 72 h (and then only rarely) in the 14 day-old pups. Even two weeks after instillation of the conidia, the fungus could still be detected in the lung homogenates, both by dilution plating and quantitative PCR analysis.
In the 4 day-old pups, pulmonary inflammation with hemorrhagic exudates was observed and resulted in about 15% mortality rate compared to 0% for the controls instilled with phosphate buffered saline. In the 4 day-old pups, after 3 days, acute neutrophilic inflammation and intense interstitial pneumonia with poorly formed granulomas associated with fungal hyphae and conidia were obvious. The surviving experimental pups showed significantly slower weight gain for seven days. However, 14 day-old rat pups showed neither the lethal effects of exposures to instilled S. chartarum conidia nor the slower weight gain. Rat pup age may determine the response to exposures to S. chartarum conidia with the youngest pups being less able to clear the conidia resulting in more severe health effects.
JOURNAL Evaluation of An Alternative Ims Dissociation Procedure for Use With Method 1622: Detection of Cryptosporidium in Water 12/01/2003
Ware, M. W., L. Wymer, H. A. Lindquist, AND F. W. Schaefer III. Evaluation of An Alternative Ims Dissociation Procedure for Use With Method 1622: Detection of Cryptosporidium in Water. WATER RESEARCH. Elsevier Science Ltd, New York, NY, 55(3):575-583, (2003).
Abstract: U.S. EPA Method 1623 is used to detect and quantify Cruptosporidum spp. oocysts in ater. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). Confirmation is required because some algae appear to be oocysts when using this fluorescent antibody method. The purpose fo this study was to develop and evaluate a new IMS dissociation using a 10 minute incubation at 80 degrees C. Using this adaptation, the average oocyst recovery rate improved from 41% to 71% in seeded reagent water samples, and from 10% to 51% in seeded river samples. The average oocyst confirmation rate by DAPI improved from 49% to 93% in reagent water samples and from 48% to 73% in river water samples. This modification improved Method 1623 by increasing both the oocyst recovery and DAPI confirmation rates.
JOURNAL Evaluation of a Rapid, Quantitative Real-Time Pcr Method for Enumeration of Pathogenic Candida Cells in Water 03/01/2003
Brinkman, N., R. A. Haugland, L. J. Wymer, B. Muruleedhara, R. L. Whitman, AND S. J. Vesper. Evaluation of a Rapid, Quantitative Real-Time Pcr Method for Enumeration of Pathogenic Candida Cells in Water. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69(3):1775-1782, (2003).
Abstract: Quantitative Real-Time PCR (QRT-PCR) technology, incorporating fluorigenic 5' nuclease (TaqMan?) chemistry, was developed for the specific detection and quantification of six pathogenic species of Candida (C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaneae) in water (Brinkman et al, 2001). Known numbers of target cells were added to distilled and tap water samples, filtered and the DNA extracted. The assays sensitivities were between one and three cells with a two-fold accuracy around the mean at a 95% confidence interval. In similar tests with surface water samples, the presence of QRT-PCR inhibitory compounds necessitated further purification and/or dilution of the DNA extracts with resultant reductions in sensitivity but not quantitative accuracy. Analyses of a series of freshwater samples collected from a recreational beach showed positive correlations between the QRT-PCR results and colony forming counts of the corresponding target species. Positive correlations were also seen between the cell quantities of the target Candida species detected in these analyses and colony counts of Enterococcus. With a sample processing time of less than three hours, this method shows great promise as a tool for rapidly assessing potential exposures to waterborne pathogenic Candida species from drinking or recreational water and which may be an alternative indicator of fecal pollution._______

Yeasts are a significant component of the micro biota of most natural water systems and can also occur in drinking water distribution systems as a result of their ability to survive treatment practices and become incorporated into biofilms (Woollett and Hedrick, 1970). The majority of these organisms have no known human health effect, however, a small number of species, primarily within the anamorphic genus Candida, are important opportunistic pathogens (Hurley, de Louvi and ).

The importance of pathogenic Candida as agents of nocosomial infections has led to the development of a number of modern molecular diagnostic methods to facilitate their detection and identification in clinical samples (Reiss et al ). Methods based on the polymerase chain reaction (PCR) and DNA hybridization probes have received particular attention (Mannarelli ??; Martin??; Widjojoatmodjo et al 1999 ). The more recent advent of fluorescent probe-based, real-time PCR (RT-PCR) technology (Heid et al 1996) has led to the development of homogeneous methods for detecting these organisms that require relatively short periods of time to perform (Guiver, Levi Oppenhaimer ).

Quantitative Real-Time PCR (QRT-PCR) has been demonstrated to be useful for quantitative analysis of microorganisms (Roe et al 2001), but, to our knowledge, this approach has not been used in the analysis of yeasts in water. Analyses for pathogenic yeasts in drinking or recreational water systems have the potential to expedite the identification of possible health hazards resulting either directly from the presence of these organisms or as indicators of other waterborne pathogens.

The first objective of this study was to develop QRT-PCR technology, incorporating fluorigenic 5' nuclease (TaqMan) chemistry, for specifically detecting and quantifying six common pathogenic species of Candida including: C. albicans, C. tropicalis, C. krusei, C. parapsilosis, C. glabrata and C. lusitaneae. The second objective was to evaluate a simple and rapid method, using QRT-PCR, for the detection and enumeration of these organisms in different types of water samples. Finally, the technology was tested by comparing this methodology with conventional plating and culturing methods in the analysis of a series of freshwater samples collected from a recreational beach on Lake Michigan.
JOURNAL Determination of Metals in Composite Diet Samples By ICP-MS 03/01/2003
Melnyk, L. J., J. N. Morgan, R. Fernando, O. Akinbo, AND E. D. Pellizzari. Determination of Metals in Composite Diet Samples By ICP-MS. JOURNAL OF AOAC INTERNATIONAL 86(2):439-447, (2003).
Abstract: In order to assess an individual's total exposure to contaminants in the environment, it is essential that the contribution of dietary exposure be quantified. As a result, USEPA's National Exposure Research Laboratory has initiated a program to develop methods to measure chemical pollutants in dietary samples collected from individuals. Previous efforts have utilized inductively coupled plasma (ICP)-atomic emission spectrometry (AES) and graphite furnace atomic absorption spectroscopy (GFAAS) techniques for determination of metals in composite diets. However, there is often a trade-off between sensitivity and sample throughput with these techniques. ICP-mass spectrometry (MS) offers sensitvity comparable to or better than GFAAS while retaining sample throughput comparable to ICP-AES. This study evaluated the applicability of ICP-MS techniques for the determination of metals in composite diets. An ICP-MS method for the determination of aluminum, arsenic, barium, cadmium, chromium, copper, lead, manganese, nickel, vanadium, and zinc is presented. The procedure utilizes atmospheric pressure microwave digestion to solubilize analytes in homogenized composite diet samples followed by ICP-MS analysis. Recovery of certified elements from standard reference materials (SRMs) ranged from 92-119% with relative standard deviation (RSD) ranging from 0.4-1.9%. Recovery of elements from fortified composite diet samples ranged from 75-129% with RSDs ranging from 1-11.3%. LODs ranged from 1 - 1700 ng/g, with high values due to significant amounts of certain elements naturally present in composite diets. Results of this study demonstrate that low resolution ICP-MS provides precise and accurate measurements of the elements tested in composite diet samples.
JOURNAL A Waterborne Outbreak of Norwalk-Like Virus Among Snowmobilers Wyoming, 2001 01/15/2003
Anderson, A. D., A. G. Heryford, J. P. Sarisky, C. Higgins, S. S. Monroe, S. R. Beard, C. Newport, J. L. Cashdollar, G. S. Fout, D. Robbins, S. A. Seys, K. J. Musgrave, J. Bartkus, J. Vinje, J. S. Bresee, H. M. Mainzer, AND R. Glass. A Waterborne Outbreak of Norwalk-Like Virus Among Snowmobilers Wyoming, 2001. JOURNAL OF INFECTIOUS DISEASES 187(2):303-306, (2003).
Abstract: In February 2001, episodes of acute gastroenteritis were reported to the Wyoming Department of Health from persons who had recently vacationed at a snowmobile lodge in Wyoming. A retrospective cohort study found a significant association between water consumption and illness, and testing identified Norwalk-like virus (NLV) in 8 of 13 stool samples and 1 well. Nucleotide sequences from the positive well-water specimen and 6 of the positive stool samples were identical. This multistrain NLV outbreak investigation illustrates the importance of NLV as a cause of waterborne illness and should encourage monitoring for NLVs in drinking water.
JOURNAL A Multiplex Reverse Transciption-Pcr Method for Detection of Human Enteric Viruses in Groundwater 06/01/2003
Fout, G. S., B. C. Martinson, M. N. Moyer, D. R. Dahling, AND L. J. Wymer. A Multiplex Reverse Transciption-Pcr Method for Detection of Human Enteric Viruses in Groundwater. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69(6):3158-3164, (2003).
Abstract: Untreated groundwater is responsible for about half of the waterborne disease outbreaks in the United States. Human enteric viruses are thought to be leading etiological agents of many of these outbreaks, but there is relatively little information on the types and levels of viruses found in groundwater. To address this problem, monthly samples from 29 groundwater sites were analyzed for one year for enteroviruses, hepatitis A virus (HAV), Norwalk virus, reovirouses and rotaviruses by multiplex reverse transcription-PCR (RT-PCR). A procedure was developed to remove environmental inhibitors of RT-PCR from groundwater samples. The procedure allowed an average of 71 liters of the original groundwater to be asssayed per RT-PCR reaction with an average virus recovery rate of 74%, based upon seeded samples. Human enteric viruses were detected in 16% of the groundwater samples analyzed with reoviruses being the most frequently detected virus group.
JOURNAL Research Note: Autofluorescence of Toxoplasma Gondii Oocysts 09/01/2003
Lindquist, H. A., J. W. Bennett, J. D. Hester, M. W. Ware, J. P. Dubey, AND W. V. Everson. Research Note: Autofluorescence of Toxoplasma Gondii Oocysts. JOURNAL OF PARASITOLOGY 89(4):865-867, (2003).
Abstract: This is the first report of a blue autofluorescence as a useful characteristic in the microscopic identification of Toxoplasma gondii oocysts. This autofluorescence appears to be of high intensity. Similar to the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet light source and viewed with the correct (UV) excitation and emission filter set.
JOURNAL Reversetransciption-Pcr Assays for the Detection of Bovine Enteric Caliciviruses (Bec) and Analysis of the Genetic Relationships Among Bec and Human Caliciviruses 07/01/2003
Smiley, J. R., A. E. Hoet, M. Traven, H. Tsunemitsu, AND L. J. Saif. Reversetransciption-Pcr Assays for the Detection of Bovine Enteric Caliciviruses (Bec) and Analysis of the Genetic Relationships Among Bec and Human Caliciviruses. JOURNAL OF CLINICAL MICROBIOLOGY 41(7):3089-3099, (2003).
Abstract: Two genetically distinct bovine enteric caliciviruses (BEC) have been identified: the Norwalk-like-viruses (NLV), which are genetically related to human NLV, and the distinct NB-like BEC, which is most closely related to Sapporo-like viruses and lagoviruses, but potentially may represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Also, although RT-PCR primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In this study, veal calf fecal samples were assayed for enteric caliciviruses using six RT/PCR primer sets designed for the detection of human NLV or BEC. Caliciviruses genetically related to the NLV-BEC Jena and Newbury-agent-2 (NA-2) strains, or the recently characterized NB BEC strain, were identified in 3 of 4, and 4 of 4 sampled veal herds, respectively. Extended 3' terminal genome sequences of two NLV-BECs, designed CV95-OH and CV186-OH, encoding the RdRp (ORF-1), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome regoin demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289/290 primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positvie fecal pools (using other assays) led us to design two new primer sets, CBEC-UFP/URP and NB-UFP/URP, for the sensifitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2), and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed using the new assays identified 72% (54/75) of veal calf fecal samples positive, with 21 of 21 sequenced reaction products specific for the target RdRP gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-huCVs, but with the NLV-BECs comprising two clusters within a third NLV genogroup.
JOURNAL A New Species of Memnoniella 03/01/2003
Li, D., C. S. Yang, R. A. Haugland, AND S. J. Vesper. A New Species of Memnoniella. MYCOTAXON 85(Jan-Mar):253-257, (2003).
Abstract: A new species, Stachybotrys longistipitata sp. nov is described and illustrated. This fungus was originally isolated from forest soil in Japan and deposited as Memnoniella subsimplex.
Introduction

