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Microbiological & Chemical Exposure Assessment Research Division Publications: 2001

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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 2001, organized by Publication Type. Your search has returned 66 Matching Entries.

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

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

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Presented/Published
BOOK Analytical Mass Spectrometry; Strategies for Environmental and Related Applications 05/01/2001
Budde, W L. Analytical Mass Spectrometry; Strategies for Environmental and Related Applications. American Chemical Society, Washington, DC, (2001).
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.

BOOK CHAPTER Detection of Protozoan Parasites in Source and Finished Water 11/01/2001
Schaefer III, F W. Detection of Protozoan Parasites in Source and Finished Water. , 2nd., Chapter 19, Manual of Environmental Microbiology. American Society for Microbiology, Washington, DC, 220-233, (2001).
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.

JOURNAL The Development of Iodine Based Impinger Solutions for the Efficient Capture of Hg Using Direct Injection Nebulization Inductively Coupled Plasma Mass Spectrometry Analysis 09/15/2001
Hedrick, E J., T. G. Lee, P. Biswas, AND Y. Zhuang. The Development of Iodine Based Impinger Solutions for the Efficient Capture of Hg Using Direct Injection Nebulization Inductively Coupled Plasma Mass Spectrometry Analysis. ENVIRONMENTAL SCIENCE & TECHNOLOGY 35(18):3764-3773, (2001).
Abstract: Inductively coupled plasma mass spectrometry (ICP/MS) with direct injection nebulization (DIN) was used to evaluate novel impinger solution compositions capable of capturing elemental mercury (Hgo) in EPA Method 5 type sampling. An iodine based impinger solutoin proved to be very efficient for Hgo capture and was amenable to direct analysis by DIN-ICP/MS. Hgo capture efficiency using aqueous iodine (I3-) was comparable to Hgo capture using acidified potassium permanganate impinger solutions which were analyzed by cold vapor atomic absorption (CVAA), with greater than 98% capture of Hgo in the first oxidizing impinger. Using DIN-ICP/MS, it was demonstrated for the first time that iodine can be generated just prior to impinger sampling for efficiently oxidizing Hgo and retaining it in solution as HgI42-. Due to the increased interest Hg speciation from combustion sources, and the potential for using DIN-ICP/MS for mulitple metals analyses, an impinger sampling train for gaseous Hg speciation and multiple metals analyses using DIN-ICP/MS analyses is presented. The unique feature of such a sampling train is that each impinger solution in the series is amenable to direct analysis by DIN-ICP/MS. A bituminous coal was combusted in a bench scale coal system and gaseous Hg species (oxidized and elemental) were determined using the proposed impinger train. The DIN-ICP/MS instrumental detection limit was 0.003 ppb and MDLs ranged from 0.007 - 0.116 ug/L (ppb) in a variety of impinger solutions used for Hg capture.

JOURNAL Hplc Determination of Cyanuric Acid in Swimming Pool Waters Using Phenyl and Confirmatory Porous Graphitic Carbon Columns 07/15/2001
Cantu, R, O M. Evans, L J. Wymer, A P. Dufour, AND F K. Kawahara. Hplc Determination of Cyanuric Acid in Swimming Pool Waters Using Phenyl and Confirmatory Porous Graphitic Carbon Columns. Analytical Chemistry 73(14):3358-3364, (2001).
Abstract: The chlorinated salts of cyanuric acid have found an important role in recreational swimming pool waters across the United States. Upon application to pool water, they can (1) release disinfectant chlorine or (2) stabilize the free available chlorine by acting as chlorine reservoirs in the form of cyanuric acid, preventing the photolytic destruction of residual chlorine by sunlight. Recommended levels of the cyanuric acid stabilizer are in the 10-100 mg/L concentration range according to the National Swimming Pool Foundation (San Antonio, TX). Two isocratic HPLC methods with UV detection (213 nm) employing Phenyl and Porous Graphitic Carbon (PGC) columns and phosphate buffer eluents (pH 6.7 and pH 9.1, respectively) were developed to accurately measure cyanuric acid in swimming pools. The two methods showed fast separation and detection of the stabilizer in 4 (phenyl) and 8 (PGC) minutes. Both methods offered practical sensitivities with method detection limits of 0.07 (Phenyl) and 0.02 mg/L (PGC). Neither one of the two methods requires the use of sample cleanup cartridges. They exhibited chromatograms with excellent baseline stability enabling low level quantitaiton. Most important, the PGC column showed good ruggedness with a useful lifetime of five months and 500 sample analyses per column. Finally, the statistical analysis of the relative precisions of the two methods indicated equivalence at the 0.05 critical level. Eleven pool water samples were fortified with 4.8 to 50.0 mg/L of the stabilizer, and the averagre recovery was 99.8%.

JOURNAL Contribution of Chlidren's Activities to Lead Contamination of Food 06/01/2001
Freeman, N. G., L S. Sheldon, L J. Melnyk, E. D. Pellizzari, M R. Berry Jr., AND M. Jimenez. Contribution of Chlidren's Activities to Lead Contamination of Food. JOURNAL OF EXPOSURE ANALYSIS AND ENVIRONMENTAL EPIDEMIOLOGY 11(5):407-413, (2001).
Abstract: This study evaluates the relationship of children's hygiene habits and food handling behaviors on lead levels on hands and handled foods for toddlers living in lead contaminated homes. Forty eight inner city toddlers who had previously been identified as having elevated blood lead levels participated in this study. The children participated in 3 consecutive days of activities. During the visits duplicate diets were obtained, the child handled a banana, a hot dog and had his/her hands wiped with a moist towellette. In addition, wipe samples were collected from the kitchen floor, and food items were collected from the kitchen floor. All samples were analyzed for lead. The child's caregiver completed a questionnaire which addressed the child's hygiene and eating behaviors. It demonstrated that children's contact with residential lead can transfer lead to food. Both lead in the home and on the children's hands can contribute to the contamination of food, and hence excess dietary exposure. In addition, child's hygiene habits as reported by the parent indicate that lack of basic hygiene patterns within a high lead environment can contribute to children's dietary exposure to lead.

JOURNAL Determination of Pesticides in Composite Dietary Samples By Gas Chromatography/Mass Spectrometry in the Selected Ion Monitoring Mode Using a Temperature Programmable Large Volume Injector With Pre-Separation Column 06/01/2001
Rosenblum, L, T Hieber, AND J N. Morgan. Determination of Pesticides in Composite Dietary Samples By Gas Chromatography/Mass Spectrometry in the Selected Ion Monitoring Mode Using a Temperature Programmable Large Volume Injector With Pre-Separation Column. JOURNAL OF AOAC INTERNATIONAL 84(3):891-900, (2001).
Abstract: Use of a temperature-programmable pre-separation column in the gas chromatographic injection port permits determination of a wide range of semi-volatile pesticides including organochlorines, organophosphates, triazines, and anilines in fatty composite dietary samples while reducing sample preparation time and solvent consumption. Dietary samples are mixed with diatomaceous earth and Soxhlet extracted with an azeotropic solution of hexane and acetone. Sample preparation uses liquid-liquid partitioning over diatomaceous earth followed by normal phase chromatography over partially deactivated alumina. The final clean-up step occurs in a pre-separation column in the GC injector, which is able to perform splitless transfer of the analytes to the analytical column and purge 99% of the high molecular weight residue. Detection is performed by gas chromatography/mass spectrometry in the selected ion monitoring mode. Method detection limits are at or below 2 ng/g for 24 of 35 pesticides studied, with recovery between 70 and 125% for 27 pesticides in samples fortified at 10 ng/g. Recovery was not dependent on fat content when measured in laboratory fortified samples containing 1%, 5%, and 10% fat by weight. Precision over multiple injections was acceptable with a relative standard deviation of 2.6-15% for 25 analytes.

JOURNAL Speciation and Preservation of Inorganic Arsenic in Drinking Water Sources Using Ic-ICP-MS 05/31/2001
Gallagher, P A., C A. Schwegel, J A. Shoemaker, J T. Creed, AND X. Wei. Speciation and Preservation of Inorganic Arsenic in Drinking Water Sources Using Ic-ICP-MS. JOURNAL OF ENVIRONMENTAL MONITORING 3(4):371-376, (2001).
Abstract: The native distribution of As(III) and As(V) in drinking water supplies can influence the treatment removal strategy. The stability of As(III) and As(V) in iron rich drinking waters can be affected by the formation of Fe precipitates (Fe oxides and/or hydroxides designated by "FeOOH"). These precipitates (PPT) can form during the transportation of the sample to the laboratory for arsenic speciation analysis. The analysis of the precipitate indicates considerable loss of the aqueous arsenic species (Asaq) to the solid phase "FeOOH" PPT. Studies of laboratory reagent water containing both As(III) and Fe(III) indicate that the reslting "FeOOH" PPT contained a mixture of As(III) and As(V) with near quantitative removal of the Asaq in 18hrs. The corresponding aqueous fraction after filtration through a 0.45u filter was composed primarily of As(V). The formation of "FeOOH" PPT and the loss of Asaq to the PPT can be virtually eliminated by the use of EDTA which sequesters the Fe(III). Reagent water fortified with Fe(III), As(III) and EDTA produced less than a 1ppb change in the As(III)aq concentration over 16 days.
The EDTA treatment was also tested on three well waters with different native As(III)/As(V) ratios. The native distribution of As(III)/As(V) was stabilized over a period of 10 days with worst case conversion of As(III) to As(V) of 2 ppb over a 30 day period. All well waters not treated with EDTA had dramatic losses (a factor of 2-5) of Asaq in less than one day. These results indicated that EDTA preservation treatment can be used to preserve Asaq in waters wherever the predominate species is the reduced form [As(III)] or in waters which the predominate species is the oxidized form [As(V)]. This preliminary investigation of EDTA to preserve As species in Fe rich waters indicates stability can be achieved for greater than 14 days.

JOURNAL Rapid Analysis of Cynanuric Acid in Swimming Pool Waters By High Performance Liquid Chromatography Using Porous Graphitic Carbon Column 04/01/2001
Cantu, R, O M. Evans, AND M L. Magnuson. Rapid Analysis of Cynanuric Acid in Swimming Pool Waters By High Performance Liquid Chromatography Using Porous Graphitic Carbon Column. , 2001. CHROMATOGRAPHIA 53(7/8):454-456, (2001).
Abstract: An innovative approach is presented for reducing analysis times of cyanuric acid in swimming pool waters by high performance liquid chromatography (HPLC). The HPLC method exploits the unique selectivity of porous graphitic carbon (PGC) to fully resolve cyanuric acid from other pool interferences within 10 minutes. By carefully timing the injections, multiple injections can be made before the end of the initial chromatographic run, more than doubling sample throughout. The method utilizes 95% of a 50 mM phosphate buffer solution (pH 9.1) and 5% methanol (%, v/v) with UV detection at 213 nm. This approach yielded run times rivaling those of the fastest methods using silica columns, and with the benefits of increased sensitivity.

JOURNAL Comparison of Mercury Capture Efficiencies of Three Different in Situ Generated Sorbents 04/01/2001
Lee, T. G., E J. Hedrick, AND P. Biswas. Comparison of Mercury Capture Efficiencies of Three Different in Situ Generated Sorbents. AMERICAN INSTITUTE OF CHEMICAL ENGINEERS JOURNAL 47(4):954-961, (2001).
Abstract: Three different sorbent materials (Ti, Si and Ca based) were compared for their mercury capture efficiencies in an entrained flow reactor. Agglomerated particles with a high specific surface area were generated in situ by injecting gas phase sorbent precursors into a high temperature furnace reactor. Titania particles in the presence of uv irradiation were most effective at mercury capture (>98%). In situ generated CaO particles had a capture efficiency of 33% (without any uv irradiation), while SiO2 was completely ineffective at mercury capture. The efficiency of elemental mercury capture of both CaO and Tio2 particles decreased with increasing SO2 concentration. The increase in the feed rate of the Ti sorbent resulted in a recovery of the higher mercury capture efficiency due to the availability of more active sites. The in situ generated titania sorbents were also effective at mercury capture (>87%) at elevated temperatures (160oC).