Four species have been described in the mitosporic genus Memnoniella Hvhnel: M. echinata (Rivolta) Galloway, M. levispora Subramanian, M. subsimplex (Cooke) Deighton, and M. zingiberis Rao. Members of this genus did not attract much attention until the health effects of indoor fungi, like Stachybotrys chartarum, became an issue. At that point, the similarities of these two genera in mycotoxin production (Jarvis et al. 1995) and growth on water saturated, cellulose based building materials demanded investigation of their phylogenetic relationship.
A comprehensive, comparative sequence analysis of species of Stachybotrys and Memnoniella lead Haugland et al. (2001) to consider these genera synonymous under the epithet Stachybotrys, as previously recognized by Smith (1962), Kendrick and Carmichael (1973) and Carmichael et al. (1980). However, one isolate, ATCC 22699 collected by T. Matsushima and deposited under the name of M. subsimplex, was found to be morphologically and genetically different from all described species of these genera (Haugland and Heckman, 1998; Haugland, et al. 2001). This isolate is described here as a new species of Stachybotrys.
JOURNAL Waterborne Outbreak of Gastroenteritis Associated With a Norovirus 09/15/2003
Parshionikar, S., S. WillianTrue, G. S. Fout, D. Robbins, S. A. Seys, J. D. Cassady, AND R. Harris. Waterborne Outbreak of Gastroenteritis Associated With a Norovirus. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69(9):5263-5268, (2003).
Abstract: The Wyoming Department of Health investigated an outbreak of acute gastroenteritis among persons who dined at a tourist saloon in central Wyoming during October 2001. Human caliciviruses (HuCVs) were suspected as the etiological agent of the outbreak based upon the incubation period, duration of illness and symptoms observed in ill patrons. A retrospective cohort study demonstrated that ill patrons were 4.5 times as likely to have exposure to drinking water and/or ice than non-ill patrons. No food items were associated with illness. An environmental investigation gave evidence that the saloon's groundwater was contaminated with septage. Water from the saloon's only well was processed for viruses. The processed water sample and stool samples collected from three ill patrons were analyzed by reverse transcription- polymerase chain reaction (RT-PCR) for the presence of HuCV. All positive RT-PCR results were confirmed by sequence and phylogenetic analysis of cloned RT-PCR products. A genogroup I, subtype 3 HuCV stain was found to be present in the well water sample and two stool samples. In addition, a genogroup II, subtype 6 strain was detected in one stool sample. The identification of the same HuCV strain in both the well water and stool samples strongly suggests a link between exposure to well water and the outbreak of gastroenteritis. The presence of genogroup II, subtype 6 strain in one of the stool samples suggests that multiple HuCV strains may have been involved in this outbreak. The laboratory isolation of HuCV strains from outbreak-associated drinking water is relatively novel in the US. This investigation outlines the procedure for virus isolation and illustrates the utility of the RT-PCR for the identification of HuCV in large volumes of water and stool samples obtained during outbreaks of acute nonbacterial gastroenteritis.
JOURNAL Molecular Characterization of Microsporidia Indicates That Fur-Bearing Wild Mammals Can Be a Source of Human Pathogenic Enterocytozoon Bieneusi 08/01/2003
Sulaiman, I. M., R. Fayer, A. A. Lal, J. Trout, F. W. Schaefer III, AND L. Xiao. Molecular Characterization of Microsporidia Indicates That Fur-Bearing Wild Mammals Can Be a Source of Human Pathogenic Enterocytozoon Bieneusi. JOURNAL OF INFECTIOUS DISEASES 69(8):4495-4501, (2003).
Abstract: Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392 bp fragment of the internal transcribed spacer (ITS) region of the rRNA gene of Enterocytozoon bieneusi, using primers complementary to the conserved regions of published E. bieneusi nucleotide sequences. Fifty-nine PCR positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of E. bieneusi (WL1-WL17), of these 15 E. bieneusi genotypes have not been reported before. Most of the E. bieneusi genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described E. bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water can be a potential source of human-pathogenic E. bieneusi.
JOURNAL Low Pressure Ultraveiolet Studies for Inactivation of Giardia Muris Cysts 01/01/2003
Hayes, S. L., E. W. Rice, M. W. Ware, AND F. W. Schaefer III. Low Pressure Ultraveiolet Studies for Inactivation of Giardia Muris Cysts. JOURNAL OF APPLIED MICROBIOLOGY 94(1):54-59, (2003).
Abstract: Cysts of Giardia muris were inactivated using a low pressure ultravolet (UV) light source. Cyst viability was detemined by both in vitro excystation and animal infectivity. Cyst doeses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ/cm(2) did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, UV fluences of 1.4, 1.9 and 2.3 mJ/cm(2) yielded log(10) reductions ranging from 0.3 to greater than or equal to 4.4. Results indicate in vitro excystation is not a reliable indicator of G. muris cyst viability after UV disinfection.
JOURNAL E. Coli and Public Health. Monitoring the Quality of Recreational Waters 06/01/2003
Dufour, A. P. E. Coli and Public Health. Monitoring the Quality of Recreational Waters. LAKELINE 23(2):13-15, (2003).
Abstract: The responsibility for protecting the health of swimmers who may be exposed to microbial hazards at our nations beaches falls on state, municipal or community authorities. They accomplish this by measuring a microorganism called E. coli in beach water samples. We call these microorganisms indicator bacteria because they indicate to us something about the quality of the water. If there are too many of the E. coli in the water sample, the beach water is determined to be unsuitable for swimming. Although we see the names E. coli and indicator bacteria in the newspapers and on television and we know that it is not good to find this microorganism in food or water, there are many misconceptions about why this bacterium is measured and what it means to find it in water.
JOURNAL An Investigation of the Chemical Stability of Arsenosugars in Basic Environments Using Ic-ICP-MS and Ic-Esi-MS/MS 11/05/2003
Gamble, B. M., P. A. Gallagher, J. A. Shoemaker, A. N. Parks, D. M. Freeman, C. A. Schwegel, AND J. T. Creed. An Investigation of the Chemical Stability of Arsenosugars in Basic Environments Using Ic-ICP-MS and Ic-Esi-MS/MS. ANALYST 128(12):1458-1461, (2003).
Abstract: This paper evaluates the chemical stability of four arsenosugars using tetramethylammonium hydroxide (TMAOH) as an extraction solvent. This solvent was chosen because of the near quantitative removal of these arsenicals from difficult to extract seafood (oysters and shellfish). Four arsenosugars (3-[5'-deoxy-5'-(dimethylarsinoyl)-b-ribofuranosyloxy]-2-hydroxypropylene glycol - As(328), 3-[5'-deoxy-5'-(dimethylarsinoyl)-b-ribofuranosyloxy]-2-hydroxypropanesulfonic acid - As(392), 3-[5'-deoxy-5'-(dimethylarsinoyl)-b-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulphate - As(408), and 3-[5'-deoxy-5'-(dimethylarsinoyl)-b-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate - As(482)) were evaluated. The stability of these four arsenosugars were studied independently in a solution of 2.5% TMAOH at 60 0C over a period of up to 8 hours. Two arsenosugars, As(328) and As(392), were found to be relatively stable in this solution for up to 8 hours. However, As(408) and As(482) formed detectable quantities of dimethylarsinic acid (DMAA) and As(328) within 0.5 and 2 hours, respectively. It was found that 97% of As(408) degrades after 8 hours of treatment producing 3.4 times as much DMAA as As(328). This is contrary to As(482), which produces 13 times as much As(328) as DMAA and only 37% of the As(482) was converted by the 8 hour treatment at 60 0C. These degradation products led to the investigation of weaker TMAOH extraction solvents. Three different concentrations (2.5%, 0.83% and 0.25%) were used to determine the effect of TMAOH concentration on degradation rate of As(408). By reducing the TMAOH concentration to 0.83%, the conversion of the arsenosugar to As(328) and DMAA is nearly eliminated (less than 5% loss).
Arsenosugars, As(408) and As(482), were also studied in 253 mM NaOH to verify the degradation products. The NaOH experiments were conducted to investigate a possible hydroxide based reaction mechanism. Similar degradation plots were found for each arsenosugar when compared to the 2.5% TMAOH data. A mechanism has been proposed for the formation of As(328) from As(408) and As(482) in base via an SN2 reaction (hydroxide attack) at the side chain carbon adjacent to the inorganic ester. The formation of DMAA is observed in all arsenosugars after prolonged exposure. This probably occurs via an SN2 attack at the arsenic atom.
JOURNAL 21st Century Mold Analysis in Food 12/01/2003
Vesper, S. J. AND R. A. Haughland. 21st Century Mold Analysis in Food. FOOD QUALITY, 39-40, (2003).
Abstract: Traditionally, the indoor air community has relied on mold analysis performed by either microscopic observations or the culturing of molds on various media to assess indoor air quality. These techniques were developed in the 19th century and are very laborious and time consuming with distinct limitations in standardization. Today, the analytical laboratory has a 21st century solution to mold identification and enumeration. In 1993, the Nobel Prize was awarded for the development of a method to amplify DNA sequences using the enzyme DNA polymerase. DNA sequences are those strings of A, C, G and Ts that form the genomic blueprint that make each creature, including each mold species, unique. This process of DNA amplification became known as the polymerase chain reaction or PCR.
The PCR process is initiated by short, specific sequences of DNA called the forward and reverse primers. As PCR amplification proceeds, copies of DNA accumulate and the number of accumulated copies can be estimated. PCR technology, as practiced before 1997, had great specificity but it lacked good quantitation and was relatively slow to perform. In 1997, Applied Biosystems, Inc. in Foster City CA announced the release of a new instrument called the Sequence Detector which provided a very accurate way to measure the accumulated PCR product. This measurement was accomplished by adding a species specific, fluorescently labeled probe in the sequence between the primers used in PCR. This process now goes by the name quantitative PCR (QPCR).
The QPCR analysis begins by extracting the DNA from an environmental sample. For example, a dust sample is weighed and added to a 2 ml extraction tube that contains tiny glass beads and an extraction fluid. The extraction tube is then shaken for 1 minute at 5,000 rpms which releases the DNA from the cells. For an air sample collected on a filter, the entire filter is added to the extraction tube and shaken. The extracted DNA is then ready for analysis or it made need some additional purification depending on how "dirty" it is. With a few simple steps, the purified sample is ready for analysis. Next the analyst needs the mold specific primers and probe.
In May of 2002, EPA was granted a patent (6,387,652) on the DNA sequences of primers and probe needed for analyzing well over 100 of the most important or numerous indoor molds. The EPA has now licensed 10 companies (Table 1) in the US and UK to use these primers and probes in QPCR analysis. Now an analyst from one of these licensed analytical laboratories can go to the freezer, where the primers and probes are stored, and select the set for each of the target mold species.
Of course, by this time, the analytical laboratory has already invested $40 to $100 K to purchase a Sequence Detector instrument. (There are now a number of manufactures of these instruments and price depends on many factors including the number of analyses that can be run at the same time). By this time, the analytical laboratory has also prepared standard spore suspensions, containing known numbers of spores, of each of the molds.
The analyst adds the primers and probe for a particular species together with the amplification solution that contains the PCR polymerase to form the reaction mixture. To this mixture is added a small aliquot of the purified sample DNA extract. This process is repeated for each of the target species. At the same time, the analyst prepares an extract of the standard spore suspension for the particular target mold(s).
The reaction mixtures are then placed in the sequence detector instrument which causes the mixture to heat which opens the double helix of the sample DNA and allows the primers and probe to hybridize or bind, if a match is found in the sample. If there is an exact match, fluorescence from the probe is measured. The sequence detector records and sums this fluorescence from each reaction mixture. At the same time, this analysis is performed with the standard spore suspension with known number of spores of the target mold. By comparing the fluorescence from the standard spore suspension analysis to the fluorescence of the DNA extracted from the environmental sample, the analyst can determine the number of target spores that was in the environmental sample. This process is repeated for as many molds as desired.
QPCR-based mold analysis has many advantages. QPCR provides simple, standardized speciation of molds. Air sampling can be extended for hours to days, if desired. And QPCR results can be obtained in a matter of hours not weeks. At this time, QPCR is more expensive than other methods of mold analysis but, like an new technology, costs will decline with greater volume of use. If we are ever going to understand the role of molds in human health, we need the standardized method of analysis which QPCR provides.
JOURNAL Sporulation and Survival of Toxoplasma Gondii Oocysts in Sea Water 07/01/2003
Lindsay, D. S., M. V. Collins, S. M. Mitchell, R. Cole, G. J. Flick, C. N. Wetch, H. A. Lindquist, AND J. P. Dubey. Sporulation and Survival of Toxoplasma Gondii Oocysts in Sea Water. JOURNAL OF EUKARYOTIC MICROBIOLOGY 50(S1):687-688, (2003).
Abstract: Since 1992, we have been collaborating in studies on southern sea otters (Enhdyra lutris nereis) as part of a program to define factors which may be responsible for limiting the growth of the southern sea otter population. We previously demonstrated Toxoplasma gondii in sea otters. We postulated that cat feces containing oocysts could be entering the marine environment through storm run-off or through municipal sewage since cat feces are often disposed down toilets by cat owners. The present study addressed the sporulation of T. gondii oocysts in sea water and the survival of sporulated oocysts in sea water. Unsporulated oocysts were placed in 15 ppt artificial sea water, 32 ppt artificial sea water or 2% sulfuric acid (positive control) at 24 C in an incubator. Samples were examined daily for 3 days and development monitored by counting 100 oocysts from each sample. From 75 to 80% of the oocysts were sporulated by 3 days postinoculation in all treatments. Groups of 2 mice were fed 10,000 oocysts each from each of the 3 treatments. All inoculated mice developed toxoplasmosis indicating that oocysts were capable of sporulating in sea water. Survival of sporulated oocysts was examined by placing sporulated T. gondii oocysts in 15 ppt sea water at room temperature 22-24 C (RT) or in a refrigerator kept at 4 C. Mice fed oocysts that had been stored at 4C or RT for 6 months became infected. These results indicate that T. gondii oocysts can sporulate in and remain viable in sea water for several months.Toxoplasma gondii is acquired by the ingestion of tissue cysts in raw or under cooked meat, by ingestion of oocysts, or by transplacental infection in mammalian species [6,9]. Clinical and subclinial toxoplasmosis has been reported in marine mammals [1-4,10-12,14-16,18,19]. Cole et al. [2] suggested that marine mammals acquired T. gondii infection by ingesting invertebrates which were acting as phoretic agents for T. gondii oocysts. They believed that cat feces containing T. gondii oocysts could be entering the marine environment through storm run-off and were picked up by invertebrate food items of Southern sea otters. Miller et al. [16] have shown a positive correlation between the presence of costal storm run off water and the presence of antibodies to T. gondii in sea otters. The present study was done to determine if nonsporulated T. gondii oocysts would sporulated in sea water and to determine if sporulated oocysts would survive in sea water.
JOURNAL Real-Time Pcr Method to Detect Enterococcus Faecalis in Water 02/01/2003
Santo Domingo, J., S. D. Siefring, AND R. A. Haugland. Real-Time Pcr Method to Detect Enterococcus Faecalis in Water. BIOTECHNOLOGY LETTERS 25(3):261-265, (2003).
Abstract: A 16S rDNA real-time PCR method was developed to detect Enterococcus faecalis in water samples. The dynamic range for cell detection spanned five logs and the detection limit was determined to be 6 cfu/reaction. The assay was capable of detecting E. faecalis cells added to biofilms from a simulator of a water distribution system and in freshwater samples. Nucleic acid extraction was not required, permitting the detection of E. faecalis cells in less than 3 h.
JOURNAL Survival of Helicobacter Pylori in a Natural Freshwater Environment 12/01/2003
Adams, B. L., T. C. Bates, AND J. D. Oliver. Survival of Helicobacter Pylori in a Natural Freshwater Environment. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 69(12):7462-7466, (2003).
Abstract: The mode by which Helicobacter pylori, the causative agent of most gastric ulcers, is transmitted remains undetermined. Epidemiological evidence suggests these organisms are waterborne; however, H. pylori has rarely been grown from potential water sources. This may be due to the ability of this organism to rapidly enter the viable but nonculturable (VBNC) state. Our investigation examines the entrace of H. pylori into this state in laboratory cultures and a natural freshwater environment as well as the relationship between morphology and culturability. To this end, membrane diffusion chambers were utilized to expose the cells to the natural fluctuations of a freshwater stream. In both the laboratory and environment, samples were assayed for culturability using plate counts and stained using a LIVE/DEAD BacLight assay for viability and morphological determinations. Additionally, water samples were collected, six environmental parameters were measured, and resuscitation conditions were examined, H. pylori was observed to lose culturability in the laboratory and stream, although viability was maintained. While the results of our study agree with those of previous studies which suggested that there is a transition in morphology from rods to cocci as culturability is lost, the morphological distribution of cells did not change as culturability was lost in the environment. The majority of cells in the VBNC state in the laboratory are cocci; however, all morphological forms were present in the environment. The results of these studies suggest that H. pylori persists in laboratory cultures and the environment in the VBNC state and that cells in this state represent a public health hazard.
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 Information Management and Related Quality Assurance for a Large Scale, Multi-Site Research Project 04/14/2003
Martinson, J., D. Lewis, K. Brenner, L. Wymer, AND S. Brown. Information Management and Related Quality Assurance for a Large Scale, Multi-Site Research Project. Presented at The U.S. EPA 20th Annual Conference on Managing Environmental Quality Systems: Quality Management Solutions for Today's Environmental Challenges, New Orleans, LA, April 14-17, 2003.
Abstract: During the summer of 2000, as part of a U.S. Environmental Protection Agency study designed to improve microbial water quality monitoring protocols at public beaches, over 11,000 water samples were collected at five selected beaches across the country. At each beach, samples were collected at least twice daily according to one or four sampling schemes. Samples were delivered to near-by laboratories and analyzed the same day for either E. coli or Enterococcus by established microbiological methods. Locational data, data describing ambient conditions at the beaches, and other supporting environmental data were also collected. The staff at each of the five study sites reported their data daily to a central location.
The collective effort generated close to a half-million data points during the months of July and August. Managing the steady and complex flow of informatiton and assuring the quality required a well planned, well-organized, and innovative approach. This approach included:

. standardized paper and electronic data entry forms,
. centrally-provided waterproof sample labels with pre-printed sample identifiers,
. independent, double entry of data with automated comparison,
. adherence to basic relational database rules, and careful attention to good data structure design, and data management.

This presentation will report in detail the data management approach, explain the reasoning behind the approach, and explain how it was integrated with the QA Project Plan. The speaker will describe procedures used to assess data quality in both the electornic forms completed at the sampling locations and the central database. The speaker will analyze the costs and benefits associated with the chosen approach, and will evaluate its success. Future plans (long-term archival, public availability, etc.) for the data will be described.
PRESENTATION An Investigation of Arsenic Mobility from Iron Oxide Solids Produced During Drinking Water Treatment 03/09/2003
Parks, A. N., P. A. Gallagher, C. A. Schwegel, A. H. Ackerman, AND J. T. Creed. An Investigation of Arsenic Mobility from Iron Oxide Solids Produced During Drinking Water Treatment. Presented at 2003 Pittsburgh Conference, Orlando, FL, March 9-14, 2003.
Abstract: The Arsenic Rule under the Safe Drinking Water Act will require certain drinking water suppliers to add to or modify their existing treatment in order to comply with the regulations. One of the treatment options is iron co-precipitation. This treatment is attractive because arsenic and iron are geologically correlated so that well waters containing arsenic have a propensity to contain iron. Iron can be precipitated from water via aeration and in the process the iron co-precipitates some of the arsenic leading to arsenic removal. The aeration can take place during the treatment process or occur as the water is pumped from the supply through the distribution system. The presence of these iron oxide solids can alter the distribution of the naturally occurring As(III) and As(V) in the water. The stability of the resulting iron oxide/arsenic complex ("FeOOHAs") is not well characterized. This research will attempt to estimate the distribution of As(III) and As(V) on these iron oxide solids and estimate the stability of these complexes produced by changing water quality parameter. The arsenic distribution on the solids will be estimated by extraction with acetic acid followed by IC-ICP-MS separation and detection. The stability will be estimated by conducting total metal measurements on solids in contact with waters prior to and after changes in pH, nitrate and chlorine concentrations
PRESENTATION Transport of Chemical and Microbial Contaminants from Known Wastewater Discharges 03/19/2003
Glassmeyer, S., I. Ferrer, E. T. Furlong, J. D. Cahill, S. D. Zaugg, S. L. Werner, D. W. Kolpin, AND D. D. Kryak. Transport of Chemical and Microbial Contaminants from Known Wastewater Discharges. Presented at National Groundwater Association 3rd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water, Minneapolis, MN, March 19-21, 2003.
Abstract: The quality of drinking and recreational water is currently ascertained using indicator bacteria, such as Escherichia coli and fecal enterococci. However, 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. One solution to these problems is to use chemicals that are commonly found in human wastewater as supplementary tracer compounds. The chemicals have the advantage of requiring shorter analysis times, and they can be chosen to be human specific markers.
For this project, we focused on a variety of compounds to determine their efficacy as chemical indicators of human fecal contamination. This list included compounds that are produced and excreted by humans (coprostanol, urobilin), that are consumed and pass easily through humans (pharmaceuticals and caffeine), and that are associated with humans and deposited into the combined graywater/ blackwater household septic waste stream (surfactants). Stream samples were collected upstream, at the point of discharge, and at two points downstream from wastewater treatment facilities at ten locations. This longitudinal sampling scheme was developed to determine the persistence of the compounds in streams. Compounds that are quickly removed or degraded may not be persistent enough to serve as tracers; those that are too recalcitrant would similarly not be suitable as they would be present after the pathogens have been eliminated. To estimate the desired duration, the water samples were analyzed for E. coli and fecal enterococci in addition to the suite of chemicals. For the chemicals, the water samples were extracted using either solid phase extraction (for the pharmaceuticals) or liquid-liquid extraction (for the other wastewater contaminants) and were analyzed using either high-performance liquid chromatography/mass spectrometry (HPLC/MS; pharmaceuticals) or gas chromatography/mass spectrometry (GC/MS; other wastewater contaminants; Kolpin et al., 2002). To analyze for the microbial contaminants, three aliquots of water (100, 10, and 1 mL) were filtered through cellulose disks and placed on modified mTEC (E. coli, ) or mEI (enterococci) media and incubated (USEPA, 2000).
PRESENTATION Distribution of Organic Wastewater Contaminants Between Water and Sediment in Surface Waters of the United States 03/19/2003
Furlong, E. T., I. Ferrer, S. Glassmeyer, J. D. Cahill, S. D. Zaugg, S. L. Werner, D. W. Kolpin, AND D. D. Kryak. Distribution of Organic Wastewater Contaminants Between Water and Sediment in Surface Waters of the United States. Presented at National Groundwater Association 3rd International Conference on Pharmaceuticals and Endocrine Disrupting Chemicals in Water, Minneapolis, MN, March 19-21, 2003.
Abstract: Trace concentrations of pharmaceuticals and other organic wastewater contaminants have been determined in the surface waters of Europe and the United States. A preliminary report of substantially higher concentrations of pharmaceuticals in sediment suggests that bottom sediment may be an important reservoir of pharmaceuticals discharged to surface water.
The U.S. Geological Survey undertook a systematic examination of bottom sediment collected from a dozen sites across the United States, in order to determine the range of compositions and concentrations of pharmaceuticals and other organic wastewater contaminants present in those sediments. Composite sediment samples were collected at one point upstream and two points downstream of a point source discharge. Surface water samples were collected concurrently at the same sites, as well as at the point discharge. The samples were shipped by overnight express and water samples were analyzed within 48 hours of receipt. Sediments were held frozen until extraction and analysis. Pharmaceuticals were extracted from filtered water samples by solid-phase extraction and the concentrated extract analyzed by high-performance liquid chromatography/mass spectrometry (HPLC/MS). Other organic wastewater contaminants were extracted from whole-water samples by continuous liquid-liquid extraction followed by gas chromatography/mass spectrometry (GC/MS; Kolpin and others, 2002). Sediments were extracted by accelerated solvent extraction and the extracts analysed by HPLC/MS (pharmaceuticals) or GC/MS (other wastewater constituents).

Preliminary results indicate that a wide range of pharmaceuticals and other wastewater constituents are present in sediments at concentrations substantially higher than in surface water. For example, caffeine, a highly water soluble contaminant, has estimated partitioning coefficient of 0.85, suggesting that concentrations should be higher in water than in sediment. However, the ratio observed for caffeine in environmental samples ranges between 4 and 136. Similarly, the predicted partitioning coefficient for trimethoprim is 8.2, while the observed partitioning coefficients range between 17 and 1,100. Also, as water and sediment concentrations increase, the observed partitioning coefficient more closely approaches the predicted partitioning coefficient. Further, pharmaceuticals were not detected in pore-water samples, suggesting that pharmaceuticals are intimately associated with the sediment phase. Taken together, these data suggest that sediments are potentially important reservoir for sequestering pharmaceuticals in surface water systems. Determining the mechanisms of association between water-soluble pharmaceuticals and sediment will be an important new area of research.
PRESENTATION The Liberation of Arsenosugars from Matrix Components in Difficult to Extract Seafood Samples Utilizing Tmaoh/Acetic Acid Sequentially in a Two-Stage Extraction Process 01/12/2003
Parks, A. N., P. A. Gallagher, C. A. Schwegel, A. H. Ackerman, AND J. T. Creed. The Liberation of Arsenosugars from Matrix Components in Difficult to Extract Seafood Samples Utilizing Tmaoh/Acetic Acid Sequentially in a Two-Stage Extraction Process. Presented at 2003 European Winter Conference on Plasma Spectrochemistry, Garmisch-Partenkirchen, Germany, January 12-17, 2003.
Abstract: Sample extraction is one of the most important steps in arsenic speciation analysis of solid dietary samples. One of the problem areas in this analysis is the partial extraction of arsenicals from seafood samples. The partial extraction allows the toxicity of the extracted arsenicals to be determined but the unextracted fraction's toxicity can not be determined. This inability to determine the toxicity of the unextracted arsenicals creates a relatively large source of uncertainty in estimating the risk from seafood exposures. Seafood exposures can produce a relatively large source of uncertainty because the arsenic concentrations in seafoods can be orders of magnitude higher than those associated with other food groups and therefore, a small error in quantitating toxicity (unextracted arsenicals imply unknown toxicity) in seafoods produces larger uncertainties in the overall exposure assessment. An additional problem can be generated by non-quantitative extractions in which the extraction solvent preferentially removes the non-toxic species while leaving the toxic species unextracted. The interpretation of these data would be that the sample contains only non-toxic arsenicals when in reality the extraction solvent has selectively removed only the non-toxic species. In this case the solvent selectivity produces an analytical negative bias which underestimates the concentration of the toxic arsenicals. Thus, a quantitative extraction is needed to minimize the unextractable arsenicals (which should provide the maximum species specific information) and eliminate the question about analytical bias induced by solvent selectivity (since all the arsenic is accounted for).
This presentation will focus on the use of tetramethylammonium hydroxide (TMAOH) as an extraction solvent for difficult-to-extract seafoods. The extraction utilizes a 0.83% TMAOH extraction solvent which is heated in a convection oven at 60oC for 3hrs. The sample is then neutralized with acetic acid to remove proteins and returned to the oven for 21hrs at 80oC. Data will be presented which indicates that the neutralization step is essential because arsenosugars are converted during this process from an unchromatographable species to a chromatographable species. Chromatographic and total arsenic data will be presented to document this conversion. Finally, chromatographic data will be summarized utilizing the two-stage (base followed by acid) extraction procedure.
PRESENTATION Speciation of Non-Pesticidal Organotin Compounds Using Gas Chromatography With Inductively Coupled Plasma-Mass Spectrometry 01/12/2003
Gallagher, P. A., O. M. Evans, A. N. Parks, C. A. Schwegel, A. H. Ackerman, J. T. Creed, AND S. Wilbur. Speciation of Non-Pesticidal Organotin Compounds Using Gas Chromatography With Inductively Coupled Plasma-Mass Spectrometry. Presented at 2003 European Winter Conference on Plasma Spectrochemistry, Garmisch-Partenkirchen, Germany, January 12-17, 2003.
Abstract: Organotins can be classified into a broad class of compounds referred to as endocrine disruptors and are presently being used in many applications such as UV stabilizers in polyvinyl chloride (PVC) pipes, antifoulants in marine paints, and as fungicides to name a few. The organotins used in PVC pipes are a growing concern given the increased use of PVC pipe 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. These data raise two issues: initial exposure severity and long-term low dose exposure effects of organotins. The first issue can be addressed rather easily using existing analytical methodologies. However, addressing the second issue requires the measurement of organotins at sub-ppb to fractional ppt, which is considerably more challenging. One of the most promising analytical techniques for the detection of organotins at extremely low concentrations involves gas chromatographic (GC) separation and inductively coupled plasma mass spectrometry (ICP-MS).
This presentation will discuss the use of an Agilent GC-ICP-MS system for the detection of dimethyltin, monomethyltin, monobutyltin and dibutyltin. Preliminary performance data including detection limits and reproducibility at 5-10 times the method detection will be reported. This presentation will also provide isotope ratio performance data at low level concentrations utilizing the GC interface. The research findings will be presented in the context of the analytical requirements for PVC pipe leaching studies. Finally, data will be presented on the use of a liquid-liquid extraction as a means of pre-concentration prior to GC-ICP-MS detection.
PRESENTATION A Mass Balance Approach to Determine Arsenic Absorption Rates from Contaminated Water By Rice During the Food Preparation Process 01/12/2003
Ackerman, A. H., P. A. Gallagher, A. N. Parks, C. A. Schwegel, J. T. Creed, D. T. Heitkemper, AND N. Vela. A Mass Balance Approach to Determine Arsenic Absorption Rates from Contaminated Water By Rice During the Food Preparation Process. Presented at 2003 European Winter Conference on Plasma Spectrochemistry, Garmisch-Partenkirchen, Germany, January 12-17, 2003.
Abstract: Rice represents a unique set of arsenic exposure assessment challenges in that it does contain a relatively high concentration of arsenic and it does absorb about 100% of its dry weight during food preparation. Arsenic exposure from consumption of rice can conceptually be divided into arsenic native to the rice [As(III), DMA, MMA and As(V)] and arsenic absorbed from the water [As(III) and As(V)] during food preparation. The arsenic exposure associated with the rice is a relative constant while the arsenic absorbed from the water may depend on the concentration of arsenic in the water used in food preparation and the way the rice is cooked (boiled or steamed). The actual exposure becomes difficult to calculate without knowing the native rice arsenic concentration, the arsenic concentration in the water and the percentage of arsenic absorbed during the food preparation process. The ability to estimate the native arsenic concentration in rice using a nearly quantitative extraction process has been reported [1] and the concentration of arsenic in the water is analytically fairly simple to determine. The unknown quantity is the absorption rates of the arsenic from the water to the rice. Given that rice is a common staple and that the water used for food preparation could represent a large percentage of the exposure dose, it is important to estimate the absorption rates in order to better characterize rice as an arsenic exposure source.
This presentation will use an arsenic mass balance approach throughout the food preparation process in order to estimate arsenic absorption rates from water. Native arsenic concentrations will be determined for the rice, water and cooked rice. These data will be utilized along with percent moisture increase for the cooked rice to calculate the arsenic absorption rates. Total arsenic concentrations and speciation data will be acquired for individual components (rice, water, cooked rice) and the quality of the mass balance for boiled vs steamed rice will be discussed. All results will be based on replicate IC-ICP-MS analysis and the absorption rates will be estimated.
PRESENTATION Changes in Mouse Circulating T-Lymphocyte Numbers During a Normal Cryptosporidium Muris Infection and After a Single Injection of Methylprednisolone Acetate 04/11/2003
Miller, T. A. AND F. W. Schaefer. Changes in Mouse Circulating T-Lymphocyte Numbers During a Normal Cryptosporidium Muris Infection and After a Single Injection of Methylprednisolone Acetate. Presented at Experimental Biology Meeting, San Diego, CA, April 11-15, 2003.
Abstract: Cryptosporidium species are of public health significance in both developing nations and the industrialized nations of the world. Persons with immature or compromised immune systems are said to have increased risk or mortality once infected. The objective of this study was to chart circulating lymphocyte numbers before, during and after a natural C. muris infection and to analyze the effects of a single dose of methylprednisolone acetate (MPA) on oocyst shedding and T cell numbers. Thirty-two day old female CF-1 mice were exposed and monitored for oocyst shedding. CD3, CD4, and CD8 numbers were obtained using flow cytometry from sacrificed mice over the course of the infection. In a subset of mice MPA was injected on day 15 post exposure and both T-lymphocytes and oocyst shedding were monitored. Results showed a significant increase in the duration and numbers of oocyst shed after MPA. T-lymphocytes radically decreased by greater than 90% after MPA.
PRESENTATION Fingerprinting of Fecal Enterococci By Matrix Assisted Laser Desorption Ionization Mass Spectrometry 05/18/2003
Glassmeyer, S., C. A. Kelty, AND J. Santo Domingo. Fingerprinting of Fecal Enterococci By Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Presented at American Society of Microbiology, Washington, DC, May 18-22, 2003.
Abstract: The fecal enterococci group has been suggested as an indicator of fecal contamination in freshwater and marine water systems and as a potential target for bacterial source tracking of fecal pollution. While many studies have described the diversity of enterococci in environmental waters, most of these studies have relied on biochemical characteristics that can not discriminate between some of the different species of fecal enterococci. In this study, we evaluated the use of whole cell protein profiles using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a tool to identify different enterococci species. Seven enterococci species were used in this study, namely, Enterococcus faecalis, E. faecium, E. durans, E. gallinarum, E. avium, E. mundtii, and E. casseliflavus. Analysis of protein spectral profiles of masses lower than 2000 mass-to-charge (m/z) were identical between all enterococci species tested suggesting that these might be characteristic of the enterococci group. While many peaks were also shared among the different enterococci species in the 4,000 to 11,000 m/z range, each species showed distinctive peaks, primarily in the 6,000 to 7,000 m/z region. When environmental isolates were tested, the signature peaks were observed in many of the different isolates, suggesting that these peaks could be used for species identification. Sequence analysis of the environmental isolates 16S rDNA confirmed the identity of the strains tested. The results from this study indicate that the analysis of whole cell by MALDI generates a protein profile which can be used for the rapid identification of fecal enterococci environmental isolates. Since not all environmental isolates that belong to the same species had an identical protein profile, these results suggest that MALDI may also be used to discriminate between different strains of the same enterococci species.
PRESENTATION Sensitivity of Different Aeromonas Species to Copper and Silver 05/18/2003
Rodgers, M. R. AND A. W. Smallwood. Sensitivity of Different Aeromonas Species to Copper and Silver. Presented at American Society for Microbiology, Washington, DC, May 18-22, 2003.
Abstract: Aeromonas bacteria are common flora in surface and ground waters and are considered to be human pathogens. They can also be found in municipally treated drinking water, likely as a component of biofilms, as found in distribution system pipes and point of use water filters. It has been previously reported that Aeromonas bacteria are very sensitive to certain heavy metals, such as copper, found in treated drinking water and that this sensitivity affects the recovery of these bacteria from water samples. In addition, copper and silver ions are frequently employed as bactericidal agents in water filters to limit biofilm formation. Earlier studies on metal sensitivity of aeromonads did not investigate differences among species. We have undertaken the present study to investigate possible differences among Aeromonas species with regard to heavy metal sensitivities. Eleven Aeromonas species, including A. hydrophila, A. caviae, A. veronii biovar veronii and A. bestiarum (species most commonly encountered in both clinical and environmental samples) were used in the sensitivity studies. Heavy metal sensitivities of E. coli and Vibrio cholerae were also measured for comparison. Bacteria were exposed to varying concentrations of copper and silver ions in saline. Our copper sensitivity results support previous studies, indicating that Aeromonas bacteria, regardless of species, are more sensitive than E. coli to copper. V. cholerae displayed a similar copper sensitivity. Differences among Aeromonas species were seen however at exposure to 20 micromolar copper, providing a possible explanation for why certain species may be isolated more frequently than others from water. Silver, at concentration as low as 5 micromolar, had a pronounced effect on all strains tested, with the Aeromonas strains more sensitive than either E. coli or V. cholerae.
PRESENTATION Genetic Fingerprinting of Mycobacterium Avium Complex (Mac) Organisms Isolated from Hospital Patients and the Environment 05/18/2003
Pfaller, S. L. AND T. C. Covert. Genetic Fingerprinting of Mycobacterium Avium Complex (Mac) Organisms Isolated from Hospital Patients and the Environment. Presented at American Society for Microbiology, Washington, DC, May 18-22, 2003.
Abstract: A particularly pathogenic group of mycobacteria belong to the Mycobacterium avium complex (MAC), which includes M. avium and M. intracellulare. MAC organisms cause disease in children, the elderly, and immuno-compromised individuals. A critical step in preventing MAC infections is identifying the source of infection and preventing exposure to that source. The standard method for tracking MAC infection outbreaks is long restriction fragment length polymorphism analysis using pulsed-field gel electrophoresis (PFGE). The method is labor-intensive and may lack sensitivity. The purpose of this study was to develop a rapid, reproducible, and efficient method for fingerprinting mycobacteria at the strain level. Mycobacteria isolates were obtained from AIDS and non-AIDS patients, as well as drinking water and food sources. The strains were identified as M. avium, M. intracellulare, or "MX" using AccuProbe (GenProbe). One hundred and sixty-six isolates were typed using Amplified Fragment Length Polymorphism (AFLP) analysis. Phylogenetic analysis using maximum parsimony was utilized to determine the genetic relatedness of the isolates. AFLP was able to distinguish between MAC species and differentiate between strains within each species. Furthermore, the method was rapid and highly reproducible. None of the isolates were genetically identical. Several "MX" strains clustered with M. intracellulare, and their identities were confirmed with 16S sequence analysis. For both species of Mycobacterium, most drinking water isolates clustered more closely with each other than with patient or food isolates, suggesting that the harsh decontamination procedures used to isolate mycobacteria from the environment select for a subset of the organisms present. Patient isolates were more genetically diverse.
PRESENTATION Identification and Characterization of Aeromonas Isolates from Drinking Water Distribution Systems 05/18/2003
Birkenhauer, J. M. AND M. R. Rodgers. Identification and Characterization of Aeromonas Isolates from Drinking Water Distribution Systems. Presented at American Society for Microbiology, Washington, DC, May 18-22, 2003.
Abstract: Members of the bacterial genus Aeromonas are commonly isolated from both fresh and salt waters worldwide and some are believed to cause infections in humans, including gastroenteritis and wound infections. Currently, aeromonads are on the United States Environmental Protection Agency's Contaminant Candidate List, and are suspected of contaminating drinking water distribution systems. Identification of aeromonads to the species level is difficult as new species, taxa, and biogroups continue to be proposed. In this study, we employ both metabolic and genomic fingerprinting identification methods to obtain an understanding of the occurrence and types of aeromonads in drinking water distribution systems in the US.
Water samples were analyzed from 18 drinking water distribution systems across the US, eight of which were found to contain aeromonads. All colonies were isolated from ADA-V medium and were confirmed to be aeromonads as recommended in EPA Method 1605. Confirmed isolates, 212 in total, were then subjected both a Restriction Fragment Length Polymorphism (RFLP) analysis (Borrell et al, 1997) and to a carbon source utilization assay employing the BIOLOG microbial identification system.

The BIOLOG microbial identification system offers a straightforward approach to identifying environmental microbes. However, we found that only after compiling our own database were we able to gain confidence in the system's ability to correctly identify each isolate. The RFLP analysis, while requiring much more time and technical skill, was able to give a more consistent identification of each isolate, with the exception to certain biotypes.

Based on both the metabolic and genomic fingerprinting of these organisms we were able to identify several different biotypes, including A. hydrophila, A. bestiarum, and A. salmoncida from drinking water distribution systems. Since some of the species that were isolated have been implicated in human disease, the results from this study indicate that a more comprehensive survey of drinking water utilities is warranted to determine if aeromonads in drinking water pose a threat to public health.
PRESENTATION Development of a Molecular Method to Detect Astrovirus 07/12/2003
Grimm, A. C., G. S. Fout, J. L. Cashdollar, AND F. P. Williams. Development of a Molecular Method to Detect Astrovirus. Presented at American Society for Virology Annual Meeting, Davis, CA, July 12-16, 2003.
Abstract: Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed to detect all known astrovirus. When tested, this assay was able to detect strains from all eight serotypes. In addition, an internal control was developed so that it will be possible to determine if the sample being tested contains PCR inhibitors. Most probable number analysis determined that when amplified with the developed assay, a single DNA molecule of the internal control could be detected if inhibitors were not present. The assay was successfully adapted to real-time PCR and this method was then used for integrated cell culture RT-PCR detection of live virus. The methods were successfully used to detect astrovirus present in clinical samples and spiked water samples.
PRESENTATION The Key Viral Players 01/15/2003
Fout, G. S. The Key Viral Players. Presented at EPA Workshop for Development of Protocols for Reliable Genetic Methods for Viruses for Use in EPA's Drinking Water Program, Cincinnati, OH, January 15-16, 2003.
Abstract: A number of different types of human enteric viruses cause waterborne outbreaks when individuals are exposed to contaminated drinking and recreational waters. Members of the enterovirus group cause numerous diseases, including gastroenteritis, encephalitis, meningitis, myocarditis, temporary paralysis and perhaps diabetes and chronic fatigue syndrome. Hepatitis A and more recently hepatitis E have caused large waterborne hepatitis outbreaks. The second leading cause of illness in the United States is acute nonbacterial gastroenteritis. This disease results from infection of susceptible individuals with members of the Caliciviridae, Astroviridae, Reoviridae and Adenoviridae families. The characteristics of these enteric virus groups will be discussed.
PRESENTATION EPA Methods for Virus Detection in Water 03/28/2003
Fout, G. S. EPA Methods for Virus Detection in Water. Presented at University of Surrey - Site Visit, Guildford, UK, March 28, 2003.
Abstract: A number of different types of human enteric viruses cause waterborne outbreaks when individuals are exposed to contaminated drinking and recreational waters. Members of the enterovirus group cause numerous diseases, including gastroenteritis, encephalitis, meningitis, myocarditis, temporary paralysis and perhaps diabetes and chronic fatigue syndrome. Hepatitis A and more recently hepatitis E have caused large waterborne hepatitis outbreaks. The second leading cause of illness in the United States is acute nonbacterial gastroenteritis. This disease results from infection of susceptible individuals with members of the Caliciviridae, Astroviridae, Reoviridae and Adenoviridae families. The characteristics of these enteric virus groups will be discussed.
Viruses are recovered and concentrated from water by passage through a positively charged cartridge filter. Following virus elution from the cartridge filter with beef extract and concentration of the beef extract solution, viruses are usually assayed by cell culture. However, cultural methods are too time consuming and expensive for routine use and many of the viruses that cause waterborne disease are either very difficult to culture or cannot be cultured.

Rapid polymerase chain reaction (PCR) methods have been developed to overcome these problems. While PCR methods are rapid and can detect all the virus groups known to cause waterborne disease, the methods have several unique problems that can cause false negative and false positive results. The presence of potent environmental inhibitors of PCR that are co-concentrated along with viruses during sample processing can result in false negative results. In addition, aerosols containing previously amplified products can cause false-positive results. The quality control measures needed to prevent these problems are much more complex than those used in traditional microbiology or chemistry laboratories.