JOURNAL Determination of Carbamate, Urea, and Thiourea Pesticides and Herbicides in Water 03/01/2001
Wang, N. AND W L. Budde. Determination of Carbamate, Urea, and Thiourea Pesticides and Herbicides in Water. Analytical Chemistry 73(5):997-1006, (2001).
Abstract: Microbe liquid chromatography and positive ion electrospray mass spectrometry are applied to the determination of 16 carbamate, urea, and thiourea pesticides and herbicides in water. The electrospray mass spectra of the analytes were measured and are discussed and mobile phase matrix effects were evaluated. Analyte positive ion abundances are generally inversely related to the concentration of acetic acid in the acetonitrile-water mobile phase in the range of 0.001% to 0.1% (V/V) acetic acid. Using an internal standard for quantitative analyses and no acid in the mobile phase, retention time precision, peak width precision, concentration measurement precision, mean recoveries, and instrument detection limits were determined in reagent water. The 16 analytes were also measured in fortified environmental water samples from a recreational lake, a ground water well, a cistern, a farm pond, and drinking water. These measurements were at 5ng/mL of each analyte which is within the range expected for environmental pesticide and herbicide contaminants. The analytes were separated from the environmental water matrices with an online extraction and concentration to provide rapid sample analyses without a slow off-line liquid-liquid or liquid-solid-liquid extraction and extract concentration. Recoveries of 12 of the analytes from four environmental water samples were in the range of 75-124% with relative standard deviations in the range of 11-16%.

JOURNAL Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples By Method 1622 Using Ultrafiltration and Capsule Filtration 03/01/2001
Simmons, O. D., M. D. Sobsey, C. D. Heaney, F W. Schaefer, AND D. S. Francy. Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples By Method 1622 Using Ultrafiltration and Capsule Filtration. , 2001. APPLIED AND ENVIRONMENTAL MICROBIOLOGY 67(3):1123-1127, (2001).
Abstract: The protozoan parasite cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the USEPA developed Method 1622 for isolation and detection of cryptosporidim oocysts in water. Method 1622 is performance-based and involves filtration, concentration, immunomagnetic separation, fluorescent antibody and 4',6-diamidino-2-penylindole (DAPI) counter staining and microscopic evaluation. The capsule filter system currently recommended for Method 1622 was compared to a hollow fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed acccording to Method 1622. Cryptosporidium parvum oocyst recoveries from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (S.D.=24%) and 46% (S.D.=18%) for hollow fiber ultrafilters and capsule filters, respectively. Mean oocyst recoveries in experiments testing both filters on seeded surface water samples were 42% (S.D.=27%) and 15% (S.D.=12%) for hollow fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters using the approved filter of Method 1622, recoveries were significantly lower and more variable than from reagent grade water. In contrast, the disposable hollow fiber ultrafilter system was compatible with subsequent Method 1622 processing steps and recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the currently approved filter.

JOURNAL Letter to the Editor 03/01/2001
Simmons, O. D., M. D. Sobsey, D. S. Francy, AND F W. Schaefer III. Letter to the Editor. JOURNAL OF THE AMERICAN WATER WORKS ASSOCIATION 93(3):108-109, (2001).
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.

JOURNAL Testing Methods for Detection of Cryptosporidium Spp. in Water Samples 02/01/2001
Lindquist, H.D A., J W. Bennett, M W. Ware, R E. Stetler, M. Gauci, AND F W. Schaefer III. Testing Methods for Detection of Cryptosporidium Spp. in Water Samples. SOUTHEAST ASIAN JOURNAL OF TROPICAL MEDICINE AND PUBLIC HEALTH 32(2):190-194, (2001).
Abstract: A large waterborne outbreak of cryptosporidiosis in Milwaukee, Wisconsin, U.S.A. in 1993 prompted a search for ways to prevent large-scale waterborne outbreaks of protozoan parasitoses. Methods for detecting Cryptosporidium parvum play an integral role in strategies that lead to appropriate treatment of surface water, but are criticize because they produce results that are highly variable.
The U.S. Environmental Protection Agency developed a set of criteria to evaluate detection methods for protozoan parasites in water. As a consequence, the Agency has had to develop approaches to reducing uncertainty of evaluations. The variability and accuracy of various methods of producing small numbers of Cryptosporidium spp. oocysts were tested. The least variable and most accurate method was used to spike seven surface water, and one tap water sample to compare 4 detection methods that had been reported in the literature. The least variable and most accurate method for spiking specified numbers of oocysts into samples was found to be flow cytometry. The most effective of the methods tested for detection in both environmental and reagent water was solid phase cytometry.

JOURNAL Characterization of the Hemolysin, Stachylysin, from Stachybotrys Chartarum 02/01/2001
Vesper, S J., D. G. Dearborn, M L. Magnuson, I. Yike, AND R A. Haugland. Characterization of the Hemolysin, Stachylysin, from Stachybotrys Chartarum. INFECTION AND IMMUNITY 69(2):912-916, (2001).
Abstract: Stachybotrys chartarum is a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage/hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its monomeric form, has a molecular wieght of 11,920 daltons as determined by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-MS). Heat treatment at 60oC for 30 min inactivated the protein. Stachylysin is composed of about 40% hydrophobic amino acids and contains 2 cysteine residues. Purified stachylysin requires more than 6 h to begin lysing sheep's red blood cells, but by 48 h the treated sheep's red blood cells show pores on the surface.

JOURNAL Caffeine Selectivity of Divinylbenzene Crosslinked Polymers in Aqueous Media 01/31/2001
Villamena, F. A. AND A A. de la Cruz. Caffeine Selectivity of Divinylbenzene Crosslinked Polymers in Aqueous Media. JOURNAL OF APPLIED POLYMER SCIENCE 82(1):195-205, (2001).
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.

JOURNAL A Comparison of Four Fluorescent Antibody Based Methods for Purifying, Detecting and Confirming Cryptosporidium Parvum in Surface Waters 01/31/2001
Lindquist, H.D A., M W. Ware, R E. Stetler, L J. Wymer, AND F W. Schaefer III. A Comparison of Four Fluorescent Antibody Based Methods for Purifying, Detecting and Confirming Cryptosporidium Parvum in Surface Waters. JOURNAL OF PARASITOLOGY 87(5):1124-1131, (2001).
Abstract: Cryptosporidiosis has been traced to drinking contaminated surface water, either not treated, or ineffectively treated. Testing to detect Cryptosporidium parvum in surface water has been suggested to help prevent future outbreaks. This study compared purifications and detection steps from 4 testing methods. The same sampling and filtration were used for all methods. The methods were: modified information collection rule (ICR) method, method 1623 (both developed by the U.S. Environmental Protection Agency), a flow cytometric method, and a solid phase cytometric method. Fluorescent antibody staining is only a presumptive indication of the presence of this parasite, confirmation requires another assay. Methods were evaluated for both presumptive and confirmed detection. Solid phase cytometry had the highest presumptive and confirmed detection rates. Flow cytometry had the next highest presumptive detection rate in reagent water, but was third in spiked environmental waters, with no confirmation procedure. The ICR method had the third highest presumptive detection rate in reagent water and the second highest in spiked environmental waters, but failed to confirm any oocysts. Method 1623 had significantly lower presumptive detection than any other method, and significantly lower confirmation rate than the solid phase cytometry method.

JOURNAL A Comparison of Urinary Arsenic Speciation Via Direct Nebulization and on-Line Photooxidation-Hydride Generation With Detection By Inductively Coupled Plasma Mass Spectrometry 01/01/2001
Wei, X., C A. Schwegel, AND J T. Creed. A Comparison of Urinary Arsenic Speciation Via Direct Nebulization and on-Line Photooxidation-Hydride Generation With Detection By Inductively Coupled Plasma Mass Spectrometry. JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY 16(1):12-19, (2001).
Abstract: Arsenic speciation continues to be important in assessing human and environmental exposure risk. Urinary arsenic analysis provides information on recent arsenic exposure. In this study, two sample introduction pathways: direct nebulization (DN) and hydride generation (HG) were utilized and compared for urinary arsenic analysis via IC-ICP-MS. In the DIN method, the retention characteristics of arsenobetaine (AsB), arsenite (As(III)), dimethylarsinic acid (DMA), monomethylarsonic acid (MMA), arsenate (As(V)) and Cl were systematically evaluated with respect to column temperature and the (NH4)2CO3 eluent molarity. This evaluation indicated that the three early eluters (AsB, As(III) and DMA) were best separated at a higher column temperature and lower eluent molarity, whereas MMA, As(V) and Cl were best separated at a lower column temperature and higher eluent molarity. From this obsdervation, a gradient elution program was developed using 40mM and 70mM (NH4)2CO3(pH 10.5) at 69oC. This gradient condition produced satisfactory resolution for all five arsenic species with a Cl tolerance up to 0.3% (w/w). In the hydride generation (HG) configuration, a membrane based liquid-gas separator was used. A photo reactor interface was installed between the ion chromatography and the hydride gneration device to facilitate the oxidation and detection of non-hydride active arsenic species. Optimum photo-oxidation efficiency was achieved using a thin walled PTFE reaction coil braided around the UV lamp. Isocratic elution using 40mM (NH4)2CO3 was found adequate to resolve all five arsenic species while 40Ar35Cl nterference was eliminated. The HG method offers high sensitivity and wide eluent selection compared to DN, but has added experimental complexity and reagent background contamination. NIST SRM 2670 freeze dried urine was analyzed via both methods and the resuls from DN were within the 95% confidence bound calculated for HG.

JOURNAL Detection of Cryptosporidium Oocysts in Stream Water Samples Using U.S. Environmental Protection Agency Method 1622 01/01/2001
Simmons, O. D., C. D. Heaney, D. S. Francy, R. Nally, F W. Schaefer, AND M. D. Sobsey. Detection of Cryptosporidium Oocysts in Stream Water Samples Using U.S. Environmental Protection Agency Method 1622. JOURNAL OF THE AMERICAN WATER WORKS ASSOCIATION 67(3):1123-1127, (2001).
Abstract: To improve surveillance for cryptosporidium oocysts in water the USEPA developed Method 1622 which consists of filtration, concentration, immunomagnetic separation, fluorescent antibody and DAPI counter staining, and microscopic evaluation. Two filters were compared for analysis of 11 stream water samples collected throughout the U.S. Replicate 10--L stream water samples, unspiked and spiked with 100-250 oocysts, were tested to evaluate matrix effects. Oocyst recoveries from the stream water samples averaged 22% and 12% with a membrane disk and a capsule filter, respectively. These results demonstrate that cryptosporidium oocysts can be recovered from stream waters using Method 1622 but recoveries are lower than from reagent-grade water. Concentrations of indicator bacteria also were evaluated in these steam water samples. Because few samples were oocyst-positive, relationships between detections of oocysts and concentrations of indicator organisms could not be determined.