The number and types of environmental inhibitors vary among different water types and even within a single water type. Thus PCR methods must be designed to remove as many inhibitors as possible from different water types without affecting virus recovery. A multiplex PCR method was developed at the U.S. EPA to measure the occurrence of enteroviruses, reoviruses, rotaviruses, hepatitis A virus and Norwalk virus in water. The method uses a celite-based elution/reconcentration procedure. It results in concentrating a portion of the filter eluate by greater than 800 fold while providing a 74% recovery of poliovirus. Although the method was very effective, false negative and positive results still occur. In one study false negative results were observed in 14% of the samples and false positive results in 6%. The method and appropriate quality controls will be described.
PRESENTATION Detection By Pcr of Human Enteric Viruses Concentrated from Large Volumes of Water 01/15/2003
Fout, G. S. Detection By Pcr of Human Enteric Viruses Concentrated from Large Volumes of Water. Presented at EPA Workshop for Development of Protocols for Reliable Genetic Methods for Viruses for Use in EPA's Drinking Water Program, Cincinnati, OH, January 15-16, 2003.
Abstract: Viruses are recovered and concentrated from water by passage through a positively charged cartridge filter. Following virus elution from the cartridge filter with beef extract and concentration of the beef extract solution, viruses are usually assayed by cell culture. However, cultural methods are too time consuming and expensive for routine use and many of the viruses that cause waterborne disease are either very difficult to culture or cannot be cultured.
Rapid polymerase chain reaction (PCR) methods have been developed to overcome these problems. While PCR methods are rapid and can detect all the virus groups known to cause waterborne disease, the methods have several unique problems that can cause false negative and false positive results. The presence of potent environmental inhibitors of PCR that are co-concentrated along with viruses during sample processing can result in false negative results. In addition, aerosols containing previously amplified products can cause false-positive results. The quality control measures needed to prevent these problems are much more complex than those used in traditional microbiology or chemistry laboratories.

The number and types of environmental inhibitors vary among different water types and even within a single water type. Thus PCR methods must be designed to remove as many inhibitors as possible from different water types without affecting virus recovery. A multiplex PCR method was developed at the U.S. EPA to measure the occurrence of enteroviruses, reoviruses, rotaviruses, hepatitis A virus and Norwalk virus in water. The method uses a celite-based elution/reconcentration procedure. It results in concentrating a portion of the filter eluate by greater than 800 fold while providing a 74% recovery of poliovirus. Although the method was very effective, false negative and positive results still occur. In one study false negative results were observed in 14% of the samples and false positive results in 6%. The method and appropriate quality controls will be described.
PRESENTATION Characterization of a Mouse Model of Iimmunosuppression Using a Single Does of Methylprednisolone Acetate 06/01/2003
Miller, T. A. AND F. W. Schaefer. Characterization of a Mouse Model of Iimmunosuppression Using a Single Does of Methylprednisolone Acetate. Presented at Society of Comparative Endocrinology, Asheville, NC, June 1-4, 2003.
Abstract: Immunosuppression is a widely used but rarely defined term. To the transplant specialist it is the state at which the transplanted organ is not rejected. For the HIV patient it is the point when AIDS begins. In research, immunosuppression is achieved through the use of glucocorticoids, usualy for prolong period of time. In this model a single dose of methylprednisolone acetate (Depomedrol) was used to produce immunosuppression. The circulating CD3, CD4 and CD8 lymphocyte levels were analyzed to characterize the immunologic changes induced. A drop of greater than 80% was needed to re-establish a Cryptosporidium muris infection in mice who had fully recovered from an initial infection. When compared, the ID50 for mice who were immunosuppressed was three times higher than the ID50 for normal mice, showing immunosuppressed mice were less susceptible to C. muris invasion.
PRESENTATION Proteomic Analysis of Allergens from Metarhizium Anisopliae 06/08/2003
Donohue, M. J., J. A. Shoemaker, M. J. Selgrade, M. W. Ward, AND L. B. Copeland. Proteomic Analysis of Allergens from Metarhizium Anisopliae. Presented at 51st ASMS Conference, Montreal, Canada, June 8-12, 2003.
Abstract: Introduction
The goal of this project is the identification and characterization of allergens from the fungus Metarhizium anisopliae, using mass spectrometry (MS). The US EPA, under the "Children at Risk" program, is currently addressing the problem of indoor fungal bioaerosol contamination. One of the research objectives is to develop a basic understanding of IgE inducing proteins from fungi, using advanced proteomics. The fungus M. anisopliae has been used as a bio-pesticide for insect control since the 1800's. Recent studies have shown that exposure to this microorganism can cause an immediate hypersensitivity or Type I allergic responses in mice1. Matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) were used to analyze the fungal peptides and proteins isolated by 2-D gel electrophoresis and immuno-blot analysis. The MS data on molecular weight, peptide profiles, and amino acid sequence were used to mine databases for potential sequence or domain homology to known allergens. This information will provide better characterization of the M. anisopliae proteins that induce IgE responses, eventually paving the way to a better understanding as well as the prevention and/or treatment of protein allergies.

Method

Sample Preparation
The fungal extract preparation was preformed following a modified method described by Stankus and O'Neil2. Metarhizium anisopliae was grown under three different preparations. The proteins were extracted, and assayed for total protein concentration. Equal protein concentrations of each growth condition were combined to create the M. anisopliae crude antigen (MACA) preparation.

2D gel Electrophoresis and Immuno-blot Analysis
Total protein extracts of M. anisopliae were separated using 2-D gel electrophoresis. Allergenic proteins were identified by immuno-blot analysis, using hyperimmune mouse serum against M. anisopliae extract as the primary antibody and anti-mouse IgE as the secondary antibody. Protein spots identified as inducing IgE were excised and in-gel digested. The peptides were extracted and analyzed by MALDI-TOF-MS and ESI-MS/MS. Peptide sequences were analyzed using the MASCOT data mining algorithm.

Mass Spectrometry

All MALDI analyses were acquired on a Bruker Biflex III. Samples were diluted 1:1 in matrix (a -HCCA, 10mg/ml; ACN 50% (v/v); TFA 0.1% (v/v). One microliter of this solution was spotted onto the MALDI target.

All nanospray ESI/MS/MS data were acquired using an Applied Biosystem QSTAR XL equipped with a Proxeon nano-ESI source. Low-energy collision induced dissociation (CID) was preformed using nitrogen as the collision gas.

Results and Discussion

Eight proteins were identified as allergenic proteins, based on IgE reactivity in the immuno-blot assay. Five of the eight proteins had apparent molecular weights greater than 75 kDa. All the eight proteins have acidic pIs in the range between 4.8 and 5.5. At this time, all eight spots have been excised from the Coomassie stained gel. Protein fingerprints have been obtained for Spots 1, 2, and 5. The mass values have been used to mine the non-redundant databases to identify these allergenic proteins. Based on database mining, the proteins have been determined to be novel and are currently being sequenced by ESI-MS/MS.

Conclusion
In this study, we have clearly demonstrated that exposures to M. anisopliae can induce an allergenic response by the identification of eight proteins that are reactive to IgE antibodies. Three of the identified proteins appear to be novel due to the lack of significant matches when mining the non-redundant databases. Efforts are ongoing to verify that the proteins are novel by amino acid sequencing the observed peptides.

Reference
1. Ward, M.D.; Sailstad, D.M.; Selgrade MJ.K. Toxicol. Sci. 1998, 4,195-203.
2. Stankus, R.P.; O'Neil, C.E. J. Allergy Clin. Immunol. 1988, 81, 563-570.
PRESENTATION Low-Level Determination of Perchlorate in Drinking Water Using Ion Chromatography Mass Spectrometry 06/08/2003
Hedrick, E. J., R. Slingsby, D. J. Munch, AND D. P. Hautman. Low-Level Determination of Perchlorate in Drinking Water Using Ion Chromatography Mass Spectrometry. Presented at 51st ASMS Conference, Montreal, Canada, June 8-12, 2003.
Abstract: Perchlorate is a drinking water contaminant originating from the dissolution of the salts of ammonium, potassium, magnesium, or sodium in water. It is used primarily as an oxidant in solid propellant for rockets, missiles, pyrotechnics, as a component in air bag inflators, and in highway safety flares. Based on EPA Information Request Responses, there are 44 states that have reported perchlorate manufacturers or users. From accidental releases and disposal, perchlorate has become a contaminant in surface and ground waters where it is highly mobile and, due to its chemical stability, persists for decades. The primary human health effect is inhibition of iodide uptake by the thyroid gland. By disrupting thyroid hormone production, perchlorate interferes with metabolism and can affect brain development in fetuses and children, leading to mental impairment. The perchlorate anion (ClO4 -) has been found in numerous drinking water supplies at concentrations that recent studies indicate may adversely affect human health. There is an urgent need to be able to confirm and quantify perchlorate at lower concentrations than the currently approved U.S. EPA method allows. In this work, sub-ppb quantitation of perchlorate in drinking waters using ion chromatography with conductivity suppression, electrospray ionization mass spectrometry (IC-ESI-MS) was demonstrated. The primary mass of interest is 99 based on the 75.77% relative abundance of the chlorine-35 isotope. Mass 101, derived from the 24.23% abundance of chlorine-37, is a secondary mass that was also utilized for quantitation and confirmation.
Low-level linear calibrations from 0.01 - 1.0 ppb, yielded R2 >0.999. Method detection limits (MDLs) in deionized and high ionic waters (up to 1000 ppm common anions sulfate, chloride and carbonate) were from 0.03 - 0.11 ppb with no significant difference, at alpha=0.01, between mass 99 and mass 101 MDLs. Precision of replicate injections at 1 ppb, yielded <5% relative standard deviation on mass 99 and <5% on mass 101 on a daily basis. Accuracy as determined by analysis of a certified reference material was +5% of the certified value in deionized water. Ruggedness, as determined by the reproducibility of area counts of 1.0 ppb check standards analyzed periodically over a day of continuous analysis of high ionic matrices, revealed some deterioration of signal intensity (~15% drop).

The major cause of a loss of sensitivity was fouling of the MS sampling cone. Rapid fouling occurred during the first five minutes of analysis due to the elution of cations and common anions such as chloride, sulfate and nitrate. It is also during this time frame that the eluate pH dips to pH 1 due to H+ exchange with monovalent cations that occurs in the electrolytic conductivity suppressor. This highly acidic eluate can damage the stainless steel capillary and sampling cone. To slow column fouling, mass spectrometer manufacturers often recommend diverting the LC flow to the MS until just prior to the elution of the analyte of interest (matrix cutting). Matrix cutting proved to be beneficial for extending the period between cone cleanings and in maintaining signal intensity over the course of a day. A chromatographic problem observed was tailing of large concentrations of the anions sulfate, chloride and nitrate into the elution time of ClO4-. Due to a minor isotope of sulfur, HSO4 - (mass 99) interferes spectrally with ClO4-. The best way to eliminate the problem is to remove the sulfate prior to sample analysis using precipitation with barium.

Recoveries in three different tap waters ranged from 99 -102% based on the average recoveries from quantitation at masses 83, 99 and 101. In-source collisionally activated dissociation of ClO4- to yield ClO3- was done by increasing the cone voltage and was used as additional confirmation of ClO4 - .

Contaminated ground waters with suspect concentrations of ClO4- (based upon previous analyses using IC with conductivity detection) were analyzed by the IC-MS method. The IC-MS method revealed that the suspect contaminants were not perchlorate. One site yielded low spike recovery which is believed to be due to ionization suppression from the combined effect of high sulfate and another contaminant with the same retention time as ClO4 -

Future work will focus on finding a suitable internal standard and in exploring whether other mobile phases and separator columns can achieve the same performance. Isotope dilution calibration using an enriched ClO4- standard (enriched on 18-O) will be tested in the near future.

Overall, IC-suppressed conductivity-ESI-MS proved to be sensitive and specific for ClO4- in drinking and ground waters at sub-ppb concentrations.
PRESENTATION A Comparison of Three Assay Procedures for Determining Chlorine Inactivation of Waterborne Pathogenic Bacteria 05/18/2003
McDaniels, A. E., E. W. Rice, R. A. Tally, L. J. Wymer, AND G. N. Stelma Jr. A Comparison of Three Assay Procedures for Determining Chlorine Inactivation of Waterborne Pathogenic Bacteria. Presented at 103rd General Meeting American Society for Microbiology, Washington, DC, May 18-22, 2003.
Abstract: One criterion on which chlorine treatment of water may be based is the concentration (C) in mg/l multiplied by the time (t) in min of exposure or Ct values. We compared different Ct values on waterborne pathogenic bacteria by cultural assay for viability and 2 assays that measured metabolic activity. Total counts were measured by 4'6-diamidino-2-phenylindole dihydrochloride (DAPI). Metabolic activity was based on the presence of esterase as detected by a modified fluorescein diacetate fluorophore designated V6 and respiration by the fluorophore 5-cyano-2,4-ditoyl tetrazolium chloride (CTC). V6 results were obtained using a solid phase cytometer and counts were validated with a microscope. CTC and DAPI results were obtained by applying a mathematical formula to determine microscopic counts of cells per ml. The bacteria included Escherichia coli 0157:H7, Legionella pneumophila and Helicobacter pylori. Each bacterium was added to 3 separate chlorine demand free water samples. Three different Ct values per sample were examined at 5oC and pH 7.0. Counts were obtained before and after chlorine treatment.
No significant differences were found between the DAPI controls and the corresponding chlorine treated samples. In the Chick-Watson theory of disinfection the rate of die-off of microorganisms is hypothesized to be proportional to the number of organisms remaining and a power function of the disinfectant concentration. This theory was shown to be reasonable for E. coli 0157:H7, L.
pneumophila and H. pylori where no plate growth or CTC counts were found after exposure to Ct

ranges of 7 to 95. V6 counts for these 3 treated bacteria were present but as Ct values increased,

counts were significantly reduced. After chlorine treatment each bacterium decreased by at least 2 to 3 log10 for all assays. V6 activity persisted when other assays were negative and would thus be

more apt to indicate any lingering activity within the cells. It may be a conservative indicator of

metabolic activity within bacteria.
PRESENTATION Developing Analytical Methods for Gathering Nationwide Occurrence Data for Chemicals on the Drinking Water Contaminant Candidate List (Ccl) 05/05/2003
Munch, J. W. Developing Analytical Methods for Gathering Nationwide Occurrence Data for Chemicals on the Drinking Water Contaminant Candidate List (Ccl). Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: Amendments to the Safe Drinking Water Act (SDWA) require the United States Environmental Protection Agency (USEPA) to publish a list of contaminants that are known or anticipated to occur in public water systems, and which may require regulation under the SDWA. In response to this requirement, and after extensive consultation with the scientific community, the USEPA's Office of Ground Water and Drinking Water (OGWDW) published a Drinking Water Contaminant Candidate List (CCL) of 50 chemicals and 10 microorganisms in March 1998. A critical piece of information that OGWDW must have for making a regulatory determination on these contaminants is the frequency and magnitude of their occurrence in public drinking water systems across the country. This information will be collected by OGWDW through a nationwide chemical monitoring program at drinking water utilities. Because of the importance of the regulatory determination process, it is essential that the test methods used to gather this occurrence data be both sensitive and specific.
NERL collaborated with OGWDW to determine which of the 50 chemicals under consideration for regulatory action needed new, improved analytical methods prior to collection of occurrence data. From the list of chemicals for which method development was required, NERL has completed the development of two analytical methods that together will test for five chemicals of concern, and meet the data quality objectives of OGWDW. Method 528 is currently being used in a nationwide monitoring study of public drinking water supplies to obtain data on four substituted phenols listed on the CCL, and on an additional eight substituted phenols that are priority pollutants. Phenols are of concern as drinking water contaminants because of their widespread use as intermediates in the manufacture of pesticides, bactericides, and other industrial chemicals. Chlorinated phenols are also a by-product of the paper manufacturing industry. Method 529 was developed to monitor for hexahydro-1,3,5-trinitro-1,3,5-triazine, also known as Royal Demolition Explosive (RDX), and an additional 13 explosives and related chemicals. Recent studies of groundwaters near military installations have identified RDX and other munitions as an emerging drinking water concern. Method 529 will be used in the next nationwide monitoring study to gather data on the occurrence of RDX and other explosives.

NERL is currently working with OGWDW to develop three additional methods for chemicals listed on the CCL. These methods will be used to monitor perchlorate, organotins, alachlor-ESA and other acetanalide pesticide degradation products in a nationwide study beginning in 2006.
PRESENTATION The Need for Speed-Rapid Methodologies to Determine Bathing Beach Water Quality 05/05/2003
Haugland, R. A. The Need for Speed-Rapid Methodologies to Determine Bathing Beach Water Quality. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: Current methods for determining fecal contamination of recreational waters rely on the culture of bacterial indicators and require at least 24 hours to determine whether the water is unsafe for use. By the time monitoring results are available, exposures have already occurred. New methods are needed that will allow near real-time determination of water quality, such that public notifications can be made and hazardous exposures avoided. The U.S. EPA, National Exposure Research Laboratory (NERL) has evaluated several new antibody and molecular-based approaches for obtaining timely measurements of recreational water quality within a two-three hour time period. These approaches include: 1) flow cytometric detection of indicator organisms using fluorescently labeled antibody-paramagnetic bead complexes; 2) antibody capture and fiber optic detection of indicator organisms; and 3) quantitative polymerase chain reaction amplification of nucleic acids from specific fecal indicator organisms. These methods will be used in a joint study by NERL, the National Health and Environmental Effects Laboratory and the Centers for Disease Control to develop new water quality-health effects relationships that can be used for establishing scientifically defendable guidelines for recreational waters.
The objectives of this research are: (1) to obtain in a timely manner water quality data using the new rapid, state-of-the-art methods and the new EMPACT monitoring protocol in conjunction with epidemiological studies that will produce water quality-health data; and (2) to provide the information to the Office of Water so they can promulgate new state and/or federal guidelines and limits for water quality indicators of fecal contamination so that beach managers and public health officials can alert the public about the potential health hazards before exposure to unsafe water can occur.
PRESENTATION The Need for Speed Rapid Methodologies to Determine Bathing Beach Water Quality 05/05/2003
Haugland, R. A., K. P. Brenner, A. P. Dufour, AND S. D. Siefring. The Need for Speed Rapid Methodologies to Determine Bathing Beach Water Quality. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.
PRESENTATION Environmental Monitoring for Public Access and Community Tracking (Empact) Program Microbiological Monitoring of Recreational Water 05/05/2003
Brenner, K. Environmental Monitoring for Public Access and Community Tracking (Empact) Program Microbiological Monitoring of Recreational Water. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: Current Environmental Protection Agency (EPA) recommended microbiological monitoring practices for bathing beach water quality were suggested in 1968, as a part of the fecal coliform guideline developed by the Federal Water Pollution Control Administration. The guideline stated that the geometric mean of the fecal coliform counts from five water samples taken over a thirty-day period would be used to determine the beach water quality. This level would be compared with an established limit beyond which the risk of illness was unacceptable. Although EPA has developed much better health guidelines for bathing beach waters that were recommended to the states in 1986, the old methods for monitoring continue to be used by many states and local public health authorities. This approach does not provide timely, accurate information for risk managers or the public, nor does it provide results that are easily interpreted. This shortcoming can be overcome by developing a statistically valid monitoring protocol that takes into account the sampling and environmental factors that vary considerably and, hence, contribute to the uncertainty on how and when to sample and how to interpret the results.
This research study examined five representative beaches from various sections of the United States in depth. The beaches were selected to obtain data on a variety of pollution sources, population density, type of swimming water (fresh or marine), and the type of beach (large coastal beaches, small lake or impoundment beaches, river recreational areas). An appropriate sampling design was developed to account for variation associated with spatial factors, e.g., depth of water, length of the beach and its distance from shore to permissible swimming limits, and temporal factors, such as hourly, daily and seasonal variation. By considering these factors, more appropriate site-specific monitoring protocols can be developed that are based on sound science and will result in better protection of the public health.