JOURNAL Extraction and Detection of Arsenicals in Seaweed Via Accelerated Solvent Extraction With Ion Chromatographic Separation and ICP-MS Detection 01/01/2001
Gallagher, P A., J A. Shoemaker, X. Wei, C A. Schwegel, AND J T. Creed. Extraction and Detection of Arsenicals in Seaweed Via Accelerated Solvent Extraction With Ion Chromatographic Separation and ICP-MS Detection. FRESENIUS'JOURNAL OF ANALYTICAL CHEMISTRY 369(1):71-80, (2001).
Abstract: An accelerated solvent extraction (ASE) device was evaluated as a semi-automated means of extracting arsenicals from ribbon kelp. Objective was to investigate effect of experimentally controllable ASE parameters (pressure, temperature, static time and solvent composition) on extraction efficiencies of arsenicals from seaweed. Changes in pressure (500-3000psi) and static time (1min-5min) parameters resulted in increase in extraction efficiency (after 3 ASEcycles) of <3% and 1% respectively. A solvent (50/50 MeOH/H2O) temperature increase from ambient to 60oC produced increase in extraction efficiency of 19% after first ASE extrac- tion. This difference was reduced to 9% after 3 ASE cycles. An extraction temperature of 120oC using 50/50 MeOH/H2O mixture produced improved extraction efficiencies (~11% after 3 ASE cycles) but increase in co-extractants was evident by dark ring in C18 sample preparation cartridge. Solvent compositions containing 50/50-0/100 MeOH/H2O produced extraction efficiencies within 5% after 3 ASE cycles. Finally particle size was evaluated by comparing a standard homogenized sample with a cryogenically ground sample. The cryogenically ground sample produced higher (72.4% cryogenic; 59.0% conventional mill) extraction efficiencies after first ASE cycle but difference decreased to less than 2% after 3 ASE cycles. In general extraction efficiencies for ribbon kelp (approximately 75%) using ASE was fairly independent (+/-3%) of pressure, static time and particle size after 3 ASE cycles.

PRESENTATION A Pilot Study to Determine the Water Volume Injested By Recreational Swimmers 12/02/2001
Evans, O M., R Cantu, T D. Behymer, D D. Kryak, AND A P. Dufour. A Pilot Study to Determine the Water Volume Injested By Recreational Swimmers. Presented at Society for Risk Analysis 2001 Annual Meeting, Seattle, WA, December 2-5, 2001.
Abstract: The volume of water ingested by recreational swimmers is unknown. Previous estimates by a number of investigators range from 10mL to 100mL. These estimates, however, are unsupported by empirical data. Many outdoor swimming pools are disinfected using cyanuric acid stabilized chlorine. This compound, when ingested by a swimmer, is not metabolized by the human body and is excreted quantitatively in the urine. The volume of water ingested can be calculated by collecting 24-hour urine sample, and subsequently determining the total concentration of the stabilizer in the sample and in the swimming pool water. A pilot study, involving about 100 swimmers, was conducted to evaluate: (1) the study design, (2) the analytical method for quantifying the chlorine stabilizer, and (3) the response of the study population. The results of the pilot study indicate that a full-scale study using current design may, in part, address key questions relating water quality to swimmer illnesses.

PRESENTATION An Efficient Immunomagnetic Capture System for Enterococcus Faecalis and Enterococcus Faecium 11/14/2001
AzconaOlivera, J. I. AND B G. Smith. An Efficient Immunomagnetic Capture System for Enterococcus Faecalis and Enterococcus Faecium. Presented at Water Quality Technology Conference, Nashville, TN, November 14, 2001.
Abstract: Enterococci detection is one of the two approved procedures by the US Environmental Protection Agency (EPA) used for the assessment of the microbiological quality of recreational waters. The action levels established by the EPA for enterococci are 35 pr 100 ml in marine recreational waters and 33 per 100 ml in fresh water. Turn-around time of the method is over 24 hr, and thus there is a clear need to reduce that time to allow a faster and reliable assessment of the safety/quality of the waters. The most abundant and prevalent species among the fecal enterococci (>90%) are E. faecalis and E. faecium. Our objective is to devise an efficient bacterial capture/concentration system in conjunction with a rapid detection method that will make possible the assessment of very low levels of fecal enterococci within a working day. In the first phase of our project described in this paper our objectives have been twofold (1) the procurement and/or production of antibodies able to capture efficiently the above mentioned microorganisms and (2) the evaluation of varoius tagging alternatives (nucleic acid based generic dyes, specific antibody fluorochrome-conjugates, etc) for the detection of enterococci with the RBD2000, a specialized flow cytometer designed for bacterial detection. This initial part of the project has been done in clean samples spiked with various bacterial loads. The next phase of our project is to test the reagents produced and the methods developed in real samples, being our final goal the full development and validation of a flow cytometry based method that will enable the detection and enumeration of low levels of fecal enterococci in recreational waters in less than 8 hr.

PRESENTATION Analysis of Rdx and Other Explosives By Solid Phase Extraction and Gc/MS 11/11/2001
Munch, J W. Analysis of Rdx and Other Explosives By Solid Phase Extraction and Gc/MS. Presented at The American Water Works Water Quality Technology Conference, Nashville, TN, November 11-15, 2001.
Abstract: Amendments to the Safe Drinking Water Act (1996) require the USEPA to publish a list of contaminants that are known or anticipated to occur in public water sysytems, and which may require regulation under the Safe Drinking Water Act (SDWA). In response to this requirement, and after extensive consultation with the scientific community, USEPA published a Drinking Water Contaminant Candidate List (CCL) of fifty chemicals and ten microorganisms in March 1998. One of the chemicals listed on the CCL is hexahydro-1,3,5-trinitro-1,3,5-triazine, also known as Royal Demolition Explosive (RDX). Before a regulatory detemination can be made concerning RDX, USEPA must assess the extent of its occurrence in public water systems. This will be accomplished through a nationwide monitoring program of drinking water utilities.
A new USEPA analytical method is being developed for monitoring drinking water for RDX and other explosive compounds. In addition to RDX, approximately 12 other explosives and related compounds of environmental interest will be evaluated for inclusion into the analytical method. This new method uses solid phase extraction to isolate and concentrate the target analytes. Preliminary data show greater than 85% recovery of 13 target analytes from 0.5 L of water using a divinylbenzene and N-vinylpyrrolidone solid phase. The concentrate extract is analyzed by gas chromatography/mass spectrometry (GC/MS) with programmed temperature vaporizing (PTV) injection.

PRESENTATION Analytical Method Readiness for the Contaminant Candidate List 11/11/2001
Pepich, B., S. D. Winslow, M V. Bassett, D. J. Munch, D. P. Hautman, J. L. Sinclair, J W. Munch, J A. Shoemaker, AND O M. Evans. Analytical Method Readiness for the Contaminant Candidate List. Presented at American Water Works Water Technology Conference, Nashville, TN, November 11-15, 2001.
Abstract: The Contaminant Candidate List (CCL), which was promulgated in March 1998, includes 50 chemical and 10 microbiological contaminants/contaminant groups. At the time of promulgation, analytical methods were available for 6 inorganic and 28 organic contaminants. Since then, 4 analytical methods have been pubished encompassing 12 additional analytes leaving only 4 chemical contaminant groups that still require method development. Method development work for microbiological contaminants will result in a method for Aeromonas later this year. Much research still remains in this area. This paper summarizes some of the technical challenges encountered during the method development projects, and updates the current status of the ongoing efforts to develop analytical methods for CCL contaminants.

PRESENTATION Fingerprinting of C. Parvum By Matrix Assisted Laser Desorption Ionization Mass Spectrometry 11/11/2001
Glassmeyer, S, J A. Shoemaker, F W. Schaefer III, AND D D. Kryak. Fingerprinting of C. Parvum By Matrix Assisted Laser Desorption Ionization Mass Spectrometry. Presented at Society of Environmental Toxicology and Chemistry Annual Meeting, Baltimore, MD, November 11-15, 2001.
Abstract: The oocysts of Cryptosporidium parvum, an enteric protozoan pathogen, are responsible for the worst microbial waterborne outbreak of gastroenteritis in recent history. The 1993 outbreak in Milwaukee, WI, sickened approximately 403,000 individuals, resulting in the hospitalization of over 4,000, and the death of nearly 100 people. The current assay for identifying C. parvum is labor intensive and time consuming. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is one tool that may eventually help create a faster, easier assay for Cryptosporidium and other microbial pathogens (including other protozoa, bacteria, and/or viruses). Similar gas chromatography/mass spectrometry (GC/MS), MALDI-TOF MS produces a characteristic "fingerprint" of the proteins contained within a sample. By comparing the mass spectral fingerprints of whole, viable oocysts, to oocysts that have been made non-viable (by means such as heating, freezing, exposure to UV light, and ozonation), as well as isolated oocyst walls, the specific (and potentially unique) proteins that compromise the outer wall of the viable oocysts can be determined. Further analysis using MALDI-TOF MS or liquid chromatography/electrospray/mass spectrometry (LC/ESI/MS), can yield the peptide sequence for these specific proteins. This information can be used to develop more sensitive and precise immunological techniques, such as ELISA and IFA, that focus in on these external parasite proteins in drinking water samples.

PRESENTATION An Overview for the Technical Session: Exposure Medium Food 11/05/2001
Berry Jr., M R. An Overview for the Technical Session: Exposure Medium Food. Presented at ISEA Annual Meeting, Charleston, SC, November 5, 2001.
Abstract: Historically, research on food as a source of human exposure to environmental contaminants has focused on a few chemical categories. Crops are treated with insecticides, fungicides and herbicides to enhance production and market appeal. Pestidice residues persist and are routinely found in foods at the market level. Other contaminants like metals accumulate in foods through uptake from soil or air, or as a result of the food chain process. Over the last decade, aggregate or multimedia exposure evaluations, such as the National Human Exposure Assessment Survey (NHEXAS), have included daily dietary intake of food as a potential pathway of human exposure. Often, diet has proved to be a significant contributor for a wider variety of compounds, a few of which were previously considered to be limited to unique pathways, such as air, water, dust or soil. The Food Quality Protectoin Act of 1996 has also had a major impact on the way pesticides are evaluated before they can be applied to foods. Both aggregate and cumulative exposures are now evaluated to establish safe levels for agricultural and residential pesticide applications. In some cases, it is the synergism of foods in contact with other contaminated media that has proven to be of significance. We are beginning to understand that the way children behave while they eat can be a dominant factor in exposure evaluation. The presentations in this and other dietary exposure sessions and posters are indeed varied. Such diverse research activities and exposure evaluations reflect that food is now more generally recognized throughout the exposure community as a potentially significant source of exposure to a broader range of environmental contaminants.

PRESENTATION Measuring Excess Dietary Exposures Caused By Eating Activities of Young Children 11/04/2001
Akland, G G., E. D. Pellizzari, Y. Hu, D. Whitaker, L J. Melnyk, AND M R. Berry Jr. Measuring Excess Dietary Exposures Caused By Eating Activities of Young Children. Presented at ISEA Annual Meeting, Charleston, SC, November 4-8, 2001.
Abstract: A small, pilot field study was conducted to measure dietary exposures of young children which included contamination of foods while eating. Samples were collected to estimate the amount of a pesticide recently applied within the home which was transferred from contaminated surfaces or hands to foods.
Three homes were selected for study. Each home had a young child (1-3 years old), and diazinon had recently been applied, which was confirmed by surface wipe samples. Sampling occurred over six consecutive days following pesticide application. During the monitoring period, enviornmental samples (e.g. air, surface wipe and surface press), personal exposure samples (e.g. hand wipe), biological samples (e.g. morning urine samples), food samples (e.g. duplicate diet, leftover handled food, and foods contacted with surfaces), questionnaires and activity video were collected. A continuous dust (particle) monitoring device with data logger was used to measure air particulate concentrations throughout the monitoring period as an indicator of aerosol generation and/or resuspension related to on-going activities occurring within the home. Twenty-four hour indoor and outdoor integrated air samples were collected each of six consecutive days of monitoring. Samples for surface pesticide loading were collected at the beginning, middle and end of the monitoring period. Duplicate diet, leftover and handled food and morning urine samples were collected daily. Results from the pilot study illustrate the following:
. children handle food items, on average, about 50 times/eating event
. contacts of foods with the child's hands were a greater source of contamination than direct contact with other surfaces
. handling food items can contribute 10 times more than the residue values measured in food items that had not been handled
. diazinon metabolites measured in the urine of the children from the homes studied were elevated above the comparative reference range for other children of this age

This study demonstrates that additional contamination of foods caused by pesticides found in homes on surfaces contacted by children's foods do contribute to excess dietary exposures. Additional research is needed to more fully characterize the parameters that influence food handling by the young child so that this potentially significant contribution to dietary exposure may be incorporated in risk assessments.

PRESENTATION Pesticide Surface Residue Measurements By a Press Sampler 11/04/2001
Melnyk, L J., C. Rohrer, T Hieber, AND M R. Berry Jr. Pesticide Surface Residue Measurements By a Press Sampler. Presented at ISEA Annual Meeting, Charleston, NC, November 4-8, 2001.
Abstract: Pesticides on household surfaces are a source of exposure to children. Accurate measurements of residues on surfaces are needed to determine amounts available for transfer to foods and other objects handled or eaten by a child. Wiping the surface with a solvent has been the acceptable measurement method, but sing solvents can mar the surface which would be unacceptable for field sampling. A surface press sampler capable of using C18 or polyurethane foam (PUF) transfer disks pressed onto a surface for a specified time with constant force was compared to surface wipe measurements. The transfer disks, which adsorb and/or absorb the residue during contact of the surface, are then removed and extracted for analysis and quantified for surface residue concentration or loading.
The objective of this study was to determine if the surface press sampler with dry C18 and PUF disks could be used to obtain residue information from household surfaces comparable to isopropanol surface wipes. The surfaces tested were ceramic tile, hardwood flooring, and carpet. Each surface was contaminated with an aqueous solution of the pesticides commonly found in homes (diazinon, malathion, chlorpyrifos, isofenphos, heptachlor, and cis- and trans- permethrin) at levels previously measured (20 to 55 ng/cm2) and dried for 2-1/2 hours. Duplicate contaminated surfaces were wiped and pressed for 5 sec, 1, 2, 5, 10, and 60 min and percentages of pesticides transferred based on wipes were compared.

Recoveries of pesticides wiped were variable, ranging from trace to near applied levels, and depended on surface type. Only a fraction of the pesticides were transferred to either press material when compared to wipes. This varied by pesticide and was highly dependent on duration of the press. On average, less than 50% of the pesticide were transferred from tile to C18 and PUF for the various times, except for 60% of the pesticides transferred from tile C18 after 60 minutes of pressing. For the average of all pesticides, C18 was slightly more efficient in the transfer of pesticides than PUF. In comparing the various times for both press materials, the average pesticide transferred to C18 was between 6 to 60% and for PUF was 2 to 50%. Transfer of pesticides from hard surfaces (tile and hardwood flooring) occurred more readily than from carpet. Negligible pesticides were transferred to either of the press materials from carpet.

PRESENTATION The Importance of Arsenic Species Specific Mass Balance on the Evaluation of Arsenic Speciation Results in Seafood Matrices 11/04/2001
Creed, J T., P A. Gallagher, B. M. Gamble, A. Heck, D M. Freeman, AND C A. Schwegel. The Importance of Arsenic Species Specific Mass Balance on the Evaluation of Arsenic Speciation Results in Seafood Matrices. Presented at International Society of Exposure Analysis 2001, Charleston, SC, November 4-8, 2001.
Abstract: The two predominant pathways to arsenic exposure are drinking water and dietary ingestion. A large percentage of the dietary exposure component is associated with a few food groups. For example, seafood alone represents over 50% of the total dietary exposure. From a daily dose perspective, these dietary arsenic exposures can easily exceed those from drinking water, especially in populations with high seafood consumption rates. Unlike drinking water, dietary arsenic is a mixture of tox and non-toxic arsenicals and to assess toxicity from dietary exposures, it is necessary to extract and speciate (separate) the arsenic. The quantitative nature of this extraction can dramatically affect risk assessment associated with seafood exposure and thus, the extraction process also needs to be species non-specific. This is essential because some extraction techniques can selectively extract the non-toxic (neutrally charged) species, while allowing the toxic species (anionic) to remain unextracted/undetected. However, the selectivity of an extraction technique is difficult to assess in unknown samples. Therefore, a quantitative extraction is often pursued in which the concentration of each arsenic species are added together and compared to the total digestion (i.e., total arsenic) concentration for that sample. This comparison measures extraction efficiency and is the foundation for species specific mass balance. This mass balance provides some data quality criteria to aid in assessing the reliability of dietary arsenic speciation.
Recently, our laboratory has been investigating various alternatives to quantitatively extracting arsenicals from seafoods. Some of these extractions have been found to be very matrix dependent, with oysters and clams producing extraction efficiencies of only aproximately 50-60%. However, these extraction efficiencies can be improved to greater than 90% by using tetramethylammonium hydroxide as an extraction solvent. In addition, these high extraction efficiencies show little matrix dependence, but the extract has been found to contain unknown arsenic species which do not elute from conventional anion exchange columns until treated with mild acid. The species specific mass balance approach is used to monitor the reappearance of the unknown arsenic species after treatment with the mild acid. Currently, the unchromatographable species are believed to be arsenosugars bound to proteins, lipids or other sugar substrates. Size exclusion chromatography indicates that the unchromatographable species have a higher molecular weight than the acid treated species. The nature of these initially unchromatographable species will be investigated using both atomic and molecular instrumental techniques. These results will be summarized in the context of minimizing uncertainty in assessing risk from seafood exposures.

PRESENTATION Method 1623 for Cryptosporidium and Giardia Detection 11/01/2001
Schaefer, F W. Method 1623 for Cryptosporidium and Giardia Detection. Presented at Safe Drinking Water Act Seminar (AWWA), Columbus, OH, November 1, 2001.
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 Can We Believe Our Results? 10/25/2001
Schaefer III, F W. Can We Believe Our Results? Presented at Royal Society of Chemistry Water Chemistry Forum. Cryptosporidium: The Analytical Challenge, Warwick, UK, October 25-26, 1999.
Abstract: Numerous waterborne outbreaks of cryptosporidiosis have occurred recently with the most notable being the 1993 episode in Milwaukee. Due to these outbreaks and the concern for public health, the past decade has seen a massive effort expended on the development of methods to detect Cryptosporidium parvum oocysts in source and finished water. Initial analyses for C. parvum oocysts were based on methods developed for detecting Giardia cysts in water. In short, the procedure involved sampling either 100 liters of source water or 1,000 liters of finished water using a fiber wound filter with a nominal porosity of 1.0um. After washing the filter fibers with buffer, the extracted particulates containing the parasites were further concentrated by centrifugation. Buoyant density centrifugation was then used to separate the parasites from the particulates. Because buoyant density centrifugation is not a selective purification procedure, the parasites still were associated with and masked by particulates. To overcome this problem, the parasites were selectively stained with fluorescently labeled monoclonal antibodies. The results of the assay were microscopially determined using epifluorescence microscopy to detect the presumptive parasites and differential interference contrast microscopy to confirm the identity of the parasites by demonstration of internal morphological characteristics. In the case of Cryptosporidium, recovery rates ranged from 0 to 11% in seeded samples.
Method 1622 for Cryptosporidium was developed using off the shelf reagents and equipment in an attempt to improve on oocyst recovery rates. Instead of sampling large volumes of water, 10 liter samples are concentrated by passing the water through a 1.0 um absolute porosity filter. Immunomagnetic separation, a more selective procedure employing monoclonal antibodies, is used in place of buoyant density centrifugation to separate the parasites from the particulates. Like the prevoius method, staining is done with fluorescently labeled monoclonal antibodies for detection. However, in addition to differential interference contrast microscopy for confirmation, the samples are counter stained with 4',6=diamidino-2-phenyllindole (DAPI) which helps to demonstrate sporozoite nuclei. Even with these improvements, the recovery results for the Method 1622 only average around 38%.

PRESENTATION Detection of Protozoa in Water 10/21/2001
Lindquist, H.D A. Detection of Protozoa in Water. Presented at XXI Congresso Brasileiro de Mimcrobiologia (XXI Brazilian Society of Microbiology Congress), Foz do Iguassu, Brazil, October 21-25, 2001.
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 Mini Symposium on Alachlor Esa 10/16/2001
Shoemaker, J A. Mini Symposium on Alachlor Esa. Presented at Mini Symposium on Alachlor ESA, St. Louis, MO, October 16, 2001.
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 Detection of Cryptosporidium Oocysts in Water Matrices 10/07/2001
Schaefer, F W. Detection of Cryptosporidium Oocysts in Water Matrices. Presented at Cryptosporidium: From Molecules to Disease, Fremantle, Western Australia, Australia, October 7-12, 2001.
Abstract: Since the advent and recognition of waterborne outbreaks of cryptosporidiosis great effort has been expended on development of methods for detecting Cryptosporidium oocysts in water. Oocysts recovery rates using a method originally developed for detecting Giardia cysts ranged from 0 to 11% in seeded samples. The poor oocyst recovery was based on the fact that up to 60% of the seeded oocysts passed through the fiber filter. Method 1622 for Cryptosporidium was developed using off the shelf reagents and equipment in an attempt to improve on oocyst recovery. Even with these improvements, the recovery results for the Method 1622 are only on average around 38%. Besides undefined parameters in water matrices, a number of other factors, such as laboratory proficiency and commercial reagent and equipment quality, are now known to compromise Cryptosporidium oocyst recovery. Fluorescent in situ hybridization probes have been suggested as assay additions for speciation of detected oocysts and for providing an indication of viability. Cryptosporidium cell culture has also been suggested as a mechanism for determining viability and infectivity of detected oocysts. Sensitivity, specificity, and the correlation with animal infectivity must be established for these new methods. At this point these new approaches are just research methods and remain to be multi-laboratory validated.

PRESENTATION Arsenic Speciation in Water and Dietary Samples By Ic-ICP-MS With Structural Verification Via Ic-Esi-MS/MS 10/07/2001
Creed, J T., P A. Gallagher, B. M. Gamble, A. Heck, AND C A. Schwegel. Arsenic Speciation in Water and Dietary Samples By Ic-ICP-MS With Structural Verification Via Ic-Esi-MS/MS. Presented at Federation of Analytical Chemistry and Spectroscopy Society Meeting, Detroit, MI, October 7-12, 2001.
Abstract: The two predominate sources of arsenic exposure are water and dietary ingestion. Dietary sources can easily exceed drinking water exposures based on "total" arsenic measurements. This can be deceiving because arsenic's toxicity is strongly dependent on its chemical form and therefore, "total" arsenic measurements do not accurately reflect risk. The limited availability of species specific data in target foods induces a fair amount of uncertainty in the arsenic risk assessment. Thus, species specific data is essential for accurate risk assessment. The ability to quantitatively extract, separate and detect arsenicals from dietary sources has been the subject of numerous publications. Recently, our laboratory has been conducting research in a number of these areas including: 1) utilizing accelerated solvent extraction (ASE) as a potential semi-automated means to extract arsenicals from seafoods; 2) the effect of acidic and basic extraction solvents on the stability of certain arsenicals; 3) the bio-transformatoin of arsenosugars in synthetic stomach type environments; 4) improvement of extraction efficiencies in problrematic seafood matrices; and 5) preservation of AS(III) and number of analytical techniques including IC-ICP-MS and IC-ESI-MS-MS. This presentation will focus on the research highlights and some of the unique capabilities generated in order to better understand species specific stability.