The EMPACT project was a collaborative effort between a team of NERL-Cincinnati scientists, two contractors, Lockheed Martin and Battelle, and the collaborating EMPACT cities and laboratories. In addition, outside experts helped with the planning process through their involvement in a Data Quality Objectives Workshop, provided input during the statistical analysis, and participated in a final workshop to review the statistical analysis of the study data. The study report will be used by the Office of Water to develop official monitoring guidelines and a mechanism for translating technical monitoring data into a simple system the public can use to make personal decisions about risks associated with swimming activities.
PRESENTATION The Establishment of Laboratory Guidelines for Analysis of Bioterrorism Samples 05/05/2003
Schaefer III, F. W. The Establishment of Laboratory Guidelines for Analysis of Bioterrorism Samples. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: After the attack on the World Trade Center on September 11, 2002, and the subsequent deaths associated with Bacillus anthracis spore contaminated mail, a worldwide need was apparent for increased laboratory capacity to safely analyze bioterrorism samples. The U.S. Department of Health and Human Services has furnished guidelines for microbiological and biomedical laboratory safety. These guidelines encompass laboratory practices and techniques, facility design, safety equipment, monitoring the analyst's health, vermin and insect control, as well as government control of select agents and specialized reagents.
Before work is initiated, the laboratory must have protocols which cover all standard operating procedures, quality assurance, chain of custody, and a detailed biosafety plan. Special emphasis is placed on the approach used in the event of either an accidental spill or accidental exposure. Extensive training in all aspects of the protocols is required for each analyst. Great care also is used to document both how and who processed the samples in case they have forensic significance.

Design of the laboratory facility centers around containment and segregation of the sample analyses, so as few people as possible are involved. Persons who are immunocompromised should not under any circumstances be permitted access to such a hazardous facility. Key card access through an airlock allowing only authorized personnel into the laboratory helps ensure such a policy. All laboratory benches must have impervious surfaces. The walls and floors also should be sealed, so liquids cannot penetrate them. The laboratory equipment and benches should be set up in a fashion to allow routine cleaning and disinfection. There must be a sink in each laboratory to facilitate hand washing. Interlocking double door autoclaves, a specialized negative pressure ventilation system, and waste stream treatment must all be part of the design.

The required safety equipment depends upon the type of analysis being performed. Generally gloves, laboratory coat or gown, shoe covers or boots, and eye protection are required. If the protocol entails analysis of a sample that is prone to the production of an aerosol, then a respirator is mandated. Most if not all procedures should be conducted in a biosafety cabinet. When centrifugations are necessary, they should be done with capped containers that in turn are placed in safety centrifuge cups. These cups are designed to prevent aerosols from being released during centrifugation. A biohazard warning sign naming the organism and incorporating the international biohazard symbol must be posted on all laboratory doors.
PRESENTATION Detection of Toxicant(s) on Building Surfaces Following Chemical Attack 05/05/2003
Bernard, C. E., J. N. Morgan, AND L. J. Melnyk. Detection of Toxicant(s) on Building Surfaces Following Chemical Attack. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: A critical step prior to reoccupation of any facility following a chemical attack is monitoring for toxic compounds on surfaces within that facility. Low level detection of toxicant(s) is necessary to ensure that these compounds have been eliminated after building decontamination. This requires developing a sensitive, rapid, and systematic sampling protocol that can be used to measure low levels of toxic compounds on building surfaces. Although the military and other organizations have performed research on traditional chemical weapons, many industrial chemicals may be used as nontraditional chemical agents. For example, many pesticides are readily available to almost anyone, including potential terrorists. Although pesticides are typically not as potent as traditional chemical weapons, the release of such compounds into a building would certainly cause panic to the occupants and would present a chronic health concern if not properly removed. However, limited research has been performed with respect to such misuse of these compounds.
Since the compounds that might be encountered in a chemical attack are diverse, the surface sampling protocol will be developed using a variety of industrial chemicals including pesticides. The selection of these compounds will be based on factors such as amounts manufactured worldwide, usage, and availability. Some classes of pesticides (e.g., organophosphate insecticides) will also be used because of their structural similarity to compounds that have been developed as chemical weapons. The procedure also needs to be applicable to a variety of building surfaces, such as walls, floors, ceilings, office furniture, and duct work. Therefore, different types of building surfaces will be contaminated with the study compounds at known concentrations and subsequently sampled. Surface sampling will consist of wipes with organic solvent moistened gauze pads, which were developed from previous EPA-NERL sponsored pesticide surface monitoring studies. Analytical methods will be optimized for detecting and measuring the study compounds in extracts from the gauze wipes.

This project will result in the production of protocols for conducting surface wipes to collect toxicant(s) from contaminated building surfaces, as well as the detection and quantification of compounds captured in these surface wipes. Since the selected toxic compounds in this study will encompass a wide range of physical and chemical properties, the techniques could be applied to a wide variety of industrial chemicals and other chemical agents. These protocols and analytical procedures will be available for use by EPA and its partners in an emergency response to chemical terrorism.
PRESENTATION The Discovery of Biomarkers of Viral Infectivity By Mass Spectrometry 05/05/2003
Shoemaker, J. A., J. M. Talley, D. R. Dahling, AND G. S. Fout. The Discovery of Biomarkers of Viral Infectivity By Mass Spectrometry. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: Over the past three decades, the CDC and the U.S. EPA have collected and reported data relating to occurrences and causes of waterborne-disease outbreaks in the United States. Thirty nine outbreaks associated with drinking water were reported during 1999-2000. According to CDC's 1999-2000 water surveillance report, of the 20 outbreaks with known infectious causes, four were caused by viruses, which indicates that viral contamination of drinking water is a problem. A number of these viruses (e.g. coxsackie) are now listed on the U. S. EPA 1998 Contaminant Candidate List as needing additional research (i.e., analytical methods, occurrence, treatment and health effects). Coxsackievirus causes flu-like symptoms including diarrhea and fever and can cause complications such as diabetes mellitus and myocarditis. Thus, NERL is striving to develop methods for viruses listed on the CCL. This research project is a collaborative effort between NERL microbiologists and chemists to use analytical chemistry to differentiate between virus strains and to identify potential biomarkers of viral infectivity. Typical microbiological methods require months to determine the virus strain. Through the use of mass spectrometry, we have identified potential viral biomarkers which can be used to differentiate rapidly between the strains of coxsackievirus and can potentially indicate whether the virus is infectious. A unique peak was observed in an infectious coxsackievirus, but was not observed in a non-infectious coxsackievirus. This unique peak may be responsible for viral infectivity, thus, be a potential biomarker of infectivity. This type of information, gathered through the use of mass spectrometric techniques is cutting edge, ground breaking basic research, and can have a significant impact on the field of virology and play a role in the future development of drinking water methods to detect viruses and their infectivity.
PRESENTATION The Good, the Bad and the Ugly Determination of Bacterial Virulence Using Animal Models and Microarray Technology 05/05/2003
Rodgers, M. R., S. J. Vesper, AND D. Lye. The Good, the Bad and the Ugly Determination of Bacterial Virulence Using Animal Models and Microarray Technology. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: In its Computational Toxicology Program, EPA/ORD proposes to integrate genomics and computational methods to provide a mechanistic basis for the prediction of toxicity of chemicals and the pathogenicity of microorganisms. The goal of microbiological water testing is to be able to determine with scientifically sound probabilities that the consumption of and/ or exposure to a given volume of water is safe. At present, the US EPA relies on indirect methods to determine the probabilities of the safety of drinking water, for example, the detection and enumeration of indicator microorganisms (e.g. E. coli ). Although the concept of indicator organisms as a measure of risk has served to reduce water-borne disease significantly over the years, public health professionals generally agree that the presence of pathogens does sometimes escape detection and at an unknown frequency.
Municipally treated drinking water is known to contain many bacteria, whose identity and potential human virulence is poorly understood. In some cases pathogens may not be culturable by our standard methods. In addition, polymicrobial disease agents may be present. Polymicrobial diseases are infections that result from multiple co-infecting organisms as opposed to a single microorganism. Application of computational-toxicologically based DNA microarrays offers a totally new approach to address the issue of water-borne pathogens.

There are many potential human pathogens in potable water, some identified and some not yet identified. In order to cause disease, a pathogen must possess "virulence factors" that interact with a host cell to cause damage that results in disease. The interaction with a host cell results in a change in the metabolism and thus the messenger RNAs (mRNAs) that are produced by the human cell(s). These changes can be monitored using Human DNA microarrays which will reveal whether a pathogen is present and whether it is virulent.

Ultimately, we will build a database of these mRNA responses to more and more known pathogens and the virulence factors associated with inducing the mRNAs. It may be possible to then use this database to predict the potential pathogenicity of unknown or non-culturable pathogens based on their producing similar mRNA responses found in known pathogens. Some animal experiments may then be used to confirm these predictions. However, ultimately this type of analysis should dramatically reduce the EPA's need for animals in experimentation.

PRESENTATION The Application of Emerging Technologies to Virus Detection in Water 05/05/2003
Fout, G. S. The Application of Emerging Technologies to Virus Detection in Water. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: Human enteric viruses belonging to many different viral genera cause waterborne disease when susceptible individuals are exposed to contaminated drinking and recreational waters. Diseases resulting from infection with these viruses include gastroenteritis, hepatitis, encephalitis, meningitis, myocarditis, temporary paralysis and possibly diabetes. Symptoms are often mild, but can be severe in sensitive target groups, such as the very young and elderly.
It is important that methods based upon emerging technologies are able to detect many of the viral groups that cause waterborne disease. Specific virus methods, based upon the polymerase chain reaction (PCR), have been developed by the National Exposure Research Laboratory (NERL). While PCR methods are rapid and can detect all the virus groups known to cause waterborne disease, the methods have several unique problems that require a much higher level of quality assurance than that used in traditional microbiology or chemistry laboratories. A quality assurance guide for using PCR methods in support of EPA's mission is being developed through a collaborative partnership of NERL and the Technical Support Center (TSC) of the Office of Ground Water and Drinking Water. This QA guide is a key outcome of a recent, jointly sponsored workshop on PCR methods.

NERL's PCR methods were originally developed as part of a national groundwater survey that was conducted through the partnering of NERL, TSC and the American Water Works Association Research Foundation. In order to determine whether the methods would detect viruses in surface waters and test the level of quality assurance that had been developed, further collaboration was sought with another partner, the U.S. Geological Survey. The studies on ground and surface waters demonstrated that the methods could be used on a majority of water types covering most of the conditions typically found in the U.S. Methods were further tested in collaborative studies to determine the cause of two independent waterborne outbreaks of gastroenteritis in Wyoming during 2001. These studies involved personnel from Wyoming, the Centers for Disease Control, EPA's Region VIII and the University of North Carolina. In both cases NERL's methods successfully identified the agent in water that was responsible for the outbreak. NERL's collaborative efforts to develop and evaluate emerging virus detection methodology have clearly demonstrated the usefulness of the partnering process.
PRESENTATION Rapid Virus Detection in Water 05/05/2003
Fout, G. S., A. C. Grimm, D. R. Dahling, J. L. Cashdollar, C. Newport, S. Parshionikar, AND S. WillianTrue. Rapid Virus Detection in Water. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.
PRESENTATION Are All Arsenic Exposures Toxic? Supporting Regional Risk Assessments Through Improved Arsenic Speciation Methodology 05/05/2003
Creed, J. T. Are All Arsenic Exposures Toxic? Supporting Regional Risk Assessments Through Improved Arsenic Speciation Methodology. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: Arsenic exposure assessments require the evaluation of the relative contribution of both media (water, food, etc.) and routes of exposure (ingestion, inhalation, dermal). For arsenic, the important media are predominately water and food and therefore, the route of concern for exposure is ingestion. In addition, the toxicity of an exposure is strongly dependent on the chemical form of the arsenic ingested. Water contains predominately toxic arsenic while foods contain a mixture of both toxic and non-toxic arsenicals. Thus, an accurate risk assessment must assess the exposure from water and food and must differentiate (or speciate) the toxic and non-toxic arsenicals present in foods. Furthermore, speciation of arsenicals in foods will aid in: 1) formulating an accurate relative source contribution (water vs. food), 2) conducting exposure studies to determine dose vs. response, and 3) help identify sub-populations which are highly exposed. These types of information become part of the scientific foundation used in formulating drinking water regulations Seafood has been identified as the major dietary contributor to arsenic exposure by the US FDA. US EPA's Region 10 (Alaska, Washington, Oregon, Idaho) seafood consumption rates are often well above average national values. For example, Native American (Washington and Oregon) and Alaska Native studies have indicated average seafood consumption rates up to ten times greater than the US EPA average estimate of 6.5 g/day. Thus, improved analytical methodology is needed to determine whether these sub-populations are being exposed to toxic or non-toxic arsenic species. As a result, NERL has developed an analytical procedure for the speciation of arsenic in seafoods and transferred it to the Region 10 laboratory. This procedure is being used to generate a preliminary speciation based database for arsenic in seafoods and should aid regulators in estimating the relative source contribution of food vs. water. Ultimately, this will aid the Region in assessing the impact of their elevated seafood consumption rates.
PRESENTATION Investigatoin of Cyanobacteria Toxins in Water 06/08/2003
Budde, W. L. AND M. Maizels. Investigatoin of Cyanobacteria Toxins in Water. Presented at Annual Conference of the American Society for Mass Spectrometry, Montreal, CA, June 8-13, 2003.
Abstract: Introduction:
Approximately 80 alkaloid and cyclic peptide toxins produced by various freshwater and marine cyanobacteria (blue-green algae) have been identified and their structures determined. The U. S. Environmental Protection Agency has identified two neurotoxin alkaloids and four cyclic peptide toxins that have the potential for serious contamination of sources of drinking water for humans and animals. The purpose of this research is to develop a convenient, rapid, but definitive analytical method for these toxins in water.

Methods and Instrumentation:

Four of the six toxins of interest, and three of lessor interest, were available for this work. Anatoxin-a, microcystins RR, LR, and YR, and nodularin were added to laboratory reagent water and to samples of natural waters at environmentally significant concentrations. The analytes were separated from the water by liquid-solid extraction using C-18 silica impregnated filter disks, eluted from the disks with methanol, and the eluate was analyzed using microbore (1 mm ID) LC combined with electrospray and time-of-flight mass spectrometry.

Preliminary Data:

The analytes anatoxin-a, microcystins RR, LR, and YR, and the cyclic peptide nodularin in methanol are separated in about 25 minutes. With about 50 ng of each injected, the LC peaks in the TIC have very good S/N. Microcystin RR gives mainly the (M+2H]2+ ion and the other analyes give the corresponding singly charged ions. In source CID gives a characteristic m/z 135 ion for the microcystins and nodularin, but anatoxin-a does not have the necessary structural feature to give this ion. Mean recoveries from laboratory water for the microcystins and nodularin were in the 85-98% range with RSDs in the 5- 17% range. The mean recovery of anatoxin-a was just 68% (RSD 11%). A suitable internal standard has not been identified and external standardization may account for some of the recoveries and analytical presicion.
PRESENTATION Inactivation of Giardia Muris By Low Pressure Ultraviolet Light 05/05/2003
Ware, M. W., F. W. Schaefer III, S. L. Hayes, AND E. W. Rice. Inactivation of Giardia Muris By Low Pressure Ultraviolet Light. Presented at Science Forum 2003, Washington, DC, May 5-7, 2003.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.
PRESENTATION A Revolution in Mold Identification and Enumeration 04/10/2003
Vesper, S. J. A Revolution in Mold Identification and Enumeration. Presented at Building Environment Council of Ohio Spring Conference, Columbus, OH, April 10, 2003.
Abstract: More than 100 assay were developed to identify and quantify indoor molds using quantitiative PCR (QPCR) assays. This technology incorporates fluorigenic 5' nuclease (TaqMan�) chemistry directed at the nuclear ribosomal RNA operon internal transcribed spacer regions (ITS1 or ITS2). The assays varied in specificity from species to closely related groups of species, subject to the amount of nucleotide sequence variation in the different organisms. Estimated conidia detection limits ranged from less than one to several hundred per sample for the different assays, using a previously reported glass bead milling and glass filter purification DNA extraction method. The precision, accuracy and sensitivity of the method were determined from analyses of replicate mixed conidia suspensions and different dust samples spiked with known quantities of target organisms.
PRESENTATION Rapid Measurement of Bacterial Fecal Indicators in Surface Waters By Quantitative Polymerase Chain Reaction (Qpcr) Analysis 05/14/2003
Haugland, R. A. Rapid Measurement of Bacterial Fecal Indicators in Surface Waters By Quantitative Polymerase Chain Reaction (Qpcr) Analysis. Presented at Rapid Microbiological Measurement Method Workshop, Monterey, CA, May 14-16, 2003.
Abstract: Current methods for determining fecal contamination of recreational waters rely on the culture of bacterial indicators and require at least 24 hours to determine whether the water is unsafe for use. By the time monitoring results are available, exposures have already occurred. New methods are needed that will allow near real-time determination of water quality, such that public notifications can be made and hazardous exposures avoided. With assistance from the U.S. Geological Survey Laboratory in Porter IN, the U.S. EPA, National Exposure Research Laboratory has conducted a two year pilot study to evaluate the use of quantitative PCR (QPCR) analysis for measuring water-borne fecal indicator microorganisms at two recreational beaches on Lake Michigan.
A rapid, simple and generally applicable method for the recovery of total DNA from various microorganisms in water samples has been developed. The method involves filtration of water samples on polycarbonate membrane filters and disruption of the collected cells directly on the membranes by glass bead milling. Recovered DNAs are subjected to QPCR analysis using the TaqManTM PCR product detection system in a real time PCR product detection instrument. Procedures have also been developed for the use of cycle threshold (CT) values generated by the instrument to enumerate cells in the water samples. The approach is based on the comparative cycle threshold (CT) method, which employs an arithmetic formula to determine target sequence quantities in DNA extracts from test samples relative to those in similarly-prepared DNA extracts from calibrator samples containing a known quantity of target organism cells. Assay CT values for a DNA sequence from an exogenous reference organism, added in equal quantities to both the test and calibrator samples before extraction, are used to normalize results for differences in the amount of total DNA added to each reaction (e.g., caused by differences in DNA extraction efficiency between samples) or to signal potential PCR inhibition in test samples. The entire analysis process can be performed in approximately two to three hours.