PRESENTATION Detection of Cryptosporidium Oocysts in Source and Finished Waters 10/07/2001
Schaefer, F W. Detection of Cryptosporidium Oocysts in Source and Finished Waters. Presented at Cryptosporidium: From Molecules to Disease, Fremantle, Western Australia, Australia, October 7-12, 2001.
Abstract: Numerous waterborne outbreaks of cryptosporidiosis have occurred with the most notable being the 1993 episode in Milwaukee. As a result, the past decade has seen a massive effort expended on the development of methods to detect Cryptosporidium parvum oocysts in source and finished water. Initial analyses for C. parvum oocysts were based on methods originally developed for detecting Giardia cysts in water. This procedure involved sampling either 100 liters of source water or 1,000 liters or finished water using a fiber wound filter with a nominal porosity of 1.0 um. After washing the filter fibers with buffer, the particulates containing the parasites were concentrated by centrifugation. Further purification of the parasites was done by buoyant density centrifugation. Because buoyant density centrifugation is not a selective purification procedure, the parasites still were associated with and masked by particulates. To overcome this problem, the parasites were selectively stained with fluorescently labeled monoclonal antibodies. The stained samples were then microscopically read using epifluoresdcence microscopy to detect the presumptive parasites and differential interference contrast microscopy to confirm the identity of the parasites by demonstration of internal morphological characteristics. In the case of Cryptosporidium, recovery rates ranged from 0 to 11% in seeded samples. The poor oocyst recovery was based on the fact that up to 60% of the seeded oocysts passed through the fiber filter.
Method 1622 for Cryptosporidium was developed using off the shelf reagents and equipment in an attempt to improve on oocyst recovery. Instead of sampling large volumes of water, 10 liter samples are concentrated by passing the water through a 1.0 um absolute porosity filter. Immunomagnetic separation is a selective procedure employing monoclonal antibodies. To separate the parasites from the concentrated particulates, immunomagnetic separation now is used in place of buoyant density centrifugation. Like the previous method, oocysts are detected by staining with fluorescently labeled monoclonal antibodies. However, in addition to differential interference contrast microscopy for confirmation of internal morphological characteristics, the samples are counter stained with 4',6-diamidino-2-phenylindole (DAPI) which helps demonstrate sporozoite nuclei. Even with these improvements, the recovery results for the Method 1622 is only on average around 38%. Besides undefined parameters in water matrices, a number of other factors, such as laboatory proficiency and commercial reagent and equipment quality, are now known to compromise Cryptosporidium oocyst recovery. A modified version of the Information Collection Rule method, the ChemScan RDI solid phase cytometry method, and the AusFlow method were compared against Method 1622. The oocyst recovery results for Method 1622 in this compartive study were not as good as expected. Modification to immunomagnetic bead dissociation and DAPI staining are required, when these techniques are used in conjunction with any method in the future. Fluorescent in situ hybridization probes have veen suggested as assay additions for speciation of detected oocysts and for providing an indication of viability. Cryptosporidium cell culture has also been suggested as a mechanism for determining viability and infectivity of detected oocysts. Sensitivity, specificity, and the correlation with animal infectivity must be established for these new methods. At this point these new approaches are just research methods and remain to be multi-laboratory validated.

PRESENTATION Cyclospora Cayetanensis 10/03/2001
Lindquist, H.D A. Cyclospora Cayetanensis. Presented at Ohio Environmental Health Association Southwest District Fall Conference, Dayton, OH, October 3-4, 2001.
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 Stability: An Investigation of As(iii)/As(v) Stability in Iron Rich Drinking Water Matrices 09/12/2001
Gallagher, P A., J T. Creed, C A. Schwegel, A. Heck, AND B. M. Gamble. Stability: An Investigation of As(iii)/As(v) Stability in Iron Rich Drinking Water Matrices. Presented at International Ion Chromatography Symposium, Chicago, IL, September 9-12, 2001.
Abstract: Arsenic in drinking water is predominantly inorganic arsenic. The two oxidation states of inorganic arsenic are As(III)(pKa=9.3) and As(V)(pKa2=6.9). The distribution of As(III) and AS(V) in a water is dependent on the redox potential of the water. The actual distribution can vary considerably with a general rule that high percentages of As(III) are ususally associated with ground wqters while high percentages of As(V) are generally associated with surface waters. Current drinking water regulatins do not differentiate between these two oxidation states when setting the maximum contaminate level (MCL) for arsenic. The need to differentiate As(III) and As(V) arises from a treatment/removal perspective within the drinking water regulations. Certain treatment strategies remove As(V) more efficiently than As(III). Therefore, treatment engineers usually require a speciation based analysis before designing a treatment strategy. The accuracy of this analysis is strongly dependent on how well the native distribution of As(III) and As(V) is preserved prior to analysis. Factors which can influence the preservation are dissolved oxygen, iron preciption, temperature, time and biological actvity. The precipitation of iron is an important factor because it can change the distribution and the contration of aqueous arsenic. In addition, there is a geological correlation between arsenic and iron, thus the iron precipitaiton factor is quite common. In an attempt to preserve the native arsenic distribution in iron rich waters we have been investigating the use of EDTA in combination with acidification with acetic acid (HAc). This presentation will focus on the use of EDTA/HAc in a number of well waters from across the US. The stability of the EDTA/HAc treated samples will be investigated over time and compared to controls and non-treated samples. The overall change in the distribution will be reported with a special emphasis on how these changes affect the treatment decision making process.

PRESENTATION Automated Pressurized Fluid Extraction With in-Line Clean-Up for Determination of Pesticides in Composite Diets. 09/09/2001
Morgan, J N. Automated Pressurized Fluid Extraction With in-Line Clean-Up for Determination of Pesticides in Composite Diets. Presented at AOAC International Annual Meeting, Kansas City, MO, September 9-13, 2001.
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 organophosphate 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 determiantion of thirty-seven organophosphate pesticides in composite diet samples. The current study evaluated an automated system for performing the extraction and cleanup in one step. C18 (5g), diatomaceous earth (5g) and composite diet sample (5g) mixed with diatomaceous earth (3g) were layered in a 33 mL pressurized fluid extractoin was performed at 80 degree C and 1000 psi. This technique was applied to composite diet samples varying in fat content and fortified with organophosphate pesticides. Organophosphate pesticides were quantititated by capillary GC using a DB-17 column with flame photometric detection (phosphorous mode). Results of this study demonstrated that the procedure separated the pesticides from greater than 95% of the lipid material. Pesticide recoveries fell within the target range of 60 to 140%. Results obtained with this automated technique compare favorably with the more laborious procedure utilizing PFE and column clean-up.

PRESENTATION Development of Molecular Methods to Detect Emerging Viruses 09/05/2001
Grimm, A C., C Newport, AND G S. Fout. Development of Molecular Methods to Detect Emerging Viruses. Presented at ORD/Regional Training Workshop on "Emerging Issues" Associated with Aquatic Enviornmental Pathogens, Ft. Meade, MD, September 5-7, 2001.
Abstract: A large number of human enteric viruses are known to cause gastrointestinal illness and waterborne outbreaks. Many of these are emerging viruses that do not grow or grow poorly in cell culture and so molecular detectoin methods based on the polymerase chain reaction (PCR) are being developed. Current studies focus on detecting two virus groups, the caliciviruses and the hepatitis E virus strains, both of which have been found to cause significant outbraks via contaminated drinking water. Once developed, these methods will be used to collect occurrence data for risk assessment studies.

PRESENTATION Iodide Aerosol Sorbents for Mercury Capture in Combustion Exhausts 08/20/2001
Rodriguez, S., T. G. Lee, E J. Hedrick, AND P. Biswas. Iodide Aerosol Sorbents for Mercury Capture in Combustion Exhausts. Presented at EPA-DOE-PRI Mega Symposium Mercury Emissions Fate, Effects and Control, Chicago, IL, August 20-24, 2001.
Abstract: Several sorbent processes are being studied for their feasibility for mercury capture. Mercury is different from the other heavy metals as it is not as chemically reactive (due to a filled outer electronic shell), thus making it difficult for sorbents to chemically trap it (a). A titania based sorbent was found to be effective due to its potential to oxidize the mercury on irradiation with uv light(b). During the development of impinger solutions for efficient capture of mercury for measurement by direct injection nebulization inductively coupled plasma mass spectrometry (c), we found out that in situ generated aqueous iodine, starting with potassium iodide solution, was very effective at capturing elemental mercury. In this study we demonstrate the effectivenss of potassium iodide aerosols at room temperature for capturing elemental mercury in gas streams.
The system consisted of a mercury feed unit, an atomizer for nebulizing potassium iodide solutions to generate a high surface area sorbent aerosol, a filter for effectively capturing the particles, followed by an impinger train to caputre the gase phase mercury species. Results of a series of experiments that were conducted will be discussed. The first phase was to characterize the KI generated sorbent particles, and the second phase focused on the caputre efficiencies. Rather high capture efficiencies (>99%) for elemental mercury were obtained using the potassium iodide sorbent particles. The capture mechanism will be discussed, along with the potential to scale up for applicability in pilot systems.

PRESENTATION Sampling Design for Assessing Recreational Water Quality 08/13/2001
Wymer, L J. Sampling Design for Assessing Recreational Water Quality. Presented at The International Environmetrics Society Conference 2001, Portland, OR, August 13-17, 2001.
Abstract: Current U.S. EPA guidelines for monitoring recreatoinal water quality refer to the geometric mean density of indicator organisms, enterococci and E. coli in marine and fresh water, respectively, from at least five samples collected over a four-week period. In order to expand this guidance to account for spatial and temporal variation in the contamination of bathing waters, sampling studies were conducted at five marine and freshwater beaches during the summer of 2000, these studies invovling twice-a-day, and sometimes hourly, sampling at several locations in the water at each beach. Implications for a sampling design for monitoring warer quality are examined, considering factors such as sample volume, number of locations sampled, and the compositing of samples. Variance components are estimated, and operating characteristic curves for sampling schemes designed to detect alternative in excess of some standard level of microbial contamination are constructed via generalized linear modeling and bootstrapping on the results from these studies. Principles of sampling design developed from these five bathing areas, all of which were limited to a fixed length of beach, may be adapted to varying stretches of beaches through consideration of spatial relationships of variation among sampling locations.