Target DNA sequences for QPCR detection in the pilot study included the large subunit ribosomal RNA gene of Enterococcus spp. and the small subunit ribosomal RNA gene of Bacteroides spp. Tests with pure cultures of representative species within these two genera gave extrapolated detection limits of approximately two cells per sample for the Enterococcus assay and 25 cells per sample for the Bacteroides assay. Tests on a subset of the Lake Michigan water samples spiked with ~1000 cells of Enterococcus cells gave an overall mean value of 0.96 for the ratio of measured to added cells and a 95% occurrence range for individual sample ratios of ~0.3 to 3, based on analyses of three replicates of each sample. Mean QPCR-measured quantities of native enterococci in the 100 ml water samples ranged from less than 10 to ~1000 cells whereas mean quantities of Bacteroides ranged from less than 100 to ~100,000 cells. DNA extracts of the water samples were routinely diluted 10-fold prior to analysis to eliminate the effects of PCR inhibitors. The distributions of QPCR-measured cell quantities of both Enterococcus and Bacteroides in the water samples paralleled those of culturable enterococci measured in corresponding water samples by the currently accepted mEI filter plating method.

These results indicate that the QPCR method has the potential to detect a broad range of fecal indicator densities in recreational water samples. Past findings of a correlation between the quantities of culturable enterococci in water samples and illness rates among bathers have provided the basis for establishing recreational water quality guidelines. New studies will be initiated this summer to establish whether similar correlations exist between fecal indicator measurements by QPCR and other rapid methods and rates of illness among bathers.
PRESENTATION Transfer Efficiencies of Household Pesticides from Surfaces to Foods 07/20/2003
Bernard, C. E., L. J. Melnyk, AND M. R. Berry Jr. Transfer Efficiencies of Household Pesticides from Surfaces to Foods. Presented at 40th Annual AFDOSS Florida Pesticide Residue Workshop, St. Petersburg, FL, July 20-23, 2003.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.
PRESENTATION Determination of Pesticides in Comosite Diets Using Large Volume Pressurized Fluid Extraction With in-Line Sample Preparation 07/20/2003
Morgan, J. N., T. Hieber, P. Kauffman, AND J. Brisbin. Determination of Pesticides in Comosite Diets Using Large Volume Pressurized Fluid Extraction With in-Line Sample Preparation. Presented at 40th Annual AFDOSS Florida Pesticide Residue Workshop, St. Petersburg, FL, July 20-23, 2003.
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 various classes of pesticides in composite diet samples. In previous work, pressurized fluid extraction (PFE) followed by diatomaceous earth and C18 reversed phase column chromatography was used in the determination of organophosphate pesticides in composite diet samples. PFE followed by diatomaceous earth and alumina column chromatography was used for a diverse mix of organochlorine and other pesticides. The current study evaluated an automated system for performing the extraction and cleanup in one step in a 100 mL PFE cell. Various combinations of sample amounts, extraction solvents and adsorbents were tested. Organophosphate pesticides were quantitated by gas chromatography with pulsed flame photometric detection. Organochlorine pesticides were quantified by gas chromatography/mass spectrometry in the selected ion monitoring mode. Results of this study demonstrated this automated procedure, using acetonitrile, a super absorbent polymer and C18, was comparable to the more laborious PFE/column chromatography methods used previously. Acetonitrile replaced a mixed solvent system of acetone/methylene chloride used in previous studies. Recoveries for most pesticides fell within the target range of 60 to 140%. Results obtained for organochlorine pesticides are preliminary and additional work is needed to optimize this system for those analytes.
PRESENTATION Detection of Toxicants on Building Surfaces Following Chemical Attack 09/07/2003
Melnyk, L. J., C. E. Bernard, AND J. N. Morgan. Detection of Toxicants on Building Surfaces Following Chemical Attack. Presented at ACS Meeting, New York City, NY, September 7-11, 2003.
Abstract: A critical step prior to reoccupation of any facility following a chemical attack will be the monitoring of toxic compounds on surfaces within that facility. Low level detection of toxicant(s) is necessary to ensure that these compounds have been eliminated after decontamination. Since the compounds that might be encountered in a chemical attack are diverse, surface sampling protocols will be developed using a variety of industrial chemicals including pesticides. Selection will be based on factors such as amounts manufactured worldwide, usage, and availability. The procedure also needs to be applicable to a variety of building surfaces, such as walls, floors, ceilings, office furniture, and duct work. Therefore, different types of building surfaces will be contaminated with the study compounds at known concentrations then sampled with wipes of organic solvent moistened gauze pads. Analytical methods will be optimized for detecting and measuring the study compounds in extracts from the gauze wipes.
PRESENTATION Determination of a Standard Food Item for Analysis of Pesticide Consumption in the Dietary Intake of Young Children 09/21/2003
Raymer, J., T. Marrero, G. G. Akland, Y. Hu, M. Berry, C. E. Bernard, AND L. J. Melnyk. Determination of a Standard Food Item for Analysis of Pesticide Consumption in the Dietary Intake of Young Children. Presented at ISEA Meeting, Stresa, Italy, September 21-25, 2003.
Abstract: The objective of this study was to establish a standard food item for the collection of residential use pesticides from household surfaces commonly encountered by young children while eating. The amount of a pesticide that young children ingest during eating is influenced by the residue in/on foods and the excess intake caused by food handling. The latter is not easily measured. If a young child allows food to contact hands, tabletops, or flooring, pesticides may transfer to the food and then be ingested by the child. A standard food item will create a uniform measure of excess food contamination from child handling with known transfer characteristics for target pesticides from multiple surfaces. Three food items (bologna, cheese, and fruit roll-up) were evaluated for transfer of pesticides from three types of pesticide-treated surfaces (hardwood flooring, vinyl flooring, and a plastic high chair tray). Seven pesticides (permethrin, cypermethrin, esfenvalerate, cyfluthrin, deltamethrin, malathion, and chlorpyrifos) were mixed in an aqueous solution and uniformly sprayed across the various surfaces. Each food item was placed on the three surfaces. All surfaces were wiped with isopropanol moistened gauze pads following the removal of the food items or after a similar time period for unchallenged surfaces. A GC/MS-based analytical method was established for the extraction and analysis of the foods and wipes. Transfer efficiencies were based on the amount of the pesticides in the foods as compared to the amount wiped off the surfaces. Transfer efficiencies of bologna, cheese, and fruit roll-ups ranged from less than 1% to 23% of the applied concentration for the three surfaces. Bologna transferred the organophosphate pesticides (5% - 22%) more efficiently than the pyrethroids (1% - 7%). Cheese showed consistent transfer efficiencies for the pesticides on all surfaces; pyrethroids at ~3% and the organophosphates at ~7%. Fruit roll-ups transferred all classes of pesticides with similar efficiency (3% - 7%), with the exception of the vinyl flooring where values were less than 1% for 4 of the 7 pesticides. Based on the transfer results, all three choices would be acceptable for use as a standard food item.

This study was funded by EPA contract 68D-99-012, Task 8. The EPA has not reviewed the results of this study. The use of trade names does not imply official endorsement.
PRESENTATION Sensitivity of Different Aeromonas Biotypes to Copper and Silver 05/18/2003
Rodgers, M. R. AND A. W. Smallwood. Sensitivity of Different Aeromonas Biotypes to Copper and Silver. Presented at American Society for Microbiology, Washington, DC, May 18-22, 2003.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.
PRESENTATION Development of a Molecular Method to Identify Astrovirus in Water. 06/04/2003
Grimm, A. C., J. L. Cashdollar, F. P. Williams, AND G. S. Fout. Development of a Molecular Method to Identify Astrovirus in Water. Presented at Science and Mission Club, Cincinnati, OH, June 4, 2003.
Abstract: Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus and we have developed a sensitive RT-PCR assay that is designed to detect all known astrovirus. When tested, this assay was able to detect strains from all eight serotypes. In addition, an internal control was developed so that it will be possible to determine if the sample being tested contains PCR inhibitors. Most probable number analysis determined that when amplified with the developed assay, a single DNA molecule of the internal control could be detected if inhibitors were not present. The assay was successfully adapted to real-time PCR and this method was then used for integrated cell culture RT-PCR detection of infectious virus. The methods were successfully used to detect astrovirus present in clinical samples and spiked water samples.
PRESENTATION Approaches to Inactivation and Federal Regulations 05/28/2003
Schaefer III, F. W. Approaches to Inactivation and Federal Regulations. Presented at Workshop on Transport and Disposal of Wastes from Facilities Contaminated with Chemical and Biological Agents, Cincinnati, OH, May 28-30, 2003.
Abstract: There is no abstract available for this product. If further information is requested, please refer to the bibliographic citation and contact the person listed under Contact field.
PRESENTATION Methods Development to Improve Low-Level Perchlorate Detection in Drinking Water By Conductivity and Mass Spectrometry Issues and Impact 06/17/2003
Hedrick, E. J. Methods Development to Improve Low-Level Perchlorate Detection in Drinking Water By Conductivity and Mass Spectrometry Issues and Impact. Presented at Severn Trent 4th annual Analytical Program Compliance, Louisville, KY, June 17-18, 2003.
Abstract: The goal of this research is to develop a USEPA method for the determination of sub-ppb concentrations of the perchlorate anion in ground and surface drinking waters. To date, ion chromatography using a KOH mobile phase, electrolytic conductivity suppression and electrospray ionization mass spectrometric detection has been explored. Perchlorate is a drinking water contaminant originating from the dissolution of the salts of ammonium, potassium, magnesium or sodium in water. It is used primarily as an oxidant in solid propellant for rockets, missiles, pyrotechnics, as a component in air bag inflators, and in highway safety flares. Based on EPA Information Request Responses and occurrence monitoring, there are 95 confrmed perchlorate releases in 25 states and 230 users or manufacturers in 40 states. From accidental releases and disposal, perchlorate has become a contaminant in surface and ground waters where it is highly mobile and, due to its chemical stability, persists for decades. The primary human health effect is inhibition of iodide uptake by the thyroid gland. By disrupting the thyroid hormone production, perchlorate interferes with metabolism and can affect brain development in fetuses and children, leading to mental impairment. The perchlorate anion has been found in numerous drinking water supplies across the United States. There is an urgent need to be able to confirm and quantify perchlorate at lower concentrations than the currently approved USEPA method allows which use ion chromatography with suppressed conductivity detection. In this work, sub-ppb quantitation of perchlorate in drinking waters and contaminated ground waters using ion chromatography with electrolytic conductivity suppression, electrospray ionization mass spectrometry (IC-ESI-MS) is demonstrated.
PRESENTATION New Laboratory Methods Dna Immunology 07/31/2003
Haugland, R. A. AND S. J. Vesper. New Laboratory Methods Dna Immunology. Presented at Expert Indoor Mycology Workshop, Asilomar, CA, July 31, 2003.
Abstract: Indoor fungi present potential, although as yet not fully defined, health risks to the occupants of heavily contaminated buildings due to their production of allergens, and a wide range of mycotoxins. A better understanding of the health risks posed by these organisms will require accurate, quantitative estimates of the occurrence of individual species or groups of species with common allergenic and/or biologically active compound production characteristics as well as the development of mechanistic biomarkers of human exposure. The same culture-based, microscopic and chemical methods have been used for decades with few improvements for identifying fungi and measuring their occurrence in environmental and clinical samples. Molecular technologies now offer the opportunity to provide more rapid, accurate and standardized measurements of fungi and fungal biomarkers. This presentation will review recent nucleic acid and immunology based methods that have been developed for the detection of indoor fungi with emphasis on real time PCR and ELISA methods, developed by the U.S. EPA, Office of Research and Development, for quantifying the occurrence of and human exposure to these organisms.
PRESENTATION The Application of Mass Spectrometry to the Study of Microorganisms 08/05/2003
Shoemaker, J. A. AND S. Glassmeyer. The Application of Mass Spectrometry to the Study of Microorganisms. Presented at U.S. EPA's Research on Micoorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
Abstract: The purpose of this research project is to use state-of-the-art mass spectrometric techniques, such as electrospray ionization (ESI) and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS), to provide "protein mass fingerprinting" and protein sequencing information for microorgansims listed on the 1998 Contaminant Candidate List (CCL) that cause waterborne disease. The responsibility of characterizing and investigating microorganisms has traditionally fallen to microbiologists, but recent advances in mass spectrometry have allowed analytical chemists to also enter the realm of microorganisms.Protein mass fingerprinting libraries will be developed and evaluated to determine whether MS techniques can identify protein fingerprints related to the infectivity/viability of selected microorganisms and whether they can differentiate between species and strains of selected microorganisms. Sequence information for proteins which are found to be specific or unique to species/strain and infectivity/viability can also be obtained with these MS techniques.
This global proteomic project has a number of subtasks for which preliminary results have been obtained on microorganisms such as coxsackievirus, Cryptosporidium parvum, and enterococci. Through the use of mass spectrometry, a potential viral biomarker of coxsackievirus has been identified which may indicate whether the virus is infectious. A unique mass spectral peak was observed in an infectious coxsackievirus, but was not observed in a non-infectious coxsackievirus. This unique peak may be responsible for viral infectivity, thus, be a potential biomarker.
In addition to viruses, initial experiments were performed to determine the ability of MALDI to analyze C. parvum both in an intact form, as well as oocysts that have been rendered nonviable. MALDI analysis was performed on several different harvests of the intact oocysts, as well as the separated cell walls and sporozoites that make up the oocysts. The analysis of the oocysts walls was inconclusive due to lack of discernable mass spectral peaks, but MALDI analysis of the sporozoites yielded reproducible mass spectra.
Whole enterococci cell protein profiles were evaluated using MALDI as a tool to identify seven different enterococci species. Many mass spectral peaks were shared among the different enterococci species, however, each species showed unique peaks, primarily in the 6,000 to 7,000 m/z region. When environmental isolates were tested, the signature peaks were observed in many of the different isolates, suggesting that these peaks could be used for species identification. Sequence analysis of the environmental isolates by 16S rDNA confirmed the identity of the strains tested, and matched the MALDI identity prediction in 75 % of the samples. The results from this study indicate that the analysis of whole enterococci cells by MALDI generate unique protein profiles which can be used for the rapid identification of fecal enterococci environmental isolates.
Although mass spectrometry currently is not sensitive enough to detect single cells in drinking water, the basic proteomic information obtained with these mass spectrometric techniques can be used to develop more sensitive and precise microbiological techniques that focus on these unique proteins in drinking water samples. These conventional microbiological methods can then be used to gather the occurrence data that will be used to create better EPA regulations for protecting humans from microbiological contaminants in U.S. drinking water supplies.
PRESENTATION Micobacterium Paratuberculosis and Nontuberculous Mycobacterial in Potable Water 08/05/2003
Pfaller, S. L. AND T. C. Covert. Micobacterium Paratuberculosis and Nontuberculous Mycobacterial in Potable Water. Presented at U.S. EPA's Research on Micoorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
Abstract: Nontuberculous mycobacteria (NTM) include Mycobacterium species that are not members of the Mycobacterium tuberculosis Complex. Members of the NTM group are important causes of disease in birds and mammals. Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium paratuberculosis are NTM and members of the Mycobacterium avium Complex (MAC). These organisms are found in a variety of environments including soil and water and are included on the Contaminant Candidate List (CCL). Earlier exploratory occurrence studies suggest that NTM have widespread occurrence in potable water throughout the U.S. M. paratuberculosis is the causative agent for Johne's disease in cattle. In addition to well - documented evidence of M. paratuberculosis as the causative agent of Johne's disease in cattle, there has been evidence linking M. paratuberculosis with Crohn's disease, a chronic inflammatory disease in of the intestinal tract in humans. Transmission of M. paratuberculosis via water contaminated with cattle feces may be one route of infection.
Current NTM research focuses on three areas: (1) Development of an improved cultural method for isolation of NTM in drinking water. (2) Development of a rapid PCR multiplex method for detection of MAC organisms in drinking water and (3) development of a molecular method for detection of M. paratuberculosis in water.

Improved Cultural Method

Goals/Objectives: Current methods for isolating NTM from environmental samples require harsh decontamination techniques to reduce the levels of background organisms often leading to loss of 50 -70% of the target NTM. The goal of this research is to develop improved selective method(s) which do not use classical decontamination procedures.

Approach: The use of antibiotics, dyes, detergents and other growth inhibitors are being examined for their ability to reduce background organisms and permit growth of NTM. A membrane filter method approach has been selected. Screening studies with spiked drinking water samples comparing candidate methods to classical decontamination techniques have been initiated. Candidate methods which permit better recovery of NTM and better reduction of background organisms will be tested with additional recovery studies and analyses of drinking water samples.

Preliminary Findings: Various antibiotics, dyes, detergents have been examined using a membrane filter cultural method approach. Thus far an oxidizer has shown promise for better recovery (80%) and reduction of background organisms than the standard accepted cultural method.

Significance: A improved cultural method would lead to better estimates of the occurrence of NTM, better estimates of the numbers of NTM in positive samples, and the possibility of recovering NTM unusually sensitive to decontaminating agents.

Next Steps: Future research will entail additional NTM recovery studies followed by comparison studies with the standard cultural approach and the improved method with distribution samples.

PCR Multiplex Method

Goals/Objectives: Current methods for detection of MAC organisms in drinking water typically take 3 - 8 weeks for completion of analyses with additional time for identification of the organisms. The goal of this research is to develop a rapid PCR multiplex method for detection of M. avium and M. intracellulare.

Approach: Drinking water samples (500 ml) are membrane filtered and the filters placed in modified 7H9 broth for seven day enrichment. After enrichment the cells are centrifuged and lysed to harvest the genomic DNA. The DNA is amplified (PCR) using primers specific for M. avium and M. intracellulare and all Mycobacteria. The PCR product is visualized by gel electrophoresis.

Preliminary Findings: Sixty samples (reservoir and drinking water) have been analyzed by the standard culture method and the multiplex PCR method. Nine samples were positive by both methods, seven were positive only by multiplex PCR and three were positive only by the cultural method.

Significance: The use of multiplex PCR significantly decreases the time for analyses for these organisms, and is able to detect MAC organisms not detected by the culture method.

Next Steps: Completion of detection limit studies and additional comparison studies with the standard culture technique using drinking water samples.

Method for detection of M. paratuberculosis

Goals/Objectives: A new project in our lab involves the development of a molecular detection and quantification method for M. paratuberculosis (MAP) in water. The method will be an important step in determining the significance of exposure to MAP in contaminated water, and may help to establish the link between contaminated water and Crohn's disease.