PRESENTATION Extraction of Pesticides from Fatty and Non-Fatty Foods in Composite Dietary Samples 07/15/2001
Rosenblum, L, J N. Morgan, AND S. Garris. Extraction of Pesticides from Fatty and Non-Fatty Foods in Composite Dietary Samples. Presented at Florida Pesticide Residue Workshop, St. Pete Beach, FL, July 15-18, 2001.
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 Characterization of Arsenosugars and Associated Degradation Products Following An Aggressive Acid/Base Extraction Procedure 07/01/2001
Gallagher, P A., B. M. Gamble, A. Heck, C A. Schwegel, AND J T. Creed. Characterization of Arsenosugars and Associated Degradation Products Following An Aggressive Acid/Base Extraction Procedure. Presented at International Symposium of Environmental Toxicology of Metals and Metallloids - Environmental Chemistry, Toxicology and Health, South Mole Island, Australia, July 1-5, 2001.
Abstract: The speciation of arsenic in seafood products is important for the determination of an improved toxicity based relative source (water vs. diet) contribution estimate. The two major sources of arsenic are drinking water and seafood ingestion. Drinking water contains predominately inorganic or toxic arsenic while arsenic exposures from seafood products are thought to be predominantly non-toxic. The daily dose of arsenic from drinking water is generally less than 20ug/day (i.e., 2L/day at 10ng/mL) while from a total metal standpoint, seafood ingestion of arsenic can easily exceed 1000ug/day (i.e., 227[8oz] seafood ingestion at 5ug/g; some fish contain 10-50ug/g). The toxicity from this arsenic dose is considered non-toxic. However, this generalization comes from speciation analyses of seafood products like Atlantic Cod, halibut and English sole. The arsenicals in these types of seafood are easily extracted once the sample has been freeze dried. The subsequent speciation analysis indicates that 99% of the arsenic is AsB. For these seafood products, the current assumption that arsenic in seafood is non-toxic is accurate. But in many types of seafood the extraction efficiencies may be as low as 50%. Thus, the subsequent speciation analysis can then only provide speciation-based information on one-half of the arsenic present. The speciated fraction can contain dimethylarsinic acid [DMA] (a suspected cancer promoter), As(V) (suspected carcinogen) and a variety of arsenosugars (which have been shown to degrade to DMA in the body, a potential conversion to a more toxic spcies). This in combination with the poor extraction efficiency creates considerable uncertainty in estimating the risk from these exposures. In this case, the assumption that arsenicals in these seafood types are non-toxic can produce a misleading risk assessment. Finally, there are seafood products (i.e., kelp, sushi, etc.) which produce extraction efficiencies of less than 50% and half of which is As(V). The saving grace here is that large quantities are not typically ingested, but even a 5g portion can easily exceed a typical exposure from drinking water. Again, the risk assessment with these seafood products should not be lumped together with the Atlantic cod, halibut and English sole analysis due to the fact that the extraction efficiencies are poor and that the species extracted are more toxic.
The objective of this research is to evaluate extraction techniques, which provide near quantitative extraction of the arsenicals from a wide variety of seafood products. Some "aggressive" extraction techniques have already been shown to produce by-products from arsenosugars. These by-products will be characterized chromatographically and identified using electrospray [ESI]-MS/MS. This poster will identify extraction conditions that tend to produce degradation products and describe analytical conditions that can be used to minimize their formation. Structural identfication of these degradation products in standard solutions will also be used to evaluate chromatographic recovery of the arsenicals extracted from seafood matrices.

PRESENTATION Enhanced Dapi Staining for Cryptosporidium in Water Samples 06/29/2001
Ware, M W., F W. Schaefer III, AND H.D A. Lindquist. Enhanced Dapi Staining for Cryptosporidium in Water Samples. Presented at American Society for Parasitologists Annual Meeting, Albuquerque, NM, June 29-July 3, 2001.
Abstract: The U.S. Environmental Protection Agency's Method 1623 is used to detect and quantify the presence of {ital Cryptosporidium} spp. oocysts in water. The protocol consists of concentrating a sample, staining this concentrate with a fluorescent antibody, and examining the sample microscopically. Since algae can be misinterpreted as positives using this procedure, confirmation may be done by either differential interference contrast microscopy, or by observation of the staining pattern when stained with 4,6-diamidino 2-phenyl-indole dihydrochloride (DAPI). Our research has shown that differential interference contrast is only able to confirm about 2% of oocysts with sporozoites in a fresh preparation. The rate of confirmation is improved to around 30% with DAPI staining when following the Method 1623 protocol. By a slight modification of the DAPI protocol, the confirmation rate improves to nearly 100%. A number of formats for optimizing slide preparation have been tested. Confirmation by both fluorescent antibody and DAPI staining is also possible using flow cytometry.

PRESENTATION Comparing Methods for Estimating {ital Cryptosporidium} Spp. Oocyst Concentrations in Water 06/29/2001
Lindquist, H.D A., J W. Bennett, M W. Ware, R E. Stetler, AND F W. Schaefer III. Comparing Methods for Estimating {ital Cryptosporidium} Spp. Oocyst Concentrations in Water. Presented at Annual Meeting of the American Society of Parasitologists, Albuquerque, NM, June 29-July 3, 2001.
Abstract: {ital Cryptosporidium parvum} in drinking water is a threat to public health. Estimating the parasite burden of {ital C. parvum} in water to be treated for use as drinking water constitutes an integral element to developing strategies for protecting public health in a cost effective manner. These estimations must be made using methods that are as unbiased toward under reporting and have high visability. The high levels of bias and variability call into question the way in which methods are tested, and not just the methods themselves. To overcome this criticism, a set of criteria for evaluating detection methods were developed. The critical parameters in methods evaluation were determined to be the sample matrix, and the precisoin and accuracy of the enumeration of organisms seeded into this sample. A series of comparisons were made that indicate that flow cytometry is superior to other methods for seeding oocysts into samples. Flow cytometrically enumerated organisms, spiked into reagent and experimental sample water were detected and counted by four methods. Recovery ranged from 11.5 to 48.6 with highest recovery being obtained using a solid phase cytometry method.

PRESENTATION The Determination of Mercury Species and Multiple Metals in Coal Combustion Emissions Using Iodine-Based Impingers and Direct Injection Nebulization Inductively Coupled Plasma Mass Spectrometry Analysis 06/24/2001
Hedrick, E J., P. Biswas, T. G. Lee, AND Y. Zhuang. The Determination of Mercury Species and Multiple Metals in Coal Combustion Emissions Using Iodine-Based Impingers and Direct Injection Nebulization Inductively Coupled Plasma Mass Spectrometry Analysis. Presented at 94th Annual Air and Waste Management Association, Orlando, FL, June 24-28, 2001.
Abstract: Mercury (Hg) emissions from coal utilities are difficult to control. Hg eludes capture by most air pollution control devices (APCDs). To determine the gaseous Hg species in stack gases, U.S. EPA Method 5 type sampling is used. In this type of sampling a hole is drilled into the stack wall and a volume of gas is isokenetically drawn through a heated probe, through a filter to remove particulate, and finally through a series of gas-scrubbing solutions designed to selectively capture the gaseous Hg species of interest. Gaseous speciation is achieved by selective, sequential capture in the impinger solutions. Analysis of Hg on the filter and in the impinger solutions is done by oxidation of all Hg species followed by cold vapor atomic absorption spectormetry (CVAA) analysis. A drawback to this type of analysis scheme is that there are no reference methods capable of both Hg speciation and multiple metals analysis since the solutions typically used for Hg capture are not amenable to low level metals techniques like ICP-MS. This can double the cost of study designed to gather Hg and multiple metals data since two separate sampling and analysis methods must be used. In previous research we developed an impinger sampling train for Hg speciation using inductively coupled plasma mass spectrometry (ICP/MS) with direct injection nebulization (DIN) with the potential for performing multiple metals determinations on the same solutions. In the current work we (1) demonstrate the Hg speciation sampling and analysis method using a bench scale coal combustor and compare the results to the Ontario Hydro Method, (2) demonstrate the capability of performing low-level Hg determination in addition to low-level analysis of Pb, Cd, Ba, Cr, Cu, Mn, Ni, Se, V and Zn, and (3) demonstate the proposed methodology in real world stack sampling of a coal utility.

PRESENTATION A Quantitative Real-Time Pcr Assay for Detection of Cyclospora Cayetanesis 06/13/2001
Varma, M, H.D A. Lindquist, F W. Schaefer III, M W. Ware, AND J. D. Hester. A Quantitative Real-Time Pcr Assay for Detection of Cyclospora Cayetanesis. Presented at VII International Workshops on Opportunistic Protists (IWOP), Cincinnati, OH, June 13-16, 2001.
Abstract: Cyclospora cayetanensis, a coccidian parasite with a direct fecal-oral life cycle, has become recognized world-wide as an emerging pathogen in both immunocompromised and immunocompetent individuals. Clinical manifestations include prolonged diarrhea, nausea, abdominal cramps, anorexia, weight loss and other symptoms of gastroenteritis. While most cases of infection with C. cayetanensis have been associated with food-borne transmission, contamination of water has also been observd. Until now, there has been no standardized protocol for the detection of C. cayetanensis oocysts in the environment.
We report for the first time the development and application of a real-time, quantitative PCR assay for the detection of C. cayetanensis in a variety of human clinical speciments as well as in water concentrates seeded with C. cayetanensis oocysts. The specificity of this oligonucleotide primer set and probe is derived from the genetic variability between the rDNA of C. cayetanensis and other organisms. The real-time PCR assay has been optimized specifically to detect a single C. cayetanensis oocyst. This molecular technique can be easily adapted to detect this parasite from a wide variety of food, clinical and environmental samples, as it is robust, reliable, and sensitive.

PRESENTATION Wet Demonstration Workshop on the Identification of Cryptosporidium, Microsporidia, and Giardia in Drinking Water 06/13/2001
Schaefer III, F W. AND H.D A. Lindquist. Wet Demonstration Workshop on the Identification of Cryptosporidium, Microsporidia, and Giardia in Drinking Water. Presented at VII International Workshop on Opportunistic Protists, Cincinnati, OH, June 13, 2001.
Abstract: This method is for determination of the identity and concentration of Cryptosporidium (CAS Registry number 137259-50-8) and Giardia (CAS Registry number 137259-49-5) in untreated surface water and in other waters by filtration, immunomagnetic separation (IMS), and immunofluorescence assay (FA) microscopy. Cryptosporidium and Giardia may be confirmed using 4',6-diamidino-2-phenylindole (DAPI) staining and differential interference contract (D.I.C.) microscopy. This method is designed to meet the survey and monitoring requirements of the U.S. Environmental Protection Agency (EPA). It is based on laboratory testing of recommendations by a panel of experts convened by EPA. The panel was charged with recommending an improved protocol for recovery and detection of protozoa that could be tested and implemented with minimal additional research. This method will not identify the species Cryptosporidium or Giardia or the host species of origin, nor can it determine the viability or infectivity of detected oocysts and cysts. This method is for use only by persons experienced in the determination of Cryptosporidum and Giardia by filtration, IMS, and FA. experienced persons are defined in Section 22.0 at the end of this method as the principal analyst/supervisor, analyst, and technician. Laboratories unfamiliar with analyses of environmental samples by the techniques in this method should gain experience using water filtration techniques, IMS, fluorescent antibody staining with monoclonal antibodies, and microscopic examination of biological particulates using bright-field and D.I.C. microscopy.

PRESENTATION Use of Long Acting Steroid Methylprednisolone Acetate for the Prolongation of Cryptosporidium Sp. in Mice 06/13/2001
Miller, T. A. AND F W. Schaefer III. Use of Long Acting Steroid Methylprednisolone Acetate for the Prolongation of Cryptosporidium Sp. in Mice. Presented at VII International Workshops on Opportunistic Protists (IWOP), Cincinnati, OH, June 13-16, 2001.
Abstract: Current protocols for Cryptosporidium sp. propagation in mice vary according to the strain and age of mouse as well as the species of Cryptosporidium. Cryptosporidium muris is a natural parasite of mice, but only neonatal mice are susceptible to C. parvum infection. Published C. parvum protocols for adult mice usually involve the use of the water soluble form of dexamethazone, administered daily or every other day, either ad libitum in the drinking water or by intra peritoneal injection. Uniformity of doses a problem with any water bottle delivery system. Also, excessive animal handling and labor costs are significant with daily injections.
In an attempt to decrease handling problems and dosing variations a single injection method was developed. A single injection of Methylprednisolone Acetate (DepoMedrol - Pharmacia-UpJohn) is sufficient to allow a Cryptosporidium parvum infection in adult CF-1 mice. The effective dose appears to increase with the age and weight of the mouse. In mice naturally infected with Cryptosporidium muris a single dose prior to infection will prolong the shedding of oocysts. A single dose after the prepatient period will radically incrase the number of oocysts produced per mouse as well as prolong the shedding interval.