Approach: Current methods of detection, which include culture-based methods, are inadequate. A sixteen to twenty week incubation time is required to grow the organism, during which other microorganisms overgrow the medium. Harsh decontamination procedures used to reduce background organisms also kill a portion of MAP. This work proposes to develop a rapid molecular method to detect and quantify MAP in environmental samples by targeting a genetic molecule specific to MAP. One potential target is the MAP-specific insertion sequence IS900. The element is found only in MAP, and is present in fourteen to eighteen copies per cell. Other possible targets include seven recently discovered MAP-specific gene segments. A quantitative PCR-based method would significantly reduce detection times from approximately sixteen weeks to a few hours.
PRESENTATION Transport of Chemical and Microbial Contaminants from Known Wstewater Discharges: Potential Chemical Indicators of Human Fecal Contamination 08/05/2003
Glassmeyer, S., I. Ferrer, E. T. Furlong, J. D. Cahill, S. D. Zaugg, S. L. Werner, M. Meyer, D. W. Kolpin, AND D. D. Kryak. Transport of Chemical and Microbial Contaminants from Known Wstewater Discharges: Potential Chemical Indicators of Human Fecal Contamination. Presented at U.S. EPA's Research on Microorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
Abstract: The quality of drinking and recreational water is currently ascertained using indicator bacteria, such as Escherichia coli and fecal enterococci. However, the tests to analyze for these bacteria require 24 to 48 hours to complete, and do not discriminate between human and animal fecal material sources. One solution to these problems is to use chemicals that are commonly found in human wastewater as supplementary tracer compounds. The chemicals have the advantage of requiring shorter analysis times (3-4 hours), and a suite of human specific markers can be selected that are unique to human wastewater. For this project, compounds includes those that are produced and excreted by humans (e.g. coprostanol), that are consumed and pass easily through humans (e.g. pharmaceuticals and caffeine), and that are associated with humans and deposited into the combined graywater/ blackwater household septic waste stream (e.g. surfactants). At ten wastewater treatment facilities, a treated effluent sample, as well as surface water samples from upstream, and at two successive points downstream from the facility were collected. This longitudinal sampling scheme was used to determine the persistence of the target compounds in streams. Compounds that are quickly removed or degraded may not be persistent enough to serve as tracers; those that are too recalcitrant would similarly not be suitable as they would be present after the pathogens have been eliminated. To estimate the environmental persistence of pathogens, the water samples were analyzed for E. coli and fecal enterococci in addition to the suite of chemicals being measured. For chemical analysis, the water samples were extracted using either solid phase extraction (for the pharmaceuticals) or liquid-liquid extraction (for the other wastewater contaminants) and were analyzed using either high-performance liquid chromatography/mass spectrometry (HPLC/MS; pharmaceuticals) or gas chromatography/mass spectrometry (GC/MS; other wastewater contaminants). The concentration of microbial indicators was determined using modified mTEC (E. coli) or mEI (enterococci) media. Of the 114 chemical analytes investigated in this project, more than 80 were found in at least one sample. While most concentrations 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 the expected trend: they were either non-existent or at only trace levels in the upstream samples, had their maximum values in the wastewater effluent samples, and then declined in the two downstream samples. However, at most locations, there were indicator bacteria in the upstream samples, illustrating some of the difficulty in using bacteria to monitor water quality. This work indicates that these human wastewater constituents do have utility as tracers of human wastewater discharge. However, until the behavior of these chemical analytes is evaluated in a rigorous epidemiological study, their true potential as chemical indicators of human fecal contamination will not be determined. To begin this assessment, samples are currently being analyzed as part of the National Epidemiological and Environmental Assessment of Recreational Water Study, which should determine if there is a correlation between concentration of any of the chemicals and incidence of illness.
PRESENTATION Application of USEPA's Drinking Water Regulations Towards Rainwater Catchment Systems 08/21/2003
Lye, D. J. Application of USEPA's Drinking Water Regulations Towards Rainwater Catchment Systems. Presented at ARCSA Conference, Austin, TX, August 21-23, 2003.
Abstract: Rainwater harvesting is receiving increased attention worldwide as an alternative source of drinking water. Although federal agencies such as the USEPA acknowledge the existence of rainwater collection systems, the monitoring of this water source is still typically carried out by individual state or regional health agencies. States such as Texas, Ohio, and Hawaii are developing guidelines for the use and maintenance of these types of systems. It is most likely that this water source will eventually be regulated like other public drinking water sources according to the U.S. Safe Drinking Water Act of 1986. Whenever governmental agencies become involved in regulating systems, there are a number of challenges in complying with their sometimes complex regulations. Under present guidelines, collected rainwater will be characterized as a type of surface water. Existing EPA regulations for surface water sources will be discussed along with steps users can take to understand what will be needed to meet this type of regulatory activity.
This is an abstract of a proposed presentation and does not necessarily reflect the United States Environmental Protection Agency (EPA) policy. The actual presentation has not been peer reviewed by EPA. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
PRESENTATION The Application of Mass Spectrometry to Protein Analysis 07/22/2003
Shoemaker, J. A. The Application of Mass Spectrometry to Protein Analysis. Presented at NHEERL Presenation on Proteomics, Research Triangle Park, NC, July 22, 2003.
Abstract: The purpose of this presentation is to give our NHEERL collaborators a brief introduction to the use of mass spectrometric (MS) techniques in the analysis of proteins. The basic principles of electrospray ionization and matrix-assisted laser desorption ionization will be discussed, as well as the use of tandem mass spectormetry for de novo sequencing of novel proteins. This information will aid our collaborators in understanding the proteomic data we will be providing them on allergenic fungal proteins extracted from Metarhizium Anisopliae.
PRESENTATION Pathogenicity of Biofilm Bacteria 08/05/2003
Lye, D. J. Pathogenicity of Biofilm Bacteria. Presented at USEPA's Research on Micoorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
Abstract: There is a paucity of information concerning any link between the microorganisms commonly found in biofilms of drinking water systems and their impacts on human health. For bacteria, culture-based techniques detect only a limited number of the total microorganisms associated with biofilms. The possibility of unknown opportunistic pathogens occurring in potable water and biofilms within drinking water systems still exists but it is unlikely that pathogenic microorganisms will be found using individual in vivo culture-based techniques or by screening large numbers of isolates using the currently available in vitro virulence tests. A combination of molecular-based techniques and animal-exposure studies will provide the information necessary to fully characterize the pathogenicity of microorganisms commonly associated with biofilms.
This is an abstract of a proposed presentation and does not necessarily reflect the United States Environmental Protection Agency (EPA) policy. The actual presentation has not been peer reviewed by EPA. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.
PRESENTATION Proteomic Analysis of Allergens from Metarhizium Anisopliea 07/22/2003
Donohue, M. J. Proteomic Analysis of Allergens from Metarhizium Anisopliea. Presented at NHEERL Presentation on Proteomics, Research Triangle Park, NC, July 22, 2003.
Abstract: The goal of this project is the identification and characterization of allergens from the fungus M. Anisopliae, using mass spectrometry (MS). The US EPA, under the "Children at Risk" program, is currently addressing the problem of indoor fungal bioaerosol contamination. One of the research objectives is to develop a basic understanding of IgE inducing proteins from fungi, using advanced proteomic. The Fungus M. Anisopliae has been used as a bio-pesticide for insect control sijnce the 1800's . Recent studies have shown that exposure to this micro-organism can cause an immediate hyper-immunosensitivity or Type I allergenic rsponse. Matrix assisted laser desorption ionization time-of=flight mass spectrometry (MALDI-TOF-MS) and electrospray ionization mass spectrometry (ESI-MS) were used to analyze the fungal peptides and proteins isolated by 2-D gel electrophoresis and immuno-blot analysis. The MS data on molecular weight, peptide profiles, and amino acid sequence or domain homology to known allergens. This information could lead to the identification of the M. Anisopliae proteins that induce IgE responses, eventually paving the way to prevention or treatment of allergies.
PRESENTATION Development of a Molecular Method to Identify Astrovirus in Water 08/05/2003
Grimm, A. C., J. L. Cashdollar, F. P. Williams, AND G. S. Fout. Development of a Molecular Method to Identify Astrovirus in Water. Presented at USEPA Microorganisms in Drinking Water Workshop, Cincinnati, OH, August 5-7, 2003.
Abstract: Astrovirus is a common cause of gastroenteritis that has been determined to be responsible for several outbreaks. Since astrovirus can be waterborne, there is interest in testing environmental water for astrovirus. We have developed a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay that is designed to detect all known astrovirus strains.
The assay was based on a primer set that contained multiple upper and lower primers as well as multiple probes. This would allow for amplification of all of the known strains of astrovirus using a single reaction. When tested, this assay was able to detect strains from all eight serotypes. In addition, an internal control was developed so that it will be possible to determine if the sample being tested contains PCR inhibitors. Most probable number analysis determined that when amplified with the developed assay, a single DNA molecule of the internal control could be detected if inhibitors were not present. The assay was successfully adapted to real-time PCR and this method was then used for integrated cell culture/RT-PCR detection of infectious virus. The methods were successfully used to detect astrovirus present in clinical samples and spiked water samples.

A simple, sensitive method for detecting all known astrovirus strains has been developed that can be used to detect this virus in water. This assay will be field tested by analyzing environmental water samples.
PRESENTATION Methods Used to Analyze a Norovirus Outbreak 08/05/2003
Cashdollar, J. L., S. Parshionikar, C. Newport, S. WillianTrue, D. R. Dahling, AND G. S. Fout. Methods Used to Analyze a Norovirus Outbreak. Presented at USEPA's Research on Micoorganisms in Drinking Water, Cincinnati, OH, August 5-7, 2003.
Abstract: Project Goals and Objectives:To isolate and identify the viral agents in well water samples associated with two outbreaks of acute gastroenteritis reported to the Wyoming Department of Health in February 2001 and October 2001. To isolate and identify the viral agents in patient stool samples and to determine the link between water consumption and illness.

Approach:
The project had a three way approach:
An epidemiological investigation was performed to identify any common routes of exposure among those afflicted with gastroenteritis.
An environmental survey was done of the premises involved in each outbreak to determine possible sources of contamination.
Laboratory analysis was performed on well water samples for coliform and viral detection using RT-PCR and DNA sequencing. Stool samples were also analyzed for the presence of noroviruses.

Preliminary Findings:
Epidemiological studies revealed a close association between water consumption and illness. Environmental surveys in both outbreaks determined that the water supply was vulnerable to fecal contamination. Well water samples in both cases were positive for coliforms, and RT-PCR and DNA sequencing revealed noroviruses as the causative agents of acute gastroenteritis.

Significance of Findings:
This investigation demonstrates that EPA's viral concentration and molecular methods in conjunction with epidemiological and environmental analysis are very useful in outbreak studies.

Next Steps:
The methods used in this study can be performed in most laboratories with trained personnel and appropriate equipment, which would allow for routine monitoring of enteric viruses in drinking water, thus preventing any future outbreaks from occurring.
PRESENTATION Method to Classify Environmental Samples Based on Mold Analyses By Qpcr 09/10/2003
Haugland, R. A., T. Meklin, M. Varma, L. J. Wymer, D. G. Dearborn, I. Yike, AND S. J. Vesper. Method to Classify Environmental Samples Based on Mold Analyses By Qpcr. Presented at Bioaerosols Meeting, Saratoga, NY, September 10-12, 2003.
Abstract: A total of 82 quantitative PCR (QPCR) assays were used to identify and quantify different indoor molds in dust samples from the homes of six infants suffering from pulmonary hemorrhage and 26 reference homes in Cleveland, Ohio. No significant difference was seen in the total cell quantities (presumed to be primarily spores and conidia) of these molds in the reference home (RH) and pulmonary hemorrhage home (PHH) samples. The assay groups were categorized based on whether the ratio of measured organisms in the PHH and RH samples were <2 or > 2. Twenty-two groups were identified in the >2 category. The combined cell quantities of these groups were significantly higher in the PHH samples than in the RH samples (p=0.001) and comprised 4% of all detected organisms in the PHH samples.
PRESENTATION Measurement of Perchlorate in Water Using An Oxygen-18 Enriched Isotope Standard and Ion Chromatography Mass Spectrometric Detection 09/21/2003
Hedrick, E. J. AND D. J. Munch. Measurement of Perchlorate in Water Using An Oxygen-18 Enriched Isotope Standard and Ion Chromatography Mass Spectrometric Detection. Presented at International Ion Chromatography Symposium, San Diego, CA, September 21-24, 2003.
Abstract: Perchlorate (ClO4 -) is a drinking water contaminant originating from the dissolution of the salts of ammonium, potassium, magnesium, or sodium in water. It is used primarily as an oxidant in solid propellant for rockets, missiles, pyrotechnics, as a component in air bag inflators, and in highway safety flares. Based on EPA Information Request Responses and occurrence monitoring, there have been 95 confirmed ClO4 - releases in 25 states and 230 users or manufacturers in 40 states. From accidental releases and improper disposal practices of the past, ClO4 - has become a contaminant in surface and ground waters where it is highly mobile and, due to its chemical stability, persists for decades. The primary human health effect is inhibition of iodide uptake by the thyroid gland. By disrupting thyroid hormone production, ClO4 - interferes with metabolism and can affect brain development in fetuses and children, leading to mental impairment. There is now a need for a method that can confirm and quantify ClO4 - at concentrations lower than what is achievable using ion chromatography with suppressed conductivity detection. Coupled with ion chromatographic separation, mass spectrometry is by far the most promising analytical tool available today for low-level identification and quantitation of ClO4 - in drinking water. In this work, sub-ppb quantitation of ClO4 - in drinking waters using ion chromatography electrospray ionization mass spectrometry (IC-ESI-MS) is demonstrated.
The primary mass of interest for ClO4 - is 99 based on the 75.77% relative abundance of the chlorine-35 isotope. Mass 101 is a secondary mass of interest based on the 24.23% abundance of chlorine-37. In this work we demonstrate the feasibility of using oxygen-18 enriched perchlorate as an internal standard. The greatest benefit of such an internal standard is for the improvement of accuracy in the determination of perchlorate in waters with high total dissolved solids such as sulfate, carbonate and chloride. In the event that ClO4 - is regulated in drinking water or that a second national occurrence survey is conducted, this research will lead to an inherently more specific and sensitive U.S. EPA method than is currently available.
PRESENTATION Methods for Emerging Contaminant Groups: Explosives and Nitrosamines 11/02/2003
Munch, J. W. AND M. V. Bassett. Methods for Emerging Contaminant Groups: Explosives and Nitrosamines. Presented at American Water Works Association Water Quality Technology Conference, Philadelphia, PA, November 2-6, 2003.
Abstract: BackgroundIn support of requirements in the Safe Drinking Water Act, the National Exposure Research Laboratory (NERL), has been developing analytical methods for chemicals on the 1998 contaminant candidate list (CCL) and for other chemicals of emerging interest. The purpose behind this effort is to provide analytical testing methods that can be used by the United States Environmental Protection Agency (USEPA) in unregulated contaminant monitoring (UCM) to gather nationwide occurrence data of these chemicals in drinking water. This occurrence data will support the Agency=s decision making process regarding future regulation of these chemicals in drinking water. Ideally, these methods will be specific, accurate, sensitive, and suitable for compliance monitoring if the chemical is regulated in the future. Specificity is of special concern when collecting UCM data, because with non-specific methods, there is the potential of regulating a chemical based on false positive occurrence data. Because many of these chemicals are not easy to measure at low concentrations in water matrices, method development has been an on-going challenge. Adding to the challenge is the fact that often the health effects information available for a chemical at the time of the method development are insufficient to provide a target detection limit. Two chemical groups of interest to EPA that fall into the category of emerging contaminants are explosives and nitrosamines. A new analytical method for explosives and related compounds, USEPA Method 529, was completed in 2002 (1). A method for nitrosamines is expected to be completed early in 2004.

Explosives
The interest in developing a method for explosives began with the inclusion of RDX on the 1998 CCL. Hexahydro-1,3,5-trinitro-1,3,5-triazine is commonly known as RDX (Royal Demolition eXplosive). It is for the most part a military explosive, although it has a few commercial uses in demolition and in fireworks. At its peak production, the average volume of RDX produced in the U.S. was 180 million pounds per year (1969-1971) (2). RDX enters the environment from military installations where it is used and stored. Current groundwater contamination is attributed to historical disposal practices, which included open burning (3).


A survey of available information on analytical methods for, and occurrence of, RDX indicated that there was a specific grouping of military explosives and related chemicals that were typically analyzed for, and often found together in the environment. Existing methods were based on high performance liquid chromatography with ultraviolet detection (HPLC/UV) or gas chromatography with electron capture detection (GC/ECD). These methods were not selected for UCM because of their potential for false positives. However, the analyte list from existing methods was selected as the initial analyte list for a new gas chromatography/mass spectrometry (GC/MS) method (Table1). The objective for the new method development was to include as many of the analytes in Table 1 as possible. The procedure would use solid phase extraction (SPE) to concentrate the samples, followed by GC/MS detection. This process would provide a large concentration factor, minimal solvent usage, and excellent selectivity. The initial target reporting limit for RDX was 2 Fg/L or less based on EPA=s current Lifetime Health Advisory. Subsequent health information developed by USEPA indicated a theoretical 10-6 lifetime risk level of cancer from exposures at 0.3 Fg/L (4). A summary of the final method procedure is as follows:

1. One-liter samples are collected in amber glass bottles. Samples are preserved with Trizma pH 7.0 buffer and copper sulfate.
2. Samples are extracted by passing the 1-L sample through either a Waters RDX SPE cartridge or Varian RPS SPE disk.
3. After a short drying period, the SPE media is eluted with a small volume of ethyl acetate. Extracts are dried, and then evaporated to 1 mL with a stream of nitrogen gas.
4. Concentrated extracts are analyzed by GC/MS using a programmed temperature vaporizing (PTV) injector. The use of a PTV injector is required to minimize thermal decomposition of analytes in the GC injection port.
5. Quantitate the sample concentration by comparison to a calibration curve prepared from calibration standards in the range of 0.1-10 Fg/mL.

Data collected in full scan GC/MS mode resulted in a calculated method detection limit (MDL) for RDX of 0.082 Fg/L (SPE disk) or 0.12 Fg/L (SPE cartridge). MDLs for the other method analytes ranged from 0.021- 0.18 Fg/L depending on the analyte and the type of SPE used. However, there was no apparent difference in the precision or accuracy of the data between the two SPE types. Replicate sample analysis (N=5 for each SPE type) of reagent water fortified with method analytes at a concentration of 5 Fg/L resulted in mean recoveries of 87-109%, with an RSD for each analyte of less than 12%. Replicate sample analysis (N=8 for SPE cartridge, N=7 for SPE disk) of reagent water fortified with method analytes at concentrations of 0.1-1.0 Fg/L resulted in mean recoveries of 71-134%, with an RSD of less than 8% for 12 of the 14 analytes. Nitrobenzene and 2-nitrotoluene had RSDs of 15-18% respectively, with either cartridge or disk extraction, probably due to their volatility and possible loss during extract evaporation. Full scan data were obtained using a Varian Saturn 4D GC/MS with an Agilent 15m H 0.25 mm i.d. DB-5ms GC column, with a 0.25 Fm film.


An option to use selected ion monitoring (SIM) was included in the method to obtain a lower detection limit. Three ions in the proper relative abundance ratio were used for identification, and one ion was used for quantitation. Data collected in the SIM GC/MS mode resulted in a calculated method detection limit (MDL) for RDX of 0.006 Fg/L (SPE disk) or 0.010 Fg/L (SPE cartridge). MDLs for other method analytes ranged from 0.004 to 0.090 Fg/L depending on the analyte and the type of SPE used. Again, there was no apparent difference in the precision or accuracy of the data between the two SPE types. Replicate sample analysis (N=4 for each SPE type) of reagent water fortified with method analytes at a concentration of 1 Fg/L resulted in mean recoveries of 87-122%, with an RSD for each analyte of less than 9%. Replicate sample analysis (N=8 for each SPE type) of reagent water fortified with method analytes at concentrations of 0.05 to 0.25 Fg/L resulted in mean recoveries of 80-134%, with an RSD for each analyte of less than 10%. SIM data were obtained using a Shimadzu QP5050A GC/MS with an Agilent 15 m H 0.25 mm i.d. DB-5ms GC column with a 0.25 Fm film thickness.

Nitrosamines
The interest in developing a method for nitrosamines began with the information that N-nitrosodimethylamine (NDMA) is a likely disinfection by-product. This is of concern to USEPA because NDMA is an extremely potent carcinogen, with a theoretical 10-6 lifetime cancer risk level at exposures of 0.7 ng/L (5,6). Although NDMA is not on the 1998 CCL, the decision was made to start a method development effort because of its emerging importance as a drinking water contaminant. In addition to NDMA, it was decided to try to include seven additional nitrosamines that are on USEPA=s Resource Conservation and Recovery Act (RCRA) Groundwater Monitoring List to the method (Table 2). Other sources of NDMA and other nitrosamines in the environment are: production of rocket fuel, rubber manufacture and tanneries.

Analytical challenges associated with developing a method for NDMA centered around two issues. The first issue is that NDMA is miscible with water in all proportions, and therefore very difficult to extract from water efficiently. The approach to solving that issue was to investigate the use of carbon as an SPE sorbent. The second issue is the need for an extremely low detection limit combined with the need for very specific detection. In light of the fact that NDMA and other nitrosamines are very insensitive to conventional electron ionization MS, a less conventional approach was needed. The initial approach to detection was chemical ionization (CI) GC/MS using methanol as the CI reagent. As the method development progressed it became clear that the molecular ion for NDMA (m/z 75) may not be specific enough to preclude false positives. At this point, tandem mass spectrometry (MS/MS) was investigated as a possible option. Although method development is still in progress, a summary of the preliminary extraction and detection procedures is as follows:

1. Collect a 0.5-L water sample in an amber glass bottle. Dechlorinate with sodium thiosulfate.
2. Extract a 0.5-L water sample by passing it through an SPE column (6-mL volume) containing 2 g of coconut charcoal (80-120 mesh).
3. After a short drying period, elute the SPE column with a small volume of methylene chloride.
4. Dry the extract and concentrate it to 1 mL with a stream of nitrogen gas.
5. Analyze the extract by CI GC/MS or GC/MS/MS using methanol as the CI reagent. For the data presented here, an 8-FL injection was made using a PTV injector.
6. Quantitate the sample concentration by comparison to a calibration curve prepared from calibration standards in the range of 0.5 to 200 ng/mL.

Data obtained in the GC/MS mode from extracts of reagent water fortified with NDMA and seven additional nitrosamines at a concentration of 20 ng/L (N=3) showed 94% recovery of NDMA with an RSD of 3%. Results for six of the other nitrosamines in Table 2, showed 87-106% recovery with RSDs less than 4%. N-nitrosomorpholine could not be determined in this data set due to background contamination. Linear calibration was achieved over the concentration range representing sample concentrations of 1 to 400 ng/L (r 2 = 0.999 or 1.000 for all analytes). Preliminary data indicate that this procedure will produce a detection limit for NDMA near 1ng/L, with similar sensitivity for the other nitrosamines. Extracts were analyzed on a Varian Saturn 4D GC/MS/MS, using a Restek 30 m H 0.25 mm Rtx-5Sil MS GC column with a 1.0 Fm film thickness. No data are presented for GC/MS/MS because MS/MS parameters are still being optimized.

Future work on the nitrosamine method will include sample and extract preservation and holding time studies, a study of potential matrix effects, further optimization of MS/MS parameters, and selection of an internal standard and surrogate analyte.

Table 1. Method Analytes for USEPA Method 529

ANALYTE
CAS NUMBER

2-amino-4,6-dinitrotoluene
35572-78-2

4-amino-2,6-dinitrotoluene
1946-51-0

3,5-dinitroanaline
618-87-1

1,3-dinitrobenzene
99-65-0

2,4-dinitrotoluene
121-14-2

2,6-dinitrotoluene
606-20-2

hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)
121-82-4

nitrobenzene
98-95-3

2-nitrotoluene
88-72-2

3-nitrotoluene
99-08-1

4-nitrotoluene
99-99-0

1,3,5-trinitrobenzene
99-35-4

2,4,6-trinitrophenylmethylnitramine (Tetryl)
479-45-8

2,4,6-trinitrotoluene (TNT)
118-96-7


Table 2. Proposed Method Analytes for Nitrosamine Method

ANALYTE
CAS NUMBER

N-Nitrosodimethylamine
62-75-9

N-Nitrosomethylethylamine
10595-95-6

N-Nitrosodiethylamine
55-18-5

N-nitrosodi-n-propylamine
621-64-7

N-Nitrosodi-n-butylamine
924-16-3

N-nitrosomorpholine
59-89-2

N-nitrosopyrrollidine
930-55-2

N-Nitrosopiperdine
100-75-4
PRESENTATION Acetanilide Herbicide Degradation Products By LC/MS 11/02/2003
Shoemaker, J. A. Acetanilide Herbicide Degradation Products By LC/MS. Presented at American Water Works Association Water Quality Technology Conference, Philadelphia, PA, November 2-6, 2003.
Abstract: Acetanilide herbicides are frequently applied in the U.S. on crops (corn, soybeans, popcorn, etc.) to control broadleaf and annual weeds. The acetanilide and acetamide herbicides currently registered for use in the U.S. are alachlor, acetochlor, metolachlor, propachlor, flufenacet and dimethenamid. Acetanilide degradation products are generally more water soluble and mobile than the parent herbicide, thus there is greater potential for these degradates to be found in ground waters and surface waters. The most common acetanilide degradation products are the ethanesulfonic acid (ESA) and oxanilic acid (OA) derivatives of the parent herbicides. The ESA and OA degradates of alachlor, metolachlor, and acetochlor have been reported in U.S. Midwestern surface and ground waters at typical concentrations of 0.1- 20 �g/L [1-5].
High priority has been given to this research project in EPA's Office of Research and Development because acetanilide degradation products are identified on the 1998 Drinking Water Contaminant Candidate List (CCL) [6]. Specifically, analytical methodology is needed to gather occurrence data on the acetanilide degradation products. While several methods have been reported in the literature [3,7], these methods do not address issues specific to analyzing compounds in drinking water, such as preservatives and internal and surrogate standards. In addition, the reported methods do not contain all 12 target analytes (the ESA and OA of the six U.S. registered acetanilide/acetamide herbicides). The objective of this research is to develop an accurate and precise analytical method to detect and quantitate the 12 ESA and OA acetanilide pesticide degradates in drinking water matrices. This will include two steps: 1) evaluating the capability of solid phase extraction (SPE) techniques to concentrate the acetanilide ESA and OA degradates from drinking water, and 2) evaluating the capability of liquid chromatography/mass spectrometry (LC/MS) techniques to separate and detect ESA and OA acetanilide herbicide degradates in the concentrated sample extracts. Future occurrence data gathered with this developed method can then be used in determining whether to study the health effects of the acetanilide herbicide degradation products, and ultimately whether to regulate these compounds or remove them from the CCL.

Approach

LC/MS: Since the EPA methodology developed will be used to determine the occurrence of acetanilide degradates in drinking water, positive confirmation by LC/MS with negative ion electrospray ionization (ESI) is desired. However, two structural isomer pairs, alachlor ESA/alachlor ESA and alachlor OA/acetochlor OA present a difficult challenge. Not only are alachlor ESA and acetochlor ESA not separated on conventional LC columns, they also have the same molecular weight. LC/MS typically only provides a protonated molecule [M+H]+ or deprotonated molecule [M-H]-; therefore, alachlor ESA and acetochlor ESA would be indistinguishable by LC/MS. Alachlor OA and acetochlor OA would also be indistinguishable by LC/MS (without fragmentation) for the same reason. In-source CAD (collisionally activated dissociation) on LC/MS instruments or tandem mass spectrometry (MS/MS) has been used to produce unique product ions for the ESA and OA degradates of alachlor and acetochlor [7,8]. However, the in-source CAD and MS/MS fragment ions unique to acetochlor ESA and alachlor ESA are not very abundant, thus compromising sensitivity. To overcome this problem, chromatographic approaches, such as mobile phase modifiers and column heating, were investigated to achieve separation of the structural isomers.

SPE: Another challenge in methods development is the ability to concentrate the target analytes from water using a suitable SPE sorbent. The goal is to find an SPE procedure that, combined with LC/MS analysis, will produce a method that will meet our data quality objectives of 70-130% mean recovery (% of true value) and <30% relative standard deviation (RSD). Polystyrenedivinylbenzene or C18 SPE sorbents are most commonly used to extract the ESA and OA degradates of acetochlor, alachlor, metolachlor and dimethenamid ESA from water [2,7,9]. However, there were no literature reports indicating recoveries of propachlor or flufenacet degradates. Thus, experiments were conducted on all 12 target analytes to determine recoveries on C18 SPE cartridges using a procedure similar to Thurman [2], as well as on other types of SPE sorbents, such as C18OH and carbon.

Internal and Surrogate Standards: Internal standards (IS) and surrogates (SURR) are an important part of MS methods development research in order to achieve the highest quality data. Ideally, the IS and SURR should be similar in properties and structure to the target analytes and perform similarly in the extraction and detection as the target analytes. Unfortunately, obtaining an IS with these similarities is not always achievable. A number of acids were evaluated for use as potential internal standards and surrogates, but many were not chromatographically suitable or did not mimic the target analytes in the extraction process.

Preservatives: EPA drinking water regulatory methods typically use preservatives to prevent microbial degradation (e.g., acid, copper(II) sulfate, DZU, trinitro) and to dechlorinate (e.g., sodium sulfite, trizma) the sample. Microbial degradation of the target analytes cannot be predicted in all types of matrices containing various types of microbiological contaminants, thus an anti-microbial agent is desirable. While chlorine may not adversely affect the stability of acetanilide degradates, it can interfere in the solid phase extraction, thus the residual chlorine should be removed. A number of preservative combinations were investigated based on research conducted by Winslow and colleagues [10] (e.g., copper(II) sulfate/trizma, DZU/trizma, trisnitro/trizma, hydrochloric acid (pH=2)/sodium sulfite).

Results

The methods development for these acetanilide ESA and OA degradates is nearing completion. Chromatographic separation of alachlor ESA and acetachlor ESA has been achieved using a 5 mM ammonium acetate/methanol gradient and heating the analytical column to 65�5�C [11]. SPE extraction of the target analytes from drinking water using carbon sorbents produced typical recoveries of 85-115% and relative standard deviations <12%. Studies were conducted to determine the effect of water quality parameters, such as humic/fulvic material and hardness, on the recovery of these analytes. Humic/fulvic material, measured as total organic carbon (TOC), is retained on the carbon sorbent causing low recoveries (<70%) for propachlor OA at TOC levels above 5 mg/L. Many of the preservative combinations tested interfered in the extraction process. Ammonium chloride is currently being evaluated as a potential dechlorination agent, but no anti-microbial agent, which does not affect the analyte recoveries, has been found. Holding time studies will be performed to ensure the target analytes will survive typical shipping conditions and times with the chosen preservative. Currently, 4-phenoxybenzoic acid (PBA) is the internal standard and butachlor ESA and dimethachlor ESA are the surrogate standards. The internal calibration using PBA is linear with r2>0.995 for all target analytes. Butachlor and dimethachlor are not registered for use in the U.S., thus the potential for environmental contamination of their ESA degradates is minimal. Finally, the minimum reporting level (MRL) will be determined and is expected to be = 0.1 �g/L.

Disclaimer: This work has been conducted by the United States Environmental Protection Agency. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products does not constitute endorsement of recommendation for use.
PRESENTATION The Chemistry of Arsine Oxides Related to the Synthesis of Arsenosugars 12/03/2003
Fricke, M., H. Sun, N. Gorman, J. T. Creed, J. Thayer, J. A. Caruso, AND W. R. Cullen. The Chemistry of Arsine Oxides Related to the Synthesis of Arsenosugars. Presented at International Conference on Environmental and Biological Aspects of Main-Group Organomettalics, Pau, France, December 3-5, 2003.
Abstract: Ongoing toxicokinetic and biogenesis investigations require gram quantities of the naturally occurring dimethylarsinoylribofuranosides. The principal synthetic route to these compounds involves the hydrogen peroxide oxidation of the parent arsine in ether. This reaction is hazardous; at least one laboratory explosion has occurred involving these conditions. We wish to report a new route to arsine oxides involving the activation of dimethylarsinic (cacodylic) acid by reaction with carbonyldiimidazole. The resulting dimethylarsinoylimidazolide reacts with Grignard or organolithium reagent to produce arsine oxides. This route approaches arsine oxides through a reduction and avoids the dangerous oxidation reaction. Mass spectroscopic evidence for a mechanism of this reaction will be discussed.
The 19th century Meyer reaction has been examined as a one-step alternate procedure for attaching the dimethylarsinoyl group to sugar substrates. Yields and methods for the Meyer synthesis of trimethylarsine oxide will be reported.
The final point of discussion will be the esterification of the arsine oxide group with pinacol to yield 5-coordinate dioxarsolanes. Arsine oxide protection in this manner permits the chemically orthogonal derivatization of side chains in these molecules. This strategy has considerable potential for preparing a variety of unavailable complex arsenosugars. Protection and deprotection chemistry will be described for model arsine oxides.

This work has been conducted by the United States Environmental Protection Agency. It has been subjected to Agency review and approved for publication. Mention of trade names or commercial products do not constitute endorsement or recommendation for use.
PRESENTATION National Epidemiological and Environmental Assessment of Recreational Water Study 10/21/2003
Dufour, A. P., R. L. Calderon, M. Beach, T. J. Wade, AND E. A. Sams. National Epidemiological and Environmental Assessment of Recreational Water Study. Presented at The Annual Meeting of the Great Lakes Beach Association, Muskegon, MI, October 21-22, 2003.
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, "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.
The National Epidemiologic and Environmental Assessment of Recreational (NEEAR) Water Study is a 5-year research project that will document human health effects associated with recreational water use. Data collected from this study will be useful in identifying new water quality indicators and rapid methods for measuring water quality.

The NEEAR Water Study is a prospective cohort study that will include 9-11 marine and freshwater beaches in the nation. Approximately 5000-8000 persons from each beach will be surveyed to determine swimming exposure and risk factors for illness. Follow-up interviews at one and then two weeks 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 quantitative PCR and optical fiber/fluoroimmunoassay technology. The latter two tests 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. Water quality guidelines will be developed from the data showing the best relationship between swimmer health effects and water quality measurements.
PRESENTATION Determination of Perchlorate By Ion Chromatography, Suppressed Conductivity and Mass Spectrometric Detection Using An Oxygen-18 Enriched Isotropic Internal Standard 10/22/2003
Hedrick, E. J. AND D. J. Munch. Determination of Perchlorate By Ion Chromatography, Suppressed Conductivity and Mass Spectrometric Detection Using An Oxygen-18 Enriched Isotropic Internal Standard. Presented at DoD Environmental Data Quality Workgroup/Intergovernmental Data Quality Task Force Perchlorate Testing Roundtable, Dallas, TX, October 22-23, 2003.
Abstract: Perchlorate (ClO4 -) is a drinking water contaminant originating from the dissolution of the salts of ammonium, potassium, magnesium, or sodium in water. It is used primarily as an oxidant in solid propellant for rockets, missiles, pyrotechnics, as a component in air bag inflators, and in highway safety flares. Based on EPA Information Request Responses and occurrence monitoring, there have been 95 confirmed ClO4 - releases in 25 states and 230 users or manufacturers in 40 states. From accidental releases and improper disposal practices of the past, ClO4 - has become a contaminant in surface and ground waters where it is highly mobile and, due to its chemical stability, persists for decades. The primary human health effect is inhibition of iodide uptake by the thyroid gland. By disrupting thyroid hormone production, ClO4 - interferes with metabolism and can affect brain development in fetuses and children, leading to mental impairment. There is now a need for a method that can confirm and quantify ClO4 - at concentrations lower than what is achievable using ion chromatography with suppressed conductivity detection. Coupled with ion chromatographic separation, mass spectrometry is by far the most promising analytical tool available today for low-level identification and quantitation of ClO4 - in drinking water. In this work, sub-ppb quantitation of ClO4 - in drinking waters using ion chromatography electrospray ionization mass spectrometry (IC-ESI-MS) is demonstrated.
The primary mass of interest for ClO4 - is 99 based on the 75.77% relative abundance of the chlorine-35 isotope. Mass 101 is a secondary mass of interest based on the 24.23% abundance of chlorine-37. In this work we demonstrate the feasibility of using oxygen-18 enriched perchlorate as an internal standard. The greatest benefit of such an internal standard is for the improvement of accuracy in the determination of perchlorate in waters with high total dissolved solids such as sulfate, carbonate and chloride. In the event that ClO4 - is regulated in drinking water or that a second national occurrence survey is conducted, this research will lead to an inherently more specific and sensitive U.S. EPA method than is currently available.
PRESENTATION Quality Controls for Pcr 11/02/2003
Fout, G. S. Quality Controls for Pcr. Presented at Water Quality Technology Conference, Philadelphia, PA, November 2-5, 2003.
Abstract: The purpose of this presentation is to present an overview of the quality control (QC) sections of a draft EPA document entitled, "Quality Assurance/Quality Control Guidance for Laboratories Performing PCR Analyses on Environmental Samples." This document has been prepared by the Office of Water and the Office of Research and Development to serve as guidance for environmental sample analyses involving PCR techniques, for incorporation into new EPA PCR-based methods, for use with EPA grants and assistance agreements involving PCR methods and as guidance to evaluate PCR data in technical papers. This presentation will describe when and how to use four positive controls and two negative controls with PCR methods and will describe the proper controls for assays used to confirm PCR results. The four positive controls consist of a "PCR Positive Control," which is used to verify that reagents have been prepared properly; the "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; the "Method Positive Control," which is used to demonstrate that the entire method (sampling, concentration, inhibitor removal, PCR, confirmation) is working; and the "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 corrective actions will be described.

 

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