PRESENTATION Real-Time Pcr Detection of Three Human-Pathogenic Species from the Microsporidial Genus Encephalitozoon 06/13/2001
Hester, J. D., M Varma, A. M. Bobst, M W. Ware, H.D A. Lindquist, AND F W. Schaefer III. Real-Time Pcr Detection of Three Human-Pathogenic Species from the Microsporidial Genus Encephalitozoon. Presented at VII International Workshops on Opportunistic Protists (IWOP), Cincinnati, OH, June 13-16, 2001.
Abstract: Three microsporidial species from the genus Encephalitozoon, E. hellem, E. cuniculi and E. intestinalis, have emerged as important opportunistic pathogens of humans affecting organ transplant recipients, AIDS patients, and other immunocompromised patients. Even though these three microsporidial species have the potential to be waterborne via body secretions (i.e., feces, urine, or respiratory secretions), little is known about the occurrence of these protozoan parasites in water matrices. Methods capable of detecting these microsporidial species in surface waters have been severely limited because of complex matrices and low organism density.
We report here the development of a 5'-nuclease assay for the quantitative detection of E. hellem, E. cuniculi, and E. intestinalis. Species-specific oligonucleotide primer sets and a genus-specific, fluorescent-labeled Taqman probe were designed to target the rDNA gene for all three Encephalitozoon species. DNA isolation from microsporidial spores that were quantitated using both flow cytometric and hemacytometer counts permitted an extremely accurate assessment of this assay's level of sensitivity. After optimization of the assay conditions, we were able to specifically detect a single infectious spore as well as obtain a standard curve with a linear range across 5 logs of microsporidial spore counts for all three human-pathogenic microsporidia in surface waters and may serve as a sensitive and specific diagnostic tool in clinical analysis.

PRESENTATION Analytical Method Development for Alachlor Esa and Other Acetanilide Herbicide Degradation Products 05/27/2001
Shoemaker, J A. Analytical Method Development for Alachlor Esa and Other Acetanilide Herbicide Degradation Products. Presented at 49th ASMS Conference on Mass Spectrometry and Allied Topics, Chicago, IL, May 27-31, 2001.
Abstract: In 1998, USEPA published a Drinking Water Contaminant Candidate List (CCL) of 50 chemicals and 10 microorganisms. "Alachlor ESA and other acetanilide herbicide degradation products" is listed on the the 1998 CCL. Acetanilide degradation products are generally more water soluble and mobile, thus there is greater potential for these degradates to be found in groundwater and surface water. Ethanessulfonic acid (ESA) and oxanilic acid (OA) degradates of the parent herbicides have been reported in US Midwestern surface and ground waters in concentrations as high as 20 micrograms/L. Before a regulatory determination can be made concerning these chemicals, EPA must assess the extent of their occurrence in public water systems. Thus, a reliable EPA occurrence method is needed.

PRESENTATION An Alternative Eluent to Beef Extract for Eluting Poliovirus from Electropositive Filters 05/22/2001
Cashdollar, J L., D R. Dahling, AND G S. Fout. An Alternative Eluent to Beef Extract for Eluting Poliovirus from Electropositive Filters. Presented at 101st General Meeting of the American Society for Microbiology, Orlando, FL, May 20-24, 2001.
Abstract: Traditional methods for enteric virus removal from waters involve filtering the water through a positively charged filter followed by elution with beef extract, second step concentration by flocculation, and assay in cell culture. Two of the problems associated with this method are closely related. First, many enteric viruses are non-culturable, and thus are not detected by traditional cell culture methods. Alternative molecular procedures such as those based upon the polymerase chain reaction (PCR) are often used to detect these viruses. Second, it has been reported that beef extract may directly inhibit and concentrate inhibitors of the molecular procedures. This study describes the development of a method which substitutes amino acides for beef extract to elute poliovirus from 1MDS electropositive filters and the subsequent analysis by cell culture and RT-PCR. Initial experiments examined the ability of several different amino acides to elute poliovirus from 47 mm electropositive filters. From there, a concentratoin technique was developed using aluminum sulfate. To test the procedure, 20 L volumes of Ohio River water were seeded with poliovirus and eluted by beef extract, a single amino acid, or a combination of amino acids. Results based upon the plaque assay procedures indicate that alanine, serine, and aminobenzoic acid can elute virus, although poliovirus does not enumerate to the same levels in aminobenzoic acid as beef extract. Poliovirus was also detected in samples eluted by both beef extract and amino acids. In addition, we have been unable to detect any inhibitory RT-PCR reactions with beef extract. Further research is necessary to study viral enumeratoin in aminobenzoic acid and to evaluate the method using larger sample volumes and other enteric viruses.

PRESENTATION A Pilot Study to Compare Microbial and Chemical Indicators of Human Fecal Contamination in Water 05/20/2001
de la Cruz, A A., J W. Messer, C C. Rankin, AND G N. Stelma Jr. A Pilot Study to Compare Microbial and Chemical Indicators of Human Fecal Contamination in Water. Presented at American Society for Microbiology 101st General Meeting, Orlando, FL, May 20-24, 2001.
Abstract: Limitations exist in applying traditional microbial methods for the detection of human fecal contamination of water. A pilot study was undertaken to compare the microbial and chemical indicators of human fecal contamination of water. Sixty-four water samples were collected in Ohio in June-August 2000. Solid-phased extraction (SPE) was performed to preconcentrate cafffeine and urobilin using 100-200 ml of each water samples followed by high-performance liquid chromatography (HPLC) with photodiode array and fluorescence detection methods to simultaneously detect caffeine and urobilin. Enterococci, total coliform (TC), and Escherichia coli (E. coli) were performed on each sample using standard procedures. Caffeine was detected in 33% (21/64) of samples and urobilin in 5% (3/64) of samples. Caffeine level and the presence of E. coli showed strong correlation (r=0.98), whereas enterococci and total coliform levels were poorly correlated with caffeine (r=.022 and -0.01, respectively). This study demonstrated a need for further investigation of the feasibility of using caffeine, a chemical by-product of human activities, as an alternate marker of human fecal contamination in water.

PRESENTATION The Effect of Temperature on the Growth of Mycobacterium Avium Complex (Mac) Organisms 05/20/2001
Covert, T C., A. L. Reyes, M R. Rodgers, AND A P. Dufour. The Effect of Temperature on the Growth of Mycobacterium Avium Complex (Mac) Organisms. Presented at American Society of Microbiology 101st General Meeting, Orlando, FL, May 20-24, 2001.
Abstract: MAC organisms are able to grow, persist, and colonize in water distribution systems and may amplify in hospital hot water systems. This study examined the response of MAC organisms (M. avium, M. intracellulare, and MX) to a range of temperatures commonly associated with drinking water and hot water distribution systems. Clinical and environmental isolates (n=30) in 7H9 broth were exposed to temperatures at 2 degree increments between 13 degrees C and 65 degrees C for two weeks. Relative growth rates, minimum, maximum, and optimum growth temperatures for all MAC organisms ranged from 28 degrees C to 38.5 degrees C. M. avium strains showed a mean optimum growth at 34.5 degrees C whereas M. intracellulare and MX grew best at mean temperatures of 31.5 degrees C and 30 degrees C, respectively. Ninety per cent of M. avium isolates were capable of survival at temperatures of 49 degrees C or greater, the temperature used in many institutional hot water systems. Only 10% of the M. intracellulare and MX isolates were capable of survival at 49 degrees C or greater. Growth temperature profiles for MAC organisms indicate that most M. avium strains may survive in hot water plumbing systems whereas the majority of M. intracellulare and MX strains may not survive temperatures greater than 49 degrees C. The optimum growth temperature of MX differed significantly from M. avium but not from M. intracellulare.

PRESENTATION Recovery of Helicobacter Pylori from Water By Immunomagnetic Capture 05/20/2001
McDaniels, A E. AND S G. Lynn. Recovery of Helicobacter Pylori from Water By Immunomagnetic Capture. Presented at American Society for Microbiology, Orlando, FL, May 20-24, 2001.
Abstract: A few reports have been written stating that H. pylori can be found in waters. However, detection and identification of H. pylori from water samples remains a very difficult task. One method that seems to work successfully is immunomagnetic capture. Water samples were concentrated to low volumes without significant loss of the bacteria by stirred cells. This was followed by an indirect method of capture in which monoclonal mouse anti-H. pylori antibodies were added to H. pylori cells and incubated at 4 degrees C for approximately 18 h with gentle mixing. Goat anti-mouse IgG coated superparamagnetic beads were then added and mixed at 4 degrees C for 60 min. The bead-cell-antibody complex was separated from the supernatant by a magnet. The supernatant was removed and the bead complex gently rinsed. The bead complex and the supernatant were each filtered. A fluorescein isothiocyanate tagged rabbit anti-H. pylori was added to the filters which were incubated for 60 min. at room temperature. Detection and identification were by a solid phase cytometer where the presence of only one cell is counted. Validation of the counts was completed by microscopy. Capture of H. pylori cells ranged between 90% and 96%. Filters containing fluorescent antibody-antigen complexes were scanned by a solid phase cytometer within a 3 min. period. Another 15 min. were required to validate the presence of the bacteria. The immunomagnetic capture method was shown to reliably concentrate low numbers of H. pylori from water samples and to produce high recoveries. Solid phase cytometry is a rapid and efficient method to detect and identify targets labeled with a fluorescent tag that has an excitation wave length of 488nm.

PRESENTATION Use of Taqman to Enumerate Enterococcus Faecalis in Water 05/19/2001
Brown, S, J Santo Domingo, S D. Siefring, AND R A. Haugland. Use of Taqman to Enumerate Enterococcus Faecalis in Water. Presented at American Society of Microbiology, Orlando, FL, May 19-24, 2001.
Abstract: The Polymerase Chain Reaction (PCR) has become a useful tool in the detection of microorganisms. However, conventional PCR is somewhat time-consuming considering that additional steps (e.g., gel electrophoresis and gene sequencing) are required to confirm the presence of the target organism. To circumvent these steps a real time PCR (TaqMan) approach was developed to monitor the presence of Enterococcus faecalis in water samples. The TaqMan primers and probes used in this study align to regions wihtin the 16S rDNA of E. faecalis. When the method was empirically tested against 14 different enterococci species only E. faecalis cells produced cycle threshhold (Ct) values that were significantly different than negative controls. No differences in Ct values were obtained regardless if E. faecalis cells were innoculated in sterile distilled water, in untreated river samples, or in a background of biofilm material from a water distribution system. In contrast, a 100 fold dilution was necessary to detect E. faecalis in human feces. The detection limit was determined to be approximately 60 dfu/ml. The method was successfully used to identify E. faecalis strains from a culture collection of environmental fecal enterococi isolates. Biochemical profiles and 16S rDNA sequencing analysis of presumptive E. faecalis strains confirmed the TaqMan identification. Since no nucleic acid extraction was required, the presence of E. faecalis was detected in less than three hours.

PRESENTATION Detection of Fecal Enterococci Using a Real Time Pcr Method 05/19/2001
Siefring, S D., J Santo Domingo, N Brinkman, AND R A. Haugland. Detection of Fecal Enterococci Using a Real Time Pcr Method. Presented at American Society of Microbiology, Orlando, FL, May 19-24, 2001.
Abstract: In spite of their importance in public health, the detection of fecal enterococci is performed via culturing methods that are time consuming and that are subject to inaccuracies that relate to their culturable status. In order to address these problems, a real time PCR (TaqMan) method was used to monitor the presence of several fecal enterococci species in water samples. Using 16S rDNA sequencing data, three different TaqMan assays were designed to detect Enterococus faecalis, E. faecium, and E. casseliflavus. The specificity of each assay was empirically tested using known cultures. The results from these studies indicated that assay I was specific to E. faecalis, assay II specific to E. facium, E. hirae, E. durans, and E. dispar, while assay III was specific to E. casseliflavus and E. gallinarum. These assays were then used in analyses of environmental isolates to determine the composition of fecal enterococci from a freshwater pond with history of fecal contamination. Isolates obtained during three different months in each case gave signals in only one of the three TaqMan assays, further suggesting the specificity of the assays. TaqMan studies with total biomass harvested from membrane filtration media specific for fecal enterococci indicated the presence of each of the above described enterococci groups in the pond. The relative abundance of the enterococci groups in these samples was different each month suggesting changes in fecal enterococci composition. This result suggests the possible presence of different sources of fecal pollution during each month. The results from this study indicate that these assays could be used to potentially detect the most relevant enterococci species associated with fecal pollution.

PRESENTATION Occurrence of Enteric Viruses in Waters 05/07/2001
Fout, G S. Occurrence of Enteric Viruses in Waters. Presented at Safe Drinking Water Meeting, Saskatoon, Saskatchewan, Canada, May 4-12, 2001.
Abstract: A number of different types of human enteric viruses cause waterborne outbreaks when individuals are exposed to contaminated drinking and recreational waters. Vaccination against poliovirus has virtually eliminated poliomyelitis from the planet, but other members of the enterovirus group to which poliovirus belongs cause numerous diseases, including gastronenteritis, encephalitis, meningitis, myocarditis and perhaps diabetes and chronic fatigue syndrome. Because of these diseases and the uncertainty about their waterborne spread the coxsackie and echovirus members of the enterovirus group have been placed on EPA's Contaminant Candidate List. Hepatitis A and more recently hepatitis E have caused larged 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 first step in establishing the risk of waterborne disease from these viruses is to determine the levels of their occurrence in contaminated waters. Occurrence measurement requires methods to recover, identify and measure the concentration of viruses in affected waters. 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.

To overcome these problems, we developed a rapid multiplex polymerase chain reaction (PCR) method that can detect many of the virus groups known to cause waterborne disease. This method has been effectively used to measure virus levels in surface and drinking waters. The results of these studies will be discussed.

PRESENTATION Occurrence of Heterotrophic Bacteria With Virulence Characteristics in Potable Water 05/01/2001
Smith, B G., D J. Lye, AND J W. Messer. Occurrence of Heterotrophic Bacteria With Virulence Characteristics in Potable Water. Presented at American Society for Microbiology 101st General Meeting, Orlando, FL, May 20-24, 2001.
Abstract: Treated potable water contains a variety of heterotrophic bacteria that survive current treatment processes. There is evidence that these bacteria are not hazardous to the healthy population, however, the possibility exists that some of them may be opportunistic pathogens capable of infecting individuals with impaired immune systems. Our laboratory is interested in possible health risk from naturally occurring levels of heterotrophic bacteria levels when they increase to high levels in water environments. Increases in heterotrophic bacteria sometimes occur at specific point-of-use elements within a distribution system, such as a water line used for hemodialysis. A battery of in vitro tests for putative virulence factors was used to screen 45 isolates from potable water cultured on TSA with sheep blood. Virulence tested included extracelluar enzyme production, cytotoxicity for Hep-2 cells and animal studies using outbred Swiss mice immunocompromised with carrageenan (200 mg/kg) to induce susceptibility to facultative intracellular pathogens. Plate-based assays for extracellular activities such as hemolysis, elastase and collagenase were not good indicators of potential virulence in the animal studies for the isolates tested. Only the present/absence of cytotoxicity correlated well with animal study results.

PRESENTATION Simple Sample Clean Up Procedure and High Performance Liquid Chromatographic Method for the Analysis of Cyanuric Acid in Human Urine 04/01/2001
Cantu, R, O M. Evans, T D. Behymer, J A. Shoemaker, A P. Dufour, AND F K. Kawahara. Simple Sample Clean Up Procedure and High Performance Liquid Chromatographic Method for the Analysis of Cyanuric Acid in Human Urine. Presented at ACS Meeting, San Diego, CA, April 1-5, 2001.
Abstract: Cyanuric acide (CA) is widely used as a chlorine stabilizer in outdoor pools. No simple method exists for CA measurement in the urine of exposed swimmers. The high hydrophilicity of CA makes usage of solid phase sorbents to extract it from urine nearly impossible because of sample breakthrough. However, octadecyl silica cartridges retain most urinary interferences while allowing CA to pass through unretained. Using this simple approach, CA recoveries of 89-112% were obtained using isocratic high performance liquid chromatographic conditions (graphitic carbon column, 95% phosphate buffer: 5% methanol eluent, and UV detection at 213 nm). The resulting method offers effective CA separation from urinary interferences including creatinine (identified by electrospray ionization mass spectrometry), and detectability of 0.02 mg CA per liter of urine. The acidic nature of CA permits its preservation in urine for 3 weeks through the addition of 1% perchloric acid and 0.1% metaphosphoric acid.

PRESENTATION Low Pathogenic Potential in Heterotrophic Bacteria from Potable Water 03/29/2001
Stelma Jr., G N., J W. Messer, D J. Lye, AND B G. Smith. Low Pathogenic Potential in Heterotrophic Bacteria from Potable Water. Presented at Annual Meeting of the Water Quality Association, Orlando, FL, March 29, 2001.
Abstract: Forty-five isolates of HPC bacteria, most of which express virulence-related characteristics are being tested for pathogenicity in immunocompromised mice. All forty-five were negative for facultative intracellular pathogenicity. All twenty-three isolates tested thus far were also negative for extracellular pathogenicity.

PRESENTATION NERL Microbial Program With Emphasis on Protozoan Methods 02/22/2001
Lindquist, H.D A. NERL Microbial Program With Emphasis on Protozoan Methods. Presented at NCER STAR Drinking Water Progress Review Meeting, Silver Springs, MD, February 22-23, 2001.
Abstract: The National Exposure Research Laboratory's facility in Cincinnati is engated in a variety of microbiological research projects that include studies on bacteria, viruses, fungi and protozoa. One of these was a protzoology project to determine the best way to evaluate methods reported for detecting Cryptosporidium parvum in water. The technology used in preliminary surveys to determine the incidence of this parasite in watersheds has had obvious shortcomings, notably low recovery of organisms and high inherent variability. To improve this technology, a large number of possible alternatives are available. Without a framework to compare these alternative methods, there is no way to select a method or battery of methods that will address basic question of the underlying distribution of C. parvum in the environment. Procedures for evaluating methods have inherent variability and thus, it is difficult to determine if the method, or the procedures for evaluating the method, is the source of variability. To meet this challenge, a set of criteria were developed and tested for use in methods evaluation. One of the primary tools, counting of absolute numbers of organisms by flow cytometry, has enabled more precise method evaluation. An example of an evaluation of four methods using this technique is presented.

PRESENTATION An Investigation of Chemical Stability of Arsenosugars in Extration Solvents Utilized to Quantitatively Extract Arsenicals from Seafood Products Using Ic-ICP-MS and Ic-Esi-MS/MS 02/05/2001
Gallagher, P A., C A. Schwegel, J T. Creed, B. M. Gamble, X. Wei, AND A. Heck. An Investigation of Chemical Stability of Arsenosugars in Extration Solvents Utilized to Quantitatively Extract Arsenicals from Seafood Products Using Ic-ICP-MS and Ic-Esi-MS/MS. Presented at European Winter Conference on Plasma Spectrochemistry, Lillehammer, Norway, February 4-8, 2001.
Abstract: Arsenosugars are commonly associated with seaweed products which have total arsenic concentrations that can exceed 50 ppm on a dry weight basis. Arsenosugars are also present in other seafood products but the associated concentrations are usually considerably lower. The analytical problems generated by the extraction of these sugars from seafood matrices prior to arsenic speciation are two fold. The first problem is that the sugars require an analyte free chromatographic retention window if an ICP-MS is used as the chromatographic detector. For some seafood, the sugars can be extracted, separated, and detected without degradation by-products when relatively mild extraction conditions are utilized. However, due to poor extraction efficiencies in certain seafood products a more chemically aggressive extraction solvent needs to be investigated in order to assure a more quantitative extraction procedure. The second analytical problem is that the need to use a more agressive extraction solvent may produce arsenosugar degradation by-products. These by-products could further complicate the chromatographic separation. Therefore, characterizing the extent to which the arsenosugars degrade under a more aggressive extraction condition is essential to developing an arsenic speciation methodology for seafood. Thus, the goal is to establish a set of extraction conditions which allows for quantitative extraction of arsenicals from seafood and does not produce arsenosugar degradation by-products. This presentation will report on the stability of arsenosugars (purified from kelp xtracts) in acidic extraction environments (below pH 3). The effect of heat and overall extraction time will be investigated. The degradation by-products will be characterized via IC-ESI-MS/MS and their retention times will be reported using ICP-MS detection. Preliminary extraction conditions will be reported which maximize the quantitative nature of the extraction process while minimizing unwant arsenosugar degradation by-products.

PUBLISHED REPORT USEPA Manual of Methods for Virology 06/01/2001
Williams, F P., R E. Stetler, AND R S. Safferman. USEPA Manual of Methods for Virology. U.S. Environmental Protection Agency, Washington, DC, EPA/600/4-84/013 (N16), 2001.
Abstract: This chapter describes procedures for the detection of coliphases in water matrices. These procedures are based on those presented in the Supplement to the 20th Edition of Standard Methods for the Examination of Water and Eastewater and EPA Methods 1601 and 1602. Two quantitative procedures and one qualitative, presence-absence procedures are presented. The procedures can be used, without supplementary methods, to assay small volumes of water (10 mL to 1L). For larger volumes (>100L), large-scale concentration methods such as described in Chapter 14 may be incorporated into the assay scheme. However, as some concentration procedures may result in appreciable loss or inactivation of coliphage, it is recommended that the suitability of any large volume concentration method be evaluated in measured recovery trials before implementation.

PUBLISHED REPORT Total Culturable Virus Quantal Assay 04/01/2001
Fout, G S., D R. Dahling, AND R S. Safferman. Total Culturable Virus Quantal Assay. U.S. Environmental Protection Agency, Washington, DC, EPA/600/4-84/013, 2001.
Abstract: This chapter describes a quantal method for assaying culturable human enteric viruses from water matrices. The assay differs from the plaque assay described in Chapter 10 (December 1987 Revision) in that it is based upon the direct microscopic viewing of cells for virus-induced cytopathic effects. The quantal method can be used to assay viruses concentrated from sewages, effluents and waters using the methods described in Chapters 5, 6 and 14 (February 1999), Dahling and Wright (1986b), Fout et al. (1996) or from sludges and other solids (Chapter 7, September 1989 Revision; Fout, 1999).

PUBLISHED REPORT Concentration and Processing of Waterborne Viruses By Positive Charge 1mds Cartridge Filters and Organic Flocculation 04/01/2001
Fout, G S., D R. Dahling, AND R S. Safferman. Concentration and Processing of Waterborne Viruses By Positive Charge 1mds Cartridge Filters and Organic Flocculation. U.S. Environmental Protection Agency, Washington, DC, EPA/600/4-84/013 (N14), 2001.
Abstract: This chapter describes the most widely used virus adsorption-elution (VIRADEL) method for recovering human enteric viruses from water matrices (Fout et al., 1996). The method takes advantage of postively charged cartridge filters to concentrate viruses from water. The major advantage of methods that use these filters over those described in Chapters 5 and 6 that use negatively charged filters is that additives are not normally needed to achieve virus adsorption to filters.

 

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